1 Klinik für Epileptologie, Universität Bonn, Sigmund-Freud-Str. 25, 53105 Bonn ,Germany 2 Institut für Physiologie und Pathophysiologie, Ruprecht-Karls-Universität Heidelberg, Im Neuenheimer Feld 326, 69120 Heidelberg, Germany; [email protected]
1
PRESYNAPTIC IONOTROPIC GABA RECEPTORS
A homeostatic feedback mechanism at axon terminals of inhibitory interneurons
Nikolai Axmacher1, Kristin Hartmann2, Andreas Draguhn2
1. INTRODUCTION Neuronal network activity and -synchrony can vary widely between different
functional states of the brain. This makes it necessary to maintain stability by homeostatic
mechanisms. One common stabilizing mechanism at most chemical synapses is
comprised by the negative feedback of transmitters on further vesicular release through
autoreceptors. Most of these presynaptically located receptors are G-protein coupled and
act in the time frame of tens to hundreds of milliseconds. The inhibitory transmitter
GABA is known to suppress vesicle release via presynaptic GABABR which are present
at most inhibitory- and even at many excitatory- synapses (Misgeld et al., 1995). Being
formed as dimers of proteins with the typical 7-transmembrane domain motive they
belong to the family of metabotropic transmitter receptors (Kaupmann et al., 1997).
There is, however, increasing evidence for ligand-gated ion channels in presynaptic
terminal membranes which could exert much faster feedback than metabolically coupled
receptors (MacDermott et al., 1999). Such fast acting channels might be of special
importance at inhibitory synapses which can be activated at very high frequencies,
depending on the state of the network. Some subtypes of hippocampal interneurons, for
example, fire action potentials at frequencies around 200 Hz during fast network
B
N. Axmacher, K. Hartmann, A. Draguhn 2
oscillations (Csicsvari et al., 1999; Klausberger et al., 2003). In order to cope with inter-
spike intervals of few milliseconds, regulatory mechanisms thus have to be similarly fast.
Our recent work has focused on presynaptic GABA-gated ion channels which
regulate the release of GABA from hippocampal interneurons onto their (principal) target
cells. It should be noted, however, that GABAA- or GABAC-receptors have also been
identified at the endings of non-GABAergic cells, where GABA thus has paracrine,
rather than autocrine, functions. In the rodent hippocampus, GABAA receptors are
present at Schaffer collateral terminals (Stasheff et al., 1993) and at mossy fibers (Ruiz et
al., 2003), i.e. two types of glutamatergic axons. These and other examples show an
enormous functional heterogeneity of GABAergic signaling at axons: at the
glutamatergic endings of the CA3-to-CA1 projection GABA triggers action potentials
which travel antidromically into CA3 pyramidal cells (Stasheff et al., 1993). At mossy
fibers, in contrast, GABA reduces excitability by shunting action potentials, much more
similar to its "standard" inhibitory function (Ruiz et al., 2003). At the calyx of Held,
glutamate release is facilitated by presynaptic glycine- or GABA-receptors (Turecek and
Trussell, 2001; 2002). In the spinal cord, axo-axonal synapses inhibit transmission from
muscle spindle afferents onto motor neurons by depolarizing GABA responses which
drive sodium channels into inactivation - the classical afferent depolarizing shift by
Eccles and coworkers (Eccles, 1964). More recent work by the Akaike-group revealed
that presynaptic GABAA receptors facilitate action potential-independent release of
glycine from spinal cord neurons but suppress action potential-dependent release (Jang et
al., 2002). These examples show that the heterogeneous effects of presynaptic GABA
receptors depend on several factors: axonal membrane potential, transmembrane chloride
gradient, the molecular nature, density and functional state of voltage-gated ion channels
etc. Unfortunately, these parameters are difficult to assess at small synaptic endings.
Most urgently, one would need reliable data on [Cl-] within the axon and its endings (see
below). This experimental program, however, has to await refinement of available
imaging or electrical recording techniques.
