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The distribution of lamprey gonadotropin-releasing hormone (GnRH)-I and -III has been extensively characterized by immunocytochemistry in the forebrain of the sea lamprey, Petromyzon marinus. However, the cellular location of lamprey GnRH-III mRNA expression by in situ hybridization in the lamprey brain has not been determined. We show for the first time the location of expression of lamprey GnRH-III, as well as provide a more comprehensive in situ study of lamprey GnRH-I and glutamic acid decarboxylase (GAD; GABA-synthesizing enzyme) mRNA expression in the brain of the lamprey in different reproductive life stages. Colorimetric and dual-label fluorescent amplification methods of in situ hybridization were used on brain tissue sections of adult, juvenile, and larval sea lamprey. In each life stage of the lamprey, expression of lamprey GnRH-I was shown in the preoptic area (POA) and the hypothalamus forming the characteristic arc-like cell population extending from the preoptic nucleus (NPO) to the neurohypophysis. Lamprey GnRH-III expression was also seen in the POA of each life stage in close proximity to lamprey GnRH-I mRNA containing neurons. GAD expression was shown in distinct cell clusters in and around the POA, in the olfactory bulb, in the dorsal thalamus beneath the habenular region, and also in the ventral-medial hypothalamus stretching from the periventricular region to the anterior portion of the rhombencephalon. Using dual-label in situ hybridization, we have shown that lamprey GnRH-I and -III mRNA are colocalized in the same cells in the POA in adult lampreys. Dual-label in situ hybridization also showed close proximity of GAD mRNA containing neurons and GnRH containing neurons in the POA. These data suggest that gamma-aminobutyric acid (GABA) may directly affect GnRH release in the brain of the sea lamprey.

In this study we examined the spatial relationship of GABA-containing and GnRH-containing neurons by immunocytochemistry and in situ hybridization in larval and adult brains of sea lamprey, Petromyzon marinus. In immunocytochemical studies, GABA-containing neurons were detected early in lamprey development, by day 20 post-fertilization. At this time point, one population of GABA-containing neurons was visualized in the hypothalamus and preoptic area, and another population was located in the olfactory bulb of the telencephalon. By day 30 after fertilization, after the GABA neurons were detected, GnRH-containing neurons were visualized in the preoptic area/rostral hypothalamus region, adjacent to the GABA-containing neurons in the wall of the third ventricle. Similarly, in adult lamprey brains distinct populations of both GABA- and GnRH-containing neurons were located in the hypothalamus adjacent to the third ventricle. To further establish a proximate relationship between GABA and GnRH, the mRNA for glutamate decarboxylase (GAD), the enzyme catalyzing GABA synthesis from glutamate, and GnRH were examined by in situ hybridization in the brains of larval lamprey. These studies also showed that GnRH and GAD are produced in cell populations in and around the third ventricle of the hypothalamus. This close spatial relationship of GABA neurons and GnRH neurons provides a basis for a regulatory role of GABA on GnRH neurons in the sea lamprey., (Copyright 2002 S. Karger AG, Basel)