The Journal of Neuroscience, April 1993, 13(4): 1636-l 641

Activation of Metabotropic Glutamate Receptors Produces Reciprocal Regulation of lonotropic Glutamate and GABA Responses in the Nucleus of the Tractus Solitarius of the Rat

Steven R. Glaum and Richard J. Miller Department of Pharmacological and Physiological Sciences, The University of Chicago, Chicago, Illinois 60637

Whole-cell voltage-clamp recordings were made in thin effect was found to be independent of changes in [Ca”], and transverse slices from of the dorsomeclial subdi- could be mimicked by endogenous glutamate released from af- vision of the nucleus of the tractus solitarius (NTS) of the ferent projections to the NTS by high-frequency stimulation of rat. Cells were exposed to either the ionotropic glutamate the tractus solitarius (TS). Furthermore, lS,3R-ACPD reduced receptor (R,S)-a-amino+hydroxy-5methylisoxa- the EPSC and GABAergic IPSC evoked by low- zole-4-propionate (AMPA) or the GABA, receptor agonist frequency stimulation in the region of the TS. These effects via pressure ejection directed at the cell soma. support the view that the action of glutamate at metabotropic The metabotropic agonist 1 SJR-1 -ami- receptors plays a key role in synaptic transmission from baro- nocyclopentane-1,3-dicarboxylate (1 S,3/+ACPD; 2-l 00 PM) receptor afferents (Leone and Gordon, 1989; Meeley et al., 1989; reversibly depressed muscimol-evoked currents. Converse- Drewe et al., 1990; Mifflin and Felder, 1990). ly, 1 S,BR-ACPDreversiblypotentiatedAMPA-evokedcurrents. In our previous report, we suggested that the ability of lS,3R- High-frequency stimulation of the tractus solitarius in the ACPD to inhibit synaptic transmission in the NTS might be presence of 6,7-dinitroquinoxaline-2,3-dione and o-2-amino- due to a reduction in the presynaptic release of glutamate and 5phosphonopentanoic acid also produced a reversible de- GABA. However, in the present investigation we have identified pression of muscimol-evoked currents that was occluded in additional postsynaptic effects of lS,3R-ACPD-an inhibitory the presence of 100 PM 1 S,3/&ACPD. 6-Br-cGMP or brain- action on postsynaptic GABA, responses and a simultaneous derived natriuretic peptide mimicked the effects of 1 S,3R- potentiation of postsynaptic (R,S)-a-amino-3-hydroxy-5-meth- ACPD on AMPA and muscimol currents. However, agents ylisoxazole-4-propionate (AMPA)-activated currents. These re- that mimicked the actions of CAMP or diacylglycerol did not. sults help to explain the potent reduction of inhibitory trans- These findings indicate that metabotropic glutamate recep- mission by lS,3R-ACPD and also provide another example of tors may mediate multiple components of excitatory trans- the multifaceted signal transduction mechanisms linked to the mission in the NTS including modulation of glutamate and activation of metabotropic glutamate receptors (Miller, 199 1). GABA-activated ion channels. [Key words: cardiovascular, medulla, solitary tract, baro- Materials and Methods receptor, lS,3R-ACPD, patch clamp] Preparation of slices and recordings were made as previously described (Glaum and Miller, 1992). Briefly, Sprague-Dawley rats of either sex, aged between 18 and 36 d, were given overdoses of ether and the whole brains, including the cervical spinal cord, were rapidly removed and Recent evidence has indicated that stimulation of metabotropic placed in ice-cold artificial cerebrospinal fluid (aCSF), which contained glutamate receptors within the nucleus of the tractus solitarius (in rnM) NaCI. 126: NaHCO,. 26.2: NaH,PO,. 1: KCl. 3: M&O,. 