This study was supported by the NIH grant (R15DA029863), the Florida Atlantic University undergraduate research grant (M30014) and the Ross University School of Veterinary Medicine research grant. We would like to thank the National Institute on Drug Abuse (Rockville, MD) for providing (&#177;)3,4-methylenedioxymethamphetamine (&#177;MDMA) for this work.

Animal use procedures described below were approved by The Institutional Animal Care and Use Committee (IACUC) at Ross University School of Veterinary Medicine and Florida Atlantic University. Although sterile techniques and gloves are required, the RNase-free environment is not necessary while using this protocol.
1. Preparation
<ol>
	<li>Tissue preparation and sectioning
	<ol>
		<li>Assign rats randomly into three groups: fresh/saline (SAL; 0.9% NaCl), prefixed/SAL and prefixed/3,4-methylenedioxymethamphetamine (MDMA; see details in table 1).</li>
		<li>Administer three doses of 1 ml/kg SAL and 10 mg/kg MDMA intraperitoneally, respectively, to the SAL and MDMA groups at 2 hr interval. Allow animals to survive the treatment for 6 days.</li>
		<li>On day 7, administer 100 mg/kg ketamine in combination of 5 mg/kg xylazine intraperitoneally to the rats for a deep anesthesia (i.e., loss of corneal blink and tail pinch reflexes).</li>
		<li>For the fresh/SAL group, quickly decapitate rats and remove the brain for the fresh brain test (see details in the reference5).</li>
		<li>For the prefixed/SAL and prefixed/MDMA groups, make a cut along the sternum about 10 -12 cm and then expose the end of the sternum.</li>
		<li>Hold the end of the sternum with a hemostat and cut the diaphragm laterally on both sides with sharp scissors and then cut upward across the ribs and parallel to the lungs.</li>
		<li>With one hand, hold the ventral tip of the heart with small forceps. With the other hand, pierce the left ventricle with a 18 G&#160;syringe needle (note the adapter side of the needle is connected with a Y-shaped silicon hose to ice-cold SAL and paraformaldehyde containers and peristaltic pumps; see the reference9).</li>
		<li>Turn on the SAL pump at the medium level of the flow rate (~5 ml/min) and puncture the right atrium with the forceps to allow the escape of return circulation. Maintain SAL perfusion for 30 min.</li>
		<li>Turn on the paraformaldehyde pump at the medium level and allow the ice-cold 4% paraformaldehyde to perfuse the animal for 30 min.</li>
		<li>Remove perfusion instrument and decapitate the animal. Make a posteroanterior cut through the midline of the skull, and then flap the skull to expose the skull cavity 10.</li>
		<li>Remove the brain with a spatula from the skull cavity and place the brain in a 50&#160;ml centrifuge tube containing 25 ml of 4% paraformaldehyde. Keep the brain within the paraformaldehyde-containing tube at 4 &#186;C refrigerator for 24 hr.</li>
		<li>Move the brain into a 50&#160;ml centrifuge tube containing 25 ml of 30% sucrose solution at 4 &#186;C for 3 days. Finally, store the brain within plastic bags at -80 &#176;C till use.</li>
		<li>Mount the brain on a chuck with embedding media and freeze the embedding media containing brain in a cryostat at temperature of -26 &#186;C. Cut the fresh frozen brain into 20&#160;&#181;m sections and paraformaldehyde-prefixed brain into 10-&#181;m sections. Transfer the section to a RT microscope slide by touching the slide to the section.</li>
		<li>Air-dry at RT for 4 hr. Store the section slides in a tight sealed bag at -80 &#176;C till use.</li>
	</ol>
	</li>
	<li>Dehydration
	<ol>
		<li>Take slides from the -80 &#176;C freezer and assign sections into one of three tests: ht2a, ppiB (positive control) or dapB (negative control); mark and label sections with a ball pen (do not use a pencil). Submerge slides in 100% alcohol at RT for 5 min. Dry the slides for 5 min in a fume hood.</li>
	</ol>
	</li>
	<li>Pretreatment
	<ol>
		<li>Pipette 20 &#181;l of the Pretreatment-1 reagent (inactivation of alkaline phosphatase) to the sections on slides. Place the slides on a rack rail in a moisture tray (note that the tray is covered with a lid to maintain a high humidity for preventing evaporation from the reaction solution).</li>
