Neuroscience Vol. 46, No. I, pp. 211-223, Printed in Great Britain
0306-4522/92 $5.00 + 0.00 Pergamon Press plc
1992
IBRO
CHARACTERIZATION OF TACHYKININ-INDUCED VENTRAL ROOT DEPOLARIZATION IN THE NEONATAL RAT ISOLATED SPINAL CORD S. J. IRELAND,* I. K. WRIGHT and C. C. JORDAN Department
of Neuropharmacology,
Glaxo Group Research SG12 ODP, U.K.
Ltd, Park
Road,
Ware,
Hertfordshire
Abstract-Depolarization responses to tachykinin receptor agonists were recorded extracellularly from lumbar ventral roots of spinal cord isolated from neonatal rats (one to eight days posf purfum). All spinal cords were hemisected in the sagittal plane. In addition, in some hemisected cords, the dorsal horns were removed by means of a further cut, perpendicular to the first. In both hemisected and quadrisected spinal cords, reproducible depolarization responses were induced by low concentrations of the neurokinin-lselective agonist substance P methylester (10 nM-1 p M) or of the neurokinin-3-selective agonist senktide (3-300nM). On both types of preparation, responses to substance P methylester (1 FM) or senktide (300 nM) were of comparable size. The amplitude of the response to senktide (300 nM) was reduced by at least 88% in spinal cord preparations exposed to tetrodotoxin (0.5 p M) or to physiological medium containing magnesium chloride (20mM). In contrast, under either of these conditions, concentration-response curves to substance P methylester were shifted rightward by 2.8+8.5-fold, with little effect on the maximum response. Responses to senktide were blocked selectively by the N-methyl-D-aspartate antagonist 3-[( +)-2-carboxypiperazine-4-yllpropyl-I-phosphonic acid (100 PM); the antagonist had little effect on substance P methylester-induced depolarization (mean concentration ratio 2.0). These results suggest that in the neonatal rat spinal cord, application of exogenous tachykinin agonists can induce ventral root depolarization by activation of neurokinin- 1 and/or neurokinin-3 receptors. The response to stimulation of neurokinin-1 receptors has a major component likely to be due to a direct action at motoneurons. In contrast, ventral root depolarization evoked by stimulation of neurokinin-3 receptors is due, almost exclusively, to an indirect action. The observation that depolarization induced by the neurokinin-3 agonist senktide was not abolished by removal of the dorsal horns is at variance with the finding that in the adult rat, neurokinin-3 binding sites are confined to this region of the spinal cord. An investigation of this paradox is described in the companion paper (Beresford I. J. M., et al. (1991) Neuroscience 46, 225-232.)
The mammalian tachykinins substance P (SP), neurokinin A (NKA) and neurokinin B (NKB) are present in the spinal cord of the adult rat.‘4.2’,26 For all three tachykinins, the concentration in the dorsal horns is higher than in the ventral horns. It seems likely that SP and NKA are present in, although not restricted to, afferent nerves (possibly small diameter non-myelinated fibres) while NKB is confined to neurons intrinsic to the spinal cord.‘4.21.26 The neonatal rat spinal cord in vitro has been used extensively to investigate the possible roles of tachykinins in spinal function. The effects of afferent nerve stimulation or of application of exogenous agonist can be measured from the motoneurons of hemisected preparations (see Refs 1,2,22,29 and 33). Ventral root depolarization responses to SP or NKA appear to comprise at least two components that differ in sensitivity to tetrodotoxin (TTX).‘.” The two components of the response to SP differ also in sensitivity to the tachykinin antagonist spantide’
*To whom correspondence should be addressed. Abbreviations : CPP. 3-[( &)-2-carboxypiperazine-4-ly]propyl-I-phosphonic acid; NKA, neurokinin A; NKB, neurokinin B; NMDA, N-methyl-D-aspartate; SP, substance P; SPOMe, substance P methylester; TTX, tetrodotoxin. 217
raising the possibility that they are mediated by different tachykinin receptors. Responses mediated by neurokinin-I and/or neurokinin-3 receptors have been recorded from neonatal rat spinal cord”,28 and SP, NKA and NKB each has appreciable activity at these receptors (see Refs 13,23 and 24). There is no evidence that neurokinin-2 receptors can mediate tachykinin-induced ventral root depolarization3’ despite the possible existence in adult rat spinal cord of neurokinin-2 binding sites.32 Evidence from autoradiographic studies prompted the hypothesis that, in the rat spinal cord, the characteristics of ventral root depolarization responses mediated by neurokinin-1 and neurokinin-3 receptors might differ. Thus, in adult rats, binding sites resembling neurokinin- 1 receptors are present in the ventral as well as the dorsal horns and so could be present on motoneurons (see Ref. 12), while sites resembling neurokinin-3 receptors appear confined to superficial laminae of the dorsal horns only.8.‘o,‘5,20 Therefore, in the neonatal rat spinal cord, we have measured ventral root depolarization responses induced by a neurokinin-l-selective agonist, substance P methylester (SPOMe)27 or a neurokinin-3selective agonist, senktide,30 and compared their sensitivity to agents that block synaptic transmission or the propagation of action potentials. We have also
examined whether responses to the two agonists exhibited differential sensitivity to removal of the dorsal horns. A preliminary account of this study has been communicated to the British Pharmacological Society.”
at 4pM for 3 min and then at O.SpM for the remainder of the experiment. Its effects were measured after a minimum of 60min exposure. The influence of MgCI, was tested 30-40 min after starting application. This period was required to allow the recorded ventral root potential to stabilize. The effects of CPP were measured after 5 min pre-equilibration.
