Photoperiodic regulation of substance P immunoreactivity in the mating behavior pathway of the male golden hamster

Brain Research, 590 (1992) 29-38

29

© 1992 Elsevier Science Publishers B.V. All rights reserved 0006-8993/92/$05.00

BRES 18016

Photoperiodic regulation of substance P immunoreactivity in the mating behavior pathway of the male golden hamster J e n n i f e r Marie S w a n n a n d Nick M a c c h i o n e Department of Biological Sciences, Rutgers State U~irersity, Newark, NJ 07102 (USA) (Accepted 31 March 1992)

Key words: Medial nucleus of the amygdala; Bed nucleus of the stria terminalis; Medial preoptic area; Neuropeptide; Testosterone; Seasonal

Mating behavior in the male golden hamster is regulated by both gonadal steroids and photoperiod. Gonadal steroids may regulate mating behavior by actions on the medial nucleus of the amygdala, bed nucleus of the stria terminalis, and medial preoptic area. Neurons in these areas actively accumulate gonadal steroids and lesions of these nuclei disrupt mating behavior in male hamsters. Photoperiodic regulation of mating behavior is regulated, at least in part, by decreased responsiveness to gonadal steroids. Therefore, we sought to determine if the changes induced by changes in gonadal steroids would mimic those induced by changes in photoperiod. The number of substance P-containing neurons in these areas decrease following castration and are restored with testosterone treatment suggesting that this peptide may mediate steroidal regulation of male mating behavior. To determine the effect of photoperiod on substance P, peptide containing neurons were counted in (1) enucleates (a -- 6), (2) ¢nudeated castrates treated with testosterone (n = 6), (3) castrates treated with testosterone (n -- 4), and (4) intact controls (n = 6). Bilateral enucleation caused a decrease in the number of substance P neurons in the medial nucleus, bed nucleus of the stria terminalis, and medial preoptic area (P < 0.05), Testosterone treatment prevented this decrease (P < 0.05). Thus, a decrease in daylength causes a decrease in substance P in the medial nucleus of the amygdala, the medial bed nucleus of the stria terminalis :rod the medial preoptic area that is mediated by changes in testosterone levels.

INTRODUCTION In the male golden hamster, as in other mammals, copulatory behavior is regulated by both internal and external factors. The most significant internal factor is the level of circulating hormones, primarily androgens. Important external factors include those related to the proximity of the female and the time of year. Specifically, reproductive behavior in the male hamster is stimulated by pheromones secreted by the female ~7 and testosterone 3 and inhibited by decreasing daylength t. Pheromones secreted by the female stimulate receptors in the vomeronasal organ and olfactory mucosa of the male 17'26 which project to the accessory and main olfactory bulbs, respectively 49. Neurons in both areas project directly and indirectly to the medial nucleus of the amygdala 43, which in turn projects to the bed nucleus of the stria terminalis, and the medial preoptic

area 2°. This chemosensory system plays a crucial role in the regulation of copulatory behavior in the male hamster. Destruction of input from both vomeronasal organ and mucosa s') or ablation of the main and accessot), olfactory bulbs 2"s'at'eliminates male mating behavior. Similarly, bilateral lesions of the medial nucleus of the amygdala, the bed nucleus of the stria terminalis or the medial preoptic area abolish or severely disrupt normal copulation in the male hamster 2x38. The medial nucleus of the amygdala, the bed nucleus of the stria terminalis and the medial preoptic area may serve as sites for hormonal regulation of copulatory activity. Neurons in these areas concentrate gonadal steroids in a number of mammalian species 44 including the hamster '~'35. Androgen pellets implanted in these sites maintain or restore copulation in castrated hamsters 24. Similarly, a pellet of dihydrotestosterone implanted in the medial nucleus of the amygdala of castrated, estrogen-primed male rats facilitates

Correspondence: J.M. Swann, Department of Biological Sciences, 135 Smith Hall, Rutgers Univ°rsity, Newark, NJ 07102, USA. Fax (1) (2{)1) 648-1007.

