Kainic acid induces 14-3-3 zeta expression in distinct regions of rat brain

Areas of the limbic system of adult male Wistar rats were screened for kainic-acid-induced gene expression. Polymerase-chain-reactionbased differential display identified a 147-bp cDNA fragment, which represented an mRNA that was upregulated in the entorhinal cortex and hippocampus in the kainic-acid-treated animals. The sequence was 97.8% homologous to rat 14-3-3 zeta isoform mRNA. Detailed Northern analysis revealed increased mRNA levels in the entorhinal cortex I h after kainic acid exposure and continued elevation 24 h post-injection in both the entorhinal cortex and hippocampus. Western blot analyses confirmed that the protein product of this gene was also present in increased amounts over the same time period. Immunohistochemistry and terminal transferase-mediated dUTP nick end labelling (TUNEL) detected expression of 14-3-3 protein exclusively in the entorhinal cortex and hippocampus, and only in TUNEL-positive neuronal cells. Expression of the tumor suppressor protein, p53 was also induced by kainate injection, and was co-localized with 14-3-3 zeta protein in selected cells only in the affected brain regions. The increase gene expression of 14-3-3 represents a transcription-mediated response associated with region selective neuronal damage induced by kainic acid. (C) 2002 Elsevier Science B.V. All rights reserved.

Neonatal ethanol exposure reduces AMPA but not NMDA receptor levels in the rat neocortex

Fetal alcohol syndrome (FAS) is the leading cause of mental retardation in western society. We investigated possible changes in glutamate receptor levels in neonatal animals following ethanol exposure using radioligand binding and western blot analysis. We used a vapor chamber to administer ethanol to neonatal Wistar rats 3 h a day from postnatal day (PND) 4-9. A separation control group was separated from their mothers for the same time and duration as the vapor treatment, while a normal control group was left to develop normally. Daily ethanol administrations resulted in decreased brain weight and body weight, as well as microencephaly (decreased brain:body weight ratio). Neither the affinity nor maximum binding of [H-3]MK-801 (dizoclipine maleate) in the cortex of PND10 rats differed between treatment groups. Western blot analysis also failed to reveal any changes in NMDAR1, NMDAR2A, or NMDAR2B receptor levels. In contrast, the AMPA receptor subunit GluR1 was greatly reduced in vapor-treated pups compared with control pups, as revealed by western blot analysis. A similar reduction was found in westerns with an antibody recognizing the GluR2 and 4 subunits. These results indicate that ethanol reduces AMPA rather than NMDA receptors in the developing neocortex, possibly by blocking NMDA receptors during development. (C) 2002 Elsevier Science B.V. All rights reserved.

Expression of hNP22 is altered in the frontal cortex and hippocampus of the alcoholic human brain

Background: Human neuronal protein (hNP22) is a gene with elevated messenger RNA expression in the prefrontal cortex of the human alcoholic brain. hNP22 has high homology with a rat protein (rNP22). These proteins also share homology with a number of cytoskeleton-interacting proteins. Methods: A rabbit polyclonal antibody to an 18-amino acid epitope was produced for use in Western and immunohistochemical analysis. Samples from the human frontal and motor cortices were used for Western blots (n = 10), whereas a different group of frontal cortex and hippocampal samples were obtained for immunohistochemistry (n = 12). Results: The hNP22 antibody detected a single protein in both rat and human brain. Western blots revealed a significant increase in hNP22 protein levels in the frontal cortex but not the motor cortex of alcoholic cases. Immunohistochemical studies confirmed the increased hNP22 protein expression in all cortical layers. This is consistent with results previously obtained using Northern analysis. Immunohistochemical analysis also revealed a significant increase of hNP22 immunoreactivity in the CA3 and CA4 but not other regions of the hippocampus. Conclusions: It is possible that this protein may play a role in the morphological or plastic changes observed after chronic alcohol exposure and withdrawal, either as a cytoskeleton-interacting protein or as a signaling molecule.

