CaBP D-28k and NADPH-diaphorase coexistence in the magnocellular neurosecretory nuclei.

Coexistence of the calcium binding protein calbindin D-28k and NADPH-diaphorase activity was studied in the magnocellular secretory nuclei of the rat hypothalamus using both immunocytochemical and histochemical techniques. Coexistence was found in all the nuclei considered (supraoptic, paraventricular, circularis and fornicals nuclei) with the exception of the hypothalamic area situated between the supraoptic and the paraventricular nuclei. Since both stainings are reliable markers, not based upon the physiological characteristics at a given moment, our study provides a further characterization of the neurons in the magnocellular neurosecretory nuclei.     

NADPH-diaphorase activity in the hypothalamic magnocellular neurosecretory nuclei of the rat

A histochemical study of the distribution of NADPH diaphorase activity in the hypothalamus of normal rats was carried out. Our study demonstrates the presence of NADPH-diaphorase activity in the circularis and anterior and posterior fornicals nuclei for the first time. Additionally, we confirm the presence of NADPH-diaphorase-stained neurons in the paraventricular (both magno- and parvicellular neurons) and supraoptic nuclei, as well as a population of isolated positive neurons (not included in any hypothalamic nuclei) located among the different nuclei. Because NADPH diaphorase has recently been shown to be a nitric oxide synthase, our study reveals a wide presence of this enzymatic activity in the hypothalamus of the rat.     

Magnocellular neurosecretory neurons with ferritin-like immunoreactivity in the hypothalamic supraoptic and paraventricular nuclei of the rat.

Immunohistochemistry for rat liver ferritin (FRT) revealed an intensive labeling in some structures of the rat brain. In the supraoptic (SON) and paraventricular (PVN) hypothalamic nuclei, almost all neurosecretory neurons with vasopressin (AVP)-like immunoreactivity were immunostained with FRT. After water deprivation, a marked enlargement of cell body and an immunoreactivity to transferrin receptors were found in AVP-, FRT- and double (AVP+FRT)-labeled neurons in the SON and PVN.     

Calbindin D-28k immunoreactivity in the rat accessory olfactory bulb

The distribution pattern and the morphology of calbindin D-28k-immunoreactive neurons were studied in the accessory olfactory bulb of the rat using a monoclonal antibody and the avidin-biotin-immunoperoxidase method. Positive neurons were observed in all layers but the vomeronasal nerve layer. Scarce monodendritic periglomerular neurons were calbindin D-28k-immunoreactive. Different morphological types of short-axon cells were calbindin D-28k-immunostained, with different degrees of intensity, in the boundary between the internal and external plexiform layer. In addition, deep short-axon cells located in the granule cell layer were calbindin D-28k-immunopositive. By contrast, previous studies described all cells in the rat accessory olfactory bulb as calbindin D-28k-immunonegative. The staining pattern in the rat accessory olfactory bulb showed both similarities and differences with the distribution pattern of the same calcium-binding protein in the main olfactory bulb.     

Comparison of the efficacy of four viral vectors for transducing hypothalamic magnocellular neurosecretory neurons in the rat supraoptic nucleus

Since transgenes were first cloned into recombinant adenoviruses almost 30 years ago, a variety of viral vectors have become important tools in genetic research. Viruses adeptly transport genetic material into eukaryotic cells, and replacing all or part of the viral genome with genes of interest or silencing sequences creates a method of gene expression modulation in which the timing and location of manipulations can be specific. The hypothalamo-neurohypophyseal system (HNS), consisting of the paraventricular (PVN) and supraoptic (SON) nuclei in the hypothalamus, regulates fluid balance homeostasis and is highly plastic, yet tightly regulated by extracellular fluid (ECF) osmolality and volume. Its reversible plasticity and physiological relevance make it a good system for studying interactions between gene expression and physiology. Here, four viral vectors were compared for their ability to transduce magnocellular neurosecretory neurons (MNCs) of the SON in adult rats. The vectors included an adenovirus, a lentivirus (HIV) and two serotypes of adeno-associated viruses (AAV5 and AAV2). Though adenovirus and AAV2 vectors have previously been used to transduce SON neurons, HIV and AAV5 have not. All four vectors transduced MNCs, but the AAV vectors were the most effective, transducing large numbers of MNCs, with minimal or no glial transduction. The AAV vectors were injected using a convection enhanced delivery protocol to maximize dispersal through the tissue, resulting in the transduction of neurons throughout the anterior to posterior length of the SON (～1.5mm). AAV5, but not AAV2, showed some selectivity for SON neurons relative to those in the surrounding hypothalamus.     

