Lack of placental neurosteroid alters cortical development and female somatosensory function.

Placental endocrine function is essential to fetal brain development. Placental hormones include neurosteroids such as allopregnanolone (ALLO), a regulator of neurodevelopmental processes via positive allosteric modulation of the GABAA receptor (GABAA-R). Using a mouse model (plKO) in which the gene encoding the ALLO synthesis enzyme is specifically deleted in trophoblasts, we previously showed that placental ALLO insufficiency alters cerebellar white matter development and leads to male-specific autistic-like behavior. We now demonstrate that the lack of placental ALLO causes female-predominant alterations of cortical development and function. Placental ALLO insufficiency disrupts cell proliferation in the primary somatosensory cortex (S1) in a sex-linked manner. Early changes are seen in plKO embryos of both sexes, but persist primarily in female offspring after birth. Adolescent plKO females show significant reduction in pyramidal neuron density, as well as somatosensory behavioral deficits as compared with plKO males and control littermates. Assessment of layer-specific markers in human postmortem cortices suggests that preterm infants may also have female-biased abnormalities in cortical layer specification as compared with term infants. This study establishes a novel and fundamental link between placental function and sex-linked long-term neurological outcomes, emphasizing the importance of the growing field of neuroplacentology.

Oxidative Stress-Induced Damage to the Developing Hippocampus Is Mediated by GSK3ß.

Neonatal brain injury renders the developing brain vulnerable to oxidative stress, leading to cognitive deficit. However, oxidative stress-induced damage to hippocampal circuits and the mechanisms underlying long-term changes in memory and learning are poorly understood. We used high oxygen tension or hyperoxia (HO) in neonatal mice of both sexes to investigate the role of oxidative stress in hippocampal damage. Perinatal HO induces reactive oxygen species and cell death, together with reduced interneuron maturation, inhibitory postsynaptic currents, and dentate progenitor proliferation. Postinjury interneuron stimulation surprisingly improved inhibitory activity and memory tasks, indicating reversibility. With decreased hippocampal levels of Wnt signaling components and somatostatin, HO aberrantly activated glycogen synthase kinase 3 ß activity. Pharmacological inhibition or ablation of interneuron glycogen synthase kinase 3 ß during HO challenge restored progenitor cell proliferation, interneuron development, inhibitory/excitatory balance, as well as hippocampal-dependent behavior. Biochemical targeting of interneuron function may benefit learning deficits caused by oxidative damage.SIGNIFICANCE STATEMENT Premature infants are especially vulnerable to oxidative stress, as their antioxidant defenses are underdeveloped. Indeed, high oxygen tension is associated with poor neurologic outcomes. Because of its sustained postnatal development and role in learning and memory, the hippocampus is especially vulnerable to oxidative damage in premature infants. However, the role of oxidative stress in the developing hippocampus has yet to be explored. With ever-rising rates of neonatal brain injury and no universally viable approach to maximize functional recovery, a better understanding of the mechanisms underlying neonatal brain injury is needed. Addressing this need, this study uses perinatal hyperoxia to study cognitive deficits, pathophysiology, and molecular mechanisms of oxidative damage in the developing hippocampus.

Placental endocrine function shapes cerebellar development and social behavior.

Compromised placental function or premature loss has been linked to diverse neurodevelopmental disorders. Here we show that placenta allopregnanolone (ALLO), a progesterone-derived GABA-A receptor (GABAAR) modulator, reduction alters neurodevelopment in a sex-linked manner. A new conditional mouse model, in which the gene encoding ALLO's synthetic enzyme (akr1c14) is specifically deleted in trophoblasts, directly demonstrated that placental ALLO insufficiency led to cerebellar white matter abnormalities that correlated with autistic-like behavior only in male offspring. A single injection of ALLO or muscimol, a GABAAR agonist, during late gestation abolished these alterations. Comparison of male and female human preterm infant cerebellum also showed sex-linked myelination marker alteration, suggesting similarities between mouse placental ALLO insufficiency and human preterm brain development. This study reveals a new role for a placental hormone in shaping brain regions and behaviors in a sex-linked manner. Placental hormone replacement might offer novel therapeutic opportunities to prevent later neurobehavioral disorders.

Preterm Birth Alters the Maturation of the GABAergic System in the Human Prefrontal Cortex.

