Nucleus Suprachiasmaticus Research Articles

P96. New laboratory method to mirror individual circadian rhythms and sleep preference in idiopathic hypersomnia

Introduction The circadian regulation is based on a molecular genetic network of transcriptional feedback loops of clock genes. The nucleus suprachiasmaticus is the central pacemaker and integrates environmental information and synchronizes the phase of oscillators in peripheral cells. Numerous studies had proven that the molecular clock mechanism is cell-autonomous and that circadian period and phase is associated with circadian and sleep preference. Material and methods We measured clock gene expression in fibroblasts from healthy controls and patients suffering from idiopathic hypersomnia who were polysomnographically screened in our sleep laboratory and diagnosed according to the ICSD2-criteria. Two complementary molecular genetic methods to quantify the dynamic of clock gene expression were applied. Human fibroblasts derived from punch biopsies were transfected with a circadian reporter gene, the Bmal1 promoter-driven luciferase gene, using a lentiviral system. The luciferase activity showed a circadian rhythm of lumincescence expression. The individual period length over a time course of 5days was detectable by measuring the bioluminescence every ten minutes in vitro. Previous studies had proven that the physiological period length of human circadian clock in vivo is directly proportional to period in human fibroblast. To test whether differences in the magnitude of oscillatory amplitude of the clock genes Bmal1, Per1 and Per2 might account for period and phase differences, we quantified the transcriptional gene expression of clock genes in dermal fibroblasts by a different method because the measurement of the bioluminescent oscillation of clock genes which is sensitive to the degree of viral infection is not suitable to quantify the transcriptional amplitude. Thus we measured RNA levels of clock genes by means of quantitative real-time PCR (qPCR) and complemented these data with the bioluminescence assay. Total RNA was extracted from fibroblast cell lines every 6h over 72h and totalRNA was reverse transcribed into cDNA followed by qPCR using the primer for the clock genes. Results In fibroblasts from patients suffering from idiopathic hypersomnia clock gene expression exhibit a diminished amplitude of BMAL1 which was significantly reduced by 63% ( P =0.004) compared to healthy controls. The amplitude of PER1 was reduced by 45% ( P =0.048) compared to the control group so was the amplitude of PER2 reduced by 32% ( P =0.032). In the luciferase assay using the same fibroblast cell lines,an averaged period length of 24.6h could be detected in dermal fibroblasts obtained from healthy volunteers. Conclusion Due to the significantly diminished amplitude of clock gene expression in the group of idiopathic hypersomniacs, our data suggest a disturbed chronobiology as a heterogeneous aspect in a primary sleep disorder. The implementation of this combined strategy to measure the dynamics of clock gene expression permits a reliable assessment – in objective statistical terms – of both the period length and the absolute magnitude of oscillatory amplitude. The coupling of the circadian gene regulation with individual EEG-parameter following the principles of the two-process-model of sleep regulation can be a useful tool to quantify therapeutic agents and regimens modifying circadian disruption (e.g. period length, phase and amplitude of circadian rhythms) and to develop possible personalized therapeutic options.

