Neuroscienc~" Letters, 137 (1992) 119 122 (~ 1992 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/92/$ 05.00

119

NSL 08474

A sexually dimorphic population of galanin-like neurons in the rat lumbar spinal cord: functional implications Bruce W. N e w t o n Department (~[ Anatomy, Universi O' of Arkansasji~r Medical Sciences, Little Rock, A R 72205 ([\S'A (Received 15 August 1991; Revised version received 9 December 1991 : Accepted 11 December 1991 )

Key word~: Dorsolateral nucleus: Lumbar spinal cord: Nociception: Parasympathetic nervous system: Sexual reflex: Spinothalamic: Sympathetic nervous system The rat lumbar spinal cord contains a population of galanin- and cholecystokinin-containing neurons which are located dorsolateral to the central canal and project to the thalamus. New data are presented herein which reveal that the number of these neurons, as shown by galanin-like immunostaining, is sexually dimorphic with males containing 62% more of these neurons than females. This is the first demonstration of a sexually dimorphic population of intraspinal neurons which projects to higher CNS centers rather than to peripheral targets.

Galanin is a recently discovered 29 amino acid peptide [22] that has a widespread distribution throughout the central nervous system [10]. This biologically active peptide is thought to play an inhibitory role in the modulation of spinal cord nociceptive reflexes [24]. In 1987 a population of neurons which either co-contained galanin- and cholecystokinin-like immunoreactivities (GAL-LI and CCK-LI), or contained GAL-LI and CCK-LI separately, was described in the female rat lumbar (~ 5) spinal cord [6]. The same study revealed that these G A L / C C K - L I neurons projected to the thalamus. During a recent investigation, our laboratory found that the amount of GAL-LI in fibers found in the lumbosacral spinal cord was sexually dimorphic: males contain more GAL-LI fibers than females [14-16]. Furthermore, GAL-LI fibers could be followed from the caudal aspect of this L~ 5 column of neurons to the medial aspect of the dorsolateral nucleus (DEN) and the spinal nucleus of the bulbocavernosus (SNB) [16]. Both of these motor neuron pools are found in the L5.6 spinal cord segments [5], and innervate perineal muscles which carry out reflex penile movements [19]. Together, these data suggested that the lumbar column of G A L / C C K - L I neurons may be one of several CNS regions involved in controlling sexual reflexes. Therefore, since the DLN and SNB are sexually dimorphic [5], and the lumbosacral GAL-L! fibers are sexually dimorphic [14-16], this labC'orre~v)ondence: B.W. Newton, Department of Anatomy, Slot 510. University of Arkansas for Medical Sciences, 4301 W. Markham St., Little Rock. A R 72205, USA. Fax: (1) (501) 686-6382.

oratoy sought to find if GAL-LI neurons in lumbar laminae VII and X are also sexually dimorphic. Six adult (60 120 days of age) Sprague Dawley rats (SASCO, Inc., Omaha, NE) of each sex were deeply anesthetized with sodium pentobarbital (65 mg/kg, i.p.) and perfused for the free-floating indirect antibody peroxidase-antiperoxidase (PAP) technique of Sternberger as previously described [15]. Colchicine was n o t used to enhance the numbers of GAL-LI neurons. Following perfusion, the lumbosacral region of the spinal cord was sectioned in the horizontal plane at 40/~m, with the aid of a Vibratome, and the tissue was exposed overnight to a galanin specific antiserum (RAS 7153N, lot 008504-10: Peninsula Laboratories, Inc., Belmont, CA) diluted to 1:10,000. Sections from the male and female lumbosacral spinal cords were processed together through the PAP technique in order to help eliminate variation in immunostaining between reaction vessels which may confound the interpretation of sexual dimorphism. With the aid of a camera lucida, the sections were projected onto drawing paper and the number of laminae VII and X, Ll 5, GAL-LI neurons was noted. Only neurons which presented a full perikaryon that contained a non-GAL-LI nucleus were counted. Since these neurons are small, and the sections are 40 ~m thick, a correction factor was not used to compensate for 'double-counting' GAL-LI neurons in the initial statistical evaluation of these data. However, the post hoc use of a correction factor [21] did not alter the statistical outcome of the data. The statistical determination of the presence of sexual dimorphism in the number of laminae