EXPERIMENTAL PROCEDURES RESULTS
Preparation of spinul c0rd.s
Experiments were performed using spinal cords excised from CD. rat pups (Glaxo) at one to eight dayspos~ partum. Isolated cords with lumbar ventral roots L3-L5 attached were placed in modified Krebs-Henseleit medium at room temperature (18-22°C) and hemisected sagittally under a dissecting microscope. In some hemiseeted cords, the dorsal horn was removed by making a further single cut at right angles to the first. Accuracy of preparation of such quadrisected spinal cords was checked by microscopic examination of paraffin-embedded sections.
Fundamental
charucteristics
In both hemisected and quadrisected spinal cords, rapid ventral root depolarization responses were induced by low concentrations of SPOMe (10 nM-I PM) or senktide (3-300 nM). Using
Exiraee~lu~ar recording
Hemisected or quadrisected spinal cords were placed on an inclined plane covered with absorbent material (nappy liner, Boots) and superfused at a rate of approximately 2 ml/min with modified Krebs-Henseleit medium at room temperature. A ventral root (L3-L5) was placed on a cotton wick which was connected via an agar-saiine bridge to a silver-silver chloride electrode. A grease seal was created by applying a mixture of Vaseline and liquid paraffin (I : I w/v) to insulate the root from the rest of the spinal cord preparation, The d.c. potential between the root electrode and a second silver-silver chloride electrode placed in contact with the body of the cord was recorded continuously and displayed on a potentiometric chart recorder (Servogor SE 130 or 220). Drugs were applied at known con~entratjon via the superfusion stream. The composition of the modified Krebs-Henseleit medium was (in mM): NaCl, 118; NaHCO,, 25; KCl, 4.7; MgSO,,7H,O, 0.7; KH,PO,, 1.2; CaCl,, 1.2; glucose, 11.1. The solution was prepared using gIass-distilled water and analytical grade materials (BDH). The medium was gassed continuously with 95% 0, and 5% CO,. Medium containing a high concentration of magnesium ions was prepared by adding M&l,, 20 mM but no CaCI,; the small change in osmolarity was ignored. SPOMe and senktide were purchased from Bachem U.K.. Cambridge Research Biochemicals or Peninsula. N-Methylr,-aspartate (NMDA), quisqualic acid and ‘ITX were purchased from Sigma. 3-[( f )-2-Carboxypiperazine-4-y& propyl- 1-phosphonic acid (CPP) was obtained from Tocris Neuramin. SPOMe was dissolved in 0.01 M acetic acid, senktide in dimethylsulphoxide to give stock solutions of S--IOmM. These were stored frozen (-20°C) under nitrogen until they were diiuted with Krebs-Henseleit medium for use. The identity of peptides was verified by fast atom bombardment mass sp~trometry and concentrations confirmed by amino acid analysis (see Ref. 6). Solutions of NMDA, quisqualic acid, CPP and TTX were prepared in Krebs-Henseleit medium immediately before use. Experimental design
Al1 preparations were exposed to senktide and SPOMe applied alternately at 15min intervals. Concentrationresponse curves were constructed non-~umulativeiy using serially-increasing concentrations applied for approximately I min. A minimum period of 1h was left after completion of control curves before testing the effect of any given treatment on responses to the two agonists. In experiments in which TTX was used, the toxin was applied initially
10-S
10‘8
10-7
1
o-6
Agonist CM) Fig. 1,Comparison of ventral root depolarization responses induced by senktide (m) or SPOMe (0) on the isolated spinal cord of the neonatal rat. Results were obtained from hemisected cords (a) or quadrisected cords from which the dorsal horns had been removed (b). Bach point is the mean with vertical lines indicating the S.E.M. from four individual preparations. Preparations were exposed to both senktide and SPOMe applied alternately at 15min intervals. Depolarization is expressed as a percentage of the response to senktide. 300 nM.
Tachykinin-induced
219
depolarization of spinal cord
these concentrations, tachyphylaxis was avoided by applying agonists alternately at 15min intervals. No attempt was made to define the maxima of concentration-response curves since long-lasting desensitization was observed following application of concentrations of senktide greater than 300nM. Responses to SPOMe (1 p M) and senktide (300 nM) were of comparable amplitude, both being about 1 mV. For convenience, in each preparation, the response to senktide (300 nM) was termed, arbitrarily, 100%. Senktide was more active than SPOMe although concentration-response curves were approximately parallel (Fig. 1). The mean (k S.E.M.) of equi-potent molar ratio (senktide/SPOMe) calculated in each preparation at the 50% response level was 0.34 f 0.42 (n = 4) in hemisected cords and 0.24 f 0.45 (n = 4) in quadrisected cords.
Eflects of tetrodotoxin or MgCl, Exposure of spinal cord preparations to TTX or MgCl, produced similar effects: in both hemisected or quadrisected cords, the maximum evoked response to senktide was inhibited by a mean of at least 88% (Figs 2 and 3, Table 1). In contrast, concentration-response curves to SPOMe were shifted to the right by 2.8-8.5-fold with little effect on maximum evoked response (Figs 2 and 3, Table 1). Effects of 3-[( f )-2-carboxypiperazine-4-lyl-propyl-lphosphonic acid Application of CPP (30-100 p M) caused approximately parallel and concentration-dependent rightward displacement of concentration-response curves to senktide and NMDA (Table 2). CPP had essen-
b
control
Conuol
P
h+
8 t
9
SC
b c
T-l-x
/
w--z 1 104
10-e
lo-' SPoMe
10-s
lo-'
(M)
Fig. 2. Effect of TTX (0 0) on ventral root depolarization responses induced by senktide (m) or SPOMe (0) in the neonatal rat spinal cord in vitro. Results are shown for hemisected spinal cords (a, b) or quadrisected cords from which the dorsal horns had been removed (c, d). Each point is the mean with vertical lines indicating the S.E.M. of single determinations in four separate preparations. Depolarization is expressed as a percentage of the response to senktide, 300 nM.
