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.

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.

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.

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.

Enhanced Synaptic Activity and Epileptiform Events in the Embryonic KCC2 Deficient Hippocampus.

The neuronal potassium-chloride co-transporter 2 [indicated thereafter as KCC2 (for protein) and Kcc2 (for gene)] is thought to play an important role in the post natal excitatory to inhibitory switch of GABA actions in the rodent hippocampus. Here, by studying hippocampi of wild-type (Kcc2(+/+)) and Kcc2 deficient (Kcc2(-/-)) mouse embryos, we unexpectedly found increased spontaneous neuronal network activity at E18.5, a developmental stage when KCC2 is thought not to be functional in the hippocampus. Embryonic Kcc2(-/-) hippocampi have also an augmented synapse density and a higher frequency of spontaneous glutamatergic and GABA-ergic postsynaptic currents than naïve age matched neurons. However, intracellular chloride concentration ([Cl(-)](i)) and the reversal potential of GABA-mediated currents (E(GABA)) were similar in embryonic Kcc2(+/+) and Kcc2(-/-) CA3 neurons. In addition, KCC2 immunolabeling was cytoplasmic in the majority of neurons suggesting that the molecule is not functional as a plasma membrane chloride co-transporter. Collectively, our results show that already at an embryonic stage, KCC2 controls the formation of synapses and, when deleted, the hippocampus has a higher density of GABA-ergic and glutamatergic synapses and generates spontaneous and evoked epileptiform activities. These results may be explained either by a small population of orchestrating neurons in which KCC2 operates early as a chloride exporter or by transporter independent actions of KCC2 that are instrumental in synapse formation and networks construction.

Neuronal chloride accumulation and excitatory GABA underlie aggravation of neonatal epileptiform activities by phenobarbital.

Phenobarbital produces its anti-epileptic actions by increasing the inhibitory drive of γ-aminobutyric acid. However, following recurrent seizures, γ-aminobutyric acid excites neurons because of a persistent increase of chloride raising the important issue of whether phenobarbital could aggravate persistent seizures. Here we compared the actions of phenobarbital on initial and established ictal-like events in an in vitro model of mirror focus. Using the in vitro three-compartment chamber preparation with the two hippocampi and their commissural fibres placed in three different chambers, kainate was applied to one hippocampus and phenobarbital contralaterally, either after one ictal-like event or after many recurrent ictal-like events that produce an epileptogenic mirror focus. Field, perforated patch and single-channel recordings were used to determine the effects of γ-aminobutyric acid and their modulation by phenobarbital, and alterations of the chloride cotransporters were investigated using sodium-potassium-chloride cotransporter 1 and potassium chloride cotransporter 2 antagonists, potassium chloride cotransporter 2 immunocytochemistry and sodium-potassium-chloride cotransporter 1 knockouts. Phenobarbital reduced initial ictal-like events and prevented the formation of a mirror focus when applied from the start. In contrast, phenobarbital aggravated epileptiform activities when applied after many ictal-like events by enhancing the excitatory actions of γ-aminobutyric acid due to increased chloride. The accumulation of chloride and the excitatory actions of γ-aminobutyric acid in mirror foci neurons are mediated by the sodium-potassium-chloride cotransporter 1 chloride importer and by downregulation and internalization of the chloride-exporter potassium-chloride cotransporter 2. Finally, concomitant applications of the sodium-potassium-chloride cotransporter 1 antagonist bumetanide and phenobarbital decreased excitatory actions of γ-aminobutyric acid and prevented its paradoxical actions on mirror focus. Therefore, the history of seizures prior to phenobarbital applications determines its effects and rapid treatment of severe potentially epileptogenic-neonatal seizures is recommended to prevent secondary epileptogenesis associated with potassium chloride cotransporter 2 downregulation and acquisition of the excitatory γ-aminobutyric acid phenotype.

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.

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.

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.

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.

Changes in vesicular transporters for gamma-aminobutyric acid and glutamate reveal vulnerability and reorganization of hippocampal neurons following pilocarpine-induced seizures.

The reorganizations of the overall intrinsic glutamatergic and gamma-aminobutyric acid (GABA)-ergic hippocampal networks as well as the time course of these reorganizations during development of pilocarpine-induced temporal lobe epilepsy were studied with in situ hybridization and immunohistochemistry experiments for the vesicular glutamate transporter 1 (VGLUT1) and the vesicular GABA transporter (VGAT). These transporters are particularly interesting as specific markers for glutamatergic and GABAergic neurons, respectively, whose expression levels could reflect the demand for synaptic transmission and their average activity. We report that 1) concomitantly with the loss of some subpopulations of VGAT-containing neurons, there was an up-regulation of VGAT synthesis in all remaining GABA neurons as early as 1 week after pilocarpine injection. This enhanced synthesis is characterized by marked increases in the relative amount of VGAT mRNAs in interneurons associated with increased intensity of axon terminal labeling for VGAT in all hippocampal layers. 2) There was a striking loss of mossy cells during the latent period, demonstrated by a long-term decrease of VGLUT1 mRNA-containing hilar neurons and associated loss of VGLUT1-containing terminals in the dentate gyrus inner molecular layer. 3) There were aberrant VGLUT1-containing terminals at the chronic stage resulting from axonal sprouting of granule and pyramidal cells. This is illustrated by a recovery of VGLUT1 immunoreactivity in the inner molecular layer and an increased VGLUT1 immunolabeling in the CA1-CA3 dendritic layers. These data indicate that an increased activity of remaining GABAergic interneurons occurs during the latent period, in parallel with the loss of vulnerable glutamatergic and GABAergic neurons preceding the reorganization of glutamatergic networks.