120 VII and X, L~ s, G A L - L I neurons was verified using a Wilcoxon two-sample test. Fig. 1 shows the location of the G A L - L I neurons (arrowheads) dorsal to the central canal in spinal segments L3.4. Note that the male (Fig, la) spinal cord contains a greater number of both G A L - L I neurons and fibers than the female (Fig. lb) spinal cord. This column of G A L - L I neurons extends from spinal segments L~ s, with the position of the neurons slowly changing such that they are predominately dorsal to the central canal in rostral lumbar segments and become progressively more lateral and slightly ventral to the central canal in caudal lumbar segments (see ref. 6 for details). An estimation of the number of G A L - L I neurons in laminae VI1 and X of the L~ .~ spinal segments reveals that males contain significantly more (P=0,01) G A L - L I neurons than females (averages = 612 G A L - L I neurons/male vs. 382 G A L - L I neurons/female). (Although not yet examined, this may also imply that C C K - L I neurons in these spinal segments may be sexually dimorphic.) Previous studies [14-16] indicated that the number of G A L - L I fibers in females could be arbitrarily divided into two classes: an "average' amount of G A L - L I fibers and a 'heavy' amount of G A L LI fibers. These new data indicate that there is no statisti-

cal difference in the number of lumbar G A L - L I neurons in either 'class' of female (372 G A L - L I neurons/average female vs. 394 G A L - L I neurons/heavy temale). These data reveal that the L~ 5 column of G A L - L I neurons is sexually dimorphic with males having 62% more of these neurons than females. This is the second population of intraspinal neurons, which are not motor [5] or sympathetic [11] in nature, that has been shown to be sexually dimorphic [17]: and it is the first demonstration of sexual dimorphism for spinal neurons which have been clearly shown to project to higher CNS regions [6], Although this is a unique finding, what is also interesting is that these G A L / C C K - L I neurons are anatomically positioned to act as a spinal integrative center for sexual reflexes in male and female rats [1, 8]. These reflexes involve the perineal muscles in males and females, which are innervated by the SNB and D L N , and the female lordotic response. The hypothesis that this L~ 5 column of spinothalamicprojecting neurons is a spinal integrative center for sexual reflexes is supported by 5 findings: (1) these L~ neurons not only project to higher CNS centers, such as the thalamus, hypothalamus, and telencephalon [2, 6, 12]; (2) they also receive efferents from CNS regions

Fig. 1. a and b show GAL-L1 neurons and fibers in the L3, a segments of the male (a) and female (b) spinal cord. These GAL-LI neurons (arrowheads) lie dorsolateral to the central canal and are 62% more numerous in males than females. Note that the male also contains more GAL-LI fibers than the female. Arrowhead in upper right corner of (B) indicates rostral. Bar --- 100 tim.

