Brain Research, 561 (1991) 236-251 (~ 1991 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/91/$03.50 ADONIS 000689939117033U

236

BRES 17033

Participation of excitatory amino acid receptors in the slow excitatory synaptic transmission in rat spinal dorsal horn G. Gerber*, R. Cerne and M. Randi6 Department of Veterinary Physiology and Pharmacology, Iowa State University, Ames, IA 50011 (U.S.A.) (Accepted 21 May 1991) Key words: N-Methyl-o-aspartate receptor; Non-N-methyl-D-aspartate receptor; Slow excitatory synaptic transmission; Spinal slice; Dorsal horn neuron

In a rat spinal slice preparation the participation of excitatory amino acid (EAA) receptors in the responses of deep dorsal horn neurons repetitive stimulation of lumbar dorsal roots was investigated using 3 EAA receptor antagonists, kynurenic acid, D-(-)-2-amino-4phosphonovaleric acid (D-APV) and 6-cyano-2,3-dihydroxy-7-nitroquinoxaline (CNQX) and current-clamp and voltage-clamp techniques. We found that the slow excitatory synaptic response evoked by 10-20 Hz electrical stimulation of primary afferent fibers consisted of two depolarizing components: an initial component lasting 1-5 s and a late one of 1-3 min duration. The initial and late components of the slow excitatory synaptic response can also be distinguished on the basis of their voltage-dependence and sensitivity to Mg 2+ ions, kynurenate, D-APV and CNQX. In the presence of Mg 2÷, the initial component of the slow excitatory synaptic response increased with membrane hyperpolarization, whereas the late component decreased in most of the cells examined. In a zero-Mg 2+ medium, the initial component was potentiated, but the late component was reduced. In both transverse and longitudinal spinal cord slices perfused with 1.2 mM Mg 2+-containing medium, bath application of kynurenic acid (0.1-0.5 mM), D-APV (0.05-0.1 raM) and CNQX (5-7/~M) caused a reversible reduction of the peak amplitude of the initial slow depolarizing component that was greater in transverse (kynurenic acid: by 92.6 -+ 5.0%; D-APV: by 69.1 -+ 7.8%; CNQX: by 76.6 -+ 9.8%) than in longitudinal slices (kynurenic acid: by 53.3 -+ 1.3% ; D-APV: by 31.5 -+ 9.1%; CNQX: by 35.3 -- 11.1%). In contrast, all 3 antagonists of EAA receptors produced no consistent change in the peak amplitude or half-duration of the late depolarizing component of the slow excitatory synaptic response. Our results obtained with EAA receptor antagonists, at resting membrane potentials, in the absence and presence of Mg 2+ and synaptic inhibition, indicate that the synaptic activation of the NMDA- and non-NMDA-receptor systems of deep spinal dorsal horn neurons by repetitive stimulation of primary afferent fibers may be selectively involved in the mediation of the initial, but not the late depolarizing component of the slow excitatory synaptic response. to

INTRODUCTION

dent release of G L U from electrically activated primary afferent fibers has been d e m o n s t r a t e d 4°'45'66'67'75. G L U

Synaptic transmitters released during activation of primary sensory neurons in the dorsal horn ( D H ) of the rat spinal cord m a y elicit b o t h fast and slow excitatory synaptic responses in a single neuron 52'76. G l u t a m a t e ( G L U ) , or a related amino acid, appears to be the major candidate for the fast excitatory neurotransmitter in the m a m m a l i a n central nervous system 5°'8°, including the spinal D H 14'21,24,27,28,34,38,40-44,72,73,86,87. Tachykinins, sub-

binding sites are found in high densities in the superficial laminae of the rat spinal D H 3°'51. G L U was found

stance P (SP) and neurokinin A ( N K A ) , and perhaps also o t h e r peptides in the dorsal root ganglia ( D R G ) 3' 11,32,39, a p p e a r to be functionally involved in the slow excitatory synaptic transmission 28"61"63"64"69'7°'76. Immunocytochemical studies have shown that about 70% of both large and small D R G neurons are labeled for G L U 79, and G L U immunoreactivity has been detected within myelinated and unmyelinated primary afferent terminals in the superficial D H 82'83. Ca2+-depen -

to excite and depolarize almost all spinal D H neurons in vivo 7"8° and a p r o p o r t i o n of D H neurons in vitro 72"86"87. The actions of G L U are m e d i a t e d by at least 3 pharmacologically distinct receptor subtypes characterized on the basis of their responsiveness to selective agonists, quisqualate ( Q A ) , kainate ( K A ) and N-methyl-D-aspartate (NMDA), and antagonists21,80,81. D-2-Amino-5-phosphonovalerate ( D - A P V ) is a specific and potent antagonist of N M D A receptors ~3, whereas 6-cyano2,3-dihydroxy-7-nitroquinoxaline ( C N Q X ) has been r e p o r t e d to be a potent competitive antagonist at nonN M D A receptors, especially kainate receptors 19"33. Although the present view is that n o n - N M D A receptors (quisqualate, kainate) mediate most of the fast excitatory postsynaptic potentials (EPSPs) in the spinal cord 1,

* Present address: Second Department of Anatomy, Semmelweis University Medical School, Budapest, H-1450, Hungary. Correspondence: M. Randir, Department of Veterinary Physiology and Pharmacology, Iowa State University, Ames, IA 50011, U.S.A.

237 5,19,33-35,71.80.81 e v i d e n c e is a c c u m u l a t i n g that e x c i t a t o r y a m i n o acid ( E A A ) r e c e p t o r s o f the N M D A class are inv o l v e d in a slow e x c i t a t o r y s y n a p t i c t r a n s m i s s i o n in s o m e spinal i n t e r n e u r o n s r e c e i v i n g i n p u t f r o m p r i m a r y s e n s o r y n e u r o n s 6.8,10,13,22,27,28,36,42,46,74. T h e possibility o f f u n c t i o n a l i n v o l v e m e n t o f E A A rec e p t o r s also in t h e d o r s a l r o o t ( D R ) r e p e t i t i v e stimulat i o n - e v o k e d slow e x c i t a t o r y synaptic r e s p o n s e s o f d e e p D H n e u r o n s that e x p r e s s r e c e p t o r s for E A A and p e p tides has n o t b e e n i n v e s t i g a t e d until r e c e n t l y 14'16'28. T h e p r e s e n t study was u n d e r t a k e n

10"-4-10-2 M for 0.1-0.5 s) with tip diameters of 5-10 ~m. Positioning of these micropipettes within 100/~m of the cell body reliably produced excitatory amino acid responses. Pressure application of bath solution alone had no effect. Kynurenate (Sigma), D-APV (CRB, Sigma), and CNQX (Tokris) were administered by addition to the perfusate. Dorsal horn neurons were voltageclamped using a single electrode-mode of voltage-clamp amplifier (Axoclamp 2). The sampling frequency was 3-4 kHz, 30% duty cycle. Microelectrodes filled with cesium-acetate were exclusively used for the voltage-clamp experiments in order to reduce the potassium currents and improve the space clamp. Statistical significance of data has been assessed relative to control responses by use of either a paired or unpaired Student's t-test, as appropriate.

