Pathway-Specific Targeting of GABAA Receptor Subtypes to Somatic and Dendritic Synapses in the Central Amygdala

Andrew J. Delaney and Pankaj Sah

Division of Neuroscience, John Curtin School of Medical Research, Australian National University, Canberra, ACT 2601, Australia


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Delaney, Andrew J. and Pankaj Sah. Pathway-Specific Targeting of GABAA Receptor Subtypes to Somatic and Dendritic Synapses in the Central Amygdala. J. Neurophysiol. 86: 717-723, 2001. Neurons in the central amygdala express two distinct types of ionotropic GABA receptor. One is the classical GABAA receptor that is blocked by low concentrations of bicuculline and positively modulated by benzodiazepines. The other is a novel type of ionotropic GABA receptor that is less sensitive to bicuculline but blocked by the GABAC receptor antagonist (1,2,5,6-tetrohydropyridine-4-yl) methylphosphinic acid (TPMPA) and by benzodiazepines. In this study, we examine the distribution of these two receptor types. Recordings of GABAergic miniature inhibitory postsynaptic currents (mIPSCs) showed a wide variation in amplitude. Most events had amplitudes of <50 pA, but a small minority had amplitudes >100 pA. Large-amplitude events also had rise times faster than small-amplitude events. Large-amplitude events were fully blocked by 10 µM bicuculline but unaffected by TPMPA. Small amplitude events were partially blocked by both bicuculline and TPMPA. Focal application of hypertonic sucrose to the soma evoked large-amplitude mIPSCs, whereas focal dendritic application of sucrose evoked small-amplitude mIPSCs. Thus inhibitory synapses on the dendrites of neurons in the central amygdala express both types of GABA receptor, but somatic synapses expressed purely GABAA receptors. Minimal stimulation revealed that inhibitory inputs arising from the laterally located intercalated cells innervate dendritic synapses, whereas inhibitory inputs of medial origin innervated somatic inhibitory synapses. These results show that different types of ionotropic GABA receptors are targeted to spatially and functionally distinct synapses. Thus benzodiazepines will have different modulatory effects on different inhibitory pathways in the central amygdala.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

gamma -Aminobutyric acid (GABA) is the principal inhibitory transmitter in the CNS and activates ionotropic GABAA and GABAC receptors, and metabotropic GABAB receptors. Ionotropic GABA receptors are heterooligomeric proteins of which 20 subunits have so far been identified: alpha 1-6, beta 1-4, gamma 1-3, delta , epsilon , pi , theta , and rho 1-3. Of these, the rho  subunits assemble to form GABAC receptors, while pi  subunits are only found outside the CNS. The other subunits coassemble as heteromultimers to form a number of different types of GABA receptor (Barnard et al. 1998). The subunit composition of GABA receptors determines their biophysical and pharmacological properties (MacDonald and Olsen 1994). Thus considerable GABA receptor heterogeneity is expected to be present within the CNS. Immunohistochemical studies have shown that different subunits can be targeted to different regions of the neuronal membrane (Nusser et al. 1996, 1998). It has therefore been suggested that functionally different receptors may be targeted to different synapses. However, there is little direct evidence to support this proposal.

