Long-Term Potentiation and Long-Term Depression of Primary Afferent Neurotransmission in the Rat Spinal Cord

The Journal of Neuroscience, December 1993. 13(12): 52286241

Long-term Potentiation and Long-term Depression of Primary Afferent Neurotransmission in the Rat Spinal Cord

M. RandiC, M. C. Jiang, and R. Cerne Department of Veterinary Physiology and Pharmacology, Iowa State University, Ames, Iowa 50011

Synaptic transmission between dorsal root afferents and ably mediated by L-glutamate, or a related amino acid (Jahr and neurons in the superficial laminae of the spinal dorsal horn Jessell, 1985; Gerber and RandiC, 1989; Kangrga and Randic, (laminae I-III) was examined by intracellular recording in a 1990, 1991; Yoshimura and Jessell, 1990; Ceme et al., 1991). transverse slice preparation of rat spinal cord. Brief high- Neuronal excitatory amino acids (EAAs), including gluta- frequency electrical stimulation (300 pulses at 100 Hz) of mate, produce their effects through two broad categoriesof re- primary afferent fibers produced a long-term potentiation ceptorscalled ionotropic and metabotropic (Honor6 et al., 1988; (LTP) or a long-term depression (LTD) of fast (monosynaptic Schoepp et al., 1991; Watkins et al., 1990). The ionotropic and polysynaptic) EPSPs in a high proportion of dorsal horn NMDA, a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic neurons. Both the AMPA and the NMDA receptor-mediated acid (AMPA)/quisqualate (QA), and kainate receptors directly components of synaptic transmission at the primary afferent regulate the opening of ion channelsto Na, K+, and, in the case synapses with neurons in the dorsal horn can exhibit LTP of NMDA receptors, CaZ+as well (Mayer and Westbrook, 1987; and LTD of the synaptic responses. In normal and neonatally Ascher and Nowak, 1987). In addition, the activation of ion- capsaicin-treated rats, the induction of LTP requires the ac- otropic receptors can induce Ca2+ influx through voltage-de- tivation of NMDA receptor-gated conductances. The induc- pendent Ca*+ channels activated as a result of cell depolarization of LTP or LTD, however, was not abolished in the pres- tion. The metabotropic receptors (activated by glutamate/QA) ence of bicuculline, a GABA, receptor antagonist. appear to be coupled to phospholipaseC through G-proteins. The results demonstrate that distinct and long-lasting Their activation causesan increase in turnover of polyphos- modulation in synaptic efficiency can be induced at primary phoinositidesand releaseofCa2+ from intracellular stores.Thus, afferent synapses with neurons in the superficial laminae of the activation of both classesof EAA receptors can result in spinal dorsal horn by high-frequency stimulation of dorsal elevation of intracellular free Ca2+concentration ([CaZ+]) (May- root afferents and that these changes may be physiologically er and Miller, 199 1). The increasein [Ca2+],, in turn, may lead relevant for transmission and integration of sensory infor- to activation of other second messengersystems with conse- mation, including pain. quent changesin the properties of EAA receptor channel com- [Key words: spinal dorsal horn neuron, synaptic plasticity, plexes that contribute to long-term influenceson the fast excit- spinal dorsal horn, long-term potentiation, long-term de- atory synaptic transmission (Collingridge and Singer, 1990; pression, EPSPs] Madison et al., 1991; Siegelbaumand Kandel, 1991; Johnston et al., 1992). The superficial spinal dorsal horn (SDH), including substantia The efficiency of synaptic transmissionin the CNS, including gelatinosa(SG), is an area where primary afferent fibers arising spinal cord, is not constant and can be modulated by the rate predominantly from skin, but also the viscera and muscles, of activity in presynapticpathways (Mendell, 1984;Burke, 1987). terminate and form the first synaptic relay with dendrites of In a variety of brain structures, repetitive activation of synaptic dorsal horn (DH) neurons. For this reason, the SDH has been connections can lead to long-term potentiation (LTP) or long- regardedas an important site for the initial processingof afferent term depression(LTD) of synaptic transmission(Ito, 1989; Col- signalsdirectly related to the transmission and modulation of lingridge and Singer, 1990; Madison et al., 1991; Siegelbaum cutaneous information, including pain. and Kandel, 1991; Johnston et al., 1992). Although a great deal Previous studies using spinal cord slice preparations from is known about LTP and LTD in the mammalian brain, the immature (Urban and Rand@ 1984; Gerber and RandiC, 1989; existence of similar synaptic plasticity at primary afferent syn- Gerber et al., 1991) and adult (Yoshimura and Jessell, 1989, apseswith DH neuronshas not until recently been demonstrated 1990) rats had demonstrated that primary afferent stimulation (Ceme et al., 199 1; Jiang and Rand@ 1991). evokes both fast and slow EPSPs in SDH neurons, including The present work was aimed at studying long-term modifi- SG. Pharmacologic evidence has indicated that fast EPSPs at cations of primary afferent neurotransmissionfollowing high- synapsesbetween A6 and C afferents and SG neuronsare prob- frequency stimulation of dorsal roots in spinal cord slicesobtained from young rats. Our major findings were that in a high Received Jan. 4, 1993; revised June 1, 1993; accepted June 10, 1993. proportion of DH neurons(laminae I-III), a prolonged increase We thank Dr. T. Honor6 for kindly providing us with NBQX. This effort was or decreaseof amplitude of monosynaptic EPSPscan be induced supported by NIH Grant NS-26352, Grants BNS 8418042 and IBN-9209462 by brief repetitive stimulation of primary afferent fibers in a from the National Science Foundation, and the U.S. Department of Agriculture. Correspondence should be addressed to M. Rand% at the above address. dorsal root. In both normal and capsaicin-treated rats the in- Copyright 0 I993 Society for Neuroscience 0270-6474/931135228-14$05.00/O duction of the potentiation requires the activation of NMDA The Journal of Neuroscience, December 1993, 73(12) 5229 receptor-gated conductances. The effect is, however, not abol- A B ished by blockade ofGABA, receptors in polysynaptic pathways to primary afferents. Long-lasting depression of the primary NBQX afferent input to DH neurons, however, does not involve activation of postsynaptic NMDA or GABA, receptors. The un- AL derlying molecular mechanisms responsible for positive or neg- -I I 20ms ative amplitude modulation of EPSPs in spinal DH neurons have yet to be identified. Part of theseresults have been presented elsewhere (Ceme et APV al., 1991; Jiang and RandiC, 1991; RandiC et al., 1993; Rusin et al., 199313). k _--.-.. 5mV L J Materials and Methods 5ms

