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.

Neuronal carbonic anhydrase VII provides GABAergic excitatory drive to exacerbate febrile seizures.

Brain carbonic anhydrases (CAs) are known to modulate neuronal signalling. Using a novel CA VII (Car7) knockout (KO) mouse as well as a CA II (Car2) KO and a CA II/VII double KO, we show that mature hippocampal pyramidal neurons are endowed with two cytosolic isoforms. CA VII is predominantly expressed by neurons starting around postnatal day 10 (P10). The ubiquitous isoform II is expressed in neurons at P20. Both isoforms enhance bicarbonate-driven GABAergic excitation during intense GABAA-receptor activation. P13-14 CA VII KO mice show behavioural manifestations atypical of experimental febrile seizures (eFS) and a complete absence of electrographic seizures. A low dose of diazepam promotes eFS in P13-P14 rat pups, whereas seizures are blocked at higher concentrations that suppress breathing. Thus, the respiratory alkalosis-dependent eFS are exacerbated by GABAergic excitation. We found that CA VII mRNA is expressed in the human cerebral cortex before the age when febrile seizures (FS) occur in children. Our data indicate that CA VII is a key molecule in age-dependent neuronal pH regulation with consequent effects on generation of FS.

K-Cl Cotransporter 2-mediated Cl- Extrusion Determines Developmental Stage-dependent Impact of Propofol Anesthesia on Dendritic Spines.

BACKGROUND: General anesthetics potentiating γ-aminobutyric acid (GABA)-mediated signaling are known to induce a persistent decrement in excitatory synapse number in the cerebral cortex when applied during early postnatal development, while an opposite action is produced at later stages. Here, the authors test the hypothesis that the effect of general anesthetics on synaptogenesis depends upon the efficacy of GABA receptor type A (GABAA)-mediated inhibition controlled by the developmental up-regulation of the potassium-chloride (K-Cl) cotransporter 2 (KCC2). METHODS: In utero electroporation of KCC2 was used to prematurely increase the efficacy of (GABAA)-mediated inhibition in layer 2/3 pyramidal neurons in the immature rat somatosensory cortex. Parallel experiments with expression of the inward-rectifier potassium channel Kir2.1 were done to reduce intrinsic neuronal excitability. The effects of these genetic manipulations (n = 3 to 4 animals per experimental group) were evaluated using iontophoretic injection of Lucifer Yellow (n = 8 to 12 cells per animal). The total number of spines analyzed per group ranged between 907 and 3,371. RESULTS: The authors found a robust effect of the developmental up-regulation of KCC2-mediated Cl transport on the age-dependent action of propofol on dendritic spines. Premature expression of KCC2, unlike expression of a transport-inactive KCC2 variant, prevented a propofol-induced decrease in spine density. In line with a reduction in neuronal excitability, the above result was qualitatively replicated by overexpression of Kir2.1. CONCLUSIONS: The KCC2-dependent developmental increase in the efficacy of GABAA-mediated inhibition is a major determinant of the age-dependent actions of propofol on dendritic spinogenesis.

µ-Opioid Receptor-Mediated Inhibition of Intercalated Neurons and Effect on Synaptic Transmission to the Central Amygdala.