121 which are either targets for gonadal steroids and/or are sexual reflex control centers [2, 7, 8, 20, 23]. (3) These G A L / C C K - L I neurons, as well as the whole lumbosacral spinal cord, receive a large number of afferents of dorsal root ganglion origin which carry information from the genitofemoral, pudendal, hypogastric and pelvic nerves. These nerves have been shown to innervate the pelvic viscera and somatic and smooth musculature involved with sexual reflexes and functions [3, 9, 11, 18]. (4) The rostral aspect of this population of G A L / C C K - L I neurons overlaps the position of the El, 2 preganglionic sympathetic neurons [4]: while the G A L - L I fibers which extend caudally from these neurons project through the dendritic fields of the L:SL preganglionic parasympathetic neurons. (5) G A L - L I fibers which arise from these G A L / C C K - L I neurons project to the medial portion of the DLN. and possibly to the SNB, [16]: two nuclei which innervate perineal muscles involved in sexual reflexes [5, 19]. If these findings are considered together, the evidence suggests that G A L / C C K - L I neurons are in a perfect anatomical position to integrate descending sexual reflex information, from the brain and brainstem [7], with visceral and somatic afferent information, of dorsal root ganglion origin, concerning sexual and pelvic functions. In addition, input from the preganglionic sympathetic and parasympathetic nervous system can be added to the afferent in|\~rmation impinging upon this L~ 5 column of neurons. After integrating inputs from the higher CNS, dorsal root ganglion afferents, and the autonomic nervous system, the processed information can either be relayed back to the higher CNS, for further conscious or unconscious interpretation and processing, or it can be relayed to spinal preganglionic autonomic neurons and the D L N and SNB for peripheral distribution. This latter pathway could coordinate all of the incoming afferent sexual/pelvic information in order to orchestrate the appropriate timing of the opening and closing of involuntary sphincters with the clonic contractions of somatic and smooth musculature. Coordination of these activities is needed for appropriate emission, ejaculation, and orgasmic responses. In female rats, the LL 5 column of G A L / C C K - L I neurons may nol only integrate information concerning sexual reflexes involving the perineal musculature [1] and the lordotic response [8], they may also be involved in parturition. In this regard, galanin may be involved in reducing the pain of parturition (see ref. 18) by antagonizing the excitatory effect of other nociceptive neuropeptides such as calcitonin gene-related peptide [24]. Previous work by others has shown that lamina X receives nociceptive input [13] and that decreases in pain perception accompany sexual arousal (see ref. 7).

In conclusion, this newly described sexually dimorphic population of L~ 5 G A L / C C K - L I neurons in rats is anatomically positioned to integrate autonomic and somatic reflex mechanisms. By acting as a spinal relay and integrative center, these G A L / C C K - L I neurons can coordinate somatic and visceral sensory afferents with autonomic and higher CNS inputs which are involved with sexual reflexes. In addition, as suggested by Ju et al. [6], the position of these G A L / C C K - L I neurons in lamina X [12], and the fact that both G A L and C C K are involved in nociception, infer that they may be involved in modulating pain input to the spinal cord before sending it to higher CNS centers. Furthermore, the presence of this population of L~ 5 neurons provides a sexually dimorphic connection between sexually dimorphic brain regions and sexually dimorphic spinal motoneuron pools. Therefore, sexual dimorphism extends, in a continuous fashion, from discrete centers in the brain, to the spinal cord, and to the perineal musculature. As a sexually dimorphic link, these L~ , G A L / C C K - L I neurons may be unique, since other populations of spinothalamic neurons [2, 12] may not differ between the sexes. An abstact of this material was presented at the 21st meeting of the Society for Neuroscience, New Orleans, LA, USA, I wish to thank Teresa Wilson for her expert technical assistance, Dr. Cheryl Puskarich tk~r the statistical interpretation of the data, and Dr. Patrick Tank for critical comments concerning the manuscript. This research was supported by BRSG RR05350 and NSF Grant RII-8922108. The Olympus BH-2 microscope was purchased via an Arkansas Caduceus Club Junior Faculty Research Award. I Chung. S.K., McVary, K.T. and McKenna, K.E., Sexual reflexes in male and l'emalerats, Neurosci. Lett., 94 (1988) 34,:; 348. 2 Cliffer, K.D.. Burstein, R. and Oieslcr Jr.. G..I., Distributions of spinothalamic, spinohypothalamic, and spinotelencephalic libers revealed by anterograde transport of PHA-[, ill rats, J. Ncurosci., II (1991)852 868. 3 de Groat, W.C., Spinal cord projections and ncuropeptides in visceral aflerent neurons, Prog. Brain Rcs., 67 (1986) 165 187. 4 Hancock, M.B. and Peveto, C.A., A preganglhmic autonomic nucleus in the dorsal gray commissure o( the hlmbar spinal cord of the rat. ]. Comp. Neurol., 183 (1979) 65 72. 5 Jordan. C.L., Beedlove,S.M. and Arnold, A.P., Sexual dimorphism in the dorsolateral motor nucleus of the rat lumbar spinal cord and its response to neonatal androgen. Brain Res.,249(l~)82)309 314. 6 Ju, G., Melander, T., Ceccatelli, S., HOkfelt.T. and t:rey, P., hnmunohistochemical evidence for a spinolhalamic pathway co-containing cholecystokinin-and galanin-like immunoreactivitiesin the rat, Neuroscience, 20 (1987) 439 456. 7 Marson, L. and McKenna, K.E., The identitication of a bramstem site controlling spinal sexual reflexes in male rats, Brain Res., 515 (1990) 303 308,