Preparation ofthespinalcordslice. Transverse slices were obtained from Figure 1. Schematic arrangement for intracellular recording and dorsal Sprague-Dawley rats of both sexes (14-40 d old) using a technique that root stimulation. A, Dorsal root was stimulated by a coaxial stainless has been described elsewhere (Murase and Randit, 1983, 1984; Gerber steel stimulating electrode (arrow). B, Presumed monosynaptic EPSPs et al., 1989, 199 1). Briefly, after the animal was anesthetized with ether, evoked by single electrical shocks (a and b) are shown before and during a segment of the lumbosacral (L,,-S,) spinal cord was dissected out and bath application of 5 PM NBQX (a) and 100 PM APV (b). sectioned with a Vibratome to yield several transverse slices 300400 pm thick with short (3-6 mm) dorsal rootlets (DRs) (Fig. 1A). After incubation for 1 hr in a solution equilibrated with 95% 0,, 5% CO, (mM: NaCl, 124; KCl, 5; KH,PO,, 1.2; CaCl,, 2.4; MgSO,, 1.3; Na- change in size of EPSPs, and the synaptic responses remained stable, HCO,, 26; glucose, 10; pH 7.4, at 36 + l°C), a slice was transferred then subsequent tetani utilized pulses of increased intensity. The test into a recording chamber, where it was submerged beneath an oxygen- stimuli were always the same before and after tetani. ated superfusing medium (flow rate of about 3 ml/min) containing a Data analysis. The chief dependent variable in the intracellular studies lowered concentration of potassium ions (1.9 mM KCl). The use of a was the EPSP amplitude. LTP or LTD magnitude was quantified from high-K+ solution during cutting and incubation of the slices seemed to the peak amplitude of a single or averaged EPSP (n = 4) measured at improve their viability as assessed electrophysiologically in the same the time of maximal change (21.8 f 1.8 min, mean ? SEM, n = 53) preparation. posttetanus with respect to the pretetanus baseline EPSP. LTP was Dorsal root stimulation and intracellular recording of synaptic re- defined as at least a 20% increase in amplitude of the synaptic response sponses. Conventional electrophysiological techniques were used for that was maintained for a minimum of 20 min following brief high- intracellular recordinn from DH neurons (laminae I-III). includina SG frequency stimulation. Conversely, LTD was defined as at least a 20% cells, as described (Mirase and RandiC, 1983, 1984; Gerber et al., 1589, decrease in amplitude of EPSP lasting at least 20 min. Statistical sig- 1991). SG neurons were identified by their location in the spinal DH. nificance of data has been assessed relative to control responses by use When viewed under a dissecting microscope with transmitted illumi- of either a paired or unpaired Student’s t test, as appropriate. For sta- nation, the SG was distinguishable as a translucent bend in the super- tistical analysis of data seen in Figure 4 we used one-way ANOVA, and ficial DH, although it was difficult to discern with certainty the border statistical significance between means was determined by a Student- between laminae I and II. Under visual control, a single fiber-glass Newman-Keuls test. All values are expressed as means + SEM. The (#6010: o.d. and i.d., 1.0 and 0.58 mm. resuectivelv: AM Svstems. significance level for all statistical tests was P 5 0.05. Everett, WA) microelectrode filled with either 4 M potassium acetate or Application of drugs.Drugs were applied by superfusing slices with 4 M potassium chloride (pH 7.2) (DC impedance, 105-l 50 MB) was solutions of known drug concentration. Drugs used were 2,3-dihydroxy- placed in the DH (Fig. lA), and neurons were impaled by oscillating 6-nitro-7-sulfamoyl-benzo(F)quinoxaline (NBQX; 0.5-20 ELM;a gift from the capacity compensation circuit of a high-input impedance bridge Dr. T. Honor& A/S Ferrosan, Soeborg, Denmark), 6-cyano-7-nitro- amplifier (Axoclamp 2). Cells were activated synaptically by electrical quinoxaline-2,3-dione (CNQX; 5-10 PM; To&s), D-2-amino-5-phos- stimulation of primary afferent fibers in the dorsal roots. A coaxial phonovaleric acid (APV, 50-100 PM; Cambridge Research Biochemi- stainless steel stimulating electrode (o.d. of inner and outer electrodes, cals), bicuculline methiodide (5-10 PM; Sigma), L-glutamate (Peptides 25 and 200 pm, respectively; Frederick Haer Co.) positioned on a lum- International, Sigma), and N-methyl-D-aspartic acid (NMDA, CRB). bar DR was used in transverse slices (Fig. 1A). A DC pen-recorder was Capsaicin experiments. Sixteen rats of both sexes taken from two used to record membrane potential continuously; the synaptic responses different litters were injected subcutaneously with 50 mg/kg capsaicin were stored on diskettes of a digital oscilloscope. (Sigma) in vehicle [ 10% ethanol, 10% Tween (v/v) in 0.9% (w/v) saline] Experimental arrangement. The protocol for assessing the effects of 48 hr after birth. Ten control littermates received equal volumes of tetanic stimulation of primary afferents on excitatory synaptic responses vehicle alone. After a survival time of 14-29 d, the animals were sub- was as follows. Once the electrode position was optimized on a DR, jected to the experimental procedure described above. sampling of excitatory postsynaptic potentials (referred to as “test” EPSPs) began. Single shocks at a fixed suprathreshold strength (0.05- 0.2 msec pulses, 5-30 V), repeated at 30 set or 3 min intervals, were Results given through a stimulating electrode for 1O-20 min before tetanic stim- Stable intracellular recordings of up to 5 hr were obtained from ulation. This frequency of stimulation was chosen for sampling data because it did not result in response facilitation or depression. A stim- 92 DH neurons, including 8 I SG cells, that received fast EPSPs ulus intensity that yielded a 5-l 5 mV EPSP was chosen to standardize elicited by stimulation of primary afferent fibersin the DR. The the baseline svnautic strength across slices. and it was below threshold resting membrane potential and action potential amnlitude of for eliciting an action potential in most of the slices chosen for study. DH n&rons examined were -73.0 ~fr0.8 mV (mean -t SEM, However, in some neurons, in order to obtain EPSPs of 5-15 mV n = 83) and 78.5 -t 1.8 mV, respectively. amplitude without evoking action potentials in the postsynaptic cell, hyperpolarizing DC current (up to 0.1 nA) was passed into the cell for the duration of the experiment, resulting in measured resting membrane EPSPs evoked by primary afferent stimulation and their potentials of - 70 to - 85 mV. After the baseline period of 1O-20 min, antagonism by NBQX and APV a high-frequency train (three tetani of 1 set duration, each at 100 Hz Primary afferent stimulation produces several distinct types of and 10 set intervals) was delivered at the test or greater intensity (0.05- 0.2 msec pulses, 20-35 V), as used for the baseline responses. Normally EPSPsin superficial laminae of the SDH, including SG neurons, the first tetanization utilized stimulus pulses of the same intensity and as has been reported previously (Yoshimura and Jessell,1990). duration as during test stimulation. If this treatment did not induce In 69 of 92 (75%) DH neurons, the poststimulus latency of DR- 5230 RandiC et al. l Modulation of Primary Afferent Neurotransmission

Table 1. The effects of repetitive stimulation of primary afferent fibers on EPSPs in SC neurons

Type of Resting change Maximal change Peak change potential Type of EPSP in EPSP n (% control) (min) WV) Monosynaptic LTP 22 173.6 + 10.8 20.0 k 3.7 70.5 + 1.4 EPSP LTD 20 44.5 + 4.7 21.4 k 1.6 74.3 + 1.5 Poly-synaptic EPSP LTP 17 177.1 * 17.3 18.9 * 2.1 73.4 + 1.7 LTD 6 42.5 z!z 9.8 23.5 5 3.3 74.2 f 3.2 EPSP in capsaicin- LTP 6 182.8 k 12.0 16.8 + 2.7 70.7 +- 7.8 treated rats LTD 7 39.2 k 4.9 18.6 + 1.6 70.6 -+ 4.4 NMDA component LTP 4 237.0 + 89.3 12.8 IL 5.2 59.0 + 1.9 of EPSP LTD 5 43.1 3r 13.4 14.2 + 3.3 70.2 + 3.4 Changes in the amplitude of synaptic responses produced by electrical stimulation of DRs are presented as mean percentagesof their respective controls f SEM. initiated EPSPsremained constant when repetitive stimulation examined the sensitivity of monosynaptic EPSPs to EAA re- (10 Hz) was usedand failures were not observed, although the ceptor antagonists.Bath-applied NBQX (1-20~~ for 2-l 3 min), amplitude of EPSPs was significantly decreasedduring stimu- the novel and selective non-NMDA receptor antagonist (Shear- lation. The latency of these EPSPs also remained constant in down et al., 1990), causedpotent and reversible antagonism of the presenceof a high concentration of divalent cations (4 mM EPSPs. The amplitude of EPSPs was significantly reduced (to Ca2+,8 mM MgZ+; data not shown). On the basisof thesecriteria 3.0 f 0.8%, n = 26) with 1 PM NBQX, and the EPSPs were we assumedthat these DH neurons are monosynaptically ac- completely blocked with 5-10 PM NBQX (n = 6; Fig. l&z). tivated by primary afferent fibers. In a smaller proportion of APV (50-100 PM, 7-14 min), the selective NMDA receptor DH neurons(25%, n = 23) the latency of the DR-evoked EPSPs antagonist (Davies et al., 198 1; Watkins and Evans, 1981) pro- was variable, suggestinga polysynaptic input from primary af- duced a small decrease(to 75.3 f 7.3% of control, n = 10; Fig. ferents. Increasing the intensity of primary afferent stimulation leb) in the amplitude of most EPSPs,although in two cells with usually increasedthe amplitude and duration of the polysynaptic long-latency EPSPs almost complete block occurred. In addi- EPSPs,whereas perfusion with Krebs solution containing 4 mM tion, almost all cells showed a decreasein the half-decay time Ca2+, 8 mM Mg2+ markedly depressedvariable latency EPSPs. of the EPSP. Our resultsobtained with NBQX and APV indicate To test further the possibility that L-glutamate or a related that the synaptic activation of the non-NMDA receptorsof SDH amino acid is the transmitter at primary afferent synapses,we neurons predominantly mediates the DR-evoked, presumably

