Localization of arginine vasotocin (AVT) mRNA in extrasomal compartments of magnocellular neurons in the chicken hypothalamo-neurohypophysial system

In the chicken, arginine vasotocin (AVT) is produced in and secreted by magnocellular neurons in the supraoptic (SON) and paraventricular (PVN) nuclei. To test the hypothesis of axonally transported AVT mRNA, the localization of AVT mRNA within extrasomal, axonal/dendritic compartments in the chicken hypothalamo-neurohypophysial system (HNS) were examined using AVT specific in situ hybridization histochemistry (ISHH) and RT-PCR. Many perikarya in the PVN and external--but none in the ventral subgroup of the SON show ISHH signals clearly extended into one or two processes, some with branching collaterals, traceable over a distance of more than 100 microns. Furthermore by using RT-PCR, AVT mRNA was detected in the median eminence and neurohypophysis representing the distal parts of the HNS, mainly consisting of axons and/or axon terminals. These observations of axonal mRNA offer new insights to the organization and function of the avian HNS.     

Reactivity pattern of vasopressin-containing neurons and its relation to the antipyretic reaction in the pregnant guinea pig

Summary Changes in the content of vasopressin-immunoreactive material in neurons and their projections were examined in pregnant and nonpregnant guinea pigs as well as in mother and newborn animals. Before sacrifice all animals used in the present study were submitted to a pyrogen test, during which the pregnant animals displayed a reduced fever response to exogenous pyrogen. The unlabeled enzyme-immunoperoxidase method was used in the present study. Light microscopic examination showed that, in comparison to all other groups examined, the pregnant animals exhibited a reduced content of the vasopressin-immunoreactive substance in the supraoptic nucleus (SON), in the neuronal pathways extending between the paraventricular nucleus (PVN) and the SON, as well as in the axons projecting to the neural lobe of the pituitary. An increased amount of vasopressin-immunoreactive material was observed during pregnancy especially in the medial portion of the PVN, in axonal distensions in the external zone of the median eminence and in the extrahypothalamic projection sites of the PVN in the lateral septum and in the amygdala. In the pregnant animals neurovascular contacts of vasopressinergic perikarya and fibers were abundant in the PVN; in the lateral septum and in the amygdala vasopressinergic terminals appeared to contact neurons of other types. It is suggested from the present immunocytochemical results that activation of neurons in the medial portion of the PVN and the increased number of vasopressinergic terminals and preterminals in the lateral septum and in the amygdala might be functionally involved in fever suppression at the term of pregnancy.     

The kallikrein-kinin system in the rat hypothalamus

Summary High molecular weight kininogen (HKg) and T kininogen (TKg) were detected and localized by immunocytochemistry in adult rat hypothalamus. In addition, kininogens were measured by their direct radioimmunoassay (RIA) or by indirect estimation of kinins released after trypsin hydrolysis and high pressure liquid chromatography (HPLC) separation of bradykinin (BK) and T kinin. A specific HKg immunoreactivity demonstrated with antibodies directed against the light chain (LC) of HKg was colocated with SRIF in neurons of hypothalamic periventricular area (PVA) projecting to external zone (ZE) of median eminence (ME). Heavy chain (HC) immunoreactivity which could be related to HKg or to low molecular weight kininogen (LKg) was detected in some other systems: i) parvocellular neurons of suprachiasmatic (SCN) and arcuate nuclei containing SRIF, ii) magnocellular neurons (mostly oxytocinergic) of paraventricular (PVN) and supraoptic (SON) nuclei, iii) neurons of dorsomedian and lateral hypothalamic areas. TKg immunostaining was restricted to magnocellular neurons of PVN, SON, accessory nuclei (mostly vasopressinergic) and to parvocellular neurons of SCN (vasopressinergic). TKg projections are directed towards the internal zone (ZI) of ME, but very few immunoreactive terminals are detectable in neurohypophysis. TKg staining parallels with vasopressin during water deprivation, and is undetectable in homozygous Brattleboro rats. In some magnocellular neurons, TKg and HC (related to HKg or LKg) are coexpressed. TKg, was also detected in hypothalamus and cerebellum extracts by direct RIA, and BK and T kinin were identified after trypsin hydrolysis. HKg and LKg can act as precursor of BK which can play a physiological role as releasing factor, neuromodulator — neurotransmitter, — or modulator of local microcirculation in hypothalamus. The three kininogens are also potent thiolprotease inhibitors which could modulate both the maturation processes of peptidic hormones and their inactivation and catabolism.     