30 mating:. Castration causes an increase in the refractory periods of medial nucleus of the amygdala neuons that project to the medial preoptic area via the stria terminalis ts. This effect is reversed by testosterone treatment ~'~ indicating that neurons in these areas respond to changes in circulating gonadal steroids. Taken together this information suggests that the medial nucleus of the amygdala, medial preoptic area, and bed nuc!eus of the stria terminalis may integrate hormonal and chemosensory information to regulate male mating behavior. The length of day also plays an important role in the regulation of male hamster copulatory behavior. Following prolonged exposure to short days, i.e., less than 12 h of light per day, adult, male hamsters show a gradual decline in copulatory behavior t''~'~. Six to 10 weeks of exposure to short days eliminates ejaculations, intromissions, mounts and anogenital investigation in this species 7''~°'-~'~'~'~.The decline in reproductive behavior is preceded by a decline in reproductive hormones. Exposure to an inhibitory photoperiod causes a reduction in testicular function resulting in a decrease in testosterone 4. Exposure to an inhibitory photoperiod also causes an increase in the negative feedback effects of testosterone on gonadotropin release such that these low levels of testosterone completely suppress gonadotropin secretion s:,s.~. The low levels of testosterone induced by exposure to the inhibitory photoperiod is only partially responsiblt.' for the decrease in the copulatory behavior. Castrated males maintained on short days take longer to display normal mating behav. ior when treated with testosterone than castrates maintained on long days 7,'~". Thus, exposure to short days appears to decrease the sensitivity of the neural substrates to the stimulatory effects of testosterone on copulatory behavior. The pathway which mediates photoperiodic regulation of gonadotropin release has been well documented. Light stimulates photoreceptors in the retina which project to the suprachiasmatic nucleus ~7. This nucleus is the master circadian oscillator in vertebrates which, among other things, regulates the timing and duration of the nocturnal release of melatonin from the pineal dand -~". The timing and/or duration of melatonin release appears to serve as a measure of daylength in golden hamsters 1.4". Melatonin receptors have been located in the median eminence and the pituitary - areas that directly regulate gonadotropin release sT. Thus, melatonin may regulate hormonal responses to photoperiod via direct actions on these structures. The pathway by which daylength regulates male reproductive behavior has not been thoroughly investi-

gated. Destruction of the suprachiasmatic nuclei 4~ or the pineal gland 3~ prevents the loss of mating behavior that follows exposure to an inhibitory photoperiod. Pinealectomy destroys the normal timed release of melatonin and may influence behavior by preventing its actions on the suprachiasmatic nuclei or efferents from these nuclei. The suprachiasmatic nucleus is also densely invested with melatonin receptors 57 and projects to the bed nucleus of the stria terminalis and the medial preoptic area 37'56. As the medial preoptic area, and bed nucleus of the stria terminalis appear to mediate steroidal effects on copulation in the male hamster, we reasoned that these areas may also mediate photoperiodic effects on male hamster sexual behavior. The neurotransmitters that mediate steroidal regulation of mating behavior have not been identified. There is mounting evidence that substance P plays a role. Substance P appears to serve as a neuromodulator in several CNS regions 42. High concentrations of the peptide, its receptor and immunoreactive neurons have been described in the medial nucleus of the amygdala, medial preoptic area, and bed nucleus of the stria terminalis ~'45. lontophoretic application of substance P increases the firing rate of neurons in the medial nucleus :2 and medial preoptic area 27. Injections of substance P into the medial preoptic area facilitate mating behavior in the male rat t". in addition, substance P levels are regulated by gonadal steroids in the rat s':'~'~4 and the hamster :t'4N. These results suggest that substance P may mediate steroidal effects on copulation in the male hamster as well as the male rat. The purpose of the present study was to determine if: (l) enueleation which mimics a short photoperiod It' would decrease substance P immunoreaetivity in the bed nucleus of the stria terminalis, medial preoptic area, and medial nucleus of the amygdala and if (2) the effects of enuclcation on substance P levels are mediated by testosterone. MATERIALS AND METHODS Animals

