Dual modulation of excitatory synaptic transmission by agonists at group I metabotropic glutamate receptors in the rat spinal dorsal horn

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1 Brain Research 887 (2000) locate/ bres Research report Dual modulation of excitatory synaptic transmission by agonists at group I metabotropic glutamate receptors in the rat spinal dorsal horn c a b c, Jie Zhong, Gabor Gerber, Ljubomir Kojic, Mirjana Randic * a Department of Anatomy, Histology and Embryology, Semmelweis University of Medicine, Tuzolto utca 58., 1094 Budapest, Hungary b Department of Ophthalmology, Faculty of Medicine, University of British Columbia, 2550 Willow Street, Vancouver, BC, Canada V5Z 3N9 c Department of Biomedical Sciences, Iowa State University, Ames, IA 50011, USA Accepted 3 October 2000 Abstract The effects of group I metabotropic glutamate (mglu) receptors on excitatory transmission in the rat dorsal horn, but mostly substantia gelatinosa, neurons were investigated using conventional intracellular recording in slices. The broad spectrum mglu receptor agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD), the group I mglu receptor selective agonist (S)-3,5-dihydroxyphenylglycine (DHPG), and the selective mglu subtype 5 agonist (RS)-2-chloro-5-hydroxyphenylglycine (CHPG), all induce longlasting depression of A primary afferent fibers-mediated monosynaptic excitatory postsynaptic potential (EPSP), and long-lasting potentiation of polysynaptic EPSP, and EPSP in cells receiving C-afferent fiber input. The DHPG potentiation of polysynaptic EPSP was partially or fully reversed by (S)-4-carboxyphenylglycine (S-4CPG), the mglu subtype 1 preferring antagonist. 2-Methyl-6- (phenylethynyl)-pyridine, the potent and selective mglu subtype 5 antagonist, partially reversed the CHPG potentiation of polysynaptic EPSP. The effects of DHPG on monosynaptic and polysynaptic EPSPs were reduced, or abolished, by the N-methyl-D-aspartate (NMDA) receptor antagonist D(2)-2-amino-5-phosphonopentanoic acid (AP5). A clear and pronounced facilitation of the expression of DHPG- and CHPG-induced enhancement of polysynaptic EPSP, and EPSP evoked at C-fiber strength, was seen in the absence of gammaaminobutyric acid subtype A receptor- and glycine-mediated synaptic inhibition. Besides dual modulation of excitatory synaptic transmission, DHPG induces depression of inhibitory postsynaptic potentials evoked by primary afferent stimulation in dorsal horn neurons. In addition, group I mglu receptor agonists produced a direct persistent excitatory postsynaptic effect consisting of a slow membrane depolarization, an increase in input resistance, and an intense neuronal discharge. Cyclothiazide and (S)-4-CPG, the mglu receptor subtype 1 preferring antagonists, significantly attenuated the DHPG-induced depolarization. These results demonstrate that the pharmacological activation of group I metabotropic glutamate receptors induces long-term depression (LTD) and long-term potentiation (LTP) of synaptic transmission in the spinal dorsal horn. These types of long-term synaptic plasticity may play a functional role in the generation of post-injury hypersensitivity (LTP) or antinociception (LTD) Elsevier Science B.V. All rights reserved. Theme: Excitable membranes and synaptic transmission Topic: Long-term potentiation: physiology Keywords: Excitatory postsynaptic potential; Group I metabotropic glutamate receptors; Spinal cord dorsal horn; Slice-intracellular recording technique 1. Introduction Glutamate acts through two broad classes of receptors, ion channel-linked (ionotropic) receptors, which include It is now well established that primary afferent fibers use a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid glutamate as a principal fast excitatory transmitter in the (AMPA), kainate, and N-methyl-D-aspartate (NMDA) redorsal horn (DH) of the spinal cord, the first modulatory site in the relay of sensory information to the brain [,98]. ceptors, and metabotropic receptors (mglurs) which cou- ple via G-proteins to the intracellular second messenger cascades and ion channels. Eight mglu receptor subtypes have been cloned to date and are classified into three *Corresponding author. Tel.: ; fax: groups based on structural homology, pharmacology and address: mrandic@iastate.edu (M. Randic). signal transduction mechanisms: group I (mglu receptors / 00/ $ see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S (00)

