Cutting edge: neuronal recognition by CD8 T cells elicits central diabetes insipidus.

An increasing number of neurologic diseases is associated with autoimmunity. The immune effectors contributing to the pathogenesis of such diseases are often unclear. To explore whether self-reactive CD8 T cells could attack CNS neurons in vivo, we generated a mouse model in which the influenza virus hemagglutinin (HA) is expressed specifically in CNS neurons. Transfer of cytotoxic anti-HA CD8 T cells induced an acute but reversible encephalomyelitis in HA-expressing recipient mice. Unexpectedly, diabetes insipidus developed in surviving animals. This robust phenotype was associated with preferential accumulation of cytotoxic CD8 T cells in the hypothalamus, upregulation of MHC class I molecules, and destruction of vasopressin-expressing neurons. IFN-γ production by the pathogenic CD8 T cells was necessary for MHC class I upregulation by hypothalamic neurons and their destruction. This novel mouse model, in combination with related human data, supports the concept that autoreactive CD8 T cells can trigger central diabetes insipidus.

Reactive oxygen species are required for the hypothalamic osmoregulatory response.

Free radicals, or reactive oxygen species (ROS), are highly reactive byproducts of oxygen degradation. They are well known for their cellular toxicity, but few studies have analyzed their potential role in homeostatic processes. We investigated ROS production and function during the arginine vasopressin (AVP) hypothalamic response to hyperosmolarity. Six-week-old male C3H/HeJ mice were subjected to salt loading for 2 or 8 d. The osmotic axis was progressively activated and reached a new steady-state status at 8 d as demonstrated by monitoring of plasmatic osmolality and c-Fos and AVP expression in the supraoptic nucleus (SON). Free radicals, visualized by dihydroethidine staining and measured by 2'-7'dichlorofluorescein diacetate assays, were detected after 2 d of salt loading. The activity and expression of superoxide dismutase 2 and catalase were concomitantly up-regulated in the SON, suggesting that free radicals are detoxified by endogenous antioxidant systems, thereby avoiding their deleterious effects. The early phase of the osmoregulatory response has been investigated using an acute hyperosmotic model; free radicals were produced 45 min after an ip injection of 1.5 m NaCl. This was followed by an increase in c-Fos and AVP expression and an increase in superoxide dismutase 2 and catalase activities. α-Lipoic acid, a ROS scavenger, administrated during the 3 d before the hypertonic ip injection, abolished the increase of AVP. These findings establish that hyperosmolarity causes ROS production in the SON, which is essential for AVP increase. This demonstrates the importance of free radicals as physiological signaling molecules in the regulation of body-fluid balance.

Architecture of the hypothalamo-posthypophyseal complex is controlled by monoamines.

The hypothalamo-neurohypophyseal system displays significant plasticity when subjected to physiological stimuli, such as dehydration, parturition, or lactation. This plasticity arises at the neurochemical and electrophysiological levels but also at a structural level. Several studies have demonstrated the role of monoaminergic afferents in controlling neurochemical and electrophysiological plasticity of the supraoptic nucleus (SON) and of the neurohypophysis (NH), but little is known about how the changes in structural plasticity are triggered. We used Tg8 mice, disrupted for the monoamine oxidase A gene, to study monamine involvement in the architecture of the SON and of the NH. SON astrocytes in Tg8 mice displayed an active status, characterized by an increase in S100ß expression and a significant decrease in vimentin expression, with no modification in glial fibrillary acidic protein (GFAP) levels. Astrocytes showed a decrease in glutamate dehydrogenase (GDH) levels, whereas glutamine synthetase (GS) levels remained constant, suggesting a reduction in astrocyte glutamate catabolism. Tenascin C and polysialic acid-neural cell adhesion molecule (PSA-NCAM) expressions were also elevated in the SON of Tg8 mice, suggesting an increased capacity for structural remodelling in the SON. In the NH, similar date were obtained with a stability in GFAP expression and an increase in PSA-NCAM immunostaining. These results establish monoamine (serotonin and noradrenaline) involvement in SON and NH structural arrangement. Monoamines therefore appear to be crucial for the coordination of the neurochemical and structural aspects of neuroendocrine plasticity, allowing the hypothalamo-neurohypopyseal system to respond appropriately when stimulated.

