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Immunocytochemical localization of LHRH in central olfactory pathways of hamster
574
Brain Research, 193 (1980) 574 579 !: Elsevier:North-Hollalld Biomedical Press
Immunocytochemical localization of LHRH in central olfactory pathways of hamster
H. S. PHILLIPS*, G. HOSTETTER, B. KERDELHUE and G. P. KOZLOWSKI Department of Neurobiology and Anatomy, University of Texas Medical School at Houston, P.O. Box 20708, Houston, Texas 77025 and Department of Anatomy, University of Oregon Health Sciences Center, School of Medicine, Portland, Oregon 97201 ( U.S.A.) and Laboratoire des Hormones Polypeptidiques, CNRS, 91190 Gi]-sur- Yvette (France)
(Accepted March 6th, 1980) Key words': hamster --- olfactory - - immunocytochemical luteinizing hormone - releasing hormone
Olfactory stimuli associated with mating are capable of eliciting hormonal and behavioral changes in a variety of mammalian species. In the golden hamster (Mesocricetus auratus), exposure of a male to female odor may result in sexual behavior directed towards an inappropriate objectS, 15, and increased plasma testosterone 13. Since luteinizing hormone-releasing hormone ( L H R H ) may enhance sexual behavior 14,17 as well as luteinizing hormone release, it was of interest to examine the anatomical relationship between central olfactory pathways and LHRH-containing neurons. The male golden hamster was chosen for study due to its critical dependence on olfactory pathways for sexual arousaP 6,22. Since there are no previous comprehensive reports on L H R H localization in central olfactory pathways, a second species, the rat, was included for comparison. Four male golden hamsters (Lak:LVG, 3-4 months old) and 4 male Long-Evans rats (3-5 months old) were used for study. Animals were housed in colony cages and kept on a 24 h light/dark cycle (14 h light : 10 h dark for hamsters; 12 h light : 12 h dark for rats). Each animal was anesthetized with sodium pentobarbital and perfused intracardially with 2 ~ glutaraldehyde and 3 ~,, paraformaldehyde in 0.1 M phosphate buffer, p H 7.4. Brains were removed, immersed in fixative for two days, and then vibratome sectioned at 50 #m. Sections were treated for L H R H localization using the unlabeled antibody PAP technique 9. Three a n t i - L H R H sera were utilized: Kerdelhu6 4152-8, 4150-11 at 1:1000, and Nilaver-Silverman 3 at l:2000. Primary antiserum incubations were carried out for 24 h in 0.1 M tris buffer, p H 7.2 containing 0.9 ~/,~ saline, 0.1 ~ gelatin, and 0.1 ~ or 0.25 ~ Triton X-100. All staining was abolished by the addition of 1 /~g L H R H (Abbott) to 1 ml of antiserum at final dilution. Although staining intensities did vary between antisera, the distribution of reactive elements visualized was identical. The localization of LHRH-positive cell bodies and fibers associated with olfactory regions of the hamster is summarized in Fig. 1. Fibers present in hypothala* Present address: TRIMS, 1300 Moursund Ave., Houston, Texas 77030 U.S.A.