2 360 J. Zhong et al. / Brain Research 887 (2000) and 5) are coupled to phospholipase C and stimulate mm thick) were cut with attached dorsal roots in an phosphoinositide hydrolysis and intracellular Ca signal oxygenated (95% O 2, 5% CO 2) Krebs-bicarbonate solutransduction, whereas group II (mglu receptors 2 and 3) tion (48C) on a vibratome and placed in a holding chamber and group III (mglu receptors 4 and 6 8) are negatively (36618C) to recover for at least 1 h. A single slice was coupled to adenyl cyclase [1,16,79]. These receptors then transferred into an interface-type recording chamber modulate synaptic transmission and neuronal excitability. where it was submerged beneath an oxygenated superfus- Class I mglu receptors have been postulated to play a role ing medium (flow-rate of about 3 ml/ min, C) in synaptic plasticity such as long-term potentiation (LTP) containing (in mm): NaCl, 124; KCl, 1.9; KH2PO 4, 1.2; [3,9,106] and long-term depression (LTD) of synaptic CaCl, 2.4; MgSO, 1.3; NaHCO, 26; glucose, 10; ph transmission [53,55,71] in the brain, as well as in the 7.4, mosm, and was equilibrated with 95% O, 2 spinal cord [32,82]. 5% CO 2. Although the presence of multiple mglu receptors (subtypes 1 5 and 7) in the spinal cord DH has been 2.2. Dorsal root stimulation and intracellular recording shown [10,46,54,70,89,92 94], their roles in physiology and pathophysiology of synaptic transmission are not clear Intracellular recordings with sharp microelectrodes were and are complicated by the diversity of pre- and postsynap- made from DH neurons (laminae I V), including subtic receptors. There is behavioral and electrophysiological stantia gelatinosa (SG, lamina II) cells. When viewed evidence that the activation of mglu receptors, in par- under a dissecting microscope at a magnification of 10 ticular, mglu subtypes 1/ 5, increases the excitability of the 403 with transmitted illumination, the SG was distinguishrat spinal DH neurons [51,64,65] and facilitates responses able as a translucent bend in the superficial DH, although it to NMDA and AMPA receptor activation was difficult to discern with certainty the border between [5,8,12,24,25,48,61,69, ]. Studies of the actions of laminae I and II. Under visual control, a single fiberglass mglu receptor agonists and antagonists on responses of ([6010; O.D. and I.D., 1.0 and 0.58 mm, respectively; AM deep DH neurons [68,69, ] to noxious and non- Systems) microelectrode filled with 4 M potassium acetate noxious stimuli indicate that mglu receptors are involved (ph 7.2) (DC resistance: MV) was placed in the in mediating nociceptive inputs. In particular, group I SG or deep DH (DDH), and neurons were impaled by mglu 1/ 5 receptors have been implicated in mediating oscillating the capacity compensation circuit of a highnociception following a sustained noxious input [24 input impedance bridge amplifier (Axoclamp 2A, Axon 26,28,29,104,105]. However, the synaptic and cellular Instruments). A DC pen-recorder was used to record mechanisms underlying functional plasticity following membrane potentials continuously and a Digidata 1200 tissue or nerve injury are not known. Moreover, the data system with PCLAMP (version 6) software (Axon Instrudescribed above are largely based on observations obtained ments) was used for data acquisition and analysis. Most from wide-dynamic-range neurons in deep DH laminae of recordings were obtained from cells with a stable resting rats and primates in vivo. membrane potential (more negative than 255 mv) and In the present study we examined the effects of group I with overshooting action potentials. The protocol for mglu receptor activation by the group I and II mglu assessing the effects of mglu receptor agonists and antagoagonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylate nists on EPSPs was as follows. Monosynaptic and poly- (1S,3R-ACPD), (S)-3,5-dihydroxyphenylglycine (DHPG), synaptic EPSPs in DH neurons were evoked by orthoa selective agonist for the group I mglu receptors [88], and dromic electrical stimulation of primary afferent fibers in (RS)-2-chloro-5-hydroxyphenylglycine (CHPG), a selec- the lumbar dorsal root (L4 and/ or L5) using a bipolar tive mglu subtype 5 agonist [20] on the synaptic responses platinum wire electrode or glass suction electrode (with the mostly of rat superficial dorsal horn neurons to primary cathode internal). Single shocks ( ms pulses, 2 35 afferent stimulation in vitro. Some of the results have been V), repeated at 2-min intervals, were given for at least 10 reported previously in abstract form [51,84,108]. min before, during (10 min), and for a min period after bath administrations of chemicals. This frequency of stimulation was chosen for sampling data because it did 2. Materials and methods not result in response facilitation or depression. A stimulus intensity that yielded a 5 15 mv EPSP was chosen to 2.1. Spinal slice preparation standardize the baseline synaptic strength across slices, and it was below threshold for eliciting an action potential in Experiments were performed on spinal cord slices most of the slices chosen for study. The stimulus intensity obtained from young Sprague Dawley rats (17 26 days necessary to activate Ad and C fibers and the afferent fiber old) of either sex, as described previously [33,83]. Under conduction velocity were determined by extracellular deep ether anesthesia segments of the lumbosacral (L 4 S 1) recording of compound action potentials from longitudinal spinal cord were removed with long (8 15 mm) dorsal spinal slice-dorsal root-dorsal root ganglia preparations in roots. Longitudinal or several transverse slices ( the previous experiments [49]. The classification of EPSPs