Developmental changes in GABAergic and glutamatergic systems during frontal lobe development have been hypothesized to play a key role in neurodevelopmental disorders seen in children born very preterm or at/with low birth weight, but the associated cellular changes have not yet been identified. Here we studied the molecular development of the GABAergic system specifically in the dorsolateral prefrontal cortex, a region that has been implicated in neurodevelopmental and psychiatric disorders. The maturation state of the GABAergic system in this region was assessed in human post-mortem brain samples, from term infants ranging in age from 0 to 8 months (n = 17 male, 9 female). Gene expression was measured for 47 GABAergic genes and used to calculate a maturation index. This maturation index was significantly more dynamic in male than female infants. To evaluate the impact of premature birth on the GABAergic system development, samples from 1-month-old term (n = 9 male, 4 female) and 1-month corrected-age very preterm (n = 8 male, 6 female) infants, were compared using the same gene list and methodology. The maturation index for the GABAergic system was significantly lower (-50%, p < 0.05) in male preterm infants, with major alterations in genes linked to GABAergic function in astrocytes, suggesting astrocytic GABAergic developmental changes as a new cellular mechanism underlying preterm brain injury.

Impaired Interneuron Development in a Novel Model of Neonatal Brain Injury.

Prematurity is associated with significantly increased risk of neurobehavioral pathologies, including autism and schizophrenia. A common feature of these psychiatric disorders is prefrontal cortex (PFC) inhibitory circuit disruption due to GABAergic interneuron alteration. Cortical interneurons are generated and migrate throughout late gestation and early infancy, making them highly susceptible to perinatal insults such as preterm birth. Term and preterm PFC pathology specimens were assessed using immunohistochemical markers for interneurons. Based on the changes seen, a new preterm encephalopathy mouse model was developed to produce similar PFC interneuron loss. Maternal immune activation (MIA; modeling chorioamnionitis, associated with 85% of extremely preterm births) was combined with chronic sublethal hypoxia (CSH; modeling preterm respiratory failure), with offspring of both sexes assessed anatomically, molecularly and neurobehaviorally. In the PFC examined from the human preterm samples compared to matched term samples at corrected age, a decrease in somatostatin (SST) and calbindin (CLB) interneurons was seen in upper cortical layers. This pattern of interneuron loss in upper cortical layers was mimicked in the mouse PFC following the combination of MIA and CSH, but not after either insult alone. This persistent interneuron loss is associated with postnatal microglial activation that occurs during CSH only after MIA. The combined insults lead to long-term neurobehavioral deficits which parallel human psychopathologies that may be seen after extremely preterm birth. This new preclinical model supports a paradigm in which specific cellular alterations seen in preterm encephalopathy can be linked with a risk of neuropsychiatric sequela. Specific interneuron subtypes may provide therapeutic targets to prevent or ameliorate these neurodevelopmental risks.

miRNA Long-Term Response to Early Metabolic Environmental Challenge in Hypothalamic Arcuate Nucleus.

Epidemiological reports and studies using rodent models indicate that early exposure to nutrient and/or hormonal challenges can reprogram metabolism at adulthood. Hypothalamic arcuate nucleus (ARC) integrates peripheral and central signals to adequately regulate energy homeostasis. microRNAs (miRNAs) participate in the control of gene expression of large regulatory networks including many signaling pathways involved in epigenetics regulations. Here, we have characterized and compared the miRNA population of ARC of adult male rats continuously exposed to a balanced metabolic environment to the one of adult male rats exposed to an unbalanced high-fat/high-carbohydrate/moderate-protein metabolic environment during the perinatal period and/or at adulthood that consequently displayed hyperinsulinemia and/or hyperleptinemia. We identified more than 400 miRNA species in ARC of adult male rats. By comparing the miRNA content of six biological replicates in each of the four perinatal/adult environments/rat groups, we identified the 10 miRNAs specified by clusters miR-96/182/183, miR-141/200c, and miR-200a/200b/429 as miRNAs of systematic and uncommonly high variation of expression. This uncommon variation of expression may underlie high individual differences in aging disease susceptibilities. By comparing the miRNA content of the adult ARC between the rat groups, we showed that the miRNA population was not affected by the unbalanced adult environment while, in contrast, the expression of 11 miRNAs was repeatedly impacted by the perinatal unbalanced environment. Our data revealed a miRNA response of adult ARC to early metabolic environmental challenge.

Substantial and robust changes in microRNA transcriptome support postnatal development of the hypothalamus in rat.