230
E
T
-9
*
0-8
-fs’=l,
10-7 104 Senkcide(Ml
10-J
-9
10-a
10-6
10-r SPOMe
10-s
CM)
Fig. 3. Effect of magnesium chloride (MgCI,) (Do) on ventral root depolarization responses induced by senktide (a) or SPOMe (0) in the neonatal rat spinal cord in vitro. Results are shown for hemisected spinai cords (a, b) or quadrisected cords from which the dorsal horns had been removed (c, d). Each point is the mean with vertical lines indicating the S.E.M. of single dete~inations in four separate preparations. Medium containing MgCI, (20 mM) had no added CaCi,. Depolarization is expressed as a percentage of the response to senktide. 300nM.
Table I. Effects of tetrodotoxin or magnesium chloride (MgCI,, 20 mM) on ventral root depolarization responses induced by senktide or substance P methylester in the neonatal rat spinal cord
Treatment TTX
MgC&
Mean concentration Hemisected spinal cord SPOMe Senktide Maximum reduced to 12.0 + 4.7% Maximum reduced to 1.3 rf- 2.3%
4.7 * 1.3
8.5 + 1.4
ratio (IrS.E.M.) Quadrisected spinal cord SPOMe Senktide Maximum reduced to 6.3 + 2.0% MaGmum reduced to 4.8 + 2.4%
2.8 + 0.5
4.6 f 0.5
Results are shown for both hemisected spinal cords and quadrisected cords from which the dorsal horns had been removed. Each preparation was exposed to senktide and SPQMe applied alternately. Effects on responses to SPOMe are expressed as concentration ratios measured at a level of response approximately equal to half that induced by senktide, 300 nM. Against senktide, reduction of maximum response was estimated by comparing the response to senktide, 300 nM, before and after application of TTX or MgCl,. Each value is the mean (+ S.E.M.) of single determinations in four separate preparations.
Tachykinin-indu~d
depola~~tion
221
of spinat cord
Table 2. Effect of 3-[( k)-2-carboxypiperazine-4-iy]-propyl-I-phosphonic acid (30 or 100 PM) on ventral root depolarization of neonatal rat spinal cord CPP (FM) 30 100
Senktide
Mean concentration ratio (+ S.E.M.) Quisquahc acid NMDA SPQMe
5.2 f 1.2 46.4 & 12.9
1.4 + 0.4 2.0 + 0.7
19.9 * 2.6 52.7 * 5.2
1.0 kO.0 1.1 +0.4
Results are shown for isolated hemisected spinal cord preparations from I-S-day-old rats. Each preparation was exposed to senktide and SPOMe or NMDA and quisqualic acid applied alternately. Concentration ratios were measured at a level of response approximately equal to half that induced by senktide, 300 nM, or NMDA, 30 PM. Each value is the mean (k S.E.M.) of single dete~inations in four separate preparations. tially
no
effect
responses induced (Table 2).
ventral root depolarization by SPOMe or quisqualic acid
on
DISCUSSION
SPOMe and senktide are highly selective agonists at neurokinin-1 or neurokinin-3 receptors, respectively.27*30 In the neonatal rat spinal cord, at nanomolar concentration, either agonist caused ventral root depolarization. This confirms suggestions that, in this preparation, activation of either neurokinin-1 or neurokinin-3 receptors is able to influence the resting membrane potential of motoneurons.‘* However, it is likely that the mechanisms involved can be different. Ventral root depolarization induced by senktide was very substantially reduced by TTX at a concentration sufficient to block conduction of action potentials (see Ref. 31). The response was also virtually abolished by MgCl, (20 mM): magnesium ions at such concentrations are adequate to prevent synaptic transmission.3.16.zs In contrast, against SPOMeinduced de~larization, TTX or MgCl, had only modest effect, each causing a small rightward displacement of the concentration-response curve to the agonist with little effect on the maximum response. These resufts suggest that in neonatal rat spinal cord, ventral root depolarization induced by exogenously applied tachykinin agonists and mediated by neurokinin-3 receptors is predominantly indirect, being initiated at sites remote from the motoneurons. In contrast, a substantial component of the ventral root depolarization mediated by neurokinin-1 receptors is direct although there appears to be an indirect, TTXor magnesium ion-sensitive component too. Responses to senktide were antagonized by CPP at concentrations that appeared selective for NMDA receptors since quisqualate-induced depolarization was unaffected. This prompts the hypothesis that in neonatal rat spinal cord, neurokinin-3 receptorevoked ventral root depolarization is mediated, at least partially, by the release of excitatory amino acid(s). CPP had negligible effect against SPOMeinduced ventral root depolarization suggesting that even the apparently indirect component of this response does not have a significant NMDA receptor-
mediated component. The present data do not rule out the involvement of excitatory amino acid receptors of non-NMDA type in responses to exogenously applied tachykinins nor, indeed, do they exclude other potential chemical mediators. Both SP and NKA are reported to increase the release of the excitatory amino acids glutamate and aspartate from slices of the spinal cords of 234%day-old rats; unfortunately it is not clear what type of tachykinin receptor is involved. i8.i9 The source of the amino acids too is uncertain: both neurons and glia are possible.4,‘8,‘9 In the neonatal rat spinal cord, the precise location of both the neurokinin-I and the neurokinin-3 receptors mediating tachykinin-induced ventral root depolarization remains to be elucidated. Although it is likely that a population of neurokinin-1 receptors occurs on motoneurons, the apparently indirect effect of neurokinin-1 receptor stimulation may be initiated elsewhere albeit within the ventral horn. Binding sites resembling neurokinin-I receptors are found throughout the ventral and dorsal horns of the spinal cord of both adult and neonatal rats.’ The distribution of neurokinin-3 receptors in the rat spinal cord is also of interest. In adults, autoradiographic studies suggest that neurokinin-3 receptors are confined to superficial laminae of the dorsal horns only. 8~‘o,‘5.20 However, in the present study on the neonatal rat spinal cord, ventral root depolarization induced by stimulation of neurokinin-3 receptors, although apparently indirect, was not abolished by removal of the dorsal horns. This result is consistent with new evidence presented in the companion paper that, in neonatal rat, binding sites resembling neurokinin-3 receptors are found throughout both the ventral and dorsal horns5 As yet, it is not clear whether age-related changes in the distribution of neurokinin-3 receptors reflects maturation of spinal function.
