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>Decapitate the rat and remove the brain.</li>
	<li>Submerge the rat brain in liquid nitrogen for 15 s within 5&#160;min of tissue harvest.</li>
	<li>Equilibrate the brain to -20 &#176;C in a cryostat for 1&#160;h.</li>
	<li>Cut 30 &#181;m sections and transfer onto slides (see Table of Materials).</li>
	<li>Choose sections from the nucleus accumbens region, approximately 2.76 mm to 2.28 mm anterior to Bregma14.</li>
	<li>Continuously slice the rat brain from the olfactory bulb through the nucleus accumbens region according to the stereotaxic brain structure of the rat.</li>
	<li>Mount samples onto the slides. Keep the sections at -20 &#176;C for 10 min to dry.</li>
	<li>Immediately immerse slides in the pre-chilled 4% paraformaldehyde for 1 h at 4 &#176;C.</li>
	<li>Place the slides in an increasing ethanol gradient at room temperature (RT): 50% EtOH for 5&#160;min; 70% EtOH for 5&#160;min; and 100% EtOH for 5&#160;min.</li>
	<li>Repeat with fresh 100% EtOH.</li>
	<li>Place slides on absorbent paper, and air dry.</li>
	<li>Draw a barrier around each section with a barrier pen (see Table of Materials). Let the barrier dry completely for 1 min.</li>
</ol>
2. Pretreatment of Brain Sections
<ol>
	<li>Turn on the oven and set the temperature to 40 &#176;C.</li>
	<li>Add 3 drops (90 &#181;L) of Pretreatment 4 reagent (see Table of Materials) on each brain section (one section per slide).</li>
	<li>Incubate the sections for 30 min at RT.</li>
	<li>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.</li>
</ol>
3. RNA In Situ Hybridization Fluorescent Multiplex Assay
<ol>
	<li>Warm the target probe for 10 min at 40 &#176;C in a water bath, and then cool to RT.</li>
	<li>Place the RNA reagent (e.g., Amp 1-4 FL) at RT.</li>
	<li>Remove excess liquid from slides with absorbent paper and place back on the slide rack (see Table of Materials).</li>
	<li>Add 3 drops (90 &#181;L) of the Drd1&#945; probe (C1) on the sample slice. Incubate for 2 h at 40 &#176;C.</li>
	<li>Submerge slides in 1x wash buffer for 2 minutes at RT. Repeat with fresh 1x wash buffer.</li>
	<li>Remove excess liquid from the slides with absorbent paper and place back on the slide rack (see Table of Materials).</li>
	<li>Add 3 drops (90 &#181;L) of Amp 1-FL on the sample slice. Incubate for 30 min at 40 &#176;C.</li>
	<li>Submerge slides in 1 wash buffer for 2 min at RT. Repeat with fresh 1wash buffer.</li>
	<li>Remove excess liquid from the slides with absorbent paper and place back on the slide rack (see Table of Materials).</li>
	<li>Add 3 drops (90 &#181;L) of Amp 2-FL on the sample slice. Incubate for 15 min at 40 &#176;C.</li>
	<li>Submerge slides in 1x wash buffer for 2 minutes at RT. Repeat with fresh 1x wash buffer.</li>
	<li>Remove excess liquid from the slides with absorbent paper and place back on the slide rack (see Table of Materials).</li>
	<li>Add 3 drops (90 &#181;L) of Amp 3-FL on the sample slice. Incubate for 30 min at 40 &#176;C.</li>
	<li>Submerge slides in 1x wash buffer for 2 min at RT. Repeat with fresh 1x wash buffer.</li>
	<li>Remove excess liquid from the slides with absorbent paper and place back on the slide rack (see Table of Materials).</li>
	<li>Add 3 drops (90 &#181;L) of Amp 4-FL-Alt A on the sample slice. Incubate for 15 min at 40 &#176;C.</li>
	<li>Submerge slides in 1x wash buffer for 2 minutes at RT. Repeat with fresh 1x wash buffer.</li>
	<li>Remove excess liquid from slides and immediately place 2 drops of mounting reagent (see Table of Materials) onto each section.</li>
	<li>Place a 22 mm 22 mm coverslip (see Table of Materials) over each brain section.</li>
	<li>Store slides in the dark at 2-8 &#176;C until dry.</li>
	<li>Turn on the confocal microscope and switch to a 60X objective.</li>
	<li>Obtain Z-stack images with a confocal microscope. See the supplemental video for a detail procedure of confocal imaging.</li>
	<li>Acquire images of Drd1&#945; 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.</li>
</ol>
4. Data Analysis
<ol>
	<li>Semi-quantitatively analyze the staining pattern: &#34;discrete dots&#34;.
	<ol>
		<li>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.</li>
		<li>Visually score each cell within the representative confocal images based on the number of dots per cell using the specific criteria (Figure 1B).</li>
		<li>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).</li>
		<li>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).</li>
	</ol>
	</li>
	<li>Statistically analyze the &#34;discrete dots&#34; staining pattern (Optional).
	<ol>
		<li>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.</li>
		<li>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&#39;s analytical approach.</li>
	</ol>
	</li>
	<li>Quantify the signal intensity in the region of interest (ROI) for staining pattern: &#34;clusters&#34;.
	<ol>
		<li>Calculate the signal intensity of each image using the following equation: 
		Signal intensity = Total intensity of ROI image - (Average background intensity &#215; Total image area)</li>
		<li>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).</li>
	</ol>
	</li>
	<li>Statistically analyze the &#34;clusters&#34; staining pattern (Optional).
	<ol>
		<li>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.</li>
	</ol>
	</li>
</ol>

