Activation of vasopressin neurons in the human supraoptic and paraventricular nucleus in senescence and senile dementia

A recent study has shown that vasopressin (AVP) cells in the human supraoptic (SON) and paraventricular (PVN) nuclei increase in size after 60 years of age, suggesting that AVP production is increased in senescence. In the present study, the same brain material was used for the determination of nucleolar size in immunocytochemically identified AVP and oxytocin (OXT) neurons as an additional parameter for peptide production.A strong correlation was found between nucleolar size and cell size, both in AVP and OXT neurons. Nucleolar size of AVP but not of OXT neurons increased significantly in senescence. Observations in brains from patients with senile dementia of the Alzheimer type (SDAT) were commensurate with their ages. These results strongly support the hypothesis that AVP neurons in the SON and PVN are activated in old age.     

Specific expression of secretogranin II in magnocellular vasopressin neurons of the rat supraoptic and paraventricular nucleus in response to osmotic stimulation

As secretogranin II is considered to be a marker for the regulated secretory pathway, its distribution in the hypothalamo-neurohypophyseal system of salt-loaded Wistar rats was studied in detail by immunocytochemistry. Although after an osmotic challenge both vasopressin and oxytocin neurons are stimulated, secretogranin II was exclusively expressed in a subpopulation of vasopressinergic magnocellular neurons in the supraoptic and paraventricular nucleus of Wistar rats. Secretogranin II was only surely visualized after a combination of osmotic challenge and blockade of axonal transport by colchicine treatment. When these pre-treatments were not performed, only punctate fibers situated around the magnocellular neurons within the paraventricular and supraoptic nucleus were observed. Oxytocinergic magnocellular neurons never displayed any secretogranin II immunoreactivity, not even during lactation and after colchicine treatment. These findings suggest that secretogranin II is of functional importance during enhanced secretory activity within vasopressinergic neurons.     

Prolonged alcohol intake leads to irreversible loss of vasopressin and oxytocin neurons in the paraventricular nucleus of the hypothalamus

Previous data revealed that numerous neurons in the supraoptic nucleus degenerate after prolonged ethanol exposure, and that the surviving neurons increase their activity in order to prevent dramatic changes in water metabolism. Conversely, excess alcohol does not induce cell death in the suprachiasmatic nucleus, but leads to depression of neuropeptide synthesis that is further aggravated by withdrawal. The aim of the present study is to characterize the effects of prolonged ethanol exposure on the magnocellular neurons of the paraventricular nucleus (PVN) in order to establish whether or not magnocellular neurons display a common pattern of reaction to excess alcohol, irrespective of the hypothalamic cell group they belong. Using conventional histological techniques, immunohistochemistry and in situ hybridization, the structural organization and the synthesis and expression of vasopressin (VP) and oxytocin (OXT) in the magnocellular component of the PVN were studied under normal conditions and following chronic ethanol treatment (6 or 10 months) and withdrawal (4 months after 6 months of alcohol intake). After ethanol treatment, there was a marked decrease in the number of VP- and OXT-immunoreactive magnocellular neurons that was attributable to cell death. The surviving neurons were hypertrophied and the VP and OXT mRNA levels in the PVN unchanged. Withdrawal did not alter the number of VP- and OXT-producing neurons or the gene expression of these peptides. These results substantiate the view that after prolonged ethanol exposure numerous neurons of the hypothalamic magnocellular system degenerate, but the mRNA levels of VP and OXT are not decreased due to compensatory changes undergone by the surviving neurons.     