575
o~
Fig. 1. Distribution of LHRH-containing cell bodies and fibers in olfactory pathways. Areas found to contain cell bodies are represented by dots, while areas in which fibers reside are indicated by dashes. Modified from K6nig and Klippel8. ac, anterior commissure; am, medial nucleus of amygdala; aob, accessory olfactory bulb; aom, medial division of anterior olfactory nucleus; dbb, diagonal band of Broca; g, granular layer of olfactory bulb; p, periventricular layer of olfactory bulb; gcc, genu of corpus callosum; icm, Insula Calleja Magna; lot, lateral olfactory tract; lv, lateral ventricle; na, nucleus accumbens; oc, optic chiasm; ot, optic tract; sm, medial nucleus of septum; tdd, dorsal division of tenia tecta. mic nuclei and areas not associated with olfaction are not represented in this diagram and will not be discussed. Anteriorly, clusters of perikarya form a band along the medial surface of each hemisphere (Fig. 21). This band extends rostrally from the level of the genu of the corpus callosum into the olfactory peduncle at the medial division of the anterior olfactory nucleus. This group of cell bodies occupies the plexiform region ventral to the dorsal and superior ventral divisions of the tenia tecta. The majority of these perikarya display one prominent process oriented dorsocaudally in alignment with the medial olfactory tract. A more dispersed collection of L H R H perikarya appears posterior to the parolfactory group just described. These cell bodies occupy a region that includes: the medial septum and the region just lateral to it, the vertical limb of the diagonal band of Broca, the bed nucleus of the stria terminalis, the triangular nucleus of the septum, and medial preoptic area. The greatest concentration of positive cell bodies appears in the vertical limb of the diagonal band of Broca at the level of the organum vasculosum of the lamina terminalis. The distribution of LHRH-containing cell bodies in the hamster is similar to that which we observed in the rat, and to that reported by Barry et al. 1 in the guinea pig. LHRH-containing fibers associated with components of central olfactory
576 p a t h w a y s in the h a m s t e r may be discussed in 4 groups. These g r o u p s consist o f fibers projecting to or t h r o u g h (1) superficial layers o f the main and accessory olfactory bulbs; (2) deep layers o f the olfactory b u l b ; (3) p o r t i o n s o f the a m y g d a l a ; and (4) olfactory tubercle and related structures.
Fig. 2. 1 : sagittal section displaying cluster of cell bodies just caudal to olfactory peduncle, x 610. 2: coronal section through accessory olfactory bulb (AOB) displaying a dense bundle of fibers in the region of the vomeronasal nerve. Counterstained with hematoxylin, x 300. 3 : sagittal section through olfactory bulb with positive fibers along lateral ventricle (LV), and in periventricular (P) and granular (G) layers, x 460.4: coronal section through medial nucleus of amygdala (AM) with reactive fibers in close association with optic tract (OT). × 650. 5: sagittal section through amygdala demonstrating LHRH-containing fibers along the lateral ventricle (LV) and medial nucleus (AM). x 540. 6: coronal section displaying reactive fibers in lnsula Calleja Magna (ICM), which lies between diagonal band of Broca (DBB) and nucleus accumbens (NA). / 530.
577 Two systems of LHRH-containing fibers are visible in the olfactory bulbs of hamsters. One group of fibers occupies the superficial layers of the bulb, while the other plexus extends through the deepest layers. Bundles of reactive fibers which appear to originate in the anterior (parolfactory) collection of cell bodies (Fig. 21) may be traced rostrally along the medial surface of the olfactory peduncle into portions of the glomerular layer of the main olfactory bulb (MOB). Positive fibers are also present along the outer layers of the accessory olfactory bulb (AOB). A very dense bundle of fibers occupies the AOB in the region of the vomeronasal nerve (Fig. 22). These strongly reactive fibers are arranged in parallel and cannot be distinguished from the vomeronasal nerve. A deeper network of stained fibers traverses along the rostral extension of the lateral ventricle into the periventricular and granular layers of the AOB and MOB (Fig. 2~). A preliminary report by other investigators 7 described a similar distribution of fibers to olfactory bulb in hamsters. In rats, our observations indicate that no reactive fibers occupy the deep layers of the MOB, but parolfactory perikarya do send projections to the superficial layers of the main and accessory olfactory bulbs. The origin and course of fibers to the superficial layers of MOB and AOB in both rat and hamster coincides with descriptions of the nervus terminalis 4 in several respects. The nervus terminalis arises from the septal area and the region just caudal to the olfactory peduncle, courses along the medial surface of olfactory peduncle and bulb, and joins with fibers of the vomeronasal nerve before passing through the cribiform plate. As the nervus terminalis is said to innervate