| Budget Amount *help |
¥4,010,000 (Direct Cost: ¥3,500,000、Indirect Cost: ¥510,000)
Fiscal Year 2007: ¥2,210,000 (Direct Cost: ¥1,700,000、Indirect Cost: ¥510,000)
Fiscal Year 2006: ¥1,800,000 (Direct Cost: ¥1,800,000)
|
|---|
| Research Abstract |
Law conductance voltage-and calcium-activated potassium (BK) channels and A-type γ-aminobutyric acid receptor (GABAAR) expressed in rat GnRH neurons were investigated. Brain was removed from adult GnRH-EGFP transgenic rat under anesthesia, and neurons were dispersed enzymatically and plated on coverslips and cultured overnight. GnRH neurons were identified by EGFP-fluorescence and patch-clamped to record membrane current and membrane potentials by means of perforated patch-clamp. Rat GnRH neurons exhibited BK currents (Hiraizumi, et. al., 2008) in addition to the SK currents (Kato, et. al., 2006). The 60-mV pulse-evoked BK current occupied 30% of total outward potassium current. This BK current was blocked by 60% by BK channel blocker charybdotoxin (1 μM . Total outward potassium current evoked by 60-mV pulse was not suppressed by SK channel blocker apamin. Moreover, soma membrane of rat GnRH neuron does not show A current. So that the total outward current evoked by 60-mV pulse is com
… More
posed with BK current and delayed rectifier potassium current. We further investigated calcium channels involved in the activation of BK channels and revealed that R-type and L-type channels contributed to activate BK channels in rat GnRH neurons. Multi-cell RT-PCR of BK channel subunits revealed the expressions of α, β1, β2 and β4. GABAAR current was investigated by means of gramicidin perforated patch-clamp method (Yin, et. al., 2008). The current was blocked by GABAAR channel blocker picrotoxin and only weakly blocked by gabazine. The reversal potential of GABAAR currents were around-30 mV in adult rat GnRH neurons and around-57 mV in unidentified neurons. GABAAR currents in GnRH neurons were augmented by neurosteroids allopregnanolone and THDOC. Results of RT-PCR of GABAAR subunits mRNA indicate that GABAAR in rat GnRH neurons is composed with α3, β3, γ1 and γ2. These results were not influenced by either sex or estrous cycle. In conclusion, rat GnRH neurons isolated from adult animals functionally express BK channels and GABAARs. Activation of GABAAR depolarizes the cell and neurosteroids further modulate the depolarization. Less
|
