Granular Layer Of Dentate Gyrus Research Articles

NeuN distribution in brain structures of normal and Zika-infected suckling mice.

Microcephaly is the more severe brain malformation because of Zika virus infection. Increased vulnerability of neural stem and progenitor cells to Zika infection during prenatal neurodevelopment impairs the complete formation of cortical layers. Normal development of cerebellum is also affected. However, the follow-up of apparently healthy children born to Zika exposed mothers during pregnancy has revealed other neurological sequelae. This suggests Zika infection susceptibility remains in nervous tissue after neurogenesis end, when differentiated neuronal populations predominate. The neuronal nuclear protein (NeuN) is an exclusive marker of postmitotic neurons. Changes in NeuN expression are associated with neuronal degeneration. We have evaluated immunohistochemical expression of NeuN protein in cerebral cortex, hippocampus, and cerebellum of normal and Zika-infected neonatal Balb/c mice. The highest NeuN immunoreactivity was found mainly in neurons of all cortical layers, pyramidal layer of hippocampus, granular layer of dentate gyrus and in internal granular layer of cerebellum. Viral infection caused marked loss of NeuN immunostaining in all these brain areas. This suggests neurodegenerative effects of Zika virus infection during postmitotic neuron maturation and contribute to interpretation of neuropathogenic mechanisms of Zika.

Dynamics of neurodegeneration in the hippocampus of Krushinsky-Molodkina rats correlates with the progression of limbic seizures

Audiogenic seizures (AGS) (audiogenic kindling) in genetically selected audiogenic rodents are a reliable model of temporal lobe epilepsy (TLE). Temporal lobe epilepsy is accompanied with neurodegeneration in the hippocampus, but how the cells die is not fully understood. We analyzed the dynamics and mechanisms of cell loss in the hippocampus of audiogenic Krushinsky-Molodkina (KM) rats during the development of TLE. Audiogenic kindling of different durations was carried out to reproduce TLE progression in KM rats. Behavioral analysis showed the development of post-tonic clonus, the main indicator of TLE, by the 14th AGS. The severity and duration of post-tonic clonus positively correlated with the increase in the number of AGS. Temporal lobe epilepsy development was accompanied with two peaks of cell loss. The first peak was detected after 7 AGS in the dentate gyrus (DG) granular layer and associated with activation of p53- and mitochondria-dependent apoptosis. After a 7-day rest period, activation of autophagy and restoration of cell number were revealed. The second peak occurred after 14 AGS, affected both granular and hilar mossy cells and persisted further after 21 AGS, but no compensation was observed. Thus, activation of autophagy probably plays a neuroprotective role and supports survival of hippocampal cells at the beginning of epileptogenesis, but exacerbation of limbic seizures during TLE development causes irreversible neurodegeneration.

Interrelation structural and functional alterations in the dentate gyrus of rats with aging.

The process of natural aging is considered as a risk factor for the development of neurodegenerative pathology, in which decrease of physiological cognitive functions of the body is found. 2 groups were formed: group 1 - rats at the age of 12 months (n = 10), group 2 - rats at the age of 24 months (n = 10). We evaluated the neuropsychiatric state of animals and determined the degree injury of neurons. It was established that the motor and orientational-research activity of old rats (24 months) was lower than that of rats of mature age (12 months). Microscopic examination of rat brain sections stained with thionin according to the Nissl method in mature animals in the granular layer of dentate gyrus showed that the most of neuronal perikarya had a close to round shape with a centrally located nucleus and nucleolus, characterized by normochromic cytoplasm and compact arrangement. There were single neurons with hyperchromatosis of the cytoplasm and wrinkled hyperchromic neurons, which were mainly located on the border of granular and subgranular layers of dentate gyrus. Sites of neuronal loss were observed in senile age rats, a significant increase in both the specific number of hyperchromic shrunken neurons (p <0.05) and neurons with cytoplasm hyperchromatosis without shrinkage (p <0.05) was observed. Such neurons prevailed at the boundary of the granular and subgranular layers, as well as in the crest region. During aging in rats aged 24 months, pronounced changes in neurons of granular layer of dentate gyrus in the form of an increase in the number of hyperchromic shrunken neurons are found at the boundary of granular and subgranular layers and in the crest region. These changes are accompanied by more severe functional disorders in 24-month-old rats which may accelerate the process of aging.

