- Email: [email protected]
Noxious heating of the skin releases immunoreactive substance P in the substantia gelatinosa of the cat: A study with antibody microprobes
BrainResearch, 403(1987)345-349 Elsevier
345
BRE22027
Noxious heating of the skin releases immunoreactive substance P in the substantia gelatinosa of the cat: a study with antibody microprobes A.W. Duggan, C.R. Morton, Z.Q. Zhao and I.A. Hendry Department of Pharmacology, John Curtin School of Medical Research, The Australian National University, Canberra (Australia) (Accepted 30 September 1986) Key words: Substance P release; Noxious heat; Substantia gelatinosa
Using a new method, the antibody microprobe technique, the release of immunoreactive substance P (SPiR) in the dorsal horn in response to noxious heating of the skin, was studied in barbiturate anaesthetized spinal cats. Release of SPiR was not produced by immersing the ipsilateral hind paw in water at 37 °C. With water at 50 and 52 °C, however release was consistently detected in the region of the substantia gelatinosa. These results directly show a central release of SPiR with excitation of nociceptors by heat.
The demonstration of the release of a substance from the axon terminals of neurones is a crucial piece of evidence that it is involved in transmission of information. Such evidence is needed to assign functional significance to the many complexities revealed by immunohistochemical studies of dorsal root ganglion ( D R G ) neurones 5,9'10'13'16. This is particularly so in the case of substance P (SP) and excitation of nociceptors by heat. Early studies associated immunoreactive substance P (SPiR) with small diameter D R G neurones with the suggestion that many of these were nociceptors 1°. The effects of microelectrophoretic ejection of SP near neurones of the dorsal horn of the cat lead to the proposal that this peptide was released from the central terminals of thermal nociceptors s. Depleting D R G neurones of substance P by the administration of capsaicin however, has given discordant results when using thermal tests of nociception. For example using the tail flick test, capsaicin-treated rodents have been variously found to have normal responses 1'6'7, reduced responses 11'19or impaired visceral but normal somatic responses 2. Supporting the association between SP and the function of thermal nociceptors is the finding that thermal nociception is im-
paired in familial dysautonomia of humans in which the major spinal site of termination of nociceptors, the substantia gelatinosa, is severely depleted of SP 15. In the rat, however, depleting t h e s u b s t a n t i a gelatinosa of substance P by peripheral lesions of the sciatic nerve was not associated with obvious changes in the spinal transmission of impulses produced by stimulating unmyelinated fibres in the central end of the cut nerve 17. To resolve this controversy there is a need to demonstrate whether SP is or is not released centrally by impulses in thermal nociceptors. In a recent study in which the dorsal horn of the rabbit spinal cord was perfused with a push-pull cannula, noxious heating of the skin of the periphery was found to release immunoreactive somatostatin but not SP 12. The use of the push-pull cannula, however, is traumatic and cannot localize sites of release with any precision. The release of neuropeptides in the central nervous system can now be detected to within 100 ,urn resolution using the antibody microprobe technique of Duggan and Hendry 4. This technique has already demonstrated a release of SPiR in the substantia gelatinosa of the cat with electi~al stimulation of peripheral nerve 4. Because of its spatial precision and lack of
Correspondence: A.W. Duggan, Department of Pharmacology, John Curtin School of Medical Research, G.P.O. Box 334, Canberra, A.C.T., Australia. 0006-8993/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
346 trauma this method seemed an ideal means of resolving the uncertainties associated with the release of substance P in the spinal cord. Using antibody microprobes, the present experiments have shown that noxious heating of the skin of the anaesthetized cat does result in the release of SPiR in the substantia gelatinosa of the dorsal horn. Experiments were performed on 7 cats anaesthetized with pentobarbitone sodium (35 mg/kg intraperitoneally initially) and maintained with a continuous i.v. infusion of 3 mg/kg/h. Following lumbar laminectomy, the spinal cord was transected at the thoracolumbar junction and covered with a Ringer-agar layer. All animals were artificially ventilated with air under neuromuscular paralysis with gallamine triethiodide (4 mg/kg/h) and end tidal CO 2 