Measuring In Vivo Changes in Extracellular Neurotransmitters During Naturally Rewarding Behaviors in Female Syrian Hamsters

The overall goal of this experimental procedure is to measure neurotransmitter release in awake behaving animals. Although this video demonstrates application to behavioral neuroscience, with the ability to characterize specific neurotransmitter release, in relation to specific behavioral events, this technique can be employed in a variety of other neuro science sub-disciplines, such as with drug injection. The main advantage of this technique is it's turnkey nature to measure near real time release of neurotransmitters in vivo.

Begin by injecting 500 micrograms of progesterone in 0.1 milliliters of cottonseed oil, subcutaneously, four hours prior to the introduction of the male to induce sexual receptivity. Be sure to test each animal at the start of the dark phase of their daily cycle, as social behaviors of rodents are subject to alteration under white light and red light testing conditions. Make the calibration solutions fresh on the day of testing in 20 milliliter glass centrifuge tubes.

For the analyte solution, dissolve 7.4 milligrams of L-glutamic acid into 10 milliliters of ultra pure water by gently inverting the tube. Then, make the interference solution by dissolving 176.1 milligrams AA in 10 milliliters of ultra pure water. Set up the calibration by placing a magnetic stirrer on the base of a basic lab ring stand.

Place a 20 milliliter jacketed beaker on top of the magnetic stirrer and clamp in place using a medium two prong clamp. Connect the jacketed beaker to a circulating water bath to heat the buffer solution to 37 degrees Celsius. Add a magnetic stir bar and 20 milliliters of 100 millimolar phosphate buffered saline, or PBS, into the jacketed beaker.

Next, place the sensor calibration holder on top of the jacketed beaker, and set a four channel calibration preamplifier on top, securing it in place using a right angle clamp. Then, connect the preamplifier to a data conditioning and acquisition device to record calibration data. Test the sensitivity of each enzymatic biosensor to glutamate before the experimental recording by placing the probe into the calibration holder, atop a jacketed beaker, submerging the sensing cavity and some portion of the silver chloride reference wire in the buffer solution.

Initiate a new recording on the computer interface utilizing free downloadable acquisition software. And then, connect each sensor being tested to a port on a four channel calibration preamplifier. Allow the sensors to reach a stable baseline.

Begin adding the analyte injections when the sensors have reached a stable baseline. Use the hole in the calibration holder to introduce a pipet tip to the buffer solution. Then, use the quick key annotation tool in the recording software to annotate when an injection is made.

Next, make 10 micromolar injections of glutamate by pipetting 40 microliters of the analyte solution into the buffer solution. Wait for the sensor to stabilize between additions. Then, add three injections of analyte solution before adding a single injection from the interferent AA solution.

Line the test chamber with bedding taken from the animal's home cage. Remove the occlusive obdurator from the guide cannula and insert either the carbon fiber electrode or enzymatic probe through the guide shaft. Connect the sensor to the recording system.

Then stabilize the sensor connection by screwing a pin attachment onto the head mount. Then, place the animal in the testing chamber. Begin recording the video and time locked amperometric signal in the software program.

Allow the sensor to equilibrate in the brain before experimental testing. Then, introduce a stimulus male into the testing chamber. Following the first mount with penile insertion by the stimulus male, continue recording for an additional 10 to 30 minutes, depending on the experimental goals.

Finally, after behavioral testing, disconnect the female sensor. Remove both hamsters from the testing chamber, as well as the bedding, to clean the chamber using 70%ethanol. Begin by viewing and annotating the videos, using free commercially downloadable software, in slow motion to precisely code behaviors time locked to the amperometric signal.

Annotate Start Lordosis in the frame when the female initiates a dorsiflexion of her back, and deflects her tail upwards. Annotate End Lordosis when the female terminates this posture, which often occurs when the female readjusts to another location in the testing chamber. Then, annotate Start AI when the female is in the lordosis posture and the male moves his snout towards the female's anogenital region, where sniffing, licking, or nuzzling of her perineal region may occur.

Annotate End AI when the male removes his snout from close proximity to the female's anogenital region. Next, annotate Start Mount when the male approaches and places his forepaws on the female in a mounting posture, regardless of the orientation of the mount attempt. Likewise, annotate Start Intromission when successful thrusting gains the mounted male penile access to the female's vagina.

When applicable, annotate Ejaculate, which can be noted by a treading motion with the male's hind foot. Lastly, annotate End Intromission and End Mount simultaneously in the frame when the male has removed his front two paws, and thus has no contact with the female. Due to the frequent inability to visualize his withdrawal, simultaneously code these two behaviors to allow for consistency and reliability across mating bouts.

Under the file tab, select the Export function and choose the TSV tab to save the voltage measurements as a TSV extension file. Open each file with a spreadsheet program and save as XLS/XLSX. Finally, manually add annotations for the start of the premating bout lordosis at the same time as lordosis behavior begins that also includes a mount behavior, and also at the start of a new mating bout if the female remains in the lordosis posture.

During mating bouts, dopaminergic transience in the nucleus incumbents arise during vaginal insertion, or intermission, by the male. A similar pattern has been observed in other animals with regard to glutamate release. With rapid transience that correspond to individual intermissions in the dorsal nucleus accumbens core during a copulatory bout.

Once mastered, this technique, not including the preparatory sterotactic surgeries, can be completed in one day. While attempting this procedure, it's important to ensure that each sensor is functional. Although this video demonstrates a single probe recording, this approach can be extended to simultaneous dual recording.

But this adds a layer of complexity that is not trivial. This video is demonstrated under light conditions for easy of visibility. However, we carry our behavioral experiments under dark conditions, and this parameter can be altered depending on specific experimental needs.

Following this procedure, other methods like subsequent histological analysis of brain slices can be performed in order to identify and confirm exact sensor placement. Additionally, further data analysis, using the coded behavioral data, can be performed with regard to whatever parameters are of interest. After it's development, this technique paved the way for researchers in the field of neuroscience to explore neurotransmitter release, including nonelectro active neurotransmitters like glutamate in near real time in awake behaving animals.

After watching this video, you should have a good understanding of how to calibrate glutamatergic probes, implant probes, run a behavioral experiment measuring changes in neurotransmitter release to different components of behavior, and code that data for subsequent analysis. Don't forget that when working with live animals to be sensitive to their comfort and overall wellbeing to ensure your best results and the animals'