Materials and methods not result in response facilitation or depression. A stimulus

360 J and 5 are coupled to phospholipase C and stimulate mm thick were cut with attached dorsal roots in an 21 phosphoinositide hydrolysis and intracellular Ca signal oxygenated 95 O , 5 CO Krebs-bicarbonate solu- 2 2 transduction, 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, 34–358C in synaptic plasticity such as long-term potentiation LTP containing in mM: NaCl, 124; KCl, 1.9; KH PO , 1.2; 2 4 [3,9,106] and long-term depression LTD of synaptic CaCl , 2.4; MgSO , 1.3; NaHCO , 26; glucose, 10; pH 2 4 3 transmission [53,55,71] in the brain, as well as in the 7.4, 310–320 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 sub- tic 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 distinguish- rat 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 [5,8,12,24,25,48,61,69,102–105]. 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 [68,69,103–105] 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: 140–220 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 high- nociception 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 Instru- described 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 antago- agonist 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 ortho- a 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 0.01–0.5 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 30–60 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 –S recording of compound action potentials from longitudinal 4 1 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 300–400 the previous experiments [49]. The classification of EPSPs J . Zhong et al. Brain Research 887 2000 359 –377 361 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 10–20 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. Statisti- vate 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 components 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 30–45 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 200–300 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-phosphonopen- subsequent action potential firing due to the removal of tanoate D -AP5, S -4-carboxyphenylglycine 4-CPG, 21 synaptic inhibition, the Mg concentration in the super- RS -2-chloro-5-hydroxyphenylglycine CHPG, 6-cyano- fusing 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- GABA and glycine receptors. 7-sulphanoylbenzo[ f ]quinoxaline-2,3-dione NBQX, all A obtained from Tocris Cookson Ltd. Bristol, UK, 2- 2.3. Data analysis methyl-6-phenylethynyl-pyridine MPEP kindly pro- vided 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 imme- diately prior to drug application V to the averaged 3. Results control 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 . V was typically determined obtained from 119 DH 94 in LI and II; 25 in LIII–V treatment treatment 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 these neurons was 270.060.7 mV mean6S.E.M, and the treatment V 3 100. Moreover, an additional measure of poly- input resistance 133.067.7 MV, in agreement with previ- control 362 J ous results [83,100]. Single shock electrical stimulation of applications of 10–100 mM 1S,3R-ACPD for 10 min the primary afferent fibers in a L4 or L5 dorsal root caused a slow membrane depolarization 7.060.9 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, n 526, suggesting that with nonsignificant changes in input resistance they were principally mediated by the AMPA subtype of 100.263.5 of control at 10 min of agonist application; glutamate receptors. A small component sensitive to n 512. 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]. 3.2. 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 am- prototypic 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 62.2621.6 of 100 mM, 10 min the amplitude of the presumed A-fiber- control; n 54 followed by a late reversible potentiation to evoked monosynaptic EPSP was reduced in a dose-depen- 164.8613.8 of control; n 54; 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 n 57 and evoked synaptic responses also received A-fiber-mediated transverse n 57 spinal slices Fig. 1A, Table 1. How- input, and often polysynaptic EPSPs, it was usually ever, in three of 17 DH cells 25–100 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 subtypes 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 dif- by 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 J . Zhong et al. Brain Research 887 2000 359 –377 363 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 25–100 mM, 10 min; n 53 25 mM, n 56 50 mM, n 55 100 mM caused a depression of apparently monosynaptic Ad fiber-evoked EPSPs c.v. 1.6–5.8 m s in 14 of 17 neurons tested, which was sustained after the drug washout. V 5 262 to 286 mV, 18–23 day-old rats. B In a SG neuron receiving monosynaptic m 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. V 5 273 mV, 23 day-old rat. C Summarized data from m 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; n 51 10 mM, n52 25 mM, n55 50 mM. 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. V 5 270 to 278 mV, 20–26 day-old rats. In this and in all subsequent figures the summary graphs m 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. V 5 270 mV, 24 day-old rat. m 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 n 53. 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 139.964.9 of baseline; DHPG14CPG: 96.966.7, n 5 in the peak amplitude Fig. 3C, Table 2, and the area 3; P ,0.02 on co-application of S -4-CPG Fig. 3E. 364 J Table 1 a Modulation of primary afferent fiber-evoked monosynaptic EPSPs by mGlu receptor agonists in different perfusion media mGluR agonists Control solution Control1 D -AP5 Depression Potentiation Depression 1S,3R-ACPD n 14 3 25–100 mM During application 72.266.6 166.2653.6 Washout 75.067.3 126.463.9 DHPG n 9 4 6 10 mM During application 70.367.3 129.9610.8 92.763.7 Washout 79.569.7 130.8613.9 97.066.8 CHPG n 7 500 mM During application 85.764.0 Washout 75.968.3 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 16–22 min after removal of the agonist. Statistical significance of data is indicated by asterisks: P ,0.05; P ,0.01. 