CGMP kinase: past, presence and future

Signaling via cGMP-dependent protein kinase is the major pathway of the NO/cGMP cascade in vascular smooth muscle (SM), heart, CNS, and other cells. cGMP-dependent protein kinases (cGK) are serine/threonine kinases that are widely distributed in eukaryotes. Two genes – prkg1 and prkg2 - code for cGKs, namely cGKI and cGKII. In mammals, two isozymes, cGKIα and cGKIβ, are generated from the prkg1 gene. The cGKI isozymes are prominent in all types of smooth muscle, platelets and specific neuronal areas such as cerebellar Purkinje cells, hippocampal neurons, and the lateral amygdala. The cGKII prevails in the secretory epithelium of the small intestine, the juxta-glomerular cells, the adrenal cortex, the chondrocytes, and in the nucleus suprachiasmaticus. Both cGKs are major down- stream effectors of many, but not all signalling events of the NO/cGMP and the ANP/BNP/CNP/cGMP pathways. cGKI relaxes smooth muscle tone and prevents platelet aggregation, whereas cGKII inhibits renin secretion, chloride/water secretion in the small intestine, the resetting of the clock during early night and endochondreal bone growth. cGKs are involved in cardiovascular and non-cardiovascular processes and are modulators of cell growth and many other functions. Recent evidence suggest that vascular smooth muscle relaxation is not affected by cGK-dependent phosphorylation of smooth muscle expressed TRPC channels, but by cGK-dependent inhibition of TRPC-6 in endothelial cells and attenuation of endothelial release of smooth muscle relaxing factors. Angiotensin increases blood pressure and induces cardiac hypertrophy. Hypertrophy is reduced in sildenafil treated wild type mice but not in beta rescue mice. This suggests that the sildenafil effects are mediated by inhibition of a cGMP hydrolysing phosphodiesterase. Restricted evidence supports the notion that sildenafil affects cGMP levels in cardiomyofibroblasts and/or in cardiac endothelial cells. It will be shown that cGK are essential modulators of many regulatory processes.

The control of brain mitochondrial energization by cytosolic calcium: The mitochondrial gas pedal

This review focuses on problems of the intracellular regulation of mitochondrial function in the brain via the (i) supply of mitochondria with ADP by means of ADP shuttles and channels and (ii) the Ca(2+) control of mitochondrial substrate supply. The permeability of the mitochondrial outer membrane for adenine nucleotides is low. Therefore rate dependent concentration gradients exist between the mitochondrial intermembrane space and the cytosol. The existence of dynamic ADP gradients is an important precondition for the functioning of ADP shuttles, for example CrP-shuttle. Cr at mM concentrations instead of ADP diffuses from the cytosol through the porin pores into the intermembrane space. The CrP-shuttle isoenzymes work in different directions which requires different metabolite concentrations mainly caused by dynamic ADP compartmentation. The ADP shuttle mechanisms alone cannot explain the load dependent changes in mitochondrial energization, and a complete model of mitochondrial regulation have to account the Ca(2+) -dependent substrate supply too. According to the old paradigmatic view, Ca(2+) (cyt) taken up by the mitochondrial Ca(2+) uniporter activates dehydrogenases within the matrix. However, recently it was found that Ca(2+) (cyt) at low nM concentrations exclusively activates the state 3 respiration via aralar, the mitochondrial glutamate/aspartate carrier. At higher Ca(2+) (cyt) (> 500 nM), brain mitochondria take up Ca(2+) for activation of substrate oxidation rates. Since brain mitochondrial pyruvate oxidation is only slightly influenced by Ca(2+) (cyt) , it was proposed that the cytosolic formation of pyruvate from its precursors is tightly controlled by the Ca(2+) dependent malate/aspartate shuttle. At low (50-100 nM) Ca(2+) (cyt) the pyruvate formation is suppressed, providing a substrate limitation control in neurons. This so called "gas pedal" mechanism explains why the energy metabolism of neurons in the nucleus suprachiasmaticus could be down-regulated at night but activated at day as a basis for the circadian changes in Ca(2+) (cyt) . It also could explain the energetic disadvantages caused by altered Ca(2+) (cyt) at mitochondrial diseases and neurodegeneration.

α The generation and importance of circadian rhythms through the action of clock genes in the endocrine pancreas

Higher vertebrates have evolved mechanisms to measure time and to adopt body functions to the physical change of night and day. These so-called „biological clocks“ generate a rhythm with a phase length (τ) of approximately 24 hr. To adjust these „circa-dian“ clocks to the exact 24h day, these endogen biological clocks need to be adjusted, or synchronized. In mammals, the central circadian oscillator is localized in specialized neurons of the hypothalamic nucleus suprachiasmaticus (SCN). On a molecular level, the circadian rhythm is generated due to the activity of so-called „clock“ genes. These genes code for either transcription promoting (e.g. BMAL1/CLOCK) or suppressing (e.g. PER1/CRY1) factors which are interrelated through transcriptional/translational feedback loops acting on so-called E-box promoter sequences. The circadian clock impinges on other transcription factors like Dbp or RevErbα on the output side of the circadian system, carrying circadian information and activating many other genes. In addition, it has recently become evident that clock genes function even on the level of single cells, displaying a circadian rhythm.

