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Huntington's disease (HD) is classically characterized as a movement disorder, however cognitive impairments precede the motor symptoms by ∼15 y. Based on proteomic and bioinformatic data linking the Huntingtin protein (Htt) and KCC2, which is required for hyperpolarizing GABAergic inhibition and the important role of inhibition in learning and memory, we hypothesized that aberrant KCC2 function contributes to the hippocampal-associated learning and memory deficits in HD. We discovered that Htt and KCC2 interact in the hippocampi of wild-type and R6/2-HD mice, with a decrease in KCC2 expression in the hippocampus of R6/2 and YAC128 mice. The reduced expression of the Cl-extruding cotransporter KCC2 is accompanied by an increase in the Cl-importing cotransporter NKCC1, which together result in excitatory GABA in the hippocampi of HD mice. NKCC1 inhibition by the FDA-approved NKCC1 inhibitor bumetanide abolished the excitatory action of GABA and rescued the performance of R6/2 mice on hippocampalassociated behavioral tests. [ABSTRACT FROM AUTHOR]

Currently available inducible Cre/loxP systems, despite their considerable utility in gene manipulation, have pitfalls in certain scenarios, such as unsatisfactory recombination rates and deleterious effects on physiology and behavior. To overcome these limitations, we designed a new, inducible gene-targeting system by introducing an in-frame nonsense mutation into the coding sequence of Cre recombinase (nsCre). Mutant mRNAs transcribed from nsCre transgene can be efficiently translated into full-length, functional Cre recombinase in the presence of nonsense suppressors such as aminoglycosides. In a proof-of-concept model, GABA signaling from hypothalamic neurons expressing agouti-related peptide (AgRP) was genetically inactivated within 4 d after treatment with a synthetic aminoglycoside. Disruption of GABA synthesis in AgRP neurons in young adult mice led to a dramatic loss of body weight due to reduced food intake and elevated energy expenditure; they also manifested glucose intolerance. In contrast, older mice with genetic inactivation of GABA signaling by AgRP neurons had only transient reduction of feeding and body weight; their energy expenditure and glucose tolerance were unaffected. These results indicate that GABAergic signaling from AgRP neurons plays a key role in the control of feeding and metabolism through an age-dependent mechanism. This new genetic technique will augment current tools used to elucidate mechanisms underlying many physiological and neurological processes. [ABSTRACT FROM AUTHOR]

Stressful life events increase the susceptibility to developing psychiatric disorders such as depression; however, many individuals are resilient to such negative effects of stress. Determining the neurobiology underlying this resilience is instrumental to the development of novel and more effective treatments for stress-related psychiatric disorders. GABAB receptors are emerging therapeutic targets for the treatment of stress-related disorders such as depression. These receptors are predominantly expressed as heterodimers of a GABAB(2) subunit with either a GABAB(1a) or a GABAB(1b) subunit. Here we show that mice lacking the GABAB(1b) receptor isoform are more resilient to both early-life stress and chronic psychosocial stress in adulthood, whereas mice lacking GABAB(1a) receptors are more susceptible to stress-induced anhedonia and social avoidance compared with wild-type mice. In addition, increased hippocampal expression of the GABAB(1b) receptor subunit is associated with a depression-like phenotype in the helpless H/Rouen genetic mouse model of depression. Stress resilience in GABAB(1b)-/- mice is coupled with increased proliferation and survival of newly born cells in the adult ventral hippocampus and increased stress-induced c-Fos activation in the hippocampus following early-life stress. Taken together, the data suggest that GABAB(1) receptor subunit isoforms differentially regulate the deleterious effects of stress and, thus, may be important therapeutic targets for the treatment of depression. [ABSTRACT FROM AUTHOR]

The dorsal raphe nucleus (DR) controls forebrain serotonin neurotransmission to influence emotional states. GABA neurotransmission in the DR has been implicated in regulating sleep/wake states and influencing anxiety and aggression. To gain insight into how GABA regulates DR activity, we analyzed the organization of both GABA and glutamate axons in the rat DR using a high-resolution immunofluorescence technique, array tomography, as well as EM. This analysis revealed that a third or more of GABA-containing axons are organized in synaptic triads with a glutamatergic axon and a common postsynaptic target. Electrophysiological recordings showed that GABA has the capacity to presynaptically gate glutamate release in the DR through a combination of GABA-A and GABA-B receptor-mediated effects. Thus, GABA-glutamate synaptic triads are a common feature of the network architecture of the DR with the potential to regulate excitation of the nucleus. [ABSTRACT FROM AUTHOR]