Immunocytochemical observations of corticotropin-releasing-factor-containing neurons in the rat hypothalamus with special reference to neuronal communication

Synapses between neurons with corticotropin-releasing-factor-(CRF)-like immunoreactivities and other immunonegative neurons in the hypothalamus of colchicine-treated rats, especially in the paraventricular nucleus (PVN) and the supraoptic nucleus (SON) were observed by immunocytochemistry using CRF antiserum. The immunoreactive nerve cell bodies and fibers were numerous in both the PVN and the SON. The CRF-containing neurons had synaptic contacts with immunonegative axon terminals containing a large number of clear synaptic vesicles alone or combined with a few dense-cored vesicles. We also found CRF-like immunoreactive axon terminals making synaptic contacts with other immunonegative neuronal cell bodies and fibers. And since some postsynaptic immunonegative neurons contained many large neurosecretory granules, they are considered to be magnocellular neurosecretory cells. These findings suggest that CRF functions as a neurotransmitter and/or modulator in addition to its function as a hormone.     

The kallikrein-kinin system in the rat hypothalamus. Immunohistochemical localization of high molecular weight kininogen and T kininogen in different neuronal systems

High molecular weight kininogen (HKg) and T kininogen (TKg) were detected and localized by immunocytochemistry in adult rat hypothalamus. In addition, kininogens were measured by their direct radioimmunoassay (RIA) or by indirect estimation of kinins released after trypsin hydrolysis and high pressure liquid chromatography (HPLC) separation of bradykinin (BK) and T kinin. A specific HKg immunoreactivity demonstrated with antibodies directed against the light chain (LC) of HKg was colocated with SRIF in neurons of hypothalamic periventricular area (PVA) projecting to external zone (ZE) of median eminence (ME). Heavy chain (HC) immunoreactivity which could be related to HKg or to low molecular weight kininogen (LKg) was detected in some other systems: i) parvocellular neurons of suprachiasmatic (SCN) and arcuate nuclei containing SRIF, ii) magnocellular neurons (mostly oxytocinergic) of paraventricular (PVN) and supraoptic (SON) nuclei, iii) neurons of dorsomedian and lateral hypothalamic areas. TKg immunostaining was restricted to magnocellular neurons of PVN, SON, accessory nuclei (mostly vasopressinergic) and to parvocellular neurons of SCN (vasopressinergic). TKg projections are directed towards the internal zone (ZI) of ME, but very few immunoreactive terminals are detectable in neurohypophysis. TKg staining parallels with vasopressin during water deprivation, and is undetectable in homozygous Brattleboro rats. In some magnocellular neurons, TKg and HC (related to HKg or LKg) are coexpressed. TKg, was also detected in hypothalamus and cerebellum extracts by direct RIA, and BK and T kinin were identified after trypsin hydrolysis. HKg and LKg can act as precursor of BK which can play a physiological role as releasing factor, neuromodulator--neurotransmitter,--or modulator of local microcirculation in hypothalamus. The three kininogens are also potent thiolprotease inhibitors which could modulate both the maturation processes of peptidic hormones and their inactivation and catabolism.     

Participation of neuronal NO-synthase in regulation of hypothalamus vasopressinergic neurons of rat pups at early stages of postnatal ontogeny

