Brain Research, 107 (1976) 623-627

623

© Elsevier ScientificPublishing Company, Amsterdam - Printed in The Netherlands

Substance P in the substantia nigra

J. DAVIES AND A. DRAY Department of Pharmacology, The School of Pharmacy, University of London, London WC1N lAX (Great Britain)

(Accepted February 4th, 1976)

Considerable interest has recently been shown in the action of substance P in the central nervous system. This undecapeptide is present in various brain regions. In the spinal cord its concentration in the dorsal grey matter is 4-14 times higher than in the ventral grey matter 14. This finding, together with reports that substance P is 200-900 times more potent than L-glutamate in depolarizing motor neurones in the isolated frog and newborn rat spinal cordT,S, 13 has led to the suggestion that substance P may be the transmitter released from sensory nerve terminals in the spinal cord ~0. However, there is little evidence that substance P serves a neurotransmitter function in the central nervous system. The highest concentration of substance P of any brain region is found in the substantia nigra where it has been shown to be concentrated in nerve ending particles 5,12, 15. In view of the suggestion that substance P may be a neurotransmitter or a modulator of transmission in this brain region 5 we have investigated the effects of this substance on single neurones in the substantia nigra of the rat using standard microelectrophoretic techniques. Electrophoretically administered substance P has previously been shown to excite single neurones in the cerebral cortex, cuneate nucleus and spinal cord6,9,11. Albino rats (220-270 g), anaesthetized with urethane 1.4 g/kg, were used during this study. Multibarrelled glass micropipettes (tip diameter 6/~m) were used to record action potentials extracellularly and to administer substances by electrophoresis onto spontaneously firing neurones in the substantia nigra. Micropipettes were filled immediately before use by a glass-fibre method or by centrifugation. The recording barrel contained 3 M sodium chloride and another containing 1 M sodium chloride or 20 m M sodium acetate (pH 5.5) was used to balance the net current at the electrode tip to zero or to test for electrophoretic current effects. Other barrels contained substance P (Beckman) 0.7 m M made up in 20 m M acetic acid, pH 5.5; eledoisin dihydrochloride (Calbiochem) 24 mM, pH 4.5; fl-p-chlorophenyl-GABA (Lloresal, fl-pCPG) 50 mM, in 165 m M NaCI, pH 3.5; acetylcholine chloride 0.5 mM, pH 4.5; sodium glutamate 0.2 M, pH 6.8-7.2; GABA, 0.2 M, pH 3.5. Action potentials were recorded and displayed using conventional techniques. The location of the micropipette tip within the substantia nigra was marked by ejec-

624

I

I sP lOO

--mTn

-"

,

I sP 80hA

sP

o

I

sP

0

GI 10

I El 15

~

I

.

I

U

Eledo~,n75

]

I

a 75hA

Fig. 1. Ratemeter records of the responses of substantm nigra neurones to electrophoretlcally apphed substance P (sP) and eledoisin (El). The duration of electrophoretic applications are indicated by bars and the expellingcurrents are shown in nA. Substance P produced a prolonged increase (top left), or occasionally a reduction (top right) m neuronal firing. The lower record shows (left) excitation by glutamate (GI) and eledoisin and (right) reduction of neuronal firing by eledoisin but not by the same current of Na +. tion of pontamine-blue dye and subsequently confirmed histologicallya. In addition, substantia nigra neurones were identified by their constant latency inhibitory response following stimulation of the ipsilateral caudate nucleus 4. Substance P (25-300 nA) was ejected with a cationic current near 34 cells in the substantia nigra. Of these 26 showed a significant response to substance P (at least 20 % change in firing rate). Excitation was the predominant response observed (23 cells) and this was characteristically slow in onset, commencing 10-20 sec after the beginning of the ejection and continuing for 0.5-3 rain after the termination of the substance P ejection (Fig. 1). Although excitations were prolonged, the absolute increase m neuronal discharge rate was small, usually only 20-50 % above the background rate, even after substance P expulsion at 300 nA. However, a few cells showed > 100% increases in firing. Occasionally responses to substance P were accompanied by a small reduction in spike amplitude. Inhibition of neuronal firing was observed more rarely (3 cells) but like the excitations was slow in onset and of prolonged duration (Fig. 1). A further two neurones were inhibited during the ejection of substance P but this was rapid in onset and offset and was difficult to distinguish from the effects of the same current of Na ~. Overall it was difficult to reproduce identical excitatory responses to a given current of substance P despite strict adherence to a rigid time cycle of drug applications and exploration of various periods of application or intervals between applications. During such tests subsequent responses to substance P rapidly decreased. However, responses were considered to be genuine since they were not mimicked by applications of Na + from solutions of sodium acetate at the same pH. This indicates the possible development of tachyphylaxis to the excitatory effects of substance P. All the cells tested could be excited by both acetylcholine and glutamate and depressed by GABA, and consequently it was difficult to correlate the effects of substance P with those produced by these agents. Eledoisin, an undecapeptide with structural similarity to substance P, also ex-

