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Inhibitory effects of galanin on the isolated spinal cord of the newborn rat
Neuroscience Letters, 70 (I 986) 278 282
278
Elsevier NSL 04181
Inhibitory effects of galanin on the isolated spinal cord of the newborn rat Mitsuhiko Yanagisawa l, Nobuyuki Yagi 2, Masanori Otsuka 1, Chizuko Yanaihara 2 and N o b o r u Yanaihara 2 IDepartment of Pharmacology, Faculty of Medicine, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo 113, and eLaboratory of Bioorganic Chemistry, Shizuoka College of Pharmacy, Shizuoka 422 (Japan) (Received 26 March 1986; Revised version received and accepted 30 June 1986)
Key words: Galanin --- Spinal cord - - Monosynaptic reflex - - Nociceptive reflex - - Brain-gut peptide The effects of galanin, a 29-amino-acid peptide, on spinal reflexes were studied. In the isolated hemisected spinal cord of the newborn rat, galanin (0.1-5/~M) depressed the monosynaptic reflex that was induced by dorsal root stimulation and recorded from the corresponding ventral root. In the isolated spinal cord-tail preparation of the newborn rat, galanin (0.3~).6 aM) depressed the nociceptive reflex that was induced by application ofcapsaicin to the tail and recorded from a lumbar ventral root. In both preparations the inhibitory effects of galanin were reversible and the full recovery of the reflexes was observed within 3-20 min after removal of the peptide. The mechanisms of action of galanin on the spinal reflexes and the physiological role of the peptide in the spinal cord are discussed.
Galanin is a 29-amino-acid peptide isolated from porcine upper small intestine [15]. Immunohistochemical [1, 12, 13] and radioimmunological [1, 12] studies showed that galanin-like immunoreactivity is widely distributed in the mammalian central nervous system (CNS). Thus galanin appears to be a novel brain-gut peptide. Ch'ng et al. recently studied the distribution of galanin immunoreactivity in rat spinal cord and, based on the effects of dorsal rhizotomy and capsaicin treatment [1, 13], suggested that galanin occurs in unmyelinated primary afferent fibers. The physiological role of galanin in the CNS, however, is still unknown. In the present study, therefore, we studied the effects of galanin on spinal neurons by the use of two preparations; first, the isolated spinal cord of the newborn rat [10], and second, the isolated spinal cord-tail preparation of the same animal [16, 17]. Galanin was synthesized by solid-phase technology [8] using a peptide synthesizer (Beckman model 990B) according to the method previously described [9]. The pept i d e w a s p u r i f i e d b y gel f i l t r a t i o n o n a B i o G e l P - 6 c o l u m n f o l l o w e d b y r e v e r s e - p h a s e
Correspondence." M. Yanagisawa, Department of Pharmacology, Faculty of Medicine, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo 1 t 3, Japan. 0304-3940/86/$ 03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
279
high performance liquid chromatography (HPLC). Purity of the product was assessed by routine analytical criteria. The synthetic peptide was eluted as a singlepeak at a retention time of 16.3 rain in HPLC on a TSK G E L ODS-120T column (Toyo Soda Co., Tokyo) (0.46 x 25 cm) in 0.01 N HC1/CH3CN (80/20-60/40, v/v) over 30 rain at a flow rate of 1.0 ml/min. The purified product was identified by Dr. K. Tatemoto and Prof. V. Mutt (Karolinska Institute) with a natural galanin preparation in HPLC on a / t B o n d a p a k C-18 column (0.39 x 30 cm) in a linear gradient solvent system of 0.1% trifluoroacetic acid (TFA)/HzO 0.1% TFA/CH3CN (80/20 50/50, v/v) over 30 min at a flow rate of 1.0 ml/min. Both were eluted at a retention time of 20.9 min. Amino acid ratios of an acid hydrolysate (6 N HCI, I IOC, 24 h) of synthetic galanin were: Asp(4)3.94, Thr(1) 1.06, Set(2)2.10, Pro(1 )0.8 I, Gly(4)4.25, Ala(3)3.04, Ile(1)0.95, Leu(4)3.91, Tyr(2)1.96, Phe(l )0.97, Lys(l) I. 13, His(3)2.83, Arg(l)l.01 (recovery 91%); Trp(1) was not determined. Details of the synthesis will be described elsewhere. The isolated spinal cord of 0 4-day-old Wistar rats was prepared as described previously [10. 