CONCLUSION
In the spinal cord of the neonatal rat, ventral root depolarization induced by activation of neurokinin-3 receptors was substantially inhibited by TTX or
222
S.J.
IRELAND YI al.
MgClz. In contrast, the response to activation of neurokinin-1 receptors had a major component res&ant to these agents. The results suggest that blockade of neuronal transmission with TTX or MgCl, could, in effect, alter the spectrum of receptors able to mediate ventral root depolarization in response to
the application of non-selective tachykinin agonists (cf. Refs 7, 31). The finding that neurokinin-I receptor-mediated depolarization appeared to have a component which was sensitive to TTX or MgCI, indicates that caution must be exercised before using these agents to define receptors.
REFERENCES I.Akagi H., Konishi S., Otsuka M. and Yanagisawa M. (1985) The role of substance Pas a neurotransmitter in the reflexes of slow time courses in the neonatal rat spinal cord. Br. J. Pharmac. 84, 6633673. 2. Akagi H. and Yanagisawa M. (1987) GABAergic modulation of a substance P-mediated reflex of slow time course in the isolated rat spinal cord. Br. J. Pharmac. 91, 189-197. 3. Ault B., Evans R. H., Francis A. A., Oakes D. J. and Watkins J. C. (1980) Selective depression of excitatory amino acid induced depolarizations by magnesium ions in isolated spinal cord preparations. J. Physiol. (Lond.) 307, 413-428. 4. Beaujouan J. C., Daguet de Montety M. C., Torrens Y., Saffroy M., Diet1 M. and Glowinski J. (1990) Marked regional
heterogeneity of ‘*SI-Bolton Hunter substance P binding and substance P-induced activation of phospholipase C in astrocyte cultures from the embryonic or newborn rat. J. Neurochem. 54, 669-675. 5. Beresford I. J. M., Ireland S. J., Stables J. and Hagan R. M. (1991) Ontogeny and characterization of [“‘IJBolton Hunter-eledoisin binding sites in the rat spinal cord by quantitative autoradiography. Neuroscience 46, 225 232. 6. Brown J. R., Hunter J. C., Jordan C. C., Tyers M. B., Ward P. and Whittington A. R. (1986) Problems with peptides-all that glisters is not gold. Trends Pharmac. Sci. 9, 100-102. 7. Brugger F., Evans R. H. and Hawkins N. S. (1990) Effects of N-methyl-n-aspartate antagonists and spantide on spinal reflexes and responses to substance P and capsaicin in isolated spinal cord preparations from mouse and rat. Neuroscience 36, 6 1 I-622. 8. Buck S. H., Helke C. J., Burcher E., Shults C. W. and O’Donohue T. L. (1986) Pharmacologic characterization and autoradiographic distribution of binding sites for iodinated tachykinins in the rat central nervous system. Peptides 7, 110991120. 9. Charlton C. G. and Helke C. J. (1986) Ontogeny of substance P receptors in rat spinal cord: quantitative changes in receptor number and differential expression in specific loci. Deul Brain Res. 29, 81-91. IO. Danks J. A., Rothman R. B., Cascieri M. A., Chicchi G. G., Liang T. and Herkenham M. (1986) A comparative autoradiographic study of the distributions of substance P and eledoisin binding sites in rat brain. Brain Res. 385, 273-28 I. Il. Guard S., Watling K J. and Watson S. P. (1989) NK-3 tachykinin receptors are coupled to inositol phospholipid hydrolysis in neonatal rat spinal cord. Br. J. Pharmac. 98, 908P. 12. Helke C. J., Charlton C. G. and Wiley R. G. (1986) Studies on the cellular localization of spinal cord substance P receptors. Neuroscience 19, 5233533. 13. Henry J. L. (1988) Discussions of nomenclature for tdchykinins and tachykinin receptors. In Substance P and Neurokinins (eds Henry J. L., Couture R.. Cuello A. C., Pelletier G., Quirion R. and Regoli R.). pp. xvii-xviii. Springer. New York. 14. Hiikfelt T., Kellerth J.-O., Nilsson G. and Pernow B. (1975) Experimental immunohistochemical studies on the localization and distribution of substance P in cat primary sensory neurons, Brain Res. 100, 235-252. 15. Hunter J. C., Kilpatrick G. J. and Brown J. R. (1987) Pharmacological analysis of [‘251]-Bolton and Hunter labelled eledoisin binding sites in rat spinal cord by quantitative autoradiography. Neurosci. Lelt. 78, 12-16. J. Physiol. 16. Hutter 0. F. and Hostill K. (1954) Effect of magnesium and calcium ions on the release of acetylcholine. (Lo&) 124, 234424 I. receptor agonists may depolarize spinal motoneurones 17. Ireland S. J., Jordan C. C. and Wright 1. K. (1988) Neurokinin via two distinct mechanisms, Br. J. Pharmac. 93, 85P. I., Larew J. S. and Randic M. (1990) The effects of substance P and calcitonin gene-related peptide on the 18. Kangrga efflux of endogenous glutamate and aspartate from the rat spinal dorsal horn in vitro. Neurosci. Let?. 108, 155- 160. I. and Randic M. (1990) Tachykinins and calcitonin gene-related peptide enhance release of endogenous 19. Kangrga glutamate and aspartate from the rat spinal dorsal horn slice. J. Neurosci. 10,20262038. 20. Nincovic M., Beaujouan J. C., Torrens Y., Saffroy M., Hall M. D. and Glowinski J. (1985) Differential localization of tachykinin receptors in rat spinal cord. Eur. J. Pharmac. 106,4633464. 