Acute neuroinflammation leads to disruption of neuronal chloride regulation and consequent hyperexcitability in the dentate gyrus.

Neuroinflammation is a salient part of diverse neurological and psychiatric pathologies that associate with neuronal hyperexcitability, but the underlying molecular and cellular mechanisms remain to be identified. Here, we show that peripheral injection of lipopolysaccharide (LPS) renders the dentate gyrus (DG) hyperexcitable to perforant pathway stimulation in vivo and increases the internal spiking propensity of dentate granule cells (DGCs) in vitro 24 h post-injection (hpi). In parallel, LPS leads to a prominent downregulation of chloride extrusion via KCC2 and to the emergence of NKCC1-mediated chloride uptake in DGCs under experimental conditions optimized to detect specific changes in transporter efficacy. These data show that acute neuroinflammation leads to disruption of neuronal chloride regulation, which unequivocally results in a loss of GABAergic inhibition in the DGCs, collapsing the gating function of the DG. The present work provides a mechanistic explanation for neuroinflammation-driven hyperexcitability and consequent cognitive disturbance.

Expression patterns of NKCC1 in neurons and non-neuronal cells during cortico-hippocampal development.

The Na-K-2Cl cotransporter NKCC1 is widely expressed in cells within and outside the brain. However, our understanding of its roles in brain functions throughout development, as well as in neuropsychiatric and neurological disorders, has been severely hindered by the lack of reliable data on its developmental and (sub)cellular expression patterns. We provide here the first properly controlled analysis of NKCC1 protein expression in various cell types of the mouse brain using custom-made antibodies and an NKCC1 knock-out validated immunohistochemical procedure, with parallel data based on advanced mRNA approaches. NKCC1 protein and mRNA are expressed at remarkably high levels in oligodendrocytes. In immature neurons, NKCC1 protein was located in the somata, whereas in adult neurons, only NKCC1 mRNA could be clearly detected. NKCC1 immunoreactivity is also seen in microglia, astrocytes, developing pericytes, and in progenitor cells of the dentate gyrus. Finally, a differential expression of NKCC1 splice variants was observed, with NKCC1a predominating in non-neuronal cells and NKCC1b in neurons. Taken together, our data provide a cellular basis for understanding NKCC1 functions in the brain and enable the identification of major limitations and promises in the development of neuron-targeting NKCC1-blockers.

Carbonic anhydrase seven bundles filamentous actin and regulates dendritic spine morphology and density.

Intracellular pH is a potent modulator of neuronal functions. By catalyzing (de)hydration of CO2 , intracellular carbonic anhydrase (CAi ) isoforms CA2 and CA7 contribute to neuronal pH buffering and dynamics. The presence of two highly active isoforms in neurons suggests that they may serve isozyme-specific functions unrelated to CO2 -(de)hydration. Here, we show that CA7, unlike CA2, binds to filamentous actin, and its overexpression induces formation of thick actin bundles and membrane protrusions in fibroblasts. In CA7-overexpressing neurons, CA7 is enriched in dendritic spines, which leads to aberrant spine morphology. We identified amino acids unique to CA7 that are required for direct actin interactions, promoting actin filament bundling and spine targeting. Disruption of CA7 expression in neocortical neurons leads to higher spine density due to increased proportion of small spines. Thus, our work demonstrates highly distinct subcellular expression patterns of CA7 and CA2, and a novel, structural role of CA7.

The Multifaceted Roles of KCC2 in Cortical Development.

KCC2, best known as the neuron-specific chloride-extruder that sets the strength and polarity of GABAergic currents during neuronal maturation, is a multifunctional molecule that can regulate cytoskeletal dynamics via its C-terminal domain (CTD). We describe the molecular and cellular mechanisms involved in the multiple functions of KCC2 and its splice variants, ranging from developmental apoptosis and the control of early network events to the formation and plasticity of cortical dendritic spines. The versatility of KCC2 actions at the cellular and subcellular levels is also evident in mature neurons during plasticity, disease, and aging. Thus, KCC2 has emerged as one of the most important molecules that shape the overall neuronal phenotype.

NKCC1, an Elusive Molecular Target in Brain Development: Making Sense of the Existing Data.