both olfactory and vomeronasal mucosa 4, the possibility of LHRH-containing neural elements in these regions deserves attention. In the amygdala of hamster, LHRH-containing processes are present in the medial nucleus and medial portions of the cortical nucleus. Bundles of fibers are located in close association with the stria terminalis, the optic tract (Fig. 24), and the lateral ventricle (Fig. 25). Fibers are seen at all levels of the stria terminalis, from the amygdala to their presumed origin in the bed nucleus. Positive fibers are also seen in the ventral amygdalafugal pathway, and in the superficial plexiform layer of the ventral surface between the optic tract and the lateral olfactory tract. In the present study, the distribution of reactive elements in the amygdala of rat and hamster are similar, with clusters of fibers appearing in both species. Other investigators have reported a few scattered LHRH-containing fibers in the amygdala of guinea pigs 11 and rats z0. The long antibody incubations employed in the present study may have enhanced the appearance of these projections and account for the higher concentration of fibers reported here. It is intriguing that positive fibers are located in regions of the amygdala reported to receive direct input from the AOB TM and to project to the preoptic area, a region implicated in the control of both hormonal and behavioral processes. A loosely organized system of stained processes is visible in the olfactory tubercle of both rat and hamster. Scattered, lightly stained fibers are visible in and around the lateral olfactory tract and prepyriform cortex. A dense plexus of reactive fibers occupies the Insula Calleja Magna in hamster (Fig. 2~). These fibers stain intensely, and some appear to terminate in swollen varicosities. While some fibers may also be seen in the medial islands of Calleja, there was no visible reaction product in
578 the lateral islands. Very few reactive fibers were seen in any of the islands of Calleja in rats. There is abundant evidence to suggest that the olfactory regions reported here to contain L H R H may modulate both hormonal and behavioral events. Androgen-concentrating cells have been located in the rat in: bed nucleus of stria terminalis, medial nucleus of the amygdala, nucleus of the diagonal band of Broca, medial preoptic area, and lateral septum adjacent to the lateral ventricle 19. The overlap of LHRHcontaining regions with testosterone-concentrating areas is particularly interesting since testosterone is necessary for LHRH-enhanced sexual behavior in male ratslL Luteinizing hormone release may be elicited in rats by electrical stimulation of AOB 3, or of amygdala 2. In the male hamster, mating behavior is severely compromised or eliminated by ablation of both MOB and AOB 16, section of the lateral olfactory tract 6, or lesions of the medial and cortical nuclei of the amygdala ~°, while mating deficits are observed in male rats following lesions of bed nucleus of stria terminalis 2~ or medial preoptic area 1~. The present finding of overlap in central olfactory pathways and LHRH projections in both hamster and rat suggests that L H R H may play a role in olfactoryinitiated behaviors. The observation of a more extensive system of L H R H projections to the olfactory bulb in hamster is intriguing in light of the critical role olfaction plays in reproductive behavior of this species ~6,22. While it was not possible to determine the regions in which LHRH-containing axons form synaptic connections, the present results suggest that LHRH-containing neurons may send projections to areas from which they receive olfactory impulses, i.e. the main and accessory olfactory bulbs and the medial nucleus of the amygdala. These LHRH-projections might act to modulate olfactory impulses as they ascend towards septal, preoptic, or hypothalamic regions controlling behavioral and hormonal events. The action of L H R H in enhancing sexual behavior might arise by modulation of neural activity in sensory pathways as it ascends from peripheral receptors towards diencephalic and telencephalic sites. The possibility that L H R H pathways may form feedback loops in the olfactory system of the male hamster to enhance the effectiveness of olfactory stimuli in elicting sexual behavior deserves further attention. The authors wish to express their appreciation to Drs. G. Nilaver and E. A. Zimmerman for the generous gift of antiserum. The advice and assistance of Dr. Beng T. Ho, Joe Phillips, and Lena Chu is gratefully acknowledged. This work was completed in partial fulfillment of the Doctor of Philosophy Degree in the University of Texas Graduate School of Biomedical Sciences at Houston. Heidi S. Phillips is a recipient of a predoctoral fellowship from the Texas Research Institute of Mental Sciences. This work was supported by U.S.P.H.S. Grant HD-1278 I.
1 Barry, J., Dubois, M. P. and Carette, B., lmmunofluorescencestudy of the preoptico-infundibular LRF neurosecretory pathway in the normal, castrated, or testosterone-treated male guinea pig, Endocrinology, 95 (1974) 1416-1423.