Transient induction of c-fos in rat magnocellular hypothalamic neurons after hypophysectomy.

Using immunofluorescence histochemistry, the paraventricular and supraoptic hypothalamic nuclei of normal control and hypophysectomized rats were studied in double labelling experiments with antibodies against the protein c-fos (Fos) and against vasopressin or oxytocin in order to characterize the activated neurons chemically. Normal controls showed no expression of Fos, whereas in hypophysectomized animals an intense induction of Fos-like immunoreactivity (-LI) was observed 12 h and 24 h post hypophysectomy but not beyond this survival time. Both vasopressinergic and oxytocinergic magnocellular neurons were labelled with Fos-LI. Thus Fos-LI can be induced in magnocellular hypothalamic neurons by injury, suggesting that this protein may be involved in adaptive mechanisms following axotomy.     

Simultaneous monoamine histofluorescence and neuropeptide immunocytochemistry: II. Correlative distribution of catecholamine varicosities and magnocellular neurosecretory neurons in the rat supraoptic and paraventricular nuclei

The comparative morphology of catecholamine (CA) varicosities and neurophysin (NP)-containing perikarya of the supraoptic (SON) and paraventricular nuclei (PVN) was examined. The major CA innervation to the SON and PVN did not coexist with the major distribution of magnocellular perikarya, but was located peripheral to the nuclei. A dense distribution of CA varicosities was found ventral to the neurosecretory perikarya of the SON and overlapped numerous immunoreactive oxytocin- and vasopressin-containing neuritic profiles. Examination of Golgi-stained sections revealed that dendrites from SON perikarya projected to the CA zone and were likely candidates for the processes identified immunocytochemically. In addition, a heterogenous distribution of axosomatic contacts was found within the SON which suggested a preferential innervation of VP-containing neurons. The densest concentration of CA varicosities in the PVN occurred in the periventricular region adjacent to the third ventricle and in the contiguous parvocellular portion of the PVN. These CA varicosities overlapped scattered oxytocinerigic perikarya in both areas. In addition the ventromedial as well as the dorsolateral subnuclei of the PVN were contacted by CA varicosities; this heterogeneous distribution suggests that the each subnucleus of the PVN with its individual hypothalamic, neurohypophyseal, brainstem, or cortical projections may possibly receive a catecholaminergic innervation by a select group of CA cells or nuclear groups from the brain stem.     

Calbindin D-28k- and substance P-immunoreactive primary sensory neurons: Peripheral projections in chick hindlimbs

The peripheral projections of two distinct subpopulations of primary sensory neurons, expressing either calbindin D-28k or substance P, were studied in chick hindlimbs by immunodetecting calbindin D-28k with a rabbit antiserum and substance P with a mouse monoclonal antibody. Calbindin D-28k-immunoreactive axons provided an innervation restricted to specific mechanoreceptors such as muscle spindles, Herbst and Merkel corpuscles, or collars of feather follicles but were absent from Golgi tendon organs. In contrast, substance P-positive axons spread out diffusely in muscles and skin, formed loose plexuses, and extended free branches to the endomysium, arteries, superficial dermis, or dermal pulp of feather follicles. The present results show that calbindin D-28k- and substance P-immunoreactive primary sensory neurons provide distinct modes of innervation to selective targets in peripheral tissues. The results suggest a possible correlation between CaBP-expressing nerve endings and rapidly adapting mechanoreceptors. © 1993 Wiley-Liss, Inc.     