3 J. Zhong et al. / Brain Research 887 (2000) in relation to the primary afferents activated was done synaptic responses, EPSP area, was used. The area was solely on the basis of conduction velocity which was calculated under the curve of polysynaptic EPSPs in a calculated either by measuring the distance between the 180-ms time window starting at the onset of the EPSP. We stimulating electrode and the recording site on the dorsal first tested the stability of the synaptic and passive root and dividing by the conduction latencies of action membrane properties of DH neurons in slices over a period potentials recorded, or from the latency of evoked EPSPs of min and next examined the changes in these and the distance from the stimulating electrode to the properties as a result of mglu receptor-ligand treatment. recording site. Primary afferents conducting at velocity Over a recording period of 1 2 h, resting membrane above 15 m/s were classified as Ab [76], whereas those potential, input resistance, and the peak amplitude of the conducting between 1.5 and 15 m/s were classified as Ad, EPSP did not change significantly (#10% change in peak and those conducting below 1.5 m/ s as C fibers. The amplitude) in DH neurons of untreated slices used in this minimum stimulus intensities and durations used to acti- study. All values are expressed as means6s.e.m. Statistivate Ad and C fibers were 3 V/ 0.1 ms and 5 V/ 0.5 ms, cal significance of data (P#0.05) has been assessed respectively. Stimulation of dorsal roots led to generation relative to control responses by use of either paired or of an EPSP. With small stimulus strength this EPSP was unpaired Student s t-test, as appropriate. graded in amplitude, had a fixed latency and monophasic decay. As the stimulus strength was increased, however, a 2.4. Application of chemicals later slow polysynaptic component(s) was apparent. In order to discriminate between monosynaptic and poly- Drugs were dissolved in oxygenated recording solution synaptic EPSPs, two criteria were used: (1) EPSPs latency immediately prior to use, and applied to the slices in should not change with increasing intensities of electrical known concentrations by addition to the superfusing stimulation; and (2) EPSPs should follow high frequency medium. All compounds were applied via the bath, and stimulation (50 Hz) with reduced amplitude but no change each neuron served as its own control. Drug-containing in latency. The polysynaptic EPSPs have variable latencies solution entered the recording chamber within s of and show failures with high frequency stimulation. More- changing solutions, with complete exchange occurring over, the shapes and amplitudes of polysynaptic EPSPs are within 3 min. Drugs were administered for a sufficient variable in different trials when dorsal roots are stimulated period (5 10 min) to allow their full equilibration. Only at a constant intensity. Input resistance was measured at one cell in a slice was subject to one trial with mglu 2-min intervals by passing a hyperpolarizing current pulse receptor agonist, the exception being the experiments using of 0.05 na across the cell membrane and measuring the mglu receptor antagonists where each cell was subjected voltage deflection produced. The current values were of to two or three trials. sufficient duration ( ms) to fully charge the Chemicals used and their sources were as follows: membrane capacitance. Bridge balance was monitored (2)-bicuculline methiodide and strychnine hydrochloride throughout experiments and corrected when necessary. To from Sigma (St. Louis), (1S,3R)-1-aminocyclopentane-1,3- reduce the increased spontaneous synaptic activity and dicarboxylate (1S,3R-ACPD), D-2-amino-5-phosphonopensubsequent action potential firing due to the removal of tanoate (D-AP5), (S)-4-carboxyphenylglycine (4-CPG), synaptic inhibition, the Mg concentration in the super- (RS)-2-chloro-5-hydroxyphenylglycine (CHPG), 6-cyanofusing solution was increased to 3 mm in the experiments 7-nitroquinoxaline-2,3-dione (CNQX), cyclothiazide where bicuculline and strychnine were applied to block the (CTZ), (S)-3,5-dihydroxyphenylglycine (DHPG), 6-nitro- GABAA and glycine receptors. 