Chemically defined collateral projections from the pons to the central nucleus of the amygdala and hypothalamic paraventricular nucleus in the rat

Triple fluorescence labelling was employed to reveal the distribution of chemically identified neurons within the pontine laterodorsal tegmental nucleus and dorsal raphe nucleus which supply branching collateral input to the central nucleus of the amygdala and hypothalamic paraventricular nucleus. The chemical identity of neurons in the laterodorsal tegmental nucleus was revealed by immunocytochemical detection of choline-acetyltransferase or substance P; in the dorsal raphe nucleus, the chemical content of the neurons was revealed with antibody recognizing serotonin. The projections were defined by injections of two retrograde tracers, rhodamine-and fluorescein-labelled latex microspheres, in the central nucleus of the amygdala and paraventricular nucleus, respectively. Neurons projecting to both the central nucleus of the amygdala and the paraventricular nucleus were distributed primarily within the caudal extensions of the laterodorsal tegmental nucleus and dorsal raphe nucleus. Approximately 11% and 7% of the labelled cells in the laterodorsal tegmental nucleus and dorsal raphe nucleus projected via branching collaterals to the paraventricular nucleus and central nucleus of the amygdala. About half of these neurons in the laterodorsal tegmental nucleus were cholinergic, and one-third were substance-P-ergic; in the dorsal raphe nucleus, approximately half of the neurons containing both retrograde tracers were serotonergic. These results indicate that pontine neurons may simultaneously transmit signals to the central nucleus of the amygdala and paraventricular nucleus and that several different neuroactive substances are found in the neurons participating in these pathways. This coordinated signalling may lead to synchronized responses of the central nucleus of the amygdala and paraventricular nucleus for the maintenance of homeostasis. Interactions between different neuroactive substances at the target site may serve to modulate the responses of individual neurons.     

The implantation of puromycin into the neurosecretory nuclei of the rat

Summary Following the bilateral implantation of puromycin into the paraventricular nuclei of rats, the neurosecretory cells became atrophic and the amount of aldehyde-fuchsin (AF) positive material in the neural lobe decreased. In these rats, urine excretion and water intake increased remarkably. The supraoptic nuclei of the rats were not affected by this treatment. After the unilateral implantation of puromycin in the paraventricular nucleus, the neurosecretory cells of the implanted side became atrophic, while those of the unimplanted side hypertrophied. The neural lobe contained similar amounts of AF-positive material to those of the control rats with unilateral cholesterol implants. In the rats implanted bilaterally with puromycin immediately above the supraoptic nucleus, the neurosecretory cells of this nucleus contained little or no AF-positive material, and urine excretion and water intake increased greatly. The cells of the paraventricular nucleus remained unchanged in these rats.     

A Golgi study on the cerebrospinal fluid (CSF)-contacting neurons in the paraventricular nucleus of the Pekin duck

The present investigation based on classical neurohistological techniques (Nissl-staining, Golgi-impregnation) was focussed on the cytoarchitecture of the periventricular layer of the paraventricular nucleus in the Pekin duck. This region is endowed with intraependymal neurons, the perikarya of which are mostly covered by a thin ependymal lamella. Several of the intraependymal neurons were shown to give rise to dendrites extending into the third ventricle. An additional population of nerve cells located in the deeper layers of the periventricular region also gained direct access to the cerebrospinal fluid by means of long dendrites terminating with a bulbous-like swelling in the third ventricle. This cerebrospinal fluid (CSF)-contacting dendrite branched off several times in a rectangular fashion to give rise to collaterals running in the subependymal or periventricular layers. The axons of these CSF-contacting neurons were followed into the magnocellular portion of the paraventricular nucleus. Small bipolar nerve cells with processes parallel to the surface of the third ventricle occupied a subependymal position. The isodendritic magnocellular neurons of the paraventricular nucleus emitted dendritic processes that reached the basal pole of the ependymal cells. The complex arrangement of the periventricular layer of the paraventricular nucleus might provide the structural basis for the mechanisms of cerebral osmoreception defined by means of physiological parameters.     