Besides the examples given above, presynaptic ionotropic GABA receptors have
been identified at cultured hippocampal neurons (Vautrin et al., 1994). A direct
observation of GABAA- or GABAC-receptor mediated effects of GABA at interneurons
in situ has, however, not been reported prior to our work. We did therefore look for such
PRESYNAPTIC IONOTROPIC GABA RECEPTORS 3
receptors in acutely prepared rat hippocampal brain slices, using inhibitory postsynaptic
currents (IPSCs) and destaining of fluorescence-labeled vesicles as functional assays
(Axmacher and Draguhn, 2004a, Axmacher et al., 2004b). Our observations suggest that
GABAergic inhibition is indeed regulated by feedback of GABA through axonally
expressed autoreceptors at the endings of CA3 hippocampal interneurons. These
receptors exert massive effects on action potential-dependent and -independent release of
GABA and it is therefore likely that they provide a mechanism for fast, frequency-
dependent regulation of GABA release.
2. EXPERIMENTAL
Horizontal hippocampal slices were prepared from juvenile (second postnatal
week) rats using standard methods. We recorded from CA3 pyramidal cells with patch
clamp techniques in the whole cell configuration (Hamill et al., 1981). Our electrode
solution contained a high chloride concentration (140 mM CsCl) leading to depolarizing
GABA-induced potentials or inward currents, respectively. It should be noted that IPSCs
were recorded as an indicator of GABA release from presynaptic terminals. We did not
aim at analyzing the native postsynaptic effects of GABA which would require recording
techniques which do not interfere with the internal chloride concentration (Ebihara et al.,
1995) or imaging of intracellular [Cl-] (Kuner et al., 2000). We isolated GABAergic
IPSCs pharmacologically by addition of CNQX and APV (each 30 µM) and suppressed
effects of GABAB receptors by adding 2 µM CGP 55845A. Measurements were taken at
room temperature (20 - 25 °C) under visual control with an upright high-magnification
microscope. We did carefully monitor input and access resistance throughout the
experiments, especially when comparing miniature IPSCs before and after application of
GABAergic drugs. Evoked IPSCs were elicited by stimulation (200 µs, < 50 V) with a
locally positioned patch electrode. The software package developed by John Dempster
(University of Glasgow, Scotland) was used for analysis of miniature IPSCs (mIPSCs)
which were detected using a threshold detection algorithm. Parameters were adjusted by
comparison with hand-evaluated data.
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N. Axmacher, K. Hartmann, A. Draguhn 4
In a previous series of experiments we had observed changes in the frequency of
mIPSCs after manipulating GABA metabolism in cultured hippocampal slices (Engel et
al., 2001) (see below). Slice cultures were prepared and maintained as described by
Yamamoto and coworkers (Yamamoto et al., 1989, 1992) following the protocol by
Stoppini and coworkers (Stoppini et al., 1991).
For imaging, we prepared horizontal hippocampal slices from 9-14 days old
Wistar rats. Slices were transferred into a recording chamber under submerged conditions
and at room temperature. In order to monitor vesicular release we loaded the readily
releasable pool of vesicles with the fluorescent dye FM1-43 by brief (25 s) superfusion
with hypertonic solution (ACSF supplemented with sucrose to 800 mOsmol) according to
the protocol of Stanton et al. (2001). We blocked action potentials by addition of 1 µM
TTX and suppressed glutamate-evoked GABA release by adding 30 µM CNQX and 10
µM APV. Modulation of transmitter release by GABABR was excluded by 2 µM CGP
55845A. Two-photon confocal imaging was performed using a Leica microscope and a
Ti:sapphire laser (Spectra-Physics, Freemont, USA). Regions of interest were selected at
bright fluorescent spots (putative synaptic boutons) in the perisomatic region of CA3
pyramidal cells. Control regions showing no puncta were selected in each slice and were
used for subtraction of background and bleaching. As a control, we also monitored
destaining of putative excitatory synapses located further outward in the dendritic layers.
Destaining was monitored over 40 minutes.
