Alterations in Neurokinin A-, Substance P- and Calcitonin Gene- Related

Neuroscience Letters 261 (1999) 179–182

Alterations in neurokinin A-, substance P- and calcitonin gene- related peptide immunoreactivities in the caudal trigeminal nucleus of the rat following electrical stimulation of the trigeminal ganglion

M. Samsama,b, R. Coven˜asa,*, R. Ahangaria,b, J. Yajeyaa,c, J.A. Narva´ezd, G. Tramue

aInstituto de Neurociencias de Castilla y Leoń, Laboratorio de Neuroanatomıá de los Sistemas Peptide´rgicos, Facultad de Medicina, Dpto. de Biologıá Celular y Patologıá, C/Alfonso X El Sabio, s/n 37007 Salamanca, Spain bDepartamento de Biologıá Celular y Patologıá, Facultad de Medicina, Universidad de Salamanca, Salamanca, Spain cDepartamento de Fisiologıá y Farmacologıá, Facultad de Medicina, Universidad de Salamanca, Salamanca, Spain dDepartamento de Fisiologıá, Facultad de Medicina, Universidad de Ma´laga, Ma´laga, Spain eUniversite´ de Bordeaux I, Laboratoire de Neurocytochimie Fonctionnelle, CNRS, URA 339, Talence, France

Received 30 November 1998; received in revised form 6 January 1999; accepted 6 January 1999

Abstract

We have carried out an immunohistochemical study on the presence of neurokinin A (NKA) and substance P (SP) in the rat caudal trigeminal nucleus (CTN) after electrical stimulation of the trigeminal ganglion (TG), used as an experimental model to induce alterations, some of which may occur during migraine attacks (release of vasoactive peptides from perivascular trigeminal axons and neurogenic inflammation). Both unilateral, 30 min electrical stimulation (5 Hz, 5 ms, 0.1–1 mA) of the TG and 5 min stimulation with a slight increase in the stimulating parameters (7.5 Hz, 5 ms, 1.4 mA) caused a significant depletion of the NKA and SP immunoreactivities (-IR) of the TG nerve central terminals in the ipsilateral CTN. Calcitonin gene-related peptide (CGRP)-IR of the ipsilateral CTN was also studied in the CTN using the increased stimulating parameters and a marked depletion of CGRP-IR was also observed following TG stimulation. Such depletion may be due to the release of neuropeptides from the trigeminal central terminals. These findings suggest that NKA, SP and CGRP could act as neurotransmitters at the first central synapses of the trigeminal nociceptive pathway to transmit the sensory stimuli to the higher brain centers.  1999 Elsevier Science Ireland Ltd. All rights reserved.

Keywords: Neurokinin A; Substance P; Calcitonin gene-related peptide; Migraine; Trigeminal ganglion; Electrical stimulation; Caudal trigeminal nucleus

It is known that the central terminals of the rat trigeminal with neurokinin A (NKA) [4] exist in TG cells and their ganglion (TG) sensory neurons reach the brain stem, mainly ﬁbers. It has also been proved that neuropeptides are the trigeminal sensory nuclei [16], and that the peripheral involved in the pathogenesis of migraine headaches axons of some trigeminal ganglion neurons are responsible [13,19]. In this respect, neurogenic inﬂammation in the for the main innervation of the dura mater [15]. Moreover, dural blood vessels has been suggested as a model to there is evidence that neuropeptides such as substance P mimic vascular head pain [13]. In keeping with this, elec- (SP) and calcitonin gene-related peptide (CGRP) together trical stimulation of the TG promotes the extravasation of albumin from plasma in the postcapillary venules of the dura mater, possibly through the release of neuropeptides * Corresponding author. Tel.: +34-923-294400 ext. 1856; fax: +34- [12]. Moreover, electrical stimulation of the TG increases 923-294549; e-mail: [email protected] CGRP levels in the rat superior sagittal sinus [2]. This is in