The amygdala is intimately involved in emotional behavior (Kluver and Bucy 1939; LeDoux 2000), and its role in the generation of anxiety and conditioned fear is well known (Davis 1992; LeDoux 1995). Studies of Pavlovian fear conditioning have suggested that the basolateral amygdala is the site for convergence of neural pathways conveying information about conditioned and unconditioned stimuli. This information is processed locally and then transmitted to the central amygdala. Neurons in the central nucleus project to the hypothalamus and brain stem regions, which are important in the behavioral, hormonal, and autonomic aspects of the fear response (LeDoux 2000). Neurons in the central amygdala express two types of ionotropic GABA receptor (Delaney and Sah 1999). One is a typical GABAA-receptor that is blocked by low doses of bicuculline and positively modulated by benzodiazepines, anesthetics, and barbiturates. The other is a novel type of receptor, similar to GABAC receptors first described in the retina (Enz et al. 1995; Qian and Dowling 1994). These receptors are less sensitive to bicuculline and are little affected by anesthetics and barbiturates. However, unlike GABAC receptors, they are inhibited by 1,4 benzodiazepines such as diazepam (Delaney and Sah 1999). In this study, we examine the distribution of these two types of ionotropic GABA receptors. We find that GABAC-like receptors are present on dendritic synapses where they colocalize with GABAA receptors. These synapses are innervated by axons of the intercalated cell masses (ICMs). In contrast, somatic synapses express only GABAA receptors and are innervated by axons of different origin.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Wistar rats (17- to 20-days old) were anesthetized with intraperitoneal pentobarbitone (50 mg/kg) and decapitated, and the brain was removed and immersed in ice-cold Ringer. Coronal brain slices (400 µm) were prepared using standard methods. All procedures were in accordance with the Institutional Animal Care and Ethics Committee guidelines. Slices were superfused at 200 ml/h with oxygenated external solution containing (in mM) 118 NaCl, 2.5 KCl, 25 NaHCO3, 10 glucose, 1.2 NaH2PO4, 1.3 MgCl2, and 2.25 CaCl2 in a bath volume of 1 ml. Kynurenic acid (2 mM) was included in the external solution to block excitatory glutamatergic transmission. Tetrodotoxin (0.5 µM) was added to block synaptic transmission when recording mIPSCs. Recordings were made from neurons in the lateral division of central amygdala (CeL) using the whole cell patch-clamp method with either the blind approach or infrared differential interference contrast techniques. Borosilicate glass electrodes (3-5 MOmega ) were filled with high chloride internal solution containing (in mM) 130 CsCl2, 1 MgCl2.6H2O, 10 EGTA, 10 HEPES, 2 Mg2ATP, and 0.2 Na3GTP (pH 7.3 with CsOH, 290 mOsM). In experiments where the sucrose solution was applied to the soma and dendrites, Lucifer yellow was also added to the internal solution. Neurons were held in voltage-clamp mode at -60 mV.

Drugs used were bicuculline methiodide, (1,2,5,6-tetrohydropyridine-4-yl) methylphosphinic acid (TPMPA; RBI Research Chemicals), kynurenic acid (Sigma), tetrodotoxin (Alamone), and diazepam (gift from Professor P. Gage). Inhibitory postsynaptic currents (IPSCs) were evoked electrically using stainless steel bipolar stimulating electrodes (Frederick Haer) placed laterally along the edge of CeL or medially in central nucleus medial sector (CeM). Stimuli were 50 µs in duration. Sucrose stimulation of miniature IPSCs (mIPSCs) was performed by pressure ejection of external solution containing 0.5 M sucrose through a 3 MOmega patch pipette to the surface of the slice or by low pressure injection through a 3-5 MOmega patch pipette under visual guidance. Lucifer yellow was included in the sucrose solution for visualization of the spread of the ejected sucrose solution. Signals were recorded using an Axopatch 200B amplifier (Axon Instruments), filtered at 5 kHz, and digitized at 10 kHz (Instrutech, ITC 16). Data were acquired with Axograph (Axon Instruments) on a Macintosh G3 computer. Series resistance (10-30 MOmega ) was monitored on-line throughout the experiment, and experiments were rejected if resistance changed by 10%. No series resistance compensation was used. IPSC peak amplitude, 10-90% rise time, half-peak width, and decay time constants were analyzed using Axograph 4.0 and compiled and statistically analyzed using Microsoft Excel. All values are expressed as means ± SE, and all statistical comparisons were done using Student's t-test. Spontaneous mIPSC were detected using the variable-amplitude template event-detection program included in Axograph. Measurements of amplitude and kinetics and tracer of mIPSCs shown are averages of between 4 and 10 events for large mIPSCs and between 30 and 50 events for the small-amplitude mIPSCs under control conditions. All experiments were done at room temperature (21-24°C).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Whole cell recordings were made from neurons in the lateral division of the central amygdala (Jolkkonen and Pitkanen 1998). We have shown that locally evoked inhibitory synaptic inputs to these neurons activate both GABAA and GABAC-like ionotropic receptors (Delaney and Sah 1999). To test if both types of GABA receptor were colocalized at all inhibitory synapses, we examined spontaneously occurring mIPSCs that reflect the response to single quanta of transmitter. At a holding potential of -60 mV, mIPSCs in CeL neurons showed a wide variation in amplitude (Fig. 1). Most responses were of small amplitude (<50 pA; mean amplitude, 19 ± 2 pA; n = 8). However, a small number of large-amplitude events (>100 pA; mean amplitude, 123 ± 9 pA; n = 7) were also detected. These large-amplitude events had significantly faster (P < 0.01) 10-90% rise times (0.91 ± 0.09 ms) compared with the rise times of the smaller events (1.51 ± 0.12 ms; n = 4; Fig. 1, C and D).



