NMDA receptor subunit expression in GABAergic interneurons in the prefrontal cortex: Application of laser microdissection technique

doi:10.1016/j.jneumeth.2008.09.013

Journal of Neuroscience Methods

Volume 176, Issue 2, 30 January 2009, Pages 172-181

https://doi.org/10.1016/j.jneumeth.2008.09.013 Get rights and content

Abstract

The selective involvement of a subset of neurons in many psychiatric disorders, such as gamma-aminobutyric acid (GABA)-ergic interneurons in schizophrenia, creates a significant need for in-depth analysis of these cells. Here we introduce a combination of techniques to examine the relative gene expression of N-methyl-d-aspartic acid (NMDA) receptor subtypes in GABAergic interneurons from the rat prefrontal cortex. Neurons were identified by immunostaining, isolated by laser microdissection and RNA was prepared for reverse transcription polymerase chain reaction (RT-PCR) and real-time PCR. These experimental procedures have been described individually; however, we found that this combination of techniques is powerful for the analysis of gene expression in individual identified neurons. This approach provides the means to analyze relevant molecular mechanisms that are involved in the neuropathological process of a devastating brain disorder.

Section snippets

Animals and tissue preparation

Twelve female adult rats (90 days of age) were used in our experiments. The animals were cared for under National Institute of Health (NIH) animal use guidelines, and the experimental protocol was approved by the Institutional Animal Care and Use Committee (IACUC) at Drexel University College of Medicine. All rats were anesthetized by an intraperitoneal (i.p.) injection of 0.2 ml/kg Euthasol (Henry Schein, Indianapolis, IN) and sacrificed by cervical dislocation. The brain region containing PFC

NovaRed immunostaining of PV in adult rat PFC and LMD

To identify different types of cortical interneurons and at the same time to preserve the integrity of RNA, we used rapid immunocytochemistry of fresh tissue. Subpopulations of interneurons expressing PV were visualized with NovaRed staining. Fig. 1A–D shows the brain region of the medial PFC at low magnifications. However, only under higher magnification of 40× (Fig. 1E) could the PV-ir interneurons be identified. The final staining product of the PV-ir neurons in the cortex is brown/red in

Discussion

Here we introduce a combination of techniques to examine the expression of mRNA for NMDA receptor subtypes in PV-ir GABAergic interneurons in rat PFC. Although techniques such as rapid immunostaining, LMD, RT-PCR and real-time PCR, have been used elsewhere (Burbach et al., 2003, Espina et al., 2006, Ginsberg et al., 2004, Hemby et al., 2002, Hinkle et al., 2004, Kerman et al., 2006), we found that this combination is very useful for analyzing gene expression in an identified subpopulation of

Funding sources

This study was supported by a grant from Drexel University College of Medicine, a NARSAD young investigator award and NIH R21 grant MH232307 to W.-J. Gao; and NIH R37 NS26380 to J.D. Houle. The NIH had no further role in study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the paper for publication.

Conflict of interest

None.

Acknowledgement

We thank Mr. George Stradtman for comments on the manuscript.

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¹: These authors contribute equally.

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NMDA receptor subunit expression in GABAergic interneurons in the prefrontal cortex: Application of laser microdissection technique

Abstract

Section snippets

Animals and tissue preparation

NovaRed immunostaining of PV in adult rat PFC and LMD

Discussion

Funding sources

Conflict of interest

Acknowledgement

J Chem Neuroanat

Schizophr Res

Neuropsychopharmacology

Schizophr Res

J Neurosci Methods

J Neurosci Methods

Schizophrenia Res

Life Sci

Neurochem Int

Lab Invest

Am J Pathol

Am J Pathol

Lab Invest

Biol Psychiatry

Prog Neurobiol

Ageing Res Rev

J Neurosci Methods

Brain Res Protoc

Adv Clin Chem

Curr Opin Pharmacol

Mol Aspects Med

Brain Res

Methods

Brain Res Protoc

Neuron

Cell

J Neurosci Methods

Acta Histochem

Schizophrenia Res

J Psychiatr Res

Biol Psychiatry

Mol Brain Res

Trends Genet

Biol Psychiatry

J Neurosci Methods

J Neurosci Methods

Prenatal exposure to an NMDA receptor antagonist, MK-801 reduces density of parvalbumin-immunoreactive GABAergic neurons in the medial prefrontal cortex and enhances phencyclidine-induced hyperlocomotion but not behavioral sensitization to methamphetamine in postpubertal rats

Psychopharmacology (Berl)

Gene expression for glutamic acid decarboxylase is reduced without loss of neurons in prefrontal cortex of schizophrenics

Arch Gen Psychiatry

Selective alterations in gene expression for NMDA receptor subunits in prefrontal cortex of schizophrenics

J Neurosci

Deficits in small interneurons in prefrontal and cingulate cortices of schizophrenic and schizoaffective patients

Arch Gen Psychiatry

Lamina-specific abnormalities of NMDA receptor-associated postsynaptic protein transcripts in the prefrontal cortex in schizophrenia and bipolar disorder

Neuropsychopharmacology

Molecular diversity of neocortical GABAergic interneurones

J Physiol

Quantitative real-time RT-PCR—a perspective

J Mol Endocrinol

Pitfalls of quantitative real-time reverse-transcription polymerase chain reaction

J Biomol Tech

Abnormalities of the NMDA receptor and associated intracellular molecules in the thalamus in schizophrenia and bipolar disorder

Neuropsychopharmacology

Glutamate and schizophrenia: beyond the dopamine hypothesis

Cell Mol Neurobiol

An introduction to laser-based tissue microdissection techniques

Methods Mol Biol

Quantitative analysis of nucleic acids—the last few years of progress

J Biochem Mol Biol

N-methyl-d-aspartic acid receptor expression in the dorsolateral prefrontal cortex of elderly patients with schizophrenia

Am J Psychiatry

Microdissection of histologic sections: past, present, and future

Adv Anat Pathol