Interactions between neurotensin and GnRH neurons in the positive feedback control of GnRH/LH secretion in the mouse.

In female mammals, increased ovarian estradiol (E(2)) secretion triggers GnRH release from neurons in the basal forebrain, which drives LH secretion from the pituitary and subsequently induces ovulation. However, the neural circuits that activate this preovulatory GnRH/LH surge remain unidentified. Neurotensin is expressed in neurons of the anteroventral periventricular nucleus (AVPV), a region thought to be critical for generating the preovulatory GnRH/LH surge. E(2) induces neurotensin (Nts) gene expression in this region, and blockade of neurotensin signaling reduces the LH surge in the rat. We postulated that neurotensin signaling plays a similar role in generating the E(2)-induced GnRH/LH surge in mice. We used in situ hybridization (ISH) to determine whether E(2) induces Nts expression in the mouse and found evidence to support this proposition. Next, we determined that the neurotensin receptor (Ntsr2) is present in many GnRH-expressing neurons. Since the kisspeptin gene (Kiss1) is expressed in the AVPV and is responsive to E(2), we predicted that some neurons in this region express both Kiss1 and Nts; however, by double-label ISH, we observed no coexpression of the two mRNAs. We also postulated that Nts mRNA expression would increase in parallel with the E(2)-induced LH surge and that the central (icv) administration of neurotensin would stimulate LH secretion and activation of GnRH neurons but found no evidence to support either of these hypotheses. Together, these findings suggest that, although neurotensin neurons in the AVPV are targets for regulation by E(2), neurotensin does not appear to play a direct role in generating the GnRH/LH surge in the mouse.

Microwave-assisted antigen retrieval and incubation with cox-2 antibody of archival paraffin-embedded human oligodendroglioma and astrocytomas.

Immunohistochemistry is an important tool that is often used for the diagnosis of pathologies; however, the length of time required to process the tissue is relatively long. Furthermore, the quality and sensitivity of immunohistochemical staining is affected by formalin fixation which results in variable loss of antigenicity, known as masking effect. Here we assess the effect of microwave irradiation on the incubation time required to obtain high quality immunohistochemical staining for cox-2 using archival formalin-fixed, paraffin-embedded human oligodendrogliomas and astrocytomas. The results show that intermittent microwave irradiation during the incubation with the primary antibody reduced the time requirement to 5 min while the staining quality was indistinguishable from 1 or 24 h long incubations. Thus, the use of this procedure results in a significant saving of time which is important for a timely diagnosis of pathological conditions that await treatment.

Localization of kainate receptor subunit GluR5-immunoreactive cells in the rat hypothalamus.

Glutamate is the major excitatory neurotransmitter in the hypothalamus, which exerts its effects by activating ion channel-forming (ionotropic) or G-protein-coupled (metabotropic) receptors. Kainate-preferring glutamate receptor subunits (GluR5, GluR6, GluR7, KA1, and KA2) form one of the three ionotropic receptor families. In the present study, we analyzed the distribution of GluR5 subunit protein in the rat hypothalamus with immunohistochemistry. GluR5 immunoreactivity was observed in perikarya and processes of many hypothalamic cells some of which, based upon their morphological differentiation by size and structure, appeared to be neurons and others glial cells. Analyses revealed that higher number of glial cells were GluR5 positive when compared to the moderate number of GluR5-labeled neurons in the anteroventral periventricular nucleus. Numerous GluR5-expressing neurons and similar number of glia were detected in the suprachiasmatic nucleus. In the arcuate nucleus more glial cells were identified with GluR5 immunoreactivity than the number of labeled neurons. Scattered GluR5-positive cells were present in the periventricular nucleus. Specific immunostaining was not seen in the ventromedial nucleus or dorsomedial nucleus. In conclusion, it is suggested that the GluR5 subunits participate in the glutamatergic regulation of several neuroendocrine systems, such as the tubero-infundibular systems as well as in the control of circadian output through neuron-to-neuron and/or neuron-to-glia interactions.

Distribution of vesicular glutamate transporter-2 messenger ribonucleic Acid and protein in the septum-hypothalamus of the rat.

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.

Expression of estrogen receptor-alpha and cFos in norepinephrine and epinephrine neurons of young and middle-aged rats during the steroid-induced luteinizing hormone surge.

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.