to f u r t h e r e x p l o r e t h e

p h a r m a c o l o g y o f the slow e x c i t a t o r y synaptic transmis-

RESULTS

sion in the rat spinal D H and especially t h e possibility of t h e i n v o l v e m e n t o f n o n - N M D A a n d N M D A - r e c e p t o r s

General observations

in the m e d i a t i o n o f b o t h d e p o l a r i z i n g c o m p o n e n t s of t h e

Stable i n t r a c e l l u l a r r e c o r d i n g s o f u p to 5 h w e r e ob-

slow e x c i t a t o r y synaptic r e s p o n s e o f d e e p D H n e u r o n s

t a i n e d f r o m 114 D H n e u r o n s l o c a t e d in l a m i n a e I I I - V I

using in v i t r o spinal c o r d slice p r e p a r a t i o n a n d c u r r e n t

of t h e spinal cord. D H n e u r o n s e x h i b i t e d m e a n resting

clamp

voltage-clamp techniques.

p o t e n t i a l , m e m b r a n e i n p u t resistance and action p o t e n -

P r e l i m i n a r y r e p o r t s o f s o m e aspects o f this w o r k h a v e b e e n p u b l i s h e d 28.

- 5 5 to - 8 0 m V ) , 75.4 --+ 11.7 M f l (range: 2 6 - 1 4 0 Mf~)

and

single-electrode

tial a m p l i t u d e o f - 6 5 . 7

-- 0.8 m V ( m -+ S . E . M . ; r a n g e :

and 75.7 -- 1.9 m V ( r a n g e 6 0 - 8 6 m V ) . Single a n d r e p e t i t i v e stimuli a p p l i e d to t h e l u m b a r

MATERIALS AND METHODS

dorsal r o o t l e t e v o k e d in t h e d e e p D H n e u r o n s 4 distinct Horizontal and transverse slices were obtained from SpragueDawley rats of both sexes (17-31 days old) by using a technique that has been described elsewhere 26'52'53. Briefly, after the animal was anesthetized with ether, a segment of the lumbosacral (L4-$1) spinal cord was dissected out and sectioned with a Vibratome to yield several transverse slices with short (3-5 mm) dorsal rootlets or one horizontal slice, 300-400/~m thick, with attached DRs and ganglia. After the incubation for 1 h in oxygenated (95% O 2 + 5% CO2) control solution (in mM: NaCl 124, KCl 5, KH2PO 4 1.2, CaCl 2 2.4, MgSO 4 1.3, NaHCO 3 26, glucose 10, pH 7.4 at 30 -+ 1 °C), a slice was transferred into a recording chamber, where it was submerged beneath an oxygenated superfusing medium (flow rate about 3 ml/min) containing lowered concentration of potassium ions (1.9 mM KC1). The use of a high-K + solution during cutting and incubation of the slices seemed to improve their viability as assessed electrophysiologically in the same preparation. Conventional electrophysiological current and voltage-clamp techniques were useed for intracellular recording from DH neurons (laminae III-VI), as described 52"55"68. Under visual control, a single fiberglass microelectrode filled with either 4 M potassium acetate or 3 M cesium-acetate (DC impedance: 90-120 MQ) was placed in the DH, and neurons impaled by oscillating the capacity compensation circuit of a high input impedance bridge amplifier (DAGAN 8100 or Axoclamp 2). Cells were activated synaptically by electrical stimulation of primary afferent fibers. A coaxial stainless steel stimulating electrode (o.d. of inner and outer electrodes being 25 and 200/~m, respectively; Frederick Haer Co.) positioned on a lumbar dorsal rootlet was used in transverse slices and a bipolar platinum wire electrode in longitudinal spinal cord slice with attached DRG. Single (0.02-0.5 ms pulses, 1-25 V) or repetitive (10-20 Hz 1-2 s) stimuli to lumbar DRs were used to elicit slow excitatory synaptic response. The synaptic responses were stored on diskettes of a digital oscilloscope (Nicolet, model 4092) or Axolab 1100 system with pCLAMP software was used on-line. A DC pen-recorder (Gould 2600S) was used to record membrane potential continuously. QA (Cambridge Research Biochemicals, CRB; Sigma), KA (Sigma), L-glutamate, L-aspartate (Peptides International, Sigma) and NMDA (CRB), were applied extracellularly by positive pressure ejection (1-5 kPa) from micropipettes (drug concentration:

types o f d e p o l a r i z i n g p o t e n t i a l s o f v a r i e d d u r a t i o n (Fig. 1). T h e y w e r e r e v e r s i b l y b l o c k e d by t e t r o d o t o x i n ( T r x ,

A

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10Hz lmin

Fig. 1. A: the upper trace is the computer average of 6 successive, presumably monosynaptic, EPSPs elicited by a low intensity stimulation of a lumbar (L6) dorsal rootlet (2V, 0.2 ms) at the resting membrane potential of the cell (V m = -68 mV), and the lower trace is the average of 12, presumably polysynaptic, EPSPs evoked by a single shock stimulus (4 V, 50/~s) to a lumbar (Ls) dorsal root at Vm of -64 mV. B,C: high-intensity repetitive stimulation (upper traces: 15 V, 0.2 ms at 8 Hz for 2 s; lower traces: 15 V, 0.5 ms at 10 Hz for 1 s) of a lumbar (Ls) dorsal rootlet evoked a slow excitatory synaptic response consisting of an initial slow depolarizing component (upper and lower trace in B) in the both cells and a late slow depolarizing component (lower trace in C) only in the cell responding with a polysynaptic EPSP (lower trace in A). Horizontal spinal slices. Upper row, 19-day-old rat; lower row, 21-day-old rat. In this and subsequent figures stimulus artifacts have been blanked for purpose of clarity and the times of stimulation are indicated by arrowheads.