0304-3940/99/$ - see front matter  1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S0304-3940(98)00989-6 180 M. Samsam et al. / Neuroscience Letters 261 (1999) 179–182 agreement with the increased levels of CGRP in blood this work showed immunohistochemical cross-reactivities obtained from the jugular vein of human subjects during with neurokinin B, eledoisin, and kassinin. Finally, possible migraine [6]. Consistent with this, thermocoagulation of interference by endogenous peroxidases was ruled out by the TG in human and also electrical stimulation of the TG staining some sections beginning with the diaminobenzi- in cat increased SP and CGRP levels in plasma [5]. Appar- dine step. Immunohistochemical assays were carried out ently electrical stimulation of the TG induces a series of as described previously, the tissue-bound peroxidase was cellular and chemical changes consistent with some findings revealed by using the 3,3′ diaminobenzidine method [11]. observed during migraine attacks. In view of this, we were The central terminals of primary sensory TG cells exhibit prompted to investigate NKA-, SP- and CGRP-IRs of the a strong NKA-, SP- and CGRP-IR and form a curved lami- central trigeminal terminals in the rat caudal trigeminal nar pattern in the rat CTN on both sides close to the dorsal nucleus (CTN) following electrical stimulation of the TG surface of the medulla, as observed by light microscopic to get further insight into the role and action of these neu- examination under normal condition. No differences in the ropeptides in nociception. immunoreactivity of either side were observed in the control Twenty seven young adult Wistar albino rats of both animals. Five minutes electrical stimulation of the TG in sexes (weight 200–250 g) were used in this study. The group 1 animals (reference parameter), did not cause any animals were deeply anesthetized with chloral hydrate apparent change in the NKA- or SP-IR of the CTN of both (4%) and were placed in a stereotactic apparatus. One side sides, but 30 min stimulation of the TG in the second group TG of the animals were electrically stimulated with square induced a marked decrease in neuropeptide-IR in the ipsi- pulses using a concentric bipolar electrode (0.5 mm in dia- lateral (stimulated side) CTN (Fig. 1A,B). Stimulation of meter). The stimulation coordinates used to reach the ganglion were according to Schneider et al. [18]: bregma = zero point, 3.2–3.4 mm posteriorly and 2.8–3.2 mm lateral to the bregma and 9.3 mm depth from the external surface of the dura. The animals were divided into four groups (six animals/groups I, II or IV, and nine animals/group III). Group 1, stimulated according to the following parameters: 5 Hertz (Hz) frequency, 5 ms duration and 0.1–1 milliamps (mA) amplitude (reference parameter) for 5 min. Group 2: stimulated with the same parameters for 30 min. Group 3: stimulated with 7.5 Hz, 5 ms, and 1.4 mA (new parameter) for 5 min. Group 4: used as control animals without any stimulation. Every two rats from groups I, II or IV were investigated for only one neuropeptide (SP or NKA or CGRP) and in group III, every three rats were investigated for either neuropeptide. Immediately following stimulation, the animals were subjected to transcardial flushing with 100 ml cold physiologic saline (NaCl 0.9%) followed by 500 ml cold fixative solution (4% paraformaldehyde), then, medulla was removed and postfixed overnight. Fifty-milli- meter thick frozen sections from medulla (between Obex and the cervical segment 1) were obtained using a cryostat. Sections were treated with a mixture of methanol and H2O2 for 30 min and then transferred into the SP or NKA or Fig. 1. Neuropeptide immunoreactivity (-IR) in the caudal trigeminal nucleus (CTN) of the rat after a unilateral electrical stimulation of the CGRP primary antibody (1/1500, 1/2500, 1/1500 dilution trigeminal ganglion (TG). (A,B) are parts of the same section of the respectively) for 16 h. Polyclonal primary antibodies were medulla; (A) Substance P (SP)-IR in the CTN (arrows) in the normal raised in rabbits against their respective immunogens. These or non-stimulated side (NS). (B) Depletion of the SP-IR (arrows) in were prepared by coupling the antigens (the whole synthetic the CTN of the stimulated (S) side following a unilateral 30 min peptides, SP, CGRP or NKA) to a carrier protein (human electrical stimulation (reference parameters) of the TG. (C,D) are two sides of the same section of the medulla; (C) SP-IR in the serum albumin) with glutaraldehyde. Immunostaining was CTN (arrows) in the non-stimulated (NS) side and (D) shows a sig- controlled by treating each of the three antisera with an nificant depletion of the SP-IR in the CTN of the stimulated (S) side excess (100 mg/ml of diluted antiserum at 1/1500, 1/ after 5 min unilateral stimulation of the TG with the new parameters. 2500) of the homologous antigen, and by omitting the first The arrow in (D) is pointing to a few immunoreactive nerve terminals. antisera in the first incubation bath. In both cases no residual (E,F) Are also parts of the same section of the medulla; using the new parameters, a unilateral 5 min electrical stimulation of the TG immunoreactivity was found. Preabsorption with heterolo- caused a marked disappearance of the CGRP-IR in the CTN gous antigens (e.g. SP or CGRP to antineurokinin A serum) (arrows) of the stimulated (S) side (E) comparing to the non-stimu- did not block specific staining. None of the antisera used in lated (NS) side of the same section (F). Scale bar, 100 mm. M. Samsam et al. / Neuroscience Letters 261 (1999) 179–182 181

