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.

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.

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.

Comparative localization of corticotropin and corticotropin releasing factor-like peptides in the brain and hypophysis of a primitive vertebrate, the sturgeon Acipenser ruthenus L.

The sturgeon is a primitive actinopterigian fish that, unlike modern teleosts, possess a portal vascular system that connects a true median eminence with the anterior pituitary as in mammals. The occurrence and localization of corticotropin and corticotropin releasing factor-like immunoreactivies were examined in the brain of the sturgeon (Acipenser ruthenus L.) by immunocytochemistry with antisera raised against synthetic non-conjugated human corticotropin, and rat/human corticotropin releasing factor. In the hypothalamus, corticotropin-immunoreactive parvicellular perikarya were found in the infundibular nucleus and in dendritic projections to the infundibular recess. In addition, ependymofugal corticotropin-immunoreactive fibres were found to terminate in the ventral hypothalamus. Corticotropin releasing factor-immunoreactive neurons were found in the rostral portion of the ventral hypothalamus (tuberal nucleus), and in the vicinity of the rostral aspect of the lateral recess. These cells projected to the dorsal hypothalamus, the ventral hypothalamus, the median eminence, the anterior and posterior telencephalon, the tegmentum mesencephali, and the pars nervosa of the pituitary. An affinity-purified UI antiserum failed to stain the sturgeon hypothalamus. Corticotrophs in the rostral pars distalis of the pituitary were also corticotropin-immunoreactive. In the neurointermediate lobe, only about 50% of cells of the pars intermedia appeared to be corticotropin-positive, the rest appeared unstained. These results suggest that the presence of corticotropin-like and corticotropin releasing factor-like peptides in the brain is a relatively early event in vertebrate evolution, already occurring in Chondrostean/Actinopterigian fishes, as exemplified by A. ruthenus. The close spatial relationship between corticotropin releasing factor immunoreactivity and corticotropin immunoreactivity in the ventral hypothalamus of A. ruthenus supports a possible interaction between the two systems in that area of the sturgeon brain. The pars intermedia might be an important site for corticotropin synthesis, even though the possibility cannot be excluded that the antiserum was recognizing the proopiomelanocortin molecule. The occurrence of corticotropin releasing factor immunoreactivity in the region of median eminence/pars intermedia of the sturgeon suggests that the sturgeon corticotropin releasing factor might regulate the adenohypophyseal release of proopiomelanocortin products in the same manner as in other vertebrates. The presence of extrahypothalamic corticotropin releasing factor-immunoreactive projections suggests further neuromodulatory functions for this peptide in A. ruthenus.

Isolation and amino acid sequence of urotensin II from the sturgeon Acipenser ruthenus.

Urotensin II (UII) peptides have previously been isolated from the urophysis (the neurohemal organ of the caudal neurosecretory system) of several teleost fish, and the UII amino acid sequences have been determined. Chondrostean fish, such as the Acipenseridae (sturgeon), though without a distinct urophysis, also have a caudal neurosecretory system, which has been indicated by bioassay and immunological evidence to contain UII-like peptides. In the present studies, we investigated by UII radioimmunoassay the UII-like peptides in the spinal cord of three Acipenser species, and isolated and sequenced UII from one of them. As expected, much more UII immunoreactivity (UII-IR) was found in caudal than in anterior spinal cord extracts. In addition, caudal extracts from A. ruthenus were found to contain much more UII-IR (whether determined on a UII-IR/weight or UII-IR/fish basis) than those from the larger A. stellatus and A. guldenstadti. UII was therefore isolated from A. ruthenus and its amino acid sequence was shown to be H-Gly-Ser-Thr-Ser-Glu-Cys-Phe-Trp-Lys-Tyr-Cys-Val-OH. This sequence is identical at positions 6-11 (the disulfide ring) with the known teleost UII peptides, and has acidic and hydrophobic amino acids at positions 5 and 12, respectively, as do the teleost UII peptides. Overall sequence identity with the various forms of teleost UII was 58-83%.