Compensatory Hippocampal Neurogenesis in the Absence of Cognitive Impairment Following Experimental Hippocampectomy in Adult Rats.

Temporal lobe epilepsy (TLE) is the commonest type of focal epilepsy in adult humans, and hippocampal sclerosis (HS) is the main pathological finding in this type of epilepsy. In refractory TLE, patients are indicated for unilateral resection of the affected hippocampus by a surgical procedure called hippocampectomy which generally does not cause any cognitive impairment. Once adult hippocampus is a region of endogenous neurogenesis, even in elderly people, we have hypothesized that a compensatory increase in hippocampal neurogenesis might occur in the remaining hippocampus after unilateral hippocampectomy. To test this hypothesis, we performed unilateral hippocampectomy in adult Wistar rats, which were perfused at 15 (G15) and 30 (G30) days post-surgery. Eighteen Wistar rats were randomly distributed in the following experimental groups: control (no surgery, N = 6), G15 (N = 6), and G30 (N = 6). Adjacent cortex and hippocampus of the left hemisphere were completely removed. Behavioral procedures were performed to address possible cognitive impairments. Brains were collected and fixed from animals belonging to all experimental groups. Gross histopathology was performed using thionine staining. Neuroblasts and mature neurons were immunolabeled using anti-doublecortin (DCX) and anti-NeuN antibodies, respectively. Numbers of DCX and NeuN positive cells were quantified for all experimental groups. Animals submitted to hippocampectomy did not present any cognitive impairment as evaluated by eight-arm radial maze behavioral test. The remaining hippocampus presented a higher number of DCX positive cells compared to control (p < 0.001, ANOVA-Tukey) at both G15 and G30. A higher number of NeuN positive cells were present in the granular layer of dentate gyrus at G30 compared to control and G15 (p < 0.001, ANOVA-Tukey). The data suggest that unilateral hippocampectomy induces compensatory neurogenic effect in the contralateral hippocampus. This may underlie the reported absence of significant cognitive impairment and parallels the findings in human patients submitted to unilateral hippocampectomy to treat refractory TLE.

Immunophenotypic characteristics of inducible NO synthase expression in dentate gyrus of mature rats in modeling depression and its pharmacological correction

AIM: The work aimed to investigate inducible NO synthase (iNOS) expression in dentate gyrus in mature rats when modeling depression, as well as the establish the pharmacological correction possibility of detected changes with Phenibut and compounds under laboratory codes of RSPU-189, RSPU-135.&#x0D; MATERIALS AND METHODS: Depressive-like behavior in animals was modeled by combining stressful stimuli such as loud sound, pulsating bright light, and vibration simultaneous with constant restriction of mobility and fluctuations in temperature of environment for 7 days (daily for 30 minutes). Changes in level of iNOS expression in dentate gyrus were assessed by calculating relative area of immunoreactive material (IRM) and staining intensity in points from 0 to 3.&#x0D; RESULTS: Compared with the control group, rats with experimental depression showed an increase in expression of iNOS-IRM in cytoplasm of neuronal perikarya in granular layer of dentate gyrus, as well as an increase in relative area of iNOS-IRM in neuropil and nerve cells. The use of the compound RSPU-189 (salifen) demonstrated to a greater extent the corrective effect, since in the cytoplasm of neuronal perikarya in granular layer of dentate gyrus of rats, there was a decrease in the expression of iNOS-IRM, as well as a decrease in the relative area of iNOS-IRM in neuropil and nerve cells, which corresponded to values of these parameters in the control group of animals.&#x0D; CONCLUSIONS: An experimental modeling of depression in dentate gyrus of mature rats revealed an increase of iNOS-IRM expression, the decrease of which was noted in its pharmacological correction with the compound RSPU-189 (salifen), which may indicate the predominant neuroprotective effect of this compound on GABAergic neurotransmission mechanisms.