625 bO

[ so

50

• ~ ACh 25

I

I

~

I

sP 60

t

I

/

sP 40

I

I

L___I

/

p-pCPG 40

~'i'

ACh 25

t

~

mm

~I-pCPG

i

t

Fig. 2. Continuous ratemeter records showing the effects of fl-pCPG on neuronal responses to substance P (sP) and acetylchohne (ACh). Top: this cell was reproducibly excited by alternate electrophoretic applications of sP (40 nA) and ACh (25 nA). During the administration of fl-pCPG (40 nA) spontaneous neuronal firing rate and reponses to both excitants were reversibly reduced. Bottom: reproducible responses of a substantia nigra neurone to sP (60 nA) and ACh (25 nA). As before fl-pCPG (40 nA) produced a gradual reduction in neuronal firing and also a reduction in the excitatory response to sP but not to ACh. Complete recovery of the sP response occurred some 6 min later.

cited (Fig. 1) the majority of substantia nigra neurones tested (12 of 18) when ejected as a cation (50--100 nA), though some neurones (4 of 18) were depressed by this substance (Fig. 1). The time course and magnitude of these effects were similar to those observed with substance P (Fig. 1). A recent report la suggests that the centrally acting muscle relaxant fl-pCPG specifically antagonizes substance P-induced depolarization of rat spinal motoneurones. In the present study fl-pCPG (10-75 nA) reversibly reduced the spontaneous firing rate of all 20 neurones tested (Fig. 2) and also generally reduced the excitatory effects of glutamate and acetylcholine. The inhibition of both spontaneous firing and drug-evoked excitation by fl-pCPG was reproducible and often prolonged (0.5-5 min). The excitatory effects of substance P were reversibly reduced in 4 of 5 cells by fl-pCPG. In two of these cells the effect of fl-pCPG was selective for substance P but in the remaining two cells the excitatory effects of acetylcholine and glutamate were also reduced (Fig. 2). On one neurone neither substance P nor acetylcholine excitations were modified and fl-pCPG did not affect the inhibitory response to substance P (two cells tested). The present results show that neurones in the substantia nigra were dearly re-