Simultaneous monoamine histofluorescence and neuropeptide immunocytochemistry: VI. Catecholamine innervation of vasopressin and oxytocin neurons in the rhesus monkey hypothalamus

The co-localization patterns of catecholamine varicosities and peptide-specific neuronal perikarya were assessed within the supraoptic and paraventricular nuclei in the rhesus monkey, Macaca mulatta. Formaldehyde-induced histofluorescence was coupled with the unlabelled antibody technique for the demonstration of neuropeptides. Hormone-specific neurophysin staining served to identify vasopressin and oxytocin-containing neurons in these hypothalamic nuclei. Catecholamine varicosities were seen in juxtaposition to vasopressin- and oxytocin-containing perikarya and proximal dendrites. The densest catecholamine innervation patterns were seen in the ventrolateral portion of the supraoptic nucleus; the dorsomedial portion of this nucleus received a considerably less dense innervation pattern. Oxytocin neurons were clustered in this relatively catecholamine poor region, whereas the vasopressin-containing neurons were more abundantly found in the Catecholamine rich region. The paraventricular nucleus presented a considerably more complex pattern, perhaps reflecting the more diverse organization of this nucleus. Nevertheless, some separation of the oxytocin neurons, in a region less densely innervated by catecholamine varicosities, was noted. These observations confirm our earlier reports, in rat hypothalamus, that the norepinephrine innervation of the hypothalamic magnocellular neurons as seen with catecholamine histofluorescence favors the vasopressin-containing neurons over those located within the same nuclei which synthesize another neurohyphysial principal, oxytocin.     

Branching projections of catecholaminergic brainstem neurons to the paraventricular hypothalamic nucleus and the central nucleus of the amygdala in the rat

In this study, we have employed triple fluorescent-labelling to reveal the distribution of catecholaminergic neurons within three brainstem areas which supply branching collateral input to the central nucleus of the amygdala (CNA) and the hypothalamic paraventricular nucleus (PVN): the ventrolateral medulla (VLM), the nucleus of the solitary tract (NTS) and the locus coeruleus (LC). The catecholaminergic identity of the neurons was revealed by immunocytochemical detection of the biosynthetic enzyme, tyrosine hydroxylase. The projections were defined by injections of two retrograde tracers, rhodamine- and fluorescein-labelled latex microspheres, in the CNA and PVN, respectively. In the VLM and NTS, the greatest incidence of neurons which contained both retrograde tracers was found at the level of the area postrema. These neurons were mainly located within the confines of the A1/C1 (VLM) and A2 (NTS) catecholaminergic neuronal groups. Double-projecting neurons in the LC (A6) were distributed randomly within the nucleus. It was found that 15% in the VLM, 10% in the NTS and 5% in the LC of the retrogradely labelled cells projected via branching collaterals to the PVN and CNA. One half of these neurons in the VLM and NTS were catecholaminergic, in contrast to the LC where virtually all double-retrogradely labelled neurons revealed tyrosine hydroxylase immunoreactivity. In the other brainstem catecholaminergic cell groups (A5, A7, C3), no catecholaminergic neurons were found that supplied branching collaterals to the CNA and PVN. Our results indicate that brainstem neurons may be involved in the simultaneous transmission of autonomic-related signals to the CNA and the PVN. Catecholamines are involved in these pathways as chemical messengers. Brainstem catecholaminergic and non-catecholaminergic neurons, through collateral branching inputs may provide coordinated signalling of visceral input to rostral forebrain sites. This may lead to a synchronized response of the CNA and PVN for the maintenance of homeostasis.     

Local projections of GABAergic neurons in the dentate gyrus and CA1 region in the rat hippocampal formation

Using retrograde transport of wheat germ agglutinin conjugated colloidal gold (WGA-gold) combined with immunoreactivity for glutamate decarboxylase (GAD), a specific synthesizing enzyme for γ-aminobutyric acid (GABA), local projections of GABAergic neurons in the dentate gyrus and CAI were examined. In the hilus of the dentate gyrus, it was found that GABAergic neurons in the granule cell layer projected to the ipsilateral upper leaf of the molecular layer, with a mediolateral extension of more than 1.2 mm and a rostrocaudal extension of over 0.8 mm. Non-GABAergic neurons in nearly the entire hilar area were found to project to the ipsilateral upper leaf of the molecular layer. In the dorsal CAI region, GABAergic neurons in the stratum pyramidale and radiatum converged onto the ipsilateral stratum pyramidal/oriens, with a mediolateral extension of over 1 mm and a rostrocaudal extension of over 0.7 mm. These results provide direct evidence that in both the dentate gyrus and CAI, GABAergic interneurons from a fairly large field converge onto a very small target area. This suggests that the output signals from GABAergic neurons in the dentate gyrus and CAI, and non-GABAergic neurons in the dentate gyrus, may propagate beyond the anatomical limits contained in conventional slice preparations of the hippocampal formation.     