The increase in the number of astrocytes in the total cerebral ischemia model in rats

Astrocytes are the most abundant cell class in the CNS. Astrocytic therapies have a huge potential for neuronal repair after stroke. The majority of brain stroke studies address the damage to neurons. Modern studies turn to the usage of morphological and functional changes in astroglial cells after stroke in regenerative medicine. Our study is focused on the changes in the number of astrocytes in the hippocampus (where new glia cells divide) after brain ischemia. Ischemia was modeled by occlusion of tr. brachiocephalicus, a. subclavia sin., a. carotis communis sin. Astrocytes were determined using immunohistochemical labeling with anti GFAP antibody. We found out that the number of astrocytes increased on the 10th and 30th days after stroke in the CA1, CA2 fields, the granular layer of dentate gyrus (GrDG) and hilus. The morphology of astrocytes became reactive in these regions. Therefore, our results revealed long-term reactive astrogliosis in the hippocampus region after total ischemia in rats.

Comparison of the influence of two models of mild stress on hippocampal brain-derived neurotrophin factor (BDNF) immunoreactivity in old age rats.

The way hippocampal neurons function during stress in old age (critical times of life) is dependent on brain derived neurotrophin factor (BDNF). This study examined the influence of acute and chronic forced swim (FS) or high-light open field (HL‑OF) stimulation on the density of BDNF immunoreactive (ir) neurons in the hippocampal pyramidal layers of CA1, CA2, CA3 regions and the granular layer of dentate gyrus (DG) in old (postnatal day 720; P720) Wistar Han rats. Our data showed that in comparison with non-stressed rats, acute FS caused a significant increase in the density of BDNF-ir neurons in CA2 and CA3, while acute HL-OF led to an increase in this factor in all hippocampal subfields with the exception of DG. However, the density of BDNF-ir cells remained unchanged after exposure to chronic FS or HL‑OF in the hippocampal regions in relation to the control rats. These results indicate that acute FS or HL-OF proved to be a stressor that induces an increase in the density of BDNF-ir pyramidal neurons, which was probably connected with up-regulation of HPA axis activity and short‑time memory processing of the stressful situation. Moreover, as far as the influence on BDNF-ir cells in hippocampus is concerned, chronic FS or HL-OF was not an aggravating factor for rats in the ontogenetic periods studied.

Hippocampal distribution of IL-1β and IL-1RI following lithium-pilocarpine-induced status epilepticus in the developing rat.

The contribution of Interleukin-1β (IL-1β) to neuronal injury induced by status epilepticus (SE) in the immature brain remains unclear. The goal of this study was to determine the hippocampal expression of IL-1β and its type 1 receptor (IL-1RI) following SE induced by the lithium-pilocarpine model in fourteen-days-old rat pups; control animals were given an equal volume of saline instead of the convulsant. IL-1β and IL-1RI mRNA hippocampal levels were assessed by qRT-PCR 6 and 24 h after SE or control conditions. IL-1β and IL-1RI expression was detected in the dorsal hippocampus by immunohistochemical procedures; Fluoro-Jade B staining was carried out in parallel sections in order to detect neuronal cell death. IL-1β mRNA expression was increased 6 h following SE, but not at 24 h; however IL-1RI mRNA expression was unaffected when comparing with the control group. IL-1β and IL-1RI immunoreactivity was not detected in control animals. IL-1β and IL-1RI were expressed in the CA1 pyramidal layer, the dentate gyrus granular layer and the hilus 6 h after SE, whereas injured cells were detected 24 h following seizures. Early expression of IL-1β and IL-1RI in the hippocampus could be associated with SE-induced neuronal cell death mechanisms in the developing rat.