Fig. 2. (A) NKA-IR in the CTN of the rat after a 5 min unilateral electrical stimulation of the TG; a marked depletion of the NKA-IR can be observed in the stimulated (S) side comparing to the normal or non-stimulated (NS) side. (B) and (C) are the higher magnification of the CTN in (A) which have been indicated in squares; (B) a few immunoreactive nerve terminals (arrow heads) can be seen in the stimulated (S) side comparing to the non-stimulated (NS) side (C). The darker spots in (C) are due to the massive NKA-IR of the central processes and terminals of the TG primary sensory neurons which terminate in the CTN (arrow heads) in the non-stimulated (NS) side. Such immunoreactivity has been markedly decreased (B) after a 5 min stimulation of the TG with the new parameters. Scale bar, 100 mm. the third group of animals with the new parameter for 5 min but kept the stimulating time constant (5 min). Increased caused a significant decrease in SP, NKA or CGRP-IR of the frequency could produce a more rapid firing of the cell ipsilateral CTN in comparison with the other side which was (greater depletion of neuropeptide from the central term- not stimulated (Figs. 1C–F and 2A). The difference in inals in 5 min). Moreover, increasing the intensity of the immunoreactivity is more evident at higher magnification stimulus would increase the area under stimulation (in this (Fig. 2B,C). case, number of the cells in the TG) and consequently more The depletion of NKA- and SP-IR in the CTN after elec- central terminals might be depleted in the CTN. On the other trical stimulation of the TG might reflect the changes in the hand, a complete depletion of the neuropeptide content of central trigeminal terminals during migraine attack. This the central trigeminal terminals may not occur. This might depletion is due to the release of neuropeptides from trigem- be due to the overlapping of the terminals in the CTN. inal central terminals following activation of TG cells. Although there is a somatotopic presentation of the central However, such depletion was not very clear after 5 min trigeminal terminals in the CTN, terminals at each level of stimulation with the reference parameters, possibly because: CTN may belong to different sensory division and different (1) depletion of the neuropeptide content of the central tri- sensory neurons of the ipsilateral or even the contralateral geminal terminals could occur later than peripheral changes TG [16], which may not have been affected sufficiently and/or (2) the immunohistochemical technique used here enough to release their neuropeptide content. These term- was not sensitive enough to reveal possible changes. Elec- inals are situated beside, over or under each other, produ- trical stimulation of the TG increased the plasma CGRP cing the dense immunoreaction of the CTN; supposing a levels in the superior sagittal sinus within 1–3 min [2]. few dark spots over each other and subtracting one spot This suggests neuropeptide release from peripheral trigem- would not change the overall darkness so much. Thus the inal axons. Such CGRP release is preceded by an increase in terminals which are not depleted effectively (probably far the size of the perivascular nerve terminals following TG from the stimulating electrode), may intensify the immunor- stimulation and their disintegration shortly in the dura mater eactivity of the terminals that have been depleted and also [8]. Release of CGRP from the trigeminal central terminals the immunoreactivity of the whole CTN of that side; this is a following 30 min electrical stimulation of the TG (reference false positive color. Hence masking the real changes