7-sulphanoylbenzo[ f ]quinoxaline-2,3-dione (NBQX), all obtained from Tocris Cookson Ltd. (Bristol, UK), Data analysis methyl-6-(phenylethynyl)-pyridine (MPEP) kindly provided by Novartis, Switzerland, tetrodotoxin (TTX; The mglu receptor agonists were applied in the perfu- Alomone Labs, Jerusalem, Israel). All solutions were sate for 5 10 min in the absence or continuous presence of freshly prepared every day from stock solutions that were the antagonist. The magnitude of their effects in any stored at 2208C. individual cell was determined by comparing the averaged peak amplitude of three consecutive EPSPs evoked immediately prior to drug application (V control) to the averaged 3. Results peak amplitude of three consecutive EPSPs measured at the time of maximal change induced by mglu receptor Stable intracellular recordings of up to 5 h were agonists (V treatment). Vtreatment was typically determined obtained from 119 DH (94 in LI and II; 25 in LIII V) during application of mglu receptor ligand, and also 20 neurons in the longitudinal (19) and transverse (86) spinal 22 min following the washout of mglu receptor agonist, cord slices. The average resting membrane potential of and was expressed as percentage of control: V treatment / these neurons was mv (mean6s.e.m), and the Vcontrol Moreover, an additional measure of poly- input resistance MV, in agreement with previ-

4 362 J. Zhong et al. / Brain Research 887 (2000) ous results [83,100]. Single shock electrical stimulation of applications of mm (1S,3R)-ACPD for 10 min the primary afferent fibers in a L4 or L5 dorsal root caused a slow membrane depolarization ( mv) in elicited monosynaptic and/or polysynaptic EPSPs in DH 20 (13 SDH and seven DDH cells) of 28 DH neurons as cells that were suppressed by 1 10 mm NBQX in a described previously [51,64]. This effect was associated reversible manner (to 360.8%, n526), suggesting that with nonsignificant changes in input resistance they were principally mediated by the AMPA subtype of ( % of control at 10 min of agonist application; glutamate receptors. A small component sensitive to n512). The depression of the EPSP amplitude was not application of the NMDA antagonist D-AP5 (100 mm) was due to (1S,3R)-ACPD-induced depolarization since the also observed, such that co-application of NBQX (10 mm) membrane potential was always adjusted to its control and D-AP5 (100 mm) led to an almost complete block of level prior to dorsal root stimulation. the EPSP [31,83,100] Potentiation of EPSPs in DH cells receiving inputs 3.1. Depression of monosynaptic EPSP and potentiation from C-afferent fibers of polysynaptic EPSP by (1S,3R)-ACPD Superfusion of spinal slices with (1S,3R)-ACPD (25 The activation of group I/ II mglu receptors by the 100 mm, 10 min) caused variable changes in peak amprototypic broad-spectrum agonist (1S,3R)-ACPD resulted plitude of the EPSPs in DH cells (six SG and two DDH in a depression of A-fiber-mediated monosynaptic EPSP neurons) receiving input from C-fibers, that when averaged and potentiation of polysynaptic EPSP in DH neurons. showed an initial, statistically non-significant depression During the superfusion of slices with (1S,3R)-ACPD (25 during (1S,3R)-ACPD administration (to % of 100 mm, 10 min) the amplitude of the presumed A-fiber- control; n54) followed by a late reversible potentiation (to evoked monosynaptic EPSP was reduced in a dose-depen % of control; n54; P,0.05) upon washout dent manner in 14 (12 SG and two deep DH neurons) of (Fig. 2B). Because neurons that exhibited C-fiber strength- 17 DH neurons studied both in longitudinal (n57) and evoked synaptic responses also received A-fiber-mediated transverse (n57) spinal slices (Fig. 1A, Table 1). How- input, and often polysynaptic EPSPs, it was usually ever, in three of 17 DH cells mm (1S,3R)-ACPD possible to analyze C-fiber-evoked input only in the subset reversibly increased monosynaptic EPSPs both during and of neurons that did not receive a preceding A-fiber input. after washout of the agonist (Table 1). The peak depres- To investigate further the actions of (1S,3R)-ACPD on DH sion had a latency of 6 8 min and the effect persisted for cells receiving input only from C-afferents, our approach more than 25 min after the application of (1S,3R)-ACPD was to selectively block the action potential generation in was terminated (Fig. 1A). The depressant effect of large primary afferent fibers by using the sodium channel (1S,3R)-ACPD (100 mm) was reduced (from 55.4 to blocker TTX [97,99]. Fig. 2A illustrates a typical experi- 79.1% of baseline) by the mglu subtype 1 preferential ment in which TTX (0.5 mm) when applied to DRG and antagonist 4-CPG (500 mm for 20 min), as illustrated in dorsal roots blocked the fast component of the synaptic Fig. 1B. response induced by low threshold DR stimulation, but did Besides depressing monosynaptic EPSP, (1S,3R)-ACPD not block the component induced by activation of high (10 50 mm, 10 min) produced a reversible increase of the threshold, slowly (C) conducting afferents. The isolated peak amplitude of A-fiber-evoked polysynaptic EPSP, and C-fiber strength-evoked EPSP was almost completely the area under the curve of EPSPs (Fig. 1C) in eight cells blocked (Fig. 2B, trace 2 inset) during 6 min superfusion (four SG and four deep DH cells) examined (Table 2). The with 50 mm (1S,3R)-ACPD. Following removal of maximal effect had a latency of 8 10 min and it persisted (1S,3R)-ACPD from the bath, a sustained potentiation of for approximately 25 min (Fig. 1C). The potentiating the synaptic response (to about 200% of control) was effects of (1S,3R)-ACPD (100 mm) on a polysynaptic observed. EPSP evoked by low-intensity stimuli (Fig. 1D, left column), and a DR-evoked EPSP suprathreshold for action 3.3. Dual modulation of excitatory transmission and potential firing followed by longer-lasting polysynaptic synaptic plasticity following activation of the group I activity evoked by high-intensity stimuli (Fig. 1D, right mglu receptors column), are illustrated in Fig. 1D. A significant increase in the EPSP size was evident for the polysynaptic component To characterize the spinal mglu receptor subtype(s) evoked by high-intensity stimuli after application of responsible for dual effects of (1S,3R)-ACPD on synaptic (1S,3R)-ACPD. As illustrated in Fig. 1D, the underlying responses of DH neurons, we next examined the actions of increase in the peak amplitude of the EPSP is sufficient to two group I mglu receptor selective agonists DHPG and induce the spike activity, and the number of action CHPG, as well as mglu receptor subtype 1 preferring potentials in response to high-threshold current increased antagonist 4-CPG. Group I mglu receptors may be difby almost 6-fold. ferentiated pharmacologically using CHPG, which is active When recording at resting membrane potential, bath at mglu subtype 5, but not mglu subtype 1 [20], and

5 J. Zhong et al. / Brain Research 887 (2000) Fig. 1. Dual modulation of dorsal root-evoked EPSPs by the broad spectrum mglur agonist (1S,3R)-ACPD in the spinal DH, and reduction of (1S,3R)-ACPD-induced depression of monosynaptic EPSP by the group I mglur antagonist 4-CPG. (A) Bath application of (1S,3R)-ACPD ( mm, 10 min; n53 (25mM), n56 (50mM), n55 (100 mm)) caused a depression of apparently monosynaptic Ad fiber-evoked EPSPs (c.v m/ s) in 14 of 17 neurons tested, which was sustained after the drug washout. Vm 5262 to 286 mv, day-old rats. (B) In a SG neuron receiving monosynaptic input from Ad primary afferent fibers (c.v. 2.5 m/ s), superfusion of 100 mm (1S,3R)-ACPD reduced the amplitude of the EPSPs evoked by orthodromic stimulation of a DR. 4-CPG (500 mm), the mglur I preferential antagonist, produced no significant changes in the EPSPs by itself. However, the depressant effect of (1S,3R)-ACPD was reduced in the presence of 4-CPG in a reversible manner. Vm 5273 mv, 23 day-old rat. (C) Summarized data from eight DH neurons showing the time course of the potentiation of the DR-evoked polysynaptic EPSPs by (1S,3R)-ACPD (10 50 mm, 6 10 min; n51 (10 mm), n52 (25mM), n55 (50mM)). The area under the curve of EPSPs showed a faster and greater increase following (1S,3R)-ACPD application in comparison with the EPSP amplitude (not shown). Vm 5270 to 278 mv, day-old rats. In this and in all subsequent figures the summary graphs (means6s.e.m.) show the time course of changes in the peak amplitude/ area of EPSPs, whereas above the graph are displayed individual EPSPs taken at the time marked by the corresponding number, the solid bar above the graph indicates the time at which drug application occurred. (D) (1S,3R)-ACPD (100 mm, 5 min) increased the amplitude and duration of EPSP and action potential firing (right panel, middle trace) evoked by high (25 V, 0.5 ms) intensity stimulation of primary afferents. Upper row shows control responses, middle row responses obtained 5 min after the onset of (1S,3R)-ACPD application, lower row recovery following 12 min washout. Vm 5270 mv, 24 day-old rat. MPEP, the potent and selective mglu subtype 5 antagonist under the curve of polysynaptic EPSPs (Fig. 3D, Table 2), [30]. in a dose-dependent manner (Fig. 3D inset-graph). The EPSP depression/ potentiation persisted without recovery 3.4. Dual modulation of DR-evoked EPSPs by DHPG for more than 30 min. To determine the subtype of the group I mglu receptor involved in mediating potentiation In a normal medium, the S-isomer of DHPG (10 mm, 10 of A-fiber-evoked monosynaptic EPSP by DHPG, we min) produced a sustained depression in the amplitude of examined the action of mglu subtype 1 preferential the A-fiber-mediated monosynaptic EPSP in nine of 13 antagonist (S)-4-CPG. Bath application of (S)-4-CPG at cells examined (Fig. 3A, Table 1). However, in four DH 200 mm, by itself, had no effect on the peak amplitude of cells DHPG produced a persistent enhancement of the EPSP (n53). However, the EPSP potentiation caused by monosynaptic EPSPs (Fig. 3B, Table 1). In addition, DHPG (10 mm) was partially or fully reversed (DHPG: DHPG (1 100 mm, 10 min) induced a prolonged increase % of baseline; DHPG14CPG: %, n5 in the peak amplitude (Fig. 3C, Table 2), and the area 3; P,0.02) on co-application of (S)-4-CPG (Fig. 3E).