The effect of vitamin E on neuronal apoptosis in hippocampal dentate gyrus in rabbits fed with high-cholesterol diets

Background:Hypercholesterolemia that can increase stress oxidative has destructive effects on brain functions. Vitamin E is a powerful antioxidant and its effects on decrement of oxidative stress in the diseases such as Alzheimer's and hypercholesterolemia are demonstrated. The aim of this study was evaluation of the effects of vitamin E on the level of neuronal apoptosis in granular layer of dentate gyrus in the rabbits that fed with high-cholesterol diet.Materials and Methods:Male New Zealand white rabbits were divided into the control, the Vitamin E (50 mg/kg; gavage), the high-cholesterol diet (containing 2% cholesterol), and the high-cholesterol diet-vitamin E groups. Serum levels of cholesterol, LDL, and HDL, before and after the regimen for 6 weeks, were measured. Then, the rabbits for immunohistochemical staining (TUNEL Test) and evaluation of neuronal apoptosis in dentate gyrus of hippocampal formation were anesthetized and brains were dissected.Results:Results showed that after the regimens, serum levels of cholesterol, LDL, and HDL in the cholesterol receiving groups were increased significantly (P < 0.05). Histological results demonstrated that neuronal apoptosis in the dentate gyrus of the high-cholesterol diet group was increased significantly (P < 0.05) comparing to the control group; however, vitamin E decreased apoptosis as there wasn’t any significant differences between the high-cholesterol diet-vitamin E and control groups.Conclusions:Present results showed that consumption of high-cholesterol diets through hypercholesterolemia and its complication can induce neuronal death in hippocampus and probable resulting cognition disorders; however, vitamin E has neuroprotective effects and prevents neuronal apoptosis significantly.

Baicalin and deferoxamine alleviate iron accumulation in different brain regions of Parkinson's disease rats.

Previous studies found that iron accumulates in the substantia nigra of Parkinson’s disease patients. However, it is still unclear whether other brain regions have iron accumulation as well. In this experiment, rats with rotenone-induced Parkinson’s disease were treated by gastric perfusion of baicalin or intraperitoneal injection of deferoxamine. Immunohistochemical staining demonstrated that iron accumulated not only in the substantia nigra pars compacta, but also significantly in the striatum globus pallidus, the dentate gyrus granular layer of the hippocampus, the dentate-interpositus and the facial nucleus of the cerebellum. Both baicalin and deferoxamine, which are iron chelating agents, significantly inhibited iron deposition in these brain areas, and substantially reduced the loss of tyrosine hydroxylase-positive cells. These chelators also reduced iron content in the substantia nigra. In addition to the substantia nigra, iron deposition was observed in other brain regions as well. Both baicalin and deferoxamine significantly inhibited iron accumulation in different brain regions, and had a protective effect on dopaminergic neurons.

Hyperoxia-induced changes in morphometric parameters of postnatal neurogenic sites in rat