11]. The spinal cord below the thoracic portion (Ti0) was hemisected, placed in a 0.3-ml bath at 27°C, and perfused at a rate of 6 ml/min with artificial cerebrospinal fluid (CSF) equilibrated with 95% 02 5% CO2 (see ref. 11 for composition). Galanin and other drugs were applied by perfusion. The potential changes were recorded extracellularly from a lumbar ventral root (L3 5) with a tight-fitting suction electrode. The dorsal root of the corresponding segment was stimulated with a suction electrode. The isolated spinal cord-tail preparation of 1 3-day-old Wistar rats was made as described previously [16]. After the superficial layer of the skin was mechanically removed, the tail was placed in a 0.2-ml chamber and perfused with artificial CSF at a rate of 5 ml/min. For stimulation of the tail a small amount of capsaicin (0.5 itM, 50-200 ~1) was injected into the perfusion solution of the tail by brief pressure pulses [17]. Occasionally, prostaglandin E1 or E2 (1/~M) was added to the perfusion medium of the tail for 2 min before applying capsaicin, to potentiate the action of capsaicin [18]. The spinal cord below the thoracic portion was placed without hemisection in an adjacent recording bath and perfused with artificial CSF at a rate of 6 ml/min, Temperature was kept at 2T'C. Recording from the lumbar ventral root and stimulation of the dorsal root were made in the same way as described for the isolated spinal cord. In the isolated hemisected spinal cord preparation of the newborn rat, a single shock stimulation of a lumbar dorsal root induces in the corresponding ventral root an early sharp spike followed by later asynchronous waves (Fig. I B). There is good evidence that the early spike represents the monosynaptic reflex [5, 6]. Application of galanin in concentrations ranging from 0.1 to 5.0/~M did not produce any noticeable change in DC potential level recorded from the ventral root, but exerted a definite inhibitory effect on the monosynaptic reflex (Fig. !). The depressant effect of galanin was augmented in a dose-dependent manner when the concentration was increased up to 2.5/tM. The amplitude of the monosynaptic reflex was reduced by about 50% with 2.5 ~M galanin. Further increase in galanin concentration did not
280
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Fig. 1. The effect of galanin on the monosynaptic reflex. A: continuous AC records of monosynaptic reflexes. The L4 dorsal root was stimulated with a single shock every 20 s and the spinal reflexes were recorded extracellularly from the corresponding ventral root in a hemisected spinal cord of a l-day-old rat. The reflex response during 20 ms post-stimulus was stored in a transient memory device, and displayed on a pen recorder with a time base expanded 500-fold. During the period indicated by the black horizontal bar, galanin (2.5/tM) was applied to the bath. During the recovery, the record was interrupted for 10 min (shown by a gap). B: sample records on an oscilloscope from the same experiment as shown in A. a..-c were recorded at the times shown by arrows in A. Depolarization was upwards. C: a dose-response curve derived from the same preparation as in A and B. Percent inhibition of the amplitude of the monosynaptic reflex was plotted against the concentration of galanin,
result in any further decrease in the amplitude of the monosynaptic reflex (Fig. IC) but a prolongation of the inhibitory effect. The depressant effect of galanin on the monosynaptic reflex was reversible and the. full recovery of the reflex was observed within 3-20 rain after removal of the peptide. The polysynaptic reflexes of fast time courses were not markedly affected by galanin (Fig. 1B). Some attempts were made to examine the mechanisms of action of galanin. It is conceivable that galanin inhibits the monosynaptic reflex by activating spinal inhibitory interneurons that release GABA, glycine or enkephalins as a neurotransmitter. This is, however, unlikely because the depressant effect ofgalanin on the monosynaptic reflex was similarly observed in the presence of bicuculline (0.5/iM), strychnine (1 /~M) or naloxone (1 /tM). There is evidence that the transmitter of group Ia primary afferent fibers eliciting the monosynaptic reflex is L-glutamate, or a similar compound, which activates the L-glutamate receptor on motoneurons [3, 4]. We therefore