Stress induced risk-aversion is reverted by D2/D3 agonist in the rat

Stress exposure triggers cognitive and behavioral impairments that influence decision-making processes. Decisions under a context of uncertainty require complex reward-prediction processes that are known to be mediated by the mesocorticolimbic dopamine (DA) system in brain areas sensitive to the deleterious effects of chronic stress, in particular the orbitofrontal cortex (OFC). Using a decision-making task, we show that chronic stress biases risk-based decision-making to safer behaviors. This decision-making pattern is associated with an increased activation of the lateral part of the OFC and with morphological changes in pyramidal neurons specifically recruited by this task. Additionally, stress exposure induces a hypodopaminergic status accompanied by increased mRNA levels of the dopamine receptor type 2 (Drd2) in the OFC; importantly, treatment with a D2/D3 agonist quinpirole reverts the shift to safer behaviors induced by stress on risky decision-making. These results suggest that the brain mechanisms related to risk-based decision-making are altered after chronic stress, but can be modulated by manipulation of dopaminergic transmission.

Glutathione deficit during development induces anomalies in the rat anterior cingulate GABAergic neurons: Relevance to schizophrenia.

A series of studies in schizophrenic patients report a decrease of glutathione (GSH) in prefrontal cortex (PFC) and cerebrospinal fluid, a decrease in mRNA levels for two GSH synthesizing enzymes and a deficit in parvalbumin (PV) expression in a subclass of GABA neurons in PFC. GSH is an important redox regulator, and its deficit could be responsible for cortical anomalies, particularly in regions rich in dopamine innervation. We tested in an animal model if redox imbalance (GSH deficit and excess extracellular dopamine) during postnatal development would affect PV-expressing neurons. Three populations of interneurons immunolabeled for calcium-binding proteins were analyzed quantitatively in 16-day-old rat brain sections. Treated rats showed specific reduction in parvalbumin immunoreactivity in the anterior cingulate cortex, but not for calbindin and calretinin. These results provide experimental evidence for the critical role of redox regulation in cortical development and validate this animal model used in schizophrenia research.

Temporal and spatial appearance of the membrane cytoskeleton and perineuronal nets in the rat neocortex.

Parvalbumin-immunoreactive interneurons are surrounded by perineuronal nets, containing molecules of the extracellular matrix (e.g. tenascin-R). Furthermore, they seem to have a special cytoskeleton composed of, among others, ankyrinR and beta Rspectrin. In the present developmental study we showed that the intracellular markers parvalbumin, ankyrinR and beta Rspectrin as well as Vicia Villosa agglutinin, an extracellular marker for perineuronal nets, appeared in the second postnatal week. In the third postnatal week, ankyrinR and beta R spectrin were present in the parvalbumin-positive interneurons. Tenascin-R appeared in a similar topographic distribution as the intracellular markers. The adult pattern was established upon the end of the fourth postnatal week. Our results indicate that cytoskeletal maturity maybe a prerequisite for the organization of perineuronal nets of extracellular matrix.

DIFFERENTIAL EXPRESSION OF GLUTARYL-CoA DEHYDROGENASE IN ADULT RAT CNS, PERIPHERAL TISSUES AND DURING EMBRYONIC DEVELOPMENT

Glutaryl-CoA dehydrogenase (GCDH, EC 1.3.99.7) deficiency, known as glutaric acidemia type I, is one of the more common organic acidurias. To investigate the role of this pathway in different organs we studied the tissue-specific expression pattern of rat Gcdh. The open reading frame cDNA of the rat Gcdh gene was cloned from rat brain mRNA by RT-PCR, allowing the synthesis of digoxigenin-labeled in situ hybridization (ISH) riboprobes. Gcdh mRNA expression was analyzed by ISH on cryosections of adult rat brain, kidney, liver, spleen and heart muscle, as well as on E15 and E18 rat embryos. Gcdh was found expressed in the whole rat brain, almost exclusively in neurons. Gcdh was absent from astrocytes but expressed in rare oligodendrocytes. Strong Gcdh expression was found in liver and spleen, where expression appears predominant to lymphatic nodules. In kidney, the highest Gcdh expression is found in the juxtamedullar cortex (but not in glomerula), and at lower levels in medulla. Heart muscle was negative. During embryonic development, Gcdh was found well expressed in liver, intestinal mucosa and skin, as well as at lower levels in CNS. Further studies are ongoing to provide evidence on the presence of the entire pathway in CNS in order to understand the mechanisms leading to neurotoxicity in glutaric aciduria. The high expression of Gcdh in kidney may explain why certain patients with residual enzyme activity are low excretors at the urine metabolite level.