1.5: (NTS) mimics the effects of baroreceptor activation in viva CaCl,, i.5; aid glucose, 10. TransGerse 550 ;rn brain&m &ces’con: (Pawlowski-Dahm and Gordon, 1992). We have demonstrated taining the NTS were prepared and submerged in a recording chamber where they were continuously perfused (4-5 ml.min-I) with oxygenated that activation of metabotropic glutamate receptors within the aCSF at room temperature. dorsomedial subdivision of the NTS, a principle region for the Whole-cell recordings were obtained from 63 visually identified neu- termination of baroreceptor afferents, produces a variety of cel- rons in the dorsomedial subdivision of the NTS adjacent to the area lular effects (Glaum and Miller, 1992; Glaum et al., 1993). lS,3R- postrema. Patch electrodes were filled with a solution containing (in mM) K+-gluconate, 145; MgCl,, 2; HEPES, 5; EGTA, 1.1; CaCl,, 0.1; 1-aminocyclopentane- 1,3-dicarboxylate (1 S,3R-ACPD), a se- and K,ATP, 5. Where indicated, GTP-7-S was dissolved in the intra- lective agonist at metabotropic glutamate receptors, depolarized cellular solution to a final concentration of 400 PM and used within 1 some NTS neurons due to the closure of a K+ channel. This hr for recording. Neurons were voltage clamped at V,,,, = - 50 mV. A single experiment was performed in each slice. AMPA or muscimol(l0 NM) was applied by a brief (20-45 msec) pressure pulse (2-10 psi) via a blunt patch pipette (approximate tip diameter, 5 pm) directed toward Received July 10, 1992; revised Sept. 14, 1992; accepted Oct. 16, 1992. the soma under visual guidance. Recordings were obtained in the pres- S.R.G. was supported by National Institutes of Health T32HL7237-14. Ad- ence ofD-2-amino-5-phosphonopentanoic acid (AP5; 50 PM) and either ditional support came from the Brain Research Foundation, Digestive Disease Care Center Award lP30DK42086-01, and U.S. Public Health Service Grants (10 PM) or 6,7-dinitroquinoxaline-2,3-dione (DNQX; 10 DA-02575, DA-02121, and MH-40165 to R.J.M. PM), respectively. Tetrodotoxin (TTX; 0.5 PM) was included in all ex- Correspondence should be addressed to Steven R. Glaum, Ph.D., Department periments examining ZAMPAand where indicated in experiments ex- of Pharmacological and Physiological Sciences, The University of Chicago, 947 amining the muscimol-activated current (I,,,,). The absence of pres- East 58th Street, Chicago, IL 60637. sure ejection artifacts was confirmed at the conclusion of the experiments Copyright 0 1993 Society for Neuroscience 0270-6474/93/131636-06$05.00/0 by the addition of the remaining antagonist (DNQX or bicuculline). The Journal of Neuroscience, April 1993, 73(4) 1637

A w/fl JlOpA ACPD 30s . . . . . AMPA

OSmm B r--

. . STIM . . . . . MUSC \ 1 S,SR-ACPb 20pA VContol 5ms 14OpA 10s Figure 1. Excitatory and inhibitory transmission were inhibited by lS,3R-ACPD in the dorsomedial NTS. A, Transverse schematic of the NTS at the level of the area postrema (AZ’) shows the dorsomedial subnucleus (dmNTS) where recordings were made. Also shown is the 160 dorsal motor nucleus of the vagus (ZLW’). Electrical stimulation in the region of the TS evoked a monosynaptic EPSC in the presence of bi- d cuculline (10 PM) and AP5 (50 PM). In the presence of DNQX (10 PM) E 120 and AP5 (50 UM). a monosvnantic IPSC is recorded in the maioritv of neurons in this region (seeGlaum and Miller, 1992). B, The metabo- 5 ,” 80 tropic agonist lS,3R-ACPD (50 PM) inhibited the evoked EPSC (Vhold = -80 mV). C, The monosynaptic IPSC was similarly inhibited by lS,3R-ACPD (50 PM) (V,,,, = -50 mV). 0 40 80 120