Figure 2. LTP of fast excitatory synaptic transmission at primary afferent synapses with neurons in the superficial ,.L I-“cg SDH. A, The graph shows the time A I- C lb- course of LTP of EPSP recorded intra- 25m. cellularly from an SDH neuron (inset) in response to electrical stimulation (20 V, 0.1 msec) of a lumbar dorsal root. At time 0 (arrow) the dorsal root was given three tetani (at the same intensity as the test stimulus) of 1 set duration, each at 100 Hz and 10 set intervals. Above the graph are displayed individual, apparently monosynaptic EPSPs taken before (truce 1) and during (trace i 0 5 10 15 20 25 2) the potentiation. B, Summarized data (mean + SEM) showing the time course of the long-lasting potentiation for this and two other neurons (inset) following the tetanic stimulation at the intensity of the test stimulus. C, Even longer- lasting potentiation ofdorsal root stimulation (9 V, 0.1 msec)-evoked EPSPs, recorded from another SDH neuron (inset), was obtained when the tetanic stimulation was twice the test intensity. D, Responses for this and five other neurons are summarized below. A, V,, = -70 mV, 33-d-old rat; f?, k’,, = -72 mV, 20-d-old rat; C, V,, = -64 to -74 501 -10 0 10 20 50 -15 0 15 90 45 50 mV, 21-33-d-old rats; D, V,,, = -65 to TIME (ml@ -84 mV, 2240-d-old rats. TIME (mh) The Journal of Neuroscience, December 1993, 13(12) 5231

25ms

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i 251 0-c . ’ -15 0 15 30 -15 0 15 30 45 60 75 90 TIME (min) TlME(min)

Figure 3. LTD of fast excitatory synaptic transmission in the superficial SDH. A, The graph shows the time course of LTD of an apparent monosynaptic EPSP recorded in an SDH neuron in response to stimulation (11 V, 0.1 msec) of a dorsal root. At time 0 (arrow), the dorsal root was given three tetani (test intensity) of 1 set duration, each at 100 Hz and 10 set intervals. Above the graphare displayed individual EPSPs taken before (trace I) and during (truce 2) the LTD. B, The summarized data showing the time course for nine SDH neurons that expressed LTD when given the tetanic stimulation at the test stimulus intensity. Inset shows positions of the cells in SDH. C, The graph shows the time course of LTD of an apparent monosynaptic EPSP recorded in response to stimulation (15 V, 0.3 msec) of a dorsal root. At time 0 (arrow), the dorsal root was given three tetani (35 V, 0.3 msec) of 1 set duration, each at 100 Hz and 10 set intervals. Posttetanic depression of the EPSP’s amplitude to 40% of the control value was followed by long-lasting depression to 70% of the control EPSP’s amplitude. 0, The graph shows summarized results for nine SDH neurons that upon tetanic stimulation of high intensity (twice or three times the intensity of the test stimuli for a single EPSP) developed LTD. A, V,,, = -73 mV, 22-d-old rat; B, V, = -83 mV, 20-d-old rat; C, V,,, = -69 to -90 mV, 19-25d-old rats; D, V,,, = -63 to -75 mV, 18- 28-d-old rats.

monosynaptic, EPSP. These findings are consistent with other It appearsthat the sametetanic stimulation can induce either published reports for spinal neurons (Dale and Roberts, 1985; LTP or LTD of the evoked EPSP depending on the level of Forsythe and Westbrook, 1988; Schneider and Perl, 1988; Ger- membrane potential of the postsynaptic neuron (n = 5) and ber and RandiC, 1989; Yoshimura and Jessell, 1990; Cerne et intensity ofthe conditioning train (n = 7). As illustrated in Figure al., 199 1; Jiang and RandiC, 1991; Yoshimura and Nishi, 1993). 5, when the membrane potential was regulated during synaptic use by directly passinga depolarizing or hyperpolarizing current Changesin synaptic eflcacy induced by tetanization of the through the recording electrode in the same DH cell kept at primary afferents -70 mV, tetanic stimulation induced LTP (Fig. 5A), whereas We have investigated in the present experiments the excitatory at -85 mV the same train led to LTD (Fig. 5C). At the same synapsebetween primary afferent fibers and neuronsin the su- membrane potential, the sametetani never resultedin alternate perficial laminae (I-III) of the SDH. The strength of primary LTP or LTD. When tetanic stimulation given at test intensity afferent transmissionwas assayedby conventional intracellular for singleEPSP evoked LTP, a subsequenttrain of higher strength recording of the size of the monosynaptic EPSPsthat result from also induced LTP. However, in the cellswhere LTD wasevoked stimulation of primary afferent fibers with electrical shocks(Fig. with the first train, a successivetrain, given at a higher intensity, IB). With singlestimuli delivered at a frequency of 0.002-0.033 produced either LTP or LTD. The latter result suggeststhat the Hz, the synaptic strength varies little over 1 hr of testing. How- sign of use-dependentsynaptic modification in a proportion of ever, when high-frequency stimulation (typically these trains DH neuronsswitches from negative to positive as a function of consistedof electrical shocksat the sameor greater intensity as the activation level. the test stimulus for single EPSP, delivered at 100 Hz for 1 set, repeated three times at 10 set intervals) was applied to a dorsal LTP of the monosynapticEPSPs root, two distinct types of changesin the monosynaptic EPSP Brief, high-frequency (tetanic) stimulation of primary afferent amplitude resulted (Table 1). Of the 49 neurons having stable fibers resulted in the following sequenceof results: (1) a rapid resting membrane potential and stable baselineEPSP size, 22 buildup of the slow depolarization during the tetanic stimula- (45%) showed a long-term enhancement of EPSP amplitude tion, (2) a brief period of EPSP potentiation or depression im- (Figs. 2, 4A; Table l), 20 (41%) showed a long-term decrease mediately after the tetanic stimulation, and (3) a prolongedphase (Figs. 3, 4B; Table l), and 7 (14%) were not influenced. of EPSP potentiation involving both monosynaptic and poly- 5232 RandiC et al. * Modulation of Primary Afferent Neurotransmission

