Discussion reported [50]. Class I mGlu receptors have also been

368 J elevation of intracellular calcium [83,87]. In addition, recent studies suggest the potential role of neurokinin 1 NK1 and opioid receptors in the generation of LTP and LTD [56,57,82,84,108]. The results of this study provide the first demonstration of de novo long-lasting synaptic plasticity LTP and LTD in the spinal cord dorsal horn that is induced by activation of group I metabotropic glutamate receptors in the absence of repetitive presynaptic activity. The present study addressed electrophysiologically and pharmacologically how group I mGlu receptors influence primary afferent-mediated synaptic transmission and plas- ticity in the rat spinal DH. This is a particularly important question in view of an emerging evidence that implicates group I mGlu receptors in nociception and hyperalgesia. The principal findings are that the activation of group I mGlu receptors by 1S,3R-ACPD, DHPG and CHPG induced: 1 a long-lasting depression of Ad-fiber-mediated monosynaptic EPSP in neurons of both substantia gelatin- osa and deep dorsal horn, and 2 a long-lasting potentiation of polysynaptic EPSP. The effects of DHPG on mono- synaptic and polysynaptic EPSPs were inhibited by D -AP5. The increases in polysynaptic EPSPs produced by DHPG and CHPG are greatly augmented after synaptic inhibition Fig. 6. S-DHPG decreased the dorsal root-evoked inhibitory postsynaptic is blocked. In addition, we confirmed previous demonstra- potentials IPSPs in DH neurons. A S-DHPG 1 mM, 10 min blocked the DR-evoked IPSP in a SG neuron. V 5 264 mV, 19 day-old rat. B tion of a direct increase in excitability of DH neurons by m The graph shows the time course of the reversible depression of DR- the mGlu receptor agonist 1S,3R-ACPD [51,64] and evoked IPSPs following bath application of S-DHPG 10 mM, 10 min in demonstrated that the effect is exerted via the activation of a deep DH neuron. The individual IPSPs from the same experiment are group I mGlu receptors. shown above the graph. V 5 254 mV, 18 day-old rat. m 4.1. Long-lasting depression of Ad-fiber-mediated monosynaptic EPSPs by activation of group I mGlu control solution Fig. 9C, Table 2. Moreover, under receptors conditions of blockade of GABA - and glycine-mediated A synaptic inhibition, the EPSP-evoked at C-fiber strength 1S,3R-ACPD, the group I and II mGlu receptor was substantially increased Fig. 9D. agonist, primarily caused a sustained decrease in the Application of 0.5 mM CHPG for 5 min had no amplitude of the DR-evoked monosynaptic EPSP. This significant effect on the resting membrane potential n 5 effect appears to be specific for group I mGlu receptors, as 13, or membrane input resistance n 515. However, 1 it could be mimicked by DHPG and CHPG, the selective mM CHPG produced a prolonged depolarization 7.862.7 group I agonists, and was attenuated by 4-CPG, the group I mV, n 55, lasting 9–32 min. In the presence of TTX 0.5 antagonist. Depression of the monosynaptic and, to a lesser mM, NBQX 10 mM and D -AP5 50 mM, the CHPG- extent, polysynaptic components of the dorsal root-evoked induced depolarization was not modified, the finding ventral root potential VRP of the neonatal rat spinal cord suggesting a postsynaptic site of action. by 1S,3R-ACPD [23,43–45,80, but see 11] and also EPSPs evoked by low or high intensity dorsal root stimulation in immature ventral horn neurons in vitro was 4. Discussion reported [50]. Class I mGlu receptors have also been shown to play a role in the long-term depression in the We have previously reported long-term potentiation brain [7,53,55,60,71–73,75]. LTP and long-term depression LTD of both mono- One of the most prominent physiological effects of synaptic and polysynaptic EPSPs in the spinal cord DH mGlu receptor agonists that is consistent throughout the neurons in vitro following high- and low-frequency repeti- brain is reduction of transmission at glutamatergic tive stimulation of primary afferents [82,83,87]. The synapses [1,16,79]. The present concept is that this effect cellular mechanisms underlying