A variety of procedures are conducted in the laboratory

v In situ hybridization histochemistry is employed to detect and quantify messenger ribonucleic acids (mRNA) in specific areas of the brain.

Example of in situ hybridization histochemistry against c-fos mRNA

(reversed picture from autoradiogram)

v Double fluorescent in situ hybridization (Fish) histochemistry is also employed to help the determination of colocalized mRNAs in the central nervous system.

Example of double FISH against c-fos mRNA (Fluorescein – green) and

the vesicular glutamate transporter 2 mRNA (CY3 – red),

also counterstained with DAPI in the auditory thalamus (blue)

(Courtesy, Sarah K. Sasse, 2008)

v Immunohistochemistry is employed to detect specific proteins in the brain.

Example of double immunohistochemical staining against the protein CRH (blue)

and the anterograde tracer PHA-l (brown; brightfield view)

v Anterograde and retrograde tract tracing techniques are used extensively to assess the connections between different parts of the brain.

Example of anterograde tract tracing (Biotinylated Dextran-amines)

(darkfield view)

v Sophisticated microscopes (Nikon E800, Zeiss AxioImager Z1) coupled with digital cameras and associated software allow resolution of mRNA, protein, and neural tracing obtained by in situ hybridization, immunohistochemistry, and various neuronal tracers.

v Brain inactivation is produced acutely by direct brain injections of local anesthetics (bupivocaine, lidocaine, tetrodotoxin, muscimol). Chronic inactivation of specific brain areas is also obtained by injecting neurotoxic agents (high doses of ibotenic acid or NMDA).

v Radioimmunoassays are employed to measure the release of various hormones in blood plasma.

v Different behavioral stress procedures are employed to study the brain circuits associated with psychological stress. Some examples include loud noises, very bright strobe lights, and body restraint.