A NOVEL NEURAL PATHWAY CONTROLS TESTOSTERONE RELEASE

我们最近发现了一种神经脑-睾丸通路的存在，这种通路独立于脑垂体会干扰间质细胞的功能。具体地说，脑室注射促肾上腺皮质激素释放因子(CRF)或异丙肾上腺素(ISO)后5分钟内，可阻断人绒毛膜促性腺激素(HCG)对睾酮(T)释放的刺激作用。在hCG前15分钟全身注射酒精也有类似的效果。静脉注射GnRH拮抗剂既不能模拟或逆转侧脑室CRF、ISO或酒精阻断T反应的能力，表明它们的抑制作用不是由于低水平的黄体生成素所致。相反，侧脑室注射肾上腺素能拮抗剂可部分逆转ICV ISO、ICV CRF或全身酒精的作用。这些结果支持存在一种通过肾上腺素能机制迅速抑制间质细胞功能的神经通路。目前，关于这一途径的神经学证据尚不清楚。在具体目标1下，我们将使用病毒跨神经元标记方法来识别中枢神经系统(CNS)中参与这一途径的位置。这一强大的神经解剖学工具包括向睾丸内注射伪狂犬病病毒(PRV)。在复制之后，PRV以逆行的方式被运送到支配性腺的一级神经元的核膜，然后随着感染进一步进入突触连接的细胞体，被运送到二级、三级和四级神经元。使用多克隆抗体通过免疫细胞化学的方法鉴定病毒标记的神经元。标记的特异性将取决于精索去神经和/或脊髓T1段的能力，以消除更高层结构中的PRV标记。在我们确定了属于所提出的通路的大脑区域之后，我们将进行功能实验来确定它的生理重要性。在特定目标2下，我们将在选定的脑区微量注射CRF或ISO。我们预计，通过激活所提出的抑制途径，这些治疗将减少hCG诱导的T分泌，与在注射该载体的动物中获得的结果相比。在特定目标3下，我们将切断被认为与所提出的通路有关的T1水平的脊髓或病变特定的下丘脑区域，以确定这些程序是否阻断了脑室CRF或全身酒精对hCG诱导的T分泌的抑制作用。这些实验将提供第一个形态和功能证据，证明在睾丸和中枢神经系统之间存在受酒精影响的多突触神经通路。

We recently uncovered the existence of a neural brain-testicular pathway that interferes with Leydig cell function independently of the pituitary. Specifically, we showed that within 5 min of their intracerebroventricular (icv) injection, corticotropin- releasing factor (CRF) or isoproterenol (ISO) blocked the stimulatory effect of human chorionic gonadotropin (hCG) on testosterone (T) release. Systemic alcohol injected 15 min prior to hCG exerted a similar effect. The ability of icv CRF or ISO, or of alcohol, to block the T response was neither mimicked nor reversed by iv pretreatment with a GnRH antagonist, indicating that their inhibitory effect was not due to low LH levels. In contrast, the icv injection of an adrenergic antagonist partially reversed the effect of icv ISO, icv CRF or systemic alcohol. These results support the existence of a neural pathway that rapidly inhibits Leydig cell function through an adrenergic mechanism. At present, neuromorphological evidence for this pathway is missing. Under Specific Aim 1, we will use a viral transneuronal labeling method to identify sites in the central nervous system (CNS) that are involved in this pathway. This powerful neuroanatomical tool consists of the injection of pseudorabies virus (PRV) into the testis. Following replication, PRV is transported in a retrograde fashion to the perikarya of first-order neurons innervating the gonad, then to second-, third- and fourth-order neurons as the infection proceeds to further synaptically linked cell bodies. Virus-labeled neurons are identified by means of immunocytochemistry using polyclonal antibodies. Specificity of labeling will be determined by the ability of spermatic denervation and/or spinal cord T1 section, to eliminate PRV labeling in higher structures. After we have identified brain areas that belong to the proposed pathway, we will conduct functional experiments to determine its physiological importance. Under Specific Aim 2, we will microinfuse CRF or ISO in selected brain areas. We anticipate that by activating the proposed inhibitory pathway, these treatments will decrease hCG-induced T secretion, compared to results obtained in animals infused with the vehicle. Under Specific Aim 3, we will sever the spinal cord at the T1 level or lesion specific hypothalamic areas thought to be involved in the proposed pathway, to determine whether these procedures block the inhibitory effect of icv CRF or systemic alcohol on hCG-induced T secretion. These experiments will provide the first morphological and functional evidence of the existence of a multisynaptic neural pathway between the testes and the CNS, that is influenced by alcohol.