What is microdialysis?
A dialysis probe is inserted into the brain area of interest, and artificial cerebrospinal fluid (aCSF) is pumped very slowly through it. The probe consists of stainless steel tubing, with a tiny (210 microns in diameter, 1 mm long) “sock” of dialysis membrane glued to the end. During the time that the aCSF is inside the “sock,” small molecules of neurotransmitters and metabolites can diffuse into the fluid, which flows out via a silicon tube inside the probe. The fluid is then collected and analyzed, using high performance liquid chromatography with electrochemical detection (HPLC-EC).
What is immunocytochemistry (immunohistochemistry)?
Antibodies to specific proteins are grown in various species of animals (for example, rabbits). Antibodies that recognize a protein of interest (for example, nitric oxide synthase, NOS) are incubated on slices of brain. The antibodies bind to the proteins of interest, and then secondary antibodies that recognize the species of the original host animal (rabbit) are incubated on the brain slices. The secondary antibodies carry visible tags, allowing cells containing the proteins (NOS) to be seen under a microscope. The figure below shows one NOS immunoreactive cell under high magnification and numerous cells in the MPOA under low magnification.
Microinjections
Rationale. Microdialysis provides evidence that a neurotransmitter is, in fact, released in the brain area of interest before and/or during a behavior. However, the data are correlational; it is not clear whether the transmitter release was a causative factor in eliciting the behavior or a response to the behavior. Microinjections are used to introduce an agonist or antagonist of the neurotransmitter of interest into the brain area immediately before a behavioral test. Such experiments also allow one to determine which receptor subtype mediates the effect of the transmitter. For example, we have shown that D1 dopamine agonists in the MPOA facilitate the early stages of copulation, including increasing erections. Higher amounts of dopamine, acting on D2 receptors shift the autonomic balance to favor ejaculation (see below).
Procedure. A stainless steel cannula is surgically implanted, ending just above the structure of interest. After recovery, a drug or vehicle is injected just before a behavioral test, and the effects on behavior can be analyzed.
Care of animals
Great care is taken to ensure the health and well-being of our animals. They are handled and weighed every day, in order to accustom them to being handled and to check their health. All surgeries are done under anesthesia, with additional local anesthetic, followed by an analgesic. Because we are interested in brain mechanisms of behavior, it is critical that our animals are healthy, so that their behavior is normal. All procedures are approved by the local Animal Care and Use Committee, and are overseen by our AAALAC-approved Laboratory Animal Resources (LAR). All members of the lab are required to take a filmed course in animal care, offered by the LAR.