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Anatomy and Human Biology Group, King’s College London (S.L.D.), The Strand, London WC2R 2LS; and Department of Neurobiology, The Babraham Institute (S.M.L.), Babraham, Cambridge CB2 4AT, United Kingdom
Address all correspondence and requests for reprints to: Suzanne L. Dickson, The Department of Physiology, University of Cambridge, Downing Street, Cambridge CB2 3EG, United Kingdom. E-mail: sld20{at}cam.ac.uk
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Abstract |
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Introduction |
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It is unlikely that the sole action of GHRP-6 within the central nervous system is to activate GRF neurons because there is a large synergistic release of GH when GHRP-6 and GRF are administered concomitantly (1). Both our electrophysiological studies and the functional mapping studies with Fos immunoreactivity have led us to speculate that the cells activated by GHRP-6 may be a heterogeneous population, including cells other than the GRF-containing cells. Notably, in GHRP-6-injected rats, there is a large cluster of Fos-positive cells in the ventromedial aspects of the arcuate nucleus, where few GRF cells have been described (9, 10). Studies using the retrograde tracer Fluorogold indicate that between 68% and 82% of the cells excited by GHRP-6 project outside the blood-brain barrier, probably at the median eminence and are therefore likely to be neurosecretory neurons (11), leaving a further 18–32% that could not be identified as neurosecretory cells. In electrophysiological studies, GHRP-6 has been shown to elicit a predominantly excitatory response in putative neurosecretory cells (that is, cells antidromically identified as projecting to the median eminence); however, of the cells that did not fulfill the criteria for antidromic identification, the predominant response was inhibitory (5).
In this study, we investigated further the neurochemical identity of arcuate neurons activated by GHRP-6. To maintain the integrity of the response, we chose to avoid immunocytochemistry because this would require pretreating the animals with the axonal transport inhibitor colchicine to bring levels of neuropeptides to detectable levels. Instead, we used an in situ hybridization protocol in which alternate sections were probed for c-fos messenger RNA (mRNA), and consecutive sections were probed for mRNAs for either GRF, neuropeptide Y (NPY), tyrosine hydroxylase (TH; a marker of dopaminergic cells in this region), POMC, or somatostatin.
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Materials and Methods |
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On the day of the experiment, conscious rats were injected iv with either 50 µg GHRP-6 (Bachem, Saffron Walden, UK; n = 5) or an equal volume (0.2 ml) saline vehicle (n = 5). Thirty minutes later they were terminally anesthetized with sodium pentobarbitone (60 mg/kg BW; ip) and were perfused transcardially with heparinized isotonic saline (5 min) followed by 4% paraformaldehyde in 0.1 M phosphate buffer (PB) (pH 7.4; 15–20 min). This time point was chosen because it is the expected time of c-fos mRNA induction following GHRP-6 injection, corresponding to the subsequent synthesis of the Fos protein that can be detected at 90 min following GHRP-6 injection (4). Brains were removed and dehydrated before paraffin embedding. For each brain, three sets of 10 consecutive coronal sections, 7 µm thick, were cut and collected onto subbed slides.
In situ hybridization procedure
For in situ hybridization, single-stranded antisense
oligonucleotide probes were made using previously tested sequences. The
probe sequences were complimentary to the nucleotides spanning the
following coding regions: c-fos (45-mer) bases 141–185
(12), GRF (30-mer) bases 184–213 (13), NPY (30-mer) bases 192–221
(14), TH (30-mer) bases 1223–1252 (15), POMC (48-mer) bases 96–111
(16), and somatostatin (30-mer) bases 98–127 (17). The probes were 3'
end-labeled using terminal deoxynucleotidyl transferase (Pharmacia,
Milton Keynes, UK) and [
-35S]deoxycytidine ATP (NEN Du
Pont, Stevenage, UK) to specific activities of 3–5 x
106 dpm/pmol and purified using a Sephadex G50 column.