Differential involvement of noradrenaline and nitric oxide in the regulation of vasopressin and oxytocin expression in rat supraoptic nucleus.

The hydroosmotic balance of the body is controlled by supraoptic nuclei and paraventricular nuclei of the hypothalamo-posthypophyseal complex. In response to a physiological stimulation such as an osmotic stress, the supraoptic nuclei (SON) and the paraventricular nuclei undergo remarkable neurochemical and morphological changes. Therefore, the neuroendocrine hypothalamus is a particularly relevant model for studying the molecular and cellular mechanisms that govern these plasticity phenomena. Slices of rat hypothalamus maintained ex vivo by perfusion were used to study the short-term involvement of noradrenaline (NA) and nitric oxide (NO) in the mechanisms of chemical plasticity of the SON. NA is involved early in the regulation of the expression of neuropeptides, including vasopressin (AVP) and oxytocin (OT). NO appears to be a key molecule in noradrenergic control of the chemical plasticity of the endocrine neurons: in the SON, NO is involved in the signaling pathway regulating the expression of AVP but not that of OT.

A deficit of brain dystrophin 71 impairs hypothalamic osmostat.

Patients with Duchenne muscular dystrophy (DMD) and mdx mice, devoid of dystrophin proteins, show altered ionic homeostasis. To clarify dystrophin's involvement in the central control of osmotic stimuli, we investigated the effect of the disruption of Dp71, the major form of dystrophin in the brain, on the hypothalamoneurohypophysis system (HNHS) osmoregulatory response. Dp71 and Dp140 are the principal DMD gene products in the supraoptic nucleus (SON) and neurohypophysis (NH). They are present in astrocyte and pituicyte end-feet, suggesting involvement in both intrinsic osmosensitivity of the SON and vasopressin (AVP) release from the NH. In Dp71-null mice, the cellular distribution of Dp140 was modified, this protein being detected on the membrane of magnocellular soma. The plasma osmolality of Dp71-null mice was lower than that of wild-type mice under normal conditions, and this difference was maintained after salt loading, indicating a change in the set point for osmoregulation in the absence of Dp71. The increase in AVP levels detected in the SON and NH of the wild-type was not observed in Dp71-null mice following salt loading, and the increase in AVP mRNA levels in the SON was smaller in Dp71-null than in wild-type mice. This suggests that Dp71 may be involved in the functional activity of the HNHS. Its astrocyte end-feet localization emphasizes the importance of neuronal-vascular-glial interactions for the central detection of osmolality. In the SON, Dp71 may be involved in osmosensitivity and definition of the "osmostat," whereas, in the neurohypophysis, it may be involved in fine-tuning AVP release.

Cellular and subcellular evidence for neuronal interaction between the chemokine stromal cell-derived factor-1/CXCL 12 and vasopressin: regulation in the hypothalamo-neurohypophysial system of the Brattleboro rats.

We previously described a colocalization between arginine vasopressin (AVP) and the chemokine stromal cell-derived factor-1alpha (SDF-1) in the magnocellular neurons of both the hypothalamic supraoptic and paraventricular nucleus as well as the posterior pituitary. SDF-1 physiologically affects the electrophysiological properties of AVP neurons and consequently AVP release. In the present study, we confirm by confocal and electron microscopy that AVP and SDF-1 have a similar cellular distribution inside the neuronal cell and can be found in dense core vesicles in the nerve terminals in the posterior pituitary. Because the Brattleboro rats represent a good model of AVP deficiency, we tested in these animals the fate of SDF-1 and its receptor CXCR4. We identified by immunohistochemistry that both SDF-1 and CXCR4 immunoreactivity were strongly decreased in Brattleboro rats and were strictly correlated with the expression of AVP protein in supraoptic nucleus, paraventricular nucleus, and the posterior pituitary. We observed by real-time PCR an increase in SDF-1 mRNA in both heterozygous and homozygous rats. The effect on the SDF-1/CXCR4 system was not linked to peripheral modifications of kidney water balance because it could not be restored by chronic infusion of deamino-8D-ariginine-vasopressin, an AVP V2-receptor agonist. These original data further suggest that SDF-1 may play an essential role in the regulation of water balance.