<ol><li>Goldman-Rakic, P. S., Castner, S. A., Svensson, T. H., Siever, L. J., Williams, G. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Targeting+the+dopamine+D1+receptor+in+schizophrenia%3A+insights+for+cognitive+dysfunction%5BTitle%5D)+AND+%22Psychopharmacology+%28Berl%29%22%5BJournal%5D)">Targeting the dopamine D1 receptor in schizophrenia: insights for cognitive dysfunction.</a> Psychopharmacology (Berl). 174 (1), 3-16 (2004).</li>
<li>Beaulieu, J. M., Gainetdinov, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+physiology%2C+signaling%2C+and+pharmacology+of+dopamine+receptors%5BTitle%5D)+AND+%22Pharmacol+Rev%22%5BJournal%5D)">The physiology, signaling, and pharmacology of dopamine receptors.</a> Pharmacol Rev. 63, 182-217 (2011).</li>
<li>Zahrt, J., Taylor, J. R., Mathew, R. G., Arnsten, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Supranormal+stimulation+of+D1+dopamine+receptors+in+the+rodent+prefrontal+cortex+impairs+spatial+working+memory+performance%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">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/pubmed?term=((Multiple+dopamine+receptor+subtypes+in+the+medial+prefrontal+cortex+of+the+rat+regulate+set-shifting%5BTitle%5D)+AND+%22Neuropsychopharmacology%22%5BJournal%5D)">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/pubmed?term=((Novel+dopamine+therapeutics+for+cognitive+deficits+in+schizophrenia%5BTitle%5D)+AND+%22Biol+Psychiatry%22%5BJournal%5D)">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/pubmed?term=((Prepulse+inhibition+and+latent+inhibition%3A+the+role+of+dopamine+in+the+medial+prefrontal+cortex%5BTitle%5D)+AND+%22Neuroscience%22%5BJournal%5D)">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. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Prefrontal+D1+dopamine+signaling+is+necessary+for+temporal+expectation+during+reaction+time+performance%5BTitle%5D)+AND+%22Neuroscience%22%5BJournal%5D)">Prefrontal D1 dopamine signaling is necessary for temporal expectation during reaction time performance.</a> Neuroscience. 255, 246-254 (2013).</li>
<li>Manduca, A., Servadio, M., Damsteegt, R., Campolongo, P., Vanderschuren, L. J., Trezza, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dopaminergic+Neurotransmission+in+the+Nucleus+Accumbens+Modulates+Social+Play+Behavior+in+Rats%5BTitle%5D)+AND+%22Neuropsychopharmacology%22%5BJournal%5D)">Dopaminergic Neurotransmission in the Nucleus Accumbens Modulates Social Play Behavior in Rats.</a> Neuropsychopharmacology. 41 (9), 2215-2223 (2016).</li>
<li>Okubo, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Decreased+prefrontal+dopamine+D1+receptors+in+schizophrenia+revealed+by+PET%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Decreased prefrontal dopamine D1 receptors in schizophrenia revealed by PET.</a> Nature. 385 (6617), 634-636 (1997).</li>
<li>Abi-Dargham, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Prefrontal+dopamine+D1+receptors+and+working+memory+in+schizophrenia%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">Prefrontal dopamine D1 receptors and working memory in schizophrenia.</a> J Neurosci. 22 (9), 3708-3719 (2002).</li>
<li>Shinohara, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dopamine+D1+receptor+subtype+mediates+acute+stress-induced+dendritic+growth+in+excitatory+neurons+of+the+medial+prefrontal+cortex+and+contributes+to+suppression+of+stress+susceptibility+in+mice%5BTitle%5D)+AND+%22Mol+Psychiatry%22%5BJournal%5D)">Dopamine D1 receptor subtype mediates acute stress-induced dendritic growth in excitatory neurons of the medial prefrontal cortex and contributes to suppression of stress susceptibility in mice.</a> Mol Psychiatry. 19, (2017).</li>
<li>Okubo, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Decreased+prefrontal+dopamine+D1+receptors+in+schizophrenia+revealed+by+PET%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Decreased prefrontal dopamine D1 receptors in schizophrenia revealed by PET.</a> Nature. 385, 634-636 (1997).</li>
<li>Wang, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((RNAscope%3A+a+novel+in+situ+RNA+analysis+platform+for+formalin-fixed%2C+paraffin-embedded+tissues%5BTitle%5D)+AND+%22J+Mol+Diagn%22%5BJournal%5D)">RNAscope: a novel in situ RNA analysis platform for formalin-fixed, paraffin-embedded tissues.</a> J Mol Diagn. 14 (1), 22-29 (2012).</li>
<li>Paxinos, G., Watson, C. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aPaxinos%2C+G+%22The+rat+brain+in+stereotaxic+coordinates%22">The rat brain in stereotaxic coordinates</a>. , 7th ed, Elsevier Academic Press. Burlington. (2014).</li>
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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;.