579 2 Beltramino, C. and Taleisnik, S., Facilitatory and inhibitory effects of electrochemical stimulation of the amygdala on the release of luteinizing hormone, Brain Research, 144 (1978) 95-107. 3 Beltramino, C. and Taleisnik, S., Effect of electrochemical stimulation in the olfactory bulbs on the release of gonadotropin hormones in rats, Neuroendocrinology, 28 (1979) 320-328. 4 Bosjen-Moller, F., Demonstration of terminalis, olfactory, trigeminat, and perivascular nerves in the rat nasal septum, J. comp. Neurol., 159 (1975) 245-256. 5 Darby, E. M., Devor, M. and Chorover, S. L., A presumptive sex pheromone in the hamster: some behavioral effects, J. comp. physiol. Psychol., 88 (1975) 496-502. 6 Devor, M., Components of mating dissociated by lateral olfactory tract transection in male hamsters, Brain Research, 64 (1973) 437--441. 7 Hoffman, G. E., Davis, B. J. and Macrides, F., LHRH perikarya send axons to the olfactory bulb in the hamster, Neurosci. Abstr., 5 (1979) 528. 8 K6nig, J. F. R. and Klippel, R. A., The Rat Brain, R. E. Krieger, Huntington, N.Y., 1967. 9 Kozlowski, G. P. and Hostetter, G., Cellular and Subcellular Localization and Behavioral Effects of Gonadotropin-releasingHormone (Gn-RH) in the rat. In D. E. Scott, G. P. Kozlowski, and A. Weindl (Eds.), Brain Endocrine Interaction IlL Neural Hormones and Reproduction, Third Int. Syrup., Wurzburg, 1977, S. Karger, Basel, 1978, pp. 138-153. 10 Lehman, M. N., Kevetter, G. A. and Powers, J. B., Sexual and aggressive behavior in male hamster after lesions of corticomedial amygdala, Neurosci. Abstr.., 4 (1978) 11 Leonardelli, J. and Poulain, P., About a ventral LH-RH preoptico-amygdaloid pathway in the guinea pig, Brain Research, 124 (1977) 538-543. 12 Lisk, R. D., Copulatory activity of the male rat following placement of preopticoanterior hypothalamic lesions, Exp. Brain Res., 5 (1968) 306-313. 13 Macrides, F., Bartke, A., Fernandez, F. and D'Angelo, W., Effects of exposure to vaginal odor and receptive females on plasma testosterone in the male hamster, Neuroendocrinology, 15 (1974) 355-364. 14 Moss, R. L., McCann, S. M. and Dudley, C. A., Releasing hormones and sexual behavior. In W. H. Gispen, Tj. B. van Wimmersma Greidanus, B. Bohus and D. de Wied (Eds.), Hormones, Homeostasis and the Brain, Progress in Brain Research, Vol. 42, Elsevier, Amsterdam, 1975, pp. 37-46. 15 Murphy, M. R., Effects of female hamster vaginal discharge on the behavior of male hamsters, Behav. BioL, 9 (1973) 367-375. 16 Murphy, M. R. and Schneider, G. E., Olfactory bulb removal eliminates mating behavior in the male golden hamster, Science, 167 (1970) 302-303. 17 Pfaff, D. W., Luteinizing hormone-releasing factor potentiates lordosis behavior in hypophysectomized, ovariectomized female rats, Science, 182 (1973) 1148-1149. 18 Scalia, F. and Winans, S. S., The differential projections of the olfactory bulb and accessory olfactory bulb in mammals, J. comp. Neurol., 161 (1975) 31-56. 19 Sheridan, P. J., The nucleus interstitialis striae terminalis and the nucleus amygdaloideus medialis: prime targets for androgen in the rat forebrain, Endocrinology, 104 (1979) 130-136. 20 Silverman, A. J., Krey, L. C. and Zimmerman, E. A., A comparative study of the luteinizing hormone releasing hormone (LHRH) neuronal networks in mammals, Biol. Reproduction, 20 (1979) 98-110. 21 Valcourt, R. J. and Sachs, B. D., Penile reflexes and copulatory behavior in male rats following lesions in the bed nucleus of the stria terminalis, Brain Res. Bull., 4 (1979) 131-133. 22 Winans, S. S. and Powers, J. B., Olfactory and vomeronasal deafferentation of male hamsters: histological and behavioral analyses, Brain Research, 126 (1977) 325-344.
