CALCIUM CONDUCTANCE IN AN IDENTIFIEDCHOLINERGIC SYNAPTIC TERMINAL IN THE CENTRAL
NERVOUS SYSTEM OF THE COCKROACH
BY JONATHAN M. BLAGBURN AND DAVID B. SATTELLE
AFRC Unit of Insect Neurophysiology and Pharmacology, Department of Zoology,University of Cambridge, Downing Street, Cambridge CB2 3EJ, UK
Accepted 20 January 1987
SUMMARY
Intracellular microelectrodes were used to study a cholinergic synapse betweentwo identified neurones: the lateral filiform hair sensory neurone (LFHSN) andgiant interneurone 3 (GI 3) in the terminal ganglion of the first-instar cockroachPeriplaneta americana. The presynaptic cell, LFHSN, was impaled in a region ofthe axon which forms large numbers of output synapses.
The sign and magnitude of the LFHSN spike afterpotential were shown to dependon [Ca2+]o. ljumoir1 tetrodotoxin (TTX) abolished LFHSN spikes but theaddition of 0-1 mmoir 1 4-aminopyridine (4-AP) enabled regenerative depolariz-ations to be evoked which were followed by large EPSPs in GI3. Addition of20mmoir1 tetraethylammonium ions (TEA+) abolished the cholinergic EPSPs butresulted in long-duration LFHSN spikes. Intracellular injection of caesium ions(Cs+) into LFHSN enabled long-duration spikes to be evoked and had no effect onsynaptic transmission. Long-duration LFHSN spikes were (1) increased in ampli-tude by increased [Caz+]o; (2) accompanied by an increase in conductance; (3) notabolished by replacement of external Na+ with Tris+ or choline+; (4) blocked by1 mmoir 1 Cd2+ and lOmmolT1 Co2+; (5) not supported by substitution of Mg2+
for Ca2+; and (6) supported by Baz+ substitution. They are thus considered to beCaz+ spikes. The Ca2+ spikes were blocked by organic Ca2+ channel blockers at0-5-1 mmoir 1 .
The putative Ca2+ spike was followed by a hyperpolarizing afterpotential (HAP),the duration of which was proportional to the amplitude and duration of the Ca2+
spike. The HAP was (1) accompanied by a conductance increase; (2) reversed atpotentials 30mV more negative than resting potential; (3) not supported bysubstituting Ba2+ for Ca2+; and (4) partially blocked by 150mmoir1 TEA+. TheHAP is considered to result from an increase in Ca2+-dependent K+ conductance.
It is concluded that, in addition to Na+ channels and delayed rectifying K+
channels, Ca2+ channels and Ca2+-dependent K+ channels are present in the axonalmembrane of LFHSN, in a region which forms many output synapses.
Many neurobiological preparations have been developed in which intracellularrecording from identified pre- and postsynaptic elements of a chemical synapse ispossible, but only a few allow direct access to the junctional site, particularly thepresynaptic region. Examples include: the squid giant axon synapse (Bullock &Hagiwara, 1957; Llinas, Steinberg & Walton, 1981a,b), the chick ciliary ganglion(Martin & Pilar, 1963), the Mauthner cell-giant fibre synapse of the hatchetfish(Auerbach & Bennett, 1969), the lamprey Muller neurone—interneurone synapse(Martin & Ringham, 1975), the crab muscle receptor-motoneurone synapse (Blight& Llinas, 1980), the crayfish neuromuscular junction (Wojtowicz & Atwood, 1984)and the lobula giant movement detector (LGMD)-descending contralateral move-ment detector (LCMD) synapse in the locust (Rind, 1984).
For pharmacological studies of synaptic transmission in insect CNS the cockroachPeriplaneta americana is particularly suitable, since the blood-brain barrier can beremoved without impairing synaptic function, allowing access to pre- and post-synaptic elements of identifiable pathways. The cerci of orthopterous insects such asthe cockroach are a pair of conical appendages on the posterior end of the abdomen.Each cercus bears several types of sensillum, one of which, the filiform hair, respondsto air movements (Dagan & Camhi, 1979; Tobias & Murphey, 1979). Sensory axonswithin the cerci join to form the cereal nerves which enter the terminal abdominalganglion. The largest neurones in this ganglion are the giant interneurones, so calledbecause they send large-diameter axons up the nerve cord towards the head. Thegiant interneurones receive sensory input from the cereal sensory neurones (Callec,Guillet, Pichon & Boistel, 1971; Matsumoto & Murphey, 1977).
In the cockroach Periplaneta americana cereal afferent—giant interneuronesynapses have been shown to be cholinergic (Callec, 1974; Sattelle, 1980) and thenicotinic antagonist ar-bungarotoxin blocks transmission (Sattelle et al. 1983). Thegiant interneurones of Periplaneta americana mediate the escape response to airmovements (Westin, Langberg & Camhi, 1977; Camhi & Tom, 1978; Camhi, Tom& Volman, 1978: Ritzmann & Camhi, 1978).