[l S.3R-ACPD] (FM) lS,3R-ACPD (2-100 PM) was delivered in the perfusate for 0.5-4 min. In 14 experiments, cells were also exposed to 8-Br-cGMP (100 /AM), delivered in the perfusate for 2-4 min. In nine occlusion experiments, Figure 2. lS,3R-ACPD reversibly potentiated AMPA-evoked cur- rents and reversibly inhibited muscimol currents. A, AMPA currents control responses for lS,3R-ACPD (100 FM, l-4 min) and 8-Br-cGMP (2-4 min) were determined prior to the combined application of drugs evoked by brief pressure ejection of AMPA onto neurons in the dor- applied for the control intervals. All other drugs were delivered in the somedial NTS were potent&ted in the presence of lS,3R-ACPD (ACPD; perfusate for the indicated intervals. In all cases, the flow rate of the 10 PM). AMPA currents were evoked from I’,,,,, = -50 mV in the perfusate was carefully regulated to avoid artifactual changes in the presence of TTX (0.5 PM), AP5 (50 FM), and bicuculline (10 PM) in this amplitudes of the AMPA and muscimol currents. and all subsequent figures. B, The outward current evoked by pressure- High-frequency stimulation was carried out by placing a bipolar tung- applied muscimol (MZLSC) was reversibly inhibited in the presence of sten stimulating electrode in the region of the ipsilateral tractus solitarius lS,3R-ACPD (ACPD; 100 PM) or by 50 Hz (100 msec, 4.3 V) stimu- (TS), as previously described (Glaum and Miller, 1992). Recordings of lation in the region of the TS (STZMJ An identical stimulation in the muscimol currents were made as described above, but in the absence vresence of lS,3R-ACPD vroduced no further inhibition of the mus- of TTX. Single stimulus trains (50 Hz, 100-300 msec, 3.0-5.2 V) were cimol current. Recording was made from Vhold= - 50 mV in the presence delivered 5-10 set prior to the application of muscimol. The effects of of DNQX (10 PM) and AP5 (50 PM) in this and all subsequent figures. stimulation and 100 PM lS,3R-ACPD (l-3 min) on ZMusc were ex- C, Dose-response relationship for the effects of lS,3R-ACPD on AMPA amined independently in seven cells. The effects of stimulation in the (ZAMpA)and muscimol (I vuKIMoL ) currents in the NTS. Values shown presence of lS,3R-ACPD were examined in six of these cells. are mean f SEM for three or more cells, as indicated. Data were filtered at 3 kHz and stored on a chart recorder (Gould), digitized at 125 kHz by a computer-driven analog-to-digital converter (INDEC Systems), and stored on hard disk for subsequent analysis. In order to examine the possible contribution of postsynaptic The peak amplitudes of ZMuscand ZAMPAwere evaluated immediately actions of lS,3R-ACPD on this inhibition of afferent synaptic prior to the addition of drugs (I,,,,,,) and compared to the current at transmission in the NTS, neurons were exposed to brief pressure the end of the drug application (I,,,,,,,,). The effect of high-frequency pulses of selective at ionotropic glutamate and GABA, stimulation on ZMvscwas evaluated immediately following the stimulus. Data were normalized to the control value and expressed as (I,,,,,,,,/ receptors. Figure 2A illustrates the response to pressure-applied ZEOn,rDl )* 100. Values were then pooled and expressed as the mean ? SEM. AMPA in a single NTS in the absence and presence of 1S,3R-ACPD (10 PM). 1S,3R-ACPD produced a rapid and re- Results versible potentiation of the AMPA current (ZAMPA). Maximum Previous investigations (Champagnat et al., 1985; Donoghue et potentiation of ZAMPA was observed within 1 min of lS,3R- al., 1985; Miles, 1987; Brooks et al., 1992; Glaum and Miller, ACPD application, and this effect was not diminished in the 1992) have shown that a single low-intensity stimulus in the continuous presence of lS,3R-ACPD. Z,,,, was observed to region of the TS produces a monosynaptic DNQX-sensitive recover to control values rapidly (1-2 min) upon washout of EPSC and, in many NTS neurons, a mixed mono- and poly- lS,3R-ACPD. Curiously, AMPA currents evoked following synaptically evoked bicuculline-sensitive IPSC. Figure 1 shows washout of lS,3R-ACPD were sometimestransiently smaller the region of the NTS examined in both our previous (Glaum than the control value. The effects of lS,3R-ACPD on ZAMPA and Miller, 1992; Glaum et al., 1993) and present studies. did not diminish with repeated application over the course of Figure 1 also illustrates the inhibitory effects of 50 FM lS,3R- an experiment (1-5 hr). ACPD on the monosynaptic EPSC and IPSC recorded from In contrast to the findings with AMPA, the current activated these neurons following TS stimulation (see also Glaum and by the GABA, agonistmuscimol (ZMUsc)was potently depressed Miller, 1992). in the presenceof lS,3R-ACPD (Fig. 2B). As with the regulation 1636 Glaum and Miller - Metabotropic Receptors in the NTS A 40pA * 30s ACPD . . . . AMPA

i ...... n MUX

120pA A- 6opA B-12OpA -I 305 ACPD . STIM Figure 4. The cell-permeant cGMP analog S-Br-cGMP (100 PM) pro- . . . n MUSC duced qualitatively similar effects to those of lS,3R-ACPD (100 PM) on AMPA and muscimol currents. A, AMPA-evoked currents from an Figure 3. The potentiating effects of lS,3R-ACPD on AMPA currents NTS neuron were reversibly potentiated by lS,3R-ACPD (ACPD) and and the inhibition of muscimol currents were unaffected by intracellular %Br-cGMP. The application of 8-Br-cGMP in the presence of lS,3R- dialysis with GTP--r-S. A, AMPA-evoked currents in an NTS neuron ACPD produced no further potentiation of the AMPA current. B, Qual- dialyzed with 400 PM GTP-7-S were reversibly potentiated by 20 FM itatively similar results on muscimol (MUSC)-evoked currents were lS,3R-ACPD (ACPD). Currents were evoked as described in Figure 2. obtained in another cell. Currents were evoked in A, and B as described B, Muscimol (MUSC) currents were inhibited by either 100 PM lS,3R- in Figure 2 and Materials and Methods; however, both AMPA and ACPD (ACPD) or 50 Hz (150 msec, 3.2 V) stimulation ofthe TS (STIM) muscimol currents were evoked in the presence of 0.5 FM TTX. The in a neuron dialyzed with 400 PM GTP-7-S. Currents were evoked as durations of drug applications were identical in the control and occlusion described in Figure 2. parts of the experiments shown in A and B as described in Materials and Methods, but are truncated in the figure. ofLMPA, the depression of I,,,, was rapid in onset, nondesen- sitizing, and readily reversible. In the samecell, 50 Hz stimu- current (5-20 PA) was observed in the present investigation in lation of the TS (100 msec, 4.3 V, in the presenceof DNQX 12 of 37 cells exposedto 1S,3R-ACPD at a concentration of 25 and AP5) also reduced the amplitude of the ZMUsc.In the con- PM or greater. Furthermore, in addition to the ability of high- tinuous presenceof lS,3R-ACPD, no further reduction in Z,,,, frequency stimulation of the TS to reduce ZhlUsc,a stimulus- was observed following an identical stimulus train. Such occlu- induced inward current was observed in five of seven cells. sion of the synaptically mediated depressionof ZMuscby lS,3R- lS,3R-ACPD occluded not only the stimulus-mediatedreduc- ACPD is consistentwith a common mechanismbeing involved tion in ZMUscbut, in keeping with our earlier findings, it also in the two phenomena.This would not be surprisingas we have occluded the stimulus-inducedinward current. However, given previously demonstrated(Glaum and Miller, 1992) that high- the presentobservation that low concentrationsof lS,3R-ACPD frequency stimulation of the TS can produce synaptic activation (< 25 PM; Fig. 2C) modulated Z,,,, and I,,,, without inducing of metabotropic glutamate receptorsthrough the releaseof en- an inward current, it seemslikely that the two types of effects dogenousglutamate. High-frequency stimulation of the TS in- are unrelated. Thus, the NTS may contain several types of me- hibited I,,,, in sevenof sevenneurons. In five of six cellstested, tabotropic glutamate receptors(Tanabe