The amygdala is a key region for the processing of information underlying fear, anxiety, and fear extinction. Within the local neuronal networks of the amygdala, a population of inhibitory, intercalated neurons (ITCs) modulates the flow of information among various nuclei of amygdala, including the basal nucleus (BA) and the centromedial nucleus (CeM) of the amygdala. These ITCs have been shown to be important during fear extinction and are target of a variety of neurotransmitters and neuropeptides. Here we provide evidence that the activation of µ-opioid receptors (MORs) by the specific agonist DAMGO ([D-Ala2,N-Me-Phe4,Gly5-ol]-Enkephalin) hyperpolarizes medially located ITCs (mITCs) in acute brain slices of mice. Moreover, we use whole-cell patch-clamp recordings in combination with local electrical stimulation or glutamate uncaging to analyze the effect of MOR activation on local microcircuits. We show that the GABAergic transmission between mITCs and CeM neurons is attenuated by DAMGO, whereas the glutamatergic transmission on CeM neurons and mITCs is unaffected. Furthermore, MOR activation induced by theta burst stimulation in BA suppresses plastic changes of feedforward inhibitory transmission onto CeM neurons as revealed by the MOR antagonist CTAP d-Phe-Cys-Tyr-d-Trp-Arg-Thr-Pen-Thr-NH2. In summary, the mITCs constitute a target for the opioid system, and therefore, the activation of MOR in ITCs might play a central role in the modulation of the information processing between the basolateral complex of the amygdala and central nuclei of the amygdala.

A variant of KCC2 from patients with febrile seizures impairs neuronal Cl- extrusion and dendritic spine formation.

Genetic variation in SLC12A5 which encodes KCC2, the neuron-specific cation-chloride cotransporter that is essential for hyperpolarizing GABAergic signaling and formation of cortical dendritic spines, has not been reported in human disease. Screening of SLC12A5 revealed a co-segregating variant (KCC2-R952H) in an Australian family with febrile seizures. We show that KCC2-R952H reduces neuronal Cl(-) extrusion and has a compromised ability to induce dendritic spines in vivo and in vitro. Biochemical analyses indicate a reduced surface expression of KCC2-R952H which likely contributes to the functional deficits. Our data suggest that KCC2-R952H is a bona fide susceptibility variant for febrile seizures.

K-Cl cotransporter KCC2--a moonlighting protein in excitatory and inhibitory synapse development and function.

The K-Cl cotransporter KCC2 has two entirely independent biological actions as either an ion transporter or a structural protein orchestrating the organization of the cytoskeleton in neuronal structures. The K-Cl cotransport by KCC2 is central for hyperpolarizing inhibitory signaling, which is based on chloride currents mediated by γ-aminobutyric acid (GABA)- or glycine-gated receptor channels. In contrast, the structural role of KCC2 seems to be crucially involved in the maturation and regulation of excitatory glutamatergic synapses. This dual role at GABAergic/glycinergic and glutamatergic synapses makes KCC2 a key molecule in the regulation of inhibitory and excitatory signaling. Therefore, KCC2 is most likely involved in the synchronization of the two types of activity during network formation in the immature system and a similar synchronizing role might also be important under physiological and pathological conditions in mature neuronal networks. In this review, we explore new findings on the regulation of KCC2 by protease-mediated cleavage and on the structural role of KCC2 in spine morphogenesis and glutamate receptor clustering. We then discuss the implications of the putative interaction between the independent functions of the transporter and overlapping regulatory mechanisms in a neurophysiological context. In addition, we look at the multifunctional properties of KCC2 in the light of evolution and propose that KCC2 belongs to the group of moonlighting (multifunctional) proteins.

An ion transport-independent role for the cation-chloride cotransporter KCC2 in dendritic spinogenesis in vivo.

The neuron-specific K-Cl cotransporter, KCC2, is highly expressed in the vicinity of excitatory synapses in pyramidal neurons, and recent in vitro data suggest that this protein plays a role in the development of dendritic spines. The in vivo relevance of these observations is, however, unknown. Using in utero electroporation combined with post hoc iontophoretic injection of Lucifer Yellow, we show that premature expression of KCC2 induces a highly significant and permanent increase in dendritic spine density of layer 2/3 pyramidal neurons in the somatosensory cortex. Whole-cell recordings revealed that this increased spine density is correlated with an enhanced spontaneous excitatory activity in KCC2-transfected neurons. Precocious expression of the N-terminal deleted form of KCC2, which lacks the chloride transporter function, also increased spine density. In contrast, no effect on spine density was observed following in utero electroporation of a point mutant of KCC2 (KCC2-C568A) where both the cotransporter function and the interaction with the cytoskeleton are disrupted. Transfection of the C-terminal domain of KCC2, a region involved in the interaction with the dendritic cytoskeleton, also increased spine density. Collectively, these results demonstrate a role for KCC2 in excitatory synaptogenesis in vivo through a mechanism that is independent of its ion transport function.

