Activation of postsynaptic metabotropic glutamate receptors by trans- ACPD hyperpolarizes neurons of the basolateral amygdala

Glutamate has traditionally been regarded as an excitatory neurotransmitter. Synaptic activation of ionotropic glutamate receptors mediates fast EPSPs in the CNS. Moreover, activation of metabotropic glutamate receptors (mGluRs), which are coupled to second messenger effector systems via GTP-binding proteins (G-proteins), results in the expression of slow EPSPs. We have now examined the response of basolateral amygdala (BLA) neurons to activation of postsynaptic mGluRs. In approximately 78% of BLA neurons examined, activation of postsynaptic mGluRs results in membrane hyperpolarization and an associated decrease in membrane input resistance or a hyperpolarization followed by a depolarization associated with an increase in input resistance. The purpose of this study was to address the mechanisms underlying the membrane hyperpolarization. Here, we report that the ACPD-induced hyperpolarization is insensitive to TTX, is dependent on extracellular K+ concentrations, and has a reversal potential (-84 mV) close to that estimated from the Nernst equation for an increase in a K+ conductance. In addition, the ACPD response is resistant to (1) intracellular chloride loading, (2) the GABAB receptor antagonist CGP55845A, (3) the ACh receptor antagonist atropine, and (4) the ionotropic glutamate receptor antagonists CNQX and APV. These data suggest that the hyperpolarization results from a direct activation of postsynaptic mGluRs on neurons of the BLA. Furthermore, we performed studies that suggest that the hyperpolarization is G-protein mediated and results from activation of a TEA-sensitive, calcium-dependent potassium conductance. The sensitivity of this conductance to thapsigargin further suggests that this response requires the release of calcium from intracellular stores. In summary, these data suggest a role for glutamate as an inhibitory transmitter in the BLA during periods of metabotropic glutamate receptor activation. In nuclei such as the BLA that are exquisitely sensitive to seizure induction, an inhibitory response to glutamate may act to delay the onset of epileptogenesis.


Glutamate
has traditionally been regarded as an excitatory neurotransmitter.
Synaptic activation of ionotropic glutamate receptors mediates fast EPSPs in the CNS. Moreover, activation of metabotropic glutamate receptors (mGluRs), which are coupled to second messenger effector systems via GTPbinding proteins (G-proteins), results in the expression of slow EPSPs. We have now examined the response of basolateral amygdala (BLA) neurons to activation of postsynaptic mGluRs. In approximately 78% of BLA neurons examined, activation of postsynaptic mGluRs results in membrane hyperpolarization and an associated decrease in membrane input resistance or a hyperpolariration followed by a depolarization associated with an increase in input resistance.
The purpose of this study was to address the mechanisms underlying the membrane hyperpolarization. Here, we report that the ACPD-induced hyperpolarization is insensitive to TTX, is dependent on extracellular K+ concentrations, and has a reversal potential (-84 mV) close to that estimated from the Nernst equation for an increase in a K+ conductance.
In addition, the ACPD response is resistant to (1) intracellular chloride loading, (2) the GABA, receptor antagonist CGP55845A, (3) the ACh receptor antagonist atropine, and (4) the ionotropic glutamate receptor antagonists CNQX and APV. These data suggest that the hyperpolarization results from a direct activation of postsynaptic mGluRs on neurons of the BLA. Furthermore, we performed studies that suggest that the hyperpolarization is G-protein mediated and results from activation of a TEAsensitive, calcium-dependent potassium conductance. The sensitivity of this conductance to thapsigargin further suggests that this response requires the release of calcium from intracellular stores. In summary, these data suggest a role for glutamate as an inhibitory transmitter in the BLA during periods of metabotropic glutamate receptor activation. In nuclei such as the BLA that are exquisitely sensitive to seizure induction, an inhibitory response to glutamate may act to delay the onset of epileptogenesis.