		<li>Gently shake the moisture tray on a horizontal shaker at a low speed for 10 min. Wash the slides twice with double-distilled water (ddH2O) for 2 min. Submerge the slide in a 200 ml beaker containing 150 ml of the Pretreatment-2 reagent (breaking of the cross-linkages induced by paraformaldehyde fixation). Boil the slides for a total of 5 min at 100 &#186;C (heat-induced restoration of RNA structure11).</li>
		<li>Wash the slides twice with ddH2O for 2 min. Place the slides in 100% alcohol for 5 min. Dry the slides for 5 min in the fume hood. Use a hydrophobic pen to draw a circle around the selected area in the tissue section (e.g., non-cortical structures, including thalamus and hypothalamus as outlined in Figure 2).</li>
		<li>Pipette 10 &#181;l of the Pretreatment-3 reagent (increases in probe penetration) to each selected area, and cover the moisture tray with the lid. Place the tray in the hybridization oven equipped with the horizontal shaker and set the oven temperature at 40 &#186;C; gently shake the tray for 30 min. Wash the slides twice with ddH2O for 2 min.</li>
	</ol>
	</li>
</ol>
2. Hybridization and Amplification
<ol>
	<li>Pipette 10 &#181;l of htr2a, ppiB and dapB probe reagents, respectively, on the selected areas. Cover the moisture tray with the lid to prevent evaporation of the reagent. Gently shake on the horizontal shaker set at a low speed. Incubate the slides at 40 &#186;C in the oven for 2 hr.</li>
	<li>Wash the slides twice with a washing buffer for 2 min. Pipette 10 &#181;l of the Amplification-1 reagent on each selected area, shake and incubate the slides at 40 &#186;C for 30 min.</li>
	<li>Wash the slides twice with a washing buffer for 2 min. Pipette 10 &#181;l of the Amplification-2 reagent on each selected area, shake and incubate the slides at 40 &#186;C for 15 min.</li>
	<li>Wash the slides twice with a washing buffer for 2 min. Pipette 10 &#181;l of the Amplification-3 reagent on each selected area, shake and incubate the slides at 40 &#186;C for 30 min.</li>
	<li>Wash the slides twice with a washing buffer for 2 min. Pipette 10 &#181;l of the Amplification-4 reagent on each selected area, shake and incubate the slides at 40 &#186;C for 15 min.</li>
	<li>Wash the slides twice with a washing buffer for 2 min. Pipette 10 &#181;l of the Amplification-5 reagent on each selected area, shake and incubate the slides at RT for 30 min.</li>
	<li>Wash the slides twice with a washing buffer for 2 min. Pipette 10 &#181;l of the Amplification-6 reagent on each selected area, shake and incubate the slides at RT for 15 min. Wash the slides twice with a washing buffer for 2 min.</li>
</ol>
3. Signal Detection
<ol>
	<li>Pipette 10 &#181;l of the Red reagent to each selected area (Note that the reagent is a mixture of Red-B and Red-A at a 1:60 ratio and should be used immediately). Place the slide in the moisture tray on the horizontal shaker at RT and shake gently at a low speed for 10 min.</li>
	<li>Wash the slides twice with (ddH2O) for 10 min. Dry the slides at 60 &#186;C in the oven for 15 min. Place the slides in xylene under the fume hood for 5 min. Pipette 20 &#181;l of xylene-based mounting media on each selected area and cover with a coverslip.</li>
</ol>
4. Image Capture
<ol>
	<li>Take digital microphotographs of the selected areas (e.g., hypothalamus, Figures 1 and 3) with a magnification of 4&#215; and 20&#215; objective lenses. Save the image file.</li>
</ol>
5. Image Particle Analysis
<ol>
	<li>Drag and drop the image file into the main ImageJ window. Go to the menu bar and select &#8216;Image&#8217;, next select &#8216;Type&#8217; from the drop-down menu, and select &#8216;8-bit&#8217;.</li>
	<li>Select &#8216;Adjust&#8217;, then select &#8216;Threshold&#8217; to open the dialog box. Adjust the lower bar value in the dialog box so that the unwanted background is removed. Go to the menu bar and select &#8216;Analyze&#8217;, then select &#8216;Analyze Particles&#8217; to open a second dialog box.</li>
	<li>Set particle size at 10, next select &#8216;Show outlines&#8217; and select &#8216;Display results&#8221; and &#8216;Summarize&#8217; boxes. Lastly, click &#8216;OK&#8217; to view particle data in the dialog boxes.</li>
</ol>
6. Spreadsheet Calculation
<ol>
	<li>Enter the data into the Excel sheet and average the numbers of particles in the SAL group as a baseline. Calculate the percentage level of each section and make a graph accordingly (Figure 4).</li>
</ol>