The effects of osmotic stimulation and water availability on c-Fos and FosB staining in the supraoptic and paraventricular nuclei of the hypothalamus

We studied the effects of osmotic stimulation on the expression of FosB and c-Fos in the supraoptic nucleus (SON) and paraventricular nucleus (PVN). Adult male rats were divided into two groups that were injected with lidocaine (0.1-0.2 ml sc) followed by either 0.9% or 6% NaCl (1 ml/100 g bw sc). After the NaCl injections, the rats were anesthetized and perfused 2, 6, or 8 h after the injections. Their brains were prepared for immunocytochemistry and stained with FosB and c-Fos antibodies. The number of c-Fos-positive cells was significantly increased only at 2 h in the SON and PVN. In contrast, the number of FosB-positive cells was significantly increased at 6, and 8 h in both the SON and PVN. In a second experiment, the effect of water availability on FosB staining 8 h after injections of 6% NaCl was tested in 3 groups of rats: water ad libitum, rats that had no access to water, and rats that were given water 2 h prior to perfusion. FosB staining was significantly reduced in both the SON and the PVN of rats that had ad libitum water compared to the two water-restricted groups. In the third experiment, rats were injected with either 0.9% NaCl or 6% NaCl and were either given ad libitum access to water or water restricted for 6 h after the injections and perfused 24 h after the saline injections. FosB staining was not increased when water was available ad libitum. FosB staining was significantly increased at 24 h in the rats injected with 6% NaCl when water was restricted. Thus, FosB may continue to influence protein expression in the SON and PVN for at least 24 h following acute osmotic stimulation.     

Median preoptic nucleus projections to vasopressin-containing neurones of the supraoptic nucleus in sheep. A light and electron microscopic study

It has been postulated that osmoreceptors are situated in either or both of two components of the lamina terminalis, the subfornical organ (sfo) and organum vasculosum laminae terminalis (ovlt) and that information from these sites may be relayed to the hypothalamus directly or via a synapse in the median preoptic nucleus (mnpo). We have investigated the nature of projections from the mnpo to vasopressin (AVP)-containing neurones in the hypothalamus. Microinjections of horseradish peroxidase-wheat germ agglutinin (HRP-WGA) have been made into the mnpo and supraoptic nucleus (son) of the sheep. These injections indicated that in the sheep, as in the rat, the mnpo shares a reciprocal innervation with the sfo and ovlt. Furthermore, the most extensive efferent outflow of the mnpo is to the son, with lesser projections directed to the pvn and other hypothalamic sites. When examined at the electron microscopic level, fibres projecting from the mnpo to the son were found to form synapses with immunocytochemically identified AVP neurones. It is suggested that this pathway is one of the major routes by which information from putative osmoreceptors in the lamina terminalis is conveyed to AVP neurones in the hypothalamus.     

Parabrachial nucleus neurons providing axons to both the thalamus and the spinal cord in the rat

After injecting Fluoro-gold (FG) and tetramethylrhodamine-dextran amine (TMR-DA), respectively, into the medial part of the ventrobasal thalamus and the upper segments of the cervical cord of the rat, a small number of neuronal cell bodies were double-labeled retrogradely with both FG and TMR-DA in the parabrachial nuclear complex (PBN) ipsilateral to the injection into the thalamus. The cell bodies double-labeled with FG/TMR-DA were seen mainly in the Kölliker-Fuse subnucleus and additionally in the external medial subnucleus.     