All solutions used until posthybridization washes were made using buffers or water pretreated with 0.1% diethylpyrocarbonate and autoclaved. Following fixation for 30 min with 4% paraformaldehyde in 0.1 M PB (pH 7.4) sections were rinsed twice in 0.1 M PBS before treatment three times with 0.2% glycin followed by 0.25% acetic anhydride in 0.1 M triethanolamine/0.9% NaCl for 10 min at room temperature. Sections were dehydrated through a series of ethanol solutions ending with absolute ethanol. Dried sections were hybridized in the following buffer: 4x SSC (standard sodium chloride-sodium citrate), 50% formamide, 1x Denhardt’s solution, 500 µg/ml sheared salmon testis DNA, 10% dextran sulphate, and 0.3% ß-mercaptoethanol. 35S-labeled probes were used at an approximate concentration of 4 fmol/µl, which gave a total of 3 x 105 dpm per slide. Each of the probes were successfully competed for by a 100 times excess of the relevant unlabeled oligonucleotide. Hybridization was carried out in a moist chamber overnight at 37 C. Hybridized slides were dipped briefly in 3 washes of 1x SSC at 55 C for 30 min and finally in 1x SSC for 60 min at room temperature. Slides were then washed briefly in 300 mM ammonium acetate, then 70% ethanol and air dried before processing for emulsion autoradiography. Ilford K-5 nuclear track emulsion (Ilford, Knutsford, Cheshire, UK) was used and the slides were exposed in the dark for 9 weeks (c-fos), 5 weeks (NPY), 3 weeks (GRF, somatostatin, and TH), or 2 weeks (POMC). Slides were developed with Kodak D-19 developer (Kodak, Hemel Hempstead, UK) for 2.5 min at 20 C, fixed with Ilfohypam (Ilford) for 10 min, and washed in tap water. Before coverslipping, the sections were counterstained with methylene blue.
Cell counting and analysis
For each brain, three sets of 10 consecutive sections, including
the anterior, medial, and posterior extent of arcuate nucleus, were
examined using a bright field microscope coupled to a camera
lucida. A statistical comparison (Mann-Whitney U test)
was made of the mean number of cells per section expressing
c-fos mRNA in the GHRP-6-treated brains with the control
brains; for each of the three sets of sections, cells expressing
c-fos mRNA were counted unilaterally in the arcuate nucleus
in the five sections hybridized with the c-fos probe. The
cellular colocalization of c-fos mRNA to neurons expressing
mRNAs for the other neurochemical markers was determined by
superimposing serial maps of pairs of sections. Colabelled neurons in
the adjacent section were identified as those displaying a partial or
total overlap in the cluster of silver grains covering the cell
cytoplasm. The data are expressed as mean number of cells per section
per brain.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). The majority of cells expressing c-fos in
GHRP-6-injected rats were located in the ventral portions of the
arcuate nucleus, in agreement with previous studies using centrally or
systemically administered GHRP-6 followed by Fos immunocytochemistry
(4, 5). Occasional scattered cells expressing c-fos mRNA
were present in other hypothalamic structures examined, including the
neighboring ventromedial nucleus and the periventricular nucleus but
there was no evidence of selective activation of discrete populations
of cells in these regions by GHRP-6.
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). NPY cells were scattered throughout the
arcuate nucleus with a dense cluster in the most ventromedial aspects.
Cells expressing somatostatin mRNA were located predominantly in the
dorsomedial and ventromedial arcuate nucleus. A separate population of
somatostatin neurons were clearly visible in the periventricular
nucleus of the hypothalamus in some rostral sections not included in
this analysis. TH mRNA-positive neurons were clustered in dorsomedial
and ventrolateral aspects of the arcuate nucleus. The distribution of
these neurochemically defined cells was in agreement with those
previously described (18, 19 and references therein).
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). Despite the similar distribution and greater number
per section (38 ± 3 cells/section) of POMC mRNA-containing
neurons, only 6 ± 1% of these also expressed c-fos
mRNA (Fig. 3C). The largest population of neurochemically identified
neurons in this study were those containing NPY mRNA (41 ± 3
cells/section). Of these, almost one third (30 ± 3%) also
contained c-fos mRNA in the consecutive section (Fig. 3B).