MicroRNAs (miRNAs) modulate gene expression in male germ cells and somatic tissues of mammals on a genome-wide scale. Hundreds of miRNAs are encoded by mammalian genomes, a large fraction of which is expressed in brain. Here we have investigated the complexity and dynamics of miRNA transcriptomes that associate with neuronal network maturation of hypothalamic arcuate nucleus and median eminence (ARC/ME) in rat by analysing more than 300 miRNAs from 3-7 biological replicates at 5 postnatal time-points. The network connecting ARC/ME to other hypothalamic and extra-hypothalamic regions maturates in an environment-dependent manner. We therefore analyzed miRNA transcriptomes of progeny of dams fed either a balanced or unbalanced diet during gestation and lactation. More than 30% of the miRNAs displayed significative changes of expression between stages P8 and P14, and P21 and P28; half of the changes were greater than 3-fold. Among those miRNAs were well-known and dozens of still poorly documented miRNAs. Progeny of dams fed an unbanced diet displayed a severe growth retardation phenotype, lower levels of plasma leptin but almost identical miRNA transcriptomes. Together these data demonstrate that two substantial and robust changes in miRNA transcriptome of ARC/ME occur at a period crucial for neuronal network functional organization.

Hippocampal GSK3ß as a Molecular Link Between Obesity and Depression.

Obesity is considered as a risk factor for mood disorders including depression. Nevertheless, the mechanisms underlying this association are not clearly understood. To address this issue, we investigated the impact of high-fat (HF)-diet-induced obesity on depressive-like behavior and on serotonin (5-HT)-dependent Akt/glycogen synthase kinase 3ß (GSK3ß) signaling in the dentate gyrus (DG) of the hippocampus, which has been associated with mood regulation. We first showed that a HF diet induced significant overweight and hyperglycemia as well as a depressive-like behavior in adult Wistar rats. By using an ex vivo approach on brain slices, we demonstrated that 5-HT activates the Akt/GSK3ß cascade in the DG of control chow (C) diet-fed animals and that a 16-week HF diet feeding abolishes this activation, concurrently with a desensitization of leptin and insulin signaling in the same region. Furthermore, depressive-like behavior inversely correlated with 5-HT-induced phosphorylation of GSK3ß in the subgranular neurons of the DG. Interestingly, a substitution of HF with C diet for 6 weeks induced a total loss of depressive symptoms, whereas body weight and glycemia remained significantly higher compared to control rats. In addition, food restoration led to a recovery of the Akt/GSK3ß signaling pathway activation in the DG. In parallel, we observed a negative correlation between body weight and cell proliferation in the subgranular zone of the DG. To conclude, we provide evidence for a desensitization of 5-HT-induced Akt/GSK3ß signaling and an impaired cell proliferation in the DG by HF diet, suggesting novel molecular mechanisms linking obesity to depression.

Cerebral cortex and hippocampus respond differently after post-natal exposure to uranium.

The central nervous system (CNS) is known to be sensitive to pollutants during its development. Uranium (U) is a heavy metal that occurs naturally in the environment as a component of the earth's crust, and populations may therefore be chronically exposed to U through drinking water and food. Previous studies have shown that the CNS is a target of U in rats exposed in adulthood. We assessed the effects of U on behavior and cholinergic system of rats exposed from birth for 10 weeks at 10 mg.L⁻¹ or 40 mg.L⁻¹. For behavioral analysis, the sleep/wake cycle (recorded by telemetry), the object recognition memory and the spatial working memory (Y-maze) were evaluated. Acetylcholine (ACh) and acetylcholinesterase (AChE) levels were evaluated in the entorhinal cortex and hippocampus. At 40 mg.L⁻¹, U exposure impaired object recognition memory (-20%), but neither spatial working memory nor the sleep/wake cycle was impaired. A significant decrease was observed in both the ACh concentration (-14%) and AChE activity (-14%) in the entorhinal cortex, but not in the hippocampus. Any significant effect on behaviour and cholinergic system was observed at 10 mg U.L⁻¹. These results demonstrate that early exposure to U during postnatal life induces a structure cerebral-dependant cholinergic response and modifies such memory process in rats. This exposure to U early in life could have potential delayed effects in adulthood.

The anorexigenic cytokine ciliary neurotrophic factor stimulates POMC gene expression via receptors localized in the nucleus of arcuate neurons.

