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Ghrelin stimulation of growth hormone-releasing hormone neurons is direct in the arcuate nucleus.

Osterstock G, Escobar P, Mitutsova V, Gouty-Colomer LA, Fontanaud P, Molino F, Fehrentz JA, Carmignac D, Martinez J, Guerineau NC, Robinson IC, Mollard P, Méry PF - PLoS ONE (2010)

Bottom Line: Indeed, ghrelin does not modify synaptic currents of GHRH neurons.However, ghrelin exerts a strong and direct depolarizing effect on GHRH neurons, which supports their increased firing rate.These results support the view that ghrelin related therapeutic approaches could be directed separately towards GH deficiency or feeding disorders.

View Article: PubMed Central - PubMed

Affiliation: Inserm U-661, Montpellier, France.

ABSTRACT

Background: Ghrelin targets the arcuate nucleus, from where growth hormone releasing hormone (GHRH) neurones trigger GH secretion. This hypothalamic nucleus also contains neuropeptide Y (NPY) neurons which play a master role in the effect of ghrelin on feeding. Interestingly, connections between NPY and GHRH neurons have been reported, leading to the hypothesis that the GH axis and the feeding circuits might be co-regulated by ghrelin.

Principal findings: Here, we show that ghrelin stimulates the firing rate of identified GHRH neurons, in transgenic GHRH-GFP mice. This stimulation is prevented by growth hormone secretagogue receptor-1 antagonism as well as by U-73122, a phospholipase C inhibitor and by calcium channels blockers. The effect of ghrelin does not require synaptic transmission, as it is not antagonized by gamma-aminobutyric acid, glutamate and NPY receptor antagonists. In addition, this hypothalamic effect of ghrelin is independent of somatostatin, the inhibitor of the GH axis, since it is also found in somatostatin knockout mice. Indeed, ghrelin does not modify synaptic currents of GHRH neurons. However, ghrelin exerts a strong and direct depolarizing effect on GHRH neurons, which supports their increased firing rate.

Conclusion: Thus, GHRH neurons are a specific target for ghrelin within the brain, and not activated secondary to altered activity in feeding circuits. These results support the view that ghrelin related therapeutic approaches could be directed separately towards GH deficiency or feeding disorders.

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Ghrelin did not synchronize the activity of GHRH neurons in dual patch-clamp epxeriments.A, stimulatory effects of ghrelin (10 nM) on the firing rate of two GHRH neurons recorded simultaneously. Action potential rates were calculated every 30 s. B, cumulative distributions of the frequency of the action potentials of the GHRH neurons from panel A, showing the extent of the rightward shifts induced by ghrelin. C, intervals between action potentials of the GHRH neurons from panel A, under control conditions and in the presence of ghrelin, were then used in generating the cross-correlograms shown in D. The correlations of activity were calculated within consecutive bins of 100 ms during 60 s (see Methods for further details). Dotted lines indicate the 95% confidence boundaries within which the distributions behave as random, in the absence and presence of ghrelin. E&F, same as C&D, except that random distributions of instantaneous frequencies of action potentials were generated using the properties of the experimental data, in the absence and in the presence of ghrelin. The shapes of these cross-correlograms characterizing de-correlated series of events were almost undistinguishable from the experimental curves.
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pone-0009159-g002: Ghrelin did not synchronize the activity of GHRH neurons in dual patch-clamp epxeriments.A, stimulatory effects of ghrelin (10 nM) on the firing rate of two GHRH neurons recorded simultaneously. Action potential rates were calculated every 30 s. B, cumulative distributions of the frequency of the action potentials of the GHRH neurons from panel A, showing the extent of the rightward shifts induced by ghrelin. C, intervals between action potentials of the GHRH neurons from panel A, under control conditions and in the presence of ghrelin, were then used in generating the cross-correlograms shown in D. The correlations of activity were calculated within consecutive bins of 100 ms during 60 s (see Methods for further details). Dotted lines indicate the 95% confidence boundaries within which the distributions behave as random, in the absence and presence of ghrelin. E&F, same as C&D, except that random distributions of instantaneous frequencies of action potentials were generated using the properties of the experimental data, in the absence and in the presence of ghrelin. The shapes of these cross-correlograms characterizing de-correlated series of events were almost undistinguishable from the experimental curves.

