The present works were supported by National Institutes of Health (NIH) grants HD043680, MH106392, DA013137, and NS100624. Partial fund was provided by a NIH T32 training grant in Biomedical-Behavioral science.

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
<ol>
	<li>Use the F344/N rat strain: three rats of each sex, 13 months of age, body weight approximately 320&#160;g.</li>
	<li>Adjust the sevoflurane concentration to 5% (overdose of sevoflurane). Continue sevoflurane exposure after breathing stops for an additional minute.</li>
	<li>Decapit...

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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Supranormal+stimulation+of+D1+dopamine+receptors+in+the+rodent+prefrontal+cortex+impairs+spatial+working+memory+performance.">Supranormal stimulation of D1 dopamine receptors in the rodent prefrontal cortex impairs spatial working memory performance.</a> J Neurosci. 17, 8528-8535 (1997).</li><li>Floresco, S. B., Magyar, O., Ghods-Sharifi, S., Vexelman, C., Tse, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+dopamine+receptor+subtypes+in+the+medial+prefrontal+cortex+of+the+rat+regulate+set-shifting.">Multiple dopamine receptor subtypes in the medial prefrontal cortex of the rat regulate set-shifting.</a> Neuropsychopharmacology. 31, 297-309 (2006).</li><li>Arnsten, A. F., Girgis, R. R., Gray, D. L., Mailman, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+dopamine+therapeutics+for+cognitive+deficits+in+schizophrenia.">Novel dopamine therapeutics for cognitive deficits in schizophrenia.</a> Biol Psychiatry. 81, 67-77 (2017).</li><li>Ellenbroek, B. A., Budde, S., Cools, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prepulse+inhibition+and+latent+inhibition:+the+role+of+dopamine+in+the+medial+prefrontal+cortex.">Prepulse inhibition and latent inhibition: the role of dopamine in the medial prefrontal cortex.</a> Neuroscience. 75 (2), 535-542 (1996).</li><li>Parker, K. L., Alberico, S. L., Miller, A. D., Narayanan, N. 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There are no conflicts of interest to declare.

In this protocol, we describe a novel technique of in situ hybridization for fresh-frozen brain slices to evaluate Drd1&#945; 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: &#34;discrete dot&#34; or &#34;clusters&#34;.
The critical steps for a successful multiplex fluorescence RNA in situ hybridization assay are included....

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 behavior2,3,4,5,6,7. 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 schizophrenia9,10 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 Drd1&#945; 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&#39;s complexities. Here, we describe the procedure for fresh-frozen brain slices and provide methods of data analysis for different staining patterns: &#34;discrete dot&#34; or &#34;clusters&#34;.

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			<td height="23" style="height:23px;">4% paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>158127-500G</td>
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identification of dopamine d1-alpha receptor within rodent nucleus accumbens by an innovative rna in situ detection technology

In the central nervous system, the D1-alpha subtype receptor (Drd1&#945;) is the most abundant dopamine (DA) receptor, which plays a vital role in regulating neuronal growth and development. However, the mechanisms underlying Drd1&#945; 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 Drd1&#945; receptor and tyrosine hydroxylase (TH) RNA expression from DA-related circuitry in the nucleus accumbens (NAc) area and substantia nigra region (SNR), respectively. Drd1&#945; expression in the NAc shows a &#34;discrete dot&#34; staining pattern. Clear sex differences in Drd1&#945; expression were observed. In contrast, TH shows a &#34;clustered&#34; 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.

The current study observed a &#34;discrete dots&#34; staining pattern for RNA expression in the dopamine D1-alpha receptors (Drd1&#945;) of the NAc in F344/N rats (Figure 1A). Individual fluorescence signals were easily identified and can be seen as single &#34;dots,&#34; each of which represents a single RNA transcript within the cell. For images from the NAc that display the &#34;discrete dots&#34; staining pattern, we assessed the dynamic range of expressi...

Watch this Scientific Journal Video about Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology at JoVE.com

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

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.

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

In the central nervous system, the D1-alpha subtype receptor (Drd1&#945;) is the most abundant dopamine (DA) receptor, which plays a vital role in ...