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The authors declare that they have no competing financial interests.

One of the major concerns of in situ hybridization test is to choose appropriate techniques used for RNA preservation because of RNase enzymes. It is well known that these enzymes are widely present in the cells and environment which can affect the results. However, enzyme activity can be quickly distinguished by placing the tissue in the dry ice/alcohol solution5,12. Although quick preservation is critical for hybridization using a riboprobe13, little is known about experimental conditions for oligonucleotide probes. In this study using oligonucleotide probes, we found that there was no difference between the paraformaldehyde-prefixed and fresh brains quickly preserved in the ice/alcohol solution. This suggests that brain RNA prefixed by paraformaldehyde had no significant effect on the quality of hybridization using the oligonucleotide probe. Since the prefixed brain is easily sectioned compared to the fresh brain, this protocol provides an alternative tissue preparation for the hybridization test. 
We believe that keeping the brain tissue sections inside the sealed moisture tray with constant shaking on the horizontal shaker is essential for successful hybridization. Failure to follow those steps may result in uneven hybridization or high background. To eliminate possibilities of false-negative or false-positive results, tests should include necessary control slides. We found that the dapB probe did not show a visible signal. This is not surprising since dapB is a bacterial gene that is unlikely found in mammalian cells. In contrast, the area hybridized with the ppiB probe showed signals homogenously distributed throughout the region examined. This is predictable since the housekeeping gene should be present in any kind of mammalian cells. It has been suggested that 5-HT2A mRNA is distributed heterogeneously in the brain nuclei, including the hypothalamus14,15. As expected, htr2a signals are present in certain nuclei in the region of the hypothalamus although the probe has been applied to all of the selected areas. It should be kept in mind that the use of alcohol should be avoided after the Red reagent (step 3.1) since red signals can be easily decolored by alcohol. We found that, after cellular RNA stained with the Red reagent, cells cannot be counterstained with hematoxylin or crystal violet although the exact cause of counterstaining failure is unknown. In summary, a characteristic distribution of htr2a mRNA supports the idea that these oligonucleotide probes are gene-specific despite the shorter sequence relative to other probes16,17. 
Background signals are another major concern, which likely affects the quality and faithfulness of data analysis. Unlike the high background produced by riboprobes13, oligonucleotide probes normally show a very low background. Thus, image can be easily and reasonably quantified using publicly-available NIH ImageJ software. In this study, we showed an example of this technique by examining changes in htr2a expression in response to systemic MDMA at 10 mg/kg. We demonstrated that MDMA caused a 20% reduction in htr2a expression, supporting previous reports18. 
In addition to the three genes outlined above, this protocol has been validated with two other gene probes (trh, thyrotropin-release hormone gene, and trhr, thyrotropin-releasing hormone receptor gene; data not shown), demonstrating a high sensitivity and accuracy in detecting mRNA levels. Since most reactions require only 10 &#181;l of reagents, the cost of an assay is also significantly reduced, compared to radioactive riboprobes. Furthermore, it is important to note that this protocol does not require the RNase-free environment. In conclusion, this protocol improves the speed of in situ hybridization assay, and increases reproducibility with relatively low cost.

