This research was supported by grants from NIH/NIAAA (AA11852 and AA007471).

1. Stereotaxic Surgery
<ol>
 <li>All procedures follow the NIH Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee. </li>
 <li>Using a stereotaxic apparatus, well-handled Long-Evans rats, anesthetized with isoflurane, are implanted with a 21-gauge cannula (for later microdialysis probe insertion) (Plastics One, Roanoke, VA) above the brain region of interest, and a tether bolt is built into the head stage (used to support microdialysis equipment). </li>
 <li>Monitor rats carefully during and after surgery. Make sure that all animals receive at least one week of post-surgical care and recovery and are healthy before beginning the following procedures. A JoVE video of the stereotaxic surgery method is available.1 </li>
</ol>
2. Operant Training
<ol>
 <li>After a week of post-surgical recovery, animals are trained to lever press for 10% sucrose solution in a Med Associates operant chamber (MedAssociates, Inc., Vermont, USA) outfitted with a lickometer, retractable lever, and sipper tube (as previously described in Howard et al., 2009).2 Software from MedAssociates is used to design all operant programs (MedAssociates, Inc., Vermont, USA). </li>
 <li>Once trained to respond for sucrose, begin rats on an appropriate training schedule during which ethanol is gradually added into the solution over multiple drinking sessions. </li>
 <li>For example, our lab currently uses an 8-day training schedule where ethanol is faded into the solution culminating in a 10% ethanol/ 10% sucrose drinking solution. Control animals only receive 10% sucrose or no drinking solution. The specific type of operant schedule to be used can vary widely, but the general procedures described below for microdialysis sampling can still be carried out. 3 </li>
</ol>
3. Pre-microdialysis Procedures: Tethering
<ol>
 <li>The night before the second to last training session (in our example this is the 7th training session), animals are habituated to the tether spring, which attaches to the tether bolt on their head stage. The spring attaches to the microdialysis swivel and counter balance lever arm, which is suspended next to the cage with a ring stand and clamps so that the animal can move freely within its cage. Tethered animals spend the night in the operant room, in their home cage with ad libitum food and water, to habituate them to the tethering set-up. </li>
 <li>The following day, the rat completes its operant program with the tether in place, to habituate the animal to the feeling of the tether while performing its behavioral tasks. Mount the tethering apparatus to the wall of the operant chamber near the top to allow suspension of the tether and swivel above the center of the roof of the operant chamber. All of this is placed within the sound attenuating chamber. After the session, return the rat to its home cage in the operant room to await microdialysis probe insertion. </li>
</ol>
4. Pre-microdialysis Procedures: The Microdialysis Probe is Inserted the Day Before the Microdialysis Experiment, after the Rat has Completed Behavioral Training for the Day
<ol>
 <li>The day before the microdialysis experiment, after the rat has completed an operant session while tethered, anesthetize the rat with isoflurane and remove the obturator from the guide cannula. Slowly (over ~ 5 min) insert a microdialysis probe, perfused with artificial cerebral spinal fluid (ACSF), through the cranial guide cannula into the brain region of interest. We use laboratory-constructed probes and microdialysis procedures modeled after Doyon et al., 2003, and Pettit and Justice, 1991.3, 4 </li>
 <li>Use the previously discussed tethering apparatus to suspend the inlet and outlet lines of the probe above the animal. </li>
 <li>Turn the probe flow rate down to 0.2 &mu;l/min. Once again, the rat spends the night in the operant room. </li>
</ol>
5. Microdialysis Procedures: Collection of Samples During Self-administration Session with Appetitive and Consummatory Phases Separated
<ol>
 <li>At least 2 hr before the experiment begins, turn up the probe to its working flow rate. We use either 1.0 or 2.0 &mu;l/min depending on the brain region. Check that the probe flow rate is consistent, and at least 90% of the set flow rate by using a Hamilton syringe to measure volume over time. </li>
 <li>Dialysis samples (5 or 7 min) are taken before, during, and after the operant session. The sampling interval depends on the brain region, neurotransmitter being analyzed, dialysate concentration of the analyte, and sensitivity of the analytical chemistry equipment to be used for the analysis. The behavioral phases are as follows:
 <ol>
 <li>Baseline: At beginning of experiment, collect dialysis samples in the animal's home cage (4 samples). </li>
 <li>Transfer: After collection of home cage baseline samples, transfer the rat to the operant chamber. Transfer of the tethered rat requires extreme care to make sure that the microdialysis fluid transfer line does not become tangled, and that the rat remains calm. Immediately after the transfer, activate the operant program as you change from the baseline/transfer sample to the first wait sample. </li>
 <li>Wait: Continue to collect samples as the rat waits for the lever to extend into the chamber (3-4 samples depending on the brain region). </li>
 <li>Drink: After the lever is presented and pressed, a bottle of drinking solution (10% sucrose/10% ethanol or 10% sucrose) is made available to the rat for around 20 min (3-4 samples).</li>
 <li>Post-drink: After drink period, the bottle retracts, but the rat remains in the operant chamber for around 20 min (3-4 samples). </li>
 </ol>
 </li>
</ol>
6. Microdialysis Procedures: Preparation of Microdialysis Sample for Ethanol Analysis
<ol>
 <li>Two samples before animals self-administer the ethanol solution, and all samples after, are evaluated for ethanol concentration. Pipette either a 1 or 2 &mu;l aliquot from the sample of interest into a 2 ml glass vial. Then seal the vial with an air tight septum (9 mm Red Poly Screw Cap, PTFE/Sil Septa, Agilent Technologies). The volume of the ethanol analysis aliquot (1 or 2 &mu;l) depends on the total sample volume, and the sample volume required for any additional analyses. </li>
 <li>If the samples will be used for later neurochemical analysis, store the sample appropriately after pipetting the aliquot for ethanol quantification. </li>
 <li>For example, our lab analyzes the samples for dopamine. To accomplish this, place the samples on dry ice during the experiment and then, store the samples at -80 &deg;C after the experiment. We use 2 &mu;l aliquots for ethanol analysis of a 5 min sample collected using a flow rate of 2.0 &mu;l/min for probes placed in the nucleus accumbens. This allows at least 7 &mu;l remaining for later analysis of dopamine by high performance liquid chromatography with electrochemical detection. </li>
 <li>For samples taken from the prefrontal cortex, which has much lower extracellular dopamine concentrations, we use a 1 &mu;l aliquot for ethanol analysis taken from a 7-min sample using a flow rate of 1.0 &mu;l/min. </li>
</ol>
7. Post-microdialysis Procedures
<ol>
 <li>After the conclusion of the microdialysis experiment, anesthetize the rat and remove the probe. Replace the obturator if the animal is not immediately euthanized. Collect the brain within three days of the experiment. Otherwise, visualization of the probe tract may not be possible. </li>
 <li>The brain should be removed in accordance with approved animal use protocols. We use sodium pentobarbital (150 mg/kg, ip) overdose, followed by cardiac perfusion with saline and then formalin in saline. Submerge brain in a formalin-saline solution for at least 12 hr before sectioning. </li>
 <li>Coronally section the brain into 100 &mu;m slices. Stain slices with cresyl violet, and examine for correct probe placement.5, 6 </li>
</ol>
8. Analysis of Samples for Ethanol Concentration
<ol>
 <li>The collected samples as well as external standards (0.3125 - 20 mM ethanol) are run on our gas chromatograph with flame ionization detection system. This system is comprised of a Varian CP 3,800 gas chromatograph with a flame ionization detector, a Bruker (Varian) 8,400 headspace autosampler heated to 50 &deg;C, and an HP Innowax capillary column (30 m x 0.53 mm x 1.0 &mu;m film thick), with helium as mobile phase. </li>
 <li>Chromatograms are recorded and analyzed with chromatography software such as the Varian Star Chromatography Workstation software that will be specifically discussed here. </li>
 <li>To prepare the system for ethanol analysis, heat the autosampler plate using a recirculating water bath, and open the two additional gas tanks (air and hydrogen; helium is always left running to preserve the wax column).</li>
 <li>Record the gas tank pressures, as well as the number of samples you intend to run so that you can keep count of the number of times the fiber and septum have been used so that they can be changed when appropriate (fiber-500 injections; septum-100 punctures). </li>
 <li>Initiate the start-up method, which prepares the system to run samples (program parameters in Table 2). Wait for the system to report that it is "Ready". </li>