Polyamines (PAs) are ubiquitous, polycationic compounds that are essential for the growth and survival of all organisms. Although the PA-uptake system plays a key role in mammalian cancer and in plant survival, the underlying molecular mechanisms are not well understood. Here, we identified an Arabidopsis L-type amino acid transporter (LAT) family transporter, named RMV1 (resistant to methyl viologen 1), responsible for uptake of PA and its analog paraquat (PQ). The natural variation in PQ tolerance was determined in 22 Arabidopsis thaliana accessions based on the polymorphic variation of RMV1. An RMV1-GFP fusion protein localized to the plasma membrane in transformed cells. The Arabidopsis rmv1 mutant was highly resistant to PQ because of the reduction of PQ uptake activity. Uptake studies indicated that RMV1 mediates proton gradient-driven PQ transport. RMV1 overexpressing plants were hypersensitive to PA and PQ and showed elevated PA/PQ uptake activity, supporting the notion that PQ enters plant cells via a carrier system that inherently functions in PA transport. Furthermore, we demonstrated that polymorphic variation in RMV1 controls PA/PQ uptake activity. Our identification of a molecular entity for PA/PQ uptake and sensitivity provides an important clue for our understanding of the mechanism and biological significance of PA uptake. [ABSTRACT FROM AUTHOR]

Melanocortin-4 receptor (MC4R) is critical for energy homeostasis, and the paraventricular nucleus of the hypothalamus (PVN) is a key site of MC4R action. Most studies suggest that leptin regulates PVN neurons indirectly, by binding to receptors in the arcuate nucleus or ventromedial hypothalamus and regulating release of products like α-melanocyte-stimulating hormone (α-MSH), neuropeptide Y (NPY), glutamate, and GABA from firstorder neurons onto the MC4R PVN cells. Here, we investigate mechanisms underlying regulation of activity of these neurons under various metabolic states by using hypothalamic slices from a transgenic MC4R-GFP mouse to record directly from MC4R neurons. First, we show that in vivo leptin levels regulate the tonic firing rate of second-order MC4R PVN neurons, with fasting increasing firing frequency in a leptin-dependent manner. We also show that, although leptin inhibits these neurons directly at the posisynaptic membrane, α-MSH and NPY potently stimulate and inhibit the cells, respectively. Thus, in contrast with the conventional model of leptin action, the primary control of MC4R PVN neurons is unlikely to be mediated by leptin action on arcuate NPY/agouti-related protein and proopiomelanocortin neurons. We also show that the activity of MC4R PVN neurons is controlled by the constitutive activity of the MC4R and that expression of the receptor mRNA and α-MSH sensitivity are both stimulated by leptin. Thus, leptin acts multinodally on arcuate nucleus/PVN circuits to regulate energy homeostasis, with prominent mechanisms involving direct control of both membrane conductances and gene expression in the MC4R PVN neuron. [ABSTRACT FROM AUTHOR]

Neurexins (NRXs) and neuroligins are key synaptic adhesion molecules that also recruit synaptic signaling machineries. Neurexins consist of α- and β-isoforms, but how they couple synaptic transmission and adhesion to regulate activity-dependent synapse development remains unclear, in part because of poor understanding of their cell biology and regulation in the relevant neurons. Here, we examined the subaxonal localization, dynamics, and regulation of NRX1α and NRX1β in cortical perisomatic inhibitory synapses. Both isoforms are delivered to presynaptic terminals but show significant and different turnover rate at the membrane. Although NRX1α is highly diffuse along developing axons and filopodia, NRX1β is strictly anchored at terminals through binding to postsynaptic ligands. The turnover rate of NRX1β is attenuated by neural activity and presynaptic GABAB receptors. NRXs, thus, are intrinsically dynamic but are stabilized by local transmitter release. Such an activity-adjusted adhesion system seems ideally suited to rapidly explore and validate synaptic partners guided by synaptic transmission . [ABSTRACT FROM AUTHOR]

Neuregulin 1 (NRG1) is a trophic factor that acts by stimulating ErbB receptor tyrosine kinases and has been implicated in neural development and synaptic plasticity. In this study, we investigated mechanisms of its suppression of longterm potentiation (LTP) in the hippocampus. We found that NRG1 did not alter glutamatergic transmission at SC-CAl synapses but increased the GABAA receptormediated synaptic currents in CAl pyramidal cells via a presynaptic mechanism. Inhibition of GABAA receptors blocked the suppressing effect of NRG1 on LTP and prevented ecto-ErbB4 from enhancing LIP, implicating a role of GABAergic transmission. To test this hypothesis further, we generated parvalbumin (PV)-Cre;ErbB4-/- mice in which ErbB4, an NRG1 receptor in the brain, is ablated specifically in PV-positive interneurons. NRG1 was no longer able to increase inhibitory postsynaptic currents and to suppress LTP in PV-Cre; ErbB4-/- hippocampus. Accordingly, contextual fear conditioning, a hippocampus-dependent test, was impaired in PV-Cre;ErbB4-/- mice. In contrast, ablation of ErbB4 in pyramidal neurons had no effect on NRG1 regulation of hippocampal LIP or contextual fear conditioning. These results demonstrate a critical role of ErbB4 in PV-positive interneurons but not in pyramidal neurons in synaptic plasticity and support a working model that NRG1 suppresses LIP by enhancing GABA release. Considering that NRG1 and ErbB4 are susceptibility genes of schizophrenia, these observations contribute to a better understanding of how abnormal NRG1/ErbB4 signaling may be involved in the pathogenesis of schizophrenia. [ABSTRACT FROM AUTHOR]

