Light Stimulation of the Hypothalamic Neuroendocrine System

Transcription

1 Arch. Histol. Cytol., Vol. 55, No. 1 (1992) p Light Stimulation of the Hypothalamic Neuroendocrine System Shigeo DAIKOKU, Ryoji YOKOTE, Tohru AIZAWA and Hitoshi KAWANO Department of Anatomy, The University of Tokushima School of Medicine, Tokushima, Japan Received November 13, 1991 Summary. This paper demonstrates that, in the mediation of light, the suprachiasmatic nucleus (SCN) functionally associates with the anterior periventricular and parvocellular paraventricular neuron systems in rats. Intact rats (group 1) and rats undergoing a hemicomplete cutting of the SCN (group 2) were housed in a dark room (2-3 weeks) and killed after an exposure to light for 10, 30 or 60 min. Other intact animals (group 3) kept in a dark room (2 weeks) were exposed to light for 10 min, then stored 60 min in the dark room, and killed in darkness. The SCN, anterior periventricular nucleus, and parvocellular paraventricular nucleus were examined immunohistochemically using antisera for vasoactive intestinal polypeptide (VIP), arginine vasopressin, somatostatin, rat corticotropin releasing factor (rcrf), and c-fos protein. In comparison with animals kept in darkness, animals exposed for 10 and 30 min to light indicated a remarkable reduction of VIP immunoreactivity in the SCN and some increase of CRF immunoreactivity in the parvocellular paraventricular nucleus. The diminution of VIP immunoreactivity did not occur in the isolated SCN of group 2 animals. In group 3, a 10 min-light exposure induced a remarkable enhancement of nuclear c-fos immunoreactivity in neurons in the ventrolateral region of the SCN, in the anterior penventricular nucleus, and in the parvocellular paraventricular nucleus, most strongly in the SCN. Double immunolabeling methods have shown that VIP, somatostatin, and CRF neurons in the respective nuclei were c-fos positive. For years, many neuroendocrinologists have focused on the suprachiasmatic nucleus (SCN) as the pacemaker of the circadian rhythms in hormonal secretion, metabolic dynamics, or behavioral attitudes of animals. In this context, many studies have been performed to determine how activities of the SCN are conducted to other parts of the brain. Widespread efferent projections of the SCN have been demonstrated (SWANSON and COWAN, 1975; SOFRONIEW and WEINDL, 1978; BERK and FINKELSTEIN, 1981; CARD et al., 1981; STEPHAN et al., 1981; HOOR- NEMAN and BUIJS,1982; WATTS and SWANSON, 1987; WATTS et al., 1987). These authors employed orthograde tracing labeling with radioactive amino acids, retrograde tracing, and immunohistochemical staining for arginine vasopressin (AVP) and vasoactive intestinal polypetide (VIP), the peptides of which are known to be synthesized in the SCN neurons. The efferent fibers appeared to terminate throughout the hypothalamus, amygdaloid complex, midbrain, thalamus, septopreoptic area, and rhombencephalon. Immunohistochemical approaches have demonstrated that the efferent AVP and VIP fibers ascend dorsocaudally along the lateral wall of the third ventricle and then turn laterally beneath the paraventricular nucleus (PVN). However, evidence indicating distinctive neuronal associations between these efferent fibers and neurons in other parts of the brain is still insufficient. Recently, c-fos proto-oncogene (c-fos) was found to participate in the process of the transsynaptic regulation of gene expression by neurotransmitters or neuromodulators (BERRIDGE, 1986; GOELET et al., 1986). Fos-related protein immunoreactivity in the SCN appeared to be regulated by light exposure (REA,1989; ARONIN et al., 1990; EARNEST et a1.,1990; KORNHAUSER et al., 1990; RUSAK et a!.,1990). In these studies, the expression of c-fos immunoreactivity occurred in the ventrolateral region of the SCN, where VIP neurons are dominant. In the present study, we have attempted to demonstrate neurons, which indicate c-fos immunoreactivity after an exposure to light, by employing double immunostaining for c-fos and for other peptides which are synthesized in neurons. With this, we expected to determine which hypothalamic neuron systems are responsive to light stimuli. 67