The overall goal of this novel RNA in-situ hybridization assay is to visualize single RNA molecules in a specific brain area which are critical for clarifying dopaminergic dysfunction during central nervous system disease. This method can help answer key questions in the neuroscience field such as dopaminergic abnormalities mediating behavioral responses and modulating working memory function. The main advantage of this method is that it provides a novel in-situ hybridization technique to investigate dopamine system dysfunction during the progression of central nervous system diseases.Though this method can provide insight into overall expression levels of a target mRNA in a given brain region, it can also be applied to determine how mRNA transcripts are distributed throughout the cells of a given region. To begin this procedure, within five minutes after harvesting an adult rat brain, submerge it in liquid nitrogen for 15 seconds. Next, equilibrate the brain at minus 20 degrees Celsius in a cryostat for one hour.After an hour, cut 30 micron sections containing nucleus accumbens from 2.76 millimeters to 2.28 millimeters anterior to bregma and mount them onto the slides. Then dry the sections at minus 20 degrees Celsius for 10 minutes. Subsequently, immerse the slides in pre-chilled 4%paraformaldehyde for one hour at four degrees Celsius.Next, place the slides in an increasing ethanol gradient at room temperature then transfer the slides to fresh 100%ethanol for another five minutes. Afterward, place the slides on the absorbent paper to allow air dry. Draw a barrier around each section with a barrier pen.Let the barrier dry completely for one minute. To pre-treat the brain sections, turn on the oven and set the temperature to 40 degrees Celsius. Add three drops of pre-treatment reagent on each brain section.Then incubate the sections for 30 minutes at room temperature. Next, submerge the slides in 1X PBS for one minute at room temperature and transfer the slides to fresh 1X PBS for another minute. Slides should not stay in 1X PBS for longer than 15 minutes.To run the multiplex assay, warm the target probe for 10 minutes at 40 degrees Celsius in a water bath and then cool to room temperature. Place the RNA reagent at room temperature. Remove excess liquid from the slides with absorbent paper and place them back on the slide rack.Then add three drops of the Drd1alpha as probe on each sample slice and incubate the samples for two hours at 40 degrees Celsius. Afterward, wash the slides in 1X wash buffer for two minutes at room temperature twice. Then remove excess liquid from the slides with absorbent paper and place them back on the slide rack.Do not let brain sections dry out between incubation steps. Add three drops of AMP 1FL on each sample slice and incubate the samples for 30 minutes at 40 degrees Celsius. Afterward, wash the slides twice in 1X wash buffer for two minutes each time at room temperature.Then remove excess liquid from the slides with absorbent paper and place them back on the slide rack. Subsequently, add three drops of AMP 2FL on each sample slice. Incubate the samples for 15 minutes at 40 degrees Celsius.Next, submerge the slides in 1X wash buffer for two minutes at room temperature twice. Remove excess liquid from the slides with absorbent paper and place them back on the slide rack. Add three drops of AMP 3FL on each sample slice and incubate the samples for 30 minutes at 40 degrees Celsius.Next, wash the slides in 1X wash buffer for two minutes at room temperature twice. Remove excess liquid from the slides with absorbent paper and place them back on the slide rack. Then add three drops of AMP 4FL ALT A on each sample slice and incubate the samples for 15 minutes at 40 degrees Celsius.Wash the slide in 1X wash buffer for two minutes at room temperature twice. Remove excess liquid from the slides and immediately place two drops of mounting reagent onto each section. Next, place a 22 millimeter by 22 millimeter coverslip over each brain section.Then store the slides in the dark at two to eight degrees Celsius until dry. For imaging, turn on the confocal microscope and switch to a 60X objective. Obtain Z stack images and acquire images of Drd1alpha-labeled cells in the nucleus accumbens region.For images from the nucleus accumbens that displayed the discrete dots staining pattern, the dynamic range of expression was assessed using a semi-quantitative analysis. Each cell was scored from zero to nine based on the number of dots per cell. This hybridized signal score method can thus assess the extent and variability for Drd1alpha expression across individual cells.Here are the representative confocal images of Drd1alpha expression in male and female rats which have the discrete dots staining pattern. To account for the nested data structure, average count values were calculated for each category and used for statistical analysis. A Mann-Whitney U test was conducted to assess differences in median cell count in male and female rats.The frequency of cells in each category is analyzed using relative frequency and cumulative frequency. Female animals displayed a significant shift in the distribution with the greater frequency of cells in lower ordered categories relative to male animals. Overall, the results suggest clear sex differences in Drd1alpha expression in the nucleus accumbens.Once mastered, this technique can be done in seven hours if it is performed properly. While attempting this procedure, it's important to remember to work quickly as temperature changes or drying out of the tissue can damage the mRNA and hinder our ability to obtain a reliable signal. After watching this video, you should have a good understanding of how to perform the in-situ hybridization technique in cortical tissues, fluorescently image signal mRNA transcripts within chosen brain regions, and characterize the distribution of mRNA transcripts for statistical analysis.

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8.5K vistas.  University of South Carolina. The overall goal of this novel RNA in-situ hybridization assay is to visualize single RNA molecules in a specific brain area which are critical for clarifying dopaminergic dysfunction during central nervous system disease. This method can help answer key questions in the neuroscience field such as dopaminergic abnormalities mediating behavioral responses and modulating working memory function. The main advantage of this method is that it provides a novel in-situ hybridization technique to inv...