To reveal character of interaction of catecholamines (CA) and NO in regulation of development and of the functional state of vasopressinergic (VP-ergic) neurons of supraoptic (SON) and paraventricular (PVN) nuclei, the female rats were injected intraperitoneally with the inhibitor of CA synthesis α-methyl-p-tyrosine, daily, from the 13th to the 20th days of pregnancy. Rat pups born by the females administered with saline at the same period of pregnancy as well as intact pups and adult rats were used as control. Expression of neuronal NO-synthase (nNOS) in neurons of SON and PVN of rat pups at early stages of postnatal development was found to be significantly higher than the definitive level, which allows suggesting participation of NO in development of hypothalamic VP-ergic neurons. The revealed differences of periods of the maximal nNOS expression in the SON and PVN neurons have permitted suggesting development of SON to be completed earlier than that of PVN. The pups exposed to stress at the last third of embryonic development had a long-lasting effect on the state of VP-ergic neurons of the pups after birth. The nNOS expression in neurons does not change, which suggests that NO is not involved in regulation of VP-ergic neurons after exposure to stress at early stages of ontogenesis. A decrease of CA level in the brain at the last third of embryogenesis led to a long preserved decrease of the functional activity of VP-ergic neurons. The nNOS expression in VP-ergic neurons of SON and PVN rose substantially under effect of a compensatory enhancement of tyrosine hydroxylase (TH) expression in neurons of SON and of an increase of the level of CA-ergic innervation of PVN. Thus, we have shown that a decrease of CA level in the embryonic brain leads to an increase of nNOS expression of hypothalamic VP-ergic neurons of rat pups after birth and that the character of NO action on function of VP-ergic neurons does not differ from that of adult animals as soon as at early stages of ontogenesis.     

Oxytocinergic circuit from paraventricular and supraoptic nuclei to arcuate POMC neurons in hypothalamus

Intracerebroventricular injection of oxytocin (Oxt), a neuropeptide produced in hypothalamic paraventricular (PVN) and supraoptic nuclei (SON), melanocortin-dependently suppresses feeding. However, the underlying neuronal pathway is unclear. This study aimed to determine whether Oxt regulates propiomelanocortin (POMC) neurons in the arcuate nucleus (ARC) of the hypothalamus. Intra-ARC injection of Oxt decreased food intake. Oxt increased cytosolic Ca²⁺ in POMC neurons isolated from ARC. ARC POMC neurons expressed Oxt receptors and were contacted by Oxt terminals. Retrograde tracer study revealed the projection of PVN and SON Oxt neurons to ARC. These results demonstrate the novel oxytocinergic signaling from PVN/SON to ARC POMC, possibly regulating feeding.     

Evidence for local corticotropin releasing factor (CRF)-immunoreactive neuronal circuits in the paraventricular nucleus of the rat hypothalamus

Summary The interrelationships of corticotropin-releasing factor (CRF) immunoreactive neuronal cell bodies and processes have been examined in the paraventricular nucleus (PVN) of adrenalectomized-dexamethesone treated rats. Antisera generated against ovine CRF (oCRF) were used in the peroxidase-anti-peroxidase-complex (PAP)-immunocytochemical method at both the light and electron microscopic levels. In this experimental model, a great number of CRF-immunoreactive neurons were detected in the parvocellular subdivisions of the PVN and a few scattered labelled parvocellular neurons were also observed within the magnocellular subunits. Characteristic features of immunolabeled perikarya included hypertrophied rough endoplasmic reticulum with dilated endoplasmic cisternae, well developed Golgi complexes and increased numbers of neurosecretory granules. These features are interpreted to indicate accelerated hormone synthesis as a result of adrenalectomy. Afferent fibers communicated with dendrites and somata of CRF-immunoreactive neurons via both symmetrical and asymmetrical synapses. Some neurons exhibited somatic appendages and these structures were also observed to receive synaptic terminals. Within both the PVN and its adjacent neuropil, CRF-immunoreactive axons demonstrated varicosites which contained accumulations of densecore vesicles. CRF-containing axons were observed to branch into axon collaterals. These axons or axon collaterals established axo-somatic synapses on CRF-producing neurons in the parvocellular regions of the PVN, while in the magnocellular area of the nucleus they were found in juxtaposition with unlabeled magnocellular neuronal cell bodies or in synaptic contact with their dendrites. The presence of CRF-immunoreactive material in presynaptic structures suggests that the neurohormone may participate in mechanisms of synaptic transfer.These ultrastructural data indicate that the function of the paraventricular CRF-synthesizing neurons is adrenal steroid hormone dependent. They also provide morphological evidence for the existence of a neuronal ultrashort feedback mechanism within the PVN for the regulation of CRF production and possibly that of other peptide hormones contained within this complex.Supported by NIH grant NS 19266 to WKP     

Evidence for local corticotropin releasing factor (CRF)-immunoreactive neuronal circuits in the paraventricular nucleus of the rat hypothalamus. An electron microscopic immunohistochemical analysis