We have previously demonstrated that low-frequency stimulation of the stria terminalis, an afferent input to the basolateral amygdala (BLA), results in postsynaptic activation of both NMDA and KA/AMPA ionotropic glutamate receptors on BLA neurons (Rainnie et al., I99 1 a) and that a long-lasting increase in glutamatergic excitatory synaptic transmission occurs in the BLA following kindling-induced epileptogenesis . Kindling has been reported to cause a prolonged increase in phosphoinositide (PI) hydrolysis in the amygdala/pyriform cortex (Akiyama et al., , 1992Yamada et al., 1989) suggesting a possible role for the mGluR 1, and/or mGluR5 subgroup of glutamate receptors in epileptogenesis. Indeed, local infusion of lS,3R-ACPD into the hippocampus leads to seizure expression in vivo (Sacaan and Schoepp, 1992). At present the mGluR subtypes mediating this action are unknown. It is possible, however, that several mGluR subtypes may be expressed by neurons in the same amygdaloid nuclei and subserve different physiological functions. This hypothesis is supported by the observation that the amygdala is immunoreactive against the antibody for the mGluR la receptor protein (Martin et al., 1992), and expresses mRNA for both mGluR1 and mGluR2 receptor proteins Ohishi et al., 1993). Moreover, low micromolar concentrations (150 PM) of truns-ACPD act presynaptically to reduce glutamatergic transmission in the BLA with no apparent effect on membrane potential . In the present report we have extended our investigation of metabotropic glutamate receptor responses by examining the effects of activation of postsynaptic mGluRs mediating a membrane hyperpolarization in BLA neurons.

Materials and Methods
Male Sprague-Dawley rats (11 O-l 50 gm) were decapitated and the brains rapidly removed and placed in cold oxygenated artificial cerebrospinal fluid (aCSF) solution. The brain was then hemisected and cut transversely posterior to the first branch and anterior to the last branch of the superior cerebral vein. The resulting section was glued to the chuck of a Vibroslice tissue slicer (Campden Instruments). Transverse slices of 500 pm thickness were cut and the appropriate slices placed in a beaker of oxygenated aCSF at room temperature for at least 1 hr prior to recording.-The aCSF was composed of (in mM) NaCl, 117; KCli 4.7; CaCl,. 2.5: MeCl,. 1.2: NaHCO,. 25: NaH,PO,. 1.2: and ducose. 11: and was bubbikd with 45% O,, 5% CG, (pH*= 7.4). In low &high Mg solutions, Ca was reduced to 0.2 mM and Mg was raised to 10 mM. For the Cd experiments, HEPES (10 mM) was substituted for bicarbonate and the phosphates were removed; this solution was bubbled with 100% 02.
The slice was fully submerged in the recording chamber and maintained at 32°C f 2°C with continuously superfused aCSF. Microelectrodes were pulled from fiber-filled capillary tubing of borosilicate glass with a Flaming-Brown micropipette puller (Sutter Instruments, model P-80). The resistance of the microelectrodes filled with 4 M K-acetate ranged between 70 and 150 MB. On-and off-line data acquisition and analysis was performed using an Axolab 1100 interface (Axon Instruments) between an Axoclamp-2A preamplifier and a Dell 3 10 personal computer utilizing ~CLAMP 5.5.1 software programs (Axon Instruments). Analog signals were also stored for later analysis on video cassettes using an adapted video recorder (A. R. Vetter Co., model 420B) as well as on a Gould (model 3400) chart recorder. Intracellular recordings were considered acceptable if neurons exhibited overshooting action potentials and showed stable membrane potentials more negative than -60 mV in the absence of a DC holding current. The bridge balance was carefully monitored throughout the experiments and adjusted when necessary. All data are expressed as mean + standard error of the mean; in all cases, n = number of neurons. Statistical analyses used in these studies were the paired one-tailed Student's t test and where appropriate ANOVA with Bonferroni post hoc t tests. Unpaired Student's t tests were used only when indicated. Statistical significance was determined at the level ofg 5 0.05.

Results
Eflects of trans-ACPD and IS,3R-ACPD on membrane properties of BLA neurons Intracellular recordings were obtained from 126 neurons of the basolateral amygdala in vitro and the effect of mGluR activation examined. As previously reported, application of the specific mGluR agonist truns-ACPD at low micromolar concentrations (< 50 PM) had no effect on resting membrane properties of BLA neurons . In higher concentrations, ACPD affected membrane potential in 78% of BLA neurons. Application of truns-ACPD (100-200 PM) or its active isomer lS,3R-ACPD (100 PM) evoked either membrane hyperpolarization (54 of 98 neurons) alone (Fig. lA,B) or a hyperpolarization and subsequent depolarization (44 of 98 neurons) (Fig. 1C). In an additional 28 neurons ACPD either had no effect (n = 6) or induced only a membrane depolarization (n = 22).
