Somatostatin Inhibits Gonadotropin Releasing Hormone Neuronal Activities in Juvenile Mice

Seon Ah Park; Janardhan P. Bhattarai; Seong Kyu Han

doi:10.3803/EnM.2011.26.3.210

Journal List > Endocrinol Metab > v.26(3) > 1085887

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Park, Bhattarai, and Han: Somatostatin Inhibits Gonadotropin Releasing Hormone Neuronal Activities in Juvenile Mice

Original Article

Endocrinol Metab 2011;26(3):210-217.

Published online: 5 September 2011

DOI: https://doi.org/10.3803/EnM.2011.26.3.210

Somatostatin Inhibits Gonadotropin Releasing Hormone Neuronal Activities in Juvenile Mice

Seon Ah Park, Janardhan P. Bhattarai, Seong Kyu Han

Department of Oral Physiology, School of Dentistry & Institute of Oral Bioscience, Chonbuk National University, Jeonju, Korea

Corresponding author: Seong Kyu Han Department of Oral Physiology & Institute of Oral Bioscience, School of Dentistry, Chonbuk National University, Deokjindong 1-ga, Deokjin-gu, Jeonju 561-756, Korea Tel: +82-63-270-4030, Fax: +82-63-270-4028, E-mail: skhan@jbnu.ac.kr

Received 29 December 2010 Accepted 19 April 2011

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

초록

Background

The gonadotropin releasing hormone (GnRH) neurons perform a pivotal function in the central regulation of fertility. Somatostatin (SST) is an important neuromodulatory peptide in the central nervous system and alters neuronal activities via G protein-coupled SST receptors. A number of studies have shown that SST modulates the reproductive axis at the hypothalamic level. However, the precise action mechanisms of SST and related receptor subtypes have yet to be fully understood. In this study, we evaluated the direct effects of SST on GnRH neurons in juvenile mice.

Methods

Juvenile (postnatal days, < PND 30) GnRH-GFP transgenic mice expressing green fluorescent protein were used in this study. Acute coronal brain slices containing the preoptic area were prepared and all identified GnRH neurons were recorded using the gramicidin perforated-patch clamp technique; type II SST receptor (SSTR2) mRNA expression was evaluated via single cell reverse transcription-polymerase chain reaction (RT-PCR).

Results

SST caused membrane hyperpolarization, depolarization, no response, or membrane hyperpolarization with a reduction of action potential. Most (57.7%, 30/52) of the GnRH neurons tested were hyperpolarized by SST and this SST-induced hyperpolarization was found to be concentration-dependent. The percentage of responses, membrane potential changes (MPC), and resting membrane potential (RMP) by SST were not significantly different in juvenile male and female GnRH neurons. The SST-induced hyperpolarization was maintained in the presence of tetrodotoxin (TTX), a sodium channel blocker, and an amino acid blocking cocktail (AABC) containing AP-5 (NMDA receptor antagonist), CNQX (non-NMDA glutamate receptor antagonist), picrotoxin (GABAA receptor antagonist), and strychnine (glycine receptor antagonist). SSTR2 mRNA was expressed on 10 (38%) among 26 GnRH neurons. Seglitide, an SSTR2 agonist, mimicked this SST-induced hyperpolarization (11/23 47.8%) and this response was maintained in the presence of TTX and AABC.

Conclusion

Our data show that SST can exert potent inhibitory action against GnRH neuronal excitability via SSTR2 activation in juvenile mice.

Keywords: GnRH neuron, Somatostatin, Perforated-patch clamp

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Fig. 1.

Effects of somatostatin (SST) on juvenile gonadotropin releasing hormone (GnRH) neurons in mice at gramicidin perforated current clamp mode. Representative traces showing membrane hyperpolarization (A. PND18, RMP = -72 mV), membrane depolarization (B. PND28, RMP = -63 mV), no response (C. PND28, RMP = -55 mV) and surcease of action potential followed by membrane hyper-polarization (D. PND14, RMP = -62 mV) by bath application of 300 nM SST. E. Stag col-umn showing percentage response by SST (H, hyperpolarization; N, no response; D, depolarization).

Fig. 2.

Concentration-dependent hyperpolarizing effect of SST on juvenile GnRH neurons. A. Representative trace showing concentration dependent membrane hyperpolarization by application of 10, 30, 100, and 300 nM SST (PND28, RMP = -62.0 mV). B. Concentration-response relationship. ∗represents P < 0.05.

Fig. 3.

A. Percentage response by 300 nM SST in juvenile males and females. R, responded; NR, not responded. B. Comparison of the mean membrane potential changes between males and females by 300 nM SST. Found no significant difference (P > 0.05; one way ANOVA).

Fig. 4.

SST acts on the postsynaptic GnRH neurons directly. A. A representative trace showing hyperpolarization by successive application of 300 nM SST (PND27, RMP = -57.3 mV). Break bar depicts a time interval of 15-20 minutes. B. A representative trace showing hyperpolarization by SST and re-mains persisted in the presence of amino acid blocking cocktail (AABC) including AP-5 (20 µM, an NMDA receptor antagonist), CNQX (10 µM, a non-NMDA glutamate receptor antagonist), picrotoxin (50 µM, a GABA A receptor antag-onist), and strychnine (2 µM, a glycine receptor antagonist) with TTX (0.5 µM, a voltage-gated Na⁺ channel blocker) in a juvenile GnRH neuron resting at -59.6 mV. PND29. C. Relative potential changes of the 2nd response and response in the presence of AABC. ∗ and NS represent P < 0.05 and not significant, respectively (one sample t-test).

Fig. 5.

A. An example of SSTR2 mRNA expression on GnRH neurons. GAPDH, a housekeeping gene was used to conform the harvested GnRH neurons (n = 8). L (100 bp ladder), 1-8 (harvested GnRH neurons), NC (negative control, harvested GnRH neurons reacted without reverse transcriptase), PC (positive control, cDNA of total brain extract). B. A representative trace showing hyperpolarization by seglitide (SEG) (1 µM), a SSTR2 agonist from a GnRH neuron resting at -70 mV depicting mimicry of SST-induced membrane hyperpolarization (PND28). C. A representative traces showing hyperpolarization by SEG in the presence of AABC. PND13, RMP = -72 mV. D. Bar graph showing the mean membrane hyperpolarization to SEG (1 µM) and with AABC (P > 0.05).

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