Ion-Regulatory Proteins in Neuronal Development and Communication Eva Ruusuvuori Department of Biological and Environmental Sciences Faculty of Biosciences University of Helsinki and Finnish Graduate School of Neuroscience Academic Dissertation To be presented for public examination with the permission of the Faculty of Biosciences, University of Helsinki, in the lecture hall 1041 of Biocenter 2, Viikinkaari 5, Helsinki, on the 28 th of November, 2008 at 12 o’clock noon Helsinki 2008
Transcript
Ion-Regulatory Proteins in NeuronalDevelopment and Communication
Eva Ruusuvuori
Department of Biological and Environmental SciencesFaculty of BiosciencesUniversity of Helsinki
and
Finnish Graduate School of Neuroscience
Academic Dissertation
To be presented for public examination with the permission of the Faculty ofBiosciences, University of Helsinki, in the lecture hall 1041 of Biocenter 2,Viikinkaari 5, Helsinki, on the 28th of November, 2008 at 12 o’clock noon
Helsinki 2008
ii
Supervised byProfessor Kai KailaDepartment of Biological and Environmental SciencesFaculty of BiosciencesUniversity of Helsinki, Finland
and
Professor Juha VoipioDepartment of Biological and Environmental SciencesFaculty of BiosciencesUniversity of Helsinki, Finland
Reviewed byProfessor Joachim W. DeitmerDivision of General ZoologyUniversity of Kaiserslautern, Germany
and
Docent Irma HolopainenDepartment of Pharmacology, Drug Development and TherapeuticsInstitute of BiomedicineUniversity of Turku, Finland
OpponentProfessor Mitchell CheslerDepartment of Physiology and Neuroscience and Department of NeurosurgeryNew York University School of Medicine, USA
Cover picture by Aaro Pallasmaa.
ISBN 978-952-10-5091-6 (paperback)ISBN 978-952-10-5121-0 (pdf, http://ethesis.helsinki.fi)ISSN 1795-7079
Contents .................................................................................................................. iiiList of original publications .....................................................................................vAbbreviations ......................................................................................................... vi1 Summary.............................................................................................................. 12 Review of the literature ....................................................................................... 2
2.1. Ion levels in the brain...................................................................................... 22.1.1 Intra- and extracellular compartments of the brain..................................... 2
2.1.2 Basic mechanisms of cellular ion regulation.............................................. 2
2.1.2.1 Thermodynamics of transmembrane ion distribution....................... 22.1.2.2 Regulation of steady state concentrations: transport mechanisms .... 42.1.2.3 Thermodynamics of secondary active, electroneutral transport ....... 42.1.2.4 Buffering ........................................................................................ 52.1.2.5 Intracellular pH .............................................................................. 6
2.1.3 Ionic homeostasis of brain extracellular fluids........................................... 6
2.1.4 Ionic basis of GABAA receptor-mediated synaptic inhibition .................... 8
2.2 Molecular mechanisms of proton and anion regulation in the brain .................. 82.2.1 Members of cation-chloride cotransporter gene family .............................10
2.2.3.1 Intracellular carbonic anhydrases and their expression inhippocampus.............................................................................................162.2.3.2 Extracellular carbonic anhydrases and their expression inhippocampus.............................................................................................16
2.3 Developmental changes in ion regulation ........................................................172.3.1 Developmental changes in neuronal chloride regulation ...........................17
2.3.2 Developmental changes in neuronal pH regulation ...................................18
2.3.2.1 Changes in acid-base transporter expression and function..............182.3.2.2 Changes in intracellular buffering..................................................19
2.3.3 Developmental changes in extracellular ion homeostasis..........................20
2.4 Functional significance of ion regulation.........................................................212.4.1 Neuronal ion regulation modulates GABAA receptor-mediated signaling..21
2.4.1.1 GABAA receptor-mediated transmission in developing hippocampalneurons .....................................................................................................212.4.1.2 Depolarizing GABAergic transmission in the mature hippocampus.................................................................................................................22
2.4.2 Neuronal excitability is modulated by pH.................................................23
iv
2.4.2.1 Activity-induced pH transients in neurons and in the extracellularspace.........................................................................................................252.4.2.2 CA activity facilitates transmembrane ion fluxes ...........................26
2.4.3 Studies on mouse models with genetically impaired chloride and pH
2.5 Ion measurements in the mammalian brain .....................................................282.5.1 Ion-sensitive microelectrodes ...................................................................28
2.5.2.1 Sensitivity and ratiometric quantitation of the indicator .................292.5.2.2 Fluorescent indicators within the cell.............................................30
3 Aims of the study .................................................................................................324 Experimental procedures ...................................................................................335 Results and discussion .........................................................................................36
5.1 The K+-Cl- cotransporter KCC2 renders GABA hyperpolarizing duringneuronal maturation (I) .........................................................................................365.2. CA isoform VII acts as a molecular switch in the development of synchronousgamma-frequency firing of hippocampal CA1 pyramidal cells (II)........................385.3. Mice with targeted Slc4a10 gene disruption have small brain ventricles andshow reduced neuronal excitability (III)................................................................41
This Thesis is based on the following publications, referred to in the text by theirRoman numerals.
I. Rivera C, Voipio J, Payne JA, 1Ruusuvuori E, Lahtinen H, Lamsa K, PirvolaU, Saarma M, Kaila K.(1999) The K+-Cl- co-transporter KCC2 renders GABAhyperpolarizing during neuronal maturation. Nature 397:251–255.
II. 2Ruusuvuori E, Li H, Huttu K, Palva JM, Smirnov S, Rivera C, Kaila K,Voipio J. (2004) Carbonic anhydrase isoform VII acts as a molecular switch inthe development of synchronous gamma-frequency firing of hippocampalCA1 pyramidal cells. J Neurosci 24:2699-26707.
III. Jacobs S*, 3Ruusuvuori E*, Sipilä ST*, Haapanen A, Damkier HH, Kurth I,Hentschke M, Schweizer M, Rudhard Y, Laatikainen L, Tyynelä J, PraetoriusJ, Voipio J, Hübner CA. (2008) Mice with targeted Slc4a10 gene disruptionhave small brain ventricles and show reduced neuronal excitability. Proc NatlAcad Sci U S A. 105:311-316.*These authors contributed equally to this work.
1 The author contributed to designing the research, performing intracellular Cl- measurementson hippocampal slices, analyzing data, and writing the manuscript.2 The author designed and performed all intracellular pH measurements from isolated
pyramidal neurons and hippocampal slices, designed and performed extracellular fieldpotential measurements, extracellular potassium measurements and intracellular sharpelectrode recordings. The author organized and wrote the manuscript with K.K with inputfrom co-authors.3 The author performed intracellular pH measurement from hippocampal pyramidal neuronsand analyzed the data, was responsible for the breeding of the knockout animals, andperformed and analyzed field potential measurements. The author organized and wrote themanuscript with S.T.S., J.P., J.V. and C.A.H.
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Abbreviations
AE anion exchangerAIS axon initial segmentAMPA -amino-3-hydroxy-5-methylisoxazole-4-propionic acid4-AP 4-aminopyridineASIC acid sensing ion channelATP adenosine triphosphateAQP-1 aquaporin-1 water channelBA benzolamideBBB blood-brain barrierBCECF 2’,7’-bis(carboxyethyl)-5(6)-carboxyfluoresceinBT bicarbonate transporterCA1-3 cornu ammonis areas 1-3 of hippocampusCAI-XV carbonic anhydrase isoforms I-XVCARP carbonic anhydrase related proteinCCC cation-chloride cotransporterCNS central nervous systemCSF cerebrospinal fluidDIA depolarization-induced alkalinizationDIV days in vitroDRG dorsal-root ganglionE embryonic dayECl equilibrium potential of Cl-
Brain function is critically dependent on the ionic homeostasis in both the extra- andintracellular compartment. The regulation of brain extracellular ionic compositionmainly relies on active transport at blood–brain and at blood–cerebrospinal fluidinterfaces whereas intracellular ion regulation is based on plasmalemmal transportersof neurons and glia. In addition, the latter mechanisms can generate physiologically aswell as pathophysiologically significant extracellular ion transients. In this work Ihave studied molecular mechanisms and development of ion regulation and how thesefactors alter neuronal excitability and affect synaptic and non-synaptic transmissionwith a particular emphasis on intracellular pH and chloride (Cl-) regulation.
Why is the regulation of acid-base equivalents (H+ and HCO3-) and Cl- of such
interest and importance? First of all, GABAA-receptors are permeable to both HCO3-
and Cl-. In the adult mammalian central nervous system (CNS) fast postsynapticinhibition relies on GABAA-receptor mediated transmission. Today, excitatory effectsof GABAA-receptors, both in mature neurons and during the early development, havebeen recognized and the significance of the “dual actions” of GABA on neuronalcommunication has become an interesting field of research. The transmembranegradients of Cl- and HCO3
- determine the reversal potential of GABAA-receptormediated postsynaptic potentials and hence, the function of pH and Cl- regulatoryproteins have profound consequences on GABAergic signaling and neuronalexcitability. Secondly, perturbations in pH can cause a variety of changes in cellularfunction, many of them resulting from the interaction of protons with ionizable sidechains of proteins. pH-mediated alterations of protein conformation in e.g. ionchannels, transporters, and enzymes can powerfully modulate neurotransmission. Inthe context of pH homeostasis, the enzyme carbonic anhydrase (CA) needs to betaken into account in parallel with ion transporters: for CO2/HCO3
- buffering to act ina fast manner, CO2 (de)hydration must be catalyzed by this enzyme. The acid-baseequivalents that serve as substrates in the CO2 dehydration-hydration reaction are alsoengaged in many carrier and channel mediated ion movements. In such processes, CAactivity is in key position to modulate transmembrane solute fluxes and theirconsequences.
The bicarbonate transporters (BTs; SLC4) and the electroneutral cation-chloridecotransporters (CCCs; SLC12) belong the to large gene family of solute carriers(SLCs). In my work I have studied the physiological roles of the K+-Cl- cotransporterKCC2 (Slc12a5) and the Na+-driven Cl--HCO3
- exchanger NCBE (Slc4a10) and theroles of these two ion transporters in the modualtion of neuronal communication andexcitability in the rodent hippocampus. I have also examined the cellular localizationand molecular basis of intracellular CA that has been shown to be essential for thegeneration of prolonged GABAergic excitation in the mature hippocampus.
The results in my Thesis provide direct evidence for the view that the postnatal up-regulation of KCC2 accounts for the developmental shift from depolarizing tohyperpolarizing postsynaptic EGABA-A responses in rat hippocampal pyramidalneurons. The results also indicate that after KCC2 expression the developmental onsetof excitatory GABAergic transmission upon intense GABAA-receptor stimulationdepend on the expression of intrapyramidal CA, identified as the CA isoform VII.Studies on mice with targeted Slc4a10 gene disruption revealed an important role forNCBE in neuronal pH regulation and in pH-dependent modulation of neuronalexcitability. Furthermore, this ion transporter is involved in the basolateral Na+ andHCO3
- uptake in choroid plexus epithelial cells, and is thus likely to contribute tocerebrospinal fluid production.
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2 Review of theliterature
2.1. Ion levels in the brain
2.1.1 Intra- and extracellularcompartments of the brain
Mammalian brain tissue is composedof two types of cells, neurons and glia.Neurons, which are highly specializedfor electrical signal transmission, aresupported both structurally andfunctionally by glial cells. Mostneurons in cortical structures can befurther classified on the basis of theirsynaptic transmitters e.g. inglutamatergic principal neurons and inGABAergic interneurons. The twomain types of CNS glial cells areoligodendrocytes and astrocytes. Theformer are responsible for axonalmyelinization and the latter contributeto the maintenance of a chemicalenvironment suitable for neuronalsignalling. Together neurons and glialcells form the physiologically relevantintracellular compartment of the brain(see Table 1 for intraneuronal ionlevels). The densely packed cells areseparated from each other by theextracellular space. The distancebetween brain cells varies but onaverage it is estimated to be no morethan 20 nm (see Nicholson, 2001).Despite these tiny dimensions, thisextracellular space constitutes roughly20 % of the volume of brain tissue. Inaddition, ~10 % of the total brainvolume is taken by the space of brainventricles and subdural space. Thesecompartments are filled with anaqueous solution; brain cells are bathedin the extracellular fluid (ECF) whilethe cerebrospinal fluid (CSF) fills theventricular system and covers theexternal surfaces of the brain. Theependymal lining of cerebral ventricles(and of pia mater) permits a relativelyfree diffusion of ions between thesetwo compartments. There is also aconstant, but slow bulk flow from ECFto CSF, suggesting that thecomposition of these two fluids is
largely similar. Samples taken fromCSF have shown that, in comparison toblood plasma, CSF is slightlyhypertonic and contains less proteins,glucose and amino acids. Also theionic composition differs from that ofplasma (Table 1).The intracellular compartment isseparated from the extracellular spaceby a thin lipid bilayer, the plasmamembrane. The tight regulation ofcytoplasmic inorganic cations, such assodium (Na+), potassium (K+), calcium(Ca2+), and protons (H+), and anionschloride (Cl-) and bicarbonate (HCO3
-),mainly relies on a variety ofplasmalemmal ion transporters.Organellar compartmentalization ofions e.g. into the endoplasmicreticulum, via organellar transporters,further contributes to the regulation ofthe cytoplasmic ion levels.On larger scale, compartments withdifferent ionic milieus are, in a similarmanner, separated from each other bycell membranes. The blood-brainbarrier (BBB), formed of brainendothelial cells lining the cerebralvasculature, protects the mammalianbrain from fluctuations in bloodplasma composition. Two additionalselective barriers are formed betweenblood and CSF by the choroid plexusepithelium (between blood andventricular CSF) and by the arachnoidepithelium (between blood andsubarachnoid CSF) (see Abbott et al.,2006).
2.1.2 Basic mechanisms ofcellular ion regulation
2.1.2.1 Thermodynamics oftransmembrane ion distribution
The transmembrane distribution of anion species is influenced by two forcesacting across cell membrane: (1) Theconcentration gradient creates achemical driving force, (2) since ionscarry electric charge, an electricaldriving force results from themembrane potential (Vm). When theconcentration gradient of the ion and
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Free ion concentrations in body fluids
Ion (unit) Arterial Plasma CSF CNS neurons
Na+ (mM) 148 152 10
K+ (mM) 5.3 3.4 125Ca2+(mM) 1.5 1.0 0.00006
Mg2+ (mM) 0.44 0.88 0.5H+ (nM) 40 50 80
pH 7.4 7.3 7.1Cl- (mM) 121 132 6.6 (25-40)*
HCO3- (mM) 31 28 18
Table 1. The free ion concentrations in mature rodent arterial plasma, cerebrospinalfluid (CSF), and central nervous system (CNS) neurons. *Reported Cl- concentrationsfor immature CNS neurons is given in brackets. The concentrations shown representtypical values that can be found in references cited in the ‘Review of the literature’.H+ concentration was calculated using pH = -log[H+].
the electrical gradient balance eachother exactly, the ion is atelectrochemical equilibrium and therewill be no net driving force (see below)for conductive fluxes of the ion.Consequently, the ion will exhibit nonet flux in either direction across themembrane. The membrane potential atthe equilibrium is obtained from theNernst equation
i
o
sS S
Sz
TE lnF
R
which defines the relationship betweenthe chemical gradient and theequilibrium potential (Es) for an ion S,with extra- and intracellularconcentrations [S]o and [S]i ,respectively, and a charge zS. R, T, andF are the gas constant, absolutetemperature, and Faraday’s constant,respectively.If Es does not equal the membranepotential, ion S is not atelectrochemical equilibrium and therewill exist an electrochemical driving
force that is commonly quantified asVm-Es. Provided that the membranepotential dependence of a channel-mediated current of ion S (Is) issufficiently linear, IS driven by theelectrochemical driving force may begiven by a modified version of Ohm’slaw
Is = gs(Vm - Es)
where gs is the membrane conductanceof S. The reversal potential (Erev) of achannel-mediated conductive current isdefined as the membrane potential atwhich current is zero and changes itspolarity. Measured Erev values oftendiffer from equilibrium potentials ofions, because of finite selectivity ofconductive pathways.Here it is worth noting that very smallchanges in the transmembrane chargedistribution generate significantchanges in the membrane potential.Therefore, very small net currents arerequired to generate shifts in Vm.However, in reality cells in neuronal
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networks receive overlappinginhibitory- and excitatory inputsresulting in large current componentsin opposite directions mediated bysimultaneous influx of anions throughGABAergic channels and cationsthrough glutamatergic channels. Underthese circumstances the net fluxes ofdifferent ion species exceed thecapacitive current by orders ofmagnitude (Buzsaki et al., 2007).
2.1.2.2 Regulation of steady stateconcentrations: transportmechanisms
Inorganic ions are virtually insoluble inlipid bilayers and must therefore moveacross the cell membrane throughchannels or transporters. Thenomenclature of functionally distincttransporter subtypes reflects thedirection of transported ions as well asthe movement of charges.Cotransporters (also calledsymporters) move two or more ions inthe same direction whereascountertransporters (also calledantiporters or exchangers) move two ormore ions in opposite directions. Whileco- and countertransporters move atleast two different ions, uniporters (=facilitated diffusion carriers) enable themovement of a single substance downits concentration gradient. Electrogenictransport drives a net current across themembrane while transport is said to beelectroneutral if the transport processdoes not produce any current.At steady-state, the passive (“down-hill”) flux of ions in one direction iscounteracted by active transport of ionsin the opposite (”up-hill”) direction.Two types of active transportmechanisms mediate regulation of ionconcentrations. Primary activetransport is fuelled by hydrolysis ofadenosine triphosphate (ATP). Aconsiderable portion, 20-40%, of theenergy turnover in mammalian brain isaccounted for by the activity of asingle type of ATPase pump, namelythe Na+-K+-ATPase (Mellergård andSiesjö, 1998; Attwell and Laughlin,2001). By moving 3 Na+ ions out inexchange to 2 inwardly transported K+
ions the Na+-K+-ATPase creates and
maintains the transmembrane gradientsof Na+ and K+. Other ATPases, likeCa2+-H+-ATPase and H+-ATPase, arealso present in the brain and functionin cytoplasmic control and/ororganellar compartmentalization ofions (Bevensee and Boron, 1998; Roseand Ransom, 1998; Mata andSepulveda, 2005). The energy stored inion gradients created by the primaryactive, ATP-fuelled transporters isused by secondary active transporters.Most often it is the energetically“down-hill” influx of Na+ that is usedto transport other ions against theirelectrochemical gradients. Cellular pHregulation serves as a good examplefor a process that is largely dependenton Na+-coupled ion transport.Transport of acid-base equivalents ismediated by e.g. the Na+-H+
exchanger, the Na+-driven Cl-- HCO3-
exchange and the Na+-HCO3- co-
transporters. The outward gradient ofK+, instead, serves as the driving forcefor Cl- extrusion via K+-Cl-
cotransporters with a 1:1stoichiometry. Secondary activetransporters can also couple the energyderived from an electrochemicalgradient of an ion to the transport ofe.g. amino acids, sugars, andnucleotides. The present thesis willfocus on transmembrane movements ofinorganic ions in the brain, with aparticular emphasis on Cl- and HCO3
For an electroneutral ion transporter,the thermodynamic driving force isgiven by the sum of changes in thechemical potential of the transportedions. At equilibrium, the sum of thechemical potential differences is zero,i.e. the free energy change associatedwith the transport proteins is zero.Thus, the net influx and efflux of thetransported ions are equal and, eventhough unidirectional ion fluxes mayexist, there is no net flux of ions. Usingthese principles, it is straightforward toshow that, for instance, a K+-Cl-
cotransporter operating with a 1:1
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stoichiometry is at thermodynamicequilibrium when EK = ECl, i.e. when
[K+]o [Cl-]o = [K+]i [Cl-]i
If such a transporter is constitutivelyactive in the absence of significantconductive leaks, it would operateclose to its thermodynamicequilibrium. Under such conditions,minor changes in the driving force willmarkedly affect the net fluxes, or evenchange their direction.The functional activity of an iontransporter is not dictated only by theprevailing electrochemical gradients astransporters are not necessarily activeeven if they are facing a driving forcethat favours ion transport (Rocha-Gonzalez et al., 2008). The transportercan undergo fast allosteric modulatione.g. by phosphorylation or de-phosphorylation of the transporterprotein. The rate of ion fluxes depends,in addition to the transporter kinetics,on the number of functionaltransporters located on the plasmamembrane. Hence, on a longer time-scale, transport kinetics can bemodulated by altering the trafficking oftransporters to and from the plasmamembrane or by changing theexpression of transport protein genes.
2.1.2.4 Buffering
Buffering is a major determinant of pHchanges when a solution is challengedby an acid or alkaline load. Thechemical buffering power results frombuffer pairs of conjugated weakacid(s)/base(s). These buffer pairs arecapable of reversibly releasing/bindinga proton, and thereby act to minimizeand to slow the rates of pH changes.Hence, buffers determine the ability ofthe solution to resist pH transientswithout contribution of activetransporters. Buffering power ( ) isdefined as
pHpHAcidStrongBaseStrong
where Strong Base (or Acid) is theamount of strong base (or acid) added(in mM) and pH is the resulting
change in pH. The unit of is mM ormmol/l. The total buffering capacity( T) of a cell’s cytoplasm is due both tothe intrinsic buffering capacity of thecell’s cytoplasm ( i) and to thebuffering provided by the extrinsic,CO2/HCO3
- buffer system ( CO2).
T = i + CO2
i mainly arises from the titratableimidazole groups of proteins and fromphosphates. These buffers can notcross the plasma membrane andtherefore they form a closed buffersystem (Burton, 1978; Roos and Boron,1981). The buffering power of theclosed system buffers is maximal whenpH equals pKa. i is the sum of thecontribution of individual intrinsicbuffers and it can be determinedexperimentally by recordingintracellular pH (pHi) changes uponaddition/removal of a strong acid orbase in the absence of CO2/HCO3
-.Buffering provided by the CO2/HCO3
-
buffer system is usually considered asan open buffer system, i.e. the cellularpartial pressure of CO2 (PCO2) ismaintained constant because itequilibrates with the extracellularcompartment that serves as a fixed,infinite source of CO2. However, withinstantaneous acid/base loads, theimmediate pH response is that of aclosed system, since the equilibrationof CO2 is not immediate. In a systemwhich is open with respect to PCO2, the
CO2 is given by
CO2= 2.3[HCO3-]
Because at a fixed PCO2 [HCO3-] rises
with pH, also CO2 increases at higherpH.Unless catalyzed by the enzymecarbonic anhydrase (CA) the hydrationof CO2 is slow, having a time constantof 20-30 seconds at room temperature(Maren, 1967). Thus, the ability of theCO2/HCO3
- -system to efficientlybuffer fast H+ fluxes depends on CAactivity. Ignoring the intermediate stepof carbonic acid, and furtherdissociation to carbonate, both ofwhich are present at lowconcentrations at physiological pH
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levels, the hydration-dehydrationreaction of CO2 is
HHCOOHCO CA322
The fast CA-dependent CO2/HCO3- -
buffer system attenuates pH changesthat originate from net fluxes ofprotons. However, if the hydration-dehydration reaction of CO2 is broughtout of equilibrium by a substance otherthan H+, CA will facilitate rapid shiftsin [H+]. The above reaction results indistinct pH responses in open andclosed buffer systems depending on thetype of the acid or base load as well ason the presence of CA and intrinsicbuffers (Voipio, 1998). The large andconstantly growing number ofidentified members of the CA genefamily is described in chapter 2.2.3.
2.1.2.5 Intracellular pH
Without active regulation of pHi,passive equilibration of protons drivenby the resting membrane potentialwould make pHi significantly lowerthan extracellular pH (pHo). However,neuronal pHi is typically only slightlymore acidic than pHo (pHi = 7.1 vs.pHo= 7.3). Provided that the hydration-dehydration reaction of CO2 and thetransmembrane distribution of CO2 areat equilibrium, the transmembraneHCO3
- distribution is set by the pHgradient
o-
3i-
3 ][HCO10][HCO oi pHpH
Because, under these conditions, theequilibrium potential of protons (EH+,which has the value of -12 mV withthe pH values given above) equalsEHCO3- (see Kaila and Voipio, 1990) theelectrochemical gradient tends to driveH+ ions into, and OH- and HCO3
- outof the cell. As a result, any conductiveleaks of these ionic species impose anacid load on the cell.
2.1.3 Ionic homeostasis of brainextracellular fluids
On the systemic level arterial blood pHis maintained within very narrow limits(7.35-7.45). Respiratory and renalregulatory mechanisms stabilize thearterial pH by excretion or retention ofacid/base equivalents. Abnormalities inmaintaining blood pH result insystemic acid–base balance disorders.A respiratory acidosis results in a fallin pHo that is caused by an increase inPCO2 whereas in metabolic acidosis afall in pHo is caused by a decrease in[HCO3
-]o. Likewise, respiratory ormetabolic alkalosis causes a rise in pHodue to a decrease in PCO2 or to anincrease in HCO3
-, respectively.Arterial PCO2 is the most powerfulstimulus for ventilation, acting throughperipheral and central chemoreceptors(for review see Nattie, 1999).In chronic acid-base disturbances pHois maintained as close to normal aspossible by compensatory changes inventilation (in response to metabolicdisorders) and in secretorymechanisms of kidneys (in response torespiratory disorders). The iontransporters of brain barriers areresponsible for the short-termCSF/ECF pH normalization, in case ofan acute change in PCO2. Combinedmeasurements of CSF and plasma pH,PCO2 and [HCO3
-] have shown that anacute elevation of PCO2 is paralleled bya pHo decrease (Messeter and Siesjö,1971; Pavlin and Hornbein, 1975;Nattie and Edwards, 1981). With time,the pHo was restored close to normalby the ion transporters located inchoroid plexus and BBB epithelia(Messeter and Siesjö, 1971). Thesetransient CSF/ECF pHo changes arereflected in pHi.A relevant question is how wellneurons can maintain their pHi whenfaced with extracellular acid-basedisturbances. Sustained pHo changesmimicking respiratory and metabolicacid-base disturbances resulted inpersistent pHi changes in non-chemosensitive neurons both in vivo(Katsura et al., 1994) and in vitro(Bouyer et al., 2004). These findingschallenge the proposal that during the
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pHo manipulations described above, asustained change in pHi is specific forchemosensitive neurons (Ritucci et al.,1997; Ritucci et al., 1998).
2.1.3.1 Cerebrospinal fluid
The tight junctions between thechoroid plexus epithelial cells form adiffusion barrier between blood andventricular CSF (Zlokovic, 2008). Theepithelial cells of the choroid plexusare remarkably efficient secretory cellsthat express a variety of solute carriers(Bouzinova et al., 2005; Praetorius andNielsen, 2006; Praetorius, 2007). Withthe exception of aquaporin-1 waterchannels (AQP1), the transportproteins have a highly polarizedexpression pattern either on theluminal (ventricular) or on thebasolateral membrane. The ioniccomposition of the secreted CSF is setby the function of these transporters.The production rate of CSF is criticallydependent on the rate of Na+ secretion.The high luminal extrusion of Na+ ismainly due to the Na+-K+-ATPasewhereas the question what is the mainmechanism of basolateral Na+ uptakeremained to be identified untilrecently. Due to their basolaterallocalization, the Na+-proton exchanger(NHE) and Na+-driven HCO3
-
transporters (NCBE and theelectroneutral Na+-bicarbonatecotransporter NBCn1) have beenconsidered as possible contributors inthe net transport of Na+ that drives thesecretion of CSF (Bouzinova et al.,2005; Praetorius, 2007). Theinvolvement of NCBE in basolateralsolute uptake was examined in StudyIII.
