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Excitatory amino acids increase glycogen phosphorylase activity in the rat spinal cord
Neuroscienee Letters, 73 (1987) 209 214 Elsevier Scientific Publishers Ireland Ltd.
209
NSL 04377
Excitatory amino acids increase glycogen phosphorylase activity in the rat spinal cord Clifford J. Woolf ('erehral Functions Research Group, Department ~?['Anatomv. University ColleK,e London, London ~L:.K. } (Received 28 September 1986: Accepted 1 October 1986) Key wor~ZY." Glutamate: Substance P; 5-Aminophosphonovaleric acid: C-afferent fiber Glycogen phosphorylase is present in nervous tissue in an active and inactive form. Using a histochemJcal technique, an investigation into which putative neurotransmitters have the capacity to modify the activity of the enzyme, has been performed on the rat spinal cord. lntrathecal injections of L-glutamate and L-aspartatc elevate glycogen phosphorylase activity in the dorsal horn, while substance P has no eITect and only high doses of adenosine triphosphate (ATP) increase the enzyme activity. In addition the Nmethyl-I)-aspartate receptor antagonist, 5-amino-phosphonovaleric acid was found to block the elevation of glycogen phosphorylase activity in the dorsal horn produced by the peripheral activation of chemosensitive primary affcrcnts. Excitatory amino-acid ncurotransminers can thereli~re, acting via second messengers and protein kinases, modify glycogen metabolism in the spinal cord.
Although the levels of glycogen in nervous tissue are relatively low they provide a potential source of glucose during conditions of stress, hypoxia, and electrical activity [1,9, 12]. The glycogen is mobilized by the activation of the inactive dephosphorylated b form of glycogen phosphorylase to the catalytically active a form [2]. Under normal circumstances the enzyme is in the inactive state [1, 2], but it can be activated by phosphorylase-b kinase [10] which in turn is activated by calcium and cyclic adenosine monophosphate (AMP)-dependent protein kinase [5, 14, 15]. Changes in the levels of second messengers in neurones produced by neurotransmitters/neuromodulators may therefore control the activity of glycogen phosphorylase. In the rat lumbar spinal cord all laminae, with the exception of lamina I1, contain the enzyme, most of which is in the inactive form [20]. Noxious mechanical, chemical and thermal stimuli when applied to the hindlimb of rats increase the activity of glycogen phosphorylase in the ipsilateral dorsal horn within minutes, while innocuous stimuli have no effect [20]. Stimulation of the sciatic nerve also increases the activity of glycogen phosphorylase both in the dorsal horn and in dorsal root ganglia, provided that either A6- or C-afferent fibres are stimulated. The present experiments have
('orrespondence." C.J. Woolf, Cerebral Functions Research Group, Department of Anatomy, University College London, Gower Street, London WC1E 6BT, U.K. 0304-394(),'87,'5 03.50 @ 1987 Elsevier Scientific Publishers Ireland Lid.