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

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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8.6K Views.  University of South Carolina. 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.

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

Laser Capture Microdissection - A Demonstration of the Isolation of Individual Dopamine Neurons and the Entire Ventral Tegmental Area

Laser capture microdissection (LCM) is used to isolate a concentrated population of individual cells or precise anatomical regions of tissue from tissue sections on a microscope slide. When combined with immunohistochemistry, LCM can be used to isolate individual cells types based on a specific protein marker. Here, the LCM technique is described for collecting a specific population of dopamine neurons directly labeled with&#160;tyrosine hydroxylase immunohistochemistry and for isolation of the dopamine neuron containing region of the ventral tegmental area using indirect tyrosine hydroxylase immunohistochemistry on a section adjacent to those used for LCM. An infrared (IR) capture laser is used to both dissect individual neurons as well as the ventral tegmental area off glass slides and onto an LCM cap for analysis. Complete dehydration of the tissue with 100% ethanol and xylene is critical. The combination of the IR capture laser and the ultraviolet (UV) cutting laser is used to isolate individual dopamine neurons or the ventral tegmental area when using PEN membrane slides. A PEN membrane slide has significant advantages over a glass slide as it offers better consistency in capturing and collecting cells, is faster collecting large pieces of tissue, is less reliant on dehydration and results in complete removal of the tissue from the slide. Although removal of large areas of tissue from a glass slide is feasible, it is considerably more time consuming and frequently leaves some residual tissue behind. Data shown here demonstrate that RNA of sufficient quantity and quality can be obtained using these procedures for quantitative PCR measurements. Although RNA and DNA are the most commonly isolated molecules from tissue and cells collected with LCM, isolation and measurement of microRNA, protein and epigenetic changes in DNA can also benefit from the enhanced anatomical and cellular resolution obtained using LCM.