Ionotropic GABA transmission is mediated by anion (mainly Cl-)-permeable GABAA receptors (GABAARs). In immature neurons, GABA exerts depolarizing and sometimes functionally excitatory actions, based on active uptake of Cl- by the Na-K-2Cl cotransporter NKCC1. While functional evidence firmly shows NKCC1-mediated ion transport in immature and diseased neurons, molecular detection of NKCC1 in the brain has turned out to be extremely difficult. In this review, we describe the highly inconsistent data that are available on the cell type-specific expression patterns of the NKCC1 mRNA and protein in the CNS. We discuss the major technical caveats, including a lack of knock-out-controlled immunohistochemistry in the forebrain, possible effects of alternative splicing on the binding of antibodies and RNA probes, and the wide expression of NKCC1 in different cell types, which make whole-tissue analyses of NKCC1 useless for studying its neuronal expression. We also review novel single-cell RNAseq data showing that most of the NKCC1 in the adult CNS may, in fact, be expressed in non-neuronal cells, especially in glia. As future directions, we suggest single-cell NKCC1 mRNA and protein analyses and the use of genetically tagged endogenous proteins or systematically designed novel antibodies, together with proper knock-out controls, for the visualization of endogenous NKCC1 in distinct brain cell types and their subcellular compartments.

Development of inhibitory synaptic inputs on layer 2/3 pyramidal neurons in the rat medial prefrontal cortex.

Inhibitory control of pyramidal neurons plays a major role in governing the excitability in the brain. While spatial mapping of inhibitory inputs onto pyramidal neurons would provide important structural data on neuronal signaling, studying their distribution at the single cell level is difficult due to the lack of easily identifiable anatomical proxies. Here, we describe an approach where in utero electroporation of a plasmid encoding for fluorescently tagged gephyrin into the precursors of pyramidal cells along with ionotophoretic injection of Lucifer Yellow can reliably and specifically detect GABAergic synapses on the dendritic arbour of single pyramidal neurons. Using this technique and focusing on the basal dendritic arbour of layer 2/3 pyramidal cells of the medial prefrontal cortex, we demonstrate an intense development of GABAergic inputs onto these cells between postnatal days 10 and 20. While the spatial distribution of gephyrin clusters was not affected by the distance from the cell body at postnatal day 10, we found that distal dendritic segments appeared to have a higher gephyrin density at later developmental stages. We also show a transient increase around postnatal day 20 in the percentage of spines that are carrying a gephyrin cluster, indicative of innervation by a GABAergic terminal. Since the precise spatial arrangement of synaptic inputs is an important determinant of neuronal responses, we believe that the method described in this work may allow a better understanding of how inhibition settles together with excitation, and serve as basics for further modelling studies focusing on the geometry of dendritic inhibition during development.

Vasopressin excites interneurons to suppress hippocampal network activity across a broad span of brain maturity at birth.

During birth in mammals, a pronounced surge of fetal peripheral stress hormones takes place to promote survival in the transition to the extrauterine environment. However, it is not known whether the hormonal signaling involves central pathways with direct protective effects on the perinatal brain. Here, we show that arginine vasopressin specifically activates interneurons to suppress spontaneous network events in the perinatal hippocampus. Experiments done on the altricial rat and precocial guinea pig neonate demonstrated that the effect of vasopressin is not dependent on the level of maturation (depolarizing vs. hyperpolarizing) of postsynaptic GABAA receptor actions. Thus, the fetal mammalian brain is equipped with an evolutionarily conserved mechanism well-suited to suppress energetically expensive correlated network events under conditions of reduced oxygen supply at birth.

Developmental stage-dependent impact of midazolam on calbindin, calretinin and parvalbumin expression in the immature rat medial prefrontal cortex during the brain growth spurt.

BACKGROUND: Human epidemiological data suggest a link between anesthesia exposure in early postnatal life and subsequent lasting neurocognitive alterations. Understanding the underlying mechanisms of this potential association is of paramount importance in an attempt to develop protective strategies. While general anesthetics are powerful modulators of GABAergic neurotransmission, little is known about the impact of these drugs on developing GABAergic networks. Here we addressed this issue by evaluating the impact of a 6-h-long midazolam exposure on the development of calbindin-, calretinin- and parvalbumin-expressing GABAergic interneurons. METHODS: Physiological expression patterns of calbindin-, calretinin-, and parvalbumin-positive neurons as well as the impact of a 6-h-long midazolam exposure on these cell populations were evaluated in the medial prefrontal cortex of Wistar rats at defined stages of the brain growth spurt using stereological analysis. Activated caspase-3 immunohistochemistry was used to quantify apoptotic death in controls and midazolam-treated subjects. RESULTS: In control animals, the number of parvalbumin expressing cells significantly (p<0.01) increased while those of calbindin positive populations significantly (p<0.01) decreased between postnatal day 10 and 20. Expression of calretinin remained constant during this period. Immediately following exposure, midazolam induced neuroapoptosis at both early (postnatal day 5, p=0.016) and later (postnatal day 15, p=0.025) stages of brain development. While this did not diminish overall neuronal density in the medial prefrontal cortex, exposure at P5 led to a subsequent increase in the number of parvalbumin positive neurons in lower cortical layers, and midazolam administration at P15 increased the number of both parvalbumin and calretinin expressing neurons 5 days following exposure. CONCLUSION: These observations demonstrate that midazolam exposure can impair the physiological differentiation patterns of GABAergic interneurons during the brain growth spurt. Considering the important role of GABAergic networks in neuronal physiology, these data provide us with one potential mechanism that could account for the lasting neurobehavioral and cognitive deficits observed in the context of anesthesia exposure in the early postnatal period.