Activity-dependent cleavage of the K-Cl cotransporter KCC2 mediated by calcium-activated protease calpain.

The K-Cl cotransporter KCC2 plays a crucial role in neuronal chloride regulation. In mature central neurons, KCC2 is responsible for the low intracellular Cl(-) concentration ([Cl(-)](i)) that forms the basis for hyperpolarizing GABA(A) receptor-mediated responses. Fast changes in KCC2 function and expression have been observed under various physiological and pathophysiological conditions. Here, we show that the application of protein synthesis inhibitors cycloheximide and emetine to acute rat hippocampal slices have no effect on total KCC2 protein level and K-Cl cotransporter function. Furthermore, blocking constitutive lysosomal degradation with leupeptin did not induce significant changes in KCC2 protein levels. These findings indicate a low basal turnover rate of the total KCC2 protein pool. In the presence of the glutamate receptor agonist NMDA, the total KCC2 protein level decreased to about 30% within 4 h, and this effect was blocked by calpeptin and MDL-28170, inhibitors of the calcium-activated protease calpain. Interictal-like activity induced by incubation of hippocampal slices in an Mg(2+)-free solution led to a fast reduction in KCC2-mediated Cl(-) transport efficacy in CA1 pyramidal neurons, which was paralleled by a decrease in both total and plasmalemmal KCC2 protein. These effects were blocked by the calpain inhibitor MDL-28170. Taken together, these findings show that calpain activation leads to cleavage of KCC2, thereby modulating GABAergic signaling.

KCC2 interacts with the dendritic cytoskeleton to promote spine development.

The neuron-specific K-Cl cotransporter, KCC2, induces a developmental shift to render GABAergic transmission from depolarizing to hyperpolarizing. Now we demonstrate that KCC2, independently of its Cl(-) transport function, is a key factor in the maturation of dendritic spines. This morphogenic role of KCC2 in the development of excitatory synapses is mediated by structural interactions between KCC2 and the spine cytoskeleton. Here, the binding of KCC2 C-terminal domain to the cytoskeleton-associated protein 4.1N may play an important role. A more general conclusion based on our data is that KCC2 acts as a synchronizing factor in the functional development of glutamatergic and GABAergic synapses in cortical neurons and networks.

A single seizure episode leads to rapid functional activation of KCC2 in the neonatal rat hippocampus.

Functional expression of the K-Cl cotransporter KCC2 in developing central neurons is crucial for the maturation of Cl(-)-dependent, GABA(A) receptor-mediated inhibitory responses. In pyramidal neurons of the rodent hippocampus, GABAergic postsynaptic responses are typically depolarizing and often excitatory during the first postnatal week. Here, we show that a single neonatal seizure episode induced by kainate injection during postnatal days 5-7 results in a fast increase in the Cl(-) extrusion capacity of rat hippocampal CA1 neurons, with a consequent hyperpolarizing shift of the reversal potential of GABA(A)-mediated currents (E(GABA)). A significant increase in the surface expression of KCC2 as well as the alpha2 subunit of the Na-K-ATPase parallels the seizure-induced increase in the Cl(-) extrusion capacity. Exposing hippocampal slices to kainate resulted in a similar increase in the neuronal Cl(-) extrusion and in the surface expression of KCC2. Both effects were blocked by the kinase inhibitor K252a. Hence, in the neonatal hippocampus the overall KCC2 expression level is high enough to promote a rapid functional activation of K-Cl cotransport and a consequent negative shift in E(GABA) close to the adult level. The activity-dependent regulation of KCC2 function and its effect on GABAergic transmission may represent an intrinsic antiepileptogenic mechanism.

