Substance P Activates a TRP-like Channel to Increase Excitability of Neurons in A7 Catecholamine Cell Group in Rats

下傳止痛神經迴路(descending antinociceptive neuronal circuitry)中的數個核區，包括PAG、VMM及LH，皆為腦部控制脊髓背角痛覺傳遞的重要構造。各項研究結果證實此三核區均會投射到腦幹A7兒茶酚胺細胞群 (catecholamine cell group)，且投射細胞多為可分泌物質P (substance P) 的神經元，暗示物質P在A7核區極有可能扮演了相當重要的角色，進而影響下傳止痛神經迴路。我們的研究結果顯示在A7核區中，依照分泌正腎上腺素 (norepinephrine) 與否可以概分為兩群細胞，分別是”正腎上腺素神經元” (noradrenergic) 與”非正腎上腺素神經元”(non-noradrenergic)。染色結果顯示在A7核區中，不論在正腎上腺素神經元之間或是正腎上腺素神經元與非正腎上腺素神經元之間均具有連結的存在，暗示A7核區中神經元之間具有複雜的調控機制。在具有A7核區的腦薄片 (brain slice) 給予物質P後，可以觀察到正腎上腺素神經元及部分的非正腎上腺素神經元均產生自發性動作電位頻率加快及膜電位去極化的現象。在voltage-clamp下，物質P所造成的膜電位去極化可以表示為箝制電流 (holding current) 的增大。藉由RP67580, suramin, U73122之藥理測試，證實物質P所造成的箝制電流變化是透過”物質P-NK1受體 (neurokinin-1 receptor)-G蛋白-磷脂酶C (phospholipase C)”此一路徑所造成的。相對的，三磷酸肌 (inositol triphosphate) 、雙酸甘油酯 (diacyl glycerol)及蛋白質激酶A (protein kinase A) 路徑的相關藥物則無法抑制物質P造成的箝制電流變化。值得注意的是，雙酸甘油酯興奮劑 (OAG) 反而能增進物質P造成的箝制電流，顯示雙酸甘油酯對於物質P的作用具有促進的功能。另外蛋白質激酶C (protein kinase C) 則可以調控物質P箝制電流的去敏感化 (desensitization)，而此一去敏感化效應需要有細胞外鈣離子的參與。物質P最終會透過開啟一無選擇性陽離子通道 (nonselective cation channe) 而造成箝制電流的變化。此一通道的反轉電位接近於-2 mV；且在三種常用的transient receptor potential (TRP) 通道抑制劑下，物質P的反應皆會被顯著抑制。綜合以上結果，此篇研究證實了物質P在A7正腎上腺素神經元的作用主要是透過磷脂酶C開啟一”類TRP通道” (TRP-like channel)而導致細胞興奮性的提高，可能進而調控下傳止痛神經迴路而影響痛覺在脊髓背角的傳遞。Previous morphological studies showed that neurons in A7 catecholamine cell group receive nerve terminals releasing substance-P (SP) from several descending pain modulation nuclei (PAG, VMM and LH), A7 neurons are therefore believed to play important roles in regulating nociception. In this study, we provided physiological evidence for this argument. Firstly, two kinds of neurons in A7 nucleus were identified based on norepinephrine production. Physical contacts observed between these noradrenergic (NAergic) neurons and non-noradrenergic (non-NAergic) neurons displayed the complex regulation among neurons in A7 nucleus. Application of SP consistently increased the firing frequency of NAergic neurons and some of the non-NAergic neurons. The mechanism under SP function in A7 NAergic neurons was then explored. Holding current of these NAergic neurons were significantly elevated upon bath application of 0.2 μM SP. This response can be blocked by RP 67580 (NK1 receptor antagonist), by internally applied 10 μM suramine (uncoupling G protein from the receptor), and by U73122 (phospholipase C blocker), therefore showed that the effect of SP was mediated through NK1 receptor, G protein, and phospholipase C-dependent mechanism. BAPTA, heparin and inositol 1,4,5-triphosphate via the patch pipette had no effect on SP-induced current. Addition of DAG analogue (OAG) and DAG lipase inhibitor (RHC80267) had no effect on SP-induced response either. Notably, SP-induced current in the presence of OAG were larger than SP alone suggested that generation of DAG alone may not fully account for activation of SP-induced current. In addition, PKC inhibitor significantly attenuated the desensitization of SP-induced current; calcium influx was also an essential component of this PKC regulated desensitization. Three evidences showed that SP-induced current was caused by open a transient receptor potential (TRP)-like channel. The conductance was increased during SP application, the reversal potential of this current was around -2mV, and adding three TRP channel blockers could consistently depress this inward current. Taken together, the electrophysiological and pharmacological properties of the SP-induced inward current are consistent with the involvement of TRP-like channels whereas the identification and function of this TRP-like channel still needs further investigation.Introduction……………………………………………………………………….-1- A7: the noradrenergic nucleus in the rostral brainstem………...……………..-1- The role of A7 in pain modulation………...…………………..………………..-2- What is Pain?……………………………………………………………...…-2- How is Pain Regulated?..................................................................................-3- The Ascending Pathway……………………...…………………...…………-4- The Descending Pathway…………………………………………..………..-5- Higher Order Neurons- Lateral Hypothalamus (LH)……………………..-6- Periaqueductal Grey (PAG)……………………………………………….-6- Ventromedial Medulla (VMM)…………………………………………….-8- A7 Noradrenergic Cell Group…………………………………………….-9- Substance P Role in Descending Pain Transmission………….……………...-10- Substance P: First Tachykinin………………………………………………-11- Major Biological Actions of SP in CNS……………………………………-11- The Pain-Modulation Role of Substance P in Brainstem…………………..-12- Two Major Mechanisms Under Substance P Activation………………….-13- Objective………………………………………………………………………..-14- Materials and Methods…………………………………………………………-16- Results……………………………………………………………………………...-22- Two Major Neuron Types in A7 Region: Noradrenergic and Non-noradrenergic Neurons………………………………………………………………………-22- Location and Interaction of Noradrenergic Neurons in A7 Region...............-22- Non-noradrenergic Neurons in A7 Region....................................................-24- Contacts between Noradrenergic and Non-noradrenergic neurons………...-24- VIII Substance P Action on Neurons in A7 Region……………………………….-25- Excitatory Responses Induced by Substance P…………………………….-25- NK1R, G Protein and Phospholipase C Dependence………………………-26- DAG Potentiates the Substance P Response………………………………..-28- IP3 and PKA Pathway Independence……………………………………….-29- PKC Mediates Desensitization of Substance P-Induced Response………...-30- TRP-like Channels Mediate the Inward Current Induced by Substance P…-31- Slow Substance P Postsynaptic Currents Induced by Electrical Stimulus…-33- Discussion………………………………………………………………………..-35- Cellular Interactions Between Neurons in A7………………………………….-35- Cellular Mechanisms Underlying SP Activation……………………………….-38- Target Channel of SP Activation………………………………………………..-39- PKC Determines the Desensitization Response………………………………..-42- Functional Role of SP in A7 NAergic neurons…………………………………-43- Reference………………………………………………………………………...-45- Figures…………………………………………………………………………...-57