Compensatory sprouting of uninjured magnocellular neurosecretory axons in the rat neural lobe following unilateral hypothalamic lesion

Axonal sprouting of intact neurons of the magnocellular neurosecretory system was investigated using a unilateral hypothalamic knife cut of the hypothalamoneurohypophysial tract to partially denervate the rat neural lobe (NL). Densitometric, morphometric, ultrastructural, and metabolic measures were utilized to demonstrate the compensatory response to denervation in this system. Densitometric analysis revealed a transient reduction in the intensity of vasopressin staining in the NL at 10 days postsurgery (PS) with a subsequent recovery by 20 days PS. There was a comparable initial reduction in the cross-sectional area of the NL followed by a more gradual recovery to normal by 90 days PS. Ultrastructural investigation revealed a reduction in total axon number in the NL at 10 days PS similar to the declines in vasopressin immunoreactivity and size of the NL. A subsequent partial recovery of axon number occurred, paralleling the return to normal NL size between 30 and 90 days PS. Hypertrophy of both somata and cell nuclei of magnocellular neurons in the supraoptic and paraventricular hypothalamic nuclei contralateral to the lesion was also apparent during this period. Daily measurements of urine osmolality revealed an initial transient hypoosmolality followed by a chronic hyperosmolality which persisted throughout the 90 day postsurgical period. There was a concomitant chronic decrease in both daily drinking and urine excretion volumes which began immediately following surgery. These results suggest that intact, contralateral magnocellular vasopressinergic efferents undergo compensatory sprouting as a result of partial denervation of the NL in the absence of a functional deficit in vasopressin.     

Calbindin D-28k Protein and mRNA Localization in the Rat Brain

After the discovery of calretinin, a protein with high sequence homology to calbindin D-28k, the validity of immunohistochemical results obtained using polyclonal antibodies for this protein, was in question. In order to validate the previous results on the localization of calbindin D-28k in the brain, we localized the protein by highly specific monoclonal antibodies and revealed its mRNA histochemically by in situ hybridization. In general there was good agreement between the results obtained using these two different techniques and those reported in previous publications. The concordance was particularly impressive for the cerebral cortex, basal ganglia, basal nucleus of Meynert, hippocampus, thalamus, cerebellum and superior colliculus. In the amygdala and hypothalamus the low spatial resolution of in situ hybridization did not allow precise definition of some nuclei displaying a positive reaction for the protein. In the rhombencephalon, cells of the parabrachial nuclei and the dorsal raphe nucleus expressed calbindin D-28k. Neurons in the dorsal horn of the spinal cord and some horizontal cells of the retina were tagged with both methods. The only discrepancy was the presence of immunoreactive ependymal cells, whereas mRNA never occurred in cells lining the ventricles. Thus, the combined approach has established the widespread distribution of cells expressing calbindin D-28k in the rat brain.     

Calbindin D28k-containing splanchnic and cutaneous dorsal root ganglion neurons of the rat

Calbindin D28k (CaBP)-containing splanchnic and cutaneous sensory neurons in the rat dorsal root ganglia (DRGs) were investigated immunocytochemically in combination with a fluorescent dye tracer (Fluoro gold). About 15% of the DRG neurons at Th9-10 levels showed CaBP-like immunoreactivity. Eighty-four % of the splanchnic sensory neurons were immunoreactive to CaBP, while only 3% of the cutaneous sensory neurons were. The diameters of the splanchnic and cutaneous sensory neurons containing CaBP were 23.4 +/- 6.3 microns and 38.4 +/- 8.8 microns, respectively. Splanchnic sensory neurons containing CaBP were sensitive to capsaicin while cutaneous ones were not. These findings suggest that CaBP-containing splanchnic and cutaneous sensory neurons constitute different subgroups among the DRG neurons at the lower thoracic level.     

Parvalbumin- and Calbindin D-28k-Immunoreactive Innervation of Orofacial Tissues in the Rat

Parvalbumin- and calbindin D-28k-immunoreactive (-ir) innervation was examined in orofacial tissues of the rat. Labial and facial skins were devoid of the calcium-binding protein (CaBP)-ir nerve endings, while the infraorbital and mental nerves contained numerous parvalbumin-ir axons. Labial and gingival mucosae were also devoid of the CaBP-ir nerve endings. The buccal mucosa and incisive papilla contained both encapsulated and unencapsulated endings, while the hard palate mucosa excluding the incisive papilla contained only unencapsulated endings. Encapsulated endings were found just beneath the epithelium or attached to the cartilaginous core of the incisive papilla. Unencapsulated endings in the lamina propria were subdivided into two types: simple (unramified) and complex (ramified). Neurites of simple endings were straight, curved, or coiled, while those of complex endings exhibited a bush-like appearance due to the ramification. In addition, palatal rugae contained intraepithelial endings. The unencapsulated complex endings in palatal rugae coexpressed parvalbumin- and calbindin D-28k-irs, whereas other endings were immunoreactive for parvalbumin alone. The pterygopalatine ganglion contained calbindin D-28k-ir pericellular fibers but not the ir cell bodies. A subpopulation of trigeminal ganglion neurons coexpressed both CaBPs. CaBP-ir encapsulated and unencapsulated endings in the oral mucosa probably include low-threshold mechanoreceptors, while parvalbumin-ir intraepithelial endings in the palatal mucosa may be involved in nociception.     