Ciliary neurotrophic factor (CNTF) is a neural cytokine that reduces appetite and body weight when administrated to rodents or humans. We have demonstrated recently that the level of CNTF in the arcuate nucleus (ARC), a key hypothalamic region involved in food intake regulation, is positively correlated with protection against diet-induced obesity. However, the comprehension of the physiological significance of neural CNTF action was still incomplete because CNTF lacks a signal peptide and thus may not be secreted by the classical exocytosis pathways. Knowing that CNTF distribution shares similarities with that of its receptor subunits in the rat ARC, we hypothesized that CNTF could exert a direct intracrine effect in ARC cells. Here, we demonstrate that CNTF, together with its receptor subunits, translocates to the cell nucleus of anorexigenic POMC neurons in the rat ARC. Furthermore, the stimulation of hypothalamic nuclear fractions with CNTF induces the phosphorylation of several signaling proteins, including Akt, as well as the transcription of the POMC gene. These data strongly suggest that intracellular CNTF may directly modulate POMC gene expression via the activation of receptors localized in the cell nucleus, providing a novel plausible mechanism of CNTF action in regulating energy homeostasis.

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

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

Unexpected long-term protection of adult offspring born to high-fat fed dams against obesity induced by a sucrose-rich diet.

BACKGROUND: Metabolic and endocrine environment during early life is crucial for metabolic imprinting. When dams were fed a high fat diet (HF diet), rat offspring developed hypothalamic leptin resistance with lean phenotype when weaned on a normal diet. Interestingly, when grown on the HF diet, they appeared to be protected against the effects of HF diet as compared to offspring of normally fed dams. The mechanisms involved in the protective effect of maternal HF diet are unclear. METHODOLOGY/PRINCIPAL FINDINGS: We thus investigated the impact of maternal high fat diet on offspring subjected to normal or high palatable diet (P diet) on metabolic and endocrine parameters. We compared offspring born to dams fed P or HF diet. Offspring born to dams fed control or P diet, when fed P diet exhibited a higher body weight, altered hypothalamic leptin sensitivity and metabolic parameters suggesting that maternal P diet has no protective effect on offspring. Whereas, maternal HF diet reduces body weight gain and circulating triglycerides, and ameliorates corpulence index of offspring, even when subjected to P diet. Interestingly, this protective effect is differently expressed in male and female offspring. Male offspring exhibited higher energy expenditure as mirrored by increased hypothalamic UCP-2 and liver AdipoR1/R2 expression, and a profound change in the arcuate nucleus astrocytic organization. In female offspring, the most striking impact of maternal HF diet is the reduced hypothalamic expression of NPY and POMC. CONCLUSIONS/SIGNIFICANCE: HF diet given during gestation and lactation protects, at least partially, offspring from excessive weight gain through several mechanisms depending upon gender including changes in arcuate nucleus astrocytic organization and increased hypothalamic UCP-2 and liver AdipoR1/2 expression in males and reduced hypothalamic expression of NPY and POMC in females. Taken together our results reveal new mechanisms involved in the protective effect of maternal HF diet.

Heavy metal uranium affects the brain cholinergic system in rat following sub-chronic and chronic exposure.

Uranium is a heavy metal naturally present in the environment that may be chronically ingested by the population. Previous studies have shown that uranium is present in the brain and alters behaviour, notably locomotor activity, sensorimotor ability, sleep/wake cycle and the memory process, but also metabolism of neurotransmitters. The cholinergic system mediates many cognitive systems, including those disturbed after chronic exposure to uranium i.e., spatial memory, sleep/wake cycle and locomotor activity. The objective of this study was to assess whether these disorders follow uranium-induced alteration of the cholinergic system. In comparison with 40 control rats, 40 rats drank 40 mg/L uranyl nitrate for 1.5 or 9 months. Cortex and hippocampus were removed and gene expression and protein level were analysed to determine potential changes in cholinergic receptors and acetylcholine levels. The expression of genes showed various alterations in the two brain areas after short- and long-term exposure. Nevertheless, protein levels of the choline acetyltransferase enzyme (ChAT), the vesicular transporter of acetylcholine (VAChT) and the nicotinic receptor beta2 sub-unit (nAChRbeta2) were unmodified in all cases of the experiment and muscarinic receptor type 1 (m1AChR) protein level was disturbed only after 9 months of exposure in the cortex (-30%). Acetylcholine levels were unchanged in the hippocampus after 1.5 and 9 months, but were decreased in the cortex after 1.5 months only (-22%). Acetylcholinesterase (AChE) activity was also unchanged in the hippocampus but decreased in the cortex after 1.5 and 9 months (-16% and -18%, respectively). Taken together, these data indicate that the cholinergic system is a target of uranium exposure in a structure-dependent and time-dependent manner. These cholinergic alterations could participate in behavioural impairments.