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 tran- by 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, long- term 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 depres- sion 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 a Modulation of primary afferent fiber-evoked polysynaptic EPSPs by mGlu receptor agonists in different perfusion media mGluR agonist Control solution Control1bicuculline1strychnine Control1 D -AP5 Amplitude Area Amplitude Area Amplitude 1S,3R-ACPD n 8 25–100 mM During application 140.6623.3 205.0633.7 Washout 123.069.4 128.7615.6 DHPG n 8 4 7 10 mM During application 119.4618.0 116.669.6 220.2639.1 267.9652.8 90.7613.6 Washout 133.067.6 127.1614.5 201.9640.3 240.1624.2 101.467.6 CHPG n 6 6 500 mM During application 104.267.9 113.965.1 114.768.9 139.8639 Washout 115.963.7 123.564.8 164.6632.6 191.9636.3 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 16–22 min after removal of the agonist. Statistical significance of data is indicated by asterisks: P,0.05; P,0.01. J . Zhong et al. Brain Research 887 2000 359 –377 365 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, n 53 and DDH Fig. 6B was observed. 3.7. Effects of DHPG on membrane properties Bath application of 0.1–100 mM DHPG for 10 min caused a slow, dose-dependent and reversible membrane depolarization 6.660.6 mV, Fig. 7 associated with an increase in membrane input resistance 116.966.0 of control, measured at peak response; n 517, P ,0.05 in 28 of 38 DH neurons examined, or a hyperpolarization 23.860.9 mV accompanied by a decrease in input resistance 68.6613.2 of control in ten of 38 cells 31 SDH and seven DDH cells. The depolarizing and hy- perpolarizing responses outlasted the period of DHPG application for 11.062.2 min, n 524 and 5.861.1 min, n 56, 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 6.060.5 mV, n 521 was accompanied by an increase in baseline noise, whereas in the deep DH neurons, DHPG often produced a larger depolarization 8.661.8 mV, n 57 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. eliminate the possibility that DHPG activated ionotropic A The A-fiber component of the EPSP evoked by high intensity glutamate receptors, the agonist was applied after blockade stimulation 25 V, 0.5 ms of a dorsal root was blocked by 0.5 mM TTX 1 revealing the C-fiber component c.v. 0.3 m s. V 5 278 mV, 26 day-old of voltage-dependent Na channels with TTX 0.5 mM m 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. 0.3–1.4 m s by 1S,3R-ACPD 25–100 a depolarization Fig. 7B, right trace that was similar to mM, 10 min n51 25 mM, n52 50 mM, n5l 100 mM. Above the that observed under control conditions Fig. 7B, left trace. graph are displayed individual EPSPs from an experiment where the stimulated dorsal root was bathed in 0.5 mM TTX to block A-fiber inputs. These data indicate that the depolarization is independent V 5 270 to 278 mV, 22–26 day-old rats. m 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 de- in 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 GABA and glycine neurons is caused by DHPG acting on postsynaptic mGlu1 A 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 10–100 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 char- that 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 con- pression 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 10–100 mM DHPG 366 J 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: 3–9.6 m s for nine SG neurons produced by 10 mM 10 min S-DHPG. V 5 262 to 277 mV, 18–22 day-old rats. B Long-lasting potentiation of monosynaptic EPSPs was observed in m 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, n 5l; 1 mM, n 56; 10 mM, n 54; 100 mM; n 52 measured at 20 min after the offset of DHPG application at the maximum of the EPSP potentiation. V 5 259 to 276 mV, 17–22 day-old rats. C The graph shows the time course of the long-lasting potentiation m of A-primary afferent fiber-evoked polysynaptic EPSPs for eight SG neurons produced by S-DHPG 10 mM, 10 min. V 5 265 to 283 mV, 17–21 day-old m 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. V 5 253 to 273 mV, 18–22 day-old rats. E In a SG neuron, DHPG 10 mM, 5 min m 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. V 5 274 mV, 22 day-old rat. m J . Zhong et al. Brain Research 887 2000 359 –377 367 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: n 56, 2.8–10.9 m s was reduced and later long-lasting depression abolished in Fig. 5. Blockade of GABA - and glycine-mediated synaptic inhibition the presence of 50 mM D -AP5, the NMDA receptor antagonist. V 5 264 A m enhances the S-DHPG-induced potentiation of polysynaptic EPSPs. A to 277 mV, 17–22 day-old rats. B Summary graph n 57 showing the Summarized data showing the enhanced potentiation in the peak am- antagonism of the S-DHPG-induced 10 mM, 10 min potentiation of the plitude of polysynaptic EPSPs induced by S-DHPG 10 mM, 10 min in polysynaptic EPSPs by DAP5 50 mM. V 5 255 to 277 mV, 18–22 m four SG neurons in the presence of bicuculline 5 mM, strychnine 2 day-old rats. 21 mM and a high concentration of Mg 3 mM. V 5 261 to 289 mV, m 17–19 day-old rats. B, The graph shows the time course of changes in the was 5.161.1 mV and 0.0560.02 Hz n 511, respectively. peak amplitude of EPSPs caused by S-DHPG 10 mM, 10 min in a SG The induction of oscillations was concentration-dependent 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 in that the number and frequency of oscillations increased as shown on the individual EPSPs above the graph V 5 261 mV, 17 m with higher concentrations or time of exposure to DHPG. day-old rat. Experiments were also undertaken to determine if inhibi- tion plays a significant role in producing the DHPG- induced 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-fiber- presence of bicuculline 5 mM and strychnine 2 mM, the evoked monosynaptic EPSP and prolonged potentiation of GABA and glycine receptor antagonists, in a cell ex- the polysynaptic EPSP Fig. 9B, Table 2. Both effects A 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 antago- inhibitory synaptic noise Fig. 8D, left trace. nist [30], almost completely reversed the polysynaptic EPSP potentiation Fig. 9E caused by CHPG n 53. In 3.8. Dual modulation of DR-evoked EPSPs by CHPG the presence of bicuculline 5 mM, strychnine 2 mM, 21 and 3 mM Mg , the potentiation of polysynaptic EPSP by In a normal medium, CHPG 0.5–1 mM, 5 min CHPG was significantly greater than that recorded in a 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