210 examined which putative transmitters have the capacity to modify glycogen phosphorylase activity in the spinal cord. Experiments were performed on Sprague-Dawley rats (200-300 g). The techniques used for the rapid removal and freezing of the tissue and the histochemical demonstration of the active form of glycogen phosphorylase were identical to that previously described in detail [20]. Essentially this consists of the incubation of unfixed sections of spinal cord with an excess of glucose-l-phosphate which is converted by the active form of the enzyme to glycogen which can then be stained with iodine. All experiments were performed on acutely decerebrated rats. The rats were initially anaesthetized with Althesin (alphaxalone, alphadalone, Gtaxo) until decerebration by aspiration of all cranial contents rostral to the mesencephalon was completed. The anaesthetic was then discontinued, the animals paralyzed with Ftaxedil (gallamine, May and Baker) and ventilated. To administer drugs directly to the lumbar spinal cord a small polyethylene cannula (PPI0) was inserted into the subarachnoid space via a small laminectomy made at T5 or T6 and fed back so that its tip lay on the dorsolateral surface of one side of the spinal cord at the 4th lumbar segment. All injections were made using a volume of 10/11, The drugs were dissolved in artificial cerebrospinal fluid (CSF), to which, in the case of substance P, 0.01 N acetic acid was added. The spinal cords were removed 10 min following the injections. To test whether intrathecally administered drugs have the capacity to modify glycogen phosphorylase activity, dibutyryl (db) cyclic AMP (cAMP) was injected, This compound crosses cell membranes and activates cAMP-dependent protein kinase which would be expected to activate phosphorylase kinase [14]. Fig. 1A shows that an injection of 50 nmol of db cAMP produces a substantial elevation in glycogen phosphorylase activity in that half of the spinal cord adjacent to the tip of the cannula. The intrathecal injection of both L-glutamate and L-aspartate also elevated glycogen phosphorylase activity in a dose-dependent manner (n = 10), although in these cases the elevated enzyme activity was restricted to the dorsal horn (Fig. 1B). The threshold dose of L-glutamate for producing a detectable change in glycogen phosphorylase activity was i nmol and that for L-aspartate, 5 nmol. For both amino acids the elevated enzyme activity of the low doses was restricted to the lateral third of laminae Ill and IV. High doses ( > 100 nmol) produced a more medial and ventral spread of the enzyme's activity increase. Intrathecal substance P at doses that elevate the excitability of cutaneous evoked flexion reflexes (0.5 50 nmol) [19] failed to produce any change in glycogen phosphorylase activity (n = 6) while ATP which has been suggested as primary afferent transmitter [6] did produce elevated activity but only at doses that were substantially higher than the excitatory amino acids ( > 150 nmol) (17 = 5).
Glycogen phosphorylase activity can be increased in the dorsal horn by stimulating Ac~ and C primary afferents [20]. Chronic nerve section is known to produce a substantial depletion in primary afferent neuropeptide content [8], although the capacity of the sectioned afferents to excite dorsal horn neurones appears unimpaired [16]. We have now found that the elevation in glycogen phosphorylase activity produced by
211
B
Fig. 1. In A the effect of an intrathecal injection of 50 nmol of dibutyryl cAMP in 10 iA on the activity of glycogen phosphorylase in the lumbar spinal cord is illustrated on a transverse section of the 4th lumbar segment while in B the staining pattern resulting from a 2 nmol injection of L-glutamate is shown. Note that the tip of the cannula lay on the opposite sides of the cord for the two experiments. The injections were administered 10 min before sacrifice. s t i m u l a t i n g c h r o n i c a l l y (4, 10, 14 a n d 20 d a y s ) s e c t i o n e d s c i a t i c n e r v e s a t C - f i b r e s t r e n g t h ( F i g . 2 A ) is i d e n t i c a l t o t h a t p r o d u c e d
b y s t i m u l a t i n g i n t a c t n e r v e s [20].
