| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2000: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 1999: ¥2,800,000 (Direct Cost: ¥2,800,000)
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| Research Abstract |
1. Effects of tetrodotoxin (TTX) injected into the medial preoptic area (MPO) on electrical stimulation-induced luteinizing hormone (LH) surge in pentobarbital (PB)-anesthetized proestrous rats
Rats exhibiting regular 4-days estrous cycle were injected with 35 mg/kg PB, which is known to block the LH surge, on the day of proestrus at 13 : 45. Thereafter, rats were injected with 1, 2, or 5 μM TTX dissolved in 0.5 μl saline into the MPO and they received an electrical stimulation for 30 min, which is known to induce the LH surge in PB-blocked proestrous rats. Some rats were injected with saline as a control. The result showed that injection of 1, 2, or 5 μM TTX into the MPO blocked the electrical stimulation-induced LH surge in PB-blocked proestrous rats.
2. Effects of TTX injected into the MPO on the spontaneous LH surge in proestrous rats
Rats exhibiting regular 4-days estrous cycle were injected into the bilateral MPO with 5 μM TTX dissolved in 1.5 μl each on the day of proestrus at 14 :
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30. Some rats were injected with saline as control. Bilateral injection of TTX into the MPO completely blocked spontaneous LH surge.
3. Effects of Ca^<2+> channel blocker injected into the MPO on the spontaneous LH surge in proestrous rats
Rats exhibiting regular 4-days estrous cycle were injected into the bilateral MPO with 5 μM nifedipine, a L-type Ca^<2+> channel blocker, dissolved in 1.5 ml each side on the day of proestrus at 14 : 30. Some rats were injected with saline as control. Bilateral injection of nifedipine into the MPO completely blocked spontaneous LH surge.
4. Macro Ca^<2+> imaging analysis in MPO slices obtained from proestrous rats in association with the activity of GnRH surge generator
On the morning of proestrus, rats were killed between 10 : 00 and 11 : 00 and 200-μm-thick PDA slices were cut with a brain slicer. After 1 h of incubation in a standard artificial cerebrospinal fluid (aCSF) containing 10 mM Calcium green-1, POA slices were perfused with aCSF at a flow rate of 500 μl/min. Changes in [Ca^<2+>]I were determined by a macro imaging analysis. We could not see spontaneous fluctuation of [Ca^<2+>]I but 100 μM bicuculline induced an increase in [Ca^<2+>]I and GnRH secretion in POA slices. In a separate experiment, we found that infusion of 100 μM bicuculline significantly increased GnRH secretion but infusion of 100 μM naloxone was without effect.
In summary, results suggests that the activation of TTX-sensitive voltage-dependent Na^+ channel and L-type Ca^<2+> channel are important for the activity of GnRH surge generator. Further, as we hypothesized, it is suggested that a disinhibition of inhibitory GABA tone on GnRH neurons in the POA elicits a surge of GnRH secretion but that a disinhibition of inhibitory opioid tone does not. Less
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