The great advantage of the first-instar nymphal cockroach as an experimentalsystem compared to the adult, or to nymphs and adults of other orthopterous insects,lies in the small number of cereal sensory axons. There are only two filiform hairs oneach cercus compared to approximately 220 in the adult, and each filiform hairsensory neurone (FHSN) is identifiable (Blagburn & Beadle, 1982; Dagan &Volman, 1982). The small size of the terminal ganglion allows it to be viewed withNomarski optics. The FHSN axons and GI 2 and GI 3 can be located visually in theliving ganglion, which greatly facilitates impalement with intracellular microelec-trodes (Blagburn, Beadle & Sattelle, 1986). The first-instar FHSN-GI pathway isparticularly suitable for developmental studies of, for example, synapse formation(Blagburn et al. 1985a) and chemosensitivity (Blagburn et al. 19856).
It has been shown by electron microscopy (Blagburn et al. 1984) that the site ofimpalement of the LFHSN axon is also the site of large numbers of output synapses
Calcium spikes in an insect synaptic terminal 349
(approximately 8/zm~2: Blagburn et al. 1985a). This gives us the opportunity toinvestigate the physiology of a large (6-lOjUm diameter) presynaptic terminal of anidentified insect sensory neurone, while monitoring postsynaptic potential changes.
MATERIALS AND METHODS
Cockroaches (Periplaneta americana) were reared at 27 °C with food and waterfreely available. Newly hatched first-instar nymphs were placed in a saline solution ofthe following composition (in mmoll"1): NaCl, 150-0; KC1, 3-1; CaCl2, 5-4;MgCl2, 1-0; Hepes buffer, 5-0; sucrose, 50-0; pH7-4 (based on Callec & Sattelle,1973).
The legs and antennae were removed with fine dissecting scissors and a ventralstrip of cuticle was excised, bearing the head, nerve cord and cerci. The rectum wasremoved and all but the most posterior abdominal cuticle segments were pulled awayfrom the nerve cord. The isolated CNS and cerci were then transferred to a rubber-walled chamber constructed on a glass microscope slide and anchored, ventral sideup, using petroleum jelly. The connective tissue sheath around the terminal ganglionwas softened by brief (5 s) exposure to saline containing 1 -0 mg ml~' protease (TypeXIV, Sigma, UK) then removed using fine forceps. All four filiform hairs wereimmobilized by covering the cerci with petroleum jelly.
Isolated preparations were viewed with Nomarski optics, using a 40x water-immersion objective lens (Zeiss, FDR), electrically isolated from the body of themicroscope with a Perspex insert. The cell body, axon and primary dendrite of GI 3could be identified reliably using the criteria of size, morphology, position andappearance (Fig. IB). The LFHSN axon was located visually near the ventralmargin of the neuropile, following the lateral neuropile boundary (Fig. IB). Theaxon was characteristically large in this region (6— 10,um diameter) and its manymitochondria gave it a granular appearance.
For intracellular recording, glass capillary microelectrodes were made using'Kwik-fiT 1 mm diameter, standard-bore capillary tubing (Clark ElectromedicalInstruments, UK) in a vertical electrode puller (Narishige, Japan) and filled with1-Omoir1 KC1 and 5-Ommoir1 Hepes, adjusted to pH7-2 with KOH. Alterna-tively, electrodes were filled with 1-0moll"1 potassium acetate, pH-adjusted withacetic acid. For intracellular ionophoresis of caesium ions, microelectrodes werefilled with the following solution: KC1, 0-75moll"1; CsCl2, 0-25moll"1; Hepes,3-8 mmol P 1 , adjusted to pH 7-2 with HC1. The microelectrodes were 30-60 MQ inresistance, and exhibited some degree of Type II nonlinearity (Purves, 1981) whenpassing current of more than 2nA. Current flow in the bath was monitored using avirtual earth circuit (Fig. 1C). Oscilloscope traces were recorded on a Racal Store4DS tape recorder and permanent records were made using a Medelec storageoscilloscope.
350 J. M. BLAGBURN AND D. B. SATTELLE
B
Connective
Nerve 7
Nerve 8
1 mm
• Filiform hairCercus
Objective lens
Nerves 11 and 10
100 //m
Perfusion in •
Saline ( r Ganglion
Illumination IFig. 1. Microelectrode recording from pre- and postsynaptic elements of an identifiedcentral synapse in an insect. (A) Dorsal view of first-instar cockroach (Periplanetaamericana) showing position of abdominal ganglia. (B) Camera lucida tracing of theterminal abdominal ganglion seen under Nomarski optics. One lateral filiform hairsensory neurone (LFHSN) and one giant interneurone 3 (GI 3) are shown impaled withmicroelectrodes; each neurone has a homologous contralateral partner which is notshown here. Stippling indicates the characteristic granular appearance of the cytoplasm.(C) Experimental system.