2.1.3.2 Extracellular fluid
While CSF provides the macro-environment for the brain and acts bothas a fluid cushion and as a drainageroute for solutes (Davson and Segal,1969), the ECF fills the extracellularspace in the immediate vicinity of thebrain cells. The brain capillaryendothelial cells are involved insecretory mechanisms producing fluidacross the BBB into the brain
interstitial space. Because of the bulkflow of ECF from the endothelialsecretory site to the ventricular system,ECF is estimated to make acontribution of one third to the CSFproduction (Cserr, 1974; Abbott,2004). The tight junctions between theneighbouring endothelial cells form a‘physical barrier’ that preventsparacellular movement of mostmolecules. Instead, moleculartrafficking across the BBB is carriedout by active transport systems orchannel-mediated passive diffusion viathe transcellular route. An exception ismade by the gaseous molecules (likeO2 and CO2) and small lipophilicagents that can freely diffuse throughthe epithelial plasma membranes. Theelectrical potential difference of a fewmillivolts that prevails between theCSF/ECF and blood is sensitive tochanges in PCO2 and pH (Woody et al.,1970; Voipio et al., 2003). Thissuggests that the transporters involvedin the generation of thetransendothelial potential differencehave a marked sensitivity to pH and/orthat the acid-base transporters make aconsiderable contribution to thetransendothelial transport.Even though under normalcircumstances the regulatorymechanisms of BBB (together with theblood-CSF barrier) maintain the globalion homeostasis of brain extracellularfluids, local fluctuations in ECF ionconcentrations occur as a result ofneuronal activity (Somjen, 2002).Extracellular ion concentrations arerestored in co-operation withextracellular buffering and diffusion,and by transmembrane movements ofions. Astrocytes make a significantcontribution to extracellular ionregulation. These glial cells have beenshown to mediate both spatialbuffering/siphoning and net uptake ofK+ (Newman, 1996; Kofuji andNewman, 2004), and a role in pHoregulation has been suggested(Deitmer and Rose, 1996).Under physiological conditions,neuronal activity elicits only modestchanges in the ionic composition ofECF (Sykova et al., 1974; Singer andLux, 1975). The available data mightpartially reflect the limitations of the
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methods established so far. Hence, it ispossible that transients with higheramplitude take place within spatiallyrestricted microdomains in theextracellular space, allowing e.g. Ca2+
and protons to serve secondmessenger-like actions (Chesler andKaila, 1992). Under pathologicalconditions, ion fluctuations can belarge. For example, marked K+
(Heinemann and Louvel, 1983;McNamara, 1994; Avoli, 1996) andproton (Urbanics et al., 1978;Kraig etal., 1983; Somjen, 1984; Silver andErecinska, 1992) transients have beenmeasured in vivo during seizures orafter direct electrical stimulation. Theion transients evoked in manyexperiments performed in vitro arepronounced and can even exceed thoseseen in vivo under pathologicalconditions (Kaila and Chesler, 1998;Somjen, 2001; Avoli et al., 2005), butthey provide a useful approach instudies of the molecular and cellularmechanisms underlying ion transientsand ion-based signalling in the brain.
2.1.4 Ionic basis of GABAAreceptor-mediated synapticinhibition
The special importance of neuronal Cl-
regulation in neurotransmission arisesfrom the fact that GABAA (andglycine) receptor channels arepermeable to Cl-. Together withglycine, GABA is the mainneurotransmitter responsible forsynaptic inhibition in the CNS.Hyperpolarizing inhibition mediatedby ionotropic GABA receptors inmature neurons is based onplasmalemmal transporters that extrudeCl- and thereby maintain an ECl morenegative than the resting Vm (Eccles,1966; Deisz and Lux, 1982). Thiscreates a driving force for an inwardCl- flux, i.e. an outward current, whichaccounts for conventionalhyperpolarizing postsynapticinhibition. The GABAA channel-mediated currents depend also onHCO3
- because GABAA channels arepermeable to both HCO3
- and Cl-, witha HCO3
-:Cl- permeability ratio of 0.2-
0.4 (Kaila and Voipio, 1987; Bormannet al., 1987; Kaila et al., 1993). In acell with a typical negative restingmembrane potential around -60 mVand an EHCO3- close to -12 mV there isa deep electrochemical gradientfavoring HCO3
- efflux. The HCO3-
efflux creates an inwardly directed,depolarizing current, which in Cl-
extruding cells where the intracellularCl- concentrations is kept low, canmake a significant contribution to thenet GABAergic current. This HCO3
-
current keeps EGABA-A more positivethan ECl and in some cases can evenlead to a HCO3
- -dependentdepolarization (Kaila et al., 1989b;Kaila et al., 1993; Gulledge and Stuart,2003).In addition to the GABA mediatedeffect on the membrane potential, theinput conductance of a cell increasessignificantly upon GABAA receptorchannel activation. The consequentlocal decrease in the membrane timeand space constant efficientlysuppresses changes in Vm generated bysimultaneous excitatory currents(Staley and Mody, 1992). Thisshunting inhibition is effective even atslightly depolarizing GABAA channel-mediated potentials seen e.g. in adultrat dentate granule cells (Staley andMody, 1992) and neocortical neurons(Kaila et al., 1993).
2.2 Molecular mechanismsof proton and anionregulation in the brainThe Human genome organisationnomenclature committee databaseprovides a list of transporter familiesof the SLC gene series, whichcurrently covers 43 families, most ofwhich have several transportersubtypes (Hediger et al., 2004). TheSLC series includes genes encodingpassive and coupled ion transporterslocated both on cell and organellarmembranes. The members of eachSLC family share at least 20–25%amino acid sequence identity betweeneach other.
9
Identified members of the Slc4 and Slc12 gene families in mice
Table 2. The identified members of the bicarbonate transporter (Slc4) and cation-chloride cotransporter (Slc12) gene families. The expression of the Slc4and Slc12proteins and their known splice variants (given in brackets after the protein name) inthe rodent central nervous system. The data in the table are from Gamba et al., 2004and Romero et al., 2004.
10
2.2.1 Members of cation-chloride cotransporter genefamily
Members of the CCC family aresecondary active, electroneutraltransporters that are largely responsiblefor neuronal Cl- regulation (Mercado etal., 2004; Gamba, 2005). The CCCfamily (encoded by the genes Slc12a1-9, see Table 2) includes transportersthat mediate either Na+-driven Cl-
uptake (Na+-K+-Cl- cotransporters,NKCCs, isoforms NKCC1 andNKCC2, and a Na+-Cl- cotransporter,NCC) or K+-driven Cl- extrusion (K+-Cl+ cotransporters, KCCs; isoformsKCC1-4). The functions of the twomost recent CCC family members(cation-chloride cotransporterinteraction protein, CIP, and CCC9)have not yet been identified (Caron etal., 2000; Gamba, 2005).
2.2.1.1 Potassium-driven chloridecotransporters
In the adult brain transporter-mediatedCl- extrusion is mainly carried out bythe KCCs (Mercado et al., 2004;Gamba, 2005) (Fig. 1). From the fourKCC isoforms (KCC1-4, encoded byfour separate genes Slc12a4-7) KCC1,KCC3, and KCC4 have diverse andwidespread expression patterns. KCC1and KCC4 isoforms are both present inthe brain, especially in the choroidplexus, but show little expression inmature CNS neurons (Study I of thisThesis; Mercado et al., 2004).Compared to KCC1 and KCC4, KCC3is abundantly expressed in the brain,including the hippocampus, and it hasbeen shown to contribute in neuronalvolume and Cl- regulation (Mount etal., 1999; Boettger et al., 2003; LeRouzic et al., 2006). In addition to theneuronal expression, an association ofKCC3 with myelin sheet has beenreported (Pearson et al., 2001). Incomparison to the three widespreadKCC isoforms, KCC2 is found only inthe CNS where its expression is strictlylimited to neurons (Payne et al., 1996;Williams et al., 1999). In the adult rathippocampus KCC2 immunoreactivityis observed in the somatic and
dendritic membranes but it is mostprominent in dendritic spines ofprincipal cells and of (parvalbuminpositive) interneurons (Gulyas et al.,2001). KCC2 has two splice variants,KCC2a and KCC2b, with the KCC2bisoform prevailing over that of KCC2ain adult cortical neurons (Uvarov et al.,2007).Even though KCCs underphysiological conditions operate as netefflux pathways, in mature corticalprincipal neurons the process for Cl-
export is actually very near itsthermodynamic equilibrium and even asmall increase in [K+]o may change thedirection of net K+-Cl- transport(Payne, 1997). Intense neuronalactivity can result in elevation of [K+]o,especially under pathologicalconditions (Traynelis and Dingledine,1989;Avoli, 1996; Voipio and Kaila,2000) and experimental work has,indeed, provided support for KCC2mediated K+-Cl- influx uponexperimentally elevated [K+]o(Jarolimek et al., 1999; DeFazio et al.,2000; Kakazu et al., 2000).In addition to the Cl- transporters andligand-gated Cl- channels (e.g.GABAA- and glycine receptors), themembers of the CLC family of Cl-
channels can also mediate Cl- fluxes(Jentsch et al., 2005). These channelshave been shown to have a variety offunctions including stabilization ofmembrane potential, synapticinhibition, cell volume regulation, andtransepithelial transport. A channel-mediated outward net flux of Cl- ispossible only when the membranepotential is more negative than ECl.
Unlike the other K+-dependent Cl-
cotransporters, KCC2 is not activatedby cellular swelling and it is notdirectly involved in cell volumeregulation (Payne, 1997). KCC3 is agenuinely volume sensitive and itstransport activity is sensitive to cellularswelling (Race et al., 1999; Boettger etal., 2003) and both KCC1 and KCC4are activated by volume increase underhypotonic conditions (Race et al.,1999: Mercado et al., 2000). Neuronalvolume regulation is further assisted byNHEs (Rotin and Grinstein, 1989)which also play a major role in HCO3
-
11
-independent pH regulation(Schwiening and Boron, 1994;Bevensee et al., 1996; Chesler, 2003).
2.2.1.2 Sodium-driven chloridecotransporters
Of the three Cl- uptake-mediatingCCCs, only NKCC1 is expressed in thebrain, both by neurons and glia(Plotkin et al., 1997; Clayton et al.,1998; Mercado et al., 2004). There aretwo functional NKCC1 splice variants,NKCC1a and NKCC1b, whose relativeproportions vary among differenttissues (Randall et al., 1997; Vibat etal., 2001). Transcripts of both variantsare present in the adult (human) brain(Vibat et al., 2001). In contrast to
KCCs, NKCC1 is driven by the Na+-gradient and only functions toaccumulate Cl- into cells. In allmammalian cells studied thestoichiometry of the cotransporter isNa+:K+:2Cl- (Russell, 2000).NKCC1 and KCC1-4 are blocked bythe ‘loop’ diuretics furosemide andbumetanide (Payne et al., 2003). Theformer drug inhibits the transporterswith equal potency but the latter can beused as a selective blocker of NKCC1at a concentration of 1-10 µm (Gillenet al., 1996, Williams et al., 1999). Thesomewhat contradictory data onNKCC1 expression in the brain isdiscussed in chapter 2.3.1.
Figure 1. Transporter and GABAA channel-mediated Cl- movements. Theelectroneutral cation-chloride cotransporters KCCs and NKCC1 function as neuronalCl- extruders and loaders, respectively. The Na+-dependent and –independent anion-exchangers may contribute in the control of intracellular Cl- levels in addition to theirrole as pHi regulators. The electrochemical gradient of Cl- determines the direction ofGABAA channel-mediated Cl- fluxes. In (juvenile) neurons with high [Cl-]i, openingof GABAA channels results in a net Cl- efflux whereas in (mature) neurons with low[Cl-]i there is a net influx of Cl-.
12
2.2.2 Members of bicarbonatetransporter gene family
The transporters involved in neuronalpH regulation can be divided into acid-extruding (transporters mediatingefflux of H+ or influx of HCO3
- orCO3
2-) and acid-loading transporters(transporters resulting in an influx ofH+, or efflux of HCO3
- or CO32-). The
secondary active BTs of the SLC4gene family are, in co-operation withthe NHEs (the SLC9 gene family),largely responsible for neuronal pHregulation (Romero et al., 2004;Orlowski and Grinstein, 2004). Out ofthe ten SLC4 gene family members,the physiological function of eighttransporter subtypes has beenestablished (Table 2). They can bedivided into two major subfamilies: theanion exchangers (AEs) and the Na+-driven HCO3
- transporters. Thetransport mode and the predominantsubstrate of the two remainingtransporters, SLC4a9 and SLC4a11,respectively, are uncertain (Romero etal., 2004). Most of the members of theSLC4 gene family are inhibited bydisulfonic stilbene derivatives such asDIDS and SITS. It should bementioned that Cl- is a substrate ofsome transporters that are typicallyclassified as pH regulators. AEs andthe Na+-driven Cl--HCO3
- exchanger(s)mediate HCO3
- coupled Cl- fluxes.Future work will show how muchthese exchangers actually contribute toCl- regulation in various kinds ofneurons.Several other SLC gene familiesinclude members that, even though notconsidered as pH regulators, mediatetransmembrane movements of acid-base equivalents (see Hediger et al.,2004). Primary active transporters arealso involved in neuronal pHregulation. Neuronal acid extrusion inthe nominal absence of Na+ andCO2/HCO3
- is accomplished by aputative H+ pump (Bevensee et al.,1996) whereas the Ca2+-H+-ATPasefunctions as an acid loader whileextruding Ca2+ (Paalasmaa et al., 1994;Smith et al., 1994; Trapp et al., 1996)(Fig. 2).
- andCl-. In adult neurons with a low [Cl-]i,it is the inward Cl- chemical gradientthat dominates and drives the exchangeof extracellular Cl- for intracellularHCO3
-. The Cl--HCO3- exchange thus
functions as Na+-independent acid (andCl-) loader that is assumed to largelybe responsible for cellular recoveryfrom an alkaline load (Chesler, 2003).Both AE3 and AE2 are detected in thebrain: AE3 has a neuron-specificexpression pattern (Hentschke et al.,2006) whereas AE2 localizes solely tothe basolateral membrane of choroidplexus (Lindsey et al., 1990). AE1expression is most pronounced inerythrocytes and kidney (Romero etal., 2004).Na+-independent Cl--HCO3
- exchangeractivity has been demonstrated in adultrodent hippocampal neurons (Raley-Susman et al., 1993; Hentschke et al.,2006) and in cortical astrocytes(Shrode and Putnam, 1994). As AE3 isstrictly neuronal in the CNS(Hentschke et al., 2006), the anionexchanger responsible for the glialrecovery from alkalosis, described byShrode and Putnam (1994) remains tobe characterized. In experiments onAE3 knockout mice (AE3 KO), thehippocampal pyramidal cell recoveryfrom alkalosis was impaired but notabolished (Ruusuvuori et al., 2007).These results imply that also someother acidifying mechanism(s) that isindependent of Cl- and HCO3
-
contributes to pyramidal cell pHregulation during intracellularalkalosis.
2.2.2.2 Sodium-driven bicarbonatetransporters
The Na+-driven HCO3- transporters can
be classified into electrogenic andelectroneutral transporters.The three electroneutral Na+-drivenHCO3
- transporters function as acidextruders. The NBCn1 (Slc4a7), theonly stilbene-insensitive transporter in
13
the SLC4 gene family, mediates a 1:1influx of Na+ and HCO3
- (Bevensee etal., 2000) where as NDCBE (Slc4a8)functions as an Na+-driven Cl--HCO3
-
exchanger (Grichtchenko et al., 2001).Both transporters have recenly beenshown to localize to the soma anddendrites of hippocampal pyramidal
neurons (Cooper et al., 2005;Damkieret al., 2007;Boedtkjer et al., 2008;Chenet al., 2008a). NCBE (Slc4a10) wasalso initially thought to be a Na+-driven Cl--HCO3
- exchanger (Wang etal., 2000) but the Cl- dependence of the(rodent) transporter is still debated(Choi et al., 2002). It was
Figure 2. Acid extruders and loaders that contribute in pHi regulation. The cellularsteady-state pHi depends on the balance of acid-base equivalent movements mediatedby the transporters, and from the acid load generated by metabolism and by thepassive entry/exit of H+, OH-, and HCO3
-. Modified from Boron (2004).
recently shown that the Na+-HCO3-
transport activity of the humanSLC4A10 is, under physiologicalconditions, independent of Cl-countertransport and thus thetransporter should rather be calledNBCn2 (Parker et al., 2008). Twovariants of the NCBE, rb1NCBE andrb2NCBE, have been identified in theadult rat brain (Giffard et al., 2003).The rb2NCBE terminates in a PDZmotif which is absent from therb1NCBE. RT-PCR showed that bothvariants were present in RNA isolatedfrom rat (and mouse) brain. In cultured
brain cells both variants were presentin neurons but the rb2NCBE was moreprominent in astrocytes. However, inthe embryonic mouse brain NCBEexpression was suggested to follow amainly neuronal pattern (Hübner et al.,2004). In the rodent hippocampusNCBE mRNA has been detected(Wang et al., 2000; Giffard et al.,2003; Hübner et al., 2004) but theprotein distribution has not beenpreviously assessed.Functional studies have shown thatunder physiological conditions acidextrusion in adult rat hippocampal
14
CA1 pyramidal neurons is governed byan (amiloride insensitive) Na+-H+
exchanger and by Na+-driven Cl--HCO3
- exchanger(s) (Schwiening andBoron, 1994; Bevensee et al., 1996).These findings suggest that, in additionto the NHEs, NCBE and/or NDCBEare critically involved in pyramidal cellpH regulation. The localization ofNCBE at the protein level and itscontribution to pyramidal cell pHregulation and neuronal excitability areassessed in Study III.
The last subgroup of the BTs consistsof the electrogenic Na+-HCO3
-
cotransporters. In brain cells andchoroid plexus epithelial cells (NBCe1and NBCe2; Slc4a4 and Slc4a5) thesetransporters most likely have astoichiometry of 1:2 and hence importHCO3
- (Romero et al., 2004, but seeBoussouf et al., 1997) but the numberand the direction of transported HCO3
-
ions appear to be cell-type specific. Inthe renal proximal tubule whereNBCe1-A is involved in the HCO3
-
reabsorption the transporter operateswith a stoichiometry of 1:3 andextrudes HCO3
- into the interstitialspace (Gross et al., 2001), whereas ifexpressed in Xenopus oocytes thestoichiometry is 1:2 (Heyer et al.,1999).The NBCe expressed in brain cells(NBCe1; Giffard et al., 2000; Schmittet al., 2000; Rickmann et al., 2007) hasclassically been considered as a ‘glialtransporter’ that has a prominent rolein intraglial pH regulation (Deitmerand Schlue, 1989; Brune et al., 1994;O'Connor et al., 1994; Shrode andPutnam, 1994; Giffard et al., 2000).NBCe-mediated transport of HCO3
- isinvolved in the generation ofdepolarization-induced alkalinization(DIA) of glial cells, a form of neuron-glia signalling where changes in glialmembrane potential caused byneuronal activity are converted intoglial pHi changes (Chesler and Kraig,1987,Chesler and Kraig, 1989;Deitmer and Schlue, 1989; Pappas andRansom, 1994). The transmembranemovements of HCO3
- that give rise toDIA produce a simultaneous acidtransient in the brain extracellularspace (Chesler and Kraig, 1989;
Deitmer and Szatkowski, 1990;Grichtchenko and Chesler, 1994) andthus contribute to activity-induced pHochanges (Deitmer, 1992; Deitmer andRose, 1996). Today there isaccumulating molecular biological datasuggesting that in addition to glial cellssome neuronal subpopulations,including dentate granule cells andhippocampal pyramidal neurons,express NBCe1 (Bevensee et al., 2000,Schmitt et al., 2000, Rickmann et al.,2007; Majumdar et al., 2008). Theexpression of NBCe2 in the brain islimited to the luminal membrane ofchoroid plexus epithelial cells(Bouzinova et al., 2005).
2.2.3 Carbonic anhydraseisoforms
CAs are zinc-metalloenzymes thatcatalyze the reversible hydration ofCO2 (Maren, 1967;Sly and Hu, 1995;Supuran et al., 2004). The rate-limitingstep in the process, schematicallyrepresented by Equations (2-1) and (2-2), is the regeneration of thecatalytically active, basic form of theenzyme (EZn2+-OH-). It requires aproton-transfer reaction from the activesite to the environment (Equation 2-2),a process that is in the isoforms withthe highest turn-over rates (CAII,CAIV, CAV, CAVII, CAIX) assistedby several histidine residues.
322 2 HCOEZnOHEZn CO
222 OHEZnOH (2-1)
OHEZnOHEZn H 22
2
(2-2)
So far, 15 distinct isozymes or CA-related proteins (CARP) with diversesubcellular localization have beencharacterized (Supuran et al., 2003;Hilvo et al., 2005) (see Table 3). Fromthe twelve enzymatically activeisoforms, five are cytosolic (CAI-III,VII, and XIII), five are extracellular(CAIV, IX, XII, XIV, and XV), one ismitochondrial (CAVa,b), and one is
15
secreted (CAVI). CAs are among thefastest enzymes: the substrate turnovergoes up to 1.4x106 s-1, (at +25 °C);thus approaching the 5x106 s-1 (at +20°C) of catalase, the fastest enzymeknown (Hille, 2001).The three CARPs (CAVIII, X, and XI)are evolutionarily conservedcytoplasmic proteins that lack catalytic
activity because at least one of thethree zinc-binding histidine residues isreplaced with other amino acids(Supuran et al., 2004). Even thoughroles in e.g. protein complex formationand cell proliferation have beensuggested for CARPs, there is not yetmuch knowledge about theirinvolvement in biological functions.
Identified ( -)carbonic anhydrase isoforms
Isoform Catalyticactivity
Subcellularlocalization
Expression in theCNS
CAI Low Cytosolic -CAII High Cytosolic WidespreadCAIII Very Low Cytosolic Some glia, choroid
plexusCAIV High Membrane bound WidespreadCAVA Moderate-high Mitochondrial Some neurons and
CAIX High Transmembrane Low expression*CAX-CARP Acatalytic Cytosolic Some expressionCAXI-CARP Acatalytic Cytosolic Moderate expressionCAXII Low Transmembrane Low expression*CAXIII Moderate Cytosolic Some gliaCAXIV High Transmembrane WidespreadCAXV Low Membrane bound Some expression
Table 3. The ( -)carbonic anhydrase isoforms and their catalytic activity, subcellularlocalization, and expression in the rodent central nervous system (CNS).* Isoformpresent in normal brain at low levels but is over-expressed in certain carcinomas. Thedata in the table are from Supuran et al., 2003 and Hilvo et al., 2005.
16
2.2.3.1 Intracellular carbonicanhydrases and their expression inhippocampus
It is not long ago when intracellularcarbonic anhydrase (CAi) activity inthe rodent CNS was thought to berestricted to glial cells (Cammer andTansey, 1988; Agnati et al., 1995;Nogradi et al., 1997) and CAIIexpression was considered a reliablemarker for oligodendrocytes(Ghandour et al., 1980; Ghandour etal., 1992). Even though CA is highlyexpressed in glial cells and in themyelin compartment, there is by nowboth functional (Pasternack et al.,1993; Munsch and Pape, 1999;Schwiening and Willoughby, 2002)and molecular biological evidence(Nogradi et al., 1989;Lakkis et al.,1997; Nogradi et al., 1997; Wang etal., 2002b; Kida et al., 2006) for thepresence of intraneuronal CA.In addition to the well-describedlocalization of CAII in glial cells,Halmi et al. (2006) reported a diffuseexpression of CAII mRNA in stratumpyramidale and dentate gyrus. At theprotein level, staining with a CAII-specific antibody suggested awidespread neuronal expression (Wanget al., 2002b; Kida et al., 2006). In thehippocampus, CAII immunoreactivitywas most prominent within the somataand proximal dendrites of pyramidalneurones. Furthermore, there is astrong transcriptional signal for CAVIIin the juvenile and adult rodenthippocampal pyramidal cell layer(Lakkis et al., 1997; Halmi et al.,2006). Besides these two cytosolicisoforms, adult mouse hippocampalneurons show a strong positiveimmunostaining for CAV (Ghandouret al., 2000). However, CAV is amitochondrial CA isoform (Nagao etal., 1994) which is unlikely to play adirect role in reactions associated withtransmembrane movements of H+,CO2, and HCO3
-.
2.2.3.2 Extracellular carbonicanhydrases and their expression inhippocampus
In the adult rodent CNS theextracellular CA (CAo) activity isassociated with the transmembrane(CAXIV, IX and XII) and membrane-attached (CAIV and XV) CA isoformsthat have their active site oriented tothe extracellular side. CAIV andCAXV are attached to the plasmamembrane with a glycosyl-phosphatidylinositol anchor (Zhu andSly, 1990; Hilvo et al., 2005) whereasCAIX, XII, and XIV have atransmembrane segment (Pastorek etal., 1994; Tureci et al., 1998; Mori etal., 1999).By using in situ hybridization and RT-PCR Hilvo et al. (2005) showed thatCAXV is expressed, among othertissues, in the mouse brain. However,based on the sequence data, it seemsthat in humans and chimpanzees thisisoform had become a non-processedpseudogene. The lack of CAXVexpression in several human tissues,including the brain, was confirmedwith RT-PCR. CAIX and XII areexpressed at low levels in the normalrodent brain, CAXII being mostabundant in the choroid plexus (Ivanovet al., 2001; Hilvo et al., 2004; Kallioet al., 2006). These two isoforms havean exceptional expression pattern asthey are markedly up-regulated incertain human carcinoma cells in e.g.kidney, lung, and CNS (Tureci et al.,1998; Ivanov et al., 2001). CAIV(Carter et al., 1990; Ghandour et al.,1992; Tong et al., 2000; Wang et al.,2002b) and CAXIV (Parkkila et al.,2001) are both present in rodent andhuman brain at the mRNA and proteinlevel. At least in the rodenthippocampus the CAIX, XII, and XVmake a minor contribution to the totalCAo activity. Direct monitoring ofextracellular pH transients with H+
sensitive microelectrodes inhippocampal slices from rats (Tong etal., 2000) and from CAIV and XIVdouble knock-out mice (Shah et al.,2005) demonstrated that these two CAisoforms are largely responsible forCAo activity in the hippocampus.
The ion whose intracellularconcentration changes the most duringdevelopment of the mammalian CNS isCl-. No other ion undergoes such adramatic ontogenic alteration in itsintraneuronal concentration (Luhmannand Prince, 1991, Zhang et al., 1991;Owens et al., 1996;Tyzio et al., 2008;Blaesse et al., 2008). In immature CNSneurons Cl- concentration is typicallyin the range of 25-40 mM (see e.g.Owens et al., 1996; Balakrishnan et al.,2003; Yamada et al., 2004; Sipilä etal., 2006b; Achilles et al., 2007) whilstin the soma of mature neurons the Cl-
concentration is kept around 5-7 mM(for recent reviews see Farrant andKaila, 2007;Blaesse et al., 2008). Thischange reflects major differences in thefunctional expression of Cl-
transporters during neuronalmaturation.KCCs show distinct spatiotemporalexpression patterns in the embryonicbrain. Li et al. (2002) have shown thatall four KCC isoforms are expressed inthe embryonic brain but their regionalexpression was not overlapping.Rather, their expression reflected thedevelopmental stage of the individualbrain region with KCC4 beingexpressed in undifferentiated regionswith proliferating cells, KCC3 inrecently differentiated regions, andKCC2 in differentiating regionsfollowing the functional maturation ofneurons. Because of the strictlyneuronal localization of KCC2 and itsvast expression in the adult rodentbrain (Payne et al., 1996, Williams etal., 1999 but see Gulacsi et al., 2003;Bartho et al., 2004), increased KCC2mediated cellular Cl- extrusion is anobvious candidate to contribute to themaintenance of low intracellular Cl-,characteristic for most mature neurons.Before Study I of this Thesis theexpression pattern of KCC2 indifferent brain regions had beenstudied both in the embryonic and
adult brain but no such studies hadbeen performed during the postnataldevelopment of the rodent brain. Someindication of increased postnatalexpression of KCC2, occurring inparallel with the decrease in [Cl-]i seenin cortical neurons (Owens et al., 1996;Tyzio et al., 2008) had been providedby Lu et al. (1999). Using RT-PCR,immunoblotting, and immuno-fluorescence Lu et al. (1999) showedthat KCC2 transcript and protein levelsincreased during the two first postnatalweeks. However, the specificity of theKCC2 antibody and cDNA probe usedin these experiments might bequestioned because the authors report astrong KCC2 expression in the dorsalroot ganglion (DRG). According toStudy I of this Thesis, the DRG can beused as a negative control for KCC2.NKCC1 mRNA is detected in thehippocampus at birth (Plotkin et al.,1997; Li et al., 2002; Wang et al.,2002a). Indeed, electrophysiologicalrecordings from juvenile hippocampalneurons suggest that NKCC1 acts asthe main Cl- uptake mechanismresponsible for GABAA-mediateddepolarizing currents (Yamada et al.,2004;Sipilä et al., 2006b; Achilles etal., 2007). Some groups have reporteda decline in NKCC1 expression duringlater developmental stages (Plotkin etal., 1997; Hübner et al., 2001a;Yamada et al., 2004) but there is alsocontrasting data showing NKKC1expression in adult animals (Wang etal., 2002a; Clayton et al., 1998). Ifthere is no consensus on the postnatalNKCC1 mRNA expression, the over-all picture of the developmentalexpression of NKCC1 protein is notmuch clearer. The conflicting data onthe protein expression can be at leastpartially explained by the use of anNKCC antibody (T4) that seems to bereliable in immunoblots (Zhang et al.,2006) but not in immuno-histochemistry (see Kaila et al., 2008).Electrophysiological data providesupport for the finding that NKCC1expression continues into theadulthood. Szabadics et al. (2006)reported that in the axon initialsegment (AIS) of mature neocorticallayer 2/3 neurons EGABA-A is at a muchmore depolarized level than the resting
18
membrane potential. By usingimmunoelectronmicroscopy, theauthors also demonstrated an unevensubcellular distribution of KCC2between the soma and AIS. However,impaired Cl- extrusion alone cannotexplain the highly (20 mV)depolarizing reversal potential ofGABA. To study the role of Cl- uptakemechanisms in setting the EGABA-A,Khirug et al. (2008) performedexperiments on NKCC1 KO animalsand in neurons in which NKCC1 wasinhibited by bumetanide. Thedepolarizing GABAA responses seen atthe AIS of mature neocorticalpyramids was shown to be caused byan axo-somatic Cl- gradient that isexplained by distinct subcellularexpression patterns of NKCC1 andKCC2.It can be concluded that the interplaybetween KCCs and NKCC1 dominatesneuronal Cl- regulation both duringdevelopment and in adulthood. Thefunctional importance of the postnatalup-regulation of KCC2 on GABAergicsignalling is the subject of Study I.
