Name: identification of dopamine d1-alpha receptor within rodent nucleus accumbens by an innovative rna in situ detection technology
Uploaded: 2018-03-27T15:00:00
Description: 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

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...

<ol>
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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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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 ...

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