Sleep Disturbances and Glucose Homeostasis

Sleep disturbances, induced by either lifestyle, shift work or sleeping disorders, have become more prevalent in our 24/7 Western society. Sleep disturbances are associated with impaired health including metabolic diseases such as obesity and type 2 diabetes. The question remains whether there is a direct effect of disturbed sleep on glucose homeostasis. Experimental studies under controlled laboratory conditions in both humans and experimental animals revealed that there are differences between the effects of acute or chronic sleep disturbance. Acute sleep restriction clearly leads to glucose intolerance, often combined with insulin resistance. Although glucose intolerance does also occur after chronic sleep disturbance, the changes in insulin can vary, dependent on the body weight changes in the various studies. The underlying mechanism that might cause the changes in glucose homeostasis after sleep disturbance remains unclear, but both the biological clock located in the nucleus suprachiasmaticus as well as orexinergic mechanisms in brain and periphery seem to be involved.

Orexin-A immunoreactive cells and fibers in the central nervous system of the axolotl brain and their association with tyrosine hydroxylase and serotonin immunoreactive somata

Orexin-A-like immunoreactivity in the axolotl brain was investigated by immunohistochemistry. Immunoreactive somata formed a single group in the hypothalamus, but were distributed beyond several nuclei, namely, the ventral aspect of the nucleus preopticus posterior, dorsal aspect of the nucleus suprachiasmaticus and anterior aspect of the pars ventralis hypothalami. Immunoreactive fibers were distributed throughout the brain from the olfactory bulb to the spinal cord except the cerebellum. The densest immunoreactive fibers were seen in the medial forebrain bundle and caudal lateral forebrain bundle. The largest number of immunoreactive puncta were seen in the mesencephalic tectum in addition to the hypothalamus. Immunoelectron microscopic analysis revealed the presence of synaptoid connections of immunoreactive fibers on neuronal somata in the tectum. The function of the mesencephalic system in the urodele seems to be sensory integration, suggesting that the orexin-A nervous system is associated with the modulation of sensory inputs. Orexin-A immunoreactive puncta were also observed on catecholaminergic and serotonergic somata. In view of the restricted somatic distribution in the hypothalamus, wide distribution of fibers throughout the central nervous system (CNS), and intimate association with monoaminergic somata, the orexin nervous system in the axolotl CNS is similar to those of other vertebrates, suggesting that this system is essential for brain functions throughout vertebrates.

Rhythm‐Dependent Light Induction of the c‐fos Gene in the Turkey Hypothalamus

Day length (photoperiod) is a powerful synchroniser of seasonal changes in the reproductive neuroendocrine activity in temperate-zone birds. When exposed to light during the photoinducible phase, reproductive neuroendocrine responses occur. However, the neuroendocrine systems involved in avian reproduction are poorly understood. We investigated the effect of light exposure at different circadian times upon the hypothalamus and components of the circadian system, using c-fos mRNA expression, measured by in situ hybridisation, as an indicator of light-induced neuronal activity. Levels of c-fos mRNA in these areas were compared after turkey hens (on a daily 6-h light period) had been exposed to a 30-min period of light occurring at 8, 14, or 20 h after the onset of first light of the day (subjective dawn). Non-photostimulated control birds were harvested at the same times. In birds, photostimulated within the photoinducibile phase (14 h), in contrast to before or after, c-fos mRNA was significantly increased in the nucleus commissurae pallii (nCPa), nucleus premamillaris (PMM), eminentia mediana (ME), and organum vasculosum lamina terminalis (OVLT). Photostimulation increased c-fos mRNA expression in the pineal gland, nucleus suprachiasmaticus, pars visualis (vSCN) and nucleus inferioris hypothalami compared to that of their corresponding nonphotostimulated controls. However, the magnitudes of the responses in these areas were similar irrespective of where in the dark period the pulses occurred. No c-fos mRNA was induced in the nucleus infundibulari, in response to the 30-min light period at any of the circadian times tested. The lack of c-fos up-regulation in the pineal gland and vSCN following photostimulation during the photoinducible phase lends credence to the hypothesis that these areas are not involved in the photic initiation of avian reproduction. On the other hand, c-fos mRNA increases in the nCPa, ME, and OVLT support other studies showing that these areas are involved in the onset of reproductive behaviour initiated by long day lengths. The present study provides novel data showing that the PMM in the caudal hypothalamus is involved in the neuronally mediated, light-induced initiation of reproductive activity in the turkey hen.

