Luteinizing hormone-releasing hormone (LH-RH) cells and their projections in the forebrain of the batMyotis lucifugus lucifugus

Brain Research, 298 (1984) 289-301

289

Elsevier

Luteinizing Hormone-Releasing Hormone (LH-RH) Cells and Their Projections in the Forebrain of the Bat Myotis lucifugus lucifugus JOAN C. KING, EDYTHE L. P. ANTHONY, ALVAR W. GUSTAFSON and DAVID A. DAMASSA

Department of Anatomy and Cellular Biology, Tufts University Schools of Medicine, 136 Harrison Avenue, Boston, MA 02111 (U.S.A.) (Accepted September 6th, 1983)

Key words: luteinizing hormone-releasing hormone - - bat - - immunocytochemistry - - releasing hormones - hypothalamus --epithalamus - - limbic system

Luteinizing hormone-releasing hormone (LH-RH) neurons and their projections were studied by immunocytochemistry in the brains of little brown bats (Myotis lucifugus lucifugus: Chiroptera: Vespertilionidae) as a first step in the study of relationships between these neurons and the seasonal reproductive events characteristic of this species. The majority of immunoreactive neurons in adult male, adult female, and fetal bats were ovoid bipolar cells with one thin and one thicker process, both of which gave rise to fine varicose fibers. LH-RH-immunoreactive perikarya were concentrated in the region of the arcuate nuclei in all bats examined. Perikarya were also consistently found dispersed in the mammillary region, anterior hypothalamus, preoptic areas, septum, diagonal band of Broca, and olfactory tracts; they were occasionally observed in the dorsal hypothalamus, organum vasculosum of the lamina terminalis (OVLT), habenula, amygdala, and cingulate gyrus. LH-RH-immunoreactive fibers projected heavily to the median eminence, infundibularstalk, and posterior pituitary. In extrahypothalamic areas, these fibers were especially abundant in the stria medullaris/habenula and stria terminalis/amygdala, but also contributed to the diagonal band of Broca and the olfactory tracts. Immunoreactive fibers that may be components of many different pathways clustered in the rostral septum and permeated the medial hypothalamus. LH-RH-containing fibers frequently entered the subfornical organ, but were observed less often in the OVLT and only occasionally in the pineal. The organization of the LH-RH system in the little brown bat resembles that of primates, but differs considerably from that in the rat. Anatomical characteristics of the LH-RH system in bats thus suggest that this animal may be a particularly suitable species for further study of neuroendocrine control of reproductive function as it may relate to primates, including humans. INTRODUCTION

bat speciesa9,35,50,51 and is now rather well characterizeda7,36.

The control of reproductive function in vertebrates is intimately linked to the production of the neuropeptide luteinizing hormone-releasing h o r m o n e (LH-RH). The structure of this decapeptide is conserved among mammals, and exhibits only minor amino acid substitutions in some lower vertebrate groups23, 25. Whereas the distribution of neurons con-

Reproductive events in this species are relatively synchronous and show strong seasonality in large populations. A d u l t females develop a mature Graafian follicle in early fall and remain preovulatory

taining L H - R H has been examined in several orders of mammals4AS,39,43, this neurosecretory system has not been described in bats, mammals which exhibit unusual reproductive specializations. The reproductive cycle of the little brown bat, Myotis lucifugus lucifugus, has been the most thoroughly studied of all

throughout hibernation. W h e n they emerge from their hibernacula the following spring, ovulation occurs, the ovum is fertilized by sperm stored in the uterus from fall or winter copulation, pregnancy ensues, and birth of a single fetus occurs in early summer. In the male, spermatogenic activity is limited to the summer months, with regression of testes occurring in late summer. The gonads then remain in a prepubertal-like state throughout the fall and winter and

Correspondence: Dr. J. C. King, Department of Anatomy and Cellular Biology, Tufts University Schools of Medicine, 136 Harrison Avenue, Boston, MA 02111, U.S.A. 0006-8993/84/$03.00 (~ 1984 Elsevier Science Publishers B.V.