The hyperpolarization was associated with a decrease in membrane input resistance and the depolarization associated with an increase. In all cases, bath application of truns-ACPD evoked a membrane hyperpolarization that far outlasted the period of drug application. A typical response is shown in Figure 1 A; here truns-ACPD (100 PM) evoked a prolonged hyperpolarization and subsequent depolarization. The long duration of action of the mGluR agonist (> 10 min) made repeated testing with bath application difficult. Consequently, we applied truns-ACPD and lS,3R-ACPD by drop application (10 or 50 ~1, 20 PM to 20 mM, respectively). Using this method, reproducible responses could be obtained with repeated application. We calculated an approximate 1:20 dilution factor for the 50 ~1 drop and 1: 100 dilution for the 10 ~1 drop application. A concentration-dependent increase in membrane hyperpolarization resulted from topical application of both trans-ACPD and lS,3R-ACPD over (5 mV) and concomitant decrease in input resistance that persists for the duration of the superfusion (8 min). The effect was reversible on washout. B, Drop application of trans-ACPD (100 PM) induced a membrane hyperpolarization (6 mV) and decrease in input resistance of similar magnitude to that induced by trans-ACPD superfusion. Both the membrane potential and the input resistance gradually returned to the predrug values. C, In another neuron application of trans-ACPD induced an initial membrane hyperpolarization followed by a depolarization and spontaneous action potential generation. The depolarization was often associated with an increase in input resistance. Membrane potential in A-C was -65 mV.  Figure 1B. In another neuron, drop application of truns-ACPD evoked a hyperpolarization followed by a pronounced depolarization resulting in action potential generation (Fig. 1C). The hyperpolarization evoked by bath superfusion of 1 S,3R-ACPD (100 FM) from a holding potential of -65 mV was -4.8 + 1.8 mV (n = 6). Similarly, drop application of truns-ACPD (100 KM) or lS,3R-ACPD (50 PM) evoked membrane hyperpolarizations of -4.2 + 0.4 mV (n = 20) and -3.8 ? 0.5 mV (n = 17), respectively. In all subsequent experiments, lS,3R-ACPD or trans-ACPD was applied by drop except where noted, numbers in brackets reflect the final bath concentrations. Preliminary data suggest that the membrane hyperpolarization can be mimicked by glutamate (500 PM; n = 3), an effect that can be recorded in the presence of D-APV (50 PM) and CNQX (30 FM, n = 1; kvanov, Holmes and Shinnick-Gallagher, unpublished observations). In the hippocampus the depolarizing response to mGluR activation is attributed to a reduction of potassium currents (Baskys et al., 1990;Charpak et al., 1990;. In this study, however, we were concerned with the mechanisms un-derlying the hyperpolarizing response and so did not address the mechanisms mediating the depolarization. The ACPD-induced membrane hyperpolarization was observed in 78% of all BLA neurons recorded. Morphologically, neurons of the rat BLA consist of a heterogeneous population and have been divided into two main classes: class I, pyramidal and stellate spiny neurons, and class II, multipolar spine-sparse neurons. Moreover, the electrophysiological characteristics of identified BLA cells have been reported (Washburn and Moises, 1992;Rainnie et al., 1993). We first tested whether the hyperpolarizing response to ACPD was present in the different classes of BLA neurons. Application of lS,3R-ACPD induced a membrane hyperpolarization in class I pyramidal ( Fig. 2A,,A,) and multipolar spiny neurons (not shown) and class II multipolar aspiny neurons (Fig. 2B,,B,). Thus, the hyperpolarizing action of ACPD was not confined to a particular neuronal cell type.
The ACPD-induced hyperpolarization does not result from an indirect release of secondary neurotransmitter The delayed depolarizing response to ACPD seen in some BLA neurons suggested that the hyperpolarizing response to trans- . The lS,3R-ACPD (100 pr+induced hyperpolarization can be recorded in BLA neurons of differing morphology, is unaffected by chloride loading, and is resistant to the muscarinic receptor antagonist atropine. A,, Biocytin-filled class I spiny pyramidal neuron of the BLA, the surrounding cells were counterstained with II neutral red. A,, Preloading the neuron shown in A, with chloride during biocytin injection did not affect the hyperpolarizing response to lS,3R-ACPD application. Resting membrane potential, -68 mV. B,, Biocytin-filled class II aspiny multipolar neuron of the BLA. Bz, lS,3R-ACPD also caused a hyperpolarization and decrease in input resistance in this class II neuron. B,, Superfusion with the muscarinic antagonist atropine (5 PM) did not block the hyperpolarizing response to lS,3R-ACPD in the same neuron. Resting membrane potential, -67 mV. Downward deflections represent electrotonic potentials generated in response to injecting 200 msec, 0.1 nA current pulses across the membrane as a measure of membrane resistance ACPD and 1 S,3R-ACPD could be due to release of an inhibitory transmitter "upstream" from the recorded neuron. Consequently, we tested whether the ACPD hyperpolarization could be due to release of another transmitter by examining ACPD action in the presence of TTX. The ACPD-evoked hyperpolarization (3.0 -+ 0.3 mV), however, was resistant to prior superfusion with TTX (0.5 MM; 2.9 f 0.2 mV, n = 4, p > 0.05) suggesting that indirect, action potentialdependent release was not responsible for the recorded response.