238 5 × 10 -7 M), or by superfusing the slices with a low Ca2+-high Mg2+-containing Krebs solution, suggesting that they have synaptic origin (not illustrated). Responses to single stimuli. As shown in Fig. 1, low intensity stimulation of a lumbar D R evoked a presumed monosynaptic (Fig. 1A, top trace) or polysynaptic (Fig. 1A, bottom trace) EPSP in deep D H neurons. In 5 of 80 neurons studied, the post-stimulus latency of DR-initiated EPSP remained constant when higher-intensity repetitive stimuli (10 Hz) were used, and also in the presence of high concentration of divalent cations (4 mM Ca2+-8 m M Mg2+), suggesting a monosynaptic input from primary afferents (Fig. 1A, top trace). However, in most of the deep dorsal horn neurons the post-stimulus latency of the DR-elicited EPSPs was variable, suggesting a polysynaptic input from primary afferents. Increasing the intensity of primary afferent stimulation usually increased the amplitude and duration of the polysynaptic EPSP (Fig. 1A, bottom trace), whereas perfusion with Krebs solution containing 4 m M CaZ+-8 mM Mg 2+ markedly depressed variable latency EPSPs. Responses to repetitive stimulation. When repetitive stimulation (trains of 10-40 stimuli at a rate of 10-20/s) at higher intensity (2-25 V pulses of 0.2-0.5 ms duration) was applied to primary afferent fibers in the dorsal root, two additional slow excitatory responses were noted. During the stimulus train, the m e m b r a n e depolarized (an initial slow depolarization) and summation of evoked fast EPSPs usually occurred (Figs. 1B and 3A) that frequently led to action potential discharge 28. The initial depolarizing component of the slow excitatory synaptic response was of longer duration (half-duration: 1.47 --- 0.2 s; mean --- S.E.M., n -- 53; range 0.3-3.4 s) and had slower rise and decay times than the fast EPSPs (Fig. 1B). A long-duration depolarization (a late slow depolarization) frequently associated with an increase in frequency and amplitude of spontaneous E P S P s 61'64'76 followed a stimulus train after 1-4 s, or more (Fig. 1C, bottom trace; Fig. 2C,D). The late slow depolarization was more frequently encountered in transverse (about 76%, n = 75) than in longitudinal slices (about 31%, n = 39). The shape, the incidence and the distribution of recording sites at which the various types of the late slow depolarization were observed in the transverse slices are shown in Fig. 2 D - I . In the first group which constituted about 53% of the cells (n = 57) in transverse slices and 25% of the cells (n = 12) in longitudinal slices, the late slow depolarization was monophasic (Fig. 2D,G), and it followed an initial orthodromic burst of action potentials (vertical tracings have been truncated). The late slow depolarization had an average peak amplitude of 6.6 --- 0.7 mV, and a half-duration of 1.3 _-_ 0.1 min

(n = 35). In the second group, which represented about 16% of the cells in transverse slices and 8% of cells in longitudinal slices (Fig. 2E,H), the initial orthodromic burst was followed by a hyperpolarization which in turn was followed by a late slow depolarization. The average peak amplitude of the late slow depolarization was 4.1 - 0.4 mV and a half-duration of 1.0 --- 0.2 min (n = 9). The third group (Fig. 2F, I), which amounted to about 32% (n = 57) of the cells in the transverse slices and 67% (n = 12) of the cells examined in the longitudinal slices, the initial orthodromic burst was followed by a slowlydecaying late depolarization having average peak amplitude of 4.5 - 0.8 m V and a half-duration of 0.6 --- 0.05 min (n = 26). There is a significant difference in the amplitude and half-duration between Groups I and II (amplitude: P < 0.007; half-duration: P < 0.01), Groups I and III (amplitude: P < 0.05; half-duration: P < 0.0001) and Groups II and III (amplitude: P < 0.02; half-duration: P < 0.05). Primary afferent fibers activated by single volleys (6-V pulses of 0.2 ms duration) elicited a long-lasting membrane depolarization (30-60 s) in a proportion of dorsal horn cells (Fig. 2A).

Dependence of the initial and the late slow depolarizations on the type of slice and intensity and frequency of primary afferent volleys The amplitude of the initial and the late depolarizing

A

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Fig. 2. Late slow depolarizing potentials recorded from dorsal horn neurons after electrical stimulation of primary afferent fibers. Recordings from 4 different neurons (A-F) are depicted. A: a single dorsal root stimulus (10 V, 0.2 ms) produced a late slow depolarizing potential (A) which was enhanced both in amplitude and, especially in duration (C), following repetitive dorsal root stimulation (10 V, 0.2 ms at 10 Hz for 2 s). Inset (B) shows the location of the examined cell in the spinal dorsal horn. D-F: 3 different types of the late slow depolarizing responses of dorsal horn neurons following repetitive stimulation of dorsal roots are shown. G-I: distribution of recording loci from the dorsal horn neurons for the different types of the late slow depolarizing potentials illustrated in D-F are shown for the larger sample of examined cells.

239 components of the slow excitatory synaptic response was a function of the type of slice used and the frequency and intensity of electrical stimulation of the lumbar dorsal root (Fig. 3). Whereas the initial depolarization was larger in amplitude in longitudinal slices, the late depolarization was more prominent in transverse slices. As shown in Fig. 3, repetitive stimulation of primary afferents at a frequency between 1-50 Hz and 2-16 V intensity increased the peak amplitude of the initial (Fig. 3B,C) and the late slow depolarizations (Fig. 3D,E). In the example illustrated in Fig. 3 A - B it is evident that the initial depolarizing component was detectable at 2.5 Hz and increased in amplitude as the frequency and number of shocks increased (Fig. 3B). There was a positive correlation (r = 0.96) between the amplitude of this component and the frequency of stimulation. T h e peak amplitude of this component was also dependent on intensity of stimulation of the dorsal roots. However, it reached the peak at different times in different cells

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(Figs. 3A and 8A). The lowest stimulus intensity at which accurate measurements could be made was 3 V for pulses of 0.2 ms duration (Fig. 3C). The size of the initial depolarizing component increased with stimulus intensity and the maximum observed amplitude was approximately 24 mV (Fig. 3C). The average amplitude (12.2 --- 0.9 mV, n = 53) of this component was usually smaller than the peak amplitude of the first fast EPSP in the stimulus train (Fig. 3A). In those cells in which a late slow depolarizing potential evoked by single pulses could be demonstrated (Fig. 2A), repetitive stimulation of the dorsal root especially at frequencies between 5 and 20 Hz for 1-2 s increased the amplitude and duration of the late depolarization (Figs. 2C and 3D). The peak amplitude of the late slow depolarization also increased with stimulus intensity (Fig. 3E). It ranged from 2 to 18 mV when recorded at membrane potentials between -55 and -75 mV, with a mean of 5.5 - 0.5 mV (mean - S.E.M., n = 52). The duration of the late slow depolarizaton ranged from 0.3 to 7 min (2.7 --- 0.2 min; n = 66). The stimulus threshold necessary to elicit the late slow depolarizing potential appeared to be two or three times higher (about 6 V, 0.2 ms) than that required to evoke a fast EPSP.

Voltage dependence of the dorsal root-evoked slow excitatory response 1Hz

2.5

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We used the single electrode voltage-clamp technique to examine the voltage dependence and the sensitivity to Mg 2÷ of the initial and the late depolarizing components of the slow excitatory synaptic response of dorsal horn neurons recorded when train stimuli were applied to primary afferents.

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Fig. 3. A: the initial slow depolarizing potentials of a single dorsal horn neuron recorded in response to three different frequencies of dorsal root stimulation: 1, 2.5 and 8 Hz. B-E: plots of amplitude of the initial slow depolarization (B,C) and late slow depolarization (D,E) as a function of frequency (B,D) and intensity of dorsal root stimuli (C,E). Each point represents a single measurement of the peak amplitude (D,E) or the amplitude at times indicated by the arrows in A (B,C). Vm = -68 mV, 19-day-old rat (A); Vm = --70 mV, 26-day-old rat (B,C); Vm = -66 mV, 20-day-old rat (D); Vm = -70 mV, 31-day-old rat (E).

