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Substance P modulates glutamate-induced currents in acutely isolated rat spinal dorsal horn neurones
74
Neuro.vcience Letlers. 117 (1990) 74 80 Elsevier Scientific Publishers Ireland Ltd
NSL 07086
Substance P modulates glutamate-induced currents in acutely isolated rat spinal dorsal horn neurones M. Randi6, H. Hedimovi6 and P.D. Ryu Department q[" Veterinary Physiology and Pharmacology Iowa State University, Ames, IA . 5001l (U.S.A.) (Received 19 April 1990: Revised version received 22 April 1990; Accepted 23 April 1990) Key words." Rat spinal dorsal horn neuron; Substance P; Excitatory amino acid; Whole-cell voltageclamp The whole-cell patch-clamp technique was used to examine the effect of substance P (SP) on glutamateinduced currents in freshly dissociated rat spinal dorsal horn neurons (LI-III). In 48% of examined cells SP (10 ~0 10 6 M) at - 7 0 mV, induced an inward current that desensitized in the continued presence of SP. When applied simultaneously with, or prior to L-glutamate, SP caused a potentiation of L-glutamate-induced current in 65% of the tested cells. Since glutamate activates both N-methyl-D-aspartate (NMDA) and n o n - N M D A receptors in rat dorsal horn neurons, selective agonists, kainate, quisqualate, ~-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) and N M D A were used to determine which subtype of excitatory amino acid receptors interacted with SP. We found that the responses to quisqualate, kainate, and A M P A were not significantly affected by SP ( < 20% increase). In contrast, the inward currents induced by N M D A (30-300/IM) appear to be reduced and potentiated after the administration of 2 200 nM of SP. These results suggest that post-synaptic mechanisms of action of tachykinins may contribute to the regulation of the strength of glutamate-mediated excitatory transmission in the rat spinal dorsal horn.
Glutamate appears to be the major candidate for the fast excitatory neurotransmitter in the mammalian central nervous system [13] including the spinal dorsal horn [4, 8, 9]. Tachykinins, substance P (SP) and neurokinin A (NKA), appear to be functionally involved in the slow excitatory synaptic transmission [23]. Although the coexistence of SP and glutamate in some small primary afferent neurons and their terminals in the superficial dorsal horn has been reported [3] our understanding of physiological implications of this phenomenon is still unclear. In an attempt to determine whether tachykinins modulate the sensitivity of L-glutamate receptors of dorsal horn neurons we have investigated the effects of SP on membrane currents elicited by Lglutamate (Glu) and selective agonists of excitatory amino acid (EAA) receptors such as quisqualate (QA), kainate (KA), N-methyl-D-aspartate (NMDA) and ~-amino-3Correspondence: M. Randic, Department of Veterinary Physiology and Pharmacology, Iowa State University, Ames, IA 50011, U.S.A. 0304-3940/90/$ 03.50 (C) 1990 Elsevier Scientific Publishers Ireland Ltd.
75 hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA). Preliminary report has appeared in an abstract form [19]. Experiments were carried out on acutely isolated spinal dorsal horn neurons (laminae I-IV) from 9 to 15-days-old rats using published procedures [1, 16]. The wholecell voltage-clamp technique [6] was used to record membrane currents of dorsal horn neurons at room temperature (22-25°C). Currents elicited by EAA and SP were monitored with a List L/M-EPC7 patch-clamp amplifier and recorded on a GouldBrush pen recorder. The solution perfusing the outside of the cell had the following composition (mM): NaC1 150, KC1 5, CaC12 x2, H20 2, HEPES 10, D-glucose 10, MgCI2 1 (or zero when L-glutamate and N M D A tested), BSA (0.1 mg/ml) and NaOH to adjust pH to 7.4. Two intracellular solutions (A, B) were used (mM). Solution A: potassium-aspartate 120, KCI 20, MgCI2 1, CaCI2 0.5, HEPES 10, EGTA 5, Na-ATP 3, GTP 0.1 and leupeptin 0.1. The pH was adjusted with KOH to 7.2. Solution B: potassium aspartate, 100 or 120, KC1 20, NaC1 5, MgC12 1, HEPES 10, EGTA or BAPTA 1-5, Na-ATP 3; GTP 0.1 and leupeptin 0.1, pH was adjusted with Tris base to 7.4. EAA and SP, dissolved