Expression and developmental regulation of Ehk-1, a neuronal Elk-like receptor tyrosine kinase in brain.

Protein tyrosine kinases are pivotal in central nervous tissue development and maintenance. Here we focus on the expression of Ehk-1, a novel Elk-related receptor tyrosine kinase. Ehk-1 gene expression is observed in the developing and adult central nervous system and is highly regulated throughout development at both the messenger RNA and protein levels. Three messenger RNA transcripts of 8.5, 5.9 and 5.1 kb are detectable in the rat brain and a variety of splice possibilities have been identified. However, a major protein species of around M(r) 120,000 predominates throughout development. Ehk-1 messenger RNA and protein levels are highest in the first postnatal week. By in situ messenger RNA hybridization the gene is expressed by all neurons of the adult brain, but mostly in the hippocampus, cerebral cortex and large neurons of the deep cerebellar nuclei, as well as the Purkinje and granular cells of the cerebellum. At earlier stages of development, transcripts are most prominent in the periventricular germinal layers of the brain. Immunohistochemistry reveals a pronounced membrane associated protein expression in immature neurons. In the adult animal, peak reactivity was found in the neuropil with sparing of most perikarya. The spatial and temporal pattern of ehk-1 gene expression suggests a role in both the development and maintenance of differentiated neurons of the central nervous system.

Direct in vivo measurement of glycine and the neurochemical profile in the rat medulla oblongata.

The medulla oblongata (MO) contains a high density of glycinergic synapses and a particularly high concentration of glycine. The aims of this study were to measure directly in vivo the neurochemical profile, including glycine, in MO using a spin-echo-based (1)H MRS sequence at TE?=?2.8 ms and to compare it with three other brain regions (cortex, striatum and hippocampus) in the rat. Glycine was quantified in MO at TE?=?2.8 ms with a Cramér-Rao lower bound (CRLB) of approximately 5%. As a result of the relatively low level of glycine in the other three regions, the measurement of glycine was performed at TE?=?20 ms, which provides a favorable J-modulation of overlapping myo-inositol resonance. The other 14 metabolites composing the neurochemical profile were quantified in vivo in MO with CRLBs below 25%. Absolute concentrations of metabolites in MO, such as glutamate, glutamine, ?-aminobutyrate, taurine and glycine, were in the range of previous in vitro quantifications in tissue extracts. Compared with the other regions, MO had a three-fold higher glycine concentration, and was characterised by reduced (p?<?0.001) concentrations of glutamate (-50?±?4%), glutamine (-54?±?3%) and taurine (-78?±?3%). This study suggests that the functional specialisation of distinct brain regions is reflected in the neurochemical profile.

Immunocytochemical localization of the glucose transporter 2 (GLUT2) in the adult rat brain. II. Electron microscopic study.

Following a former immunohistochemical study in the rat brain [Arluison, M., Quignon, M., Nguyen, P., Thorens, B., Leloup, C., Penicaud, L. Distribution and anatomical localization of the glucose transporter 2 (GLUT2) in the adult rat brain. I. Immunohistochemical study. J. Chem. Neuroanat., in press], we have analyzed the ultrastructural localization of GLUT2 in representative and/or critical areas of the forebrain and hindbrain. In agreement with previous results, we observe few oligodendrocyte and astrocyte cell bodies discretely labeled for GLUT2 in large myelinated fibre bundles and most brain areas examined, whereas the reactive glial processes are more numerous and often localized in the vicinity of nerve terminals and/or dendrites or dendritic spines forming synaptic contacts. Only some of them appear closely bound to unlabeled nerve cell bodies and dendrites. Furthermore, the nerve cell bodies prominently immunostained for GLUT2 are scarce in the brain nuclei examined, whereas the labeled dendrites and dendritic spines are relatively numerous and frequently engaged in synaptic junctions. In conformity with the observation of GLUT2-immunoreactive rings at the periphery of numerous nerve cell bodies in various brain areas (see previous paper), we report here that some neuronal perikarya of the dorsal endopiriform nucleus/perirhinal cortex exhibit some patches of immunostaining just below the plasma membrane. However, the presence of many GLUT2-immunoreactive nerve terminals and/or astrocyte processes, some of them being occasionally attached to nerve cell bodies and dendrites, could also explain the pericellular labeling observed. The results here reported support the idea that GLUT2 may be expressed by some cerebral neurones possibly involved in glucose sensing, as previously discussed. However, it is also possible that this transporter participate in the regulation of neurotransmitter release and, perhaps, in the release of glucose by glial cells.

