Increasing GABA concentration to 1 1 mm produced significantly larger amplitudes (?631 489 pA, related to 298 276 pA/pF, = 10) while the desensitization time constants remained unchanged (fast = 95.1 60.0 ms, amplitude element 0.19 0.03; sluggish = 1.63 1.32 s, = 7). of NG2 cells. Judging from your zolpidem Cortisone level of sensitivity, postsynaptic GABAA receptors in NG2 cells contain the 2-subunit, in contrast to extrasynaptic receptors, which were not modulated by zolpidem. To determine the effect of GABAA receptor activation on membrane potential, perforated patch recordings were from NG2 cells. In the current-clamp mode, GABA depolarized the cells to approximately ?30 mV, indicating a higher intracellular Cl? concentration (50 mm) than previously reported. GABA-induced depolarization in NG2 cells might result in Ca2+ influx through voltage-activated Ca2+ channels. Intro Glial cells communicate a similar set of ion channels and receptors as neurons (Verkhratsky and Steinh?user, 2000). Recently, NG2 cells have emerged as a separate type of neuroglia. While in white matter the majority of NG2-positve cells differentiate into oligodendrocytes, many NG2 cells in gray matter keep their phenotype throughout postnatal existence (Dimou et al., 2008). NG2 cells receive direct synaptic input from glutamatergic and GABAergic neurons, a property whose physiological effect is not yet recognized (Nishiyama et al., 2009; Bergles et al., 2010). While the AMPA receptors of NG2 cells (previously termed complex Cortisone cells, immature astrocytes, or GluR cells; for review, observe Bergles et al., 2010) have been investigated in much fine detail (Seifert and Steinh?user, 1995; Matthias et al., 2003; Seifert et al., 2003), less info is definitely available on the subject of properties and identity of GABAA receptors indicated by these cells. GABAA receptors are ionotropic pentameric receptors composed of two -, two -, and one -subunit, with the second option being replaced by a – or -subunit in some cells (Olsen and Sieghart, 2008). Fourteen GABAA receptor subunits (6, 3, 3, , ) are known to form receptors with unique practical properties. Cell type-specific variations in subunit composition can be distinguished by using modulators and blockers that bind at the numerous allosteric ligand binding sites of GABAA receptors, e.g., benzodiazepines, barbiturates, and Zn2+ (Hevers and Lddens, 1998; Mehta and Ticku, 1999; Sieghart, 2006). Rabbit Polyclonal to Cytochrome P450 2A7 GABAA receptors form Cl?-permeable and was performed with an Octaflow system (ALA Medical Instruments). To analyze receptor kinetics, we used freshly isolated cells and used a rapid concentration-clamp technique for drug application, permitting answer exchanges within 1 ms (Seifert and Steinh?user, 1995). The pipette answer for whole-cell recordings consisted of the following (in mm): 130 KCl, 2 MgCl2, 0.5 CaCl2, 3 Na2-ATP, 5 BAPTA, and 10 HEPES. For dedication of receptor current reversal potentials, perforated patch recordings were performed with the following pipette answer (in mm): 125 K-gluconate, 20 KCl, 3 NaCl, 2 MgCl2, 0.5 EGTA, and 10 HEPES, pH 7.25. In these experiments, to block voltage-activated K+-, Na+-, and Ca2+-currents and AMPA receptor-mediated currents, bath solutions with 135 mm NaCl supplemented with the following (in mm): 10 BaCl2, 4 4-aminopyridine, 0.03 CdCl2, 0.001 tetrodotoxin (TTX), and 0.025 CNQX (300 mOsm) were used. Voltage was corrected for liquid junction potential (13 mV for K-gluconate answer vs blocking answer). Zolpidem, diazepam, DMCM, and SNAP 5114 were dissolved in dimethylsulfoxide (DMSO; final DMSO concentration 0.1%). Gramicidin A solution (20 mg/ml in DMSO) was freshly prepared every day and added to the pipette answer at a final concentration of 40C60 g/ml. Tonic GABAA receptor currents were recorded using the following CsCl-based pipette answer (in mm): 130 CsCl, 2 MgCl2, 3 Na2-ATP, 0.5 CaCl2, 5 BAPTA, and 10 HEPES. Substances were purchased from Sigma and Tocris Bioscience. Single cell reverse transcription-PCR. After recording, the cytoplasm of individual cells was harvested under microscopic control and reverse transcription (RT) was performed (Matthias et al., 2003). A multiplex two-round single-cell PCR was performed with primers for , , and Cortisone GABAA receptor subunits, respectively (Berger et al., 1998; Table 1). The 1st PCR was performed after adding PCR buffer, MgCl2 (2.5 mm), primers (200 nm each), and Taq polymerase (3.5 U; Invitrogen) to the RT product (final volume 50 l). Forty-five cycles were performed (denaturation at 94C, 25 s; annealing at 49C, 2 min for the 1st five cycles, and 45 s for the remaining.
Increasing GABA concentration to 1 1 mm produced significantly larger amplitudes (?631 489 pA, related to 298 276 pA/pF, = 10) while the desensitization time constants remained unchanged (fast = 95
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