Serotonin (5-hydroxytryptamine; 5-HT) syndrome is an acute neurologic disorder caused by 5-HT-promoting drugs such as antidepressants1, while also occurring in situations of MDMA use for recreational purposes2. Molecular mechanisms responsible for mood swings, learning and memory deficits that occur in association with the acute syndrome are not well understood3,4. In situ hybridization is a powerful research tool allowing the detection and quantification of specific mRNAs expressed potentially at a single-cell level. The conventional way to perform in situ hybridization is to utilize a radioactive-labeled riboprobe that specifically hybridizes the gene of interest. However, a major drawback is that the method requires complicated and time-consuming probe preparation and hybridization steps, as well as access to fresh frozen tissues maintained in an RNase-free environment5,6. 
Oligonucleotide probes have been recently developed to hybridize shorter RNA fragments than those required for riboprobes7. Furthermore, the probes produce a low background signal without sacrificing specificity8. This newly-developed probe technology can be used in situ hybridization on paraformaldehyde-prefixed brain tissues commonly available in immunocytochemical laboratories.
Here, we describe a protocol for in situ hybridization using oligonucleotide probes on paraformaldehyde-prefixed rat brain and compare findings with those noted in a fresh frozen brain5,6. This protocol is used to test the hypothesis that MDMA substance abuse causes changes in 5-HT2A receptor gene htr2a mRNA in the brain. We began the procedure with MDMA treatment followed by paraformaldehyde tissue perfusion of the animal, in situ hybridization of the thr2a probe, and data analysis. Note that dapB (the bacterial gene coding for dihydrodipicolinate reductase) is used as a negative control, and ppiB (housekeeping gene coding for peptidylprolyl isomerase B) as a positive control.

<table><tbody><tr><td>RNAscope Negative Control Probe-DapB</td><td>Advanced cell diagnostic, INC</td><td>310098</td><td></td></tr><tr><td>RNAscope Pretreat 4</td><td>Advanced cell diagnostic, INC</td><td>320046</td><td></td></tr><tr><td>RNAscope 2.0 HD Reagent Kit - Red</td><td>Advanced cell diagnostic, INC</td><td>310036</td><td></td></tr><tr><td>RNAscope Probe - Rn-Ppib</td><td>Advanced cell diagnostic, INC</td><td>313921</td><td></td></tr><tr><td>Rat Htr2a</td><td>Advanced cell diagnostic, INC</td><td>300031</td><td></td></tr><tr><td>Cryostat</td><td>Leica</td><td>CM 1850</td><td></td></tr><tr><td>Horizontal shaker</td><td>VWR</td><td>88032-088</td><td></td></tr><tr><td>Hybridization oven</td><td>Thermo Fisher Scientific</td><td>222000</td><td></td></tr><tr><td>Superfrost Plus microscope slide</td><td>Fisher Scientific</td><td>12-550-15</td><td></td></tr><tr><td>Hydrophobic pen</td><td>Vector</td><td>H-4000</td><td></td></tr><tr><td>Microscope</td><td>Olympus</td><td>Provis AX70</td><td></td></tr></tbody></table>

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.