 <li>Initiate a 20-min "burn," which prepares the fiber for sample analysis by subjecting it to a high temperature to desorb any contaminates it has absorbed while at rest. </li>
 <li>Standards are made by diluting 238 &mu;l of 95% ethanol with water to a final volume of 100 ml using a volumetric flask in a chemical fume hood. This creates a 40 mM ethanol solution. We use a 1:1 serial dilution to create 20, 10, 5, 2.5, 1.25, 0.625, 0.3125 mM standards. For each concentration of standard, use the same volume aliquot that will be taken from the dialysis samples. Pipette the aliquot into a 2 ml glass vial, and then seal the vial with an air tight septum.</li>
 <li>All ethanol samples are heated in the autosampler until the entire liquid aliquot has been vaporized. We heat our samples for about 30 min. </li>
 <li>This section describes the Star Workstation software that we use with our gas chromatograph. Other software may be used, but the description below may not be applicable. </li>
 <li>To run samples, create a sample list noting which sample is in which autosampler slot, and how many times the sample should be punctured. Be sure to route the data files to your selected folder. Then, select the method of choice for your samples, and begin the run. We use the running parameters noted in Table 2. </li>
 <li>Our dialysate ethanol program absorbs the sample for 3 min and desorbs into the helium stream, which feeds into the wax column, for 1 min. However, programs can be written to accommodate your specific needs. </li>
 <li>The sample components separate in the wax column, and then go through the flame ionization detector where the carbon containing compounds, such as ethanol, combust and release ions. This results in increased electron flow from the detector's anode to the cathode creating a current, which is converted to voltage and recorded resulting in chromatograms like the one shown below (Figure 1). The change in voltage is proportional to the amount of carbon passing through the detector across time.7 </li>
 <li>After the system is finished running the samples you will need to check the integration of each peak. For Star Workstation software, click on the blue peak icon in the tool bar. Click on each peak's color and drag the arrows to adjust the baseline. Re-integrate each peak before moving onto the next group of peaks. The peak analysis software can be any general chromatography software. </li>
 <li>For Star Workstation software, click on the batch report icon in your tool bar. Drag each sample from your specified folder to the batch report to print the sample reports. Reporting software can be customized with most common chromatography software systems. </li>
 <li>The reports can be programmed to show the information of your choice. We currently use peak height, but the reports can also be programmed to use peak area.</li>
 <li>To shut down the system, initiate the shut-down method (parameters noted in Table 2), turn off the water bath, and close the hydrogen and air tanks when the column oven temperature reaches 30 &deg;C. </li>
</ol>
9. Ethanol Data
<ol>
 <li>Plot the peak height as a function of each known external standard ethanol concentration (Figure 2A). Use the linear function given by these points to calculate the ethanol concentration from the peak height given by each dialysate sample.</li>
 <li>By plotting the concentration of ethanol in the dialysate over time, we can see the pattern of ethanol levels in the brain region of interest during our behavioral session. Example data, shown below (Figure 2B), are represented as the concentration of ethanol in the dialysate across time during the self-administration session. </li>
</ol>
10. General Maintenance: The Fiber Should be Changed Every 500 punctures, and the Septum Every 100 Punctures
To change the fiber
<ol>
 <li>On the gas chromatograph key pad press Menu, select 8,400, press enter; select change syringe, press enter. The autosampler will position itself to allow the fiber (SPME Fiber Assembly, 75 &mu;m Carboxen-PDMS, Supelco Analytical, Bellefonte, PA) to be removed. </li>
 <li>Open the door covering the fiber. Unscrew the lock nut, and move the latch to allow the fiber assembly to be removed. Take the fiber assembly out. Unscrew the nut that holds the fiber in the assembly, and then unscrew the fiber. </li>
 <li>Replace the old fiber with a new one, and reassemble the fiber assembly. Replace the assembly in the autosampler, re-latching and screwing the assembly into place. When you are finished, press "change done" on the key pad, and the autosampler will ready itself for use. </li>
</ol>
To change the septum
<ol>
 <li>First, unscrew the cap covering the septum (Hi-Temp .450 Dia. Generic Conditioned Septa, Varian) and then remove the old septum. Seat the new septum down into the fitting. Re-screw the cap and tighten it with the wrench. Then, use an injection needle to puncture the septum so that the fiber will not break the first time the septum is punctured. </li>
</ol>
11. Representative Results
Figure 1 shows example chromatograms for three concentrations of ethanol standards and 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 &gt; 10. Failure to meet these criteria means that your system requires maintenance. Quality chromatography and correctly prepared standards produce a linear standard curve (R2 &ge; 0.99; Figure 2A) that is used to calculate the ethanol concentration of dialysate samples collected from ethanol self-administering animals over the course of their self-administration session (Figure 2B). 
<img src="/files/ftp_upload/4142/4142fig1.jpg" alt="Figure 1" /> 
Figure 1. Example chromatograms. One &mu;l of ethanol standard or dialysate sample was loaded into a gas chromatograph vial and analyzed as described in the text. A) Overlay of peaks generated from 2.5, 5.0 and 10 mM ethanol standards. B) Peak generated from a dialysate sample from a rat that has self-administered ethanol. 
<img src="/files/ftp_upload/4142/4142fig2.jpg" alt="Figure 2" /> 
Figure 2. Graphical results from example experiment shown in Figure 1. A) Ethanol standard curve. B) Time course of dialysate ethanol concentration across an ethanol self-administration session. 
<table border="1">
 <tbody>
 <tr>
 <td>Hardware</td>
 <td>Parameter</td>
 <td>Start-up setting</td>
 <td>Running setting</td>
 <td>Resting setting</td>
 </tr>
 <tr>
 <td colspan="5">8,400 Bruker (Varian) Autosampler</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>Injection mode</td>
 <td>Spme</td>
 <td>Spme</td>
 <td>n/a</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>Solvent penetration depth</td>
 <td>0%</td>
 <td>20%</td>
 <td>n/a</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>Sample penetration depth</td>
 <td>20%</td>
 <td>20%</td>
 <td>n/a</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>Absorption time</td>
 <td>0.01 min</td>
 <td>3 min</td>
 <td>n/a</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>Desorption time</td>
 <td>19 min</td>
 <td>1 min</td>
 <td>n/a</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>Clean mode solvent source</td>
 <td>I</td>
 <td>I</td>
 <td>n/a</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>Clean mode adsorption and desorption time</td>
 <td>0.01 min</td>
 <td>0.01 min</td>
 <td>n/a</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>Water bath (heats autosampler)*</td>
 <td>50 &deg;C</td>
 <td>50 &deg;C</td>
 <td>Off</td>
 </tr>
 <tr>
 <td colspan="5">CP-3800 Varian Gas Chromatograph</td>
 </tr>
 <tr>
 <td>Injector Oven</td>
 <td>Oven power</td>
 <td>On</td>
 <td>On</td>
 <td>On</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>Oven temperature</td>
 <td>250 &deg;C</td>
 <td>220 &deg;C</td>
 <td>30 &deg;C</td>
 </tr>
 <tr>
 <td>Column Oven</td>
 <td>Stabilization time</td>
 <td>0.10 min</td>
 <td>0.10 min</td>
 <td>0.10 min</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>Temperature</td>
 <td>65 &deg;C</td>
 <td>65 &deg;C</td>
 <td>30 &deg;C</td>
 </tr>
 <tr>
 <td>Column</td>
 <td>Mobile phase flow rate</td>
 <td>8.5 ml/min</td>
 <td>8.5 ml/min</td>
 <td>n/a</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>Column Pressure</td>
 <td>~ 6 psi</td>
 <td>~ 6 psi</td>
 <td>&ge; 0.1 psi</td>
 </tr>
 <tr>
 <td>FID detector</td>
 <td>Oven power</td>
 <td>On</td>
 <td>On</td>
 <td>On</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>Temperature</td>
 <td>220 &deg;C</td>
 <td>220 &deg;C</td>
 <td>120 &deg;C</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>Electronics</td>
 <td>On</td>
 <td>On</td>
 <td>Off</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>Time constant</td>
 <td>Fast</td>
 <td>Fast</td>
 <td>Fast</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>Range</td>
 <td>11</td>
 <td>11</td>
 <td>11</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>Autozero</td>
 <td>Yes</td>
 <td>Yes</td>
 <td>Yes</td>
 </tr>
 </tbody>
</table>
Table 2. Gas chromatograph with flame ionization detection system parameters. This table shows the parameters for the three programs used to prepare (start-up settings), run (running settings) and maintain the system while not in use (resting settings).