MS - EPSP PS - EPSP NMDA CAPSAICIN MS - EPSP PS - EPSP NMDA CAPSAICIN

Figure 4. Summarized effects of tetanic stimulation of primary afferents on various forms of EPSPs in SDH neurons. Tetanic stimulation (100 Hz, 3 x 1 set) of primary afferents produced long-lasting modulation of EPSPs evoked by the low-frequency (0.0024.033 Hz) dorsal root stimulation in a proportion of SDH neurons. The bar graphs show the maxima1 potentiation and depression of the amplitude of EPSPs, expressed as percentage change of control EPSP taken as the average of two to four EPSPs preceding the tetanus (mean + SEM; *, P < 0.05). A, The potentiation was maximal 20 min after the tetanus and was present in both monosynaptic (MS-EPSP, n = 22) and polysynaptic (PS-EPSP, n = 17) EPSPs. The effect was somewhat greater with tetani of higher stimulus intensity (diagonal hatched bar) than with tetani of the test stimulus intensity for single EPSP (open bar) and was observed also in the presence of 2-5 PM NBQX (NMDA; n = 4) and in neurons obtained from capsaicin-pretreated rats (n = 6). B, The long-lasting depression was maximal 2 1 min after the tetanic stimulation. There was no significant difference in the effects observed on monosynaptic (MS-EPSP, n = 31) and polysynaptic (PS-EPSP, n = 6) EPSPs and between tetani of the test or higher intensity. LTD was observed also in the presenceof NBQX at 2-10 NM(NMDA; n = 5) and in capsaicin-pretreatedrats (n = 7). V,,,= -56 to -90 mV, 1440-d-old rats. synaptic componentsof the evoked EPSP. This sequencewas a by every stimulus in the train, in others failures were observed. characteristic feature of the experiments, although there was It was not possible,however, to perform precisemeasurements notable variation in the extent and duration of thesethree phases. of their amplitude becauseof the distortion of records caused During each of the short trains of the tetanus, the E, depo- by the high-frequency stimulation. Tetanization was followed larized up to 20 mV. Whereasin somecells EPSPswere evoked in many casesby a short-lasting (from 2 to 5 min) increasein

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\ *-*-a . 0’ , j...... ‘J-;i- _ ...... j 85 ” \*/. \ :./ Figure 5. The effects of prolonged de- 0’ polarizing and hyperpolarizing DC current injectionson inductionof LTP (A) 70. and LTD (C) of EPSPs. Tetanic stim- -10 0 10 20 30 -10 0 10 20 30 ulus was given at the test intensity (10 V, 0.1 msec). Time course of posttetanic changes of EPSP amplitude in the same SDH neuron (location shown in B inset) conditioned with depolarizing (A, 3 V,, = - 70 mV) and hyperpolarizing (B, B V,, = -85 mV) currents is shown. g 200 Above the graphs are displayed indi- TET 1 (A, C, vidual EPSPs obtained before ! LI 1 truce I) and during LTP (A, truce 2) or -150 + I/*- +,-a LTD (C, truce 2). Band D, Summarized x 2 data showing the time course of the 3 100 0-a J ...... SO posttetanic changes of EPSPs in the 0_ . TET same cells (n = 3; inset) conditioned 50 1 with depolarizing (B) and hyperpolar- -10 0 10 m 30 izing (D) pulses. A and C, 2 1-d-old rat: w B and D, 2 l-25-d-old rats. nhia(mln) TiME (mh) The Journal of Neuroscience, December 1993, 73(12) 5233

Figure 6. Bath application of glutamate (GLU) and NMDA can induce

orolonaedincrease in EPSP.A. V- = 5 10 15 20 -68 kV, 28-d-old rat; B, V,, = :72 TIME (min) TIME (min) mV, 25-d-old rat.

EPSP amplitude (posttetanic potentiation, or PTP), probably bath on the DR-evoked EPSPs in DH (laminae I-III) neurons. resulting from an increase in transmitter releasefrom presyn- We found that glutamate ( 1O-5 to 1O-4 M for 1 min) in five aptic terminals. LTP of the evoked EPSP, as manifestedby an spinal slices produced a transient depressionof the monosyn- increasein slope and amplitude of EPSP, was induced in 7 of aptic EPSPfollowed by a potentiation (Fig. 6A). The depression 49 (14%) SDH cellswhen tetanic stimulation at the test intensity was usually associatedwith a reduction in the membrane po- for single EPSP was employed. The LTP varied in magnitude, tential. Furthermore, NMDA ( 1O-4M for 1 min) potentiated an resultingin EPSPsthat were between 120%(control being 100%) apparent monosynaptic EPSP (Fig. 6B). Primary afferent fiber and 2 10% of the amplitude of EPSPs during the control period stimulation in the presenceof the agonist was not required for (Figs. 2B, 4A; Table 1). The potentiation always lasted for the the potentiation and NMDA could elicit potentiation repeatedly duration of the recording period, which ranged from 25 min to in the same slice if sufficient time (about 30 min) was allowed 90 min (Fig. 2A,C). No changewas detected in the evoked EPSP between applications. This agonist-inducedpotentiation is dec- latency during LTP. The result of one of the experiments is remental and of a relatively short duration (1 O-30 min) if com- shown in Figure 2A. Immediately following tetanic stimulus, pared with a high-frequency tetanus-induced potentiation of the the strength of the tetanized synaptic connections, as testedwith synaptic response(Fig. 2). The present resultsindicate that pre- singleshock stimuli, increased(PTP) up to about 2.5-fold. Most synaptic stimulation provides an essentialcomponent for de- of this increase decays to a level of about 200% of baseline velopment of LTP and that this form of information storage within a few minutes after the tetanus. The graph in Figure 28 may be usedunder physiological conditions. The resultsare in showsthe time course of the EPSP potentiation summarized agreementwith previous studiesdemonstrating that glutamate for three cells. and NMDA application does potentiate synaptic transmission In the next seriesof experiments we increasedpostsynaptic in the hippocampus (Collingridge et al., 1983; Kauer et al., activation by raising the strength of tetanic stimulus two- to 1988). fourfold the test intensity for single EPSP and obtained LTP of the monosynaptic EPSP that lasted up to 90 min in 15 of 49 Pharmacology of the induction and expression of the LTP of (3 1%) cells tested (Fig. 2C). The potentiation of the EPSP can EPSPs: actions of EAA receptor antagonists be seennot only as an increase in the peak amplitude but also We next examined the contribution of NMDA and non-NMDA as an increasein the initial slopeof EPSP. Of 13 cells examined, receptors to the induction and expressionof LTP of EPSPsby 11 showed no change in EPSP latency, whereas in two cells a using selective antagonistsof NMDA and non-NMDA recep- decreasewas detected. tors. We first examined whether LTP of the AMPA receptor- mediated responserequires NMDA receptor activation during Glutamate and NMDA potentiate synaptic transmission in DH tetanus. The selective and competitive antagonistof the NMDA neurons subtype of glutamate receptors, APV (So-100 PM), caused a The present concept is that the site of induction of LTP in the small decreasein the amplitude of EPSP but prevented the CA1 region of hippocampusappears to be the postsynaptic cell, induction of LTP when presentat the time of tetanic stimulation and induction requires both activation of NMDA receptors by (Fig. 7; n = 5). Moreover, in three of five cells, a small but synaptically releasedglutamate (Collingridge et al., 1983) and prolonged depressionoccurred following perfusion with APV depolarization of the postsynaptic membrane (Gustafssonand during high-frequency stimulation (Fig. 7A). Perfusion of spinal Wigstrom, 1986). Moreover, it is thought that this depolariza- sliceswith APV after the potentiation has been establishedhas tion relieves a voltage-dependent Mg2+ block of the NMDA no effect; that is, APV does not prevent expressionof LTP of receptor-ion channel complex, resulting in increasedcalcium the non-NMDA receptor-mediated EPSP (n = 2; not shown). influx (Mayer et al., 1984; Nowak et al., 1984) the latter being Experimental evidence so far presentedsuggests that the cur- the trigger for the induction of LTP (Madison et al., 1991; Sie- rents initiated by the activation of NMDA receptors are nec- gelbaum and Kandel, 1991). This model predicts that appli- essaryfor the induction of LTP, but there is no information as cation of a largedepolarizing doseof NMDA or glutamate should to the contribution of the NMDA receptors to the expression also evoke LTP. In order to determine the effects of glutamate of this effect. The present concept is that the expressionof LTP and NMDA on primary afferent neurotransmission, we have appears to be dependent predominantly on AMPA receptor- examined the long-term effects of these agents applied to the mediated transmission. However, a pharmacologically isolated 5234 RandiC et al. l Modulation of Primary Afferent Neurotransmission