Retinal projections in two crocodilian species, Caiman crocodilus and Crocodylus niloticus.

The retinal projections of Caiman crocodilus and Crocodylus niloticus were investigated by means of the orthograde axonal transport of either rhodamine beta-isothiocyanate or tritiated proline. In these two species, each tracer revealed contralateral retinal projections to three hypothalamic regions (subventricular gray matter, nucleus suprachiasmaticus, and area optica hypothalami lateralis), five thalamic regions (nuclei ovalis, dorsolateralis anterior, ventrolateralis and ventrobasalis, and lateral geniculate complex, of which six subcomponents can be distinguished), six pretectal regions (nuclei posterodorsalis, lentiformis mesencephali, griseus tectalis, geniculatus pretectalis, area optica commissurae posterior and area optica pretectalis lateroventralis), six outermost layers of the optic tectum, and the nucleus opticus tegmenti. Weak ipsilateral retinal projections have been observed in two hypothalamic nuclei and in the nucleus opticus tegmenti. Comparative analysis with other data show that the contralateral retinal projections of crocodiles are considerably more reptilian than avian. Moreover, crocodiles share with birds an extremely poor contingent of ipsilateral retinal projections.

Urinary 6-sulfatoxymelatonin profiles in male Djungarian hamsters (Phodopus sungorus) responding and not responding to short-day photoperiods: possible role of elevated daytime levels.

The lack of endocrine and physiological responses of some Djungarian hamsters (Phodopus sungorus) to the transition from long to short photoperiods (L:D 16:8 --> L:D 8:16) has been known for a long time but is not yet understood. We investigated the role of melatonin synthesis in this context because melatonin, as part of the circadian system, may play a role in non-responsiveness. In ten responding and ten non-responding male hamsters, the urinary 24 hr 6-sulfatoxymelatonin (aMT6s) profiles under L:D 8:16 and L:D 16:8 were measured. Both short day responding and non-responding hamsters showed diurnal aMT6s excretion rhythms. Whereas responders reacted to the transition L:D 16:8 --> L:D 8:16 with a marked elevation of aMT6s excretion, in non-responders no adjustment of the melatonin rhythm to the change of the photoperiod was seen. Furthermore, under L:D 16:8 the daytime levels of aMT6s were significantly (P<0.001) lower in responders compared to non-responders whereas under L:D 8:16 these levels were higher (P<0.01). It is speculated that high daytime levels of aMT6s under long-day photoperiods in non-responders result in down-regulation of melatonin receptors of the nucleus suprachiasmaticus, the pacemaker for the pineal gland, leading to a lack of response to the transition to short-day photoperiods.

Rest-activity rhythm of the blind mole rat Spalax ehrenbergi under different lighting conditions