levels kept at 4%. SPiR release was measured with antibody microprobes prepared as described previously 4. Briefly, glass micropipettes were immersed in a 10% solution of v-aminopropyltriethoxysilane in toluene. This produced a coating of a siloxane polymer containing free amino groups. Glutaraldehyde (2.5%) was then used to couple, covalently, protein A to the alkylamine glass surface• Antibody to SP (UCB Bioproducts) was then bound by protein A by allowing 24 h incubation at 6 °C using a 1 in 400 dilution of the original serum. As reported previously 4, the antibody used had negligible cross-reactivity with a number of neuropeptides including neurokinin A. Half of the antibody microprobes were used for extracellular recording from neurones and for this, the tips were broken back to 5-10/zm diameter and the probes filled with a 2% solution of Pontamine sky blue in 1.2 M sodium acetate. Dye deposits were used to locate probe tips histologically. Small openings were made in the spinal cord pia for the entry of microprobes and the dorsal surface of the cord was continually irrigated with warm Ringer's solution at 37 °C. Microprobes were inserted into the spinal cord to depths of 1.5 to 3 mm. Since noxious stimuli were subsequently applied to the ipsilateral hind paw it was important to ensure that microprobes were amongst neurones excited by primary afferents innervating the hind paw. Extracellular recordings from neurones encountered during the passage of the dye filled microprobes ensured that this was so. Probes were kept in the spinal cord for
10-30 min with either (1) no peripheral stimulation (2) during immersion of the ipsilateral hind paw in warm water (37 °C) and (3) during immersion of the ipsilateral hind paw in hot water (50 or 52 °C). Extracellular recordings of multi-unit activity were obtained before and during immersion of the limb in hot water (see Fig. 2B). When using hot water, the limb was removed after 10 min of immersion and allowed to cool in room air (23 °C) for 1-2 min before reimmersion. The increases in firing produced by hot water returned to pre-immersion levels with each period of cooling indicating that any possible tissue damage was insufficient to produce sustained firing of nociceptors. After removal from the spinal cord, antibody microprobes were washed and incubated in phosphatebuffered saline containing 1500-2000 cpm BoltonHunter 125I-SP (Amersham) and bovine serum albumin (5 mg/ml) for 24 h. They were then washed and the tips broken off and placed on X-ray film (Kodak • XRP5) for 24-72 h. In vitro assays 4 were performed in parallel with all in vivo experiments. Microprobes consistently detected SP 10 -7 M in vitro, but failed to detect neurokinin A 10 -5 M, the tachykinin which occurs in comparable amounts to SP in mammalian tissues 14. In previous experiments using antibody microprobes, electrical stimulation of unmyelinated primary afferents of the tibial nerve of the cat produced localized bands of inhibition of binding of 125I-SP and the evidence that these bands represent focal release of SP has been discussed previously 4. The X-ray film image of each microprobe was printed photographically with a linear enlargement of X12. Three grades of inhibition of binding were recognized and given a weighting factor. These were: complete inhibition equivalent to >10 -7 SP in vitro (weighting factor, 3), just detectable inhibition equivalent to < 1 0 .7 M SP in vitro (weighting 1) and partial inhibition (weighting 2). The length, in mm, of the zones of inhibition were measured on the x12 prints of microprobe images and these lengths were multiplied by the relevant weighting factor to give an inhibition of binding index. For example in Fig. 2A, probe 2, was determined as having 2.5 mm of complete inhibition at x12 enlargement giving an inhibition index of 7.5. With water at 50 °C for 15 min there was usually no difficulty in assigning inhibition of binding to a spinal
347 WATER 52 ° C n-19
1
A
2
3
4
5
14
WATER 520 C
12
Q 10 Z =
g -F--
(5 z 5 2: n3
8
~n=14 ~ ;I WATER 50oc n=2 ~ WATER n=14
4
t E~z
~ t
CONTROL
WATEF 50 ° £ ~.~': n=7 ~ * WATER n=13
:,
CONTROL_ rlmt
PROBES 1 0 - 1 5 MIN
PROBES 2 0 - 3 0 MIN
IN SPINAL CORD
IN SPrNAL CORD
Fig. 1. Histograms of the effect of noxious heat in causing inhibition of binding of 125I-SP to zones of microprobes corresponding to the substantia gelatinosa of the spinal cord. The inhibition indices were calculated as described the text. The means and S.E.M.s have been graphed for probes which remained either 10-15 or 20-30 min in the spinal cord. There were only 2 probes in the 10-15 min, water at 50 °C, category and hence no standard error is plotted for these.