et al., 1992). Altema- 100 PM lS,3R-ACPD fully occluded the ability of high-fre- tively, a single type of receptor may be linked to several signal quency stimulation to reduce ZMUsc.In the other cell, high-fre- transduction events (Aramori and Nakanishi, 1992). quency TS stimulation in the presenceof lS,3R-ACPD resulted In order to addressthe question of the mechanismby which in a further 6% decreasein ZMUsc.lS,3R-ACPD produced an lS,3R-ACPD modulates ZAMPAand ZMusc,the effectsof lS,3R- identical reversible depressionof ZMUscin 13 additional cells ACPD were examined in seven cells dialyzed with GTP-7-S. If recorded in the presenceof TTX. As with ZAMPA,the effects of the effects of IS,3R-ACPD were mediated via a G-protein, lS,3R-ACPD did not desensitizewith continuous or repeated dialysis with GTP-7-S might be expectedto produce irreversible application over the duration of the recordings. The dose-re- effects of lS,3R-ACPD and to occlude its subsequentactions. sponserelationship for 1S,3R-ACPD effectson ZAMPAand IMuse In this and our earlier investigations (Brooks et al., 1992), di- are summarized in Figure 2C. Basedupon these observations, alysis with GTP-y-S produced a slow outward current in NTS we routinely employed a 100PM concentration of 1S,3R-ACPD neuronsand blocked the hyperpolarizing effects of the GABA, as a supramaximal dosein all occlusion experiments. It should agonistbaclofen. However, as illustrated in Figure 3, the effects be noted that the effectsof lS,3R-ACPD (20 PM) on excitatory of lS,3R-ACPD on Z,usc (n = 4) and ZAMPA(n = 3) were indis- and inhibitory transmission(Glaum and Miller, 1992) as well tinguishable from those observed in cells without added GTP- as on LpA and Lsc were unaffected in the presence of y-S, suggestingthat a G-protein intermediate is not an integral L-aminophosphonopentanoicacid (200 PM) (n = 3) which blocks part of the signal transduction pathway. metabotropic glutamate receptor-mediated [Ca2+li mobiliza- The effects of lS,3R-ACPD were neither mimicked nor tion in some systems(Schoepp and Johnson, 1989). The effects blocked by the addition to the perfusate for 8 min of phorbol of high-frequency stimulation on ZAMPAwere not examined as myristate acetate (20 PM; n = 3), arachidonic acid (100 FM; n the resultswould not be readily interpretable (seeDiscussion). = 3), or sodium nitroprusside (200 PM; n = 3). We previously reported that lS,3R-ACPD produced an in- Recently, Lewis et al. (1991) have provided evidence that ward current in some neurons in the dorsomedial NTS as the activation of guanylate cyclasemay play a role in the processing result of a decreasein a K+ current (Glaum and Miller, 1992). of afferent baroreceptor information at the level of the NTS. High-frequency stimulation of the TS in the presenceof DNQX We therefore examined the effects of a cell-permeant form of and AP5 produced a similar inward current that was occluded cGMP on ZAMPAand Z,,,,. As illustrated in Figure 4, addition by lS,3R-ACPD. In keepingwith theseobservations, an inward of 100 PM 8-Br-cGMP reversibly potentiated ZAMPA(123 f 6.2% The Journal of Neuroscience, April 1993, f3(4) 1639

of control, n = 9) and depressed ZMusc(75.0 f 4.1% of control, n = 5) in a similar fashion to 1S,3R-ACPD (128.7 f 7.2% and 77.9 f 3.4% of control, respectively). We then performed oc- clusion experiments to compare the effects of 1S,3R-ACPD and 8-Br-cGMP when separately and coapplied. lS,3R-ACPD (100 n . . MUSC - ACPD PM) or 8-Br-cGMP (100 PM) potentiated ZAMPAto a similar extent when applied separately (134.6 * 8.6% and 130.9 +- 9.4% of control, respectively) or when coapplied (131.6 f 10.6% of G,x”h , control, n = 4). Similar results were obtained in experiments examining inhibition of ZMUsc(lS,3R-ACPDalone, 83.7 * 