Co-localization of enkephalin and TRH in perifornical neurons of the rat hypothalamus that project to the lateral septum

Neurons of the lateral septal nucleus are surrounded by terminals immunoreactive for thyrotropin-releasing hormone (TRH) and enkephalin (Enk). Retrograde labeling from the lateral septum in combination with immunocytochemical analyses for Enk and TRH in colchicine-treated rats has revealed that nearly all Enk- (and TRH-) containing perifornical neurons project to the lateral septum. Immunostaining of adjacent, thin paraffin sections for either TRH or Enk and double staining of thick vibratome sections for the two peptides have shown that TRH and Enk immunoreactivities co-exist within the same neurons in the perifornical region of the hypothalamus.     

Chronic intermittent salt loading enhances functional recovery from polydipsia and survival of vasopressinergic cells in the hypothalamic supraoptic nucleus following transection of the hypophysial stalk

Hypophysial stalk-transected (ST) and sham-operated animals were subjected to a chronic intermittent salt loading regimen (CISL) for 14 days beginning 1 day post surgery (dps). Animals were sacrificed at 15 and 36 dps. Three days after the termination of CISL, water consumption in ST + CISL animals decreased to the same level as that of sham-operated animals, while that of ST + water animals was maintained at a significantly higher level. The number of the surviving vasopressinergic neurons in the supraoptic nuclei of the ST + CISL group was significantly higher than that of ST + water group. CISL induced vasopressinergic axonal sprouting into the external zone of the median eminence, and formation of subependymal perivascular plexus. While CISL also enhanced regeneration of oxytocinergic axons into the external zone, it did not, however, have any effect on the number of oxytocinergic neurons surviving axotomy.     

Electrophysiological identification of neurons in ventrolateral medulla sending collateral axons to paraventricular and supraoptic nuclei in the cat

In recent studies, it has been reported that high-frequency stimulation restricted to A alpha beta fibers in man can be perceived as painful and evoke a nociceptive flexion reflex. These results would indicate that some patterns of activity in low-threshold mechanoreceptors can lead to painful sensations. Because of the theoretical importance of this question, the above studies were extended by recording the evoked neural activity with the technique of percutaneous microneurography. Painful sensations and the nociceptive reflex did not appear unless the evoked nerve response contained activity in A delta fibers. The results support the theory that painful sensations occur in normal man only when nociceptor afferents are activated.     

Serotonergic projections from the midbrain periaqueductal gray and nucleus raphe dorsalis to the nucleus parafasccicularis of the thalamus

By a double-labeling method combining the retrograde tracing of horseradish peroxidase and the immunocytochemical technique, serotonin-like immunoreactive neurons in the midbrain periaqueductal gray (PAG) and nucleus raphe dorsalis (DR) of the rat were observed to send projection fibers to the nucleus parafascicularis of the thalamus bilaterally with an ipsilateral dominance. These serotonin-containing projecting neurons were observed mainly at the middle-caudal levels of the ventrolateral subdivision of the PAG and less at the middle-rostral levels of the DR.     

Synaptic inputs of neuropeptide Y-immunoreactive noradrenergic nerve terminals to neurons in the nucleus preopticus medianus which project to the paraventricular nucleus of the hypothalamus of the rat: a combined immunohistochemical and retrograde tracing method

The nucleus preopticus medianus (POMe) is known to serve as a relay site in the neural pathway, from the subfornical organ to the paraventricular nucleus of the hypothalamus (PVN), and to play an important role in the regulation of fluid balance and caridovascular control. A neural connection of noradrenergic nerve terminals in the POMe was examined using electron microscopic immunohistochemistry with the retrograde tract tracing method. Double immunofluorescent labelling revealed nerve terminals immunoreactive to both tyrosine hydroxylase (TH) and neuropeptide Y (NPY) and those immunoreactive to both TH and noradrenaline in the POMe. This indicates that there is an NPY-immunoreactive noradrenergic innervation in the POMe. At the electron microscopic level, nerve terminals immunoreactive to TH or NPY in the POMe formed synapses with dendrites or cell bodies of neurons which were retrogradely labeled after injection of the retrograde tracer, WGA-HRP-colloidal gold, in the PVN. These observations suggest that neurons in the POMe with projections to the PVN may be directly affected by NPY-immunoreactive noradrenergic afferent fibers which presumably originate in the brainstem.     