in extracellular solution, were applied by a pressure application system [16]. QA, AMPA, NMDA, and SP were obtained from Cambridge Research Biochemicals (CRB), KA from Sigma, and Glu was obtained from Peptide International. When applied by fast application method using U-tube [16], L-glutamate (5-30 FtM) produced an inward current response in 83% of the dorsal horn neurons voltageclamped to - 7 0 mV. The peak currents produced by 10/IM Glu ranged from - 3 0 to - 4 0 0 pA with a mean value of -208.8 + 28.6 ( + S.E.M.; n = 12). The inward currents activated by this agent at a concentration of less than 10/tM were non-desensitizing, whereas the responses elicited with higher concentrations of Glu (10-30/zM) showed fast desensitization [22], as shown in Fig. 1. Fig. 1A illustrates inward current responses of a dorsal horn neuron to application of Glu (10/zM) alone and Glu plus SP (20 nM). The cell was clamped to a holding potential of - 7 0 mV. The standard protocol used for bath application of Glu and SP was as follows: the U-tube [16] was brought to within 2 mm of the cell, and 8 s pulses of Glu were given at 90 s intervals for 15 min before switching the content of the U-tube to SP plus Glu solution. The U-tube solution was then switched back to Glu solution in order to follow the reversibility of the response. As shown in Fig. 1A, perfusion of a cell with Glu resulted in an inward current of 120 pA that quickly desensitized. SP (20 riM) applied simultaneously with Glu caused a reversible increase of the Glu-elicited inward current. SP potentiated both initial and desensitised states of the Glu response. Since Glu and SP were applied simultaneously, the time course of the effect could be influenced by a slow onset of action of SP in relation to Glu. Therefore, additional experiments were performed in which SP perfused the cell for 20-60 s before application of Glu. Using this protocol there was no noticeable difference in our results (Fig. 1B). The potentiation of Glu conductance (peak amplitude of the fast component: 184.4 + 37.2%, mean + S.E.M.) by 2-100 nM of SP or N K A was observed in 15 out of 23 cells examined. Increase in the desensitised component of the Glu response ranged between 105 and 200%. However, in 4 cells a small decrease (78.1 __.3.7%) in
76
A. Glu
G~ +SP
Glu 3 rain
Glu 7.5
Glu
5.5
T B. 411t?Glu'
G~
SP
Glu t mln
o--o
G~ o
Glu
3.5
300
~
__j
200-
100-
O'
0
SP
I
I
I
5
10
15
Tlme (mln)
Fig. I. A: pressure application of 10/tM of Glu onto a dorsal horn neuron (inset) whole-cell voltageclamped at - 70 mV elicited inward currents consisting of an initial transient and later more slowly decaying components. Both, responses were potentiated in a reversible manner by the application of SP (20 nM) simultaneously with Glu. Individual Glu responses after SP application, at the times indicated, are shown. Calibration bars: 20 s and 20 pA. B: the potentiation of Glu (10/tM) conductance by 20 nM SP was also observed in another dorsal horn neuron in which SP perfused the cell for 45s before application of Glu. Time course of peak Glu responses recorded before and after SP administration is shown in the graph. Tetrodotoxin (TTX, 5 x 10 v M) was present throughout. Calibration bars: 20 s, 20 pA (SP), 50 pA (Glu). A 12-day-old rat; B, 13-day-old rat. the G l u - i n d u c e d c u r r e n t w a s o b s e r v e d w i t h 2 - 2 0 n M o f SP (Fig. 3B). A l t h o u g h in s o m e cells the G l u - e v o k e d c u r r e n t s s h o w e d c o m p l e t e r e c o v e r y a f t e r SP a d m i n i s t r a t i o n (Fig. 1) in o t h e r cells the SP effect was o n l y in p a r t reversible. A s e c o n d a p p l i c a tion o f SP to the s a m e cell f r e q u e n t l y elicited a s m a l l e r r e s p o n s e i.e. the SP r e s p o n s e showed desensitization. In a b o u t h a l f o f tested cells, SP ( 1 0 - l 0 to 10 - 6 M ) by itself i n d u c e d a n i n w a r d c u r rent (-9.2+2.6
pA, n=21)
t h a t r e a c h e d a p e a k w i t h i n 2 - 1 5 s a n d d e s e n s i t i z e d in
the c o n t i n u e d p r e s e n c e o f SP (Fig. IB). T h e S P - i n d u c e d i n w a r d c u r r e n t w a s in s o m e
77
Ao AMPA NMDA
SP
.J
B. NMDA AMPA
~ly
+gly
I
[]
SP .