2 68 S. DAIKOKU et al.: MATERIALS AND METHODS We used adult male Sprague-Dawley rats weighing g. The animals, divided into three groups, were kept under a regular condition at L: D=12 h:12 h (starting 7: 00), and then stored in a dark room for days before the experiments began. They received water and food ad libitum. Thirty animals were cut in the SCN with Halasz knives of various radii and lengths of knife edge. A knife with 1.2 mm in radius and 1.2 mm in height was suitable for performing a hemicomplete cutting of the SCN. One week after the operation, the animals were placed in the dark room. Some of the operated rats and eleven intact animals housed in the dark room were injected with sodium pentobarbital intraperitoneally in darkness at 20: 00 (one hour after the beginning of the prescribed night). A sack of heavy black cloth was placed over the head of each anesthetized animal, and tied at their necks to prevent light from entering. The animals were then fixed by perfusing Bouin's solution-free of acetic acid-from the ascending aorta, proceeding to immunohistochemical labeling for neuropeptides. Other operated and seven intact animals were exposed to light (600 lux) for 10, 30 or 60 min at 20:00 and then killed in the same manner as mentioned above. Five other intact animals housed in the dark room were exposed to light for 10 min at 20: 00, then sent to the dark room, killed 60 min later in the darkness by perfusing them with Zamboni's fixative for the staining of c-fos. The hypothalamus was excised, fixed with Bouin's fixative, embedded in paraffin, and serially cut at 5 um-thick on the frontal plane. Serial paraffin sections were mounted separately on different glass slides and stained with anti-vip (1:40,000), -AVP (1:30,000), -somatostatin (1:6,000) or -rat corticotropin releasing factor (rcrf) (1:8,000) serum using Vectastain Elite ABC kit (Vector Laboratories). Using a cameralucida, the locations of immunoreactive cells were drawn on white paper to examine their topographical relations. The staining specificities and characteristics of the sera for the peptides have been previously examined (VIP, HIsANO et al., 1987; AVP, HISANO et al., 1985; somatostatin, OHTSUKA et al., 1983; rcrf, DAIKOKU et al., 1984). To demonstrate the presence of c-fos, we cut the hypothalamus serially at pm thickness on a vibratome along the frontal plane. The vibratome sections were stained with anti-c-fos (Ab-2, Oncogene Science, Inc., Manhasset, NY). The staining specificity of the serum was examined by a preabsorption test: The serum was diluted 1,000 times with phosphate buffered saline (PBS) and mixed with c-fos antigen (1pM); sections stained with this preabsorbed serum exhibited no immunoreaction. The vibraome sections immunostained for c-fos were further stained with anti-vip, -AVP, -somatostatin, or -rcrf. We were able thereby to determine the nature of c-fos positive neurons. The double immunolabeling procedures of vibratome sections were as follows: 1) treatment of the vibratome sections with 0.1% Triton X-100 and 0.5% H2O2 in PBS for 20 min at room temperature; 2) incubation in anti-c-fos (1:1,000) in PBS containing 0.5% skimmed milk overnight at 4C; 3) incubation in biotinylated goat anti-rabbit IgG (1: 200) for 1 h at 32t; 4) incubation in Elite ABC solution for 1 h at 32C; 5) visualization of the immunoreaction incubating in Tris-HCl buffer (0.05M, ph 7.6) containing 0.01% 3,3'-diaminobenzidine tetrahydrochloride (DAB) and 0.01% hydrogen peroxide for 10 min at 37C; 6) washing the sections vigorously in PBS; 7) incubation of the sections with anti-somatostatin (1: 6,000), anti-vip (1:20,000), anti-avp (1:10,000), or anti-rcrf (1:8,000) in PBS containing 0.5% skimmed milk for 15 h at 4C; 8) incubation in biotinylated goat anti-rabbit IgG (1: 200) for 1 h at 32t; 9) incubation in Elite ABC solution for 1 h at 32t; 10) visualization of the immunoreactions with DAB-cobalt ion solution (HSU and SOBAN,1982) containing 10 mg of DAB in 40 ml of Tris-HCl buffer (0.05 M, ph 7.6) and 0.2 ml of 1% CoCl2 for 5 min at 35C ;11) further incubation for 2-3 min after adding 0.1 ml of 0.3% H202 at 35C ;12) washing in distilled water; and 13) coverslipping with Canada balsam. Immunoreaction for c-fos was localized in the nucleus as brown DAB chromogen, and that for the peptides appeared as blue pigments in the perikarya. When we stained the sections in reverse order, the immunoreaction appeared blue for c-fos and brown for peptides. Additionally, we embedded the vibratome sections immunostained previously for c-fos in an agar-gelatin mixture (4% Bacto-Agar and 2% gelatin), and further embedded these sections in paraffin, which were serially cut at a 5 pm thickness. The details of this procedure have been shown previously (YOKOTE et al., 1991). The paraffin sections were stained with anti-vip, -AVP, -rcrf, or -somatostatin. RESULTS Since this experiment was performed using rats without pretreatment with colchicine, immunoreactive cell bodies-especially those of AVP and