1. and Kimura S. (1985) Regional distribution of substance P, neurokinin a and neurokinin fi in 21. Ogawa T., Kanazawa rat spinal cord, nerve roots and dorsal root ganglia, and the effects of dorsal root section or spinal transection. Bruin Res. 359, 152-157. M. (1988) Effect of a tachykinin antagonist on a nociceptive reflex in the isolated spinal 22. Otsuka M. and Yanagisawa cord-tail preparation of the newborn rat. J. Physiol. (Lund.) 395, 2555270. receptors: recent developments. Regul. Pepl. 22, 18. 25. 23. Quirion R. and Dam T. V. (1988) Multiple neurokinin P. (1987) Recent developments in neurokinins pharmacology. 24. Regoli D., Drapeau G., Dion S. and D’Orleans-Juste Life Sci. 40, 109-117. 25. Richards C. D. and Sercombe R. (1970) Calcium, magnesium and the electrical activity of guinea-pig olfactory cortex in &r-o. J. Physiol. (Lond.) 211, 571-584. R. and Uddman R. (1985) Sensory nerve fibres: distribution of substance 26. Sundler F., Brodin E., Elkbald E., Hakanson P, neurokinin A and calcitonin gene-related peptide. In Tachykinin Antagonists (eds Hakanson R. and Sundler F.). pp. 3314. Elsevier, Amsterdam. 27. Watson S. P., Sandberg B. E. B., Hanley M. R. and Iversen L. L. (1983) Tissue selectivity of substance P alkyl esters: suggesting multiple receptors. Eur. J. Pharmac. 87, 77784. 28. Wienrich M., Lues I., Sandberg B. E. B. and Harting J. (1987) Evidence for at least two tachykinin receptor subtypes in the mammalian spinal cord. In Substance P and Neurokinins (eds Henry J. L., Couture R., Cue110 A. C., Pehetier G.. Quirion R. and Regoli D.), pp. 328-330. Springer. New York.
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depolarization of spinal cord
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29. Wiemich M. and Harting 3. (1985) Effects of putative tachykinin antagonists on the slow ipsilateral ventral root potential in the isolated spinai cord of the neonatal rat. In ~uc~~~i~j~ ~nfago~jsr~ (eds Hakanson R. and Sundler F.), DD. 367-376. Elsevier. Amsterdam. 30. Wormser U., Laufer &Hart Y., Chorev M., Gilon C. and Sehnger Z. (1986) Highly selective agonists for substance P receptor subtypes. Eur. molec. Bid. Ora. J. 5. 2805-2808. 31. YanaGsawa M.-and Otsuka M. (1990) Pharmacological profile of a tachykinin antagonist, spantide, as examined on rat spinal motoneurones. Br. J. Pharmac. 100, 711-716. 32. Yashpal K., Dam T. V. and Quirion R. (1990) Quantitative autoradiographic distribution of multiple neurokinin binding sites in rat spinal cord. Bruin Res. 506, 259-266. 33. Yoshioka K., Sakuma M. and Otsuka M. (1990) Cutaneous nerve evoked chohnergic inhibition of monosynaptic reflex in the neonatal rat spinal cord: involvement of M2 receptors and tachykinin primary afferents. Neuroscience 38, 195-203. (Atcepted
I July 1991)
0306-4522/92 $5.00 + 0.00 Pergamon Press plc
1992
IBRO
CHARACTERIZATION OF TACHYKININ-INDUCED VENTRAL ROOT DEPOLARIZATION IN THE NEONATAL RAT ISOLATED SPINAL CORD S. J. IRELAND,* I. K. WRIGHT and C. C. JORDAN Department
of Neuropharmacology,
Glaxo Group Research SG12 ODP, U.K.
Ltd, Park
Road,
Ware,
Hertfordshire
Abstract-Depolarization responses to tachykinin receptor agonists were recorded extracellularly from lumbar ventral roots of spinal cord isolated from neonatal rats (one to eight days posf purfum). All spinal cords were hemisected in the sagittal plane. In addition, in some hemisected cords, the dorsal horns were removed by means of a further cut, perpendicular to the first. In both hemisected and quadrisected spinal cords, reproducible depolarization responses were induced by low concentrations of the neurokinin-lselective agonist substance P methylester (10 nM-1 p M) or of the neurokinin-3-selective agonist senktide (3-300nM). On both types of preparation, responses to substance P methylester (1 FM) or senktide (300 nM) were of comparable size. The amplitude of the response to senktide (300 nM) was reduced by at least 88% in spinal cord preparations exposed to tetrodotoxin (0.5 p M) or to physiological medium containing magnesium chloride (20mM). In contrast, under either of these conditions, concentration-response curves to substance P methylester were shifted rightward by 2.8+8.5-fold, with little effect on the maximum response. Responses to senktide were blocked selectively by the N-methyl-D-aspartate antagonist 3-[( +)-2-carboxypiperazine-4-yllpropyl-I-phosphonic acid (100 PM); the antagonist had little effect on substance P methylester-induced depolarization (mean concentration ratio 2.0). These results suggest that in the neonatal rat spinal cord, application of exogenous tachykinin agonists can induce ventral root depolarization by activation of neurokinin- 1 and/or neurokinin-3 receptors. The response to stimulation of neurokinin-1 receptors has a major component likely to be due to a direct action at motoneurons. In contrast, ventral root depolarization evoked by stimulation of neurokinin-3 receptors is due, almost exclusively, to an indirect action. The observation that depolarization induced by the neurokinin-3 agonist senktide was not abolished by removal of the dorsal horns is at variance with the finding that in the adult rat, neurokinin-3 binding sites are confined to this region of the spinal cord. An investigation of this paradox is described in the companion paper (Beresford I. J. M., et al. (1991) Neuroscience 46, 225-232.)
