The extracellular matrix glycoprotein tenascin-R regulates neurogenesis during development and in the adult dentate gyrus of mice.

Abnormal generation of inhibitory neurons that synthesize γ-aminobutyric acid (GABAergic) is characteristic of neuropsychological disorders. We provide evidence that the extracellular matrix molecule tenascin-R (TNR) - which is predominantly expressed by a subpopulation of interneurons - plays a role in the generation of GABAergic and granule neurons in the murine dentate gyrus by regulating fate determination of neural stem or progenitor cells (NSCs). During development, absence of TNR in constitutively TNR-deficient (TNR(-/-)) mice results in increased numbers of dentate gyrus GABAergic neurons, decreased expression of its receptor ß1 integrin, increased activation of p38 MAPK and increased expression of the GABAergic specification gene Ascl1. Postnatally, increased GABAergic input to adult hippocampal NSCs in TNR(-/-) mice is associated not only with increased numbers of GABAergic and, particularly, parvalbumin-immunoreactive neurons, as seen during development, but also with increased numbers of granule neurons, thus contributing to the increased differentiation of NSCs into granule cells. These findings indicate the importance of TNR in the regulation of hippocampal neurogenesis and suggest that TNR acts through distinct direct and indirect mechanisms during development and in the adult.

Intrinsic expression of transcortin in neural cells of the mouse brain: a histochemical and molecular study.

Corticosteroid binding globulin (CBG, transcortin) has been shown to be expressed in the brain of rat and human species. In this study, we examined the CBG brain expression and cDNA structure in mice, comparing wild-type (Cbg(+/+)) and Cbg knockout mice (Cbg(-/-), obtained by genetic disruption of the SerpinA6 alias Cbg gene). We used double immunofluorescence labeling with specific neuronal and glial markers to analyze the cellular localization of CBG in various regions of the mouse brain. In wild-type (Cbg(+/+)) mice, we found CBG immunoreactivity in neuronal perikarya of the magnocellular hypothalamic nuclei, amygdala, hippocampus, cerebral cortex, cerebellum and pituitary. A portion of glial cells (astrocytes, oligodendrocytes) contained CBG immunoreactivity, including some of the ependymal cells and choroid plexus cells. No CBG immunoreactivity was detected in Cbg(-/-) brain tissues. Using RT-PCR, we showed that the full-length Cbg mRNA is present in those regions, indicating an intrinsic expression of the steroid-binding globulin. Furthermore, sequencing analysis showed that Cbg cDNA obtained from the mouse hypothalamus was homologous to Cbg cDNA obtained from the liver. Finally, we have evaluated the relative levels of CBG expression in various brain regions and in the liver by quantitative PCR. We found that brain levels of Cbg mRNA are low compared with the liver but significantly higher than in CBG-deficient mice. Although derived from the same gene as liver CBG, brain CBG protein may play a specific or complementary role that requires the production and analysis of brain-specific Cbg knockout models.

Patients with non-insulin depending diabetes mellitus and metabolic syndrome are suboptimal treated in Swiss primary care.

The prevalence of complicated hypertension is increasing in America and Europe. This survey was undertaken to assess the status quo of primary care management of hypertension in patients with the high-risk comorbid diseases metabolic syndrome (MetS) and/or type 2 diabetes mellitus (non-insulin depending diabetes mellitus (NIDDM)). Data of anti-hypertensive treatment of 4594 Swiss patients were collected over 1 week. We identified patients with exclusively NIDDM (N = 95), MetS (N = 168), and both (N = 768). Target blood pressure (TBP) attainment, frequency of prescribed substance-classes, and correlations to comorbidities/end-organ damages were assessed. In addition, we analyzed the prescription of unfavorable beta-blockers (BB) and high-dose diuretics (Ds). In NIDDM, Ds (61%), angiotensin receptor blockers (ARBs) (40%), and angiotensin converting enzyme inhibitors (ACEIs) (31%) were mostly prescribed, while in MetS, drugs prevalence was Ds (68%), ARBs (48%), and BB (41%). Polypharmacy in patients with MetS correlated with body mass index; older patients (>65 years) were more likely to receive dual-free combinations. TBP was attained in 25.2% of NIDDM and in 28.7% of MetS patients. In general, low-dose Ds use was more prevalent in NIDDM and MetS, however, overall, Ds were used excessively (NIDDM: 61%, MetS: 68%), especially in single-pill combination. Patients with MetS were more likely to receive ARBs, ACEIs, CCBs, and low-dose Ds than BBs and/or high-dose Ds. Physicians recognize DM and MetS as high-risk patients, but select inappropriate drugs. Because the majority of patients may have both, MetS and NIDDM, there is an unmet need to define TBP for this specific population considering the increased risk in comparison to patients with MetS or NIDDM alone.

