Oxytocin administration in neonates shapes hippocampal circuitry and restores social behavior in a mouse model of autism.

Oxytocin is an important regulator of the social brain. In some animal models of autism, notably in Magel2tm1.1Mus-deficient mice, peripheral administration of oxytocin in infancy improves social behaviors until adulthood. However, neither the mechanisms responsible for social deficits nor the mechanisms by which such oxytocin administration has long-term effects are known. Here, we aimed to clarify these oxytocin-dependent mechanisms, focusing on social memory performance. Using in situ hybridization (RNAscope), we have established that Magel2 and oxytocin receptor are co-expressed in the dentate gyrus and CA2/CA3 hippocampal regions involved in the circuitry underlying social memory. Then, we have shown that Magel2tm1.1Mus-deficient mice, evaluated in a three-chamber test, present a deficit in social memory. Next, in hippocampus, we conducted neuroanatomical and functional studies using immunostaining, oxytocin-binding experiments, ex vivo electrophysiological recordings, calcium imaging and biochemical studies. We demonstrated: an increase of the GABAergic activity of CA3-pyramidal cells associated with an increase in the quantity of oxytocin receptors and of somatostatin interneurons in both DG and CA2/CA3 regions. We also revealed a delay in the GABAergic development sequence in Magel2tm1.1Mus-deficient pups, linked to phosphorylation modifications of KCC2. Above all, we demonstrated the positive effects of subcutaneous administration of oxytocin in the mutant neonates, restoring hippocampal alterations and social memory at adulthood. Although clinical trials are debated, this study highlights the mechanisms by which peripheral oxytocin administration in neonates impacts the brain and demonstrates the therapeutic value of oxytocin to treat infants with autism spectrum disorders.

NMDA-dependent switch of proBDNF actions on developing GABAergic synapses.

The brain-derived neurotrophic factor (BDNF) has emerged as an important messenger for activity-dependent development of neuronal network. Recent findings have suggested that a significant proportion of BDNF can be secreted as a precursor (proBDNF) and cleaved by extracellular proteases to yield the mature form. While the actions of proBDNF on maturation and plasticity of excitatory synapses have been studied, the effect of the precursor on developing GABAergic synapses remains largely unknown. Here, we show that regulated secretion of proBDNF exerts a bidirectional control of GABAergic synaptic activity with NMDA receptors driving the polarity of the plasticity. When NMDA receptors are activated during ongoing synaptic activity, regulated Ca(2+)-dependent secretion of proBDNF signals via p75(NTR) to depress GABAergic synaptic activity, while in the absence of NMDA receptors activation, secreted proBDNF induces a p75(NTR)-dependent potentiation of GABAergic synaptic activity. These results revealed a new function for proBDNF-p75(NTR) signaling in synaptic plasticity and a novel mechanism by which synaptic activity can modulate the development of GABAergic synaptic connections.

Leptin antagonism improves Rett syndrome phenotype in symptomatic male Mecp2-null mice.

Rett syndrome (RTT) is a severe neurodevelopmental disorder that arise from de novo mutations in the X-linked gene MECP2 (methyl-CpG-binding protein 2). Circulating levels of the adipocyte hormone leptin are elevated in RTT patients and rodent models of the disease. Leptin targets a large number of brain structures and regulates a wide range of developmental and physiological functions which are altered in RTT. We hypothesized that elevated leptin levels might contribute to RTT pathogenesis. Accordingly, we show that pharmacological antagonism of leptin or genetic reduction of leptin production prevents the degradation of health status, weight loss and the progression of breathing and locomotor deficits. At the neuronal level, the anti-leptin strategies rescue the hippocampal excitatory/inhibitory imbalance and synaptic plasticity impairment. Targeting leptin might therefore represent a new approach for RTT treatment.

The Chloride Homeostasis of CA3 Hippocampal Neurons Is Not Altered in Fully Symptomatic Mepc2-null Mice.