Drugs used in the experiments were: tetrodotoxin (TTX, Sankyo Co., Japan,distributed by Koch-Light Laboratories, UK), 4-aminopyridine (4-AP, Sigma,UK), tetraethylammonium bromide (TEABr, Kodak, UK). The highly purifiedbromide salt of TEA was used to avoid possible contamination by triethylamine(Zucker, 1981). The organic calcium channel blockers diltiazem, verapamil (Sigma,UK), D600 and nifedipine (Bayer, FDR) were also used. Saline solution wasperfused continuously at a rate of lmlmin"1, into a bath of 0-3 ml volume.Experiments were performed at a temperature of 20-22°C.
Calcium spikes in an insect synaptic terminal 351
RESULTS
Electrical properties of the lateral filiform hair sensory neurone
The resting potential recorded from the LFHSN axon was — 54 ± 1 mV (mean± S.E.M., N= 48), and the resting potential recorded from the cell body of GI 3 was- 7 8 ± l m V (iV = 41). The input resistance of LFHSN (Rin) was 20±2MQ(N = 20) for a negative current pulse of 1 nA, and the axon exhibited pronounceddelayed rectification (Fig. 2A). With the filiform hair immobilized, LFHSNspontaneously produced action potentials at a frequency of 40—60 Hz. In the majorityof preparations the overall amplitude of the spikes was between 30 and 70 mV(51 ± lmV, Ar = 46), peaking at a potential of —4±2mV (iV=46). Preparationswith a resting potential more positive than — 40 mV, an action potential amplitude ofless than 30 mV, or R;n of less than 10 MQ were discarded.
LFHSN spikes had a duration of approximately 1 ms, measured at a potential10 mV positive to resting potential. Measurement of the spike duration wascomplicated by the variable nature of the spike afterpotential (AP), which rangedfrom a hyperpolarizing afterpotential (HAP) of up to —10 mV from resting potential,to a depolarizing afterpotential (DAP) of up to +12mV from resting potential. Thereversal potential for the afterpotential (EAP) varied from —45 to — 70 mV ( —58 ± 1,7V=43).
Current (nA)
-50
> -60
- 8 0
5mV
Bi Biiu3
Biii
. — - — _ •
2X
Ci
10 ms
Fig. 2. (A) Averaged current-voltage relationship of nine LFHSN axons. The ampli-tude of the depolarization was measured 20 ms after the onset of the pulse. Delayedrectification is present in the depolarizing direction. Vertical bars represent ±S.E.M.(B) Calcium-dependence of afterpotentials (APs) following spikes recorded from thelateral filiform hair sensory neurone (LFHSN). Dependence on [Ca2+]o of the size andpolarity of the AP. The APs are shown at (Bi) resting, (Bii) depolarized and (Biii)hyperpolarized membrane potentials, with superimposition of those recordings obtainedin (1) 200^1011"' Caz+, (2) S^mmoll"1 Ca2+, and (3) Ommoll"1 Ca2+ plus2 0 m m o i r ' Mg2"1". (Ci) Calcium-dependent voltage change [AV(Ca2+)] calculated bysubtracting the records of spikes in zero external Ca2+ from those in normal and highCa2+. (Cii) The GI3 EPSP in (1) high [Ca2+]o and (2) normal [Ca2+]o is shown.
352 J. M. BLAGBURN AND D. B. SATTELLE
Monosynaptic connection with giant interneurone 3
Earlier electron microscope and physiological studies have demonstrated that theLFHSN—GI3 synapse is a monosynaptic connection (Blagburn et al. 1984;Blagburn & Sattelle, 1987).
Synchronous recording from LFHSN and the contralateral GI 3 cell body showedthat spikes in the sensory axon give rise to depolarizing postsynaptic potentials, in theinterneurone. At high frequency these potentials may summate to give rise tointerneurone spikes; they are therefore termed excitatory postsynaptic potentials(EPSPs). These EPSPs had an amplitude of 6-4±0-3mV (N=9l), a latency of1-37 ± 0-06ms (N = 21), measured from lOmV positive to resting potential on thespike rising phase to the start of the EPSP rise, a time to peak of 2-05 ± 0 0 8 ms(N = 24) and a decay time constant of 4-55 ± 0-13ms (N = 24). These values wereobtained from cell body recordings and, although it has been shown that depolariz-ations are attenuated to 75 % of their original value during electrotonic conductionfrom the primary dendrite to the cell body (J. M. Blagburn & D. B. Sattelle,unpublished observation), the degree to which the EPSPs were attenuated duringconduction from their points of origin in the fine dendritic branches is not known.
Evidence for a calcium component of the afterpotential following sensory axonspikes
It was noted that the sign of the spike afterpotential (hyperpolarizing ordepolarizing) varied according to the level of the resting membrane potential. Theeffects of changing [Ca2+]o on the AP were then investigated (Fig. 2B). With a[Ca2+]o of S^mmoll"1 the EAP was 9mV more positive than with a [Ca2+]o ofOmmolP1 (with the addition of 20mmolP1 Mg2+ to produce complete block ofCa2+ channels). With a [Ca2+]o of 20mmolP1 the EAP was 14 mV more positive thanin OmmolP1 Ca2+. The spike amplitude was not affected by changes in [Ca2+]o.Changes in [Ca2+]o took effect in 5min and were reversible on perfusion of normalsaline.