The interrelationships of corticotropin-releasing factor (CRF) immunoreactive neuronal cell bodies and processes have been examined in the paraventricular nucleus (PVN) of adrenalectomized-dexamethasone treated rats. Antisera generated against ovine CRF (oCRF) were used in the peroxidase-anti-peroxidase-complex (PAP)-immunocytochemical method at both the light and electron microscopic levels. In this experimental model, a great number of CRF-immunoreactive neurons were detected in the parvocellular subdivisions of the PVN and a few scattered labelled parvocellular neurons were also observed within the magnocellular subunits. Characteristic features of immunolabeled perikarya included hypertrophied rough endoplasmic reticulum with dilated endoplasmic cisternae, well developed Golgi complexes and increased numbers of neurosecretory granules. These features are interpreted to indicate accelerated hormone synthesis as a result of adrenalectomy. Afferent fibers communicated with dendrites and somata of CRF-immunoreactive neurons via both symmetrical and asymmetrical synapses. Some neurons exhibited somatic appendages and these structures were also observed to receive synaptic terminals. Within both the PVN and its adjacent neuropil, CRF-immunoreactive axons demonstrated varicosites which contained accumulations of densecore vesicles. CRF-containing axons were observed to branch into axon collaterals. These axons or axon collaterals established axo-somatic synapses on CRF-producing neurons in the parvocellular regions of the PVN, while in the magnocellular area of the nucleus they were found in juxtaposition with unlabeled magnocellular neuronal cell bodies or in synaptic contact with their dendrites. The presence of CRF-immunoreactive material in presynaptic structures suggests that the neurohormone may participate in mechanisms of synaptic transfer. These ultrastructural data indicate that the function of the paraventricular CRF-synthesizing neurons is adrenal steroid hormone dependent. They also provide morphological evidence for the existence of a neuronal ultrashort feed-back mechanism within the PVN for the regulation of CRF production and possibly that of other peptide hormones contained within this complex.     

Neuronal organization of the avian paraventricular nucleus: intrinsic, afferent, and efferent connections

The heterogeneous paraventricular nucleus (PVN) of birds offers favorable conditions for the analysis of intrinsic, afferent, and efferent connections of neuroendocrine systems. Paraventricular neurons are successfully impregnated with the Golgi-technique. The findings indicate a direct influence of the cerebrospinal fluid (CSF) on the magnocellular neurons that, via their axon terminals in the neural lobe of the pituitary, are also exposed to the hemal milieu. The magnocellular neurons are intermingled with parvocellular elements which may represent local interneurons. A group of parvocellular nerve cells is identified as CSF-contacting neurons. This type of cell forms a basic morphologic component of the avian neuroendocrine apparatus. Immunocytochemical and ultrastructural studies further support the concept of neuronal interactions between parvocellular and magnocellular elements. Moreover, these findings speak in favor of the existence of recurrent collaterals of the magnocellular neurons. Nerve cells giving rise to afferent connections to the PVN are located in the limbic system and autonomic areas of the upper and lower brainstem. Further afferents may originate from the subfornical organ, the organon vasculosum laminae terminalis, the ventral tegmentum, and the area postrema. Via efferent projections, the PVN is connected to the nucleus accumbens, lateral septum, several hypothalamic nuclei, the neural lobe of the pituitary, the organon vasculosum laminae terminalis, the subfornical organ, the pineal organ, the area postrema, the lateral habenular complex, and various autonomic areas of the reticular formation in the upper and lower brainstem and the spinal cord. In conclusion, the PVN may be regarded as an integral component of the neuroendocrine apparatus reciprocally coupled to the limbic system, several circumventricular organs, and various autonomic centers of the brain.     

Participation of catecholamines and NO in regulation of apoptosis of nonapeptidergic neurons of neonatal rat pups