B
NOXIOUS
100 a Z 0 0 LU m
HEAT
50 ° C TOUCH
50 iii
E
¢O 0-
MiN l a m i n a but with the m o r e intense n o x i o u s t h e r m a l stimuli a p p l i e d for l o n g e r times, a p p a r e n t m a s s i v e release could e x t e n d o v e r several spinal l a m i n a e (see Fig. 2 A , p r o b e 3). In t h e s e cases r e l e a s e was assigned to the l a m i n a o v e r which the z o n e of i n h i b i t i o n of binding was c e n t e r e d . Results w e r e o b t a i n e d f r o m 105 m i c r o p r o b e s . O f these, 33 w e r e controls with the l o w e r limb n o t imm e r s e d in w a t e r , 27 with t h e l o w e r l i m b in w a t e r at 37 °C, 9 with the l i m b in w a t e r at 50 °C and 36 w i t h the limb in w a t e r at 52 °C. Fig. 1 illustrates t h e m e a n inhibition indices calculated for t h e s e p r o b e s for release c e n t e r e d on t h e substantia gelatinosa. It is clear that i m m e r s i o n of the limb in w a r m w a t e r did n o t increase release o v e r that with the limb in r o o m air
Fig. 2. A: inhibition of binding of t25I-SP to antibody microprobes with noxious heating of the skin of the ipsilateral hindlimb. Photographic enlargements of X-ray images of microprobes have been superimposed on a similarly enlarged section of the lumbar spinal cord. The location of a deposit of Pontamine sky blue was the basis for positioning the tips of the microprobes. Microprobe 1 was 30 min in the spinal cord with the ipsilateral hind paw in water at 37 °C. Microprobe 2 was 20 min in the spinal cord with the ipsilateral hind paw in water at 50 °C. Microprobe 3 was 30 min in the spinal cord with the ipsilateral hind paw in water at 52 °C. Microprobe 4 was 30 min in the spinal cord with the limb in air at 23 °C. As the images of microprobes show considerable scattering the actual dimensions of one (5) are shown diagrammatically to the right of the spinal cord. Actual tip sizes were 5-10/~m. Bar = 1 mm. B: ratemeter records of multi-unit firing recorded with microprobe 2. Note the increases produced by touching the skin of the lateral hind limb digits 4 and 5, the large increase produced by handling the limb and placing it in water at 50 °C. and the sustained firing which followed.
(23 °C), but that r e l e a s e was significantly i n c r e a s e d by i m m e r s i o n in w a t e r at 50 °C and f u r t h e r i n c r e a s e d
dices of 0.9 _+ 0.4 S . E . M . (n = 29) for air, 0.4 + 0.2
by w a t e r at 52 °C. E x a m p l e s of t h e s e effects are
S . E . M . (n = 27) for w a r m w a t e r , 0.9 _+ 0.5 S . E . M .
s h o w n in Fig. 2 A in w h i c h m i c r o p r o b e i m a g e s h a v e
(n = 9) for w a t e r at 50 °C and 2.8 _+ 0.7 S . E . M . (n =
b e e n s u p e r i m p o s e d on a spinal c o r d section. R e l e a s e of S P i R c e n t e r e d on l a m i n a V was v e r y
c e n t e r e d on l a m i n a I V ( a l t h o u g h in s o m e cases t h e r e
32) for w a t e r at 52 °C. T h e r e was negligible r e l e a s e
small c o m p a r e d with that c e n t e r e d o n t h e SG. P o o l -
was c o n s i d e r a b l e o v e r f l o w f r o m t h e S G ) and l a m i n a e
ing d a t a f r o m all p r o b e s g a v e i n h i b i t i o n of b i n d i n g in-
VI-VII.
348 A s in previous experiments 4 inhibition of binding was sometimes seen at the site of electrode p e n e t r a tion (for example p r o b e 3, Fig. 2 A ) , and the source of this inhibition is uncertain. It was found however that increasing the rate of irrigation of the surface of the spinal cord abolished this zone of inhibition without affecting that seen within the spinal cord. The methods of the present experiments cannot differentiate b e t w e e n release of SPiR from p r i m a r y afferents and that from neurones of the spinal cord, but since SPiR in dorsal horn is heavily d e p l e t e d by dorsal root section 3'17 release from p r i m a r y afferents is likely to be the m a j o r c o m p o n e n t . The result that SPiR is released by impulses in thermal nociceptors differs from that of Kuraishi et al. 12, in which release of somatostatin but not SP followed radiant heating of the skin of the rabbit. Kuraishi et al. 12 used a pushpull cannula of 600 ~ m d i a m e t e r and the depth in the dorsal horn was not stated. A l t h o u g h it is possible that damage to the substantia gelatinosa could explain the failure to detect SP release, the reason is p r o b a b l y the use of i n a d e q u a t e skin temperatures. It was stated that a subcutaneous t h e r m o c o u p l e showed that t e m p e r a t u r e s during heating rose to a maximum of 48.5 °C and were above 44 °C for 11.5 min of the 20-min p e r i o d of heating. H i g h e r t e m p e r a tures, such as those of the present experiments, m a y have p r o d u c e d a release of SP. Of interest is the finding that somatostatin was released by these lower skin t e m p e r a t u r e s , suggesting a role in the transmis-