Effects of unilateral or bilateral lesions within the anteroventral third ventricular region on c-fos expression induced by dehydration or angiotensin II in the supraoptic and paraventricular nuclei of the hypothalamus

This paper reports the effects of AV3V lesions on the pattern of c-fos induced by 24 h dehydration. As expected, bilateral electrolytic lesions within the AV3V region (the ventral median preoptic nucleus) suppressed water intake following 24 h water deprivation. C-fos expression was also suppressed in the supraoptic (SON) and (less completely) in the paraventricular (PVN) nuclei, but not in the subfornical organ (SFO). Unilateral lesions of the AV3V region suppressed c fos expression in the ipsilateral SON, but this selective ipsilateral effect was less in the PVN. The SFO was again unaffected. Unilateral lesions also suppressed c-fos expression in the ipsilateral SON and PVN (to a lesser degree) following intraventricular infusions of angiotensin 11 (250 pmol). These results suggest that the cellular response of supraoptic neurons to osmotic stimuli require inputs from the AV3V region, but that this is less absolute for the PVN; that the projection from the ventral AV3V area to the SON is ipsilateral, but that to the PVN may be less lateralised. Activation of the SFO by dehydration is not dependent upon the integrity of the ventral AV3V region. These results are closely comparable to the effects of similar lesions on c-fos expression following intraventricular infusions of angiotensin 11, and suggest that the effect of dehydration on forebrain c-fos expression may be related to the central actions of angiotensin II.     

Dual projections of gonadotropin releasing hormone containing neurons to the interpeduncular nucleus and to the vasculature in the female rat

Immunofluorescence for gonadotropin releasing hormone (GnRH) in combination with retrograde labeling from the interpeduncular nucleus, as well as from the vasculature confirms that, in the rat, certain GnRH neurons project from the septum-diagonal band to the interpeduncular nucleus. However, about one half of these GnRH neurons also project to fenestrated capillaries as evidenced by uptake and retrograde transport of both peripherally injected Fluoro-Gold and centrally injected rhodamine labeled microspheres. The results indicate that the endocrine effects of GnRH are exerted in part by neurons which simultaneously project to neurohemal contact zones and to areas in the brain which are involved in the regulation of certain behaviors. It is suggested that certain GnRH neurons can directly couple endocrine events with other intracerebral events, such as regulation of lordosis behavior.     

The effect of morphine on responses of mediodorsal thalamic nuclei and nucleus submedius neurons to colorectal distension in the rat

In halothane-anesthetized rats, we characterized the responses of single neurons in the nuclei of medial thalamus (MT), specifically the mediodorsal thalamic nucleus (MD) and the nucleus submedius (Sm), to a noxious visceral stimulus (colorectal balloon distension, CRD), and studied the effects of intravenous morphine (Mor) on these responses using standard extracellular microelectrode recording techniques. 62 MD and 46 Sm neurons were isolated on the basis of spontaneous activity. 47 of the MD neurons (76%) responded to CRD, of which 70% had excitatory and 30% had inhibitory responses. 38 of the Sm neurons (83%) responded to CRD, of which 89% had excitatory and 11% had inhibitory responses. Responses of MD and Sm neurons excited by CRD were related significantly to distension pressure (20–100 mmHg), with maximum excitation occurring at 60 and 100 mmHg, respectively. MD neurons inhibited by CRD also had graded responses to graded CRD, with maximum inhibition occurring at 80 mmHg. The responses to noxious (pinch, heat) and nonnoxious (tap, brush) cutaneous stimuli were studied in 59 of the MD and 44 of the Sm neurons isolated. 22 of the MD neurons (37%) studied had cutaneous receptive fields, of which 59% were large and bilateral, 41% were small and usually contralateral receptive fields. 55% of these neurons were nociceptive-specific, 45% responded to both noxious and nonnoxious cutaneous stimulation. 29 of the Sm neurons (66%) studied had cutaneous receptive fields, of which 72% were large and usually bilateral, 14% were small and bilateral, 14% were small and contralateral receptive fields. 90% of these neurons were nociceptive-specific, 10% responded to both noxious and nonnoxious stimulation. No MD or Sm neurons responded exclusively to nonnoxious cutaneous stimulation. Mor (0.125, 0.25, 0.5 and 1 mg/kg IV) attenuated MD and Sm neuronal excitatory responses to CRD in a dose-dependent fashion, abolishing evoked activity with a dose of 0.5 mg/kg (p<0.05) and 1 mg/kg (p<0.05), respectively. Naloxone (0.4 mg/kg IV) reversed the effects of Mor. Mor and naloxone had no effects on spontaneous activity. These data support the involvement of MD and Sm neurons in visceral nociception, and are consistent with a role of Sm in affective-motivational, and MD in both sensory-discriminative and affective-motivational aspects of nociception.     

