presynaptic ionotropic gaba receptors

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.

B


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).


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.


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.


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


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


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


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


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


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


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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