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Fig. 1. Spontaneous GABAergic inhibitory postsynaptic currents in the central amygdala show wide variation in amplitude. A and B: spontaneous miniature inhibitory postsynaptic currents (mIPSCs) recorded in the presence of tetrodotoxin are shown from 1 cell in A and their peak amplitudes are plotted as a histogram in B. C: examples of events with small amplitude (<50 pA, top traces) and those with large amplitude (>100 pA, bottom traces). D: average data for small- and large-amplitude events. Events with large amplitudes had faster rise times than the smaller events.

To increase the frequency of large-amplitude events, we applied a hypertonic Ringer solution containing 0.5 M sucrose, which causes asynchronous release of minis (Fatt and Katz 1952). Application of hypertonic solution to the slice caused a large increase in mIPSC frequency (Fig. 2A) and revealed a much larger proportion of fast large-amplitude mIPSCs. The frequency of large-amplitude events increased from 0.02 ± 0.01 to 0.31 ± 0.01 Hz (n = 6; P < 0.01). Large-amplitude mIPSCs evoked in sucrose had amplitudes and rise times identical to those of large-amplitude events under control conditions (Fig. 2B). The higher frequency of large-amplitude events following application of sucrose increased the amplitude of the average mIPSC from 19.4 ± 1.5 to 35.5 ± 3.4 pA (n = 9; Fig. 3, A and B). Application of the GABAA receptor antagonist bicuculline methiodide (BIC, 10 µM) markedly reduced the amplitude (71 ± 6% reduction; n = 6) and frequency of small mIPSCs but completely abolished the large events (Fig. 3A). Furthermore in the presence of BIC, application of sucrose increased the frequency of mIPSCs with no change in their average amplitude (Fig. 3A). In contrast, application of the GABAC-receptor antagonist TPMPA (60 µM) (Ragozzino et al. 1996) reduced the amplitude of the small events (P < 0.01) while having no significant effect (P > 0.05) on the large-amplitude IPSCs (Fig. 3, C and D; n = 3). Application of hypertonic Ringer in the presence of TPMPA increased the frequency of the large-amplitude events (Fig. 3B). In the presence of TPMPA, mIPSCs were completely blocked by 10 µM BIC and application of sucrose did not evoke any IPSCs (data not shown). These results indicate that two types of GABAergic mIPSCs are present in CeL neurons. One consists of large-amplitude fast-rising events that are fully blocked by low doses of BIC but are unaffected by TPMPA, suggesting that they are due to activation of pure GABAA receptors. The second population consists of small-amplitude slow-rising events that are partially blocked by BIC and TPMPA, indicating that both GABAA receptors and GABAC-like receptors (Delaney and Sah 1999) are present at these synapses.



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Fig. 2. Application of hypertonic Ringer increases the frequency of large amplitude mIPSCs. A: application of hypertonic (0.5 M) sucrose increased the frequency of large-amplitude events. B: events recorded before (left) and during sucrose application (right) are shown as histograms. Note the increase in number of large-amplitude events in sucrose. C: large-amplitude events evoked by sucrose application have properties identical to those recorded under control conditions (n = 6).