These data together with the results of the intrathecal injections suggest that neuro-
212
Fig. 2. A: illustrates the increased glycogen phosphorylase activity that follows stimulation of the chronically sectioned (14 days) sciatic nerve for 10 min on the right side at a strength (5 mA, 500 l~s, 1 lqz) that activates all afferents. B: the effect of the application of mustard oil to the skin of the hindpaw on the activity of glycogen phosphorylase is illustrated. The increased enzyme activity is restricted to lamina I. Ill and III of the medial two thirds of the ipsilateral dorsal horn. p e p t i d e s a r e n o t r e s p o n s i b l e f o r t h e a f f e r e n t e v o k e d c h a n g e s in g l y c o g e n p h o s p h o r y lase a c t i v i t y . T h e l a s t series o f e x p e r i m e n t s h a v e i n v o l v e d a n a t t e m p t u s i n g e x c i t a t o r y a m i n o
213
acid receptor antagonists to reduce afferent-induced glycogen phosphorylase activity changes. Mustard oil, a highly irritant chemical that only activates chemosensitive C-afferent fibres when applied to the skin [18], also produces an increase in glycogen phosphorylase activity in the dorsal horn (Fig. 2B) [20]. The intrathecal injection of the non-specific excitatory amino acid receptor antagonist ~,-t)-glutamyl glycine [4] reduced the mustard oil-evoked changes in glycogen phosphorylase at doses of 50 100 nmol and abolished them at doses of > 2.5/~mol. The specific N-methyl-o-aspartate (NMDA) receptor antagonist 5-aminophosphonovaleric acid (APV) [17] was even more potent, reducing mustard oil induced elevations in phosphorylase activity at doses of 0.5 nmol ( n = 4 ) and abolishing it at l0 nmol ( n = 4 ) (Fig. 3). At relatively high doses ( > 50 nmol), APV produced some increased activity in the enzyme itself. The excitatory amino acids have been proposed as candidates for fast excitatory transmitters in the spinal cord [4, 7]. The present findings are consistent with the view that C-afferent fibres either themselves release excitatory amino acids or activate neurons that do so. Certainly, small-diameter dorsal root ganglion cells contain glutaminase [3], and N M D A receptors are concentrated in lamina II [13]. A possible mechanism for an NMDA-mediated increase in glycogen phosphorylase activity is the recent finding that activation of NM DA receptors increases intracellular calcium levels [11]. Further work is required to establish the extent to which different neuro-
Fig. 3. The effect of the intrathecal administration of increasing doses of APV (in 10/zl) on mustard oilevoked changes in glycogen phosphorylase activity in the dorsal horn. The doses administered were: A 0. I nmol, B 0.5 nmol, C [.0 nmol and D 5 nmol. In each case the drug was administered 5 min before the application of the mustard oil, I0 rnin after which the cord was rapidly removed and frozen.
214
transmitters/neuromodulators modify the internal chemical environment of their target neurons in addition to changing the movement of ions across the membrane and how this provides an activity increase in metabolism, as with glycogen phosphorylase, or prolonged changes in membrane properties. The financial support of the Wellcome Trust and M.R.C. is gratefully acknowledged. I thank Jacqueta Middleton for technical assistance. 1 Breckenridge, B.M. and Norman, J,N., Glycogen phosphorylase in brain, J. Neurochem., 9 (1962~ 383 392. 2 Breckenridge, B.M. and Norman, J.N., The conversion of phosphorylase-b to phosphorylase-a in brain, J. Neurochem., 12 (1965) 51-57. 3 Cangro, C.B., Sweetnam, P.M., Wrathall, J.R., Haser, W.B., Curthoys, N.P. and Neale, J.H., Localization of elevated glutaminase immunoreactivity in small DRG neurons, Brain Res., 336 (1985) 158 161. 4 Davies, J. and Watkins, J.C., Role of excitatory amino acid receptors in mono- and polysynaptic excitation in the cat spinal cord, Exp. Brain Res., 49 (1983) 280-290. 