examined whether galanin affects the sensitivity of motoneurons to agonists of the L-glutamate receptor. The isolated spinal cord was treated with 0.3 ~M tetrodotoxin. Bath application of L-glutamate (4 mM) or N-methyl-D-aspartate (NMDA; 0.5 mM) with brief pulses (1--2 s) produced depolarizing responses, the amplitudes of which were not altered by 2/~M galanin (for experimental procedures see ref. I 1). Immunohistochemical studies [1, 13] showed that galanin-like immunoreactivity in the dorsal horn is depleted by capsaicin treatment, suggesting that galanin occurs in C-type primary afferent fibers which are known to play an important role in nociception. We therefore studied the effect of galanin on nociceptive transmission in the spinal cord. In the isolated spinal cord-tail preparation of the newborn rat, brief
281
pulse application of capsaicin to the tail induced a depolarizing response of the lumbar ventral root, whose amplitude was 0.5 3 mV and duration 5-30 s [17]. This nociceptive reflex was slightly and reversibly depressed by galanin at a concentration of 0.3 ltM. The higher concentration (0.6/tM) of galanin abolished the nociceptive reflex, and after the removal of galanin the reflex recovered in 15 min (Fig. 2). The depressant effect of galanin on the monosynaptic reflex as observed in the present study is much stronger than that of [MetS]-enkephalin or somatostatin. Both peptides, in a concentration of 1 /tM, depressed the monosynaptic reflex by only 5-10% in the isolated rat spinal cord (ref. 14 and unpublished observations). Since galanin did not affect the responses of motoneurons to L-glutamate and NMDA, a possible mechanism of the galanin action is to inhibit the release of the transmitter from group Ia primary afferent fibers, although further experiments are needed to examine this possibility. In this connection, recent studies suggested that galanin inhibits the secretion of insulin from pancreatic//-cells [7] as well as the release of acetylcholine and substance P from nerve fibers in the guinea pig taenia coli [2]. Immunohistochemical study of Ch'ng et al. [1] showed that galanin-immunorcactire fibers are most abundant in laminae I I I of the dorsal horn of the rat spinal cord. Since galanin probably occurs in C-type primary afferent fibers [1, 13], it seems reasonable to suppose that galanin plays a physiological role in nociception. In combination with the immunohistochemical data [1, 13], the present results suggest that galanin is released, either alone or together with some excitatory transmitter(s), t¥om C-type primary afferent terminals in the superficial layers of the dorsal horn and exerts an inhibitory action on spinal nociceptive neural pathways. We wish to thank Drs. S. Konishi and T. Murakoshi for helpful discussions. The work was supported by research grants from the Ministry of Education, Science. and Culture of Japan, and from the Mitsubishi Foundation.
b
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e
30 sec
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i
---] lmV
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•
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Fig. 2. The effect of galanin on capsaicin-induced nociceptive reflex in an isolated spinal cord tail preparalion of a 2-day-old rat. Extracellular DC recording from an L5 ventral root. A constant amounl (about 100 ILl) of capsaicin solution (0.5 l/M) was injected into the solution perfusing the tail with a brief pressure pulse (0.2 s) every 15 rain. The tail was pretreated with prostaglandin E~ ( 1 /lM) for 2 min before capsaicin application. To suppress the spontaneous activity, the ipsilaleral L4 dorsal root was stimulated wilh a single shock every 45 s ti)r 12 min during the interwd, and then capsaicin was applied to the tail 45 s alter the last stimulus given to the dorsal root. a, control; b, 5 min alter addition of galanin (0.3/tM) to the solution perfusing the spinal cord; c, I I min after removal of galanin: d, 5 min after addition of 0.6 BM galanin to the solution perfusing the spinal cord; e, 11 rain after removal of galanin. At the times indicated by open triangles the dorsal root was stimulated, and at the times indicated by closed triangles, capsaicin was injected.
