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.

Pro-Brain-Derived Neurotrophic Factor (proBDNF)-Mediated p75NTR Activation Promotes Depolarizing Actions of GABA and Increases Susceptibility to Epileptic Seizures.

The brain-derived neurotrophic factor (BDNF) is synthesized as a precursor, namely proBDNF, which can be processed into mature BDNF (mBDNF). Evidences suggest that proBDNF signaling through p75NTR may account for the emergence of neurological disorders. These findings support the view that the relative availability of mBDNF and proBDNF forms is an important mechanism underlying brain circuit formation and cognitive functions. Here we describe novel insights into the proBDNF/p75NTR mechanisms and function in vivo in modulating neuronal circuit and synaptic plasticity during the first postnatal weeks in rats. Our results showed that increased proBDNF/p75NTR signaling during development maintains a depolarizing γ-aminobutyric acid (GABA) response in a KCC2-dependent manner in mature neuronal cells. This resulted in altered excitation/inhibition balance and enhanced neuronal network activity. The enhanced proBDNF/p75NTR signaling ultimately led to increased seizure susceptibility that was abolished by in vivo injection of function blocking p75NTR antibody. Altogether, our study shed new light on how proBDNF/p75NTR signaling can orchestrate the GABA excitatory/inhibitory developmental sequence leading to depolarizing and excitatory actions of GABA in adulthood and subsequent epileptic disorders.

Depolarizing γ-aminobutyric acid contributes to glutamatergic network rewiring in epilepsy.

OBJECTIVE: Rewiring of excitatory glutamatergic neuronal circuits is a major abnormality in epilepsy. Besides the rewiring of excitatory circuits, an abnormal depolarizing γ-aminobutyric acidergic (GABAergic) drive has been hypothesized to participate in the epileptogenic processes. However, a remaining clinically relevant question is whether early post-status epilepticus (SE) evoked chloride dysregulation is important for the remodeling of aberrant glutamatergic neuronal circuits. METHODS: Osmotic minipumps were used to infuse intracerebrally a specific inhibitor of depolarizing GABAergic transmission as well as a functionally blocking antibody toward the pan-neurotrophin receptor p75 (p75NTR ). The compounds were infused between 2 and 5 days after pilocarpine-induced SE. Immunohistochemistry for NKCC1, KCC2, and ectopic recurrent mossy fiber (rMF) sprouting as well as telemetric electroencephalographic and electrophysiological recordings were performed at day 5 and 2 months post-SE. RESULTS: Blockade of NKCC1 after SE with the specific inhibitor bumetanide restored NKCC1 and KCC2 expression, normalized chloride homeostasis, and significantly reduced the glutamatergic rMF sprouting within the dentate gyrus. This mechanism partially involves p75NTR signaling, as bumetanide application reduced SE-induced p75NTR expression and functional blockade of p75NTR decreased rMF sprouting. The early transient (3 days) post-SE infusion of bumetanide reduced rMF sprouting and recurrent seizures in the chronic epileptic phase. INTERPRETATION: Our findings show that early post-SE abnormal depolarizing GABA and p75NTR signaling fosters a long-lasting rearrangement of glutamatergic network that contributes to the epileptogenic process. This finding defines promising and novel targets to constrain reactive glutamatergic network rewiring in adult epilepsy. Ann Neurol 2017;81:251-265.

Pro-brain-derived neurotrophic factor inhibits GABAergic neurotransmission by activating endocytosis and repression of GABAA receptors.