1 Buck, S.H., Deshmukh, P.P., Yamamura, H.I. and Burks, T.F., Thermal analgesia and substance P depletion induced by capsaicin in guinea pigs, Neuroscience, 6 (1981) 2217. 2 Cervero, F. and McRitchie, H.A., Neonatal capsaicin and thermal nociception: a paradox, Brain Research, 215 (1981) 414-418. 3 Di Giulio, A.M., Mantegazza, P. and Gorio, A., Peripheral nerve lesions cause simultaneous alterations of substance P and enkephalin levels in the spinal cord, Brain Research, 342 (1985) 405-408. 4 Duggan, A.W. and Hendry, I.A., Laminar localization of the sites of release of immunoreactive substance P in the dorsal horn with antibody-coated microelectrodes, Neurosei. Lett., 68 (1986) 134-140. 5 Gibson, S.J., Polak, J.M., Bloom, S.R. and Wall, P.D., The distribution of nine peptides in rat spinal cord with special emphasis on the substantia gelatinosa and on the area around the central canal (lamina X), J. Comp. Neurol., 201 (1981) 65-74. 6 Hayes, A.G., Skingle, M. and Tyers, M.B., Effects of single doses of capsaicin on nociceptive thresholds in the to-
sion of impulses in thermal receptors. A n indirect a p p r o a c h led Wiesenfeld Hallin 18 to propose that both SP and somatostatin are released by thermal nociceptors. The flexor reflex of decerebrate rats to thermal p e r i p h e r a l stimuli was increased by both SP and somatostatin applied topically to the spinal cord. Reflexes to mechanical stimuli were increased only by SP. A l t h o u g h this reflex may be complex with receptors for the two peptides on m a n y involved neurones, the result is particularly interesting in implying a release of both SP and somatostatin with noxious thermal stimuli. Kuraishi et al. 12 believed their results were consistent with different populations of D R G cells containing either substance P or somatostatin as originally r e p o r t e d for the rat by H6kfelt et al. 9. This appears not to be generally true however, as in the cat it has been found that 83% of D R G neurones which contained somatostatin-iR also contained SPiR a3. A n o t h e r difficulty with the results of Kuraishi et a1.12 is that most nociceptors are polymodal responding to noxious mechanical and thermal stimuli and to associate a p e p t i d e with a particular stimulus implies that the m a j o r nociceptor releases neither SP nor somatostatin. W h e t h e r SP mediates transmission between thermal nociceptors and dorsal horn neurones or has some other role is uncertain. W h a t is clear from the present experiments is that SPiR is released centrally when cutaneous nociceptors are excited by heat.
dent, Neuropharmacology, 20 (1981) 505-511. 7 Hayes, A.G. and Tyers, M.B., Effects of capsaicin on nociceptive heat, pressure and chemical thresholds and on substance P levels in the rat, Brain Research, 189 (1980) 561-564, 8 Henry, J.L., Effects of substance P on functionally identified units in cat spinal cord, Brain Research, 114 (1976) 9-451. 9 H6kfelt, T., Elde, R., Johansson, O., Luft, R., Nilsson, G. and Arimura, A., Immunohistochemical evidence for separate populations of somatostatin-containing and substance P-containing primary afferent neurons in the rat, Neuroscience, 1 (1976) 131-136. 10 H6kfelt, T., Ljundahl, A., Terenius, L., Elde, R. and Nilsson, G., Immunohistochemical analysis of peptide pathways possibly related to pain and analgesia; enkephalin and substance P, Proc. Natl. Acad. Sci. U.S.A., 74 (1977) 3081-3085. 11 Holzer, P., Jurna, I., Gamse, R. and Lembeck, F., Nociceptive threshold after neonatal capsaicin, Eur. J. Pharmacol., 58 (1979) 511-514.
349 12 Kuraishi, Y., Hirota, N., Sato, Y., Hino, Y., Satoh, M. and Takagi, H., Evidence that substance P and somatostatin transmit separate information related to pain in the spinal dorsal horn, Brain Research, 325 (1985) 294-298. 13 Leah, J.D., Cameron, A.A., Kelly, W.L. and Snow, P.J., Coexistence of peptide immunoreactivity in sensory neurons in the cat, Neuroscience, 16 (1985) 683-690. 14 Ogawa, T., Kanazawa, I. and Kimura, S., Regional distribution of substance P, neurokinin a and neurokinin fl in rat spinal cord, nerve roots and dorsal root ganglia and the effects of dorsal root section or spinal transection, Brain Research, 359 (1985) 152-157. 15 Pearson, J., Brandeis, L. and Cuello, A., Depletion of substance P-containing axons in substantia gelatinosa of patients with diminished pain sensitivity, Nature (London),
295 (1982) 61-63. 16 Price, J., An immunohistochemical and quantitative examination of dorsal root ganglion neuronal subpopulations, Neuroscience, 5 (1985) 2051-2059. 17 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. 18 Wiesenfeld-Hallin, Z., Substance P and somatostatin modulate spinal cord excitability via physiologically different sensory pathways, Brain Research, 372 (1986) 172-175. 19 Yaksh, T.L., Farle, D.H., Leeman, S.E. and Jessell, T.M., Intrathecal capsaicin depletes substance P in the rat spinal cord and produces prolonged thermal analgesia, Science, 206 (1979) 481-483.