282 I Ch'ng, J.L.C., Christofides, N.D., Anand, P., Gibson, S.J., Allen, Y.S., Su, H.C., Tatemoto, K., Morrison, J.F.B., Polak, J.M. and Bloom, S.R., Distribution of galanin immunoreactivity in the central nervous system and the responses of galanin-containing neuronal pathways to injury, Neuroscience, 16 (1985) 343- 354. 2 Ekblad, E., H/ikanson, R., Sundler, F. and Wahlestedt, C., Galanin: neuromodulatory and direct contractile effects on smooth muscle preparations, Br. J. Pharmacol., 86 (1985) 241-246. 3 Jahr, C.E. and Yoshioka, K., la afferent excitation ofmotoneurones in the in vitro new-born rat spinal cord is selectively antagonized by kynurenate, J. Physiol. (London), 370 (1986) 515-530. 4 Kawagoe, R., Onodera, K. and Takeuehi, A., Release of endogenous glutamate from the frog spinal cord following dorsal root stimulation, Biomed. Res., 6 (1985) 239-245. 5 Konishi, S., Electrophysioiogy of mammalian spinal cord and sympathetic ganglia in vitro. In It. Yoshida, Y. Hagihara and S. Ebashi (Eds.), Advances in Pharmacology and Therapeutics II, Pergamon Press, Oxford, 1982, pp. 256-260. 6 Kudo, N. and Yamada, T., Development of the monosynaptic stretch reflex in the rat: an in vitro study, J. Physiol. (London), 369 (1985) 127 144. 7 McDonald, T.J., Dupre, J., Tatemoto, K., Greenberg, G.R., Radziuk, J. and Mutt, V., Gatanin inhibits insulin secretion and induces hyperglycemia in dogs, Diabetes, 34 (1985) 192 196. 8 Merrifield, R.B., Solid phase peptide synthesis. I. The synthesis of a tetrapeptide, J. Am. Chem. Sot., 85 (1963) 2149 2154. 9 Mochizuki, T., Yamamoto, Y., Yanaihara, C., lmura, H. and Yanaihara, N., Synthesis of human leumorphin and its biological and immunochemical characterization. In N. lzumiya (Ed.), Peptide Chemistry 1984, Protein Research Foundation, Osaka, 1985, pp. 211 216. 10 Otsuka, M. and Konishi, S., Electrophysiology of mammalian spinal cord in vitro, Nature (London), 252 (1974) 733 734. 11 Otsuka, M. and Yanagisawa, M., The effects of substance P and baclofen on motoneurones of isolated spinal cord of the newborn rat, J. Exp. Biol., 89 (1980) 201 214. 12 R6kaeus, A., Melander, T., H6kfelt, T., Lundberg, J.M,, Tatemoto, K., Carlquist, M. and Mutt, V., A galanin-like peptide in the central nervous system and intestine of the rat, Neurosci. Lett., 47 (1984) 161 166. 13 Skofitsch, G. and Jacobowitz, D.M., Galanin-like immunoreactivity in capsaicin sensitive sensory neurons and ganglia, Brain Res. Bull., 15 (1985) 191 195. 14 Suzue, T. and Jessell, T.. Opiate analgesics and endorphins inhibit rat dorsal root potential in vitro, Neurosci. Lett., 16 (1980) 161 166. 15 Tatemoto, K., R6kaeus, A., J6rnvalt, H., McDonald, T.J. and Mutt, V., Galanin - - a novel biologically active peptide from porcine intestine, FEBS Lett., 164 (1983) 124-128. 16 Yanagisawa, M., Murakoshi, T., Tamai, S. and Otsuka, M., Tail-pinch method in vitro and the effects of some antinociceptive compounds, Eur. J. Pharmaeol., 106 (1984) 231 -239. 17 Yanagisawa, M. and Otsuka, M., The effect of a substance P antagonist on chemically induced nociceptive reflex in the isolated spinal cord-tail preparation of the newborn rat, Proc. Jpn. Acad,, 60, Ser. B (1984) 427 430. 18 Yanagisawa, M., Otsuka, M. and Garcia-Arrarfis, ,I.E., E-type prostaglandins depolarize primary afferent neurons of the neonatal rat, Neurosci. Lett., 68 (1986) 351-355.