GABA is the canonical inhibitory neurotransmitter in the CNS. This inhibitory action is largely mediated by GABA type A receptors (GABAARs). Among the many factors controlling GABAergic transmission, brain-derived neurotrophic factor (BDNF) appears to play a major role in regulating synaptic inhibition. Recent findings have demonstrated that BDNF can be released as a precursor (proBDNF). Although the role of mature BDNF on GABAergic synaptogenesis and maintenance has been well studied, an important question still unanswered is whether secreted proBDNF might affect GABAergic neurotransmission. Here, we have used 14 d in vitro primary culture of hippocampal neurons and ex vivo preparations from rats to study the function of proBDNF in regulation of GABAAR trafficking and activity. We demonstrate that proBDNF impairs GABAergic transmission by the activation of two distinct pathways: (1) a RhoA-Rock-PTEN pathway that decreases the phosphorylation levels of GABAAR, thus affecting receptor function and triggering endocytosis and degradation of internalized receptors, and (2) a JAK-STAT-ICER pathway leading to the repression of GABAARs synthesis. These effects lead to the diminution of GABAergic synapses and are correlated with a decrease in GABAergic synaptic currents. These results revealed new functions for proBDNF-p75 neurotrophin receptor signaling pathway in the control of the efficacy of GABAergic synaptic activity by regulating the trafficking and synthesis of GABAARs at inhibitory synapses.

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.

Early life oxytocin treatment improves thermo-sensory reactivity and maternal behavior in neonates lacking the autism-associated gene Magel2.

Atypical responses to sensory stimuli are considered as a core aspect and early life marker of autism spectrum disorders (ASD). Although recent findings performed in mouse ASD genetic models report sensory deficits, these were explored exclusively during juvenile or adult period. Whether sensory dysfunctions might be present at the early life stage and rescued by therapeutic strategy are fairly uninvestigated. Here we found that under cool environment neonatal mice lacking the autism-associated gene Magel2 present pup calls hypo-reactivity and are retrieved with delay by their wild-type dam. This neonatal atypical sensory reactivity to cool stimuli was not associated with autonomic thermoregulatory alteration but with a deficit of the oxytocinergic system. Indeed, we show in control neonates that pharmacogenetic inactivation of hypothalamic oxytocin neurons mimicked atypical thermosensory reactivity found in Magel2 mutants. Furthermore, pharmacological intranasal administration of oxytocin to Magel2 neonates was able to rescue both the atypical thermosensory response and the maternal pup retrieval. This preclinical study establishes for the first-time early life impairments in thermosensory integration and suggest a therapeutic potential benefit of intranasal oxytocin treatment on neonatal atypical sensory reactivity for autism.

Mechanism of GABAB receptor-induced BDNF secretion and promotion of GABAA receptor membrane expression.

Recent studies have shown that GABA(B) receptors play more than a classical inhibitory role and can function as an important synaptic maturation signal early in life. In a previous study, we reported that GABA(B) receptor activation triggers secretion of brain-derived neurotrophic factor (BDNF) and promotes the functional maturation of GABAergic synapses in the developing rat hippocampus. To identify the signalling pathway linking GABA(B) receptor activation to BDNF secretion in these cells, we have now used the phosphorylated form of the cAMP response element-binding protein as a biological sensor for endogenous BDNF release. In the present study, we show that GABA(B) receptor-induced secretion of BDNF relies on the activation of phospholipase C, followed by the formation of diacylglycerol, activation of protein kinase C, and the opening of L-type voltage-dependent Ca(2+) channels. We further show that once released by GABA(B) receptor activation, BDNF increases the membrane expression of ß(2/3) -containing GABA(A) receptors in neuronal cultures. These results reveal a novel function of GABA(B) receptors in regulating the expression of GABA(A) receptor through BDNF-tropomyosin-related kinase B receptor dependent signalling pathway.

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.

Leptin increases GABAergic synaptogenesis through the Rho guanine exchange factor ß-PIX in developing hippocampal neurons.

Developing hippocampal neurons undergo rapid synaptogenesis in response to neurotrophic signals to form and refine circuit connections. The adipokine leptin is a satiety factor with neurotrophic actions, which potentiates both glutamatergic and GABAergic synaptogenesis in the hippocampus during neonatal development. Brief exposure to leptin enhances GABAA receptor-dependent synaptic currents in hippocampal neurons. Here, using molecular and electrophysiological techniques, we found that leptin increased the surface localization of GABAA receptors and the number of functional GABAergic synapses in hippocampal cultures from male and female rat pups. Leptin increased the interaction between GABAA receptors and the Rho guanine exchange factor ß-PIX (a scaffolding protein at GABAergic postsynaptic sites) in a manner dependent on the kinase CaMKK. We also found that the leptin receptor and ß-PIX formed a complex, the amount of which transiently increased upon leptin receptor activation. Furthermore, Tyr985 in the leptin receptor and the SH3 domain of ß-PIX are crucial for this interaction, which was required for the developmental increase in GABAergic synaptogenesis. Our results suggest a mechanism by which leptin promotes GABAergic synaptogenesis in hippocampal neurons and reveal further complexity in leptin receptor signaling and its interactome.