2.3.2.1 Changes in acid-basetransporter expression and function
pHi values measured fromhippocampal neurons with thefluorescent pH indicator 2’,7’-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) aredistributed over a broad range and canvary even by one pH unit in a singlestudy (Schwiening and Boron, 1994;Bevensee et al., 1996; Baxter andChurch, 1996; Kelly and Church,2006). The postnatal development ofsteady-state pHi values in hippocampalneurons has been followed in twoseparate studies. In both cases themean initial pHi of acutely isolatedadult and embryonic/juvenilehippocampal pyramids did not differsignificantly between the two agegroups studied (Raley-Susman et al.,1993; Bevensee et al., 1996). Bevenseeet al. (1996) also reported that the rateof pHi recovery from an acute acid
load, in the nominal absence ofCO2/HCO3
-, was similar in adult andjuvenile neurons.There is little molecular biologicalevidence for postnatal up- (or down)regulation of BTs or other transportersinvolved in pH regulation in rodenthippocampal neurons. Douglas et al.(2001) used immunoblotting to detectvarious transporters involved in pHiregulation at several different postnatalages. In protein lysates from the wholerat cerebral cortex NBCe expressionincreased significantly from embryonicday 16 (E16) to adulthood (postnatalday 105; P105). NBCe mRNA has alsobeen shown to be up-regulated duringthe two first postnatal weeks in the ratbrain (Giffard et al., 2000). The latterresult, however, does not permitconclusions on neuronal NBCe up-regulation because the expression ofthe transporter was reported to berestricted to astrocytes. Other groupshave reported NBCe expression inseveral neuronal subpopulations in theadult brain, including hippocampalgranule cells and pyramidal neurons(Bevensee et al., 2000; Schmitt et al.,2000; Rickmann et al., 2007;Majumdar et al., 2008). In the work byDouglas et al., 2001) the expression ofthe NHE isoforms 1, 2, and 4 wasassessed in addition to NBCeexpression. As with the NBCeexpression, the levels of all three NHEisoforms increased in the cerebralcortex during the postnataldevelopment. These results, both onNBCe and NHE expression patterns,should be considered with somereservation because no referenceproteins were used in the densitometryanalyses.The studies on the developmentalexpression of other BTs have mainlybeen limited to individual time points.The NCBE mRNA is present in thehippocampal pyramidal cell layeralready at prenatal stages (Hübner etal., 2004; Chen et al., 2008a) and thetranscriptional signal remained fairlystable during the early postnataldevelopment (Giffard et al., 2003). Theexpression of NDCBE and NBCn1have not been thoroughly followed butimmunohistochemical and single-cellPCR data show that both transporters
19
are present in embryonic and adultmouse hippocampal pyramidal neurons(Cooper et al., 2005; Chen et al.,2008b). Experiments using in situhybridization have revealed that theNa+-independent Cl--HCO3
- exchangerAE3 is present in the embryonic rat(Raley-Susman et al., 1993) and mousebrain (Hentschke et al., 2006).However, the results from studiesaddressing the functional activity ofthe exchanger in embryonic neuronsare somewhat contradictory.Intracellular pH recordings fromacutely isolated and cultured (10 daysin vitro, DIV) fetal hippocampalpyramidal neurons were performedusing a pH-sensitive fluorescent dye(Raley-Susman et al., 1993). Theseauthors did not detect functionalactivity of CO2/HCO3
- exchange uponremoval of Cl- from the extracellularsolution whereas according to (Baxterand Church, 1996) Cl--HCO3
-
exchange is functionally active incultured rat fetal neurons (6-14 DIV).In adult CA1 pyramidal neuronswashout of Cl- results in anintracellular alkalinization indicatingreversal of Cl--HCO3
- exchange(Raley-Susman et al., 1993; Hentschkeet al., 2006; Ruusuvuori et al., 2007).
2.3.2.2 Changes in intracellularbuffering
Even though steady state cytoplasmicpH doesn’t seem to undergo markedchanges during development, a changein the intrinsic buffering capacity or inthe activity of CA could significantlymodulate the amplitude, and thus thefunctional impact, of activity-inducedpH transients. The pHi dependence ofintrinsic buffering power of CA1pyramidal neurons is kept relativelystable during postnatal development(Bevensee et al., 1996). This resultimplies that significant qualitativechanges in i capacity are also unlikelyto take place during this time window.Hence, developmental changes in CAexpression would be highly relevant assuch a change would be in a keyposition to modulate CO2 with aconsequent, age-dependent, effect onthe kinetics of pHi transients.
In the rodent CNS CAII mRNA isexpressed at a very low level at birth(Cammer and Zhang, 1996) but thelevels increase several-fold during thefirst postnatal months (Nogradi et al.,1997; see Kida et al., 2006 for CAIIexpression in humans). Because of thevast expression of CAII inoligodendrocytes, this increase mightlargely reflect myelin formation whichtakes place within the same timewindow (Suzuki and Raisman, 1994;Savaskan et al., 1999). Developmentalchanges in the expression of otherintracellular CA isoforms have notbeen studied before Study II in whichwe assessed the postnatal developmentof intrapyramidal CA activity and theexpression profiles of both CAII andCAVII, the two putative intraneuronalisoforms in adult rodent hippocampalpyramidal neurons.Here it is tempting to speculate that thedevelopmental decrease in Cl-
concentration is a specific adjustmentof adult neurons to GABA- (andglycine-) mediated fast synapticinhibition (see Blaesse et al., 2008)whereas the relatively constant restinglevel of somatic pH values reflect thepH sensitivity of several elementarycytoplasmic processes and proteinsthat are kept unchanged duringpostnatal development. Onlymodulation of pH transients, duringdevelopment or within subcellularcompartments, would permit the cell tomodify proton-mediated signaling.An intriguing question is also how theelectroneutrality within a cell ismaintained when intracellular anionconcentration is reduced as theintracellular Cl- concentrationdecreases. Assuming that theosmolarity is kept constant, the amountof intracellular charges could bemaintained if (1) the concentration ofother anions is accordingly increased,(2) the net loss of Cl- is accompaniedby an equal loss of a cation (the likelycandidate being K+), which wouldrequire that the osmolarity ismaintained by an increase in someneutral macromolecules or low-weightorganic molecules, or (3) the chargecarried by intracellular proteins ischanged.
20
Because of the shortage of data on thetwo latter options, only the first will bediscussed further. It is obvious that acorresponding increase in HCO3
-
concentration does not occur as, atfixed PCO2, such a change woulddisturb cellular pH balance. A ~30 mMelevation in phosphate concentration isalso unlikely to take place as thiswould result in a concomitant increasein the intrinsic buffering capacity. Asalready discussed, the results ofBevensee et al. (1996) did not suggestchanges in the intrinsic bufferingcapacity in hippocampal neuronsduring postnatal development,although the age window of theanimals may not be optimal for thepresent examination (P2-10 vs. P21-30). Hence, the question whatmechanisms compensate for the loss ofCl- under constant osmolarity remainsunanswered.
2.3.3 Developmental changes inextracellular ion homeostasis
Whether the fetal mammalian BBB isleaky is still a matter of debate(Saunders et al., 2000; Engelhardt,2003). Tight junctions, themorphological basis of brain barriers,are present from the very early stagesof development both betweenendothelial cells of blood vessels andbetween epithelial cells of the choroidplexus (Mollgård et al., 1979). Thejunctions are impermeable to smallmolecules from early development andare thus able to restrict diffusionalready in fetal and juvenile animals(Johansson et al., 2006; Ek et al.,2006). Actually, there are additionalmorphological barriers unique to thefetal brain at the interface betweenCSF and brain tissue (Fossan et al.,1985). The transient expression of thespecific intercellular junctions, “thestrap junctions”, prevents themovement of macromolecules at theinner neuroependymal surface ofventricles (Mollgård et al.,1987;Saunders et al., 2000). However,indications of developmentaltightening of the BBB have beenpresented. The transendothelialresistance of pial microvessels,
reflecting the permeability of the BBBto ions, was measured in fetal andadult rats (Butt et al., 1990; Keep et al.,1995). A marked increase in theresistance was found to take place justbefore birth.The distribution of ions between CSFand plasma in prenatal (Jones andKeep, 1987) and juvenile rat pups(Amtorp and Sorensen, 1974) issimilar to what is seen in olderanimals. This indicates that thetransport systems responsible formaintenance of concentrationdifferences of ions across the blood-CSF barrier are functional in newbornmammals (Ferguson and Woodbury,1969; Bradbury et al., 1972; Amtorpand Sorensen, 1974). The most strikingdevelopmental change in CSFcomposition is the considerably higherconcentration of proteins present in thefetal and newborn CSF (Amtorp andSorensen, 1974; Dziegielewska et al.,2000). As the leakiness of theimmature BBB can be excluded (Ek etal., 2006; Johansson et al., 2006) thedevelopmental changes in the proteinconcentration likely reflect changes inthe transepithelial transport (Habgoodet al., 1992; Johansson et al., 2008).The significance of the finding is notclear but the high protein concentrationof CSF appears to be maintainedduring the period of ventricularexpansion thus contributing to specificfeatures of CNS development.Like the intracellular pH of brain cells,also the CSF pH is maintained constantduring postnatal development (Pavlinand Hornbein, 1975; Nattie andEdwards, 1981; Johanson et al., 1992).Because the buffering of pH in brainfluids is dominated by CO2, CAactivity has a central role inmodulating activity-dependent pHtransients (Chesler and Kaila, 1992).In the developing rat hippocampusmeasurements of ECF CAo activitywith OH- injections and pH sensitivemicroelectrodes have demonstratedthat CAo activity commenced at theend of the first postnatal week (Voipioet al., 1999) most likely reflecting theexpression of CAIV and/or CAXIV(Shah et al., 2005).
21
2.4 Functional significanceof ion regulation
2.4.1 Neuronal ion regulationmodulates GABAA receptor-mediated signaling
2.4.1.1 GABAA receptor-mediatedtransmission in developinghippocampal neurons
The high intracellular Cl- of theimmature neurons results in an EClmore positive than resting Vm (Payneet al., 2003) and GABAA receptor-mediated responses are oftenconsidered to be depolarizing enoughto trigger neuronal spiking, openvoltage-gated Ca2+ channels, andfacilitate the activation of NMDAchannels (Janigro and Schwartzkroin,1988; Ben-Ari et al., 1989; Cherubiniet al., 1990; Yuste and Katz 1991;Leinekugel et al., 1997; Fukuda et al.,1998). The depolarizing GABAAreceptor-mediated currents have beensuggested to represent a generalmechanism of the developing brainthat controls different aspects ofneuronal development e.g. migration,differentiation, synaptogenesis, andrefinement of the emerging neuronalnetworks (LoTurco et al., 1995;Owensand Kriegstein, 2002;Demarque et al.,2002; Chudotvorova et al.,2005;Akerman and Cline, 2006).GABAA receptor-mediated signalingbegins before synaptic connectionsemerge (Valeyev et al., 1993; Tyzio etal., 1999; Demarque et al., 2002). Atthis early developmental stage theanion currents evoked by GABA aremediated by tonic activation of non-synaptic GABAA receptors. Only afterneuronal migration is completed,synaptic transmission starts tocontribute to GABAA-receptoractivation. The establishment ofGABAergic and glutamatergicsynapses in developing rathippocampus is sequential, withGABAergic transmission precedingthat of glutamatergic neurons (Tyzio etal., 1999; Hennou et al., 2002).In the developing nervous systemspontaneous network activity is
characteristic for different brainregions, including the hippocampus(Yuste et al., 1992; Katz, 1993; Ben-Ari et al., 1989; O'Donovan, 1999).Although the CA3 subregion ofhippocampus often functions as apacemaker, spontaneous networkevents can be generated in other areas(i.e. CA1 and dentate gyrus) evenwhen isolated from other regions(Khazipov et al., 1997; Garaschuk etal., 1998; Ben-Ari, 2001). Experimentsusing Ca2+ imaging have revealed thatspontaneous network activity in theimmature hippocampus is associatedwith synchronous intracellular Ca2+
oscillations mediated by voltage-dependent Ca2+- and NMDA-channels(Leinekugel et al., 1997;Garaschuk etal., 1998;Garaschuk et al., 2000). Thisjoint presynaptic activity andpostsynaptic [Ca2+]i influx couldmediate Hebbian type of activity-dependent plasticity of developingsynapses and be involved in activity-dependent synaptogenesis and networkformation (Feller, 1999;Spitzer, 2006).
When Ben-Ari et al. (1989) firstdescribed spontaneous oscillations inthe neonatal rat hippocampal CA3pyramidal cells (named as giantdepolarizing potentials, GDPs) theevents were described as network-driven synaptic events mediated byGABA. The GABAergic nature ofGDPs was suggested by intracellularmeasurements which showed thatGDPs were blocked by the GABAAreceptor antagonist bicuculline andreversed at the same membranepotential as exogenously appliedGABA. The contribution of pyramidalneurons, i.e. glutamatergictransmission, in GDP generation wasalso observed and the importance ofthe synergistic action of GABAA andionotropic glutamatergic transmissionin synchronizing neuronal networkdischarges was underlined in laterstudies (Khazipov et al., 1997;Leinekugel et al., 1997). However, thecentral role of depolarizing GABAA-channel mediated postsynaptic currentsin providing the excitatory drive forGDP generation was still stronglyemphasized (Ben-Ari, 2002; Ben-Ari
22
et al., 2007; see also Strata et al.,1997).Recently Sipilä et al., 2005, 2006a)have presented a different mechanisticexplanation for GDP generation. Theauthors demonstrated that immatureCA3 pyramidal neurons are able toproduce bursts of action potentials inthe absence of ionotropic glutamateand GABA mediated transmission. Theintrinsic bursting was triggered by aslow regenerative depolarizationmediated by a persistent Na+ currentand terminated by a slow, Ca2+-activated K+-current (Sipilä et al.,2006a). In line with previousobservations (Ben-Ari et al., 1989,Bolea et al., 1999; Lamsa et al., 2000)CA3 network activity was fullyblocked by ionotropic glutamate-receptor antagonists. According toSipilä et al. (2005, 2006a) thisendogenous bursting activity ofimmature CA3 pyramids acts togenerate GDPs while the depolarizingGABAA mediated transmission has apermissive role facilitating thespontaneous activity of the immatureneurons.The gradual disappearance of GDPsduring the second postnatal week(Ben-Ari et al., 1989; Khazipov et al.,2004) occurs in parallel with thetransition to more negative ECl valuesand to hyperpolarizing GABAAresponses. This developmentalincrease in the Cl- gradient is readilyexplained by a major reorganization ofintracellular Cl- regulation (Study I ofthis Thesis).
2.4.1.2 Depolarizing GABAergictransmission in the maturehippocampus
It has been known for a long time thatactivation of dendritic GABAAreceptors using exogenous agonists canresult in depolarizing responses inmature hippocampal pyramidalneurons (Alger and Nicoll, 1982a;Alger and Nicoll, 1982b; Huguenardand Alger, 1986; Kaila et al., 1993;Voipio and Kaila, 2000). Previousresults from our laboratory have shownthat large-scale activation of GABAAchannels, by using high-frequency
stimulation (HFS), can evoke a HCO3--
dependent depolarization of adulthippocampal CA1 pyramidal (Kaila etal., 1997; see also Grover et al., 1993).The GABAergic response is biphasicwith an early hyperpolarization(representing fused individualhyperpolarizing inhibitory postsynapticpotentials) followed by a prolongeddepolarization that is often associatedwith pronounced spiking (Perreaultand Avoli, 1988; Grover et al., 1993;Staley et al., 1995;Kaila et al., 1997;Kaila et al., 1997; Smirnov et al.,1999). The depolarization is generatedby a fast initial anionic redistributionand of a long-lasting increase in [K+]o(Voipio and Kaila, 2000). The anionredistribution is caused by thedepolarizing HCO3
– current that leadsto accumulation of intraneuronal Cl–and consequently, to a large positiveshift in EGABA-A (Kaila et al., 1987;Kaila et al., 1989a; Voipio et al.,1991). Inhibition of intra- but notextracellular CA has been shown toattenuate the post-tetanicdepolarization (Kaila et al., 1997) butbefore Study II of this Thesis thecellular subtype expressing theintracellular CA activity that plays acrucial role here had not beenidentified.Synchronous firing of principalneurons is seen under bothphysiological and pathophysiologicalconditions: it plays a central role inneuronal plasticity (Traub et al., 1998;Linden, 1999) as well as in the genesisand maintenance of epileptic activity(McCormick and Contreras, 2001).Therefore it is intriguing that the HFS-induced neuronal firing can be locallysynchronized into gamma frequencyrange oscillations (afterdischarges) ofconfined pyramidal-cell andinterneuronal networks (e.g. Traub etal., 1996a; Colling et al., 1998; Bracciet al., 1999). The HFS-induced gammaoscillations are characterized bymassive, synchronous firing ofprinciple cells, a property that morelikely represents epileptiform activitythan the small-amplitude, theta-modulated gamma oscillationsmeasured in vivo, which are suggestedto be involved in cognition andperception (Ylinen et al., 1995;
23
Penttonen et al., 1998; Gray, 1999). Inaddition to the intrinsic properties ofneurons (Connors and Gutnick, 1990;Stanford et al., 1998; Hille, 2001), themechanisms that have been shown tocontribute to the temporal patterns ofneuronal firing include GABAAreceptor-mediated inhibition(Whittington et al., 1995), ionotropicglutamatergic transmission (Traub etal., 1996a), gap junctions (Draguhn etal., 1998; Schmitz et al., 2001), andephaptic effects (Bracci et al., 1999).Intracellular recordings from CA1pyramidal neurons have shown that theHFS-induced prolonged excitation ofthe principal cells depends on theGABAA channel-mediateddepolarization described above (Kailaet al., 1997; Bracci et al., 1999;Smirnov et al., 1999). Furthermore,HFS-induced population activity in thegamma frequency range can be evokedin the absence of ionotropic glutamatereceptor activity and blocked byGABAA antagonist bicuculline(Colling et al., 1998; Bracci et al.,1999). In light of the important role ofthe GABAA-channel mediated, K+
o-dependent depolarization of pyramidalcells (Perreault and Avoli, 1988;Grover et al., 1993; Kaila et al., 1997)it is interesting that oscillations atgamma frequencies can also be evokedby brief K+ application in the CA1(and CA3) region of rat hippocampus(LeBeau et al., 2002). The activation ofinterneuronal metabotropic glutamatereceptors (mGluRs) has also beensuggested to be involved in the HFS-induced gamma oscillations(Whittington et al., 1995; Whittingtonet al., 1997; Traub et al., 1996a; Traubet al., 1996b; Whittington et al., 1997;but see Bracci et al., 1999) althoughthe role of mGluRs has been shown tobe most significant in the ‘pure’interneuronal network oscillations(Whittington et al., 1995).A shift to depolarizing GABAergicresponses is also detected in severaltrauma models of adult neurons e.g.after injury of neurites oranoxia/ischemia (Katchman et al.,1994; van den Pol et al., 1996;Nabekura et al., 2002). There isincreasing evidence suggesting that thedepolarizing EGABA-A seen after
neuronal trauma depends on changes inthe expression of the CCCs (Nabekuraet al., 2002; Fukuda et al., 1998; Pondet al., 2006). It has been proposed thatthese expressional changes mightfunction to “turn back the time” of theinjured neurons in order to restore theirdevelopmental flexibility, which wouldable to rearrange the neuronalconnectivity by e.g. axonal sproutingand retargeting (see Payne et al.,2003).The study by Cohen et al., 2002)assessed the role of GABAA receptor-mediated transmission in hippocampalpreparations from human epilepsypatients. Depolarizing GABAergictransmission was shown to underlie theinterictal activity in a subpopulation ofsubicular neurons. In a follow-uppaper, this finding was explained by adown regulation of KCC2 (Huberfeldet al., 2007). A pronounced change inneuronal EGABA-A, with a concomitantreduction in KCC2 levels, has alsobeen shown to take place in animalmodels of epilepsy both in vitro and invivo (Rivera et al., 2002; Rivera et al.,2004; Pathak et al., 2007). Hence itmight be that expressional changes ofCCCs are a general response ofneurons to different kind of trauma.
2.4.2 Neuronal excitability ismodulated by pH
A common observation both in vitroand in vivo has been that a fall in pHleads to a decrease in neuronal activity.Manipulations that aim to reduce tissuepH decrease hippocampal neuronalexcitability and suppress evokedseizure activity in vitro (Aram andLodge 1987; Jarolimek et al., 1989;Lee et al., 1996; de Curtis et al., 1998;Bonnet et al., 2000; Xiong et al., 2000;Dulla et al., 2005) and in vivo(Balestrino and Somjen, 1988;Ziemann et al., 2008). From ateleological point of view, such pHdependence provides a negativefeedback control of network activitythat would limit the generation andpropagation of seizure activity.Furthermore, such a feedback loopwould assist in seizure termination(Velisek et al., 1994; de Curtis et al.,
24
1998; Ziemann et al., 2008). In mostneurons protons suppress electricalactivity. However, the pH dependenceof intrinsic excitability appears to beopposite in central chemosensoryneurons. These cells detect changes inCO2/H+ and respond to a raise inplasma CO2/H+ (i.e. to respiratoryacidosis) by increased activity (Nattie,2001, Putnam et al., 2004). Via thesecells, an increase in CO2/H+ serves asthe major stimulus for increasedventilation in response to respiratoryacidosis. This forms a “higher level ofexcitability control” where the brainregulates breathing, and thus its ownCO2 levels, thereby affecting its ownexcitability.In contrast to acidification, a rise in pHleads to increased neuronal excitabilitythat may even lead to epileptiformactivity in vitro (Aram and Lodge,1987; Jarolimek et al., 1989; Lee et al.,1996; de Curtis et al., 1998; Dulla etal., 2005). In vivo experiments providesupport for the findings done in vitro.Balestrino and Somjen, 1988) showedthat rat hippocampal neurons werehighly sensitive to small deviationsfrom normal pH. Reduction in PCO2concentration enhanced neuronal firing(and increased PCO2 depressed it).Recently, hyperthermia-inducedrespiratory alkalosis was demonstratedto trigger ictal activity in a febrileseizure model (Schuchmann et al.,2006).The effect of pH on neuronalexcitability is commonly believed toreflect changes in the activity of ionchannels (voltage-, ligand-, or proton-gated) and gap junctions. The intrinsicexcitability of a neuron is suppressedby a decline in pH probably becausethe currents through voltage-gated Na+
and especially Ca2+ channels are oftendepressed by protons (Tombaugh andSomjen, 1996; Tombaugh and Somjen,1997). Notably, the proton-mediatedsuppression of currents flowingthrough voltage gated K+ channelstend to mitigate the stabilizing effect ofK+ currents on resting membranepotential and to reduce after–hyperpolarization and thus to enhanceneuronal excitability.The acid sensing ion channels (ASICs)are non-selective cation channels that
are directly gated by extracellularprotons (Waldmann et al., 1997).ASICs are widely expressed in theCNS (Waldmann et al., 1997; de laRosa et al., 2003) where they modulateneuronal excitability (Vukicevic andKellenberger, 2004). Recently, ASICswere shown to be involved in seizuretermination in an animal model wherethe gene encoding ASIC1a protein wasdisrupted (Ziemann et al., 2008).The high sensitivity of ligand-gated ionchannels to extracellular pH furthersupports the important modulatory roleof protons in synaptic transmission.NMDA receptor channels are ofteninhibited (Tang et al., 1990; Traynelisand Cull-Candy, 1990) whereasGABAA responses are increased upona fall in pHo (Takeuchi and Takeuchi,1967; Pasternack et al., 1996). The pHsensitivity of glutamate- and GABA-mediated transmission can differmarkedly because different subunits ofpostsynaptic ligand-gated channelsshow varying sensitivity to pH(Traynelis et al., 1995; Krishek et al.,1996; Wegelius et al., 1996). Eventhough the steep pHo dependency ofGABAA and NMDA channels has beenrecognized for several decades, theamount of data supporting thefunctional impact of e.g. activity-evoked pHo transients on synaptictransmission is strikingly sparse (Tairaet al., 1993;Gottfried and Chesler,1994;Tong et al., 2006;Fedirko et al.,2007; Makani and Chesler, 2007).Nevertheless, because the pHodependence of synaptic transmissiontakes place at proton concentrationsclose to the physiological pH range,the ability of endogenous pH transientsto modulate GABAA and NMDAreceptor mediated synaptictransmission is of much potentialimpact (Chesler and Kaila, 1992;Gottfried and Chesler, 1993; Makaniand Chesler, 2007).Cellular communication via electricalsynapses, gap junctions, is affected byintracellular pH with cytoplasmicacidification closing and alkalinizationopening the channels (Spray et al.,1981;Spray and Scemes, 1998; Churchand Baimbridge, 1991). During earlydevelopment principal neurons in e.g.rat neocortex are extensively coupled
25
via gap junctions (LoTurco andKriegstein, 1991). However,experiments on the adult rathippocampus have repeatedly revealedelectrical synapses among inhibitoryinterneurons but not betweenexcitatory cells (Michelson and Wong,1994; Hormuzdi et al., 2001;Connorsand Long, 2004 but see Schmitz et al.,2001).