Leptin but not ciliary neurotrophic factor (CNTF) induces phosphotyrosine phosphatase-1B expression in human neuronal cells (SH-SY5Y): putative explanation of CNTF efficacy in leptin-resistant state.

Growing evidences suggest that obesity is associated with hypothalamic leptin resistance, leading to the alteration of food intake control. Alternative treatment using ciliary neurotrophic factor (CNTF) has been suggested because CNTF exerts a leptin-like effect, even in leptin-resistant states, but the mechanisms by which CNTF maintains this effect are not yet understood. Both leptin and CNTF act in the hypothalamus through similar signaling pathways including janus kinase-2/signal transducer and activator of transcription (STAT)-3 pathway. To explore the differences and interactions between leptin and CNTF signaling pathways, differentiated human neuroblastoma cells (SH-SY5Y) were exposed to either leptin or CNTF and then challenged for each cytokine. Leptin pretreatment completely abolished leptin-dependent STAT-3 and ERK 1/2 phosphorylations without affecting CNTF action. The lack of cross-desensitization between leptin and CNTF signaling pathways occurred despite the induction of suppressor of cytokine signaling-3 in response to both cytokines. Interestingly, leptin as well as insulin induced the expression of phosphotyrosine phosphatase (PTP)-1B, whereas CNTF treatment did not affect its expression. In addition, acute leptin treatment but not CNTF induced PTP-1B expression in mouse hypothalamic arcuate nucleus. Furthermore, the overexpression of human PTP-1B in SH-SY5Y cells completely abolished leptin- and insulin-dependent janus kinase-2, STAT-3, and ERK 1/2 phosphorylations, but CNTF action was not altered. Collectively, our results suggest that PTP-1B constitutes a key divergent element between leptin/insulin and CNTF signaling pathways at the neuronal level, which may constitute a possible mechanism that explains the efficacy of CNTF in leptin-resistant states.

Differential compartmentalization and distinct functions of GABAB receptor variants.

GABAB receptors are the G protein-coupled receptors for the main inhibitory neurotransmitter in the brain, gamma-aminobutyric acid (GABA). Molecular diversity in the GABAB system arises from the GABAB1a and GABAB1b subunit isoforms that solely differ in their ectodomains by a pair of sushi repeats that is unique to GABAB1a. Using a combined genetic, physiological, and morphological approach, we now demonstrate that GABAB1 isoforms localize to distinct synaptic sites and convey separate functions in vivo. At hippocampal CA3-to-CA1 synapses, GABAB1a assembles heteroreceptors inhibiting glutamate release, while predominantly GABAB1b mediates postsynaptic inhibition. Electron microscopy reveals a synaptic distribution of GABAB1 isoforms that agrees with the observed functional differences. Transfected CA3 neurons selectively express GABAB1a in distal axons, suggesting that the sushi repeats, a conserved protein interaction motif, specify heteroreceptor localization. The constitutive absence of GABAB1a but not GABAB1b results in impaired synaptic plasticity and hippocampus-dependent memory, emphasizing molecular differences in synaptic GABAB functions.

Hyperdopaminergia and altered locomotor activity in GABAB1-deficient mice.

GABAB1-/- mice, which are devoid of functional GABAB receptors, consistently exhibit marked hyperlocomotion when exposed to a novel environment. Telemetry recordings now revealed that, in a familiar environment, GABAB1-/- mice display an altered pattern of circadian activity but no hyperlocomotion. This indicates that hyperlocomotion is only triggered when GABAB1-/- mice are aroused by novelty. In microdialysis experiments, GABAB1-/- mice exhibited a 2-fold increased extracellular level of dopamine in the striatum. Following D-amphetamine administration, GABAB1-/- mice released less dopamine than wild-type mice, indicative of a reduced cytoplasmic dopamine pool. The hyperdopaminergic state of GABAB1-/- mice is accompanied by molecular changes, including reduced levels of tyrosine hydroxylase mRNA, D1 receptor binding-sites and Ser40 phosphorylation of tyrosine hydroxylase. Tyrosine hydroxylase activity, tissue dopamine content and dopamine metabolism do not appear to be measurably altered. Pharmacological and electrophysiological experiments support that the hyperdopaminergic state of GABAB1-/- mice is not severe enough to inactivate dopamine D2 receptors and to disrupt D2-mediated feedback inhibition of tyrosine hydroxylase activity. The data support that loss of GABAB activity results in a sustained moderate hyperdopaminergic state, which is phenotypically revealed by contextual hyperlocomotor activity. Importantly, the presence of an inhibitory GABA tone on the dopaminergic system mediated by GABAB receptors provides an opportunity for therapeutic intervention.