Q
m
1:3
m
D
-70 mV
/
Fig. 2. A: the traces show inward current responses evoked by 3 gM AMPA (El) or 300/~M NMDA (11) recorded at one-min intervals from a dorsal horn neuron of a 14-day-old rat (inset) held at - 7 0 mV prior to and after SP (200 nM for 60 s) application. First two traces show control responses to AMPA and NMDA. The responses to AMPA recorded at 90 s, 3.5 and 5 min after SP, and to NMDA at 2.5, 4 and 6 min after SP. B: the SP- (200 nM for 60 s) induced potentiation of NMDA conductance, but not that of AMPA, was observed in the same neuron in the presence of glycine (10 6 M). The response to NMDA+Gly (11) recorded at I, 2.5 and 7.5 min after SP; to AMPA+Gly (El) at 1, 5, 6.5 and 8.5 min after SP. TTX (5 × 10-~ M) was present throughout. Calibration bars: 20 s, 50 pA (A), 100 pA (B). cells a c c o m p a n i e d b y a n increase in m e m b r a n e c u r r e n t noise (Figs. 1B a n d 2A). T h e e n h a n c i n g effect o f SP on the G l u - i n d u c e d c u r r e n t o u t l a s t e d for 5-50 min the time course o f the S P - i n d u c e d i n w a r d c u r r e n t (Fig. 1). T h e p o t e n t i a t i o n o f the G l u - r e s p o n s e was o b s e r v e d even in the a p p a r e n t , absence o f a n y d e t e c t a b l e i n w a r d c u r r e n t elicited by SP. Since G l u activates b o t h N - m e t h y l - D - a s p a r t a t e ( N M D A ) a n d n o n - N M D A receptors in the r a t spinal d o r s a l h o r n n e u r o n s [16], Q A , K A , A M P A a n d N M D A were used in o r d e r to d e t e r m i n e which r e c e p t o r s u b t y p e SP interacted with. W e f o u n d t h a t the i n w a r d c u r r e n t responses o f d o r s a l h o r n cells to Q A (n = 54), A M P A (n = 8; Fig. 2) a n d K A (n = 16) were n o t significantly affected by SP ( < 20% c h a n g e o n average, h o w e v e r r a n g e for Q A : 105-156%). In c o n t r a s t , in 35% o f the e x a m i n e d cells (Fig. 3), the p e a k a m p l i t u d e o f the transient c o m p o n e n t o f N M D A ( 1 0 0 - 3 0 0 / t M ) was frequently d e p r e s s e d d u r i n g a p p l i c a t i o n o f SP. ( 6 4 . 2 _ 3 . 7 % , n = 17) SP ( 0 . 0 2 - 2 / z M ) p o t e n t i a t e d (183.9-I-24.1, n = 21) the transient a n d s u s t a i n e d responses o f 44% o f cells
78
A. NMDA
SP
1rnin
2
3
5
NMDA
NMDA
NMDA
+ SP
4.5
Bo Glu
Glu + SP
Glu 3 rain
i Fig. 3. A: the traces show currents evoked by 300/tM N M D A recorded at 1-min intervals from a dorsal horn neuron of I 1-day-old rat (inset) held at - 70 mV prior to and after SP application. SP (20 n M for 60 s) reversibly reduced the initial component of the N M D A response. Calibration bars: 20 s, 20 pA (SP), 100 pA (NMDA). B: suppression of the initial transient component of Glu (10/zM, left traces: 1 and 3) and N M D A (100/IM, right traces: I and 3) responses by SP (6 n M ) in the same dorsal horn neuron of 13-day-old rat. T T X (5 × 10 -7 M) was present throughout. Calibration bars: 20 s, 20 pA.
to N M D A (Fig. 2A). In the spinal dorsal neurons N M D A responses desensitize [16] as shown in other preparations [14]. SP potentiated both initial and desensitized states of the N M D A response (Fig. 2). The present concept is that glycine is required for activation of N M D A receptors expressed by brain m R N A in oocytes [10]. In isolated dorsal horn neurons glycine at concentrations of as low as 10 nM potentiated the NMDA-induced current [16]. In the presence of glycine (0.03-1/~M) both a decrease of NMDA-current responses with 0.2-20 nM of SP (76.5 +4.5%, n = 5) and an increase (Fig. 2B) with higher doses of SP (130.1 _ 5.6%, n = 8) was still observed. The exact molecular mechanisms underlying the enhancement of Glu receptoractivated conductance by SP have yet to be elucidated. The possibility that SP directly modifies kinetic properties of single N M D A channels, or unmasks silent Glu receptor-channel complexes, seems unlikely since the NMDA-activated single channel currents recorded in the outside-out configuration of the patch-clamp in excised patches of cultured cerebral cortical neurons were not significantly modified by 200 nM of SP (n = 3). It is of interest, however, that SP was found to reduce ACh-induced depolarization of chromaffin cells either by increasing the rate of desensitization or by inducing channel blockade [2].
