Increased glutamic acid decarboxylase expression in the hypothalamic suprachiasmatic nucleus in depression.

In depression, disrupted circadian rhythms reflect abnormalities in the central circadian pacemaker, the hypothalamic suprachiasmatic nucleus (SCN). Although many SCN neurons are said to be GABAergic, it was not yet known whether and how SCN GABA changes occur in the SCN in depression. We, therefore, studied GABA in the SCN in relation to the changes in arginine vasopressin (AVP), which is one of the major SCN output systems. Postmortem hypothalamus specimens of 13 subjects suffering from depression and of 13 well-matched controls were collected. Quantitative immunocytochemistry was used to analyze the protein levels of glutamic acid decarboxylase (GAD)65/67 and AVP, and quantitative in situ hybridization was used to measure transcript levels of GAD67 in the SCN. There were a significant 58% increase of SCN GAD65/67-ir and a significant 169% increase of SCN GAD67-mRNA in the depression group. In addition, there were a significant 253% increase of AVP-ir in female depression subjects but not in male depression patients. This sex difference was supported by a re-analysis of SCN AVP-ir data of a previous study of our group. Moreover, SCN-AVP-ir showed a significant negative correlation with age in the control group and in the male, but not in the female depression group. Given the crucial role of GABA in mediating SCN function, our finding of increased SCN GABA expression may significantly contribute to the disordered circadian rhythms in depression. The increased SCN AVP-ir in female-but not in male-depression patients-may reflect the higher vulnerability for depression in women.

Localization and expression of GABA transporters in the suprachiasmatic nucleus.

GABA is a principal neurotransmitter in the suprachiasmatic hypothalamic nucleus (SCN), the master circadian clock. Despite the importance of GABA and GABA uptake for functioning of the circadian pacemaker, the localization and expression of GABA transporters (GATs) in the SCN has not been investigated. The present studies used Western blot analysis, immunohistochemistry and electron microscopy to demonstrate the presence of GABA transporter 1 (GAT1) and GAT3 in the SCN. By using light microscopy, GAT1 and GAT3 were co-localized throughout the SCN, but were not expressed in the perikarya of arginine vasopressin- or vasoactive intestinal peptide-immunoreactive (-ir) neurons of adult rats, nor in the neuronal processes labelled with the neurofilament heavy chain. Using electron microscopy, GAT1- and GAT3-ir was found in glial processes surrounding unlabelled neuronal perikarya, axons, dendrites, and enveloped symmetric and asymmetric axo-dendritic synapses. Glial fibrillary acidic protein-ir astrocytes grown in cell culture were immunopositive for GAT1 and GAT3 and both GATs could be observed in the same glial cell. These data demonstrate that synapses in the SCN function as 'tripartite' synapses consisting of presynaptic axon terminals, postsynaptic membranes and astrocytes that contain GABA transporters. This model suggests that astrocytes expressing both GATs may regulate the extracellular GABA, and thereby modulate the activity of neuronal networks in the SCN.

Selective Distribution of Retinal Input to Mouse SCN Revealed in Analysis of Sagittal Sections.

The suprachiasmatic nucleus (SCN) is the locus of the master circadian clock, setting the daily rhythms in physiology and behavior and synchronizing these responses to the local environment. The most important of these phase-setting cues derive from the light-dark cycle and reach the SCN directly via the retinohypothalamic tract (RHT). The SCN contains anatomically and functionally heterogeneous populations of cells. Understanding how these neurons access information about the photic environment so as to set the phase of daily oscillation requires knowledge of SCN innervation by the RHT. While retinal innervation of the SCN has long been a topic of interest, the information is incomplete. In some instances, studies have focused on the caudal aspect of the nucleus, which contains the core region. In other instances, subregions of the nucleus have been delineated based on projections of where specific peptidergic cell types lie, rather than based on double or triple immunochemical staining of distinct populations of cells. Here, we examine the full extent of the mouse SCN using cholera toxin ß (CTß) as a tracer to analyze RHT innervation in triple-labeled sagittal sections. Using specific peptidergic markers to identify clusters of SCN cells, we find 3 distinct patterns. First is an area of dense RHT innervation to the core region, delineated by gastrin-releasing peptide (GRP) and vasoactive intestinal peptide (VIP) immunoreactive cells. Second is an area of moderate RHT fiber clusters, bearing arginine-vasopressin (AVP)-positive cells that lie close to the core. Finally, the outermost, shell, and rostral AVP-containing regions of the SCN have few to no detectable retinal fibers. These results point to a diversity of inputs to individual SCN cell populations and suggest variation in the responses that underlie photic phase resetting.