2.4.2.1 Activity-induced pHtransients in neurons and in theextracellular space
In addition to the metabolic activityand the constant tendency ofconductive leaks to impose a chronicintracellular acid load, electricalactivity of neurons has been shown toinduce rapid and robust pH changes.The underlying mechanisms vary, butrapid pH shifts are largely generatedby transmembrane fluxes of acidequivalents.Intracellular pH measurements fromcultured mammalian neurons haveshown that glutamate-inducedmembrane depolarization results in anintracellular acidification (Hartley andDubinsky, 1993). In experiments withprolonged-glutamate exposure it wassuggested that the glutamate-inducedpHi decline may, in concert with theincreased calcium levels, mediateexitotoxicity. Furthermore, bursts ofaction potentials result in anintracellular acidification in neurons ofrat brain stem slices (Trapp et al.,1996). The effect of membranedepolarization on pHi occurs in theabsence of CO2/HCO3
- and is probablydirectly linked to the depolarization-induced entry of Ca2+ ions. Thesubsequent extrusion of Ca2+ by Ca2+-H+-ATPase has been suggested toexplain the activity-induced decreaseof pHi (Paalasmaa et al., 1994; Smithet al., 1994; Trapp et al., 1996).The pHi shifts associated withinhibitory GABA-mediatedneurotransmission are caused by anentirely different mechanism. Asdiscussed earlier on, GABAA andglycine receptor channels arepermeable to HCO3
-, and it is theefflux of HCO3
- and influx of CO2 that
produce the intracellular acidification(and extracellular alkalinization) uponGABAA receptor channel opening(Kaila and Voipio, 1987; Chen andChesler, 1990, Kaila et al., 1990; Chenand Chesler, 1991; Voipio et al., 1991;Pasternack et al., 1993; Luckermann etal., 1997).The pH shifts evoked by synaptictransmission and neuronal activity arenot limited to the intracellular space. Apronounced extracellular alkalinizationis the immediate pH response uponhippocampal Schaffer collateralstimulation (Jarolimek et al., 1989;Walz, 1989; Chen and Chesler, 1992a;Chesler and Kaila, 1992). The initial,fast alkalinization evoked by neuronalactivity is frequently followed by aslower and longer lasting acidification(Jarolimek et al., 1989; Chen andChesler, 1992a; Kaila et al., 1992) thatcan be, at least under certainexperimental conditions, accounted forby a rise in tissue PCO2 (Voipio andKaila, 1993).Both excitatory and inhibitoryneurotransmission are involved in thegeneration of the extracellular alkalinetransients, the relative contribution ofglutamatergic and GABAergiccomponents depending on the patternof stimulation (Taira et al., 1995). Inthe rat hippocampus exogenouslyapplied GABA or direct stimulation ofpharmacologically-isolated inhibitorypathways produced a fast, HCO3--dependent alkalosis (Chen and Chesler,1992a; Kaila et al., 1992; Voipio et al.,1995). The pHo transients wereabolished with GABAA-channelblockers and after inhibition ofextracellular CA activity. Thus, theGABAergic extracellular alkaline shiftis strictly dependent on the presence ofCO2/HCO3
- and on interstitial CAactivity (Kaila and Voipio, 1987;Saarikoski and Kaila, 1992), and it iscaused by a transmembrane GABAAreceptor channel mediated CO2/HCO3
-
shuttle (Kaila et al., 1990; Chen andChesler, 1991; Kaila et al., 1992).Extracellular alkalinization evoked byexcitatory synaptic transmission isfundamentally different from theGABA-mediated pHo increases.Repetitive stimulation of Schaffercollaterals or pressure injection of
26
glutamate (or its agonists) inhippocampal CA1 area evoked aCO2/HCO3
- independent pHo increasethat was amplified upon CAo inhibition(Chen and Chesler, 1992b; Voipio etal., 1995; Taira et al., 1995). In thisHCO3
--independent, glutamate-mediated alkaline shift the role ofglutamate uptake (Amato et al., 1994),Ca2+- H+ -ATPase (Paalasmaa et al.,1994; Smith et al., 1994; Grichtchenkoand Chesler, 1996) and H+ channel(Thomas and Meech, 1982; Thomas,1988) activity have been studied, butthe mechanism behind thisalkalinization has not been firmlyestablished.
2.4.2.2 CA activity facilitatestransmembrane ion fluxes
Proton diffusion can be rate limiting inprocesses involving a molecular sinkor source of protons and in movementsof other acid-base equivalents (Geerset al., 1985; Wetzel and Gros, 1998;Voipio, 1998). In line with this, animportant role of CAo activity in lacticacid transport was reported in ratskeletal muscle cells (Wetzel et al.,2001) and later in hippocampalneurons and astrocytes (Svichar andChesler, 2003). In view of thepostulated astrocyte-neuron lactate-shuttle (Magistretti and Pellerin, 1999),brain CAos may turn out to be of greatimportance for neuronal energy supply.
CAo activity affects the function ofHCO3
--transport proteins (Seki andFromter, 1992; Alvarez et al., 2005;Becker and Deitmer, 2007; Svichar etal., 2007). The coupling of CA activityto BTs is suggested to result in moreefficient HCO3
- transport. The closelyassociated CA and BT are thought toform a complex, a transport metabolon,where the sequential pathway providesan efficient mechanism for BTmediated HCO3
- fluxes (Sterling et al.,2001; Sterling et al., 2002; but see Luet al., 2006; Piermarini et al., 2007).
2.4.3 Studies on mouse modelswith genetically impairedchloride and pH regulation
KO mice have provided useful modelsin studies of the specific roles ofdifferent ion regulatory proteins in theCNS. Notably, abnormalities in eitherthe function and/or membranetargeting of certain members of theSLC4, as well from the SLC12A andCA, gene families cause geneticdiseases in humans (see below).The outcomes of the CCC KO mice arein most cases far more deleterious thanthose of the BTs or CAs, which ingeneral have an apparently normalphenotype. At the time when Hübner etal. (2001) and Woo et al. (2002)generated their KCC2 KO mousestrains, only one KCC2 isoform (nowtermed KCC2b) had beencharacterized. The recent finding of thenovel KCC2a isoform (Uvarov et al.,2007) provides an explanation to thedifferent outcome of the two KOstrains and indicate distinct functionsof the two isoforms. Full KCC2 KOmice (i.e. KCC2a and KCC2b KO) dieimmediately after birth due torespiratory failure (Hübner et al.,2001b) while the selective KCC2b KOhas a life span of about two weeks anddies possibly because of spontaneousgeneralized seizures (Woo et al.,2002). Comparison of these two KCC2KO mouse strains implies that KCC2aexpression is vital for motor controlwhile KCC2b is responsible for thehyperpolarizing GABAergic responsesseen in adult cortical neurons (but seealso Balakrishnan et al., 2003). KCC2hypomorphic mice that have 15-20%of the normal KCC2a and KCC2bprotein levels are viable but showreduced seizure susceptibility andaltered behaviour in tests measuringlearning, anxiety, and nociception(Vilen et al., 2001, Tornberg et al.,2005).The two KCC3 KO mouse linesdeveloped by Howard et al (2002) andBoettger et al. (2003) both showmotor dysfunction caused by theprogressive degeneration of peripheralnervous system (PNS). The KCC3 KOmice generated by Boettger et al.
27
(2003) also show degeneration ofCNS, inner ear defects, abnormalelectrocortico-gram, and reducedseizure threshold. The degeneration ofcells is likely due to the impairment ofcellular volume and Cl- regulation aswell as to the changes in the relativeextra- and intracellular ion levels(Boettger et al., 2003). Apart from thelack of corpus callosum degeneration,the KCC3 KO phenotype replicates thesymptoms of the human Anderman’ssyndrome (Howard et al., 2002).KCC4 (Boettger et al., 2002) andNKCC1 (Delpire et al., 1999; Dixon etal., 1999; Flagella et al., 1999; Pace etal., 2000) KO mice both have non-neuronal inner ear defects and sufferfrom distinct physiologicalmalfunctions but the gross morphologyof the CNS is surprisingly normal.
The relevance of AE3 in seizuresusceptibility was addressed inexperiments where the Slc4a3 genewas disrupted (Hentschke et al., 2006).Adult AE3 KO mouse had anapparently normal phenotype butshowed a reduced susceptibility topharmacologically-induced seizures.However, in the febrile-seizure model(Schuchmann et al., 2006) the time oftonic-clonic seizure onset in juvenile,P14-15, AE3 KO pups was similar tothat seen in the wild type littermates(E.Tolner, J.Voipio, and K.Kaila,unpublished observations). AE3 isactivated at high pHi values and iscommonly suggested to mediatecellular recovery from alkaline load(Chesler, 2003). Since the predominantpHi response to neuronal activity is anintracellular acidosis, the degree towhich the reduced seizuresusceptibility seen in adults is due toimpaired, AE3-mediated pHiregulation is not known. Deletion ofanother BT, the NBCn1, resulted in aspecific visual and auditory phenotypeof the animal model (Bok et al., 2003).The selective degeneration of sensoryreceptors in the eye and inner ear in theNBCn1 KO mice are characteristic forUsher syndrome in humans (Petit,2001).
Many cells express more than one ofthe 12 enzymatically active CA
isozymes (Supuran et al., 2004). Toclarify their individual contributions,five selective KOs and one double KOhave been developed. Because CAIIIand CAIX are mainly expressed inskeletal muscle (Kim et al., 2004) cellsand gastrointestinal tract (Hilvo et al.,2004), respectively, these KO micewill not be discussed.CAIV and CAXIV have beensuggested to be the two predominantCAo isoforms expressed in the brain(Tong et al., 2000; Parkkila et al.,2001). The development of CAIV andCAXIV KO mice, and byintercrossing, a CAIV/CAXIV double-KO, provided further support for theprevious findings (Shah et al., 2005).The double KO animals had noobvious phenotype and appearednormal except for growth retardationand slightly distorted sex ratio of theoffspring. Knowing the high pHsensitivity of ligand-gated ionchannels, an interesting subject forfuture studies is to examine whetherthe impaired ECF buffering of theseanimals has significant effects on e.g.NMDA- and GABAA-channelmediated transmission.A mouse mutant lacking CA II wasdeveloped by Lewis et al., 1988). Inthis animal model for human CAII-deficiency syndrome, the symptomsinclude systemic and renal tubularacidosis, and growth retardation. Nosigns of osteopetrosis or braincalcification, as seen in CAII-deficientpatients, were noticed (Sly et al.,1983). This probably reflects speciesdifferences in the physiological rolesof CAII in osteoclast function and inthe maintenance of the acid-basebalance in the brain. It also points tolimitations of modelling humandisorders using transgenic mice.The antiepileptic effect of CAinhibitors has been proposed to dependon a PCO2 increase that results in extra-and intracellular acidification (e.g.Millichap et al., 1955; Bickler et al.,1988; Leniger et al., 2002). Thus, thepossible effect of the systemic acidosisobserved in the CAII KO animals onseizure susceptibility was tested(Velisek et al., 1993). Mice fromheterozygous breeding pairs were usedin experiments in which the time of
28
seizure onset was measured, and theseverity of the pharmacologically-induced and audiogenic seizures wererated. In comparison to the wild typeand heterozygote mice, CAII KOanimals had an increased latency to theonset of pharmacologically inducedseizures and showed reduced incidenceof both clonic and tonic-clonicseizures. A decrease in the incidence ofaudiogenic seizures in KO animals wasseen only if animals were primed at anearlier developmental stage.Surprisingly, a similar seizureresistance was not observed in in vitroexperiments, where spontaneousepileptiform activity was induced inbrain slices by withdrawal ofextracellular Mg2+ (Velisek et al.,1995). In contrast, slices from CAIIKO mice had a shorter latency to theonset of epileptiform activity whichalso developed faster into sustained,status-like epileptiform activity thatwas not inhibited by increasing theCO2 concentration.
2.5 Ion measurements inthe mammalian brainThere are several means for monitoringof ion concentrations in livepreparations (i.e. radio-isotopic tracerand atomic absorption methods) butonly two techniques enable direct,continuous recordings of ionic changesat the cellular level. These methods areion-sensitive microelectrodes (ISMEs)and optical indicators.The technical advancement of both ofthe methods during the last decadeshas enabled researchers to performmeasurements with higher selectivityand with better temporal and spatialresolution and hence, to examine ionhomeostasis in the brain moreaccurately. Especially fluorescentindicators (e.g. the genetically encodedCl- indicator Clomeleon; Kuner andAugustine, 2000) and the equipmentfor detection of optical signals (e.g.two-photon imaging; Williams et al.,1994) have been a rapidly developingfield. Despite the great advances, bothtechniques have their limitations andrecordings of ion transients from e.g.
extracellular microdomains are achallenge for the future.
2.5.1 Ion-sensitivemicroelectrodes
The commonly used ISMEs are glassmicroelectrodes with an organic liquid-membrane solution forming aselectivity barrier at the very tip of theelectrode (Voipio, 1998). The liquid-membrane at tip of the microelectrodebehaves as an ion-selective barrier sothat the voltage-difference across itdepends only on the permeant ion.Today there are commerciallyavailable, ready-to-use membranesolutions for many different ions,including H+, K+, and Cl-. Ion-sensitiveelectrodes can also be made usingglass that has ion-sensitive properties.However, ion-selective glass is onlyavailable for measurements of H+ andNa+. The use of ion-sensitive glass isalso limited because these electrodeshave considerably large tip-diameteror, if the recessed tip configuration isused (Thomas, 1974), the resistance ofthe electrodes is high and the responsetime long.Because of the small dimensions ofmammalian neurons and glial cells, theuse of liquid-membranemicroelectrodes is under in vitroconditions mainly limited toextracellular ion measurements inbrain tissue preparations. Neurons andglial cells seem to tolerate ionmeasurements with ISMEs somewhatbetter under in vivo conditions, andbrief recordings of intracellular Ca2+
and H+ concentrations have beenperformed in rats (Chesler and Kraig,1987; Chesler and Kraig, 1989; Silverand Erecinska, 1990) and cats(Ballanyi et al., 1994). However, e.g.many invertebrate nerve cells and thegiant axon of the squid are so large andeasily accessible that ISMEs can beused for long-lasting intracellular ionmeasurements in them (Voipio, 1998).ISMEs provide a valuable tool forintracellular ion measurements as theyare highly sensitive and selective forthe permeant ion. The calibration ofISMEs is usually fairly simple and theion activity is measured strictly from
29
the cytoplasm with no “contamination”from possible organellar ioncompartments. The major limitationsof the ISMEs include the possibleinterference of lipophilicpharmacological substances with theorganic liquid-membrane (and hencewith the calibration), the susceptibilityof the electrodes to noise due to thehigh electrical resistance of theselectively permeable membrane, andtheir slow response time. Recently,Fedirko et al., 2006) modified thepreviously established low impedancecoaxial liquid ion-exchangermicroelectrodes to prepare concentricCa2+ and H+ sensitive microelectrodes.These electrodes had significantlyshorter time constants than theconventional ISMEs and hence providea better accuracy for detection of iontransients with rapid kinetics. Theability of the conventional K+
sensitive, liquid-membranemicroelectrodes, used for instance inthe Study II in this Thesis, to detectfast K+ transients has been previouslyverified. The response time of the K+
sensitive microelectrodes has beenevaluated by Santhakumar et al.,2003). These authors used a fastcalibration system and showed that theK+ sensitive microelectrodes coulddetect changes in [K+]o lasting lessthan 500 ms. Kaila et al. (1997)measured glial membrane potential,known to selectively reflect changes in[K+]o (Lothman and Somjen, 1975), toconfirm the temporal resolution ofISMEs used in HFS-induced K+
transient measurements. ISMEs willinevitably cause some damage whenplaced into the preparation. A deadspace surrounding the tip of theelectrode will lead to a diffusion delayand false averaging of the signal.Reduction of the tip diameter to, orbelow, 2-4 m effectively minimizesthe damage and prevents the distortionof rapid K+ signals (Ransom et al.,1987; K Lamsa, K Kaila and J Voipio,unpublished observations).
2.5.2 Fluorescent indicators
The ability of fluorescent indicators tovisualize changes in ion activities is
based on the specific sensitivity oftheir fluorescence excitation oremission-spectra to a certain ion. Sincethe development of modern fluorescentindicators in the early 1980s (Rink etal., 1982), the use of ion-sensitive dyeshas become a more and more popularmethod for continuous measurementsof ions in small cells and in cellpopulations. The response time of thisrecording technique is usually notlimited by the properties of theindicators but rather by the propertiesof the hardware. Thus, fluorescent dyesprovide a selective and very fast (downto a millisecond time-scale) method forintracellular ion measurements.Because fluorescent indicators arechemical substances that areintroduced into the cell(s), there areseveral aspects that are essential for agood ion-sensitive fluorescent dye. Theproperties of the widely used pHindicator BCECF, introduced by RogerTsien (Rink et al., 1982), will be takenas an example.
2.5.2.1 Sensitivity and ratiometricquantitation of the indicator
Ion-induced spectral shifts are moreinformative for detection andquantification of ion transients thanwhat are the changes in magnitude ofpeaks (Yuste et al., 2000). In the caseof BCECF fluorescence emission issteeply dependent on pH atwavelengths around 500 nm but ispractically insensitive at onewavelength (at 440 nm). The ion-insensitive wavelength is called theisosbestic point. The ratio of these twointensities (I) is sensitive to pH but,because I440 nm depends only on theconcentration of the dye, otherparameters e.g. volume changes,leakage or photobleaching of the dye,are excluded from the ratio. In somedyes the ratio can also be taken fromtwo intensities with opposite ion-sensitive responses (e.g. the Ca2+
sensitive indicator FURA) as this givesa better signal-to-noise ratio. If anisosbestic point can not be used,pseudoratiometric measurements aresometimes possible. This protocolcombines two fluorescent indicators,
30
one of which is sensitive to the ion andone only to the dye concentration assuch.Here it should be emphasized thatthere is a clear need for a selective andratiometric Cl- dye. The problems withthe quinoline-based, non-ratiometricCl- dyes available at the moment aretheir instability (Lucigenin, MQAE),sensitivity to other anions (SPQ),required processing before non-invasive loading (MEQ), and most ofall, the movement of water associatedwith Cl- fluxes. The movement ofwater unavoidably results in cellularvolume changes that will affectfluorescence signal due to changes inion concentrations and movement ofthe cell with respect to the region ofinterest in the fluorescent measurement(E.Ruusuvuori, K.Kaila, and J.Voipiounpublished results). The recentlydeveloped approach using geneticallyencoded fluorescent proteins sensitiveto Cl- have so far not provided thedesired resolution to the problem. Theyellow fluorescent protein (Wachterand Remington, 1999) and itsderivatives (Jayaraman et al., 2000;Galietta et al., 2001) have been usedfor intraneuronal Cl- monitoring(Slemmer et al., 2004; Kruger et al.,2005) but they are markedly sensitiveto other anions and protons, and theyare non-ratiometric. The constructionof a fusion protein (Clomeleon), theyellow fluorescent protein with the Cl-
insensitive cyan fluorescent protein byKuner and Augustine (2000) hasallowed fluorescence-resonance-energy-transfer (FRET) -basedratiometric measurements of [Cl]i inneurons and retinal cells of thetransgenic animals (Kuner andAugustine, 2000; Duebel et al., 2006).Markova et al., 2008) have modifiedthe fusion protein in order to improvethe low Cl--sensitivity of the indicator.However, the construct still exhibits asignificant pH-sensitivity over a broadrange of pH values (see also Kuner andAugustine, 2000). This property shouldbe recognized, especially if theindicator is used in measurements ofGABAA-channel or AE mediated Cl-
fluxes, both of which are associatedwith pHi changes.
2.5.2.2 Fluorescent indicators withinthe cell
One of the major advantages of somefluorescent indicators is that they canbe loaded into cells non-invasively.Converting the indicator into itselectrically neutral ester derivative andbathing the cells in this solutionenables the compound to cross the cellmembrane. Intracellular esterasescleave the ester groups and release thedye (with its charges now exposed)within the cell. In this way it ispossible to measure ionic changes notonly in one cell but even in a largepopulation of cells. If ester derivativesare not available the dye can beintroduced into the cell via a patch-pipette or using a single-cellelectroporation technique (Khirug etal., 2008). The retention of the chargedform of the indicator within the cell(detected on the basis of fluorescenceintensity at the isosbestic wavelength)can be used as an indicator ofmembrane integrity if the possibleleakage and photobleaching are takeninto account (Bevensee et al., 1995).There are some clear weaknesses offluorescent indicators, in comparisonto ISMEs. The cell membranepermeable ester form of the indicatorcan also move through membranes ofcellular organelles and hence, themeasured signal will be a combinationof the cytoplasmic and organellar ionconcentrations. Furthermore, eventhough the high selectivity of theindicator is its fundamental property,the selective binding of the measuredion to the indicator can result inbuffering of the measured ion (Neher,2000). With ions with low absoluteconcentrations (like Ca2+ and protons)fluorescent indicators can function asmobile buffers and facilitate thediffusion of the measured ion. Thecalibration of ratiometric fluorescentindicators is not as straightforward asthe calibration of ISMEs and with thenon-ratiometric indicators a reliablecalibration is often impossible toobtain (Thomas et al., 1979; Eisner etal., 1989). Finally, the use of high-intensity excitation light in a livepreparation (e.g. in confocal laser
31
microscopy) can induce photodamagedue to phototoxic products, such as thehighly reactive singlet oxygen andother free radicals.
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3 Aims of the study
The present work aimed in specific:
1) To characterize the molecular mechanism that renders GABAA receptor-mediated responses hyperpolarizing during maturation in rat hippocampalpyramidal neurons. First we studied the developmental expression pattern ofthe putative Cl- extruder KCC2 on both mRNA and protein level. After thisprimary finding we aimed to show that the ontogenetic change in EGABA-A fromdepolarizing to hyperpolarizing has a causal link to the developmental up-regulation of KCC2 (I)
2) To identify the cell type expressing the intracellular CA activity that isessential for the HFS-induced, depolarizing GABAA-receptor mediatedresponses in mature hippocampus. Thereafter, the aim was to examine thepostnatal development of intrapyramidal CA activity, to identify the cytosolicCA isoform(s), and to study its (their) role as key molecule(s) in thegeneration of the tonic GABAergic excitation and the underlying [K+]otransients that provide the drive for the HFS-induced afterdischarges (II)
3) To examine the localization and function of the Na+-driven Cl--HCO3-
exchanger NCBE in the mouse CNS. In order to address the role of NCBE inphysiological and pathophysiological processes we generated mice withtargeted Slc4a10 gene disruption (III)
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4 Experimental procedures
Table 4. The methods used in the Studies I-III of this Thesis. The methods are listedhere in order of appearance, and described in detail in the original publications. Thecontribution of the author in the experimental work is described on the page where theoriginal publications are listed.
All experiments were approved by the Ethics Committee for Animal Research at theUniversity of Helsinki. In the three original publications a total number of 356 rats(Studies I and II), 24 guinea pigs (Study I), and 305 mice (Study III) were used. Thefunctioning and development of ion-regulatory proteins of hippocampal CA1 andCA3 pyramidal neurons was the main focus of this Thesis (see Fig 3.).
Methods
Northern blot analysis I IIIRT-PCR I III(Anti)sense oligodeoxynucleotide-exposed hippocampal slice cultures IImmunoblotting I IIIIn situ hybridization I II IIIIntracellular recordings with sharp electrodes I IIExtracellular field potential measurements II IIIExtracellular K+ measurements with ion-selective microelectrodes IIIntracellular ion measurements with fluorescent indicators I II IIIGeneration of Slc4a10 knockout mice IIIGeneration of Slc4a10 antibody IIIImmunohistochemistry IIIBehavioral testing IIIEpileptiform seizure induction IIIMRI analysis III
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The evaluation of the applicability offluorescent chloride indicators for thepresent study
In Study I of this Thesis we aimed tomeasure the intracellular Cl-
concentration before and after KCC2down regulation. However, because ofthe calibration-limitations of thesingle-wavelength Cl- dyes suchexperiments were not possible toperform reliably. Hence, we tried tocarry out measurements to demonstratethat the intracellular Cl- fluxes inpyramidal neurons were affected bythe down-regulation of KCC2 inphosphorothonate-protectedoligonucleotides A (PODN A) treatedhippocampal slices. We tookadvantage of the fact that KCC2operates close to its thermodynamicequilibrium; by raising the [K+]o wecould reverse the transporter andmeasure the change in the [Cl-]i. Forthis purpose we loaded the pyramidalcells with the Cl- sensitive dye MEQ(and in some experiments withMQAE) (see e.g. Galeffi et al., 2004;Chub et al., 2006). The detection of Cl-in both of these dyes is basedcollisional quenching and thus, an
increase in [Cl-] will bee seen as adecrease in the fluorescence.Raising [K+]o from the 3 mMconcentration used in the controlsolution to 9 mM resulted in a decreasein the fluorescence that was larger inthe control slices than in the slicestreated with PODN A (Figure 4a).These results suggested that an KCC2mediated increase in [Cl-]i could beevoked by raising extracellular K+
concentration. Unfortunatelymeasurements using the pH sensitivedye BCECF showed that this approachcaused significant volume changes inpyramidal neurons (Figure 4b). Thefluorescence detected at the isosbesticpoint (I440 nm) decreased markedlyupon increase in [K+]o. It is likely, thatthe change in the direction of KCC2transport results in an influx of Cl- thatis accompanied by an influx of water.Thus, the signal from the Cl- sensitiveindicator is reduced both due tocellular swelling and increased [Cl-]i.As already mentioned, cellularswelling will affect the fluorescencesignal due to changes in ionconcentrations and because of themovement of the cell with respect tothe region of interest used in analysis
Figure 3. Line drawing of a transverse section of the hippocampus illustrating theorientation of granule cells in the dentate gyrus (DG) and pyramidal cells in theCA3 and CA1 fields of the hippocampus. Modified from Fig. 6 in Witter andAmaral, 2004, courtesy of Elsvier Academic Press, San Diego.
35
of fluorescence intensity in themeasurement. Quenching of thequinoline-based Cl- indicators is notaccompanied by a spectral shift,excluding the possibility of directratiometric measurements.Pseudoratiometric measurements withan ion/pH insensitive indicator likecalcein (see e.g. Yamada et al., 2001)could be an option, but the instabilityof many Cl- dyes (Biwersi andVerkman, 1991; Chub et al., 2006)complicates reliable estimation on therelative contributions of volume andCl- changes to the signal obtained,
especially in long-lasting recordings.Furthermore, because the relativechange in the fluorescence intensitywas roughly similar in amplitude withCl- sensitive dye and BCECF (I440)(Fig. 4), the signal-to-noise ratio in thepseudoratiometric signal was too lowfor detection of a change in [Cl-]i. Forthese reasons the Cl- indicators turnedout to be unsuitable for the presentstudy.
.
Figure 3. Intracellular fluorescence measurements from hippocampal pyramidalcells with the Cl- and pH indicators MQAE and BCECF, respectively.A) Raising [K+]oto 9 mM induced a more prominent change in MQAEfluorescence (apparent increase in[Cl.-]i, excitation at 350 nm, emission at 460 nm)in semi-organotypic control slices (dark grey trace) than in slices exposed toPODN A (light grey trace). B) A recording from acute hipopcampal slice with theisosbestic wavelength of BCECF (excitation at 440 nm, emission at 520 nm)revealed a response upon an increase in [K+]o that was strikingly similar to thatshown in (A) and most likely reflects a change in cellular volume (E.Ruusuvuori,unpublished results). The values on the y-axis are the absolute emission intensities.Time calibration 500 s (A) and 250 s (B).