In literature many works address the effects of hypoxia exposure on postnatal neurogenesis but few data are available about hyperoxia effects, although high oxygen concentrations are frequently used for ventilation of premature newborns. Thus, the aim of the present study was to compare with controls the morphometrical parameters of the main neurogenic sites (subventricular zone and dentate gyrus) in newborn Sprague-Dawley rats exposed to 60% or 95% oxygen for the first 14 postnatal days. Six rats were studied for each of the three groups. The unbiased quantitative method of the optical disector was applied to analyze neuronal densities, nuclear volumes, and total neuron numbers of the subventricular zone and hippocampal dentate gyrus. Apoptosis (terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling, TUNEL) and proliferation (Ki67) were also studied. The subventricular zone of newborn rats exposed to 95% hyperoxia showed statistically significant higher volume (mean value ± coefficient of variation: 0.40 ± 0.20 mm3) than subventricular zone of rats raised in normoxia (0.20 ± 0.11 mm3) or 60% hyperoxia (0.26 ± 0.18 mm3). Total neuron number was also significantly higher in 95% hyperoxia while neuronal densities did not reach statistically significant differences. TUNEL showed increased apoptotic indexes in hyperoxic rats. The percentage of proliferating KI67 positive cells was also higher in hyperoxia. The dentate gyrus granular layer of the normoxic rats showed higher volume (0.65 ± 0.11 mm3) than both the hyperoxic groups (60% hyperoxia: 0.39 ± 0.14 mm3; 95% hyperoxia: 0.36 ± 0.16 mm3). Total neuron numbers of hyperoxic dentate gyrus were also significantly reduced; neuronal densities were not modified. Hyperoxia-exposed rats also showed higher apoptotic and proliferating indexes in the dentate gyrus. Hyperoxia exposure in the first postnatal period may affect the main neurogenic areas (subventricular zone and dentate gyrus) increasing apoptosis but also inducing a certain reparative response consisting of increased proliferation. In particular, the increased volume of the subventricular zone may be ascribed to compensatory neurogenic response to the hyperoxic damage. Conversely, the decreased volume of the dentate gyrus granular layer could derive by a non sufficient neurogenic response to counterbalance the hyperoxic neuronal injury.

Is the dentate gyrus an independent generator of in vitro recorded theta rhythm?

In this study we investigated the ability of the dentate gyrus to independently generate cholinergically induced theta rhythm in vitro. Two different experiments were performed. In Experiment I, new laminar profiles of theta phase, amplitude, and current sources and sinks were constructed. In this experiment a gradual phase shift of theta waves in the CA1 stratum radiatum was observed. Simultaneously, two amplitude maxima were detected: the first in the CA1 stratum oriens, and the second in the CA1 stratum lacunosum-moleculare. Moreover, during the positive peak of theta in the CA1 stratum oriens, two large sinks were observed: the first localized in the stratum oriens and the second in the stratum lacunosum-moleculare. In Experiment II the EEG activity of three different transected hippocampal slices (CA1 transected slices, CA3c transected slices, and DG transected slices) was recorded. It was demonstrated that the dentate gyrus granular cell body layer was not able to independently produce in vitro theta rhythm. Data obtained in both experiments provide strong evidence that in cholinegically treated hippocampal formation maintained in vitro there is no independent generator of theta rhythm in the region of the dentate gyrus granular cell layer.

Study of Molecular and Granular Layer of Dentate Gyrus of Female Rats Neonatally Treated with Estrogen

The proliferation of hippocampal dentate gyrus granule cells was investigated using 3H-thymidine incorporation in control and estrogen-treated rats. Newborn 3-day old female Wistar rats were treated with a single dose of 1 mg of estradiol and 30 μCi 3H-thymidine, and were sacrificed when 10 days old. The total number of neurons and the number of labeled granule cells in the granular layer and its subdivisions of both suprapyramidal and infrapyramidal limbs were analyzed using a stereological method. In both limbs, the total number of neurons as well as the total number of labeled granule cells in the granular layer were significantly increased in treated rats compared to corresponding controls. The thicknesses of the molecular and the granular layers and their subdivisions of both suprapyramidal and infrapyramidal limbs were analyzed using a stereological method. In treated female rats the molecular layer (ML) in both limbs was significantly decreased, and the granular layer (GL) was significantly increased in suprapyramidal limb. However, in the infrapyramidal limb an increased number of labeled cells in treated animals were significant in all particular zones of the granular layer. In the suprapyramidal limb's granular layer a significant increase in labeled cells was observed in subgranular zone (SGZ). Our results suggest a differential effect of estradiol on thicknesses of the ML and the GL, and dentate gyrus granule cells proliferation through the early rat life.