Using the oligonucleotide RNA probes (&#60;25 nt), hybridization can be detected as red dots in hypothalamic cells prepared from the paraformaldehyde-prefixed and fresh frozen brains. The htr2a mRNA molecules are present in some cells (indicated by solid arrows), but not others (open arrows). We observed that there is no difference between the paraformaldehyde-prefixed and the fresh frozen tissues (Figure 1). A successful hybridization can be evaluated first by the naked eye (Figure 2). We found that the selected area (marked with circles) hybridized with the dapB probe did not show a visible signal to the naked eye (Figures 2A-B). In contrast, the area hybridized with the ppiB probe showed signals homogenously distributed throughout the region examined (Figures 2C-D). While in the htr2a test, signals are present in certain nuclei (indicated by arrows; Figures 2E-F), although all the circled area has been hybridized with the htr2a probe. Interestingly, the signals in MDMA slides were relatively weak compared to SAL slides. Substantial details of hybridization signals can be evaluated under a microscopic instrument. There is no red dot (Figure 3A-B) in the cells hybridized with the dapB probe whereas signals can be seen throughout the microscopic field hybridized with the ppiB probe (Figure 3C-D). The htr2a signals are mainly located in certain nuclei of the hypothalamus (Figure 3E-F). We observed a reduction in htr2a signals in the brain tissues previously treated with systemic MDMA at 10 mg/kg, which can be quantitatively determined using the ImageJ analysis. As shown in Figure 4, we found a 20% reduction in htr2a expression.
In conclusion, our results showed that using oligonucleotide probes is specific, and suitable for the prefixed brain tissue. Furthermore, protocol procedures, including in situ hybridization, signal detection and data analysis can be completed within two days.
<img alt="Figure 1" src="/files/ftp_upload/53165/53165fig1.jpg" /> 
Figure 1.&#160;Comparison of the fresh and prefixed frozen tissues. Procedures used for fresh frozen brains have been described elsewhere5. Note that the fresh frozen sections were cut at 20 &#181;m in thickness while the fixed frozen sections at 10 &#181;m. The oligonucleotide probe was htr2a that hybridizes the 5-HT2A receptor mRNA in the hypothalamus. Solid arrows indicate cells containing htr2a mRNA molecules in the fresh frozen (A) and prefixed tissues (B). Open arrows, no htr2a mRNA molecules detected. Bar: 20 &#181;m. Using NIH ImageJ analysis, htr2a was counted. Numbers of htr2a counts are not different between the fresh and paraformaldehyde-prefixed frozen tissues (C). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53165/53165fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53165/53165fig2.jpg" /> 
Figure 2.&#160;A naked-eye view of hybridization signals. Tissues were prefixed with paraformaldehyde in deeply anesthetized rats previously treated with saline (SAL) and 3,4-methylenedioxymethamphetamine (MDMA). The white-dashed lines indicate the region hybridized with oligonucleotide probes. The red color denotes mRNA signals to the naked eye. No red signal is observed while using the dapB probe hybridized to sections of SAL-pretreated (A) or MDMA-pretreated rats (B). In contrast, red signals are homogeneously distributed throughout the hypothalamic regions while using the ppiB probe (C-D). Interestingly, red signals produced by the htr2a probe are only in some regions (E-F; Arrows indicate the hypothalamic area). Stereotaxic coordinates: -3.50 ~ -3.80 relative to the bregma. 3V, the third ventricle. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53165/53165fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53165/53165fig3.jpg" /> 
Figure 3.&#160;A light microscopic view of hybridization signals. Tissues were prefixed with paraformaldehyde in deeply anesthetized rats previously treated with saline (SAL) and 3,4-methylenedioxymethamphetamine (MDMA). Oligonucleotide probes are dapB (A-B), ppiB (C-D) and htr2a (E-F). Hybridization signals are identified as red dots using objective magnification powers ranging from 4&#215; (Bars: 200 &#181;m) to 20&#215; (inset microphotographs). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53165/53165fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/53165/53165fig4.jpg" /> 
Figure 4.&#160;Effect on hypothalamic htr2a expression in rats previously treated with MDMA. Samples were assayed in duplicates and repeated three times. Data are expressed as a mean percentage &#177; s.e.m. of the SAL group (a total of 8 rats used in this study). *P &#60;0.05 vs. the SAL group using Student&#8217;s t test.
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td></td>
			<td>Fresh</td>
			<td></td>
			<td colspan="2">Paraformaldehyde-prefixeda</td>
		</tr>
		<tr>
			<td></td>
			<td>SALb</td>
			<td></td>
			<td>SALb</td>
			<td>MDMAc</td>
		</tr>
		<tr>
			<td>Target probe (htr2a)</td>
			<td>&#8730;</td>
			<td></td>
			<td>&#8730;</td>
			<td>&#8730;</td>
		</tr>
		<tr>
			<td>Positive probe (ppiB)</td>
			<td>&#8211;</td>
			<td></td>
			<td>&#8730;</td>
			<td>&#8730;</td>
		</tr>
		<tr>
			<td>Negative probe (dapB)</td>
			<td>&#8211;</td>
			<td></td>
			<td>&#8730;</td>
			<td>&#8730;</td>
		</tr>
	</tbody>
</table>
&#8730;, indicates the group examined with oligonucleotide probe in this study. 
&#8211;, not examined; 
a, each group contains 4 different animals; 
b, the group pretreated intraperitoneally with physical saline (SAL; 0.9% NaCl); 
c, pretreated intraperitoneally with 10 mg/kg MDMA.
Table 1.&#160;Experimental design