Postsynaptic gephyrin clustering controls the development of adult-born granule cells in the olfactory bulb.

In adult rodent olfactory bulb, GABAergic signaling regulates migration, differentiation, and synaptic integration of newborn granule cells (GCs), migrating from the subventricular zone. Here we show that these effects depend on the formation of a postsynaptic scaffold organized by gephyrin-the main scaffolding protein of GABAergic synapses, which anchors receptors and signaling molecules to the postsynaptic density-and are regulated by the phosphorylation status of gephyrin. Using lentiviral vectors to selectively transfect adult-born GCs, we observed that overexpression of the phospho-deficient gephyrin mutant eGFP-gephyrin(S270A), which facilitates the formation of supernumerary GABAergic synapses in vitro, favors dendritic branching and the formation of transient GABAergic synapses on spines, identified by the presence of α2-GABAA Rs. In contrast, overexpression of the dominant-negative eGFP-gephyrin(L2B) (a chimera that is enzymatically active but clustering defective), curtailed dendritic growth, spine formation, and long-term survival of GCs, pointing to the essential role of gephyrin cluster formation for its function. We could exclude any gephyrin overexpression artifacts, as GCs infected with eGFP-gephyrin were comparable to those infected with eGFP alone. The opposite effects induced by the two gephyrin mutant constructs indicate that the gephyrin scaffold at GABAergic synapses orchestrates signaling cascades acting on the cytoskeleton to regulate neuronal growth and synapse formation. Specifically, gephyrin phosphorylation emerges as a novel mechanism regulating morphological differentiation and long-term survival of adult-born olfactory bulb neurons.

Pre-existing astrocytes form functional perisynaptic processes on neurons generated in the adult hippocampus.

The adult dentate gyrus produces new neurons that morphologically and functionally integrate into the hippocampal network. In the adult brain, most excitatory synapses are ensheathed by astrocytic perisynaptic processes that regulate synaptic structure and function. However, these processes are formed during embryonic or early postnatal development and it is unknown whether astrocytes can also ensheathe synapses of neurons born during adulthood and, if so, whether they play a role in their synaptic transmission. Here, we used a combination of serial-section immuno-electron microscopy, confocal microscopy, and electrophysiology to examine the formation of perisynaptic processes on adult-born neurons. We found that the afferent and efferent synapses of newborn neurons are ensheathed by astrocytic processes, irrespective of the age of the neurons or the size of their synapses. The quantification of gliogenesis and the distribution of astrocytic processes on synapses formed by adult-born neurons suggest that the majority of these processes are recruited from pre-existing astrocytes. Furthermore, the inhibition of astrocytic glutamate re-uptake significantly reduced postsynaptic currents and increased paired-pulse facilitation in adult-born neurons, suggesting that perisynaptic processes modulate synaptic transmission on these cells. Finally, some processes were found intercalated between newly formed dendritic spines and potential presynaptic partners, suggesting that they may also play a structural role in the connectivity of new spines. Together, these results indicate that pre-existing astrocytes remodel their processes to ensheathe synapses of adult-born neurons and participate to the functional and structural integration of these cells into the hippocampal network.

Glycine transporter-1 controls nonsynaptic inhibitory actions of glycine receptors in the neonatal rat hippocampus.

Although functional glycinergic synapses have not been identified in the hippocampus, neurons in this area express Cl(-) permeable extrasynaptic glycine receptors (GlyRs). In experiments on CA3 pyramidal neurons on postnatal day 0-6 rat hippocampal slices, we detected robust GlyR activity as a tonic current and as single-channel events. Glycine release was independent of neuronal activity or extracellular Ca(2+). The endogenous GlyR activity was strongly enhanced by inhibition of the glycine-transporter-1 (GlyT1). Blockade of GlyT1 also caused a profound increase in the baseline current induced by exogenous glycine. Inhibition of GlyT1 reduced the frequency of spontaneous network events known as field giant depolarizing potentials (fGDPs) and of the unit activity in the absence of synaptic transmission. This inhibitory action on fGDPs was mimicked by applying 2 µm glycine or 0.1 µm isoguvacine, a GABAA-receptor agonist. Furthermore, 2 µm glycine suppressed unit spiking in the absence of synaptic transmission. Hence, despite the well known depolarizing Cl(-) equilibrium potential of neonatal hippocampal neurons, physiologically relevant extracellular glycine concentrations can exert an inhibitory action. The present data show that, akin to GABA uptake, GlyT1 exerts a powerful modulatory action on network events in the newborn hippocampus.