The mammalian tachykinins substance P (SP), neurokinin A (NKA) and neurokinin B (NKB) are present in the spinal cord of the adult rat.‘4.2’,26 For all three tachykinins, the concentration in the dorsal horns is higher than in the ventral horns. It seems likely that SP and NKA are present in, although not restricted to, afferent nerves (possibly small diameter non-myelinated fibres) while NKB is confined to neurons intrinsic to the spinal cord.‘4.21.26 The neonatal rat spinal cord in vitro has been used extensively to investigate the possible roles of tachykinins in spinal function. The effects of afferent nerve stimulation or of application of exogenous agonist can be measured from the motoneurons of hemisected preparations (see Refs 1,2,22,29 and 33). Ventral root depolarization responses to SP or NKA appear to comprise at least two components that differ in sensitivity to tetrodotoxin (TTX).‘.” The two components of the response to SP differ also in sensitivity to the tachykinin antagonist spantide’
*To whom correspondence should be addressed. Abbreviations : CPP. 3-[( &)-2-carboxypiperazine-4-ly]propyl-I-phosphonic acid; NKA, neurokinin A; NKB, neurokinin B; NMDA, N-methyl-D-aspartate; SP, substance P; SPOMe, substance P methylester; TTX, tetrodotoxin. 217
raising the possibility that they are mediated by different tachykinin receptors. Responses mediated by neurokinin-I and/or neurokinin-3 receptors have been recorded from neonatal rat spinal cord”,28 and SP, NKA and NKB each has appreciable activity at these receptors (see Refs 13,23 and 24). There is no evidence that neurokinin-2 receptors can mediate tachykinin-induced ventral root depolarization3’ despite the possible existence in adult rat spinal cord of neurokinin-2 binding sites.32 Evidence from autoradiographic studies prompted the hypothesis that, in the rat spinal cord, the characteristics of ventral root depolarization responses mediated by neurokinin-1 and neurokinin-3 receptors might differ. Thus, in adult rats, binding sites resembling neurokinin- 1 receptors are present in the ventral as well as the dorsal horns and so could be present on motoneurons (see Ref. 12), while sites resembling neurokinin-3 receptors appear confined to superficial laminae of the dorsal horns only.8.‘o,‘5,20 Therefore, in the neonatal rat spinal cord, we have measured ventral root depolarization responses induced by a neurokinin-l-selective agonist, substance P methylester (SPOMe)27 or a neurokinin-3selective agonist, senktide,30 and compared their sensitivity to agents that block synaptic transmission or the propagation of action potentials. We have also
examined whether responses to the two agonists exhibited differential sensitivity to removal of the dorsal horns. A preliminary account of this study has been communicated to the British Pharmacological Society.”
at 4pM for 3 min and then at O.SpM for the remainder of the experiment. Its effects were measured after a minimum of 60min exposure. The influence of MgCI, was tested 30-40 min after starting application. This period was required to allow the recorded ventral root potential to stabilize. The effects of CPP were measured after 5 min pre-equilibration.
EXPERIMENTAL PROCEDURES RESULTS
Preparation of spinul c0rd.s
Experiments were performed using spinal cords excised from CD. rat pups (Glaxo) at one to eight dayspos~ partum. Isolated cords with lumbar ventral roots L3-L5 attached were placed in modified Krebs-Henseleit medium at room temperature (18-22°C) and hemisected sagittally under a dissecting microscope. In some hemiseeted cords, the dorsal horn was removed by making a further single cut at right angles to the first. Accuracy of preparation of such quadrisected spinal cords was checked by microscopic examination of paraffin-embedded sections.