B
Our model of presynaptic vesicle dynamics (Axmacher et al., 2004c) was based on
a reduced version of the vesicle cycle as described by Sudhof (1995). We divided
vesicles into three pools, namely the readily releasable pool (RRP), the reserve pool and
the pool of fused vesicles. Transitions between the pools were modeled by a set of
ordinary differential equations. Filling of vesicles was assumed to be a bi-directional flux
of transmitter into and out of the vesicle with saturation when influx and efflux are equal.
Numerical simulation was performed using the Matlab program (Mathworks, Natick,
USA).
PRESYNAPTIC IONOTROPIC GABA RECEPTORS 5
3. RESULTS AND DISCUSSION
We examined the GABAergic modulation of GABA-release onto CA3 pyramidal
neurons using both electrophysiological and imaging techniques. GABAergic
postsynaptic currents were pharmacologically isolated and GABABR were blocked to
exclude confounding effects by this well-established negative feedback loop (see
Methods). In our electrophysiological approach we recorded synaptic currents from CA3
pyramidal cells in whole cell configuration. IPSCs were evoked by local electrical
stimulation with an extracellularly positioned patch pipette, adjusting parameters such
that about 75% of our stimuli caused postsynaptic responses. Miniature IPSCs were
recorded as spontaneous GABAergic events in the presence of TTX.
B
After recording 20-30 stimulus-triggered traces under baseline conditions, we
applied muscimol at 1 µM via bath perfusion. Our aim was to tonically stimulate
presynaptic GABAAR, if present, and to test for their effects on stimulus-evoked IPSCs.
It is obvious that perfusion of the recording chamber with a GABA receptor agonist will
not selectively activate presynaptic GABA receptors but will also open GABA-gated ion
channels in the postsynaptic membrane. This can massively distort the results by
desensitizing postsynaptic receptors or by changing the recording conditions (increased
membrane conductance). Such effects could, however, be attenuated by recording at 5
minutes after washout of muscimol. While, at this time, the acute effects of the substance
on membrane conductance and noise were eliminated, the presynaptic effects were still
present. This prolonged action of muscimol at presynaptic terminals is an interesting
finding in itself and has not yet been understood (see below). Furthermore, muscimol did
not change the responses to iontophoretically applied GABA nor did it suppress
amplitudes of mIPSCs. These findings do largely exclude that any effects of muscimol on
stimulus-induced or miniature IPSCs are confounded by receptor desensitization or by
deterioration of the experimental conditions.
N. Axmacher, K. Hartmann, A. Draguhn 6
Figure 1. Stimulus-induced IPSCs are suppressed by muscimol. Middle traces were recorded 5 min after washout of muscimol, right traces after 25 min of wash. Bottom traces are averages from the single events shown above. Modified from Axmacher and Draguhn, 2004a.
Evoked IPSCs were massively reduced by muscimol, both in amplitude and in
response rate (i.e., failure rate increased; see Figure 1). We suggest that this effect reflects
a reduced probability of transmitter release from endings of inhibitory interneurons in the
presence of muscimol. The experiment does, however, not clarify whether the underlying
mechanism is located at the bouton itself or whether more indirect mechanisms, e.g.
shunting of the axonal membrane, cause the suppression of evoked release. Recent work
by Ruiz and colleagues has indeed shown that glutamatergic transmission from granule
cells to CA3 pyramids is regulated by GABAAR located at the axons, rather than the
synaptic boutons, of mossy fibers (Ruiz et al., 2003). In order to find out whether the
effects are restricted to the endings we recorded action potential-independent IPSCs in
the presence of TTX. Similar to the evoked responses these mIPSCs were reduced in
frequency after application of muscimol. Their amplitude, however, remained unaltered,
confirming the presynaptic origin of the action of muscimol. Similar effects were
obtained by bath-application of isoguvacine, an agonist with specificity for GABAAR
over GABACR (Figure 2). Thus, we assume that presynaptically located GABAA
receptors mediate a negative feedback on vesicular release of GABA.