Somatostatin (14 ± 1 cells/section) and TH (14 ± 2
cells/section) mRNA-containing neurons only colocalized
c-fos in 7 ± 2% and 14 ± 3% of the cells,
respectively (Fig. 4).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recently, Kamegai and colleagues (20) used a similar approach to determine whether cells expressing c-fos mRNA in rats injected with KP-102 (a peptidyl GHRP-6 mimetic) could be identified as expressing GRF mRNA on the consecutive section. Although the proportion of cells expressing c-fos mRNA which were identified as GRF-containing cells correspond to that observed in the present study (23% in both GHRP-6 and KP-102-injected rats), the proportion of GRF cells expressing c-fos mRNA was very different in each case (38% for GHRP-6 injected rats and 20% in KP-102-injected rats) (20). Thus, it would appear that GHRP-6 and KP-102 activate a subpopulation of GRF cells, that more GRF neurons are activated by GHRP-6 than by KP-102, and that both of these GH secretagogues also activate non-GRF cells in the arcuate nucleus.
The GRF- and TH-containing cells in the arcuate nucleus have accepted adenohypophysiotropic status, and both of these systems project to the median eminence where they release factors into the portal system of capillaries for transport to the anterior pituitary. Thus, activation of GRF- and, to a lesser extent, TH-containing neurons by GHRP-6 is consistent with a previous study demonstrating increased expression of Fos protein in neurons that project outside of the blood-brain barrier and are therefore likely to be neurosecretory (11). Because as many as 82% of the cells activated by GHRP-6 were found to project outside of the blood-brain barrier, it seems unlikely that the GRF- (and the TH-) containing cells are the only neurosecretory cells activated by GHRP-6. Although NPY is present in portal blood in concentrations that are higher than in the peripheral circulation (21), there appears to be some doubt as to whether the arcuate NPY cells are true neurosecretory adenohypophysiotropic cells that project to the external zone of the median eminence (22, 23). In rats treated parenterally with monosodium glutamate (this causes cytotoxic lesion of 80–90% of arcuate cells that project outside of the blood-brain barrier), there was a depletion of NPY-immunoreactive cell bodies in this region, consistent with the idea that NPY cells project outside the blood-brain barrier. However, monosodium glutamate treatment did not alter the NPY fibers in the external layer of the median eminence, suggesting that these fibers may originate from elsewhere, perhaps as ascending noradrenergic fibers from the brain stem that colocalize NPY (18). Thus, although the majority of cells activated by GHRP-6 project outside the blood-brain barrier (11), it remains to be determined whether the NPY population project to the median eminence or whether they are open to the circulation in some other way. In this respect, it is interesting that arcuate NPY neurons are believed to detect circulating levels of leptin (24). Because the majority of cells activated by GHRP-6 project outside of the blood-brain barrier, it is not surprising that populations of cells in the arcuate nucleus such as the POMC and somatostatin-containing cells (which have not been identified as projecting to the external layer of the median eminence) (18, 19), did not express c-fos mRNA following GHRP-6 injection.
In this study we provide neurochemical evidence to corroborate our hypothesis that the arcuate cells activated by GHRP-6 are likely to be a heterogeneous population of neurons. This hypothesis was first suggested from our electrophysiological studies (in vivo), because it was evident that GHRP-6 injection resulted in a variety of different responses recorded at the cell bodies of arcuate neurons: of the cells antidromically identified as projecting to the median eminence (the putative neurosecretory cells) the predominant response was excitatory and for the arcuate cells not antidromically identified as projecting to the median eminence (the majority of which are unlikely to be neurosecretory cells), the predominant response was inhibitory (5).
One of the limitations of the present study is that it is not possible to determine to what extent the arcuate cells expressing c-fos mRNA following GHRP-6 injection express more than one of the neuropeptide markers. For example, TH and NPY appear to colocalize with GRF in a subpopulation of arcuate neurons (25, 26). Thus, activation of GRF neurons by GHRP-6 could account for a small proportion of the c-fos-positive cells expressing NPY mRNA and/or the TH mRNA in the present study. The latter is consistent with a previous double immunocytochemical study, demonstrating that approximately 7% of the cells expressing Fos protein following GHRP-6 injection are TH-positive (11).