Human-Specific Neuropeptide S Receptor Variants Regulate Fear Extinction in the Basal Amygdala of Male and Female Mice Depending on Threat Salience.

BACKGROUND: A nonsynonymous single nucleotide polymorphism in the neuropeptide S receptor 1 (NPSR1) gene (rs324981) results in isoleucine-to-asparagine substitution at amino acid 107. In humans, the ancestral variant (NPSR1 I107) is associated with increased anxiety sensitivity and risk of panic disorder, while the human-specific variant (NPSR1 N107) is considered protective against excessive anxiety. In rodents, neurobiological constituents of the NPS system have been analyzed in detail and their anxiolytic-like effects have been endorsed. However, their implication for anxiety and related disorders in humans remains unclear, as rodents carry only the ancestral NPSR1 I107 variant. METHODS: We hypothesized that phenotypic correlates of NPSR1 variants manifest in fear-related circuits in the amygdala. We used CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/Cas9)-mediated gene editing to generate a "humanized" mouse strain, in which individuals express either NPSR1 I107 or NPSR1 N107. RESULTS: Stimulation of NPSR1 evoked excitatory responses in principal neurons of the anterior basal amygdala with significant differences in magnitude between genotypes, resulting in synaptic disinhibition of putative extinction neurons in the posterior basal amygdala in mice expressing the human-specific hypofunctional N107 but not the ancestral I107 variant. N107 mice displayed improved extinction of conditioned fear, which was phenocopied after pharmacological antagonism of NPSR1 in the anterior basal amygdala of I107 mice. Differences in fear extinction between male and female mice were related to an interaction of Npsr1 genotype and salience of fear training. CONCLUSIONS: The NPS system regulates extinction circuits in the amygdala depending on the Npsr1 genotype, contributing to sex-specific differences in fear extinction and high anxiety sensitivity of individuals bearing the ancestral NPSR1 I107 variant.

Compensatory enhancement of intrinsic spiking upon NKCC1 disruption in neonatal hippocampus.

Depolarizing and excitatory GABA actions are thought to be important in cortical development. We show here that GABA has no excitatory action on CA3 pyramidal neurons in hippocampal slices from neonatal NKCC1(-/-) mice that lack the Na-K-2Cl cotransporter isoform 1. Strikingly, NKCC1(-/-) slices generated endogenous network events similar to giant depolarizing potentials (GDPs), but, unlike in wild-type slices, the GDPs were not facilitated by the GABA(A) agonist isoguvacine or blocked by the NKCC1 inhibitor bumetanide. The developmental upregulation of the K-Cl cotransporter 2 (KCC2) was unperturbed, whereas the pharmacologically isolated glutamatergic network activity and the intrinsic excitability of CA3 pyramidal neurons were enhanced in the NKCC1(-/-) hippocampus. Hence, developmental expression of KCC2, unsilencing of AMPA-type synapses, and early network events can take place in the absence of excitatory GABAergic signaling in the neonatal hippocampus. Furthermore, we show that genetic as well as pharmacologically induced loss of NKCC1-dependent excitatory actions of GABA results in a dramatic compensatory increase in the intrinsic excitability of glutamatergic neurons, pointing to powerful homeostatic regulation of neuronal activity in the developing hippocampal circuitry.

Premature expression of KCC2 in embryonic mice perturbs neural development by an ion transport-independent mechanism.