Immunohistochemical study on the development of CRF-containing neurons in the hypothalamus of the rat

Summary Appearance of immunoreactive corticotropin-releasing factor (CRF)-containing neurons was studied in developing hypothalamus of the rat by use of antisera against rat- and ovine CRF. These neurons were first recognized in the lateral and paraventricular nuclei on days 15.5 and 16.5 of gestation, respectively, when antiserum against rat CRF was employed. Antiserum against ovine CRF revealed the cells two days later exclusively in the latter nucleus. In both nuclei, the neurons increased in number with development. The neurons in the paraventricular nucleus appeared to project their immunoreactive processes to the median eminence via the periventricular and lateral pathways. In the median eminence, the immunoreaction with antiserum to rat CRF was first recognized in its anterior portion in the form of dots on day 16.5 of gestation but as beaded fibers in the external layer on day 17.5; these structures increased in amount with development in rostro-caudal direction. Although antiserum to ovine CRF was less potent in immunostainability than antiserum to rat CRF, it also revealed the beaded fibers in the median eminence on day 17.5 of gestation. Since evidence is available that the paraventricular nucleus is involved in corticotropin release, it is concluded that, in rats, the hypothalamic regulatory mechanism controlling the release of corticotropin initially appears on days 16.5–17.5 of gestation.     

Evidence for the presence of nerve growth factor (NGF) and NGF receptors in human testis

Summary The co-localization of arginine vasopressin-and enkephalin-like immunoreactivities in nerve cells of the rat paraventricular hypothalamic nucleus and adjacent areas was investigated by the simultaneous application of immuno--galactosidase staining and the peroxidase-antiperoxidase method to sections. Arginine vasopressin-like immunoreactive cells were stained blue with immuno--galactosidase staining and enkephalin-like immunoreactive cells brown with the peroxidase-antiperoxidase method. Double-labeled cells with overlap of blue and brown immunoreaction products were identified in the anterior, medial, and lateral parvocellular parts of the paraventricular hypothalamic nucleus as well as in the previously indicated posterior magnocellular part. Other regions that contained double-labeled cells were the lateral hypothalamic area, anterior hypothalamic nucleus, area between the lateral hypothalamic area and anterior hypothalamic nucleus, suprachiasmatic nucleus, and bed nucleus of the stria terminalis, medial division, posterolateral part. These findings suggest that nerve cells with both arginine vasopressin- and enkephalin-like immunoreactivities may be more actively involved in neuroendocrine regulation and neural transmission than previously considered. They may provide a morphological basis for an increase in enkephalin-like immunoreactivity within the anterior pituitary in cases of hemorrhagic shock which is presumably accompanied by arginine vasopressin hypersecretion.Abbreviations AH anterior hypothalamic nucleus - ap anterior parvocellular part of the paraventricular hypothalamic nucleus - BSTMPL bed nucleus of the stria terminalis, medial division, posterolateral part - dp dorsal parvocellular part of the paraventricular hypothalamic nucleus - f fornix - LH lateral hypothalamic area - lp lateral paryocellular part of the paraventricular hypothalamic nuclcus - mp medial parvocellular part of the paraventricular hypothalamic nucleus - MPA medial preoptic area - pm posterior magnocellular part of the paraventricular hypothalamic nucleus - pv periventricular part of the paraventricular hypothalamic nucleus - SC suprachiasmatic nucleus - Zi zona incerta     

Histochemical demonstration of acetylcholinesterase in the hypothalamus of the female guinea-pig

Summary The distribution of acetylcholinesterase (AChE) in the hypothalamus of the female guinea pig has been examined histochemically.Activity was found in neurones of the supraoptic, paraventricular and infundibular nuclei; in the lateral hypothalamic area, in cells dorsomedial to the fornix at the level of the paraventricular nucleus and in a large group of cells surrounding the fornix at the level of the in-fundibular nucleus. A small well-stained group of cells, not identified in histological preparations, was present ventral to the paraventricular nucleus. The neuropil stained at the lateral border of the paraventricular nucleus and in the medial mammillary, suprachiasmatic and dorsomedial nuclei. The walls of some blood vessels stained; activity was particularly strong in vessels in the preoptic area and at the base of the median eminence. Nerve fibres on blood vessels ventral to the hypothalamus also stained.The degree of enzyme activity in the cell groups was compared in immature, pregnant, lactating, ovariectomized and hysterectomized animals. The greatest variation occurred in the infundibular nucleus. Hypophysectomy markedly reduced staining in the supraoptic nucleus. The possibility that AChE may be involved in the elaboration or transport of releasing factors is discussed.The authors are indebted to Dr. J. S. Perry for doing the surgery involved in this work. They are also grateful to him and to Dr. R. B. Heap for helpful discussions and to Miss M. Hamon for excellent technical assistance.     