PRESYNAPTIC IONOTROPIC GABA RECEPTORS 7
Figure 2. Reduction of mIPSC frequency by the GABAAR agonist isoguvacine (10 µM). Left part of the figure shows original recordings of mIPSCs from a CA3 pyramidal cell. Summary graph (right) shows mean normalized frequency (solid diamonds) and amplitude (open squares) from 5 cells. Note stability of amplitude while frequency is reduced. Modified from Axmacher and Draguhn, 2004a.
Our approach, however, did rely on the application of GABAergic drugs while
measuring postsynaptic GABAergic currents as an indicator of the presynaptic
modulatory effects. This experimental paradigm impedes a clear separation between the
hypothesized mechanism and the test signal. Therefore, we searched for an alternative
approach which would make us independent from the postsynaptic IPSCs. We recruited
to the method developed by W. Muller and P. Stanton who had used two-photon confocal
video-microscopy for FM1-43 imaging of vesicular release in hippocampal slices
(Stanton et al., 2003, 2001). We focused on perisomatic synapses of CA3 pyramidal cells
which can be assumed to be preferentially GABAergic, as compared to more distal
dendritic synapses. Indeed, basal destaining in the presence of TTX was faster in the
former, compatible with the known higher frequency of mIPSCs as compared to
miniature excitatory postsynaptic currents. In the somatic region, synapses destained at a
rate of roughly 10% per 10 minutes. Muscimol was applied after 12 minutes of
spontaneous destaining in control solution. In these experiments, we left muscimol in the
bath solution for 10 minutes (as compared to 1 minute in our electrophysiological
studies), since postsynaptic receptor desensitization was no concern. Muscimol did
clearly slow down the rate of destaining of perisomatic synapses (Figure 3). This effect
was irreversible within the time of observation, well compatible with the long-lasting
N. Axmacher, K. Hartmann, A. Draguhn 8
effects of muscimol in our previous study on mIPSCs. In contrast to perisomatic boutons,
distal (dendritic) synapses showed a slower destaining which was resistant to modulation
by muscimol.
Figure 3. Fluorescence signals of FM1-43 stained putative inhibitory synapses in the perisomatic region of CA3 pyramidal cells. Top images show the slow decay observed when muscimol was added after 12 min, bottom images show faster decay in the absence of muscimol. Quantitative data (right panel) show continuous decrease of vesicle-staining in the absence of muscimol (open squares) and plateau formation after addition of muscimol (solid diamonds). Modified from Axmacher et al., 2004c.
Together, our data show that perisomatic inhibition in rat hippocampal CA3
pyramidal cells is negatively regulated by presynaptic feedback of GABA on GABAAR.
This constitutes a potential mechanism of short-term plasticity and is of obvious
importance for the dynamics of synaptic inhibition and for the function of interneuron-
networks, especially during oscillations (Whittington and Traub, 2003). Ionotropic
receptors can act faster than the metabotropically coupled GABABR and therefore might
influence release at a millisecond time-scale, although this has not yet been directly
proven. A caveat is given by the observation that the effects of muscimol outlasted the
presence of the substance by several minutes. This suggests that plastic effects of
presynaptic ionotropic GABA receptors last longer than the typical time course of
B
PRESYNAPTIC IONOTROPIC GABA RECEPTORS 9
postsynaptic IPSCs, constituting a more sustained modulation of synaptic inhibition. The
underlying mechanisms are, as to yet, unclear. An intriguing possibility would be that
ionic fluxes induce volume changes at the presynaptic terminal which are known to
influence release probability, hence the use of hypertonic solution for massive release of
the RRP (Stanton et al., 2003). The tiny volume of presynaptic boutons can be heavily
affected even by a rather small ionic load, e.g. influx of chloride through GABAAR.
Direct observation of the volume of presynaptic boutons in living tissue is beyond present
technical possibilities. We are also lacking information about the expression of volume-
regulatory proteins at the presynaptic boutons of interneurons. It would be of special
importance to know whether the chloride extruding transporter KCC-2 is present at these
boutons. This would also help to clarify the chloride reversal potential which, in
conjunction with resting potential, defines the direction and size of the GABA-induced
chloride flux.
