Refined anatomical isolation of functional sleep circuits exhibits distinctive regional and circadian gene transcriptional profiles

doi:10.1016/j.brainres.2009.02.083

Brain Research

Volume 1271, 19 May 2009, Pages 1-17

https://doi.org/10.1016/j.brainres.2009.02.083 Get rights and content

Abstract

Powerful new approaches to study molecular variation in distinct neuronal populations have recently been developed enabling a more precise investigation of the control of neural circuits involved in complex behaviors such as wake and sleep. We applied laser capture microdissection (LCM) to isolate precise brain nuclei from rat CNS at opposing circadian time points associated with wake and sleep. Discrete anatomical and temporal analysis was performed to examine the extent of variation in the transcriptional control associated with both identifiable anatomical nuclei and with light/dark cycle. Precise isolation of specific brain nuclei regulating sleep and arousal, including the LC, SCN, TMN, VTA, and VLPO, demonstrated robust changes in gene expression. Many of these differences were not observed in previous studies where whole brain lysates or gross dissections were used to probe for changes in gene expression. The robust and differential profiles of genomic data obtained from the approaches used herein underscore the requirement for careful anatomical refinement in CNS gene expression studies designed to understand genomic control within behaviorally-linked, but functionally isolated brain nuclei.

Introduction

Sleep and wake behavioral arousal states are established through very disparate levels of neuronal activity among functionally interconnected and well-characterized brain nuclei (Saper et al., 2005). A major challenge for understanding genetic control points that could shape complex sleep and wake behavior arises from the hierarchical interaction of behaviorally-related brain nuclei within complex neural networks modulated by unique molecular divergence within each nucleus to modulate the overall behavior. This complexity presents challenges for many of the technologies typically utilized to unravel these neural networks, including microarray-based mRNA profiling, which typically requires several micrograms of RNA, and thus relatively large brain tissue samples for analysis. To overcome this requirement experimentally, more tissue, and thus more anatomically and functionally diverse tissues are often utilized (Stansberg et al., 2007). Under this condition, though the anatomically localized fold-changes in mRNA expression may be large (several fold), no change or minor fold-changes (< 2-fold) are traditionally detected by microarray profiling of whole brain regions, such as hypothalamus (Letwin et al., 2006, Pavlidis and Noble, 2001). This difficulty is a wide-spread concern when profiling brain tissue, given the extensive functional and cellular heterogeneity throughout the brain.

Here we describe results of transcriptional profiling obtained through the application of laser capture microscopy (LCM) combined with ultra low input (1 ng) RNA amplification (ULI), which overcomes the difficulties of profiling brain tissue by allowing mRNA profiling of individual brain nuclei and cell populations. We report the light/dark cycle transcriptional variation of 5 different components of a neuronal circuit that controls aspects of sleep and wakefulness, namely the locus coeruleus (LC), suprachiasmatic nucleus (SCN), tuberomammillary nucleus (TMN), ventrolateral preoptic nucleus (VLPO) and the ventral tegmental area (VTA). These studies demonstrate both the specificity and sensitivity of this approach for differentiating brain nuclei according to their mRNA profiles and show that transcriptional changes are associated with physiological activities relevant to the light/dark cycle. Importantly, the characterization of regional and light/dark-responsive signature genes in this study reveals previously unrecognized genomic regulation within these discrete brain nuclei. The substantial gene expression changes detected in each discrete nucleus highlight the complexity of transcriptional regulation in the network of components governing the sleep–wake system and underscore the need for more refined efforts when addressing functional-anatomical circuit oriented gene profiling studies.

Section snippets

Isolation, amplification and profiling of RNA from specific nuclei involved in sleep

Laser capture microdissection was used to isolate LC, SCN, TMN, VLPO and VTA (Fig. 1A) from rats sacrificed during the light period (7 h after lights-on, referred to as ZT-7) and during the dark period (7 h after lights-off, referred to as ZT-19). These times correspond with the middle of normal inactive phase (ZT-7) and mid active phase (ZT-19) of the rat. Regions were identified based on standard anatomical landmarks and stereotaxic coordinates (see Supplemental Table 3) (Paxinos and Watson,

Discussion

Analysis of transcriptional changes within gross brain regions has substantially increased the understanding of key factors governing sleep, circadian signaling and regional differences including recent investigations of CNS gene expression in normal and sleep deprived models (Cirelli and Tononi, 1999, Cirelli and Tononi, 2000, Cirelli et al., 2004, Fukuhara and Tosini, 2008, Greco et al., 1999, Kilduff et al., 2008, Mackiewicz et al., 2008, Tafti and Franken, 2007, Terao et al., 2003a, Terao

Animals

All experimental animals were housed and handled according to the Federal Animal Welfare guidelines and all studies were approved in advance by the Animal Care and Use Committee at Merck & Co., Inc. Male Sprague Dawley rats at 8 weeks of age were individually housed in 12:12 light:dark conditions with water and food available ad libitum prior to sacrifice by conscious decapitation at ZT-7 and ZT-19 time points to correspond with the middle of the inactive and active phases (n = 4 per time point).