Subtraction of the action potential record obtained for 0 mmol I"1 Ca2+ from thosefor 5-4 and 20mmolP1 Ca2+ enabled examination of the effect of [Ca2+]o on theshape of the spike (Fig. 2C). The calcium-dependent potential changes thusobtained were termed AV(Ca + ) . It is not known why the time course and amplitudeof the AV(Ca2+) and the EPSP are similar. The above results suggest that there maybe a significant involvement of calcium ions in producing a depolarization which,superimposed on the normal HAP due to K+ efflux, reduces or reverses it. Furtherevidence was sought with the use of pharmacological agents which block Na+ and K+
channels.
Effects on the nerve terminal action potential of sodium and potassium channel-blocking agents
Tetrodotoxin (TTX) has been shown to block insect sodium channels atmicromolar concentrations (Sattelle, Pelhate & Hue, 1979). After 5 min exposure to
Calcium spikes in an insect synoptic terminal 353
1 ' TTX, LFHSN spikes were reduced in amplitude to 3—10 mV; these spikeremnants persisted for 5—10 min. Exposure to 1 \m\o\ P 1 TTX completely abolishedLFHSN spikes and GI 3 EPSPs in 10-15 min. The small spikes may have beenremnants of electrotonically conducted spikes originating in distal regions of thecereal nerve in which the blood-brain barrier remained intact.
As the LFHSN spikes were blocked, EPSPs recorded from GI 3 progressivelydecreased in size (Fig. 3), with the transmission threshold being reached when thespike amplitude was approximately 30 mV (33 ± 3mV, N= 6). Some depolarizingslow PSPs remained in both LFHSN and GI 3 long after complete spike block.These may have resulted from incomplete penetration of the ganglionic neuropile bythe toxin. Alternatively, they may represent synaptic inputs from neurones withTTX-insensitive spikes or inputs from non-spiking neurones.
Addition of 0-l mmolP1 4-AP alone caused a doubling of LFHSN spike durationand a 30 % increase in GI 3 EPSP amplitude (as measured in the cell body). LFHSNdelayed rectification was also reduced. 4-AP blocks K+ channels at membranepotentials of up to -20 mV (Pelhate & Pichon, 1974; Pelhate & Sattelle, 1982).
Addition of 0-lmmolP1 4-AP to a preparation in which spikes had previouslybeen blocked with ljUmoll"1 TTX often resulted in a small decrease in restingpotential (approximately +4mV), and allowed regenerative depolarizations to beelicited by 20-ms positive current pulses (Fig. 4). These regenerative spike-likedepolarizations took place when the membrane was depolarized beyond a thresholdof 12mV above resting potential (threshold potential —38 ± 1 mV, N= 18), were3-1 ± 0-1 ms (N — 18) in duration, and reached a peak of 40 ± 2 mV (N = 18) positiveto resting potential.
LFHSN depolarizations were followed, at a latency of 2-5—3-0 ms, by large (upto 35 mV) EPSPs in GI 3 (Figs 4, 5). Addition of lmmoll"1 Cd2+, lOmmolP1
10 20 30 40 50LFHSN spike amplitude (mV)
60
Fig. 3. Transfer function for the synapse between the lateral filiform hair sensoryneurone (LFHSN) and giant interneurone 3 (GI 3). Amplitude of GI 3 EPSPs plotted asa function of LFHSN spike amplitude during progressive spike block by l ^ m o l l "tetrodotoxin. Resting potential of LFHSN was — 60 mV.
354 J. M. BLAGBURN AND D. B. SATTELLE
Co2+ or substitution of Ca2+ with Mg2+ abolished the regenerative depolarization,suggesting that it is dependent upon the entry of Ca2+ through voltage-dependentchannels (Hagiwara, 1973; Kostyuk, 1980). Injection of a 2-5 ms positive currentpulse of approximately 3 nA elicited a rapid depolarization in LFHSN which was
I.OmV
LFHSN
Mg-2 +
20 ms
Fig. 4. Putative calcium spikes recorded in the presence of sodium and potassiumchannel blockers. Regenerative potentials elicited in the lateral filiform hair sensoryneurone (LFHSN) in the presence of 1 /anoll"1 tetrodotoxin (TTX) and O-lmmoll"1
4-aminopyridine (4-AP). Upper trace, current and zero potential; second trace, LFHSNpotential; third trace, GI 3 potential; fourth trace, LFHSN potential in the presence ofOmmolF1 Ca2+ plus 5 -4mmoir ' Mg2+. 20ms current pulses of increasing intensitywere followed by a 3 nA, 3 ms pulse.