3.3%; . . n MUSC A-50pA 8-Br-cGMP, 80.3 f 2.3%; coapplication, 78.6 f 5.9% of con- B-l OOpA trol; II = 5). The effect of 8-Br-cGMP was also compared to the B 30s cell-permeant CAMP analog dibutryl-CAMP (100 PM). In three - ACPD of four cells, dibutryl-CAMP produced no discernable effect on I AMPAor IMu,,. However, in one cell, I,,, was reduced to 83.3% of control, an effect opposite to that produced by lS,3R-ACPD . n AMPA or 8-Br-cGMP. Endogenous G-protein-independent activators of such as brain-derived natriuretic peptide (BNP) (Chang et al., 1989; Ermirio et al., 1989; Lewis et al., 1991) are found . AMPA in the NTS (Standaert et al., 1986) and appear to be colocalized Figure 5. Addition to the bath of BNP (3 PM) producedqualitatively with glutamate in peripheral afferent projections to the NTS (K. similareffects to thoseof lS,3R-ACPD (25 PM) on AMPA and mus- Hurley, personal communication). In order to examine further cimol (MUSC)currents. A, The muscimolcurrent from an NTS neuron the potential role of cGMP, we next examined the effects of was reversiblyreduced by bath-appliedlS,3R-ACPD (ACPD). BNP similarly reducedthe muscimolcurrent. However, recovery to control exogenous BNP (3 PM) on ZAMPAand I,,,,. As illustrated in levelsrequired approximately 17 min of washing.B, 1&3R-ACPDand Figure 5, BNP produced qualitatively similar effects on ZAMPA BNP similarlyenhanced AMPA currents.As in A, recoveryfrom BNP (122.7 f 1.0% of control, IZ = 3) and IMuse (80.6 * 11.4% of wasfar slowerthan that of lS,SR-ACPD. Currentsin A and B were control, IZ = 4) compared with those produced by lS,3R-ACPD evokedin the presenceof TTX (0.5 PM) and glutamateor GABA re- (25 PM) (119.0 f 3.0% and 8 1.2 + 7.1% of control, respectively) ceptorantagonists, as described in Materialsand Methods. in the same group of cells. Although rapid in onset, the effects of BNP differed from those of lS,3R-ACPD in two notable the dorsal horn of the spinal cord, this effect of lS,3R-ACPD ways. Recovery of ZAMPAor Z,,,, to control levels following is clearly independent of a changein [Ca*+], (Bleakman et al., washout of BNP was much more prolonged than that following 1992). We have now demonstratedthat 1S,3R-ACPD can also lS,3R-ACPD, requiring up to 20 min. Further addition of the modulate postsynaptic receptors for AMPA and GABA in the peptide within 30 min of the initial application failed to affect NTS and in the latter case that these effectsare mimicked by AMPA or muscimol currents (n = 6). Notably, lS,3R-ACPD endogenouslyreleased transmitter. applied during this period of BNP desensitization (either alone Our previous experiments demonstratedthat lS,3R-ACPD or in the presence of BNP) was fully effective in five of six cells produced potent inhibition of pharmacologicallyisolated EPSCs at potentiating I,,,, or reducing IMusc, indicating an absence and IPSCs evoked by electrical stimulation in the region of the of heterologousdesensitization. TS in the dorsomedial subdivision of the NTS (Glaum and Miller, 1992). We also observed direct postsynaptic excitatory Discussion effects of lS,3R-ACPD on some NTS neurons that could be Metabotropic glutamate receptors represent a large family of mimicked by high-frequency stimulation of the TS in the pres- related proteins that are linked to several signal transduction ence of ionotropic glutamate receptor antagonists.This inward systems(Aramori and Nakananishi, 1992; Tanabe et al., 1992). current evoked by TS stimulation was indistinguishablefrom Initially, activation of metabotropic glutamate receptors was that produced by lS,3R-ACPD. These excitatory effects of shownto produce mobilization of Ca*+from intracellular stores lS,3R-ACPD appearedto be unrelatedto mobilization of [Ca*+], in neuronsand glia through an