The Journal of Neuroscience, June 1992, I.?‘(6): 2043-2050

L-AP4 Inhibits Calcium Currents and Synaptic Transmission via a G-Protein-coupled Glutamate

Paul Q. Trombley ‘.a and Gary L. Westbrook ‘Department of Biology and Institute of Neuroscience, University of Oregon, Eugene, Oregon 97403 and 2Vollum Institute and Department of Neurology, Oregon Health Sciences University, Portland, Oregon 97201

The AP4 (2-amino-4-phosphonobutyrate) receptor is a pre- phosphatidylinositol 4,5-bisphosphate hydrolysis with subse- synaptic that inhibits transmitter release quent generation of the second messengersdiacylglycerol and via an unknown mechanism. We examined the action of 1,4,5-inositol trisphosphate (IP,; Sugiyama et al., 1987; Slade- L-AP4 on voltage-dependent calcium currents and excitatory czek et al., 1988). The recent cloning of the metabotropic re- synaptic transmission on cultured olfactory bulb ceptor revealed a primary sequenceconsistent with a G-protein- using whole-cell voltage-clamp methods. In neurons dia- coupled receptor (Houamed et al., 199 1; Masu et al., 199 1). lyzed with GTP, L-AP4 inhibited high-threshold calcium cur- The coupling mechanism for the L-AP4 receptor is not de- rents evoked in barium solutions. The inhibition was irre- fined. Except in the retina (Miller and Slaughter, 1986; Nawy versible in the presence of GTP-y-S and blocked by removing and Jahr, 1990), the L-AP4 receptor appearsto be located pri- intracellular Mg2+ or by preincubation with pertussis toxin marily on presynaptic nerve terminals, suggestingan action as (PTX), consistent with the involvement of a PTX-sensitive an autoreceptor controlling glutamate release.L-AP4 inhibits G-protein. Dialysis with staurosporine or buffering of intra- glutamate-mediated EPSPs in the hippocampus, olfactory cor- cellular calcium to pCa <8 did not block the action of L-AP4, tex, and spinal cord (Davies and Watkins, 1982; Harris and suggesting that protein phosphorylation or release of intra- Cotman, 1983; Anson and Collins, 1987), as well as EPSPsin cellular calcium stores was not involved in calcium current cultures of hippocampal neurons(Forsythe and Clement& 1990) inhibition under these experimental conditions. PTX also and olfactory bulb (Trombley and Westbrook, 1990). There are, blocked the L-AP4-induced inhibition of monosynaptic EPSPs however, marked differencesin the sensitivity of synaptic path- evoked by intracellular stimulation of cultured mitral cells. ways to L-AP4. The lateral perforant path input to the hippo- These results suggest that the presynaptic AP4 receptor is campal dentate gyrus and the lateral olfactory tract, the major a G-protein-coupled glutamate receptor, and that inhibition output pathway of the olfactory bulb, are among the most sen- of calcium influx by a membrane-delimited action of a G-pro- sitive (for review, seeMayer and Westbrook, 1987). tein may account for L-AP4-induced presynaptic inhibition. G-proteins regulate a large array of cellular processesinclud- ing both regulation of ion channels and modulation of trans- Many excitatory synaptic pathways in the mammalian CNS use mitter releaseby presynaptic receptors (Dunlap et al., 1987; L-glutamate as a transmitter (Cotman et al., 1987; Mayer and Gilman, 1987; Ross, 1989). Several presynaptic receptors in- Westbrook, 1987). Glutamate activates ion channelsin the post- cluding GABA,, A 1 adenosine,and (Yeadrenergic receptors may synaptic membrane linked to NMDA and AMPA (oc-amino-3- inhibit synaptic transmissionby G-protein regulation of calcium hydroxy-5-methyl-4-isoxazoleproprionic acid) receptors. The channels (Holz et al., 1989; Lipscombe et al., 1989; Scholz and contributions of thesereceptor/channels to the excitatory post- Miller, 1991). This can occur via diffusible second messengers synaptic current can be distinguishedon the basisof time course or membrane-delimited actions of G-proteins (Brown and Birn- and single-channel properties (e.g., Forsythe and Westbrook, baumer, 1988). To determine the coupling mechanismof AP4 1988; Lester et al., 1990). In addition to -gated ion chan- receptors, we examined voltage-dependentcalcium currents and nels, two other glutamate receptors have been described, the EPSPsin mitral/tufted cells of the olfactory bulb in short-term metabotropic receptor and the L-2-amino-4-phosphonobutyrate culture. L-AP4 inhibited high-threshold calcium currents via a (L-AP4) receptor (e.g., seeCollingridge and Lester, 1989). The pertussistoxin (PTX)-sensitive G-protein. As L-AP4 does not metabotropic receptor, first identified in mRNA-injected oo- stimulate production of IP, via activation of the metabotropic cytes, couples to a GTP-binding (G)-protein and stimulates receptor (Sladeczek et al., 1988; Sugiyama et al., 1989), our results are consistent with a distinct G-protein-coupled gluta- mate receptor that can act directly (i.e., in a membrane-delim- Received Oct. 11, 199 I ; revised Dec. 19, 199 1; accepted Dec. 30, 199 1. ited manner) on high-threshold calcium channels. L-AP4-me- This work was supported by a predoctoral training grant from the University diated inhibition of monosynapticEPSPs was also PTX sensitive, of Oregon to P.Q.T. and grants from the National Institutes of Health (NS26494) and the Klingenstein Fund for the Neurosciences. We thank Drs. J. Williams and suggestingthat AP4 receptors on nerve terminals are coupled C. Jahr for reading earlier versions of the manuscript. to a similar G-protein. Correspondence should be addressed to Gary L. Westbrook, Vollum Institute, L474, 3 181 SW Sam Jackson Park Road, Oregon Health Sciences University, Materials and Methods Portland, OR 9720 1. = Present address: Section of Neurobiology, Yale University School of Medicine, Cell culture. Olfactory bulbswere dissected from 5-7-d-old rat pups 333 Cedar Street, New Haven, CT 065 10. that wereanesthetized with halothaneand killed by decapitation.Neu- Copyright 0 1992 Society for Neuroscience 0270-6474/92/122043-08$05.00/O ronswere incubated in activatedpapain, dissociated by gentletritura- 2044 Trombley and Westbrook - AP4 Receptors in Cultured Olfactory Bulb Neurons