long-term synaptic plas- is mediated by presynaptic mGlu receptors that serve as ticity in the DH are still not well understood. Induction of autoreceptors to reduce glutamate release. In recent years, LTP and LTD requires NMDA receptor activation and an it has become clear that multiple mGlu receptor subtypes, J . Zhong et al. Brain Research 887 2000 359 –377 369 Fig. 7. S-DHPG-induced depolarization of DH neurons. The group I mGluR agonist, S-DHPG 10 mM, 10 min induced a depolarization and increase in the baseline noise in both superficial A and deep B DH neurons. In the deep DH neuron the larger depolarization was associated with spontaneous action potential firing B, left trace. In the presence of TTX 0.5 mM, and ionotropic glutamate receptor antagonists D -AP5 30 mM and NBQX 10 mM, the depolarization was not modified, the finding suggesting a postsynaptic site of action B, right trace. V 5 267 mV A, 273 mV B, 19 day-old rats. m C In the presence of ionotropic glutamate receptor antagonist CNQX 20 mM, bath-applied S-DHPG 5 mM for 90 s to a deep DH neuron caused a large membrane depolarization associated with increased baseline noise and action potential firing left trace. This effect is reversibly blocked by cyclothiazide CTZ, 25 mM, an antagonist at mGluR1 middle trace. The inhibitory response was recorded 30 min after exposure of the slice to CTZ. Partial recovery of the S-DHPG-induced depolarization occurred 30 min after removal of CTZ right trace. V 5 262 mV, 21 day-old rat. m which belong to each of the three major groups, can serve 4.2. Activation of group I mGlu receptors induces long- as autoreceptors. Although the pharmacological profiles of lasting potentiation of DR-evoked polysynaptic EPSPs mGlu autoreceptors at synapses between primary afferent fibers and DH neurons have not been vigorously examined Besides inhibitory, a sustained excitatory action of [32,108], there is anatomical evidence indicating that in group I agonists on afferent A-fiber-evoked polysynaptic, primary afferent neurons multiple mGlu receptors mGlu1- some monosynaptic EPSPs Tables 1 and 2, and EPSPs of 5 and 7 subtypes may serve as autoreceptors cells receiving C-fiber afferent input, has been observed in [46,70,92,93]. Although we did not specifically address the the DH region. 1S,3R-ACPD induced potentiation of locus of the synaptic depression of monosynaptic EPSP, monosynaptic EPSPs, evoked by stimulation of the de- and the group I mGlu receptor subtype involved, it can be scending lateral column fibers, in frog spinal motoneurons assumed on the basis of presynaptic localization of was reported [36]. Moreover, it was found that trans- mGlu1a and 5 subtypes in the perikarya of small or ACPD enhances the responsiveness of primate medium diameter dorsal root ganglion neurons [92], the spinothalamic tract cells to innocuous stimuli [74], where- mGluR5 presence in vesicle-containing profiles apposed to as DHPG potentiates the responses to both innocuous and afferent terminals in glomeruli of the superficial DH [46], noxious stimuli at low concentrations, but had inhibitory failure of 1S,3R-ACPD and CHPG to alter input resist- effects at higher concentrations [68]. There are relatively ance of postsynaptic neurons in the present study, and in few reports suggesting potentiation of excitatory transmis- the lamprey spinal cord [52], the persistence of depression sion via group I and II mGlu receptors in the brain, as of monosynaptic EPSP despite offsetting mGlu receptor- compared with their inhibitory actions [1,16,79]. At pres- agonist-induced depolarization, that it is presynaptic. How- ent, the mechanisms by which group I mGlu receptor ever, because of the postsynaptic depolarizing effect of agonists elicit excitatory action on EPSPs in the CNS is group I agonists and evidence for predominant localization unknown. While mGlu receptor-induced inhibition of of mGlu1 5 subtypes postsynaptically [46,89,94], it is excitatory transmission has been explained mostly by more difficult to conclude that the group I mGlu receptor- presynaptic mechanisms [16,79], postsynaptic