<ol>
	<li>Geiger, B. M., Frank, L. E., Caldera-Siu, A. D., Pothos, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survivable+stereotaxic+surgery+in+rodents.">Survivable stereotaxic surgery in rodents.</a> J. Vis. Exp. (20), e880 (2008).</li><li>Howard, E. C., Schier, C. J., Wetzel, J. S., Gonzales, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dopamine+response+in+the+nucleus+accumbens+core-shell+border+differs+from+that+in+the+core+and+shell+during+operant+ethanol+self-administration.">The dopamine response in the nucleus accumbens core-shell border differs from that in the core and shell during operant ethanol self-administration.</a> Alcohol Clin. Exp. Res. 33, 1355-1365 (2009).</li><li>Doyon, W. M., York, J. L., Diaz, L. M., Samson, H. H., Czachowski, C. L., Gonzales, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dopamine+activity+in+the+nucleus+accumbens+during+consummatory+phases+of+oral+ethanol+self-administration.">Dopamine activity in the nucleus accumbens during consummatory phases of oral ethanol self-administration.</a> Alcohol Clin. Exp. Res. 27, 1573-1582 (2003).</li><li>Pettit, H. O., Justice, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+dose+on+cocaine+self-administration+behavior+and+dopamine+levels+in+the+nucleus+accumbens.">Effect of dose on cocaine self-administration behavior and dopamine levels in the nucleus accumbens.</a> Brain Res. 539, 94-102 (1991).</li><li>Paxinos, G., Kus, L., Ashwell, K. W. S., Watson, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Chemoarchitectonic Atlas of The Rat Forebrain. , (1998).</li><li>Paxinos, G., Watson, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Rat Brain in Stereotaxic Coordinates. , (1986).</li><li>Skoog, D. A., Holler, F. J., Nieman, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Principles of Instrumental Analysis. , (1998).</li><li>Doyon, W. M., Anders, S. K., Ramachandra, V. S., Czachowski, C. L., Gonzales, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+operant+self-administration+of+10%+ethanol+plus+10%+sucrose+on+dopamine+and+ethanol+concentrations+in+the+nucleus+accumbens.">Effect of operant self-administration of 10% ethanol plus 10% sucrose on dopamine and ethanol concentrations in the nucleus accumbens.</a> J Neurochem. 93, 1469-1481 (2005).</li><li>Carrillo, J., Gonzales, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+single+exposure+to+voluntary+ethanol+self-administration+produces+adaptations+in+ethanol+consumption+and+accumbal+dopamine+signaling.">A single exposure to voluntary ethanol self-administration produces adaptations in ethanol consumption and accumbal dopamine signaling.</a> Alcohol. 45, 559-5566 (2011).</li><li>Howard, E. C., Schier, C. J., Wetzel, J. S., Duvauchelle, C. L., Gonzales, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+shell+of+the+nucleus+accumbens+has+a+higher+dopamine+response+compared+with+the+core+after+non-contingent+intravenous+ethanol+administration.">The shell of the nucleus accumbens has a higher dopamine response compared with the core after non-contingent intravenous ethanol administration.</a> Neuroscience. 154, 1042-1053 (2008).</li><li>Robinson, D. L., Lara, J. A., Brunner, L. J., Gonzales, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+Ethanol+Concentrations+in+the+Extracellular+Fluid+of+the+Rat+Brain.">Quantification of Ethanol Concentrations in the Extracellular Fluid of the Rat Brain.</a> J. Neurochem. 75, 1685-1693 (2000).</li><li>Gonzales, R. A., McNabb, J., Yim, H. J., Ripley, T., Bungay, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+Microdialysis+of+Ethanol+in+Rat+Striatum.">Quantitative Microdialysis of Ethanol in Rat Striatum.</a> Alcohol Clin. Exp. Res. 22, 858-867 (1998).</li></ol>

This article was funded by Med Associates, Inc.

