N-Methyl-D-aspartate receptor gene expression in the hamster hypothalamus and in immortalized luteinizing hormone-releasing hormone neurones

Abstract

Although the excitatory amino acid (EAA) receptor agonist N-methyl-D-aspartate (NMDA) can exert profound stimulatory effects on the neuroendocrine reproductive axis of Syrian hamsters, the exact relationship between NMDA receptors and LHRH neurones is unclear. In the present study, in situ hybridization histochemistry was performed on sections of hamster brain using an 35S-labelled riboprobe to the EAA receptor gene, NMDAR1. A high content of NMDA receptor mRNA was detected not only in brain areas classically associated with specific NMDA binding (for example, hippocampus and cerebral cortex) but also in the hypothalamus, in particular the ventromedial-arcuate area; diffuse hybridization of the riboprobe also occurred in the medial-septal area and diagonal band of Broca, regions of the hamster brain in which the LHRH neuronal perikarya are primarily located. In a separate experiment, RNA was extracted from immortalized LHRH neurones (GT1-1 and GT1-7 cells) and used for northern analysis with a 32P-labelled NMDAR1 riboprobe. Clear-cut hybridization occurred with RNA bands of approximately 4.2 and 4.4 kb from the two LHRH neuronal subtypes. These findings suggest that at least some of the stimulatory action of EAAs on LHRH secretion is likely to be exerted directly at the level of the LHRH neurones rather than being mediated through interneurones. Furthermore, the demonstration of abundant NMDA receptor gene expression within hypothalamic areas that lie outside the blood-brain barrier adds plausibility to the concern that EAAs of dietary origin, such as monosodium glutamate, have the capacity to perturb the normal secretory activity of neuroendocrine circuits of the hypothalamus.

Characterization of Ionotropic Glutamate Receptors in Rat Hypothalamus, Pituitary and Immortalized Gonadotropin-Releasing Hormone (GnRH) Neurons (GT1-7 Cells)

“…Functional studies also support the absence or extremely low expression of ionotropic glutamate receptors in GT1-7 cells, as GT1-7 cells were shown to be resistant to glutamate toxicity, and patch-clamp studies revealed that NMDA was unable to induce currents in GT1-7 cells. Native GnRH neurons have also been reported to be resistant to glutamate toxicity [47], which agrees with no or low expression of ionotropic glutamate receptors in native GnRH neurons [17, 18, 19, 20]. Likewise, Seeburg and Spergel [personal commun.]…”

“…Furthermore, there is abundant evidence that glutamate acts through an indirect mechanism to stimulate GnRH secretion. For instance, the aforementioned lack or low expression of NMDAR1 mRNA in GnRH neurons in male hamsters and male and female rats argues for an indirect mechanism of action [17, 18, 19, 20]. In agreement with a lack or low expression of glutamate ionotropic receptors in GnRH neurons in vivo, Ebling et al [47]recently reported that NMDA and kainate injected into the cortical area, striatum and preoptic area caused massive neuronal loss, but GnRH neurons were spared.…”

“…While the stimulatory effects on GnRH release are clear, less clear is whether glutamate acts directly or indirectly on the GnRH neuron to elicit GnRH release. The concept of an indirect action of glutamate on the GnRH neuron is supported by the absence of NMDAR1 and GluR1 mRNA or protein in GnRH neurons in male hamsters [17, 18], a finding of less than 5% colocalization of NMDAR1 mRNA in GnRH neurons in adult male and female rats [19, 20], and the absence of c- fos activation in GnRH neurons by NMDA [10, 21]. On the other hand, Gore et al [22]showed an increase in GnRH neurons containing NMDAR1 at puberty.…”

“…Using this cell line, investigators have reported the presence of NMDAR1 mRNA levels [18, 25], as well as changes in intracellular calcium and GnRH release following NMDA, kainate and AMPA stimulation [25, 26]. Thus, while in vivo studies suggest an indirect mechanism of action, results from the GT1-7 cell line suggest a possible direct action.…”

Glutamate: A Major Excitatory Transmitter in Neuroendocrine Regulation

“…These findings suggests that EAA receptors may in fact be present on GnRH neurons and that EAAs may regulate GnRH release directly. Along these lines, Urbanski et al [96] have reported that the immortalized GT1-7 GnRH neurons grown in culture contain high levels of NMDA receptor mRNA. This is at variance with the in vivo findings described earlier where less than 5% of GnRH neurons possessed NMDA recep tor mRNA [93], Clearly, more work is necessary to resolve this issue.…”