3 Photic Responsiveness of Hypothalamic Neurons 69 Fig. 1. Light responsive VIP immunoreactivity in the SCN. Animals were kept in darkness for 2 weeks (a) and killed 10 min (b), 30 min (c), and 60 min (d) after exposure to light. The rat indicated in c bears a hemicomplete cutting of the left SCN (arrowheads). Remarkable reduction of VIP immunoreactivity in the SCN is evident in b and in the intact SCN (right) in c but not in the isolated SCN (left). Ch optic chiasm, V third ventricle. x 76

4 70 S. DAIKOKU et al.: somatostatin Constant neurons in the SCN-were scarce. darkness Immunoreactive VIP neurons appeared in the ventrolateral portion of the nucleus (Figs. 1, 2). Fine VIP fibers made a dense plexus spreading over the major part of the nucleus, and further extended dorsally, forming a bundle of fibers along the lateral wall of the third ventricle. AVP neurons were very scarce, being confined in the dorsomedial portion of the nucleus. These fibers were more loosely dispersed than the VIP fibers, but were distributed throughout the dorsomedial portion of the nucleus. They further appeared to ascend dorsally, medial to the VIP fiber bundle, making a bundle along the lateral wall of the third ventricle. The fiber bundles of AVP and VIP neurons ascended through the periventricular region; most of the fibers turned laterally at the sub-paraventricular region, sending some fibers to the PVN. Somatostatin neurons were few in number, scattering along the border between the area containing AVP neurons and that containing VIP neurons. Somatostatin fibers were distributed widely throughout the nucleus, but did not show any fiber bundle projecting out of the nucleus. Light exposure Fig. 2. VIP cells in the SCN indicated in Figure la, b and d, are shown with a larger magnification in a, b and c, respectively. The cell-size in b is smaller than others (a, c). x 80 VIP neurons displayed the most dramatic alteration in appearance. Light exposure induced a remarkable diminution of VIP immunoreactivity in the SCN (Figs. 1, 2). Such a reduction was most prominent in animals exposed to light for 30 min, but not evident in animals exposed to light for 60 min. If the SCN was completely isolated, this reduction did not occur even in the 30 min group (Fig. 1). These findings suggest that VIP synthesized in cell bodies may be conveyed dorsally in the ascending fibers by light stimuli. For somatostatin and AVP neurons, we could not recognize any noticeable alterations by exposure to light. In the animals exposed to light for 10 min and killed 60 min later in darkness, a strong immunoreaction for c-fos was evident in cells localized in the ventrolateral region of the SCN but not in cells within the dorsomedial region (Fig. 3). However, in the animals killed at the same time (20:00) in darkness, the c-fos reaction did not occur (Fig. 3). Double Fig. 3. PVN (a, c) and SCN (b, d) of a rat housed during 2 weeks in a dark room (c, d) and of a rat exposed to light for 10 min followed by 60 min-darkness (a, b). Cell nuclei in the parvocellular PVN (arrow in a) and in the ventrolateral division of the SCN (arrow in b) indicate enhanced c-fos immunoreaction. X 80