>B
40-
30-
B
CuUJ
20 -
10-
40-
30-
20-
10-
10 20 30 40 50 0 10LFHSN depolarization (mV)
20 30 40 50
Fig. 5. Relationship between size of presynaptic depolarization and postsynaptic EPSPamplitude for the lateral filiform hair sensory neurone (LFHSN) - giant interneurone 3(GI 3) synapse. (A) Postsynaptic depolarization plotted against presynaptic depolariz-ation recorded in the presence of l^moll"1 tetrodotoxin (TTX) and O-lmmoll"1
4-aminopyridine. (B) Postsynaptic depolarization plotted against presynaptic depolariz-ation in the presence of 1 ^molF 1 TTX and with Cs+ injected into LFHSN.
Calcium spikes in an insect synaptic terminal 355
terminated before a full regenerative potential was elicited. This depolarization gaverise to an EPSP which was similar in shape to those produced by normal LFHSNspikes (Fig. 4).
Subsequent addition of 20 mmol I"1 TEA+ greatly increased the amplitude andduration of the regenerative depolarization, or putative Ca2+ spike. External TEA+
blocks K+ channels in Helix neurones with weak voltage-dependence (Hermann &Gorman, 1981), when compared to the strongly voltage-dependent blocker 4-AP,and external TEA+ and 4-AP block pharmacologically distinct K+ channels inTritonia (Thompson, 1977). In saline containing 5-4 mmol P 1 Ca2+ the putativeCa2+ spike depolarized the membrane to near OmV potential (—2 ± 2mV, N = 13)and lasted for 15-20ms (18 ± 2ms, N=7). The spike was followed by an HAPapproximately 400 ms in duration and 9-23 mV peak hyperpolarization from restingpotential (15±3mV, N=5). A second spike, elicited 700ms after the first, wasreduced in duration, as was the HAP (Fig. 6A,C). Hyperpolarization of LFHSNshowed that the HAP was reversed at membrane potentials approximately 30 mVmore negative than resting potential (HAP reversal potential —78±3mV, N = 5)(Fig. 7A).
Increasing the TEA+ concentration from 5 to 50mmoll 1 did not result in anincrease in duration of the putative Ca2+ spikes. However, increasing the concen-tration to 150 mmol I"1 resulted in a partial block of the HAP and an increase in spikeduration.
5-4 mmol r 10mmoir 'Ca2 +
I.OmV-
LFHSN-
I,0mV-
LFHSN-
5-4 mmol I Ca-lr\,2+D
10mmoir 'Ca2 +
Fig. 6. Calcium spike elicited in the presence of 1/imoll ' tetrodotoxin (TTX),O-lmmolP1 4-aminopyridine (4-AP) and 20mmoll~' tetraethylammonium (TEA+).The first and second spikes in a series are superimposed, the first being of longerduration. (A,C) Spikes in S^mmoll"1 Ca2+. (B,D) Larger-amplitude and longer-duration spikes recorded in 10 mmol I"1 Ca2+. C and D are on a slower timebase to showthe hyperpolarizing afterpotentials. Upper trace: current and zero potential; lower trace:lateral filiform hair sensory neurone (LFHSN) potential.
356 J. M. BLAGBURN AND D. B. SATTELLE
Increasing the [Ca2+]o to 10 mmol 1 l increased the amplitude and duration of theputative Ca2+ spike (Fig. 6B). The first spike in a series peaked at about +5 mV andlasted for 100 ms, with an HAP reaching —18 mV from resting potential and lastingfor 800 ms. Putative Ca2+ spikes could not be elicited during the HAP but the nextspike peaked at OmV and lasted for 30 ms, with a proportionate reduction in theduration of the HAP (Fig. 6D). In both 5-4 and 10 mmol I"1 Ca2+ there was littlefurther change in spike duration after the second spike.
20 mmol 1~' TEA+ also had the effect of enhancing the size and duration of EPSPsin LFHSN (Fig. 6C,D) to the extent that putative Ca2+ spikes could be elicited bythese EPSPs. TEA+ also enhanced some PSPs in GI 3 but the EPSPs elicited by thecholinergic LFHSN were blocked. This reversible blocking of cholinergic synapticinputs (see Twarog & Roeder, 1957) by the action of TEA+ as a quaternaryammonium ion (Adler et al. 1979) means that extracellular TEA+ cannot be used instudies of both the pre- and postsynaptic sides of the synapse.
LFHSN
-15mV
-30mV-40mV
2 n A ( I ) |20 mV
100 ms (A)20 ms (B)
Fig. 7. (A) Four superimposed lateral filiform hair sensory neurone (LFHSN)calcium spikes elicited in the presence of ljumolF1 tetrodotoxin (TTX), 0-1 mmol T1
4-aminopyridine (4-AP) and 20 mmol I"1 tetraethylammonium (TEA+). Hyperpolariz-ation of LFHSN reverses the hyperpolarizing afterpotential. (B) LFHSN calcium spikeand GI 3 EPSP elicited in the presence of 1 /imol I"1 TTX and with Cs+ injected into theLFHSN axon. Top trace in A, current; top trace in B, current and zero potential; secondtrace, LFHSN potential; lower trace in B, GI 3 potential.