Adult, male, golden hamsters (Mesocricetus auratus) w e r e obtained from Charles River Laboratories of New Jersey. All hamsters were pro- and postoperativelygroup housed on a tong day photoperiod (14 h of light-10 h of darkness). All surgical procedures were perft;rmed on anesthetized animals(0.9 mg sodium pentobarbital per 100 g b.wt., i.p,), The hamsters were divided into 4 groups: intacts (n = 6), enucleates (n = 6), testosterone-treated castrates (n = 4), and enucleated, testosterone-treated castrates (n = 6). Three groups of hamsters underwent surgery at day 0 of the experiment. Hamsters in the testosterone-treated castrate group were anesthetized, bilaterally castrated via scrotal incision, and implanted subcutaneously with one 20-mm,

testosterone-filled, silastic capsule. Animals in the enucleated group were anesthetized and both eyes were retracted, the optic nerves ligated, and the eyes removed. Hamsters in the enucleated, testes-

31 terone-treated castrate group were subjected to castration followed by enucleation 3 days later. Intact animals served as controls and did not undergo surgery. The width of the left testis of each enucleated and control animal were measured every two weeks after day 0 of the experiment (Fig. 2). In addition paired testes' weights were obtained at the time of sacrifice. This experiment was conducted at two different times. In the initial experiment surgery was conducted in March of 1988 and the animals were sacrificed 7 weeks later, in the replicate experiment, surgery was conducted in August of 1988 and animals were sacrificed 10 weeks after surgery. Animals from all 4 groups were included in both parts of the experiment.

Tissue preparation Forty-eight h prior to perfusion all hamsters were anesthetized and injected with colchicine (320 p,g/2 p,I) in either the right or left cerebral, lateral ventricle. Colchicine treatment prevents microtubule formation and, thereby, stops axonal transport of st, bstance P. in the absence of colchicine treatment substance P cell bodies are not readily visible. As the duration between colchicine treatment and perfusion was similar for all animals the subsequent accumulation of peptide should normalize any circadian differences in substance P content. Each hamster was sacrificed with an overdose of anesthesia (20 mg sodium pentobarbital per 100 g b.wt., i.p.) and perfused transcardially with 0.1% sodium nitrite in phosphate buffered saline (PBS; 0.1 M KPO 4, PH 7.4) followed by 200 ml of 2% paraformaldehyde with 0.25% benzoquinone, Each brain was removed, postfixed in the same fixative for 6-12 h, and immersed in 211% sucrose in PBS before being cut into 40-#m sections on a freezing microtome. Free-floating sections were stored in 20% PBS with 0.01% sodium azide at 4"C or cryoprotectant 54 at -2I)°C until they were processed for substance P immunoreactivity.

bmmmocytochemistry (ICC) Sections were placed in I% sodium horohydride solution for 30 rain to remove excess aldehydes from the tissue. The sections were incubated in monoclonal antisera to substance P generated in rat (Accurate Scientific) at 4°C. Forty-eight to 72 h later tile sections were washed 3 times (5 rain per wash) in PBS and placed in goat anti-rat IGg (Cappel Lahor,lory) for I h, then washed in PBS and placed in peroxidase anti-peroxidase (PAP; Miles Scie,ltific) for an additional hour at room temperature. Primary and secondary antisera were diluted to 1:500 and 1:50 respectively in 0.1 M PBS co,taining 0.3% Triton X.100 and 0.001% sodium azide, and PAP was diluted to I: 100 in (1.1 M PBS containing 0.3% Triton X-100 without sodium azide. The sections were then placed in 0.125% diaminobenzidine hydrochloride (DAB) containing 0.03% hydrogen peroxide, and 0.0125% nickel chloride in PBS for exactly 15 rain. The sections were washed in PBS to remove excess DAB. The sections were later mounted on gelatin-coated slides, allowed to dry, dehydrated in ascending alcohol concentrations, cleared in xylenes, and coverslipped with Permount. Every third section was reacted for immunocytochemistry. Adjacent sections were mounted, stained with Cresyl violet, dehydrated, and coverslipped to use as reference.