Applications and limitations
Drug self-administration is used in rodents to model drug addiction. Many drugs of abuse that are modeled in this fashion can be administered intravenously, in which the drug is delivered directly to the central compartment. This allows for close monitoring of dose over a self-administration session. Since ethanol is usually orally self-administered, it is much harder to monitor drug levels due to individual differences in absorption and metabolism. By using microdialysis to sample from the brain region of interest, we are not only able to monitor the pattern of ethanol reaching the region, but we are also able to simultaneously monitor neurotransmitter changes in the same region over time during each self-administration phase. 
Neurochemical alterations and drug-induced responses in the brain are associated with drug abuse and dependence; thus, the ability to concurrently measure neurochemical and drug concentrations during specific self-administration phases provides a very powerful and unique tool. One issue to keep in mind for correlating dialysate concentrations of analytes with behavior is the physical characteristics of the microdialysis plumbing. Specifically, the time it takes for fluid to be transferred from the lumen of the microdialysis probe to the collection tube is critical, and shorter times are better. In our lab, we construct the probes so that the time is about 90 sec. Using these conditions, we have found that there is an accumbal dopamine increase at the initiation of ethanol consumption, which decreases over the course of the drink and post-drink periods as the dialysate ethanol concentration increases.2,3,8,9 These experiments, when combined with data from pharmacological studies, have allowed us to parse out the strictly pharmacological effects of ethanol and self-administration associated environmental cues on changes in neurotransmitter concentrations. 
It should be noted that this particular application of combined behavioral-microdialysis techniques is suited to the current research interests of our laboratory. It is designed to evaluate the temporal pattern of ethanol reaching the brain in comparison to the pattern of neurotransmitter changes in the same region, so that we can relate these measures to synchronous self-administration behaviors. 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 should be experimentally determined. See previous publications from our laboratory for methods and further discussion.10,11,12 
Although this protocol illustrates the use of gas chromatography along with solid phase microextraction of ethanol from the headspace of microdialysis samples, other methods for analysis of the ethanol content of the microdialysis sample could be used. However, alternative methods may suffer from some disadvantages. For example, less sensitive analytical methods may require a larger sample volume which necessitates sampling times greater than the 5-7 min illustrated here. The type of system discussed here uses a solid phase microextraction that concentrates the ethanol in the vapor phase in the sealed sample vial by allowing absorption to the fiber placed in the vial headspace. This improves the detection limit compared with direct headspace sampling which typically allows 50-100 &mu;l of the vapor to be injected. Another major advantage of the headspace method is that the sample injected for analysis is extremely clean and free of salts. Direct injection of the liquid microdialysis sample may also be used with higher sensitivity, but this will require more instrument down time due to regular maintenance required for cleaning out the injected salts. 
Trouble-shooting and other notes
<ol>
 <li>Before your experiment begins, give yourself plenty of time to check that your microdialysis set-ups are functioning properly and to trouble-shoot any issues. We suggest that you have an extra set-up, perfused with ACSF, ready to switch out with a malfunctioning set-up to save yourself time, as most operant sessions tend to occur at a specific time every day. </li>
 <li>Be sure that the microdialysis probes are inserted over a period of about 5 min to minimize the tissue damage produced by shearing of the tissue as the probe penetrates the brain.</li>
 <li> When transferring the rat into the operant chamber it is helpful to have a second person assist so that the transfer line (part of the microdialysis set-up) does not becoming tangled, and so that the operant program can be initiated on time. </li>
 <li>Make sure that you give the autosampler water bath plenty of time to reach the appropriate temperature. Our system typically requires two hr to reach 50 &deg;C. Also, assure that your samples are fully vaporized before analyzing the samples. We typically visually confirm that this has occurred before initiating sample analysis. </li>
 <li>Standard good analytical chemistry practices should be followed. These include, but are not limited to, validation of individuals, equipment, and procedures. In brief, we employ the following guidelines: Individual users should demonstrate the ability to generate reproducible peak height values for standard concentrations and linear standard curves (R2 &ge; 0.99) across multiple days. To verify the GC-FID system is working correctly, standards should be always be analyzed before any dialysate samples are injected. The standard curve generated should have an R2 &ge; 0.99. </li>
 <li>Most of the equipment we use was purchased through Varian, Inc., which was acquired by Agilent Technologies in 2010. At this time, Bruker Scientific Instruments obtained Varian's laboratory gas chromatography instruments business. For future purchases, consult either company. </li>
 <li>The present protocol shows operant behavior with rats, but the viewer should be aware that more disruption of behavior is likely to occur with the smaller mouse model. Another issue to be aware of is that placement of a microdialysis probe into brain regions other than the medial prefrontal cortex or nucleus accumbens may disrupt operant behavior to a greater degree than shown here. It is important to closely examine numerous behavioral parameters to determine if the damage caused by probe placement produces severe changes in behavior. </li>
</ol>

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
		</tr>
		<tr>
			<td>95% Ethanol</td>
			<td>AAPER Alcohol and Chemical Co., Shelbyville, KY</td>
			<td>E190, 111000190</td>
		</tr>
		<tr>
			<td>Ultra-Pure Sucrose</td>
			<td>MP Biomedicals, LLC, Solon, OH</td>
			<td>821721</td>
		</tr>
		<tr>
			<td>Ultra High Purity Helium</td>
			<td>Air Gas</td>
			<td>HE UHP300</td>
		</tr>
		<tr>
			<td>Air</td>
			<td>Air Gas</td>
			<td>AIZ300</td>
		</tr>
		<tr>
			<td>Hydrogen</td>
			<td>Air Gas</td>
			<td>AIZ300</td>
		</tr>
		<tr>
			<td>GC 2 ml vials</td>
			<td>Agilent</td>
			<td>#8010-0015</td>
		</tr>
		<tr>
			<td>GC vial caps with PTFE/silicone septa</td>
			<td>Agilent</td>
			<td>#8010-0084</td>
		</tr>
	</tbody>
</table>
Table 1. Specific reagents

microdialysis of ethanol during operant ethanol self-administration and ethanol determination by gas chromatography

Operant self-administration methods are commonly used to study the behavioral and pharmacological effects of many drugs of abuse, including ethanol. However, ethanol is typically self-administered orally, rather than intravenously like many other drugs of abuse. The pharmacokinetics of orally administered drugs are more complex than intravenously administered drugs. Because understanding the relationship between the pharmacological and behavioral effects of ethanol requires knowledge of the time course of ethanol reaching the brain during and after drinking, we use in vivo microdialysis and gas chromatography with flame ionization detection to monitor brain dialysate ethanol concentrations over time. 
Combined microdialysis-behavioral experiments involve the use of several techniques. In this article, stereotaxic surgery, behavioral training and microdialysis, which can be adapted to test a multitude of self-administration and neurochemical centered hypotheses, are included only to illustrate how they relate to the subsequent phases of sample collection and dialysate ethanol analysis. Dialysate ethanol concentration analysis via gas chromatography with flame-ionization detection, which is specific to ethanol studies, is described in detail. Data produced by these methods reveal the pattern of ethanol reaching the brain during the self-administration procedure, and when paired with neurochemical analysis of the same dialysate samples, allows conclusions to be made regarding the pharmacological and behavioral effects of ethanol.

Watch this Scientific Journal Video about Microdialysis of Ethanol During Operant Ethanol Self-administration and Ethanol Determination by Gas Chromatography at JoVE.com

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

A method to determine the time course of ethanol concentration in the brains of rats during operant ethanol self-administration is described. Gas chromatography with flame ionization detection is used to quantify ethanol in the dialysate samples, because it has the sensitivity required for the small volumes that are generated.