Calcium spikes in an insect synoptic terminal 357
In order to circumvent the cholinergic receptor-blocking action of external TEA+
and the synapse-blocking action of internal TEA+ (Blagburn & Sattelle, 1987a),LFHSN was impaled with microelectrodes containing caesium ions. Internal Cs+
blocks outward K+ currents at depolarized potentials (Hille, 1984). This wasfollowed by total block of action potentials with ljUmolF1 TTX, and subsequentinjection of positive current pulses into the presynaptic terminal for 2-5 min (1 nAamplitude, 1 Hz frequency, 500ms duration). Injection of K+ alone had nosignificant effect.
Ionophoretic injection of Cs+ into LFHSN allowed putative Ca2+ spikes to beevoked (Fig. 7B). These were similar in amplitude to those evoked in the presence ofexternal TEA+ and were 10 ms in duration. The putative Ca2+ spikes were followedby HAPs of proportionate duration, and elicited EPSPs in GI3, similar to thoseproduced by the shorter-duration putative Ca + spikes seen in the presence of TTXand 4-AP. Using internal Cs+ it was possible to determine the relationship betweenthe amplitude of the presynaptic putative Ca2+ spike and that of the GI 3 EPSP inthe absence of any possible effects on the membrane properties of GI 3 resulting from4-AP (Fig. 5B).
Fast hyperpolarizing current pulses into LFHSN were used to monitor Rm duringputative Ca2+ spikes evoked by enlarged non-cholinergic EPSPs (Fig. 8A). R;n wasreduced, i.e. there was an increase in membrane conductance, during the initialEPSP, then Rin dropped to near zero during the spike. A conductance increasepersisted during the HAP.
Because TTX-resistant spikes have been reported in vertebrate sensory cells(Heyer & MacDonald, 1982), an increase in Na+ conductance was ruled out byreplacing all the Na+ in the saline with either Tris+ or choline+. The putative Ca2+
spikes were not affected by removal of Na+ (Fig. 8B), suggesting that the spike is notthe result of an increased TTX-insensitive Na+ conductance. Zero-Na+ salineabolished LFHSN action potentials within 5 min so it is unlikely that sodium ionsremained in the intercellular spaces surrounding the axon. Lengthy treatment withzero-Na+ saline (over 20 min) resulted in a loss of membrane resistance.
Barium ions carry current through Ca2+ channels (Hagiwara & Byerly, 1981).Substitution of Ba2+ for Ca2+ had little effect upon the amplitude or duration ofnormal LFHSN action potentials, but abolished the EPSPs elicited in GI 3 byLFHSN spikes. In contrast, other background synaptic activity in both neuroneswas enhanced.
Substitution of Ba2+ for Ca2+ had little effect upon the amplitude of the putativeCa2+ spike, but prolonged it to 1-5s (Fig. 8C). No HAP was observed. Aconductance increase took place during the first half of the extended spike, and R|nreturned to its original value before the termination of the spike. Ba2+ spikes wereblocked with lmmoll"1 Cd2+ or lOmmolP1 Co2+.
Effects of calcium channel-blocking compounds
The phenylalkylamine Ca2+ channel blockers D600 and verapamil had little effecton Ca2+ spikes at 0-1 mmolP1 but blocked spikes reversibly at 1 mmoll"1. Similarly,
J. M. BLAGBURN AND D. B. SATTELLE
B Ommoir'Na"1"
I.OmV
2nA (I)20 mV
Ommoir' Ca2+, 5-4mmoll"' Ba220ms (A,B)200 ms (C)
Fig. 8. Calcium spikes in the lateral filiform hair sensory neurone (LFHSN) axonrecorded in the presence of l^moll"1 tetrodotoxin (TTX), O-lmmoll"1 4-amino-pyridine (4-AP) and 20mmoll~' tetraethylammonium (TEA+). TEA+ blocks thecholinergic EPSPs in GI 3. (A) Changes in the membrane input resistance of the LFHSNaxon were monitored by injection of 1 nA hyperpolarizing pulses. There is a conductanceincrease during the spike-initiating EPSP and during the hyperpolarizing afterpotential(HAP), while the input resistance drops to near zero during the spike. (B) Ca2+ spikeelicited in the absence of external sodium (replaced by Tris+). (C) Barium substituted forcalcium supports long-duration spike with no HAP. Upper traces, current and zeropotential; centre traces, LFHSN potential; lower traces, GI3 potential.
the benzodiazepine diltiazem blocked the Ca2+ spikes reversibly at 1 mmoll"1, butnot at (Mmmoll~1. The dihydropyridine nifedipine blocked the Ca2+ spikesirreversibly at O-Smmoll"1 but not at lO/xmoll"1.
DISCUSSION
The combination of Nomarski optics with conventional electrophysiologicaltechniques has enabled us to record intracellularly from an identified insect synapticterminal while monitoring the postsynaptic potential changes. The lateral filiformhair sensory neurone (LFHSN) can be impaled in a region of the axon which formshundreds of output synapses with giant interneurone 3 (GI 3). The closest synapsesto the recording site are less than 5jum from the microelectrode, while the furthestare less than 100 (Um; to our knowledge this is considerably closer than in otherpreparations, even the squid giant synapse.