During neuronal maturation, the neuron-specific K-Cl co-transporter KCC2 lowers the intracellular chloride and thereby renders GABAergic transmission hyperpolarizing. Independently of its role as a co-transporter, KCC2 plays a crucial role in the maturation of dendritic spines, most probably via an interaction with the cytoskeleton-associated protein 4.1N. In this study, we show that neural-specific overexpression of KCC2 impairs the development of the neural tube- and neural crest-related structures in mouse embryos. At early stages (E9.5-11.5), the transgenic embryos had a thinner neural tube and abnormal body curvature. They displayed a reduced neuronal differentiation and altered neural crest cell pattern. At later stages (E11.5-15.5), the transgenic embryos had smaller brain structures and a distinctive cleft palate. Similar results were obtained using overexpression of a transport-inactive N-terminal-deleted variant of KCC2, implying that the effects were not dependent on KCC2's role as a K-Cl co-transporter. Interestingly, the neural tube of transgenic embryos had an aberrant cytoplasmic distribution of 4.1N and actin. This was corroborated in a neural stem cell line with ectopic expression of KCC2. Embryo phenotype and cell morphology were unaffected by a mutated variant of KCC2 which is unable to bind 4.1N. These results point to a role of KCC2 in neuronal differentiation and migration during early development mediated by its direct structural interactions with the neuronal cytoskeleton.

The µ-opioid system in midline thalamic nuclei modulates defence strategies towards a conditioned fear stimulus in male mice.

BACKGROUND: Nuclei located in the dorsal midline thalamus, such as the paraventricular nucleus of the thalamus (PVT), are crucial to modulate fear and aversive behaviour. In addition, the PVT shows a dense expression of µ-opioid receptors (MORs) and could mediate the anxiolytic effects of opioids. METHODS: We analysed the contribution of MORs in the dorsal midline thalamus (i.e. the PVT) to the performance of mice in a classical fear conditioning paradigm. We locally injected a specific agonist (DAMGO), an antagonist (CTAP) of MOR or saline as a control into the dorsal midline thalamus of male mice, prior to fear extinction training. We assessed freezing as a typical measure of fear and extended our analysis by evaluation of aversive, non-aversive and neutral behavioural features using compositional data analysis. RESULTS: Pharmacological blockade of MORs through CTAP in the dorsal midline thalamus induced a fear memory extinction deficit, as evidenced by maintained freezing during extinction sessions. Stimulation of MORs by DAMGO resulted in an overall increase in locomotor activity, associated with decreased freezing during recall of extinction. Compositional data analysis confirmed the freezing-related pharmacological effects and revealed specific differences in basic behavioural states. CTAP-treated mice remained in an aversive state, whereas DAMGO-treated mice displayed predominantly neutral behaviour. CONCLUSIONS: Fear extinction requires the integrity of the µ-opioid system in the dorsal midline thalamus. Pharmacological stimulation of MOR and associated facilitation of fear extinction recall suggest a potential therapeutic avenue for stress-related or anxiety disorders.

Opposite effect of membrane raft perturbation on transport activity of KCC2 and NKCC1.

In the majority of neurons, the intracellular Cl(-) concentration is set by the activity of the Na(+)-K(+)-2Cl(-) cotransporter (NKCC1) and the K(+)-Cl(-) cotransporter (KCC2). Here, we investigated the cotransporters' functional dependence on membrane rafts. In the mature rat brain, NKCC1 was mainly insoluble in Brij 58 and co-distributed with the membrane raft marker flotillin-1 in sucrose density flotation experiments. In contrast, KCC2 was found in the insoluble fraction as well as in the soluble fraction, where it co-distributed with the non-raft marker transferrin receptor. Both KCC2 populations displayed a mature glycosylation pattern. Disrupting membrane rafts with methyl-beta-cyclodextrin (MbetaCD) increased the solubility of KCC2, yet had no effect on NKCC1. In human embryonic kidney-293 cells, KCC2 was strongly activated by a combined treatment with MbetaCD and sphingomyelinase, while NKCC1 was inhibited. These data indicate that membrane rafts render KCC2 inactive and NKCC1 active. In agreement with this, inactive KCC2 of the perinatal rat brainstem largely partitioned into membrane rafts. In addition, the exposure of the transporters to MbetaCD and sphingomyelinase showed that the two transporters differentially interact with the membrane rafts. Taken together, membrane raft association appears to represent a mechanism for co-ordinated regulation of chloride transporter function.