Voltage dependence and Mg 2+ sensitivity of the initial depolarizing component of the slow excitatory synaptic response As shown in Fig. 4B, the peak (Fig. 4Aa) and the steady-state (Fig. 4Ab) components of the inward synaptic current behaved differently when the membrane potential was varied between -110 and -50 mV, and in the presence or absence of added Mg 2÷ to the external solution. Irrespective of the presence of Mg 2÷ ions in the perfusion medium, the peak inward synaptic current increased with hyperpolarization in a manner expected for a response generated by a relatively voltage- and Mg 2÷independent conductance. In contrast, in MgE÷-contain ing medium, the steady-state component of the inward synaptic current was only slightly modified when the postsynaptic membrane potential, at which the cell was clamped, was varied between -110 and -50 mV. However, when external Mg 2÷ was removed, the amplitude of the steady-state component of the synaptic current

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Fig. 4. A: Superimposed records of the initial inward component of the slow excitatory postsynaptic current (EPSC) recorded at -114 mV in

the presence (trace 1) and in the absence of Mg2+ (trace 2). Arrows indicate the points of measurements of peak inward current component (a) and steady-state inward current component (b) used in the construction of the graphs shown in B-D. B: removal of external Mg 2+ has no significant effect on the peak inward current component but causes a progressive increase of the amplitude of the steady-state current component with membrane hyperpolarization. C-D: the effects of D-APV (50/~M for 12 min) on the peak and steady-state components of the inward current recorded at 4 different membrane potentials in 1.2 mM Mg 2+ (C) and in a nominally zero-Mg 2+ solution (D). Note the voltage-dependence of the blocking action of D-APV against the steady-state component but not the peak component of the slow EPSC, in 1.2 mM Mg2+ (C), and the absence of this behavior in a zero-Mg 2+ solution (D). Vm = -73 mV, 21-day-old rat.

increased with hyperpolarization (Fig. 4B), the effect being larger in transverse (190.6 --- 18.0%, n = 8) than in longitudinal slices (138.9 --- 7.5%, n = 11). The latter result suggested the involvement of the N M D A receptor-coupled ionophore in the steady-state component of the synaptic response, since the N M D A ionophore is gated by Mg 2+ ions in a voltage-dependent manner and this block is partially relieved by removing extracellular Mg 2+ (Refs. 2,59).

Voltage dependence and Mg 2+ sensitivity of the late depolarizing component of the slow excitatory synaptic response When in the present study we used the single electrode voltage-clamp technique, we confirmed the earlier finding 28 that the peak of the late component of the slow excitatory postsynaptic current varied non-linearly with clamped membrane potential in all cells examined. Moreover, the form of the non-linearity differed among cells examined. Three types of responses by dorsal horn neu-

rons could be distinguished as shown in Fig. 5. The first type of response, observed in the majority of tested cells (n = 8), exhibited a decrease in the peak inward synaptic current when the cells were clamped at membrane potentials between -52 and -100 mV (Fig. 5A). Although the slow synaptic current remained inward at the membrane potentials between -52 and -100 mV, in none of the cells was it possible to reverse the polarity of synaptic current (Fig. 5A). In one cell, the synaptic current increased at hyperpolarized potentials (Fig. 5B). In the third type of response (Fig. 5C), the slow EPSC amplitude remained unchanged when the cell was clamped at hyperpolarized membrane potentials exceeding the equilibrium potential for potassium (EK). The voltage dependency of the late slow depolarization in a proportion of dorsal horn neurons (Fig. 5A), as well as the depolarizing effects of N M D A on these neurons (Figs. 8C and 9C), raised the possibility that the late slow depolarization is mediated, at least in part, by N M D A receptors. Therefore, we examined the effects of

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1rain Fig. 5. Voltage-dependence of the late inward component of the slow excitatory synaptic response evoked by repetitive stimulation (15 V, 0.5 ms at 20 Hz for 2 s) of a dorsal rootlet. Three types of responses could be distinguished as shown in A-C. A: decrease in the peak inward current occurred with membrane hyperpolarization. B: an increase in the late inward component is shown at hyperpolarized potentials. C: the size of the late inward current component remains unchanged when the cell was clamped at membrane potentials negative to the equilibrium potential for potassium (EK). Inset shows the approximate locations of the examined cells. V m = -74 mV, 21-day-old rat (A); V~ = -60 mV, 20-day-old rat (B); Vm = -72 mV, 16-day-old rat (C).

removing Mg 2+ from the bathing solution on this component using both current- and voltage-clamp techniques. Within 20-30 min superfusion with a nominally Mg 2+free medium the late slow depolarizing response decreased in peak amplitude (by 60.1 _ 5.7% in 6 of 8 cells) and duration (by 60.3 --- 4.0% in 8 of 9 cells). The depressant effect of a zero-Mg 2+ medium on the late inward synaptic current was also evident when the postsynaptic membrane potential, at which the cell was clamped, was varied between -110 and - 5 0 m V 28. These results indicate that the activation of N M D A receptors contributes only in a small degree to the generation of the late depolarizating component of the slow excitatory synaptic response.

Effects of Mg2+-free medium on passive membrane properties of rat dorsal horn neurons During the period in which Mg 2+ was washed out of the slice, i.e. when we changed from a solution containing 1.2 m M Mg 2÷ to one with a nominally zero-Mg 2+, the membrane potential depolarized (Fig. 6Aa) in 13 of 19 cells examined, and hyperpolarized (Fig. 6Ba) in 2

cells. The removal of Mg 2+ from the external medium was frequently associated with an increase in the spontaneous synaptic activity (Fig. 6Aa). There was, however, no consistent change in neuronal membrane input resistance; both increase and decrease (Fig. 6Bb, right trace) were recorded. Out of 14 dorsal horn neurons, we observed an increase in 8 cells (115.6 -+ 2.3%), and a decrease in 3 cells (86.0 -- 9.5%). In a Mg2+-free medium, there was an increase in the excitability of almost all tested neurons, as indicated by a decrease in the threshold for action potential generation evoked by intracellular current injection (Fig. 6ABc, fight traces). Both depolarizing and hyperpolarizing responses of dorsal horn neurons to bath administration of N M D A were enhanced in a Mg2+-free medium (Fig. 6ABd, fight traces).

Possible involvement of intracellular metabolic process in the mechanism of generation of both components of the slow excitatory synaptic response A n important problem, which is not as yet investigated about the slow excitatory synaptic response of the

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~

15rnV

lmin Fig. 6. The effects of Mg2+-removal from the bathing solution on two different dorsal horn neurons, one with a predominantly excitatory input (A) and another one, with inhibitory input (B). A: when we changed from a solution containing 1.2 mM Mg 2+ to one with a nominally zero-Mg2+, the membrane potential depolarized and the synaptic activity was enhanced (a). Membrane input resistance measured by a hyperpolarizing pulse was not changed (b), but the excitability of the cell was increased (c) as indicated by a decrease in the threshold for action potential generation lowered by a depolarizing intracellular current pulse. B: Mg 2+ removal from the external solution led to the membrane potential hyperpolarization and a reduction in the membrane synaptic noise (a). In addition, there is a decrease in neuronal membrane input resistance (b), but the increase in excitability was observed (c). The depolarizing component of the NMDA response (Ad: 5 mM, 0.03 s, 5 kPa; Bd: 1 mM, 0.15 s, 2 kPa) was enhanced in A, whereas both depolarizing, but especially hyperpolarizing component, were enhanced in cell B. Vm = -76 mV, 18-day-old rat (A); Vm = -58 mV, 18-day-old rat (B).