9 mM K+ 9 mM K+
MQAE BCECF, I440
155
165
115
125A
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5 Results and Discussion
5.1 The K+-Cl-
cotransporter KCC2renders GABAhyperpolarizing duringneuronal maturation (I)From previous studies it was knownthat the K+-Cl- cotransporter KCC1was ubiquitously expressed in differentmammalian tissues (Gillen et al., 1996)while KCC2 expression was restrictedto brain (Payne et al., 1996). In theirarticle Payne et al. (1996) alsoanalyzed the expression of KCC1 andKCC2 mRNAs in cultured glial cellsas well as in tissue samples from wholerat brain. KCC1 was present in pureglial samples whereas co-expression ofthe two transporters was seen in wholebrain samples. This result indicatedthat KCC2 expression was most likelyabsent from glia.The spatiotemporal expression patternof KCC2 was studied in the postnatalrat brain and the role of this transporterin setting the hyperpolarizing GABAA-receptor mediated responses inhippocampal pyramidal neurons wasassessed. The expression of KCC1 andKCC2 mRNA in postnatal rathippocampus was followed usingNorthern blot analysis. There washardly any KCC2 present in the P0 rathippocampus but a marked increase inthe KCC2 mRNA levels occurredbetween P0 and P5. The KCC2expression continued to increase, andby the end of the second postnatalweek it had attained a level similar tothat seen in the mature animals. Incontrast to the steep postnatalupregulation of the KCC2, aconsiderable KCC1 expression wasdetected already at birth. There was nomarked increase in the brain’s KCC1expression levels during the postnataldevelopment.Southern blot analysis of single-cellRT-PCR was used to examine theexpression of KCC1 and KCC2 mRNAin 16 neurons, identified by theirexpression of light neurofilament. All
16 neurons were strongly positive forKCC2. Notably, in the two cells aweak KCC1 signal was evident onlyafter increasing the exposure time ofthe film from 4 hours to 48 hours.Later studies have confirmed the glialexpression of KCC1 and in accordancewith our results, suggested that there islittle, if any, KCC1 in mature neurons(Kanaka et al., 2001; Li et al., 2002).From the two remaining KCCisoforms, KCC3 and KCC4 (Mount etal., 1999), only KCC3 has been shownto be abundantly present in thepostnatal rodent CNS (Pearson et al.,2001; Boettger et al., 2003). Studies onKCC3 KO mice have pointed to a roleof this KCC isoform in themaintenance of Cl- homeostasis andcellular volume in some adult neurons(Boettger et al., 2003).The GABAA-receptor responses inadult hippocampal pyramidal neuronsare predominantly hyperpolarizing(Kaila, 1994; Ben-Ari et al., 1989;Khazipov et al., 2004). The suggestedrole of a KCC mediated Cl- extrusionin the maintenance of low [Cl-]i inthese cells (Misgeld et al., 1986;Thompson et al., 1988a; Thompson etal., 1988b) is in good agreement withour results showing a high expressionof KCC2 in adult rat hippocampus. Itshould be noticed however, that a lowintraneuronal Cl- level is notnecessarily associated withhyperpolarizing IPSPs. First, theHCO3
- permeability of GABAA-receptors makes EGABA-A less negativethan ECl, and second, the polarity ofIPSPs is dependent not only on thelevel of EGABA-A but also on the levelof resting Vm (see e.g. Kaila et al.,1993).The DRG cells are a classical exampleof PNS neurons that maintain a high[Cl-]i concentration and depolarizingGABA responses into adulthood(Deschenes and Feltz, 1976). The Cl-
accumulation in these cells is at leastpartially mediated by NKCC1 (Plotkinet al., 1997; Price and Trussell,2006;Rocha-Gonzalez et al., 2008). Tosee if the KCC2 mediated intracellularCl- extrusion is strictly characteristic toadult neurons with hyperpolarizingGABAA-receptor response we
37
examined KCC2 expression in DRG.Indeed, the RT-PCR resultsdemonstrated that KCC2 expressionwas non-existing in DRG but the“house-keeping” isoform KCC1 wasstrongly expressed in DRG cells. Thereversal potential of GABAA-mediatedsynaptic responses has later beenshown to reflect the presence offunctional KCC2 also in the matureCNS. Gulacsi et al. (2003)demonstrated that within the ratsubstantia nigra, the KCC2 expressingneurons had significantly morehyperpolarizing GABAA-mediatedsynaptic responses than the KCC2-negative neurons. In this context itshould be noted that the evidence ofKCC2 expression does not necessarilyimply functional KCl extrusion(Khirug et al., 2005).From a developmental point of view itwas of interest to see if KCC2expression was a more generalindicator of neuronal maturation (seeLi et al., 2002). To test this we usedguinea pig pups because they are, ifcompared to rat pups, born at a muchmore developed stage. The newbornguinea pigs have physical features andbehavior resembling those of a roughlytwo week old rat pups. Interestingly, inNorthern blot analysis hippocampalKCC2 mRNA showed high levelsalready in E42 (full-term gestation day68) guinea pig hippocampi and thelevels did not further increase duringthe early postnatal life.To study in more detail the localizationof KCC2 mRNA in the developing ratbrain we performed in situhybridization with 35S-labelled KCC2antisense RNA probes in sagittal brainsections from E20 to P16 animals.KCC2 mRNA was absent from E20 rathippocampus. At P0 there were someindividual labeled neurons but at P5 aclear signal was seen in the wholehippocampal pyramidal cell layer.There was still a more gradual increasein the expression during the secondpostnatal week after which a steadylevel of expression was attained.KCC2 was expressed in different brainareas, or within a given area, strictlyaccording to the state of maturation,e.g. with KCC2 expression in the brainstem preceding that of cortical
structures and within the dentate gyrusKCC2 expression in the dorsal bladepreceding that of the ventral blade.Later, Li et al. (2002) have providedevidence that in prenatal rats and micethe spatiotemporal expression patternof KCC2 mRNA likewise followsneuronal maturation. Duringembryogenesis, KCC2 was shown tobe undetectable in areas whereneurogenesis or neuronal migrationtook place but co-localized with cellsthat were positive to a marker ofneuronal differentiation.The results of Study I discussed so farindicated that KCC2 is the main K+-Cl-
cotransporter in mature neurons.Consequently, KCC2 is likely tomediate the postnatally occurringreduction in neuronal [Cl-]i and hence,to be responsible for fasthyperpolarizing inhibition seen inmature hippocampal neurons. To gainfurther support to this idea wecompared the voltage responsesevoked by the GABAA-receptoragonist muscimol in acutehippocampal slices duringdevelopment as well as after a specificdown-regulation of KCC2 in short-term organotypic slice cultures.Voltage responses were measured withsharp microelectrodes filled with asolution that did not impose a Cl- loadon the neurons.In P12-30 rat pyramidal neuronsiontophoretic application of GABAA-receptor agonist muscimol resulted in ahyperpolarization with a mean drivingforce (DFGABA-A, defined here asEGABA-A- Vm) of -9.6±0.9 mV. In P0-4rat pyramidal neurons, whichaccording to the results describedabove do not express KCC2, themuscimol-evoked responses wereclearly depolarizing (7.0±3.3 mV). Insharp contrast, pyramidal neurons inP0-4 guinea pig slices hadhyperpolarizing responses (-10.6±2.4mV) similar to those seen in moremature animals (P17-40; -9.0±3.3mV). While these developmentalstudies clearly suggested a causal linkbetween the KCC2 mRNA expressionand the transition from depolarizing tohyperpolarizing GABAA-receptorresponses, we still aimed at obtainingcausal evidence for this by
38
manipulating the KCC2 mRNA, andthus the protein, levels in juvenilehippocampal slices in vitro (P11-13, 2-5 DIV). For this purpose, we generatedthree distinct antisenseoligonucleotides (ODNs A, B, and C)against KCC2 mRNA. Immunoblotanalysis of KCC2 levels in the short-term cultured hippocampal slicestreated with the phosphorothonate-protected antisenses (PODNs A-C) andthe unprotected ODN A (8-15 hours)revealed that treatment with PODN Aresulted in the largest down-regulationof KCC2 levels. This down-regulationof KCC2 was accompanied by a shiftto only slightly hyperpolarizingDFGABA-A values, confirming the strictdependency of hyperpolarizingpostsynaptic inhibition on functionalactivity of KCC2. In slices exposed toPODN A the mean DFGABA-A was (-2.8±0.8 mV) whereas in control slicesand in slices treated with sensecounterparts of PODN A the meanvalues were significantly morehyperpolarizing, -10.9±0.8 mV and -10.3±1.7 mV, respectively.Taken together, the results of Study Iprovided direct evidence for the long-postulated, critical role of K+-Cl-
cotransporter in intraneuronal Cl-
homeostasis. The increase in KCC2mRNA in the rat hippocampalpyramidal neurons during the first twopostnatal weeks reflects neuronalmaturation and is necessary for the fastGABAA-receptor mediatedhyperpolarizing inhibition.
5.2. CA isoform VII acts asa molecular switch in thedevelopment ofsynchronous gamma-frequency firing ofhippocampal CA1pyramidal cells (II) Our group had previously shown thatin mature rat hippocampus the tonicexcitation driving HFS inducedsynchronous firing of CA1 principalcells is caused by a GABAergic,CO2/HCO-
3-dependent increase in[K+]o that is critically dependent on thefunctional activity of CAi (Kaila et al.,
1997; Taira et al., 1997; Smirnov et al.,1999). However, it was not known ifthe CAi activity that plays a crucialrole here originates form a certaincellular subtype. An intriguingquestion was whether the expression ofbrain cytosolic CA isoforms isdevelopmentally regulated; and if so,would this shape the ontogeny of HFS-induced tonic GABAergic excitationand synchronous neuronal firing.
Characterization of intrapyramidalcarbonic anhydrase expression andfunctional activity during postnataldevelopmentPreliminary experiments based on pHimeasurements suggested anupregulation of CA activity inpyramidal neurons at around the end ofthe second postnatal week (Ruusuvuoriet al., 1999). Therefore, the postnataldevelopment of intracellular carbonicanhydrase activity in CA1 and CA3pyramidal cells was followed. pHimeasurements from hippocampal slicesand from acutely isolated rathippocampal CA1 and CA3 pyramidalneurons were performed withfluorescence imaging using the pHsensitive indicator BCECF.Intracellular alkalinization was inducedby withdrawal of CO2/HCO3
- in theabsence and presence of the membranepermeable CA inhibitorethoxyzolamide (EZA; 30-100 M;Fig. 3 in the original publication). AtP0-8 the initial rate of change inintracellular pH (dpHi/dt) was alwaysslow and showed no sensitivity toEZA. These results were taken asevidence for the absence of cytosolicCA activity. During the secondpostnatal week the initial slope of thepHi change became faster and, inparallel with the faster kinetics of theevoked alkalinization, EZA started tocause a decrease in the dpHi/dt. Theappearance of functional CA activitydid not change steady-state pHi valuesof the pyramidal neurons. However, itis expected that pHi shifts arising fromCO2/HCO3
- fluxes, e.g. upon GABAAreceptor activation are more prominentin cells with functional CA activity(Pasternack et al., 1993). The lack ofcytosolic CA could indeed explain the
39
miniscule pHi changes (0.0016±0.002pH) evoked in hippocampal neurons by1 mM GABA in the experimentsperformed by Kuner and Augustine(2000). These authors used culturedneurons from E16 to P4 rathippocampi after 7-14 DIV.Two cytoplasmic, catalytically activeCA isoforms had been proposed tolocalize to the hippocampal pyramidalcell layer; CAII and CAVII (Lakkis etal., 1997; Wang et al., 2002b).Notably, the nucleotide sequence thatwas used in the design of the probeused by Lakkis et al. (1997) has notbeen published. These authors alsoreported CAVII expression in thechoroid plexus, a finding that has notbeen confirmed in latter publications(unpublished data of the authors of theStudy II, see also Halmi et al., 2006).The results from both radioactive andfluorescence in situ hybridizationsindicated that the appearance of thecytoplasmic CA activity inhippocampal pyramidal neurons,detected with the pHi measurements,coincided with the postnatal up-regulation of CAVII mRNA, but notwith that of CAII. The expression ofCAVII mRNA in all brain regions,including the hippocampus, was low atbirth and increased significantly duringthe second postnatal week. In juvenileanimals the most prominent CAVIIexpression was observed, in addition tothe neurons of cornu ammonis anddentate gyrus, in cerebellar Purkinjeneurons, olfactory bulb, cerebral cortex(with an especially intense labelling inthe piriform cortex), and somethalamic nuclei (E.Ruusuvuori,C.Rivera K. Kaila, and J.Voipio,unpublished observations).In contrast to the CAVII expression,we could not detect CAII mRNAexpression in hippocampal principal(or any other) neurons during the firsttwo postnatal weeks. At thisdevelopmental stage, data fromradioactive in situ hybridizationsindicate that CAII was stronglyexpressed only in choroid plexusepithelial cells. In the white matter,e.g. in the corpus callosum and in thewhite matter of cerebellum, CAIIexpression started after the secondpostnatal week. This up-regulation of
CAII coincides with the maturation ofoligodendrocytes and myelin formation(Suzuki and Raisman, 1994; Savaskanet al., 1999). In line with the previousreports, there was a diffuse CAII signalin the P15-22 hippocampal pyramidalcell layer (mainly in the CA1; see alsoHalmi et al., 2006; Wang et al., 2002b)and a more punctuate labelling of thecerebellar Purkinje neurons (see alsoNogradi et al., 1997;Wang et al.,2002b). Together the results fromfluorescence imaging and in situhybridizations indicate a strongpostnatal up-regulation of functionallyactive CAVII expression inhippocampal pyramids at around P12.
Developmental expression of the HFS-induced GABAergic depolarizationand extracellular potassium transientPrevious studies on the GABAAreceptor-mediated tonic excitation hadbeen done on hippocampal slices fromadult rats. As our results pointed to asimultaneous up-regulation ofintrapyramidal CA expression andfunctional activity, we next studied thepostnatal development of the HFSinduced tonic GABAergic excitationand the underlying [K+]o transients. Inall experiments, HFS evoked an initialhyperpolarization of CA1 pyramidalcells but only after P12 it was followedby a prominent depolarization, oftenassociated with action potential firing.The depolarization and the neuronalfiring were markedly suppressed by themembrane permeable CA inhibitorEZA. Taken together, these resultssuggested that the rapid replenishmentof HCO3
- catalyzed by cytosolic CAactivity (Kaila et al., 1990; Pasternacket al., 1993) in pyramidal neurons isinvolved in the generation of the HFSinduced GABAergic depolarization. Infull agreement with the idea that anactivity-induced increase of [K+]omediates the tonic GABAergicexcitation, the HFS-induced K+
otransients developed from small andEZA-insensitive shifts to large, over 7mM in amplitude, transients whichwere decreased upon CAi inhibition.Both the K+
o transients and theassociated membrane depolarization ofCA1 pyramidal neurons were
40
unaffected by bath application of theCAo inhibitor benzolamide (BA).Stimulation-induced changes in theextracellular space, due to cellularswelling, can result in local interstitialion accumulation (Dietzel et al., 1980).However, simultaneous measurementsof the extracellular volume fractionand [K+]o revealed only minimal HFS-induced changes in the volume of theextracellular space in comparison tothe evoked [K+]o transient. Therefore,it can be concluded that the activity-dependent increase in [K+]o underlyingthe tonic excitation results to largeextent (~ 93 %) from cellular netrelease of K+.Remembering that a largeintraneuronal Cl- load can be createdupon GABAA receptor activation (seechapter 2.4.1.2), it is interesting topoint out that extrusion of Cl- must becoupled to other ion fluxes in order tomaintain electroneutrality. After thesecond postnatal week, hippocampalneurons show a strong expression ofKCC2 (Study I of this Thesis) andhence, it is possible that the K+
transients accounting for the tonicGABAergic excitation arise, at least tosome extent, from a net extrusion ofKCl from pyramidal cells via KCC2.
Developmental expression of HFS-induced afterdischargesWhile intracellular sharpmicroelectrode recordings providedinformation on the developmentalprofile of HFS-induced tonicGABAergic excitation, and ionsensitive microelectrodes revealed asimilar ontogeny for the K+
o transients,we used extracellular field recordingsto study the synchronization of CA1pyramidal cell firing. Notably, the HFSparadigm used in this study is similarto what has been classically used in theinduction of long-term potentiation inthe CA1 area (Bliss and Collingridge,1993). This provides an obvious link tostudies aimed at elucidatingmechanisms underlying networkactivity and synaptic plasticity (Traubet al., 1998; Traub et al., 1999). In theadult rat hippocampal CA1 area,synchronous population oscillations atgamma frequency range can be
measured close (~ 400 m) to single-site HFS (Colling et al., 1998). With atwo-site HFS the firing of principalneurons is synchronized over longerdistances and this method can be usedto study oscillatory synchronizationbetween different sites (Traub et al.,1996b; Traub et al., 1999). In thepresent experiments, the single-siteHFS-induced afterdischarges in thegamma-frequency range werequantified from the field potentialrecordings by integrating off-line theapparent power of the populationspikes (Figure 1 in the originalpublication). Experiments were doneusing both submerged slices and slicesat saline-gas interface in order toexclude the possible increase in fieldeffects caused by hypo-osmoticconditions that has been reported totake place in poorly perfused interfacechambers (see Whittington et al.,2001).Field potential recordings from rathippocampal CA1 stratum pyramidale,both in the presence and absence ofionotropic glutamate receptors,revealed a striking temporal correlationbetween the expression of pyramidalcarbonic anhydrase activity and theappearance of HFS-induced networkactivity at around P12. The likelycausal relation between these twoevents was further validated inexperiments examining the effects ofEZA. Once the extracellularlymeasured afterdischarges could beevoked, their apparent power wasalways significantly suppressed by themembrane permeable CA inhibitorEZA whereas BA did not decrease theapparent power. The strict dependencyof the HFS-induced afterdischarges onthe availability of HCO3
- wasconfirmed in experiments where thestandard CO2/HCO3
- -buffered solutionwas replaced with a nominallyCO2/HCO3
- free, HEPES-bufferedsolution. Washout of CO2/HCO3
-
rapidly and reversibly abolished theHFS-induced afterdischarges.A developmental increase inexcitability at around P13 has beenreported by Kohling et al. (2000) for 0-Mg2+ induced gamma oscillations. Ourresults readily explain their findingsand point to a crucial role of
41
intrapyramidal CAVII in thegeneration of developmentalupregulation of network excitability.Even though one should be cautious incomparing data obtained in vitro and invivo, the dependency of HFS-inducedafter discharges (as well as 0-Mg2+
induced activity; Kohling et al., 2000)on CAi activity is intriguing. Inhumans, CA inhibitors have been usedto control epileptic seizures both asmonotherapy and in combination withother antiepileptic medication (Wyllie,1997; Supuran et al., 2003; see alsoKatayama et al., 2002). Our findingthus places CAVII as a possible noveldrug target in the therapy of epilepsy.To verify that the nominal absence ofCO2/HCO3
- did not result in a generalblockage of hippocampal oscillationswe studied the effect of CO2/HCO3
-
withdrawal in another model ofnetwork activity, the GDPs of thedeveloping hippocampus (also calledthe spontaneous population oscillationsin Study II in this Thesis). While HFS-induced after discharges could not beevoked in the absence CO2/HCO3
-,GDPs readily occurred afterwithdrawal of CO2/HCO3
-. In fact, theamplitude of GDPs increasedsignificantly in the HEPES-bufferedsolution. These results, together withthe early onset of GDPs (Ben-Ari etal., 1989; Khazipov et al., 2004) whichprecedes CAVII expression, showedthat there is no universal mechanism ofhippocampal neuronal network activitythat would depend on the presence ofCO2/HCO3
- or on fast functionalintrapyramidal CA activity. Thisconclusion is further supported by theobservation that hippocampal networkactivity induced by cholinergicagonists (Fisahn et al., 1998) starts ataround P7 (A. Fisahn personalcommunication; E. Ruusuvuori, K.Kaila, and J. Voipio, results based on41 slices from eleven P6-21 rats,unpublished observations).On the basis of results of Study II itcan be concluded that intrapyramidalCA activity is a key factor in theontogeny of tonic GABAergicexcitation that drives HFS-inducedafterdischarges. It is only after theexpression of the intrapyramidal CAthat a pronounced, HFS-induced
GABAergic excitation becomesfunctional. Moreover, Study II showsthat the up-regulation of thecytoplasmic CA isoform CAVIIaccounts for the developmentalincrease in the intracellular CA activityin hippocampal pyramidal neurons.This puts CAVII in a key position ingoverning the electrophysiologicalbehavior of CA1 pyramidal neurons,the major output pathway of thehippocampus, in situations wherelarge-scale GABAA receptor activationtakes place.
5.3. Mice with targetedSlc4a10 gene disruptionhave small brain ventriclesand show reduced neuronalexcitability (III)The Na+-driven HCO3
- transporterNCBE (encoded by Slc4a10 gene)belongs to the SLC4 gene family ofHCO3
- transporters which have beenshown to be closely involved in solutetransport and pH regulation. To studythe role of NCBE in physiologicalprocesses mice with targeted Slc4a10gene disruption was generated bydeleting exon 12 of the gene.Two independent correctly targetedembryonic stem cell clones wereinjected into blastocysts and gaveheterozygous offspring that had noobvious phenotype. Fromheterozygous mating, homozygous KOmice were born in the expectedMendelian ratio. However, in theselitters NCBE KO pups had a highermortality rate than the heterozygousand wild-type (WT) pups. After thesecond postnatal week NCBE KO micedid not gain weight at the same rate astheir littermates and many of them diedaround weaning. Only if providedmoistened food at the age of P14-30,NCBE KO mice grew normally andthe increased mortality aroundweaning was avoided. In litters fromKO mating pairs the pups gainedweight and developed normally. Theseresults most likely exclude thepossibility that NCBE deletion per sewould have an effect on the viabilityand development of the animals.Rather, it seems that juvenile NCBE
42
KO mice are unable to compete formaternal care with their heterozygousand WT littermates and therefore havea higher mortality. Adult NCBE KOmice were indistinguishable from theirheterozygous and WT littermates, theywere fertile even as KO mating pairs,and had no major behaviouralabnormalities, as tested for circadianrhythm, overall locomotor activity,motor coordination and spatiallearning. The slightly delayedhabituation to novel surroundings andobjects could be taken as an indicationof a type of hippocampal-relatedmemory impairment (Malleret et al.,1999).Multiple tissue Northern blots revealeda prominent expression of NCBE inthe mouse brain, retina and spinal cord.In line with the normal phenotype ofthe NCBE KO animals, the grossmorphology of the brain tissue of adultKO mice revealed no major changesexcept for a dramatic decrease in thetotal volume of brain ventricles (seebelow). With quantitative real-timePCR it was further verified that theloss of Slc4a10 was not compensatedby an up-regulation of other solutecarriers from the BTs (Slc4), the CCCs(Slc12), the NHEs (Slc9), or form thegene family of multifunctional anionexchangers (Slc26).To detect NCBE at the protein level, anovel polyclonal antibody against anN-terminal epitope of murine NCBEwas generated. This antibody shoulddetect both of the NCBE isoforms withdistinct carboxyterminal inserts (seeGiffard et al., 2003). The specificity ofthe antibody, both in immunoblots andin immunohistochemistry, wasconfirmed by using brain tissue fromKO animals. In immunoblots theantibody detected an approximately180 kDa band in protein lysates fromwhole WT brain. To examine theexpression of NCBE in neurons andglia, protein lysates were made frommixed neuronal/glial and from pureglial cell cultures. Transcripts of aNCBE splice variant with a terminalPDZ motif had been detected incultured rat astrocytes (Giffard et al.,2003). We detected NCBE in proteinlysates from co-cultured mouse
neurons and glia but not in lysatesfrom pure glial cultures.
Localization and function of NCBE inchoroid plexusDouble staining immunofluorescenceconfirmed the basolateral localizationof NCBE in choroid plexus epithelialcells, previously described byPraetorius et al., 2004) and Bouzinovaet al., 2005). The high expression ofNCBE in the choroid plexus togetherwith the obvious change in theventricular volume seen in thehistological sections, pointed to animportant role of NCBE mediated Na+
-uptake in CSF production. Thereduction in the ventricular volumewas analyzed in more detail by in vivoMRI scans. These measurementsshowed that the total ventricularvolume was diminished from 10.49 ±1.8 mm3 (mean ± SEM) in WT mice to2.31 ± 0.39 mm3 in KO mice.An elevated intracranial pressure dueto brain edema could perhaps alsoresult in collapsing of ventricles.However, this would be accompaniedwith cellular edema or increased watercontent of the brain, and result in otheranatomical changes such as inprotrusion of the cerebellum towardsthe foramen magnum. No anatomicalchanges were observed and thepossibility of edema was excluded byfurther analysis of T2 relaxation timesand apparent diffusion coefficientmaps calculated from the MRI scans.The results thus strongly support theconclusion that the collapsing ofventricles in NCBE KO mice resultsfrom abnormally low CSF production.Closer examination of the NCBE KOchoroid plexus epithelia withelectronmicroscopy revealed an intactepithelial lining but with stronglyreduced apical microvilli and increasedlateral intercellular spaces.The role of NCBE in Na+-dependentpHi regulation in choroid plexusepithelial cells was studied in clustersof isolated epithelial cells (see Fig. 2 inthe original publication). pHi wasmeasured with the pH sensitivefluorescent indicator BCECF and therecovery from an exogenous acid loadwas studied using the ammonium pre-
43
pulse technique (Boron and De Weer,1976). pHi recovery was fullydependent on extracellular Na+ and itwas significantly slower in epithelialcells from KO than from the WT mice.This shows that NCBE markedlycontributes to the basolateral Na+-dependent pHi regulation in choroidplexus epithelial cells. While thecritical roles of Na+-K+-ATPase andAQP1 water channels for CSFproduction have been recognized andtheir importance in apical Na+
secretion and osmotic water flowemphasized, the basolateral Na+ uptakemechanism has so far remainedunidentified (for a recent review seePraetorius, 2007). Combining theresults from pHi measurements withthose from the enhanced MRI scans, itcan be speculated that NCBE is alikely candidate to mediate thebasolateral uptake of Na+, and therebyNCBE would make a significantcontribution to the production of CSF.
Localization of NCBE in thehippocampusThe regional distribution of NCBE inthe brain parenchyma was studied indiaminobenzidine stained, free-floatingbrain sections. The protein was broadlyexpressed in various brain regions butwas absent from fiber tracts such as thecorpus callosum or the white matter ofthe spinal cord. In the hippocampusthere was a striking regional differencein NCBE expression. The barelydetectable labeling of CA1 pyramidsand the dentate granule cells was insharp contrast to the strong signalobtained from CA3 stratum oriens,stratum pyramidale, and stratumradiatum.To examine the cell-type specificexpression of NCBE in more detail,double-staining with glial cell,interneuronal, axonal, and dendriticmarkers were performed.Immunohistochemical double-stainingrevealed no overlap of NCBE withmarkers of oligodendrocytes (CNPase)or astrocytes (glial fibrillary acidicprotein, GFAP). Interneurons,identified by their expression ofglutamic acid decarboxylase, werestrongly immunopositive for NCBE in
all regions of the cornu ammonis. Onthe subcellular level NCBE wasabundantly detected in somata andproximal and apical dendrites of CA3pyramidal cells. With the resultsobtained from immunoblots, our datapoint to a neuronal expression ofNCBE.Electron micrographs suggested that inthe CA3 area NCBE localized to thesomatic region and to dendritic spineswithin the stratum oriens and thestratum radiatum. As most somaticsynapses are inhibitory, whereasdendritic spines receive excitatoryinput, NCBE appears to be expressedin the postsynaptic membranes of bothinhibitory and excitatory synapses.These findings on ultrastructuralNCBE localization wait to be verifiedin studies using immunogold labeling.
NCBE-mediated pHi regulationcontrols neuronal network excitabilityin CA3The high expression of NCBE in CA3pyramidal neurons led us to addressthe role of NCBE in neuronal pHregulation and in the control ofnetwork excitability in the CA3 area.The contribution of NCBE inintrapyramidal pH regulation wasstudied in adult hippocampal slices.The fluorescence pH imagingexperiments showed that the steady-state pH of the CA3 pyramidal neuronswas similar in slices from NCBE KOand WT mice. However, the recoveryfrom an acid load, induced by 20 mMpropionate, was significantly slower inNCBE KO slices than in WT controls.Consequently, reflecting the faster pHirecovery, the alkaline over-shoot uponpropionate washout was moreprominent in the WT slices. Theimpaired ability to recover from animposed acid load could modify theexcitability of CA3 neurons. Thisaspect was examined in experimentswhere intense, periodic bursting ofCA3 neurons was induced by bathapplication of the non-selective K+
channel blocker 4-aminopyridine (4-AP). The activity of the CA3 networkwas measured with extracellular fieldpotential microelectrodes.