The hypothalamus in Alzheimer's disease: a Golgi and electron microscope study.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder, characterized by irreversible decline of mental faculties, emotional and behavioral changes, loss of motor skills, and dysfunction of autonomic nervous system and disruption of circadian rhythms (CRs). We attempted to describe the morphological findings of the hypothalamus in early cases of AD, focusing our study mostly on the suprachiasmatic nucleus (SCN), the supraoptic nucleus (SON), and the paraventricular nucleus (PVN). Samples were processed for electron microscopy and silver impregnation techniques. The hypothalamic nuclei demonstrated a substantial decrease in the neuronal population, which was particularly prominent in the SCN. Marked abbreviation of dendritic arborization, in association with spinal pathology, was also seen. The SON and PVN demonstrated a substantial number of dystrophic axons and abnormal spines. Alzheimer's pathology, such as deposits of amyloid-ß peptide and neurofibrillary degeneration, was minimal. Electron microscopy revealed mitochondrial alterations in the cell body and the dendritic branches. The morphological alterations of the hypothalamic nuclei in early cases of AD may be related to the gradual alteration of CRs and the instability of autonomic regulation.

Efferent projections of the suprachiasmatic nucleus based on the distribution of vasoactive intestinal peptide (VIP) and arginine vasopressin (AVP) immunoreactive fibers in the hypothalamus of Sapajus apella.

The suprachiasmatic nucleus (SCN), which is considered to be the master circadian clock in mammals, establishes biological rhythms of approximately 24 h that several organs exhibit. One aspect relevant to the study of the neurofunctional features of biological rhythmicity is the identification of communication pathways between the SCN and other brain areas. As a result, SCN efferent projections have been investigated in several species, including rodents and a few primates. The fibers originating from the two main intrinsic fiber subpopulations, one producing vasoactive intestinal peptide (VIP) and the other producing arginine vasopressin (AVP), exhibit morphological traits that distinguish them from fibers that originate from other brain areas. This distinction provides a parameter to study SCN efferent projections. In this study, we mapped VIP (VIP-ir) and AVP (AVP-ir) immunoreactive (ir) fibers and endings in the hypothalamus of the primate Sapajus apella via immunohistochemical and morphologic study. Regarding the fiber distribution pattern, AVP-ir and VIP-ir fibers were identified in regions of the tuberal hypothalamic area, retrochiasmatic area, lateral hypothalamic area, and anterior hypothalamic area. VIP-ir and AVP-ir fibers coexisted in several hypothalamic areas; however, AVP-ir fibers were predominant over VIP-ir fibers in the posterior hypothalamus and medial periventricular area. This distribution pattern and the receiving hypothalamic areas of the VIP-ir and AVP-ir fibers, which shared similar morphological features with those found in SCN, were similar to the patterns observed in diurnal and nocturnal animals. This finding supports the conservative nature of this feature among different species. Morphometric analysis of SCN intrinsic neurons indicated homogeneity in the size of VIP-ir neurons in the SCN ventral portion and heterogeneity in the size of two subpopulations of AVP-ir neurons in the SCN dorsal portion. The distribution of fibers and morphometric features of these neuronal populations are described and compared with those of other species in the present study.

Circadian rhythm of enolase in suprachiasmatic nucleus depends on mitochondrial function.