Rett syndrome (RTT) is an X-linked neurodevelopmental disorder caused mainly by mutations in the MECP2 gene. Mouse models of RTT show reduced expression of the cation-chloride cotransporter KCC2 and altered chloride homeostasis at presymptomatic stages. However, whether these alterations persist to late symptomatic stages has not been studied. Here we assess KCC2 and NKCC1 expressions and chloride homeostasis in the hippocampus of early [postnatal (P) day 30-35] and late (P50-60) symptomatic male Mecp2-null (Mecp2 -/y) mice. We found (i) no difference in the relative amount, but an over-phosphorylation, of KCC2 and NKCC1 between wild-type (WT) and Mecp2 -/y hippocampi and (ii) no difference in the inhibitory strength, nor reversal potential, of GABA A -receptor-mediated responses in Mecp2 -/y CA3 pyramidal neurons compared to WT at any stages studied. Altogether, these data indicate the presence of a functional chloride extrusion mechanism in Mecp2 -/y CA3 pyramidal neurons at symptomatic stages.

GABA(B) receptor activation triggers BDNF release and promotes the maturation of GABAergic synapses.

GABA, the main inhibitory neurotransmitter in the adult brain, has recently emerged as an important signal in network development. Most of the trophic functions of GABA have been attributed to depolarization of the embryonic and neonatal neurons via the activation of ionotropic GABA(A) receptors. Here we demonstrate a novel mechanism by which endogenous GABA selectively regulates the development of GABAergic synapses in the developing brain. Using whole-cell patch-clamp recordings on newborn mouse hippocampi lacking functional GABA(B) receptors (GABA(B)-Rs) and time-lapse fluorescence imaging on cultured hippocampal neurons expressing GFP-tagged brain-derived neurotrophic factor (BDNF), we found that activation of metabotropic GABA(B) receptors (GABA(B)-Rs) triggers secretion of BDNF and promotes the development of perisomatic GABAergic synapses in the newborn mouse hippocampus. Because activation of GABA(B)-Rs occurs during the characteristic ongoing physiological network-driven synaptic activity present in the developing hippocampus, our results reveal a new mechanism by which synaptic activity can modulate the development of local GABAergic synaptic connections in the developing brain.

Sonic Hedgehog Signaling Agonist (SAG) Triggers BDNF Secretion and Promotes the Maturation of GABAergic Networks in the Postnatal Rat Hippocampus.

Sonic hedgehog (Shh) signaling plays critical roles during early central nervous system development, such as neural cell proliferation, patterning of the neural tube and neuronal differentiation. While Shh signaling is still present in the postnatal brain, the roles it may play are, however, largely unknown. In particular, Shh signaling components are found at the synaptic junction in the maturing hippocampus during the first two postnatal weeks. This period is characterized by the presence of ongoing spontaneous synaptic activity at the cellular and network levels thought to play important roles in the onset of neuronal circuit formation and synaptic plasticity. Here, we demonstrate that non-canonical Shh signaling increases the frequency of the synchronized electrical activity called Giant Depolarizing Potentials (GDP) and enhances spontaneous GABA post-synaptic currents in the rodent hippocampus during the early postnatal period. This effect is mediated specifically through the Shh co-receptor Smoothened via intracellular Ca2+ signal and the activation of the BDNF-TrkB signaling pathway. Given the importance of these spontaneous events on neuronal network maturation and refinement, this study opens new perspectives for Shh signaling on the control of early stages of postnatal brain maturation and physiology.

Leptin down-regulates KCC2 activity and controls chloride homeostasis in the neonatal rat hippocampus.

The canonical physiological role of leptin is to regulate hunger and satiety acting on specific hypothalamic nuclei. Beyond this key metabolic function; leptin also regulates many aspects of development and functioning of neuronal hippocampal networks throughout life. Here we show that leptin controls chloride homeostasis in the developing rat hippocampus in vitro. The effect of leptin relies on the down-regulation of the potassium/chloride extruder KCC2 activity and is present during a restricted period of postnatal development. This study confirms and extends the role of leptin in the ontogenesis of functional GABAergic inhibition and helps understanding how abnormal levels of leptin may contribute to neurological disorders.

Developmental Switch of Leptin Action on Network Driven Activity in the Neonatal Rat Hippocampus.

The adipose-derived circulating hormone leptin plays a pivotal role in the control of energy balance and body weight. Sound data indicate that this hormone also acts as an important developmental signal impacting a number of brain regions during fetal and postnatal stages. Leptin levels surge during the two first postnatal weeks of life in rodents. This period is characterized by the presence of early network driven activity in the immature hippocampus, the so-called Giant Depolarizing Potentials (GDPs). GDPs are thought to contribute to the wiring of the hippocampal network. We therefore tested the effect of leptin on GDPs. Leptin increased GDPs frequency between the postnatal days (P) 1 and 3 via a calcium/Calmodulin-dependent kinase (CaMK) and extracellular signal-related kinase (ERK) dependent pathways. Between P6 and P7, leptin inhibited the frequency of GDPs through the activation of large conductance Ca2+ activated K+ (BK) channels driven by a phosphoinositol-3 kinase (PI3K) dependent pathway. These results show that leptin exerts a bi-directional and age-dependent control of GDPs and extends the scope of leptin's action in the developing brain.