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Fig. 3. Large fast mIPSCs are due to activation of purely GABAA receptors, but small mIPSCs are due to activation of 2 types of GABA receptor. A: effect of 10 µM bicuculline application on spontaneous mIPSCs evoked by sucrose. Left: traces are in control condition and average mIPSCs before and after sucrose application have been superimposed in Ai. Note the increase in mIPSC amplitude by sucrose due to the presence of more large-amplitude events. Right: traces show the effects of sucrose application in the presence of bicuculline. The average amplitude of the control mIPSCs is smaller in bicuculline, but there is no increase in their amplitude by sucrose (Aii). B and C: the GABAC antagonist (1,2,5,6-tetrohydropyridine-4-yl) methylphosphinic acid (TPMPA) reduces the amplitude of small sIPSCs but has no effect on large sIPSCs. B, left: traces show the action of sucrose under control conditions; right: traces show the effect of sucrose in the presence of 60 µM TPMPA. B, i and ii: average mIPSCs before and after sucrose have been superimposed. C: small-amplitude events are reduced in amplitude by TPMPA (P < 0.01). Average data (n = 3) are shown in the histograms on the right. The IPSC recorded in control Ringer is marked (*). D: large-amplitude mIPSCs evoked by sucrose before and after application of TPMPA have been superimposed. Note that these events are not significantly affected by TPMPA (P > 0.05). Average data are shown in the histograms on the right.

What explains the rapid rising phase and large amplitude of the pure GABAA mIPSCs? One possibility is that these synapses are located electrotonically closer to the soma resulting in less filtering of their kinetic properties (Bekkers and Stevens 1996; Maccaferri et al. 2000). To test this idea, we focally applied hypertonic sucrose solution to the soma and dendrites of CeL neurons. Focal somatic application of sucrose evoked mostly large-amplitude mIPSCs (Fig. 4, A-C). These events had a peak amplitude of 144 ± 13 pA and 10-90% rise time of 0.92 ± 0.04 ms (n = 4). Activation of these large-amplitude events led to a large increase in the average mIPSC (Fig. 4D). In contrast, dendritic application of sucrose evoked mIPSCs that had amplitudes and rise times indistinguishable from those of the small events present before application of sucrose (Fig. 4, E-G). Thus the average mIPSC was not affected by the dendritic application of sucrose (Fig. 4H). In addition, large-amplitude mIPSCs evoked by somatic application of sucrose were fully blocked by 10 µM BIC (n = 3) but unaffected by TPMPA (Fig. 5; 13% reduction in amplitude, n = 2). In contrast, mIPSCs evoked by dendritic sucrose application were reduced in amplitude by TPMPA (Fig. 5; 25% reduction in amplitude, n = 2). Together these results show that fast-rising large-amplitude mIPSCs arise from synapses on or near the soma of CeL neurons that express pure GABAA receptors. In contrast, the smaller-amplitude slower-rising mIPSCs arise from synapses of more distal dendritic origin that express both GABAA and GABAC-like receptors. We next tested if these synapses are innervated by axons of the same or different origin.



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Fig. 4. Large fast events are due to somatic synapses, while smaller-amplitude events are due to dendritic synapses. Whole cell recordings were made with the patch pipette solution and the sucrose-containing electrode containing Lucifer yellow. A and B: somatic application of sucrose evokes large-amplitude fast-rising mIPSCs. A, top: the control condition with the recording electrode attached to the soma. Bottom: application of sucrose to the soma. Scale bar: 5 µm. C: the amplitude and rise time of mIPSCs recorded before () and after (open circle ) application of sucrose are shown. D: average mIPSC before and after sucrose application have been superimposed and show that there is a large increase in peak amplitude. E and F: dendritic application of sucrose evokes only small-amplitude IPSCs. E, top: the control condition with the soma out of view to the right of the panel. Bottom: sucrose application is restricted to the dendrite. Scale bar: 5 µm. G: the amplitude and rise time of mIPSCs recorded before () and after (open circle ) application of sucrose are shown. H: average mIPSC before and after sucrose have been superimposed and show that there is no change in peak amplitude.