number of sections counted. Preliminary analysis using Bartlet's test revealed significant differences in the variance between groups t-'. These differences were eliminated when the data were co.verted to square root. Therefore, significant differences were determined for each area by a two factor analysis of variance of the square root of the mean number of SPIR neurons per area for each group. Individual group differences were determined with the use of Duncan's Multiple Range Test i.', RESULTS

Testicular measurements Enucleation caused a gradual decrease in testis width (Fig. !). Interestingly, animals enucleated in March showed testicular involution in 6 weeks whereas the testis of animals enucleated in August did not begin to regress until week 6. At the time of sacrifice each enucleated animal had paired testis weight less than 800 mg. Paired testis weights for each sighted animal was greater than 2400 mg at the time of sacrifice. SPIR neurons The medial mwleus of the amygdala. The medial nucleus of the amygdala showed the highest number of SPIR of all the nuclei of the amygdala. In intact animals the highest concentration of SPIR neurons were found in the posterior dorsal subdivision of the medial nucleus of the amygdala ~5 (Fig. 2A). The SPIR neurons were 6-10 p,m in diameter and moderately to darkly stained (Fig. 3A). The medial nucleus of the

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Data coll,,ction Substance P immunoreactive (SPIR) neurons were identified by a black to brown DAB reaction product that appeared to be evenly distributed throughout the soma and its processes. Neurons were counted using a hand-held counter. The location of SPIR neurons in the medial nucleus of the amygdala, bed ,ucleus of the stria termi.. nails and medial preoptic area were determined by comparing the DAB sections with adjacent sections stained with Cresyl violet via light microscopy. As a control, SPIR neurons were also counted in the suprachiasmatic nuclei of all animals. Neurons in the suprachiasmatic nuclei do not contain steroidal receptors'J and therefore, should not respond to hormonal manipulations. To compensate for individual differences in the numbers of sections the total number of SPIR neurons were divided by the

Time (weeks) Fig. I. Mean testis widths for intact and enucleated hamsters that were enucleated in March of 1988 (A) or August of 1988 (B) and sacrificed after the last measurement shown. Note that the hamsters enucleated in March showed faster testicular regression than those enucleated in August but that testis widths in both groups of enucleated hamsters were less than 8 mm at the time of sacrifice.

32 castrates (n = 5) displayed 123.7 + 66.5 neurons per section; twice as many as testosterone-treated sighted animals or intacts. However, this difference did not achieve significance (Figs. 3D and 4). The bed nucleus of the stria terminalis. Subdivisions for the BNST were identified using the cytoarchitecture of Moga and coworkers 3-'. In intact animals SPIR neurons were distributed mainly throughout the posteromedial subdivision of the bed nucleus of the stria terminalis (BNSTpm; Fig. 2B). These neurons were 6 - 1 0 / z m in diameter. The highest concentrations of SPIR neurons were seen in the dorsal part of this region (Fig. 5A). A few SPIR neurons were also found in the posterointermediate division of the bed nucleus of the stria terminalis. The BNSTpm of intact animals (n = 6) contained an average of 95.6 + 28.9 SPIR neurons per section (Fig. 4). Approximately 75% of these neurons were darkly stained.

amygdala of intact animals (~ = 4) contained :m average of 68.1 + 10.59 (~+S.E.M.) SPIR neurons per section (Fig. 4). Bilateral enucleation caused a 3-fold reduction in the mean number of neurons displaying SPIR. This group of animals (n = 6) contained an average of 17.4 + 14.6 SPIR neurons per section (Fig. 3B) which was significantly fewer neurons than intact, testosteronetreated castrates or enucleated, testosterone-treated castrates ( P < 0.05; Fig. 4). In addition, the SPIR neurons that were present displayed lighter staining than that of the other 3 groups suggesting a decrease in substance P co,tent. The medial nucleus of the amygdala of sighted, testosterone-treated castrates showed levels of SPIR neurons similar to that of intacts (Fig. 3C). Sighted, testosterone-treated animals (n = 3) containcd an average of 60.8 + 3~.3 substance P neurons per section (Fig. 4). Enucleated, testosterone-treated

