An electrophysiological study of Orexin A and [Ala11, D-Leu15] orexin-B effects in rat periaqueductal gray slices

Autor: Szu-Ying Fu, 傅思穎
Rok vydání: 2007
Druh dokumentu: 學位論文 ; thesis
Popis: 95
Orexins, also known as hypocretins, are a family of hypothalamic neuropeptides derived from prepro-orexin which include: orexin A (hypocretin 1) and orexin B (hypocretin 2). Orexin A and orexin B each consist of 33 and 28 amino acids, respectively, and share 46% sequence homology. Two receptors, OX1R and OX2R, were identified from G-protein coupled orphan receptors. OX1R has a higher affinity for orexin A, while OX2R displays equal affinity to both. Orexin-containing neurons are localized in the hypothalamus and project widely to numerous brain regions, including the periaqueductal gray (PAG). Orexins have been implicated in the several brain functions, including arousal, feeding, pain regulation, hyperlocomotion and autonomic stimulation. Previous reports have implicated that orexins have an antinociceptive effect in animal pain models, suggesting that orexins are involved in pain regulation. Furthermore, the PAG has been defined as a brain region with a critical role in the pain pathway. With regard to these findings, we investigated the effects of orexins in rat midbrain ventrolateral PAG (vlPAG) neurons using blind patch whole cell clamp recording. vlPAG neurons treated with orexin A (10~300 nM) were observed to dose-dependently induce membrane potential depolarization. The EC50 of orexin A was 12.3 ± 0.3 nM. Spontaneous firing activity was increased in 3/75 neurons after application of orexin A and just 45% of the recorded neurons were affected by orexin A. Orexin A-induced depolarization in vlPAG neurons was incompletely inhibited by SB-334867 (3 micromolar), a selective OX1R antagonist. Membrane potential depolarization was also induced when neurons were treated with [Ala11, D-Leu15] orexin-B (100~300 nM), an OX2R agonist. Yet this effect could not be reversed by SB-334867 (3 micromolar). The application of 300 nM [Ala11, D-Leu15] orexin-B was able to induce greater increments of membrane potential changes than the 100 nM dosage. According to a 2003 study, orexin A inhibited GIRK-mediated currents. Here, we used hyperpolarization ramps to research whether the effects of orexin A were mediated through GIRK channels in vlPAG neurons. We observed that orexin A (100 nM) did not change hyperpolarization ramp-induced currents, but the current was inhibited after treatment with 0.3 mM BaCl2. The BaCl2-inhibited currents were characterized with inward rectification and the reversal potential was near the equilibrium potential of K+ ions according to the Nernst equation. In another experiment, vlPAG neurons were first treated with N/OFQ (300 nM) to induce a GIRK-mediated current, and then treated with orexin A (100 nM). We did not observe any changes in the N/OFQ-induced current which was also a GIRK-mediated current. Furthermore, we observed the I-V relationships of orexin A-induced currents, but it was not constant. In conclusion, orexin A could induce membrane potential depolarization in a dose-dependant manner, and this effect could almost be reversed using SB-334867, a selective OX1R antagonist. Compared with orexin-A, 300 nM of [Ala11, D-Leu15] Orexin-B was more effective in altering the neuron membrane potential compared to 100 nM [Ala11, D-Leu15] orexin-B. We found that [Ala11, D-Leu15] orexin-B-induced depolarization was not affected when neurons were co-treated with [Ala11, D-Leu15] orexin-B and SB-334867. In vlPAG neurons, orexin A-induced depolarization did not seem to be via GIRK channels. In addition, I-V relationships created from orexin A-induced currents were found to be inconsistent. Thus, the mechanisms behind orexin A-induced effects in vlPAG neurons are still unclear.
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