Transitory gliosis in the CA3 hippocampal region in rats fed on a ketogenic diet.

The ketogenic diet (KD) is a high-fat, low-protein and low-carbohydrate diet included as medical practice against seizure disorders, particularly in children refractory to conventional anti-epileptic drug treatment. However, the molecular basis of its therapeutic effect remains unclear. Considering the growing evidence for the importance of glial cells for neuronal development, survival and plasticity, we investigated astrocyte protein markers from KD fed rats, in different regions of hippocampus, a brain structure commonly involved in seizure disorders. We found a transitory increment in GFAP in the CA3 hippocampal region, but not in the CA1 or dentate gyrus (DG). This change was not accompanied by changes in S100B content or glutamine synthetase activity. In order to evaluate possible hippocampal involvement we investigated spatial-cognitive behavior using the water-maze task. No changes were observed. This transitory gliosis in CA3 could be related to, or precede, other associated changes proposed to be involved in the attenuation of seizure disorders. These data reinforce the importance of hippocampal astrocytes as cell targets during KD feeding.

Habituation to stress and dexamethasone suppression in rats with selective basal forebrain cholinergic lesions.

Previous studies suggest a role for basal forebrain cholinergic neurons in enhancing the inhibitory influence of the hippocampus and medial prefrontal cortex (mPFC) on glucocorticoid stress responses mediated by the hypothalamic-pituitary-adrenocortical (HPA) axis. An inhibitory action of the basal forebrain cholinergic (BFC) system may occur through facilitation of stress-related information processing and maintenance of glucocorticoid receptor (GR) expression and negative feedback signaling in these target regions. The current study investigated the possibility that BFC input to the hippocampus contributes to habituation of the glucocorticoid response following repeated exposure to a stressor. Cholinergic lesions were made by microinjections of the immunotoxin 192 IgG-saporin into the medial septum/vertical limb of the diagonal band, and 3 weeks later rats were subjected to six daily sessions of restraint stress. Blood samples taken before, during and after acute stress revealed a significant increase in peak activation and protracted elevation of corticosterone in cholinergic lesioned rats. After 5 days of repeated stress, however, both groups habituated to the stressor, as indicated by similarly low corticosterone profiles throughout both the response and recovery period. Against that habituated background, rats were administered a dexamethasone challenge on day 6, so that feedback status could be examined. Dexamethasone-induced suppression of endogenous corticosterone before, during, and after stress was significantly attenuated in lesioned rats. The profile of dysfunction in glucocorticoid regulation after selective cholinergic lesions in young animals may be relevant to the adrenocortical hyperactivity and negative feedback deficits seen in conditions such as normal aging and Alzheimer's dementia, in which integrity of the basal forebrain cholinergic system is compromised.

Local integration of glutamate signaling in the hypothalamic paraventricular region: regulation of glucocorticoid stress responses.

The present study is a test of the hypothesis that endogenous glutamatergic input to the hypothalamic paraventricular nucleus (PVN) is involved in stress-induced activation of the hypothalamic-pituitary-adrenocortical (HPA) axis. We examined whether corticosterone (CORT) responses to restraint stress could be attenuated by bilateral 50 nl microinjections of kynurenic acid (KYN, ionotropic glutamate receptor antagonist) into the medial PVN of conscious rats. Immediately following microinjection, rats were subjected to 30 min restraint, and stress-induced plasma CORT was measured at 30, 60, 120, and 180 min following the onset of restraint. KYN (50 pmol) significantly reduced cumulative CORT responses to acute restraint stress by 24%. In contrast, microinjections centered dorsal to the PVN increased CORT responses by 31%, suggestive of a disinhibition of local PVN-inhibitory input. KYN injections immediately anterior, ventral, or posterior to the PVN had no effect, suggesting an absence of potential diffusion artifacts. These results provide evidence that endogenous glutamate in the PVN and surrounding peri-PVN region plays a physiologically significant role in both generating and limiting glucocorticoid stress responses in awake rats.

Expression of ionotropic glutamate receptor subunit mRNAs in the hypothalamic paraventricular nucleus of the rat.