The isolation of individual dopamine neurons or the ventral tegmental area with direct or indirect immunohistochemistry is demonstrated using laser capture microdissection. Parameters for isolation of tissue from a glass slide using an infrared laser and from membrane slides using the combination of an infrared and ultraviolet laser are discussed.

Laser capture microdissection (LCM) is used to isolate a concentrated population of individual cells or precise anatomical regions of tissue from tissue ...

Detection of Axonally Localized mRNAs in Brain Sections Using High-Resolution In Situ Hybridization

mRNAs are frequently localized to vertebrate axons and their local translation is required for axon pathfinding or branching during development and for maintenance, repair or neurodegeneration in postdevelopmental periods. High throughput analyses have recently revealed that axons have a more dynamic and complex transcriptome than previously expected. These analysis, however have been mostly done in cultured neurons where axons can be isolated from the somato-dendritic compartments. It is virtually impossible to achieve such isolation in whole tissues in vivo. Thus, in order to verify the recruitment of mRNAs and their functional relevance in a whole animal, transcriptome analyses should ideally be combined with techniques that allow the visualization of mRNAs in situ. Recently, novel ISH technologies that detect RNAs at a single-molecule level have been developed. This is especially important when analyzing the subcellular localization of mRNA, since localized RNAs are typically found at low levels. Here we describe two protocols for the detection of axonally-localized mRNAs using a novel ultrasensitive RNA ISH technology. We have combined RNAscope ISH with axonal counterstain using fluorescence immunohistochemistry or histological dyes to verify the recruitment of Atf4 mRNA to axons in vivo in the mature mouse and human brains.

Detection of Axonally Localized mRNAs in Brain Sections Using High-Resolution In Situ Hybridization

RNA in situ hybridization (ISH) enables the visualization of RNAs in cells and tissues. Here we show how combination of RNAscope ISH with immunohistochemistry or histological dyes can be successfully used to detect mRNAs localized to axons in sections of mouse and human brains.

mRNAs are frequently localized to vertebrate axons and their local translation is required for axon pathfinding or branching during development and for ...

Rapid In Situ Hybridization using Oligonucleotide Probes on Paraformaldehyde-prefixed Brain of Rats with Serotonin Syndrome

3,4-Methylenedioxymethamphetamine (MDMA; ecstasy) toxicity may cause region-specific changes in serotonergic mRNA expression due to acute serotonin (5-hydroxytryptamine; 5-HT) syndrome. This hypothesis can be tested using in situ hybridization to detect the serotonin 5-HT2A receptor gene htr2a. In the past, such procedures, utilizing radioactive riboprobe, were difficult because of the complicated workflow that needs several days to perform and the added difficulty that the technique required the use of fresh frozen tissues maintained in an RNase-free environment. Recently, the development of short oligonucleotide probes has simplified in situ hybridization procedures and allowed the use of paraformaldehyde-prefixed brain sections, which are more widely available in laboratories. Here, we describe a detailed protocol using non-radioactive oligonucleotide probes on the prefixed brain tissues. Hybridization probes used for this study include dapB (a bacterial gene coding for dihydrodipicolinate reductase), ppiB (a housekeeping gene coding for peptidylprolyl isomerase B), and htr2a (a serotonin gene coding for 5-HT2A receptors). This method is relatively simply, cheap, reproducible and requires less than two days to complete.

Rapid In Situ Hybridization using Oligonucleotide Probes on Paraformaldehyde-prefixed Brain of Rats with Serotonin Syndrome

This protocol describes a rapid and simplified in situ hybridization method ideal forparaformaldehyde-prefixed brain, thus reducing the need for prolonged complex steps while using fresh frozen tissues. The method is validated using the identification of the serotonin 5-HT2A receptor gene htr2a in rats.

3,4-Methylenedioxymethamphetamine (MDMA; ecstasy) toxicity may cause region-specific changes in serotonergic mRNA expression due to acute serotonin ...