Differential regulation of chloride homeostasis and GABAergic transmission in the thalamus.

The thalamus is important for sensory integration with the ventrobasal thalamus (VB) as relay controlled by GABAergic projections from the nucleus reticularis thalami (NRT). Depending on the [Cl-]i primarily set by cation-chloride-cotransporters, GABA is inhibitory or excitatory. There is evidence that VB and NRT differ in terms of GABA action, with classical hyperpolarization in VB due to the expression of the Cl- extruder KCC2 and depolarizing/excitatory GABA action in the NRT, where KCC2 expression is low and Cl- accumulation by the Cl- inward transporter NKCC1 has been postulated. However, data on NKCC1 expression and functional analysis of both transporters are missing. We show that KCC2-mediated Cl- extrusion set the [Cl-]i in VB, while NKCC1 did not contribute substantially to Cl- accumulation and depolarizing GABA action in the NRT. The finding that NKCC1 did not play a major role in NRT neurons is of high relevance for ongoing studies on the therapeutic use of NKCC1 inhibitors trying to compensate for a disease-induced up-regulation of NKCC1 that has been described for various brain regions and disease states like epilepsy and chronic pain. These data suggest that NKCC1 inhibitors might have no major effect on healthy NRT neurons due to limited NKCC1 function.

Physiological Profile of Neuropeptide Y-Expressing Neurons in Bed Nucleus of Stria Terminalis in Mice: State of High Excitability.

Both, the anterior bed nucleus of the stria terminalis (BNST) and the neuropeptide Y (NPY) system are involved in shaping fear and defensive responses that adapt the organism to potentially life-threatening conditions. NPY is expressed in the BNST but NPY-expressing neurons in this critical hub in the stress response network have not been addressed before. Therefore, we performed whole-cell patch-clamp recordings in acute slices of anterior BNST from Npy-hrGFP transgenic mice to identify and characterize NPY-expressing neurons. We show that NPY-positive and NPY-negative neurons in anterior BNST match the previous classification scheme of type I (Regular Spiking), type II (Low-Threshold Bursting), and type III (fast Inward Rectifying) cells, although the proportion of these physiological phenotypes was similar within both neuronal subpopulations. However, NPY-positive and NPY-negative neurons possessed distinct intrinsic electrophysiological properties. NPY-positive neurons displayed higher input resistance and lower membrane capacitance, corresponding to small cell bodies and shorter less ramified dendrites, as compared to their NPY-negative counterparts. Furthermore, NPY-positive neurons generated higher frequent series of action potentials upon membrane depolarization and displayed significantly lower GABAA receptor-mediated synaptic responsiveness during evoked, spontaneous, and elementary synaptic activity. Taken together, these properties indicate an overall state of high excitability in NPY-positive neurons in anterior BNST. In view of the role of the anterior BNST in anxiety- and stress-related behaviors, these findings suggest a scenario where NPY-positive neurons are preferentially active and responsive to afferent inputs, thereby contributing to adaptation of the organism to stressful environmental encounters.

Oligomerization of KCC2 correlates with development of inhibitory neurotransmission.