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Fig. 5. Somatic large IPSCs are fully blocked by bicuculline. A: mIPSCs evoked by somatic application of sucrose are shown before and after application of 10 µM bicuculline. B: averaged large (>= 100 pA) IPSCs in control and all IPSCs in the presence of bicuculline have been superimposed. The average IPSC in the presence of bicuculline is marked (*). C: mIPSCs evoked by somatic application of sucrose are shown before and after application of 100 µM TPMPA. D: averaged large IPSCs in control and in the presence of TPMPA have been superimposed. E: averaged small IPSCs (<= 50 pA) before and after application of TPMPA have been superimposed.

We have shown previously that IPSCs evoked by stimulation on the border of the CeL and the lateral amygdala activates IPSCs that contain both GABAA and GABAC-like receptors (Delaney and Sah 1999). These inhibitory inputs are thought to arise from GABAergic neurons within the intercalated cell masses of the amygdala (Paré and Smith 1993; Royer et al. 1999). To test if both types of GABA receptors were present at all synapses made by these inputs, we evoked single-fiber IPSCs by using minimal stimulation (Raastad et al. 1992). Glutamatergic inputs were blocked by inclusion of 2 mM kynurenic acid to the perfusing Ringer. Stimulation electrodes were placed on the lateral border of the CE to activate inputs arising from the ICMs (Royer et al. 1999) and medial to the CE to activate inputs that form part of the extended central amygdala (Sun and Cassell 1993) (Fig. 6A). IPSCs evoked from the ICMs had a peak amplitude of 19.3 ± 3.8 pA and a 10-90% rise time of 3.2 ± 0.4 ms (n = 10; Fig. 6B). In contrast, when the stimulating electrode was placed medially, minimal stimulation evoked a more rapidly rising (10-90% rise time 1.7 ± 0.1 ms) large-amplitude (130.8 ± 13.4 pA) IPSCs (Fig. 6, C and D). Laterally evoked IPSCs were partially inhibited by BIC (10 µM, 71 ± 4% block; n = 8;) and TPMPA (100 µM, 47 ± 10% block; n = 4), indicating that both types of GABA receptors are present at these synapses (Fig. 7, A and B). Medially evoked IPSCs in the same cells were fully blocked by 10 µM BIC (98 ± 3% block; n = 7; Fig. 7A) and not affected by TPMPA (n = 10; Fig. 7B). This result shows that IPSCs evoked by medial stimulation activate a population of pure GABAA receptors. In contrast, IPSCs evoked from the ICMs activate both GABAA and GABAC-like receptors. In further confirmation of this, application of diazepam potentiated the amplitude of minimally evoked medial IPSCs from 99 ± 6 to 108 ± 10 pA, whereas it reduced the amplitude of laterally evoked IPSCs from 23 ± 7 to 19 ± 6 pA (n = 6) (Delaney and Sah 1999) (Fig. 8).



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Fig. 6. Laterally evoked IPSCs activate dendritic inhibitory synapses, whereas medially evoked IPSCs activate somatic synapses. A: schematic diagram illustrating medial and lateral sites of stimulation. EC, external capsule; LA, lateral amygdala; BLA, basolateral amygdala; CeL, central nucleus lateral sector; CeM, central nucleus medial sector. Lateral stimulation activates cells that form the interacalated cell masses (ICM), whereas medial stimulation activates axons coursing through the medial division of the central amygdala. B: minimal stimulation of ICM cells evokes a smal-amplitude slowly rising IPSC. Right: trace shows the average of 4 sweeps at a stimulus of 4 V. C: minimal stimulation of medially located fibers evoked large-amplitude fast-rising IPSC. Right: trace shows the average of 4 sweeps at a stimulus of 3.5 V. D: average data (n = 6) illustrating the difference in the evoked IPSC from medial and lateral inputs.