In the BLA most spontaneous and evoked IPSPs are mediated by activation of either GABA, or GABA, receptors, resulting in an increase in either a chloride or potassium conductance, respectively (Rainnie et al.,199 1 b). Moreover, postsynaptic mGluR activation in hippocampal inhibitory interneurons results in an increase in spontaneous chloride-mediated IPSPs  (200 msec) hyperpolarizing current steps of increasing intensity (-0.05 to -0.5 nA, not shown) before, during, and after truns-ACPD (100 PM) superfusion. Note the decrease in the peak voltage deflection during truns-ACPD application (circle). R, A plot of the current-voltage relationship for the neuron shown in A, before (triangles) and during (circles) truns-ACPD ( 100 PM) application, indicated a reversal potential of -82.5 mV. C, In another neuron, extracellular [K+], is plotted as a function of the reversal potential for the trans-ACPD-induced hyperpolarization (E,.ACPD); the E,.ACPD shifted in a manner predicted by the Nemst equation for a response mediated by a K+ conductance. recorded from pyramidal neurons (Miles and Poncer, 1993). Consequently, we examined the effects of chloride preloading on ACPD-mediated responses. When KCl-filled electrodes were used for recording, bicuculline-sensitive PSPs were depolarizing at the resting membrane potential -66 f 1.3 mV (n = 8). However, the hyperpolarizing response to drop application of lS,3R-ACPD (50 PM) was unaffected by chloride loading ( Fig.  2A,B, see Fig. 8). In all neurons recorded with a KC1 electrode, 1 S,3R-ACPD evoked a membrane hyperpolarization (-3.7 f 0.6 mV, n = 8), from a holding potential of -65 mV. In addition, the ACPD-mediated hyperpolarization persisted in the presence of bicuculline (30 PM) and CGP55845A (10 PM; control, 3.7 f 1.2 mV; treatment, 3.8 + 0.8 mV; n = 3, p > 0.05), concentrations previously demonstrated to block postsynaptic GABA, receptor activation in the BLA (Rainnie et al.,199 1 b) and GA-BA, receptors in hippocampal neurons (Davies et al., 1993;Jarolimek et al., 1993). Washburn and Moises (1992) have reported a TTX-insensitive hyperpolarization and subsequent depolarization to occur within BLA neurons with the muscarinic agonist, oxotremorine-M. These responses were reduced by increasing extracellular potassium concentrations and were blocked by atropine (1 PM). Because of both the similarity in the BLA responses to oxotremorine-M and trans-ACPD, and dense innervation of the BLA by cholinergic fibers (Ben-Ari et al., 1977), we examined the effect of atropine (5 PM) on the ACPD-mediated response (Fig. 2B). Atropine had no effect on the hyperpolarizing response to IS,3R-ACPD (50 PM; control, 3.8 f 0.2 mV; atropine, 3.7 * 0.3 mV; n = 3, p > 0.05) recorded at a membrane potential of -65 mV.