5 Drummond, G.I. and Bellward, G., Studies on phosphorylase-b kinase from neural tissue, J. Neurochem., 17 (1970) 475 482. 6 Jahr, C.E. and Jessell, T.M., ATP excites a subpopulation of rat dorsal horn neurones, Nature (London), 304 (1983) 730 733. 7 Jahr, C.E. and Yoshida, K., la afferent excitation of motoneurones in the in vitro new born rat spinal cord is selectively antagonized by kynurenate, J. Physiol. (London), 370 (1986) 515-530. 8 Jessel, T., Tsunoo, A., Kanazawa, I. and Otsuka, M., Substance P-depletion in the dorsal horn of ral spinal cord after section of the peripheral processes of primary sensory neurones, Brain Res., 168 (1979) 247-259. 9 Knull, H.R. and Khandelwal, Glycogen metabolizing enzymes in brain, Neurochem. Res., 7 (1982) 1307-1317. 10 Krebs, E.G., Phosphorylation and dephosphorylation of glycogen phosphorylase; A prototype of reversible covalent enzyme modification, Curr. Topics Cell. Regul., 18 (1981) 401-419. 11 MacDermott, A.B., Mayer, MIL., Westbrook, G.L., Smith, D.J. and Barker, J.L., NMDA-receptor activation increases cytoplasmic calcium concentration in cultured spinal cord neurones, Nature (London), 321 (1986) 519-521. 12 Madesen, N.B., Kasvinsky, P.J. and Fletternick, R.J., Allosteric transitions of phosphorylase and the regulation of glycogen metabolism, J. Biol. Chem., 253 (1978) 9097 9101. 13 Managhan, D.T. and Cotman, C.W., Distribution of N-methyl-D-aspartate sensitive L-l~H]glutamate binding sites in rat brain, J. Neurosci., 5 (1985) 2909 2919. 14 Nestler, E.J. and Greengard, P., Protein Phosphorylation in the Nervous System, Wiley, Chichester, pp. 398. l 5 Ogawa, E., Activation of phosphorylase kinase from brain by small amounts of calcium ion, J. Neurochem., 20 (1973) 1487-1488. 16 Wall, P.D., Fitzgerald, M. and Gibson, S.J., The response of rat spinal cord cells to unmyelinated afferents after peripheral nerve section and after changes in substance P levels, Neuroscience, 6 (1981) 2205-2215. 17 Watkins, J.C. and Evans, R.H., Excitatory amino acid transmitters, Annu. Rev. Pharmacol. Toxicol., 21 (1981) 165-204. 18 Woolf, C.J. and Wall, P.D., The relative effectiveness of C-primary afferent fibres of different origins in evoking a prolonged facilitation of the flexor reflex in the rat, J. Neurosci., 6 (1986) 1433-1442. 19 Woolf, C.J. and Wiesenfeld-Hallin, Z., Substance P and calcitonin gene related peptide synergistically modulate the gain of the receptive flexor withdrawal reflex in the rat, Neurosci. Lett., 66 (1986) 226 230. 20 Woolf, C.J., Chong, M.S. and Rashdi, T.A., Mapping increased glycogen phosphorylase activity in dorsal root ganglia and in the spinal cord following peripheral stimuli, J. Comp. Neurol., 234 (1983) 60- 76.
209
NSL 04377
Excitatory amino acids increase glycogen phosphorylase activity in the rat spinal cord Clifford J. Woolf ('erehral Functions Research Group, Department ~?['Anatomv. University ColleK,e London, London ~L:.K. } (Received 28 September 1986: Accepted 1 October 1986) Key wor~ZY." Glutamate: Substance P; 5-Aminophosphonovaleric acid: C-afferent fiber Glycogen phosphorylase is present in nervous tissue in an active and inactive form. Using a histochemJcal technique, an investigation into which putative neurotransmitters have the capacity to modify the activity of the enzyme, has been performed on the rat spinal cord. lntrathecal injections of L-glutamate and L-aspartatc elevate glycogen phosphorylase activity in the dorsal horn, while substance P has no eITect and only high doses of adenosine triphosphate (ATP) increase the enzyme activity. In addition the Nmethyl-I)-aspartate receptor antagonist, 5-amino-phosphonovaleric acid was found to block the elevation of glycogen phosphorylase activity in the dorsal horn produced by the peripheral activation of chemosensitive primary affcrcnts. Excitatory amino-acid ncurotransminers can thereli~re, acting via second messengers and protein kinases, modify glycogen metabolism in the spinal cord.
