A Study On The Mechanism Of Cannabinoid/Orexin Affecting The Discharge Of Hippocampal Neurons With Patch Clamp Technique

ObjectiveEffect and mechanism of cannabinoid and orexin receptor pathway on the discharges of pyramidal neuron in hippocampal CA1 region by patch clamp technique.MethodsBrain tissues were sliced from 15-20 days Wistar rats , and single CA1 pyramidal neuron was dissociated rapidly from the tissues include CA1 pyramidal cells. Then the effects of Win55212-2, the non-selective cannabinoid receptors agonist, and Orexin A,the non-selective orexin receptors agonist, on resting potential and spontaneous firing frequency of neurons were observed using the whole-cell patch-clamp technique. In the first part, the neurons were divided into hyperpolarization or depolarization groups according to the different responses to Win55212-2. In the second part, the neurons were divided into low firing rate or high firing rate groups according to their electric characteristics. The tissue viability was evaluated using MTT staining.Results:Part 1 (Cannabinoid): The firing frequency and the membrane potential were significantly fewer/lower during the administration than pre- administration period in the hyperpolarization group[0 Hz vs. (4.3 ± 3.2) Hz, p<0.05; (-57.0 ± 4.6) mV vs. (-54.1±3.8) mV, p<0.01]; The firing frequency and the membrane potential were significantly more/higher after administration than during-administration period(p<0.01). The firing frequency was significantly fewer during the administration than pre- administration period in the depolarization group[0 Hz vs. (6.2±3.6) Hz,p<0.01],while the membrane potential was significantly higher[ (-45.0±4.7) mV vs.(-47.3±4.4) mV, p<0.01]; The firing frequency was significantly increased, the membrane potential was significantly lower after administration than during-administration period(p<0.05).These effects of Win55212-2 were attenuated by AM251, the selective antagonist for cannabinoid receptor 1, in some neurons. In addition, tetraethylammonium (TEA,2?mol/L), non-selective blocker of the potential dependent potassium channels weakened the effects of Win55212-2 in the hyperpolarization group at different levels.Part 2 (Orexin): In the low-firing rate group, Orexin A excited 58%(28/48) neurons,the firing rate was significantly more during-administration than pre-administration period (5.36±3.64 Hz vs. 1.43±0.88 Hz, p<0.05); however, there was not significantly different between after-administration and during-administration(3.00±2.85 Hz vs.5.36±3.64 Hz, p=0.052),so was after-administration and pre-administration(3.00±2.85 Hz vs. 1.43±0.88 Hz, p=0.136). In the high-firing rate group, Orexin A excited 40%(4/10) neurons, the firing rate was significantly more during-administration than pre-administration period (6.98±2.45 Hz vs. 4.93 ± 2.55 Hz, p<0.05); and significantly fewer after-administration than during-administration (3.80± 1.28 Hz vs. 6.98±2.45 Hz,p<0.05), however, there was not significantly different between after and pre-administration(3.80± 1.28 Hz vs. 4.93 ± 2.55 Hz, p=0.215). There shows no different statistically of membrane potential comparing the three stages in both groups. These effects of orexin A were attenuated by SB 334867, the selective antagonist for orexin receptor 1, in some neurons.Conclusions:These results suggest that there existed cannabinoid receptorl and orexin receptor 1 in the CA1 pyramidal neurons. Activation of orexin receptor excited neurons .The activation of cannabinoid receptors could inhibit the excitability of CA1 pyramidal neurons through different mechanisms, and partially through voltage-dependent potassium channels.