mechanisms mediated depression is exclusively a result of presynaptic have often been suggested in 1S,3R-ACPD-induced inhibition. potentiation of NMDA or non-NMDA receptor-mediated 370 J Fig. 8. S-DHPG-induced oscillatory activity recorded from DH neurons. A In a deep DH neuron S-DHPG 100 mM, 90 s produced stereotypic oscillatory activity lasting over an hour in the presence of competitive ionotropic glutamate receptor antagonists CNQX 20 mM and D -AP5 50 mM. The oscillations consisted of trains of depolarizations with overriding action potentials. V 5 263 mV, 18 day-old rat. B In a SG neuron addition of bicuculline m 5 mM and strychnine 2 mM, the GABA and glycine receptor antagonists, to the perfusion medium augmented the amplitude of the oscillatory activity A induced by S-DHPG 10 mM, 10 min. To reduce the increased spontaneous synaptic activity and subsequent action potential firing due to the removal of 21 synaptic inhibition, the Mg concentration in the perfusing solution was increased to 3 mM. V 5 279 mV, 18 day-old rat. C,D The increase in the m baseline noise and the oscillatory activity induced by 10 mM S-DHPG persisted, and oscillations were even enhanced C, right trace in the presence of bicuculline 5 mM and strychnine 2 mM in a cell exhibiting excitatory synaptic noise C, but were abolished D, right trace in the cell in which S-DHPG applied in a normal medium produced a marked increase in inhibitory synaptic noise D, left trace, inset. The insets in C, D illustrate the spontaneous synaptic activity taken at the time indicated by the arrow on a slower time scale. C V 5 274 mV, 19 day-old rat. D V 5 253 mV, 18 m m day-old rat. 21 responses [5,12,18,34,35]. In a few brain regions, however, Ca concentration, in the induction of LTP and LTD in the 1S,3R-ACPD-induced potentiation seems to be pre- the brain and spinal cord [1,6,53,55,82]. The present study synaptically controlled [15,40,41]. There is also evidence demonstrates that the induction of long-lasting depression for a role of presynaptic group I mGlu receptors in the of monosynaptic EPSP and the magnitude of long-lasting positive modulatory control of neuronal glutamate release, potentiation of polysynaptic EPSP produced by DHPG in probably from primary afferent C-fibers, in the rat spinal the spinal cord DH, appears to be dependent on co- cord [27]. activation of group I mGlu and NMDA receptors, as the effects were reduced, or abolished, by blockade of either 4.3. DHPG-induced long-lasting depression potentiation receptor class. Our finding of the NMDA receptor depen- of EPSPs is NMDA receptor-dependent dence of the long-lasting depression of primary afferent A-fiber-mediated monosynaptic EPSPs is in agreement There is evidence for a role for postsynaptic NMDA and with some reports for the CA1 region of the adult rat mGlu receptors, and a rise in postsynaptic intracellular hippocampus [39,73,75], but not for the LTD in other J . Zhong et al. Brain Research 887 2000 359 –377 371 Fig. 9. Effects of RS-CHPG on the synaptic responses of DH neurons. A Summarized data showing the time course of the depression of the monosynaptic EPSPs Ad input: n 57, 2–10 m s by bath-applied selective mGluR5 agonist RS-CHPG 0.5 mM, 5 min. V 5 264 to 280 mV, 17–23 m day-old rats. B Bath application of RS-CHPG 0.5 mM, 5 min in the control solution produced a small potentiation in the peak amplitude of the Ad-primary afferent-evoked polysynaptic EPSPs of six SG neurons inset recorded in response to electrical stimulation of a lumbar dorsal root. V 5 265 m 21 to 277 mV, 17–22 day-old rats. C In a medium containing bicuculline 5 mM, strychnine 2 mM and a high concentration of Mg 3 mM, 0.5 mM RS-CHPG-induced long-lasting potentiation in the peak amplitude of polysynaptic EPSPs recorded from SG neurons inset was increased n 56. V 5 258 to 277 mV, 20–22 day-old rats. D In a SG neuron inset receiving input from C-fibers c.v. 1.3 m s, superfusion of RS-CHPG 0.5 mM, 5 m 21 min potentiated the EPSPs during and after drug application in the presence of bicuculline 5 mM, strychnine 2 mM and high concentration