Although the levels of glycogen in nervous tissue are relatively low they provide a potential source of glucose during conditions of stress, hypoxia, and electrical activity [1,9, 12]. The glycogen is mobilized by the activation of the inactive dephosphorylated b form of glycogen phosphorylase to the catalytically active a form [2]. Under normal circumstances the enzyme is in the inactive state [1, 2], but it can be activated by phosphorylase-b kinase [10] which in turn is activated by calcium and cyclic adenosine monophosphate (AMP)-dependent protein kinase [5, 14, 15]. Changes in the levels of second messengers in neurones produced by neurotransmitters/neuromodulators may therefore control the activity of glycogen phosphorylase. In the rat lumbar spinal cord all laminae, with the exception of lamina I1, contain the enzyme, most of which is in the inactive form [20]. Noxious mechanical, chemical and thermal stimuli when applied to the hindlimb of rats increase the activity of glycogen phosphorylase in the ipsilateral dorsal horn within minutes, while innocuous stimuli have no effect [20]. Stimulation of the sciatic nerve also increases the activity of glycogen phosphorylase both in the dorsal horn and in dorsal root ganglia, provided that either A6- or C-afferent fibres are stimulated. The present experiments have
('orrespondence." C.J. Woolf, Cerebral Functions Research Group, Department of Anatomy, University College London, Gower Street, London WC1E 6BT, U.K. 0304-394(),'87,'5 03.50 @ 1987 Elsevier Scientific Publishers Ireland Lid.
210 examined which putative transmitters have the capacity to modify glycogen phosphorylase activity in the spinal cord. Experiments were performed on Sprague-Dawley rats (200-300 g). The techniques used for the rapid removal and freezing of the tissue and the histochemical demonstration of the active form of glycogen phosphorylase were identical to that previously described in detail [20]. Essentially this consists of the incubation of unfixed sections of spinal cord with an excess of glucose-l-phosphate which is converted by the active form of the enzyme to glycogen which can then be stained with iodine. All experiments were performed on acutely decerebrated rats. The rats were initially anaesthetized with Althesin (alphaxalone, alphadalone, Gtaxo) until decerebration by aspiration of all cranial contents rostral to the mesencephalon was completed. The anaesthetic was then discontinued, the animals paralyzed with Ftaxedil (gallamine, May and Baker) and ventilated. To administer drugs directly to the lumbar spinal cord a small polyethylene cannula (PPI0) was inserted into the subarachnoid space via a small laminectomy made at T5 or T6 and fed back so that its tip lay on the dorsolateral surface of one side of the spinal cord at the 4th lumbar segment. All injections were made using a volume of 10/11, The drugs were dissolved in artificial cerebrospinal fluid (CSF), to which, in the case of substance P, 0.01 N acetic acid was added. The spinal cords were removed 10 min following the injections. To test whether intrathecally administered drugs have the capacity to modify glycogen phosphorylase activity, dibutyryl (db) cyclic AMP (cAMP) was injected, This compound crosses cell membranes and activates cAMP-dependent protein kinase which would be expected to activate phosphorylase kinase [14]. Fig. 1A shows that an injection of 50 nmol of db cAMP produces a substantial elevation in glycogen phosphorylase activity in that half of the spinal cord adjacent to the tip of the cannula. The intrathecal injection of both L-glutamate and L-aspartate also elevated glycogen phosphorylase activity in a dose-dependent manner (n = 10), although in these cases the elevated enzyme activity was restricted to the dorsal horn (Fig. 1B). The threshold dose of L-glutamate for producing a detectable change in glycogen phosphorylase activity was i nmol and that for L-aspartate, 5 nmol. For both amino acids the elevated enzyme activity of the low doses was restricted to the lateral third of laminae Ill and IV. High doses ( > 100 nmol) produced a more medial and ventral spread of the enzyme's activity increase. Intrathecal substance P at doses that elevate the excitability of cutaneous evoked flexion reflexes (0.5 50 nmol) [19] failed to produce any change in glycogen phosphorylase activity (n = 6) while ATP which has been suggested as primary afferent transmitter [6] did produce elevated activity but only at doses that were substantially higher than the excitatory amino acids ( > 150 nmol) (17 = 5).