Metabolic activity in the suprachiasmatic nucleus (SCN), a center of biological rhythm, is higher during the daytime than at night. The rhythmic oscillation in the SCN is feedback controlled by the CLOCK/BMAL1 heterodimer binding to the E-box in target genes (e.g., Arg- vasopressin). Similar transcriptional regulation by NPAS2/BMAL1 heterodimer formation operates in the brain, which depends on the redox state (i.e., NAD/NADH). To clarify the metabolic function of SCN in relation to the redox state, two-dimensional electrophoresis was carried out on the mitochondrial fraction of SCN, obtained from rats kept under a light:dark cycle and constant under dim light. The electrophoretic pattern with TOF-mass spectrometry analysis revealed that enolase catalyzes the interconversion of 2-phosphoglycerate and phosphoenolpyruvate. The enolase activity, coupled with lactate dehydrogenase, was higher during the light period than that in the dark. However, enolase mRNA, analyzed by RT-PCR, showed higher levels during the dark period than in the light. The clock gene products Per2, Bmal1, Rev-erbα, and AVP mRNA in the mitochondrial fraction of SCN developed a circadian rhythm showing almost the same peak time as that in whole SCN. These mRNA rhythms ran free except for that of Rev-erbα mRNA. The results indicate that, in the glycolysis-related energy pathway, enolase might be involved in higher metabolic activity during the day than at night, at least in part.

Intrinsic regulation of spatiotemporal organization within the suprachiasmatic nucleus.

The mammalian pacemaker in the suprachiasmatic nucleus (SCN) contains a population of neural oscillators capable of sustaining cell-autonomous rhythms in gene expression and electrical firing. A critical question for understanding pacemaker function is how SCN oscillators are organized into a coherent tissue capable of coordinating circadian rhythms in behavior and physiology. Here we undertake a comprehensive analysis of oscillatory function across the SCN of the adult PER2::LUC mouse by developing a novel approach involving multi-position bioluminescence imaging and unbiased computational analyses. We demonstrate that there is phase heterogeneity across all three dimensions of the SCN that is intrinsically regulated and extrinsically modulated by light in a region-specific manner. By investigating the mechanistic bases of SCN phase heterogeneity, we show for the first time that phase differences are not systematically related to regional differences in period, waveform, amplitude, or brightness. Furthermore, phase differences are not related to regional differences in the expression of arginine vasopressin and vasoactive intestinal polypeptide, two key neuropeptides characterizing functionally distinct subdivisions of the SCN. The consistency of SCN spatiotemporal organization across individuals and across planes of section suggests that the precise phasing of oscillators is a robust feature of the pacemaker important for its function.

Cell-type specific distribution of chloride transporters in the rat suprachiasmatic nucleus.

The suprachiasmatic nucleus (SCN) is a circadian oscillator and biological clock. Cell-to-cell communication is important for synchronization among SCN neuronal oscillators and the great majority of SCN neurons use GABA as a neurotransmitter, the principal inhibitory neurotransmitter in the adult CNS. Acting via the ionotropic GABA(A) receptor, a chloride ion channel, GABA typically evokes inhibitory responses in neurons via Cl(-) influx. Within the SCN GABA evokes both inhibitory and excitatory responses although the mechanism underlying GABA-evoked excitation in the SCN is unknown. GABA-evoked depolarization in immature neurons in several regions of the brain is a function of intracellular chloride concentration, regulated largely by the cation-chloride cotransporters NKCC1 (sodium/potassium/chloride cotransporter for chloride entry) and KCC1-4 (potassium/chloride cotransporters for chloride egress). It is well established that changes in the expression of the cation-chloride cotransporters through development determines the polarity of the response to GABA. To understand the mechanisms underlying GABA-evoked excitation in the SCN, we examined the SCN expression of cation-chloride cotransporters. Previously we reported that the K(+)/Cl(-) cotransporter KCC2, a neuron-specific chloride extruder conferring GABA's more typical inhibitory effects, is expressed exclusively in vasoactive intestinal peptide (VIP) and gastrin-releasing peptide (GRP) neurons in the SCN. Here we report that the K(+)/Cl(-) cotransporter isoforms KCC4 and KCC3 are expressed solely in vasopressin (VP) neurons in the rat SCN whereas KCC1 is expressed in VIP neurons, similar to KCC2. NKCC1 is expressed in VIP, GRP and VP neurons in the SCN as is WNK3, a chloride-sensitive neuron-specific with no serine-threonine kinase which modulates intracellular chloride concentration via opposing actions on NKCC and KCC cotransporters. The heterogeneous distribution of cation-chloride cotransporters in the SCN suggests that Cl(-) levels are differentially regulated within VIP/GRP and VP neurons. We suggest that GABA's excitatory action is more likely to be evoked in VP neurons that express KCC4.