Reliable Identification of Living Dopaminergic Neurons in Midbrain Cultures Using RNA Sequencing and TH-promoter-driven eGFP Expression

In Parkinson&#39;s Disease (PD) there is widespread neuronal loss throughout the brain with pronounced degeneration of dopaminergic neurons in the SNc, leading to bradykinesia, rigidity, and tremor. The identification of living dopaminergic neurons in primary Ventral Mesencephalic (VM) cultures using a fluorescent marker provides an alternative way to study the selective vulnerability of these neurons without relying on the immunostaining of fixed cells. Here, we isolate, dissociate, and culture mouse VM neurons for 3 weeks. We then identify dopaminergic neurons in the cultures using eGFP fluorescence (driven by a Tyrosine Hydroxylase (TH) promoter). Individual neurons are harvested into microcentrifuge tubes using glass micropipettes. Next, we lyse the harvested cells, and conduct cDNA synthesis and transposon-mediated &#34;tagmentation&#34; to produce single cell RNA-Seq libraries1,2,3,4,5. After passing a quality-control check, single-cell libraries are sequenced and subsequent analysis is carried out to measure gene expression. We report transcriptome results for individual dopaminergic and GABAergic neurons isolated from midbrain cultures. We report that 100% of the live TH-eGFP cells that were harvested and sequenced were dopaminergic neurons. These techniques will have widespread applications in neuroscience and molecular biology.

In Parkinson&#39;s Disease (PD), Substantia Nigra (SNc) dopaminergic neurons degenerate, leading to motor dysfunction. Here we report a protocol for culturing ventral midbrain neurons from a mouse expressing eGFP driven by a Tyrosine Hydroxylase (TH) promoter sequence, harvesting individual fluorescent neurons from the cultures, and measuring their transcriptome using RNA-seq.

In Parkinson&#39;s Disease (PD) there is widespread neuronal loss throughout the brain with pronounced degeneration of dopaminergic neurons in the SNc, ...

3D Particle Tracking for Noninvasive In Vivo Analysis of Synaptic Microtubule Dynamics in Dendrites and Neuromuscular Junctions of Drosophila

Microtubules (MTs) play critical roles in neuronal development, but many questions remain about the molecular mechanisms of their regulation and function. Furthermore, despite progress in understanding postsynaptic MTs, much less is known about the contributions of presynaptic MTs to neuronal morphogenesis. In particular, studies of in vivo MT dynamics in Drosophila sensory dendrites yielded significant insights into polymer-level behavior. However, the technical and analytical challenges associated with live imaging of the fly neuromuscular junction (NMJ) have limited comparable studies of presynaptic MT dynamics. Moreover, while there are many highly effective software strategies for automated analysis of MT dynamics in vitro and ex vivo, in vivo data often necessitate significant operator input or entirely manual analysis due to inherently inferior signal-to-noise ratio in images and complex cellular morphology. &#160;To address this, this study optimized a new software platform for automated and unbiased in vivo particle detection. Multiparametric analysis of live time-lapse confocal images of EB1-GFP labeled MTs was performed in both dendrites and the NMJ of Drosophila larvae and found striking differences in MT behaviors. MT dynamics were furthermore analyzed following knockdown of the MT-associated protein (MAP) dTACC, a key regulator of Drosophila synapse development, and identified statistically significant changes in MT dynamics compared to wild type. These results demonstrate that this novel strategy for the automated multiparametric analysis of both pre- and postsynaptic MT dynamics at the polymer-level significantly reduces human-in-the-loop criteria. The study furthermore shows the utility of this method in detecting distinct MT behaviors upon dTACC-knockdown, indicating a possible future application for functional screens of factors that regulate MT dynamics in vivo. Future applications of this method may also focus on elucidating cell type and/or compartment-specific MT behaviors, and multicolor correlative imaging of EB1-GFP with other cellular and subcellular markers of interest.&#160;

This study presents a noninvasive intravital neuronal imaging strategy combined with a new software strategy to achieve automated, unbiased tracking and analysis of in vivo microtubule (MT) plus-end dynamics in the sensory dendrites and the neuromuscular junctions of Drosophila.

Microtubules (MTs) play critical roles in neuronal development, but many questions remain about the molecular mechanisms of their regulation and function. ...