“V% 100 pA AP4 10 msec 10 Time (min)

Figure 1. L-AP4 reversibly blocks C high-threshold calcium currents in ol- factory bulb neurons. A, Under voltage mV clamp, a 25 msec voltage step to 0 mV from a holding potential of -60 mV evoked a sustained inward barium cur- -6i.l rent through high-threshold calcium channels. B, L-AP4 (30 PM) reversibly reduced the current by 27% at 2 min and by 25% at 17 min after the begin- ning of whole-cell recording. C, I-fre- lationship for the effects of 30 PM L-AP4 on the barium current. Bath contained no added glycine and 100 PM AP5. -100’ pA

tion, and plated on confluent layers of olfactory bulb astrocytcs as pre- Current records wcrc filtered at l-3 kHz (eight-pole Bessel filter). Mem- viously described (Trombley and Westbrook, 1990). Whole-cell brane currents and EPSPs were collected as averages of three to five recordings were made after 6-48 hr in culture. Excitatory synaptic trans- trials; measurements were made at the peak. To determine the inhibition mission between olfactory bulb neurons was studied after 7-10 d from by L-AP4 or norepinephrine (NE), the control barium current amplitude cultured neurons obtained from newborn rats. was taken as the average of peak current values immediately before and Electrophysiology.Voltage- and current-clamp recordings were per- after drug application in order to correct for current rundown. Peak formed at room temperature using a discontinuous voltage-clamp am- current amplitudes and inhibition of current by L-AP4 or NE were plifier (Axoclamp 2A, Axon Instruments). During voltage clamp, the measured as the average of a 2 msec data epoch beginning 10 msec after membrane voltage was continuously monitored to ensure adequate volt- the onset of the voltage jump. Leak and capacitative currents were age control; switching frequencies were 10-l 5 kHz. The recordingcham- subtracted using a P/4 procedure; the initial 400 rsec after a voltage ber was perfused at 0.5-2.0 ml/min with a solution containing (in mM) jump had uncanceled capacitative transients and was blanked. Results NaCl, 145; KCl, 2.5; CaCl,, 2.0; HEPES, 10; glucose, 10; MgCl,, 1.0; are reported as mean ? SD. and no added glycine. The pH was adjusted to 7.3 with NaOH and the osmolarity to 325 mOsm with sucrose. During recording, neurons were Results locally perfused via an array of 400 pm i.d. glass barrels fed by gravity from drug reservoirs. Neurons were always bathed from one barrel L-AP4 reversibly blocks the high-threshold calcium current containing control solution except during application of drugs. Calcium High-threshold calcium currents were evoked in olfactory bulb currents were evoked in solutions containing tetrodotoxin (0.3 PM) and neurons dissociated from 5-7-d-old rat pups. Whole-cell re- 10 mM barium substituted for calcium. In experiments with quisqualate, cordings were made after 12-48 hr in culture; under these con- glutamate, and ibotenate, 100 PM m-aminophosphonopentanoic acid (DL-APS) and/or 100 WM 6-cyano-7-dinitroquinoxaline-2,3-dione ditions adequate space clamp was possible before extensive neu- (CNQX) was added to prevent activation of agonist-gated ion channels. ritic outgrowth had occurred. We restricted our analysis to large During recording of EPSPs, DL-AP5 (100 FM) and picrotoxin (20 PM) neurons that are putative mitral/tufted cells based on their phys- were added to block NMDA and GABA receptor-mediated synaptic iological and immunohistochemical criteria (Trombley and potentials. Patch electrodes (4-6 MB) were pulled from borosilicate glass, fire polished, and filled with solution containing (in mM) KMeSO, Westbrook, 1990). Calcium currents were isolated by blocking or