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Fig. 2. Schematic representation of substance P distrihution in the medial nucleus of the amygdala (A), bed nucleus of the stria terminalis (B), medial pret~ptic area ~E'), and the suprachiasmatic nucleus (D). Each dot represents 5-10 neurons in A, B, C, and 10-15 neurons in D. Ahhreviation~: AAA. anterior amygdaloid area', BLa, I~asolateral amygdaloid area; BM, basomedial nucleus of the amygdala; BNST(pi), posterointermediate bed nucleus ,~tria tcrminalis', BNST(pm), posteromedial bed nucleus stria terminalis; Ce, central amygdaloid nucleus; CP, caudate-putamen; Endo, endopirifl~rm nucleus; F, fornix; GP, globus pallidus; Iil, third ventricle; L, lateral nucleus of the amygdala; MePD, medial nucleus of the amyddala, posterodorsal; MI, intercalated mass cell group; MPN(maB), medial preoptic nucleus, magnoeeilular part; MPOA, medial preoptic area; NAOT, nucleus of the accessory olfactory tract; NLOT, nucleus of the lateral olfactory tract; PC, optic chiasm; OT, optic tract: PVN, paraventricular nucleus of the hypothalamus; PMCo, posteromedial cortical amygdaloid nucleus: POC, primary olfactory ct~rtcx; S('N, suprachiasnlatic nuclei; SM, stria medullaris; ST, stria terminalis; VMH, ventromedial hypothalamic nucleus. Subdivisions of the medial nucleus are descril~cd in Gomez, el al. t~. BNST subdivisions are those of Moga el al.32; MPOA subdivisions are those of Maragos et al.28.

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Fig. 3. Photographs of coronal sections through the posterodorsal part of the medial nucleus of the amygdala at the level indicated in Fig. 2A. Photographs are of representative sections from an intact hamster (A), an enucleate (B), a castrate treated wilh testosterone (C), and an enucleated castrate treated with testosterone (D) thai were immunohistochemically labelled for substance P. Note the abundance of substance P cells and Ilea~ fiber labelling in A, C and D, and the absence of cell bodies and the light fiber labelling in tile enucleated h,mster (B). In these and subsequent photographs the bar = 750 p.m.

As in the case of the medial nucleus, bilateral enucleation reduced the mean number of neurons displaying SPIR in the BNSTpm to a third of that of intacts (Fig. 5B). Animals in this group (n = 6) had an average of 34.3 + 12.8 SPIR neurons per section (Fig. 4), significantly less than that of the testosterone-treated castrates, enucleated, testosterone-treated castrates or intacts (P < 0.01). The SPIR neurons that were present in enucleated animals were lighter stained than those of the other 3 groups. The mean number of SPIR neurons in the BNSTpm of sighted, testosteronetreated castrates (n = 4) was 90.5 :t: 12.3 per section, comparable to that of intacts (Figs. 4, 5C). As in the medial nucleus of the amygdala treatment with testosterone doubled the levels of SPIR neurons in the BNSTpm (Fig. 5D) although this difference did not reach significance. Animals in the enucleated, testosterone-treated castrate group contained an average of

161.1 + 29.9 SPIR neurons p~r section through this region (Fig. 4). The medial preoptic area. Subdivisions of the medial preoptic area were identified using the criteria of Maragos and coworkers 2~. All 4 groups of animals displayed substance P neurons throughout the medial preoptic area (Fig. 2C). Two different types of immunoreactive neurons were seen. Lightly stained neurons were located in medial and ventral regions of this area (Fig. 6A). Darkly stained, large neurons (8-12 ~m in diameter) were located in the dorsal and lateral part of the medial preoptic area. The average number of SPIR neurons in the medial preoptic area of intact animals (n = 6) was 102.3:1= 8.28 per section (Fig. 4). Bilaterally enucleated hamsters (n = 5) had 61.18 :i: 10.3 SPIR neurons per section; significantly fewer SPIR neurons (P < 0.5) than that of intacts and enucleated castrates treated with testosterone (Figs. 4, 6B). Unlike

