The excitatory neurotransmitter glutamate is involved in the control of most, perhaps all, neuroendocrine systems, yet the sites of glutamatergic neurons and their processes are unknown. Here, we used in situ hybridization and immunohistochemistry for the neuron-specific vesicular glutamate transporter-2 (VGLUT2) to identify the neurons in female rats that synthesize the neurotransmitter glutamate as well as their projections throughout the septum-hypothalamus. The results show that glutamatergic neurons are present in the septum-diagonal band complex and throughout the hypothalamus. The preoptic area and ventromedial and dorsomedial nuclei are particularly rich in glutamatergic neurons, followed by the supraoptic, paraventricular, and arcuate nuclei, whereas the suprachiasmatic nucleus does not express detectable amounts of VGLUT2 mRNA. Immunoreactive neurites are seen in very high densities in all regions analyzed, particularly in the preoptic region, followed by the ventromedial, dorsomedial, and arcuate nuclei as well as the external layer of the median eminence, whereas the mammillary complex does not exhibit VGLUT2 immunoreactivity. Many VGLUT2 immunoreactive fibers also contained synaptophysin, suggesting that the transporter is indeed localized to presynaptic terminals. Together, the results identify glutamatergic cell bodies throughout the septum-hypothalamus in region-specific patterns and show that glutamatergic nerve terminals are present in very large numbers such that most neurons in these brain regions can receive glutamatergic input. We examined the GnRH system as an example of a typical neuroendocrine system and could show that the GnRH perikarya are closely apposed by many VGLUT2-immunoreactive boutons, some of which also contained synaptophysin. The presence of VGLUT2 mRNA-containing cells in specific nuclei of the hypothalamus indicates that many neuroendocrine neurons coexpress glutamate as neurotransmitter, in addition to neuropeptides. These systems include the oxytocin, vasopressin, or CRH neurons as well as many others in the periventricular and mediobasal hypothalamus. The presence of VGLUT2 mRNA in steroid-sensitive regions of the hypothalamus, such as the anteroventral periventricular, paraventricular, or ventromedial nuclei indicates that gonadal and adrenal steroid can directly alter the functions of these glutamatergic neurons.

Norepinephrine (NE) and epinephrine are important stimulators of GnRH release during the preovulatory surge in female rats. Previous studies have shown that the catecholaminergic neurons are sensitive to estradiol and that NE release in the hypothalamus is decreased in middle-aged rats at the time when the estrous cycles become irregular and later cease to exist. The aims of the present study were to determine whether the NE and epinephrine neurons continue to express estrogen receptor (ER)-alpha in middle-aged rats; temporal expression of ER-alpha and cFos changes with age during the steroid-induced surge; and tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH), and phenylethanol-N-methyltransferase mRNA content in catecholaminergic neurons of the brain stem changes during the surge with age. The results show that there was no difference in TH mRNA content; however, DBH mRNA levels in areas A1, A2, and C1 of the middle-aged animals did not rise during the surge as was observed in the young animals. Although the percentage of NE and epinephrine neurons that express ER-alpha was unchanged during the surge in both young and middle-aged animals, cFos expression was enhanced in areas A1 and A2 of the middle-aged animals but not in the young animals. Together the results suggest that NE and epinephrine neurons in the middle-aged rat continue to express appropriate basal levels of TH, DBH, and phenylethanol-N-methyltransferase mRNAs as well as ER-alpha and cFos; however, the enhancement of DBH expression, as seen in the young animals during the steroid-induced surge, was not detected in middle-aged animals. On the other hand, cFos expression in the middle-aged rat was higher in areas A1 and A2 during the surge. It is concluded that the reduced catecholamine release during the surge in middle-aged rats is caused, in part, by an altered sensitivity of the NE neurons to estradiol, which results in an aberrant cFos expression and probably not by major deficits in the expression of transmitter synthesizing enzymes or steroid receptors.

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