Calbindin D28K-immunoreactivity identifies distinct subpopulations of sympathetic pre- and postganglionic neurons in the rat

Neurons performing the same function can be identified immunohistochemically because they often share the same neurochemistry. The distribution of calcium-binding proteins, like calbindin, has been used previously to identify functional subpopulations of neurons in many parts of the nervous system. In this study we have investigated the distribution of calbindin D28K-immunoreactivity in subpopulations of sympathetic preganglionic neurons in the intermediolateral nucleus of the rat spinal cord. The majority of calbindin D28K-immunoreactive preganglionic neurons also had co-localised nitric oxide synthase, although a population of preganglionic neurons in the mid- to low thoracic intermediolateral nucleus expressed only calbindin D28K-immunoreactivity. Retrograde-tracing studies showed that calbindin D28K-immunoreactive neurons projected to the superior cervical and stellate ganglia, with smaller numbers of cells projecting to the lumbar sympathetic chain and superior mesenteric ganglia. Very few calbindin D28K-immunoreactive neurons projected to the inferior mesenteric ganglion, and none projected to the adrenal medulla. The distribution of calbindin D28K-immunoreactive terminals and postganglionic neurons in the superior cervical and stellate ganglia was also investigated. Many postganglionic neurons were calbindin D28K-immunoreactive, and most of these lacked neuropeptide Y-immunoreactivity. Calbindin D28K-immunoreactive nerve terminals were common and formed dense pericellular baskets around many postganglionic neurons, including some of those that were calbindin D28K-immunoreactive, but only rarely formed pericellular baskets around neuropeptide Y-immunoreactive neurons. The function of some of the classes of postganglionic neurons that were the target of calbindin D28K-immunoreactive preganglionic terminals was determined by combining immunohistochemistry with retrograde-tracer injections into a range of peripheral tissues. Calbindin D28K-immunoreactive nerve terminals, with co-localised nitric oxide synthase-immunoreactivity, surrounded secretomotor neurons projecting to the submandibular salivary gland and pilomotor neurons projecting to skin, but did not surround neurons projecting to brown fat or vasomotor neurons projecting to the skin, muscle, or salivary glands. J. Comp. Neurol. 386:245–259, 1997. © 1997 Wiley-Liss, Inc.     

Forced swimming stimulates the expression of vasopressin and oxytocin in magnocellular neurons of the rat hypothalamic paraventricular nucleus.

Previous studies have shown that a 10-min forced swimming session triggers the release of both vasopressin and oxytocin into the extracellular fluid of the hypothalamic paraventricular (PVN) and supraoptic nuclei (SON) in rats. At the same time oxytocin, but not vasopressin, was released from the axon terminals into the blood. Here we combined forced swimming with in situ hybridization to investigate whether (i) the stressor-induced release of vasopressin and oxytocin within the PVN originates from parvo- or magnocellular neurons of the nucleus, and (ii) central release with or without concomitant peripheral secretion is followed by changes in the synthesis of vasopressin and/or oxytocin. Adult male Wistar rats were killed 2, 4 or 8 h after a 10-min forced swimming session and their brains processed for in situ hybridization using 35S-labelled oligonucleotide probes. As measured on photo-emulsion-coated slides, cellular vasopressin mRNA concentration increased in magnocellular PVN neurons 2 and 4 h after swimming (P < 0.05). Similarly, oxytocin mRNA concentration was significantly increased in magnocellular neurons of the PVN at 2 and 8 h (P < 0.05). We failed to observe significant effects on vasopressin and oxytocin mRNA levels in the parvocellular PVN and in the SON. Taken together with results from previous studies, our data suggest that magnocellular neurons are the predominant source of vasopressin and oxytocin released within PVN in response to forced swimming. Furthermore, in the case of vasopressin, central release in the absence of peripheral secretion is followed by increased mRNA levels, implying a refill of depleted somato-dendritic vasopressin stores. Within the SON, however, mRNA levels are poor indicators of the secretory activity of magnocellular neurons during stress.     

