. Whole cell patch-clamp recordings were obtained from thalamic ventrobasal (VB) and reticular (RTN) neurons in mouse brain slices. A bicuculline-sensitive tonic current was observed in VB, but not in RTN, neurons; this current was increased by the GABA A receptor agonist 4,5,6,7-tetrahydroisothiazolo-[5,4-c]pyridine-3-ol (THIP; 0.1 M) and decreased by Zn 2ϩ (50 M) but was unaffected by zolpidem (0.3 M) or midazolam (0.2 M). The pharmacological profile of the tonic current is consistent with its generation by activation of GABA A receptors that do not contain the ␣ 1 or ␥ 2 subunits. GABA A receptors expressed in HEK 293 cells that contained ␣ 4 ␤ 2 ␦ subunits showed higher sensitivity to THIP (gaboxadol) and GABA than did receptors made up from ␣ 1 ␤ 2 ␦, ␣ 4 ␤ 2 ␥ 2s, or ␣ 1 ␤ 2 ␥ 2s subunits. Western blot analysis revealed that there is little, if any, ␣ 3 or ␣ 5 subunit protein in VB. In addition, co-immunoprecipitation studies showed that antibodies to the ␦ subunit could precipitate ␣ 4 , but not ␣ 1 subunit protein. Confocal microscopy of thalamic neurons grown in culture confirmed that ␣ 4 and ␦ subunits are extensively co-localized with one another and are found predominantly, but not exclusively, at extrasynaptic sites. We conclude that thalamic VB neurons express extrasynaptic GABA A receptors that are highly sensitive to GABA and THIP and that these receptors are most likely made up of ␣ 4 ␤ 2 ␦ subunits. In view of the critical role of thalamic neurons in the generation of oscillatory activity associated with sleep, these receptors may represent a principal site of action for the novel hypnotic agent gaboxadol. I N T R O D U C T I O NThe activation of GABA A receptors inhibits neurons in two ways-via a fast or transient inhibition after GABA binding to synaptically localized receptors and by a sustained inhibition due to GABA binding to extrasynaptic receptors (Brickley et al. 1996; Farrant and Nusser 2005;Kaneda et al. 1995;Mody 2001). Extrasynaptic receptors are excellent sensors for extracellular GABA due to their high affinity for GABA and slow rates of desensitization (Bai et al. 2001; Brickley et al. 1999;Yeung et al. 2003). The activation of these receptors regulates neuronal input resistance and, hence, excitability (Brickley et al. 2001;Semyanov et al. 2003). The ␦ subunit appears to be present in many extrasynaptic GABA A receptors. In cerebellar granule cells, these receptors likely contain ␣ 6 , ␤, and ␦ subunits (Brickley et al. 2001;Nusser et al. 1998;Pirker et al. 2000), whereas granule cells in the dentate gyrus likely contain ␣ 4 , ␤, and ␦ subunits (Nusser and Mody 2002;Sperk et al. 1997;Sun et al. 2004;Wei et al. 2003). Extrasynaptic GABA A receptors in CA1 pyramidal neurons in the hippocampus, however, likely contain ␣ 5 , ␤ 2/3 , and ␥ 2 subunits (Caraiscos et al. 2004).Several GABA A receptor subunits are expressed in the thalamus. ␣ 1 , ␣ 4 , ␤ 2 , ␥ 2 , and ␦ subunits are found in thalamocortical neurons in the ventrobasal (VB) complex, whereas ␣ 3 , ␤ 3 , and ␥ 2 subunits...

The nervous system transmits signals between neurons via neurotransmitter release during synaptic vesicle fusion. In order to observe neurotransmitter uptake and release from individual presynaptic terminals directly, we designed fluorescent false neurotransmitters as substrates for the synaptic vesicle monoamine transporter. Using these probes to image dopamine release in the striatum, we made several observations pertinent to synaptic plasticity. We found that the fraction of synaptic vesicles releasing neurotransmitter per stimulus was dependent on the stimulus frequency. A kinetically distinct "reserve" synaptic vesicle population was not observed under these experimental conditions. A frequency-dependent heterogeneity of presynaptic terminals was revealed that was dependent in part on D2 dopamine receptors, indicating a mechanism for frequency-dependent coding of presynaptic selection.