CsCl, i40; MgCl,, 5; HEPES, 10; Na-ATP, 5; Na-GTP; 0.5; and voltage-dependent sodium channels with TTX; potassium chan- EGTA. 1.1 or 11: nH 7.2.3 10 mOsm. To obtain low intracellular Ma2+ nels were blocked using cesium in the patch pipette. In most in some experiments, Mk+ was omitted from the patch solution, aid experiments, barium (10 mM) was substituted for calcium as EGTA was replaced by 11 mM EDTA. Most drugs were dissolved in the charge carrier. As shown in Figure lA, a 25 msec depolar- water and diluted in the recording solution before use. CNQX stocks were dissolved in dimethylsulfoxide (final concentration, ~0.1%) and izing voltage step to 0 mV from a holding potential of -60 mV nifedipine, in absolute ethanol (final concentration, ~0.01%). Most evoked a sustained inward current. The barium current was chemicals were obtained from Sigma. L-AP4 and excitatory amino acids completely blocked by Cd and showed little inactivation even were obtained from Cambridge Research Biochemicals or Tocris. with 150 msec voltage jumps (not shown). The inward current Voltage and current protocols were generated using PCLAMP software (version 5.5, Axon Instruments); EPSPs and membrane currents were activated near -40 mV peaked at 0 mV and reversed near + 55 digitized and stored for later analysis on an IBM-AT. Barium currents mV (Fig. 1 C). Nifedipine (1 PM) reduced the current by 38 & were evoked by 25 msec voltage jumps from - 60 mV at 30 set intervals. 14% (n = 7) from a holding potential of -80 mV, suggesting The Journal of Neuroscience, June 1992, 12(6) 2045 that at least part of the current was due to L-type calcium chan- nels. Although we did not attempt to determine the relative contribution of N- and L-type channels to the high-threshold current, which can vary considerably between neuronal cell types, the incomplete block by high concentrations of nifedipine is similar to that reported in acutely isolated hippocampal or visual cortex neurons (Regan et al., 199 1). Transient (T-type) calcium currents were small and rarely observed in these neurons even from a holding potential of - 100 mV; however, we used a holding potential of -60 mV where T-currents are largely in- activated (Fox et al., 1987). Rapid perfusion with L-AP4 (l-300 FM) reversibly inhibited the high-threshold calcium current in more than 80% of the neurons tested (Fig. IA,@. The inhibition of inward barium 100 pA current was similar for voltage jumps to membrane potentials B between -40 and +40 mV, although the effect of L-API on outward current through calcium channels was minimal as shown 10 msec in voltage jumps to +60 and +70 mV (Fig. 1C). L-AP4 had little effect at 1 FM (n = 7) and was maximal at concentrations near 30 KM. In the presence of 30 PM L-AP4, currents were reduced by 23.6 f 9.1% (n = 59). Similar concentrations of L-AP4 have been shown to be effective in inhibiting transmitter release from mitral cells in brain slices and in culture (Anson and Collins, 1987; Trombley and Westbrook, 1990). Although L-AP4 also is a weak agonist at NMDA receptors (see Mayer and Westbrook, 1987) NMDA receptors were blocked in these Figure 2. Inhibition of the bariumcurrent by NE andthe (Yeadrenergic experiments by adding Mg2+ (1 mM) and DL-APS (100 PM) to receptoragonist UK 14304.A, NE (30PM) produceda 21% reduction in the bariumcurrent. This wasmimicked by 500 nM UK-14304(B), glycine-free medium. L-AP4 also had no effect on the resting which reducedthe current by 23% in the sameneuron. The barium conductance or on potassium currents evoked by voltage steps currentwas evoked by a voltagestep to 0 mV from a holdingpotential to +20 mV (data not shown). of -60 mV. In the presence