Operant self-administration methods are commonly used to study the behavioral and pharmacological effects of many drugs of abuse, including ethanol.  ...

microdialysis-ethanol-during-operant-ethanol-self-administration

Research

JoVE Journal

Neuroscience

17.4K ビュー.  The University of Texas at Austin. オペラントエタノール自己投与時のラットの脳におけるエタノール濃度の経時変化を決定するための方法が記載されている。水素炎イオン化検出器付きガスクロマトグラフィーは、それが生成される小さなボリュームに必要な感度を持っているので、透析液サンプル中のエタノールを定量化するために使用されます。

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

A method to determine the time course of ethanol concentration in the brains of rats during operant ethanol self-administration is described.  Gas chromatography with flame ionization detection is used to quantify ethanol in the dialysate samples, because it has the sensitivity required for the small volumes that are...

quantification of ethanol levels in zebrafish embryos using head space gas chromatography

Quantification of Ethanol Levels in Zebrafish Embryos Using Head Space Gas Chromatography

This work describes a protocol to quantify ethanol levels in a zebrafish embryo using head space gas chromatography from proper exposure methods to embryo processing and ethanol...

rodent brain microinjection to study molecular substrates of motivated behavior

Rodent Brain Microinjection to Study Molecular Substrates of Motivated Behavior

Rodents are an appropriate model to investigate the molecular substrates of behavior and complex psychiatric disorders. Brain microinjection in awake rodents can be used to elucidate disease substrates. An efficient and customizable brain microinjection method as well as the execution of an operant paradigm that quantifies motivation is presented.

headspace gas chromatography: a technique to assess ethanol concentration in zebrafish embryos

Working with one sample at a time, quickly open the cap, and aspirate all residual embryo media with a p200 micropipette set to 200 microliters. Rapidly, but gently, add and remove the 50 microliters of water, then, add the 50 microliters of protease cocktail, and close the tube...

Headspace Gas Chromatography: A Technique to Assess Ethanol Concentration in Zebrafish Embryos

This video demonstrates the use of headspace gas chromatography to determine the ethanol concentrations in zebrafish embryos. This method provides a useful tool for qualitative and quantitative analysis of volatile organic...

flypub to study ethanol induced behavioral disinhibition and sensitization

Flypub To Study Ethanol Induced Behavioral Disinhibition and Sensitization

The Flypub assay measures the behaviors that the fruit fly Drosophila melanogaster displays under the influence of ethanol. The assay can be readily mastered by experimenters at all levels and applied to various vaporized stimuli, facilitating substance abuse and addiction...

a simple way to measure ethanol sensitivity in flies

A Simple Way to Measure Ethanol Sensitivity in Flies

A simple assay to measure the sedating effects of ethanol on Drosophila flies, based on the loss of righting reflex, is...

an inexpensive, scalable behavioral assay for measuring ethanol sedation sensitivity and rapid tolerance in drosophila

An Inexpensive, Scalable Behavioral Assay for Measuring Ethanol Sedation Sensitivity and Rapid Tolerance in Drosophila

Straightforward assays for measuring ethanol sensitivity and rapid tolerance in Drosophila facilitate the use of this model organism for investigating these important ethanol-related behaviors. Here, a relatively simple, scalable assay for measuring ethanol sensitivity and rapid tolerance in flies is...

An Inexpensive, Scalable Behavioral Assay for Measuring Ethanol Sedation Sensitivity and Rapid Tolerance in Drosophila

establishing a mouse model of thin endometrium

Author Spotlight: Advancing Infertility Studies with a Thin Endometrium Mouse Model Developed Using Controlled Ethanol Uterine Injection

This article describes a detailed protocol for producing a reliable and reproducible thin endometrium with a very low mortality rate and minimal intrauterine adhesions by injecting 95% ethanol into the mouse uterus within 1-3...

Optimized Thin Endometrium Mouse Model with Low Mortality and Minimal Adhesions

x-ray visualization of intraductal ethanol-based ablative treatment for prevention of breast cancer in rat models

X-Ray Visualization of Intraductal Ethanol-Based Ablative Treatment for Prevention of Breast Cancer in Rat Models

A procedure for the delivery of a chemical ablative solution to the rat mammary ductal tree for image-guided preventive treatment of breast cancer is described. Mammary epithelial cells can be targeted with minimal collateral tissue damage through cannulation directly into the nipple opening and intraductal infusion of a 70% ethanol-based ablative...

ethanol fixation and dapi staining: a method to visualize dna in c. elegans

Here, we describe a technique used to visualize genomic DNA in fixed, intact nematodes. The video includes an example protocol to count chromatin in unfertilized C. elegans...

Ethanol Fixation and DAPI Staining: A Method to Visualize DNA in C. elegans

an assay for measuring the effects of ethanol on the locomotion speed of caenorhabditis elegans

An Assay for Measuring the Effects of Ethanol on the Locomotion Speed of Caenorhabditis elegans

C. elegans is a useful model for studying the effects of ethanol on behavior. We present a behavioral assay that quantifies the effects of ethanol on the locomotion speed of crawling worms; both initial sensitivity and the development of acute functional tolerance to ethanol can be measured with this assay.

An Assay for Measuring the Effects of Ethanol on the Locomotion Speed of Caenorhabditis elegans

extraction of lignin with high &#946;-o-4 content by mild ethanol extraction and its effect on the depolymerization yield

Extraction of Lignin with High &amp;#946;-O-4 Content by Mild Ethanol Extraction and Its Effect on the Depolymerization Yield

Here, we present a protocol to perform ethanol extraction of lignin from several biomass sources. The effect of the extraction conditions on the lignin yield and &#946;-O-4 content are presented. Selective depolymerization is performed on the obtained lignins to obtain high aromatic monomer products.

Extraction of Lignin with High &#946;-O-4 Content by Mild Ethanol Extraction and Its Effect on the Depolymerization Yield

techniques for the evolution of robust pentose-fermenting yeast for bioconversion of lignocellulose to ethanol

Techniques for the Evolution of Robust Pentose-fermenting Yeast for Bioconversion of Lignocellulose to Ethanol

Adaptive evolution and isolation techniques are described and demonstrated to yield derivatives of Scheffersomyces stipitis strain NRRL Y-7124 that are able to rapidly consume hexose and pentose mixed sugars in enzyme saccharified undetoxified hydrolyzates and to accumulate over 40 g/L...

a method for evaluating the reinforcing properties of ethanol in rats without water deprivation, saccharin fading or extended access training

A Method for Evaluating the Reinforcing Properties of Ethanol in Rats without Water Deprivation, Saccharin Fading or Extended Access Training

This protocol describes a novel and efficient method to quickly initiate operant responding for ethanol in rats that, contrary to standard methods, does not require water deprivation or saccharin/sucrose fading to initiate...

chronic intermittent ethanol vapor exposure paired with two-bottle choice to model alcohol use disorder

Author Spotlight: Chronic Intermittent Ethanol Vapor Exposure Paired with Two-Bottle Choice to Model Alcohol Use Disorder

Presented here is a protocol for the 2BC/CIE model of alcohol dependence in mice to study alcohol use disorder.