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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.

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 ...

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8.8K Views.  Ross University School of Veterinary Medicine. 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.

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

Double Fluorescence in situ Hybridization in Fresh Brain Sections

Here we describe a modified version of a double fluorescence in situ hybridization (dFISH) method optimized for detecting two mRNAs of interest in fresh frozen brain sections. Our group has successfully used this approach to study gene co-regulation. More specifically, we have used this dFISH method to explore the anatomical organization, neurochemical properties, and the impact of sensory experience in central sensory circuits, at single cell resolution. This protocol has been validated in brain tissue from mice, rats and songbirds but is expected to be easily adaptable to other vertebrate species, as well as to an array of non-neural tissues. In this film we provide a detailed demonstration of the main steps of this procedure.

Double Fluorescence in situ Hybridization in Fresh Brain Sections

This protocol involves a non-radioactive in-situ hybridization procedure that enables the simultaneous identification of two transcript species, at a single cell resolution, in thin sections of the vertebrate brain.

Here we describe a modified version of a double fluorescence in situ hybridization (dFISH) method optimized for detecting two mRNAs of interest in fresh ...

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

Medicine

DetectSyn: A Rapid, Unbiased Fluorescent Method to Detect Changes in Synapse Density

Synapses are the site of communication between neurons. Neuronal circuit strength is related to synaptic density, and the breakdown of synapses is characteristic of disease states like major depressive disorder (MDD) and Alzheimer's disease. Traditional techniques to investigate synapse numbers include genetic expression of fluorescent markers (e.g., green fluorescent protein (GFP)), dyes that fill a neuron (e.g., carbocyanine dye, DiI), and immunofluorescent detection of spine markers (e.g., postsynaptic density 95 (PSD95)). A major caveat to these proxy techniques is that they only identify postsynaptic changes. Yet, a synapse is a connection between a presynaptic terminal and a postsynaptic spine. The gold standard for measuring synapse formation/elimination requires time-consuming electron microscopy or array tomography techniques. These techniques require specialized training and costly equipment. Further, only a limited number of neurons can be assessed and are used to represent changes to an entire brain region. DetectSyn is a rapid fluorescent technique that identifies changes to synapse formation or elimination due to a disease state or drug activity. DetectSyn utilizes a rapid proximity ligation assay to detect juxtaposed pre- and postsynaptic proteins and standard fluorescent microscopy, a technique readily available to most laboratories. Fluorescent detection of the resulting puncta allows for quick and unbiased analysis of experiments. DetectSyn provides more representative results than electron microscopy because larger areas can be analyzed than a limited number of fluorescent neurons. Moreover, DetectSyn works for in vitro cultured neurons and fixed tissue slices. Finally, a method is provided to analyze the data collected from this technique. Overall, DetectSyn offers a procedure for detecting relative changes in synapse density across treatments or disease states and is more accessible than traditional techniques.

DetectSyn is an unbiased, rapid fluorescent assay that measures changes in relative synapse (pre- and postsynaptic engagement) number across treatments or disease states. This technique utilizes a proximity ligation technique that can be used both in cultured neurons and fixed tissue.

Synapses are the site of communication between neurons. Neuronal circuit strength is related to synaptic density, and the breakdown of synapses is ...