The hypopthalamic paraventricular nucleus (PVN) coordinates multiple aspects of homeostatic regulation, including pituitary-adrenocortical function, cardiovascular tone, metabolic balance, fluid/electrolyte status, parturition and lactation. In all cases, a substantial component of this function is controlled by glutamate neurotransmission. In this study, the authors performed a high-resolution in situ hybridization analysis of ionotropic glutamate receptor subunit expression in the PVN and its immediate surround. N-methyl-D-aspartate (NMDA) receptor 1 (NMDAR1), NMDAR2A, and NMDAR2B mRNAs were expressed highly throughout the PVN and its perinuclear region as well as in the subparaventricular zone. NMDAR2C/2D expression was limited to subsets of neurons in magnocellular and hypophysiotrophic regions. In contrast with NMDA subunit localization, AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate)-preferring and kainate (KA)-preferring receptor subunit mRNAs were expressed heterogeneously in the PVN and surround. Glutamate receptor 1 (GluR1) mRNA labeling was most intense in preautonomic subregions, whereas GluR2, GluR4, GluR5, and KA2 were expressed in hypophysiotrophic cell groups. It is noteworthy that GluR5 mRNA expression was particularly robust in the dorsolateral region of the medial parvocellular PVN, suggesting localization in corticotropin-releasing hormone neurons. All four AMPA subunits and GluR6 and GluR7 mRNAs were expressed highly in the perinuclear PVN region and the subparaventricular zone. These data suggest the capacity for multifaceted regulation of PVN function by glutamate, with magnocellular neurons preferentially expressing NMDA subunits, preautonomic neurons preferentially expressing AMPA subunits, and hypophysiotrophic neurons preferentially expressing KA subunits. Localization of all species in the perinuclear PVN suggests that glutamate input to the immediate region of the PVN may modulate its function, perhaps by communication with local gamma-aminobutyric acid neurons.

Excitatory influence of the locus coeruleus in hypothalamic-pituitary-adrenocortical axis responses to stress.

The locus coeruleus (LC) is a key brainstem region involved in arousal and is highly responsive to alerting/stressful stimuli, including those that activate the hypothalamic-pituitary-adrenocortical (HPA) axis. It is currently unclear whether the LC exerts any regulatory influence on the HPA axis and, consequently, on neuroendocrine responses to stress. The present studies were designed to test the hypothesis that the LC promotes HPA axis responses to acute and chronic stress. Adult male rats received bilateral (6-hydroxydopamine) lesions of the LC that produced severe cell loss in the LC and 80% depletion of noradrenaline in medial prefrontal cortex. Notably, lesions did not affect dopamine-beta-hydroxylase protein content in the parvocellular paraventricular nucleus (PVN), indicating a lack of collateral damage to other ascending noradrenergic pathways. LC lesions significantly reduced peak adrenocorticotropic hormone (ACTH) and corticosterone responses to 30 min acute restraint stress. However, LC lesions did not significantly attenuate neuroendocrine or other physiological responses to a 4-week chronic variable stress regimen. LC lesions did not substantially affect basal concentrations of plasma corticosterone or corticotropin-releasing hormone mRNA expression in the hypothalamic paraventricular nucleus following chronic stress. We conclude that the LC is a HPA-excitatory brain region, promoting neuroendocrine and physiological responses primarily to acute stress. However, a potential role for the LC in the induction of HPA axis hyperactivity following chronic stress can not be ruled out.

The S100B protein inhibits phosphorylation of GFAP and vimentin in a cytoskeletal fraction from immature rat hippocampus.

The S100B protein belongs to a family of small Ca2+-binding proteins involved in several functions including cytoskeletal reorganization. The effect of S 100B on protein phosphorylation was investigated in a cytoskeletal fraction prepared from immature rat hippocampus. An inhibitory effect of 5 microM S100B on total protein phosphorylation, ranging from 25% to 40%, was observed in the presence of Ca2+ alone, Ca2+ plus calmodulin or Ca2+ plus cAMP. Analysis by two dimensional electrophoresis revealed a Ca2+/calmodulin-dependent and a Ca2+/cAMP-dependent inhibitory effect of S100B, ranging from 62% to 67% of control, on the phosphorylation of the intermediate filament proteins glial fibrillary acidic protein (GFAP) and vimentin. The fact that S100B binds to the N-terminal domain of GFAP and that the two proteins are co-localized in astrocytes suggests a potential in vivo role for S100B in modulating the phosphorylation of intermediate filament proteins in glia.

Briefly delayed reinforcement: An interresponse time analysis.

Key-peck responding of pigeons was compared under VI or DRL schedules arranging immediate reinforcement and briefly (.5 sec) delayed reinforcement. Delays were either signaled by a blackout in the chamber, unsignaled, or unsignaled with an additional requirement that responding not occur during the .5 sec interval immediately preceding reinforcement (response delay). Relative to the immediate reinforcement condition, response rates increased during the unsignaled delay, decreased during the signaled delay, and were inconsistent during the response delay condition. An analysis of interresponse times (IRTs) under the different conditions revealed a substantial increase in the frequency of short (0 to .5 sec) IRTs during the unsignaled condition and generally during the response delay conditions compared to that during the immediate reinforcement baseline. Signaled delays decreased the frequency of short (0 to .5 sec) IRTs relative to the immediate reinforcement condition. The results suggest that brief unsignaled delays and, in many instances, response delays increase the frequency of short IRTs by eliminating constraints on responding.