Chronic Intermittent Ethanol Vapor Exposure Paired with Two-Bottle Choice to Model Alcohol Use Disorder

ethanol-induced cervical sympathetic ganglion block applications for promoting canine inferior alveolar nerve regeneration using an artificial nerve

Ethanol-Induced Cervical Sympathetic Ganglion Block Applications for Promoting Canine Inferior Alveolar Nerve Regeneration Using an Artificial Nerve

We evaluated the effect of cervical sympathetic ganglion block on nerve repair using artificial nerve conduits. Male beagle dogs were each implanted with an artificial nerve across a 10-mm gap in the left inferior alveolar nerve; left cervical sympathetic ganglion was blocked by injecting 99.5% ethanol via lateral...

determination of 45 pesticides in avocado varieties by the quechers method and gas chromatography-tandem mass spectrometry

Avocado Sample Preparation Using the QuEChERS Method with Ammonium Formate for Pesticide Analysis

The present protocol describes the analysis of multiclass pesticide residues in avocado varieties using the Quick-Easy-Cheap-Effective-Rugged-Safe (QuEChERS) method with ammonium formate, followed by gas chromatography-tandem mass spectrometry.

Determination of 45 Pesticides in Avocado Varieties by the QuEChERS Method and Gas Chromatography-Tandem Mass Spectrometry

intraductal delivery and x-ray visualization of ethanol-based ablative solution for prevention and local treatment of breast cancer in mouse models

Intraductal Delivery and X-ray Visualization of Ethanol-Based Ablative Solution for Prevention and Local Treatment of Breast Cancer in Mouse Models

A method of intraductal injection of reagents for an ethanol-based ablative solution to the mouse mammary ductal tree for in vivo imaging and breast cancer prevention is described. Injection directly into the nipple opening allows for targeting mammary epithelial cells with minimal collateral tissue...

assessment of ultrasonic vocalizations during drug self-administration in rats

Assessment of Ultrasonic Vocalizations During Drug Self-administration in Rats

Drug self-administration and ultrasonic vocalizations (USV) are used as behavioral assessments in animal research, but rarely in combination. The purpose of this article is to describe the advantages of recording USVs during drug self-administration procedures to assess affective responses to drug...

Drug self-administration procedures are commonly used to study behavioral and neurochemical changes associated with human drug abuse, addiction and relapse. Various types of behavioral activity are commonly utilized as measures of drug motivation in animals. However, a crucial component of drug abuse relapse in abstinent cocaine users is "drug craving", which is difficult to model in animals, as it often occurs in the absence of overt behaviors. Yet, it is possible that a class of ultrasonic vocalizations (USVs) in rats may be a useful marker for affective responses to drug administration, drug anticipation and even drug craving. Rats vocalize in ultrasonic frequencies that serve as a communicatory function and express subjective emotional states. Several studies have shown that different call frequency ranges are associated with negative and positive emotional states. For instance, high frequency calls ("50-kHz") are associated with positive affect, whereas low frequency calls ("22-kHz") represent a negative emotional state. This article describes a procedure to assess rat USVs associated with daily cocaine self-administration. For this procedure, we utilized standard single-lever operant chambers housed within sound-attenuating boxes for cocaine self-administration sessions and utilized ultrasonic microphones, multi-channel recording hardware and specialized software programs to detect and analyze USVs. USVs measurements reflect emotionality of rats before, during and after drug availability and can be correlated with commonly assessed drug self-administration behavioral data such lever responses, inter-response intervals and locomotor activity. Since USVs can be assessed during intervals prior to drug availability (e.g., anticipatory USVs) and during drug extinction trials, changes in affect associated with drug anticipation and drug abstinence can also be determined. In addition, determining USV changes over the course of short- and long-term drug exposure can provide a more detailed interpretation of drug exposure effects on affective functioning.

Drug self-administration and ultrasonic vocalizations (USV) are used as behavioral assessments in animal research, but rarely in combination. The purpose of this article is to describe the advantages of recording USVs during drug self-administration procedures to assess affective responses to drug experience.

Drug self-administration procedures are commonly used to study behavioral and neurochemical changes associated with human drug abuse, addiction and ...

Operant Sensation Seeking in the Mouse

Operant methods are powerful behavioral tools for the study of motivated behavior. These 'self-administration' methods have been used extensively in drug addiction research due to their high construct validity. Operant studies provide researchers a tool for preclinical investigation of several aspects of the addiction process. For example, mechanisms of acute reinforcement (both drug and non-drug) can be tested using pharmacological or genetic tools to determine the ability of a molecular target to influence self-administration behavior1-6. Additionally, drug or food seeking behaviors can be studied in the absence of the primary reinforcer, and the ability of pharmacological compounds to disrupt this process is a preclinical model for discovery of molecular targets and compounds that may be useful for the treatment of addiction3,7-9. One problem with performing intravenous drug self-administration studies in the mouse is the technical difficulty of maintaining catheter patency. Attrition rates in these experiments are high and can reach 40% or higher10-15. Another general problem with drug self-administration is discerning which pharmacologically-induced effects of the reinforcer produce specific behaviors. For example, measurement of the reinforcing and neurological effects of psychostimulants can be confounded by their psychomotor effects. Operant methods using food reinforcement can avoid these pitfalls, although their utility in studying drug addiction is limited by the fact that some manipulations that alter drug self-administration have a minimal impact on food self-administration. For example, mesolimbic dopamine lesion or knockout of the D1 dopamine receptor reduce cocaine self-administration without having a significant impact on food self-administration 12,16. 
Sensory stimuli have been described for their ability to support operant responding as primary reinforcers (i.e. not conditioned reinforcers)17-22. Auditory and visual stimuli are self-administered by several species18,21,23, although surprisingly little is known about the neural mechanisms underlying this reinforcement. The operant sensation seeking (OSS) model is a robust model for obtaining sensory self-administration in the mouse, allowing the study of neural mechanisms important in sensory reinforcement24. An additional advantage of OSS is the ability to screen mutant mice for differences in operant behavior that may be relevant to addiction. We have reported that dopamine D1 receptor knockout mice, previously shown to be deficient in psychostimulant self-administration, also fail to acquire OSS24. This is a unique finding in that these mice are capable of learning an operant task when food is used as a reinforcer. While operant studies using food reinforcement can be useful in the study of general motivated behavior and the mechanisms underlying food reinforcement, as mentioned above, these studies are limited in their application to studying molecular mechanisms of drug addiction. Thus, there may be similar neural substrates mediating sensory and psychostimulant reinforcement that are distinct from food reinforcement, which would make OSS a particularly attractive model for the study of drug addiction processes. The degree of overlap between other molecular targets of OSS and drug reinforcers is unclear, but is a topic that we are currently pursuing. While some aspects of addiction such as resistance to extinction may be observed with OSS, we have found that escalation 25 is not observed in this model24. Interestingly, escalation of intake and some other aspects of addiction are observed with self-administration of sucrose26. Thus, when non-drug operant procedures are desired to study addiction-related processes, food or sensory reinforcers can be chosen to best fit the particular question being asked. 
In conclusion, both food self-administration and OSS in the mouse have the advantage of not requiring an intravenous catheter, which allows a higher throughput means to study the effects of pharmacological or genetic manipulation of neural targets involved in motivation. While operant testing using food as a reinforcer is particularly useful in the study of the regulation of food intake, OSS is particularly apt for studying reinforcement mechanisms of sensory stimuli and may have broad applicability to novelty seeking and addiction.

