Elsevier

Neuroscience

Volume 132, Issue 3, 2005, Pages 703-712
Neuroscience

Profiling neurotransmitter receptor expression in mouse gonadotropin-releasing hormone neurons using green fluorescent protein-promoter transgenics and microarrays

https://doi.org/10.1016/j.neuroscience.2005.01.035Get rights and content

Abstract

The definition of neurotransmitter receptors expressed by individual neuronal phenotypes is essential for our understanding of integrated neural regulation. We report here a single-neuron strategy using green fluorescent protein (GFP)-promoter transgenic mice and oligonucleotide microarrays that has enabled us to provide a qualitative profile of the neurotransmitter receptors expressed by the gonadotropin- releasing hormone (GnRH) neurons, critical for the neural regulation of fertility. Acute brain slices were prepared from adult female GnRH-GFP transgenic mice and single GnRH neurons identified and patched. The contents of GnRH neurons underwent reverse transcription and cDNA amplification using the switch mechanism at the 5′ end of RNA templates system, and hybridization to mouse gene oligonucleotide arrays. Fifty different neurotransmitter receptor subunit mRNAs were detected in GnRH neurons. Many of the classical amino acid and aminergic receptors were present in addition to 14 distinct, and in most cases novel, neuropeptidergic receptor signaling families. Four of the latter were selected for functional validation with gramicidin-perforated patch-clamp electrophysiology. Galanin, GnRH and neuromedin B were all found to exert direct depolarizing actions upon GnRH neurons whereas somatostatin induced a potent hyperpolarizing response. These studies demonstrate a relatively straightforward approach for transcriptome profiling of specific neuronal phenotypes. The stimulatory actions of GnRH and galanin upon GnRH neurons found here indicate that positive ultrashort feedback loops exist among the GnRH neuronal population.

Section snippets

Animals

All experimentation was approved by both the Babraham Institute and University of Otago Animal Welfare and Ethics Committees. Experiments were designed to minimize the number of mice used and any suffering. Mice were maintained under 12-h lighting conditions (lights on 07:00 h) with food and water available ad libitum. Transgenic GnRH-GFP (C57BL6/J) mice (Spergel et al., 1999) were kindly provided by Dr. Dan Spergel (Department of Paediatrics, University of Chicago, IL, USA) and bred at the

Single cell RT-PCR for GnRH

All 10 of the fluorescent cells harvested from a GnRH–GFP mouse were shown to express GnRH transcripts following nested single cell RT-PCR whereas the no-RT and water controls did not generate an amplicon (Fig. 1). This demonstrates the very high likelihood that 100% of the fluorescent cells harvested for the microarray study were GnRH neurons.

Microarray profile

The hybridizations from the four pools of GnRH neurons resulted in the positive hybridization of 1007–1975 spots on each of the microarrays. Although the

Discussion

We report here a comparatively straightforward strategy for generating a qualitative profile of the neurotransmitter receptor mRNAs expressed by a defined neuronal cell population. The present microelectrode strategy provides an alternative to laser capture microdissection and has the advantage of being able to access mRNA from living cells without the need for any prior fixation or histochemical staining that may have deleterious effects on RNA quality (Hinkle et al., 2004). A further major

Acknowledgments

We thank Tom Freeman and Debbie Williams (Medical Research Council, Hinxton, UK) for the microarrays and Dan Spergel (Department of Pediatrics, University of Chicago, USA) for generous provision of the GnRH-GFP mice. Drs. Christine Jasoni and Rebecca Campbell are thanked for critical review of the manuscript. Research supported by the Biotechnology and Biological Sciences Research Council (UK) and The Wellcome Trust.

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