Most forebrain GABAergic interneurons in rodents are born during embryonic development in the ganglionic eminences (GE) and migrate tangentially into the cortical plate. A subset, however, continues to be generated postnatally in the subventricular zone (SVZ). These interneurons populate the olfactory bulb (OB) reached via migration in the rostral migratory stream (RMS). Employing transgenic mice expressing EGFP in 5-HT3-positive neurons, we identified additional migratory pathways in the early postnatal brain. Time-lapse imaging experiments revealed massive migration of EGFP-positive cells from the SVZ into numerous forebrain regions, including cortex, striatum, and nucleus accumbens. The neuronal fate of the migratory EGFP-labeled cells was indicated by their doublecortin (DCX) expression. Birthdating experiments, by using 5-bromo-2'-deoxyuridine (BrdU) and retrovirus-based experiments, provided evidence that migrating neuroblasts were born in the SVZ postnatally and developed a distinct GABAergic phenotype. Our results demonstrate that the SVZ is a reservoir of GABAergic interneurons not only for the OB, but also for other cortical and subcortical areas. [ABSTRACT FROM AUTHOR]

Significant reductions in GABAergic cell numbers and/or activity have been demonstrated in the hippocampus of subjects with schizophrenia and bipolar disorder. To understand how different subpopulations of interneurons are regulated, laser microdissection and gene expression profiling have been used to "deconstruct" the trisynaptic pathway, so that subtypes of GABA cells could be defined by their location in various layers of CA3/2 and CA1. The results suggest that the cellular endophenotypes for SZ and BD may be determined by multiple factors that include unique susceptibility genes for the respective disorders and altered integration among hippocampal GABA cells with extrinsic and intrinsic afferent fiber systems. The extensive and intricate data that has come from this study has provided insights into how a complex circuit, like the trisynaptic pathway, may be regulated in human hippocampus in both health and disease. [ABSTRACT FROM AUTHOR]

When activity levels are altered over days, a network of cells is capable of recognizing this perturbation and triggering several distinct compensatory changes that should help to recover and maintain the original activity levels homeostatically. One feature commonly observed after activity blockade has been a compensatory increase in excitatory quantal amplitude. The sensing machinery that detects altered activity levels is a central focus of the field currently, but thus far it has been elusive. The vast majority of studies that reduce network activity also reduce neurotransmission. We address the possibility that reduced neurotransmission can trigger increases in quantal amplitude. In this work, we blocked glutamatergic or GABAA transmission in ovo for 2 days while maintaining relatively normal network activity. We found that reducing GABAA transmission triggered compensatory increases in both GABA and AMPA quantal amplitude in embryonic spinal motoneurons. Glutamatergic blockade had no effect on quantal amplitude. Therefore, GABA binding to the GABAA receptor appears to be a critical step in the sensing machinery for homeostatic synaptic plasticity. The findings suggest that homeostatic increases in quantal amplitude may normally be triggered by reduced levels of activity, which are sensed in the developing spinal cord by GABA, via the GABAA receptor. Therefore, GABA appears to be serving as a proxy for activity levels. [ABSTRACT FROM AUTHOR]

A study was made of the "rundown" of GABAA receptors, micro- transplanted to Xenopus oocytes from surgically resected brain tissues of patients afflicted with drug-resistant human mesial temporal lobe epilepsy (mTLE). Cell membranes, isolated from mTLE neocortex specimens, were injected into frog oocytes that rapidly incorporated functional GABAA receptors. Upon repetitive activation with GABA (1 mM), "epileptic" GABAA receptors exhibited a GABAA-current (IGABA) rundown that was significantly enhanced by Zn2+ (≤250 μM), and practically abolished by the high-affinity GABAA receptor inverse agonist SR95531 (gabazine; 2.5-25 μM). Conversely, lGABA generated by "control" GABAA receptors microtransplanted from nonepileptic temporal lobe, lesional TLE, or authoptic disease-free tissues remained stable during repetitive stimulation, even in oocytes treated with Zn2+. We conclude that rundown of mTLE epileptic receptors depends on the presence of "phasic GABAA receptors" that have low sensitivity to antagonism by Zn2+. Additionally, we found that GABAA receptors, microtransplanted from the cerebral cortex of adult rats exhibiting recurrent seizures, caused by pilocarpine-induced status epilepticus, showed greater rundown than control tissue, an event also occurring in patch-clamped rat pyramidal neurons. Rundown of epileptic rat receptors resembled that of human mTLE receptors, being enhanced by Zn2+ (40 μM) and sensitive to the antiepileptic agent levetiracetam, the neurotrophin brain-derived neurotrophic factor, and the phosphatase blocker okadaic acid. Our findings point to the rundown of GABAA receptors as a hallmark of TLE and suggest that modulating tonic and phasic mTLE GABAA receptor activity may represent a useful therapeutic approach to the disease. [ABSTRACT FROM AUTHOR]