Another important information at the molecular level concerns the type of
receptors expressed. Many extrasynaptic GABA receptors are composed of subunits
which confer a specially high affinity towards GABA, making them ideal detectors of
low levels of the transmitter in the extracellular space (Stell and Mody, 2002). In the
retina, this sensitive subtype of GABA receptors is the GABACR (Shields et al., 2000),
assembled from rho-subunits which belong to the same gene family as GABAAR subunits
(Bormann and Feigenspan, 1995). While the effects of isoguvacine argue against the
expression of GABACR at presynaptic endings in the CA3 region, it is feasible that other
receptor subunits with high affinity are sorted to the axons of hippocampal interneurons
(Stell and Mody, 2002).
Experimental (Engel et al., 2001; Overstreet and Westbrook, 2001; Wu et al.,
2003) and theoretical (Axmacher et al., 2004b) evidence indicates that presynaptic
ionotropic GABA receptors play a crucial role for the regulation of inhibition in a special
situation of inhibitory synaptic plasticity: the adaptation of GABA-metabolism to
changing activity within local networks. Inhibitory synapses appear to generate more
GABA when network activity is increased and down-regulate transmitter production
when network activity is lowered. Evidence for this "metabolic plasticity" comes from
several studies in tissue with increased or decreased network activity, respectively. The
GABA-synthesizing enzyme glutamate decarboxylase (GAD) is up-regulated in the
N. Axmacher, K. Hartmann, A. Draguhn 10
hippocampus of epileptic rats (Esclapez and Houser, 1999; Feldblum et al., 1990). In
contrast, GABA-production is down-regulated when network activity is reduced, e.g. in
deafferentiated areas of somatosensory cortex (Garraghty et al., 1991; Gierdalski et al.,
1999; Hendry and Carder, 1992). Thus, it appears that the production of GABA follows a
homeostatic principle (Turrigiano, 1999), adapting inhibition to the "needs" of the
network. The cellular and subcellular mechanisms of this regulation are, however, far
from trivial: several complex, non-linear steps lie between the modulation of GABA
production and inhibitory synaptic efficacy (Axmacher et al., 2004b). In order to yield
enhanced filling of synaptic vesicles upon increasing cytosolic GABA-concentration,
normally filled vesicles must have a reserve for additional uptake of the transmitter.
Given that such vesicles contain more GABA, the postsynaptic responses will only be
increased if normal IPSPs are non-saturating, i.e. there is a receptor reserve (Frerking et
al., 1995; Nusser et al., 1997; Yee et al., 1998). In addition, an increased cytosolic
GABA-concentration may enhance non-vesicular release of GABA via reverse operation
of GABA transporters. The latter mechanism might explain the observed increase in
GABA-induced membrane noise (Engel et al., 2001; Overstreet and Westbrook, 2001;
Wu et al., 2003) and GABA-release (Yee et al., 1998) in experimental situations of
increased cellular GABA content.
We have increased cellular GABA content in two different experimental systems,
namely in acutely prepared (Axmacher and Draguhn, 2004a) and in cultured (Engel et al.,
2001) rat hippocampal slices. Chronic (~4 days) incubation of cultured slices with a
blocker of the GABA-degrading enzyme GABA transaminase (γ-vinyl-GABA) led to an
increase in the amplitude of mIPSCs as well as to increased membrane noise. These
findings indicate increased vesicle filling, a postsynaptic reserve pool of GABA receptors
and increased tonic inhibition, possibly via non-vesicular release. Surprisingly, the
treatment did also increase the frequency of mIPSCs. Moreover, the events tended to
occur in bursts, rather than isolated (Engel et al., 2001). In contrast, incubation of acutely
prepared slices with γ-vinyl-GABA for 3-9 hours led to a dramatic (> 90%) decrease in
the frequency of mIPSCs in CA3 pyramidal cells (Axmacher and Draguhn, 2004a).