P2-470: Survival and differentiation of adult skin-derived neuroprecursors in a rat model of age-related cognitive impairment

mer s disease (AD). Up to now no resolutive pharmacological therapy exists and there are increasing arguments regarding the beneficial effect of natural nutrients, able to slow down the progression of the disease. Some have the ability to induce cell proliferation and/or survival. Adult neurogenesis could be a good therapeutic strategy for cognitive aging and neurodegenerative diseases(AD). The aims of this work has been to study the modulatory effect of *LMN diet (cocoa, nuts and other natural extracts) in adult mouse neurogenic brain areas. Methods: 129SV male mice, were feed during 40 days with a standard Harlan 2014 control diet and the same diet containing 9,27% of *LMN. Animals received BrdU injections. Brains were processed for inmunohistological and immunoblots studies. Results: Histological samples of mice feed with *LMN diet, showed a noticeable increasing number of proliferative cells by BrdU and PCNA staining, in the adult neurogenic areas: subventricular zone (SVZ) and subgranular layer of dentate gyrus (sgDG). Using Dcx and PSA-NCAM, an increasing in the undifferentiating neurons were determined in the granular layer of dentate gyrus and in the rostral migratory stream (RMS). The immature oligodendrocytes (Ng2) and astrocytes (GFAP) were also increasing in these brain areas. All these results were corroborated by Western-Blot analysis. Moreover, the several interneurons subpopulations of the olfactory bulb were changed. Increasing the tyrosine hydroxylase, calretinin and calbindin neuronal subpopulations, and decreasing the parvalbumin interneurons. In the dentate gyrus, the granular layer presented a high cell number when comparing with the control animals. Conclusions: All these findings showed that *LMN diet promotes the neurogenesis in the adult mouse neurogenic niches. The differences between interneurons subpopulations of olfactory bulb suggested that, *LMN diet could modulate differentiation process in the RMS. Therefore, *LMN diet could be a promising nutrient that would contribute to the neural replacement and to re-establish the brain function in order to avoid the cognitive decline, the main hallmark in Alzheimer’s disease. *Patent submitted: Reference ES2281270. Acknowledgements: This work has been financed by the Spanish Ministry of Industry, project INGENIO 2010CENIT ref MET-DEV-FUN (2006-2009).

The effect of amyloidosis-β and ageing on proliferation of neuronal progenitor cells in APP-transgenic mouse hippocampus and in culture

Stimulation of endogenous neurogenesis and transplantation of neuronal progenitors (NPs) are considered in therapy of neuronal loss associated with ageing and in neurodegenerative diseases with amyloidosis-beta, for example, Alzheimer's disease and Down syndrome. However, the influence of brain environment altered by ageing and deposits of amyloid-beta on proliferation of endogenous and transplanted NPs and their maturation into neurons is not understood. We studied the effect of ageing and development of amyloidosis-beta on proliferation of NPs (1) in the granular layer of dentate gyrus in the hippocampi of APP-transgenic mice (Tg9291) before and after development of amyloidosis-beta, that is, in mice aged 2-4 months and 9-12 months, respectively, and in age-matched controls; and (2) in culture of NPs isolated from brains of control and Tg9291 mice, aged 3 and 9 months. We found that the number of proliferating NPs was reduced in 9-12-months-old mice, in both control and Tg9291, as compared to 2-4-months-old mice. However, the 9-12-months-old Tg9291 mice with amyloid-beta deposits had significantly more proliferating NPs than the age-matched controls. NPs proliferation in culture did not depend on the age, presence of APP-transgene, and amyloidosis-beta in donors. The results indicate that the local brain environment influences proliferation of NPs, and development of amyloidosis-beta in the neurogenic regions attenuates the age-associated reduction of proliferation of NPs. Identification of the responsible mechanisms may be important for development of a successful therapy of neurodegeneration caused by amyloidosis-beta.