626 sponslve to electrophoretlc applications of substance P, the predominant effect being an increase in neuronal actwity, though more rarely inhibition of simdar time course to excitations was observed. These effects appear to be similar to the substance P responses reported m other areas of the CNS6,9,11. In addition, eledolsm, an undecapeptide with structural similarities to substance P, showed simdar patterns of activity. The possiblhty has been raised that the effects of substance P may be medmted Indirectly by the release or facditatlon of endogenous substances6, 9. Th~s explanation cannot be entirely excluded though it has been reported that the excitatory effects of substance P were unlikely to depend on chohnerglc mechanisms 6,al and that substance P inhibitions occurred by a mechanism which differed from that of GABA 9. That the effects produced by substance P were not always reproducible might md~care that desensitization or tachyphylax~s had occurred6,1L However, the uncertain release of substance P from mJcrop~pettes 6 and the rather dilute solutions employed in this and previous electrophoret~c studies could also account for these observations. Prehminary observations indicate, however, that during the occurrence of 'tachyphylaxis' to polypeptides the effects of G A B A or taurine on central neurones may be selectively reduced (Davies and Dray, unpublished results). In view of the reported selectwe interaction of fl-pCPG with substance P depolarization in the spinal cord ~3 we tested for this in the substantm nigra. Excitation by substance P was selectively reduced by fl-pCPG in some cells but not in others. Thus, although few cells were stud~ed it would seem unlikely that fl-pCPG will be a useful 'selectwe' substance P antagonist. This ~s perhaps not surprising since etectrophoret~c fl-pCPG has previously been shown to reduce acetylchohne and amino acid-reduced excitations in the cerebral cortex and spinal cord of the cat 1,'. Though substance P clearly affects neuronal activity m the substantla nigra, no firm conclusions can be drawn from the present observations regarding a possible neurotransmitter or neuromodulator role for substance P in th~s region of the CNS. It has been suggested that substance P is unlikely to be a neurotransmitter at primary afferent terminals because of ~ts prolonged onset and duration of action when ejected electrophoretically6, 9. While th~s cannot be disputed, it is noteworthy that the responses of central neurones to monoammes administered by electrophoresis may also be prolonged in onset and duration, yet these substances are considered strong neurotransmitter candidates m the CNS. This work was supported by an M R C Programme G r a n t to Professor D. W. Straughan. We would like to thank Dr. J. C. Watkins for samples of fl-pCPG.

1 CURTIS, D. R., GAME,C. J. A., JOHNSTON, G. A. R., AND McCuLLOCH, R. M., Central effects of fl-(p-chlorophenyl)-),-aminobutyric acid, Brain Research, 70 (1974) 493-500. 2 DAVIES,J., AND WATKINS, J. C., The action of fl-phenyI-GABA derivatives on neurones of the cat cerebral cortex, Brain Research, 70 (1974) 501-505. 3 DRAY, A., AND GONYE, T J, Extracellular recording in rat substantla mgra, J Phystol (Lond.), 246 (.1975) 22-23P.

627 4 DRAY, A., AND GONYE, T. J., Effects of caudate stimulation and micro-iontophoretically applied substances on neurones in the rat substantia nigra, J. PhysioL (Load.), 246 (1975) 88-89P. 5 DUFFY, M. J., MULHALL,D., AND POWELL, D., Subcellular distribution of substance P in bovine hypothalamus and substantia nigra, J. Neurochem., 25 (1975) 305-307. 6 HENRY, J. L., KRNJEVIC, g . , AND MORRIS, M. E., Substance P and spinal neurones, Canad. J. Physiol. PharmacoL, 53 (1975) 423-432. 7 KONISHI,S., AND OTSUKA, M., Excitatory action of hypothalamic substance P on spinal motoneurones of newborn rats, Nature (Lond.), 252 (1974) 734-735. 8 KONISHI,S., AND OTSUKA,M., The effects of substance P and other peptides on spinal neurons of the frog, Brain Research, 65 (1974) 397-410. 9 KRNJEVIC, K., AND MORRIS, M. E., An excitatory action of substance P on cuncate neurones, Canad. J. Physiol. PharmacoL, 52 (1974) 736-744. 10 OTSUKA, M., KONISm, S., AND TAgAHASm, T., Hypothalamic substance P as a candidate for transmitter of primary afferent neurons, Fed. Proc., 34 (1975) 1922-1928. 11 PHILLIS,J. W., AND LIMACHER,J. J., Substance P excitation of cerebral cortical Betz cells, Brain Research, 69 (1974) 158-163. 12 POWELL, D., LEEMAN, S., TREGEAR, G. W., NIALL, H. D., AND POTTS, J. T., Radioimmunoassay for substance P, Nature New Biol., 241 (1973) 252-254. 13 SAITO, K., KONISm, S., AND OTSUKO, M., Antagonism between Lioresal and substance P m rat spinal cord, Brain Research, 97 (1975) 177-180. 14 TAKAHASHI,T., AND OTSUKA,M., Regional distribution of substance P in the spinal cord and nerve roots of the cat and effect of dorsal root section, Brain Research, 87 (1975) 1-11. 15 ZETLER,G., Biologically active pcptides (substance P). In A. LA~THA(Ed.), Handbook of Neurochemistry, Plenum Press, New York, 1970, pp. 135-148.