Noradrenergic regulation in mouse supraoptic nucleus involves a nitric oxide pathway only to regulate arginine-vasopressin expression and not oxytocin expression.

Noradrenalin (NA) regulates the expression of arginine-vasopressin (AVP) and oxytocin (OT) by magnocellular neurons in the supraoptic nucleus (SON) of the hypothamalus. Nitric oxide (NO) may be one of the factors involved in the NA signaling pathway regulating AVP and OT expression. To test this possibility, we used an ex vivo experimental model of mouse hypothalamus slices. Increases in AVP and OT levels in the SON were detected by immunohistochemistry and immunoenzyme assays after 1 hr and 4 hr incubations with NA (10(-4) M). There was also an increase in the expression and activity of neuronal NOS and inducible NOS in the SON as assessed by immunohistochemical and histoenzymological analysis of NADPH-diaphorase, whereas endothelial NOS was undetectable. To specify the role of NO, the slices were treated with NA and L-arginine methyl ester (L-NAME, an NOS inhibitor; 3 microM). This treatment for 1 hr abolished the NA-induced increase in AVP. Treatment with sodium nitroprusside (SNP, an NO donor; 0.1 mM) increased AVP levels, confirming that NO regulates AVP expression. Addition of 1 mM EGTA during the incubation with NA reduced the AVP increase by half, indicating that both nNOS and iNOS activities are involved in the regulation. A 1-hr treatment with L-NAME did not prevent the increase in OT induced by NA; similarly, treatment with SNP had no effect. These findings show that NO is involved in the regulation of AVP expression by NA and that NA control of OT expression is independent of NO.

Noradrenergic regulation of galanin expression in the supraoptic nucleus in the rat hypothalamus. An ex vivo study.

Galanin is coexpressed with vasopressin and oxytocin in magnocellular neurons of the rat neuroendocrine hypothalamus. Various physiological stimuli, such as osmotic stimulation or lactation, that affect vasopressin and oxytocin expression and release also modulate galanin expression. Magnocellular neurons are highly innervated by noradrenergic inputs from the brainstem. The noradrenergic system plays a critical excitatory role in the activation of vasopressin-expressing and oxytocin-expressing neurons. Here, we have evaluated the possible regulation of Gal expression by noradrenaline in the magnocellular neurons of supraoptic nucleus in an ex vivo acute model of rat hypothalamic slices. The slices containing the supraoptic nucleus were incubated with 10(-4) M noradrenaline for 1 or 4 hr. The levels of galanin and galanin mRNA were estimated by semiquantitative immunohistochemistry and in situ hybridization, respectively. Our results show that the amount of galanin-immunopositive material in the cell bodies of the magnocellular neurons increased significantly after incubation with noradrenaline compared with control slices at the same time point and that this effect was more pronounced after 4 hr than after 1 hr. In situ hybridization showed that radiolabeling of the supraoptic nucleus with a radioactive galanin probe increased slightly after 1 hr of incubation and increased considerably after 4 hr of incubation with noradrenaline. Our study shows that galanin may be a target in the regulation of the hypothalamic magnocellular-neurohypophysial system by noradrenaline.

Noradrenaline up-regulates the neuronal and the inducible nitric oxide synthase isoforms in magnocellular neurons of rat brain slices.

Nitric oxide (NO) and noradrenaline (NA) are suggested to be implicated in the regulation of neuropeptide secretion in the supraoptic nuclei (SON) and the paraventricular nuclei (PVN) of the hypothalamus. Our study demonstrates short-term interactions between NA and the activity and expression of NO synthase (NOS) in magnocellular neurons, by using an ex vivo model of hypothalamic slices. In the SON as well as in the PVN, total NOS activity exhibited a time-dependant increase after an incubation with NA. In the SON, this increase of total NOS activity was in part the consequence of stimulation of the iNOS activity. Coimmunodetections showed that cells expressing the inducible form of NOS were not astrocytes but magnocellular neurons. Steady-state levels of iNOS and nNOS mRNA were dramatically enhanced by NA, particularly in the SON. Consequently, we provide new evidence that iNOS could play an important role in multiple physiological functions, including extracellular fluid balance, lactation, and parturition.