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The effect of GABAB and alpha-2 Adrenergic Receptor on membrane excitability in Noradrenergic Neurons of A7 Catecholamine Cell Group in Rats
brainstem,A7 neucleus,noradrenaline,GIRK channel,alpha-2 adrenoceptor,GABAB receptor,whole-cell recording,pain transmission,
|Publication Year :||2008|
|Abstract:||大鼠A7核區兒茶胺酚細胞群是由一群分泌正腎上腺素之神經元所組成，其軸突投射到脊髓背角並參與痛覺的調控。然而，型態上的分析顯示在A7核區中也有神經傳導物質釋放的軸突結構-膨體，推測在A7局部會有正腎上腺素的分泌進行自我調控。為了驗證這個假設，我們取腦幹之腦薄片，利用全細胞膜電位記錄的方式去探討正腎上腺素對A7核區正腎上腺素神經元神經細胞興奮性之影響。10 uM正腎上腺素會抑制A7正腎上腺素神經元之自主放電並造成細胞膜電位之過極化，並可被正腎上腺素alpha-2受器阻斷劑所阻斷。將膜電位箝制在-70mV，給予正腎上腺素會引發一個向外的電流，其反轉電位在-97 mV並有向內整流的性質，顯示活化alpha-2受器可能去開啟G蛋白偶合向內整流鉀離子通道(GIRK)。此外，正腎上腺素引發之電流可被G蛋白偶合向內整流鉀離子通道之阻斷劑-鋇離子以及Tertiapin-Q所阻斷。然而，以alpha-2受器阻斷劑阻斷正腎上腺素作用，並不會造成神經細胞動作電位產生的頻率增加，顯示在A7核區內生性的正腎上腺素抑制神經元的情況並不顯著。綜合以上結果，活化A7正腎上腺素alpha-2受器會開啟GIRK通道去抑制神經元之興奮性，但神經元透過釋放正腎上腺素去調控自我的興奮性的負回饋機制並不顯著。
除此之外，A7的兒茶胺酚神經元受到持續的GABA中間神經元的抑制。另外也有型態上的證據顯示，A7神經細胞表現大量的GABAB受器。GABAB 受器被廣泛的發現會去開啟GIRK通道。GIRK會影響神經細胞的膜電位及興奮性。因此我們推測，內生性的GABA 可能持續活化GABAB受器，造成細胞神經興奮性的抑制。Baclofen是GABAB 受器的促效劑，會抑制A7正腎上腺素神經元之自主放電並造成細胞膜電位之過極化。將膜電位箝制在-70mV，給予Baclofen會引發一個向外的電流，其反轉電位在-105 mV並有向內整流的性質，顯示活化GABAB受器可能去開啟G蛋白偶合向內整流鉀離子通道(GIRK)。此外，Baclofen引發之電流可被G蛋白偶合向內整流鉀離子通道之阻斷劑-鋇離子以及Tertiapin-Q所阻斷。電刺激A7神經元的周邊的組織，可引發出GABAB的電流，顯示在A7神經元上的GABAB受器會受到突觸釋放的GABA所活化。另外，當我們給予GABAB受器的阻斷劑，會造成神經細胞動作電位放射的頻率上升，顯示，A7神經元會受到持續的GABA的抑制，這個抑制是透過GABAB受器所造成的。
The A7 catecholamine cell group consists of noradrenergic (NAergic) neurons that project axonal terminals to the dorsal horn of the spinal cord. Morphological analysis revealed that there are also axonal varicosities in A7 area, suggesting a possibility of autoregulation in NAergic neurons in A7 area. To test this, we firstly examined whether NE is able to affect the neuronal excitability of A7 NAergic neurons via whole-cell patch recording in brainstem slices. Bath application of 10 uM NE resulted in suppression of spontaneous spiking and membrane hyperpolarization in A7 neurons, which is blocked by alpha-2 receptor antagonist. Under voltage-clamp, application of NE produced an outward current (INE). The INE has reversal potential of -97mV and property of inward rectification, showing that hyperpolarization by alpha-2 adrenoceptors were caused by opening of G-protein-coupled inwardly rectifying potassium (GIRK) channels. This is further confirmed by the results that INE was blocked by Barium and Tertiapin-Q, the two blockers of GIRK channel. However, under resting or exiting conditions, application of alpha-2 adrenoceptors antagonist did not increase the firing rate. Theses results suggest that activation of alpha-2 adrenoceptors suppressed neuronal excitability of NAergic neurons by opening GIRK. However, feedback mechanism mediated by alpha-2 receptor targeting on GIRK channels was not prominent in A7 NAergic neurons.
A7 catecholamine cell group have been proposed to receive tonic GABAergic inhibition from local interneurons. The GIRK channels play an important role in determining resting membrane potential (Vm) and regulation of neuronal excitability, and are well known to be activated by metabotropic GABAB receptor in brain. Since NAergic neurons express high level of GABAB receptor, the effect of GABAB receptor on NAErgic neurons and the possible relationship to GIRK channel in A7 area are tested here. Bath application of 10 uM baclofen caused suppression of spontaneous firing and hyperpolarization of Vm in A7 neurons. This effect was blocked by CGP 54626, a specific GABAB receptor antagonist. Under voltage-clamp recording, application of baclofen produced an outward current (Ibaclofen). The Ibaclofen has reversal potential of -105mV and property of inward rectification, showing that hyperpolarization by GABAB receptor was caused by opening of GIRK channels. This is further confirmed by results that Ibaclofen was blocked by Barium and Tertaipin Q, the two bockers of GIRK channel. On the other hand, GABAB-mediatd current can be evoked in A7 neurons, suggesting that the GABAB receptor can be activated by the synaptic released GABA. Firing rates of NAergic neurons significantly increased in bath appilication of GABAB antagonist CGP54626, suggesting that there was endogenous GABA tonic suppressing the neuron. Taken together, these results suggest that NAergic neurons are subjective to tonic inhibition by GABAB receptors, which open GIRK channels and regulate excitability in A7 area.
To sum up, alpha-2 adrenoceptor and GABAB receptor opens GIRK channel and regulates the excitability of A7 NAergic neruons.
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