The hypothalamo-hypophysial system in acipenseridae. X. Corticoliberin-like immunoreactivity in the hypothalamus and hypophysis of Acipenser ruthenus L.

Using the unlabeled antibody peroxidase-antiperoxidase (PAP) technique at the light microscopic level the distribution of immunoreactive corticoliberin has been studied in the hypothalamus and hypophysis of the sterlet. Corticoliberin-containing cells have been revealed in the nucleus tuberalis and to lesser degree in the nucleus preopticus of the hypothalamus. The majority of labeled cells is located subependymally or between the ependymal cells. Some dendrites of corticoliberin-containing cells can be followed toward the ventricular lumen. Their axons project to the anterior neurohypophysis (proximal neurosecretory contact region) where they terminate in contact with the primary portal capillaries. Single terminals containing immunoreactive corticoliberin are seen in the roots of the posterior neurohypophysis on the border region between the neurohypophysis and the hypophysial pars intermedia, and sometimes in contact with the capillaries of the general circulation. The possible role of corticoliberin in the regulation of the function of glandular cells of both the pars distalis and pars intermedia is discussed.

The hypothalamo-hypophysial system of the wild carp, Cyprinus carpio L. I. Structure and ultrastructure of the posterior neurohypophysis.

The posterior neurohypophysis (PNH)-pars intermedia complex of the wild and pond carp, Cyprinus carpio L., has been studied by light, fluorescence and electron microscopy. "Gomori-positive" neurosecretory fibres are abundant in the main trunk of the neurohypophysis as well as its roots penetrating the pars intermedia. Terminals of these fibres are in contact with capillaries of the general circulation and with glandular cells of the pars intermedia. Monoaminergic fibres with a weak green fluorescence, somewhat increasing after injection of nialamide into the pond carp, have largely the same distribution. Three types of neurosecretory fibres and their terminals have been recognized in the PNH-pars intermedia complex. Types-A1 and -A2 fibres, containing granules of 140-180 nm and 100-160 nm in diameter respectively, are peptidergic "Gomori-positive". Type-A2 fibres predominate in the PNH. The least frequent monoaminergic type-B fibres have granules of 60-100 nm in diameter. Numerous peptidergic and few monoaminergic neurosecretory terminals make contact with the capillaries located within the roots of the PNH as well as at the border between them and the pars intermedia. Both peptidergic and monoaminergic terminals make direct synaptoid contacts with the gland cells or end close to connective tissue septa, basal lamina or pituicytes. The PAS-positive gland cells and to a lesser degree the lead-haematoxylin-positive gland cells show these relationships with neurosecretory terminals. The question concerning the mode of interaction between peptidergic and monoaminergic structures in the dual control of the gland cells of the pars intermedia of teleosts is discussed.

The hypothalamo-hypophysial system of hypophysectomized rats. II. Structure and ultrastructure of the median eminence.

The median eminence (ME) of hypophysectomized rats was studied by means of light and electron microscopy. Paraldehyde-fuchsin (PAF)-positive material is seen in the external zone (EZ) of the ME 2--5 days after the operation. Its amount gradually increases especially in the caudal part of the ME during the following few days. Some PAF-positive fibers make contact with the subependymally located blood capillaries. In the most caudal region of the recessus infundibuli they penetrate into the third ventricle. PAF-positive material decreases markedly from the ME of rats two months after hypophysectomy and exposure to a 1% salt load. Fibers of types A1, A2 and B containing granules of 120--220 nm, 100--150 nm and 80--100 nm in diameter, respectively, are seen in the EZ of the ME in hypophysectomized rats, although almost exclusively A2- and B-type structures make contact with the primary portal capillaries in intact animals. All types of neurosecretory fibers establish contact with the subependymal nonfenestrated blood capillaries and penetrate the recessus infundibuli. Some neurosecretory terminals of different types make direct contact with the glandular cells of the pars tuberalis or are separated from them by a thin basal lamina. It is assumed that mainly neurosecretory fibers of types A2 and B are permanently connected with the primary portal capillaries in the EZ of the ME in intact mammals, while the overwhelming majority of fibers of A1-type shows ingrowth during the course of postoperative reparation. The possible physiological significance of the described changes is discussed.