122 8 McEwen, B.S., Davis, EG., Parsons, B. and PIb_ILD.W., The brain as a target for steriod hormone action, Annu. Rev. Neurosci., 2 (1979) 65 112. 9 McKenna, K.E. and Nadelhaft. I., The o,ganization of the pudendal nerve in the male and female rat, J. Comp. Neurol., 248 (1986) 532 549. 10 Melander, T., H6kfelt, T. and ROkaeus, ,&., Distribution of galanin-like immunoreactivity in the rat central nervous system. J. Comp. Neurol., 248 (1986) 475-517. 11 Nadelhaft, I. and McKenna, K.E., Sexual dimorphism in sympathetic preganglionic neurons of the rat hypogastric nerve, J. Comp. Neurol., 256 (1987) 308-315. 12 Nahin, R.L., lmmunocytochemical identification of long ascending, peptidergic lumbar spinal neurons terminating in either the medial or lateral thalamus in the rat, Brain Res., 443 (1988) 345 349. 13 Nahin, R.L., Madsen, A.M. and Giesler Jr., G.J., Anatomical and physiological studies of the gray matter surrounding the spinal cord central canal, J. Comp. Neurol., 220 (1983) 321 335. 14 Newton, B.W., Galanin immunoreactivity is sexually dimorphic in autonomic regions of the rat lumbosacral spinal cord, Soc. Neurosci. Abstr., 17 (1991) 1098. 15 Newton, B.W., The distribution of galanin-like immunoreactivity in rat spinal laminae VII and X: sexual dimorphism in lumbosacral autonomic regions, Brain Res., in press. 16 Newton, B.W., The distribution ofgalanin-like immunoreactivity in lamina IX of the rat spinal cord. In: Conference on Development and Plasticity of the Spinal Cord. The Ohio State University. Columbus, OH July 18 20, 1990.

17 Newton, B.W., Peptidergic innervation of the cremaster nucleus,. I A sexually dimorphic population of substance P-containing inlraspinal neurons exists in the substance P palhway to the rat cremaster nucleus, Brain Res,. 537 (199(t} 187 196. 18 Peters, L.C.. Kristal, M,B. and Komisaruk. BR,, Sensory mnervation of the external and internal genitalia of the female rat, Brain Res., 408 (1987} 199 2(}4. 19 Sachs, B.D.. Role of lhe striated penile muscles m penile reflexes, copulation, and induction of pregnancy in thc rat. J. Reprod. t::crt., 66 (1982) 433 443. 20 Shen, P.. Arnold, A.R and Micevych, RE., Supraspinal projections to the ventromedial lumbar spinal cord in adult male rats, J. Comp. Neurol.. 300 (1990) 263 272. 21 Smolen. A.J., Wright, L.J. and Cunningham, T.J., Neuron numbers in the superior cervical sympathetic ganglion of the rat: a critical comparison of methods tbr cell counting, .1, Neurocytol., 12 (1983 739 750, 22 Tatemoto, K,, ROkaeus, A., .lornvall, H.. McDonald, T.J. and Mutt, V.. Galanin a novel biologically active peptide from porcine intestine, FEBS Lett.. 164 (1983} 124 128, 23 Wagner, CK. and Clemens, L.G.. Projections of the paraventricular nucleus of the hypothalamus to the sexually dimorphic tumbosacral region of the spinal cord, Brain Res., 539 ( 1991 ) 254 262. 24 Xu, X,-J., Wiesenfeld-Hallin, Z., Vitlar, M.J.. Fahrenkrug, J. and H6kfelt, T., On the role of galanin, substance P and other neuropeptides in primary sensory neurons of the rat: studies on spinal reflex excitability and peripheral axotomy, Eur. J. Neurosci.. 2 (19901 733 743.