Mentions: Because GHRH neurons are such a small population [2], [29], a GHRH releasing agent such as ghrelin (or ghrelin mimetics) might trigger synchronisation between GHRH neurons [15]. This synchronicity was then studied using the dual patch clamp technique. In the example of Fig. 2A, 10 nM ghrelin simultaneously enhanced the firing rates of two GHRH neurons. The cumulative distribution of the action potential frequencies of both neurons were shifted to the right by the peptide, though to different extents (Fig. 2B). This quantitative analysis was complemented with a qualitative analysis, where crosscorrelograms were computed (Fig. 2D), as described in the Methods section, using the stretches of spike trains recorded under steady-state conditions (Fig. 2C). In brief, the correlation between these spike trains consisted in counting the spikes of the neuron “2” at the specific time delay of 100 ms with respect to the spikes of the neuron “1”. The flat shape of the crosscorrelogram obtained under control conditions indicated that neuron “2” did not fire at a preferential time before/after neuron “1”. Thus, there was no correlation between the activities of the neurons. Ghrelin induced an upward shift in the distribution as expected for a stimulatory agent, but did not induce a distinctive peak in the cross-correlogram, suggesting independence between the activities of the two neurons. Both distributions were contained within the 95%-confidence boundaries of random distributions (dotted lines, computed as stated in Methods). Furthermore, random inter-event interval distributions (Fig. 2E) were generated using the distributions of the experimental sets of data (Fig. 2D), as described in Methods. They were used to model cross-correlograms between independent series of data (Fig. 2F), which were almost undistinguishable from the experimental results (Fig. 2D). These results were typical of six similar experiments, suggesting that ghrelin induced neither a hierarchy, nor a correlation of activity, amongst GHRH neurons.


Ghrelin stimulation of growth hormone-releasing hormone neurons is direct in the arcuate nucleus.

Osterstock G, Escobar P, Mitutsova V, Gouty-Colomer LA, Fontanaud P, Molino F, Fehrentz JA, Carmignac D, Martinez J, Guerineau NC, Robinson IC, Mollard P, Méry PF - PLoS ONE (2010)

Ghrelin did not synchronize the activity of GHRH neurons in dual patch-clamp epxeriments.A, stimulatory effects of ghrelin (10 nM) on the firing rate of two GHRH neurons recorded simultaneously. Action potential rates were calculated every 30 s. B, cumulative distributions of the frequency of the action potentials of the GHRH neurons from panel A, showing the extent of the rightward shifts induced by ghrelin. C, intervals between action potentials of the GHRH neurons from panel A, under control conditions and in the presence of ghrelin, were then used in generating the cross-correlograms shown in D. The correlations of activity were calculated within consecutive bins of 100 ms during 60 s (see Methods for further details). Dotted lines indicate the 95% confidence boundaries within which the distributions behave as random, in the absence and presence of ghrelin. E&F, same as C&D, except that random distributions of instantaneous frequencies of action potentials were generated using the properties of the experimental data, in the absence and in the presence of ghrelin. The shapes of these cross-correlograms characterizing de-correlated series of events were almost undistinguishable from the experimental curves.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2820089&req=5