Effects of catecholamines (CA) and the character of interaction of CA and NO in regulation of apoptosis were studied in vasopressinergic (VP-ergic) neurons of supraoptic (SON) and paraventricular (PVN) nuclei of rat pups in early postnatal ontogenesis. To study role of CA in regulation of programmed cell death in SON and PVN in the course of embryonal development, pregnant female rats were intraperitoneally injected daily from the 13th to the 20th day with αMPT—a blocker of CA synthesis. The second group of pregnant rats was injected from the 13th to the 20th day with the same volume of saline. The third group was composed of intact animals. The born rat pups were sacrificed at the 3rd and 15th days of life. Caspase 9, Bcl-2, tyrosine hydroxylase, and neuronal NO-synthase (nNOS) in SON and PVN neurons were revealed immunohistochemically, and the amount of immunoreactive substance in neuronal bodies was estimated using the computerized digital analyzer of TV image and Video Test software. Caspase-9 was shown to play an important role in postnatal formation of cellular composition of hypothalamic nonapeptidergic centers by leading to initiation of apoptosis and rejection of “useless” postmitotic SON and PVN neurons. Survival of “useless” nonapeptidergic neurons in early postnatal ontogenesis seems to be connected with antiapoptotic action of Bcl-2. Death of postmitotic neurons, and therefore formation of cellular composition begins earlier and, accordingly, is completed earlier in SON, in which neurons were noted to have a considerable decrease of the caspase-9 expression and, therefore, also a decrease of intensity of neuronal death via caspase-9-dependent pathway. In PVN, neurons continue to die also at the 15th day of rat life, i.e., almost two weeks later than in SON. The observed high correlation between the content of nNOS, caspase-9, and Bcl-2 in the SON and PVN neurons of intact rats of both age groups allows suggesting participation of NO in realization of apoptosis in the course of early postnatal development. The increase of nNOS expression in hypothalamic neurons as a result of disturbances in CA-ergic innervation in embryogenesis might be a possible cause of the long preserved enhancement of expression of apoptosis signal proteins. It can be suggested that CA participate in morphogenesis of hypothalamic neurons by increasing expression of nNOS in neurons and thereby affecting expression of apoptotic proteins.     

Expression of Robo/Slit and Semaphorin/Plexin/Neuropilin family members in the developing hypothalamic paraventricular and supraoptic nuclei

The hypothalamic paraventricular nucleus (PVN) and supraoptic nucleus (SON) contain neuroendocrine cells that modulate pituitary secretion to maintain homeostasis. These two nuclei have a common developmental origin but they eventually form at locations distant from each other. Little is known about the molecular cues that direct the segregation of PVN and SON. As a means to identify potential factors, we have documented expression patterns of genes with known guidance roles in neural migration. Here, we focus on two groups of ligand/receptor families classified to mediate chemo-repulsion of neurons and their axons: the Slit/Robo and the Semaphorin/Plexin/Neuropilin families. Their dynamic expression patterns within and around the common PVN/SON progenitor as well as the mature PVN and SON may provide a framework for understanding the formation of these two important nuclei.     

Simultaneous monoamine histofluorescence and neuropeptide immunocytochemistry: III. Ontogeny of catecholamine varicosities and neurophysin neurons in the rat supraoptic and paraventricular nuclei

The ontogeny of the rat supraoptic (SON) and paraventricular (PVN) nuclei was studied using a combined fluorescence-immunocytochemical technique for the simultaneous localization of catecholamines (CA) and neurophysin (NP). NP neurons and CA varicosities were first detected in the SON and PVN at 17 days postcoitus. The development of NP neurons which included increases in immunoreactivity in both nuclei proceeded through fetal and neonatal stages, approaching maturity by 21–28 days postnatal; the maturation of the PVN lagged behind that of the SON. CA varicosities appeared to make contact with NP neurons beginning at 21–22 days postcoitus. An apparent increase in varicosity-perikaryal contacts with age was observed in both nuclei; by 14–21 days postnatal adult-like patterns were established. The prenatal dominance of NP stain relative to CA fluorescence may suggest a possible neurotrophic role for magnocellular neurons and/or their products upon ingrowing noradrenergic axons.     

Oxytocin and vasopressin involved in restraint water-immersion stress mediated by oxytocin receptor and vasopressin 1b receptor in rat brain

Aims

Vasopressin (AVP) and oxytocin (OT) are considered to be related to gastric functions and the regulation of stress response. The present study was to study the role of vasopressinergic and oxytocinergic neurons during the restraint water-immersion stress.

Methods

Ten male Wistar rats were divided into two groups, control and RWIS for 1h. The brain sections were treated with a dual immunohistochemistry of Fos and oxytocin (OT) or vasopressin (AVP) or OT receptor or AVP 1b receptor (V_1bR).