enantiomer of Iruns-ACPD, lS,3R-ACPD (50 PM), also induced a membrane hyperpolarization and decrease in input resistance. A,, Subsequent superfusion of the ionotropic glutamate receptor antagonists CNQX (10 no) and APV (50 PM) appeared slightly reduced but did not block the hyperpolarizing response of the BLA neuron to topical application of truns-ACPD. R, Amplitude of the lS,3R-ACPD responses is dependent on membrane potential. In another cell, the amplitude of the lS,3R-ACPD-induced hyperpolarization is greatly enhanced by holding the membrane at a more depolarized level (-59 mV vs -67 mV). C, lS,3R-ACPD (100 1~) inhibits spontaneous EPSPs. In some cells, the conductance change induced by lS,3R-ACPD was sufficient to inhibit spontaneous excitatory potentials. Membrane potentials = -67 mV in A and C. Downward . Intracellular injection of GTP-r-S occludes and GDP-&S reduces the hyperpolarlzing response to truns-ACPD (100 PM) and lS,3R-ACPD (100 PM). A, Intracellular injection of the nonhydrolyzable analog of GTP, GTP-7-S (10 mM), occludes the response to topical application of both trans-ACPD (1) and lS,3R-ACPD (3). In a slice from the same animal, a BLA neuron recorded with a conventional microelectrode containing 4 M K-acetate showed characteristic responses to topical application of truns-ACPD (2) and lS,3R-ACPD (4). Topical application of baclofen (50 PM) causes a G-protein-dependent hyperpolarization of BLA neurons recorded with K-acetate containing microelectrodes (6). In the same neuron as in I and 3, GTP-7-S also occluded the response to baclofen (5). B, Intracellular injection of the nonhydrolyzable analog of GDP, GDP+S (10 mM), reduced the response to topical application of lS,3R-ACPD (I). In a slice from the same animal, a BLA neuron recorded with a 4 M K-acetatecontaining microelectrode showed a characteristic hyperpolarizing response to topical application of lS,3R-ACPD (2). Downward deflections are electrotonic potentials generated in response to 200 msec, 0.1 nA current pulses passed across the membrane. Membrane potential was held at -65 mV with DC current injection in A and B.

The ACPD-induced hyperpolarization is mediated by an increase in K conductance
The ACPD-induced hyperpolarization was consistently associated with a decrease in membrane input resistance and corresponding increase in membrane conductance (control, 19.1 f 1.7 nS; drug, 26.3 +-2.2 nS; n = 11, p < 0.0001). A typical response is shown in Figure 4A. Here, a series of electrotonic potentials evoked in response to transient hyperpolarizing current pulses of increasing amplitude (200 msec, -0.1 to -0.6 nA) are shown before, during, and after tram-ACPD application. Tram-ACPD and lS,3R-ACPD consistently reduced the amplitude of both the peak and steady state electrotonic potentials. Current-voltage relationships constructed before and during trans-ACPD application (Fig. 4B) show the reversal poten-tial (EACPD) ofthe agonist-induced hyperpolarization to be -84.3 f 3.0 mV (n = lo), which is close to that of potassium (-87 mV) estimated by the Nemst equation for an intracellular [K+], of 140 mM and extracellular [K+], of 4.7 mM. Furthermore, E ACPD was dependent on extracellular potassium concentration (Fig. 4C). Hence, an increase in extracellular potassium shifted E ACPD to a more depolarized level whereas decreased potassium shifted E,,,, to more hyperpolarized levels. The shift in EACPD from 2 mM to 8 mM K was calculated by the Nemst equation to be 34.9 mV, whereas the measured value was about 22 mV. These data indicate that the ACPD-induced hyperpolarization is due primarily to an increased K conductance. mGluR proteins are coupled to secondary cellular transduction mechanisms via G-proteins, and hence are sensitive to intracellular manipulations of GTP or GDP (Nakanishi, 1992). blockade of the lS,3R-ACPD-induced hyperpolarization. Injection of BAPTA for 5 min had no effect on the lS,3R-ACPIXnduced hyperpolarization and subsequent depolarization. At this time point, action potential half-width was increased and the slow afterhyperpolarization following repetitive firing was abolished. In the same neuron, after 60 min of BAPTA injection, the hyperpolarization was significantly reduced, and after 90 min the hyperpolarization and membrane conductance change are abolished. Downward deflections are electrotonic potentials elicited in response to injection of 200 msec, 0.1 nA current pulses, as a measure of membrane resistance. Calibration: 10 mV, 20 msec.