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Fig. 4. Mean number of substance P-containing cells per section through the medial nucleus of the amygdala, the medial bed nucleus of the stria terminalis and the medial preoptic area of intact (l I), enuc!eated (O), castrate treated with testosterone (~]), and enucleated castrate hamsters treated with testosterone (r~). The number within each bar indicates the number of hamsters in each group. * different from enucleated hamsters within same group (P < 0.05); ** different from enucleated hamsters within the same group (P < 0,001 ).

the medial nucleus of the amygdala and the bed nucleus of the stria terminalis, the mean number of SPIR neurons in the enucleated animals did not differ from that of the sighted castrates treated with testosterone. The latter group had an average of 89.5 ± 26.3 SPIR neurons per section (n = 3) which did not differ significantly from that of any other group (Fig. 4). Enucleated, testosterone-treated castrates (n = 6) contained an average of 113.6 + 11.4 SPIR neurons per section (Fig. 4). This was within the range of testosteronetreated castrates and intact hamsters, and was greater than that of enucleated hamsters (P < 0.05). The suprachiasmatic nucleus. SPIR neurons were found in the lateral edge of the suprachlasmatic nuclei (Fig. 2D). The suprachiasmatic nuclei of intact animals (n = 6) contained an average of 40 ± 5.4 SPIR neurons per section (Fig. 4). The suprachiasmatic nuclei contained the same number of SPIR neurons in all four groups.

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Fig. 5. Photographs of coronal sections through the posteromedial subdivision of the bed nucleus of the stria terminalis at the level indicated in Fig. 2B. Photographs are of representative sections from the brains of an intact hamster (A), an enucleate (B), a castrate treated with testosterone (C), and an enucleated castrate treated with testosterone (D) that were immunohistochemically labelled for substance P. Note the abundance of substance P cells and heavy fiber labelling in A, C and D, and the absence of cell bodies and the light fiber labelling in the enucleated hamster (B),

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Fig, 6, Photographs of coronal sections through the magnocellular part of the medial preoptic nucleus at the level indicated in Fig. 2C. Photographs are of representative sections from the brains of an intact hamster (A), an enucleate (B), a castrate treated with testosterone (C), ~md an enucleated castrate treated with testosterone (D) that were immunohistochemically labelled h,r substance P. Note the decrease in the number of hlbelled cells in B compared to those in A, C and D.

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Fig. 7. Photographs of coronal sections through the suprachiasmatic nuclei at the level indicated in Fig. 2D. Photographs are of representative sections from an intact (A) and enucleated (B) hamster that were labelled for substance P. Note that the number of cells labelled and intensity of the staining is comparable for the two sections.

36 The enucleated animals (n = 3) had an average of 35 + 13.5; testosterone-treated animals (n = 2), 36.5 + 14.5, and enucleated, testosterone-treated castrates (n = 4), 39 + 4.9 SPIR neurons per section (Fig. 7). All SPIR in the suprachiasmatic nuclei were 8-11 ~tm in diameter and darkly stained. DISCUSSION Our results indicate that bilateral ent:::,.ation decreases substance P immunoreactivity in neurons in the medial nucleus of the amygdala, medial bed nucleus of the stria terminalis, and medial preoptic area of adult, male golden hamsters. Testosterone treatment prevented this decrease. Thus, our results suggest that the decrease in substance P immunoreac~ivity is mediated by the decrease in circulating testosterone that accompanies prolonged exposure to a short day 4 or enucleation ~6. Our data suggest that decrease in daylength may alter testosterone's effect on substance P production. Bilateral enucleation reduced the number of substance P neurons by two thirds in the medial nucleus of the amygdala and bed nucleus of the stria terminalis and by half in the medial preoptic area. Testosteronetreated blinded animals had twice the number of substance P neurons in the bed nucleus of the stria terminalis and medial nucleus of the amygdala as did the testosterone.treated sighted castrates. Bilateral enucleation may have a direct effect on substance P levels in these nuclei. Long term castrates also show a decrease in the level of SPIR in these areas 4x, In that study intact animals contained 2.5 times the number of immunoreactive neurons per section in the medial nucleus of the amygdala, 2.1 times the number in the bed nucleus of the stria terminalis and 2.1 times in the medial preoptic area of castrates. Results from preliminary studies suggest that this decrease occurs gradually over 13 weeks. In contrast, the results of the present study indicate that blind, gonadally intact hamsters show decreases in the levels of substaace P within 7-10 weeks. The data suggests that SPIR levels decrease faster following exposure to short photoperiods in the presence of low levels of circulating testosterone than in long photoperiods in the absence of circulating testosterone. This hypothesis should be tested by comparing the time course of changes in substance P levels in enucleated vs castrated animals. • The present data do not support the hypothesis that changes in substance P levels alone mediate photoperiod-inc~uced changes in mating behavior. Enucleated castrated hamsters, treated with testosterone had levels