Differential prefrontal-like deficit in children after cerebellar astrocytoma and medulloblastoma tumor

BACKGROUND This study was realized thanks to the collaboration of children and adolescents who had been resected from cerebellar tumors. The medulloblastoma group (CE+, n = 7) in addition to surgery received radiation and chemotherapy. The astrocytoma group (CE, n = 13) did not receive additional treatments. Each clinical group was compared in their executive functioning with a paired control group (n = 12). The performances of the clinical groups with respect to controls were compared considering the tumor's localization (vermis or hemisphere) and the affectation (or not) of the dentate nucleus. Executive variables were correlated with the age at surgery, the time between surgery-evaluation and the resected volume. METHODS The executive functioning was assessed by means of WCST, Complex Rey Figure, Controlled Oral Word Association Test (letter and animal categories), Digits span (WISC-R verbal scale) and Stroop test. These tests are very sensitive to dorsolateral PFC and/or to medial frontal cortex functions. The scores for the non-verbal Raven IQ were also obtained. Direct scores were corrected by age and transformed in standard scores using normative data. The neuropsychological evaluation was made at 3.25 (SD = 2.74) years from surgery in CE group and at 6.47 (SD = 2.77) in CE+ group. RESULTS The Medulloblastoma group showed severe executive deficit (rate to slight deficit in Stroop (word and colour) tests. In the astrocytoma group, the tumor's localization and dentate affectation showed different profile and level of impairment: moderate to slight for vermal and hemispheric patients respectively. The resected volume, age at surgery and the time between surgery-evaluation correlated with some neuropsychological executive variables. CONCLUSION Results suggest a differential prefrontal-like deficit due to cerebellar lesions and/or cerebellar-frontal diaschisis, as indicate the results in astrocytoma group (without treatments), that also can be generated and/or increased by treatments in the medulloblastoma group. The need for differential rehabilitation strategies for specific clinical groups is remarked. The results are also discussed in the context of the Cerebellar Cognitive Affective Syndrome.

Neurofilament triplet proteins are restricted to a subset of neurons in the rat neocortex.

The cellular localisation of neurofilament triplet subunits was investigated in the rat neocortex. A subset of mainly pyramidal neurons showed colocalisation of subunit immunolabelling throughout the neocortex, including labelling with the antibody SMI32, which has been used extensively in other studies of the primate cortex as a selective cellular marker. Neurofilament-labelled neurons were principally localised to two or three cell layers in most cortical regions, but dramatically reduced labelling was present in areas such as the perirhinal cortex, anterior cingulate and a strip of cortex extending from caudal motor regions through the medial parietal region to secondary visual areas. However, quantitative analysis demonstrated a similar proportion (10-20%) of cells with neurofilament triplet labelling in regions of high or low labelling. Combining retrograde tracing with immunolabelling showed that cellular content of the neurofilament proteins was not correlated with the length of projection. Double labelling immunohistochemistry demonstrated that neurofilament content in axons was closely associated with myelination. Analysis of SMI32 labelling in development indicated that content of this epitope within cell bodies was associated with relatively late maturation, between postnatal days 14 and 21. This study is further evidence of a cell type-specific regulation of neurofilament proteins within neocortical neurons. Neurofilament triplet content may be more closely related to the degree of myelination, rather than the absolute length, of the projecting axon.