Floxed allele for conditional inactivation of the GABAB(1) gene.

GABA(B) receptors are the G-protein-coupled receptors for the neurotransmitter GABA. GABA(B) receptors are broadly expressed in the nervous system. Their complete absence in mice causes premature lethality or--when mice are viable--epilepsy, impaired memory, hyperalgesia, hypothermia, and hyperactivity. A spatially and temporally restricted loss of GABA(B) function would allow addressing how the absence of GABA(B) receptors leads to these diverse phenotypes. To permit a conditional gene inactivation, we flanked critical exons of the GABA(B(1)) gene with lox511 sites. GABA(B(1)) (lox511/lox511) mice exhibit normal levels of GABA(B(1)) protein, are fertile, and do not display any behavioral phenotype. We crossed GABA(B(1)) (lox511/lox511) with Cre-deleter mice to produce mice with an unrestricted GABA(B) receptor elimination. These GABA(B(1)) (-/-) mice no longer synthesize GABA(B(1)) protein and exhibit the expected behavioral abnormalities. The conditional GABA(B(1)) allele described here is therefore suitable for generating mice with a site- and time-specific loss of GABA(B) function.

Altered hippocampal expression of calbindin-D-28k and calretinin in GABA(B(1))-deficient mice.

Balb/c GABA(B(1))(-/-) mice develop complex epileptiform activity, including spontaneous and audiogenic generalized seizures, 6-8 weeks after birth. The neuronal systems involved in these epilepsies have not been identified yet. Because the hippocampus is critically involved in epileptiform activity, we now investigated whether this brain region exhibits seizure-related alterations. Using semi-quantitative immunohistochemistry, we studied the temporal and cellular hippocampal expression pattern of two seizure-sensitive calcium-binding proteins, calbindin-D-28k and calretinin, in GABA(B(1))(-/-) mice. One month after birth, before the onset of overt epileptiform activity, wild-type (WT) and GABA(B(1))(-/-) mice exhibit comparable expression profiles for the two calcium-binding proteins. Three months after birth, once the epileptic phenotype is established, we observe clear alterations in the expression of calcium-binding proteins in the dentate gyrus area. GABA(B(1))(-/-) mice exhibit a 50% decline in the staining intensity of calbindin-D-28k expressing neurons and a 70% increase in the number of calretinin-positive neurons when compared to WT littermates. Six months after birth, the down-regulation of calbindin-D-28k protein is even more pronounced, while the calretinin expression in GABA(B(1))(-/-) mice reverts to the pattern seen in WT littermates. Our data demonstrate that the absence of functional GABA(B) receptors causes epileptiform activity through a mechanism that crucially involves dentate gyrus granule cells, and that this pathological activity is accompanied by adaptive changes.

Postnatal regulation by monoamines of vasopressin expression in the neuroendocrine hypothalamus of MAO-A-deficient mice.

We studied the influence of noradrenaline (NA) and serotonin (5-HT) on arginine-vasopressin (AVP) expression in the mouse neuroendocrine hypothalamus during the postnatal period. We used 11-day-old transgenic Tg8 mice knock-out for the monoamine oxidase A gene, which are characterized by increased amounts of NA (two-fold) and 5-HT (nine-fold) in the brain compared with wild-type littermates. AVP expression, determined by enzyme immunoassay and in situ hybridization, was increased in the suprachiasmatic nucleus (SCN), decreased in the supraoptic nucleus (SON), and unchanged in the paraventricular nucleus of Tg8 mice compared with wild-types. Inhibiting NA synthesis by injecting alpha-methylparatyrosine to Tg8 mice, AVP levels were decreased in the SCN but increased in the SON. Moreover, the administration of parachlorophenylalanine, a 5-HT synthesis inhibitor, was associated with increased AVP contents in the SCN only. Together, these data show a marked region-specific sensitivity of AVP expression to NA and 5-HT during the postnatal period in the mouse hypothalamus.