Part of the central GH-releasing action of GHRP-6 may be accounted for by increased NPY release into portal blood, because NPY stimulates GH release when administered systemically (27). When administered by icv injection, NPY inhibits GH secretion (28), and so increased central release of NPY in response to GHRP-6 injection would be expected to decrease GH secretion. Indeed, increased central release of NPY may account for the decrease in GH secretion observed following intracerebroventricular injection of GHRP-6 in the rat (29), although this response may be species specific (30).
The NPY neurons in the arcuate nucleus have been implicated in a number of important physiological mechanisms including the regulation of feeding behavior (31), sexual behavior (32) and in the regulation of various endocrine and metabolic functions (33, 34, 35). The arcuate NPY cells give rise to a major hypothalamic pathway that sends afferent fibers to the anterior hypothalamus, the preoptic area, the paraventricular nucleus (PVN), as well as the ventromedial and dorsomedial hypothalamus (36, 37). Multiple daily injections of NPY into the PVN increases daily food intake and body weight (38). Activation of NPY neurons by GHRP-6 administration may therefore provide a mechanism to explain the increase in feeding behavior in rats following GHRP-6 administration (39). NPY infused directly into the PVN has also been shown to increase the secretion of both corticosterone and ACTH (40, 41, 42, 43). In the PVN, there is ultrastructural evidence for NPY-immunoreactive contacts with CRH-immunoreactive cell bodies (44), suggesting that NPY cells make direct contacts with CRF cells within the PVN. GHRP-6 (and other peptide and nonpeptide mimetics) have also been shown to increase ACTH and cortisol secretion in many species (45, 46, 47).
Recent studies implicating the arcuate NPY neurons in neuroendocrine GH regulation have focused on their likely role in mediating GH auto-feedback control, because GH receptor mRNA has been located on the arcuate NPY cells (48, 49). In hypophysectomized rats, GH administration has been shown to induce c-fos expression in NPY neurons in the arcuate nucleus (50), raising the possibility that the induction of the c-fos gene in response to GHRP-6 could be mediated by a central action of released GH, rather than a direct response to the central action of GHRP-6. This interpretation seems unlikely because GH-deficient dw/dw rats and lit/lit mice show an increase in the number of cells expressing Fos protein in the arcuate nucleus following systemic GHRP-6 injection (51). Furthermore, icv injection of very low doses of GHRP-6 (0.1 µg) that do not release GH directly into the lateral ventricle of conscious rats elicits an increase in the number of cells expressing Fos protein in the arcuate nucleus in an identical manner to systemic injection of GHRP-6; both the number and distribution of cells activated were the same, providing evidence to suggest that GHRP-6 acts directly within the central nervous system to activate cells in this region (5). More recently, we demonstrated that GHRP-6 activates arcuate cells directly, because excitatory and inhibitory responses were recorded at the cell bodies of arcuate neurons in vitro from a slice of arcuate nucleus during bath application of GHRP-6 (52). Collectively these data suggest that GHRP-6 acts within the central nervous system (including a direct action on cells in the arcuate nucleus) to activate neurons in this region (including the NPY and GRF populations), and that the induction of the c-fos gene is not mediated secondarily by increased GH secretion. Now that the GH secretagogue receptor has been cloned (53), it will be important to establish whether the arcuate cells expressing the GH secretagogue receptor include the GRF and NPY populations, and hence, whether the GH secretagogues act directly on these cells.
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Acknowledgments |
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Footnotes |
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2 A Biotechnology and Biological Sciences Research Council
Postdoctoral Fellow.
Received August 26, 1996.
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. Three sets of consecutive sections are illustrated
that correspond to three different levels of the arcuate nucleus:
bregma -2.12 mm (left), -2.56 mm
(middle), and -3.14 mm (right) (54). 3v,
Third ventricle.