inositol trisphosphate-mediated as assessedby microfluorometric techniques.In the present in- mechanism(Murphy and Miller, 1988; Ambrosini and Mel- vestigation, lS,3R-ACPD and TS stimulation produced both dolesi, 1989; Palmer et al., 1989; Glaum et al., 1990; Manzoniet direct current responsesand, even more notably, inhibition of et al., 1990). More recently, inhibition of adenylate cyclase I M”SC even though neurons were dialyzed with EGTA. lS,3R- (Cartmell et al., 1992; Schoeppet al., 1992; Tanabe et al., 1992), ACPD also modulated ZAMPAin neurons dialyzed with EGTA. K+ (Charpak et al., 1990; Desai and Conn, 1991; McCormick In both instances, concentrations of lS,3R-ACPD that failed and Von Krosigk, 1992), and Ca*+ conductances(Sahara and to produce detectable [Caz+], changesin fura-2-loaded cells re- Westbrook, 1991; Swartz and Bean, 1992) have been demon- corded under voltage clamp (Glaum and Miller, 1992)produced strated. Importantly, the signal transduction systemslinked to profound changesin AMPA and muscimol currents. Thus, al- some types of metabotropic glutamate receptorshave not yet though undetectable changesin [Ca2+],in distal processescould been identified (Lovinger, 1991; Tanabe et al., 1992). Meta- contribute to the observedeffects of 1S,3R-ACPD, observations botropic glutamate receptorshave recently been shown to po- to date argue againstthis hypothesis. In contrast with the effects tentiate ionotropic glutamate responsesin several parts of the on Lsc, the effects of high-frequency TS stimulation on ZAMPA brain (Aniksztejn et al., 1991; Harvey et al., 1991; Bleakman were not readily determinable using the present paradigm, as et al., 1992; Glaum et al., 1992; Kinney and Slater, 1992). In the sensitivity of AMPA receptorsmay be variably decreased 1640 Glaum and Miller * Metabotropic Receptors in the NTS by synaptically released glutamate through a process of ho- beyond the detection level of the recording method could result mologous desensitization. in a small change in sensitivity to AMPA or muscimol, the How, then, are the effects of lS,3R-ACPD on I,,,, and I,,,, magnitudeand opposingnature of lS,3R-ACPD effectson these produced? We noted that a cell-permeant cGMP analog pro- currents argue againstthis hypothesis. The involvement of the duced effects that were qualitatively similar to those of lS,3R- adenylate cyclasesystem can also be ruled out by the failure of ACPD on ZAMPAand I,,,,. While the lack of effect of sodium a CAMP analog to mimic or block the effectsof lS,3R-ACPD. nitroprusside appears to suggest that a 1S,3R-ACPD-mediated The negative results with arachidonic acid and the apparent activation ofguanylate cyclase by a (NO)-dependent independenceof the effectsfrom changesin [Ca2+lisuggest that mechanism does not play a role in mediating lS,3R-ACPD’s products of phospholipid metabolism are also unlikely to me- effects, this hypothesis remains attractive for a number of rea- diate the observed actions of lS,3R-ACPD. sons. For example, NO synthase is found widely in the NTS The ability to evoke both postsynaptic 1S,3R-ACPD-like cur- (Vincent and Kimura, 1992). There is also no direct evidence rents and modulatory effectson GABA receptorsby physiolog- demonstrating that sodium nitroprusside actually generates suf- ically relevant levels of TS stimulation is intriguing. Our data ficient NO within the NTS to activate guanylate cyclase. Indeed, suggestthat synaptically releasedglutamate acting at lS,3R- Lewis et al. (199 1) have demonstrated that while sodium nitro- ACPD receptors may, under increasing levels of baroreceptor prusside failed to activate soluble guanylate cyclase in the NTS, activation, provide a positive feedforward mechanismto in- activation of this enzyme by s-nitrosocysteine could