Synaptic contacts of vesicular glutamate transporter 2 fibres on chemically identified neurons of the hypothalamic suprachiasmatic nucleus of the rat.

The hypothalamic suprachiasmatic nucleus (SCN), which plays a pivotal role in the control of circadian rhythms, consists of several neuronal subpopulations characterized by different neuroactive substances. This prominent cell group has a fairly rich glutamatergic innervation, but the cell types that are targeted by this innervation are unknown. Therefore, the purpose of the present study was to examine the relationship between the afferent glutamatergic axon terminals and the vasoactive intestinal polypeptide (VIP)-, arginine-vasopressin (AVP)- and gamma-aminobutyric acid (GABA)-positive neurons of the SCN. Glutamatergic elements were revealed via immunocytochemical double-labelling for vesicular glutamate transporter type 1 (VGluT1) and type 2 (VGluT2), and brain sections were imaged via confocal laser-scanning microscopy and electron microscopy. Numerous VGluT2-immunoreactive axons were observed to be in synaptic contact with VIP- and GABA-positive neurons, and only a few synapses were detected between VGluT2 boutons and AVP neurons. VGluT1 axon terminals exhibiting very moderate distribution in this cell group were observed to be in synaptic contact with chemically unidentified neurons. The findings provide the first morphological data on the termination of presumed glutamatergic fibres on chemically identified neurons of the rat SCN, and indicate that all three prominent cell types of the cell group receive glutamatergic afferents.

Immunoreactive vasoactive intestinal polypeptide and vasopressin cells after a protein malnutrition diet in the suprachiasmatic nucleus of the rat.

The aim of the present study was to evaluate the effects of prenatal and postnatal protein deprivation on the morphology and density of vasopressin (VP) and vasoactive intestinal polypeptide (VIP) immunoreactive neurons in the suprachiasmatic nucleus (SCN) of young rats. Female Wistar rats were fed either 6% (malnourished group) or 25% (control group) casein diet five weeks before conception, during gestation and lactation. After weaning, the pups were maintained on the same diet until sacrificed at 30 days of age. The major and minor axes, somatic area and the density of VP- and VIP-immunoreactive neurons were evaluated in the middle sections of the SCN. The present study shows that chronic protein malnutrition (ChPM) in VP neurons induces a significant decrease in number of cells (-31%,) and a significant increase in major and minor axes and somatic area (+12.2%, +21.1% and +15.0%, respectively). The VIP cells showed a significant decrease in cellular density (-41.5%) and a significant increase in minor axis (+13.5%) and somatic area (+10.1%). Our findings suggest that ChPM induces abnormalities in the density and morphology of the soma of VP and VIP neurons. These alterations may be a morphological substrate underlying circadian alterations previously observed in malnourished rats.

Heterogeneous expression of gamma-aminobutyric acid and gamma-aminobutyric acid-associated receptors and transporters in the rat suprachiasmatic nucleus.

The hypothalamic suprachiasmatic nucleus (SCN) is the primary mammalian circadian clock that regulates rhythmic physiology and behavior. The SCN is composed of a diverse set of neurons arranged in a tight intrinsic network. In the rat, vasoactive intestinal peptide (VIP)- and gastrin-releasing peptide (GRP)-containing neurons are the dominant cell phenotypes of the ventral SCN, and these cells receive photic information from the retina and the intergeniculate leaflet. Neurons expressing vasopressin (VP) are concentrated in the dorsal and medial aspects of the SCN. Although the VIP/GRP and VP cell groups are concentrated in different regions of the SCN, the separation of these cell groups is not absolute. The inhibitory neurotransmitter gamma-aminobutyric acid (GABA) is expressed in most SCN neurons irrespective of their location or peptidergic phenotype. In the present study, immunoperoxidase labeling, immunofluorescence confocal microscopy, and ultrastructural immunocytochemistry were used to examine the spatial distribution of several markers associated with SCN GABAergic neurons. Glutamate decarboxylase, a marker of GABA synthesis, and vesicular GABA transporter were more prominently observed in the ventral SCN. KCC2, a K(+)/Cl(-) cotransporter, was highly expressed in the ventral SCN in association with VIP- and GRP-producing neurons, whereas VP neurons in the dorsal SCN were devoid of KCC2. On the other hand, GABA(B) receptors were observed predominantly in VPergic neurons dorsally, whereas, in the ventral SCN, GABA(B) receptors were associated almost exclusively with retinal afferent fibers and terminals. The differential expression of GABAergic markers within the SCN suggests that GABA may play dissimilar roles in different SCN neuronal phenotypes.