Biochemistry

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 ...

Combining Double Fluorescence In Situ Hybridization with Immunolabelling for Detection of the Expression of Three Genes in Mouse Brain Sections

Detection of gene expression in different types of brain cells e.g., neurons, astrocytes, oligodendrocytes, oligodendrocyte precursors and microglia, can be hampered by the lack of specific primary or secondary antibodies for immunostaining. Here we describe a protocol to detect the expression of three different genes in the same brain section using double fluorescence in situ hybridization with two gene-specific probes followed by immunostaining with an antibody of high specificity directed against the protein encoded by a third gene. The Aspartoacyclase (ASPA) gene, mutations of which can lead to a rare human white matter disease - Canavan disease - is thought to be expressed in oligodendrocytes and microglia but not in astrocytes and neurons. However, the precise expression pattern of ASPA in the brain has yet to be established. This protocol has allowed us to determine that ASPA is expressed in a subset of mature oligodendrocytes and it can be generally applied to a wide range of gene expression pattern studies.

Combining Double Fluorescence In Situ Hybridization with Immunolabelling for Detection of the Expression of Three Genes in Mouse Brain Sections

Localizing gene expression to specific cell types can be challenging due to the lack of specific antibodies. Here we describe a protocol for simultaneous triple detection of gene expression by combining double fluorescence RNA in situ hybridization with immunostaining.

Detection of gene expression in different types of brain cells e.g., neurons, astrocytes, oligodendrocytes, oligodendrocyte precursors and microglia, can ...

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.

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.

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 ...

Using Near-infrared Fluorescence and High-resolution Scanning to Measure Protein Expression in the Rodent Brain

Neuroscience is the study of how cells in the brain mediate various functions. Measuring protein expression in neurons and glia is critical for the study of neuroscience as cellular function is determined by the composition and activity of cellular proteins. In this article, we describe how immunocytochemistry can be combined with near-infrared high-resolution scanning to provide a semi-quantitative measure of protein expression in distinct brain regions. This technique can be used for single or double protein expression in the same brain region. Measuring proteins in this fashion can be used to obtain a relative change in protein expression with an experimental manipulation, molecular signature of learning and memory, activity in molecular pathways, and neural activity in multiple brain regions. Using the correct proteins and statistical analysis, functional connectivity among brain regions can be determined as well. Given the ease of implementing immunocytochemistry in a laboratory, using immunocytochemistry with near-infrared high-resolution scanning can expand the ability of the neuroscientist to examine neurobiological processes at a systems level.

Here, we present a protocol that uses near-infrared dyes in conjunction with immunohistochemistry and high-resolution scanning to assay proteins in brain regions.

Neuroscience is the study of how cells in the brain mediate various functions. Measuring protein expression in neurons and glia is critical for the study ...

Combining Multiplex Fluorescence In Situ Hybridization with Fluorescent Immunohistochemistry on Fresh Frozen or Fixed Mouse Brain Sections

Fluorescent in situ hybridization (FISH) is a molecular technique that identifies the presence and spatial distribution of specific RNA transcripts within cells. Neurochemical phenotyping of functionally identified neurons usually requires concurrent labelling with multiple antibodies (targeting protein) using immunohistochemistry (IHC) and optimization of in situ hybridization (targeting RNA), in tandem. A &#34;neurochemical signature&#34; to characterize particular neurons may be achieved however complicating factors include the need to verify FISH and IHC targets before combining the methods, and the limited number of RNAs and proteins that may be targeted simultaneously within the same tissue section.
Here we describe a protocol, using both fresh frozen and fixed mouse brain preparations, which detects multiple mRNAs and proteins in the same brain section using RNAscope FISH followed by fluorescence immunostaining, respectively. We use the combined method to describe the expression pattern of low abundance mRNAs (e.g., galanin receptor 1) and high abundance mRNAs (e.g., glycine transporter 2), in immunohistochemically identified brainstem nuclei.
Key considerations for protein labelling downstream of the FISH assay extend beyond tissue preparation and optimization of FISH probe labelling. For example, we found that antibody binding and labelling specificity can be detrimentally affected by the protease step within the FISH probe assay. Proteases catalyze hydrolytic cleavage of peptide bonds, facilitating FISH probe entry into cells, however they may also digest the protein targeted by the subsequent IHC assay, producing off target binding. The subcellular location of the targeted protein is another factor contributing to IHC success following FISH probe assay. We observed IHC specificity to be retained when the targeted protein is membrane bound, whereas IHC targeting cytoplasmic protein required extensive troubleshooting. Finally, we found handling of slide-mounted fixed frozen tissue more challenging than fresh frozen tissue, however IHC quality was overall better with fixed frozen tissue, when combined with RNAscope.