Light and electron microscopic studies on the influence of stress on prolactin-immunoreactivity in rat anterior pituitary lobe.

An increasing number of evidence suggests an important role of prolactin in the modulation of stress response. However, the mechanisms of its action on the HPA axis are not yet understood. Glucocorticoids, liberated from adrenal cortex due to hormonal signals from pituitary corticotrophs are known to play a key role in systemic stress response. Previously we found evidence that corticosteroid-binding globulin (CBG) is involved in rapid, membrane-mediated actions of adrenal steroids. Here we studied qualitatively immunostainings for prolactin and CBG in pituitaries of male rats that had been subjected to osmotic challenge. We also examined late pregnant, parturient and early lactating rats, assuming that parturition represents a strong physiological stress. We employed double immunofluorescencent staining of semithin sections and immunoelectron microscopy. In stressed males we found increased prolactin immunofluorescence associated with membranes while in controls this staining was predominantly cytoplasmatic. CBG immunofluorescence was found in almost all prolactin cells of stressed males while such double staining was only occasionally observed in controls. Similar observations were made in females: While parturient rats showed intense membrane associated double staining for both antigens, late pregnant and early lactating animals showed patterns similar to that of male controls. Immunoelectron microscopy revealed increased exocytosis of prolactin containing vesicles in lactating rats. CBG was localized on cell membranes and additionally within prolactin vesicles. Our observations suggest prolactin liberation from pituitary lactotrophs along with CBG upon systemic stress response. Membrane effects of glucocorticoids mediated by CBG may be linked to stimulus secretion of prolactin.

Improved reversal learning and working memory and enhanced reactivity to novelty in mice with enhanced GABAergic innervation in the dentate gyrus.

The balance between excitation and inhibition controls fundamental aspects of the hippocampal function. Here, we report an increase in the ratio of inhibitory to excitatory neurons in the dentate gyrus, accompanied by γ-aminobutyric acid(A) (GABA(A)) receptor-dependent impairment of synaptic plasticity and enhancement of activity-dependent changes in excitability in anesthetized adult mice deficient for the extracellular matrix glycoprotein tenascin-R (TNR). TNR-deficient mice showed faster reversal learning, improved working memory, and enhanced reactivity to novelty than wild-type littermates. Remarkably, in wild-type and TNR-deficient mice, faster reversal learning rates correlated at the individual animal level with ratios of parvalbumin-positive interneurons to granule cells and densities of parvalbumin-positive terminals on somata of granule cells. Our data demonstrate that modification of the extracellular matrix by ablation of TNR leads to a new structural and functional design of the dentate gyrus, with enhanced GABAergic innervation, that is, enhanced ratio of inhibitory to excitatory cells, and altered plasticity, promoting working memory and reversal learning. In wild-type mice, the enhanced ratio of inhibitory to excitatory cells in the dentate gyrus also positively correlated with reversal learning, indicating that level of inhibition regulates specific aspects of learning independent of the TNR gene.

Oxytocin, but not arginine-vasopressin neurons project from the hypothalamus to amygdala in human: DiI-based tracing study in postmortem brain.

The hypothalamic neuropeptides oxytocin (OT) and arginine-vasopressin (AVP) are important factors involved in the control of socio-emotional behaviors via their modulation of amygdala functions. Since anatomical pathways of magnocellular projections to limbic structures in the human brain have not been dissected, we infused ethanol-dissolved tracer DiI into three amygdala nuclei - medial, central and lateral nuclei, and into the mammillary bodies of postmortem fixed human brains. With this modification, lipophilic diffusion of DiI occurred much faster than with conventional DiI crystals. After staining of resliced sections with antibodies against OT or AVP, we detected DiI/OT-positive neurons and their axons, specifically in the supraoptic nucleus (SON), but not in other hypothalamic nuclei producing OT or AVP. DiI fluorescence was found in the lateral portion of the paraventricular nucleus (PVN) and in the fornix columns, together with VP- immunoreactivity, only after DiI injections into the mammillary bodies. Our findings indicate that OT and AVP may have distinct neuronal pathways to the limbic system, and they are different from those previously reported in rodents.

Retarded kindling progression in mice deficient in the extracellular matrix glycoprotein tenascin-R.