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.

Activity-dependent dendritic secretion of brain-derived neurotrophic factor modulates synaptic plasticity.

During the last decade, a major role has emerged for brain-derived neurotrophic factor (BDNF) in the translation of intrinsic or sensory-driven synaptic activities into the neuronal network plasticity that sculpts neural circuits. BDNF is released from dendrites and axons in response to synaptic activity and modulates many aspects of synaptic function. Although the importance of BDNF in synaptic plasticity has been clearly established, direct evidence for a specific contribution of the activity-dependent dendritic secretion of BDNF has been difficult to obtain. This review summarizes recent advances that have established specific effects of postsynaptic BDNF secretion on synapse efficacy and development. We will also discuss these data in the light of their functional and pathological significance.

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.

Backpropagating action potentials trigger dendritic release of BDNF during spontaneous network activity.

Brain-derived neurotrophic factor (BDNF) is a major regulator of activity-dependent synapse development and plasticity. Because BDNF is a secreted protein, it has been proposed that BDNF is released from target neurons in an activity-dependent manner. However, direct evidence for postsynaptic release of BDNF triggered by ongoing network activity is still lacking. Here we transfected cultures of dissociated hippocampal neurons with green fluorescent protein (GFP)-tagged BDNF and combined whole-cell recording, time-lapse fluorescent imaging, and immunostaining to monitor activity-dependent dendritic release of BDNF. We found that spontaneous backpropagating action potentials, but not synaptic activity alone, led to a Ca2+-dependent dendritic release of BDNF-GFP. Moreover, we provide evidence that endogenous BDNF released from a single neuron can phosphorylate CREB (cAMP response element-binding protein) in neighboring neurons, an important step of immediate early gene activation. Therefore, together, our results support the hypothesis that BDNF might act as a target-derived messenger of activity-dependent synaptic plasticity and development.

Activity-dependent dendritic release of BDNF and biological consequences.

Network construction and reorganization is modulated by the level and pattern of synaptic activity generated in the nervous system. During the past decades, neurotrophins, and in particular brain-derived neurotrophic factor (BDNF), have emerged as attractive candidates for linking synaptic activity and brain plasticity. Thus, neurotrophin expression and secretion are under the control of activity-dependent mechanisms and, besides their classical role in supporting neuronal survival neurotrophins, modulate nearly all key steps of network construction from neuronal migration to experience-dependent refinement of local connections. In this paper, we provide an overview of recent findings showing that BDNF can serve as a target-derived messenger for activity-dependent synaptic plasticity and development at the single cell level.

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.

Mechanism of BDNF Modulation in GABAergic Synaptic Transmission in Healthy and Disease Brains.

In the mature healthy mammalian neuronal networks, γ-aminobutyric acid (GABA) mediates synaptic inhibition by acting on GABAA and GABAB receptors (GABAAR, GABABR). In immature networks and during numerous pathological conditions the strength of GABAergic synaptic inhibition is much less pronounced. In these neurons the activation of GABAAR produces paradoxical depolarizing action that favors neuronal network excitation. The depolarizing action of GABAAR is a consequence of deregulated chloride ion homeostasis. In addition to depolarizing action of GABAAR, the GABABR mediated inhibition is also less efficient. One of the key molecules regulating the GABAergic synaptic transmission is the brain derived neurotrophic factor (BDNF). BDNF and its precursor proBDNF, can be released in an activity-dependent manner. Mature BDNF operates via its cognate receptors tropomyosin related kinase B (TrkB) whereas proBDNF binds the p75 neurotrophin receptor (p75NTR). In this review article, we discuss recent finding illuminating how mBDNF-TrkB and proBDNF-p75NTR signaling pathways regulate GABA related neurotransmission under physiological conditions and during epilepsy.