345
BRE22027
Noxious heating of the skin releases immunoreactive substance P in the substantia gelatinosa of the cat: a study with antibody microprobes A.W. Duggan, C.R. Morton, Z.Q. Zhao and I.A. Hendry Department of Pharmacology, John Curtin School of Medical Research, The Australian National University, Canberra (Australia) (Accepted 30 September 1986) Key words: Substance P release; Noxious heat; Substantia gelatinosa
Using a new method, the antibody microprobe technique, the release of immunoreactive substance P (SPiR) in the dorsal horn in response to noxious heating of the skin, was studied in barbiturate anaesthetized spinal cats. Release of SPiR was not produced by immersing the ipsilateral hind paw in water at 37 °C. With water at 50 and 52 °C, however release was consistently detected in the region of the substantia gelatinosa. These results directly show a central release of SPiR with excitation of nociceptors by heat.
The demonstration of the release of a substance from the axon terminals of neurones is a crucial piece of evidence that it is involved in transmission of information. Such evidence is needed to assign functional significance to the many complexities revealed by immunohistochemical studies of dorsal root ganglion ( D R G ) neurones 5,9'10'13'16. This is particularly so in the case of substance P (SP) and excitation of nociceptors by heat. Early studies associated immunoreactive substance P (SPiR) with small diameter D R G neurones with the suggestion that many of these were nociceptors 1°. The effects of microelectrophoretic ejection of SP near neurones of the dorsal horn of the cat lead to the proposal that this peptide was released from the central terminals of thermal nociceptors s. Depleting D R G neurones of substance P by the administration of capsaicin however, has given discordant results when using thermal tests of nociception. For example using the tail flick test, capsaicin-treated rodents have been variously found to have normal responses 1'6'7, reduced responses 11'19or impaired visceral but normal somatic responses 2. Supporting the association between SP and the function of thermal nociceptors is the finding that thermal nociception is im-
paired in familial dysautonomia of humans in which the major spinal site of termination of nociceptors, the substantia gelatinosa, is severely depleted of SP 15. In the rat, however, depleting t h e s u b s t a n t i a gelatinosa of substance P by peripheral lesions of the sciatic nerve was not associated with obvious changes in the spinal transmission of impulses produced by stimulating unmyelinated fibres in the central end of the cut nerve 17. To resolve this controversy there is a need to demonstrate whether SP is or is not released centrally by impulses in thermal nociceptors. In a recent study in which the dorsal horn of the rabbit spinal cord was perfused with a push-pull cannula, noxious heating of the skin of the periphery was found to release immunoreactive somatostatin but not SP 12. The use of the push-pull cannula, however, is traumatic and cannot localize sites of release with any precision. The release of neuropeptides in the central nervous system can now be detected to within 100 ,urn resolution using the antibody microprobe technique of Duggan and Hendry 4. This technique has already demonstrated a release of SPiR in the substantia gelatinosa of the cat with electi~al stimulation of peripheral nerve 4. Because of its spatial precision and lack of
Correspondence: A.W. Duggan, Department of Pharmacology, John Curtin School of Medical Research, G.P.O. Box 334, Canberra, A.C.T., Australia. 0006-8993/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
346 trauma this method seemed an ideal means of resolving the uncertainties associated with the release of substance P in the spinal cord. Using antibody microprobes, the present experiments have shown that noxious heating of the skin of the anaesthetized cat does result in the release of SPiR in the substantia gelatinosa of the dorsal horn. Experiments were performed on 7 cats anaesthetized with pentobarbitone sodium (35 mg/kg intraperitoneally initially) and maintained with a continuous i.v. infusion of 3 mg/kg/h. Following lumbar laminectomy, the spinal cord was transected at the thoracolumbar junction and covered with a Ringer-agar layer. All animals were artificially ventilated with air under neuromuscular paralysis with gallamine triethiodide (4 mg/kg/h) and end tidal CO 2 levels kept at 4%. SPiR release was measured with antibody microprobes prepared as described previously 