The hypothalamo-hypophysial system of the lamprey, Lampetra fluviatilis L. III. High-resolution radioautography of monoaminergic structures in neurohemal regions.

The distribution of monoaminergic structures was studied in the proximal neurosecretory contact region and neurohypophysis of the lamprey by light and electron microscopic radioautography. Only weak radioautographic reactions were found in the proximal neurosecretory contact region 1 h after injection of 3H-dopamine. High-resolution radioautography revealed some labeled neurosecretory terminals mainly in contact with the basement membrane of the connective tissue layer separating the proximal neurosecretory contact region from the hypophysial pars distalis. The number of silver grains as well as the number of neurosecretory terminals marked by the presence of labeled dopamine was much higher in the neurohypophysis of the same species. In the latter, labeled neurosecretory terminals were found in contact with the connective tissue layer containing blood vessels of the general circulation. Some neurosecretory terminals make synaptoid contacts with tanycyte perikarya and their basal processes. According to their ultrastructure and the size of their granules, the labeled neurosecretory terminals are identical with the B type terminals described in both neurohemal regions (transmission electron microscopy). No labeled neurosecretory terminals were observed in the proximal neurosecretory contact region and the neurohypophysis of lampreys treated with the serotonin precursor, 3H-5-hydroxytryptophan.

Quantative radioautographic light and electron microscopic analysis of the localization of monoamines in the median eminence of the rat.

Serotonin containing structures in the median eminence of the rat have been studied by quantitative light and electron microscopic radioautography following intraventricular infusion of tritiated 5-hydroxytryptophan. One hour after injection of the tracer the highest density of silver grains was recorded in the ependymal and external zones, especially in the lateral palisade zone. The proportion of labelled neurosecretory terminals was also larger in the lateral palisade zone (29%) as compared with the medial palisade zone (13%), although the mean number of developed silver grains per one terminal was higher in the latter. On the average, 16% of neurosecretory terminals sequestered radiolabelled 5-hydroxytryptophan in the external zone of the rat median eminence. It is suggested that serotonin, like catecholamines, is discharged from neurosecretory terminals localized in the external zone and via the portal circulation affects the function of the anterior pituitary. The sites of origin of serotoninergic structures of the median eminence as well as the possible role of monoamine (catecholamine and indolamine) neurohormones in a dual peptidergic and monoaminergic control of anterior pituitary functions are discussed.

The hypothalamo-hypophysial system of the lamprey, Lampetra fluviatilis L. II. The proximal neurosecretory contact region.

The proximal neurosecretory contact region (PNCR) of the lamprey, a homologue of the median eminence of tetrapods, was studied by light, fluorescence and electron microscopy. Paraldehyde fuchsin-positive neurosecretory fibers are seen mainly in the central part of the rostral subdivision of the PNCR. The Falck-Hillarp technique reveals a weak, mainly diffuse yellow-green fluorescence in the PNCR. The ultrastructure of the tanycyte layer of the PNCR is very similar to that in the neurohypophysis of the same species, although the funnel-shaped protrusions of the third ventricle in the rostral part of the PNCR are more frequent than in the neurophypophysis. Peptidergic A1 and A2 neurosecretory fibers are characterized by neurosecretory granules of 120-200 nm and 100-150 nm in diameter, respectively. Monoaminergic B type fibers contain granules 80-100 nm in diameter. Neurosecretory terminals and the vascular endfeet of tanycytes make contact with the basement membrane of the avascular connective tissue layer separating the PNCR from the hypophysial pars distalis. It is suggested that both peptide and monoamine neurohormones diffuse through the thick connective tissue septa into the underlying blood vessels which supply the pars distalis and thus affect the function of its glandular cells.

Quantitative radioautographic light and electron microscopic analysis of the localization of monoamines in the median eminence of the rat. I. Catecholamines.