Immunohistochemical changes in vulnerable rat brain regions after reversible global brain ischaemia

Human global ischaemia was simulated in adult rats by inducing 20 min brain ischaemia and 60 min post-ischaemic recirculation. Immunohistochemical expression of MMP-9, TIMP-3, Bax and Bcl-2, and DNA fragmentation (with the TUNEL reaction) were investigated. The morphological data showed different neuronal responses in the hippocampus compared with the cerebral and cerebellar cortices. MMP-9 immunoreactivity was different in the hippocampus, particularly in dentate gyrus and the CA1 region, compared with these cortices. Negative TIMP-3 staining in ischaemic hippocampal neurons may indicate a loss of its inhibitory activity on MMP-9 that could enhance cell death. Bcl-2 down regulation, Bax positivity and TUNEL+ type II cells in the dentate gyrus granular layer could be responsible for induction of apoptotic death in CA1 hippocampal pyramidal cells via loss of fibre input. Results suggest differential behaviours of neural cells after 60 min reperfusion.

Hippocampal 11β-Hydroxysteroid Dehydrogenase Type 1 Messenger Ribonucleic Acid Expression Has a Diurnal Variability that Is Lost in the Obese Zucker Rat

Circulating levels of glucocorticoids show a circadian rhythm. Obesity is associated with a flattening of the diurnal rhythm; plasma cortisol levels are slightly increased during the trough, although they are normal or low in the morning. Studies in humans and in leptin-resistant Zucker rats suggest that tissue-specific alterations in glucocorticoid exposure might play a key role for development of obesity and obesity-associated dysregulation of the hypothalamic-pituitary-adrenal axis. We hypothesized that there is a circadian rhythm in prereceptor metabolism of glucocorticoids exerted by 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) in brain and/or peripheral tissues (liver, fat, and muscle) that might be abrogated in obesity. The present study demonstrates a circadian rhythm in 11beta-HSD1 mRNA expression (35-45% increase at morning vs. evening, P < 0.05) in dentate gyrus granular layer and CA1 subregions of the hippocampus in lean Zucker rats that was lost in the obese rats. Sprague Dawley rats also revealed a diurnal rhythm in hippocampal 11beta-HSD1 mRNA expression. There was no circadian variation in 11beta-HSD enzyme activity in peripheral tissues, although obese Zucker rats had a decreased enzyme activity in liver and epididymal fat (by approximately 40%, P < 0.05) compared with lean rats. In Sprague Dawley rats, 11beta-HSD activity in adipose tissue was higher in retroperitoneal and epididymal vs. sc fat (P < 0.001). In summary, obese Zucker rats lack a circadian rhythm of 11beta-HSD1 gene expression in the hippocampus, which may contribute to increased activity of the hypothalamic-pituitary-adrenal axis and altered diurnal variation of circulating corticosterone levels.

Mouse brain localization of the protein kinase C-enhanced phosphatase 1 inhibitor KEPI (Kinase C-Enhanced PP1 Inhibitor)

We recently identified the protein kinase C-enhanced protein phosphatase 1 (PP1) inhibitor KEPI based on its morphine-induced upregulation in striatum. Regulation of protein serine/threonine dephosphorylation by PP1 can modulate important brain signaling pathways. To improve understanding of KEPI’s role in the brain, we have developed anti-KEPI sera in rabbits immunized with a hemocyanin conjugate of KEPI residues 66–80, characterized the specificity that this serum provides, mapped the distribution of immunoreactive KEPI (iKEPI) in mouse brain, rat dorsal root ganglia and striatal cultures and documented KEPI binding to PP1 in vitro. Staining is found in apparently neuronal processes and, often less intensely, in neuronal perikarya in primary cultures and in neurons and neuronal elements from a number of brain regions. iKEPI fiber/terminal patterns are relatively densely distributed in striatum, nucleus accumbens, septum, bed nucleus of the stria terminalis, hippocampus, paraventricular thalamus, ventromedial hypothalamus, interpeduncular nucleus, raphe nuclei, nucleus caudalis of the spinal tract of the trigeminal and dorsal horn of the spinal cord. iKEPI-positive cell bodies lie in the nucleus accumbens, striatum, lateral septal nucleus, granular layer of dentate gyrus, interpeduncular nucleus, dorsal root ganglia and cerebellar vermis. These expression patterns point to possible roles for KEPI in regulating protein dephosphorylation by inhibiting PP1 activities in a number of brain pathways likely to use several different neurotransmitters and to participate in a number of brain functions. Dense KEPI immunoreactivity in nucleus accumbens perikarya, combined with evidence for its regulation by opiates, supports possible roles for KEPI in molecular signal transduction pathways important for drug reward and addiction.