4. Briefly, glass micropipettes were immersed in a 10% solution of v-aminopropyltriethoxysilane in toluene. This produced a coating of a siloxane polymer containing free amino groups. Glutaraldehyde (2.5%) was then used to couple, covalently, protein A to the alkylamine glass surface• Antibody to SP (UCB Bioproducts) was then bound by protein A by allowing 24 h incubation at 6 °C using a 1 in 400 dilution of the original serum. As reported previously 4, the antibody used had negligible cross-reactivity with a number of neuropeptides including neurokinin A. Half of the antibody microprobes were used for extracellular recording from neurones and for this, the tips were broken back to 5-10/zm diameter and the probes filled with a 2% solution of Pontamine sky blue in 1.2 M sodium acetate. Dye deposits were used to locate probe tips histologically. Small openings were made in the spinal cord pia for the entry of microprobes and the dorsal surface of the cord was continually irrigated with warm Ringer's solution at 37 °C. Microprobes were inserted into the spinal cord to depths of 1.5 to 3 mm. Since noxious stimuli were subsequently applied to the ipsilateral hind paw it was important to ensure that microprobes were amongst neurones excited by primary afferents innervating the hind paw. Extracellular recordings from neurones encountered during the passage of the dye filled microprobes ensured that this was so. Probes were kept in the spinal cord for
10-30 min with either (1) no peripheral stimulation (2) during immersion of the ipsilateral hind paw in warm water (37 °C) and (3) during immersion of the ipsilateral hind paw in hot water (50 or 52 °C). Extracellular recordings of multi-unit activity were obtained before and during immersion of the limb in hot water (see Fig. 2B). When using hot water, the limb was removed after 10 min of immersion and allowed to cool in room air (23 °C) for 1-2 min before reimmersion. The increases in firing produced by hot water returned to pre-immersion levels with each period of cooling indicating that any possible tissue damage was insufficient to produce sustained firing of nociceptors. After removal from the spinal cord, antibody microprobes were washed and incubated in phosphatebuffered saline containing 1500-2000 cpm BoltonHunter 125I-SP (Amersham) and bovine serum albumin (5 mg/ml) for 24 h. They were then washed and the tips broken off and placed on X-ray film (Kodak • XRP5) for 24-72 h. In vitro assays 4 were performed in parallel with all in vivo experiments. Microprobes consistently detected SP 10 -7 M in vitro, but failed to detect neurokinin A 10 -5 M, the tachykinin which occurs in comparable amounts to SP in mammalian tissues 14. In previous experiments using antibody microprobes, electrical stimulation of unmyelinated primary afferents of the tibial nerve of the cat produced localized bands of inhibition of binding of 125I-SP and the evidence that these bands represent focal release of SP has been discussed previously 4. The X-ray film image of each microprobe was printed photographically with a linear enlargement of X12. Three grades of inhibition of binding were recognized and given a weighting factor. These were: complete inhibition equivalent to >10 -7 SP in vitro (weighting factor, 3), just detectable inhibition equivalent to < 1 0 .7 M SP in vitro (weighting 1) and partial inhibition (weighting 2). The length, in mm, of the zones of inhibition were measured on the x12 prints of microprobe images and these lengths were multiplied by the relevant weighting factor to give an inhibition of binding index. For example in Fig. 2A, probe 2, was determined as having 2.5 mm of complete inhibition at x12 enlargement giving an inhibition index of 7.5. With water at 50 °C for 15 min there was usually no difficulty in assigning inhibition of binding to a spinal
347 WATER 52 ° C n-19
1
A
2
3
4
5
14
WATER 520 C
12
Q 10 Z =
g -F--
(5 z 5 2: n3
8
~n=14 ~ ;I WATER 50oc n=2 ~ WATER n=14
4
t E~z
~ t
CONTROL
WATEF 50 ° £ ~.~': n=7 ~ * WATER n=13
:,
CONTROL_ rlmt
PROBES 1 0 - 1 5 MIN
PROBES 2 0 - 3 0 MIN
IN SPINAL CORD
IN SPrNAL CORD
Fig. 1. Histograms of the effect of noxious heat in causing inhibition of binding of 125I-SP to zones of microprobes corresponding to the substantia gelatinosa of the spinal cord. The inhibition indices were calculated as described the text. The means and S.E.M.s have been graphed for probes which remained either 10-15 or 20-30 min in the spinal cord. There were only 2 probes in the 10-15 min, water at 50 °C, category and hence no standard error is plotted for these.