5 Photic Responsiveness Fig. of Hypothalamic 3. Legend on the Neurons opposite 71 page

6 72 S. DAIKOKU et al.: Fig. 4. SCN (a), parvocellular PVN (b), and anterior periventricular region (c) of a rat exposed to light for 10 min followed by 60 min-darkness. In a cell nuclei (arrowheads) are immunolabeled for c-fos (cobalt blue) and the perikarya for VIP (brown DAB-chromogen). In b and c, c-fos immunoreactivity (brown DAB-chromogen) is l

7 Photic Responsiveness of Hypothalamic Neurons 73 immunolabeling for c-fos and VIP showed that many, though not all, VIP neurons were c-fos immunoreactive (Fig. 4). Concomitant with these findings, c-fos immunoreactivity appeared strongly in neurons in the periventricular and parvocellular paraventricular nuclei (Fig. 3). CRF neurons in the parvocellular PVN seemed to increase in number and in immuno-stainability after light exposure (Fig. 5). Although the c-fos immunoreactivity in the periventricular and parvocellular paraventricular neurons was less strong than that in the SCN, these c-fos positive neurons were found to be somatostatin neurons in the anterior periventricular nucleus, and CRF neurons in the parvocellular PVN (Fig. 4). While we were not able to make an accurate statistic analysis on the appearance of these cells, about 10% of the somatostatin neurons and 40-60% of CRF neurons appeared to be c-fos positive. DISCUSSION The most salient finding in this study is that, following a short exposure to light, rats kept in complete darkness for 60 min displayed enhanced c-fos immunoreactivity in VIP neurons in the SCN, in somatostatin neurons in the anterior periventricular nucleus, and in CRF neurons in the parvocellular PVN. Because c-fos has been found to participate in the process of the transsynaptic regulation of gene expression by neuronal signal substances (BERRIDGE, 1986; GOELET et al., 1986), these findings suggest that the retinal signals evoked by the light exposure were conveyed from the retina to those neurons consecutively. Those neurons may be responding as a functional complex to the light exposure. The SCN consisted mainly of VIP neurons, AVP neurons and somatostatin neurons act as a circadian pacemaker. The VIP neurons occupy the major part of the nucleus except for a small part of the dorsomedial portion where the AVP neurons are dominant; the somatostatin neurons are located in between. Two visual projections reach the SCN, one originating in the retina (MOORE and LENN, 1972; JOHNSON et al., 1988) and the other in a retinorecipient area of the lateral geniculate nuclei and also in an adjacent part of the ventral lateral geniculate nucleus (CARD and MOORE, 1982; PICKARD, 1982; Fig. 5. The parvocellular PVNs of rats kept in darkness for 2 weeks (a) and then killed 10 min (b) and 60 min (c) after exposure to light. Immunoreactive CRF neurons appear to be increased in number in b and c. Arrows show immunonegative magnocellular PVN neurons. x 280 /located within the nuclei of cells (arrowheads), while the perikarya show immunoreactivity rcrf and for somatostatin, respectively. Arrows in b indicate c-fos negative magnocellular PVN. x1,200 (cobalt blue) for neurons in the