of L-AP4, there was a slow increase in the barium current during a voltage jump (see, e.g., Fig. 1A). This relief of inhibition has also been observed for Mg2+-freemedium, neuronswere dialyzed with a patch electrode inhibition of calcium channels in other neurons and has been containing 11 mM EDTA and no added Mgz+. As shown in termed the “reluctant” or “sleepy” channel (see Bean, 1989; Figure 3A, removal of Mg*+ completely blocked the action of Elmslie et al., 1990). The amplitude of the barium current showed L-AP4 (4.2 f 2.4% reduction; n = 5). slow rundown during intracellular dialysis even with 5 mM Mg*+/ More direct evidence for G-protein coupling of AP4 receptors ATP and 0.5 mM GTP in the pipette. However, as shown in was obtained using the nonhydrolyzable GTP analog GTP-r- Figure lB, L-AP4 was still effective in reducing the barium S. When GTP-7-S (100 FM) was substituted for GTP in the current throughout the recording period, indicating that intra- patch pipette, L-AP4 reduced the barium current by a similar cellular dialysis did not occlude the action of L-AP4. degree(inset, Fig. 3B). However, there was essentially no re- NE and the selective CQadrenergic receptor agonist UK- 14304 covery 30 min following AP4 application, and repeat applica- also reduced the peak barium current in the isolated olfactory tions of L-AP4 did not further inhibit the current (not shown). bulb neurons (Fig. 2) as has been observed in other neurons For 14 neurons,the inhibition was 24.4 + 7.9%, which wasnot (e.g., Bean, 1989; Surprenant, 1989). For NE (30 FM), the re- significantly different from neurons dialyzed with GTP. Sub- duction was 29.5 -+ 9.8% (n = 39) for voltage steps to 0 mV sequentapplication of NE usually did result in further depres- from a holding potential of -60 mV. This is similar to that seen sion of the barium current (Fig. 3B), which may suggestthat with L-AP4. Eight additional neurons showed no response to the receptorsactivated by L-AP4 and NE have accessto different NE. UK-14304 (500 nM) reduced the barium current by 22.3 pools of G-proteins. In somecells, the rundown of the calcium f 5.9% in nine neurons that also responded to NE, consistent current appearedto be enhancedwith GTP-7-S in the pipette, with activation of an a~* (Fig. 2B). consistent with direct activation of G-protein(s) acting on cal- cium channels(e.g., Toselli et al., 1989). However, the degree PTX inhibits the action of L-AP4 on barium currents ofinhibition by L-AP4 wassimilar compared to GTP-containing On sympathetic neurons, NE reduces transmitter release that pipettes, presumably becauseL-AP4 wasinitially applied within has been attributed to inhibition of high-threshold calcium cur- 5-l 0 min of achieving whole-cell recording. rents via a G-protein (Lipscombe et al., 1989). This may reflect PTX blocks coupling of G, and G, proteins to activated re- a general mechanism of action for several presynaptic receptors ceptors by ADP ribosylation (Gilman, 1987). To test the sen- (e.g., Holz et al., 1989; Scholz and Miller, 1991). In order to sitivity of the L-AP4 responses,olfactory bulb neurons were determine whether AP4 receptors are coupled to a G-protein, incubated in 250 rig/ml PTX for 15-36 hr. Following PTX we first tested the effect of removing intracellular Mg2+, which treatment, L-AP4 had no measurableeffect on the barium cur- is an absolute requirement for activation of G-proteins (Brown rent (+0.9 ? 7.2%; n = 18) whereas barium currents in un- and Birnbaumer, 1988). After a 2 hr incubation in a nominally treated sister cultures were reduced by 21.1 f 6.0% (n = 11). 2046 Trombley and Westbrook l AP4 Receptors in Cultured Olfactory Bulb Neurons