Immunocytochemical localization of somatostatin-containing neurons in the rat hypothalamus

Summary The rat hypothalamus was studied at the light microscopic level with the use of single and double immunocytochemical staining methods. It was shown that the rat supraoptic and paraventricular hypothalamic nuclei, and their accessory neurosecretory nuclei, do not contain magnocellular somatostatin neurons. The distribution of the hypothalamic parvocellular somatostatin cells is described. The parvocellular component of the rat hypothalamic paraventricular nucleus is, at least partly, composed of somatostatin cells: they form a fairly well circumscribed periventricular cell mass. The rat suprachiasmatic nuclei contain separate somatostatin neurons and vasopressin neurons. Scattered somatostatin cells are present in the entire arcuate nucleus. In addition to the periventricular somatostatin cells located in the preopticanterior hypothalamic area and in the arcuate nucleus, the rat hypothalamus also contains numerous scattered somatostatin cells located distant from the third ventricle.This investigation was supported by a grant from the Belgian Nationaal Fonds voor Geneeskundig Wetenschappelijk Onderzoek     

Changes in oxytocin content in the magnocellular neurons of the rat hypothalamus following water deprivation or estrogen treatment

Summary The effect of water deprivation or estrogen treatment on the oxytocin content of rat hypothalamic cells was examined using a quantitative immunohistological technique. Oxytocin-containing cells were visualized using the immunoperoxidase technique of Sternberger and a primary antiserum directed against oxytocin. The optical density of the darkest 3.2 m diameter spot in the cytoplasm of a cell was used as a measure of the oxytocin content of that cell. Water deprivation produced a significant decrease in anti-oxytocin staining in the anterior commissural nucleus of males and females. There was a similar decrease in the paraventricular nucleus of males, but not in the paraventricular nucleus of females or the supraoptic nucleus of either males or females. Estrogen treatment of ovariectomized female rats produced a fall in anti-oxytocin staining in the anterior commissural, but not paraventricular or supraoptic nuclei.     

NO-dependent mechanisms of the amygdalofugal modulation of the hypothalamus vegetative neurons

In neurophysiological and histochemical experiments on rats, amygdalo-fugal modulation of cells within NO-producing areas of the hypothalamus was studied. Electrical stimulation of the medial area of the central nucleus caused obvious excitatory neuronal reactions within the medial part of the paraventricular nucleus and rostral portion of the lateral hypothalamic area. The observed amygdala-induced neuronal responses were enhanced after i.v. N-nitro-1-arginine methyl ester (L-NAME, 10 mg/kg). The nistochemical study revealed that the central nucleus stimulation caused an increase in number and optical density of the NADPH-d-positive cells within the parvicellular zone of the paraventricular nucleus and in the medial part of the lateral hypothalamic area. The NO-producing cells within the ventrolateral part of the lateral hypothalamic area were inhibited. The described phenomenon may underlie the amygdalo-fugae modulation of autonomic outflow.     

Distribution of oxytocin and vasopressin neurons in the diencephalon of the Japanese horseshoe bat, Rhinolophus ferrumequinum. An immunohistochemical study.

The distribution of oxytocin (OXT) and vasopressin (VP) neurons in the diencephalon of the hibernating Japanese horseshoe bat, Rhinolophus ferrumequinum, was immunohistochemically investigated by the avidin-biotin complex method. Magnocellular OXT and VP neurons were localized mainly in the paraventricular nucleus and the supraoptic nucleus. In addition to these main nuclei, both kinds of magnocellular neurons were also found in the periventricular nucleus, perifornical area and lateral hypothalamic area. Extensively distributed parvocellular neurons containing only VP were observed in the rostral and middle portions of the suprachiasmatic nucleus. The size of OXT and VP magnocellular neurons was almost equal in the paraventricular and ventromedial supraoptic nuclei, whereas VP neurons were significantly larger than OXT neurons in the dorsolateral supraoptic nucleus. The OXT and VP cells in the ventral supraoptic nucleus showed a distinctive elliptical shape. Both OXT and VP fibers were distributed in the lateral habenular nucleus, stria medullaris thalami, lateral preoptic area, stria terminalis, and medial and supracapsular part of the bed nucleus of the stria terminalis. Moreover, OXT fibers were found in the substantia nigra, and VP fibers were noted in the nucleus reunions and the paraventricular nucleus of the thalamus.     

