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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.
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.
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.
Using the oligonucleotide RNA probes (<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.
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 μl 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.
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 (±) 3,4-methylenedioxymethamphetamine (±MDMA) for this work.
A complete version of this article that includes the video component is available at http://dx.doi.org/10.3791/53165.
Disclosures: The authors declare that they have no competing financial interests.
Ibrahim M. Shokry, Ross University School of Veterinary Medicine, St. Kitts, West Indies.
John J. Callanan, Ross University School of Veterinary Medicine, St. Kitts, West Indies.
John Sousa, Charles E. Schmidt College of Medicine, Florida Atlantic University, Boca Raton, Florida, USA.
Rui Tao, Charles E. Schmidt College of Medicine, Florida Atlantic University, Boca Raton, Florida, USA.