regulate creasethe overall synaptic afferent transmission in the NTS. the processing of baroreceptor afferent transmission. Alterna- The postsynaptic inhibition of Zhluscmay explain the potent tively, lS,3R-ACPD may generate another diffusible activator ability of lS,3R-ACPD to decreaseIPSCs evoked by stimula- of guanylate cyclase such as CO (Marks et al., 199 1). tion in the region of the TS. Conversely, according to our pre- In addition to NO-mediated activation, Ca*+ and G-protein- vious observations, lS,3R-ACPD inhibits EPSCsfar lesseffi- independent activation of guanylate cyclase can be produced by ciently (Glaum and Miller, 1992).These data suggestthat lS,3R- atria1 natriuretic peptide (ANP) and/or the related BNP (Chang ACPD inhibition of EPSCs may be the result of presynaptic et al., 1989; Ermirio et al., 1989; Lewis et al., 1991). The NTS inhibition of glutamate release.What glutamate is released, contains both ANP, derived from projections from the hypo- however, may be acting on sensitized postsynaptic ionotropic thalamus, and BNP, which is found in afferent projections to glutamate receptors. Thus, activation of postsynaptic metabo- the NTS from the nodose ganglia (Standaert et al., 1986; Hurley, tropic receptors may counteract the tendency of AMPA recep- personal communication). Exogenous glutamate or activation tors to undergo desensitization. Furthermore, given that inhib- of baroreceptor afferents has been shown to potentiate the effects itory GABAergic interneurons in the vicinity of the TS appear of ANP within the NTS (McKitrick and Calaresu, 1989). Thus, to be driven largely by excitatory synaptic inputs (Miles, 1987; it is possible that the effects of lS,3R-ACPD are mediated by Brooks et al., 1992) the net outcome of metabotropic receptor release ofa BNP-like peptide within the NTS. Indeed, exogenous activation would be predicted to be largely excitatory. Thus, BNP produces effects that are qualitatively similar to those of intrinsic neuronsofthe NTS would exhibit increasedexcitability lS,3R-ACPD. Both compounds produce a rapid potentiation via (1) a direct depolarizing influence of metabotropic receptor of ZAtmAand reduction of ZMusc. The slower kinetics of recovery occupation, (2) an increasedsensitivity of the AMPA subtype from and desensitization to BNP’s effects may reflect difficulty of ionotropic glutamate receptor, and (3) a decreasedsensitivity in washing this large peptide out of the slice following bath to the inhibitory effects of synaptically releasedGABA. These application. However, our data suggest that it is unlikely that actions may serve to potentiate vagal afferent transmissionun- the effects of lS,3R-ACPD are mediated by release of BNP as der conditions of increasedbaroreceptor activation and thus the effects of lS,3R-ACPD were preserved when those of BNP enhancedescending bradycardic influenceson the heart. were completely desensitized. The apparent similarities between the effects of BNP and lS,3R-ACPD suggest that they may be References working through parallel, cGMP-utilizing pathways, albeit via Ambrosini A, Meldolesi J (1989) Muscarinic and quisqualate receptor- different receptors. While it would be useful to examine the induced phosphoinositide hydrolysis in primary cultures of striatal effects of selective lS,3R-ACPD and BNP receptor or cGMP- and hippocampal neurons. Evidence for differential mechanisms of activation. J Neurochem 53:825-833. dependent kinase antagonists, no such compounds are widely Aniksztein L. Breeestovski P. Ben-Ari Y (199 1) Selective activation available at present. of quisqualate metabotropic receptor pbtentiates NMDA but not Although a cGMP-mediated mechanism appears to be the AMPA responses. 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