d e e p spinal dorsal horn neurons, is molecular mechanism(s) underlying the initial and the late depolarizing components of this potential. In the experiment illustrated in Fig. 7, the effect of change of t e m p e r a t u r e in

A 3it

B 23t

C 30t

~I

_

0.4s

0$mla Fig. 7. Temperature sensitivity of the initial (upper row) and the late depolarizing (lower row) components of the slow.excitatory synaptic response evoked by repetitive stimulation (15 V, 0.5 ms, 20 Hz, 2 s) of a dorsal lumbar rootlet. A: control responses recorded at 31 °C. B: lowering the temperature to 23 °C reduced the size of both components. C: recovery at 30 °C. Vm = -58 mV, 16day-old rat.

the range of 23 °C and 31 °C on the slow excitatory synaptic potential was examined. Both, the initial and the late depolarizing c o m p o n e n t s of the slow train response were significantly suppressed by cooling the slice. The initial depolarizing c o m p o n e n t was completely abolished at 23 °C (Fig. 7B, top trace), whereas the late slow depolarization was m a r k e d l y r e d u c e d (Fig. 7B, b o t t o m trace). Both slow depolarizing potentials recovered, to a substantial degree, when the t e m p e r a t u r e was brought back to 30 °C. The t e m p e r a t u r e - d e p e n d e n c e of the slow excitatory synaptic response suggests that intracellular metabolic processes m a y be involved in the generation of the slow excitatory synaptic potential, and this then may explain the slow time course of the response.

Pharmacology of the initial and the late depolarizing components of the slow excitatory synaptic response: actions of EAA receptors antagonists To further investigate the possibility28 that the activation of both N M D A and n o n - N M D A receptors may have a physiological role in the generation of the initial

243 TABLE I

fective at both n o n - N M D A and N M D A receptors 25'6°.

Effects of EAA antagonists on the initial depolarizing component of the slow excitatory synaptic response

Kyn, b a t h - a p p l i e d in concentrations of 1 0 0 - 5 0 0 / z M for 3 - 1 4 min reversibly r e d u c e d o r abolished the p e a k amplitude and duration of the initial slow depolarizing component. The degree of depression caused by Kyn was d e p e n d e n t on the type of slice used and the external Mg 2÷ concentration. In the presence of 1.2 m M Mg 2+ average reduction (by 74.5 --- 8.2%, n = 13) in the p e a k a m p l i t u d e o f the initial slow depolarizing c o m p o n e n t was greater in transverse (by 92.6 --- 5.0%, n = 7) than in longitudinal slices (by 53.3 --- 1.3%, n = 6). In addition, the depressant effect was smaller in a zero-Mg2+-solu tion (Table I and Fig. 10). The depression caused by Kyn began 1-2 min after the drug reached the tissue and achieved its maximal effect 3 - 5 min later. A full recovery was usually o b t a i n e d within 15 min after washing with a Kyn-free solution. The ability of Kyn to reversibly antagonize the depolarizing responses of dorsal horn neurons to N M D A (Figs. 8C and 9C) and glutamate (Fig. 9D), and to completely block the initial depolarizing c o m p o n e n t of the train response, is illustrated in Figs. 8 and 9. Kyn, b a t h - a p p l i e d in concentrations o f 1 0 0 - 5 0 0 / ~ M for 3 - 1 4 min, p r o d u c e d inconsistent effect on the p e a k a m p l i t u d e and half-duration of the late slow depolarizing c o m p o n e n t in 10 cells e x a m i n e d (Table II). Thus,

Size of response is given in % of control. Results are expressed as mean -+ S.E.M. EAA antagonist

Kynurenate D-APV CNQX CNQX + D-APV

1.2 mM Mg2+

zero-Mg2+

Amplitude

n

Amplitude

25.5 --- 8.2 *• 43.5 + 6.7 *• 37.0 - 7.8** 16.4 - 6.0**

13 26 15 9

60.9 37,7 68.3 37.2

_+ 14.7 +-- 6.4 •* +- 8.7* +- 10.1

4 8 4 2

**P < 0.01. *P < 0.05. and the late depolarizing c o m p o n e n t s of the slow excitatory synaptic responses, the experiments with three antagonists of E A A receptors, kynurenic acid (Kyn), D - A P V and C N Q X , were p e r f o r m e d . In the next section, the effects of these antagonists of E A A receptors on agonist-evoked and dorsal r o o t - e v o k e d synaptic responses r e c o r d e d from 63 unidentified d e e p dorsal horn neurons will be described. Effects o f K y n on the initial and the late depolarizing components o f the slow excitatory response Kyn is a b r o a d l y acting E A A r e c e p t o r antagonist el-

Control

Kynurenate lrrtvl

Wash

A l 05s

B l

l

l

5mY

C I

m/n

Fig. 8. Kynurenate (1 mM for 6 min) almost completely blocked the initial slow depolarizing potential (A, middle record) evoked by highintensity repetitive primary afferent stimulation (15 V, 0.5 ms, 10 Hz, 1 s). The late slow depolarizing potential of the same cell was, however, increased by kynurenate (B, middle record) which blocked the depolarizing response of a dorsal horn neuron to pressure-ejected NMDA (C, middle trace: 5 mM, 0.2 s, 5 kPa). Note the disappearance of the initial hyperpolarization preceding the late slow depolarizing component during the bath application of kynurenate (B, middle trace). Left column, control responses; middle column, responses obtained during the administration of kynurenate; right column shows records obtained 4 min after the drug was washed away. Longitudinal slice. V m = --62 mV, 24-day-old rat.

244 TABLE II

Effects of EAA antagonists on the late depolarizing component of the slow excitatory synaptic response Size of response is given in % of control.

EAA antagonist

Kynurenate D-APV CNQX CNQX + D-APV

Amplitude

Half-duration

Total (n)

Increased

Decreased

Increased

Decreased

140.7 -+ 5.6** (n = 4) 128.1 -+- 7.5* (n = 7) 133.0 (n = 1) -

63.0 63.2 61.3 61.3

167.1 146.4 203.5 130.9

66.5 -+ 4.5 (n = 2) 53.0 + 5.13"* (n = 3) 32.1) (n = 1) 81.5 -+ 1.8 (n = 2)

_+ 14.6 (n = 3) -+ 5.4** (n = 4) -+ 12.4 (n = 3) _+ 8.7* (n = 4)

-+ 18.7" (n = 6) -+ 7.3** (n = 8) + 23.0 (n = 2) -+ 1.4" (n = 2)

10 18 8 7

both in the presence and in the absence of added Mg 2+

plitude of the late slow depolarization (Fig. 9B) in 3 cells

to the external medium, Kyn suppressed the peak am-

(by 37.0 -+ 14.6%) and half-duration in 2 cells (by 33.5 -+ 4.5%), potentiated the amplitude (to 140.7 -+ 5.6%) in 4 cells and half-duration (to 167.1 -+ 18.7%) in 6 cells

Control KYN0.5mM

(Figs. 8B, second trace and 10B, fourth trace), and was without effect in the rest of the cells tested. It is of interest that, as shown in Fig. 8B, the e n h a n c e m e n t of the late slow depolarization was associated with a reduction of the slow hyperpolarizing potential that preceded it. Kyn (100-500 ~M for 3-14 min) markedly depressed or

