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 with the progression of attention deficit-hyperactivity disorder (ADHD)⁸, the neurocognitive symptoms in schizophrenia^9,10 and stress susceptibility¹¹. Specifically, in schizophrenia, positron emission tomography (PET) studies indicated that the binding ability of dopamine D1 receptors in the prefrontal cortices was highly related to cognitive deficits and the presence of negative symptoms¹¹. The dendritic growth of excitatory neurons in the prefrontal cortex regulated by the dopamine D1 receptor alleviates stress susceptibility. Furthermore, the knockdown of D1 receptor in medial prefrontal cortical (mPFC) neurons could enhance the social defeat stress-induced social avoidance¹².

Here, we introduce a novel technique of RNA in situ hybridization to visualize single RNA molecules in a cell with fresh-frozen tissue samples. The present technique has multiple advantages over methods that exist within the current literature. First, the current procedure preserves the spatial and morphological context of the tissue and was performed on fresh-frozen tissue samples so that other procedures requiring fresh, non-embedded tissues may be combined with the current methods. Similar procedures in formalin-fixed and paraffin-embedded tissues have illustrated that single transcription resolution can be achieved using an RNA in situ hybridization technique¹³. Detection of RNA at the single transcription level provides superior sensitivity to low copy number expression as well as the opportunity to compare gene expression at the level of individual cells that cannot be achieved by other nucleic acid detection methods, such as polymerase chain reaction (PCR) techniques. Additionally, the current method maintains images with a high signal-to-noise ratio through highly specific RNA probes that are hybridized to single target RNA transcripts, and sequentially bound with a cascade of signal amplification molecules in the detection system. Finally, the present technology provides the opportunity to evaluate multiple biological systems with its target-specific proprietary probes, rather than limiting our investigation to only one class of system-related markers such as protein detection by immunohistochemistry methods.

In our study, we used this novel RNA in situ hybridization to evaluate Drd1α receptor expression in the nucleus accumbens (NAc) and tyrosine hydroxylase (TH) expression in the substantia nigra (SNR) of both male and female F344/N rats. The innovative RNA in situ hybridization enabled us to investigate mechanisms influencing both DA uptake and DA release simultaneously, improving our understanding of the striatal DA system's complexities. Here, we describe the procedure for fresh-frozen brain slices and provide methods of data analysis for different staining patterns: "discrete dot" or "clusters".

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. Equilibrate the brain to -20 °C in a cryostat for 1 h.
6. Cut 30 µm sections and transfer onto slides (see Table of Materials).
7. Choose sections from the nucleus accumbens region, approximately 2.76 mm to 2.28 mm anterior to Bregma¹⁴.
8. Continuously slice the rat brain from the olfactory bulb through the nucleus accumbens region according to the stereotaxic brain structure of the rat.
9. Mount samples onto the slides. Keep the sections at -20 °C for 10 min to dry.
10. Immediately immerse slides in the pre-chilled 4% paraformaldehyde for 1 h at 4 °C.
11. Place the slides in an increasing ethanol gradient at room temperature (RT): 50% EtOH for 5 min; 70% EtOH for 5 min; and 100% EtOH for 5 min.
12. Repeat with fresh 100% EtOH.
13. Place slides on absorbent paper, and air dry.
14. Draw a barrier around each section with a barrier pen (see Table of Materials). Let the barrier dry completely for 1 min.

2. Pretreatment of Brain Sections

1. Turn on the oven and set the temperature to 40 °C.
2. Add 3 drops (90 µL) of Pretreatment 4 reagent (see Table of Materials) on each brain section (one section per slide).
3. Incubate the sections for 30 min at RT.
4. Submerge the slides in 1x PBS for 1 min at RT. Repeat with fresh 1x PBS.
   NOTE: Slides should not stay in 1x PBS for longer than 15 min.