Functional MRI Mapping of the Human Auditory Cortex

Mapping the human auditory cortex with standard functional imaging techniques is difficult because of its small size and angular position along the Sylvian fissure. As a result, the exact number and location of auditory cortex areas in the human remains unknown. In a first experiment, we measured the two largest tonotopic areas of primary auditory cortex (PAC, Al and R) using high-resolution functional MRI at 7 Tesla relative to the underlying anatomy of Heschl's gyrus (HG). The data reveals a clear anatomical- functional relationship that indicates the location of PAC across the range of common morphological variants of HG (single gyri, partial duplication and complete duplication). Human PAC tonotopic areas are oriented along an oblique posterior-to-anterior axis with mirror-symmetric frequency gradients perpendicular to HG, as in the macaque. In a second experiment, we tested whether these primary frequency-tuned units were modulated by selective attention to preferred vs. non-preferred sound frequencies in the dynamic manner needed to account for human listening abilities in noisy environments, such as cocktail parties or busy streets. We used a dual-stream selective attention experiment where subjects attended to one of two competing tonal streams presented simultaneously to different ears. Attention to low-frequency tones (250 Hz) enhanced neural responses within low-frequency-tuned voxels relative to high (4000 Hz), and vice versa when at-tention switched from high to low. Human PAC is able to tune into attended frequency channels and can switch frequencies on demand, like a radio. In a third experiment, we investigated repetition suppression effects to environmental sounds within primary and non-primary early-stage auditory areas, identified with the tonotopic mapping design. Repeated presentations of sounds from the same sources, as compared to different sources, gave repetition suppression effects within posterior and medial non-primary areas of the right hemisphere, reflecting their potential involvement in semantic representations. These three studies were conducted at 7 Tesla with high-resolution imaging. However, 7 Tesla scanners are, for the moment, not yet used for clinical diagnosis and mostly reside in institutions external to hospitals. Thus, hospital-based clinical functional and structural studies are mainly performed using lower field systems (1.5 or 3 Tesla). In a fourth experiment, we acquired tonotopic maps at 3 and 7 Tesla and evaluated the consistency of a tonotopic mapping paradigm between scanners. Mirror-symmetric gradients within PAC were highly similar at 7 and 3 Tesla across renderings at different spatial resolutions. We concluded that the tonotopic mapping paradigm is robust and suitable for definition of primary tonotopic areas, also at 3 Tesla. Finally, in a fifth study, we considered whether focal brain lesions alter tonotopic representations in the intact ipsi- and contralesional primary auditory cortex in three patients with hemispheric or cerebellar lesions, without and with auditory complaints. We found evidence for tonotopic reorganisation at the level of the primary auditory cortex in cases of brain lesions independently of auditory complaints. Overall, these results reflect a certain degree of plasticity within primary auditory cortex in different populations of subjects, assessed at different field strengths. - La cartographie du cortex auditif chez l'humain est difficile à réaliser avec des techniques d'imagerie fonctionnelle standard, étant donné sa petite taille et position angulaire le long de la fissure sylvienne. En conséquence, le nombre et l'emplacement exacts des différentes aires du cortex auditif restent inconnus chez l'homme. Lors d'une première expérience, nous avons mesuré, avec de l'imagerie par résonance magnétique à haute intensité (IRMf à 7 Tesla) chez des sujets humains sains, deux larges aires au sein du cortex auditif primaire (PAC; Al et R) avec une représentation spécifique des fréquences pures préférées - ou tonotopie. Nos résultats ont démontré une relation anatomico- fonctionnelle qui définit clairement la position du PAC à travers toutes les variantes du gyrus d'Heschl's (HG). Les aires tonotopiques du PAC humain sont orientées le long d'un axe postéro-antérieur oblique avec des gradients de fréquences spécifiques perpendiculaires à HG, d'une manière similaire à celles mesurées chez le singe. Dans une deuxième expérience, nous avons testé si ces aires primaires pouvaient être modulées, de façon dynamique, par une attention sélective pour des fréquences préférées par rapport à celles non-préférées. Cette modulation est primordiale lors d'interactions sociales chez l'humain en présence de bruits distracteurs tels que d'autres discussions ou un environnement sonore nuisible (comme par exemple, dans la circulation routière). Dans cette étude, nous avons utilisé une expérience d'attention sélective où le sujet devait être attentif à une des deux voies sonores présentées simultanément à chaque oreille. Lorsque le sujet portait était attentif aux sons de basses fréquences (250 Hz), la réponse neuronale relative à ces fréquences augmentait par rapport à celle des hautes fréquences (4000 Hz), et vice versa lorsque l'attention passait des hautes aux basses fréquences. De ce fait, nous pouvons dire que PAC est capable de focaliser sur la fréquence attendue et de changer de canal selon la demande, comme une radio. Lors d'une troisième expérience, nous avons étudié les effets de suppression due à la répétition de sons environnementaux dans les aires auditives primaires et non-primaires, d'abord identifiées via le protocole de la première étude. La présentation répétée de sons provenant de la même source sonore, par rapport à de sons de différentes sources sonores, a induit un effet de suppression dans les aires postérieures et médiales auditives non-primaires de l'hémisphère droite, reflétant une implication de ces aires dans la représentation de la catégorie sémantique. Ces trois études ont été réalisées avec de l'imagerie à haute résolution à 7 Tesla. Cependant, les scanners 7 Tesla ne sont pour le moment utilisés que pour de la recherche fondamentale, principalement dans des institutions externes, parfois proches du patient mais pas directement à son chevet. L'imagerie fonctionnelle et structurelle clinique se fait actuellement principalement avec des infrastructures cliniques à 1.5 ou 3 Tesla. Dans le cadre dune quatrième expérience, nous avons avons évalués la cohérence du paradigme de cartographie tonotopique à travers différents scanners (3 et 7 Tesla) chez les mêmes sujets. Nos résultats démontrent des gradients de fréquences définissant PAC très similaires à 3 et 7 Tesla. De ce fait, notre paradigme de définition des aires primaires auditives est robuste et applicable cliniquement. Finalement, nous avons évalués l'impact de lésions focales sur les représentations tonotopiques des aires auditives primaires des hémisphères intactes contralésionales et ipsilésionales chez trois patients avec des lésions hémisphériques ou cérébélleuses avec ou sans plaintes auditives. Nous avons trouvé l'évidence d'une certaine réorganisation des représentations topographiques au niveau de PAC dans le cas de lésions cérébrales indépendamment des plaintes auditives. En conclusion, nos résultats démontrent une certaine plasticité du cortex auditif primaire avec différentes populations de sujets et différents champs magnétiques.