79 Alternatively SP may act at a glycine binding site closely associated with the N M D A receptor-channel complex [7]. However, we have observed that SP enhanced the N M D A response also in the presence of glycine, although the effect appears to be smaller. Another, perhaps even more likely way, in which SP receptor activity may modify Glu and N M D A receptor conductances of dorsal horn neurons is indirectly through G-proteins or through the regulation of intracellular mechanisms [12]. A G-protein has been reported to couple SP receptors to phospholipase C in the rat parotid gland [21] and SP signal to the inward rectification channels of the rat nucleus basalis neurons [18]. It is known that there is a convergent regulation ofintracellular calcium in rat dorsal horn neurons by SP [24, 25] and Glu [11]. We have recently reported that SP enhances both low- and high-voltage-activated Ca 2+ currents in rat spinal dorsal horn neurons [20]. Although the mechanism of action of Ca 2+ is unknown, the possibilities include activation of Ca2+-dependent protein kinases or a direct action of Ca 2÷ on the Glu and N M D A receptor-ionophore complex. The hypothesis of the intracellular second messenger mechanism is supported by our recent finding that the perfusion of rat spinal dorsal horn slices with phorbol esters, potentiates Glu and N M D A responses of rat dorsal horn neurons [5], The low concentration of effective SP raises the possibility that SP may affect the strength of EAA-mediated primary afferent neurotransmission in the rat spinal dorsal horn in vivo. Thus presynaptic [8, 9] and postsynaptic mechanisms of action of tachykinins [15-17, 20] may have important physiological implications for modulation and strengthening of the synaptic connections in the spinal dorsal horn. We thank Dr. Linda N o w a k for her help in the single-channel experiments and for discussions. The work was supported in part by grants from USPHS (NS 26352) and N S F (BNS 8418042).
1 Aghajanian, G.K. and Rasmussen, K., IntraceUular studies in the facial nucleus illustrating a simple new method for obtaining viable motoneurons in adult rat brain slices, Synapse, 3 (1989) 331-338. 2 Clapham, D.E. and Neher, E., Substance P reduces acetylcholine-induced currents in isolated bovine chromaffin cells, J. Physiol., 347 (1984) 255577. 3 DeBiasi, S. and Rustioni, A., Glutamate and substance P co-exist in primary afferent terminals in the superficial laminae of spinal cord, Proc. Natl. Acad. Sci. U.S.A., 85 (1988) 7820-7824. 4 Gerber, G. and Randic, M., Excitatory amino-acid-mediatedcomponents of synapticallyevoked input from dorsal roots to deep dorsal horn neurons in the rat spinal cord slice, Neurosci. Lett., 106 (1989) 211-219. 5 Gerber, G., Kangrga, I., Ryu, P.D., Larew, J.S. and Randic, M., Multiple effects of phorbol esters in the rat spinal dorsal horn, J. Neurosci., 9 (1989) 3606-3617. 6 Hamill, O.P., Marty, A., Neher, E., Sakmann, B. and Sigworth, F.J., Improved patch-clamp techniques for high-resolution current recording from cells and cell-freemembrane patches, Pfliigers Arch., 391 (1987) 267-272. 7 Johnson, J.W. and Ascher, P., Glycine potentiates the NMDA response in cultured mouse brain neurons, Nature, 325 (1987) 529-531.