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Figure 7. APV prevented the induction of LTP of EPSP in a normal slice. Effect of tetanic stimulation on EPSP -12 0 12 24 recorded from the same SDH neuron (location shown in inset) in the presence of 50 PM APV (A) and 40 min following the removal of the drug (C). At time 0 B (arrow) the dorsal root was given three tetani at twice the intensity of the test stimulus (14 V, 0.05 msec). Above the graphs are displayed individual EPSPs obtained before (A, truce I) and 6 min (A, truce 2), 36 min (C, truce I), and 52 min (C, truce 2) after the removal of APV. Summarized data (mean C SEM) showing the time course of the postte- ,I)0 ..*.A..+..! ...... tanic changes of EPSPs in five cells (inset) in the presence of 50 PM APV (B) 2 TET + TET +’ and 30-40 min following the removal * 25 I 80 I of the drug (D). A and C, V,, = -76 & -15 0 15 30 -10 0 10 20 30 mV, 33-d-old ral; Band 0, V,. = -64 w” to -75 mV, 25-33-d-old rats. TIME (min) TIME (min)

NMDA receptor-mediated synaptic responsecan undergo syn- pressedin four DH cells, with an averageincrease ofabout 200% apse-specificLTP in the hippocampus (Bashir et al., 1991; Xie of control (Fig. 4A, Table 1). et al., 1991). We now report that the potentiation oftransmission at the primary afferent-DH cell synapses(n = 4) can also be LTD of the monosynaptic EPSPs expressedby the pharmacologically isolated NMDA receptor- Here we report that an LTD of the amplitude of monosynaptic mediated EPSP. EPSPs can occur in neurons of the superficial laminae of DH As mentioned above, synaptic responsesin the DH region of slices of the rat spinal cord after primary afferent fibers in comprise an EPSP that appearsto be mediated primarily by DRs were stimulated with three tetani of 1 set duration, each receptors of the non-NMDA class,but in addition has a small delivered at 100 Hz and 10 set intervals. When the tetanus at slow NMDA receptor-mediated synaptic component. When the the test intensity for single EPSP was employed, the depression AMPA receptor-mediatedcomponent of EPSPwas blocked with of the amplitude and initial slope of EPSPs (Figs. 3A,B, 4B; bath application of CNQX (10 PM) or NBQX (2-10 KM), the Table 1) was observed in 11 of 49 (22%) cells and persisted NMDA receptor-mediated EPSPs were revealed either when without signsof recovery for about 40 min after cessationof sliceswere perfusedwith low-Mg*+ (100 PM) medium or when conditioning stimulation. However, when in nonresponsivecells cells were depolarized to - 50 mV in normal Mg2+ (1.2 mM). we increasedpostsynaptic activation by raising the intensity of The cell depicted in Figure 8A was recorded in the presenceof the tetanic stimulus (three times the test intensity), an LTD in 100 PM external MgZ+ and 2 PM NBQX. Superfusion of NBQX 10 of 49 (20%) cells lasting between 40 min and 4 hr was ob- greatly reduced the amplitude of the EPSP. The residual syn- served. In seven neurons, the synaptic responsedisplayed LTD aptically evoked depolarization was enhancedby increasingthe when train was delivered at test intensity for single EPSP, but intensity of stimulation. After obtaining a baselineNMDA re- LTP was induced when intensity of tetanic stimulation was ceptor-mediated responseto test stimulation (0.006 Hz), teta- increased. The magnitude of LTD varied in different cells, re- nus was delivered and LTP of the synaptic responsewas resulting in EPSPsthat were between 90% and 20% of the size of corded for more than 20 min. At the end of the experiment, the test EPSPs during the control period. Compared with the cor- potentiated responsewas reversibly blocked by APV (100 PM; responding values of EPSPs recorded prior to repetitive stim- data not shown), the result indicating that the potentiation of ulation, this depressionof EPSPswas statistically significant (P EPSPs was mediated through NMDA receptor. LTP was ex- < 0.05; Fig. 4B). Figure 3 showsthe time courseand degreeof The Journal of Neuroscience, December 1993, f3(12) 5235

Figure 8. LTP and LTD of the NMDA receptor-mediated component of excitatory synaptic transmksion. A, The aranh shows the time course of LTP of NMDA receptor-mediated EPSP recorded intracellularly from an SC neuron (inset) in response to stimulation (15 V, 0.1 msec) of a lumbar dorsal root. Perfusion medium contained 0.1 mM Mg2+ and 2 FM NBQX. At time 0 (arrow) the dorsal root was given three tetani (100 Hz, 3 x 1 set) at the inten- d sity ofthe test stimulus. B, In a different SC neuron (inset) 0.1 mM Mg2+ and 10 ‘* JlOmV I.IM CNQX were used to isolate the 0.1s NMDA component of synaptic potential obtained bv stimulation of DR (15 V, 0.1 msec). Tetanic stimulation (30 V, 0.1 msec, 100 Hz, 3 x 1 set) was delivered at time 0. Above the graphs are displayed individual EPSPs obtained before (A, B, trace I) and during LTP (A, trace 2) or LTD (B, trace 2). A, V, = -76 mV, 33-d-old rat; B, V, B TIME (min) TIME (min) = -69 mV, 22-d-old rat. the prolonged depressionof EPSPs following repetitive stimu- fibers following trains of high-frequency stimulation of DR at lation of the DR in a single DH cell (Fig. 3C) and in a group test or greater intensity as for single EPSP. In addition, three of nine neurons (Fig. 30). As shown in Figure 3C, when the DH (laminae I-III) cells receiving polysynaptic input expressed baselinemeasurements following trains were resumed, the first LTP in slicesobtained from capsaicin-treated rats. Figure 10A one or two responseswere always depressed(or even abolished) illustrates the increase in the synaptic strength, as tested with even below the value attained during LTD. Although there was single shock stimuli, following tetanic stimulation of primary usually some recovery in response magnitude over the next afferent fibers at four times the test stimulus in a spinal slice several minutes, the EPSP amplitude always reached a plateau obtained from the rat neonatally treated with capsaicin. Since at a value that was significantly depressedas compared with the the similar potentiating effect cannot be induced in the same pretetanus control period. cell in the presenceof APV during tetanic stimulation (Fig. 1OB), It appearsthat the induction of LTD doesnot involve NMDA it appearsthat the processleading to the potentiation of pre- receptors, since the depressanteffect was recorded in the su- sumableA6 fiber-mediated EPSP is related to the operation of perficial laminae DH neurons in the presenceof the NMDA the NMDA receptor-ion channelcomplex. The resultsobtained receptor antagonist APV (n = 4; Fig. 7A; see also Fig. 10B). show that neonatal capsaicin treatment did not prevent the ex- This finding is in agreementwith previous studies of LTD in pression of LTP or LTD in DH neurons receiving predomi- hippocampus (Stanton and Sejnowski, 1989) and visual cortex nantly A6 afferent inputs. (Artola et al., 1990). LTD of primary afferent neurotransmission can be induced in the presenceof 10 PM CNQX or 2-10 PM Effects of GABA, receptor blockade on modulation of primary NBQX, and in low-Mg2+ (100 PM) medium (Fig. 8B), indicating aferent neurotransmission that the depression is expressed by both AMPA and NMDA Although there is little to suggestthat inhibitory mechanisms receptors. contribute to the maintenanceof synaptic LTP (Haas and Rose, 1982) there is evidence that its induction is affected by inhib- Effects of neonatal capsaicin treatment on LTP and LTD of itory influences (Douglas, 1978; Douglas et al., 1982; Wigstrom the evokedEPSPs and Gustafsson, 1985). It is known that during high-frequency Anatomical studiesof the mammalian spinal DH have provided stimulation the cell remains depolarized for a sufficient time to evidence that C fibers give the major afferent input to the SG, enable activation of the NMDA receptor system. This is made whereasA6 fibers terminate predominantly in lamina I (Rethe- possible,at least in part, by frequency-dependent depressionof lyi, 1977; Light and Perl, 1979; Sugiura et al., 1986, 1989). In the synaptic inhibition causedby GABA feeding back and de- contrast, electrophysiological studies indicated that over 70% pressingits own releaseby an action on presynaptic GABA, of SG neurons received monosynaptic input from A6 fiber af- autoreceptors (Davies et al., 1991). The latter finding demon- ferents, only 5% from C fibers, and about 20% received both strated a role for GABA, receptors in synaptic plasticity (Col- A6 and C fiber input (Yoshimura and Jessell, 1990). In order lingridge and Singer, 1990). to examine the contribution of A6 and C fiber inputs to the In order to rule out possible involvement of polysynaptic induction and expressionof the long-lasting modulation of pri- inhibitory pathways in the mechanismsaccounting for the pro- mary afferent transmission, we used neonatal treatment of rats longed EPSPpotentiation and depressionobserved in this study, with capsaicin, a neurotoxin known to causedegeneration of a we blocked the GABA,-ergic receptorsin the spinal cord slice large number of C fibers (Jancd et al., 1977). In 13 cells ex- by bicuculline. We found that both the mono- and polysynaptic amined, we found the expressionof LTP (Figs. 4A, 9A, 1OA; componentsof the EPSP of DH cells were markedly enhanced Table 1) in six and LTD (Figs. 4B, 9B, 10B; Table 1) in seven by bicuculline. The induction of LTP (Fig. 11A) or LTD (Fig. SDH cells receiving monosynaptic input from primary afferent 11B) is not abolishedwhen the slice is perfusedwith bicuculline 5236 RandiC et al. * Modulation of Primary Afferent Neurotransmission

