Microdialysis of Ethanol During Operant Ethanol Self-administration and Ethanol Determination by Gas Chromatography

The overall goal of this procedure is to monitor ethanol levels in brain dialysate during an ethanol self-administration session. In order to compare this measure to synchronous self-administration behaviors, this is accomplished by first surgically implanting a microdialysis guide, cannula and tether bolt, and then training rats to operantly self-administer ethanol. The second step is to habituate the rats to the head stage tether so that it will not impede ethanol self-administration during microdialysis.

Then microdialysis is performed during an ethanol self-administration session during which a portion of dialate is prepared for ethanol analysis. Next, the dialate samples are analyzed for ethanol concentration using a gas chromatograph with flame ionization detection. Ultimately, the results obtained show the time course of ethanol reaching the brain region of interest during an ethanol self-administration session.

The main advantage to this technique over existing methods like tail or saphenous vein puncture is that this procedure is minimally disruptive at the time of sample collection and allows for drug measurement over time in the brain region of interest while the animal is performing a behavioral task. Generally, individuals new to this method will struggle because it requires a number of specialized skills, many of which can only be acquired through repeated practice or experience. Also demonstrating the procedure will be Jeffrey Dilley, an undergraduate research assistant in our laboratory.

All animal procedures shown are approved by the University of Texas Institutional Animal Care and Use Committee and follow all guide for the care and use of Laboratory Animals regulations. The University of Texas at Austin is an ALAC accredited facility well-handled long Evans rats that have been surgically implanted with a 21 gauge cannula above the brain region of interest are used for these behavioral microdialysis experiments. All animals should receive at least one week of post-surgical care and recovery and are healthy before beginning the following procedures.

Place the rat in a med associates operant chamber, fitted with a lichter retractable lever and tipper tube and train it to press the lever for access to a 10%sucrose solution. After the rat is trained to respond for the sucrose solution, begin it on an appropriate training schedule during which ethanol is gradually added into the solution over multiple drinking sessions the night before the second to last operant session. After anesthetizing the animal and checking for level of sedation, attach the tether spring to the tether bolt on the rat's head stage to habituate it to the apparatus which consists of a counterbalance lever, arm, swivel tether, spring, and rings stand.

Allow the rat to spend the night in the operant room in its home cage with ad lido, food and water, and then complete the next day's training session with the tether and accompanying equipment in place the day before the microdialysis experiment. After anesthetizing the rat with isof fluorine, check for sedation by performing a toe pinch and insert a microdialysis probe perfused with artificial cerebral spinal fluid. Secure the probe appropriately and then adjust the probe flow rate to 0.2 microliters per minute.

Place the rat in its home cage in the operant room to spend the night with all dialysis equipment in place and add lib, food and water. The day after inserting the probe at least two hours before the experiment begins, turn up the probe to its working flow rate. Using a Hamilton syringe, check that the probe flow rate is consistent and is at least 90%of the set flow rate.

If evaluating neurotransmitters in the dialysis samples, take samples during all phases of the experiment. Start by collecting dialysis samples while the animal is in its home cage. Store the samples in an appropriate manner for later analysis.

Carefully transfer the rat to the operant chamber and start the operant program that controls the chamber. Continue taking samples while the rat waits for the lever to extend into the chamber two samples before the lever extends. Begin collecting a portion of each dialysate sample for ethanol analysis by pipetting an appropriate aliquot of dialysate into a two milliliter glass vial and sealing the vial with an airtight septum, pipette, and seal the ethanol samples quickly.

As ethanol is volatile and the sample volume is small, inconsistent or slow sample preparation will lead to inaccurate results. Store the remainder for neurotransmitter analysis. The 20 minute drink period begins after the Med Associates computer program extends the lever into the chamber and the rat presses it to gain access to a bottle of drinking solution.

Continue to take samples throughout the entire drink period after the drink period, the computer program retracts the bottle from the chamber. Keep the animal in the chamber for an additional 20 minute post drink period at the end of the microdialysis experiment. After anesthetizing the rat, remove the probe.

If the animal is not immediately humanely euthanized, replace the obterator to ensure visualization of the probe tract. Collect the brain within three days of the experiment. Analyze the samples along with external ethanol standards using a gas chromatograph with a flame ionization detector, a headspace autos sampler, heated to 50 degrees Celsius, and a capillary column with helium as the mobile phase.

For recording and analyzing the chromatograms use chromatography workstation software to prepare the system for ethanol analysis. Use a recirculating water bath to heat the autos sampler plate and open the air and hydrogen tanks. Next, initiate the startup program.

When it is ready, initiate a 20 minute burn, which decontaminates the fiber. Make standards using a 100 milliliter volumetric flask to dilute 238 microliters of 95%ethanol with water to a final volume of 100 milliliters. Use a one-to-one serial dilution to create standards.

Pipette the standards into glass vials and seal them exactly as the samples were prepared. Be sure to run a water blank along with your standards. Place the standards and samples into the autos sampler tray, and heat them until the entire liquid aliquot has been vaporized.

To run the standards and samples, create a sample list noting which sample is in which auto sampler slot, and noting that each sample should be punctured. Once route the data files to a selected folder, then select a method of choice for the samples and begin the run. After the system has run through the samples, check that the program has correctly integrated each peak.

Plot, the peak height as a function of each known external standard. Ethanol concentration. Use the linear function given by the points to interpolate the ethanol concentration of each dialysate sample.

This figure shows example chromatograms for three concentrations of ethanol standards and one for a rat dialysate sample collected at the end of the ethanol self-administration session. Ethanol peaks should be relatively symmetric, have consistent retention times and a signal to noise ratio greater than 10. Failure to meet these criteria means the system requires maintenance, quality chromatography, and correctly standards produce a linear standard curve that is used to calculate the ethanol concentration of dialysate samples collected from ethanol self-administering animals over the course of their self-administration.

The derived dialysate ethanol concentrations are not corrected for in vivo probe recovery and are only a fraction of the brain ethanol tissue concentration. If quantitative microdialysis of ethanol is required, the extraction fraction of ethanol that diffuses from the extracellular space into the probe is experimentally determined. Once mastered, this technique can be done in approximately four to five hours if it is performed properly.

This will vary based on the length of the behavioral session. Successful completion of the microdialysis phase of the experiment greatly depends upon the quality of the preparatory Phases. While attempting this procedure is important to be sure that your rats are well handled and that you have habituated them to any possibly distracting stimuli that could occur during the dialysis experiment so that the animal's self-administration behavior is not affected.