The neuron-specific K+-Cl- cotransporter KCC2 extrudes Cl- and renders GABA and glycine action hyperpolarizing. Thus, it plays a pivotal role in neuronal inhibition. Development-dependent KCC2 activation is regulated at the transcriptional level and by unknown posttranslational mechanisms. Here, we analyzed KCC2 activation at the protein level in the developing rat lateral superior olive (LSO), a prominent auditory brainstem structure. Electrophysiology demonstrated ineffective KCC2-mediated Cl- extrusion in LSO neurons at postnatal day 3 (P3). Immunohistochemical analyses by confocal and electron microscopy revealed KCC2 signals at the plasma membrane in the somata and dendrites of both immature and mature neurons. Biochemical analysis demonstrated mature glycosylation pattern of KCC2 at both stages. Immunoblot analysis of the immature brainstem demonstrated mainly monomeric KCC2. In contrast, three KCC2 oligomers with molecular masses of approximately 270, approximately 400, and approximately 500 kDa were identified in the mature brainstem. These oligomers were sensitive to sulfhydryl-reducing agents and resistant to SDS, contrary to the situation seen in the related Na+-(K+)-Cl- cotransporter. In HEK-293 cells, coexpressed hemagglutinin-tagged KCC2 assembled with histidine-tagged KCC2, demonstrating formation of homomers. Based on these findings, we conclude that the oligomers represent KCC2 dimers, trimers, and tetramers. Finally, immunoblot analysis identified a development-dependent increase in the oligomer/monomer ratio from embryonic day 18 to P30 throughout the brain that correlates with KCC2 activation. Together, our data indicate that the developmental shift from depolarization to hyperpolarization can be determined by both increased gene expression and KCC2 oligomerization.

Dynorphin-Dependent Reduction of Excitability and Attenuation of Inhibitory Afferents of NPS Neurons in the Pericoerulear Region of Mice.

The Neuropeptide S system, consisting of the 20-amino acid peptide neuropeptide S (NPS) and its G-protein coupled receptor (NPSR), modulates arousal, wakefulness, anxiety, and fear-extinction in mice. In addition, recent evidence indicates that the NPS system attenuates stress-dependent impairment of fear extinction, and that NPS-expressing neurons in close proximity to the locus coeruleus region (LC; pericoerulear, periLC) are activated by stress. Furthermore, periLC NPS neurons receive afferents from neurons of the centrolateral nucleus of the amygdala (CeL), of which a substantial population expresses the kappa opioid receptor (KOR) ligand precursor prodynorphin. This study aims to identify the effect of the dynorphinergic system on NPS neurons in the periLC via pre- and postsynaptic mechanisms. Using electrophysiological recordings in mouse brain slices, we provide evidence that NPS neurons in the periLC region are directly inhibited by dynorphin A (DynA) via activation of κ-opioid receptor 1 (KOR1) and a subsequent increase of potassium conductances. Thus, the dynorphinergic system is suited to inactivate NPS neurons in the periLC. In addition to this direct, somatic effect, DynA reduces the efficacy of GABAergic synapses on NPS neurons via KOR1 and KOR2. In conclusion, the present study provides evidence for the interaction of the NPS and the kappa opioid system in the periLC. Therefore, the endogenous opioid dynorphin is suited to inhibit NPS neurons with a subsequent decrease in NPS release in putative target regions leading to a variety of physiological consequences such as increased anxiety or vulnerability to stress exposure.

BDNF is required for seizure-induced but not developmental up-regulation of KCC2 in the neonatal hippocampus.

A robust increase in the functional expression of the neuronal K-Cl cotransporter KCC2 during CNS development is necessary for the emergence of hyperpolarizing ionotropic GABAergic transmission. BDNF-TrkB signaling has been implicated in the developmental up-regulation of KCC2 and, in mature animals, in fast activity-dependent down-regulation of KCC2 function following seizures and trauma. In contrast to the decrease in KCC2 expression observed in the adult hippocampus following trauma, seizures in the neonate trigger a TrkB-dependent up-regulation of neuronal Cl(-) extrusion capacity associated with enhanced surface expression of KCC2. Here, we show that this effect is transient, and impaired in the hippocampus of Bdnf(-/-) mice. Notably, however, a complete absence of BDNF does not compromise the increase in KCC2 protein or K-Cl transport functionality during neuronal development. Furthermore, we present data indicating that the functional up-regulation of KCC2 by neonatal seizures is temporally limited by calpain activity.