ducible in slicesobtained from both intact (n = 3) and capsaicin- treated (n = 4) rats in the presenceof bicuculline (5-10 MM). Facilitation and depressionof polysynaptic EPSPs In addition to the presenceof a high proportion of apparent monosynaptic EPSPsrecorded from the DH neuronslocated in laminae I-III, electrical stimulation of primary afferent fibers (8-20 V pulsesof 0.1 msecduration) evoked polysynaptic EPSPs in 23 of 92 cells.These EPSPshave a variable latency and exhibit failures with high-frequency stimulation and after perfusionwith solutions containing a high concentration of divalent cations. In this seriesof experiments we have confined our analysis of the effects of high-frequency stimulation of DRs to superficial DH neurons(laminae I-III) receiving polysynaptic EPSPsbased on thesecriteria. When three tetani of 1 set duration at 100 Hz at 10 set intervals, at test (n = 6) or greater intensity (n = 17) were delivered to the lumbar DR, the potentiation (Figs. 4,4, 12A; Table 1) or depression(Figs. 4B, 12B; Table 1) of poly- , synaptic EPSPamplitude resulted. In 17 (15 from intact, 2 from a.. capsaicin-treated rats) of 23 cells with stable resting membrane G potential and stable baselineEPSPs, the EPSP amplitude was \ I significantly increasedfollowing the repetitive stimulation (Fig. TET 4 ‘\ 4A, Table 1). The duration of the potentiation ranged from 15 min to 4 hr. The potentiation considerably varied in amplitude, resulting in EPSPs that were between 120% and 240% of the amplitude of test EPSPs during the control period. Similar as -10 0 10 20 30 for monosynaptic EPSPs,the induction of LTP of polysynaptic TlME (min) EPSPs requires the activation of NMDA receptor-gated con- Figure 9. Long-lasting changes in synaptic efficacy are present in SG ductance (Fig. 12B). In only six cells receiving polysynaptic neurons of capsaicin-pretreated rats. A, Summarized data for six SC inputs was an LTD observed. neurons (location shown in inset) obtained from capsaicin-pretreated rats, where tetanic stimulation (100 Hz, 3 x 1 set) induced LTP of Discussion EPSPs evoked by low-frequency stimulation of dorsal roots. B, The time course of LTD of EPSPs recorded from seven different SG neurons Use-dependentchanges in synaptic ejiciency in rat SDH in (location shown in inset)in response to electrical stimulation of dorsal vitro roots. Tetanus was delivered at time 0 (arrow). V, = -56 to -89 mV, In a variety of brain structures repetitive activation of synaptic 14-22-d-old rats. connections can lead to LTP or LTD of excitatory synaptic transmission (Madison et al., 1991; Siegelbaumand Kandel, 1991; Johnston et al., 1992). However, the presenceof the sim- (5-10 PM, n = 13). However, as shown in Figure 1 lA, after a ilar phenomena in the spinal cord has not until recently been brief period of PTP the average monosynaptic EPSPamplitudes reported (Ceme et al., 1991; Jiang and RandiC, 1991; RandiC in the presenceof bicuculline (10 PM) were smaller when com- et al., 1993; Rusin et al., 1993b). The major finding ofthe present pared with values recorded in the absenceof bicuculline. These differenceswere not statistically significant. LTD remained in-

Figure 10. Effect of APV on the induction of LTP of EPSP in the slice obtained from capsaicin-pretreated rat: effect of tetanic stimulation on synaptic b--- k---J B JL LlsmV responses recorded from SDH neuron A 2oms (location shown in inset) in a slice obtained from a neonatally capsaicin- 3 treated rat in the absence (A) and the presence (B) of APV. Intensity of the f 300, tetanic stimulus was four times the test 3 2 APV stimulus (6.4 V, 0.1 msec). Above the t 200, l ‘.‘., graphs are displayed individual EPSPs .’ @.*A obtained before (A, B, traceI) and dur- E l If! ‘0 ing LTP (A, trace 2) and 10 min after 2 loo* / removal of APV (B, truce 2). Values are ’ ‘o t TET 2 expressed as percentage of response f TET amplitude measured before the begin- 3k 0 50 ning of APV perfusion. V,, = -70 mV, -10 0 10 20 a0 -15 0 15 30 25-d-old rat. TIME (min) TIME (ml@ The Journal of Neuroscience, December 1993, 13(12) 5237