in the parameters) has been reported previously [9]. Our results depleted terminals which is more evident at higher light using NKA and SP antibodies are in agreement with those microscopic magnification. Accordingly, more terminals findings (30 min electrical stimulation of the TG). The rea- are depleted from NKA, SP and CGRP in CTN with the son for such depletion is that whether a greater neuropeptide new stimulating parameters following a 5 min electrical content of the central terminals is depleted in a longer (30 stimulation of the TG. Thus, the sensitivity of the experi- min) stimulation time or whether the central changes occur mental model has increased and further studies based on the following a massive release of neuropeptides from the per- effect of antimigraine drugs in such models may be more ipheral endings. This possibility could be due to the fact that sensitive. Release of SP from the spinal cord by electrical the bulk of the SP produced in the sensory ganglion cells is stimulation of the sciatic nerve [20] has been described. being exported towards the terminal regions of their periph- Moreover, SP has been recognized as a transmitter of noci- eral branches [1]. We increased the frequency and intensity ceptive afferents in electrical stimulation studies of C-fibers of the stimulating parameters a little more (new parameter) [7,20] or noxious stimulation of the skin [3,10], which 182 M. Samsam et al. / Neuroscience Letters 261 (1999) 179–182 increased the release of SP from the spinal cord. In agree- [6] Goadsby, P.J., Edvinsson, L. and Ekman, R., Vasoactive pep- ment with these studies, release of SP, NKA and CGRP in tide release in the extracerebral circulation of human during the CTN observed in our study, supports their role as neu- migraine headache, Ann. Neurol., 28 (1990) 183–187. [7] Kellstein, D.E., Price, D.D., Hayes, R.L. and Mayer, D.J., Evi- rotransmitters and/or neuromodulators in the first central dence that substance P selectively modulates C-fiber-evoked synapses of the trigeminal nociceptive pathway. discharges of dorsal horn nociceptive neurons, Brain Res., 526 These neuropeptides may function in vascular head pain (1990) 291–298. in a possible mechanism of migraine proposed by Mosko- [8] Knyiha´r-Csillik, E., Tajti, J., Samsam, M., Sa´ri, G. and Većsei, witz which states that activation of the trigeminovascular L., Electrical stimulation of the Gasserian ganglion induces structural alterations of calcitonin gene-related peptide-immu- system would induce the nociceptive mediators to depolar- noreactive perivascular sensory nerve terminals in the rat cere- ize the sensory perivascular nerve fibers. Such depolariza- bral dura mater: a possible model of migraine headache, tion may induce a local release of vasoactive Neurosci. Lett., 184 (1995) 189–192. neurotransmitters such as SP from peripheral trigeminal [9] Knyiha´r-Csillik, E., Tajti, J., Samsam, M., Sa´ri, G. and Većsei, fibers into the wall of the blood vessels to promote a neuro- L., Effects of short and long-term stimulation of Gasserian ganglion on the CGRP innervation pattern of the cerebral genic inflammation. Depolarization is also accompanied by dura, Cephalalgia, 15 (Suppl. 