A 0 [Mg2+]i B GTPyS

150 pA 0 I/t 10 msec 0 10 20 Time (min)

C L-AP4 NE D Staurosporine 1

100 pA L -PTx +PTx -P-Ix +PTx 10 msec

Figure 3. L-AP4 receptor is coupled via a G-protein. A, L-AP4 (30 PM) had no effect in neurons incubated for 2 hr in a nominally Mgz+-free solution and dialysed with a recording pipette containing 1 mM EDTA and no added Mg2+. B, During whole-cell dialysis with GTP-7-S (100 WM), L-AP4 and NE (both at 30 PM) caused irreversible reduction in the barium current. Inset shows barium current during 30 msec voltage step before, during, and after application of L-AP4. C, Inhibition of calcium current by L-AP4 and NE was blocked by pretreatment with PTX (250 rig/ml) for 15-24 hr. Histogram shows inhibition compared to untreated sister cultures. D, The nonselective kinase inhibitor staurosporine (1 PM) did not prevent inhibition by L-AP4.

This is consistent with the involvement of a PTX-sensitive cell recording for the compound to diffuse completely into the G-protein in coupling AP4 receptorsto calcium channels. Sim- cell. Dialysis for 5-l 0 min with staurosporine (1 PM) did not ilar results were seenfor NE. The barium current in untreated prevent L-AP4 inhibition of the barium current (Fig. 30). For neurons was reduced by 25.2 ? 4.5% (n = lo), but barium five neurons, the inhibition was 27.4 -t 9.4%, which was not currents were not significantly inhibited by NE in PTX-treated significantly different than control (seeFig. 1). This suggeststhat neurons (5.3 f 8.8%; n = 18). There were no apparent differ- the action of L-AP4 may not require protein phosphorylation. encesbetween the PTX-treated and sistercultures with respect to other membrane properties such as holding current and the Inhibition of barium currents by other excitatory amino acids magnitudeand voltage dependenceofthe calcium currents. These In addition to L-AP4, several other glutamate analogsalso in- results are summarized in Figure 3C. hibit transmitter release in the CNS (reviewed in Mayer and G-protein