Effects of two years of estrogen loss or replacement on nucleus basalis cholinergic neurons and cholinergic fibers to the dorsolateral prefrontal and inferior parietal cortex of monkeys

The present study examined the long-term (2 years) effects of estrogen loss or estrogen replacement therapy (ERT) on cholinergic neurons in the nucleus basalis of Meynert and on cholinergic fibers in the prefrontal and parietal cortex of adult female cynomolgus monkeys. Cholinergic fiber density in layer II of the prefrontal cortex was decreased in monkeys who were ovariectomized and treated with placebo for 2 years. In contrast, ovariectomized monkeys receiving ERT for 2 years had fiber densities that were comparable to those of intact controls. No differences in parietal cholinergic fiber density or nucleus basalis cholinergic neuron number or volume were found among intact, ovariectomized, or ERT monkeys. Our results suggest that ERT is effective in preventing region-specific changes in cortical cholinergic fibers that result from the loss of circulating ovarian hormones. These modest but appreciable effects on cholinergic neurobiology following long-term estrogen loss and ERT may contribute to changes in visuospatial attention function that is mediated by the prefrontal cortex.     

Differential effects of autologous peripheral nerve grafts to the corpus striatum of adult rats on the regeneration of axons of striatal and nigral neurons and on the expression of GAP-43 and the cell adhesion molecules N-CAM and L1

A segment of tibial nerve was autografted to the right corpus striatum of deeply anesthetized adult rats; the distal graft was left beneath the scalp. Horseradish peroxidase (HRP) conjugates were injected into the distal graft after 2–30 weeks, and the animals were killed 2–3 days later. Small numbers of neostriatal perikarya were HRP labeled at all survival times; most were large (ca. 20 μm in diameter), and many contained acetycholine esterase (AChE). Many more neurons were labelled in the substantia nigra pars compacta (SNpc) 4 weeks or more after grafting. When the graft encroached on the globus pallidus, numerous pallidal neurons, most of them AChE positive, were also labeled. Nigrostriatal neurons, a population of pallidal cholinergic neurons, and a subclass (or classes) of neostriatal neurons, including cholinergic interneurons, thus can be classified as central nervous system (CNS) neurons with a relatively strong regenerative response. In a second experimental series, animals were killed 1–4 weeks after grafting, and sections were probed for the expression of mRNAs encoding growth-associated protein 43 (GAP-43) and the cell adhesion molecules N-CAM and L1. Subpopulations of mostly large neurons scattered throughout the neostriatum gave moderate signals for GAP-43 and N-CAM mRNAs and a stronger signal for L1 mRNAs. Most SNpc neurons were strongly labeled with all three probes. Neostriatal grafts had no apparent effect on the expression of any of the mRNAs in the SNpc or on L1 and N-CAM mRNAs in the striatum. However, GAP-43 mRNA levels were increased in a few, mainly large neostriatal neurons around the graft tip, resembling the HRP-labeled cells. In contrast, previous work has shown upregulation (from an undetectable level) of GAP-43 and L1 mRNAs in neurons regenerating axons into grafts placed in the thalamus and cerebellum. Thus, GAP-43 and L1 mRNA expression, but not necessarily marked upregulation, may correlate with, and be intrinsic determinants of, the ability of CNS neurons to regenerate their axons. J. Comp. Neurol. 391:259–273, 1998. © 1998 Wiley-Liss, Inc.