Phasic (synaptic) and tonic (extrasynaptic) inhibition represent the two most fundamental forms of GABAA receptor-mediated transmission. Inhibitory postsynaptic currents (IPSCs) generated by GABAA receptors are typically extremely rapid synaptic events that do not last beyond a few milliseconds. Although unusually slow GABAA IPSCs, lasting for tens of milliseconds, have been observed in recordings of spontaneous events, their origin and mechanisms are not known. We show that neocortical GABAA,slow IPSCs originate from a specialized interneuron called neurogliaform cells. Compared with classical GABAA,fast IPSCs evoked by basket cells, single spikes in neurogliaform cells evoke extraordinarily prolonged GABAA responses that display tight regulation by transporters, low peak GABA concentration, unusual benzodiazepine modulation, and spillover. These results reveal a form of GABAA receptor mediated communication by a dedicated cell type that produces slow ionotropic responses with properties intermediate between phasic and tonic inhibition. [ABSTRACT FROM AUTHOR]

Phasic (synaptic) and tonic (extrasynaptic) inhibition represent the two most fundamental forms of GABAA receptor-mediated transmission. Inhibitory postsynaptic currents (IPSCs) generated by GABAA receptors are typically extremely rapid synaptic events that do not last beyond a few milliseconds. Although unusually slow GABAA IPSCs, lasting for tens of milliseconds, have been observed in recordings of spontaneous events, their origin and mechanisms are not known. We show that neocortical GABAA,slow IPSCs originate from a specialized interneuron called neurogliaform cells. Compared with classical GABAA,fast IPSCs evoked by basket cells, single spikes in neurogliaform cells evoke extraordinarily prolonged GABAA responses that display tight regulation by transporters, low peak GABA concentration, unusual benzodiazepine modulation, and spillover. These results reveal a form of GABAA receptor mediated communication by a dedicated cell type that produces slow ionotropic responses with properties intermediate between phasic and tonic inhibition. [ABSTRACT FROM AUTHOR]

The inhibitory action of glycine and GABA in adult neurons consists of both shunting incoming excitations and moving the membrane potential away from the action potential (AP) threshold. By contrast, in immature neurons, inhibitory postsynaptic potentials (IPSPs) are depolarizing; it is generally accepted that, despite their depolarizing action, these IPSPs are inhibitory because of the shunting action of the Cl- conductance increase. Here we investigated the integration of depolarizing lPSPs (dlPSPs) with excitatory inputs in the neonatal rodent spinal cord by means of both intracellular recordings from lumbar motoneurons and a simulation using the compartment model program ‘Neuron’. We show that the ability of IPSP5 to suppress suprathreshold excitatory events depends on Ecl and the location of inhibitory synapses. The depolarization outlasts the conductance changes and spreads electrotonically in the somatodendritic tree, whereas the shunting effect is restricted and local. As a consequence, dIPSPs facilitated AP generation by subthreshold excitatory events in the late phase of the response. The window of facilitation became wider as Ecl was more depolarized and started earlier as inhibitory synapses were moved away from the excitatory input. GAD65/67 immunohistochemistry demonstrated the existence of distal inhibitory synapses on motoneurons in the neonatal rodent spinal cord. This study demonstrates that small dlPSPs can either inhibit or facilitate excitatory inputs depending on timing and location. Our results raise the possibility that inhibitory synapses exert a facilitatory action on distant excitatory inputs and slight changes of Ecl may have important consequences for network processing. [ABSTRACT FROM AUTHOR]

In the olfactory bulb (OB), odorants induce oscillations in the γ range (20-80 Hz) that play an important role in the processing of sensory information. Synaptic transmission between dendrites is a major contributor to this processing. Glutamate released from mitral cell dendrites excites the dendrites of granule cells, which in turn mediate GABAergic inhibition back onto mitral cells. Although this reciprocal synapse is thought to be a key element supporting oscillatory activity, the mechanisms by which dendrodendritic inhibition induces and maintains γ oscillations remain unknown. Here, we assessed the role of the dendrodendritic inhibition, using mice lacking the GABAA receptor α1-subunit, which is specifically expressed in mitral cells but not in granule cells. The spontaneous inhibitory postsynaptic current frequency in these mutants was low and was consistent with the reduction of GABAA receptor clusters detected by immunohistochemistry. The remaining GABAA receptors in mitral cells contained the α3-subunit and supported slower decaying currents of unchanged amplitude. Overall, inhibitory-mediated interactions between mitral cells were smaller and slower in mutant than in WT mice, although the strength of sensory afferent inputs remained unchanged. Consequently, both experimental and theoretical approaches revealed slower γ oscillations in the OB network of mutant mice. We conclude, therefore, that fast oscillations in the OB circuit are strongly constrained by the precise location, subunit composition and kinetics of GABAA receptors expressed in mitral cells. [ABSTRACT FROM AUTHOR]