44
There was no difference in the base-line frequency of the 4-AP inducedinterictal-like activity in WT and KOslices, and intracellular acidificationwith propionate initially suppressed thefrequency of the bursts in a similarmanner in both genotypes. In WT theactivity recovered close to the base-line value, or slightly above, within 15minutes in the continuous presence ofpropionate. In contrast, the frequencyof neuronal bursts in KO slices showedonly little recovery during thepropionate application. The longer-lasting suppression of neuronal activityin KO slices reflects the inability of theNCBE-deficient neurons to maintainpH homeostasis when faced with anexogenous acid load. If the effect ofthe endogenous acid-load generated byneuronal activity is taken into account,the time scale of the propionate-induced suppression of networkactivity is strikingly similar with thepHi responses described earlier.Together, these results provide furtherevidence for the pHi-dependentmodulation of neuronal excitabilityand suggest a significant role forNCBE in mediating neuronal recoveryfrom acid load.The in vitro experiments provided thebackground for studying the role ofNCBE-mediated pHi regulation onneuronal excitability in vivo. Also,studies from another KO strain withimpaired pHi-regulation, the AE3 KO,have shown that epileptic-like seizuresare sensitive indicators of imbalancesin cellular pH (Hentschke et al., 2006).To test the susceptibility of the NCBEKO mice to seizures we usedproconvulsant substances(pentylenetetrazole and pilocarpine) aswell as a non-pharmacologicalapproach to provoke epileptic activity,the recently improved rat pup model offebrile seizures (Schuchmann et al.,2006). In this model ictal activity iscaused by hyperthermia-inducedhyperventilation with a concomitantrise of cortical pH.After an intraperitoneal injection ofpentylenetetrazole (40 mg/kg) threesuccessive seizure phases could bedistinguished in WT mice: myoclonicjerks which could progress to clonicseizures and then to generalized
tonic/clonic seizures. The latency tothe onset of myoclonic jerks wasalmost twice as long in NCBE KOcompared to WT mice, and theepileptic activity never progressed togeneralized seizures in the KOanimals. There was a clear influence ofthe Slc4a10 disruption on the mortalityof the mice. NCBE KOs wereprotected from lethal seizures bothafter a higher dose ofpentylenetetrazole (60 mg/kg) or afteradministration of pilocarpine (350mg/kg). Also the results from thehyperthermia model suggested thatdisruption of the Slc4a10 genesuppressed seizure susceptibility.Thus, both in vitro and in vivo findingspoint to the conclusion that NCBE hasa crucial role in modulation of networkexcitability. Neuronal activity caninduce an intracellular acid load,which, in turn, suppresses seizures. Inlight of the results presented, it isplausible that the effects of NCBE onmodulation of network excitability andseizure susceptibility are directlyattributable to its role in the regulationof intraneuronal pH.
There are several pathways by whichthe impaired HCO3
- uptake may affectnetwork excitability. The excitatoryeffects of GABA in mature neuronsand neuronal networks (Voipio andKaila, 2000) are dependent onintraneuronal HCO3
- and,consequently, on mechanisms thatregulate neuronal pHi (Bevensee andBoron, 1998): a rise in pHi enhancesGABAA-receptor mediated excitationby increasing the depolarizing HCO3
-
current component while an acid shifthas the opposite effect (Kaila et, al1993). Also, upon intense GABAAreceptor activation the depolarizingHCO3
- -efflux drives the fast anionicredistribution (Kaila, 1994; Voipio andKaila, 2000), and the rapidreplenishment of HCO3
- has beenshown to be essential for the prolongedGABAergic depolarization mediatedby increased [K+]o (Study II of thisThesis). Finally, in addition to theGABAergic transmission, theglutamatergic synaptic responses aremodified by intracellular pH. Lee et al.(1996) have shown that monosynaptic
45
AMPA receptor-mediated transmissionis suppressed by intracellularacidification. On the network level,acidification is expected to reduce gap-junctional coupling (Spray et al.,1981). Non-selective pharmacologicalblockage of gap junctions or geneticdeletion of a single, interneuronal gap-junction connexin (connexin 36) hasbeen shown to affect neuronalpopulation oscillations in the gammafrequency range (Hormuzdi et al.,2001; Traub et al., 2003).As a whole, mice with targetedSlc4a10 disruption have so far revealedtwo physiologically importantmechanisms of function for thetransporter. The impaired HCO3
-
uptake plays a key role in pH-dependent modulation of neuronalnetwork excitability. In choroid plexusepithelial cells the NCBE mediatedbasolateral Na+ and HCO3
- uptake islikely to make an significantcontribution to the transepithelialsolute transport, and hence, to thesecretion of CSF.
46
6 Conclusions
This Thesis has assessed the physiological function and development of ion regulatoryproteins that make a major contribution to the maintenance of neuronal Cl- and pHhomeostasis.Study I of this Thesis was the first report to identify the molecular mechanism thatrenders GABAA-receptor mediated transmission from depolarizing to hyperpolarizingduring the development of central neurons. In the rat, the steep upregulation of KCC2in hippocampal pyramidal neurons ends the neonatal period when depolarizingGABAergic transmission contributes in different aspects of neuronal development.Although the KCC2 mediated Cl- extrusion renders GABAA-receptor mediatedhyperpolarizing, the strictly inhibitory action of GABA is not its only mode of action.Depolarizing and excitatory actions mediated by GABAA-receptors are now well-recognized in adult neurons. In mature neurons strong GABAA-receptor activation canresult in transient ionic redistribution, and under these conditions the depolarizingphase of GABA depends on the availability of HCO3
- (Voipio and Kaila, 2000). Theextent of the activity-dependent shift in EGABA-A to more depolarizing values andhence, the power of the ‘ionic modulation’ on GABAergic transmission may differbetween neuronal subpopulations, probably arising from differences in Cl--HCO3
-
homeostasis in distinct cell types (Marty and Llano, 2005; Blaesse et al., 2008).In hippocampal pyramidal neurons the appearance of intrapyramidal CA activityparallels the developmental onset of HFS-induced tonic GABAergic depolarizationand the underlying [K+]o transients that show a strict dependency on the fastreplenishment of HCO3
-. The results of Study II of this Thesis provide a mechanisticexplanation for the depolarizing GABAergic transmission in hippocampal neuronalnetworks and urge future studies to assess the role of intrapyramidal CA activityunder conditions where its contribution to physiological processes could be furtherevaluated. Interestingly, recent findings have revealed that depolarizing GABAergicresponses in mature neocortical neurons are generated not only as a consequence ofactivity induced ionic shifts or due to the HCO3
- permeability of GABAA receptorchannels. The existence of subcellular Cl- pools within a neuron, created by spatiallycompartmentalized Cl- transporters, can also generate local, cell region-specificdifferences in EGABA-A with depolarizing IPSPs in the AIS. Therefore, both thetemporal patterning of activity in inhibitory interneurons and the subcellularlocalization of their synapses on target neurons are essential determinants that controland modulate GABAergic responses. Evidently, the traditional division ofneurotransmitter responses to excitatory and inhibitory overlooks many aspects ofGABAergic neurotransmission.The physiological significance of the Na+-driven HCO3
- transporter NCBE (Slc4a10)was the focus of the Study III. This work combined several methods at differentorganizational levels in order to elucidate the (sub)cellular localization of themolecule and to study the importance of NCBE in pH-regulation and in epithelialsolute transport. Combined together, the basic in vitro findings and the in vivo studiessuggest that NCBE has a crucial role in the pHi-dependent modulation of neuronalexcitability. They also point to a NCBE-mediated epithelial Na+ and HCO3
- uptake inchoroid plexus that is likely to make a significant contribution to CSF production.All of the Studies in this Thesis emphasize the importance of gaining mechanisticexplanations from in vitro experiments that can further be exploited to understand therole of a single molecule in a larger context, and finally to assess its function inphysiological and/or pathophysiological functions in vivo.
47
Acknowledgements
This study was carried out at the Department of Biological and EnvironmentalSciences at the University of Helsinki and generously supported by Academy ofFinland, Ella and Georg Ehrnrooth foundation, and Tekes, Finnish Funding Agencyfor Technology and Innovation.
I’m immensely thankful for my supervisors professor Kai Kaila and professor JuhaVoipio for their guidance and commitment over the years as well as for providingexcellent facilities to perform this research and for the unlimited possibilities to gatherscientific experience.
Professor Joachim Deitmer and docent Irma Holopainen, to whom no deadline wastoo daunting, are acknowledged for their valuable comments during the pre-examination of this Thesis.
I owe a great debt to professor Kristian Donner and professor Juhani Saarikoski whohad the willingness and patience to guide me when I took my first steps in science,andto professor Christian Hübner and docent Claudio Rivera for their scientific input andadvice.
As “academics spend most of their lives talking about ideas with colleagues andfriends, exchanging the gossip and information that both drives the subject on andcreates a community” I must say that I have been privileged to work in a lab wherethe enthusiastic and supporting atmosphere is created by truly talented and motivatedpeople. A warm thank to all my colleagues with whom I‘ve worked during thisjourney. The assistance provided by our technicians and the co-operation of theanimal facility have considerably eased my days in the lab.Special thanks to Matias Palva and Sampsa Sipilä with whom I’ve had the pleasure toshare my office, drink my morning coffees, and to have many thought-provokingdiscussions, to Katri Wegelius, whose ability to handle the countless administrativeduties with grace and good cheer is astonishing, to Anna-Maija Autere who hasalways found the time when I haven’t been able to stop talking, and to Karri Lämsä,whose breathtaking devotion to (scientific)life I find captivating.
I want to express my deepest gratitude to my mother and sister for all their love andencouragement, to Knut for his constant support, and to my ‘extended-family’ and‘non-scientific friends’ for the buckets of tea and joyful moments that have kept megoing.
Most of all, I’m grateful to Jari and to our beloved children Aino, Emil, and Elias fortheir love that fulfills every day of my life. Thank you for your tolerance towards“The Book”.
Helsinki, November 2008
Eva
48
References
Abbott NJ (2004) Evidence for bulk flow ofbrain interstitial fluid: significance forphysiology and pathology. Neurochem Int45: 545-552.
Abbott NJ, Ronnback L, Hansson E (2006)Astrocyte-endothelial interactions at theblood-brain barrier. Nat Rev Neurosci 7:41-53.
Achilles K, Okabe A, Ikeda M, Shimizu-Okabe C, Yamada J, Fukuda A, LuhmannHJ, Kilb W (2007) Kinetic properties ofCl uptake mediated by Na+-dependentK+-2Cl cotransport in immature ratneocortical neurons. J Neurosci 27: 8616-8627.
Agnati LF, Tinner RB, Staines WA, VaananenK, Fuxe K (1995) On the cellular-localization and distribution of CA-IIimmunoreactivity in the rat-brain. BrainRes 676: 10-24.
Akerman CJ, Cline HT (2006) DepolarizingGABAergic conductances regulate thebalance of excitation to inhibition in thedeveloping retinotectal circuit in vivo. JNeurosci 26: 5117-5130.
Alger BE, Nicoll RA (1982a) Feed-forwarddendritic inhibition in rat hippocampalpyramidal cells studied in vitro. J Physiol328: 105-123.
Alger BE, Nicoll RA (1982b) Pharmacologicalevidence for two kinds of GABA receptoron rat hippocampal pyramidal cellsstudied in vitro. J Physiol 328: 125-141.
Alvarez BV, Vilas GL, Casey JR (2005)Metabolon disruption: a mechanism thatregulates bicarbonate transport. EMBO J24: 2499-2511.
Amato A, Ballerini L, Attwell D (1994)Intracellular pH changes produced byglutamate uptake in rat hippocampalslices. J Neurophysiol 72: 1686-1696.
Amtorp O, Sorensen SC (1974) Ontogeneticdevelopment of concentration differencesfor protein and ions between plasma andCSF in rabbits and rats. J Physiol 243:387-400.
Aram JA, Lodge D (1987) Epileptiformactivity induced by alkalosis in ratneocortical slices:block by antagonists ofN-methyl-D-aspartate. Neurosci Lett 83:345-350.
Attwell D, Laughlin SB (2001) An energybudget for signaling in the grey matter ofthe brain. J Cereb Blood Flow Metab 21:1133-1145.
Avoli M (1996) GABA-mediated synchronouspotentials and seizure generation.Epilepsia 37: 1035-1042.
Avoli M, Louvel J, Pumain R, Kohling R(2005) Cellular and molecularmechanisms of epilepsy in the humanbrain. Prog Neurobiol 77: 166-200.
Balakrishnan V, Becker M, Löhrke S,Nothwang HG, Güresir E, Friauf E (2003)Expression and function of chloridetransporters during development ofinhibitory neurotransmission in theauditory brainstem. J Neurosci 23: 4134-4145.
Balestrino M, Somjen GG (1988)Concentration of carbon dioxide,interstitial pH and synaptic transmission inhippocampal formation of the rat. JPhysiol 396: 247-266.
Ballanyi K, Muckenhoff K, Bellingham MC,Okada Y, Scheid P, Richter DW (1994)Activity-related pH changes in respiratoryneurones and glial cells of cats.NeuroReport 6: 33-36.
Bartho P, Payne JA, Freund TF, Acsady L(2004) Differential distribution of the KClcotransporter KCC2 in thalamic relay andreticular nuclei. Eur J Neurosci 20: 965-975.
Baxter KA, Church J (1996) Characterizationof acid extrusion mechanisms in culturedfetal rat hippocampal neurones. J Physiol493: 457-470.
Becker HM, Deitmer JW (2007) Carbonicanhydrase II increases the activity of thehuman electrogenic Na+/HCO3-cotransporter. J Biol Chem 282: 13508-13521.
49
Ben-Ari Y (2001) Developing networks play asimilar melody. Trends Neurosci 24: 353-360.
Ben-Ari Y (2002) Excitatory actions of GABAduring development: The nature of thenurture. Nat Rev Neurosci 3: 728-739.
Ben-Ari Y, Cherubini E, Corradetti R, GaiarsaJL (1989) Giant synaptic potentials inimmature rat CA3 hippocampal neurons. JPhysiol 416: 303-325.
Ben-Ari Y, Gaiarsa JL, Tyzio R, Khazipov R(2007) GABA: a pioneer transmitter thatexcites immature neurons and generatesprimitive oscillations. Physiol Rev 87:1215-1284.
Bevensee MO, Boron WF (1998) pHregulation in mammalian neurons. In: pHand brain function (Kaila K, Ransom BR,eds), pp 211-232. Wiley-Liss.
Bevensee MO, Cummins TR, Haddad GG,Boron WF, Boyarsky G (1996) pHregulation in single CA1 neurons acutelyisolated from the hippocampi of immatureand mature rats. J Physiol 494: 315-328.
Bevensee MO, Schmitt BM, Choi I, RomeroMF, Boron WF (2000) An electrogenicNa+-HCO3- cotransporter (NBC) with anovel COOH-terminus, cloned from ratbrain. Am J Physiol 278: C1200-C1211.
Bevensee MO, Schwiening CJ, Boron WF(1995) Use of BCECF and propidiumiodide to assess membrane integrity ofacutely isolated CA1 neurons from rathippocampus. J Neurosci Meth 58: 61-75.
Bickler PE, Litt L, Banville DL, SeveringhausJW (1988) Effects of acetazolamide oncerebral acid-base balance. J Appl Physiol65: 422-427.
Biwersi J, Verkman AS (1991) Cell-permeablefluorecent indicator for cytosolic chloride.Biochemistry 30: 7879-7883.
Bliss TV, Collingridge GL (1993) A synapticmodel of memory: long-term potentiationin the hippocampus. Nature 361: 31-39.
Boedtkjer E, Praetorius J, Fuchtbauer EM,Aalkjaer C (2008) Antibody-independentlocalization of the electroneutral Na+-HCO3- cotransporter NBCn1 (slc4a7) inmice. Am J Physiol 294: C591-C603.
Boettger T, Rust MB, Maier H, SeidenbecherT, Schweizer M, Keating DJ, Faulhaber J,Ehmke H, Pfeffer C, Scheel O, Lemcke B,Horst J, Leuwer R, Pape HC, Volkl H,Hübner CA, Jentsch TJ (2003) Loss of K-Cl co-transporter KCC3 causes deafness,neurodegeneration and reduced seizurethreshold. EMBO J 22: 5422-5434.
Bok D et al., (2003) Blindness and auditoryimpairment caused by loss of the sodiumbicarbonate cotransporter NBC3. NatGenet 34: 313-319.
Bolea S, Avignone E, Berretta N, Sanchez-Andres JV, Cherubini E (1999) Glutamatecontrols the induction of GABA-mediatedgiant depolarizing potentials throughAMPA receptors in neonatal rathippocampal slices. J Neurophysiol 81:2095-2102.
Bonnet U, Leniger T, Wiemann M (2000)Alteration of intracellular pH and activityof CA3-pyramidal cells in guinea pighippocampal slices by inhibition oftransmembrane acid extrusion. Brain Res872: 116-124.
Bormann J, Hamill OP, Sakmann B (1987)Mechanism of anion permeation throughchannels gated by glycine and gamma-aminobutyric-acid in mouse culturedspinal neurons. J Physiol 385: 243-286.
Boron WF (2004) Regulation of intracellularpH. Advances in Physiology Education28: 160-179.
Boron WF, De Weer P (1976) Intracellular pHtransients in squid giant axons caused byCO2, NH3, and metabolic inhibitors. JGen Physiol 67: 91-112.
Boussouf A, Lambert RC, Gaillard S (1997)Voltage-dependent Na(+)-HCO3-cotransporter and Na+/H+ exchanger areinvolved in intracellular pH regulation of
50
cultured mature rat cerebellaroligodendrocytes. Glia 19: 74-84.
Bouyer P, Bradley SR, Zhao JH, Wang WG,Richerson GB, Boron WF (2004) Effect ofextracellular acid-base disturbances on theintracellular pH of neurones cultured fromrat medullary raphe or hippocampus. JPhysiol 559: 85-101.
Bouzinova EV, Praetorius J, Virkki LV,Nielsen S, Boron WF, Aalkjaer C (2005)Na+-dependent HCO3- uptake into the ratchoroid plexus epithelium is partiallyDIDS sensitive. Am J Physiol 289:C1448-C1456.
Bracci E, Vreugdenhil M, Hack SP, JefferysJG (1999) On the synchronizingmechanisms of tetanically inducedhippocampal oscillations. J Neurosci 19:8104-8113.
Bradbury MW, Saunders NR, Desai S,Reynolds M, Reynolds JM, Crowder J(1972) Electrolytes and water in brain andCSF of fetal sheep and guinea-pig. JPhysiol 227: 591-610.
Brune T, Fetzer S, Backus KH, Deitmer JW(1994) Evidence for electrogenic sodium-bicarbonate cotransport in cultured ratcerebellar astrocytes. Pflugers Arch 429:64-71.
Butt AM, Jones HC, Abbott NJ (1990)Electrical resistance across the blood-brainbarrier in anaesthetized rats: adevelopmental study. J Physiol 429: 47-62.
Buzsaki G, Kaila K, Raichle M (2007)Inhibition and brain work. Neuron 56:771-783.
Cammer W, Tansey FA (1988) Carbonicanhydrase immunostaining in astrocytes inthe rat cerebral cortex. J Neurochem 50:319-322.
Cammer W, Zhang H (1996) Carbonicanhydrase II in microglia in forebrains ofneonatal rats. J Neuroimmunol 67: 131-136.
Caron L, Rousseau F, Gagnon E, Isenring P(2000) Cloning and functional
characterization of a cation-Cl-cotransporter-interacting protein. J BiolChem 275: 32027-32036.
Carter ND, Fryer A, Grant AG, Hume R,Strange RG, Wistrand PJ (1990)Membrane specific carbonic anhydrase(CAIV) expression in human tissues.Biochim Biophys Acta 1026: 113-116.
Chen JC, Chesler M (1990) A bicarbonate-dependent increase in extracellular pHmediated by GABAA receptors in turtlecerebellum. Neurosci Lett 116: 130-135.
Chen JC, Chesler M (1991) Extracellularalkalinization evoked by GABA and itsrelationship to activity-dependent pHshifts in turtle cerebellum. J Physiol 442:431-446.
Chen JC, Chesler M (1992a) Modulation ofextracellular pH by glutamate and GABAin rat hippocampal slices. J Neurophysiol67: 29-36.
Chen JC, Chesler M (1992b) pH transientsevoked by excitatory synaptictransmission are increased by inhibition ofextracellular carbonic anhydrase. ProcNatl Acad Sci U S A 89: 7786-7790.
Chen LM, Kelly ML, Parker MD, Bouyer P,Gill HS, Felie JM, Davis BA, Boron WF(2008a) Expression and localization ofNa-driven Cl-HCO3- exchanger(SLC4A8) in rodent CNS. Neuroscience153: 162-174.
Chen LM, Kelly ML, Rojas JD, Parker MD,Gill HS, Davis BA, Boron WF (2008b)Use of a new polyclonal antibody to studythe distribution and glycosylation of thesodium-coupled bicarbonate transporterNCBE in rodent brain. Neuroscience 151:374-385.
Chesler M (2003) Regulation and modulationof pH in the brain. Physiol Rev 83: 1183-1221.
Chesler M, Kaila K (1992) Modulation of pHby neuronal activity. Trends Neurosci 15:396-402.
Chesler M, Kraig RP (1987) Intracellular pHof astrocytes increases rapidly withcortical stimulation. Am J Physiol 253:R666-R670.
51
Chesler M, Kraig RP (1989) Intracellular pHtransients of mammalian astrocytes. JNeurosci 9: 2011-2019.
Choi I, Rojas JD, Kobayashi C, Boron WF(2002) Functional characterization of"NCBE", a Na/HCO3 cotransporter. FasebJournal 16: A796.
Chub N, Mentis GZ, O'Donovan MJ (2006)Chloride-sensitive MEQ fluorescence inchick embryo motoneurons followingmanipulations of chloride and duringspontaneous network activity. JNeurophysiol 95: 323-330.
Chudotvorova I, Ivanov A, Rama S, HübnerCA, Pellegrino C, Ben Ari Y, Medina I(2005) Early expression of KCC2 in rathippocampal cultures augmentsexpression of functional GABA synapses.J Physiol 566: 671-679.
Church J, Baimbridge KG (1991) Exposure tohigh-pH medium increases the incidenceand extent of dye coupling between rathippocampal CA1 pyramidal neurons invitro. J Neurosci 11: 3289-3295.
Clayton GH, Owens GC, Wolff JS, Smith RL(1998) Ontogeny of cation-Cl-cotransporter expression in rat neocortex.Developmental Brain Research 109: 281-292.
Cohen I, Navarro V, Clemenceau S, Baulac M,Miles R (2002) On the origin of interictalactivity in human temporal lobe epilepsyin vitro. Science 298: 1418-1421.
Colling SB, Stanford IM, Traub RD, JefferysJGR (1998) Limbic gamma rhythms. I.Phase-locked oscillations in hippocampalCA1 and subiculum. J Neurophysiol 80:155-161.
Connors BW, Gutnick MJ (1990) Intrinsicfiring patterns of diverse neocorticalneurons. Trends Neurosci 13: 99-104.
Connors BW, Long MA (2004) Electricalsynapses in the mammalian brain. AnnuRev Neurosci 27: 393-418.
Cooper DS, Saxena NC, Yang HS, Lee HJ,Moring AG, Lee A, Choi IY (2005)Molecular and functional characterizationof the electroneutral Na/HCO3cotransporter NBCn1 in rat hippocampalneurons. J Biol Chem 280: 17823-17830.
Damkier HH, Nielsen S, Praetorius J (2007)Molecular expression of SLC4-derivedNa+-dependent anion transporters inselected human tissues. Am J PhysiolRegul Integr Comp Physiol 293: R2136-R2146.
Davson H, Segal MB (1969) Effect of CFS involume of distribution of extracellularmarkers. Brain 92: 131-136.
de Curtis M, Manfridi A, Biella G (1998)Activity-dependent pH shifts and periodicrecurrence of spontaneous interictal spikesin a model of focal epileptogenesis. JNeurosci 18: 7543-7551.
de la Rosa DA, Krueger SR, Kolar A, Shao D,Fitzsimonds RM, Canessa CM (2003)Distribution, subcellular localization andontogeny of ASIC1 in the mammaliancentral nervous system. J Physiol 546: 77-87.
Deisz RA, Lux HD (1982) The role ofintracellular chloride in hyperpolarizingpost- synaptic inhibition of crayfishstretch receptor neurones. J Physiol 326:123-138.
Deitmer JW (1992) Evidence for glial controlof extracellular pH in the leech centralnervous system. Glia 5: 43-47.
Deitmer JW, Rose CR (1996) pH regulationand proton signalling by glial cells. ProgNeurobiol 48: 73-103.
Deitmer JW, Schlue WR (1989) An inwardlydirected electrogenic sodium-bicarbonateco- transport in leech glial cells. J Physiol411: 179-194.
Deitmer JW, Szatkowski M (1990) Membranepotential dependence of intracellular pHregulation by identified glial cells in theleech central nervous system. J Physiol421: 617-631.
52
Delpire E, Lu JM, England R, Dull C, ThorneT (1999) Deafness and imbalanceassociated with inactivation of thesecretory Na-K-2Cl co-transporter. NatGenet 22: 192-195.
Demarque M, Represa A, Becq H, Khalilov I,Ben Ari Y, Aniksztejn L (2002) Paracrineintercellular communication by a Ca2+-and SNARE-independent release ofGABA and glutamate prior to synapseformation. Neuron 36: 1051-1061.
Deschenes M, Feltz P (1976) GABA-inducedrise of extracellular potassium in rat dorsalroot ganglia: an electrophysiological studyin vivo. Brain Res 118: 494-499.
Dietzel I, Heinemann U, Hofmeier G, Lux HD(1980) Transient changes in the size of theextracellular space in the sensorimotorcortex of cats in relation to stimulus-induced changes in potassiumconcentration. Exp Brain Res 40: 432-439.
Dixon MJ, Gazzard J, Chaudhry SS, SampsonN, Schulte BA, Steel KP (1999) Mutationof the Na-K-Cl co-transporter geneSlc12a2 results in deafness in mice. HumMol Genet 8: 1579-1584.
Douglas RM, Schmitt BM, Xia Y, BevenseeMO, Beimesderfer D, Boron WF, HaddadGG (2001) Sodium-hydrogen exchangersand sodium-bicarbonate co-transporters:Ontogeny of protein expression in the ratbrain. Neuroscience 102: 217-228.
Draguhn A, Traub RD, Schmitz D, Jefferys JG(1998) Electrical coupling underlies high-frequency oscillations in the hippocampusin vitro. Nature 394: 189-192.
Dulla CG, Dobelis P, Pearson T, FrenguelliBG, Staley KJ, Masino SA (2005)Adenosine and ATP link P-CO2 tocortical excitability via pH. Neuron 48:1011-1023.
Dziegielewska KM, Knott GW, Saunders NR(2000) The nature and composition of the
internal environment of the developingbrain. Cell Mol Neurobiol 20: 41-56.
Eccles JC (1966) The ionic mechanisms ofexcitatory and inhibitory synaptic action.Ann N Y Acad Sci 137: 473-494.
Eisner DA, Kenning NA, O'Neill SC, PocockG, Richards CD, Valdeolmillos M (1989)A novel method for absolute calibration ofintracellular pH indicators. Pflugers Arch413: 553-558.
Ek CJ, Dziegielewska KM, Stolp H, SaundersNR (2006) Functional effectiveness of theblood brain barrier to small water-solublemolecules in developing and adultopossum (Monodelphis domestica). JComp Neurol 496: 13-26.
Engelhardt B (2003) Development of theblood-brain barrier. Cell Tissue Res 314:119-129.
Farrant M, Kaila K (2007) The cellular,molecular and ionic basis of GABAAreceptor signalling. In: Progress in BrainResearch: GABA and the basal ganglia -from molecules to systems (Tepper JM,Abercrombie ED, Bolam JP, eds), pp 59-87. Oxford: Elsevier.
Fedirko N, Avshalumov M, Rice ME, CheslerM (2007) Regulation of postsynapticCa2+ influx in hippocampal CA1pyramidal neurons via extracellularcarbonic anhydrase. J Neurosci 27: 1167-1175.
Fedirko N, Svichar N, Chesler M (2006)Fabrication and use of high-speed,concentric H+- and Ca2+-selectivemicroelectrodes suitable for in vitroextracellular recording. J Neurophysiol96: 919-924.
Fossan G, Cavanagh ME, Evans CAN,Malinowska DH, Mollgård K, ReynoldsML, Saunders NR (1985) CSF brainpermeability in the immature sheep fetus -a CSF brain barrier. Developmental BrainResearch 18: 113-124.
Fukuda A, Muramatsu K, Okabe A, ShimanoY, Hida H, Fujimoto I, Nishino H (1998)Changes in intracellular Ca2+ induced byGABAA receptor activation and reductionin Cl- gradient in neonatal rat neocortex. JNeurophysiol 79: 439-446.
Galeffi F, Sah R, Pond BB, George A,Schwartz-Bloom RD (2004) Changes inintracellular chloride after oxygen-glucosedeprivation of the adult hippocampal slice:Effect of diazepam. J Neurosci 24: 4478-4488.
Galietta LJV, Haggie PM, Verkman AS (2001)Green fluorescent protein-based halideindicators with improved chloride andiodide affinities. FEBS Lett 499: 220-224.
Gamba G (2005) Molecular physiology andpathophysiology of electroneutral cation-chloride cotransporters. Physiol Rev 85:423-493.