A B

Figure 4. Glutamateanalogs that ac- tivate the metabotropicglutamate re- ceptoralso inhibited the high-threshold calciumcurrent. A and B, Glutamate (100PM) reducedthe bariumcurrent by 25%; however,AMPA had no effect. Histogramexpresses percent reduction of the controlbarium current for AMPA \ AMPA F- (25 PM), ibotenate(100 PM; Zbo),quis- qualate( 1PM; @is),and glutamate ( 100 PM; G/u) comparedto L-AP4 (30 PM). Bath contained1 mM Mg2+,no added glycine, 100 PM AP5, and 100 PM CNQX to block glutamatechannel ac- 10 rnsec L-AI’4 Glu Quis Ibo AMPA tivity.

in mRNA-injected oocytes (Sugiyama et al., 1989; Houamed et aptic AMPA receptors. L-AP4 reversibly inhibited the EPSP al., 1991; Masu et al., 1991). Likewise, quisqualate and gluta- amplitude by 46.5 * 18.5% (n = 9). Similar effects were seen mate have been reported to inhibit high-threshold calcium cur- with NE (n = 4). L-AP4 had no effect on the membranepotential rents in cultured hippocampal neurons (Lester and Jahr, 1990) of either or on the shapeof the presynaptic action po- and cause the release of intracellular calcium (Murphy and Mil- tential. ler, 1988, 1989). However, L-AP4 was ineffective in those ex- The top trace in Figure 5A showssuperimposed synaptic re- periments. sponsesbefore and during perfusion with L-AP4. In sister cul- We tested the effect of metabotropic receptor agonists in bar- tures pretreated with 250 &ml PTX for 15-36 hr, neither ium solutions, and with 10 mM EGTA in the patch pipette, to L-AP4 nor NE had any significant effect on the EPSPs(Fig. 5A, prevent the effects of IP,-mediated calcium release from intra- middle trace; B). AP4 (30 FM) produced a 2.4 f 5.6% increase cellular stores. To ensure that glutamate-activated ion channels (n = lo), and NE causeda 3.0 +- 2.0% decrease(n = 3). As were blocked, all drug solution contained high concentrations shownin the bottom trace of Figure 5A, the postsynaptic AMPA of DL-AP~ (100 PM) and CNQX (100 MM). This avoided the receptor antagonist CNQX (5 PM) produced a nearly complete possible action of NMDA receptor agonists on calcium currents block of the EPSP (97 -t 5.2%; n = 4), consistent with a glu- as has been reported on acutely dissociated hippocampal neu- tamate-mediated EPSP. PTX treatment had no apparent effect rons (Chernevskaya et al., 1991). AP5 and CNQX had no ap- on the leakagecurrent or action potentials of the treated cells. parent direct membrane effects. Under these conditions, L-glu- tamate reduced the barium current by 25.3 f 10.7% (n = 4), Discussion whereas AMPA (25 PM), a non-NMDA receptor agonist without Our results demonstrate that the AP4 receptor representsa activity at metabotropic receptors, had no effect (Fig. 4A). How- G-protein

A B Control/30 gM L-AP4

Figure 5. EPSPs evoked from pairs of bulb neurons are reduced by L-AP4. A: Top, Using simultaneous whole-cell re- cording from a pair of monosynapti- tally coupled bulb neurons, a 3 msec depolarizing current pulse fired an ac- tion potential in the presynaptic cell evoking an EPSP in the postsynaptic neuron. The EPSPs were reduced by PTX-treated 46.5 f 18.5% in nine pairs (30 WM L-AP4) by a presynaptic mechanism. Bottom,In sister cultures pretreated with PTX (250 &ml, 15-36 hr), EPSPs evoked from pairs of bulb neurons were not reduced by L-AP4 (n = 10). Post- synaptic receptor antagonists, however, .-. -PTX +PTX were still effective as 5 PM CNQX re- 4 \ L-Al’4 duced the EPSP by 91 ? 5.2% (n = 4). Data from nine neurons are summa- rized in B. - CNQX