Impaired regulation of KCC2 phosphorylation leads to neuronal network dysfunction and neurodevelopmental pathology.

KCC2 is a vital neuronal K+/Cl- cotransporter that is implicated in the etiology of numerous neurological diseases. In normal cells, KCC2 undergoes developmental dephosphorylation at Thr906 and Thr1007 We engineered mice with heterozygous phosphomimetic mutations T906E and T1007E (KCC2E/+ ) to prevent the normal developmental dephosphorylation of these sites. Immature (postnatal day 15) but not juvenile (postnatal day 30) KCC2E/+ mice exhibited altered GABAergic inhibition, an increased glutamate/GABA synaptic ratio, and greater susceptibility to seizure. KCC2E/+ mice also had abnormal ultrasonic vocalizations at postnatal days 10 to 12 and impaired social behavior at postnatal day 60. Postnatal bumetanide treatment restored network activity by postnatal day 15 but failed to restore social behavior by postnatal day 60. Our data indicate that posttranslational KCC2 regulation controls the GABAergic developmental sequence in vivo, indicating that deregulation of KCC2 could be a risk factor for the emergence of neurological pathology.

Spontaneous glutamatergic activity induces a BDNF-dependent potentiation of GABAergic synapses in the newborn rat hippocampus.

Spontaneous ongoing synaptic activity is thought to play an instructive role in the maturation of the neuronal circuits. However the type of synaptic activity involved and how this activity is translated into structural and functional changes is not fully understood. Here we show that ongoing glutamatergic synaptic activity triggers a long-lasting potentiation of gamma-aminobutyric acid (GABA) mediated synaptic activity (LLP(GABA-A)) in the developing rat hippocampus. LLP(GABA-A) induction requires (i) the activation of AMPA receptors and L-type voltage-dependent calcium channels, (ii) the release of endogenous brain-derived neurotrophic factor (BDNF), and (iii) the activation of postsynaptic tropomyosin-related kinase receptors B (TrkB). We found that spontaneous glutamatergic activity is required to maintain a high level of native BDNF in the newborn rat hippocampus and that application of exogenous BDNF induced LLP(GABA-A) in the absence of glutamatergic activity. These results suggest that ongoing glutamatergic synaptic activity plays a pivotal role in the functional maturation of hippocampal GABAergic synapses by means of a cascade involving BDNF release and downstream signalling through postsynaptic TrkB receptor activation.

The adipocyte hormone leptin sets the emergence of hippocampal inhibition in mice.

Brain computations rely on a proper balance between excitation and inhibition which progressively emerges during postnatal development in rodent. γ-Aminobutyric acid (GABA) neurotransmission supports inhibition in the adult brain but excites immature rodent neurons. Alterations in the timing of the GABA switch contribute to neurological disorders, so unveiling the involved regulators may be a promising strategy for treatment. Here we show that the adipocyte hormone leptin sets the tempo for the emergence of GABAergic inhibition in the newborn rodent hippocampus. In the absence of leptin signaling, hippocampal neurons show an advanced emergence of GABAergic inhibition. Conversely, maternal obesity associated with hyperleptinemia delays the excitatory to inhibitory switch of GABA action in offspring. This study uncovers a developmental function of leptin that may be linked to the pathogenesis of neurological disorders and helps understanding how maternal environment can adversely impact offspring brain development.

Leptin potentiates GABAergic synaptic transmission in the developing rodent hippocampus.