Taurine is one of the most abundant free amino acids in the brain. In a number of studies, taurine has been reported to activate glycine receptors (Gly-Rs) at moderate concentrations (Ն100 M), and to be a weak agonist at GABA A receptors (GABA A -Rs), which are usually activated at high concentrations (Ն1 mM). In this study, we show that taurine reduced the excitability of thalamocortical relay neurons and activated both extrasynaptic GABA A -Rs and Gly-Rs in neurons in the mouse ventrobasal (VB) thalamus. Low concentrations of taurine (10 -100 M) decreased neuronal input resistance and firing frequency, and elicited a steady outward current under voltage clamp, but had no effects on fast inhibitory synaptic currents. Currents elicited by 50 M taurine were abolished by gabazine, insensitive to midazolam, and partially blocked by 20 M Zn 2ϩ , consistent with the pharmacological properties of extrasynaptic GABA A -Rs (␣4␤2␦ subtype) involved in tonic inhibition in the thalamus. Tonic inhibition was enhanced by an inhibitor of taurine transport, suggesting that taurine can act as an endogenous activator of these receptors. Taurine-evoked currents were absent in relay neurons from GABA A -R ␣4 subunit knock-out mice. The amplitude of the taurine current was larger in neurons from adult mice than juvenile mice. Taurine was a more potent agonist at recombinant ␣4␤2␦ GABA A -Rs than at ␣1␤2␥2 GABA A -Rs. We conclude that physiological concentrations of taurine can inhibit VB neurons via activation of extrasynaptic GABA A -Rs and that taurine may function as an endogenous regulator of excitability and network activity in the thalamus.

Background SCN5A encodes the α-subunit (Nav1.5) of the principle Na+ channel in the human heart. Genetic lesions in SCN5A can cause congenital long QT syndrome (LQTS) variant 3 (LQT-3) in adults by disrupting inactivation of the Nav1.5 channel. Pharmacological targeting of mutation-altered Na+ channels has proven promising in developing a gene-specific therapeutic strategy to manage specifically this LQTS variant. SCN5A mutations that cause similar channel dysfunction may also contribute to sudden infant death syndrome (SIDS) and other arrhythmias in newborns, but the prevalence, impact, and therapeutic management of SCN5A mutations may be distinct in infants compared with adults.Methods and ResultsHere, in a multidisciplinary approach, we report a de novo SCN5A mutation (F1473C) discovered in a newborn presenting with extreme QT prolongation and differential responses to the Na+ channel blockers flecainide and mexiletine. Our goal was to determine the Na+ channel phenotype caused by this severe mutation and to determine whether distinct effects of different Na+ channel blockers on mutant channel activity provide a mechanistic understanding of the distinct therapeutic responsiveness of the mutation carrier. Sequence analysis of the proband revealed the novel missense SCN5A mutation (F1473C) and a common variant in KCNH2 (K897T). Patch clamp analysis of HEK 293 cells transiently transfected with wild-type or mutant Na+ channels revealed significant changes in channel biophysics, all contributing to the proband's phenotype as predicted by in silico modeling. Furthermore, subtle differences in drug action were detected in correcting mutant channel activity that, together with both the known genetic background and age of the patient, contribute to the distinct therapeutic responses observed clinically.SignificanceThe results of our study provide further evidence of the grave vulnerability of newborns to Na+ channel defects and suggest that both genetic background and age are particularly important in developing a mutation-specific therapeutic personalized approach to manage disorders in the young.

Volatile anesthetics are used clinically to produce analgesia, amnesia, unconsciousness, blunted autonomic responsiveness, and immobility. Previous work has shown that the volatile anesthetic isoflurane, at concentrations that produce unconsciousness (250 -500 M), enhances fast synaptic inhibition in the brain mediated by GABA A receptors (GABA A -Rs). In addition, isoflurane causes sedation at concentrations lower than those required to produce unconsciousness or analgesia. In this study, we found that isoflurane, at low concentrations (25-85 M) associated with its sedative actions, elicits a sustained current associated with a conductance increase in thalamocortical neurons in the mouse ventrobasal (VB) nucleus. These isoflurane-evoked currents reversed polarity close to the Cl Ϫ equilibrium potential and were totally blocked by the GABA A -R antagonist gabazine. Isoflurane (25-250 M) produced no sustained current in VB neurons from GABA A -R ␣ 4 -subunit knockout (Gabra4 Ϫ/Ϫ ) mice, although 250 M isoflurane enhanced synaptic inhibition in VB neurons from both wild-type and Gabra4Ϫ/Ϫ mice. These data indicate an obligatory requirement for ␣ 4 -subunit expression in the generation of the isoflurane-activated current. In addition, isoflurane directly activated ␣ 4 ␤ 2 ␦ GABA A -Rs expressed in human embryonic kidney 293 cells, and it was more potent at ␣ 4 ␤ 2 ␦ than at ␣ 1 ␤ 2 ␥ 2 receptors (the presumptive extrasynaptic and synaptic GABA A -R subtypes in VB neurons). We conclude that the extrasynaptic GABA A -Rs of thalamocortical neurons are sensitive to low concentrations of isoflurane. In view of the crucial role of the thalamus in sensory processing, sleep, and cognition, the modulation of these extrasynaptic GABA A -Rs by isoflurane may contribute to the sedation and hypnosis associated with low doses of this anesthetic agent.