By means of light and electron microscopic radioautography a quantitative study of the regional distribution of catecholamines in the median eminence of the rat was carried out. One hour after intraventricular injection of 3H-dopamine the highest radioautographic reaction was recorded in the external zone, especially in the lateral palisade zone where many neurosecretory terminals are separated from the basal lamina of the portal pericapillary space by a glial "cuff". This area showed the highest percentage (52%) of labelled catecholamine containing neurosecretory terminals as well as the maximal silver grain density per one terminal. In the medial palisade zone where direct neurovascular contacts with the capillary loops prevail, only 27 per cent of neurosecretory terminals were found to harbour tritiated dopamine. On the average 35 per cent of neurosecretory terminals in the median eminence of the rat contain catecholamines (both dopamine and noradrenaline). Pretreatment of animals with recretory terminals as well as grain density over terminals were decreased in both the medial and lateral palisade zones, although to a lesser degree in the latter. The site of origin of catecholamine fibers as well as the mode of catecholamine action at the level of the median eminence are discussed.

The hypothalamo-hypophysial system in acipenseridae. VI. The proximal neurosecretory contact region.

The proximal neurosecretory contact region (PCR) of Acipenseridae, a homologue of the tetrapod median eminence, has been studied by light, fluorescence and electron microscopy. It occupies the rostral and chiefly the ventral surfaces of the hypothalamic tuber cinereum. PAF-positive fibres occur in the zone of the preoptico-hypophysial tract but their terminal enlargements are concentrated mainly in the external zone. They make contact with the primary portal capillaries situated in the pia mater. Monoaminergic fibres and terminals with an intense green fluorescence are localized in the same regions. The fibres of some bipolar monoaminergic neurons of the PCR make contact both with the third ventricle and the primary portal capillaries. Three types of granule-containing neurosecretory fibres and terminals have been recognized in the PCR. Fibres of types A1 (d = 120-300 nm) and A2 (D = 100-170 nm) are peptidergic PAR-positive, although some fibres, including some of type A1, belong possibly to PAF-negative type. Monoaminergic type B fibres have granules 80-100 nm in diameter. Neurosecretory terminals and vascular "endfeet" of tanycytes make contact with the 70 nm thick outer basement membrane of the primary portal capillaries. Several laminae of thin horizontally oriented tanycyte processes form a boundary between the external zone and the preoptico-hypophysial tract. Few neuroglial cells with pale cytoplasm, numerous lysosomes and lipofuscin granules are seen in this region. It is hypothesized that, as in other vertebrates both peptide hypophysiotropic neurohormones and monoamines are discarged from the PCR into the portal circulation and affect the activity of the glandular cells of the pars distalis.

On degeneration of peptidergic neurosecretory fibres in the albino rat.

Three types of degenerating peptidergic neurosecretory fibres have been found in the posterior pituitary of chronically dehydrated albino rats. "Dark" neurosecretory fibres and their swellings contain neurosecretory granules, neurotubules, shrunken mitochondria and diffusely distributed fine dense material. Some swellings are filled with synaptic vesicles and/or conglomerations of dense membranes. The transitional forms exist between these fibres and extracellular accumulations of electron dense material. Synaptic vesicles, single neurosecretory granules, lipid-like droplets and lamellar bodies occur in the latter. Some neurosecretory fibres and swellings have numerous polymorphous inclusions arising due to degradation of secretory inclusions and organelles, mitochondria and neurotubules in particular. "Dark" neurosecretory elements and those with numerous polymorphous inclusions are enveloped by pituicyte cytoplasm. Sometimes the plasma membranes both of the pituicytes and neurosecretory fibres are destroyed or transformed into a multi-membrane complex. It is assumed that pituicytes may phagocytize degenerating neurosecretory elements. N urosecretory fibres with a locally dissolved neuroplasm and/or large lucent vacuoles seem to be due to axonal degeneration by the "light" type. These neurosecretory elements, the largest of them in particular, may transform into large cavities bordered by a membrane and containing flake-like material and single-membrane vacuoles. Degeneration of neurosecretory elements seems to occur mainly due to hyperfunction of the hypothalamo-hypophysial neurosecretory system.