PURPOSE: We investigated the role of the extracellular matrix glycoprotein tenascin-R (TNR) in formation of a hyperexcitable network in the kindling model of epilepsy. The idea that TNR may be important for this process was suggested by previous studies showing that deficiency in TNR leads to abnormalities in synaptic plasticity, perisomatic GABAergic inhibition and more astrocytes in the hippocampus of adult mice. METHODS: Constitutively TNR deficient (TNR-/-) mice and their wild-type littermates received repeated electrical stimulation in the amygdala over several days until they developed fully kindled generalized seizures at which time their brains were studied immunohistochemically. RESULTS: In TNR-/- mice, kindling progression was retarded compared with wild-type littermate controls. Morphological analysis of the mice used for the kindling studies revealed that, independently of genotype, numbers of parvalbumin-positive interneurons in the dentate gyrus correlated positively with afterdischarge threshold alterations in kindled mice. The kindling-induced increase in the number of S100 expressing astrocytes in the dentate gyrus was enhanced by TNR deficiency and correlated negatively with the kindling rate. DISCUSSION: Our data support the view that TNR promotes formation of a hyperexcitable network during kindling and suggest that an increase in S100-expressing astrocytes may contribute to retarded epileptogenesis in TNR-/- mice.

Osmotic stress induces corticosteroid-binding globulin expression in the rat hypothalamo-hypophyseal system.

Corticosteroid-binding globulin CBG is expressed in magnocellular hypothalamic nuclei, in part colocalized with vasopressin (VP) and oxytocin (OT). Here we subjected intact adult male rats to chronic osmotic stress to determine effects on distribution of CBG in VP and OT neurons and in neurons expressing corticotropin- releasing hormone (CRH). Drinking 2% NaCl solution for seven days resulted in increased CBG-immunoreactivity in magnocellular neurons. Triple immunofluorescence revealed increased colocalization with either VP, OT or CRH. Colocalization of CRH with VP was found only in a small portion of parvocellular neurons in the PVN. Most of the CBG-immunostained neurons within the magnocellular nuclei were devoid of CRH-immunoreactivity. Increased numbers of axons with colocalization of CBG and VP or OT were found in the internal zone of the median eminence (ME) of osmotically challenged rats. The external zone of the ME showed numerous CRH-positive neuronal projections. A small portion of them contained also CBG-immunofluorescence in both experimental animals and controls. Immunoassays of cerebrospinal fluid showed increased levels of CBG in osmotically stressed animals. Our observations suggest that hypothalamic CBG expression is malleable to functional status and that coexpression with the magnocellular peptide hormones may be of significance for endocrine stress response.

Magnocellular hypothalamic system and its interaction with the hypothalamo-pituitary-adrenal axis.

The hypothalamo-neurohypophyseal system plays a key role in maintaining homeostasis and in regulation of numerous adaptive reactions, e.g., endocrine stress response. Nonapeptides vasopressin and oxytocin are the major hormones of this system. They are synthesized by magnocellular neurons of the paraventricular and supraoptic hypothalamic nuclei. Magnocellular vasopressin is known to be one of the main physiological regulators of water-electrolyte balance. Its importance for control of the hypothalamo-pituitary-adrenal axis has been widely described. Magnocellular oxytocin is secreted predominantly during lactation and parturition. The complex actions of oxytocin within the brain include control of reproductive behavior and its involvement in central stress response to different stimuli. It's neuroendocrine basis is activation of the hypothalamo-pituitary-adrenal axis: corticotropin-releasing hormone is synthesized in parvocellular neurons of the paraventricular hypothalamic nuclei. The transitory coexpression of vasopressin in these cells upon stress has been described. Glucocorticoids, the end products of the hypothalamo-pituitary-adrenal axis have both central and peripheral actions. Their availability to target tissues is mainly dependent on systemic levels of corticosteroid-binding globulin. Intrinsic expression of this protein in different brain regions in neurons and glial cells has been recently demonstrated. Regulation of the hypothalamo-pituitary-adrenal axis and hypothalamo-neurohypophyseal system is highly complex. The role of both systems in the pathogenesis of various chronic ailments in humans has extensively been studied. Their disturbed functioning seems to be linked to various psychiatric, autoimmune and cardiovascular pathologies.

Adrenal steroids in the brain: role of the intrinsic expression of corticosteroid-binding globulin (CBG) in the stress response.