The mole rat is a solitary, subterranean and photoperiodic rodent. We investigated its rest activity behavior under several lighting conditions, complemented our observations with light-induced c-fos expression, and compared the activity behavior of two chromosomal forms (2n=58 and 60). The 26 mole rats had a clear overall preference for activity in the light or dark period, but prolonged recordings in five individuals showed that the initial preference was not stable in the nocturnal animals, they became diurnal. A 6-h advance of the light-dark (LD) cycle induced a shift of activity and the previous LD preference was reestablished. The large daily variability of activity onset did not allow this study to determine whether the animals were entrained to the LD cycle upon release into constant darkness (DD) or whether activity had been masked by light. The period of the motor activity rhythm in DD free ran in more than 50% of the animals. No differences in activity were observed between the two karyotypes. Immunohistochemistry for c-fos expression in the nucleus suprachiasmaticus at different circadian times showed that c-fos was induced only in animals exposed to a 1-h light pulse during the subjective night, but not during the subjective day or in control animals in the absence of a light pulse. The large intra- and inter-individual variability in daily motor activity both in LD and in DD suggest only a weak photic entrainment of the circadian clock to light of approximately 100 lux, and possibly a weak regulation of behavior by the circadian clock.

Pharmacological characterization, anatomical distribution and sex differences of the non-NMDA excitatory amino acid receptors in the quail brain as identified by CNQX binding

The distribution of non- N-methyl- d-aspartate binding sites was studied in coronal and sagittal sections through the brain of adult Japanese quail by quantitative autoradiography, using tritiated 6-cyano-7-nitroquinoxaline-2,3-dione as a radioligand. Saturation binding experiments were, in addition, carried out in areas showing high levels of binding (cerebellar molecular layer, nucleus anterior medialis and nucleus infundibularis) and demonstrated that the binding of tritiated ligand was specific and saturable. Competition studies with α-amino-3-hydroxy-methyl-4-isoxazole propionic acid and kainic acid indicated that kainic acid strongly inhibited ligand binding in all brain areas. α-Amino-3-hydroxy-methyl-4-isoxazole propionic acid was only a weak inhibitor in the hypothalamic nuclei whereas in the cerebellar molecular layer both high and low affinity inhibitions were detected. The highest binding levels of tritiated ligand were observed in the molecular layer of the cerebellum. Very high levels of binding were detected in various preoptic/hypothalamic sites including the nucleus suprachiasmaticus pars medialis, nucleus anterior medialis hypothalami, nucleus infundibularis, nucleus mammillaris medialis, nucleus posteromediale hypothalami and nucleus hypothalami ventromedialis. High levels of binding were also detected in the bulbus olfactorius, bed nucleus commissuralis anterior, bed nucleus commissuralis pallii, nucleus accumbens, bed nucleus striae terminalis and nucleus interpeduncularis. In the preoptic area/hypothalamus, high levels of binding were clearly present in all areas that contain gonadotropin releasing hormone cells or fibers. In the pons and mesencephalon, moderate levels of binding were associated with catecholaminergic areas such as the area ventralis tegmentalis (area ventralis of Tsai) and the locus coeruleus. Saturation analysis demonstrated the presence of a higher number of binding sites in females than in males in the cerebellar molecular layer, nucleus infundibularis and nucleus anterior medialis. This latter difference was confirmed in the one point assays that also identified higher levels of specific binding in the nucleus suprachiasmaticus pars medialis of males as compared with females. These anatomical data suggest a possible implication of non- N-methyl- d-aspartate receptors in the synthesis and/or release of both gonadotropin releasing hormone and catecholaminergic neurotransmitters that should now be tested by pharmacological experiments.

Distribution and expression of glucocorticoid receptor mRNA in the forebrain of the rainbow trout.

The expression and distribution of glucocorticoid receptor mRNA was studied in the forebrain of mature female and immature undifferentiated rainbow trout (Oncorhynchus mykiss) by means of Northern blotting and in situ hybridization. A single mRNA species of 7.5 kb was detected in mRNA polyA+ prepared from the anterior brain. In situ hybridization was carried out using a 35S-labelled riboprobe corresponding to the A/B-domain (between nucleotides 1224 and 1763) of the recently cloned rainbow trout glucocorticoid receptor cDNA. Comparison of adjacent sections hybridized with the sense and antisense probes allowed detection of a specific signal with a similar distribution pattern in all animals studied. In the telencephalon, a specific hybridization was detected in scattered cells of the dorsal telencephalic hemisphere, but the stronger signal was consistently observed in the dorsal nucleus, and to a lesser degree in the ventral nucleus of the ventral telencephalon. Heavy hybridization staining was consistently observed in all subdivisions of the preoptic nucleus and the nucleus lateralis tuberis, which are the main hypophysiotrophic regions in fish. A weaker signal was detected in the nucleus anterioris periventricularis, nucleus suprachiasmaticus and thalamic region. The presence of a strong signal in virtually all magnocellular neurons of the nucleus preopticus, known for producing vasotocin, isotocin and corticotropin-releasing factor favors a direct function of cortisol in regulating its own secretion.