A comparative study of cytoarchitecture and serotonergic afferents in the suprachiasmatic nucleus of primates (Cebus apella and Callithrix jacchus) and rats (Wistar and Long Evans strains).

The suprachiasmatic nucleus, an essential diencephalic component of the circadian timing system, plays a role in the generation and modulation of behavioral and neuroendocrine rhythms in mammals. Its cytoarchitecture, neurochemical and hodological characteristics have been investigated in various mammalian species, particularly in rodents. In most species, two subdivisions, based on these aspects and considered to reflect functional specialization within the nucleus, can be recognized. Many studies reveal a typical dense innervation by serotonergic fibers in this nucleus, mainly in the ventromedial area, overlapping the retinal afferents. However, a different pattern occurs in certain animals, which lead us to investigate the distribution of serotonergic afferents in the suprachiasmatic nucleus of the Capuchin monkey, Cebus apella, compared to the marmoset, Callithrix jacchus, and two Rattus norvegicus lines (Long Evans and Wistar), and to reported findings for other mammalian species. Our morphometric data show the volume and length of the suprachiasmatic nucleus along the rostrocaudal axis to be greatest in C. apella>C. jacchus>Long Evans> or =Wistar rats, in agreement with their body sizes. In C. apella, however, the serotonergic terminals occupy only some 10% of the nucleus' area, less than the 25% seen in the marmoset and rats. The distribution of the serotonergic fibers in C. apella does not follow the characteristic ventral organization pattern seen in the rodents. These findings raise questions concerning the intrinsic organization of the nucleus, as well as regarding the functional relationship between serotonergic input and retinal afferents in this diurnal species.

Ultrastructure of neurons in the hypothalamic suprachiasmatic nucleus of rats with different stress reactivity.

The rats were divided into groups demonstrating extremely high and low stress reactivity depending on the results of testing for the nociceptive threshold and thermolability in response to bacterial lipopolysaccharide administration. Specific structural features of the nucleus and mitochondria were revealed in neurons of the hypothalamic suprachiasmatic nucleus in rats with constitutionally high reactivity, which reflects high functional activity and stress-induced lability of these structures. Ultramicroscopic study revealed phenotypic differences in one of the key hypothalamic nucleus, which enables potent and rapid neurogenic response of the stress system in animals with high stress reactivity.

The thalamic paraventricular nucleus relays information from the suprachiasmatic nucleus to the amygdala: a combined anterograde and retrograde tracing study in the rat at the light and electron microscopic levels.

The relationship between efferents of the hypothalamic suprachiasmatic nucleus (SCN) and neurons of the thalamic paraventricular nucleus (PVT) projecting to the amygdala was investigated in the rat using tract tracing in light and electron microscopy. Biotinylated dextran amine was used to label anterogradely SCN efferents. These fibers were found to reach the thalamic midline, terminating in PVT, through three pathways: anterodorsally through the preoptic region, dorsally through the periventricular hypothalamus, and through the contralateral medial hypothalamic and preoptic areas after crossing the midline in the optic chiasm. Preterminal and terminal-like elements labeled from the SCN were distributed throughout the rostrocaudal extent of PVT, with an anteroposterior gradient of density. Labeled terminal elements were densest in the dorsal portion of PVT beneath the ependymal lining and some of them entered the ependyma. Anterograde tracing of SCN fibers was combined with injections of retrograde tracers in the amygdala. Numerous retrogradely labeled cell bodies were seen throughout PVT, with a prevalence in its anterodorsal portion. Overlap was detected between puncta labeled from the SCN and retrogradely labeled neurons, especially in the anterodorsal sector of PVT, where numerous puncta were in close apposition to thalamo-amygdaloid cells. Electron microscopy revealed that boutons labeled from the SCN established synaptic contacts with dendritic profiles of PVT neurons labeled from the amygdala. The findings demonstrate that information processed in the biological clock is conveyed to the amygdala through PVT, indicating that this nucleus plays a role in the transfer of circadian timing information to the limbic system.