16 )(1995) 2. antidromic (spread of inflammatory response locally) and [10] Kuraishi, Y., Hirota, N., Sato, Y., Hino, Y., Satoh, M. and orthodromic conduction of the sensory stimulus. Takagi, H., Evidence that substance P and somatostatin trans- Relevant to this idea, release of SP, NKA and CGRP in mit separate information related to pain in the spinal dorsal the CTN would transmit the orthodromic sensory informa- horn, Brain Res., 325 (1985) 294–298. [11] Marcos, P., Covenãs, R., Narvaéz, J.A., Aguirre, J.A., Tramu, tion to the CTN. SP [14] and NKA [17] have been suggested G. and Gonza´lez-Baroń, S., Mapping of enkephalins-, neuro- to function as excitatory transmitters of the primary afferent peptide Y-, calcitonin gene-related peptide-, and gastrin-releas- fibers. Thus their release activate the neuronal network in ing peptide/bombesin-like immunoreactivities in the cat CTN and would register the pain in the higher brain centers. amygdala, Peptides, (1999) in press. The current study supports the involvement of SP, NKA and [12] Markowitz, S., Saito, K. and Moskowitz, M.A., Neurogenically mediated leakage of plasma protein occurs from blood vessels CGRP during activation of the trigeminovascular system. in dura mater but not brain, J. Neurosci., 7 (1987) 4129– 4136. M.S. has been supported by the FUNDACION MAPFRE [13] Moskowitz, M.A., The neurobiology of vascular head pain, Ann. MEDICINA, (Spain). This work has been supported by the Neurol., 16 (1984) 157–168. D.G.I.C.Y.T. (PB 96/1467), Spain. We wish to thank [14] Otsuka, M. and Konishi, S., Release of substance P-like immunoreactivity from isolated spinal cord of new born rat, Nature, Professor M. Merchań for his kind advice. The assistance 264 (1976) 83–84. of Dr. P. Marcos and the technical work of Dr. A. de la [15] Penfield, W. and McNaughton, F., Dural headaches and the Fuente and Mr. Jose´ Antonio Romo are gratefully innervation of the dura mater, Am. Neurol. Ass., 64 (1983) acknowledged. 106–109. [16] Pfaller, K. and Arvidsson, J., Central distribution of trigeminal and upper cervical primary afferents in the rat studied by ante- [1] Brimijoin, S., Lundberg, J.M., Brodin, E., Ho¨kfelt, T. and rograde transport of horseradish peroxidase conjugated to Nilsson, G., Axonal transport of substance P in the vagus and wheat germ agglutinin, J. Comp. Neurol., 268 (1988) 91–108. sciatic nerves of the guinea pig, Brain Res., 191 (1980) 443– [17] Saria, A., Ma, R.C. and Dun, N.J., Neurokinin A depolarizes 457. neurons of the guinea pig inferior mesentric ganglia, Neurosci. [2] Buzzi, M.G., Carter, W.B., Shimizu, T., Heath III, H. and Lett., 60 (1985) 145–150. Moskowitz, M.A., Dihydroergotamine and Sumatriptan attenu- [18] Schneider, J.S., Denaro, F.J., Olazabal, U.E. and Leard, H.O., ate levels of CGRP in plasma in rat superior sagittal sinus dur- Stereotaxic atlas of the trigeminal ganglion in rat, cat, and ing electrical stimulation of the trigeminal ganglion, monkey, Brain Res. Bull., 7 (1981) 93–95. Neuropharmacology, 30 (1991) 1193–1200. [19] Uddman, R., Edvinsson, L., Jansen, I., Stiernholm, P., Jensen, [3] Duggan, A.W., Morton, C.R., Zhao, Z.Q. and Hendry, I.A., Nox- K., Olesen, J. and Sundler, F., Peptide-containing nerve fibers ious heating of the skin releases immunoreactive substance P in human extracranial tissue: a morphological basis for neuro- in the substantia gelatinosa of the cat: a study with antibody peptide involvement in extracranial pain?, Pain, 27 (1986) 391– microprobes, Brain Res., 403 (1987) 345–349. 399. [4] Edvinsson, L., Brodin, E., Jansen, I. and Uddman, R., Neuro- [20] Yaksh, T.L., Jessell, T.M., Gamse, R., Mudge, A.W. and kinin A in cerebral vessels: charactrization, localization and Leeman, S.E., Intrathecal morphine inhibits substance P effects in vitro, Regul. Pept., 20 (1988) 181–197. release from mammalian spinal cord in vivo, Nature, 286 [5] Goadsby, P.J., Edvinsson, L. and Ekman, R., Release of (1980) 155–157. vasoactive peptides in the extracerebral circulation of humans and the cat during activation of the trigeminovascular system, Ann. Neurol., 23 (1988) 193–196.