Distribution and anatomical localization of the glucose transporter 2 (GLUT2) in the adult rat brain—an immunohistochemical study

The aim of this work was to study the distribution and cellular localization of GLUT2 in the rat brain by light and electron microscopic immunohistochemistry, whereas our ultrastructural observations will be reported in a second paper. Confirming previous results, we show that GLUT2-immunoreactive profiles are present throughout the brain, especially in the limbic areas and related nuclei, whereas they appear most concentrated in the ventral and medial regions close to the midline. Using cresyl violet counterstaining and double immunohistochemical staining for glial or neuronal markers (GFAp, CAII and NeuN), we show that two limited populations of oligodendrocytes and astrocytes cell bodies and processes are immunoreactive for GLUT2, whereas a cross-reaction with GLUT1 cannot be ruled out. In addition, we report that the nerve cell bodies clearly immunostained for GLUT2 were scarce (although numerous in the dentate gyrus granular layer in particular), whereas the periphery of numerous nerve cells appeared labeled for this transporter. The latter were clustered in the dorsal endopiriform nucleus and neighboring temporal and perirhinal cortex, in the dorsal amygdaloid region, and in the paraventricular and reuniens thalamic nuclei, whereas they were only a few in the hypothalamus. Moreover, a group of GLUT2-immunoreactive nerve cell bodies was localized in the dorsal medulla oblongata while some large multipolar nerve cell bodies peripherally labeled for GLUT2 were scattered in the caudal ventral reticular formation. This anatomical localization of GLUT2 appears characteristic and different from that reported for the neuronal transporter GLUT3 and GLUT4. Indeed, the possibility that GLUT2 may be localized in the sub-plasmalemnal region of neurones and/or in afferent nerve fibres remains to be confirmed by ultrastructural observations. Because of the neuronal localization of GLUT2, and of its distribution relatively similar to glucokinase, it may be hypothesized that this transporter is, at least partially, involved in cerebral glucose sensing.

Expression of insulin-responsive glucose transporter GLUT4 mRNA in the rat brain and spinal cord: An in situ hybridization study

Following a previous immunocytochemical study of GLUT4 in the rat brain and spinal cord (J. Comp. Neurol. 399 (1998) 492), we now report the distribution and cellular expression of GLUT4 mRNA in the CNS using reverse transcription-polymerase chain reaction and non-radioactive in situ hybridization (ISH). The former technique demonstrated the expression of GLUT4 in the different regions examined while ISH with a specific riboprobe allowed the anatomical localization of GLUT4 mRNA. A strong hybridization signal was detected in the piriform and entorhinal cortices and in the pyramidal cell layer of the hippocampal CA1–CA3 areas. Numerous moderately labeled cells were additionally observed in the dentate gyrus granular layer, subiculum and most neocortical areas, as well as in different nuclei of the limbic and motor systems. In contrast, positive cell groups were scarce in the hypothalamus. In the hindbrain, a strong expression of GLUT4 mRNA was observed in the large cell bodies of the red nucleus and cerebellar Purkinje cell layer. Moreover, different groups of moderately labeled cells were found in the deep cerebellar and medullary motor nuclei, in various reticular fields and in the ventral horn of the spinal cord. The present results of ISH mostly agree with the immunocytochemical data reported by our group, although the immunoreactive cells were generally less numerous. However, the fact that a high expression of GLUT4 mRNA was observed in cell bodies of the piriform lobe, hippocampus and substantia nigra, whereas the immunoreactivity for GLUT4 was low in these regions, suggests the existence of post-transcriptional regulation of GLUT4 expression which may depend on the physiological conditions of the animals.