80 8 Kangrga, I., Larew, J.S.A. and Randic, M., The effects of substance P and calcitonin gene-related peptide on the efflux of endogenous glutamate and aspartate from the rat spinal dorsal horn in vitro, Neurosci. Lett., 108 (1990) 155 160. 9 Kangrga, I. and Randic, M., Tachykinins and calcitonin gene-related peptide enhance release of endogenous glutamate and aspartate from the rat spinal dorsal horn slice, J. Neurosci., 10 (1990) 2026 2038. 10 Kleckner, N.W. and Dingledine, R., Requirement for glycine in activation of NMDA-receptors expressed in Xenopus oocytes, Science, 241 (1988) 835 837. 11 MacDermott. A.B., Mayer, M.L., Westbrook, G.L., Smith, S.J. and Barker, J.L., NMDA-receptor activation increases cytoplasmatic calcium concentration in cultured spinal cord neurones, Nature, 321 (1986) 519-522. 12 Mantyh, P.W., Pinnock, R.D., Downes, C.P., Goedert, M. and Hunt, S.P., Correlation between inositol phospholipid hydrolysis and substance P receptors in rat central nervous system, Nature, 309 (1984) 795 797. 13 Mayer, M.L. and Westbrook, G.L., The physiology of excitatory amino acids in the vertebrate central nervous system, Prog. Neurobiol., 28 (1987) 197 276. 14 Mayer, M.L., Vyklicky, L. and Clements, J., Regulation of NMDA receptor desensitization in mouse hippocampal neurons by glycine, Nature, 338 (1989) 425-427. 15 Murase, K. and Randic, M., Actions of substance P on rat spinal dorsal horn neurons, J. Physiol., 346 (1984) 203 217. 16 Murase, K., Ryu, P.D. and Randic, M., Excitatory and inhibitory amino acids and peptide-induced responses in acutely isolated rat spinal dorsal horn neurons, Neurosci. Lett., 103 (1989) 5(~63. 17 Murase, K., Ryu, P.D. and Randic, M., Tachykinins modulate multiple ionic conductances in voltageclamped rat spinal dorsal horn neurons, J. Neurophysiol., 61 (1989) 854-865. 18 Nakajima, Y., Nakajima, S. and Inoue, M., Pertussis toxin-insensitive G protein mediates substance P-induced inhibition of potassium channels in brain neurons, Proc. Natl. Acad, Sci. U.S.A., 85 (1988) 3643-3647. 19 Randic, M., Ryu, P.D. and Hecimovic, H., Modulation of glutamate responses in isolated rat spinal dorsal horn neurons by substance P, Eur. J. Pharrnacol., in press. 20 Ryu, P.D. and Randic, M., Low- and high-voltage-activated calcium currents in rat spinal dorsal horn neurons, J. Neurophysiol., 63 (1990) 273--285. 21 Taylor, C.W., Merritt, J.E., Putney, J.W. and Rubin, R.P., A guanine nucleotide-dependent regulatory protein couples substance P receptors to phospholipase C in rat parotid gland, Biochem. Biophys. Res. Commun., 136(1986) 362 368. 22 Trussell, L.O., Thio, L.L., Zorumski, C.F. and Fischbach, G.D., Rapid desensitization of glutamate receptors in vertebrate central neurons, Proc. Natl. Acad. Sci., U.S.A., 85 (1988) 4562 4566. 23 Urban, L. and Randic, M., Slow excitatory transmission in rat dorsal horn: possible mediation by peptides, Brain Res., 290 (1984) 33# 34l. 24 Womack, M.D., MacDermott, A.B. and Jessell, T.M., Sensory transmitters regulate intracellular calcium in dorsal horn neurons, Nature, 334 (1988) 351 -353. 25 Womack, M.D., MacDermott, A.B. and Jessell, T.M., Substance P increases [Ca]~ in dorsal horn neurons via two distinct mechanisms, Soc. Neurosci. Abstr., 15 (1989) 184.
Neuro.vcience Letlers. 117 (1990) 74 80 Elsevier Scientific Publishers Ireland Ltd
NSL 07086
Substance P modulates glutamate-induced currents in acutely isolated rat spinal dorsal horn neurones M. Randi6, H. Hedimovi6 and P.D. Ryu Department q[" Veterinary Physiology and Pharmacology Iowa State University, Ames, IA . 5001l (U.S.A.) (Received 19 April 1990: Revised version received 22 April 1990; Accepted 23 April 1990) Key words." Rat spinal dorsal horn neuron; Substance P; Excitatory amino acid; Whole-cell voltageclamp The whole-cell patch-clamp technique was used to examine the effect of substance P (SP) on glutamateinduced currents in freshly dissociated rat spinal dorsal horn neurons (LI-III). In 48% of examined cells SP (10 ~0 10 6 M) at - 7 0 mV, induced an inward current that desensitized in the continued presence of SP. When applied simultaneously with, or prior to L-glutamate, SP caused a potentiation of L-glutamate-induced current in 65% of the tested cells. Since glutamate activates both N-methyl-D-aspartate (NMDA) and n o n - N M D A receptors in rat dorsal horn neurons, selective agonists, kainate, quisqualate, ~-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) and N M D A were used to determine which subtype of excitatory amino acid receptors interacted with SP. We found that the responses to quisqualate, kainate, and A M P A were not significantly affected by SP ( < 20% increase). In contrast, the inward currents induced by N M D A (30-300/IM) appear to be reduced and potentiated after the administration of 2 200 nM of SP. These results suggest that post-synaptic mechanisms of action of tachykinins may contribute to the regulation of the strength of glutamate-mediated excitatory transmission in the rat spinal dorsal horn.