Fundamental
charucteristics
In both hemisected and quadrisected spinal cords, rapid ventral root depolarization responses were induced by low concentrations of SPOMe (10 nM-I PM) or senktide (3-300 nM). Using
Exiraee~lu~ar recording
Hemisected or quadrisected spinal cords were placed on an inclined plane covered with absorbent material (nappy liner, Boots) and superfused at a rate of approximately 2 ml/min with modified Krebs-Henseleit medium at room temperature. A ventral root (L3-L5) was placed on a cotton wick which was connected via an agar-saiine bridge to a silver-silver chloride electrode. A grease seal was created by applying a mixture of Vaseline and liquid paraffin (I : I w/v) to insulate the root from the rest of the spinal cord preparation, The d.c. potential between the root electrode and a second silver-silver chloride electrode placed in contact with the body of the cord was recorded continuously and displayed on a potentiometric chart recorder (Servogor SE 130 or 220). Drugs were applied at known con~entratjon via the superfusion stream. The composition of the modified Krebs-Henseleit medium was (in mM): NaCl, 118; NaHCO,, 25; KCl, 4.7; MgSO,,7H,O, 0.7; KH,PO,, 1.2; CaCl,, 1.2; glucose, 11.1. The solution was prepared using gIass-distilled water and analytical grade materials (BDH). The medium was gassed continuously with 95% 0, and 5% CO,. Medium containing a high concentration of magnesium ions was prepared by adding M&l,, 20 mM but no CaCI,; the small change in osmolarity was ignored. SPOMe and senktide were purchased from Bachem U.K.. Cambridge Research Biochemicals or Peninsula. N-Methylr,-aspartate (NMDA), quisqualic acid and ‘ITX were purchased from Sigma. 3-[( f )-2-Carboxypiperazine-4-y& propyl- 1-phosphonic acid (CPP) was obtained from Tocris Neuramin. SPOMe was dissolved in 0.01 M acetic acid, senktide in dimethylsulphoxide to give stock solutions of S--IOmM. These were stored frozen (-20°C) under nitrogen until they were diiuted with Krebs-Henseleit medium for use. The identity of peptides was verified by fast atom bombardment mass sp~trometry and concentrations confirmed by amino acid analysis (see Ref. 6). Solutions of NMDA, quisqualic acid, CPP and TTX were prepared in Krebs-Henseleit medium immediately before use. Experimental design
Al1 preparations were exposed to senktide and SPOMe applied alternately at 15min intervals. Concentrationresponse curves were constructed non-~umulativeiy using serially-increasing concentrations applied for approximately I min. A minimum period of 1h was left after completion of control curves before testing the effect of any given treatment on responses to the two agonists. In experiments in which TTX was used, the toxin was applied initially
10-S
10‘8
10-7
1
o-6
Agonist CM) Fig. 1,Comparison of ventral root depolarization responses induced by senktide (m) or SPOMe (0) on the isolated spinal cord of the neonatal rat. Results were obtained from hemisected cords (a) or quadrisected cords from which the dorsal horns had been removed (b). Bach point is the mean with vertical lines indicating the S.E.M. from four individual preparations. Preparations were exposed to both senktide and SPOMe applied alternately at 15min intervals. Depolarization is expressed as a percentage of the response to senktide. 300 nM.
Tachykinin-induced
219
depolarization of spinal cord
these concentrations, tachyphylaxis was avoided by applying agonists alternately at 15min intervals. No attempt was made to define the maxima of concentration-response curves since long-lasting desensitization was observed following application of concentrations of senktide greater than 300nM. Responses to SPOMe (1 p M) and senktide (300 nM) were of comparable amplitude, both being about 1 mV. For convenience, in each preparation, the response to senktide (300 nM) was termed, arbitrarily, 100%. Senktide was more active than SPOMe although concentration-response curves were approximately parallel (Fig. 1). The mean (k S.E.M.) of equi-potent molar ratio (senktide/SPOMe) calculated in each preparation at the 50% response level was 0.34 f 0.42 (n = 4) in hemisected cords and 0.24 f 0.45 (n = 4) in quadrisected cords.
Eflects of tetrodotoxin or MgCl, Exposure of spinal cord preparations to TTX or MgCl, produced similar effects: in both hemisected or quadrisected cords, the maximum evoked response to senktide was inhibited by a mean of at least 88% (Figs 2 and 3, Table 1). In contrast, concentration-response curves to SPOMe were shifted to the right by 2.8-8.5-fold with little effect on maximum evoked response (Figs 2 and 3, Table 1). Effects of 3-[( f )-2-carboxypiperazine-4-lyl-propyl-lphosphonic acid Application of CPP (30-100 p M) caused approximately parallel and concentration-dependent rightward displacement of concentration-response curves to senktide and NMDA (Table 2). CPP had essen-
b
control
Conuol
P
h+
8 t
9
SC
b c
T-l-x
/
w--z 1 104
10-e
lo-' SPoMe
10-s
lo-'
(M)
Fig. 2. Effect of TTX (0 0) on ventral root depolarization responses induced by senktide (m) or SPOMe (0) in the neonatal rat spinal cord in vitro. Results are shown for hemisected spinal cords (a, b) or quadrisected cords from which the dorsal horns had been removed (c, d). Each point is the mean with vertical lines indicating the S.E.M. of single determinations in four separate preparations. Depolarization is expressed as a percentage of the response to senktide, 300 nM.
230
E
T
-9
*
0-8
-fs’=l,
10-7 104 Senkcide(Ml
10-J
-9
10-a
10-6
10-r SPOMe
10-s
CM)
Fig. 3. Effect of magnesium chloride (MgCI,) (Do) on ventral root depolarization responses induced by senktide (a) or SPOMe (0) in the neonatal rat spinal cord in vitro. Results are shown for hemisected spinai cords (a, b) or quadrisected cords from which the dorsal horns had been removed (c, d). Each point is the mean with vertical lines indicating the S.E.M. of single dete~inations in four separate preparations. Medium containing MgCI, (20 mM) had no added CaCi,. Depolarization is expressed as a percentage of the response to senktide. 300nM.
Table I. Effects of tetrodotoxin or magnesium chloride (MgCI,, 20 mM) on ventral root depolarization responses induced by senktide or substance P methylester in the neonatal rat spinal cord
Treatment TTX
MgC&
Mean concentration Hemisected spinal cord SPOMe Senktide Maximum reduced to 12.0 + 4.7% Maximum reduced to 1.3 rf- 2.3%
4.7 * 1.3
8.5 + 1.4
ratio (IrS.E.M.) Quadrisected spinal cord SPOMe Senktide Maximum reduced to 6.3 + 2.0% MaGmum reduced to 4.8 + 2.4%
2.8 + 0.5
4.6 f 0.5
Results are shown for both hemisected spinal cords and quadrisected cords from which the dorsal horns had been removed. Each preparation was exposed to senktide and SPQMe applied alternately. Effects on responses to SPOMe are expressed as concentration ratios measured at a level of response approximately equal to half that induced by senktide, 300 nM. Against senktide, reduction of maximum response was estimated by comparing the response to senktide, 300 nM, before and after application of TTX or MgCl,. Each value is the mean (+ S.E.M.) of single determinations in four separate preparations.