290 early spring while the sex accessory glands are stimulated, and sperm are stored in the epididymis. These unusual reproductive conditions provide a rich paradigm for assessing relationships between neural and endocrine events. It was the purpose of this study to focus on the general morphological features of LHRH neurons and their projections in M. l. lucifugus to provide a framework for examining seasonal changes in this system. MATERIALS AND METHODS

Animals The brains of 22 adult male, 14 adult female, and 6 fetal M. l. lucifugus were examined. The bats were collected at natural roosting sites in New England in hibernating (October, December, March) and active (April, May, June, July, August) conditions. Upon capture, the bats were immediately placed in special cages and transported under conditions to minimize stress, as previously described 18. Upon arrival at the laboratory, the transport cages were immediately transferred to an artificial hibernaculum (maintained at 4-5 °C and in total darkness) to keep the bats torpid. Using this procedure, all animals had a constant, controlled environment and were not handled from the time they were captured until the time that they were sacrificed. This interval did not exceed 24 h. The brains of 9 of the males were studied in paraffin sections; all other animals were studied in Vibratome sections. Dissection and tissue p reparation Each bat was terminally anesthetized using ether or an intraperitoneal injection of chloral hydrate (0.7 mg/g body wt.) and heparin. The head was removed, and following careful removal of the calvaria, the brain was gently lifted from the base of the cranium. Trauma to the hypothalamus was minimized during dissection by cutting the optic nerves and the pituitary stalk as soon as they became visible as the brain was lifted. For paraffin sectioning, brains were fixed for 24 h in Bouin's fluid, transferred to 70% ethanol for 24 h, dehydrated in 80%, 95% and 100% ethanol, cleared in Histosol, and embedded in Paraplast. Serial sections (10-20 pm) were cut in either sagittal, horizontal, or coronal planes and were mounted on chrome-

alum coated glass slides. For Vibratome sectioning, brains were fixed for 30-40 min in 5% or 10% acrolein in 0.1 M phosphate buffer, pH 7.227. After the brains were washed in the same buffer for 3 to 16 h, serial 50/~m sections were cut on a Lancer Vibratome in sagittal, horizontal, or coronal planes.

Immunocytochemistry Neurons containing immunoreactive L H - R H were identified in both paraffin and Vibratome sections using the unlabeled antibody peroxidase-antiperoxidase (PAP) technique 44. Paraffin sections were deparaffinized in xylene, hydrated in a graded ethanol series, and equilibrated in Tris-buffered saline (TBS; pH 7.6 at 21 °C). In order to eliminate endogenous peroxidase activity and minimize background coloration, these tissues were treated with 1% hydrogen peroxide/10% normal sheep serum in TBS before exposure to antisera. Acrotein-fixed Vibratome sections were pretreated sequentially with the following solutions in TBS: (1) 0.2% sodium metaperiodate; (2) 1% sodium borobydride; (3) 1% hydrogen peroxide/10% normal sheep serum; and (4) 5% dimethyl sulfoxide. The primary antiserum used most frequently in these studies was anti-LH-RH IJ29 prepared by and obtained from Dr. I. M. Jackson (Tufts New England Medical Center). This antiserum binds to C-terminal amino acids of the decapeptide 10. Other primary antisera which bind to various portions of the L H - R H molecule (R743, R744, R419, R422) were obtained from Dr. A. Arimura of Tulane University Medical School and have been fully characterized1. Finally, an antiserum which requires all 10 amino acids of LH-RH for binding (CRR11B73) was obtained from Dr. V. Ramirez (University of Illinois Medical School). In addition, an antiserum to glial filamentous acidic protein ~l was obtained from Dr. D. Dahl (Harvard Medical School). Both paraffin and Vibratome sections were incubated for 48-72 h at 4 °C in primary antiserum. All primary antisera were used at a dilution of 1:500 in TBS (pH 7.6 at 4 °C) containing 0.1% gelatin, 0.4% Triton X-100, and 0.02% sodium azide. After washing in TBS, sections were incubated in sheep or goat anti-rabbit immunoglobulin (Antibodies, Inc.) diluted 1:5, 1:20, or 1:50 (30 min for paraf-