mitter-mediated calcium channel inhibition may also involve cloned metabotropic receptor transcript (Sugiyamaet al., 1987; activation of protein kinase C (Rane et al., 1989) or releaseof Sugiyama et al., 1989; Houamed et al., 1991). Second, stauro- calcium from intracellular stores(Kramer et al., 1991). sporinedid not block the responseto L-AP4 in our experiments, Our results suggestthat L-AP4 reduction of calcium currents suggestingthat activation of protein kinaseC wasnot required. doesnot involve protein phosphorylation or a diffusible second Third, IP,-mediated calcium releaseby quisqualate is depleted messengersuch as calcium releasedfrom intracellular stores. after repeated applications in calcium-free solutions (Murphy Although calcium buffering with EGTA doesnot provide rapid and Miller, 1988). Thus, the L-AP4 response,if due to metab- control of transmembranecalcium influx, our experiments were otropic receptor stimulation, would be expected to disappear done in barium (i.e., calcium-free) solutions. Likewise, L-AP4 in barium solutions. It did not. However, in addition to was effective on repeatedapplications for periods of whole-cell L-glutamate, ibotenate and quisqualate,which both activate the dialysis of >30 min. This is inconsistent with a mechanism metabotropic receptor, also inhibited barium currents in our involving intracellular calcium stores, which should become experiments. Thus, the AP4 receptor may bind many of the depleted with repeated stimulation in calcium-free solutions same ligandsas the metabotropic receptor. By contrast, L-AP4 (e.g., Murphy and Miller, 1988; Pfaffinger et al., 1988; Kramer has been reported to antagonize IP, turnover in adult rat brain et al., 1991). slices (Nicoletti et al., 1986; Schoeppand Johnson, 1988) but PTX doesnot distinguish betweenG, and G,; thus, the iden- this wasnot seenin hippocampal slicesfrom newborn animals tity of the G-protein coupled to the AP4 receptor cannot be (Nicoletti et al., 1986)or in measurementsof quisqualate-evoked determined from our data. Although G, is much more abundant calcium transients in isolated hippocampal neurons (Murphy in brain than G,, and G, appearsto couple inhibitory receptors and Miller, 1988). Part of this discrepancy has been attributed to calcium channels in dorsal root ganglion neurons (Ewald et to developmental changesin the efficacy of metabotropic re- al., 1989) and NG108 cells (Hescheleret al., 1987; McFadzean ceptor antagonists(Schoepp et al., 1990). However, the most et al., 1989) reconstitution experiments have not demonstrated potent metabotropic receptor antagonist, 2-amino-3-phospho- clear specificity of a-subunits for every receptor (e.g., Ewald et noprioprionic acid (AP3; Schoeppand Johnson, 1989), is inef- al., 1989). Whether the presynaptic AP4 receptor is the same fective in mimicking the presynaptic action of L-AP4 in hip- asthe L-AP4-activated receptor involved in G-protein coupling pocampal slices(Koerner and Cotman, 1981). to phosphodiesterasein retinal depolarizing bipolar cells (Nawy Our resultsalso differ in severalrespects from the quisqualate- and Jahr, 1990) is also unclear. The G-protein involved in the induced inhibition of calcium currents in cultured CA1 hip- bipolar cell has not yet beencharacterized. However, the retinal pocampal neurons (Lester and Jahr, 1990). The inhibition by receptor could conceivably differ from the presynaptic AP4 re- quisqualate required the presenceof intra- or extracellular cal- ceptor only in its coupling mechanism. cium, was enhanced in GTP-7-S compared to GTP, and was not mimicked by L-AP4. The lack of L-AP4 action may be Comparison with the metabotropic glutamate receptor explained by the relative insensitivity of the Schaffercollateral- Several observations suggestthat the effects of L-AP4 do not CA 1 pathway to the presynaptic effects of L-AP4 (Koerner and result from activation of the metabotropic glutamate receptor. Cotman, 1982). Although the quisqualate-induced inhibition First, L-AP4 doesnot stimulate inositol phospholipid