Results

(1) Fos-immunoreactive (Fos-IR) neurons dramatically increased in the hypothalamic paraventricular nucleus (PVN), the supraoptic nucleus (SON), the neucleus of solitary tract (NTS) and motor nucleus of the vagus (DMV) in the RWIS rats; (2) OT-immunoreactive (OT-IR) neurons were mainly observed in the medial magnocellular part of the PVN and the dorsal portion of the SON, while AVP-immunoreactive (AVP-IR) neurons mainly distributed in the magnocellular part of the PVN and the ventral portion of the SON. In the RWIS rats, Fos-IR neurons were indentified in 31% of OT-IR neurons and 40% of AVP-IR neurons in the PVN, while in the SON it represented 28%, 53% respectively; (3) V_1bR-IR and OTR-IR neurons occupied all portions of the NTS and DMV. In the RWIS rats, more than 10% of OTR-IR and V_1bR-IR neurons were activated in the DMV, while lower ratio in the NTS.

Conclusion

RWIS activates both oxytocinergic and vasopressinergic neurons in the PVN and SON, which may project to the NTS or DMV mediating the activity of the neurons by OTR and V_1bR.     

Gastric distension-induced release of 5-HT stimulates c-fos expression in specific brain nuclei via 5-HT3 receptors in conscious rats

We examined c-fos expression in specific brain nuclei in response to gastric distension and investigated whether 5-HT released from enterochromaffin (EC) cells was involved in this response. The role of 5-HT3 receptors in this mechanism was also addressed. Release of 5-HT was examined in an ex vivo-perfused stomach model, whereas c-fos expression in brain nuclei induced by gastric distension was examined in a freely moving conscious rat model. Physiological levels of gastric distension stimulated the vascular release of 5-HT more than luminal release of 5-HT, and induced c-fos expression in the nucleus of the solitary tract (NTS), area postrema (AP), paraventricular nucleus (PVN), and supraoptic nucleus (SON). The c-fos expression in all these brain nuclei was blocked by truncal vagotomy as well as by perivagal capsaicin treatment, suggesting that vagal afferent pathways may mediate this response. Intravenous injection of 5-HT3 receptor antagonist granisetron blocked c-fos expression in all brain nuclei examined, although intracerebroventricular injection of granisetron had no effect, suggesting that 5-HT released from the stomach may activate 5-HT3 receptors located in the peripheral vagal afferent nerve terminals and then induce brain c-fos expression. c-fos Positive cells in the NTS were labeled with retrograde tracer fluorogold injected in the PVN, suggesting that neurons in the NTS activated by gastric distension project axons to the PVN. The present results suggest that gastric distension stimulates 5-HT release from the EC cells and the released 5-HT may activate 5-HT3 receptors located on the vagal afferent nerve terminals in the gastric wall leading to neuron activation in the NTS and AP and subsequent activation of neurons in the PVN and SON.     

Heterogeneous distribution of basal cyclic guanosine monophosphate within distinct neuronal populations in the hypothalamic paraventricular nucleus

The supraoptic (SON) and the paraventricular (PVN) hypothalamic nuclei constitute major neuronal substrates underlying nitric oxide (NO) effects on autonomic and neuroendocrine control. Within these nuclei, constitutively produced NO restrains the firing activity of magnocellular neurosecretory and preautonomic neurons, actions thought to be mediated by a cGMP-dependent enhancement of GABAergic inhibitory transmission. In the present study, we expanded on this knowledge by performing a detailed anatomical characterization of constitutive NO-receptive, cGMP-producing neurons within the PVN. To this end, we combined tract-tracing techniques and immunohistochemistry to visualize cGMP immunoreactivity within functionally, neurochemically, and topographically discrete PVN neuronal populations in Wistar rats. Basal cGMP immunoreactivity was readily observed in the PVN, both in neuronal and vascular profiles. The incidence of cGMP immunoreactivity was significantly higher in magnocellular (69%) compared with preautonomic ( approximately 10%) neuronal populations (P < 0.01). No differences were observed between oxytocin (OT) and vasopressin (VP) magnocellular neurons. In preautonomic neurons, the incidence of cGMP was independent of their subnuclei distribution, innervated target (i.e., intermediolateral cell column, nucleus tractus solitarii, or rostral ventrolateral medulla) or their neurochemical phenotype (i.e., OT or VP). Finally, high levels of cGMP immunoreactivity were observed in GABAergic somata and terminals within the PVN of eGFP-GAD67 transgenic mice. Altogether, these data support a highly heterogeneous distribution of basal cGMP levels within the PVN and further support the notion that constitutive NO actions in the PVN involve intricate cell-cell interactions, as well as heterogeneous signaling modalities.     