Control APVO.lmM

abolished the responses to glutamate (Fig. 9D, second

0.5s

B

I

trace), N M D A (Figs. 8C, middle trace; 9C, second trace; 10D, fourth trace) and Q A (Fig. 10C, fourth trace). Kyn (0.5-1 m M for 3-14 min) produced a small (-2 to - 4 mV), frequently transient (3-5 min) hyperpolarization in 12 of 22 cells examined, whereas in 3 cells exhibiting fast and slow inhibitory postsynaptic potentials, a small depolarization occurred. Characteristically in all cells tested a marked reduction of the spontaneous syn-

Imin Fig. 9. The effects of kynurenate (KYN, second column) and D-2amino-5-phosphonovalerate (APV, fourth column) on the initial (A) and late (B) slow depolarizing potentials (15 V, 0.5 ms at 20 Hz for I s) and on the responses of the same dorsal horn neuron to NMDA (C) and glutamic acid (D, Glu). First and third columns show control responses, second and fourth columns responses recorded 7 min (second column) and 6 min (fourth column) after the onset of the bath application of kynurenate and APV, respectively. Bath-applied kynurenate (0.5 mM for 7 min) blocked the early slow depolarizing potential (A, second trace) and significantly reduced the depolarizing responses of a dorsal horn neuron to pressureejected NMDA (C, second trace: 1 mM, 0.5 s, 5 kPa) and Glu (D, second trace: 10 mM, 0.3 s, 5 kPa). Whereas kynurenate reduced synaptic activity, the amplitude of the late slow depolarizing potential was only slightly reduced (B, second trace). D-APV (0.1 mM for 6 min) also blocked the early slow depolarizing potential (A, fourth trace), whereas the late slow depolarizing potential was not significantly modified (B, fourth trace). The responses to NMDA (C, fourth trace) and Glu (D, fourth trace) were reduced. Vm = -65 mV, 19-day-old rat.

aptic activity did occur. There was no consistent change in the m e m b r a n e input resistance accompanying Kyn application in 15 of 17 D H neurons tested. Measurements of the initial and the late slow depolarization during Kyn application were always done after restoring the membrane potential to its control value by intracellular injection of current.

Effects o f D - A P V on the initial and the late depolarizing components o f the slow excitatory synaptic response In order to determine the contribution of N M D A receptors to the generation of the initial depolarizing component of the slow excitatory synaptic response, we used D-APV. In both transverse and longitudinal spinal cord slices perfused with 1.2 mM MgZ+-containing medium, bath application of D - A P V (50-100/~M for 3-14 min) caused a reversible reduction of the amplitude of the initial depolarizing response by 56.5 -+ 6.7% (n = 24), as shown in Table I. However, the extent of the depression was dependent upon the type of slice used, the presence of extracellular Mg 2+ and the m e m b r a n e potential at which cells were examined (Fig. 4C,D). We found

245

zero-M(J* Control

APVO.lmM

Control

KYN0.5mM

A •

•

0.Ss

B

0A

0X.-Imln

Fig. 10. The effects of D-APV (second column) and kynurenate (fourth column) on the initial and the late depolarizing components of the slow excitatory synaptic response and on the responses of the same dorsal horn neuron to QA and NMDA in a nominally Mg 2+-free perfusing solution. First and third columns show control responses, second and fourth columns responses recorded 6 min (second column) and 10 min (fourth column) after the onset of the bath application of APV and Kyn, respectively. D-APV (0.1 mM for 6 min) reduced the initial slow depolarizing potential (A, second trace) recorded from a dorsal horn neurons during high intensity repetitive stimulation (15 V, 0.5 ms, 10 Hz, 1 s) of a lumbar dorsal root, whereas the amplitude and duration of the late slow depolarization was only somewhat reduced (B, second trace). The depolarizing response to pressure-ejected NMDA (D, second trace: 1 mM, 0.33 s, 5 kPa) was significantly reduced, whereas the QA-response (C, second trace: 0.1 mM, 0.33 s, 5 kPa) was little affected. Kynurenate (0.5 mM for 10 min) also reduced the initial component of the train response (A, fourth trace), while the late component was enhanced (B, fourth trace). The responses to QA (C, fourth trace) and NMDA were significantly reduced (D, fourth trace). Longitudinal slice. V m = -64 mV, 21-day-old rat. that in the presence of 1.2 m M Mg 2+, the reduction was larger in transverse (by 69.1 ± 7.8%, n = 15, Fig. 9A, fourth trace) than in longitudinal slices (by 31.5 ± 9.1%, n = 10) and also when the cells were e x a m i n e d at memb r a n e potentials positive to - 6 5 m V (V m < - 6 5 mV: by 72.0 ± 8.3%, n = 14; V m > - 6 6 mV: by 28.8 ± 6.2%, n = 12). F u r t h e r m o r e , in longitudinal slices a m e a n reduction in the amplitude of the initial depolarizing comp o n e n t of 60.5 ___ 9.3 (n = 5) was m e a s u r e d in the absence of a d d e d Mg 2÷ and 31.5 ± 9.1 (n = 10) in the presence of 1.2 m M of Mg 2+. D - A P V r e d u c e d (Fig. 9C, fourth trace) or nearly abolished (Fig. 10D, second traces) the depolarizing responses of dorsal horn neurons to N M D A but not to quisqualic acid (Fig. 10C, second trace). These results suggest that the early depolarizing c o m p o n e n t of the slow excitatory synaptic response is in a large part g e n e r a t e d by a transmitter acting at N M D A receptors. Pharmacologic evidence using substance P analogues having antagonistic properties, capsaicin and monoclonal

and polyclonal antibodies against synthetic SP has indicated that SP (or a related peptide) m a y be involved in a generation of the late slow depolarization r e c o r d e d from i m m a t u r e rat spinal dorsal horn neurons following repetitive stimulation of p r i m a r y afferents 37"61"63-65'76-78. H o w e v e r , the participation of the N M D A class of E A A receptors has b e e n also implicated in the generation of slow EPSPs and long duration synaptic excitation s'ls'2°" 41,74

Similar to Kyn, b a t h - a p p l i e d D - A P V ( 5 0 - 1 0 0 / z M for 3 - 1 4 min) p r o d u c e d no consistent change in the amplitude and half-duration of the late depolarizing component of the slow excitatory synaptic response in 18 cells e x a m i n e d in the presence of extracellular concentration of 1.2 m M Mg 2+ (Table II). Thus D - A P V increased the peak a m p l i t u d e (to 128.1 +- 7.5%), and half-duration (to 146.4 ± 7.3%; Figs. l l B and 12D) in 8 cells, r e d u c e d the amplitude in 4 cells (by 36.8 ± 4.8%) and half-duration in 2 cells (by 52.0 ± 1.4%; Figs. 9B, 10B, 12B), and it was ineffective in 6 of the remaining cells. In-