of Mg 3 mM. V 5 269 mV, 21 day-old rat. E In the presence of MPEP 10 mM, a potent mGluR antagonist, CHPG 1 mM, 5 min had no significant effect on m 5 the amplitude of A-primary afferent fibers-evoked polysynaptic EPSPs. However, after the removal of the antagonist, the same concentration of CHPG produced a long-lasting increase in the amplitude of the EPSPs. V 5 280 mV, 22 day-old rat. m 372 J regions [71,72]. The relevant question to ask here is why the superficial laminae of spinal DH [46]. In spinal DH should NMDA receptor antagonists inhibit the LTD LTP neurons, we have shown that the blockade of GABA and A of monosynaptic and polysynaptic transmission induced by glycine receptors by their selective antagonists had two DHPG? One possibility is that the requisite NMDA effects on the synaptic actions of DHPG. First, this receptor component is contributed by background NMDA treatment enhanced the size of the initial DHPG-induced receptor-mediated currents elicited by synaptically released potentiation of polysynaptic EPSPs and EPSPs evoked by glutamate and that NMDA receptor antagonists block these stimuli at C-fiber strength, and second, it greatly facilitated currents. Alternatively, tonic activation of synaptic and the likelihood and magnitude of the DHPG-induced long- extrasynaptic NMDA receptors might arise from the lasting potentiation of EPSPs. Although the mechanisms background levels of glutamate released from adjacent glia by which the absence of synaptic inhibition facilitates the cells [77]. Although the sites of interaction between DHPG-induced increase of synaptic responses has not been NMDA and group I mGlu receptors has not been examined explored in the present study, we propose that the facilita- in the present study, anatomical and other evidence tion is likely a result of a combination of direct excitatory suggests that the interactions can occur either presynapti- effects of DHPG on DH cells, and a decrease in inhibition. cally and or postsynaptically. In agreement with post- This suggestion is supported by two findings: 1 In the synaptic presence of both group I mGlu and NMDA presence of bicuculline and strychnine DHPG increases receptors in the DH [46,58,89,94], a positive postsynaptic cell excitability and magnitude of EPSPs in DH neurons, interaction between NMDA and group I mGlu receptors and 2 DHPG decreases primary afferent-evoked IPSPs has been described in DH neurons [5,8,12,32,48,96]. recorded in the absence Fig. 6 and the presence of the Besides the mutual amplification of receptor function, the ionotropic glutamate receptor antagonists 20 mM CNQX, co-activation of NMDA and mGlu receptors may also 50 mM D -AP-5. These observations are in general agree- result in the enhancement of the effects mediated by ment with a recent report that disinhibition of spinal dorsal intracellular messengers, including calcium [13,42,63]. We horn GABA-ergic and glycinergic systems may facilitate have recently shown that DHPG increases NMDA-me- recruitment of NMDA-sensitive polysynaptic circuits [2]. 21 diated Ca transients in the substantia gelatinosa neurons At present the mechanisms by which DHPG elicits [32]. Thus, the DHPG-induced depression potentiation of depressant action on IPSPs is unknown. In the hippocam- synaptic transmission could be due to increase in cytosolic pus, the mGlu receptor-induced disinhibition is mediated 21 21 [Ca ] that may have been achieved either via Ca influx by reduced transmission at excitatory synapses onto inhib- 21 through NMDA receptor channels, or Ca release from itory interneurons and inhibitory synapses onto pyramidal the internal stores following activation of PLC–IP path- cells involved in polysynaptic pathways [18,19]. However, 3 21 way. Furthermore, a Ca dependence in mGlu receptor- in the striatum, Stefani et al. [90] have shown that the stimulated phosphoinositide breakdown in synaptosomes activation of mGlu receptors inhibits GABA-mediated has also been demonstrated [95], so it is possible that synaptic potentials in the presence of ionotropic glutamate NMDA receptor and mGlu receptor