It is becoming increasingly clear that leptin is not only a hormone regulating energy homeostasis but also a neurotrophic factor impacting a number of brain regions, including the hippocampus. Although leptin promotes the development of GABAergic transmission in the hypothalamus, little is known about its action on the GABAergic system in the hippocampus. Here we show that leptin modulates GABAergic transmission onto developing CA3 pyramidal cells of newborn rats. Specifically, leptin induces a long-lasting potentiation (LLP-GABAA) of miniature GABAA receptor-mediated postsynaptic current (GABAA-PSC) frequency. Leptin also increases the amplitude of evoked GABAA-PSCs in a subset of neurons along with a decrease in the coefficient of variation and no change in the paired-pulse ratio, pointing to an increased recruitment of functional synapses. Adding pharmacological blockers to the recording pipette showed that the leptin-induced LLP-GABAA requires postsynaptic calcium released from internal stores, as well as postsynaptic MAPK/ERK kinases 1 and/or 2 (MEK1/2), phosphoinositide 3 kinase (PI3K) and calcium-calmodulin kinase kinase (CaMKK). Finally, study of CA3 pyramidal cells in leptin-deficient ob/ob mice revealed a reduction in the basal frequency of miniature GABAA-PSCs compared to wild type littermates. In addition, presynaptic GAD65 immunostaining was reduced in the CA3 stratum pyramidale of mutant animals, both results converging to suggest a decreased number of functional GABAergic synapses in ob/ob mice. Overall, these results show that leptin potentiates and promotes the development of GABAergic synaptic transmission in the developing hippocampus likely via an increase in the number of functional synapses, and provide insights into the intracellular pathways mediating this effect. This study further extends the scope of leptin's neurotrophic action to a key regulator of hippocampal development and function, namely GABAergic transmission.

Early sequential formation of functional GABA(A) and glutamatergic synapses on CA1 interneurons of the rat foetal hippocampus.

During postnatal development of CA1 pyramidal neurons, GABAergic synapses are excitatory and established prior to glutamatergic synapses. As interneurons are generated before pyramidal cells, we have tested the hypothesis that the GABAergic interneuronal network is operative before glutamate pyramidal neurons and provides the initial patterns of activity. We patch-clamp recorded interneurons in foetal (69 neurons) and neonatal P0 (162 neurons) hippocampal slices and performed a morphofunctional analysis of biocytin-filled neurons. At P0, three types of interneurons were found: (i) non-innervated "silent" interneurons (5%) with no spontaneous or evoked synaptic currents; (ii) G interneurons (17%) with GABA(A) synapses only; and (iii) GG interneurons with GABA and glutamatergic synapses (78%). Relying on the neuronal capacitance, cell body size and arborization of dendrites and axons, the three types of interneurons correspond to three stages of development with non-innervated neurons and interneurons with GABA(A) and glutamatergic synapses being, respectively, the least and the most developed. Recordings from both pyramidal neurons and interneurons in foetuses (E18-20) revealed that the majority of interneurons (65%) had functional synapses whereas nearly 90% of pyramidal neurons were quiescent. Therefore, interneurons follow the same GABA-glutamate sequence of synapse formation but earlier than the principal cells. Interneurons are the source and the target of the first synapses formed in the hippocampus and are thus in a position to modulate the development of the hippocampus in the foetal stage.

Effects of antiepileptic drugs on refractory seizures in the intact immature corticohippocampal formation in vitro.

PURPOSE: We developed a new in vitro preparation of immature rats, in which intact corticohippocampal formations (CHFs) depleted in magnesium ions become progressively epileptic. The better to characterize this model, we examined the effects of 14 antiepileptic drugs (AEDs) currently used in clinical practice. METHODS: Recurrent ictal-like seizures (ILEs, four per hour) were generated in intact CHFs of P7-8 rats, and extracellular recordings were performed in the hippocampus and neocortex. AEDs were applied at clinically relevant concentrations (at least two), during 30 min after the third ILE. Their ability to prevent or to delay the next ILE was examined. RESULTS: Valproic acid and benzodiazepines (clobazam and midazolam) but also phenobarbital and levetiracetam prevent the occurrence of seizures. In contrast, usual concentrations of carbamazepine (CBZ), phenytoin, vigabatrin, tiagabine, gabapentin, lamotrigine (LTG), topiramate, felbamate, and ethosuximide did not suppress ILEs. In addition, LTG and CBZ aggravate seizures in one third of the cases. CONCLUSIONS: This intact in vitro preparation in immature animals appears to be quite resistant to most AEDs. Blockade of seizures was achieved with drugs acting mainly at the gamma-aminobutyric acid (GABA)A-receptor site but not with those that increase the amount of GABA. Drugs with a broad spectrum of activity are efficient but not those preferentially used in partial seizures or absences. We suggest that this preparation may correspond to a model of epilepsy with generalized convulsive seizures and could be helpful to develop new AEDs for refractory infantile epilepsies.