Semi-Quantitative Determination of Dopaminergic Neuron Density in the Substantia Nigra of Rodent Models using Automated Image Analysis

Estimation of the number of dopaminergic neurons in the substantia nigra is a key method in pre-clinical Parkinson's disease research. Currently, unbiased stereological counting is the standard for quantification of these cells, but it remains a laborious and time-consuming process, which may not be feasible for all projects. Here, we describe the use of an image analysis platform, which can accurately estimate the quantity of labeled cells in a pre-defined region of interest. We describe a step-by-step protocol for this method of analysis in rat brain and demonstrate it can identify a significant reduction in tyrosine hydroxylase positive neurons due to expression of mutant &#945;-synuclein in the substantia nigra. We validated this methodology by comparing with results obtained by unbiased stereology. Taken together, this method provides a time-efficient and accurate process for detecting changes in dopaminergic neuron number, and thus is suitable for efficient determination of the effect of interventions on cell survival.

Here we present an automated method for semi-quantitative determination of dopaminergic neuron number in the rat substantia nigra pars compacta.

Estimation of the number of dopaminergic neurons in the substantia nigra is a key method in pre-clinical Parkinson's disease research. Currently, unbiased ...

Enhanced Northern Blot Detection of Small RNA Species in Drosophila Melanogaster

The last decades have witnessed the explosion of scientific interest around gene expression control mechanisms at the RNA level. This branch of molecular biology has been greatly fueled by the discovery of noncoding RNAs as major players in post-transcriptional regulation. Such a revolutionary perspective has been accompanied and triggered by the development of powerful technologies for profiling short RNAs expression, both at the high-throughput level (genome-wide identification) or as single-candidate analysis (steady state accumulation of specific species). Although several state-of-art strategies are currently available for dosing or visualizing such fleeing molecules, Northern Blot assay remains the eligible approach in molecular biology for immediate and accurate evaluation of RNA expression. It represents a first step toward the application of more sophisticated, costly technologies and, in many cases, remains a preferential method to easily gain insights into RNA biology. Here we overview an efficient protocol (Enhanced Northern Blot) for detecting weakly expressed microRNAs (or other small regulatory RNA species) from Drosophila melanogaster whole embryos, manually dissected larval/adult tissues or in vitro cultured cells. A very limited amount of RNA is required and the use of material from flow cytometry-isolated cells can be also envisaged.

Enhanced Northern Blot Detection of Small RNA Species in Drosophila Melanogaster

The aim of this publication is to visualize and discuss the operative steps of an Enhanced Northern Blot protocol on RNA extracted from Drosophila melanogaster embryos, cells, and tissues. This protocol is particularly useful for the efficient detection of small RNA species.

The last decades have witnessed the explosion of scientific interest around gene expression control mechanisms at the RNA level. This branch of molecular ...

Biology

Detection of G Protein-coupled Receptor Expression in Mouse Vagal Afferent Neurons using Multiplex In Situ Hybridization

This study describes a protocol for the multiplex in situ hybridization (ISH) of the mouse jugular-nodose ganglia, with a particular emphasis on detecting the expression of G protein-coupled receptors (GPCRs). Formalin-fixed jugular-nodose ganglia were processed with the RNAscope technology to simultaneously detect the expression of two representative GPCRs (cholecystokinin and ghrelin receptors) in combination with one marker gene of either nodose (paired-like homeobox 2b, Phox2b) or jugular afferent neurons (PR domain zinc finger protein 12, Prdm12). Labeled ganglia were imaged using confocal microscopy to determine the distribution and expression patterns of the aforementioned transcripts. Briefly, Phox2b afferent neurons were found to abundantly express the cholecystokinin receptor (Cck1r) but not the ghrelin receptor (Ghsr). A small subset of Prdm12 afferent neurons was also found to express Ghsr and/or Cck1r. Potential technical caveats in the design, processing, and interpretation of multiplex ISH are discussed. The approach described in this article may help scientists in generating accurate maps of the transcriptional profiles of vagal afferent neurons.

Detection of G Protein-coupled Receptor Expression in Mouse Vagal Afferent Neurons using Multiplex In Situ Hybridization

Multiplex in situ hybridization (ISH) was employed to simultaneously visualize the transcripts for two G protein-coupled receptors and one transcription factor in the entire vagal ganglionic complex of the adult mouse. This protocol could be used to generate accurate maps of the transcriptional profiles of vagal afferent neurons.