291

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B

L

Fig. 1. LH-RH-immunopositive perikarya in Myotis I. lucifugus. These cells are bipolar and often demonstrate a cytoplasmic swelling (arrow in A). These neuronal characteristics are observed in animals collected throughout the annual reproductive cycle. Anterior arcuate nucleus of male bats collected in April (A) and August (B, C). (Sagittal paraffin sections; antiserum IJ29; bar = 10/~m).

292 fin and 2 h for Vibratome sections). The higher concentrations were used for only one batch of antiserum, when it was necessary to compensate for loss of titer during prolonged storage. Following another wash, the sections were treated with P A P (Sternberger-Meyer Immunocytochemicals, Inc.) diluted 1:100 in TBS-gelatin (paraffin, 30 min; Vibratome, l h). After a TBS wash, reaction product was formed during a 30 min incubation using 3,3'-diaminobenzidine (25 mg%; Sigma, Grade II) and 0.05% hydrogen peroxide in 0.05 M Tris buffer (pH 7.6). Paraffin sections were then dehydrated in ethanol, cleared in xylene and coverslipped in Permount. Some Vibratome sections were mounted on glass slides, air dried, and coverslipped in Permount following the immunocytochemical procedure. The remaining Vibratome sections were osmicated (1% OsO4 in 0.1 M phosphate buffer, pH 7.2), dehydrated in ethanol, treated with propylene oxide, and embedded in an Araldite-6005/LX-112 (Ladd) resin mixture. In order to establish specificity of the L H - R H antisera used in this study, synthetic L H - R H (Beckman) was added to diluted antisera in amounts ranging from 0.6 to 1.0/~g/ml. These preparations were allowed to incubate overnight at 4 °C before being used as a control for the presence of LH-RH-specific antibodies in the primary antisera. Cell measurements

Cell length and width were measured to the nearest 0.1/~m (in 50/~m Vibratome sections) using an eyepiece reticle calibrated with a stage micrometer at a total magnification of 400x. Only immunoreactive cells having clearly delineated perikaryon and processes, and which were entirely contained within the

section, were included in the sample. In order to evaluate the possibility of differences in cell size related to sex, age, season, or reproductive condition, a heterogeneous group of animals was used for these measurements. Cells were sampled in 6 hibernating adult males, two hibernating preovulatory females, two summer active adult males, 3 pregnant females, and two fetuses. For all bats, two cells were measured in the preoptic area, in the anterior hypothalamic area, and in the region of the arcuate nucleus, for a total of 6 cells per animal. RESULTS L H - R H - i m m u n o r e a c t i v e cells

The cytoplasm in cells immunopositive with LHR H antisera was filled with reaction product, and formed a thin rim surrounding a clearly defined nucleus (Fig. 1A-C). Since these cells were small (see Tables I and II), and the reaction product dense, immunopositive material could often be seen through the unreactive nucleus in overlying or underlying cytoplasm included in the section. The majority of cells were bipolar, with reaction product extending for long distances into the processes. One of the two processes was usually thicker or wider in diameter than the other near the perikaryon (Fig. 1A, C) but both gave rise distally to fine varicose fibers. In some cells, the thicker process initially approximated the width of the perikaryon, tapering gradually to a fine beaded fiber (Fig. 1B). in other cells, this process exhibited a cytoplasmic swelling prior to narrowing to a beaded fiber (Fig. 1A). In cells that appeared unipolar, there usually was a suggestion of a second process in the plane of section, or the cell seemed to be cut

TABLE I Dimensions of LH-RH-immunoreactive perikarya in forebrains of hibernating and active Myotis I. lucifugus of varying ages, sexes, and reproductive conditions

All measurements were made in plastic-embedded Vibratome sections. In one-way analysis of variance, F = 0.26, P = 0.900 for length and F = 2.48, P = 0.053 for width. Animals

Cells (n)

~ Length (pm) +_S.E.