Glycogen phosphorylase activity can be increased in the dorsal horn by stimulating Ac~ and C primary afferents [20]. Chronic nerve section is known to produce a substantial depletion in primary afferent neuropeptide content [8], although the capacity of the sectioned afferents to excite dorsal horn neurones appears unimpaired [16]. We have now found that the elevation in glycogen phosphorylase activity produced by
211
B
Fig. 1. In A the effect of an intrathecal injection of 50 nmol of dibutyryl cAMP in 10 iA on the activity of glycogen phosphorylase in the lumbar spinal cord is illustrated on a transverse section of the 4th lumbar segment while in B the staining pattern resulting from a 2 nmol injection of L-glutamate is shown. Note that the tip of the cannula lay on the opposite sides of the cord for the two experiments. The injections were administered 10 min before sacrifice. s t i m u l a t i n g c h r o n i c a l l y (4, 10, 14 a n d 20 d a y s ) s e c t i o n e d s c i a t i c n e r v e s a t C - f i b r e s t r e n g t h ( F i g . 2 A ) is i d e n t i c a l t o t h a t p r o d u c e d
b y s t i m u l a t i n g i n t a c t n e r v e s [20].
These data together with the results of the intrathecal injections suggest that neuro-
212
Fig. 2. A: illustrates the increased glycogen phosphorylase activity that follows stimulation of the chronically sectioned (14 days) sciatic nerve for 10 min on the right side at a strength (5 mA, 500 l~s, 1 lqz) that activates all afferents. B: the effect of the application of mustard oil to the skin of the hindpaw on the activity of glycogen phosphorylase is illustrated. The increased enzyme activity is restricted to lamina I. Ill and III of the medial two thirds of the ipsilateral dorsal horn. p e p t i d e s a r e n o t r e s p o n s i b l e f o r t h e a f f e r e n t e v o k e d c h a n g e s in g l y c o g e n p h o s p h o r y lase a c t i v i t y . T h e l a s t series o f e x p e r i m e n t s h a v e i n v o l v e d a n a t t e m p t u s i n g e x c i t a t o r y a m i n o
213
acid receptor antagonists to reduce afferent-induced glycogen phosphorylase activity changes. Mustard oil, a highly irritant chemical that only activates chemosensitive C-afferent fibres when applied to the skin [18], also produces an increase in glycogen phosphorylase activity in the dorsal horn (Fig. 2B) [20]. The intrathecal injection of the non-specific excitatory amino acid receptor antagonist ~,-t)-glutamyl glycine [4] reduced the mustard oil-evoked changes in glycogen phosphorylase at doses of 50 100 nmol and abolished them at doses of > 2.5/~mol. The specific N-methyl-o-aspartate (NMDA) receptor antagonist 5-aminophosphonovaleric acid (APV) [17] was even more potent, reducing mustard oil induced elevations in phosphorylase activity at doses of 0.5 nmol ( n = 4 ) and abolishing it at l0 nmol ( n = 4 ) (Fig. 3). At relatively high doses ( > 50 nmol), APV produced some increased activity in the enzyme itself. The excitatory amino acids have been proposed as candidates for fast excitatory transmitters in the spinal cord [4, 7]. The present findings are consistent with the view that C-afferent fibres either themselves release excitatory amino acids or activate neurons that do so. Certainly, small-diameter dorsal root ganglion cells contain glutaminase [3], and N M D A receptors are concentrated in lamina II [13]. A possible mechanism for an NMDA-mediated increase in glycogen phosphorylase activity is the recent finding that activation of NM DA receptors increases intracellular calcium levels [11]. Further work is required to establish the extent to which different neuro-
Fig. 3. The effect of the intrathecal administration of increasing doses of APV (in 10/zl) on mustard oilevoked changes in glycogen phosphorylase activity in the dorsal horn. The doses administered were: A 0. I nmol, B 0.5 nmol, C [.0 nmol and D 5 nmol. In each case the drug was administered 5 min before the application of the mustard oil, I0 rnin after which the cord was rapidly removed and frozen.