pone-0009159-g002: Ghrelin did not synchronize the activity of GHRH neurons in dual patch-clamp epxeriments.A, stimulatory effects of ghrelin (10 nM) on the firing rate of two GHRH neurons recorded simultaneously. Action potential rates were calculated every 30 s. B, cumulative distributions of the frequency of the action potentials of the GHRH neurons from panel A, showing the extent of the rightward shifts induced by ghrelin. C, intervals between action potentials of the GHRH neurons from panel A, under control conditions and in the presence of ghrelin, were then used in generating the cross-correlograms shown in D. The correlations of activity were calculated within consecutive bins of 100 ms during 60 s (see Methods for further details). Dotted lines indicate the 95% confidence boundaries within which the distributions behave as random, in the absence and presence of ghrelin. E&F, same as C&D, except that random distributions of instantaneous frequencies of action potentials were generated using the properties of the experimental data, in the absence and in the presence of ghrelin. The shapes of these cross-correlograms characterizing de-correlated series of events were almost undistinguishable from the experimental curves.
Mentions: Because GHRH neurons are such a small population [2], [29], a GHRH releasing agent such as ghrelin (or ghrelin mimetics) might trigger synchronisation between GHRH neurons [15]. This synchronicity was then studied using the dual patch clamp technique. In the example of Fig. 2A, 10 nM ghrelin simultaneously enhanced the firing rates of two GHRH neurons. The cumulative distribution of the action potential frequencies of both neurons were shifted to the right by the peptide, though to different extents (Fig. 2B). This quantitative analysis was complemented with a qualitative analysis, where crosscorrelograms were computed (Fig. 2D), as described in the Methods section, using the stretches of spike trains recorded under steady-state conditions (Fig. 2C). In brief, the correlation between these spike trains consisted in counting the spikes of the neuron “2” at the specific time delay of 100 ms with respect to the spikes of the neuron “1”. The flat shape of the crosscorrelogram obtained under control conditions indicated that neuron “2” did not fire at a preferential time before/after neuron “1”. Thus, there was no correlation between the activities of the neurons. Ghrelin induced an upward shift in the distribution as expected for a stimulatory agent, but did not induce a distinctive peak in the cross-correlogram, suggesting independence between the activities of the two neurons. Both distributions were contained within the 95%-confidence boundaries of random distributions (dotted lines, computed as stated in Methods). Furthermore, random inter-event interval distributions (Fig. 2E) were generated using the distributions of the experimental sets of data (Fig. 2D), as described in Methods. They were used to model cross-correlograms between independent series of data (Fig. 2F), which were almost undistinguishable from the experimental results (Fig. 2D). These results were typical of six similar experiments, suggesting that ghrelin induced neither a hierarchy, nor a correlation of activity, amongst GHRH neurons.

Bottom Line: Indeed, ghrelin does not modify synaptic currents of GHRH neurons.However, ghrelin exerts a strong and direct depolarizing effect on GHRH neurons, which supports their increased firing rate.These results support the view that ghrelin related therapeutic approaches could be directed separately towards GH deficiency or feeding disorders.

View Article: PubMed Central - PubMed

Affiliation: Inserm U-661, Montpellier, France.

ABSTRACT

Background: Ghrelin targets the arcuate nucleus, from where growth hormone releasing hormone (GHRH) neurones trigger GH secretion. This hypothalamic nucleus also contains neuropeptide Y (NPY) neurons which play a master role in the effect of ghrelin on feeding. Interestingly, connections between NPY and GHRH neurons have been reported, leading to the hypothesis that the GH axis and the feeding circuits might be co-regulated by ghrelin.

Principal findings: Here, we show that ghrelin stimulates the firing rate of identified GHRH neurons, in transgenic GHRH-GFP mice. This stimulation is prevented by growth hormone secretagogue receptor-1 antagonism as well as by U-73122, a phospholipase C inhibitor and by calcium channels blockers. The effect of ghrelin does not require synaptic transmission, as it is not antagonized by gamma-aminobutyric acid, glutamate and NPY receptor antagonists. In addition, this hypothalamic effect of ghrelin is independent of somatostatin, the inhibitor of the GH axis, since it is also found in somatostatin knockout mice. Indeed, ghrelin does not modify synaptic currents of GHRH neurons. However, ghrelin exerts a strong and direct depolarizing effect on GHRH neurons, which supports their increased firing rate.

Conclusion: Thus, GHRH neurons are a specific target for ghrelin within the brain, and not activated secondary to altered activity in feeding circuits. These results support the view that ghrelin related therapeutic approaches could be directed separately towards GH deficiency or feeding disorders.

Show MeSH
Related in: MedlinePlus