3. RNA In Situ Hybridization Fluorescent Multiplex Assay

1. Warm the target probe for 10 min at 40 °C in a water bath, and then cool to RT.
2. Place the RNA reagent (e.g., Amp 1-4 FL) at RT.
3. Remove excess liquid from slides with absorbent paper and place back on the slide rack (see Table of Materials).
4. Add 3 drops (90 µL) of the Drd1α probe (C1) on the sample slice. Incubate for 2 h at 40 °C.
5. Submerge slides in 1x wash buffer for 2 minutes at RT. Repeat with fresh 1x wash buffer.
6. Remove excess liquid from the slides with absorbent paper and place back on the slide rack (see Table of Materials).
7. Add 3 drops (90 µL) of Amp 1-FL on the sample slice. Incubate for 30 min at 40 °C.
8. Submerge slides in 1 wash buffer for 2 min at RT. Repeat with fresh 1wash buffer.
9. Remove excess liquid from the slides with absorbent paper and place back on the slide rack (see Table of Materials).
10. Add 3 drops (90 µL) of Amp 2-FL on the sample slice. Incubate for 15 min at 40 °C.
11. Submerge slides in 1x wash buffer for 2 minutes at RT. Repeat with fresh 1x wash buffer.
12. Remove excess liquid from the slides with absorbent paper and place back on the slide rack (see Table of Materials).
13. Add 3 drops (90 µL) of Amp 3-FL on the sample slice. Incubate for 30 min at 40 °C.
14. Submerge slides in 1x wash buffer for 2 min at RT. Repeat with fresh 1x wash buffer.
15. Remove excess liquid from the slides with absorbent paper and place back on the slide rack (see Table of Materials).
16. Add 3 drops (90 µL) of Amp 4-FL-Alt A on the sample slice. Incubate for 15 min at 40 °C.
17. Submerge slides in 1x wash buffer for 2 minutes at RT. Repeat with fresh 1x wash buffer.
18. Remove excess liquid from slides and immediately place 2 drops of mounting reagent (see Table of Materials) onto each section.
19. Place a 22 mm 22 mm coverslip (see Table of Materials) over each brain section.
20. Store slides in the dark at 2-8 °C until dry.
21. Turn on the confocal microscope and switch to a 60X objective.
22. Obtain Z-stack images with a confocal microscope. See the supplemental video for a detail procedure of confocal imaging.
23. Acquire images of Drd1α labeled cells in the nucleus accumbens region (Excitation: 488 nm; Emission: 515/30 nm).
    NOTE: Do not let brain sections dry out between incubation steps.

4. Data Analysis

1. Semi-quantitatively analyze the staining pattern: "discrete dots".
   1. To identify the individual cell from the image, perform DAPI staining as a nuclear marker. However, it is still difficult to analyze certain parts of brain tissue since it has multiple types of cells with irregular shape of nuclei and/or cells. Here, two experienced researchers separately assign each cell from images to define cell region.
   2. Visually score each cell within the representative confocal images based on the number of dots per cell using the specific criteria (Figure 1B).
   3. Determine the total cell number in each category and divide by the total cell number x100 to create relative frequency graphs that display the percent frequency of cells within each category for all tissue sections (Figure 2A) and for males and females separately (Figure 2E).
   4. Divide the sum of cells within all prior categories by the total cell number x100 to determine the cumulative frequency of cells for all tissue sections (Figure 2B) and for males and females separately (Figure 2F).
2. Statistically analyze the "discrete dots" staining pattern (Optional).
   1. Examine the number of dots per cell to provide a categorical variable that is ordinal in nature (i.e., the number of cells which fall into each rank category from lowest cell expression (1) to highest cell expression (9)) for further statistical analyses.
   2. After examination of descriptive statistics, use three main approaches: a Mantel-Haenszel statistic, a Mann-Whitney U test, and a Kruskal-Wallis test. Although the precise statistical approach will be dependent upon the experimental design and research question of interest, a statistical decision tree (Figure 4) may aid in making decisions regarding one's analytical approach.
3. Quantify the signal intensity in the region of interest (ROI) for staining pattern: "clusters".
   1. Calculate the signal intensity of each image using the following equation:
      Signal intensity = Total intensity of ROI image - (Average background intensity × Total image area)
   2. Count the total number of cells within ROI image to calculate the approximate signal expression per cell. Group differences in signal intensity/image as well as the number of cells/image may be of interest to consider mechanisms that may contribute to group differences seen in signal expression/cell (Figure 3B-3D).
4. Statistically analyze the "clusters" staining pattern (Optional).
   1. Examine the signal expression/cell to provide a continuous variable for further statistical analysis. Use two approaches, including an independent sample t-test and analysis of variance (ANOVA), based on the number of between-subjects factors included in the design. A statistical decision tree (Figure 4) may aid in making decisions regarding the most appropriate statistical approach.

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