Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

Summary

Identification of dopamine D1-alpha receptor in the nucleus accumbens is critical for clarifying D1 receptor dysfunction during a central nervous system disease. We performed a novel RNA in situ hybridization assay to visualize single RNA molecules in a specific brain area.

Abstract

In the central nervous system, the D1-alpha subtype receptor (Drd1α) is the most abundant dopamine (DA) receptor, which plays a vital role in regulating neuronal growth and development. However, the mechanisms underlying Drd1α receptor abnormalities mediating behavioral responses and modulating working memory function are still unclear. Using a novel RNA in situ hybridization assay, the current study identified dopamine Drd1α receptor and tyrosine hydroxylase (TH) RNA expression from DA-related circuitry in the nucleus accumbens (NAc) area and substantia nigra region (SNR), respectively. Drd1α expression in the NAc shows a "discrete dot" staining pattern. Clear sex differences in Drd1α expression were observed. In contrast, TH shows a "clustered" staining pattern. Regarding TH expression, female rats displayed a higher signal expression per cell relative to male animals. The methods presented here provide a novel in situ hybridization technique for investigating changes in dopamine system dysfunction during the progression of central nervous system diseases.

Introduction

Dysfunction of the striatal dopamine system is involved in the progression of clinical symptoms observed in multiple neurocognitive diseases. Dopamine D1 receptors are present in the prefrontal cortex (PFC) and striatal regions of the brain and heavily influence cognitive processes¹, including working memory, temporal processing, and locomotive behavior^2,3,4,5,6,7. Previous studies elucidated that changes of dopamine D1 receptors were associated wit....

Protocol

The experimental protocol was approved by the Animal Care and Use Committee (IACUC) at the University of South Carolina (federal assurance number: A3049-01).

1. Preparation of Fresh Frozen Brain Sections

1. Use the F344/N rat strain: three rats of each sex, 13 months of age, body weight approximately 320 g.
2. Adjust the sevoflurane concentration to 5% (overdose of sevoflurane). Continue sevoflurane exposure after breathing stops for an additional minute.
3. Decapitate the rat and remove the brain.
4. Submerge the rat brain in liquid nitrogen for 15 s within 5 min of tissue harvest.
5. Equilibr....

Results

The current study observed a "discrete dots" staining pattern for RNA expression in the dopamine D1-alpha receptors (Drd1α) of the NAc in F344/N rats (Figure 1A). Individual fluorescence signals were easily identified and can be seen as single "dots," each of which represents a single RNA transcript within the cell. For images from the NAc that display the "discrete dots" staining pattern, we assessed the dynamic range of expression using a semi-quantitative .......

Discussion

In this protocol, we describe a novel technique of in situ hybridization for fresh-frozen brain slices to evaluate Drd1α receptor expression in the nucleus accumbens (NAc) and tyrosine hydroxylase (TH) expression in the substantia nigra (SNR) region. We also provide methods of data analysis for different staining patterns: "discrete dot" or "clusters".

The critical steps for a successful multiplex fluorescence RNA in situ hybridization assay are included........

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
HybEZ Oven system	Advanced Cell Diagnostics	310010
RNAscope Probe - Rn-Drd1a	Advanced Cell Diagnostics	317031	Color channel 1, Green
RNAscope Probe - Rn-Th-C2	Advanced Cell Diagnostics	314651-C2	Color channel 2, Orange
RNAscope Fluorescent Multiplex Reagent Kit	Advanced Cell Diagnostics	320850
ImmEdge Hydrophobic Barrier Pen	Vector Laboratory	H-4000
SuperFrost Plus Slides	Fisher Scientific	12-550-154%
4% paraformaldehyde	Sigma-Aldrich	158127-500G
Sevoflurane	Merritt Veterinary Supply	347075
Tissue-Tek vertical 24 slide rack	Fisher Scientific	NC9837976
Tissue-Tek staining dish	Fisher Scientific	NC0731403
Precision General Purpose Baths	ThermoFisher Scientific	TSGP28
Pretreatment 4	Advanced Cell Diagnostics	320850	parts of kits
ProLong Gold anti-fade reagent	Life Technologies	P36930
Amp 1-FL	Advanced Cell Diagnostics	320850	parts of kits
Amp 2-FL	Advanced Cell Diagnostics	320850	parts of kits
Amp 3-FL	Advanced Cell Diagnostics	320850	parts of kits
Amp 4-FL-Alt A	Advanced Cell Diagnostics	320850	parts of kits
EZ-C1 software package	Nikon Instruments		version 3.81b
SAS/STAT Software	SAS Institute, Inc.,		version 9.4