It was observed that LFHSN spikes exhibited a variable afterpotential, which, insome cases, reversed at membrane potentials more positive than resting potential.
Calcium spikes in an insect synaptic terminal 359
The size and sign of the spike afterpotential (AP) were dependent upon themembrane potential and the external calcium ion concentration. It is thought thatthis component of the spike, which is similar in time course to the GI 3 EPSP,represents the sum of the HAP due to K+ efflux and the depolarization due to Ca2+
influx. The balance of these will be depolarizing or hyperpolarizing, depending onthe resting membrane potential. A depolarizing afterpotential (DAP), which wasindependent of [Ca + ] o , has been seen after spikes in the crayfish claw-openerexcitatory motor axon and has been considered to be a passive membrane responsefollowing activation of a K+ channel (Wojtowicz & Atwood, 1984). However, in thecase of the crayfish central giant axons it has been suggested that the DAP is causedby an increase in Ca2+ conductance (Yamagishi & Grundfest, 1971).
Progressive spike block by TTX allows the input/output relationship of theLFHSN-GI 3 synapse to be determined. EPSPs in GI 3 decrease in amplitude asthe height of the LFHSN spike decreases, with a threshold spike height of 33 ± 3 mVfrom resting potential, i.e. —22 ± 3 mV. Clearly the attenuation undergone by theEPSP may render this estimate inaccurate.
The synaptic transfer curve is similar in shape to those obtained for other synapticpreparations in which phasic transmission normally takes place. The threshold value,for the presynaptic depolarization of — 22 mV (33 mV from resting potential) for theLFHSN—GI 3 synapse compares with values of —30 to —40mV (20—40mV fromresting potential) for the squid synapse (Takeuchi & Takeuchi, 1962; Katz & Miledi,1966), —35 to — 40 mV (35-40 mV from resting potential) for the lamprey (Martin &Ringham, 1975), -40 to —45 mV (25-30 mV from resting potential) for thehatchetfish (Auerbach & Bennett, 1969), — 40 mV (lOmV from resting potential) forthe locust LGMD (Rind, 1984) and -27 mV (48 mV from resting potential) for thecrayfish neuromuscular junction (Wojtowicz & Atwood, 1984).
The tonic synapses of the non-spiking barnacle photoreceptor (Ross & Stuart,1978) and the non-spiking crab T-fibre (Blight & Llinas, 1980) exhibit lowerthresholds for transmission: — 50mV (7mV from resting potential) for the barnaclephotoreceptor and — 70 mV (10 mV from resting potential) for the crab T-fibre.
The results show that LFHSN can support Ca2+ spikes in the presence of the K+
channel blockers 4-AP and TEA+. The regenerative potentials satisfy the usualcriteria for Ca2+ spikes (Hagiwara & Byerly, 1981) in that (1) regenerativedepolarization in the presence of K+ channel blockers was accompanied by anincrease in membrane conductance; (2) the spike was not blocked by TTX or byreplacement of extracellular Na+ with Tris+ or choline"1"; (3) the spike was blockedby replacement of extracellular Ca2+ with Mg2+, and by exposure to lmmolP 1
Cd2+ or lOmmoll"1 Co + ; (4) the spike height and duration were dependent uponthe external concentration of Ca2+; and (5) Ba + substituted for Ca2+ and producedspikes of longer duration. The threshold for the Ca2+ spikes of —38 mV is somewhatmore negative than the threshold for synaptic transmission.
In the presence of TTX and 4-AP only, Ca2+ spikes of relatively short durationcan be evoked in LFHSN, and these produce large EPSPs in GI 3. It is possible thatreduction of the delayed rectifier K+ conductance in GI 3 will reduce the degree of
360 J. M. BLAGBURN AND D. B. SATTELLE
attenuation undergone by large dendritic EPSPs recorded from the cell body. EPSPsevoked by LFHSN Ca2+ spikes in the presence of TTX and with Cs+ inside thepresynaptic terminal attain a maximum amplitude of 25 mV, 5 mV lower than thoseproduced with external 4-AP. This suggests that delayed rectification has a smalleffect on the size of the EPSP recorded in the cell body of GI 3.
It is possible that a regenerative Ca2+ current adds to the height of the GI 3EPSPs. This is unlikely because the EPSPs exhibited no threshold for regenerativedepolarization as did the presynaptic Ca + spikes. In addition, no TTX-insensitivedepolarizations could be evoked in GI 3 in the presence of external TEA+.