Figure 11. LTP and LTD of EPSPs do not require activation ofthe GABA, inhibitory system. A, In a slice that was constantly superfused with a recording solution containing bicuculline (10 PM), dorsal root stimulation (8 V, 0.1 msec) evoked EPSPs in a DH neuron (the truces above the graph). Tetanic stimulation (100 Hz, 3 x 1 set) of twice the test stimulus intensity induced a potentiation of EPSP as shown in the graph. B, In another neuron superfused with bicuculline (10 PM) EPSPs were induced by stimulation of a dorsal root (10 V, 0.1 msec). Tetanic stimulation with the test stimulus intensity evoked a prolonged depression of the fast synaptic transmission. Above the graphs are displayed individual EPSPs obtained before (A, B, trace I) and during LTP (A, traces 2, 3) or LTD (B, truce 2). A. V... = -85 mV. 32-d-old rat: B. TIME (min) TlME(min) -- -- G,,, 18d:old rat. study is that brief repetitive activation of primary afferent syn- lished data showing that the AMPA receptor plays a key role apseswith neurons in the superficial laminae of DH (laminae in mediating expressionof both forms of plasticity. Thus, the I-III; Rexed, 195 1) of the rat spinal cord resultsin a substantial upregulation of AMPA receptors is responsible for LTP ex- increase or decrease in synaptic strength that can last from 20 pressionin the CA1 region of the hippocampus (Davies et al., min to more than an hour. The results obtained in intact and 1989) and the AMPA receptors’ downregulation is responsible neonatally capsaicin-treated rats suggestthat these prolonged for LTD induction in the cerebellum (Linden et al., 1991). In changesin synaptic efficiency occur in a high-proportion of DH addition, we now report that the potentiation of transmission neurons receiving small-diameter primary afferent fiber inputs at the primary afferent-DH cell synapsescan also be expressed (Rethelyi, 1977; Light and Perl, 1979; Sugiuraet al., 1986, 1989). by the pharmacologically isolated NMDA receptor-mediated However, the transverse slice preparation, which retained only EPSP. This finding is in agreementwith previous reports show- 3-6 mm of attached dorsal roots, did not permit us reliable ing that the NMDA receptor-mediated synaptic responsecan measurementsof conduction velocity of the stimulated primary also undergo synapse-specificLTP in the hippocampus (Bashir afferent fibers in order to conclude which fiber groups were et al., 1991; Xie et al., 1991). responsiblefor the EPSPsin each case.Because of this problem, An interesting finding of the present study was that in a given analysis of the conduction velocity of primary afferent fibers cell the sametetanic stimulation can induce either LTP or LTD responsiblefor LTP or LTD of monosynaptic EPSPs should be of the synaptic responsedepending on the level of membrane undertaken in the future usingthe longitudinal slicepreparation potential of the postsynaptic neuron during the tetanic stimu- having intact dorsal roots and dorsal root ganglia attached. lation. Moreover, this result provides a direct proof that the The EPSP that is seento be modified is mediated by AMPA processesgenerating LTP or LTD of the primary afferent neu- receptors since it is blocked by AMPA antagonists, such as rotransmissionboth depend on postsynaptic membrane poten- NBQX (Sheardownet al., 1990; Cerne et al., 199l), and is little tial and that they have different thresholds. Previous studies affected by NMDA antagonist APV (Gerber and RandiC, 1989; have shown fhat in neocortex the sameafferent activity can lead Yoshimura and Jessell, 1990). These results agree with pub- either to LTP or LTD depending on the level of postsynaptic