interactions can occur receptor antagonists via inhibition of the GABA release. presynaptically, consistent with the location of both group In summary, it is clear from both immunohistochemical I mGlu and NMDA receptors in DRG neurons [92,93], and [46] and present data that the net facilitatory effect of on primary afferent terminals [46,58]. group I mGlu receptor agonists reported in our pharmaco- In summary, the present work has revealed a further logical experiments must emerge from complex interaction degree of complexity in the interactions between glutamate involving a variety of mediators with different and oppos- receptors at a central glutamatergic synapse. The finding ing effects. Our results suggest a potential role of the that activation of NMDA receptors can facilitate the dorsal horn GABA-ergic and glycinergic interneurons in activation of group I mGlu receptors may have implica- providing spatial and temporal conditions for modifications tions for synaptic plasticity [6,59,79,82] and central sen- of synaptic strength during plasticity in information pro- sitization of DH neurons, a phenomenon that contributes to cessing in the somatosensory system as illustrated by pain [62]. nociception-related phenomena such as sensitization, hy- peralgesia, and allodynia [78]. 4.4. Involvement of synaptic inhibition in group I mGlu receptor-induced potentiation of polysynaptic EPSP 4.5. Pharmacological identification of mGlu receptors One of the prevalent mechanisms for the inhibition of regulating glutamatergic neurone excitability : group I primary afferent transmission is through polysynaptic mGlu receptors inhibitory pathways mediated by GABA and glycine A receptors [101]. A co-release of GABA and glycine, 1S,3R-ACPD, the group I and II mGlu receptor agonist possibly from the same vesicles, has been demonstrated in [79], S isomer of the selective group I mGlu receptor the rat spinal cord [47]. There is also a recent evidence for agonist DHPG [88], and the selective mGlu5 receptor co-localization of group I mGlu5 receptor with GABA in agonist RS-CHPG [20] cause a slow depolarization, in- J . Zhong et al. Brain Research 887 2000 359 –377 373 crease in membrane noise and a burst firing, in a majority synaptic transmission through neuronal circuits and pro- of DH neurons, which effects in the case of DHPG were moting of synaptic plasticity. accompanied by an increase in membrane input resistance. In addition, both 1S,3R-ACPD and DHPG induced a spontaneous oscillatory activity in about one-third of tested 4.6. Synaptic plasticity induced by group I mGlu superficial DH and a greater proportion of DDH neurons. receptor activation : potential role in the superficial A direct excitatory effect of 1S,3R-ACPD with induction spinal dorsal horn of intense neuronal discharges [51] and oscillations was previously reported for deep DH neurons in the rat spinal Superficial spinal dorsal horn SDH, laminae I and II is slice preparation [64–66], and for DH neurons in the slices involved in modulation of nociceptive information, but of the turtle spinal cord [85]. Our results obtained in the synaptic and cellular mechanisms underlying the changes presence of TTX, CNQX, and D -APV suggest that the responsible for this function are not well understood. depolarization is independent of AMPA KA or NMDA Neuromodulation by substances released by primary affer- receptor activation, and that the effect of DHPG is likely to ents is thought to play a critical role in the development of result from activation of the group I mGlu subtypes on plasticity induced by nociceptive inputs [22,82]. The spinal the DH neurons themselves, rather than on afferent fibers mechanisms contributing to nociception-related phenom- presynaptic to these cells. ena central sensitization, hyperalgesia, allodynia, analge- The agonist and antagonist pharmacology of the mGlu sia are modifications in synaptic efficacy produced by receptors responsible for the excitatory effects in DH activity-dependent changes [22] and by modulatory effects neurons is similar to that of the mGlu receptors that