The organic Ca2+ channel blockers D600, verapamil, nifedipine and diltiazemhave been found to be potent blockers of Ca2+ currents in vertebrate smooth andheart muscle (Hagiwara & Byerly, 1981; Lee & Tsien, 1983) at concentrations of1—lOjUmolP . However, in some invertebrate preparations, such as the squid giantaxon (Baker, Meves & Ridgway, 1973) and snail neurones (Kostyuk, Krishtal &Shakhovalov, 1977), these compounds block Ca2+ currents only at high concen-trations (0T-2mmol P1) . The results of the present study, in which thesecompounds have been tested on insect Ca + spikes for the first time, are consistentwith the latter findings. Recently it has been shown that chick dorsal root ganglionneurones exhibit three types of Ca2+ channel, only one of which, the L-type, issensitive to micromolar concentrations of dihydropyridine agonists (Nowycky, Fox& Tsien, 1985). It has been postulated that N-type channels may mediatedihydropyridine-insensitive synaptic transmission (McCleskey, Fox, Feldman &Tsien, 1986), and it is possible that LFHSN synaptic Ca2+ channels are of this type.
The long duration of Ba2+ spikes is similar to results obtained from Helix andlamprey neurones (Kerkut & Gardner, 1967; Leonard & Wickelgren, 1985) and maybe due to the inability of Ba2+ to inactivate the Ca2+ channel (Eckert & Tillotson,1981) and/or to activate the Ca2+-dependent K+ conductance (Gorman & Hermann,1979; Meech & Thomas, 1980).
The HAP which followed the Ca2+ spikes in the presence of TEA+ wasproportional in duration to the amplitude and duration of the Ca + spikes. It wasaccompanied by a conductance increase and was blocked by substitution of Ba2+.The HAP reversed at potentials approximately 30 mV more negative than restingpotential. This evidence suggests that the HAP results from an increase in Ca2+-dependent K+ [K+(Ca2+)] conductance, although a possible contribution by a Cl~conductance was not ruled out. The HAP was not blocked by SOmmoll"1 TEA+,but ISOmmolP1 TEA+ caused some block of the HAP and a 10-fold increase induration of the Ca2+ spike. External TEA+ is not an efficient blocker of theK+(Ca2+) current in many vertebrate and invertebrate neurones (Stanfield, 1983)although in some molluscan neurones the K+(Ca2+) conductance is very sensitive toexternal TEA+. It has been suggested that there are two types of K+(Ca2+) currentswhich differ in their activation kinetics: fast ones, which are sensitive to TEA+ andinsensitive to apamin; and slow ones, which are insensitive to TEA+ and blocked byapamin (Mallart, 1985). The K+(Ca2+) conductance in the cockroach LFHSN may
Calcium spikes in an insect synap tic terminal 361
belong to the latter class, although it appears to be insensitive to apamin (J. M.Blagburn & D. B. Sattelle, unpublished observation).
From studies of the small number of preparations in which voltage changes in thepresynaptic terminal can be monitored, it appears that a K+(Ca2+) conductance isoften associated with the synaptic Ca2+ conductance. A K+(Ca2+) conductance hasbeen investigated by intracellular recording at the squid giant synapse (Augustine &Eckert, 1982). Indirect measurement of electrical signals has been used to studysynaptic Ca2+ and K+(Ca2+) conductances in the giant barnacle photoreceptor(Stockbridge & Ross, 1984), the frog neurohypophysis (Obaid, Orkand, Gainer &Salzberg, 1985) and the mouse neuromuscular junction (Mallart, 1985).
Both Ca2+ and K+(Ca2+) channels have been found in adult cockroach cell bodies(Pitman, 1979; Thomas, 1984), and Ca2+ conductances have been described inembryonic grasshopper neuronal cell bodies (Goodman & Spitzer, 1981) but neitherhas previously been described in synaptic regions of any insect. In other arthropods,presynaptic Ca2+ and K+(Ca2+) conductances have been described only in thebarnacle photoreceptor (Ross & Stuart, 1978), lobster neuromuscular junction(Niwa & Kawai, 1982) and crab sinus gland neurosecretory terminal (Cooke, 1985).
We have no direct evidence that the LFHSN axon within the cereal nerve does notsupport Ca2+ spikes. However, it was frequently observed that small Na+ spikeremnants, originating in the axon further down the cereal nerve, persisted after ashort period of TTX application. These spike remnants were superimposed on Ca2+
spikes, suggesting that the LFHSN axon within the nerve is not able to produceprolonged Ca2+ spikes. This property appears to be confined to the ganglionic areasof the LFHSN axon. The presence of Ca2+ and K+(Ca2+) channels in this region ofLFHSN is presumably related to it being a site of large numbers of output synapses,in effect a large synaptic terminal (Blagburn et al. 1984, 1985a).
The LFHSN-GI 3 preparation is well suited to studies of integrative synapticphysiology and presynaptic Ca + and K+(Ca +) channels, avoiding the need todeduce the properties of synaptic areas from cell body membrane properties. Thispreparation allows unrivalled access to an identified cholinergic synaptic terminal, ata distance of less than 5 jxva from the presynaptic sites.
This work was supported by a postdoctoral fellowship award from the FMCCorporation (USA) to JMB and by the Agriculture and Food Research Council ofthe UK.
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