Cholinesterase activity of the hypothalamo-hypophysial neurosecretory system in rats during the ontogenetic development

Summary The hypothalamic region and the neural lobe of rats from the 16th foetal day to adult animals have been studied for acetylcholinesterase and cholinesterase activity after Karnovsky. The attention was focused on the magnocellular nuclei-supraoptic and paraventricular, the median eminence and the neural lobe. Acetylcholinesterase activity appears in the paraventricular nucleus on the 18th foetal day, i.e. prior to that in the supraoptic nucleus. Heterochronic development and heteromorphism of paraventricular neurosecretory cells have been noticed. The median eminence shows no clear acetylcholinesterase activity. There are acetylthiocholine and butyrylthiocholine positive structures in the posterior pituitary. These structures are especially pronounced in 30–47-day rats. The cholinergic mechanism of release of neurohormones from the neural lobe is suggested. The results are discussed in functional and phylogenetic aspects.     

Ultrastructural demonstration of ovine CRF-like immunoreactivity (oCRF-LI) in the rat hypothalamus: processes of magnocellular neurons establish membrane specializations with parvocellular neurons containing oCRF-LI

Ovine corticotropin releasing factor-like (oCRF-LI) immunoreactivity was detected in the rat hypothalamus by immunocytochemistry. The unlabeled antibody peroxidase-antiperoxidase method was applied in 40 μM vibratome sections before embedding for examination under the electron microscope. Immunoreactivity was found in axons of the median eminence and the neural lobe, as well as in cell bodies and dendrites of parvocellular neurons the in paraventricular nucleus. Axon terminals in the external zone of the median eminence and in the neural lobe frequently abutted on the pericapillary space, suggesting the possible release of oCRF-LI into the fenestrated capillaries. Labeled cells in the paraventricular nucleus synapsed with unlabeled nerve terminals and were found in synaptic-like contact with protrusions of magnocellular neurons. The latter finding might represent the morphological basis for orthodromic interactions between parvocellular and magnocellular neurons of the paraventricular nucleus, which have been previously demonstrated by electrophysiological methods.     

Topography of the subnuclei of the paraventricular hypothalamic nucleus in rats and the sensitivity of their neurons to insulin deficiency

Light microscopy was used to investigate the morphology and topography of hypothalamic paraventricular subnuclei in adult male rats. The subnuclear cell reaction to experimental alloxan diabetes was studied by karyometry. The paraventricular nucleus was established to contain 10 subnuclei, 8 of which responded to disorders in carbohydrate metabolism. The data obtained are consistent with the hypothesis of the involvement of the paraventricular nucleus in the control of carbohydrate homeostasis.     

Immunocytochemical study of the hypothalamic magnocellular neurosecretory nuclei of the snake Natrix maura and the turtle Mauremys caspica

Summary An immunocytochemical study of the magnocellular neurosecretory nuclei was performed in the snake Natrix maura and the turtle Mauremys caspica by use of antisera against: (1) a mixture of both bovine neurophysins, (2) bovine oxytocin-neurophysin, (3) arginine vasotocin, and (4) mesotocin. Arginine vasotocin- and mesotocin-immunoreactivities were localized in individual neurons of the supraoptic and paraventricular nuclei, with a distinct pattern of distribution in both species. The same cells appeared to be stained by the anti-oxytocin-neurophysin and anti-mesotocin sera. The supraoptic nucleus can be subdivided into rostral medial and caudal portions. In N. maura, but not in M. caspica, neurophysin-immunoreactive neurons were found in the retrochiasmatic nucleus. No immunoreactive elements were seen in the suprachiasmatic nucleus of both species after the use of any of the antisera. A dorsolateral aggregation of neurophysin-containing cells, localized over the lateral forebrain bundle, was present in both species. Magnocellular and parvocellular neurophysin-immunoreactive neurons were present in the paraventricular nucleus of both species. In the turtle, the paraventricular neurons were arranged into four distinct layers parallel to the ependyma; these neurons were bipolar with the major axis perpendicular to the ventricle, and many of them projected processes toward the cerebrospinal-fluid compartment. In N. maura a group of large neurons of the paraventricular nucleus was found in a very lateral position. The posterior lobe of the hypophysis and the external zone of the median eminence contained arginine vasotocin- and mesotocin-immunoreactive nerve fibers. The lamina terminalis of both species was supplied with a dense bundle of fibers containing immunoreactive neurophysin. Neurophysin-immunore-active fibers were also present in the septum, some telencephalic regions, including the cortex and the olfactory tubercule, in the paraventricular organ, and the periventricular and periaqueductal gray of the brainstem.This work was partially supported by a Grant S-85-39 from the Direccion de Investigaciones, Universidad Austral de Chile to E.M. Rodriguez     