Garaschuk O, Hanse E, Konnerth A (1998)Developmental profile and synaptic originof early network oscillations in the CA1region of rat neonatal hippocampus. JPhysiol 507: 219-236.
Garaschuk O, Linn J, Eilers J, Konnerth A(2000) Large-scale oscillatory calciumwaves in the immature cortex. NatNeurosci 3: 452-459.
Geers C, Gros G, Gärtner A (1985)Extracellular carbonic anhydrase ofskeletal muscle associated with thesarcolemma. J Appl Physiol 59: 548-558.
Immunochemical and immunohisto-chemical study of carbonic anhydrase-II inadult rat cerebellum - a marker foroligodendrocytes. Neuroscience 5: 559-571.
Ghandour MS, Langley OK, Zhu XL, WaheedA, Sly WS (1992) Carbonic anhydrase IVon brain capillary endothelial cells: amarker associated with the blood-brainbarrier. Proc Natl Acad Sci U S A 89:6823-6827.
Ghandour MS, Parkkila AK, Parkkila S,Waheed A, Sly WS (2000) Mitochondrialcarbonic anhydrase in the nervous system:expression in neuronal and glial cells. JNeurochem 75: 2212-2220.
Giffard RG, Lee YS, Ouyang YB, Murphy SL,Monyer H (2003) Two variants of the ratbrain sodium-driven chloride bicarbonateexchanger (NCBE): developmentalexpression and addition of a PDZ motif.Eur J Neurosci 18: 2935-2945.
Giffard RG, Papadopoulos MC, van Hooft JA,Xu LJ, Giuffrida R, Monyer H (2000) Theelectrogenic sodium bicarbonatecotransporter: Developmental expressionin rat brain and possible role in acidvulnerability. J Neurosci 20: 1001-1008.
Gillen CM, Brill S, Payne JA, Forbush B(1996) Molecular cloning and functionalexpression of the K-CL cotransporter fromrabbit, rat, and human - a new member ofthe cation-chloride cotransporter family. JBiol Chem 271: 16237-16244.
Gottfried JA, Chesler M (1994) EndogenousH+ modulation of NMDA receptor-mediated EPSCs revealed by carbonicanhydrase inhibition in rat hippocampus. JPhysiol 478 Pt 3: 373-378.
Gray CM (1999) The temporal correlationhypothesis of visual feature integration:still alive and well. Neuron 24: 31-25.
Grichtchenko II, Chesler M (1994)Depolarization-induced acid secretion ingliotic hippocampal slices. Neuroscience62: 1057-1070.
Grichtchenko II, Chesler M (1996) Calcium-and barium-dependent extracellularalkaline shifts evoked by electrical activityin rat hippocampal slices. Neuroscience75: 1117-1126.
54
Grichtchenko II, Choi I, Zhong X, Bray-WardP, Russell JM, Boron WF (2001) Cloning,Characterization, and ChromosomalMapping of a Human Electroneutral Na+-driven Cl-HCO3 Exchanger. J Biol Chem276: 8358-8363.
Gross E, Hawkins K, Abuladze N, Pushkin A,Cotton CU, Hopfer U, Kurtz I (2001) Thestoichiometry of the electrogenic sodiumbicarbonate cotransporter NBC1 is cell-type dependent. J Physiol 531: 597-603.
Grover LM, Lambert NA, Schwartzkroin PA,Teyler TJ (1993) Role of HCO3- ions indepolarizing GABAA receptor-mediatedresponses in pyramidal cells of rathippocampus. J Neurophysiol 69: 1541-1555.
Gulacsi A, Lee CR, Sik A, Viitanen T, KailaK, Tepper JM, Freund TF (2003) Celltype-specific differences in chloride-regulatory mechanisms and GABA(A)receptor-mediated inhibition in ratsubstantia nigra. J Neurosci 23: 8237-8246.
Gulledge AT, Stuart GJ (2003) Excitatoryactions of GABA in the cortex. Neuron37: 299-309.
Gulyas AI, Sik A, Payne JA, Kaila K, FreundTF (2001) The KCI cotransporter, KCC2,is highly expressed in the vicinity ofexcitatory synapses in the rathippocampus. Eur J Neurosci 13: 2205-2217.
Habgood MD, Sedgwick JEC, DziegielewskaKM, Saunders NR (1992) Adevelopmentally regulated blood CSFtransfer mechanism for albumin inimmature rats. J Physiol 456: 181-192.
Halmi P, Parkkila S, Honkaniemi J (2006)Expression of carbonic anhydrases II, IV,VII, VIII and XII in rat brain after kainicacid induced status epilepticus.Neurochem Int 48: 24-30.
Hartley Z, Dubinsky JM (1993) Changes inintracellular pH associated with glutamateexcitotoxicity. J Neurosci 13: 4690-4699.
Hediger MA, Romero MF, Peng JB, Rolfs A,Takanaga H, Bruford EA (2004) TheABCs of solute carriers: physiological,pathological and therapeutic implications
of human membrane transport proteins -Introduction. Pflugers Arch 447: 465-468.
Heinemann U, Louvel J (1983) Changes in[Ca2+]o and [K+]o during repetitiveelectrical stimulation and duringpenteterazol induced seizure activity in thesensorimotor cortex of cats. Pflugers Arch398: 310-317.
Hennou S, Khalilov I, Diabira D, Ben Ari Y,Gozlan H (2002) Early sequentialformation of functional GABA(A) andglutamatergic synapses on CA1interneurons of the rat foetalhippocampus. Eur J Neurosci 16: 197-208.
Hentschke M, Wiemann M, Hentschke S,Kurth I, Hermans-Borgmeyer I,Seidenbecher T, Jentsch TJ, Gal A,Hübner CA (2006) Mice with a targeteddisruption of the Cl-/HCO3- exchangerAE3 display a reduced seizure threshold.Mol Cell Biol 26: 182-191.
Heyer M, Muller-Berger S, Romero MF,Boron WF, Fromter E (1999)Stoichiometry of the rat kidney Na+-HCO3- cotransporter expressed inXenopus laevis oocytes. Pflugers Arch438: 322-329.
Hille B (2001) Ionic Channels of ExcitableMembranes. Sunderland, MassachutesSinauer Associates.
Hilvo M, Rafajova M, Pastorekova S, PastorekJ, Parkkila S (2004) Expression ofcarbonic anhydrase IX in mouse tissues. JHistochem Cytochem 52: 1313-1321.
Hilvo M, Tolvanen M, Clark A, Shen BR,Shah GN, Waheed A, Halmi P, HanninenM, Hamalainen JM, Vihinen M, Sly WS,Parkkila S (2005) Characterization of CAXV, a new GPI-anchored form of carbonicanhydrase. Biochem J 392: 83-92.
Hormuzdi SG, Pais I, LeBeau FE, Towers SK,Rozov A, Buhl EH, Whittington MA,Monyer H (2001) Impaired electricalsignaling disrupts gamma frequencyoscillations in connexin 36-deficient mice.Neuron 31: 487-495.
Howard HC et al., (2002) The K-Clcotransporter KCC3 is mutant in a severeperipheral neuropathy associated withagenesis of the corpus callosum. NatGenet 32: 384-392.
55
Huberfeld G, Wittner L, Clemenceau S, BaulacM, Kaila K, Miles R, Rivera C (2007)Perturbed chloride homeostasis andGABAergic signaling in human temporallobe epilepsy. J Neurosci 27: 9866-9873.
Huguenard JR, Alger BE (1986) Whole-cellvoltage-clamp study of the fading ofGABA-activated currents in acutelydissociated hippocampal neurons. JNeurophysiol 56: 1-18.
Hübner CA, Hentschke M, Jacobs S, Hermans-Borgmeyer I (2004) Expression of thesodium-driven chloride bicarbonateexchanger NCBE during prenatal mousedevelopment. Gene Expr Patterns 5: 219-223.
Hübner CA, Lorke DE, Hermans-Borgmeyer I(2001a) Expression of the Na-K-2Cl-cotransporter NKCC1 during mousedevelopment. Mech Dev 102: 267-269.
Hübner CA, Stein V, Hermans-Borgmeyer I,Meyer T, Ballanyi K, Jentsch TJ (2001b)Disruption of KCC2 reveals an essentialrole of K-Cl cotransport already in earlysynaptic inhibition. Neuron 30: 515-524.
Ivanov S, Liao SY, Ivanova A, Danilkovitch-Miagkova A, Tarasova N, Weirich G,Merrill MJ, Proescholdt MA, Oldfield EH,Lee J, Zavada J, Waheed A, Sly W,Lerman MI, Stanbridge EJ (2001)Expression of hypoxia-lnducible cell-surface transmembrane carbonicanhydrases in human cancer. Am J Pathol158: 905-919.
Janigro D, Schwartzkroin PA (1988) Effects ofGABA and baclofen on pyramidal cells inthe developing rabbit hippocampus: an 'invitro' study. Brain Res 469: 171-184.
Jarolimek W, Lewen A, Misgeld U (1999) Afurosemide-sensitive K+-Cl- cotransportercounteracts intracellular Cl- accumulationand depletion in cultured rat midbrainneurons. J Neurosci 19: 4695-4704.
Jarolimek W, Misgeld U, Lux HD (1989)Activity dependent alkaline and acidtransients in guinea pig hippocampalslices. Brain Res 505: 225-232.
Jayaraman S, Haggie P, Wachter RM,Remington SJ, Verkman AS (2000)Mechanism and cellular applications of a
green fluorescent protein- based halidesensor. J Biol Chem 275: 6047-6050.
Jentsch TJ, Neagoe L, Scheel O (2005) CLCchloride channels and transporters. CurrOpin Neurobiol 15: 319-325.
Johanson CE, Parandoosh Z, Dyas ML (1992)Maturational differences in acetazolamidealtered pH and HCO3 of choroid plexus,CSF, and brain. Am J Physiol 262: R909-R914.
Johansson PA, Dziegielewska KM, Ek CJ,Habgood MD, Liddelow SA, Potter AM,Stolp HB, Saunders NR (2006) Blood-CSF barrier function in the rat embryo.Eur J Neurosci 24: 65-76.
Johansson PA, Dziegielewska KM, LiddelowSA, Saunders NR (2008) The blood-CSFbarrier explained: When development isnot immaturity. Bioessays 30: 237-248.
Jones HC, Keep RF (1987) The control ofpotassium concentration in thecerebrospinal fluid and brain interstitialfluid of developing rats. J Physiol 383:441-453.
Kaila K (1994) Ionic basis of GABAA receptorchannel function in the nervous system.Prog Neurobiol 42: 489-537.
Kaila K, Blaesse P, Sipilä ST (2008)Development of GABAergic signaling:from molecules to emerging networks. In:Oxford Handbook of DevelopmentalBehavioral Neuroscience (Blumberg MS,Freeman JH, Robinson SR, eds), Oxford:Oxford University Press. In press.
Kaila K, Chesler M (1998) Activity-EvokedChanges in Extracellular pH. In: pH andBrain Function (Kaila K, Ransom BR,eds), pp 309-337. Wiley-Liss, Inc.
Kaila K, Lamsa K, Smirnov S, Taira T, VoipioJ (1997) Long-lasting GABA-mediateddepolarization evoked by high- frequencystimulation in pyramidal neurons of rathippocampal slice is attributable to anetwork-driven, bicarbonate-dependentK+ transient. J Neurosci 17: 7662-7672.
Kaila K, Paalasmaa P, Taira T, Voipio J (1992)pH transients due to monosynapticactivation of GABAA receptors in rathippocampal slices. NeuroReport 3: 105-108.
56
Kaila K, Pasternack M, Saarikoski J, Voipio J(1989a) Influence of GABA-gatedbicarbonate conductance on potential,current and intracellular chloride incrayfish muscle fibres. J Physiol 416: 161-181.
Kaila K, Pasternack M, Saarikoski J, Voipio J(1989b) Influence of HCO3- on thepostsynaptic actions of GABA at thecrayfish neuromuscular synapse. ActaPhysiol Scand Suppl 582: 18.
Kaila K, Rydqvist B, Swerup C, Voipio J(1987) Stimulation-induced changes in theintracellular sodium activity of thecrayfish stretch receptor. Neurosci Lett 74:53-57.
Kaila K, Saarikoski J, Voipio J (1990)Mechanism of action of GABA onintracellular pH and on surface pH incrayfish muscle fibres. J Physiol 427: 241-260.
Kaila K, Voipio J (1987) Postsynaptic fall inintracellular pH induced by GABA-activated bicarbonate conductance. Nature330: 163-165.
Kaila K, Voipio J (1990) GABA-ActivatedBicarbonate Conductance -Influence onE(GABA) and on Postsynaptic pHRegulation. In: Chloride channels andcarriers in nerve, muscle, and glial cells(Alvarez-Leefmans FJ, Russel JM, eds),pp 331-352. Plenum PublishingCorporation.
Kaila K, Voipio J, Paalasmaa P, Pasternack M,Deisz RA (1993) The role of bicarbonatein GABAA receptor-mediated IPSPs of ratneocortical neurones. J Physiol 464: 273-289.
Kakazu Y, Uchida S, Nakagawa T, Akaike N,Nabekura J (2000) Reversibility andcation selectivity of the K+-Cl-cotransport in rat central neurons. JNeurophysiol 84: 281-288.
Kallio H, Pastorekova S, Pastorek J, WaheedA, Sly WS, Mannisto S, Heikinheimo M,Parkkila S (2006) Expression of carbonicanhydrases IX and XII during mouseembryonic development. BmcDevelopmental Biology 6:22..
Kanaka C, Ohno K, Okabe A, Kuriyama K,Itoh T, Fukuda A, Sato K (2001) The
differential expression patterns ofmessenger RNAs encoding K-Clcotransporters (KCC1,2) and Na-K-2Clcotransporter (NKCC1) in the rat nervoussystem. Neuroscience, 104: 933-946.
Katayama F, Miura H, Takanashi S (2002)Long-term effectiveness and side effectsof acetazolamide as an adjunct to otheranticonvulsants in the treatment ofrefractory epilepsies. Brain Dev 24: 150-154.
Katchman AN, Vicini S, Hershkowitz N(1994) Mechanism of early anoxia-induced suppression of the GABAA-mediated inhibitory postsynaptic current. JNeurophysiol 71: 1128-1138.
Katsura K, Kristi n T, Nair R, Siesjö BK(1994) Regulation of intra- andextracellular pH in the rat brain in acutehypercapnia: a re-appraisal. Brain Res651: 47-56.
Keep RF, Ennis SR, Beer ME, Betz AL (1995)Developmental changes-changes in blood-brain-barrier potassium permeability in therat-relation to brain growth. J Physiol 488:439-448.
Kelly T, Church J (2006) Relationshipsbetween calcium and pH in the regulationof the slow afterhyperpolarization incultured rat hippocampal neurons. JNeurophysiol 96: 2342-2353.
Khazipov R, Khalilov I, Tyzio R, Morozova E,Ben Ari Y, Holmes GL (2004)Developmental changes in GABAergicactions and seizure susceptibility in the rathippocampus. Eur J Neurosci 19: 590-600.
Khazipov R, Leinekugel X, Khalilov I, GaiarsaJL, Ben Ari Y (1997) Synchronization ofGABAergic interneuronal network in CA3subfield of neonatal rat hippocampalslices. J Physiol 498: 763-772.
Khirug S, Huttu K, Ludwig A, Smirnov S,Voipio J, Rivera C, Kaila K, Khiroug L(2005) Distinct properties of functionalKCC2 expression in immature mousehippocampal neurons in culture and inacute slices. Eur J Neurosci 21: 899-904.
57
Khirug S, Yamada J, Afzalov R, Voipio J,Khiroug L, Kaila K (2008) GABAergicdepolarization of the axon initial segmentin cortical principal neurons is caused bythe Na-K-2Cl cotransporter NKCC1. JNeurosci 28: 4635-4639.
Kida E, Palminiello S, Golabek AA, Walus M,Wierzba-Bobrowicz T, Rabe A, AlbertiniG, Wisniewski KE (2006) Carbonicanhydrase II in the developing and adulthuman brain. J Neuropathol Exp Neurol65: 664-674.
Kim G, Lee TH, Wetzel P, Geers C, RobinsonMA, Myers TG, Owens JW, Wehr NB,Eckhaus MW, Gros G, Wynshaw-Boris A,Levine RL (2004) Carbonic anhydrase IIIis not required in the mouse for normalgrowth, development, and life span. MolCell Biol 24: 9942-9947.
Kofuji P, Newman EA (2004) Potassiumbuffering in the central nervous system.Neuroscience 129: 1045-1056.
Kohling R, Vreugdenhil M, Bracci E, JefferysJG (2000) Ictal epileptiform activity isfacilitated by hippocampal GABAAreceptor-mediated oscillations. J Neurosci20: 6820-6829.
Krishek BJ, Amato A, Connolly CN, Moss SJ,Smart TG (1996) Proton sensitivity of theGABA(A) receptor is associated with thereceptor subunit composition. J Physiol492: 431-443.
Kruger W, Gilbert D, Hawthorne R, HryciwDH, Frings S, Poronnik P, Lynch JW(2005) A yellow fluorescent protein-basedassay for high-throughput screening ofglycine and GABA(A) receptor chloridechannels. Neurosci Lett 380: 340-345.
Kuner T, Augustine GJ (2000) A geneticallyencoded ratiometric indicator for chloride:capturing chloride transients in culturedhippocampal neurons. Neuron 27: 447-459.
Lakkis MM, Bergenhem NCH, OShea KS,Tashian RE (1997) Expression of theacatalytic carbonic anhydrase VIII gene,
Car8, during mouse embryonicdevelopment. Histochem J 29: 135-141.
Lamsa K, Palva JM, Ruusuvuori E, Kaila K,Taira T (2000) Synaptic GABA(A)activation inhibits AMPA-kainatereceptor-mediated bursting in the newborn(P0-P2) rat hippocampus. J Neurophysiol83: 359-366.
Le Rouzic P, Ivanov TR, Stanley PJ, BaudoinFMH, Chan F, Pinteaux E, Brown PD,Luckman SM (2006) KCC3 and KCC4expression in rat adult forebrain. BrainRes 1110: 39-45.
LeBeau FE, Towers SK, Traub RD,Whittington MA, Buhl EH (2002)Transient fast hippocampal networkoscillations in vitro by potassiumtransients. J Physiol 542: 167-179.
Lee J, Taira T, Pihlaja P, Ransom BR, Kaila K(1996) Effects of CO2 on excitatorytransmission apparently caused bychanges in intracellular pH in the rathippocampal slice. Brain Res 706: 210-216.
Leinekugel X, Medina I, Khalilov I, Ben AriY, Khazipov R (1997) Ca2+ oscillationsmediated by the synergistic excitatoryactions of GABA(A) and NMDAreceptors in the neonatal hippocampus.Neuron 18: 243-255.
Leniger T, Wiemann M, Bingmann D,Widman G, Hufnagel A, Bonnet U (2002)Carbonic anhydrase inhibitor sulthiamereduces intracellular pH and epileptiformactivity of hippocampal CA3 neurons.Epilepsia 43: 469-474.
Lewis SE, Erickson RP, Barnett LB, Venta PJ,Tashian RE (1988) N-ethyl-N-nitrosureainduced mutation at the mouse Car-2 locus- an animal model for human carbonicanhudrase II-deficiency syndrome. ProcNatl Acad Sci U S A 85: 1962-1966.
Li H, Tornberg J, Kaila K, Airaksinen MS,Rivera C (2002) Patterns of cation-chloride cotransporter expression duringembryonic rodent CNS development. EurJ Neurosci 16: 2358-2370.
Linden DJ (1999) The return of the spike:Postsynaptic action potentials and theinduction of LTP and LTD. Neuron 22:661-666.
58
Lindsey AE, Schneider K, Simmons DM,Baron P, Lee BS, Kopito RR (1990)Functional expression and subcellular-localization of an anion-exchanger clonedfrom choroid plexus. Proc Natl Acad SciU S A 87: 5278-5282.
Lothman EW, Somjen GG (1975)Extracellular potassium activity,intracellular and extracellular potentialresponses in the spinal cord. J Physiol252: 115-136.
LoTurco JJ, Kriegstein AR (1991) Clusters ofcoupled neurons in embryonic neocortex.Science 252: 563-566.
LoTurco JJ, Owens DF, Heath MJS, DavisMBE, Kriegstein AR (1995) GABA andglutamate depolarize cortical progenitorcells and inhibit DNA synthesis. Neuron15: 1287-1298.
Lu J, Daly CM, Parker MD, Gill HS,Piermarini PM, Pelletier MF, Boron WF(2006) Effect of human carbonicanhydrase II on the activity of the humanelectrogenic Na/HCO3 cotransporterNBCe1-A in Xenopus oocytes. J BiolChem 281: 19241-19250.
Luckermann M, Trapp S, Ballanyi K (1997)GABA- and glycine-mediated fall ofintracellular pH in rat medullary neuronsin situ. J Neurophysiol 77: 1844-1852.
Luhmann HJ, Prince DA (1991) Postnatalmaturation of the GABAergic system inrat neocortex. J Neurophysiol 65: 247-263.
Magistretti, P. J. and Pellerin, L. (1999)Cellular mechanisms of brain energymetabolism and their relevance tofunctional brain imaging Philos Trans RSoc Lond B Biol Sci. 354:1155-1163.
Majumdar D, Maunsbach AB, Shacka JJ,Williams JB, Berger UV, Schultz KP,Harkins LE, Boron WF, Roth KA,Bevensee MO (2008) Localization ofelectrogenic Na/bicarbonate cotransporterNBCe1 variants in rat brain. Neuroscience155: 818-832.
Makani S, Chesler M (2007) Endogenousalkaline transients boost postsynapticNMDA receptor responses in hippocampalCA1 pyramidal neurons. J Neurosci 27:7438-7446.
Malleret G, Hen R, Guillou JL, Segu L, BuhotMC (1999) 5-HT1B receptor knock-outmice exhibit increased exploratory activityand enhanced spatial memoryperformance in the Morris water maze. JNeurosci 19: 6157-6168.
Markova O, Mukhtarov M, Real E, Jacob Y,Bregestovski P (2008) Geneticallyencoded chloride indicator with improvedsensitivity. J Neurosci Meth 170: 67-76.
Marty A, Llano I (2005) Excitatory effects ofGABA in established brain networks.Trends Neurosci 28: 284-289.
Mata AM, Sepulveda MR (2005) Calciumpumps in the central nervous system.Brain Res Rev 49: 398-405.
McCormick DA, Contreras D (2001) On thecellular and network bases of epilepticseizures. Annu Rev Physiol 63: 815-846.
McNamara JO (1994) Cellular and molecularbasis of epilepsy. J Neurosci 14: 3413-3425.
Mellergård P, Siesjö BK (1998) Cerebralenergy metabolism and pH. In: pH andbrain function (Kaila K, Ransom BR, eds),pp 67-91. Wiley-Liss.
Mercado A, Mount DB, Gamba G (2004)Electroneutral cation-chloridecotransporters in the central nervoussystem. Neurochem Res 29: 17-25.
Mercado A, Song L, Vazquez N, Mount DB,Gamba G (2000) Functional comparisonof the K+-Cl- cotransporters KCC1 andKCC4. J Biol Chem 275: 30326-30334.
Messeter K, Siesjö BK (1971) Regulation ofCSF pH in acute and sustained respiratoryacidosis. Acta Physiol Scand 83: 21-30.
Michelson HB, Wong RK (1994)Synchronization of inhibitory neurones inthe guinea-pig hippocampus in vitro. JPhysiol 477 ( Pt 1): 35-45.
Millichap JG, Woodbury DM, Goodman LS(1955)Mechanisms of the anticonvulsantaction of acetazolamide a carbonic
Misgeld U, Deisz RA, Dodt HU, Lux HD(1986) The role of chloride transport inpostsynaptic inhibition of hippocampalneurons. Science 232: 1413-1415.
Mollgård K, Balslev Y, Lauritzen B, SaundersNR (1987) Cell-junctions and membranespecializations in the ventricular zone(germinal matrix) of the developíng sheepbrain- a CSFbrain-barrier. J Neurocytol16: 433-444.
Mollgård K, Lauritzen B, Saunders NR (1979)Double replica technique applied tochroid-plexus from early fetal sheep -completeness and complexity of tightjunctions. J Neurocytol 8: 139-149.
Mori K, Ogawa Y, Ebihara K, Tamura N,Tashiro K, Kuwahara T, Mukoyama M,Sugawara A, Ozaki S, Tanaka I, Nakao K(1999) Isolation and characterization ofCA XIV, a novel membrane-boundcarbonic anhydrase from mouse kidney. JBiol Chem 274: 15701-15705.
Mount DB, Mercado A, Song L, Xu J, GeorgeALJ, Delpire E, Gamba G (1999) Cloningand characterization of KCC3 and KCC4,new members of the cation-chloridecotransporter gene family. J Biol Chem274: 16355-16362.
Munsch T, Pape HC (1999) Upregulation ofthe hyperpolarization-activated cationcurrent in rat thalamic relay neurones byacetazolamide. J Physiol 519 Pt 2: 505-514.
Nabekura J, Ueno T, Okabe A, Furuta A,Iwaki T, Shimizu-Okabe C, Fukuda A,Akaike N (2002) Reduction of KCC2expression and GABA(A) receptor-mediated excitation after in vivo axonalinjury. J Neurosci 22: 4412-4417.
Nagao Y, Srinivasan M, Platero JS,Svendorowski M, Waheed A, Sly WS(1994) Mitochondrial carbonic anhydrase(isozyme-V) in mouse and rat - CDNAcloning, expression, subcellular-localization, processing and tissuedistribution. Proc Natl Acad Sci U S A 91:10330-10334.
Nattie E (1999). CO2, brainstem chemo-receptors and breathing. Prog Neurobiol59: 299-331.
Nattie EE (2001) Central chemosensitivity,sleep, and wake-fulness. Respir Physiol129: 257-268.
Nattie EE, Edwards WH (1981) CSF acid-baseregulation and ventilation during acutehypercapnia in the newborn dog. J ApplPhysiol 50: 566-574.
Neher E (2000) Some quantitavie aspects ofcalcium fluorimetry. In: Imaging neurons;Alaboratory manual (Yuste R, Lanni F,Konnerth A, eds), pp 31.1-31.11. ColdSpring Harbor Laboratory Press.
Newman EA (1996) Regulation ofextracellular K+ and pH by polarized ionfluxes in glial cells: The retinal Mullercell. Neuroscientist 2: 109-117.
Nicholson, C. (2001) Diffusion and relatedtransport mechanisms in brain tissue. Rep.Prog. Phys. 64: 815-884.
Nogradi A, Jonsson N, Walker R, Caddy K,Carter N, Kelly C (1997) Carbonicanhydrase II and carbonic anhydrase-related protein in the cerebellar cortex ofnormal and lurcher mice. DevelopmentalBrain Research 98: 91-101.
Nogradi A, Kiraly E, Mihaly A (1989)Neuronal carbonic anhydrase activity inthe central nervous system of the rat - lighthistochemical and electron histochemical-investigations of the islands of calleja.Acta Histochemica 85: 187-193.
O'Connor ER, Sontheimer H, Ransom BR(1994) Rat hippocampal astrocytes exhibitelectrogenic sodium- bicarbonate co-transport. J Neurophysiol 72: 2580-2589.
O'Donovan MJ (1999) The origin ofspontaneous activity in developingnetworks of the vertebrate nervous system.Curr Opin Neurol 9: 94-104.
Orlowski J, Grinstein S (2004) Diversity of themammalian sodium/proton exchangerSLC9 gene family. Pflugers Arch 447:549-565.
Owens DF, Boyce LH, Davis MBE, KriegsteinAR (1996) Excitatory GABA responses inembryonic and neonatal cortical slices
60
demonstrated by gramicidin perforated-patch recordings and calcium imaging. JNeurosci 16: 6414-6423.
Owens DF, Kriegstein AR (2002) Is theremore to GABA than synaptic inhibiton?Nat Rev Neurosci 3: 715-727.
Paalasmaa P, Taira T, Voipio J, Kaila K (1994)Extracellular alkaline transients mediatedby glutamate receptors in the rathippocampal slice are not due to a protonconductance. J Neurophysiol 72: 2031-2033.
Pace AJ, Lee E, Athirakul K, Coffman TM,O'Brien DA, Koller BH (2000) Failure ofspermatogenesis in mouse lines deficientin the Na+-K+-2Cl(-) cotransporter. J ClinInvest 105: 441-450.