This study describes a protocol for the multiplex in situ hybridization (ISH) of the mouse jugular-nodose ganglia, with a particular emphasis on detecting ...

Visualizing and Tracking Endogenous mRNAs in Live Drosophila melanogaster Egg Chambers

Fluorescence-based imaging techniques, in combination with developments in light microscopy, have revolutionized how cell biologists conduct live cell imaging studies. Methods for detecting RNAs have expanded greatly since seminal studies linked site-specific mRNA localization to gene expression regulation. Dynamic mRNA processes can now be visualized via approaches that detect mRNAs, coupled with microscopy set-ups that are fast enough to capture the dynamic range of molecular behavior. The molecular beacon technology is a hybridization-based approach capable of direct detection of endogenous transcripts in living cells. Molecular beacons are hairpin-shaped, internally quenched, single-nucleotide discriminating nucleic acid probes, which fluoresce only upon hybridization to a unique target sequence. When coupled with advanced fluorescence microscopy and high-resolution imaging, they enable one to perform spatial and temporal tracking of intracellular movement of mRNAs. Although this technology is the only method capable of detecting endogenous transcripts, cell biologists have not yet fully embraced this technology due to difficulties in designing such probes for live cell imaging. A new software application, PinMol, allows for enhanced and rapid design of probes best suited to efficiently hybridize to mRNA target regions within a living cell. In addition, high-resolution, real-time image acquisition and current, open source image analysis software allow for a refined data output, leading to a finer evaluation of the complexity underlying the dynamic processes involved in the mRNA&#39;s life cycle.
Here we present a comprehensive protocol for designing and delivering molecular beacons into Drosophila melanogaster egg chambers. Direct and highly specific detection and visualization of endogenous maternal mRNAs is performed via spinning disc confocal microscopy. Imaging data is processed and analyzed using object detection and tracking in Icy software to obtain details about the dynamic movement of mRNAs, which are transported and localized to specialized regions within the oocyte.

Visualizing and Tracking Endogenous mRNAs in Live Drosophila melanogaster Egg Chambers

Here, we present a protocol for the visualization, detection, analysis and tracking of endogenous mRNA trafficking in live Drosophila melanogaster egg chamber using molecular beacons, spinning disc confocal microscopy, and open-source analysis software.

Fluorescence-based imaging techniques, in combination with developments in light microscopy, have revolutionized how cell biologists conduct live cell ...

Monitoring Dopamine Release in a Rat Model with VTA Dopaminergic Neuron Receptor Manipulation

Source: Wickham, R. J., et al. <a href="https://app.jove.com/v/60886">Combined Infusion and Stimulation with Fast-Scan Cyclic Voltammetry (CIS-FSCV) to Assess Ventral Tegmental Area Receptor Regulation of Phasic Dopamine.</a> J. Vis. Exp. (2020).
This video demonstrates the implantation of electrodes and cannulas in an anesthetized rat to measure dopamine release in the nucleus accumbens using a potentiostat and the infusion of drugs to modulate dopaminergic activity in the ventral tegmental area.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
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Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

Summary

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Reliable Identification of Living Dopaminergic Neurons in Midbrain Cultures Using RNA Sequencing and TH-promoter-driven eGFP Expression

3D Particle Tracking for Noninvasive In Vivo Analysis of Synaptic Microtubule Dynamics in Dendrites and Neuromuscular Junctions of Drosophila

Semi-Quantitative Determination of Dopaminergic Neuron Density in the Substantia Nigra of Rodent Models using Automated Image Analysis

Enhanced Northern Blot Detection of Small RNA Species in Drosophila Melanogaster

Detection of G Protein-coupled Receptor Expression in Mouse Vagal Afferent Neurons using Multiplex In Situ Hybridization

Visualizing and Tracking Endogenous mRNAs in Live Drosophila melanogaster Egg Chambers

Monitoring Dopamine Release in a Rat Model with VTA Dopaminergic Neuron Receptor Manipulation