Localization of components of the renin-angiotensin system in the suprachiasmatic nucleus of normotensive Sprague-Dawley rats: part B. angiotensin II (AT1)-receptors, a light and electron microscopic study.

The dominant pacemaker of the mammalian circadian clock, located in the suprachiasmatic nucleus (SCN), is of special interest for many pharmacological, physiological and immunohistological studies on angiotensins and their receptors. Based on its role in the circadian modulation of blood pressure and vasopressin release, the distribution and function of the neuropeptide angiotensin II (ANG II) and its AT1-receptors (AT1) in the SCN became a target for several immunohistological studies. Though the distribution of ANG II and vasopressin in the SCN is well known at light microscopic level, detailed data concerning the AT1-receptor distribution in the SCN is missing. To confirm the mechanisms by which ANG II exerts its actions in the SCN, it is vital to understand how the brain renin-angiotensin system is organized at the cellular level, including the distribution of ANG II and the ANG II (AT1)-receptors as well as the protein-receptor complex. The current paper presents a light- and electron microscopic study on AT1-receptor-immunolabeling in the suprachiasmatic nucleus of normotensive Sprague-Dawley rats.

Localization of components of the renin-angiotensin system in the suprachiasmatic nucleus of normotensive Sprague-Dawley rats: part A. angiotensin I/II, a light and electron microscopic study.

The central pacemaker of the mammalian circadian clock, identified in the suprachiasmatic nucleus (SCN), is of special interest for many chronomedical studies on neuropeptides. Based on its role in the modulation of blood pressure and vasopressin release, the distribution and function of the neuropeptide angiotensin II (ANG II) in the SCN became a target for several immunohistological studies. At the light microscopic level, the distribution of ANG II in the SCN is well known, but detailed information about the localization of ANG II in the SCN at the ultrastructural level is missing. To gain further insight in the functional aspects of ANG II in the SCN, we investigated on the subcellular localization of the neuropeptide ANG II and its precursor ANG I in the SCN. The current report presents a light and electron microscopic study on ANG I/II-immunoreactivity in the suprachiasmatic nucleus of normotensive Sprague-Dawley rats.

[Functional morphology of suprachiasmatic nuclei neurons of rat hypothalamus after the combined influence of x-irradiation and light exposure]. / Funktsional'naia morfologiia neironov superkhiazmaticheskikh iader gipotalamusa krys posle kombinirovannogo vozdeistviia rentgenovskogo izlucheniia i sveta.

For estimation of light radiomodification and cellular neuroadaptation of the circadian pacemarker, the morphological changes of suprachiasmatic nuclei (SCN) neurons of rat hypothalamus after 48-hours bright light exposure, the single 5 Gy whole-body X-irradiation and their combination were subjected to analysis. The dynamics of reactive and reparative changes has got a phasic character. Temporary desynchronous changes were developed in the course of the early period. Age morphological changes after X-ray and combined irradiation were discovered in the remote period. The plastic diapason of ventrolateral division (SCNv) is more considerable in comparison with dorsomedial division (SCNd). The synergism of X-irradiation and light exposure was discovered in SCNd.

Presynaptic and postsynaptic GABAA receptors in rat suprachiasmatic nucleus.