Heterogeneity in calbindin-D28k expression in oxytocin-containing magnocellular neurons of the rat hypothalamus

We have used a double-labeling immunofluorescence method to examine whether oxytocin-containing magnocellular neurons possess a calcium-binding protein, calbindin-D28k, in the hypothalamus of the rat. In the supraoptic nucleus, most oxytocin-immunoreactive cells were also stained for calbindin-D28k. However, in the magnocellular part of the paraventricular nucleus nearly all oxytocin-labeled cells were devoid of calbindin-D28k. In the anterior commissural nucleus, approximately one-third of oxytocin-stained cells were also calbindin-D28k-immunoreactive, but the other cells were negative for calbindin-D28k. This study indicates that there may be distinct chemical features between oxytocin-containing magnocellular neurons of the supraoptic nucleus compared to those of the paraventricular nucleus.     

Fos Expression in the Rat Brain After Intraperitoneal Injection of Staphylococcus Enterotoxin B and the Effect of Vagotomy

The current study was designed to locate the neuronal activation in rat brain following intraperitoneal injection of Staphylococcus enterotoxin B (SEB) and observe the consequence of preliminary subdiaphragmatic vagotomy on SEB-induced brain Fos expression to clarify the role of the vagus nerve in sensation and transmission of abdominal SEB stimulation. The results showed that intraperitoneal SEB (1 mg/kg) induced a robust Fos expression in widespread brain areas. A significant increase of Fos immunoreactive cells were observed in the solitary tract nucleus, locus ceruleus, lateral parabrachial nucleus, ventrolateral part of central gray, medial amygdaloid nucleus, central amygdaloid nucleus, ventromedial part of thalamus, dorsomedial part of thalamus, hypothalamic paraventricular nucleus, lateral habenula, and lateral septum nucleus following SEB challenge. In hypothalamic paraventricular nucleus, in addition to the dense Fos expression in the parvocellular portion, some Fos-positive cells were also observed in the anterior magnocellular nucleus of the complex. Double immunofluorescence studies showed that these Fos-immunoreactive cells were mostly oxytocinergic. The results also showed that subdiaphragmatic vagotomy largely attenuated, but not totally abrogated, the brain Fos expression induced by abdominal administration of SEB. Our data suggest that peripheral SEB stimulation can induce activation of neurons in widespread brain areas and that the vagus plays a crucial role in transmitting the signal of abdominal immune stimulation to the brain.     

Immunoreactive tyrosine hydroxylase in the brain and pituitary gland of the platyfish

Immunoreactive tyrosine hydroxylase (ir-TH), the rate-limiting enzyme for the synthesis of dopamine and other catecholamines, was localized in the brain and pituitary gland of sexually mature platyfish (Xiphophorus maculatus). This is the first report of ir-TH in the nucleus olfactoretinalis, an LHRH-containing nucleus in the brain which plays an important role in the development and functioning of the reproductive system in platyfish. Ir-TH was also localized in the nucleus preopticus and paraventricular organ. In the pituitary gland ir-TH is found in the prolactin cells and in some fish, in some of the gonadotropin-containing cells of the pars intermedia, but not in the gonadotrops of the pars distalis. The localization of ir-TH in brain centers and pituitary cells associated with reproductive system regulation is discussed in the context of the interaction of monamines, neuropeptides and pituitary hormones during the maturation and operation of the brain-pituitary-gonadal axis.     

