The first recordings of changes in electrical activity following GH secretagogue administration were incorporated into a series of experiments in which attempts were being made to record from identified GHRH neurones in the arcuate nucleus. In addition to the GHRH population, the arcuate nucleus contains a number of different cell types, including other neuroendocrine cells (e.g. the tuberoinfundibular dopamine neurones) and many non-neuroendocrine cells. Thus, it was first necessary to determine whether the cells activated by the GH secretagogues are neuroendocrine cells. This can be achieved using in vivo electrophysiological recording techniques in which arcuate cells projecting to the median eminence are identified by antidromic identification. Since GHRH neurones are neuroendocrine cells that project from the arcuate nucleus to the median eminence, the antidromically identified cells will include GHRH-neurones.
To distinguish GHRH neurones from other neuroendocrine arcuate cells at the time of recording, several criteria were established based upon how GHRH neurones would be expected to behave following electrical stimulation of discrete brain structures. Thus, for example, stimulation of the periventricular nucleus (a region known to contain the somatostatin neurones that form part of the hypothalamic GH pulse generating mechanism) caused an inhibitory response during electrical stimulation followed by excitation following the end of stimulation. This inhibition/excitation response may account for the inhibition/rebound release of GH secretion that occurs during/following either a somatostatin infusion or, indeed, during/after electrical stimulation of the periventricular nucleus. Another criterion used in these studies was electrical stimulation of the basolateral amygdala, previously shown to stimulate GH secretion without increasing the release of any other pituitary hormone.
Thus, in summary, putative GHRH neurones were identified in these studies that project to the median eminence show an inhibition/excitation response during/after electrical stimulation of the periventricular nucleus, and were excited during electrical stimulation of the basolateral amygdala. Cells fulfilling all of these criteria for identification as putative GHRH neurones showed excitatory responses following systemic injection of GHRP-6, providing the first indication that the GH secretagogues appear to be activating the GHRH neurones. If, indeed, the GH secretagogues are targeting the GHRH neurones we might predict that their actions will be fairly selective for a sub-population of the neuroendocrine cells in the arcuate nucleus. Thus, in subsequent electrophysiological studies, we set out to characterize the effects of systemic GHRP-6 injection on the firing rate of both antidromically identified cells (the majority of which are likely to be neuroendocrine cells) and cells that did not fulfil the criteria for antidromic identification (largely non-neuroendocrine cells). The predominant response recorded at the cell bodies of the neuroendocrine cells was excitatory. By contrast, of the cells recorded that did not fulfil the criteria for antidromic identification the predominant response was inhibitory. Thus, the excitatory actions of GHRP-6 within the arcuate nucleus do indeed appear to be fairly selective for the neuroendocrine cells, consistent with the idea that the target population of cells for GH secretagogue actions include the GHRH population.
That the majority of cells activated by GH secretagogues are neuroendocrine cells was confirmed in neuroanatomical studies in which GHRP-6-induction of Fos was detected in rats given an intravenous injection five days previously with the retrograde tracer Fluorogold. When injected by this route, Fluorogold does not cross the blood-brain barrier and is not transported synaptically, rather it is taken up by neurones that project to areas supplied by fenestrated capillaries or to the periphery. Thus, retrogradely-labelled cells were identified in all hypothalamic nuclei that contain neuroendocrine cells. In the arcuate nucleus, the majority of cells expressing Fos protein following GHRP-6 injection (68-82%) were retrogradely labelled with Fluorogold. Thus, consistent with the electrophysiological data, the majority (but not all) arcuate cells activated by GHRP-6 appear to be neuroendocrine cells. If our hypothesis is correct that the neuroendocrine cells activated by GH secretagogues include the GHRH-containing cells, we might expect that these cells would also be subject to inhibitory control by central somatostatin pathways. Somatostatin receptors are present on GHRH-containing cells in this region and we have demonstrated that electrical stimulation in the region of the somatostatin cell bodies in the periventricular nucleus suppresses the activity of putative GHRH-containing cells. Indeed, in electrophysiological studies in vitro we found that the GHRP-6-responsive cells recorded from the arcuate nucleus of a hypothalamic slice preparation were also inhibited by bath application of somatostatin. Also, the induction of Fos protein by GH secretagogues is attenuated by systemic or central injection of Sandostatin, a long acting somatostatin analogue.
This suggests that the central actions of the GH secretagogues are subject to inhibition by central somatostatin pathways, possibly reflecting an inhibitory effect of somatostatin on GHRH producing cells. Interestingly this was not seen in mice with disrupted somatostatin type 2 receptor, indicating that the inhibitory effect of central somatostatin pathways on GH secretagogue-induced Fos protein expression is mediated by type 2 receptors. From both the heterogeneity of the electrophysiological responses to GHRP-6 and the demonstration that most (but not all) of the cells activated are neuroendocrine cells in the arcuate nucleus, it seems clear that the GH secretagogues are not targeting a single population of cells in the arcuate nucleus.
Direct evidence suggesting that the target cells for GH secretagogue action include the GHRH neurones was provided by a study in which we investigated the neurochemical identity of the arcuate cells expressing c-fos mRNA following systemic GHRP-6 injection. Of the cells detected that express c-fos mRNA following systemic GHRP-6 injection, almost 25% could be identified as expressing GHRH mRNA on the consecutive section, indicating that a sub-population of the GHRH cells are activated by the GH secretagogues. Consistent with this finding the GH secretagogue, hexarelin, has been shown to stimulate GHRH release into portal blood of sheep. However, the central GH-releasing activity of the GH secretagogues cannot be explained solely by increased GHRH release since this would not explain the large synergy when GHRH and GHRP-6 are administered concomitantly. Rather the central GH-releasing actions of the GH secretagogues must include GHRH-independent effects. It emerged that the GHRH neurones were not the only population of cells in the arcuate nucleus to be activated following systemic GHRP-6 injection. In this study we determined whether the cells expressing c-fos mRNA following GHRP-6 injection also express mRNAs for neurochemical markers for substances known to be expressed in discrete populations of cells within the arcuate nucleus; these include neuropeptide Y (NPY), pro-opiomelanocortin (POMC), tyrosine hydroxylase (present in dopamine neurones) and somatostatin.
The only other population of cells to show considerable activation following GH secretagogue administration in this study were the NPY-containing cells; approximately 50% of the cells expressing c-fos mRNA could be identified as containing NPY mRNA on the consecutive section. Importantly, not all NPY cells were activated by the GH secretagogues since approximately 30% of the cells expressing NPY were identified as c-95-positive on the consecutive section.
Our website is not responsible for the information contained by this article. Articleinput.com is a free articles resource thus practically any visitor can submit an article. However if you notice any copyrighted material, please contact us and we will remove the article(s) in discussion right away.
Note: This article was sent to us by: Harry S. at 12172009
1. The hypothalamus and other neuronal markers
© 2009 ArticleInput.com.