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Fig. 7. Laterally evoked IPSCs activate synapses that contain both GABAA and GABAC-like receptors, but medially evoked IPSCs activate synapses with only GABAA receptors. A: laterally evoked IPSCs were partially blocked by bicuculline, while medially evoked IPSCs were fully blocked by bicuculline. Average data are shown in the histogram. B: laterally evoked IPSCs were partially blocked by TPMPA, while medially evoked IPSCs were unaffected by TPMPA. Average data are shown in the histogram (n = 3 for TPMPA and n = 7 for bicuculline). All recording of 2 inputs are from the same cells and are averages of 6 sweeps.



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Fig. 8. Diazepam potentiates medially evoked IPSCs but reduces laterally evoked IPSCs. A: IPSCs were evoked by minimal stimulation in the region of the ICMs, and diazepam was bath applied at a concentration of 10 µM. The IPSC shows reduction in peak amplitude and a slowing of the remaining IPSC as expected for a synapse expressing both GABAC-like and GABAA receptors. B: IPSC evoked by minimal medial stimulation evokes a large-amplitude event that is potentiated by diazepam as expected for a pure GABAA synapse.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We have shown that two types of GABAergic mIPSCs are present on neurons in the CeL. One type of mIPSC is of large amplitude and has a fast rise time. These mIPSCs are fully blocked by low concentrations of BIC but are unaffected by the GABAC antagonist TPMPA, indicating that they are purely due to activation of GABAA receptors. The frequency of these mIPSCs was greatly potentiated by somatic application of hypertonic sucrose solution, indicating that they result from activation of somatic synapses. The other type of mIPSC is of smaller amplitude, has a slower rise time, and is partially blocked by both BIC and TPMPA, indicating that these synapses contain both GABAA and GABAC-like receptors. The frequency of these mIPSCs is potentiated by dendritic application of sucrose, showing that they are due to activation of dendritic synapses. Minimal electrical stimulation of inputs that have a medial-origin-evoked IPSC whose amplitude and rise time was similar to those of large mIPSCs evoked by somatic sucrose application. Medially evoked IPSCs were fully blocked by bicuculline but unaffected by TPMPA. These results indicate that inputs that have a medial origin make synapses on the soma of CeL cells, and these synapses contain only GABAA receptors. In contrast, stimulation of lateral inputs arising from the intercalated cell masses evoked IPSCs with kinetics and pharmacology similar to those of dendritically located mIPSCs. These IPSCs were partially blocked by BIC and TPMPA, indicating that both GABAA and GABAC-like receptors are present at these synapses. Together, these data show that neurons in the lateral division of the central amygdala have two types of fast inhibitory synapses. GABAergic inputs arising from the intercalated cell masses make dendritic synapses at which both GABAA and GABAC-like receptors are present. In contrast, inhibitory inputs arising medial to the CeL (Sun and Cassell 1993) make somatic synapses at which only GABAA receptors are present. Thus while GABAA receptors are present at both somatic and dendritic synapses, GABAC-like receptors appear to be selectively targeted to different synapses.

Ionotropic GABA receptors are formed from 19 different subunits that can be assembled in a variety of combinations with distinct biophysical and pharmacological properties (MacDonald and Olsen 1994; Verdoon et al. 1990). In some neurons, different subunits have been shown to be targeted to different cellular compartments (Nusser et al. 1996, 1998), and this has led to the suggestion that different inhibitory synapses may express kinetically and pharmacologically distinct types of receptor. Indeed, GABAA receptors present at synapses have been shown to have properties different from those present in the extrasynaptic membrane (Banks and Pearce 2000; Brickley et al. 1999). In hippocampal pyramidal neurons, two kinetically and pharmacologically distinct GABAA receptor-mediated IPSCs have also been described (Pearce 1993).

The mechanisms that underlie this selective targeting of different receptors are as yet not known. It has been suggested that a subsynaptic matrix of receptor-associated proteins may play a key role in selecting and anchoring particular receptor subtypes to particular synapses (Kannenberg et al. 1997; Sheng 1997). GABAA and GABAC-like receptors present in CeL neurons are pharmacologically distinct, indicating that their subunit composition is likely to be different (Delaney and Sah 1999). Thus one possibility is that the two types of receptor have different anchoring proteins. In this model, anchoring proteins that bind GABAC-like receptors would have to be present only at dendritic synapses while those for GABAA receptors would be present at somatic as well as dendritic locations.