We examined the sensitivity of the hyperpolarizing response to internal dialysis with nonhydrolyzable analogs of GTP (GTP-7-S; 10 mM) and GDP (GDP-P-S; 10 mM) contained in the recording electrode. GTP-y-S dialysis depressed the trans-ACPD and, subsequently, completely occluded the lS,3R-ACPD-induced hyperpolarization (0.3 f 0.1 mV, n = 7) as shown in Figure 5, A, and A,. In contrast, a significant hyperpolarizing response to IS,3R-ACPD was observed in other neurons from the same slices (4.0 f 0.6 mV, n = 6; ANOVA, p < 0.0001; post hoc Bonferroni, control vs GTPq-S, p < O.OOl), when recorded with an electrode filled only with 4 M K-acetate (Fig.   %I,&). Baclofen responses (0 mV, n = 3) were used as a control for G-protein activation by GTP-7-S (Fig. 5A,,A,; post hoc Bonferroni: baclofen vs GTP-+j, p > 0.05; control vs baclofen, p < 0.001) since we have previously shown that the baclofen response in the BLA is inhibited by this procedure . Dialysis with GDP-P-S (Fig. 5B), however, only partially inhibited lS,3R-ACPD-mediated responses (control, -3.5 -t0.3mV,GDP-P-S, -1.03 *0.03mV,n=3;p<0.001, unpaired t test). GTP-7-S (IO mM) is a tetralithium salt whereas GDP-P-S (10 mM) is a trilithium salt. It is unlikely that the effects of these compounds are due to a direct action of the Efect of alterations in the availability of intracellular Ca2+ on the IS,3R-ACPD-mediated hyperpolarization Apart from studies on the retina (Hirono and MacLeish, 199 l), only one previous report has linked activation of metabotropic glutamate receptors within the CNS to enhancement of K conductance (Fagni et al., 1991). In that report, a tram-ACPLL activated outward potassium current was described in cultured cerebellar granule cells that was blocked by external tetraethylammonium (TEA; 1 mM). Therefore, we first examined the sensitivity of the lS,3R-ACPD-mediated hyperpolarizing re-sponse in BLA neurons to pretreatment with TEA (Fig. 6A). In all neurons tested (n = 4), TEA (1 mM) significantly reduced the response to agonist application (control, 3.1 + 0.4 mV, TEA, 0.3 * 0.3 mV; n = 4, p < 0.001). Fagni et al. (199 1) also reported that ACPD activated a largeconductance calcium-dependent potassium channel, which was blocked by the intracellular calcium chelator BAPTA (11 mM). The sensitivity of the ACPD-mediated hyperpolarization to 1 mM TEA in our preparation suggested that activation of a similar channel may occur in BLA neurons. At NM concentrations IS,3R-ACPD potently stimulates intracellular calcium mobilization in cerebellar neurons (Irving, 1990). We therefore examined the effect of BAPTA, and depletion of intracellular calcium with thapsigargin that releases and subsequently depletes intracellular Ca by inhibiting the Ca2+ pump (Thastrup et al., 1990). Treatment of the preparation with BAPTA-AM (30 PM), an ester of BAPTA, had little effect on the lS,3R-ACPD-induced hyperpolarization even after 90 min of superfusion (Fig.   6B, n = 4; control, 2.75 f 0.6 mV; BAPTA-AM, 3.0 f 0.4 mV; p > 0.05, unpaired t test). A possible explanation for this lack of effect of BAPTA-AM is that BLA neurons do not possess sufficient esterases to cleave off the BAPTA within the cell. In contrast, however, intracellular dialysis by including BAPTA (200 mM) in the recording electrode caused a time-dependent reduction of the lS,3R-ACPD+voked hyperpolarization (Fig.  6C). In those neurons in which a stable recording lasted for greater than 90 min (n = 4) the lS,3R-ACPD response was blocked, whereas in control cells at 90 min a full hyperpolarization was elicited by lS,3R-ACPD (control, 3.0 + 0.4 mV; BAPTA, 0.8 mV * 0.3 mV; p > 0.00 1, unpaired t test; ANOVA, p > 0.007; post hoc Bonferroni: control vs BAPTA, p < 0.05; vs BAPTA-AM, NS; BAPTA vs BAPTA-AM, p < 0.05). Furthermore, the lS,3R-ACPD-evoked hyperpolarization was also reduced by pretreatment with 2.5 /IM thapsigargin (control, -5.2 + 0.6 mV; thapsigargin, -2.2 f 0.6 mV; n = 3, p < 0.006; Fig. 7), a concentration previously reported to deplete intracellular calcium stores and block the induction of LTP in hippocampal CA1 neurons (Harvey and Collingridge, 1992). In this cell thapsigargin had no effect on membrane potential or conductance; however, in two of five cells thapsigargin (2.5 PM) itselfhyperpolarized the neurons (2.5 mV, n = 2). We also tested the effects of low Ca/high Mg and Cd (200 PM), a calcium channel blocker, on the lS,3R-ACPD-induced response. The lS,3R-ACPD hyperpolarization was reduced in low Ca/high Mg (control, 3.0 mV, treatment, 0.5 mV; n = 2) or Cd (control, 3.3 f 0.3 mV, Cd*+, 1.17 + 0.6 mV; n = 3, p > 0.05) at a time when synaptic transmission was completely blocked. The response, although reduced, still persists in these solutions supporting the conclusion that lS,3R-ACPD hyperpolarization is postsynaptic and requires, in part, release of intracellular calcium stores. Furthermore, these data also suggest that voltage-gated calcium channels may contribute to the lS,3R-ACPD-induced hyperpolarizations.