Pappas CA, Ransom BR (1994)Depolarization-induced alkalinization(DIA) in rat hippocampal astrocytes. JNeurophysiol 72: 2816-2826.
Parker MD, Musa-Aziz R, Rojas JD, Choi I,Daly CM, Boron WF (2008)Characterization of human SLC4A10 asan electroneutral Na/HCO3 cotransporter(NBCn2) with Cl- self-exchange activity.J Biol Chem 283: 12777-12788.
Parkkila S, Parkkila AK, Rajaniemi H, ShahGN, Grubb JH, Waheed A, Sly WS (2001)Expression of membrane-associatedcarbonic anhydrase XIV on neurons andaxons in mouse and human brain. ProcNatl Acad Sci U S A 98: 1918-1923.
Pasternack M, Smirnov S, Kaila K (1996)Proton modulation of functionally distinctGABAA receptors in acutely isolatedpyramidal neurons of rat hippocampus.Neuropharmacology 35: 1279-1288.
Pasternack M, Voipio J, Kaila K (1993)Intracellular carbonic anhydrase activityand its role in GABA- induced acidosis inisolated rat hippocampal pyramidalneurones. Acta Physiol Scand 148: 229-231.
Pastorek J, Pastorekova S, Callebaut I, MornonJP, Zelnik V, Opavsky R, Zatovicova M.,Liao S, Portetelle D, Stanbridge, E.J,Zavada J, Burny A, Kettmann R.(1994)Cloning and characterization of MN, ahuman tumor-asociated protein with adomain homologous to carbonic
anhydrase and putative helix-loop-helixDNA-binding segment. Oncogene 9:2877-2888.
Pavlin EG, Hornbein TF (1975) Distribution ofdistribution H+ and HCO3- between CSFand blood during respiratory acidosis indogs. Am J Physiol 228: 1145-1148.
Payne JA (1997) Functional characterization ofthe neuronal-specific K-Cl cotransporter -implications for [K+](o) regulation. Am JPhysiol 42: C1516-C1525.
Payne JA, Stevenson TJ, Donaldson LF (1996)Molecular characterization of a putativeK-Cl cotransporter in rat brain. Aneuronal-specific isoform. J Biol Chem271: 16245-16252.
Pearson MM, Lu J, Mount DB, Delpire E(2001) Localization of the K-Cl-cotransporter, KCC3, in the central andperipheral nervous systems: expression inthe choroid plexus, large neurons andwhite matter tracts. Neuroscience, 103:481-491.
Penttonen M, Kamondi A, Acsady L, BuzsakiG (1998) Gamma frequency oscillation inthe hippocampus of the rat: intracellularanalysis in vivo. Eur J Neurosci 10: 718-728.
Perreault P, Avoli M (1988) A depolarizinginhibitory postsynaptic potential activatedby synaptically released gamma-aminobutyric acid under physiologicalconditions in rat hippocampal pyramidalcells. Can J Physiol Pharmacol 66: 1100-1102.
Petit C (2001) Usher syndrome: From geneticsto pathogenesis. Annu Rev GenomicsHum Genet 2: 271-297.
61
Piermarini PM, Kim EY, Boron WF (2007)Evidence against a direct interactionbetween intracellular carbonic anhydraseII and pure C-terminal domains of SLC4bicarbonate transporters. J Biol Chem 282:1409-1421.
Plotkin MD, Snyder EY, Hebert SC, Delpire E(1997) Expression of the Na-K-2Clcotransporter is developmentally regulatedin postnatal rat brains: a possiblemechanism underlying GABA's excitatoryrole in immature brain. J Neurobiol 33:781-795.
Praetorius J (2007) Water and solute secretionby the choroid plexus. Pflugers Arch 454:1-18.
Praetorius J, Nejsum LN, Nielsen S (2004) ASCL4A10 gene product maps selectivelyto the basolateral plasma membrane ofchoroid plexus epithelial cells. Am JPhysiol 286: C601-C610.
Praetorius J, Nielsen S (2006) Distribution ofsodium transporters and aquaporin-1 inthe human choroid plexus. Faseb Journal20: A1235-A1236.
Price GD, Trussell LO (2006) Estimate of thechloride concentration in a centralglutamatergic terminal: a gramicidinperforated-patch study on the Calyx ofHeld. J Neurosci 26: 11432-11436.
Putnam RW, Filosa JA, Ritucci NA (2004)Cellular mechanisms involved in CO2 andacid signaling in chemosensitive neurons.Am J Physiol 287: C1493-C1526.
Race JE, Makhlouf FN, Logue PJ, Wilson FH,Dunham PB, Holtzman EJ (1999)Molecular cloning and functionalcharacterization of KCC3, a new K-Clcotransporter. Am J Physiol 277: C1210-C1219.
Raley-Susman KM, Sapolsky RM, Kopito RR(1993) Cl-/HCO3- exchange functiondiffers in adult and fetal rat hippocampalneurons. Brain Res 614: 308-314.
Randall J, Thorne T, Delpire E (1997) Partialcloning and characterization of Slc12a2:the gene encoding the secretory Na+-K+-2Cl- cotransporter. Am J Physiol 273:C1267-C1277.
Ransom BR, Carlini WG, Yamate CL (1987)Tip size of ion-exchanger based K+ -selective microelectrodes.2. Effects onmeasurement of evoked [K+]o tranisents.Can J Physiol Pharmacol 65: 894-897.
Rink TJ, Tsien RY, Pozzan T (1982)Cytoplasmic pH and free Mg2+ inlymphocytes. J Cell Biol 95: 189-196.
Ritucci NA, Chambers-Kersh L, Dean JB,Putnam RW (1998) Intracellular pHregulation in neurons from chemosensitiveand nonchemosensitive areas of themedulla. Am J Physiol Regul Integr CompPhysiol 275: R1152-R1163.
Ritucci NA, Dean JB, Putnam RW (1997)Intracellular pH response to hypercapniain neurons from chemosensitive areas ofthe medulla. Am J Physiol Regul IntegrComp Physiol 273: R433-R441.
Rivera C, Li H, Thomas-Crusells J, LahtinenH, Viitanen T, Nanobashvili A, Kokaia Z,Airaksinen MS, Voipio J, Kaila K, SaarmaM (2002) BDNF-induced TrkB activationdown-regulates the K+-Cl- cotransporterKCC2 and impairs neuronal Cl- extrusion.J Cell Biol 159: 747-752.
Rivera C, Voipio J, Thomas-Crusells J, Li H,Emri Z, Sipilä S, Payne JA, Minichiello L,Saarma M, Kaila K (2004) Mechanism ofactivity-dependent downregulation of theneuron-specific K-Cl cotransporter KCC2.J Neurosci 24: 4683-4691.
Rocha-Gonzalez HI, Mao S, Alvarez-Leefmans FJ (2008) Na+, K+, 2Cl(-)cotransport and intracellular chlorideregulation in rat primary sensory neurons:Thermodynamic and kinetic aspects. JNeurophysiol 100: 169-184.
62
Romero MF, Fulton CM, Boron WF (2004)The SLC4 family of HCO3- transporters.Pflugers Arch 447: 495-509.
Roos A, Boron WF (1981) Intracellular pH.Physiol Rev 61: 296-434.
Rotin D, Grinstein S (1989) Impaired cell-volume regulation in Na+ - H+ exchange-deficient mutants. Am J Physiol 257:C1158-C1165.
Russell JM (2000) Sodium-potassium-chloridecotransport. Physiol Rev 80: 211-276.
Ruusuvuori, E., Hübner, C. A., and Voipio, J.(2007) The role of the Na+-independentCl-/HCO3- exchanger AE3 inhippocampal CA3 pyramidal cell pHregulation. Soc Neurosci Abstr 37:683.16.
Ruusuvuori, E., Kaila, K., and Voipio, J.(1999) Interstitial and intrapyramidalcarbonic anhydrase activity in the rathippocampus during early postnataldevelopment. Soc Neurosci Abstr25:408.3. 1999.
Saarikoski J, Kaila K (1992) Simultaneousmeasurement of intracellular andextracellular carbonic anhydrase activityin intact muscle fibers. Pflugers Arch 421:357-363.
cotransporters in rat brain: Expression inglia, neurons, and choroid plexus. JNeurosci 20: 6839-6848.
Schmitz D, Schuchmann S, Fisahn A, DraguhnA, Buhl EH, Petrasch-Parwez E,Dermietzel R, Heinemann U, Traub RD(2001) Axo-axonal coupling. a novelmechanism for ultrafast neuronalcommunication. Neuron 31: 831-840.
Schuchmann S, Schmitz D, Rivera C,Vanhatalo S, Salmen B, Mackie K, SipiläST, Voipio J, Kaila K (2006)Experimental febrile seizures areprecipitated by a hyperthermia-inducedrespiratory alkalosis. Nat Med 12: 817-823.
Schwiening CJ, Boron WF (1994) Regulationof intracellular pH in pyramidal neuronesfrom the rat hippocampus by Na+-dependent Cl-HCO3- exchange. J Physiol475: 59-67.
Schwiening CJ, Willoughby D (2002)Depolarization-induced pH microdomainsand their relationship to calcium transientsin isolated snail neurones. J Physiol 538:371-382.
Seki G, Fromter E (1992) Acetazolamideinhibition of basolateral base exit in rabbitrenal proximal tubule S2-segment.Pflugers Arch 422: 60-65.
Shah GN, Ulmasov B, Waheed A, Becker T,Makani S, Svichar N, Chesler M, Sly WS(2005) Carbonic anhydrase IV and XIVknockout mice: Roles of the respectivecarbonic anhydrases in buffering theextracellular space in brain. Proc NatlAcad Sci U S A 102: 16771-16776.
Shrode LD, Putnam RW (1994) IntracellularpH regulation in primary rat astrocytesand C6 glioma-cells. Glia 12: 196-210.
Silver IA, Erecinska M (1990) Intracellularand extracellular changes of [Ca2+] inhypoxia and ischemia in rat brain in vivo.J Gen Physiol 95: 837-866.
Silver IA, Erecinska M (1992) Ion homeostasisin rat-brain in vivo: Intracellular andextracellular [Ca2+] and [H+] in thehippocampus during recovery from short-term, transient ischemia. J Cereb BloodFlow Metab 12: 759-772.
63
Singer W, Lux HD (1975) Extracellularpotassium gradients and visual receptive-field in cat striate cortex. Brain Res 96:378-383.
Sipilä ST, Huttu K, Voipio J, Kaila K (2006a)Intrinsic bursting of immature CA3pyramidal neurons and consequent giantdepolarizing potentials are driven by apersistent Na+ current and terminated by aslow Ca2+-activated K+ current. Eur JNeurosci 23: 2330-2338.
Sipilä ST, Schuchmann S, Voipio J, Yamada J,Kaila K (2006b) The cation-chloridecotransporter NKCC1 promotes sharpwaves in the neonatal rat hippocampus. JPhysiol 573: 765-773.
Slemmer JE, Matsushita S, De Zeeuw CI,Weber JT, Knopfel T (2004) Glutamate-induced elevations in intracellular chlorideconcentration in hippocampal cell culturesderived from EYFP- expressing mice. EurJ Neurosci 19: 2915-2922.
Sly WS, Hewettemmett D, Whyte MP, YuYSL, Tashian RE (1983) Carbonicanhydrase -II deficiency identified as theprimary defect in the autosomal recessivesyndrome of osteopetrosis with renaltubular-acidosis and cerebral calcification.Proc Natl Acad Sci U S A 80: 2752-2756.
Sly WS, Hu PY (1995) Human carbonicanhydrases and carbonic anhydrasedeficiencies. Annu Rev Biochem 64: 375-401.
Smirnov S, Paalasmaa P, Uusisaari M, VoipioJ, Kaila K (1999) Pharmacologicalisolation of the synaptic and nonsynapticcomponents of the GABA-mediatedbiphasic response in rat CA1 hippocampalpyramidal cells. J Neurosci 19: 9252-9260.
Smith SE, Gottfried JA, Chen JC, Chesler M(1994) Calcium dependence of glutamatereceptor-evoked alkaline shifts inhippocampus. NeuroReport 5: 2441-2445.
Somjen GG (1984) Acidification of interstitialfluid in hippocampal formation caused byseizures and by spreading depression.Brain Res 311: 186-188.
Somjen GG (2001) Mechanisms of spreadingdepression and hypoxic spreadingdepression-like depolarization. PhysiolRev 81: 1065-1096.
Somjen GG (2002) Ion regulation in the brain:Implications for pathophysiology.Neuroscientist 8: 254-267.
Sterling D, Alvarez BV, Casey JR (2002) Theextracellular component of a transportmetabolon - Extracellular loop 4 of thehuman AE1 Cl-/HCO(3)(-)exchangerbinds carbonic anhydrase IV. J Biol Chem277: 25239-25246.
Sterling D, Reithmeier RAF, Casey JR (2001)A transport metabolon. Functionalinteraction of carbonic anhydrase II andchloride/bicarbonate exchangers. J BiolChem 276: 47886-47894.
64
Strata F, Atzori M, Molnar M, Ugolini G,Tempia F, Cherubini E (1997) Apacemaker current in dye-coupled hilarinterneurons contributes to the generationof giant GABAergic potentials indeveloping hippocampus. J Neurosci 17:1435-1446.
Supuran CT, Scozzafava A, Casini A (2003)Carbonic anhydrase inhibitors. Med ResRev 23: 146-189.
Supuran CT, Scozzafava A, Conway J (2004)Carbonic anhydrase: Its inhibitors andactivators. CRC Press.
Suzuki M, Raisman G (1994) Multificolpattern of postnatal-development of themacroglial framework of the rat fimbria.Glia 12: 294-308.
Svichar N, Chesler M (2003) Surface carbonicanhydrase activity on astrocytes andneurons facilitates lactate transport. Glia41: 415-419.
Svichar, N., Waheed, A., Sly, W. S., Hennings,J. C., Hübner, C. A., and Chesler, M.(2007) Functional link between surfacecarbonic anhydrases and chloride-bicarbonate exchange in neurons andastrocytes. Soc Neurosci Abstr 37:683.17.
Sykova E, Rothenbe S, Krekule I (1974)Changes of extracellular potassiumconcentration during spontaneous activityin mesencephalic reticular-formation ofrat. Brain Res 79: 333-337.
Taira T, Lamsa K, Kaila K (1997) Posttetanicexcitation mediated by GABA(A)receptors in rat CA1 pyramidal neurons. JNeurophysiol 77: 2213-2218.
Taira T, Paalasmaa P, Voipio J, Kaila K (1995)Relative contributions of excitatory andinhibitory neuronal activity to alkalinetransients evoked by stimulation ofSchaffer collaterals in the rat hippocampalslice. J Neurophysiol 74: 643-649.
Taira T, Smirnov S, Voipio J, Kaila K (1993)Intrinsic proton modulation of excitatorytransmission in rat hippocampal slices.NeuroReport 4: 93-96.
Takeuchi A, Takeuchi N (1967) Anionpermeability of the inhibitory post-synaptic membrane of the crayfish
neuromuscular junction. J Physiol 191:575-590.
Tang CM, Dichter M, Morad M (1990)Modulation of the N-methyl-D-aspartatechannel by extracellular H+. Proc NatlAcad Sci U S A 87: 6445-6449.
Thomas JA, Buchsbaum RN, Zimniak A,Racker E (1979) Intracellular pHmeasurements in Ehrlich ascites tumorcells utilizing spectroscopic probesgenerated in situ. Biochemistry (N Y ) 18:2210-2218.
Thomas RC (1974) Intracellular pH of snailneurones measured with a new pH-sensitive glass mirco-electrode. J Physiol238: 159-180.
Thomas RC (1988) Proton channels in snailneurons studied with surface pH glassmicroelectrodes. Ciba Found Symp 139:168-183.
Thomas RC, Meech RW (1982) Hydrogen ioncurrents and intracellular pH indepolarized voltage-clamped snailneurones. Nature 299: 826-828.
Thompson SM, Deisz RA, Prince DA (1988a)Outward chloride/cation co-transport inmammalian cortical neurons. NeurosciLett 89: 49-54.
Thompson SM, Deisz RA, Prince DA (1988b)Relative contributions of passiveequilibrium and active transport to thedistribution of chloride in mammaliancortical neurons. J Neurophysiol 60: 105-124.
Tombaugh GC, Somjen GG (1996) Effects ofextracellular pH on voltage-gated Na+, K+and Ca2+ currents in isolated rat CA1neurons. J Physiol 493 ( Pt 3): 719-732.
Tombaugh GC, Somjen GG (1997)Differential sensitivity to intracellular pHamong high- and low- threshold Ca2+currents in isolated rat CA1 neurons. JNeurophysiol 77: 639-653.
Tong CK, Brion LP, Suarez C, Chesler M(2000) Interstitial carbonic anhydrase(CA) activity in brain is attributable tomembrane-bound CA type IV. J Neurosci20: 8247-8253.
65
Tong CK, Chen K, Chesler M (2006) Kineticsof activity-evoked pH transients andextracellular pH buffering in rathippocampal slices. J Neurophysiol 95:3686-3697.
Tornberg J, Voikar V, Savilahti H, Rauvala H,Airaksinen MS (2005) Behaviouralphenotypes of hypomorphic KCC2-deficient mice. Eur J Neurosci 21: 1327-1337.
Trapp S, Luckermann M, Kaila K, Ballanyi K(1996) Acidosis of hippocampal neuronesmediated by a plasmalemmal Ca2+/H+pump. NeuroReport 7: 2000-2004.
Traub RD, Pais I, Bibbig A, LeBeau FEN,Buhl EH, Hormuzdi SG, Monyer H,Whittington MA (2003) Contrasting rolesof axonal (pyramidal cell) and dendritic(interneuron) electrical coupling in thegeneration of neuronal networkoscillations. Proc Natl Acad Sci U S A100: 1370-1374.
Traub RD, Spruston N, Soltesz I, Konnerth A,Whittington MA, Jefferys GR (1998)Gamma-frequency oscillations: a neuronalpopulation phenomenon, regulated bysynaptic and intrinsic cellular processes,and inducing synaptic plasticity. ProgNeurobiol 55: 563-575.
Traub RD, Whittington MA, Buhl EH, JefferysJG, Faulkner HJ (1999) On themechanism of the gamma --> betafrequency shift in neuronal oscillationsinduced in rat hippocampal slices bytetanic stimulation. J Neurosci 19: 1088-1105.
Traub RD, Whittington MA, Colling SB,Buzsaki G, Jefferys JG (1996a) Analysisof gamma rhythms in the rat hippocampusin vitro and in vivo. J Physiol 493: 471-484.
Traub RD, Whittington MA, Stanford IM,Jefferys JG (1996b) A mechanism forgeneration of long-range synchronous fastoscillations in the cortex. Nature 383: 621-624.
Traynelis SF, Cull-Candy SG (1990) Protoninhibition of N-methyl-D-aspartatereceptors in cerebellar neurons. Nature345: 347-350.
Traynelis SF, Dingledine R (1989)Modification of potassium-inducedinterictal bursts and electrographicseizures by divalent cations. Neurosci Lett98: 194-199.
Traynelis SF, Hartley M, Heinemann SF(1995) Control of proton sensitivity of theNMDA receptor by RNA splicing andpolyamines. Science 268: 873-876.
Tureci O, Sahin U, Vollmar E, Siemer S,Gottert E, Seitz G, Parkkila AK, Shah GN,Grubb JH, Pfreundschuh M, Sly WS(1998) Human carbonic anhydrase XII:cDNA cloning, expression, andchromosomal localization of a carbonicanhydrase gene that is overexpressed insome renal cell cancers. Proc Natl AcadSci U S A 95: 7608-7613.
Tyzio R, Minlebaev M, Rheims S, Ivanov A,Jorquera I, Holmes GL, Zilberter Y, BenAri Y, Khazipov R (2008) Postnatalchanges in somatic gamma-aminobutyricacid signalling in the rat hippocampus.Eur J Neurosci 27: 2515-2528.
Tyzio R, Represa A, Jorquera I, Ben Ari Y,Gozlan H, Aniksztejn L (1999) Theestablishment of GABAergic andglutamatergic synapses on CA1 pyramidalneurons is sequential and correlates withthe development of the apical dendrite. JNeurosci 19: 10372-10382.
Urbanics R, Leniger-Follert E, bbers DW(1978) Time course of changes ofextracellular H+ and K+ activities duringand after direct electrical stimulation ofthe brain cortex. Pflugers Arch 378: 47-53.
Uvarov P, Ludwig A, Markkanen M, PruunsildP, Kaila K, Delpire E, Timmusk T, RiveraC, Airaksinen MS (2007) A novel N-terminal isoform of the neuron-specific K-Cl cotransporter KCC2. J Biol Chem 282:30570-30576.
Valeyev AY, Cruciani RA, Lange GD,Smallwood VS, Barker JL (1993) Cl-channels are randomly activated bycontinuous GABA secretion in culturedembryonic rat hippocampal neurons.Neurosci Lett 155: 199-203.
van den Pol AN, Obrietan K, Chen G (1996)Excitatory actions of GABA after
Velisek L, Moshe SL, Stanton PK (1995)Resistance of hippocampal synaptictransmission to hypoxia in carbonicanhydrase II-deficient mice. Brain Res671: 245-253.
Velisek L, Moshe SL, Xu SG, Cammer W(1993) Reduced susceptibility to seizuresin carbonic anhydrase II deficient mutantmice. Epilepsy Res 14: 115-121.
Vibat CRT, Holland MJ, Kang JJ, Putney LK,O'Donnell ME (2001) Quantitation ofNa+-K+-2Cl(-) cotransport splice variantsin human tissues using kinetic polymerasechain reaction. Anal Biochem 298: 218-230.
Vilen H, Eerikainen S, Tornberg J, AiraksinenMS, Savilahti H (2001) Construction ofgene-targeting vectors: A rapid Mu invitro DNA transposition-based strategygenerating null, potentially hypomorphic,and conditional alleles. Transgenic Res10: 69-80.
Voipio J (1998) Diffusion and bufferingaspects of H+, HCO3-, and CO2movements in brain tissue. In: pH andbrain function (Kaila K, Ransom BR, eds),pp 45-66. Wiley-Liss.
Voipio J, Kaila K (1993) Interstitial PCO2 andpH in rat hippocampal slices measured bymeans of a novel fast CO2/H(+)-sensitivemicroelectrode based on a PVC-gelledmembrane. Pflugers Arch 423: 193-201.
Voipio J, Kaila K (2000) GABAergicexcitation and K(+)-mediated volumetransmission in the hippocampus. ProgBrain Res 125: 329-338.
Voipio J, Paalasmaa P, Taira T, Kaila K (1995)Pharmacological characterization ofextracellular pH transients evoked byselective synaptic and exogenousactivation of AMPA, NMDA, andGABAA receptors in the rat hippocampalslice. J Neurophysiol 74: 633-642.
Voipio J, Pasternack M, Rydqvist B, Kaila K(1991) Effect of gamma-aminobutyricacid on intracellular pH in the crayfishstretch-receptor neurone. J Exp Biol 156:349-360.
Voipio, J., Ruusuvuori, E., and Kaila, K.(1999) GABAA receptor channelmediated anionic load and the consequentincrease in interstitial K+: adevelopmental study sheds light on themolecular mechanisms involved. SocNeurosci Abstr 25:710.18.
Voipio J, Tallgren P, Heinonen E, VanhataloS, Kaila K (2003) Millivolt-scale DCshifts in the human scalp EEG: Evidencefor a nonneuronal generator. JNeurophysiol 89: 2208-2214.
Vukicevic M, Kellenberger S (2004)Modulatory effects of acid-sensing ionchannels on action potential generation inhippocampal neurons. Am J Physiol 287:C682-C690.
Wachter RM, Remington SJ (1999) Sensitivityof the yellow variant of green fluorescentprotein to halides and nitrate. Curr Biol 9:R628-R629.
Walz W (1989) pH shifts evoked by neuronalstimulation in slices of rat hippocampus.Can J Physiol Pharmacol 67: 577-581.
Wang C, Shimizu-Okabe C, Watanabe K,Okabe A, Matsuzaki H, Ogawa T, Mori N,Fukuda A, Sato K (2002a) Developmentalchanges in KCC1, KCC2, and NKCC1mRNA expressions in the rat brain. BrainRes Dev Brain Res 139: 59-66.
Wang WG, Bradley SR, Richerson GB(2002b) Quantification of the response ofrat medullary raphe neurones toindependent changes in pH(O) and P-CO2. J Physiol 540: 951-970.
67
Wegelius K, Reeben M, Rivera C, Kaila K,Saarma M, Pasternack M (1996) The rho 1GABA receptor cloned from rat retina isdown-modulated by protons. NeuroReport7: 2005-2009.
Wetzel P, Gros G (1998) Inhibition and kineticproperties of membrane-bound carbonicanhydrases in rabbit skeletal muscles.Arch Biochem Biophys 356: 151-158.
Wetzel P, Hasse A, Papadopouloss S, Voipio J,Kaila K, Gros G (2001) Extracellularcarbonic anhydrase activity facilitateslactic acid transport in rat skeletal musclefibres. J Physiol 531: 743-756.
Whittington MA, Doheny HC, Traub RD,LeBeau FE, Buhl EH (2001) Differentialexpression of synaptic and nonsynapticmechanisms underlying stimulus-inducedgamma oscillations in vitro. J Neurosci21: 1727-1738.
Whittington MA, Stanford IM, Colling SB,Jefferys JG, Traub RD (1997)Spatiotemporal patterns of gammafrequency oscillations tetanically inducedin the rat hippocampal slice. J Physiol502: 591-607.
Williams JR, Sharp JW, Kumari VG, WilsonM, Payne JA (1999) The neuron-specificK-Cl cotransporter, KCC2. Antibodydevelopment and initial characterization ofthe protein. J Biol Chem 274: 12656-12664.
Williams RM, Piston DW, Webb WW (1994)2-photon molecular-excitation providesintrinsic 3-dimensional resolution forlaser-microscopy and microphoto-chemistry. Faseb Journal 8: 804-813.
Woo NS, Lu JM, England R, McClellan R,Dufour S, Mount DB, Deutch AY,Lovinger DM, Delpire E (2002)Hyperexcitability and epilepsy associatedwith disruption of the mouse neuronal-specific K-Cl cotransporter gene.Hippocampus 12: 258-268.
D (1970) Brain potential shift withrespiratory acidosis in the cat and monkey.Am J Physiol 218: 275-283.
Wyllie, E. (1997) The Treatment of Epilepsy:Principles and Practice.Williams&Wilkins.
Xiong ZQ, Saggau P, Stringer JL (2000)Activity-dependent intracellularacidification correlates with the durationof seizure activity. J Neurosci 20: 1290-1296.
Yamada J, Okabe A, Toyoda H, Kilb W,Luhmann HJ, Fukuda A (2004) Cl- uptakepromoting depolarizing GABA actions inimmature rat neocortical neurones ismediated by NKCC1. J Physiol 557: 829-841.
Yamada Y, Fukuda A, Tanaka M, Shimano Y,Nishino H, Muramatsu K, Togari H, WadaY (2001) Optical imaging reveals cation-Cl- cotransporter-mediated transient rapiddecrease in intracellular Cl- concentrationinduced by oxygen-glucose deprivation inrat neocortical slices. Neurosci Res 39:269-280.
Ylinen A, Bragin A, Dasdy N, Jandó G, SzabóI, Sik A, Buzsaki G (1995) Sharp wave-associated high-frequency oscillation (200Hz) in the intact hippocampus: networkand intracellular mechanisms. J Neurosci15: 30-46.
Yuste R, Lanni F, Konnerth A (2000) Imagingneurons; A laboratory manual. ColdSpring Harbor Laboratory Press.
Yuste R, Peinado A, Katz LC (1992) Neuronaldomains in developing neocortex. Science257: 665-669.
Zhang L, Spigelman I, Carlen PL (1991)Development of GABA-mediated,chloride-dependent inhibition in CA1pyramidal neurons of immature rathippocampal slices. J Physiol 444: 25-49.
Zhang LL, Fina ME, Vardi N (2006)Regulation of KCC2 and NKCC duringdevelopment: Membrane insertion anddifferences between cell types. J CompNeurol 499: 132-143.
Zhu XL, Sly WS (1990) Carbonic anhydrase-IV from human lung-purification,
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