8 74 S. DAIKOxu et al.: MOORE et al., 1984; HARRINTON et al., 1985, 1987). These projections appear to terminate in the ventrolateral portion of the SCN (RUSAK and BouLos,1981; HARRINTON et al., 1985). In blinded rats, the presence of AVP mrna (UHL and REPPERT,1986) has indicated a diurnal variation in the SCN. Circadian rhythms of somatostatin-immunoreactivity in the SCN were recently found in blinded rats (SHINOHARA et al., 1991). Thus the intrinsic pacemaker neurons may be AVP and somatostatin neurons. In contrast, it is thought that VIP neurons of the SCN may be light receptive neurons. Diurnal variations of VIP mrna or VIP contents occur in rats kept in regular light and dark variation of the circumstance (TAKAHASHI et al., 1989; ALBERS et al., 1990). The circadian rhythmicity of VIP mrna level disappears in blinded rats (TAKEUCHI et al., submitted). A marked increase in the degree of c-fos-immunoreaction (REA, 1989; EARNEST et al., 1990; RUSAK et al., 1990) and in the content of mrna for c-fos (KORNHAUSER et al., 1990; RUSAK et al., 1990) has been reported in the SCN after light exposure. This increase occurred in cells concentrated in the ventrolateral region of the SCN. No previous studies have revealed the nature of the cells, but our double immunolabeling for c-fos and for VIP demonstrated that the cells are VIP immunoreactive. We further found an enhanced immunoreaction for c-fos in neurons existing in the periventricular region and parvocellular PVN. These neurons were determined to be immunoreactive for somatostatin or CRF. No c-fos immunoreaction, however, was evident in adjacent magnocellular neurons in the PVN. The immunoreactivity for c-fos in the periventricular nucleus and in the parvocellular PVN was less strong than in the SCN. KORNHAUSER et al. (1990) have shown that light can cause a rapid induction of c-fos mrna levels in the SCN of the hamster. The enhanced c-fos immunoreaction in the VIP, CRF, and somatostatin neurons presented here suggests that these neurons had specific gene expressions 60 min after a 10 min light stimulus. Transcriptional regulation of the specific gene that the enhanced c-fos reaction expressed might concern VIP mrna, somatostatin mrna, or CRF mrna. Our recent studies have demonstrated synaptic junctions between somatostatin neurons and CRF neurons (HISANO and DAIKOKU, 1991) and between CRF neurons and oxytocin neurons in the PVN (HISANO et al., 1992). Magnocellular PVN neurons are known to have ample projection to the hypothalamus, hippocampus, amygdala, brainstem, and spinal cord (SWANSON and SAWCHENKO, 1980,1983; WAGNER and CLEMENS, 1991). Further, as is well known, parvocellular CRF neurons and anterior periventricular somatostatin neurons project to the median eminence to secrete hormonal CRF and somatostatin. Hence, it is likely that the retinal stimulus stimulates some anterior pituitary cells. There are efferent projections of AVP neurons from the SCN; the fibers pass the periventricular nucleus, dorsomedial nucleus, and organum vasculosum laminae terminalis (HOORNEMAN and BUIJS, 1982). Recently, we have found synaptic junctions between VIP axons and AVP neurons in the SCN (DAIKOKU et al., submitted). If this is true, AVP neurons in the SCN should be also evoked by light exposure. However, as shown presently, neurons in the dorsomedial part of the SCN did not show c-fos immunoreactivity. The answer to this seeming contradiction may be obtained by examining the AVP neurons some time after light exposure. 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10 76 S. DAIKOKU et al. WATTS, A. G., L. W. SWANSON and G. SANCHEZ-WATTS: Efferent projections of the suprachiasmatic nucleus: I. Studies using anterograde transport of Phaseolus vulgaris leucoagglutinin in the rat. J. Comp. Neurol. 258: (1987). YOKOTE, R., S. HISANO and S. DAIKOKU : Immunohistochemical localization of glucocorticoid receptors in anterior pituitary cells of rats. Arch. Histol. Cytol. 54: (1991). Prof. Shigeo DAIKOKU Department of Anatomy University of Tokushima School of Medicine Kuramoto Tokushima, 770 Japan