Brain Research, 193 (1980) 574 579 !: Elsevier:North-Hollalld Biomedical Press
Immunocytochemical localization of LHRH in central olfactory pathways of hamster
H. S. PHILLIPS*, G. HOSTETTER, B. KERDELHUE and G. P. KOZLOWSKI Department of Neurobiology and Anatomy, University of Texas Medical School at Houston, P.O. Box 20708, Houston, Texas 77025 and Department of Anatomy, University of Oregon Health Sciences Center, School of Medicine, Portland, Oregon 97201 ( U.S.A.) and Laboratoire des Hormones Polypeptidiques, CNRS, 91190 Gi]-sur- Yvette (France)
(Accepted March 6th, 1980) Key words': hamster --- olfactory - - immunocytochemical luteinizing hormone - releasing hormone
Olfactory stimuli associated with mating are capable of eliciting hormonal and behavioral changes in a variety of mammalian species. In the golden hamster (Mesocricetus auratus), exposure of a male to female odor may result in sexual behavior directed towards an inappropriate objectS, 15, and increased plasma testosterone 13. Since luteinizing hormone-releasing hormone ( L H R H ) may enhance sexual behavior 14,17 as well as luteinizing hormone release, it was of interest to examine the anatomical relationship between central olfactory pathways and LHRH-containing neurons. The male golden hamster was chosen for study due to its critical dependence on olfactory pathways for sexual arousaP 6,22. Since there are no previous comprehensive reports on L H R H localization in central olfactory pathways, a second species, the rat, was included for comparison. Four male golden hamsters (Lak:LVG, 3-4 months old) and 4 male Long-Evans rats (3-5 months old) were used for study. Animals were housed in colony cages and kept on a 24 h light/dark cycle (14 h light : 10 h dark for hamsters; 12 h light : 12 h dark for rats). Each animal was anesthetized with sodium pentobarbital and perfused intracardially with 2 ~ glutaraldehyde and 3 ~,, paraformaldehyde in 0.1 M phosphate buffer, p H 7.4. Brains were removed, immersed in fixative for two days, and then vibratome sectioned at 50 #m. Sections were treated for L H R H localization using the unlabeled antibody PAP technique 9. Three a n t i - L H R H sera were utilized: Kerdelhu6 4152-8, 4150-11 at 1:1000, and Nilaver-Silverman 3 at l:2000. Primary antiserum incubations were carried out for 24 h in 0.1 M tris buffer, p H 7.2 containing 0.9 ~/,~ saline, 0.1 ~ gelatin, and 0.1 ~ or 0.25 ~ Triton X-100. All staining was abolished by the addition of 1 /~g L H R H (Abbott) to 1 ml of antiserum at final dilution. Although staining intensities did vary between antisera, the distribution of reactive elements visualized was identical. The localization of LHRH-positive cell bodies and fibers associated with olfactory regions of the hamster is summarized in Fig. 1. Fibers present in hypothala* Present address: TRIMS, 1300 Moursund Ave., Houston, Texas 77030 U.S.A.