246 crease in the late slow depolarization was seen in 4 cells with 50 #M of D-APV, and also when the slice was perfused with a high Ca 2+ (4 mM), high Mg 2+ (8 mM)-containing solution. D-APV appears to be less effective in horizontal (4 of 5 cells tested in a zero-Mg 2+ medium were not affected) than in transverse slices (3 of 12 cells not affected). In addition to being blocked by D-APV (Fig. 10D), membrane depolarizations caused by the activation of NMDA receptors can be inhibited by physiological levels of Mg z+ in a voltage-dependent manner 59. To determine if Mg 2+ present in the normal recording medium was blocking a portion of the APV-sensitive late slow depolarization, experiments were performed in a reduced Mg2+-solution in the presence and the absence of D-APV. When Mg 2+ was omitted from the bathing medium, D-APV reduced (by about 28%) the peak amplitude of the late slow depolarizing response in 1 of 5 examined cells. An increase in a half-duration (to 140.0 -+ 7.1%) occurred in 2 cells, a decrease (by about 30%) in one cell, and D-APV was ineffective in 2 cells tested. An example of the effects of D-APV and Kyn recorded in the same cell is illustrated in Fig. 10. In a nominally MgZ+-free medium, the amplitude and time course of the late slow depolarizing response was reduced in this cell by about 40% over that recorded in 1.2 mM Mg 2+. When D-APV was added to the MgE+-free medium, the peak amplitude and half-duration of the late slow depolarizing component were somewhat reduced, if compared to the size of the control response (Fig. 10B, first trace). However, in the same cell, Kyn potentiated the late slow depolarizing component, as seen in Fig. 10B (fourth trace). Since D-APV could significantly decrease (Fig. 9C) or block (Fig. 10D) the depolarizing responses to exogenous NMDA, but in the same cells had only a small suppressing effect, or even enhanced the late slow depolarization, it would seem hkely that the activation of N M D A receptors contributed only in a small part to the generation of this response. Bath administration of D-APV (50-100 #M for 3-14 min) resulted in a small hyperpolarization (2-4 mV) in 19 of 33 cells examined, whereas in 4 cells a small depolarization did occur. In 11 of 18 cells tested, the hyperpolarization was accompanied by a decrease in synaptic activity. Although membrane input resistance increased in 4 cells (20.2 +-- 7.0%), and decreased in 7 (16.9 _ 9.7%), in most of the cells tested (15 out of 26) there was no change in the input resistance. These data indicate that a tonic activation of N M D A receptors in the rat spinal dorsal horn neurons may exist at the resting membrane potential and that this factor may be an important modulator of excitability of these neurons.

Effects of CNQX on the initial and the late depolarizing components of the slow excitatory synaptic response The effect of CNQX (5-7 #M for 3-30 min) on the initial depolarizing component of the train response was investigated both in transverse and longitudinal spinal slices. In both types of slices CNQX produced a reversible decrease (Fig. 12C), or even completely abolished (Figs. 11A, 12A), the initial depolarizing component. The average decrease of the peak amplitude in the presence of 1.2 mM Mg 2+ amounted to 63 _+ 7.8% (n -- 15, Table I); CNQX was more effective in transverse (decrease by 76.6 -+ 9,8%, n = 7) than in longitudinal slices (decrease by 35.3 _+ 11.1%, n = 8). However, the suppressing effect of CNQX was significantly reduced when

Contro, I

I

CNQX

Wash I

APV l

1

5mY

Wash 1

C

Control

0.4--"~

CNQX

I

Imln

Wash

0.5s Fig. 11. The effects of CNQX and D-APV on the initial (A) and the late depolarizing (B) components of the slow excitatory synaptic response evoked by dorsal root stimulation (15 V, 0.5 ms, 10 Hz, 1 s). Both CNQX (5 #M, 10 min) and D-APV (50/~M, 9 min) enhanced the late component, in particular its duration (B, second and fourth trace), whereas the initial component was completely abolished by CNQX (A, second trace) and reduced by D-APV (A, fourth trace). C, CNQX (5/~M, 9 min; middle trace) blocked effectively the spontaneous synaptic activity in this dorsal horn neuron (inset). Vm = -72 mV, 17-day-old rat (A,B); Vm = -75 mV, 22-day-old rat (C).

247

Control

APV

CNQX C,0

h

, •

,

It

Sne

E

Control

CNOX + APV

Wash

~:-76mv

Fig. 12. Effects of D-APV (A,B middle column: 50/~M for 6 min; C,D: 50/*M for 12 rain) and CNQX (A,B right column: 5 gM for 8 min; C,D: 5 / ~ M for 11 min) on the initial (A,C) and the late (B,D) depolarizing components of the slow excitatory synaptic response evoked by repetitive stimulation of a lumbar dorsal root (A,B: 20 V, 0.5 ms at 20 Hz for 2 s; C,D: 15 V, 0.5 ms at 20 Hz for 2 s). V m = -64 mV, 18-day-oldrat (A,B); Vm = -68 mV, 24day-old rat (C,D). E: the late inward current component of the slow EPSC obtained from a dorsal horn neuron voltage-clampedat -76 mV. The combined application of CNQX (5/*M) and D-APV (50 gM) completely blocked the initial inward current component (not illustrated) of the train response (20 V, 0.5 ms at 20 Hz for 2 s), whereas the amplitude and duration of the late inward current component was not modified (E, middle trace). However, noise current associated with the late inward current component was markedly reduced (E, middle trace). Vm = -60 mV, 21-day-oldrat. Inset shows the approximate locations of the 3 examined cells.

103.5 -+ 23.0% in 2 cells, whereas in the remaining 5 cells the late slow depolarization was not modified by CNQX. When the antagonistic effects of D-APV and CNQX were compared in the same dorsal horn neurons, CNQX appeared to be a more effective depressant than D-APV of both the initial and the late depolarizing components of the slow excitatory synaptic response (Figs. l l A and 12). However, the initial depolarizing components appears to be especially sensitive to CNQX (Figs. l l A and 12A,C). A combined application of CNQX (5 #M) and D-APV (50 #M), examined in 7 cells, caused a reversible depression of the peak amplitude of the late slow depolarizing response by 20-51% (38.7 - 8.7%, n = 4) and half-duration by 16-21% (18.5 -+ 1.8%, n = 2), whereas in 2 cells the half-duration of this component increased to 130.9 - 1.4% relative to control response. An example of the effects of a combined bath application of CNQX (5 #M) and D-APV (50 #M) on the late depolarizing component of the slow excitatory synaptic response is shown in Fig. 12E. Whereas CNQX and APV abolished the initial depolarizing component in this cell (not illustrated), the late component was not affected (Fig. 12E), The deep dorsal horn neurons responded with depolarization to bath application of D,L-a-amino-3-hydroxy5-methyl-4-isoxazole-propionic acid (AMPA), quisqualate, NMDA, L-glutamate and L-aspartate. CNQX (5-7 x 10-6 M for 3-30 min) reversibly reduced, or abolished, the depolarizing responses to L-aspartate, L-glutamate, KA, Q A and AMPA, whereas responses to N M D A in a zero-Mg 2+ medium were less reduced 27'2a. The onset of the blocking action of CNQX was about twice as slow as of D-APV and recovery was frequently only partial within 20-30 min. Characteristically CNQX produced a significant decrease of, and frequently abolished, the spontaneous synaptic activity. A small depolarization (2-3 mV in 7 out of 18 cells) or hyperpolarization (n = 3) and an increase in neuronal membrane input resistance (7 out of 17 cells) was observed. DISCUSSION

slices were perfused with a nominally Mg2+-free medium (Table I). As shown in Table I, the initial depolarizing component was further reduced, but not abolished, by simultaneous administration of CNQX and D-APV (by 83.6 +_ 6.0%, n = 9). Bath application of 5-7 #M of CNQX for 3-30 min resulted in a reversible depression of the peak amplitude of the late slow depolarizing response by 14-52% (38.7 -+ 12.4%, n = 3), and of half-duration by 68% in 1 of 8 cells examined. In addition, the peak amplitude increased by about 33% in one cell and the half-duration by about