Discussion
The data presented here demonstrate for the first time that activation of postsynaptic metabotropic glutamate receptors produces a hyperpolarizing response at the resting potential in neurons of the BLA. The reversal potential of -84 mV for the ACPD-mediated hyperpolarization, its sensitivity to extracellular K+ concentrations, and its blockade by GTP-7-S and low concentrations of TEA and reduction by intracellular BAPTA, thapsigargin, and low Ca/high Mg further suggest that this hyperpolarizing response is mediated by G-proteins and involves intracellular release of calcium and subsequent activation of large-conductance calcium-dependent potassium channels.
Glutamate plays an important role in neurotransmission in both vertebrates and invertebrates. For the most part glutamate, in the vertebrate nervous system, has been assumed to have a purely excitatory role in transmission. Unlike vertebrate glu-tamate receptors, however, previous reports from the invertebrate system have shown that activation of postsynaptic glutamate receptors can also be inhibitory. In the sea hare Aplysiu californica and freshwater mollusk Planorbarius corneus, glutamate and quisqualate activate second messenger-mediated increases in a potassium conductance (Yarowsky and Carpenter, 1976;Bolshakov et al., 1991). More recently, the glutamatemediated increase in potassium conductance observed in Aplysia was mimicked by ACPD (Katz and Levitan, 1993). In the vertebrate retina glutamate-activated outward currents have been associated with both a conductance decrease (Nawy and Jahr, 1990) in slices and increase (Hirano and MacLeish, 1991) in isolated cells, having reversal potentials close to zero and to the equilibrium potential for potassium, respectively. It would seem, therefore, that increases in potassium conductance in response to glutamate agonists in invertebrate nervous systems, isolated retina cells, and mammalian brain neurons of the amygdala suggest that activation of postsynaptic metabotropic glutamate receptors may, in part, express the inhibitory responses to glutamate.
To date few researchers have reported membrane hyperpolarizations or outward currents in response to tram-ACPD in vertebrate preparations, and of these, none characteristically resemble the response in the BLA. East and Garthwaite (1992) recorded in cerebellar granule cells extracellular population responses to lS,3R-ACPD in which the hyperpolarizations usually followed the depolarizations. In cultured cerebellar granule cells, however, trans-ACPD activated a large-conductance Ca-et al. l ACPD Hyperpolarizations i n Amygdala Neurons dependent K channel but no change in the amplitude of single channel currents was recorded under cell-attached conditions; moreover, the threshold for the tvans-ACPD effect on macroscopic Kf current was -20 mV (Fagni et al.,199 1). The authors conclude that these data were not consistent with a membrane hyperpolarization at rest potential, unlike the hyperpolarization in the amygdala that is observed at resting potential. In Purkinje cells of the cerebellum, an outward current has been recorded following an inward current; this outward current, however, had a reversal potential positive to 0 mV and was accompanied by a decrease in conductance (Staub et al., 1993) whereas in the BLA the ACPD hyperpolarization reversed at -84 mV and was accompanied by an increase in conductance. The ACPD outward current in Purkinje cells is not comparable to the ACPD hyperpolarizing response in the BLA. The actions of ACPD in cerebellar neurons are clearly different from those recorded in the BLA and so the lS,3R-ACPD-induced membrane hyperpolarization in the BLA appears to be a novel response.
At low micromolar concentrations TEA specifically blocks large-conductance calcium-dependent K+ channels (BK channels) but not small-conductance calcium-dependent K+ channels (SK channels; for review, see LaTorre et al., 1989). Similar to the ACPD-mediated inhibitory response observed in this study, the inhibitory response to glutamate in Planorbarius (Bolshakov et al., 1991) was also blocked by low micromolar concentrations of TEA, suggesting that these two responses may share a common mechanism of action, that is, activation of a large-conductance Ca-dependent K channel activity, possibly BK.