.~ Width (pm) + S.E.

Hibernating adult males (October; n = 2) Hibernating preovulatory females (October; n = 2) Active adult males (June; n = 2) Pregnant adult females (June; n = 3) Fetuses (n = 2)*

12 12 12 18 12

13.7 + 0.82 13.9 -+ 1.14 12.9 + 0.58 13.3 + 0.45 13.1 -+ 0.77

7.7 + 0.35 7.5 _+0.25 7.5 +_0.36 8.5 + 0.23 7.5 _+0.40

* One male and one female, crown-rump lengths 1.9 and 2.1 cm, respectively.

293 obliquely, which would exclude one or more processes from the section (Fig. 1A, B). Tripolar cells were occcasionally observed. The mean size of LH-RH-immunopositive perikarya was 16.3/~m (S.E. = 0.54) by 8.9/~m (S.E. = 0.31; n = 24 cells) in Vibratome sections that were coverslipped in Permount. These sections were obtained from 4 hibernating adult males collected in October. In two other males also collected in October, dimensions of perikarya were slightly smaller in tissues embedded in resin, indicating that shrinkage of the cells occurred during the dehydration and embedding process (see Table I). The size and general morphological features of LH-RH-positive perikarya did not vary greatly between adult and fetal animals, or among adult males and females in various endocrine conditions (Table I). L H - R H perikarya of pregnant females were, however, slightly greater in width than those of other animals (Table I). LH-RH-immunoreactive perikarya were detected with several antisera exhibiting a variety of binding requirements: (1) the presence of all 10 amino acids (Ramirez C R R l l B 7 3 ) ; (2) the internal sequence of 2-9, and allowing C and/or N terminal modifications or extensions (Arimura R419 and R743); (3) free and unmodified carboxy terminal amino acid sequences and allowing N terminal modifications (Arimura R744 and Jackson IJ29); and (4) free amino and carboxy peptide terminals with no terminal modifications or extensions (Arimura R422). The morphological characteristics of immunopositive cells were consistent with all antisera used. No immunoreaction product was detectable when using these antisera absorbed with excess synthetic LH-RH. Distribution o f L H - R H - i m m u n o r e a c t i v e cells

Cells were scattered throughout the basal forebrain from olfactory to mammillary nuclei, but were

particularly abundant in the tuberal region of the hypothalamus (Fig. 2). In this area, L H - R H cells were located both within the boundaries of the arcuate nucleus and outside of these boundaries, extending laterally across the basal hypothalamus, dorsally into the periventricular zone, and ventrally into the median eminence. The boundaries of the arcuate nuclei were verified for this bat in sections stained with cresyl violet and also in sections in which glial filamentous protein was localized immunocytochemically. An internal glial limiting membrane was apparent in coronal section adjacent to the ependymal cells lining the dorsal two-thirds of the third ventricle, but it did not continue into the region of the arcuate nuclei. L H - R H cells were dispersed singly across nuclei of other basal forebrain areas: (1) olfactory tracts (medial and lateral); (2) diagonal band of Broca (nuclei of the vertical and horizontal limbs); (3) septum (medial and lateral septal nuclei); (4) preoptic area (medial, periventricular, and lateral nuclei, and bed nucleus of the stria terminalis); (5) anterior hypothalamic area (anterior hypothalamic and periventricular nuclei); (6) ventromedial nuclei; (7) premammillary area (ventral premammillary nucleus); and (8) mammillary body (medial mammillary nucleus). In addition, cells in the basal forebrain often appeared to lie very close to the third ventricle (Fig. 2A) or at the free surface of the brain adjacent to the pial membrane. L H - R H neurons occupied all of these sites in adult animals regardless of endocrine condition, and were also present in these areas in fetal animals. Occasional cells were evident in: (1) the organum vasculosum of the lamina terminalis (OVLT); (2) dorsal regions of the preoptic area/hypothalamus bordering the ventral thalamus; (3) the habenula (medial and lateral nuclei); (4) amygdala (medial amygdaloid nucleus); and (5) cingulate gyrus. Perikarya were also observed among fibers of the stria medullaris and