The complex interaction between hypothalamus, pituitary and adrenal glands is a key component of the neuroendocrine stress response. The major stress hormones--glucocorticoids--have both central and peripheral effects. Among the factors regulating their availability to target tissues are levels of corticosteroid-binding globulin, as the major transport protein for glucocorticoids in systemic circulation. Our recent findings demonstrated expression of corticosteroid-binding globulin in various brain regions and in different cell populations (neurons and glial cells). We showed at the cellular level the presence of corticosteroid-binding globulin in the human hypothalamus, where it was co-localized with the classical neurohypophyseal neurohormones--vasopressin and oxytocin. For the first time we demonstrated in mouse that the same gene encodes brain and liver corticosteroid-binding globulin. The full-length sequencing of hypothalamic corticosteroid-binding globulin revealed a full homology with liver corticosteroid-binding globulin cDNA. Thus, we confirmed that corticosteroid-binding globulin mRNA is produced locally within various cerebral regions and thus not transported from blood. However, the amounts of mRNA encoding corticosteroid-binding globulin are in liver about 200 times higher than in brain. The wide distribution of corticosteroid-binding globulin, distinct from the localization of glucocorticoid receptors, observed in our comparative study in rodents, led us to propose two possibilities: (1) corticosteroid-binding globulin is made in certain neurons to deliver glucocorticoids into the cell and within the cell in the absence of cytoplasmic glucocorticoid receptors or (2) is internalized into neurons specifically to deliver glucocorticoids to classical glucocorticoid receptors. Brain corticosteroid-binding globulin may be involved in the response to changing systemic glucocorticoid levels either additionally to known nuclear and membrane corticosteroid receptors or in glucocorticoid responsive brain regions devoid of these receptors. Clearly the multiple locations of corticosteroid-binding globulin within the central nervous system of humans and rodents imply multiple functional properties in normal and/or pathological conditions, which are yet to be determined. Most likely, the importance of brain corticosteroid-binding globulin exceeds the function of a mere steroid transporter.

Differences in colocalization of corticosteroid-binding globulin and glucocorticoid receptor immunoreactivity in the rat brain.

Endocrine regulation of central and systemic stress response as well as learning and memory are in part controlled by systemic glucocorticoid levels. So far steroids have been thought to act on the brain predominantly through nuclear receptors. However, some brain systems known to respond to glucocorticoids seem to be devoid of the respective receptor proteins (GR). It is likely that known central actions of adrenal steroids may also be mediated by non-genomic actions involving intrinsic binding globulins. In recent studies we described the intrinsic expression of corticosteroid-binding globulin (CBG) in rat, mouse and human brains. Here we report an immunohistochemical mapping study on the colocalization of CBG and of GR in the rat brain. In the nucleus accumbens, septum, hippocampus, globus pallidus, medial and basolateral amygdale nuclei, magnocellular preoptic nuclei, diagonal band of Broca high intensity of CBG immunoreactivity was accompanied by weak or moderate GR staining, and vice versa. In the caudate putamen, bed nucleus of stria terminalis, septohypothalamic nucleus and parvocellular subdivision of the paraventricular nucleus strong GR immunoreactivity was observed, but CBG was almost undetectable. In contrast, throughout the supraoptic nucleus and magnocellular subdivision of the paraventricular nucleus numerous strongly CBG-positive cells were observed, devoid of specific GR immunoreactivity. It is most likely that CBG in the brain may be involved in the response to changing systemic glucocorticoid levels in addition to known nuclear and membrane corticosteroid receptors, or in glucocorticoid responsive regions devoid of these receptors.

Increased hippocampal and cortical beta oscillations in mice deficient for the HNK-1 sulfotransferase.

The HNK-1 carbohydrate is detectable in perineuronal nets around inhibitory neurons in the hippocampus and neocortex. To address the functional contribution of HNK-1 to interneuron function in the adult brain, we recorded EEG and auditory-evoked potential in freely moving mice deficient for HNK-1 sulfotransferase (ST-/- mice) and in wild-type littermates. While ST-/- mice displayed normal theta oscillations, both cortical and hippocampal oscillations within the beta range were enhanced, and gamma oscillations showed an opposite trend. ST-/- mice had amplitudes of auditory-evoked potentials similar to control mice, but the latencies of their hippocampal responses were shorter. Morphological analysis revealed a decreased density of parvalbumin-positive interneurons in the hippocampal CA3 subfield of ST-/- mice, which may contribute to the observed changes in networks oscillations. These findings reveal alterations in ST-/- mice that differ from EEG abnormalities of mice deficient in the HNK-1 carrier molecule tenascin-R.