278
Elsevier NSL 04181
Inhibitory effects of galanin on the isolated spinal cord of the newborn rat Mitsuhiko Yanagisawa l, Nobuyuki Yagi 2, Masanori Otsuka 1, Chizuko Yanaihara 2 and N o b o r u Yanaihara 2 IDepartment of Pharmacology, Faculty of Medicine, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo 113, and eLaboratory of Bioorganic Chemistry, Shizuoka College of Pharmacy, Shizuoka 422 (Japan) (Received 26 March 1986; Revised version received and accepted 30 June 1986)
Key words: Galanin --- Spinal cord - - Monosynaptic reflex - - Nociceptive reflex - - Brain-gut peptide The effects of galanin, a 29-amino-acid peptide, on spinal reflexes were studied. In the isolated hemisected spinal cord of the newborn rat, galanin (0.1-5/~M) depressed the monosynaptic reflex that was induced by dorsal root stimulation and recorded from the corresponding ventral root. In the isolated spinal cord-tail preparation of the newborn rat, galanin (0.3~).6 aM) depressed the nociceptive reflex that was induced by application ofcapsaicin to the tail and recorded from a lumbar ventral root. In both preparations the inhibitory effects of galanin were reversible and the full recovery of the reflexes was observed within 3-20 min after removal of the peptide. The mechanisms of action of galanin on the spinal reflexes and the physiological role of the peptide in the spinal cord are discussed.
Galanin is a 29-amino-acid peptide isolated from porcine upper small intestine [15]. Immunohistochemical [1, 12, 13] and radioimmunological [1, 12] studies showed that galanin-like immunoreactivity is widely distributed in the mammalian central nervous system (CNS). Thus galanin appears to be a novel brain-gut peptide. Ch'ng et al. recently studied the distribution of galanin immunoreactivity in rat spinal cord and, based on the effects of dorsal rhizotomy and capsaicin treatment [1, 13], suggested that galanin occurs in unmyelinated primary afferent fibers. The physiological role of galanin in the CNS, however, is still unknown. In the present study, therefore, we studied the effects of galanin on spinal neurons by the use of two preparations; first, the isolated spinal cord of the newborn rat [10], and second, the isolated spinal cord-tail preparation of the same animal [16, 17]. Galanin was synthesized by solid-phase technology [8] using a peptide synthesizer (Beckman model 990B) according to the method previously described [9]. The pept i d e w a s p u r i f i e d b y gel f i l t r a t i o n o n a B i o G e l P - 6 c o l u m n f o l l o w e d b y r e v e r s e - p h a s e
Correspondence." M. Yanagisawa, Department of Pharmacology, Faculty of Medicine, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo 1 t 3, Japan. 0304-3940/86/$ 03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
279
high performance liquid chromatography (HPLC). Purity of the product was assessed by routine analytical criteria. The synthetic peptide was eluted as a singlepeak at a retention time of 16.3 rain in HPLC on a TSK G E L ODS-120T column (Toyo Soda Co., Tokyo) (0.46 x 25 cm) in 0.01 N HC1/CH3CN (80/20-60/40, v/v) over 30 rain at a flow rate of 1.0 ml/min. The purified product was identified by Dr. K. Tatemoto and Prof. V. Mutt (Karolinska Institute) with a natural galanin preparation in HPLC on a / t B o n d a p a k C-18 column (0.39 x 30 cm) in a linear gradient solvent system of 0.1% trifluoroacetic acid (TFA)/HzO 0.1% TFA/CH3CN (80/20 50/50, v/v) over 30 min at a flow rate of 1.0 ml/min. Both were eluted at a retention time of 20.9 min. Amino acid ratios of an acid hydrolysate (6 N HCI, I IOC, 24 h) of synthetic galanin were: Asp(4)3.94, Thr(1) 1.06, Set(2)2.10, Pro(1 )0.8 I, Gly(4)4.25, Ala(3)3.04, Ile(1)0.95, Leu(4)3.91, Tyr(2)1.96, Phe(l )0.97, Lys(l) I. 13, His(3)2.83, Arg(l)l.01 (recovery 91%); Trp(1) was not determined. Details of the synthesis will be described elsewhere. The isolated spinal cord of 0 4-day-old Wistar rats was prepared as described previously [10. 