Histochemical studies on the morphology of the Golgi apparatus in the neurons of supraoptic and paraventricular nuclei of the normal and dehydrated rabbit (application of the thiamine pyrophosphatase method)

Summary Detailed histochemical studies have been conducted on the morphology of the Golgi apparatus by applying the thiamine pyrophosphatase technique (Novikoff and Goldfisher, 1961) to the neurons of supraoptic and paraventricular nuclei of normal and dehydrated rabbits. The neurons in both nuclei were classified into five categories on the basis of the morphology of the Golgi apparatus. The number of cells in individual categories were counted to evaluate the percentage of each category in the whole nucleus.Neurons have many vesicles which show the tendency to form clusters. Such clusters are present also in the basal bodies. The Golgi apparatus is localized near one side of the nucleus in many neurons. The neurons indicate phasic activity of resting, anabolic and catabolic stages under normal conditions.During dehydration, the Golgi apparatus went through the three stages of network formation, the increase of the budding-off process and later on disintegration. The supraoptic nucleus reacted to the TPPase test more severely than the paraventricular nucleus, whereas the former went through the stages more slowly than the latter. The paraventricular nucleus also revealed sensitivity to osmotic stress.     

Distribution of FMRFamide-like immunoreactivity in the forebrain of the catfish, Clarias batrachus (Linn.).

The anatomical distribution of FMRFamide-like immunoreactivity in the forebrain and pituitary of the catfish, Clarias batrachus, was investigated. Immunoreactive cells were observed in the ganglion cells of the nervus terminalis (NT) and in the medial olfactory tracts. In the preoptic area, FMRFamide-containing perikarya were restricted to the lateral preoptic area, paraventricular subdivision of the nucleus preopticus, nucleus suprachiasmaticus and nucleus preopticus periventricularis posterior. In the postoptic area, some cells of the nucleus postopticus lateralis and nucleus of the horizontal commissure showed moderate immunoreactivity. In the tuberal area, immunoreactivity was observed in few cells of the nucleus hypothalamicus ventralis and nucleus arcuatus hypothalamicus (NAH). Nucleus ventromedialis thalami was the only thalamic nucleus with FMRFamide immunoreactivity. Immunoreactive processes were traceable from the NT through the medial as well as lateral olfactory tracts into the telencephalon and the area ventralis telencephali pars supracommissuralis (Vs). Further caudally, the immunoreactive fibers could be traced into discrete areas, including habenular and posterior commissures, neurohypophysis and pituitary; isolated fibers were also observed in the pineal stalk. A loose network of immunoreactive processes was observed in the olfactory bulbs and the entire telencephalon, with higher densities in some areas, including Vs. A dense plexus of immunoreactive fibers was seen in the pre- and postoptic areas and around the paraventricular organ, while relatively few were observed in the thalamus. A high concentration of fiber terminals was found in the caudal tuberal area.     

Neural interaction of gonadotropin-regulating hormone immunoreactive neurons and the suprachiasmatic nucleus with the paraventricular organ in the Japanese grass lizard (Takydromus tachydromoides)

Our previous study demonstrated that the paraventricular organ (PVO) in the hypothalamus of the Japanese grass lizard (Takydromus tachydromoides) showed immunoreactivity against the light signal-transducing G-protein, transducin. This finding suggested that the PVO was a candidate for the deep-brain photoreceptor in this species. To understand functions of the PVO, we investigated distributions of transducin, serotonin, gonadotropin-releasing hormone (GnRH), and gonadotropin-inhibitory hormone (GnIH) in the lizard's brain. We immunohistochemically confirmed co-localization of transducin and serotonin in PVO neurons that showed structural characteristics of cerebrospinal fluid (CSF)-contacting neurons. GnRH-immunoreactive (ir) cells were localized in the posterior commissure and lateral hypothalamic area. Some of the serotonin-ir fibers extending from the PVO to the lateral hypothalamic area contacted the GnRH-ir cell bodies. GnIH-ir cells were localized in the nucleus accumbens, paraventricular nucleus, and upper medulla, and GnIH-ir fibers from the paraventricular nucleus contacted the lateral processes of serotonin-ir neurons in the PVO. In addition, we found that serotonin-ir fibers from the PVO extended to the suprachiasmatic nucleus (SCN), and the retrograde transport method confirmed the PVO projections to the SCN. These findings suggest that the PVO, by means of innervation mediated by serotonin, plays an important role in the regulation of pituitary function and the biological clock in the Japanese grass lizard.