Similar observations have been previously reported by Overstreet and Westbrook (2001).
The reduction in mIPSC frequency may be a direct consequence of the negative feedback
PRESYNAPTIC IONOTROPIC GABA RECEPTORS 11
of GABA on its own release via ionotropic GABAAR (see Figure 4; GABABR had been
blocked in these experiments).
B
Figure 4. Simplified model of the presynaptic terminal. Vesicles are divided into three pools with different transition rates from the reserve pool to the RRP (membrane-attached vesicles, left) and to the released state. Rate of filling is labeled λ. Released GABA feeds back onto presynaptic GABA-A receptors. Right panels show the resulting simulations assuming that GABAergic feedback inhibits the transition from the reserve pool to the RRP. Note decrease release rate of vesicles with decreasing supply of vesicles into the RRP, consistent with experiments.
The feedback may be mediated by GABA released from vesicles with enhanced
transmitter content or, alternatively, by a tonically increased ambient GABA
concentration, as indicated by the enhanced GABAergic membrane noise in these
experiments (Engel et al., 2001; Overstreet and Westbrook, 2001; Wu et al., 2003). Why
did we observe an opposite effect (increase) on mIPSC frequency in cultured
hippocampal slices? One possible explanation for these apparently contradictory results
from two different preparations is that the axonal chloride gradient differs between
acutely prepared and chronically stored hippocampal slices. Depolarizing GABA
N. Axmacher, K. Hartmann, A. Draguhn 12
responses in the cultured tissue might increase presynaptic calcium levels and thereby
facilitate spontaneous vesicle release while hyperpolarizing responses in acutely prepared
tissue would inhibit vesicle release. Again, it would be important to know the chloride
equilibrium and resting potentials at presynaptic boutons. Functionally, the negative
feedback observed in acutely prepared slices will shift GABAergic inhibition from phasic
to tonic mode, i.e. postsynaptic cells receive less GABA from vesicular release but are
tonically inhibited by an increased ambient GABA concentration. While this may still
cause highly efficient inhibition, it might induce changes in interneuron-driven network
activity like gamma oscillations (Whittington and Traub, 2003). Possibly, side-effects of
GABAergic drugs may be associated with such specific disturbances of spike timing in
neuronal networks which are organized by interneurons.
Modeling the effects of altered GABA metabolism on vesicular release did indeed reveal
that positive or negative feedback of GABA on further release of GABAergic vesicles is
a likely mechanism underlying the observed changes in mIPSC frequency (Axmacher et
al., 2004b). It further turned out that the interaction between released GABA and
presynaptic vesicle dynamics takes most likely place at the transition from the reserve
pool to the RRP, providing more or less release-ready vesicles. Our model shows that this
indirect mechanism allows for sustained changes of the frequency of release, while a
direct effect of GABA release would tend to deplete the RRP.
4. CONCLUSIONS
Presynaptic autoreceptors modulate the release of GABA from terminals of
inhibitory interneurons. We have shown that in rodent hippocampal CA3 this feedback
involves the activation of ionotropic GABAAR which suppress further release in acutely
prepared tissue. Under conditions of different ECl-, the same feedback might be positively
coupled to release, giving rise to bursts of IPSCs after an initial (large) event. Our further
experimental and theoretical studies show that presynaptic ionotropic GABAAR are
specifically important during one mechanism of inhibitory synaptic plasticity, namely
changes in GABA metabolism. Besides alterations of the amount of released GABA this
homeostatic mechanism induces changes in the frequency of vesicle release which are
PRESYNAPTIC IONOTROPIC GABA RECEPTORS 13
likely to be mediated by presynaptic ionotropic GABAAR. This new feedback mechanism
will be important to understand the dynamics of inhibitory synaptic signaling and the
ratio between tonic and phasic GABAergic inhibition.
5. ACKNOWLEDGEMENTS
This work was supported by the DFG (Deutsche Forschungsgemeinschaft) grant
SFB 515/B1.
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