Neurons in the mammalian suprachiasmatic nuclei (SCN) generate daily rhythms in physiology and behavior, but it is unclear how they maintain and synchronize these rhythms in vivo. We hypothesized that parallel signaling pathways in the SCN are required to synchronize rhythms in these neurons for coherent output. We recorded firing and clock-gene expression patterns while blocking candidate signaling pathways for at least 8 days. GABAA and GABAB antagonism increased circadian peak firing rates and rhythm precision of cultured SCN neurons, but Gi/o did not impair synchrony or rhythmicity. In contrast, inhibiting Gi/o with pertussis toxin abolished rhythms in most neurons and desynchronized the population, phenocopying the loss of vasoactive intestinal polypeptide (VIP). Daily VIP receptor agonist treatment restored synchrony and rhythmicity to VIP-/- SCN cultures during continuous GABA receptor antagonism but not during Gi/o blockade. Pertussis toxin did not affect circadian cycling of the liver, suggesting that Gi/o plays a specialized role in maintaining SCN rhythmicity. We conclude that endogenous GABA controls the amplitude of SCN neuronal rhythms by reducing daytime firing, whereas Gi/o signaling suppresses nighttime firing, and it is necessary for synchrony among SCN neurons. We propose that Gi/o, not GABA activity, converges with VIP signaling to maintain and coordinate rhythms among SCN neurons. [ABSTRACT FROM AUTHOR]

Neuropathic pain is a debilitating condition affecting millions of people around the world and is defined as pain that follows a lesion or dysfunction of the nervous system. This type of pain is difficult to treat, but the novel compounds pregabalin (Lyrica) and gabapentin (Neurontin) have proven clinical efficacy. Unlike traditional analgesics such as nonsteroidal antiinflammatory drugs or narcotics, these agents have no frank antiinflammatory actions and no effect on physiological pain. Although extensive preclinical studies have led to a number of suggestions, until recently their mechanism of action has not been clearly defined. Here, we describe studies on the analgesic effects of pregabalin in a mutant mouse containing a single-point mutation within the gene encoding a specific auxiliary subunit protein (α2-δ-1) of voltage-dependent calcium channels. The mice demonstrate normal pain phenotypes and typical responses to other analgesic drugs. We show that the mutation leads to a significant reduction in the binding affinity of pregabalin in the brain and spinal cord and the loss of its analgesic efficacy. These studies show conclusively that the analgesic actions of pregabalin are mediated through the α2-δ-1 subunit of voltage-gated calcium channels and establish this subunit as a therapeutic target for pain control. [ABSTRACT FROM AUTHOR]

Allopregnanolone (ALLO) and tetrahydrodeoxycorticosterone (THDOC) are potent positive allosteric modulators of GABA action at GABAA receptors. ALLO and THDOC are synthesized in the brain from progesterone or deoxycorticosterone, respectively, by the sequential action of two enzymes: 5α-reductase (5α-R) type I and 3α-hydroxysteroid dehydrogenase (3α-HSD). This study evaluates 5α-R type 1 and 3α-HSD mRNA expression level in mouse brain by using in situ hybridization combined with glutamic acid decarboxylase 67/65, vesicular glutamate transporter 2, glial fibrillary acidic protein, and S100β immunohistochemistry. We demonstrate that 5α-R type I and 3α-HSD colocalize in cortical, hippocampal, and olfactory bulb glutamatergic principal neurons and in some output neurons of the amygdala and thalamus. Neither 5α-R type I nor 3α-HSD mRNAs are expressed in S100β- or glial fibrillary acidic protein-positive glial cells. Using glutamic acid decarboxylase 67/65 antibodies to mark GABAergic neurons, we failed to detect 5α-R type 1 and 3α-HSD in cortical and hippocampal GABAergic interneurons. However, 5α-R type 1 and 3α-HSD are significantly expressed in principal GABAergic output neurons, such as striatal medium spiny, reticular thalamic nucleus, and cerebellar Purkinje neurons. A similar distribution and cellular location of neurosteroidogenic enzymes was observed in rat brain. Taken together, these data suggest that ALLO and THDOC, which can be synthesized in principal output neurons, modulate GABA action at GABAA receptors, either with an autocrine or a paracrine mechanism or by reaching GABAA receptor intracellular sites through lateral membrane diffusion. [ABSTRACT FROM AUTHOR]

Natively unfolded phenylalanine-glycine (FG)-repeat domains are alleged to form the physical constituents of the selective barriergate in nuclear pore complexes during nucleocytoplasmic transport. Presently, the biophysical mechanism behind the selective gate remains speculative because of a lack of information regarding the nanomechanical properties of the FG domains. In this work, we have applied the atomic force microscope to measure the mechanical response of individual and clusters of FG molecules. Single-molecule force spectroscopy reveals that FG molecules are unfolded and highly flexible. To provide insight into the selective gating mechanism, an experimental platform has been constructed to study the collective behavior of surface-tethered FG molecules at the nanoscale. Measurements indicate that the collective behavior of such FG molecules gives rise to an exponentially decaying long-range steric repulsive force. This finding indicates that the molecules are thermally mobile in an extended polymer brush-like conformation. This assertion is confirmed by observing that the brush-like conformation undergoes a reversible collapse transition in less polar solvent conditions. These findings reveal how FG-repeat domains may simultaneously function as an entropic barrier and a selective trap in the near-field interaction zone of nuclear pore complexes; i.e., selective gate. [ABSTRACT FROM AUTHOR]