214
transmitters/neuromodulators modify the internal chemical environment of their target neurons in addition to changing the movement of ions across the membrane and how this provides an activity increase in metabolism, as with glycogen phosphorylase, or prolonged changes in membrane properties. The financial support of the Wellcome Trust and M.R.C. is gratefully acknowledged. I thank Jacqueta Middleton for technical assistance. 1 Breckenridge, B.M. and Norman, J,N., Glycogen phosphorylase in brain, J. Neurochem., 9 (1962~ 383 392. 2 Breckenridge, B.M. and Norman, J.N., The conversion of phosphorylase-b to phosphorylase-a in brain, J. Neurochem., 12 (1965) 51-57. 3 Cangro, C.B., Sweetnam, P.M., Wrathall, J.R., Haser, W.B., Curthoys, N.P. and Neale, J.H., Localization of elevated glutaminase immunoreactivity in small DRG neurons, Brain Res., 336 (1985) 158 161. 4 Davies, J. and Watkins, J.C., Role of excitatory amino acid receptors in mono- and polysynaptic excitation in the cat spinal cord, Exp. Brain Res., 49 (1983) 280-290. 5 Drummond, G.I. and Bellward, G., Studies on phosphorylase-b kinase from neural tissue, J. Neurochem., 17 (1970) 475 482. 6 Jahr, C.E. and Jessell, T.M., ATP excites a subpopulation of rat dorsal horn neurones, Nature (London), 304 (1983) 730 733. 7 Jahr, C.E. and Yoshida, K., la afferent excitation of motoneurones in the in vitro new born rat spinal cord is selectively antagonized by kynurenate, J. Physiol. (London), 370 (1986) 515-530. 8 Jessel, T., Tsunoo, A., Kanazawa, I. and Otsuka, M., Substance P-depletion in the dorsal horn of ral spinal cord after section of the peripheral processes of primary sensory neurones, Brain Res., 168 (1979) 247-259. 9 Knull, H.R. and Khandelwal, Glycogen metabolizing enzymes in brain, Neurochem. Res., 7 (1982) 1307-1317. 10 Krebs, E.G., Phosphorylation and dephosphorylation of glycogen phosphorylase; A prototype of reversible covalent enzyme modification, Curr. Topics Cell. Regul., 18 (1981) 401-419. 11 MacDermott, A.B., Mayer, MIL., Westbrook, G.L., Smith, D.J. and Barker, J.L., NMDA-receptor activation increases cytoplasmic calcium concentration in cultured spinal cord neurones, Nature (London), 321 (1986) 519-521. 12 Madesen, N.B., Kasvinsky, P.J. and Fletternick, R.J., Allosteric transitions of phosphorylase and the regulation of glycogen metabolism, J. Biol. Chem., 253 (1978) 9097 9101. 13 Managhan, D.T. and Cotman, C.W., Distribution of N-methyl-D-aspartate sensitive L-l~H]glutamate binding sites in rat brain, J. Neurosci., 5 (1985) 2909 2919. 14 Nestler, E.J. and Greengard, P., Protein Phosphorylation in the Nervous System, Wiley, Chichester, pp. 398. l 5 Ogawa, E., Activation of phosphorylase kinase from brain by small amounts of calcium ion, J. Neurochem., 20 (1973) 1487-1488. 16 Wall, P.D., Fitzgerald, M. and Gibson, S.J., The response of rat spinal cord cells to unmyelinated afferents after peripheral nerve section and after changes in substance P levels, Neuroscience, 6 (1981) 2205-2215. 17 Watkins, J.C. and Evans, R.H., Excitatory amino acid transmitters, Annu. Rev. Pharmacol. Toxicol., 21 (1981) 165-204. 18 Woolf, C.J. and Wall, P.D., The relative effectiveness of C-primary afferent fibres of different origins in evoking a prolonged facilitation of the flexor reflex in the rat, J. Neurosci., 6 (1986) 1433-1442. 19 Woolf, C.J. and Wiesenfeld-Hallin, Z., Substance P and calcitonin gene related peptide synergistically modulate the gain of the receptive flexor withdrawal reflex in the rat, Neurosci. Lett., 66 (1986) 226 230. 20 Woolf, C.J., Chong, M.S. and Rashdi, T.A., Mapping increased glycogen phosphorylase activity in dorsal root ganglia and in the spinal cord following peripheral stimuli, J. Comp. Neurol., 234 (1983) 60- 76.