Profiling neurotransmitter receptor expression in mouse gonadotropin-releasing hormone neurons using green fluorescent protein-promoter transgenics and microarrays.
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.
Todman
M G
MG
Laboratory of Neuroendocrinology, The Babraham Institute, Cambridge, UK.
Han
S-K
SK
Herbison
A E
AE
D000818:Animals; D004347:Drug Interactions; D005260:Female; D019004:Galanin / Q000031:analogs & derivatives / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000502:physiology*; D007987:Gonadotropin-Releasing Hormone / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D006727:Hormone Antagonists / Q000494:pharmacology; D017403:In Situ Hybridization / Q000379:methods; D066298:In Vitro Techniques; D008297:Male; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D015287:Neurokinin B / Q000031:analogs & derivatives / Q000494:pharmacology; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods; D009842:Oligopeptides / Q000494:pharmacology; D018408:Patch-Clamp Techniques / Q000379:methods; D011301:Preoptic Area / Q000166:cytology*; D012333:RNA, Messenger / Q000096:biosynthesis; D017981:Receptors, Neurotransmitter / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D013004:Somatostatin / Q000494:pharmacology; D053561:Somatostatin-28; D013373:Substance P / Q000031:analogs & derivatives / Q000494:pharmacology; D013779:Tetrodotoxin / Q000494:pharmacology
D006727:Hormone Antagonists; D009842:Oligopeptides; D012333:RNA, Messenger; D017981:Receptors, Neurotransmitter; C074975:galantide; D049452:Green Fluorescent Proteins; D013373:Substance P; D007987:Gonadotropin-Releasing Hormone; D013779:Tetrodotoxin; D013004:Somatostatin; D053561:Somatostatin-28; D015287:Neurokinin B; C038393:neuromedin B; D019004:Galanin; C057022:iturelix