Ultrastructural features of presumptive vasopressinergic synapses in the hypothalamic magnocellular secretory nuclei of the rat

Despite convincing physiological evidences for vasopressin (VP) autoregulation in the supraoptic (SON) and paraventricular (PVN) nuclei, the morphological demonstration of VP synapses has lagged behind. The present work investigates the possible existence of such synapses in the SON and PVN of the rat. Electron microscopy of sections immunostained with VP antibody (1:5,000) and conjugated with avidin-biotin demonstrated presynaptic terminals containing neurosecretory granule (NSG)-like bodies, 80-100 nm in diameter. The terminals formed axodendritic, axosomatic and axoaxonic synapses, though the postsynaptic elements remained largely unidentified. Other ultrastructural features of synaptic specialization were evident. The NSG-like bodies exhibited a varying and dynamic relationship to the presynaptic membrane, suggesting their involvement in synaptic mechanisms.     

The Notch effector gene Hes1 regulates migration of hypothalamic neurons, neuropeptide content and axon targeting to the pituitary

Proper development of the hypothalamic-pituitary axis requires precise neuronal signaling to establish a network that regulates homeostasis. The developing hypothalamus and pituitary utilize similar signaling pathways for differentiation in embryonic development. The Notch signaling effector gene Hes1 is present in the developing hypothalamus and pituitary and is required for proper formation of the pituitary, which contains axons of arginine vasopressin (AVP) neurons from the hypothalamic paraventricular nucleus (PVN) and supraoptic nucleus (SON). We hypothesized that Hes1 is necessary for the generation, placement and projection of AVP neurons. We found that Hes1 null mice show no significant difference in cell proliferation or death in the developing diencephalon at embryonic day 10.5 (e10.5) or e11.5. By e16.5, AVP cell bodies are formed in the SON and PVN, but are abnormally placed, suggesting that Hes1 may be necessary for the migration of AVP neurons. GAD67 immunoreactivity is ectopically expressed in Hes1 null mice, which may contribute to cell body misplacement. Additionally, at e18.5 Hes1 null mice show continued misplacement of AVP cell bodies in the PVN and SON and additionally exhibit abnormal axonal projection. Using mass spectrometry to characterize peptide content, we found that Hes1 null pituitaries have aberrant somatostatin (SS) peptide, which correlates with abnormal SS cells in the pituitary and misplaced SS axon tracts at e18.5. Our results indicate that Notch signaling facilitates the migration and guidance of hypothalamic neurons, as well as neuropeptide content.     

GABA-immunohistological observations, at the electron-microscopical level, of the neurons of isthmic nuclei in chicken, Gallus domesticus

Following a demonstration of Golgi-impregnated neurons and their terminal axon arborization in the optic tectum, the neurons of the nucleus parvocellularis and magnocellularis isthmi were studied by means of postembedded electron-microscopical (EM) γ-aminobutyric acid (GABA)-immunogold staining. In the parvocellular nucleus, none of the neuronal cell bodies or dendrites displayed GABA-like immunoreactivity in EM preparations stained by postembedded GABA-immunogold. However, numerous GABA-like immunoreactive and also unlabeled terminals established synapses with GABA-negative neurons. GABA-like immunoreactive terminals were usually found at the dendritic origin. Around the dendritic profiles, isolated synapses of both GABA-like immunoreactive and immunonegative terminals established glomerulus-like structures enclosed by glial processes. All giant and large neurons of the magnocellular nucleus of the isthmi displayed GABA-like immunoreactivity. Their cell surface was completely covered by GABA-like immunoreactive and unlabeled terminals that established synapses with the neurons. These neurons are thought to send axon collaterals to the parvocellular nucleus; their axons enter the tectum opticum. The morphological characteristics of neurons of both isthmic nuclei are like those of interneurons, because of their numerous axosomatic synapses with both asymmetrical and symmetrical features. These neurons are not located among their target neurons and exert their modulatory effect on optic transmission in the optic tectum at a distance.