TABLE II Dimensions of LH-RH-immunoreactive perikarya in various regions of the forebmin in the bat Myotis 1. lucifugus

All measurements were made in plastic-embedded Vibratome sections. Sample included all animals listed in Table I. In one-way analysis of variance, F = 0.67, P = 0.51 for length and F = 1.01, P = 0.37 for width. Forebrain region

Cells (n)

~ Length (/am) ++.S.E.

~ Width (lzm) + S.E.

Preoptic area Anterior hypothalamic area Region of arcuate nucleus

22 22 22

13.0 + 0.55 13.9 + 0.59 13.3 + 0.56

7.5 + 0.25 8.0 + 0.26 8.0 + 0.24

294 k. f

o

%

k

L

m

Fig. 2. LH-RH immunopositive perikarya in the middle (A) and posterior (B) arcuate nuclei of a pregnant (A) and a hibernating preovulatory female (B) bat. Immunoreactive processes are present in the median eminence and in the infundibular stalk. (Coronal Vibratome sections; antiserum IJ29; bars = 50/~m).

295

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Fig. 3. This montage is a reconstruction of a parasagittal section from a male bat collected in mid-June, during the spermatogenic period. Immunopositive fibers are directed to the median eminence (ME), pituitary stalk (S), habenular nuclei (H), and the subfornical organ (SFO). The fibers to the habenula stream along with fibers of the stria medullaris (SM). AC, anterior commissure; IR, infundibular recess of the third ventricle; M, mammillary body; OC, optic chiasm; V, third ventricle• (Paraffin section; antiserum IJ29; bar = lOOpm).

296 stria terminalis. L H - R H - i m m u n o r e a c t i v e cells did not vary in size and general m o r p h o l o g y as a function of their location in the forebrain (Table II).

LH-RH-immunoreactive fibers and their projections Fibers originating from L H - R H cells coursed through the hypothalamus and converged ventrally en route to the median eminence (Fig. 3). Only a small percentage of fibers, however, e x t e n d e d into the external zone of the median eminence (Fig. 3). Instead, the majority of fibers formed a heavy bundle which coursed down the stalk and extended into the pituitary (Fig. 4A, B). The fibers then a p p e a r e d to diverge laterally within the pars nervosa and run ad-

jacent to the posterior b o u n d a r y of the pars intermedia (Figs. 4A, B). A n o t h e r large group of immunopositive fibers clustered just dorsal to the anterior commissure to enter the stria medullaris, as can be seen in the sagittal plane in Fig. 3. These fibers then coursed dorsocaudally to reach the medial division of the lateral habenular nucleus (Figs. 3 and 5). In addition to this sizeable projection, a few L H - R H fibers were scattered in the medial h a b e n u l a r nucleus as well as in the lateral division of the lateral h a b e n u l a r nucleus (Fig. 5). The immunopositive cells that were occasionally observed in the habenula directed processes into the stria medullaris; these processes p r o b a b l y

4A

b i

Fig. 4. In the horizontal paraffin section shown in (A), LH-RH-immunopositive fibers are seen in the distal portion of the infundibular stalk (S), which crosses the pituitary pars distalis (pd). The fibers enter the pars nervosa (pn) and diverge to course along the posterior border of the pars intermedia (pi). (Pregnant female bat; antiserum IJ29; bar = 100 ~m). At higher magnification (B), the discrete nature of the varicose immunopositive fibers is more apparent. (Bar = 50/~m). Fig. 5. LH-RH immunopositive fibers concentrated in the medial division of the lateral habenular nucleus (mL). A few fibers are also seen in the medial habenular nucleus (M), the lateral division of the lateral habenular nucleus (IL), and in the habenular commissure (HC). (Pregnant female bat; coronal Vibratome section; antiserum IJ29; bar = 100~m).