11]. The spinal cord below the thoracic portion (Ti0) was hemisected, placed in a 0.3-ml bath at 27°C, and perfused at a rate of 6 ml/min with artificial cerebrospinal fluid (CSF) equilibrated with 95% 02 5% CO2 (see ref. 11 for composition). Galanin and other drugs were applied by perfusion. The potential changes were recorded extracellularly from a lumbar ventral root (L3 5) with a tight-fitting suction electrode. The dorsal root of the corresponding segment was stimulated with a suction electrode. The isolated spinal cord-tail preparation of 1 3-day-old Wistar rats was made as described previously [16]. After the superficial layer of the skin was mechanically removed, the tail was placed in a 0.2-ml chamber and perfused with artificial CSF at a rate of 5 ml/min. For stimulation of the tail a small amount of capsaicin (0.5 itM, 50-200 ~1) was injected into the perfusion solution of the tail by brief pressure pulses [17]. Occasionally, prostaglandin E1 or E2 (1/~M) was added to the perfusion medium of the tail for 2 min before applying capsaicin, to potentiate the action of capsaicin [18]. The spinal cord below the thoracic portion was placed without hemisection in an adjacent recording bath and perfused with artificial CSF at a rate of 6 ml/min, Temperature was kept at 2T'C. Recording from the lumbar ventral root and stimulation of the dorsal root were made in the same way as described for the isolated spinal cord. In the isolated hemisected spinal cord preparation of the newborn rat, a single shock stimulation of a lumbar dorsal root induces in the corresponding ventral root an early sharp spike followed by later asynchronous waves (Fig. I B). There is good evidence that the early spike represents the monosynaptic reflex [5, 6]. Application of galanin in concentrations ranging from 0.1 to 5.0/~M did not produce any noticeable change in DC potential level recorded from the ventral root, but exerted a definite inhibitory effect on the monosynaptic reflex (Fig. !). The depressant effect of galanin was augmented in a dose-dependent manner when the concentration was increased up to 2.5/tM. The amplitude of the monosynaptic reflex was reduced by about 50% with 2.5 ~M galanin. Further increase in galanin concentration did not
280
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;
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Fig. 1. The effect of galanin on the monosynaptic reflex. A: continuous AC records of monosynaptic reflexes. The L4 dorsal root was stimulated with a single shock every 20 s and the spinal reflexes were recorded extracellularly from the corresponding ventral root in a hemisected spinal cord of a l-day-old rat. The reflex response during 20 ms post-stimulus was stored in a transient memory device, and displayed on a pen recorder with a time base expanded 500-fold. During the period indicated by the black horizontal bar, galanin (2.5/tM) was applied to the bath. During the recovery, the record was interrupted for 10 min (shown by a gap). B: sample records on an oscilloscope from the same experiment as shown in A. a..-c were recorded at the times shown by arrows in A. Depolarization was upwards. C: a dose-response curve derived from the same preparation as in A and B. Percent inhibition of the amplitude of the monosynaptic reflex was plotted against the concentration of galanin,
result in any further decrease in the amplitude of the monosynaptic reflex (Fig. IC) but a prolongation of the inhibitory effect. The depressant effect of galanin on the monosynaptic reflex was reversible and the. full recovery of the reflex was observed within 3-20 rain after removal of the peptide. The polysynaptic reflexes of fast time courses were not markedly affected by galanin (Fig. 1B). Some attempts were made to examine the mechanisms of action of galanin. It is conceivable that galanin inhibits the monosynaptic reflex by activating spinal inhibitory interneurons that release GABA, glycine or enkephalins as a neurotransmitter. This is, however, unlikely because the depressant effect ofgalanin on the monosynaptic reflex was similarly observed in the presence of bicuculline (0.5/iM), strychnine (1 /~M) or naloxone (1 /tM). There is evidence that the transmitter of group Ia primary afferent fibers eliciting the monosynaptic reflex is L-glutamate, or a similar compound, which activates the L-glutamate receptor on motoneurons [3, 4]. We therefore examined whether galanin affects the sensitivity of motoneurons to agonists of the L-glutamate receptor. The