turnover required calcium, dialysis with kinaseinhibitors, tight buffering in several different mammalian preparations (see Schoepp et with BAPTA, or dialysis with IP, did not block the effect of al., 1990) or in oocytes injected with whole-brain mRNA or the quisqualate(Lester and Jahr, 1990). Therefore, if this response The Journal of Neuroscience, June 1992, 12(6) 2049 was via activation of the metabotropic receptor, it is unlikely tamate in the cleft may peak above 1 mM and take more than to result from phospholipase C activation. We have observed 10 msec to return to concentrations below 1 I.LM (Clements et in preliminary experiments that the selective metabotropic re- al., 199 1). Thus, a membrane-delimited G-protein action might ceptor agonist trans- 1-amino-cyclopentyl- 1,3-dicarboxylic acid be advantageous in eliminating the long latency that accom- (truns-ACPD, Palmer et al., 1989) can inhibit barium currents panies the activation of a diffusible second messenger (e.g., Horn on cultured hippocampal neurons with intracellular calcium buf- and Marty, 1988; Furuya et al., 1989). Transmitter release dur- fered to 10 nM (Sahara and Westbrook, 199 1). Thus, it may be ing a burst of action potentials lasting 50-100 msec could be that the metabotropic receptor activates several different trans- altered via the autoreceptor under these conditions. duction pathways. Consistent with this possibility, truns-ACPD The marked regional differences in the potency of L-AP4 as has been reported to inhibit potassium conductances in hip- a synaptic depressant could reflect differences in the affinity or pocampal neurons independent of changes in intracellular cal- numbers of presynaptic AP4 receptors or in the number of cium (Charpak et al., 1990). This may be analogous to the action available G-proteins or calcium channels. The high L-AP4 sen- of substance P and leutinizing -releasing hormone, sitivity of the lateral perfoiant path input to the dentate gyrus which stimulate IP,, but independently inhibit M-current on (IC,, = 2.5 PM; Koemer and Cotman, 198 1) contrasts with the frog sympathetic neurons (Pfaffinger et al., 1988). Schaffer collateral-CA1 pathway (IC,, > 1 mM). Functionally, this might serve to reduce excitatory input to the hippocampus, The mechanism of presynaptic inhibition perhaps contributing to a role of the dentate gyrus as a “gate” Although inhibition of calcium currents has long been an at- for synaptic activity entering the hippocampus. In addition to tractive hypothesis for the mechanism of presynaptic inhibition an action of L-AP4 in the lateral olfactory tract, the existence by , definitive evidence has been difficult to of dendrodendritic synapses in the olfactory bulb could provide obtain. The extrapolation of results obtained on somatic calcium another presynaptic site for modulation by glutamate (or ad- currents to the presynaptic terminal have been further compli- renergic) autoreceptors. Although there is no direct evidence for cated by the discovery of calcium channel subtypes (Fox et al., this at present, similar mechanisms have been observed follow- 1987). In particular, the contributions of different subtypes of ing dendritic release of in the substantia nigra (Chera- high-threshold calcium channels to transmitter release may dif- my et al., 1981). between pathways. Only a few studies have made a direct comparison of neurotransmitter effects on calcium currents and transmitter release. In dorsal root ganglion neurons, both GABA References and NE inhibit the high-threshold calcium current (Dunlap et Anson J, Collins GGS (1987) Possible presynaptic actions of 2-amino- al., 1987), and also reduce potassium-evoked substance P re- 4-phosphonobutyrate in rat olfactory cortex. Br J Pharmacol9 1:753- lease, an effect mimicked by dihydropyridines (Pemey et al., 761. 1986; Rane et al., 1987). In sympathetic neurons, N-type cur- Baskys A, Malenka RC (199 1) truns-ACPD depresses synaptic trans- mission in the hippocampus. 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