Circannual morphometric investigations of the rat suprachiasmatic nucleus after pinealectomy, ganglionectomy and thyroidectomy

We karyometrically investigated the nucleus suprachiasmaticus (SCN) which had been manipulated in several ways in order to analyze the functional importance of the pineal gland on the primary pacemaker of mammals in the course of the year. The manipulation modes were (i) pinealectomy (PX), and (ii) sympathetic denervation of the pineal by bilateral extirpation of the upper cervical ganglia (ganglion ectomy, GX). Additionally, the influence of the inactivated pineal, obtained through hypothyroidism which was realized by (iii) subtotal thyroidectomy (TX), was also investigated. With respect to annual oscillations the results of our investigations were able to illustrate the following. (1) The SCN consists of at least two parts (ventrolateral and dorsomedial) each with different functions and relationships. The nuclei of the ventrolateral cells are bigger and there are many indications both in our own research and from literature that the neurons of this part are involved in the generation of rhythms. (2) The size of the cell nuclei of the ventrolateral part shows annual patterns. In the course of the year the maxima of the nuclear volume were registered in March and September (bimodal pattern, equinox, L:D = 12:12). PX, GX or TX only negligibly changed the bimodal annual pattern. However, in comparison the smallest cell nuclei were registered in the winter (short day). The much smaller cell nuclei of the dorsomedial part likewise show bimodal patterns but only in the experimental groups. The control group of this part shows an unimodal annual curve with a minimum at long-day conditions (June. L:D = 16:8). (3) All manipulations which inactivated the pineal or reduced the content of melatonin (PX, GX, and TX) were followed by an increase (activation) of cell nuclei of the SCN. In contrast to these effects, an increase of thyroxine (by exposure to cold), has an opposite effect (not documented here). In conclusion these results indicate, without a doubt, that a negative correlation exists, functionally, between the SCN and the pineal (in the same annual experiment the nuclear size of the pinealocytes was increased, under short-day conditions in December, and decreased under long-day circumstances in June). Additionally, it could be shown that the degree of negative correlation between the pineal and the SCN was seasonally dependent. The lowest effects of PX, GX and TX were registered at short-day conditions (December, L:D = 8:16).

Distribution of 2-

Using in vitro autoradiography, the distribution of 2-[125I]iodomelatonin (IMEL) binding sites in the brain of the pied flycatcher and the zebra finch was examined. The results show IMEL binding in retinofugal, tectofugal and thalamofugal brain areas of the visual system. Additionally, IMEL binding sites were detected in the ectostriatum, the thalamus, the mesencephalon and the limbic system. No IMEL binding could be demonstrated in the pineal gland, the hippocampus, the nucleus suprachiasmaticus, the visual wulst or the pituitary.

Glutaminyl-cyclase expression in the bovine/porcine hypothalamus and pituitary.