The amygdala forms a key element of the circuit involved in emotional processing and Pavlovian fear conditioning (LeDoux 2000). Anatomically, the amygdala is divided into a number of subnuclei (McDonald 1999). Cortical (McDonald 1999) and thalamic (Turner and Herkenham 1991) inputs enter the amygdala at the level of the basolateral nuclei. These nuclei then send information to the central nucleus, which projects to hypothalamic and brain stem regions that control the expression of fear-related responses (Krettek and Price 1978; Maren and Fanselow 1996). The intercalated cells are a population of GABAergic neurons that lie in clusters between the basolateral and central amygdala (Millhouse 1986; Nitecka and Ben-Ari 1987). These cells form an inhibitory interface between the basolateral and central amygdala (Royer et al. 1999). Connections between medial and lateral groups of ICMs, which receive inputs from different regions of the basolateral complex, have been proposed to modulate the traffic of information reaching different regions of the basolateral amygdala (Royer et al. 2000). Our results indicate that the axons of ICM cells make synapses on the dendrites of CeL neurons.

We cannot be certain of the origin of the medial input to CeL neurons. It is unlikely to be a projection originating from CeM as anatomical studies have found this to be rather meager (Jolkkonen and Pitkanen 1998). The axons of GABAergic neurons in the CeL have been found to make synapses on local cells (McDonald 1982), raising the possibility that the medial input may be due to retrograde activation of neurons projecting out of the CeL. However, against this proposal, in our extensive recordings from neurons in the CeL, we have never recorded from neurons that were antidromically activated by medical stimulation (unpublished observations). The central amygdala also receives a number of extraamygdaloid inputs (Pitkänen 2000). However, as the exact nature of these inputs has not been determined, it is not possible to speculate on the identity of medially originating GABAergic inputs.

The presence of GABAC-like receptors at these synapses leads to a reduction in the amplitude of the IPSC by benzodiazepines (Fig. 7), which enhance the IPSC at most GABAergic synapses. This result suggests that processing of information between the input and output stages of the amygdala will be differently modulated by agents that modulate GABA receptors. The axons of neurons in the CeL have been found to have spines in the axon initial segment that are innervated by axons of passage (McDonald 1982). While the nature of this synapse has not been determined, it is tempting to speculate that the medial inhibitory inputs we have recorded may in part be due to the activation of these axonic synapses. The large-amplitude of these inputs would make them potent modulators of the output of CeL neurons. Agents such as benzodiazephines, which amplify these medial inputs would therefore have much larger effects on the output of these cells as compared to its effects on the integration of dendritic inputs.

Disorders of the storage or expression of fear responses are thought to underlie such mental disorders as panic attacks, anxiety, and post traumatic stress disorder. Benzodiazepines, which are commonly used for the treatment of anxiety, are thought to act by enhancing the action of the inhibitory transmitter GABA (Costa and Guidotti 1996; Tallman and Gallager 1985). The central amygdala has a particularly high density of benzodiazepine binding sites (Sibille et al. 2000). Our results show that benzodiazepines have distinct actions at different GABAergic circuits within the amygdala and highlight the complex nature of the inhibitory control over amygdaloid function. The implications of having two types of inhibitory synapse on GABAergic lateral central amygdaloid neurons must be taken into account when considering the mechanisms of action of benzodiazepines in the treatment of anxiety disorders.


    ACKNOWLEDGMENTS

We thank L. Faber, J. Power, and M. De Armentia for comments on the manuscript. P. Sah is a Charles and Sylvia Viertel Senior Medical Research Fellow.

This work was supported by grants from the National Health and Medical Research Council of Australia.


    FOOTNOTES

Address for reprint requests: P. Sah, Div. of Neuroscience, John Curtin School of Medical Research, GPO Box 334, Canberra, ACT 2601, Australia (E-mail: pankaj.sah{at}anu.edu.au).

Received 4 December 2000; accepted in final form 11 April 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

0022-3077/01 $5.00 Copyright © 2001 The American Physiological Society