Figure 12. APV prevented the induction of the potentiation of polysynaptic EPSPs in the SDH. A, The graph shows the time course of a long-lasting potentiation ofa polysynaptic EPSP recorded B _ intracellularly from an SDH neuron in response to electrical stimulation (5 V, A,f+-+2fi, 0.05 msec) of a lumbar dorsal root. At 10mr time 0 (arrow) the dorsal root was given three tetani (at twice the intensity ofthe test stimulus) of 1 set duration, each at 100 Hz and 10 set intervals. B, APV (50 PM) prevented the induction of the potentiation in the same neuron. Above the graphs are displayed individual EPSPs obtained before (A, B, truce I) and during LTP (A, truce 2) and 18 min -10 -5 0 5 10 15 20 25 after removal of APV (B, truce 2). V, TIME (mln) TIME (min) = -69 mV, 30-d-old rat. 5238 Randit: et al. - Modulation of Primary Afferent Neurotransmission depolarization obtained during the tetanus. If depolarization is effect has been establishedhas no effect; that is, APV doesnot strong enough to reach the activation threshold for NMDA prevent expressionof the enhancementof EPSP. receptor-gated channels, the tetanus causes LTP, if depolariza- Although we cannot at present be certain whether pre- or tion remains below this level, LTD is induced (Artola et al., postsynaptic factors, or both, are responsiblefor expressionof 1990). This suggests as one variable a postsynaptic voltage- a positive amplitude modulation of monosynapticEPSPs in DH dependent signal and, together with recent results of the same neurons following high-frequency stimulation, two findings ob- group (Brother et al., 1992), raises the possibility that Ca2+ may tained in our work provide evidence suggestingthe involvement actually serve as a trigger for both LTP and LTD. of presynaptic factors. First, we have demonstrateda sustained relative increase in the amount of endogenousglutamate and LTP of synaptic responsesin the rat DH neuronsfollowing aspartatereleased into the spinal slicesuperfusate following high- repetitive activation of primary aflerents frequency stimulation of primary afferent fibers in DRs (Randic Although a great deal is known about LTP in hippocampus and et al., 1993; Rusin et al., 1993b). This result is in agreement cerebral cortex (Collingridge and Singer, 1990; Madison et al., with the findings of Bliss et al. (1986) that initially provided 1991; Siegelbaumand Kandel, 1991; Johnston et al., 1992), the evidence for enhancedpresynaptic releaseof glutamate and as- cellular mechanism(s)underlying this phenomenon in the su- partate during LTP in the dentate gyrus of anesthetized rats. perficial laminae of SDH has yet to be elucidated. Second, the present study has revealed that not only AMPA There is a general consensusthat in the Schaffer collateral- receptor-mediated EPSP, but also a pharmacologically isolated commissural pathway in the CA1 region of the hippocampus NMDA receptor-mediated synaptic responsecan undergoLTP. the induction of LTP involves processeslocated in the post- LTP of NMDA receptor-mediated transmissionin hippocam- synaptic cell. The induction of LTP is presently thought to re- pal slice has been previously reported (Bashir et al., 1991; Xie quire both activation of NMDA receptors by synaptically re- et al., 1991). leasedglutamate (Collingridge et al., 1983) and depolarization Although the resultsmentioned above suggestthat the main- of the postsynapticmembrane (Gustafsson and Wigstrom, 1986). tenance of positive amplitude modulation of EPSPs following The biochemical cascadesthat lead to the associative form of high-frequency stimulation depends on properties of the pre- LTP in cortical structures appear to be triggered by a surge of synaptic terminals, they do not rule out a contribution from free Ca2+ ions in the postsynaptic neuron after activation of other mechanisms.These include increasein sensitivity of DH NMDA receptor-gated CaZ+conductances (Mayer et al., 1984; neurons to locally applied AMPA receptor ligands (Davies et Nowak et al., 1984) and voltage-dependentCaZ+ channels (Mad- al., 1989; Cemeand RandiC, 1992; Cemeet al., 1992), activation ison et al., 199 1; Siegelbaumand Kandel, 1991; Johnston et al., of parallel neural networks affecting transmissionat either pre- 1992). Ca2+influx is thought to lead to protein phosphorylation, or postsynaptic levels, co-releaseof EAAs and peptides from probably initiated by Ca2+/calmodulin kinase II (protein kinase terminals (Loechner et al., 1990) resulting in the modulation of B) and Ca*+ phospholipid-dependentkinase (protein kinase C) the amount of releasedtransmitter (Kangrga and Rand%, 1990) (Madison et al., 199 1; Siegelbaum and Kandel, 1991). More- or sensitivity of postsynaptic EAA receptors(RandiC et al., 1990; over, studies of LTP in the CA1 region of the hippocampus Rusin and RandiC, 1991; Rusin et al., 1992, 1993a,b; Kolaj et have revealed that although LTP is induced postsynaptically, al., 1993), and activation of secondmessenger systems (Gerber the maintenance of LTP may be, at least in part, presynaptic et al., 1989; Ceme et al., 1992, 1993). At present we have no due to a long-lasting enhancement of transmitter release(Dol- evidence for or against the involvement of any of these mech- phin et al., 1982; Blisset al., 1986; Davies et al., 1989; Bekkers anisms. and Stevens, 1990; Hessand Gustafsson, 1990; Malinow and It has been known for sometime that C fibers, which provide Tsien, 1990; Tsien and Malinow, 1990; Malinow, 1991) and a major primary afferent input to SG neurons (Rethelyi, 1977; delayed postsynaptic increase in the number or sensitivity of Light and Perl, 1979; Sugiura et al., 1986, 1989) have a unique postsynaptic AMPA subtypes of glutamate receptor (Davies et capacity to produce activity-dependent alterations in the excit- al., 1989). In agreementwith this model, glutamate and NMDA ability of neuronsin the spinal cord. Two such afferent-induced application wasfound to potentiate synaptic transmissionin the excitability changeshave been describedin vivo. The first is the hippocampus (Kauer et al., 1988) and, in the present study, in phenomenon of “windup,” a progressiveincrease in the number the superficial DH region of the spinal cord (Ceme et al., 199 1). of action potentials elicited per stimulus that occurs in DH As shown earlier (Schneiderand Perl, 1988; Gerber and Rand- neuronswhen C fibers are repetitively stimulated at frequencies ic, 1989; Yoshimura and Jessell,1990), and in the presentstudy, of >0.2 Hz (Mendell and Wall, 1965; Mendell, 1966; Schouen- during low-frequency transmissionNMDA receptorscontribute berg and Sjolund, 1983). The second is prolonged increasein little to a subthreshold, presumably monosynaptic EPSP re- the excitability of spinal neuronsfollowing a brief C fiber strength corded from a neuron in the superficial laminae of the spinal conditioning stimulation (Woolf, 1983; Woolf and Wall, 1986; DH at its restingmembrane potential in a standardphysiological Cook et al., 1987; Thompson et al., 1990). Both phenomena, medium. However, during high-frequency transmissionthe cell in addition to the enhancementof EPSPsdescribed in the pres- remainsdepolarized for a sufficient time to enable activation of ent study, are of interest in relation to the mechanismsinvolved the NMDA receptor system(Collingridge et al., 1988a,b;Gerber in the generation of postinjury pain hypersensitivity. and RandiC, 1989; Gerber et al., 1991). We have demonstrated in the present study that activation of NMDA receptors is a LTD of synaptic responsesin the rat DH neuronsfollowing requirement for the induction of the long-lasting enhancement repetitive activation of primary aflerents of EPSPs of DH neurons since APV, an NMDA receptor an- BesidesLTP of excitatory synaptic transmission, one mecha- tagonist, blocks this process. Moreover, whereas perfusion of nism that has recently received much attention is associative APV during high-frequency stimulation (the induction phase) LTD of synaptic transmission. Like LTP, LTD also provides a blocks the potentiation, perfusion of APV after the potentiating meansfor regulating the strength of synaptic connections in the The Journal of Neuroscience, December 1993. 13(12) 5239 mammalian brain and spinal cord. LTD has been extensively activated primary afferents: (1) activation of presynaptic inhib- studied in the cerebellum (Ito and Kano, 1982; Crepe1 and Kru- itory pathways (Nicoll and Alger, 1979; Nicoll et al., 1990), and pa, 1988; Ito, 1989; Linden et al., 199 l), the hippocampus (Stan- (2) reduced efficacy of the transmitter release machinery of the ton and Sejnowski, 1989; Pockett et al., 1990; Sejnowski, 199 1; synapse following high-frequency stimulation of DRs. The in- Dudek and Bear, 1992) and the cerebral cortex (Artola et al., volvement of GABA,-mediated presynaptic inhibition in the 1990; Hirsch and Crepe& 1990; B&her et al., 1992). Prolonged, prolonged synaptic depression is ruled out by the finding that the low-frequency (up to 5 Hz) depression of synaptic trans- blockade of the GABA, receptors by a specific antagonist bi- mission between dorsal root afferents and a-motoneurons was cuculline did not affect the level and time course ofthe prolonged recently described in the in vitro hemisected spinal cord prep- depression of EPSPs in SDH cells. aration isolated from neonatal (6-g-d-old) rats (Lev-Tov and There are several possible explanations for our results that Pinto, 1992), and during higher-frequency (18 Hz) stimulation do not involve plasticity of stimulated synapses. For example, of single group Ia fibers in motoneurons of anesthetized cats it is possible that the high-frequency stimulation produces ex- (Koerber and Mendell, 199 1). citotoxic damage in the target neurons or some generalized loss An important finding of this study is that high-frequency re- of postsynaptic excitability or damage or fatigue of the stimu- petitive stimulation of the primary afferent input to neurons in lated inputs. If this were the case then repeated stimulation in the superficial laminae of the spinal DH, including SG cells, can the same cell would be expected to always produce LTD, irre- lead to a reliable and long-lasting depression of synaptic trans- spective of the intensity of the stimulus employed and mem- mission manifested as a significant decrease in EPSP amplitude. brane potential at which tetanus was delivered. However, we The exact cellular mechanism(s) underlying this depression pro- occasionally observed (n = 7) that the synaptic response dis- cess has yet to be elucidated. In agreement with the results played LTD when train was delivered at test intensity for a obtained in the visual cortex (Artola et al., 1990), evidence has single EPSP, but LTP was induced when intensity of tetanic been obtained in our study that LTD induction at least in part stimulation was increased. In addition, the depressed input could depends on postsynaptic mechanism. It requires a critical level still undergo LTP after applying identical high-frequency stim- of membrane potential but does not depend on NMDA or GA- ulus at a more depolarized level. Although these observations BA, receptor activation. Thus, we show that LTD of primary do not rule out presynaptic damage or depletion of transmitter, afferent neurotransmission can be induced in the presence of they do indicate that depressed synapses are sufficiently viable APV, the finding that excluded a possible involvement of post- to support the mechanisms that give rise to LTP. synaptic NMDA receptors in the induction mechanism. This result is in agreement with previous studies of homosynaptic References LTD in the CA 1 region of hippocampus (Stanton and Sejnowski, Artola A, Briicher S, Singer W (1990) Different voltage-dependent 1989) and in the rat visual cortex (Artola et al., 1990). However, thresholds for inducing long-term depression and long-term poten- it differs from the results of Goldman et al. (1990) showing tiation in slices of rat visual cortex. Nature 347:69-72. Ascher P, Nowak L (1987) Electrophysiological studies of NMDA failure to reverse LTP by coupling sustained presynaptic activity receptors. Trends Neurosci 10:284-288. and NMDA receptor blockade. In this context, it should be Bashir ZI, Alford S, Davies SN, Randall AD, Collingridge CL (199 1) noted that homosynaptic LTD in area CA 1 of hippocampus was Long-term potentiation of NMDA receptor-mediated synaptic trans- recently described in response to low-frequency stimulation (l- mission in the hippocampus. Nature 349: 156-l 58. 3 Hz) of the Schaeffer collaterals (Dudek and Bear, 1992) and Bekkers JM, Stevens CF (1990) Presynaptic mechanism for long-term potentiation in the hippocampus. Nature 346:724-729. the suggestion was made that it can be triggered by prolonged Bliss TVP, Douglas RM, Errington ML, Lynch MA (1986) Correlation NMDA receptor activation that is below the threshold for in- between long-term potentiation and release of endogenous amino ducing synaptic potentiation. The involvement of prolonged acids from dentate gyrus of anaesthetized rats. J Physiol (Lond) 377: changes in the passive properties of the DH neurons due to 39 l-408. Brother S, Artola A, Singer W (1992) Intracellular injection of Ca*+ activation of postsynaptic inhibitory pathways by DR stimu- chelators blocks induction of long-term depression in rat visual cortex. lation has been excluded by the findings that the prolonged Proc Nat1 Acad Sci USA 89: 123-127. depression of EPSP was not altered by blockade of the post- Burke RE (1987) Synaptic efficacy and the control of neuronal input- synaptic GABA, receptors in the spinal cord slices with bicu- output relations. Trends Neurosci 10:42-%5. culline. Ceme R, RandiC M (1992) Modulation of AMPA and NMDA responses in rat spinal dorsal horn neurons by truns-1 -aminocyclopen- Another postsynaptic mechanism that might contribute to the tane- 1,3-dicarboxylic acid. 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