The expression of the glutaminyl cyclase (QC), an enzyme responsible for the post-translational modification of N-terminal pyroglutamyl residues of neuropeptide precursors, was examined in bovine/porcine hypothalamic and pituitary tissue by means of immunocytochemistry and in situ hybridization. In the anterior pituitary a distinct pattern of QC immunoreactivity and mRNA expression was found associated exclusively with somatotrophs. In corticotrophs of the pars intermedia QC expression was undetectable, whereas a small portion of putative pars tuberalis cells in the rostral part of the pars distalis were heavily labelled. The neurointermediate lobe was devoid of signals for QC mRNA, but showed significant QC-immunoreactivity on secretory granules of axonal nerve endings. Also nuclei of the hypothalamus were found to be positive for QC-immunoreactivity. Intense labelling was observed in the nucleus supraopticus and nucleus paraventricularis. Staining of the nucleus periventricularis was found to be moderate, whereas no labelling of perikarya in the nucleus arcuatus, the preoptic area and the nucleus suprachiasmaticus was detectable. Moreover, varicose fibers stained positive for QC-immunoreactivity and could be identified in the main transport route from the hypothalamus to the pars neuronalis (tractus hypothalamo hypophysialis). These results suggest that the enzyme is transported via the same routes as its substrate/product to the median eminence or the neural lobe. Furthermore, the mapping of the cellular QC distribution reveals a strikingly distinctive expression pattern, that should be useful for the identification of yet undiscovered places of peptide synthesis and processing.

Effects of retinal lesions upon the distribution of nicotinic acetylcholine receptor subunits in the chick visual system

Immunohistochemistry was used in this study to evaluate the effects of retinal lesions upon the distribution of neuronal nicotinic acetylcholine receptor subunits in the chick visual system. Following unilateral retinal lesions, the neuropil staining with an antibody against the beta 2 receptor subunit, a major component of alpha-bungarotoxin-insensitive nicotinic receptors, was dramatically reduced or completely eliminated in all of the contralateral retinorecipient structures. On the other hand, neuropil staining with antibodies against two alpha-bungarotoxin-sensitive receptor subunits, alpha 7 and alpha 8, was only slightly affected after retinal lesions. Decreased neuropil staining for alpha 7-like immunoreactivity was only observed in the nucleus of the basal optic root and layers 2-4 and 7 of the optic tectum. For alpha 8-like immunoreactivity, slight reduction of neuropil staining could be observed in the ventral geniculate complex, griseum tecti, nucleus lateralis anterior, nucleus lentiformis mesencephali, layers 4 and 7 of the tectum, and nucleus suprachiasmaticus. Taken together with previous data on the localization of nicotinic receptors in the retina, the present results indicate that the beta 2 subunit is transported from retinal ganglion cells to their central targets, whereas the alpha 7 and alpha 8 subunit immunoreactivity appears to have a central origin. The source of these immunoreactivities could be, at least in part, the stained perikarya that were observed to contain alpha 7 and alpha 8 subunits in all retinorecipient areas. In agreement with this hypothesis, the beta 2 subunit of the nicotinic acetylcholine receptors was not frequently found in perikarya of the same areas.

Distribution of FMRFamide-like immunoreactivity in the brain of the lizard Podarcis sicula

The distribution of FMRFamide-like immunoreactive peptides was investigated in the brain of the lizard, Podarcis sicula, using the indirect immunofluorescence technique. The main populations of FMRFamide-immunoreactive cell bodies were located in the forebrain. In the telencephalon, FMRFamide-containing neurons were found both in the pallium and subpallium, namely in the medial cortex, the anterior olfactory nucleus, the nucleus accumbens, the septal nuclei, the nucleus of the medial forebrain bundle, and the nucleus of the diagonal band of Broca. In the diencephalon, a dense accumulation of FMRFamide-immunoreactive neurons was observed in the area preoptica lateralis, the nucleus suprachiasmaticus, the nucleus periventricularis hypothalami, the area lateralis hypothalami, and the dorsal region of the nucleus geniculatus lateralis. In the midbrain, sparse immunoreactive perikarya were found in the tegmentum of the mesencephalon. FMRFamide-immunoreactive fibers were visualized in all regions containing positive cell bodies. In particular, dense bundles of immunoreactive processes were seen in the area preoptica lateralis, in the hypothalamus, and in the median eminence. The tectum and the basal mesencephalon were also densely innervated. Conversely, the caudal brain stem only exhibited scarce immunoreactive processes. The distribution pattern of FMRFamide-immunoreactive neurons in the brain of Podarcis sicula exhibits a number of similarities with that reported in mammals, but significantly differs from that reported in amphibians and fish, suggesting that the neuromodulatory functions of FMRFamide may have diverged during the emergence of terrestrial life.