6 364 J. Zhong et al. / Brain Research 887 (2000) Table 1 Modulation of primary afferent fiber-evoked monosynaptic EPSPs by mglu receptor agonists in different perfusion media mglur agonists Control solution Control1D-AP5 Depression Potentiation Depression (1S,3R)-ACPD n 14 3 ( mm) During application ** Washout * * DHPG n (10 mm) During application ** * Washout * * * CHPG n 7 (500 mm) During application ** Washout * a Values, expressed as percentage of control (means6s.e.m), show the changes induced by (1S,3R)-ACPD, DHPG and CHPG in the peak amplitude of monosynaptic EPSPs evoked by stimulation of primary afferent fibers in lumbar dorsal roots. Values represent the peak change recorded during application of respective mglu receptor agonist in control solution or in a solution containing 50 mm D-AP5. Washout values are taken min after removal of the agonist. Statistical significance of data is indicated by asterisks: * P,0.05; ** P,0.01. a Taken together, these data indicate that whereas mono- the normal medium. The mean value at 20 min following synaptic EPSPs are susceptible to both potentiation and washout was significantly different (P,0.01) from that in depression, polysynaptic EPSPs are potentiated by activa- the absence of D-AP5 (Table 1). Furthermore, DHPG (10 tion of the group I mglu receptor selective agonist DHPG. mm, 10 min) in the presence of D-AP5 produced a small, reversible decrease of the peak amplitude of DR-evoked 3.5. Depression and potentiation of synaptic responses polysynaptic EPSP during perfusion, followed by a tranby DHPG is dependent on NMDA receptor sient increase upon washout (Fig. 4B, Table 2). However, the mean value at 20 min after washout was significantly The sensitivity of DHPG-induced depression and poten- (P,0.05) lower than that from slices exposed to DHPG in tiation of EPSPs to the NMDA receptor antagonist D-AP5 the absence of D-AP5. was tested since some forms of activity-dependent, longterm changes in synaptic strength have been shown to 3.6. Effects of bicuculline and strychnine on modulation require activation of this receptor system [1]. The effect of of synaptic responses by DHPG the D-AP5 was first examined on the long-lasting depression of monosynaptic EPSP induced by DHPG. As shown Given the importance of inhibitory processes in the in Fig. 4A, DHPG (10 mm, 10 min) applied in the temporal and spatial control of sensory responses in the presence of D-AP5 (50 mm) induced a decrease in DH, and a recent evidence of co-localization of group I amplitude of monosynaptic EPSP only in two of six SG mglu5 with GABA in the superficial laminae of spinal DH cells during the application, but almost completely [46], we have investigated in the present study possible abolished a long-lasting depression induced by DHPG in interaction of DHPG with inhibitory processes within this Table 2 Modulation of primary afferent fiber-evoked polysynaptic EPSPs by mglu receptor agonists in different perfusion media mglur agonist Control solution Control1bicuculline1strychnine Control1D-AP5 Amplitude Area Amplitude Area Amplitude (1S,3R)-ACPD n 8 ( mm) During application ** Washout * DHPG n (10 mm) During application * * * Washout ** * * * * CHPG n 6 6 (500 mm) During application * Washout ** ** * * a Values, expressed as percentage of control (means6s.e.m.), show the changes induced by (1S,3R)-ACPD, DHPG and CHPG in the peak amplitude and the area of polysynaptic EPSPs evoked by stimulation of primary afferent fibers in lumbar dorsal roots. Values represent the peak change recorded during application of respective mglu receptor agonist in control solution or in a solution containing bicuculline (5 mm) and strychnine (2 mm) or D-AP5 (50 mm). Washout values are taken min after removal of the agonist. Statistical significance of data is indicated by asterisks: * P,0.05; ** P,0.01. a

7 J. Zhong et al. / Brain Research 887 (2000) dependent on the modulation of synaptic inhibition by the group I mglu receptors. In support of this possibility, a marked reduction in the amplitude of DR-evoked IPSP in SG (Fig. 6A, n53) and DDH (Fig. 6B) was observed Effects of DHPG on membrane properties Bath application of mm DHPG for 10 min caused a slow, dose-dependent and reversible membrane depolarization ( mv, Fig. 7) associated with an increase in membrane input resistance ( % of control, measured at peak response; n517, P,0.05) in 28 of 38 DH neurons examined, or a hyperpolarization ( mv) accompanied by a decrease in input resistance ( % of control) in ten of 38 cells (31 SDH and seven DDH cells). The depolarizing and hyperpolarizing responses outlasted the period of DHPG application for min, n524 and min, n56, respectively. Two types of depolarizing responses to DHPG can be recorded from DH neurons. As illustrated in Fig. 7A, in most of SDH neurons the depolarization ( mv, n5) was accompanied by an increase in baseline noise, whereas in the deep DH neurons, DHPG often produced a larger depolarization ( mv, n57) and increase in excitability, as indicated by the generation of sustained firing of spontaneous action potentials (Fig. 7B). To determine