Cell domain-dependent changes in the glutamatergic and GABAergic drives during epileptogenesis in the rat CA1 region.

An increased ratio of the glutamatergic drive to the overall glutamatergic/GABAergic drive characterizes the chronic stage of temporal lobe epilepsy (TLE), but it is unclear whether this modification is present during the latent period that often precedes the epileptic stage. Using the pilocarpine model of TLE in rats, we report that this ratio is decreased in hippocampal CA1 pyramidal cells during the early phase of the latent period (3-5 days post pilocarpine). It is, however, increased during the late phase of the latent period (7-10 days post pilocarpine), via cell domain-dependent alterations in synaptic current properties, concomitant with the occurrence of interictal-like activity in vivo. During the late latent period, the glutamatergic drive was increased in somata via an enhancement in EPSC decay time constant and in dendrites via an increase in EPSC frequency and amplitude. The GABAergic drive remained unchanged in the soma but was decreased in dendrites, since the drop off in IPSC frequency was more marked than the increase in IPSC kinetics. Theoretical considerations suggest that these modifications are sufficient to produce interictal-like activity. In epileptic animals, the ratio of the glutamatergic drive to the overall synaptic drive was not further modified, despite additional changes in synaptic current frequency and kinetics. These results show that the global changes to more glutamatergic and less GABAergic activities in the CA1 region precede the chronic stage of epilepsy, possibly facilitating the occurrence and/or the propagation of interictal activity.

Spontaneous synaptic activity is required for the formation of functional GABAergic synapses in the developing rat hippocampus.

Here we examine the role of the spontaneous synaptic activity generated by the developing rat hippocampus in the formation of functional gamma-aminobutyric acid (GABA) synapses. Intact hippocampal formations (IHFs) were dissected at birth and incubated for 1 day in control or tetrodotoxin (TTX)-supplemented medium at 25 degrees C. After the incubation, miniature GABA(A)-mediated postsynaptic currents (mGABA(A)-PSCs) were recorded in whole-cell voltage-clamped CA3 pyramidal neurones from IHF-derived slices. After 1 day in vitro in control medium, the frequency of mGABA(A)-PSCs was similar to that recorded in acute slices obtained 1 day after birth, but significantly higher than the frequency recorded from acute slices just after birth. These results suggest that the factors required in vivo for the formation of functional GABAergic synapses are preserved in the IHFs in vitro. The frequency increase was prevented when IHFs were incubated for 1 day with TTX. TTX treatment affected neither the morphology of CA3 pyramidal neurones nor cell viability. The TTX effects were reproduced when IHFs were incubated in the presence of glutamatergic or GABAergic ionotropic receptor antagonists or in high divalent cationic medium. The present results indicate that the spontaneous synaptic activity generated by the developing hippocampus is a key player in the formation of functional GABAergic synapses, possibly via network events requiring both glutamatergic and GABAergic receptors.

GABA and glycine co-release optimizes functional inhibition in rat brainstem motoneurons in vitro.

Whole-cell patch clamp recordings of miniature inhibitory postsynaptic currents (mIPSCs) were obtained in identified abducens motoneurons (aMns) from young rats (P5-P13). Three types of mIPSC were distinguished according to their kinetics and their sensitivity to receptor antagonists: faster decaying events mediated by glycine receptors (glyRs), slower decaying events mediated by GABA(A) receptors (GABA(A)Rs), and mIPSCs displaying two components corresponding to GABA and glycine co-release. Dual component events accounted for approximately 30 % of mIPSCs, independently of the rat's age and were also identified during evoked transmitter release. In contrast, the kinetics of glyR- and GABA(A)R-mediated mIPSCs became faster during development. Monosynaptic inhibitory postsynaptic potentials (IPSPs) were able to fully inhibit motoneuron discharge elicited by current pulses. When the GABA(A)R-mediated component or the glyR-mediated component of the IPSP was blocked, the inhibition of motoneuron firing was reduced. The 20-80 % rise time and duration of GABA(A)R-mediated IPSPs were significantly longer than those mediated by glyRs. The time window of inhibition for each component was determined using single postsynaptic action potentials elicited with various delays from the onset of the IPSP. GlyR-mediated IPSPs induced fast transient inhibition whereas GABA(A)R-mediated IPSPs induced slow sustained suppression of firing. Using a modelling approach, we found that the two components summated non-linearly. We conclude that in developing aMns, co-release of GABA and glycine determines the strength and timing of inhibition through non-linear interactions between the two components, thus optimizing inhibition of motoneuron function.