The mRNA levels of NKCC1, an inwardly directed Na+, K+-2Cl- cotransporter that facilitates the accumulation of intracellular Cl-, and of KCC2, an outwardly directed K+-Cl- cotransporter that extrudes Cl-, were studied in surgically resected brain specimens from drug-resistant temporal lobe (TL) epilepsy (TLE) patients. Quantitative RT-PCR analyses of the mRNAs extracted from the human TLE-associated brain regions revealed an up-regulation of NKCC1 mRNA and a down-regulation of KCC2 mRNA in the hippocampal subiculum, compared with the hippocampus proper or the TL neocortex, suggesting an abnormal transcription of Cl- transporters in the TLE subiculum. In parallel experiments, cell membranes isolated from the same TLE-associated brain regions were injected into Xenopus oocytes that rapidly incorporated human GABAA receptors into their surface membrane. The GABA currents elicited in oocytes injected with membranes from the subiculum had a more depolarized reversal potential (EGABA) compared with the hippocampus proper or the neocortex. The NKCC1 blocker bumetanide or a temperature decrease of 10°C shifted the GABA-current EGABA more negative in oocytes injected with membranes from TLE hippocampal subiculum, matching the EGABA of TL neocortex-injected oocytes. We conclude that the anomalous expression of both Cl- transporters, KCC1 and NKCC2, in TLE hippocampal subiculum probably causes altered Cl- transport in the ‘epileptic’ neurons, as revealed in the microtransplanted Xenopus oocytes, and renders GABA aberrantly ‘exciting,’ a feature that may contribute to the precipitation of epileptic seizures. [ABSTRACT FROM AUTHOR]

in adult cortical tissue, recruitment of GABAergic inhibition pre- vents the progression of synchronous population discharges to epileptic activity However, at early developmental stages, GABA is excitatory and thus unable to fulfill this role Here, we report that retrograde signaling involving endocannabinoids is responsible for the homeostatic control of synaptic transmission and the resulting network patterns in the immature hippocampus. Blockade of cannabinoid type I (CBI) receptor led to epileptic discharges, whereas overactivation of CBI reduced network activity in vivo. Endocannabinoid signaling thus is able to keep population discharge patterns within a narrow physiological time window, balancing between epilepsy on one side and sparse activity on the other, which may result in impaired developmental plasticity. Disturbing this delicate balance during pregnancy in either direction, e.g., with marijuana as a CBI agonist or with an antagonist marketed as an antiobesity drug, can have profound consequences for brain maturation even in human embryos. [ABSTRACT FROM AUTHOR]

Benzodiazepines remain widely used for the treatment of anxiety disorders despite prominent often limiting side effects including sedation, muscle relaxation, and ataxia. A compound producing a robust anxiolytic action comparable to benzodiazepines, but lacking these limiting side effects at therapeutic doses (an anxioselective agent). would represent an important advance in the treatment of generalized anxiety disorder, and perhaps other anxiety disorders. Here we report that the pyrazolo[1,5-a]-pyrimidine, ocinaplon, exhibits an anxioselective profile in both preclinical procedures and in patients with generalized anxiety disorder, the most common of the anxiety disorders. In rats, ocinaplon produces significant muscle relaxation, ataxia, and sedation only at doses >25-fold higher than the minimum effective dose (3.1 mg/kg) in the Vogel "conflict" test. This anticonflict effect is blocked by flumazenil (Ro 15-1788), indicating that like benzodiazepines, ocinaplon produces an anxiolytic action through allosteric modulation of GABAA receptors. Nonetheless, in eight recombinant GABAA receptor isoforms expressed in Xenopus oocytes, the potency and efficacy of ocinaplon to potentiate GABA responses varied with subunit composition not only in an absolute sense, but also relative to the prototypical benzodiazepine, diazepam. In a double blind, placebo controlled clinical trial, a 2-week regimen of ocinaplon (total daily dose of 180-240 mg) produced statistically significant reductions in the Hamilton rating scale for anxiety scores. In this study, the incidence of benzodiazepine-like side effects (e.g., sedation, dizziness) in ocinaplon-treated patients did not differ from placebo. These findings indicate that ocinaplon represents a unique approach both for the treatment and understanding of anxiety disorders. [ABSTRACT FROM AUTHOR]

The transcription factor Pax6 is expressed by progenitors in the ventricular zone (VZ) of dorsal telencephalon (dTel), which generate all cortical glutamatergic neurons, but not by progenitors in the medial ganglionic eminence (MGE), which generate cortical GABAergic interneurons (GABA INs), or the lateral ganglionic eminence (LGE), which generate GABA INs that normally migrate to the olfactory bulb. We show that perinatally, Pax6sey/sey mice, which lack functional Pax6 protein, have large subpial ectopias in dTel and ventral telencephalon connected by cell streams arising from an aberrant paraventricular ectopia found throughout dTel. The subpial and paraventricular ectopias and connecting cell streams are comprised of postmitotic neurons expressing markers for GABA INs characteristic of a LGE fate. Marker analyses show that dTel VZ progenitors in Pax6 mutants are progressively ventralized, acquiring expression of regulatory genes normally limited to GE progenitors; by midneurogenesis, the entire dTel VZ exhibits ventralization. This ventralization of the dTel VZ is paralleled by the expression of markers for GABA INs superficial to it, suggesting that it ectopically produces GABA INs, leading to their ectopias and a thinner cortical plate due to diminished production of glutamatergic neurons. Genetic lineage tracing demonstrates that the GABA INs comprising the ectopias are from a cortical Emx1 lineage generated in the dTeI VZ, definitively showing that dTel progenitors and progeny acquire a ventral, GE, fate in Pax6 mutants. Thus, Pax6 delimits the appropriate proliferative zone for GABA INs and regulates their numbers and distributions by repressing the ventral fates of dTel progenitors and progeny. [ABSTRACT FROM AUTHOR]