whether the depolarization resulted Fig. 2. Potentiation of EPSPs recorded from cells receiving C-fiber input from a direct postsynaptic action on DH neurons, and to by the broad spectrum mglur agonist (1S,3R)-ACPD in the spinal DH. (A) The A-fiber component of the EPSP evoked by high intensity eliminate the possibility that DHPG activated ionotropic stimulation (25 V, 0.5 ms) of a dorsal root was blocked by 0.5 mm TTX glutamate receptors, the agonist was applied after blockade 1 revealing the C-fiber component (c.v. 0.3 m/ s). Vm 5278 mv, 26 day-old of voltage-dependent Na channels with TTX (0.5 mm) rat. (B) Summarized data from four DH neurons showing the time course and ionotropic glutamate receptors by NBQX (10 mm) and of the initial depression and late potentiation of the EPSPs of cells D-AP5 (30 mm). Under these conditions, DHPG produced receiving C-fiber inputs (c.v m/ s) by (1S,3R)-ACPD ( mm, 10 min n51 (25mM), n52 (50mM), n5l (100 mm)). Above the a depolarization (Fig. 7B, right trace) that was similar to graph are displayed individual EPSPs from an experiment where the that observed under control conditions (Fig. 7B, left trace). stimulated dorsal root was bathed in 0.5 mm TTX to block A-fiber inputs. These data indicate that the depolarization is independent Vm 5270 to 278 mv, day-old rats. of AMPA or NMDA receptor activation by increased ambient levels of glutamate or a related excitatory amino spinal cord area. We tested the possibility that DHPG- acid receptor agonist. (S)-4-CPG (200 mm) (not shown) induced depression of monosynaptic EPSP and potentia- and cyclothiazide (25 mm) (Fig. 7C, middle trace), the tion of polysynaptic EPSP is caused by a long-term change preferential mglu1 receptor antagonists reversibly dein the strength of synaptic inhibition by performing pressed the DHPG-induced depolarization. Taken together experiments in the presence of bicuculline (5 mm) and these findings suggest that the depolarizing effect on DH strychnine (2 mm) to eliminate the GABAA and glycine neurons is caused by DHPG acting on postsynaptic mglu1 receptor-mediated synaptic inhibition, respectively. In each receptors on DH neurons themselves, rather than on of four slices bathed in this solution, the DHPG-induced afferent fibers presynaptic to these cells. Furthermore, as increases of the peak amplitude and area of A-fiber-evoked illustrated in Fig. 8, DHPG ( mm, 10 min) induced polysynaptic EPSP (Fig. 5A, Table 2), and EPSP-elicited a spontaneous oscillatory activity in seven out of 23 SDH at C-fiber strength (Fig. 5B), were greatly augmented after and four of seven DDH neurons. These oscillations inhibition is blocked. However, the DHPG-induced long- manifested as rhythmic oscillations in membrane potential lasting depression of monosynaptic EPSP was similar to frequently gave rise to sustained rhythmic activity charthat seen under control conditions. Thus, it can be con- acterized by burst firing of spikes (Fig. 8A). On removal of cluded that whereas the DHPG-induced long-lasting de- DHPG, the depolarizing/ hyperpolarizing episodes conpression of monosynaptic EPSP occurs independently of tinued for several minutes before the membrane potential long-term changes in synaptic inhibition, the DHPG-in- returned to control level. The mean peak amplitude and duced slow onset potentiation of the polysynaptic EPSP is frequency of oscillations induced by mm DHPG

8 366 J. Zhong et al. / Brain Research 887 (2000) Fig. 3. Dual modulation of dorsal root-evoked EPSPs by S-DHPG, and reduction of S-DHPG-induced potentiation of EPSPs by the group I mglur antagonist 4-CPG. (A) Summarized data showing the time course of the depression of monosynaptic EPSPs (Ad input: m/ s) for nine SG neurons produced by 10 mm (10 min) S-DHPG. Vm 5262 to 277 mv, day-old rats. (B) Long-lasting potentiation of monosynaptic EPSPs was observed in four SG neurons after 10 mm (10 min) DHPG application. The inset-graph shows grouped data (mean6s.e.m.) representing % of control EPSP peak amplitude vs. concentration of DHPG (0.1 mm, n5l; 1 mm, n56; 10 mm, n54; 100 mm; n52) measured at 20 min after the offset of DHPG application (at the maximum of the EPSP potentiation). Vm 5259 to 276 mv, day-old rats. (C) The graph shows the time course of the long-lasting potentiation of A-primary afferent fiber-evoked polysynaptic EPSPs for eight SG neurons produced by S-DHPG (10 mm, 10 min). Vm 5265 to 283 mv, 17 day-old rats. (D) In three SG neurons S-DHPG 100 mm (10 min) produced a large increase in the area under the curve of polysynaptic EPSP. As seen on the inset-graph, this prolonged facilitatory effect was dose-dependent. Each point is the mean6s.e.m. of between three and eight experiments. Each point was calculated at 20 min after the offset of S-DHPG application. Vm 5253 to 273 mv, day-old rats. (E) In a SG neuron, DHPG (10 mm, 5 min) produced an increase in the area under the curve of polysynaptic EPSPs-evoked by stimulation of A-primary afferent fibers. This effect war blocked by 4-CPG (200 mm), the mglur I preferential antagonist, in a reversible manner. Vm 5274 mv, 22 day-old rat.