In the present study, the reduction but not complete block of the ACPD response either by (1) intracellular injection of the calcium chelator BAPTA, (2) superfusing with low Ca/high Mg or Cd solution, or (3) the depletor of intracellular calcium stores, thapsigargin (Thastrup et al., 1990) further suggests that release of calcium from intracellular stores may contribute to activation of BK channels. The difference in time required for the BAPTAmediated blockade of the ACPD response compared to that of GTP-7-S may result from the differences in cytosolic concentration of each drug needed to affect their target regions of the cascade system. Hence, much higher intracellular levels of BAP-TA may be needed to chelate cytosolic calcium than are required for GTP-7-S to affect receptor-effecter coupling. As both drugs diffuse from the microelectrode into the cytosol, it might be expected that a longer impalement may be required to observe a full BAPTA blockade of the ACPD response. However, this time factor may also be due to differing rates of diffusion of the two substances. Recently, Takagi et al. (1992) have suggested that the metabotropic glutamate receptors mediating glutamate responses are located on the dendrites of Purkinje cells. If the mGluRs mediating the ACPD-mediated hyperpolarization are also located on the dendrites of BLA neurons, this additional spatial barrier may further contribute to delay of the full expression of BAPTA blockade.
In the vertebrate CNS, activation of either mGluRlcu, mGluRlp, or mGluR5 receptors mediate the stimulation of inositol triphosphate (IP,) metabolism (Sugiyama et al., 1987; for review, see Nakanishi, 1992;Schoepp, 1993) that can subsequently regulate intracellular calcium mobilization (Murphy and Miller, 1988). Although we did not examine the source of calcium or related mechanisms by which mGluR activation results in calcium-dependent K conductance, mRNA for mGluR 1 receptors (Shigemato et al., 1992) is expressed in mod-erate levels in the BLA, and activation of metabotropic glutamate receptors stimulates IP, formation (Aramori and Nakanishi, 1992). In addition, cytosolic IP, receptors have been identified immunohistochemically in this nucleus (Nakanishi et al., 1991). It is possible that in the BLA the ACPD-induced membrane hyperpolarization may be mediated by release of calcium from IP, stores that activates a K conductance. Alternatively, another second messenger system may be involved since mRNA for the mGluR2 receptor is found abundantly in the basolateral amygdala (Ohishi et al., 1993).
In the present study, pharmacological differentiation of the specific receptors mediating the ACPD hyperpolarization was hindered since the putative antagonists of metabotropic glutamate receptors L-AP3 and 4C3HPG had agonist actions. In hippocampal neurons, L-AP3 (1 mM) has been shown to increase IP, formation itselc it has furthermore been suggested that the L-AP3 block of lS,3R-ACPD-induced IP, formation is due to desensitization of the receptor (Lonart et al., 1992). More recently, L-AP3 was shown to mimic various 1 S,3R-ACPD effects including inhibiting CAMP formation (Schoepp and Johnson, 1993). Agonist actions of 4C3HPG have also been reported. In cerebral cortical slices of rat brain, 4C3HPG acts as an agonist in stimulating phosphoinositide hydrolysis and potentiating CAMP formation (Winder et al., 1993). 4C3HPG has also been shown to mimic metabotropic glutamate receptor-mediated responses in hippocampal slices (Gerber and Gahwiler, 1992).
In their patch-clamp study of isolated cerebellar granule cells, Fagni et al. (199 1) noted that the ACPD-mediated activation of BK channels was mimicked by the cholinergic agonist carbachol. In Aplysia neurons ACh, dopamine, and histamine evoke potassium conductances that, although acting at different receptors, may be mediated by activation of the same ion channel (Ascher and Chesnoy-Marchais, 1982). It is possible that in the vertebrate CNS multiple metabotropic receptors may also activate a common ion channel. Indeed, the similarity between responses mediated by the muscarinic subclass ofACh receptors and those mediated by the metabotropic subclass of glutamate receptors is striking (see Bonner, 1989;Nakanishi, 1992). In the BLA, Washburn and Moises (1992) have reported a biphasic potential, hyperpolarization-depolarization, evoked in response to exogenous application of the muscarinic receptor agonist oxotremorine-M that was almost identical to the biphasic ACPD response seen in this study. These authors, however, concluded that the hyperpolarization was due to an indirect release of GABA from inhibitory interneurons. In our study the ACPDevoked hyperpolarization (1) was resistant to atropine, bicuculline, and CGP55845A, (2) was unaffected by intracellular chloride loading, was (3) resistant to TTX, (4) changed reversal potential with alterations in extracellular K+ concentrations, and (5) was blocked by 1 mM extracellular TEA. We therefore conclude that the ACPD-mediated hyperpolarization results from a direct increase in a postsynaptic K+ conductance, possibly through large-conductance calcium-dependent K channels, but not from either an indirect release of ACh or GABA.
In summary, neurons in the BLA appear to possess a unique hyperpolarizing response to metabotropic glutamate receptor activation mediated through a calcium-dependent K conductance, the functional role of which may be inhibitory,