Acknowledgments

We gratefully acknowledge Eva Finney, David Levine, Duane Reiss, James Rhydderch, and Christopher Roberts for their contributions to this work.

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¹: These authors contributed equally to this work.

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Research ReportRefined anatomical isolation of functional sleep circuits exhibits distinctive regional and circadian gene transcriptional profiles

Abstract

Introduction

Section snippets

Isolation, amplification and profiling of RNA from specific nuclei involved in sleep

Discussion

Animals

Acknowledgments

Syndapin I and endophilin I bind overlapping proline-rich regions of dynamin I: role in synaptic vesicle endocytosis

J. Neurochem.

The human endogenous retrovirus envelope glycoprotein, syncytin-1, regulates neuroinflammation and its receptor expression in multiple sclerosis: a role for endoplasmic reticulum chaperones in astrocytes

J. Immunol.

Expression profiles of 50 xenobiotic transporter genes in humans and pre-clinical species: a resource for investigations into drug disposition

Xenobiotica

eIF2B and oligodendrocyte survival: where nature and nurture meet in bipolar disorder and schizophrenia?

Schizophr. Bull.

Increased cell death and delayed development in the cerebellum of mice lacking the rev-erbA(alpha) orphan receptor

Development

Differences in brain gene expression between sleep and waking as revealed by mRNA differential display and cDNA microarray technology

J. Sleep Res.

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J. Neurosci.

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Neuron

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Biol. Psychiatry

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Schizophr. Res.

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Br. Med. J.

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J. Neurosci.

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Neuroscience

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Oncogene

A manual method for the purification of fluorescently labeled neurons from the mammalian brain

Nat. Protoc.

Restoration of synapse formation in Musk mutant mice expressing a Musk/Trk chimeric receptor

Development

DNA variation and brain region-specific expression profiles exhibit different relationships between inbred mouse strains: implications for eQTL mapping studies

Genome Biol.

Expression profiling using microarrays fabricated by an ink-jet oligonucleotide synthesizer

Nat. Biotechnol.

Genomic instability and enhanced radiosensitivity in Hsp70.1- and Hsp70.3-deficient mice

Mol. Cell Biol.

Exploration, normalization, and summaries of high density oligonucleotide array probe level data

Biostatistics

Deletion of the neuron-specific protein delta-catenin leads to severe cognitive and synaptic dysfunction

Curr. Biol.

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Arthritis Res. Ther.

New developments in sleep research: molecular genetics, gene expression, and systems neurobiology

J. Neurosci.

Circadian rhythm of plasma 11-hydroxycorticosteroids in depressive illness, congestive heart failure, and Cushing's syndrome

Br. Med. J.

Sleep disturbance in psychiatric disorders: effects on function and quality of life in mood disorders, alcoholism, and schizophrenia

Ann. Clin. Psychiatry

Point mutant mice with hypersensitive alpha 4 nicotinic receptors show dopaminergic deficits and increased anxiety

Proc. Natl. Acad. Sci. U. S. A.

Phenomic, convergent functional genomic, and biomarker studies in a stress-reactive genetic animal model of bipolar disorder and co-morbid alcoholism

Am. J. Med. Genet. B Neuropsychiatr. Genet.

Combined application of behavior genetics and microarray analysis to identify regional expression themes and gene-behavior associations

J. Neurosci.

Egr3, a synaptic activity regulated transcription factor that is essential for learning and memory

Mol. Cell Neurosci.

Circadian rhythm of brain-derived neurotrophic factor in the rat suprachiasmatic nucleus

Neurosci. Lett.

The DBP gene is expressed according to a circadian rhythm in the suprachiasmatic nucleus and influences circadian behavior

EMBO J.

FRAG1, a gene that potently activates fibroblast growth factor receptor by C-terminal fusion through chromosomal rearrangement

Proc. Natl. Acad. Sci. U. S. A.

A single infusion of brain-derived neurotrophic factor into the ventral tegmental area induces long-lasting potentiation of cocaine seeking after withdrawal

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Research Report
Refined anatomical isolation of functional sleep circuits exhibits distinctive regional and circadian gene transcriptional profiles