Combining Multiplex Fluorescence In Situ Hybridization with Fluorescent Immunohistochemistry on Fresh Frozen or Fixed Mouse Brain Sections

This protocol describes a method for combining fluorescence in situ hybridization (FISH) and fluorescence immunohistochemistry (IHC) in both fresh frozen and fixed mouse brain sections, with the goal of achieving multilabel FISH and fluorescence IHC signal. IHC targeted cytoplasmic and membrane attached proteins.

Fluorescent in situ hybridization (FISH) is a molecular technique that identifies the presence and spatial distribution of specific RNA transcripts within ...

In Situ Hybridization Using Oligonucleotide Probes to Study RNA Expression in Rat Brain Sections

Source: Shokry, I. M., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/53165">Rapid In Situ Hybridization using Oligonucleotide Probes on Paraformaldehyde-prefixed Brain of Rats with Serotonin Syndrome.</a>&#160;J. Vis. Exp.&#160;(2015).
This video demonstrates the detection of serotonin receptor RNA synthesis using oligonucleotide probes in rat brain tissue affected by serotonin syndrome. The protocol includes tissue pretreatment, probe hybridization, and enzymatic detection to visualize and quantify serotonin receptor RNA expression under a microscope.

Source: Shokry, I. M., et al. Rapid In Situ Hybridization using Oligonucleotide Probes on Paraformaldehyde-prefixed Brain of Rats with Serotonin ...

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Neurodegenerative Diseases

 Performing Double Fluorescence In Situ Hybridization to Detect Gene Expression in Mouse Brain Sections

<a href='https://app-jove-com.remotexs.ntu.edu.sg/v/53976/combining-double-fluorescence-situ-hybridization-with-immunolabelling'>Source: Jolly, S., et.al. Combining Double Fluorescence In Situ Hybridization with Immunolabelling for Detection of the Expression of Three Genes in Mouse Brain Sections. J. Vis. Exp. (2016).</a>This video demonstrates the use of double fluorescence in situ hybridization to visualize the expression of two RNA targets in mouse brain sections. The protocol involves sequential detection of RNA using probes labeled with fluorescein isothiocyanate and digoxigenin, followed by enzymatic signal amplification to enhance sensitivity. The resulting fluorescence enables co-localization analysis of the target RNAs within the tissue.

Source: Jolly, S., et.al. Combining Double Fluorescence In Situ Hybridization with Immunolabelling for Detection of the Expression of Three Genes in Mouse ...

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

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Chapters in this video

Double Fluorescence in situ Hybridization in Fresh Brain Sections

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

DetectSyn: A Rapid, Unbiased Fluorescent Method to Detect Changes in Synapse Density

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

Combining Double Fluorescence In Situ Hybridization with Immunolabelling for Detection of the Expression of Three Genes in Mouse Brain Sections

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

Using Near-infrared Fluorescence and High-resolution Scanning to Measure Protein Expression in the Rodent Brain

Combining Multiplex Fluorescence In Situ Hybridization with Fluorescent Immunohistochemistry on Fresh Frozen or Fixed Mouse Brain Sections

In Situ Hybridization Using Oligonucleotide Probes to Study RNA Expression in Rat Brain Sections

Performing Double Fluorescence In Situ Hybridization to Detect Gene Expression in Mouse Brain Sections