B
NOXIOUS
100 a Z 0 0 LU m
HEAT
50 ° C TOUCH
50 iii
E
¢O 0-
MiN l a m i n a but with the m o r e intense n o x i o u s t h e r m a l stimuli a p p l i e d for l o n g e r times, a p p a r e n t m a s s i v e release could e x t e n d o v e r several spinal l a m i n a e (see Fig. 2 A , p r o b e 3). In t h e s e cases r e l e a s e was assigned to the l a m i n a o v e r which the z o n e of i n h i b i t i o n of binding was c e n t e r e d . Results w e r e o b t a i n e d f r o m 105 m i c r o p r o b e s . O f these, 33 w e r e controls with the l o w e r limb n o t imm e r s e d in w a t e r , 27 with t h e l o w e r l i m b in w a t e r at 37 °C, 9 with the l i m b in w a t e r at 50 °C and 36 w i t h the limb in w a t e r at 52 °C. Fig. 1 illustrates t h e m e a n inhibition indices calculated for t h e s e p r o b e s for release c e n t e r e d on t h e substantia gelatinosa. It is clear that i m m e r s i o n of the limb in w a r m w a t e r did n o t increase release o v e r that with the limb in r o o m air
Fig. 2. A: inhibition of binding of t25I-SP to antibody microprobes with noxious heating of the skin of the ipsilateral hindlimb. Photographic enlargements of X-ray images of microprobes have been superimposed on a similarly enlarged section of the lumbar spinal cord. The location of a deposit of Pontamine sky blue was the basis for positioning the tips of the microprobes. Microprobe 1 was 30 min in the spinal cord with the ipsilateral hind paw in water at 37 °C. Microprobe 2 was 20 min in the spinal cord with the ipsilateral hind paw in water at 50 °C. Microprobe 3 was 30 min in the spinal cord with the ipsilateral hind paw in water at 52 °C. Microprobe 4 was 30 min in the spinal cord with the limb in air at 23 °C. As the images of microprobes show considerable scattering the actual dimensions of one (5) are shown diagrammatically to the right of the spinal cord. Actual tip sizes were 5-10/~m. Bar = 1 mm. B: ratemeter records of multi-unit firing recorded with microprobe 2. Note the increases produced by touching the skin of the lateral hind limb digits 4 and 5, the large increase produced by handling the limb and placing it in water at 50 °C. and the sustained firing which followed.
(23 °C), but that r e l e a s e was significantly i n c r e a s e d by i m m e r s i o n in w a t e r at 50 °C and f u r t h e r i n c r e a s e d
dices of 0.9 _+ 0.4 S . E . M . (n = 29) for air, 0.4 + 0.2
by w a t e r at 52 °C. E x a m p l e s of t h e s e effects are
S . E . M . (n = 27) for w a r m w a t e r , 0.9 _+ 0.5 S . E . M .
s h o w n in Fig. 2 A in w h i c h m i c r o p r o b e i m a g e s h a v e
(n = 9) for w a t e r at 50 °C and 2.8 _+ 0.7 S . E . M . (n =
b e e n s u p e r i m p o s e d on a spinal c o r d section. R e l e a s e of S P i R c e n t e r e d on l a m i n a V was v e r y
c e n t e r e d on l a m i n a I V ( a l t h o u g h in s o m e cases t h e r e
32) for w a t e r at 52 °C. T h e r e was negligible r e l e a s e
small c o m p a r e d with that c e n t e r e d o n t h e SG. P o o l -
was c o n s i d e r a b l e o v e r f l o w f r o m t h e S G ) and l a m i n a e
ing d a t a f r o m all p r o b e s g a v e i n h i b i t i o n of b i n d i n g in-
VI-VII.
348 A s in previous experiments 4 inhibition of binding was sometimes seen at the site of electrode p e n e t r a tion (for example p r o b e 3, Fig. 2 A ) , and the source of this inhibition is uncertain. It was found however that increasing the rate of irrigation of the surface of the spinal cord abolished this zone of inhibition without affecting that seen within the spinal cord. The methods of the present experiments cannot differentiate b e t w e e n release of SPiR from p r i m a r y afferents and that from neurones of the spinal cord, but since SPiR in dorsal horn is heavily d e p l e t e d by dorsal root section 3'17 release from p r i m a r y afferents is likely to be the m a j o r c o m p o n e n t . The result that SPiR is released by impulses in thermal nociceptors differs from that of Kuraishi et al. 12, in which release of somatostatin but not SP followed radiant heating of the skin of the rabbit. Kuraishi et al. 12 used a pushpull cannula of 600 ~ m d i a m e t e r and the depth in the dorsal horn was not stated. A l t h o u g h it is possible that damage to the substantia gelatinosa could explain the failure to detect SP release, the reason is p r o b a b l y the use of i n a d e q u a t e skin temperatures. It was stated that a subcutaneous t h e r m o c o u p l e showed that t e m p e r a t u r e s during heating rose to a maximum of 48.5 °C and were above 44 °C for 11.5 min of the 20-min p e r i o d of heating. H i g h e r t e m p e r a tures, such as those of the present experiments, m a y have p r o d u c e d a release of SP. Of interest is the finding that somatostatin was released by these lower skin t e m p e r a t u r e s , suggesting a role in the transmis-