An experimental re-evaluation of the primary visual system of the European chameleon, Chamaeleo chameleon.

The retinofugal projections of the chameleon were investigated by means of autoradiography, horseradish peroxidase and fluorescent techniques after intraocular injection of tracers. An ipsilateral contingent of visual fibers and projections is absent. The retinal fibers decussate completely in alternating fascicles at the optic chiasma and course to terminate in two hypothalamic nuclei (nucleus suprachiasmaticus and nucleus opticus periventricularis hypothalami posterior), six thalamic nuclei (nucleus ovalis, nucleus geniculatus lateralis dorsalis partes lateralis and medialis, nucleus geniculatus lateralis ventralis, lateral part of nucleus dorsolateralis anterior, and nucleus ventrobasalis), four pretectal nuclei (nucleus griseus tectalis, nucleus lentiformis mesencephali, nucleus geniculatus pretectalis and nucleus posterodorsalis), the optic tectum (stratum griseum et fibrosum superficiale) and the tegmental nucleus opticus tegmenti. Our findings are, in general, compatible with previous descriptions of the primary visual system in other species of lizards. However, they indicate three features particular to chameleons: first, the total absence of an ipsilateral retinofugal projection; second, the existence of an additional hypothalamic visual center located in the posterior mediodorsal hypothalamus; and third, the large size of nucleus opticus tegmenti. These features are discussed in terms of the taxonomic position of chameleons with respect to other lizards.

Influence of age and hepatic branch vagotomy on the night/day distribution of food intake in rats

The aim of the present study was to investigate the influence of age and hepatic branch vagotomy on the night/day distribution of food intake in male rats. Food intake of young (age: 2 months) and aged (age: 13.5 months) hepatic branch vagotomized (HBV) and sham-vagotomized (SV) rats was measured at intervals of 6 h during the 12 h dark and the 12 h light phase. The results show that in rats the night/day ratio of food intake decreases with age and is not affected by hepatic branch vagotomy. However, the time-course of spontaneous diurnal food intake was influenced by hepatic branch vagotomy. The change in the night/day distribution of food intake might be due to age-related changes in the nucleus suprachiasmaticus of the hypothalamus.

Cytoarchitectonic pattern of the hypothalamus in the turtle, Lissemys punctata granosa

In the hypothalamus of the turtle, Lissemys punctata granosa, two magnocellular and 23 parvocellular neuronal complexes can be distinguished. The magnocellular complexes include the nucleus supraopticus and the nucleus paraventricularis; paraventricular neurons are partly arranged in rows parallel to the third ventricle. Most infundibular parvocellular nuclei display neurons disposed in rows parallel to the ventricular surface. In the preoptic region, the prominent parvocellular neuronal complexes encompass the nucleus periventricularis anterior, lateral preoptic area, the nucleus of the anterior commissure and the nucleus suprachiasmaticus. The prominent nucleus periventricularis posterior extends caudad and shows neurons arranged in vertical rows parallel to the third ventricle. Other parvocellular nuclei of the rostral hypothalamus are composed of clustered subunits. The nucleus arcuatus is a fairly large nuclear entity extending from the level marked dorsally by the nucleus paraventricularis to the area occupied by the nucleus of the paraventricular organ. A well-developed ventromedial nucleus is located ventrolateral to the paraventricular organ. The prominent paraventricular organ consists of tightly arranged neurons, some of which possess apical projections into the third ventricle; it is surrounded by the nucleus of the paraventricular organ. Nucleus hypothalamicus medialis et lateralis, nucleus hypothalamicus posterior and the nuclei recessus infundibuli are further nuclear units of the tuberal region. The caudal end of the hypothalamus is marked by the nucleus mamillaris; its neurons are scattered among the fibers of the retroinfundibular commissure. The median eminence is well developed and shows a large medial and two lateral protrusions into the infundibular recess.