Glutamate appears to be the major candidate for the fast excitatory neurotransmitter in the mammalian central nervous system [13] including the spinal dorsal horn [4, 8, 9]. Tachykinins, substance P (SP) and neurokinin A (NKA), appear to be functionally involved in the slow excitatory synaptic transmission [23]. Although the coexistence of SP and glutamate in some small primary afferent neurons and their terminals in the superficial dorsal horn has been reported [3] our understanding of physiological implications of this phenomenon is still unclear. In an attempt to determine whether tachykinins modulate the sensitivity of L-glutamate receptors of dorsal horn neurons we have investigated the effects of SP on membrane currents elicited by Lglutamate (Glu) and selective agonists of excitatory amino acid (EAA) receptors such as quisqualate (QA), kainate (KA), N-methyl-D-aspartate (NMDA) and ~-amino-3Correspondence: M. Randic, Department of Veterinary Physiology and Pharmacology, Iowa State University, Ames, IA 50011, U.S.A. 0304-3940/90/$ 03.50 (C) 1990 Elsevier Scientific Publishers Ireland Ltd.
75 hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA). Preliminary report has appeared in an abstract form [19]. Experiments were carried out on acutely isolated spinal dorsal horn neurons (laminae I-IV) from 9 to 15-days-old rats using published procedures [1, 16]. The wholecell voltage-clamp technique [6] was used to record membrane currents of dorsal horn neurons at room temperature (22-25°C). Currents elicited by EAA and SP were monitored with a List L/M-EPC7 patch-clamp amplifier and recorded on a GouldBrush pen recorder. The solution perfusing the outside of the cell had the following composition (mM): NaC1 150, KC1 5, CaC12 x2, H20 2, HEPES 10, D-glucose 10, MgCI2 1 (or zero when L-glutamate and N M D A tested), BSA (0.1 mg/ml) and NaOH to adjust pH to 7.4. Two intracellular solutions (A, B) were used (mM). Solution A: potassium-aspartate 120, KCI 20, MgCI2 1, CaCI2 0.5, HEPES 10, EGTA 5, Na-ATP 3, GTP 0.1 and leupeptin 0.1. The pH was adjusted with KOH to 7.2. Solution B: potassium aspartate, 100 or 120, KC1 20, NaC1 5, MgC12 1, HEPES 10, EGTA or BAPTA 1-5, Na-ATP 3; GTP 0.1 and leupeptin 0.1, pH was adjusted with Tris base to 7.4. EAA and SP, dissolved in extracellular solution, were applied by a pressure application system [16]. QA, AMPA, NMDA, and SP were obtained from Cambridge Research Biochemicals (CRB), KA from Sigma, and Glu was obtained from Peptide International. When applied by fast application method using U-tube [16], L-glutamate (5-30 FtM) produced an inward current response in 83% of the dorsal horn neurons voltageclamped to - 7 0 mV. The peak currents produced by 10/IM Glu ranged from - 3 0 to - 4 0 0 pA with a mean value of -208.8 + 28.6 ( + S.E.M.; n = 12). The inward currents activated by this agent at a concentration of less than 10/tM were non-desensitizing, whereas the responses elicited with higher concentrations of Glu (10-30/zM) showed fast desensitization [22], as shown in Fig. 1. Fig. 1A illustrates inward current responses of a dorsal horn neuron to application of Glu (10/zM) alone and Glu plus SP (20 nM). The cell was clamped to a holding potential of - 7 0 mV. The standard protocol used for bath application of Glu and SP was as follows: the U-tube [16] was brought to within 2 mm of the cell, and 8 s pulses of Glu were given at 90 s intervals for 15 min before switching the content of the U-tube to SP plus Glu solution. The U-tube solution was then switched back to Glu solution in order to follow the reversibility of the response. As shown in Fig. 1A, perfusion of a cell with Glu resulted in an inward current of 120 pA that quickly desensitized. SP (20 riM) applied simultaneously with Glu caused a reversible increase of the Glu-elicited inward current. SP potentiated both initial and desensitised states of the Glu response. Since Glu and SP were applied simultaneously, the time course of the effect could be influenced by a slow onset of action of SP in relation to Glu. Therefore, additional experiments were performed in which SP perfused the cell for 20-60 s before application of Glu. Using this protocol there was no noticeable difference in our results (Fig. 1B). The potentiation of Glu conductance (peak amplitude of the fast component: 184.4 + 37.2%, mean + S.E.M.) by 2-100 nM of SP or N K A was observed in 15 out of 23 cells examined. Increase in the desensitised component of the Glu response ranged between 105 and 200%. However, in 4 cells a small decrease (78.1 __.3.7%) in