In this protocol we describe a method of operant learning using sensory stimuli as a reinforcer in the mouse. It requires no prior training or food restriction, and it allows the study of motivated behavior without the use of a pharmacological or natural reinforcer such as food.

Operant methods are powerful behavioral tools for the study of motivated behavior.  These 'self-administration' methods have been used extensively in drug ...

Low doses of ethanol cause flies to become hyperactive, while high doses are sedating. The sensitivity to ethanol-induced sedation of a given fly strain is correlated with that strain s ethanol preference, and therefore sedation is a highly relevant measure to study the genetics of alcohol responses and drinking. We demonstrate a simple way to expose flies to ethanol and measure its intoxicating effects. The assay we describe can determine acute sensitivity, as well as ethanol tolerance induced by repeat exposure. It does not require a technically involved setup, and can therefore be applied in any laboratory with basic fly culture tools.

A simple assay to measure the sedating effects of ethanol on Drosophila flies, based on the loss of righting reflex, is described.

Low doses of ethanol cause flies to become hyperactive, while high doses are sedating. The sensitivity to ethanol-induced sedation of a given fly strain ...

Methods for Intravenous Self Administration in a Mouse Model

Animal models have been developed to study the reinforcing effects of drugs, including the intravenous self-administration (IVSA) paradigm. The advantages of using an IVSA paradigm to study the reinforcing properties of drugs of abuse such as cocaine include the fact that the drug is self-administered instead of experimenter-administered, the schedule of reinforcement can be altered, and accurate measurement of the quantities of drug consumed as well as the timing and pattern of IV injections can be obtained. Furthermore, the intravenous route of administration avoids potential confounds related to first pass metabolism or taste, and produces rapid increases in blood and brain drug levels. As outlined in this video, intravenous self-administration can be obtained without prior food restriction or prior drug training following careful catheter placement during surgery and meticulous daily catheter flushing and maintenance. Experimental procedures outlined in this paper include a description of animal housing and acclimation methods, operant training using sweetened milk solutions, and catheter implantation surgery.

The intravenous self-administration (IVSA) paradigm is considered to be the gold standard in examining the reinforcing properties of drugs of abuse in rodents. This manuscript outlines the experimental procedures and surgical techniques necessary to obtain reliable IVSA data. In particular, meticulous catheter implantation and maintenance are highlighted.

Animal models have been developed to study the reinforcing effects of drugs, including the intravenous self-administration (IVSA) paradigm. The advantages ...

Identification of Olfactory Volatiles using Gas Chromatography-Multi-unit Recordings (GCMR) in the Insect Antennal Lobe

All organisms inhabit a world full of sensory stimuli that determine their behavioral and physiological response to their environment. Olfaction is especially important in insects, which use their olfactory systems to respond to, and discriminate amongst, complex odor stimuli. These odors elicit behaviors that mediate processes such as reproduction and habitat selection1-3. Additionally, chemical sensing by insects mediates behaviors that are highly significant for agriculture and human health, including pollination4-6, herbivory of food crops7, and transmission of disease8,9. Identification of olfactory signals and their role in insect behavior is thus important for understanding both ecological processes and human food resources and well-being.
	To date, the identification of volatiles that drive insect behavior has been difficult and often tedious. Current techniques include gas chromatography-coupled electroantennogram recording (GC-EAG), and gas chromatography-coupled single sensillum recordings (GC-SSR)10-12. These techniques proved to be vital in the identification of bioactive compounds. We have developed a method that uses gas chromatography coupled to multi-channel electrophysiological recordings (termed 'GCMR') from neurons in the antennal lobe (AL; the insect's primary olfactory center)13,14. This state-of-the-art technique allows us to probe how odor information is represented in the insect brain. Moreover, because neural responses to odors at this level of olfactory processing are highly sensitive owing to the degree of convergence of the antenna's receptor neurons into AL neurons, AL recordings will allow the detection of active constituents of natural odors efficiently and with high sensitivity. Here we describe GCMR and give an example of its use.
	Several general steps are involved in the detection of bioactive volatiles and insect response. Volatiles first need to be collected from sources of interest (in this example we use flowers from the genus Mimulus (Phyrmaceae)) and characterized as needed using standard GC-MS techniques14-16. Insects are prepared for study using minimal dissection, after which a recording electrode is inserted into the antennal lobe and multi-channel neural recording begins. Post-processing of the neural data then reveals which particular odorants cause significant neural responses by the insect nervous system.
	Although the example we present here is specific to pollination studies, GCMR can be expanded to a wide range of study organisms and volatile sources. For instance, this method can be used in the identification of odorants attracting or repelling vector insects and crop pests. Moreover, GCMR can also be used to identify attractants for beneficial insects, such as pollinators. The technique may be expanded to non-insect subjects as well.

Identification of Olfactory Volatiles using Gas Chromatography-Multi-unit Recordings GCMR in the Insect Antennal Lobe

Olfactory cues mediate many different behaviors in insects, and are often complex mixtures comprised of tens to hundreds of volatile compounds. Using gas chromatography with multi-channel recording in the insect antennal lobe, we describe a method for the identification of bioactive compounds.

All  organisms inhabit a world full of sensory stimuli that determine their  behavioral and physiological response to their environment. Olfaction is ...

Hippocampal Insulin Microinjection and In vivo Microdialysis During Spatial Memory Testing

Glucose metabolism is a useful marker for local neural activity, forming the basis of methods such as 2-deoxyglucose and functional magnetic resonance imaging. However, use of such methods in animal models requires anesthesia and hence both alters the brain state and prevents behavioral measures. An alternative method is the use of in vivo microdialysis to take continuous measurement of brain extracellular fluid concentrations of glucose, lactate, and related metabolites in awake, unrestrained animals. This technique is especially useful when combined with tasks designed to rely on specific brain regions and/or acute pharmacological manipulation; for example, hippocampal measurements during a spatial working memory task (spontaneous alternation) show a dip in extracellular glucose and rise in lactate that are suggestive of enhanced glycolysis1-3,4-5, and intrahippocampal insulin administration both improves memory and increases hippocampal glycolysis6. Substances such as insulin can be delivered to the hippocampus via the same microdialysis probe used to measure metabolites. The use of spontaneous alternation as a measure of hippocampal function is designed to avoid any confound from stressful motivators (e.g. footshock), restraint, or rewards (e.g. food), all of which can alter both task performance and metabolism; this task also provides a measure of motor activity that permits control for nonspecific effects of treatment. Combined, these methods permit direct measurement of the neurochemical and metabolic variables regulating behavior.

Hippocampal Insulin Microinjection and In vivo Microdialysis During Spatial Memory Testing

Modulation of hippocampally-dependent spatial working memory by direct intrahippocampal microinjection, accompanied and followed by in vivo microdialysis for metabolites in conscious, behaving animals.