of neurotransmitters [82]. In addition, the modulation of mediate depolarization of spinal cord motoneurons intrinsic properties of neurons provides a potential post- [4,23,43–45,50,80]. In both sets of neurons, the effective- synaptic site of plasticity [64,66,85]. ness of the agonists tested correlates well with their Long-term synaptic plasticity LTP and LTD following capacity to generate the production of IP in brain slices stimulation of primary afferents, peripheral nerve, or 3 [88] and to activate group I mGlu receptors in transfected induced by noxious stimulation or nerve injury, has been cells [91]. Taken together, the agonist and antagonist reported both in vitro and in vivo in the rat spinal cord. pharmacology strongly implicates both subtypes 1 and 5 of There is indication that LTP of excitatory synaptic trans- group I mGlu receptors, in the mediation of postsynaptic mission may play a role in the generation of post-injury depolarization in DH neurons. Group I mGluRs are also hypersensitivity, and LTD in antinociception [32,82,86]. the predominant mGlu receptors involved in increasing The cellular mechanisms underlying LTP and LTD are still excitability of cells in various brain regions [1,16,79]. not well understood. Metabotropic glutamate receptors are Although the ionic mechanism of depolarization has not thought to play a role in modulation of neuronal excitabili- been investigated in the present study, there is a large body ty and synaptic transmission in the brain, and contribute to of evidence indicating that activation of mGlu receptors by regulation of function in neural networks [79] including stimulation of glutamatergic afferents or exogenous appli- nociceptive circuits in the spinal cord DH [11,61,102]. The cation of mGlu receptor agonists increases neuronal ex- present in vitro intracellular data, taken together with citability by modulation of a variety of ion channels [1,16]. available in vivo evidence from extracellular studies mGlu receptors can exert direct excitatory effects on [11,68,69,74,102–105] suggest the presence of functional neurons by activation of non-selective cation currents, group I mGlu receptors on young rat spinal DH neurons, 21 including activation of a Ca -activated nonspecific cation that may play a role in induction of long-term changes of current [1,17,38,66,67,81,107]. In the hippocampus, reduc- responses to innocuous and noxious stimuli resulting in 1 tion of three K conductances have been suggested to hyperalgesia, allodynia, and analgesia. Our present study mediate the 1S,3R-ACPD-induced depolarization, an M- shows that both the mono- and polysynaptic responses in like current, a calcium-dependent slow after-hyperpolariz- the superficial DH are modulated by group I mGlu 1 ing current [14], and a K leak conductance [37]. The receptors. We have demonstrated that the group I mGlu DHPG-evoked depolarization accompanied by an increase receptor activation resulted in long-term depression LTD in membrane input resistance in our study is consistent of monosynaptic EPSPs and long-term potentiation LTP with the previously described closure of potassium chan- of polysynaptic EPSPs, and EPSPs elicited at C-fiber nels [79]. However, the precise identity of ionic currents strength, both in the superficial and deep dorsal horn. underlying the group I agonist-induced depolarization of Group I mGlu receptors have been implicated in the DH neurons will remain unresolved until complementary induction of LTP and LTD in the brain [1,6,53,55,59] data becomes available using whole-cell patch-clamp where LTP and LTD are functionally associated with the technique. processes of learning and memory. The principal roles of The combined actions of mGlu receptor activation result mGlu receptors and LTP and LTD in the spinal dorsal in a marked increase in cell excitability. Physiologically horn, however, may be related to plasticity of spinal these effects may be important for improving integrity of nociception [32,33,51,86,108]. 374 J molecular switch activated by metabotropic glutamate receptors

5. Conclusion