76
A. Glu
G~ +SP
Glu 3 rain
Glu 7.5
Glu
5.5
T B. 411t?Glu'
G~
SP
Glu t mln
o--o
G~ o
Glu
3.5
300
~
__j
200-
100-
O'
0
SP
I
I
I
5
10
15
Tlme (mln)
Fig. I. A: pressure application of 10/tM of Glu onto a dorsal horn neuron (inset) whole-cell voltageclamped at - 70 mV elicited inward currents consisting of an initial transient and later more slowly decaying components. Both, responses were potentiated in a reversible manner by the application of SP (20 nM) simultaneously with Glu. Individual Glu responses after SP application, at the times indicated, are shown. Calibration bars: 20 s and 20 pA. B: the potentiation of Glu (10/tM) conductance by 20 nM SP was also observed in another dorsal horn neuron in which SP perfused the cell for 45s before application of Glu. Time course of peak Glu responses recorded before and after SP administration is shown in the graph. Tetrodotoxin (TTX, 5 x 10 v M) was present throughout. Calibration bars: 20 s, 20 pA (SP), 50 pA (Glu). A 12-day-old rat; B, 13-day-old rat. the G l u - i n d u c e d c u r r e n t w a s o b s e r v e d w i t h 2 - 2 0 n M o f SP (Fig. 3B). A l t h o u g h in s o m e cells the G l u - e v o k e d c u r r e n t s s h o w e d c o m p l e t e r e c o v e r y a f t e r SP a d m i n i s t r a t i o n (Fig. 1) in o t h e r cells the SP effect was o n l y in p a r t reversible. A s e c o n d a p p l i c a tion o f SP to the s a m e cell f r e q u e n t l y elicited a s m a l l e r r e s p o n s e i.e. the SP r e s p o n s e showed desensitization. In a b o u t h a l f o f tested cells, SP ( 1 0 - l 0 to 10 - 6 M ) by itself i n d u c e d a n i n w a r d c u r rent (-9.2+2.6
pA, n=21)
t h a t r e a c h e d a p e a k w i t h i n 2 - 1 5 s a n d d e s e n s i t i z e d in
the c o n t i n u e d p r e s e n c e o f SP (Fig. IB). T h e S P - i n d u c e d i n w a r d c u r r e n t w a s in s o m e
77
Ao AMPA NMDA
SP
.J
B. NMDA AMPA
~ly
+gly
I
[]
SP .
Q
m
1:3
m
D
-70 mV
/
Fig. 2. A: the traces show inward current responses evoked by 3 gM AMPA (El) or 300/~M NMDA (11) recorded at one-min intervals from a dorsal horn neuron of a 14-day-old rat (inset) held at - 7 0 mV prior to and after SP (200 nM for 60 s) application. First two traces show control responses to AMPA and NMDA. The responses to AMPA recorded at 90 s, 3.5 and 5 min after SP, and to NMDA at 2.5, 4 and 6 min after SP. B: the SP- (200 nM for 60 s) induced potentiation of NMDA conductance, but not that of AMPA, was observed in the same neuron in the presence of glycine (10 6 M). The response to NMDA+Gly (11) recorded at I, 2.5 and 7.5 min after SP; to AMPA+Gly (El) at 1, 5, 6.5 and 8.5 min after SP. TTX (5 × 10-~ M) was present throughout. Calibration bars: 20 s, 50 pA (A), 100 pA (B). cells a c c o m p a n i e d b y a n increase in m e m b r a n e c u r r e n t noise (Figs. 1B a n d 2A). T h e e n h a n c i n g effect o f SP on the G l u - i n d u c e d c u r r e n t o u t l a s t e d for 5-50 min the time course o f the S P - i n d u c e d i n w a r d c u r r e n t (Fig. 1). T h e p o t e n t i a t i o n o f the G l u - r e s p o n s e was o b s e r v e d even in the a p p a r e n t , absence o f a n y d e t e c t a b l e i n w a r d c u r r e n t elicited by SP. Since G l u activates b o t h N - m e t h y l - D - a s p a r t a t e ( N M D A ) a n d n o n - N M D A receptors in the r a t spinal d o r s a l h o r n n e u r o n s [16], Q A , K A , A M P A a n d N M D A were used in o r d e r to d e t e r m i n e which r e c e p t o r s u b t y p e SP interacted with. W e f o u n d t h a t the i n w a r d c u r r e n t responses o f d o r s a l h o r n cells to Q A (n = 54), A M P A (n = 8; Fig. 2) a n d K A (n = 16) were n o t significantly affected by SP ( < 20% c h a n g e o n average, h o w e v e r r a n g e for Q A : 105-156%). In c o n t r a s t , in 35% o f the e x a m i n e d cells (Fig. 3), the p e a k a m p l i t u d e o f the transient c o m p o n e n t o f N M D A ( 1 0 0 - 3 0 0 / t M ) was frequently d e p r e s s e d d u r i n g a p p l i c a t i o n o f SP. ( 6 4 . 2 _ 3 . 7 % , n = 17) SP ( 0 . 0 2 - 2 / z M ) p o t e n t i a t e d (183.9-I-24.1, n = 21) the transient a n d s u s t a i n e d responses o f 44% o f cells