297 projected back to the hypothalamus. Few fibers appeared to leave the habenular complex to enter the habenular retroflexus and project to the interpeduncular nucleus of the midbrain. Another major group of L H - R H fibers accompanied the stria terminalis and was directed toward the medial nucleus of the amygdala. Many of these fibers entered the stria terminalis in the region of its bed nucleus. In addition, LH-RH processes originating from immunopositive cells in the amygdala travelled in a reverse direction with fibers of the stria terminalis in its more commonly described trajectory. LH-RH fibers projecting to extra-hypothalamic sites via the stria medullaris and the stria terminalis, or to the median eminence, often traversed the entire hypothalamic region to reach these structures (Fig. 3). In addition, L H - R H immunopositive fibers contributed to the following pathways: (1) the vertical limb of the diagonal band of Broca; (2) the medial and lateral olfactory tracts; and (3) a tract that coursed dorsally along the anterior margin of the third ventricle to enter the subfornical organ (Fig. 3). Variously oriented immunopositive fibers not conforming to any single pathway were clustered in the lateral septum, medial to the nucleus accumbens. Finally, LH-RH-immunopositive fibers were occasionally detected in the OVLT, entering the pineal via the habenular commissure from the habenular complex, in the ventral amygdalofugal pathway, and in the supra-optic and supra-mammillary commissures. In summary, prominent groups of L H - R H fibers extended along routes which terminated in the median eminence and pituitary, the habenula, and the amygdala. While fiber projections to the habenula and amygdala followed trajectories associated with specific pathways (stria medullaris and stria terminalis), the fibers projecting to the median eminence and pituitary followed multiple and diffuse trajectories that spanned periventricular, medial, and lateral regions of the hypothalamus to reach their sites of termination. The LH-RH fiber system that funnels into the median eminence and pituitary may derive a large proportion of its fibers from cells in the region of the arcuate nuclei. A large proportion of extrahypothalamic fibers which were associated with the habenula and amygdala may have originated from cells dispersed across the rostral basal forebrain. However, all cell groups contributed to both hypothalamic

and extra-hypothalamic pathways. The diffuse character of this system is illustrated in coronal sections diagrammed in Fig. 6.

2-

/ Fig. 6. The distribution of LH-RH cells and pathways are represented diagrammatically in this drawing of 5 coronal sections. Rostral sections are at the top, with sections proceeding caudally toward the bottom of the diagram. LH-RH immunopositive cells are concentrated most heavily in the region of the arcuate nuclei, especially in the posterior arcuate (arc), although cells are also dispersed across the preoptic-hypothalamic area, i.e. the nuclei of the diagonal band of Broca (DBB), the preoptic area (poa) and anterior hypothalamicarea (aha). Fibers from these cells project to the median eminence (me), to the pituitary via the infundibular stalk (s), to the habenula (h) via the stria medullaris (SM), and to the amygdala (a) via the stria terminalis (ST). (AC, anterior commissure; ls, lateral septurn). See text for details.