Tachykinin-indu~d
depola~~tion
221
of spinat cord
Table 2. Effect of 3-[( k)-2-carboxypiperazine-4-iy]-propyl-I-phosphonic acid (30 or 100 PM) on ventral root depolarization of neonatal rat spinal cord CPP (FM) 30 100
Senktide
Mean concentration ratio (+ S.E.M.) Quisquahc acid NMDA SPQMe
5.2 f 1.2 46.4 & 12.9
1.4 + 0.4 2.0 + 0.7
19.9 * 2.6 52.7 * 5.2
1.0 kO.0 1.1 +0.4
Results are shown for isolated hemisected spinal cord preparations from I-S-day-old rats. Each preparation was exposed to senktide and SPOMe or NMDA and quisqualic acid applied alternately. Concentration ratios were measured at a level of response approximately equal to half that induced by senktide, 300 nM, or NMDA, 30 PM. Each value is the mean (k S.E.M.) of single dete~inations in four separate preparations. tially
no
effect
responses induced (Table 2).
ventral root depolarization by SPOMe or quisqualic acid
on
DISCUSSION
SPOMe and senktide are highly selective agonists at neurokinin-1 or neurokinin-3 receptors, respectively.27*30 In the neonatal rat spinal cord, at nanomolar concentration, either agonist caused ventral root depolarization. This confirms suggestions that, in this preparation, activation of either neurokinin-1 or neurokinin-3 receptors is able to influence the resting membrane potential of motoneurons.‘* However, it is likely that the mechanisms involved can be different. Ventral root depolarization induced by senktide was very substantially reduced by TTX at a concentration sufficient to block conduction of action potentials (see Ref. 31). The response was also virtually abolished by MgCl, (20 mM): magnesium ions at such concentrations are adequate to prevent synaptic transmission.3.16.zs In contrast, against SPOMeinduced de~larization, TTX or MgCl, had only modest effect, each causing a small rightward displacement of the concentration-response curve to the agonist with little effect on the maximum response. These resufts suggest that in neonatal rat spinal cord, ventral root depolarization induced by exogenously applied tachykinin agonists and mediated by neurokinin-3 receptors is predominantly indirect, being initiated at sites remote from the motoneurons. In contrast, a substantial component of the ventral root depolarization mediated by neurokinin-1 receptors is direct although there appears to be an indirect, TTXor magnesium ion-sensitive component too. Responses to senktide were antagonized by CPP at concentrations that appeared selective for NMDA receptors since quisqualate-induced depolarization was unaffected. This prompts the hypothesis that in neonatal rat spinal cord, neurokinin-3 receptorevoked ventral root depolarization is mediated, at least partially, by the release of excitatory amino acid(s). CPP had negligible effect against SPOMeinduced ventral root depolarization suggesting that even the apparently indirect component of this response does not have a significant NMDA receptor-
mediated component. The present data do not rule out the involvement of excitatory amino acid receptors of non-NMDA type in responses to exogenously applied tachykinins nor, indeed, do they exclude other potential chemical mediators. Both SP and NKA are reported to increase the release of the excitatory amino acids glutamate and aspartate from slices of the spinal cords of 234%day-old rats; unfortunately it is not clear what type of tachykinin receptor is involved. i8.i9 The source of the amino acids too is uncertain: both neurons and glia are possible.4,‘8,‘9 In the neonatal rat spinal cord, the precise location of both the neurokinin-I and the neurokinin-3 receptors mediating tachykinin-induced ventral root depolarization remains to be elucidated. Although it is likely that a population of neurokinin-1 receptors occurs on motoneurons, the apparently indirect effect of neurokinin-1 receptor stimulation may be initiated elsewhere albeit within the ventral horn. Binding sites resembling neurokinin-I receptors are found throughout the ventral and dorsal horns of the spinal cord of both adult and neonatal rats.’ The distribution of neurokinin-3 receptors in the rat spinal cord is also of interest. In adults, autoradiographic studies suggest that neurokinin-3 receptors are confined to superficial laminae of the dorsal horns only. 8~‘o,‘5.20 However, in the present study on the neonatal rat spinal cord, ventral root depolarization induced by stimulation of neurokinin-3 receptors, although apparently indirect, was not abolished by removal of the dorsal horns. This result is consistent with new evidence presented in the companion paper that, in neonatal rat, binding sites resembling neurokinin-3 receptors are found throughout both the ventral and dorsal horns5 As yet, it is not clear whether age-related changes in the distribution of neurokinin-3 receptors reflects maturation of spinal function.
CONCLUSION
In the spinal cord of the neonatal rat, ventral root depolarization induced by activation of neurokinin-3 receptors was substantially inhibited by TTX or
222
S.J.
IRELAND YI al.
MgClz. In contrast, the response to activation of neurokinin-1 receptors had a major component res&ant to these agents. The results suggest that blockade of neuronal transmission with TTX or MgCl, could, in effect, alter the spectrum of receptors able to mediate ventral root depolarization in response to
the application of non-selective tachykinin agonists (cf. Refs 7, 31). The finding that neurokinin-I receptor-mediated depolarization appeared to have a component which was sensitive to TTX or MgCI, indicates that caution must be exercised before using these agents to define receptors.
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