Neuronal integration in the adult mouse olfactory bulb is a non-selective addition process.

Adult neurogenesis in the olfactory bulb (OB) is considered as a competition in which neurons scramble during a critical selection period for integration and survival. Moreover, newborn neurons are thought to replace pre-existing ones that die. Despite indirect evidence supporting this model, systematic in vivo observations are still scarce. We used two-photon in vivo imaging to study neuronal integration and survival. We show that loss of new neurons in the OB after arrival at terminal positions occurs only at low levels. Moreover, long-term observations showed that no substantial cell death occurred at later stages. Neuronal death was induced by standard doses of thymidine analogs, but disappeared when low doses were used. Finally, we demonstrate that the OB grows throughout life. This shows that neuronal selection during OB-neurogenesis does not occur after neurons reached stable positions. Moreover, this suggests that OB neurogenesis does not represent neuronal turnover but lifelong neuronal addition.

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.

The membrane trafficking and functionality of the K+-Cl- co-transporter KCC2 is regulated by TGF-ß2.

Functional activation of the neuronal K(+)-Cl(-) co-transporter KCC2 (also known as SLC12A5) is a prerequisite for shifting GABAA responses from depolarizing to hyperpolarizing during development. Here, we introduce transforming growth factor ß2 (TGF-ß2) as a new regulator of KCC2 membrane trafficking and functional activation. TGF-ß2 controls membrane trafficking, surface expression and activity of KCC2 in developing and mature mouse primary hippocampal neurons, as determined by immunoblotting, immunofluorescence, biotinylation of surface proteins and KCC2-mediated Cl(-) extrusion. We also identify the signaling pathway from TGF-ß2 to cAMP-response-element-binding protein (CREB) and Ras-associated binding protein 11b (Rab11b) as the underlying mechanism for TGF-ß2-mediated KCC2 trafficking and functional activation. TGF-ß2 increases colocalization and interaction of KCC2 with Rab11b, as determined by 3D stimulated emission depletion (STED) microscopy and co-immunoprecipitation, respectively, induces CREB phosphorylation, and enhances Rab11b gene expression. Loss of function of either CREB1 or Rab11b suppressed TGF-ß2-dependent KCC2 trafficking, surface expression and functionality. Thus, TGF-ß2 is a new regulatory factor for KCC2 functional activation and membrane trafficking, and a putative indispensable molecular determinant for the developmental shift of GABAergic transmission.

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.

Knocking down of the KCC2 in rat hippocampal neurons increases intracellular chloride concentration and compromises neuronal survival.

KCC2 is a neuron-specific potassium-chloride co-transporter controlling intracellular chloride homeostasis in mature and developing neurons. It is implicated in the regulation of neuronal migration, dendrites outgrowth and formation of the excitatory and inhibitory synaptic connections. The function of KCC2 is suppressed under several pathological conditions including neuronal trauma, different types of epilepsies, axotomy of motoneurons, neuronal inflammations and ischaemic insults. However, it remains unclear how down-regulation of the KCC2 contributes to neuronal survival during and after toxic stress. Here we show that in primary hippocampal neuronal cultures the suppression of the KCC2 function using two different shRNAs, dominant-negative KCC2 mutant C568A or DIOA inhibitor, increased the intracellular chloride concentration [Cl⁻]i and enhanced the toxicity induced by lipofectamine-dependent oxidative stress or activation of the NMDA receptors. The rescuing of the KCC2 activity using over-expression of the active form of the KCC2, but not its non-active mutant Y1087D, effectively restored [Cl⁻]i and enhanced neuronal resistance to excitotoxicity. The reparative effects of KCC2 were mimicked by over-expression of the KCC3, a homologue transporter. These data suggest an important role of KCC2-dependent potassium/chloride homeostasis under neurototoxic conditions and reveal a novel role of endogenous KCC2 as a neuroprotective molecule.

Efficient transfection of DNA or shRNA vectors into neurons using magnetofection.

Efficient and long-lasting transfection of primary neurons is an essential tool for addressing many questions in current neuroscience using functional gene analysis. Neurons are sensitive to cytotoxicity and difficult to transfect with most methods. We provide a protocol for transfection of cDNA and RNA interference (short hairpin RNA (shRNA)) vectors, using magnetofection, into rat hippocampal neurons (embryonic day 18/19) cultured for several hours to 21 d in vitro. This protocol even allows double-transfection of DNA into a small subpopulation of hippocampal neurons (GABAergic interneurons), as well as achieving long-lasting expression of DNA and shRNA constructs without interfering with neuronal differentiation. This protocol, which uses inexpensive equipment and reagents, takes 1 h; utilizes mixed hippocampal cultures, a transfection reagent, CombiMag, and a magnetic plate; shows low toxicity and is suited for single-cell analysis. Modifications done by our three laboratories are detailed.

GABA neurotransmission and neural cation-chloride co-transporters: actions beyond ion transport.

During neuronal development, gamma-aminobutyric acid (GABA), which is the principal inhibitory neurotransmitter in the mature brain, exerts a paradoxical depolarizing action that plays an important role in the generation of neuronal synaptic activities in the immature cortical structures and in the formation of the neuronal network. The depolarizing action of GABA is due to a differential organization of the chloride homeostasis system; in immature neurons it maintains an elevated intracellular chloride concentration ([Cl-]i), whereas in mature neurons it keeps [Cl-]i at relatively low levels. Several recent studies have shown that the function of chloride transporters during neuronal development extends beyond the simple maintenance of chloride homeostasis and might play an active role in neuronal growth and formation of synaptic connections. In the present manuscript, we summarize such evidence and discuss the perspectives in the study of the functional role of ion transporters in determining the mode of GABA actions.

Early expression of KCC2 in rat hippocampal cultures augments expression of functional GABA synapses.

The development of GABAergic synapses is associated with an excitatory to inhibitory shift of the actions of GABA because of a reduction of [Cl-]i. This is due to a delayed postnatal expression of the K+ -Cl- cotransporter KCC2, which has low levels at birth and peaks during the first few postnatal weeks. Whether the expression of the cotransporter and the excitatory to inhibitory shift have other consequences on the operation of GABA(A) receptors and synapses is not yet known. We have now expressed KCC2 in immature neurones at an early developmental stage and determined the consequences on the formation of GABA and glutamate synapses. We report that early expression of the cotransporter selectively enhances GABAergic synapses: there is a significant increase of the density of GABA(A) receptors and synapses and an increase of the frequency of GABAergic miniature postsynaptic currents. The density of glutamate synapses and frequency of AMPA miniature postsynaptic currents are not affected. We conclude that the expression of KCC2 and the reduction of [Cl-]i play a critical role in the construction of GABAergic networks that extends beyond the excitatory to inhibitory shift of the actions of GABA.