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1Department of Pharmacology and Experimental Therapeutics, Tufts University
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The monitoring of extracellular neurotransmitter levels in distinct brain regions of freely moving animals offers insights on the link between neurotransmitter release and behavior. In vivo microdialysis coupled with electrochemical detection provides excellent anatomical and chemical resolution; and information on how basal neurotransmission is altered by pharmacological or physiological manipulations.
Geiger, B. M., Frank, L. E., Caldera-Siu, A. D., Pothos, E. N. Survivable Stereotaxic Surgery in Rodents . J. Vis. Exp. (20), e880, doi:10.3791/880 (2008).
The ability to measure extracellular basal levels of neurotransmitters in the brain of awake animals allows for the determination of effects of different systemic challenges (pharmacological or physiological) to the CNS. For example, one can directly measure how the animal's midbrain dopamine projections respond to dopamine-releasing drugs like d-amphetamine or natural stimuli like food. In this video, we show you how to implant guide cannulas targeting specific sites in the rat brain, how to insert and implant a microdialysis probe and how to use high performance liquid chromatography coupled with electrochemical detection (HPLC-EC) to measure extracellular levels of oxidizable neurotransmitters and metabolites. Local precise introduction of drugs through the microdialysis probe allows for refined work on site specificity in a compound s mechanism of action. This technique has excellent anatomical and chemical resolution but only modest time resolution as microdialysis samples are usually processed every 20-30 minutes to ensure detectable neurotransmitter levels. Complementary ex vivo tools (i.e., slice and cell culture electrophysiology) can assist with monitoring real-time neurotransmission.
Two-month old average age C57BL/6J mice or equivalent or three-month old average age Sprague Dawley rats or equivalent are anesthetized with ketamine (60 mg/kg i.p. for rats; 100 mg/kg i.p for mice) and xylazine (10 mg/kg, i.p. for either species). Sedation is monitored using a gentle toe pinch withdraw reflex demonstrated in Walantus et al.(JoVE, 6, 2007) and Szot et al.(JoVE, 9, 2007). Thermoregulation can be provided through a thermostatregulated heating pad (ALA Instruments Inc.) and monitored through a rectal thermometer Head is shaved of fur and cleaned with iodine before incision. After skin incision (2 cm long for rats, 1 cm long for mice) and removal of all soft tissue from the surface of the skull, placement of the guide cannula is determined in relation to bregma. A 6 mm hole is drilled through the skull with a battery-operated driller designed for rodent surgery (Fine Science Tools, Inc.). Care is taken so that the drill bit does not penetrate through meningeal membranes or blood vessels. Skull is implanted with bilateral 5 mm 21 gauge stainless steel guide shafts leading to the posterior nucleus accumbens, dorsal striatum or prefrontal cortex. The stereotaxic coordinates are established as per Franklin and Paxinos, 1997 (The Mouse Brain in Stereotaxic Coordinates, Academic Press) or Paxinos and Watson, 2006 (The Rat Brain in Stereotaxic Coordinates, Academic Press). Implants are secured by dental cement. A bolus of Lactated Ringers of the 0.9% saline is given at the end of surgery (5mls SC in rats and 1 ml SC in mice after fluids are warmed to normal body temperature) to prevent dehydration. Buprenorphine (0.1-0.5mg/kg SC) is administered twice daily and, then, on an as-needed basis, if animal appears to be in pain. Local antibiotic treatment (bacitracin ointment) and systemic antibiotic treatment (penicillin 100,000 IU/kg IM every 12 hours for the first 48 hours post-op) are administered if post-operative infections occur.
Following surgery, animals are individually housed with food and water available ad libitum. At least one week is allowed for recovery before microdialysis and euthanasia. Following recovery from surgery, the animals are transferred to a microdialysis cage and microdialysis probes are inserted and cemented in the guide shafts that have been installed during surgery. Probe insertion does not cause pain or discomfort because the probe is bypassing skin, muscle and meningeal tissue through the guide shaft. Therefore, probe insertion is done without anesthesia and any anesthesia-induced effects on neurochemistry or behavior are avoided. We let the probes stabilize for 12 hours and then we start sampling every 30 minutes for another 8-12 hours depending on the experiment. We monitor locomotor behavior of the animal through photocells or manual recording of movement by the experimenter. Microdialysate samples are injected into a High Performance Liquid Chromatography with Electrochemical Detection (HPLC-EC) instrument for neurochemical detection and analysis. We look for effects on basal neurochemistry and locomotor behavior. At the end of the experiment the animal is euthanized by an overdose of systemic ketamine (200 mg/kg i.p.) and xylazine (20 mg/kg, i.p.). Then the heart is perfused with 0.9% saline followed by 4% paraformaldehyde. The brains are removed, frozen and cut along the microdialysis probe tract to verify accurate probe placement.
In vivo microdialysis is the tool of choice for measuring multiple neurotransmitters and metabolites in distinct brain sites of a living animal. However, it only monitors extracellular levels of neurochemicals and it does not offer the time resolution to monitor neurotransmitter exocytosis in real time. Through a version of the technique called "net-flux", the actual neurotransmitter concentration at a given site can be calculated, which in turn can give accurate measurements of neurotransmitter rate of reuptake through plasma membrane transporters.
Microdialysis is ideal in illustrating differences in basal extracellular neurotransmitter levels between different groups of animals (i.e. different genotypes) and in deciphering the effects of drugs or other manipulations on neurotransmitter release.
The introduction of assays alternative to HPLC-EC like capillary zone electrophoresis (CZE) coupled with fluorescent detection has increased the time resolution of in vivo microdialysis within a few minutes per sample.
The authors have nothing to disclose.
Supported by DK065872 (ENP), a Smith Family Foundation Award of Excellence in Biomedical Research (ENP), F31 DA023760.
|Materials are described in the protocol document.|
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