575
o~
Fig. 1. Distribution of LHRH-containing cell bodies and fibers in olfactory pathways. Areas found to contain cell bodies are represented by dots, while areas in which fibers reside are indicated by dashes. Modified from K6nig and Klippel8. ac, anterior commissure; am, medial nucleus of amygdala; aob, accessory olfactory bulb; aom, medial division of anterior olfactory nucleus; dbb, diagonal band of Broca; g, granular layer of olfactory bulb; p, periventricular layer of olfactory bulb; gcc, genu of corpus callosum; icm, Insula Calleja Magna; lot, lateral olfactory tract; lv, lateral ventricle; na, nucleus accumbens; oc, optic chiasm; ot, optic tract; sm, medial nucleus of septum; tdd, dorsal division of tenia tecta. mic nuclei and areas not associated with olfaction are not represented in this diagram and will not be discussed. Anteriorly, clusters of perikarya form a band along the medial surface of each hemisphere (Fig. 21). This band extends rostrally from the level of the genu of the corpus callosum into the olfactory peduncle at the medial division of the anterior olfactory nucleus. This group of cell bodies occupies the plexiform region ventral to the dorsal and superior ventral divisions of the tenia tecta. The majority of these perikarya display one prominent process oriented dorsocaudally in alignment with the medial olfactory tract. A more dispersed collection of L H R H perikarya appears posterior to the parolfactory group just described. These cell bodies occupy a region that includes: the medial septum and the region just lateral to it, the vertical limb of the diagonal band of Broca, the bed nucleus of the stria terminalis, the triangular nucleus of the septum, and medial preoptic area. The greatest concentration of positive cell bodies appears in the vertical limb of the diagonal band of Broca at the level of the organum vasculosum of the lamina terminalis. The distribution of LHRH-containing cell bodies in the hamster is similar to that which we observed in the rat, and to that reported by Barry et al. 1 in the guinea pig. LHRH-containing fibers associated with components of central olfactory
576 p a t h w a y s in the h a m s t e r may be discussed in 4 groups. These g r o u p s consist o f fibers projecting to or t h r o u g h (1) superficial layers o f the main and accessory olfactory bulbs; (2) deep layers o f the olfactory b u l b ; (3) p o r t i o n s o f the a m y g d a l a ; and (4) olfactory tubercle and related structures.
Fig. 2. 1 : sagittal section displaying cluster of cell bodies just caudal to olfactory peduncle, x 610. 2: coronal section through accessory olfactory bulb (AOB) displaying a dense bundle of fibers in the region of the vomeronasal nerve. Counterstained with hematoxylin, x 300. 3 : sagittal section through olfactory bulb with positive fibers along lateral ventricle (LV), and in periventricular (P) and granular (G) layers, x 460.4: coronal section through medial nucleus of amygdala (AM) with reactive fibers in close association with optic tract (OT). × 650. 5: sagittal section through amygdala demonstrating LHRH-containing fibers along the lateral ventricle (LV) and medial nucleus (AM). x 540. 6: coronal section displaying reactive fibers in lnsula Calleja Magna (ICM), which lies between diagonal band of Broca (DBB) and nucleus accumbens (NA). / 530.
577 Two systems of LHRH-containing fibers are visible in the olfactory bulbs of hamsters. One group of fibers occupies the superficial layers of the bulb, while the other plexus extends through the deepest layers. Bundles of reactive fibers which appear to originate in the anterior (parolfactory) collection of cell bodies (Fig. 21) may be traced rostrally along the medial surface of the olfactory peduncle into portions of the glomerular layer of the main olfactory bulb (MOB). Positive fibers are also present along the outer layers of the accessory olfactory bulb (AOB). A very dense bundle of fibers occupies the AOB in the region of the vomeronasal nerve (Fig. 22). These strongly reactive fibers are arranged in parallel and cannot be distinguished from the vomeronasal nerve. A deeper network of stained fibers traverses along the rostral extension of the lateral ventricle into the periventricular and granular layers of the AOB and MOB (Fig. 2~). A preliminary report by other investigators 7 described a similar distribution of fibers to olfactory bulb in hamsters. In rats, our observations indicate that no reactive fibers occupy the deep layers of the MOB, but parolfactory perikarya do send projections to the superficial layers of the main and accessory olfactory bulbs. The origin and course of fibers to the superficial layers of MOB and AOB in both rat and hamster coincides with descriptions of the nervus terminalis 4 in several respects. The nervus terminalis arises from the septal area and the region just caudal to the olfactory peduncle, courses along the medial surface of olfactory peduncle and bulb, and joins with fibers of the vomeronasal nerve before passing through the cribiform plate. As the nervus terminalis is said to innervate both olfactory and vomeronasal