We studied the properties of GABAA receptors microtranspianted from the human temporal lobe epilepsy (TLE)-associated brain regions to Xenopus oocytes. Cell membranes, isolated from surgically resected brain specimens of drug-resistant TLE patients, were injected into frog oocytes, which rapidly incorporated human GABAA receptors, and any associated proteins, into their surface membrane. The receptors originating from different epileptic brain regions had a similar run-down but an affinity for GASA that was ≈60% lower for the subiculum receptors than for receptors issuing from the hippocampus proper or the temporal lobe neocortex. Moreover, GABA currents recorded in oocytes injected with membranes from the subiculum had a more depolarized reversal potential compared with the hippocampus proper or neocortex of the same patients. Quantitative RT-PCR analysis was performed of the GABAA receptor α1- to α5-, β1- to β3-, γ2- to γ3-, and β-subunit mRNAs. The levels of expression of the α3-, α5-, and flu- to p33-subunit mRNAs are significantly higher, with the exception of γ2- subunit whose expression is lower, in subiculum compared with neocortex specimens. Our results suggest that an abnormal GABA-receptor subunit transcription in the TIE subiculum leads to the expression of GABAA receptors with a relatively low affinity. This abnormal behavior of the subiculum GABAA receptors may contribute to epileptogenesis. [ABSTRACT FROM AUTHOR]

Cell membranes isolated from brain tissues, obtained surgically from six patients afflicted with drug-resistant temporal lobe epilepsy and from one nonepileptic patient afflicted with a cerebral oligodendroglioma, were injected into frog oocytes. By using this approach, the oocytes acquire human GABAA receptors, and we have shown previously that the "epileptic receptors" (receptors transplanted from epileptic brains) display a marked run-down during repetitive applications of GABA. It was found that exposure to the neurotrophin BDNF increased the amplitude of the "GABA currents" (currents elicited by GABA) generated by the epileptic receptors and decreased their run-down; both events being blocked by K252A, a neurotrophin tyrosine kinase receptor B inhibitor. These effects of BDNF were not mimicked by nerve growth factor. In contrast, the GABAA receptors transplanted from the nonepileptic human hippocampal uncus (obtained during surgical resection as part of the nontumoral tissue from the oligo- dendroglioma margins) or receptors expressed by injecting rat recombinant αβ27gamma;2 GABAA receptor subunit cDNAs generated GABA currents whose time-course and run-down were not altered by BDNF. Loading the oocytes with the Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N' -tetraacetate-acetoxymethyl ester (BAPTA-AM), or treating them with Rp-8-Br-cAMP, an inhibitor of the cAMP-dependent PKA, did not alter the GABA currents. However, staurosporine (a broad spectrum PK inhibitor), bisindolylmaleimide I (a PKC inhibitor), and U73122 (a phospholipase C inhibitor) blocked the BDNF-induced effects on the epileptic GABA currents. Our results indicate that BDNF potentiates the epileptic GABAA currents and antagonizes their use-dependent run-down, thus strengthening GABAergic inhibition, probably by means of activation of tyrosine kinase receptor B receptors and of both PLC and PKC. [ABSTRACT FROM AUTHOR]

γ-Aminobutyric acid A (GABAA) receptors are heteromeric membrane proteins formed mainly by various combinations of &alph;, β and γ subunits; and it is commonly thought that the γ2 subunit alone does not form functional receptors. In contrast, the authors found that cDNA encoding the γ2L subunit of the human GABAA receptor, injected alone into Xenopus oocytes, expressed functional GABA receptors whose properties were investigated by using the two-microelectrode voltage-clamp technique.

The fast neuronal inhibition in vertebrates is mediated mainly through γ-aminobutyric acid (GABA)-activated chloride receptor channels, which are genetically related to the nicotinic acetylcholine (nAChR), glycine, and serotonin 3 receptors. Most of the structural knowledge on ionotropic neurotransmitter receptors is based on data obtained from the Torpedo electric organ nAChR, and more recently from crystals of the soluble acetylcholine-binding protein. Thus, it is mainly by analogy to the nAChRs that the stoichiometry of ionotropic GABA receptors is assumed to be pentameric. Some electron microscope images of native GABA receptors suggested a pentameric array with a central pore.