9 J. Zhong et al. / Brain Research 887 (2000) Fig. 5. Blockade of GABAA- and glycine-mediated synaptic inhibition enhances the S-DHPG-induced potentiation of polysynaptic EPSPs. (A) Summarized data showing the enhanced potentiation in the peak am- plitude of polysynaptic EPSPs induced by S-DHPG (10 mm, 10 min) in four SG neurons in the presence of bicuculline (5 mm), strychnine (2 mm) and a high concentration of Mg (3 mm). Vm 5261 to 289 mv, day-old rats. B, The graph shows the time course of changes in the peak amplitude of EPSPs caused by S-DHPG (10 mm, 10 min) in a SG neuron receiving input from C-afferent fibers (c.v. 0.7 m/ s). Note also the long lasting potentiation of EPSPs accompanied by action potential firing as shown on the individual EPSPs above the graph Vm 5261 mv, 17 day-old rat. Fig. 4. NMDA receptor dependence of the S-DHPG-induced long-lasting depression and long-lasting potentiation of EPSPs. (A) The graph shows the pooled data from six SG cells in which the S-DHPG-induced h10 mm, 10 min) initial depression of monosynaptic EPSPs (Ad input: n56, m/ s) was reduced and later long-lasting depression abolished in the presence of 50 mm D-AP5, the NMDA receptor antagonist. Vm 5264 to 277 mv, day-old rats. (B) Summary graph (n57) showing the antagonism of the S-DHPG-induced (10 mm, 10 min) potentiation of the polysynaptic EPSPs by DAP5 (50 mm). Vm 5255 to 277 mv, day-old rats. was mv and Hz (n511), respectively. The induction of oscillations was concentration-dependent in that the number and frequency of oscillations increased with higher concentrations or time of exposure to DHPG. Experiments were also undertaken to determine if inhibition plays a significant role in producing the DHPGinduced oscillatory activity. An interesting finding was that produced a sustained depression (Fig. 9A, Table 1) or the oscillations induced by 10 mm DHPG persisted in the potentiation (in 2/3 cells with 1 mm CHPG) of Ad-fiberpresence of bicuculline (5 mm) and strychnine (2 mm), the evoked monosynaptic EPSP and prolonged potentiation of GABAA and glycine receptor antagonists, in a cell ex- the polysynaptic EPSP (Fig. 9B, Table 2). Both effects hibiting excitatory synaptic noise (Fig. 8C), but were outlasted the period of CHPG application for more than 25 abolished (Fig. 8D, right trace) in the cell in which DHPG min. a-methyl-6-(phenylethynyl)-pyridine (MPEP), the applied in a normal medium produced a marked increase in potent metabotropic glutamate receptor subtype 5 antagoinhibitory synaptic noise (Fig. 8D, left trace). nist [30], almost completely reversed the polysynaptic EPSP potentiation (Fig. 9E) caused by CHPG (n53). In 3.8. Dual modulation of DR-evoked EPSPs by CHPG the presence of bicuculline (5 mm), strychnine (2 mm), In a normal medium, CHPG (0.5 1 mm, 5 min) and 3 mm Mg, the potentiation of polysynaptic EPSP by CHPG was significantly greater than that recorded in a

10 368 J. Zhong et al. / Brain Research 887 (2000) 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 plasticity 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 gelatinosa and deep dorsal horn, and 2) a long-lasting potentiation of polysynaptic EPSP. The effects of DHPG on monosynaptic and polysynaptic EPSPs were inhibited by D-AP5. The increases in polysynaptic EPSPs produced by DHPG and CHPG are greatly augmented after synaptic inhibition is blocked. In addition, we confirmed previous demonstra- tion of a direct increase in excitability of DH neurons by the mglu receptor agonist (1S,3R)-ACPD [51,64] and demonstrated that the effect is exerted via the activation of group I mglu receptors. Fig. 6. S-DHPG decreased the dorsal root-evoked inhibitory postsynaptic potentials (IPSPs) in DH neurons. (A) S-DHPG (1 mm, 10 min) blocked the DR-evoked IPSP in a SG neuron. Vm 5264 mv, 19 day-old rat. (B) The graph shows the time course of the reversible depression of DRevoked IPSPs following bath application of S-DHPG (10 mm, 10 min) in a deep DH neuron. The individual IPSPs from the same experiment are shown above the graph. Vm 5254 mv, 18 day-old rat 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 GABAA- and glycine-mediated 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 (n5 effect appears to be specific for group I mglu receptors, as 13), or membrane input resistance (n515). However, 1 it could be mimicked by DHPG and CHPG, the selective mm CHPG produced a prolonged depolarization ( group I agonists, and was attenuated by 4-CPG, the group I mv, n55, 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,

11 J. Zhong et al. / Brain Research 887 (2000) 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). Vm 5267 mv (A), 273 mv (B), 19 day-old rats. (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). Vm 5262 mv, day-old rat. which belong to each of the three major groups, can serve 4.2. Activation of group I mglu receptors induces longas 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 deand 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 transmglu1a 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], wheremglur5 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 transmisthe 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 presagonist-induced depolarization, that it is presynaptic. How- ent, the mechanism(s) 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

12 370 J. Zhong et al. / Brain Research 887 (2000) 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. Vm 5263 mv, 18 day-old rat. (B) In a SG neuron addition of bicuculline (5 mm) and strychnine (2 mm), the GABAA and glycine receptor antagonists, to the perfusion medium augmented the amplitude of the oscillatory activity 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 synaptic inhibition, the Mg concentration in the perfusing solution was increased to 3 mm. Vm 5279 mv, 18 day-old rat. (C,D) The increase in the 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) Vm5274 mv, 19 day-old rat. (D) Vm5253 mv, 18 day-old rat. 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 cocord [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 depenof 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

13 J. Zhong et al. / Brain Research 887 (2000) 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: n57, 2 10 m/s) by bath-applied selective mglur5 agonist RS-CHPG (0.5 mm, 5 min). Vm 5264 to 280 mv, 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. Vm 5265 to 277 mv, 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 (n56). Vm 5258 to 277 mv, 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 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). Vm 5269 mv, day-old rat. (E) In the presence of MPEP (10 mm), a potent mglur5 antagonist, CHPG (1 mm, 5 min) had no significant effect on 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. Vm 5280 mv, 22 day-old rat.