mucosa 4, the possibility of LHRH-containing neural elements in these regions deserves attention. In the amygdala of hamster, LHRH-containing processes are present in the medial nucleus and medial portions of the cortical nucleus. Bundles of fibers are located in close association with the stria terminalis, the optic tract (Fig. 24), and the lateral ventricle (Fig. 25). Fibers are seen at all levels of the stria terminalis, from the amygdala to their presumed origin in the bed nucleus. Positive fibers are also seen in the ventral amygdalafugal pathway, and in the superficial plexiform layer of the ventral surface between the optic tract and the lateral olfactory tract. In the present study, the distribution of reactive elements in the amygdala of rat and hamster are similar, with clusters of fibers appearing in both species. Other investigators have reported a few scattered LHRH-containing fibers in the amygdala of guinea pigs 11 and rats z0. The long antibody incubations employed in the present study may have enhanced the appearance of these projections and account for the higher concentration of fibers reported here. It is intriguing that positive fibers are located in regions of the amygdala reported to receive direct input from the AOB TM and to project to the preoptic area, a region implicated in the control of both hormonal and behavioral processes. A loosely organized system of stained processes is visible in the olfactory tubercle of both rat and hamster. Scattered, lightly stained fibers are visible in and around the lateral olfactory tract and prepyriform cortex. A dense plexus of reactive fibers occupies the Insula Calleja Magna in hamster (Fig. 2~). These fibers stain intensely, and some appear to terminate in swollen varicosities. While some fibers may also be seen in the medial islands of Calleja, there was no visible reaction product in
578 the lateral islands. Very few reactive fibers were seen in any of the islands of Calleja in rats. There is abundant evidence to suggest that the olfactory regions reported here to contain L H R H may modulate both hormonal and behavioral events. Androgen-concentrating cells have been located in the rat in: bed nucleus of stria terminalis, medial nucleus of the amygdala, nucleus of the diagonal band of Broca, medial preoptic area, and lateral septum adjacent to the lateral ventricle 19. The overlap of LHRHcontaining regions with testosterone-concentrating areas is particularly interesting since testosterone is necessary for LHRH-enhanced sexual behavior in male ratslL Luteinizing hormone release may be elicited in rats by electrical stimulation of AOB 3, or of amygdala 2. In the male hamster, mating behavior is severely compromised or eliminated by ablation of both MOB and AOB 16, section of the lateral olfactory tract 6, or lesions of the medial and cortical nuclei of the amygdala ~°, while mating deficits are observed in male rats following lesions of bed nucleus of stria terminalis 2~ or medial preoptic area 1~. The present finding of overlap in central olfactory pathways and LHRH projections in both hamster and rat suggests that L H R H may play a role in olfactoryinitiated behaviors. The observation of a more extensive system of L H R H projections to the olfactory bulb in hamster is intriguing in light of the critical role olfaction plays in reproductive behavior of this species ~6,22. While it was not possible to determine the regions in which LHRH-containing axons form synaptic connections, the present results suggest that LHRH-containing neurons may send projections to areas from which they receive olfactory impulses, i.e. the main and accessory olfactory bulbs and the medial nucleus of the amygdala. These LHRH-projections might act to modulate olfactory impulses as they ascend towards septal, preoptic, or hypothalamic regions controlling behavioral and hormonal events. The action of L H R H in enhancing sexual behavior might arise by modulation of neural activity in sensory pathways as it ascends from peripheral receptors towards diencephalic and telencephalic sites. The possibility that L H R H pathways may form feedback loops in the olfactory system of the male hamster to enhance the effectiveness of olfactory stimuli in elicting sexual behavior deserves further attention. The authors wish to express their appreciation to Drs. G. Nilaver and E. A. Zimmerman for the generous gift of antiserum. The advice and assistance of Dr. Beng T. Ho, Joe Phillips, and Lena Chu is gratefully acknowledged. This work was completed in partial fulfillment of the Doctor of Philosophy Degree in the University of Texas Graduate School of Biomedical Sciences at Houston. Heidi S. Phillips is a recipient of a predoctoral fellowship from the Texas Research Institute of Mental Sciences. This work was supported by U.S.P.H.S. Grant HD-1278 I.
1 Barry, J., Dubois, M. P. and Carette, B., lmmunofluorescencestudy of the preoptico-infundibular LRF neurosecretory pathway in the normal, castrated, or testosterone-treated male guinea pig, Endocrinology, 95 (1974) 1416-1423.
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