NADPH-diaphorase-positive ganglion cells of the rat adrenal gland: age- and sex-related changes in their number, size, and distribution.

The rat adrenal gland contains ganglion cells able to synthesize nitric oxide (NO). This messenger molecule controls and modulates adrenal secretory activity and blood flow. The present study analyzed the number, size, and distribution of NO-producing adrenal neurons in adulthood and during postnatal development by means of beta-nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry. This method reliably visualizes the enzyme responsible for NO generation. The reactive neurons per adrenal gland were 350-400 in both male and female adult rats. The positive nerve cell bodies were mostly located in the medulla, few being detected within the cortex and the subcapsular region. Dual labeling with anti-microtubule-associated protein 2 antibody, specific for neuronal elements, confirmed this distribution. Anti-microtubule-associated protein 1b antibody identified a subset of NADPH-d-positive neurons, displaying different degrees of maturation according to their position within the adrenal gland. At birth, there were about 220 NADPH-d-labeled neurons per adrenal gland in both sexes. As confirmed by dual immunocytochemical labeling, their great majority was evenly distributed between the cortex and the subcapsular region, the medulla being practically devoid of stained neurons. After birth, the number of adrenal NADPH-d-positive ganglion cells displayed a strong postnatal increase and reached the adult-like distribution after 1-2 months. During the period of increase, there was a transient difference in the numbers of these cells in the two sexes. Thus we present here evidence of plasticity in the number, size, and distribution of NADPH-d-positive adrenal neurons between birth and adulthood; in addition, we describe transient sex-related differences in their number and distribution during the 2nd postnatal week, which are possibly related to the epigenetic action of gonadal hormones during this period.