298 In the heterogeneous group of bats studied, the pattern and density of fiber projections, especially to extrahypothalamic areas, appeared exaggerated at some times of Year; these changes may be correlated with endocrine activity. These pathways, nonetheless, were represented in all adult animals as well as in fetuses. Thus, the L H - R H system was completely established prior to birth in this bat in a pattern that seems characteristic of the species. DISCUSSION The organization of the L H - R H system in the little brown bat, M. I. lucifugus, differs in many respects from that of the rat 26,29,30 but closely resembles that of higher primates (rhesus monkey ~0,41, squirrel monkey 3,5,6, baboon34), including humans 2,7.28. A major similarity between the L H - R H systems of this bat and of primates is a significant population of LH-RHimmunoreactive perikarya in the ventral hypophysiotropic zone, specifically in the arcuate nucleus and ventral hypothalamus. In contrast, LH-RH-immunoreactive perikarya in rats are concentrated more rostrally, in septal and preoptic areas, and only a few cells are in the medial basal hypothalamus. L H - R H cells also occur in rostral regions in the little brown bat and in primates; however, they extend posteriorly in much greater numbers, even as far caudally as the mammillary region. This posterior expansion of the L H - R H cell population in the little brown bat and in primates is accompanied by an elaboration of extrahypothalamic LH-RH pathways in these animals. The largest of these pathways in the bat project to the posterior pituitary, habenula, and amygdala. A smaller, but definite pathway projects to the subfornical organ. In primates, extrahypothalamic L H - R H fibers have been described projecting to these same sites 4°.41. In rats, only a few dispersed L H - R H fibers extend to any of these regions (unpublished observations). Extrahypothalamic L H - R H pathways are, however, extensive in some other rodents, i.e., hamster 2J and guinea pig 42, which, like the little brown bat and primates, exhibit L H - R H cells extending into the medial basal hypothalamus. Hypothalamic L H - R H pathways directed to the median eminence are, of course, present in all these species, and we assume that in bats, as in other mammals, L H - R H released at this site en-

ters portal blood and stimulates gonadotropin secretion by the anterior pituitary. Extrahypothalamic L H - R H may also be involved in control of gonadotropin secretion and reproductive function, but in a more indirect manner. L H - R H neuronal processes, extending into extrahypothalamic regions, may function as indicators of neurosecretory activity of L H - R H cells, providing afferent input to several sites which have been implicated in the control of reproduction (e.g. habenula, amygdala, subfornical organ). In turn, these same forebrain sites are known to send efferent fibers back to areas where L H - R H perikarya are present 14,20,31.33. Thus, LH-RH cells may be influenced by input from these sites, and the nature of the input may be in part modulated by the L H - R H fibers themselves, in a manner analogous to a feedback loop. The region of the habenular complex that receives the vast majority of L H - R H fibers in the little brown bat, the medial division of the lateral nucleus, projects heavily to basal forebrain areas 20. These areas septal, preoptic, and anterior hypothalamic nuclei - - contain L H - R H cells. This type of reciprocal anatomical relationship is consistent with the hypothesis that the habenula may affect L H - R H and gonadotropin secretion by influencing the activity of L H - R H cells. The medial habenular nucleus, which receives only a few L H - R H fibers in this species, projects primarily to the midbrain interpeduncular nucleus via the habenular retroflexus. This system is involved in a complex way in modulating sexual receptivity of the female rat 46. Although no neuronal fibers have been described bridging the medial and lateral nuclei 20, interaction between these two divisions may occur in coordination of reproductive activities. The amygdala, another site of extrahypothalamic L H - R H fibers in the little brown bat, has a well-documented effect on LH secretion. Stimulation in the medial aspect of the amygdala has been shown to induce ovulation in phenobarbital-blocked rats22, and to advance the time of the LH surge on the afternoon of proestrus 9. Lesions of the amygdala or the stria terminalis, on the other hand, block ovulation in the proestrous rat 49 and attenuate LH elevations induced by estrogen 48. Thus, the medial amygdala is clearly involved in the modulation of gonadotropin secretion. The mechanism by which this influence is exerted may be via LH-RH fibers which course be-