Glucose metabolism is a useful marker for local neural activity, forming the basis of methods such as 2-deoxyglucose and functional magnetic resonance ...

Alcohol use disorder (AUD) is a serious health challenge. Despite a large hereditary component to AUD, few genes have been unambiguously implicated in their etiology. The fruit fly, Drosophila melanogaster, is a powerful model for exploring molecular-genetic mechanisms underlying alcohol-related behaviors and therefore holds great promise for identifying and understanding the function of genes that influence AUD. The use of the Drosophila model for these types of studies depends on the availability of assays that reliably measure behavioral responses to ethanol. This report describes an assay suitable for assessing ethanol sensitivity and rapid tolerance in flies. Ethanol sensitivity measured in this assay is influenced by the volume and concentration of ethanol used, a variety of previously reported genetic manipulations, and also the length of time the flies are housed without food immediately prior to testing. In contrast, ethanol sensitivity measured in this assay is not affected by the vigor of fly handling, sex of the flies, and supplementation of growth medium with antibiotics or live yeast. Three different methods for quantitating ethanol sensitivity are described, all leading to essentially indistinguishable ethanol sensitivity results. The scalable nature of this assay, combined with its overall simplicity to set-up and relatively low expense, make it suitable for small and large scale genetic analysis of ethanol sensitivity and rapid tolerance in Drosophila.

Straightforward assays for measuring ethanol sensitivity and rapid tolerance in Drosophila facilitate the use of this model organism for investigating these important ethanol-related behaviors. Here, a relatively simple, scalable assay for measuring ethanol sensitivity and rapid tolerance in flies is described.

Alcohol use disorder (AUD) is a serious health challenge. Despite a large hereditary component to AUD, few genes have been unambiguously implicated in ...

Alcohol use disorders are a significant public health concern, for which there are few effective treatment strategies. One difficulty that has delayed the development of more effective treatments is the relative lack of understanding of the molecular underpinnings of the effects of ethanol on behavior. The nematode, Caenorhabditis elegans (C.&#160;elegans), provides a useful model in which to generate and test hypotheses about the molecular effects of ethanol. Here, we describe an assay that has been developed and used to examine the roles of particular genes and environmental factors in behavioral responses to ethanol, in which locomotion is the behavioral output. Ethanol dose-dependently causes an acute depression of crawling on an agar surface. The effects are dynamic; animals exposed to a high concentration demonstrate an initial strong depression of crawling, referred to here as initial sensitivity, and then partially recover locomotion speed despite the continued presence of the drug. This ethanol-induced behavioral plasticity is referred to here as the development of acute functional tolerance. This assay has been used to demonstrate that these two phenotypes are distinct and genetically separable. The straightforward locomotion assay described here is suitable for examining the effects of both genetic and environmental manipulations on these acute behavioral responses to ethanol in C.&#160;elegans.

C. elegans is a useful model for studying the effects of ethanol on behavior. We present a behavioral assay that quantifies the effects of ethanol on the locomotion speed of crawling worms; both initial sensitivity and the development of acute functional tolerance to ethanol can be measured with this assay.

Alcohol use disorders are a significant public health concern, for which there are few effective treatment strategies. One difficulty that has delayed the ...

Custom-made Microdialysis Probe Design

Microdialysis is a commonly used technique in neuroscience research. Therefore commercial probes are in great demand to monitor physiological, pharmacological and pathological changes in cerebrospinal fluid. Unfortunately, commercial probes are expensive for research groups in public institutions. In this work, a probe assembly is explained in detail to build a reliable, concentric, custom-made microdialysis probe for less than $10. The microdialysis probe consists of a polysulfone membrane with a molecular cut-off of 30 kDa. Probe in vitro recoveries of substances with different molecular weight (in the range of 100-1,600 Da) and different physicochemical properties are compared. The probe yields an in vitro recovery of approximately 20% for the small compounds glucose, lactate, acetylcholine and ATP. In vitro recoveries for neuropeptides with a molecular weight between 1,000-1,600 Da amount to 2-6%. Thus, while the higher molecular weight of the neuropeptides lowered in vitro recovery values, dialysis of compounds in the lower range (up to 500 Da) of molecular weights has no great impact on the in vitro recovery rate. The present method allows utilization of a dialysis membrane with other cut-off value and membrane material. Therefore, this custom-made probe assembly has the advantage of sufficient flexibility to dialyze substances in a broad molecular weight range. Here, we introduce a microdialysis probe with an exchange length of 2 mm, which is applicable for microdialysis in mouse and rat brain regions. However, dimensions of the probe can easily be adapted for larger exchange lengths to be used in larger animals.

Microdialysis is a commonly used technique in neuroscience research. Therefore commercial probes are in great demand.In this work a probe assembly is explained in detail to build a reliable, concentric, custom-made microdialysis probe for less than $10.

Microdialysis is a commonly used technique in neuroscience research. Therefore commercial probes are in great demand to monitor physiological, ...

Assessment of Dopaminergic Homeostasis in Mice by Use of High-performance Liquid Chromatography Analysis and Synaptosomal Dopamine Uptake

Dopamine (DA) is a modulatory neurotransmitter controlling motor activity, reward processes and cognitive function. Impairment of dopaminergic (DAergic) neurotransmission is strongly associated with several central nervous system-associated diseases such as Parkinson&#39;s disease, attention-deficit-hyperactivity disorder and drug addiction1,2,3,4. Delineating disease mechanisms involving DA imbalance is critically dependent on animal models to mimic aspects of the diseases, and thus protocols that assess specific parts of the DA homeostasis are important to provide novel insights and possible therapeutic targets for these diseases.
Here, we present two useful experimental protocols that when combined provide a functional read-out of the DAergic system in mice. Biochemical and functional parameters on DA homeostasis are obtained through assessment of DA levels and dopamine transporter (DAT) functionality5. When investigating the DA system, the ability to reliably measure endogenous levels of DA from adult brain is essential. Therefore, we present how to perform high-performance liquid chromatography (HPLC) on brain tissue from mice to determine levels of DA. We perform the experiment on tissue from dorsal striatum (dStr) and nucleus accumbens (NAc), but the method is also suitable for other DA-innervated brain areas.
DAT is essential for reuptake of DA into the presynaptic terminal, thereby controlling the temporal and spatial activity of released DA. Knowing the levels and functionality of DAT in the striatum is of major importance when assessing DA homeostasis. Here, we provide a protocol that allows to simultaneously deduce information on surface levels and function using a synaptosomal6 DA uptake assay.
Current methods combined with standard immunoblotting protocols provide the researcher with relevant tools to characterize the DAergic system.

Synaptosomal dopamine uptake and high-performance liquid chromatography analysis represent experimental tools to investigate dopamine homeostasis in mice by assessing the function of the dopamine transporter and levels of dopamine in striatal tissue, respectively. Here we present protocols to measure dopamine tissue content and assess the functionality of the dopamine transporter.

Dopamine (DA) is a modulatory neurotransmitter controlling motor activity, reward processes and cognitive function. Impairment of dopaminergic (DAergic) ...