1 Buck, S.H., Deshmukh, P.P., Yamamura, H.I. and Burks, T.F., Thermal analgesia and substance P depletion induced by capsaicin in guinea pigs, Neuroscience, 6 (1981) 2217. 2 Cervero, F. and McRitchie, H.A., Neonatal capsaicin and thermal nociception: a paradox, Brain Research, 215 (1981) 414-418. 3 Di Giulio, A.M., Mantegazza, P. and Gorio, A., Peripheral nerve lesions cause simultaneous alterations of substance P and enkephalin levels in the spinal cord, Brain Research, 342 (1985) 405-408. 4 Duggan, A.W. and Hendry, I.A., Laminar localization of the sites of release of immunoreactive substance P in the dorsal horn with antibody-coated microelectrodes, Neurosei. Lett., 68 (1986) 134-140. 5 Gibson, S.J., Polak, J.M., Bloom, S.R. and Wall, P.D., The distribution of nine peptides in rat spinal cord with special emphasis on the substantia gelatinosa and on the area around the central canal (lamina X), J. Comp. Neurol., 201 (1981) 65-74. 6 Hayes, A.G., Skingle, M. and Tyers, M.B., Effects of single doses of capsaicin on nociceptive thresholds in the to-
sion of impulses in thermal receptors. A n indirect a p p r o a c h led Wiesenfeld Hallin 18 to propose that both SP and somatostatin are released by thermal nociceptors. The flexor reflex of decerebrate rats to thermal p e r i p h e r a l stimuli was increased by both SP and somatostatin applied topically to the spinal cord. Reflexes to mechanical stimuli were increased only by SP. A l t h o u g h this reflex may be complex with receptors for the two peptides on m a n y involved neurones, the result is particularly interesting in implying a release of both SP and somatostatin with noxious thermal stimuli. Kuraishi et al. 12 believed their results were consistent with different populations of D R G cells containing either substance P or somatostatin as originally r e p o r t e d for the rat by H6kfelt et al. 9. This appears not to be generally true however, as in the cat it has been found that 83% of D R G neurones which contained somatostatin-iR also contained SPiR a3. A n o t h e r difficulty with the results of Kuraishi et a1.12 is that most nociceptors are polymodal responding to noxious mechanical and thermal stimuli and to associate a p e p t i d e with a particular stimulus implies that the m a j o r nociceptor releases neither SP nor somatostatin. W h e t h e r SP mediates transmission between thermal nociceptors and dorsal horn neurones or has some other role is uncertain. W h a t is clear from the present experiments is that SPiR is released centrally when cutaneous nociceptors are excited by heat.
dent, Neuropharmacology, 20 (1981) 505-511. 7 Hayes, A.G. and Tyers, M.B., Effects of capsaicin on nociceptive heat, pressure and chemical thresholds and on substance P levels in the rat, Brain Research, 189 (1980) 561-564, 8 Henry, J.L., Effects of substance P on functionally identified units in cat spinal cord, Brain Research, 114 (1976) 9-451. 9 H6kfelt, T., Elde, R., Johansson, O., Luft, R., Nilsson, G. and Arimura, A., Immunohistochemical evidence for separate populations of somatostatin-containing and substance P-containing primary afferent neurons in the rat, Neuroscience, 1 (1976) 131-136. 10 H6kfelt, T., Ljundahl, A., Terenius, L., Elde, R. and Nilsson, G., Immunohistochemical analysis of peptide pathways possibly related to pain and analgesia; enkephalin and substance P, Proc. Natl. Acad. Sci. U.S.A., 74 (1977) 3081-3085. 11 Holzer, P., Jurna, I., Gamse, R. and Lembeck, F., Nociceptive threshold after neonatal capsaicin, Eur. J. Pharmacol., 58 (1979) 511-514.
349 12 Kuraishi, Y., Hirota, N., Sato, Y., Hino, Y., Satoh, M. and Takagi, H., Evidence that substance P and somatostatin transmit separate information related to pain in the spinal dorsal horn, Brain Research, 325 (1985) 294-298. 13 Leah, J.D., Cameron, A.A., Kelly, W.L. and Snow, P.J., Coexistence of peptide immunoreactivity in sensory neurons in the cat, Neuroscience, 16 (1985) 683-690. 14 Ogawa, T., Kanazawa, I. and Kimura, S., Regional distribution of substance P, neurokinin a and neurokinin fl in rat spinal cord, nerve roots and dorsal root ganglia and the effects of dorsal root section or spinal transection, Brain Research, 359 (1985) 152-157. 15 Pearson, J., Brandeis, L. and Cuello, A., Depletion of substance P-containing axons in substantia gelatinosa of patients with diminished pain sensitivity, Nature (London),
295 (1982) 61-63. 16 Price, J., An immunohistochemical and quantitative examination of dorsal root ganglion neuronal subpopulations, Neuroscience, 5 (1985) 2051-2059. 17 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. 18 Wiesenfeld-Hallin, Z., Substance P and somatostatin modulate spinal cord excitability via physiologically different sensory pathways, Brain Research, 372 (1986) 172-175. 19 Yaksh, T.L., Farle, D.H., Leeman, S.E. and Jessell, T.M., Intrathecal capsaicin depletes substance P in the rat spinal cord and produces prolonged thermal analgesia, Science, 206 (1979) 481-483.