isolated spinal cord was treated with 0.3 ~M tetrodotoxin. Bath application of L-glutamate (4 mM) or N-methyl-D-aspartate (NMDA; 0.5 mM) with brief pulses (1--2 s) produced depolarizing responses, the amplitudes of which were not altered by 2/~M galanin (for experimental procedures see ref. I 1). Immunohistochemical studies [1, 13] showed that galanin-like immunoreactivity in the dorsal horn is depleted by capsaicin treatment, suggesting that galanin occurs in C-type primary afferent fibers which are known to play an important role in nociception. We therefore studied the effect of galanin on nociceptive transmission in the spinal cord. In the isolated spinal cord-tail preparation of the newborn rat, brief
281
pulse application of capsaicin to the tail induced a depolarizing response of the lumbar ventral root, whose amplitude was 0.5 3 mV and duration 5-30 s [17]. This nociceptive reflex was slightly and reversibly depressed by galanin at a concentration of 0.3 ltM. The higher concentration (0.6/tM) of galanin abolished the nociceptive reflex, and after the removal of galanin the reflex recovered in 15 min (Fig. 2). The depressant effect of galanin on the monosynaptic reflex as observed in the present study is much stronger than that of [MetS]-enkephalin or somatostatin. Both peptides, in a concentration of 1 /tM, depressed the monosynaptic reflex by only 5-10% in the isolated rat spinal cord (ref. 14 and unpublished observations). Since galanin did not affect the responses of motoneurons to L-glutamate and NMDA, a possible mechanism of the galanin action is to inhibit the release of the transmitter from group Ia primary afferent fibers, although further experiments are needed to examine this possibility. In this connection, recent studies suggested that galanin inhibits the secretion of insulin from pancreatic//-cells [7] as well as the release of acetylcholine and substance P from nerve fibers in the guinea pig taenia coli [2]. Immunohistochemical study of Ch'ng et al. [1] showed that galanin-immunorcactire fibers are most abundant in laminae I I I of the dorsal horn of the rat spinal cord. Since galanin probably occurs in C-type primary afferent fibers [1, 13], it seems reasonable to suppose that galanin plays a physiological role in nociception. In combination with the immunohistochemical data [1, 13], the present results suggest that galanin is released, either alone or together with some excitatory transmitter(s), t¥om C-type primary afferent terminals in the superficial layers of the dorsal horn and exerts an inhibitory action on spinal nociceptive neural pathways. We wish to thank Drs. S. Konishi and T. Murakoshi for helpful discussions. The work was supported by research grants from the Ministry of Education, Science. and Culture of Japan, and from the Mitsubishi Foundation.
b
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I
i
---] lmV
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•
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•
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•
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Fig. 2. The effect of galanin on capsaicin-induced nociceptive reflex in an isolated spinal cord tail preparalion of a 2-day-old rat. Extracellular DC recording from an L5 ventral root. A constant amounl (about 100 ILl) of capsaicin solution (0.5 l/M) was injected into the solution perfusing the tail with a brief pressure pulse (0.2 s) every 15 rain. The tail was pretreated with prostaglandin E~ ( 1 /lM) for 2 min before capsaicin application. To suppress the spontaneous activity, the ipsilaleral L4 dorsal root was stimulated wilh a single shock every 45 s ti)r 12 min during the interwd, and then capsaicin was applied to the tail 45 s alter the last stimulus given to the dorsal root. a, control; b, 5 min alter addition of galanin (0.3/tM) to the solution perfusing the spinal cord; c, I I min after removal of galanin: d, 5 min after addition of 0.6 BM galanin to the solution perfusing the spinal cord; e, 11 rain after removal of galanin. At the times indicated by open triangles the dorsal root was stimulated, and at the times indicated by closed triangles, capsaicin was injected.
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