78
A. NMDA
SP
1rnin
2
3
5
NMDA
NMDA
NMDA
+ SP
4.5
Bo Glu
Glu + SP
Glu 3 rain
i Fig. 3. A: the traces show currents evoked by 300/tM N M D A recorded at 1-min intervals from a dorsal horn neuron of I 1-day-old rat (inset) held at - 70 mV prior to and after SP application. SP (20 n M for 60 s) reversibly reduced the initial component of the N M D A response. Calibration bars: 20 s, 20 pA (SP), 100 pA (NMDA). B: suppression of the initial transient component of Glu (10/zM, left traces: 1 and 3) and N M D A (100/IM, right traces: I and 3) responses by SP (6 n M ) in the same dorsal horn neuron of 13-day-old rat. T T X (5 × 10 -7 M) was present throughout. Calibration bars: 20 s, 20 pA.
to N M D A (Fig. 2A). In the spinal dorsal neurons N M D A responses desensitize [16] as shown in other preparations [14]. SP potentiated both initial and desensitized states of the N M D A response (Fig. 2). The present concept is that glycine is required for activation of N M D A receptors expressed by brain m R N A in oocytes [10]. In isolated dorsal horn neurons glycine at concentrations of as low as 10 nM potentiated the NMDA-induced current [16]. In the presence of glycine (0.03-1/~M) both a decrease of NMDA-current responses with 0.2-20 nM of SP (76.5 +4.5%, n = 5) and an increase (Fig. 2B) with higher doses of SP (130.1 _ 5.6%, n = 8) was still observed. The exact molecular mechanisms underlying the enhancement of Glu receptoractivated conductance by SP have yet to be elucidated. The possibility that SP directly modifies kinetic properties of single N M D A channels, or unmasks silent Glu receptor-channel complexes, seems unlikely since the NMDA-activated single channel currents recorded in the outside-out configuration of the patch-clamp in excised patches of cultured cerebral cortical neurons were not significantly modified by 200 nM of SP (n = 3). It is of interest, however, that SP was found to reduce ACh-induced depolarization of chromaffin cells either by increasing the rate of desensitization or by inducing channel blockade [2].
79 Alternatively SP may act at a glycine binding site closely associated with the N M D A receptor-channel complex [7]. However, we have observed that SP enhanced the N M D A response also in the presence of glycine, although the effect appears to be smaller. Another, perhaps even more likely way, in which SP receptor activity may modify Glu and N M D A receptor conductances of dorsal horn neurons is indirectly through G-proteins or through the regulation of intracellular mechanisms [12]. A G-protein has been reported to couple SP receptors to phospholipase C in the rat parotid gland [21] and SP signal to the inward rectification channels of the rat nucleus basalis neurons [18]. It is known that there is a convergent regulation ofintracellular calcium in rat dorsal horn neurons by SP [24, 25] and Glu [11]. We have recently reported that SP enhances both low- and high-voltage-activated Ca 2+ currents in rat spinal dorsal horn neurons [20]. Although the mechanism of action of Ca 2+ is unknown, the possibilities include activation of Ca2+-dependent protein kinases or a direct action of Ca 2÷ on the Glu and N M D A receptor-ionophore complex. The hypothesis of the intracellular second messenger mechanism is supported by our recent finding that the perfusion of rat spinal dorsal horn slices with phorbol esters, potentiates Glu and N M D A responses of rat dorsal horn neurons [5], The low concentration of effective SP raises the possibility that SP may affect the strength of EAA-mediated primary afferent neurotransmission in the rat spinal dorsal horn in vivo. Thus presynaptic [8, 9] and postsynaptic mechanisms of action of tachykinins [15-17, 20] may have important physiological implications for modulation and strengthening of the synaptic connections in the spinal dorsal horn. We thank Dr. Linda N o w a k for her help in the single-channel experiments and for discussions. The work was supported in part by grants from USPHS (NS 26352) and N S F (BNS 8418042).
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