The mammalian suprachiasmatic nucleus (SCN), the brain's circadian clock, is composed mainly of GABAergic neurons, that are interconnected via synapses with GABA(A) receptors. Here we report on the subcellular localization of these receptors in the SCN, as revealed by an extensively characterized antibody to the alpha 3 subunit of GABA(A) receptors in conjunction with pre- and postembedding electron microscopic immunocytochemistry. GABA(A) receptor immunoreactivity was observed in neuronal perikarya, dendritic processes and axonal terminals. In perikarya and proximal dendrites, GABA(A) receptor immunoreactivity was expressed mainly in endoplasmic reticulum and Golgi complexes, while in the distal part of dendrites, immunoreaction product was associated with postsynaptic plasma membrane. Many GABAergic axonal terminals, as revealed by postembedding immunogold labeling, displayed GABA(A) receptor immunoreactivity, associated mainly with the extrasynaptic portion of their plasma membrane. The function of these receptors was studied in hypothalamic slices using whole-cell patch-clamp recording of the responses to minimal stimulation of an area dorsal to the SCN. Analysis of the evoked inhibitory postsynaptic currents showed that either bath or local application of 100 microM of GABA decreased GABAergic transmission, manifested as a two-fold increase in failure rate. This presynaptic effect, which was detected in the presence of the glutamate receptor blocker 6-cyano-7-nitroquinoxaline-2,3-dione and the selective GABA(B) receptor blocker CGP55845A, appears to be mediated via activation of GABA(A) receptors. Our results thus show that GABA(A) receptors are widely distributed in the SCN and may subserve both pre- and postsynaptic roles in controlling the mammalian circadian clock.

Regulation of neurons in the suprachiasmatic nucleus of Xenopus laevis.

In the amphibian Xenopus laevis, suprachiasmatic melanotrope-inhibiting neurons (SMINs) play an important role in the regulation of the background adaptation process. In this study, we investigated the innervation of the SMINs at the light- and electron- microscopical level. Immunocytochemistry in combination with confocal laser scanning microscopy revealed co-existence of neuropeptide Y (NPY) and synaptobrevin in spots in the direct vicinity of the SMINs, suggesting the existence of NPY-containing synapses on these cells. At the ultrastructural level, the SMINs showed a high degree of plasticity, containing more electron-dense vesicles and a larger extent of RER in white- than in black-adapted animals. In black-adapted animals, symmetric synapses (Gray type II) were observed on the soma of the SMINs, suggesting an inhibitory input to these cells. The synaptic profiles contained electron-lucent and electron-dense vesicles, indicating the involvement of both a classical neurotransmitter and a neuropeptide (possibly NPY) in this input. In white-adapted animals, synapses were only found at some distance from the SMIN somata. Our findings indicate a striking plasticity of the innervation of the SMINs in relation to background adaptation and support the hypothesis that the SMINs are innervated by NPY-containing interneurons that inhibit SMIN activity in black-adapted animals.

Subcellular distribution of 5-HT(1B) and 5-HT(7) receptors in the mouse suprachiasmatic nucleus.

The suprachiasmatic nucleus (SCN), a circadian oscillator, receives glutamatergic afferents from the retina and serotonergic (5-HT) afferents from the median raphe. 5-HT(1B) and 5-HT(7) receptor agonists inhibit the effects of light on SCN circadian activity. Electron microscopic (EM) immunocytochemical procedures were used to determine the subcellular localization of 5-HT(1B) and 5-HT(7) receptors in the SCN. 5-HT(1B) receptor immunostaining was associated with the plasma membrane of thin unmyelinated axons, preterminal axons, and terminals of optic and nonoptic origin. 5-HT(1B) receptor immunostaining in terminals was almost never observed at the synaptic active zone. To a much lesser extent, 5-HT(1B) immunoreaction product was noted in dendrites and somata of SCN neurons. 5-HT(7) receptor immunoreactivity in gamma-aminobutyric acid (GABA), vasoactive intestinal polypeptide (VIP), and vasopressin (VP) neuronal elements in the SCN was examined by using double-label procedures. 5-HT(7) receptor immunoreaction product was often observed in GABA-, VIP-, and VP-immunoreactive dendrites as postsynaptic receptors and in axonal terminals as presynaptic receptors. 5-HT(7) receptor immunoreactivity in terminals and dendrites was often associated with the plasma membrane but very seldom at the active zone. In GABA-, VIP-, and VP-immunoreactive perikarya, 5-HT(7) receptor immunoreaction product was distributed throughout the cytoplasm often in association with the endoplasmic reticulum and the Golgi complex. The distribution of 5-HT(1B) receptors in presynaptic afferent terminals and postsynaptic SCN processes, as well as the distribution of 5-HT(7) receptors in both pre- and postsynaptic GABA, VIP, and VP SCN processes, suggests that serotonin plays a significant role in the regulation of circadian rhythms by modulating SCN synaptic activity.