Retrograde transport of horseradish peroxidase in the magnocellular neurosecretory system of the rat.

Horseradish peroxidase (HRP) was injected into the pituitary in adult rats. After 2-3 days, the neurones of the supraoptic nuclei, the magnocellular parts of the paraventricular nuclei, and the various accessory neurosecretory hypothalamic nuclei showed accumulation of HRP. The HRP reaction product consisted of fine, discrete cytoplasmic granules, and in electron micrographys it was seen to be located in the lysosome-like dense bodies of 0.4-0.6 mum diameter which are normally present in the cytoplasm of the neurosecretory neuron.es. Very little reaction product was found in the neurosecretory axons. Reaction product was also found in the hypothalamic arcuate nuclei. This was the result of an endogenous peroxidase-like activity, since it occurred in control animals which had not received HRP. This endogenous reaction product is non-neuronal. Morphologically, it takes the form of distinctive clusters of coarse granules which are seen in electron micrographs to be characteristic angular bodies of 0.7-1.0 mum diameter located in the cytoplasm of astrocytes or their processes.     

Chondroitin sulfate proteoglycan phosphacan/RPTPbeta in the hypothalamic magnocellular nuclei

The hypothalamo-neurohypophysial system synthesizes and releases arginine vasopressin (AVP) and oxytocin (OXT) with physiological stimulation. In the present study, we investigated localization of a chondroitin sulfate proteoglycan (CSPG), phosphacan/RPTPbeta, in the supraoptic nucleus (SON) and paraventricular nucleus (PVN) of adult rats at both the light and electron microscopic levels. Immunohistochemical analyses demonstrated stronger phosphacan/RPTPbeta immunoreactivity within the SON and PVN compared with adjacent hypothalamic areas. Double labeling experiments showed phosphacan/RPTPbeta immunoreactivity constituting punctate networks to surround the somata and dendrites of AVP- and OXT-secreting magnocellular neurons. Electron microscopic examination further revealed strong phosphacan/RPTPbeta immunoreactivity at extracellular membrane surface of some axons, somata, and dendrites of the SON, but not of synaptic junctions. Interestingly, phosphacan/RPTPbeta immunoreactivity was also observed at extracellular surface membrane between astrocytic processes and neurons rather than between magnocellular neurons. The present results indicate the high expression of the CSPG, phosphacan/RPTPbeta at the extracellular space in the hypothalamic AVP- and OXT-secreting magnocellular neurons.     

The organization of chemically characterized afferents to the perivascular neuronal groups of the hypothalamic magnocellular neurosecretory system in the rat

The hypothalamic magnocellular neurosecretory system consists of the paraventricular nucleus and supraoptic nucleus and a number of accessory nuclei. There is evidence that each of the accessory nuclei has a preferential source of afferents. Two of the accessory nuclei, namely the nucleus circularis (NC) and the lateral hypothalamic perivascular nucleus (LHPN), are particularly interesting due to their very close relationship with the blood vessels. The NC is composed of small dense clusters of neurons in the medial anterior hypothalamus. The groups of lateral hypothalamic neurons gathering around vascular branches are collectively called the LHPN. Their close topographical relationship with the blood vessels may indicate that the latter may serve as a source of input to these nuclei. As a part of the effort to investigate this issue, the present study examined in these two nuclei the distribution pattern of terminal-like elements containing 11 transmitters/modulators. Only a few, if any, terminal-like elements of the transmitters/modulators studied could be found distributed in the NC proper, although its immediate vicinity could be densely innervated. On the contrary, the LHPN proper was often densely innervated by fibers expressing the examined markers. These terminal patterns were found to be quite different from those of the paraventricular and supraoptic nuclei. The present findings further substantiate the notion of a functional differentiation among the subnuclei of the magnocellular neurosecretory system. The significance of the relationship of these two perivascular nuclei with the blood vessels is discussed.