-

299 tween the amygdala and the hypothalamus. The subfornical organ (SFO), another site of projection of L H - R H fibers, also appears to be related to induction of gonadotropin release, as lesions of the SFO have been shown to disrupt estrous cyclicity in rats, inducing a state of prolonged diestrus 32. Although the neuroendocrine mechanism whereby the SFO could alter cyclic gonadotropin secretion is unknown, its position as a circumventricular organ suggests the possibility of release of neuropeptide into the cerebrospinal fluid (CSF). However, direct secretion of L H - R H into the CSF has not been established. Alternatively, as has been postulated for the habenula and amygdala, reciprocal innervation of areas containing L H - R H perikarya by neurons in the SFO may provide a mechanism for regulating gonadotropin secretion. Such projections have been recently described in the rat 33. An additional set of extrahypothalamic L H - R H projections in the little brown bat extends into the infundibular stalk and enters the posterior pituitary. Similar fibers have been described in monkeys40,41, but have not been observed in rats. We are currently examining this tract in detail, to ascertain the precise destination and possible functions of these fibers. A few single L H - R H fibers and cells have been noted in the cingulate cortex and pineal body in this bat. L H - R H cells and fibers have been recently reported in the cingulate cortex in rats 52. The cingulate cortex appears to exert an inhibitory influence on gonadotropin secretion in this species, as electrochemical stimulation of the cingulate gyrus inhibits the LH secretion that results when the preoptic area is electrically stimulated8. The pineal also has inhibitory effects on LH secretion. Detection of L H - R H in the pineal gland has been inconsistent in the rat, but decapeptide L H - R H has recently been demonstrated in ovine pineal extracts24. The interactions that are necessary to bring about the inhibitory effects of the pineal and cingulate cortex are unknown, but they are undoubtedly complex, and may include suppression of activity in L H - R H neurons. In addition to these neuronal circuits that influence L H - R H neurons and gonadotropin secretion, gonadal steroids are also potent modulators of gonadotropin secretion. Whether L H - R H cells in the bat arcuate region concentrate estrogen is not known; however, in all species thus far examined, neurons of

the arcuate area accumulate steroids as demonstrated by autoradiography37, 45. Recently, arcuate neurons in the guinea pig that contain L H - R H have been shown also to concentrate estrogen 47. A species comparison of estrogen uptake and retention by target tissues reveals that primates, in contrast to rodents, do not concentrate estrogen in brain, uterus or pituitary, but rather demonstrate high levels in plasma ~6. This observation may relate to the presence in primates of plasma steroid-binding proteins that are not present in some rodents. Recently, we have shown that a sex steroid-binding protein is present in the plasma of the bat species examined in this study12a 3. Thus, it would be interesting to investigate the steroid concentrating capacity of target tissues, including arcuate L H - R H neurons in this animal. L H - R H cells and fibers of the little brown bat are immunoreactive with several antisera directed to specific sequences of amino acids, with an antiserum requiring free carboxy- and amino-terminals, and with an antiserum that does not tolerate substitution of any amino acid within the sequence. These data indicate that both cells and fibers contain the mature decapeptide, as that molecule would be recognized immunologically by antisera with these divergent binding requirements. Mature decapeptide is not found within L H - R H perikarya in rats 2s, but does appear to be present in perikarya of monkeys (unpublished observations) and humans 28. These data suggest that mechanisms controlling neuropeptide processing could be similar in the little brown bat and primates, but must differ from those in the rat. In this and other studies we have described several similarities between M. l. lucifugus and primates: (1) the organization of the central L H - R H neuroendocrine system; (2) the intracellular site of maturation of decapeptide LH-RH; and (3) the presence of a plasma sex steroid-binding protein with high affinity for both testosterone and estradiol. These similarities suggest that this bat could provide a useful model for study of reproductive neuroendocrine function as it may relate to primates, including man. ACKNOWLEDGEMENTS We are very grateful to Drs. A. Arimura, D. Dahl, I. M. Jackson and V. Ramirez for supplying the primary antisera used in this study. We would also like

300 to t h a n k S u s a n L e e for h e r t a l e n t e d p r e p a r a t i o n o f di-

assistance. T h i s s t u d y w a s s u p p o r t e d b y N I H G r a n t 1

a g r a m s a n d f o r a s s i s t a n c e in p h o t o g r a p h y . F. L. S n a -

K04 H D 0 0 3 5 2 , N S F G r a n t P C M - 8 1 0 3 2 4 3 a n d N I H

vely a n d E. Y. G u s t a f s o n p r o v i d e d e x p e r t t e c h n i c a l

G r a n t 1 R01 H D 1 6 5 3 5 .

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