Neuroactive steroids are potent modulators of γ-aminobutyric acid type A receptors (GABAARS), and their behavioral effects are generally viewed in terms of altered inhibitory synaptic transmission. Here we report that, at concentrations known to occur in vivo, neuroactive steroids specifically enhance a tonic inhibitory conductance in central neurons that is mediated by extrasynaptic δ subunit-containing GABAARS. The neurosteroid-induced augmentation of this tonic conductance decreases neuronal excitability. Fluctuations in the circulating concentrations of endogenous neuroactive steroids have been implicated in the genesis of premenstrual syndrome, postpartum depression, and other anxiety disorders. Recognition that δ subunit-containing GABAARs responsible for a tonic conductance are a preferential target for neuroactive steroids may lead to novel pharmacological approaches for the treatment of these common conditions. [ABSTRACT FROM AUTHOR]

Cells subjected to sustained high osmolarity almost universally respond by accumulating compatible organic osmolytes that, in contrast to inorganic ions, are not deleterious even at high intracellular concentrations. Their accumulation from the external environment by known organic osmolyte transporters, such as the four identified in mammais, occurs only slowly in response to sustained high osmolarity, by synthesis of new transporter proteins. Most cells, however, are not subject to high or varying osmolarity, and ir is not clear whether organic osmolytes are generally required at normal osmolarities or how they are regulated. The fertilized egg of the mouse is protected in the oviduct from perturbations in osmolarity. However, deleterious effects of osmotic stress were evident in vitro even at normal oviductal osmolarity. Glycine was found to protect development, indicating that early mouse embryos may use glycine as an organic osmolyte at physiological osmolarity. We have now found that GLYT1, a glycine transporter of the neurotransmitter transporter gene family, functions as the organic osmolyte transporter that mediates the osmotically regulated accumulation of glycine and regulates cell volume in early embryos. Furthermore, osmotic stimulation of GLYT1 transport was immediate, without a requirement for protein synthesis, implying regulation different from known organic osmolyte transporters. Thus, GLYT1 appears to have a previously unidentified role as an organic osmolyte transporter that functions in acute organic osmolyte and volume homeostasis near normal osmolarity. [ABSTRACT FROM AUTHOR]

Describes selective neuronal localizations of D-aspartate in discrete brain areas and endocrine structures. External plexiform layer of the olfactory bulb; Hypothalamic supraoptic and paraventricular nuclei; Medial habenula; Brainstem nuclei; Stellate and basket cells in cerebellum; Epinephrine cells in the adrenal medulla.

Presents morphological evidences for specific relationship of type II calcium/calmodulin-dependent protein kinase and subset of glutamatergic synapses in thalamus and cerebral cortex. Immunoreactivity for CAM II kinase alpha; Plasticity of gamma-aminobutyric acidergic synapses; Functional immunoreactivity of CAM II kinase; Formation of induction mechanism.

The article discusses the role of ligand-gated ion channels in fast electrochemical signaling in the brain. It mentions that receptors contain an extracellular domain that recognizes neurotransmitters and opens a transmembrane channel upon ligand binding. It further informs about a bacterial receptor ELIC which reveal the mechanism of action of mammalian neurotransmitter signaling.

The article comments on a research paper which focuses on the mechanism involved in the effects of Gamma-Hydroxybutyric acid (GHB) on functions of central nervous system. It is said that it identifies a type of extrasynaptic GABA A receptors as a target for GHB. It is said that GHB is used in treatment of alcoholism and nacrolepsy, however its availability is tightly regulated because of its abuse. The interaction of GHB with GABA receptors and GHB binding site is also discussed.

The article focuses on a study that explores the significance of plasticity of local GABAergic interneurons in stimulating olfactory habituation. It notes that habituation is a form of learning significant for normal cognition. It adds that a fruit fly Drosophila melanogaster was used to study its cellular and circuit plasticity. Furthermore, it confirms that the mechanisms underlying the olfactory habituation of the fly may also present similar habituation in sensory organs.

The article offers information on the developmental functions of the vertebrate-specific carbohydrate polysialic acid (PSA) which contains α2,8-linked sialic acids. It mentions that PSA is used to modify the neuronal cell adhesion molecule (NCAM) which is responsible for cellular interactions. Meanwhile, polysialylation has identified the synaptic cell adhesion molecule 1 (SynCAM 1) which organizes excitatory synapses in the central nervous system.

The article investigates the physiological effects of alcohol ingestion on the brain and the central nervous system by researchers from Indianapolis, Indiana. Although alcohol ingestion impacts most organ systems, its effects on the brain are of considerable interest, given alcohol's many neuropharmacological actions, including its intoxicating, sedative, anxiolytic, reinforcing and addictive properties.

The article provides information on picrotoxin (PTX), the prototypic antagonist of GABAA receptors (GABAR), which are the primary mediators of inhibitory neurotransmission in the nervous system. PTX is a polycyclic compound without a nitrogen atom. The history of PTX as a drug and research tool is discussed.

This article presents a study revealing the retrograde cellular signaling by endocannabinoids. Endocannabinoids have emerged as important intercellular signals in the nervous system. The fatty acid amide arachidonylethanolamide is the prototypical endocannabinoid, that is, an endogenous ligand of the G protein-coupled cannabinoid receptor in the brain, CB1, which binds the main psychoactive component of marijuana and other derivatives of "Cannabis sativa." Endocannabinoids often act as retrograde messengers, emanating from morphologically undifferentiated regions of postsynaptic principal cells and traveling backwards across synapses, where they transiently inhibit the release of either the inhibitory neurotransmitter y-amino-butyric acid or the excitatory transmitter glutamate.