In transverse and longitudinal spinal cord slice preparations from immature 27'2s'76 and adult ss'86 rats, D R stimulation has been shown to evoke both fast and slow EPSPs in D H neurons. Pharmacologic evidence using SP analogues with antagonistic properties 76, capsaicin 61'7s and monoclonal and polyclonal antibodies raised against synthetic SP 63, has indicated that SP, and perhaps other peptides present in the D R G , may be involved in the mediation of the slow depolarization recorded from the deep D H neurons in response to repetitive primary af-

248 ferent stimulation 64. However, the finding that glutamate and SP co-exist in primary afferent terminals 15, coupled with the recent demonstration that SP potentiates both the basal and the primary afferent stimulation-evoked outflow of glutamate and aspartate 4°'43 and the glutamate-induced current responses of acutely isolated rat spinal D H neurons 31'62, suggested the involvement of excitatory amino acid receptors, besides in the fast 2°'21'27'38'86, also in the slow excitatory synaptic transmission. Studies of excitatory amino acid receptors and associated ionic conductances have indicated that NMDA-receptor-channel complex has properties such as voltage dependence 48'49"59 and permeability to Ca 2÷ ions 47, that are suitable for mediating slow components of synaptic excitation. N M D A receptor-mediated fast EPSPs have been reported in some spinal neurons that receive input from primary afferents 8'27'36'42'74. In addition, a slow excitatory potential mediated at N M D A receptors has been recorded from ventral roots in response to supramaximal stimulation of L s D R in a hemisected spinal cord preparation of 1- to 4-day-old rats 2°. In the immature rat preparation, a long duration synaptic excitation was depressed by N M D A receptor antagonists 18'2°'36 (but cf. ref. 36). Davies and Lodge 14, and also Dickenson and Sullivan 16, have also described the involvement of N M D A receptors in a response of some of rat dorsal horn neurons following repeated synaptic stimulation of afferent input. However, the time course of most of these NMDA-mediated EPSPs 8"20'27'42'74 is much shorter than that of the late slow depolarization recorded in the deep D H neurons of young rats in response to repetitive stimulation of primary afferents 28"61'64'76. Synaptically released glutamate may also induce a slow EPSP through activation of metabotropic receptors (Proc. Roy. Soc. B, 243 (1991) 221-226). It is possible, therefore, that chemical mediators and cellular mechanisms involved in the generation of various types of the slow excitatory synaptic responses observed in the rat spinal cord may be different. We have demonstrated that the slow excitatory synaptic response evoked in the deep spinal dorsal horn neurons by repetitive stimulation of primary afferent fibers has two depolarizing components: an initial transient component that appears to require the activation of N M D A and non-NMDA receptors 28, and a late longer-lasting, possibly peptidergic, component 63'64"76. A simplified explanation for the generation of this dualcomponent slow excitatory synaptic potential assumed co-release, and/or sequential release, of E A A and peptide(s) onto deep D H neurons that express both E A A and peptide receptors. In support of this hypothesis we have recently shown that tachykinins and calcitonin

gene-related peptide enhance the basal and the D R stimulation-evoked release of endogenous glutamate and aspartate from the rat spinal D H in vitro 4°. In addition, we demonstrated that SP modulates glutamate-induced current responses of isolated rat spinal dorsal horn neurons 31,62. The present paper has described in more detail the electrophysiological properties and the pharmacology of both components of the slow depolarizing synaptic response that can be recorded from a majority of deep D H neurons during repetitive stimulation of the primary afferents. The results obtained from the study of pharmacology of the slow excitatory synaptic response using 3 antagonists of E A A receptors, Kyn D-APV and CNQX, confirmed our preliminary findings 28 that the initial depolarizing component seems to be mediated by both N M D A and non-NMDA receptors since it displayed sensitivity to Mg 2+, and is depressed by D-APV and CNQX. It appears that activation of the N M D A receptors makes a substantial contribution to the initial component of the slow excitatory synaptic response since in a MgZ+-free solution D-APV was a more effective antagonist than CNQX (Table I). The extent of blockade by the E A A antagonists was variable from cell to cell and no correlation with a specific region of the spinal dorsal horn was observed. Although our conclusion that the initial portion of the slow excitatory synaptic response is due to the activation of N M D A and non-NMDA receptors is justified by the pharmacology, we cannot assume from the present resuits that these receptors are located on the neurons from which the recordings were made. This could be true only in part. Given that both CNQX (Figs. 11 and 12) and D-APV (Fig. 9) completely blocked the initial depolarizing component, it is not unlikely that the E A A antagonists were acting 'upstream' in the polysynaptic pathway to decrease the excitability of the pathway. The variable effectiveness of APV on the initial depolarizing component (Figs. 9 and 12) could also be explained by postulating that the location of N M D A receptors was upstream in the polysynaptic pathway, by which most of the neurons were activated. However, the voltage-dependence of the steady-state initial component in the presence of magnesium, as illustrated in Fig. 4, indicates the presence of postsynaptic N M D A receptors on some D H neurons. In contrast to the effective blockade of the early depolarizing component of the slow excitatory synaptic response by Kyn, D-APV and CNQX, the late slow depolarizing component was not affected by the E A A receptor antagonists in a consistent and significant manner (Table II). The present study demonstrates that both N M D A and non-NMDA receptor antagonists are capa-

249 ble of producing both the facilitation and the depression of the late c o m p o n e n t of the slow excitatory synaptic re-

the absence and the presence of Mg 2+ and synaptic in-

sponse in a certain proportion of the deep D H neurons.

N M D A - and n o n - N M D A - r e c e p t o r systems of deep spinal dorsal horn n e u r o n s by repetitive stimulation of pri-

A n unexpected finding of this study was a p r e d o m i n a n c e of the facilitatory action produced by D - A P V and Kyn. The underlying mechanism of the E A A receptor antagonist-induced facilitation of the late slow depolarizing response of the deep D H n e u r o n s is presently not understood. In summary, our results obtained with the E A A receptor antagonists, at resting m e m b r a n e potentials, in

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hibition, indicate that the synaptic activation of the

mary afferent fibers may be predominantly involved in the mediation of the initial, but not the late depolarizing c o m p o n e n t of the slow excitatory synaptic response.

Acknowledgements. Support was provided by Grants from USPHS (NS 26352), NSF (BNS 8418042) and the United States Department of Agriculture.

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