of substance P equal to or greater than sighted intacts. Castrated hamsters maintained on short days fail to show normal mating behavior when implanted with the dose of testosterone used in the present study 7'3°. Thus, the enucleated, testosterone-treated castrated hamsters in our study probably would not have shown normal mating behavior despite the fact that their substance P levels were equal to or greater than those found in the intact sighted animals. Therefore, the level of substance P immunoreactivity in the neurons of medial nucleus of the amygdala, medial preoptic area, and bed nucleus of the stria terminalis cannot be the only fact-~r mediating photoperiod-induced changes in mating behavior. It is of interest that testicular involution occurred faster in the summer than in the winter months. Animals in both studies were housed in the same room under the same lighting- and temperature-controlled conditions suggesting that changes in the rate of regression were not due to changes in these parameters. These differences may be due to an underlying, seasonal, rhythm in sensitivity to short days that may have been synchronized to the proper season by exposure to the outside world while in transit from the supplier to the laboratory. A seasonal rhythm has also been described in an inbred strain of housemice born and raised in the laboratory suggesting that these rhythms may be entrained to the environment by other means ~~. Whether these changes reflect an endogenously driven circannual clock or variations induced by changes in environmental t~ctors remains to be determined. We have also demonstrated that substance P neurons are located within the lateral edge of the hamster suprachiasmatic nuclei. This location appears to be analogous to those in the rat suprachiasmatic nuclei 55. These neurons do not appear to play a role in the regulation of circadian rhythms but may play a role in the regulation of pupil constriction, accommodation and/or blood flow to the eye ~a. Enucleation had no significant effect on the number of substance P neurons in the suprachiasmatic nuclei. These results are somewhat surprising because enucleation reduces retinal input to these neurons. Reduction of synaptic input leads to degeneration in some systems. The suprachiasmatic nuclei contain receptors for melatonin 57, a hormone produced by the pineal which conveys information about changes in daylength to the rest of the brain 4°. Blinding increases the duration of melatonin release per day, possibly stimulating these receptors. Our results do not indicate that these changes affect substance P levels. However, as the suprachiasmatic nuclei do not contain receptors for gonadal steroids, these findings are consistent with our hypothesis that

37

changes in substance P content are restricted to steroid responsive nuclei. Thus, changes in SPIR seen in medial nucleus of the amygdala, bed nucleus of the stria terminalis, and medial preoptic area of enucleated hamsters are specific to these nuclei and not a general response to a decrease in circulating testosterone levels. In conclusion, the results of the present study add substance P to the growing list of neuroactive substances and receptors that change in response to changes in photoperiod 6.t4.46.47.St'S8.Identifying the precise role of these substances in the regulation of behavioral and hormonal components of reproduction in seasonal breeders remains one of the more challenging aspects of neurobiology. Acknowledgements. The authors would like to thank Dr. Keith Anderson for his assistance in surgical manipulations, Patria Adames for her assistance in the preparation of the tissue, Monica Cuello for assistance in data collection and Dr. Hansen for assistance in statistical analysis. The authors are also deeply indebted to Dr. Cheryl Sisk, Dr. Tony Nunez, Meri Damlama, Kevin Byrnes and Sandra Chinapen-Barletta for their critical review of the manuscript. This research was supported by funds from the Biomedical Research Support Grant PHS RR 07059-22 awarded to Rutgers University, NSF RI! 88.17677 awarded to J.M.S. and the Rutgers University Research Council.

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