Name: measuring in vivo changes in extracellular neurotransmitters during naturally rewarding behaviors in female syrian hamsters
Uploaded: 2017-09-12T14:00:00
Description: Watch this Scientific Journal Video about Measuring In Vivo Changes in Extracellular Neurotransmitters During Naturally Rewarding Behaviors in Female Syrian Hamsters at JoVE.com

The authors would like to thank undergraduate Daniel Korus for his help running the Matlab code and undergraduate Alex Boettcher for his assistance in running the behavioral experiments. This project is supported by NSF IOS 1256799 to R.L.M., and by the National Institute on Drug Abuse of the National Institutes of Health under Award Number T32DA007234.

All procedures described here were approved by the Institutional Animal Care and Use Committee (IACUC) of The University of Minnesota, and are in accordance with The Guide for the Care and Use of Laboratory Animals13.
1. Animals and Cannulation Surgery
<ol>
	<li>Obtain Syrian hamsters from a common animal supplier at approximately 55 days of age. 
	NOTE: Although the age of animals will vary due to constraints of various experimental paradigms, for sexual behavio...

<ol>
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Although relatively straightforward, some issues can arise when employing this technique. First, the stereotaxic placement of the probes must be precise: unlike microdialysis that samples a wider radius of the extracellular milieu surrounding the probe, this technique only allows the measurement of a neurotransmitter that comes into direct contact with the probe. Second, in the case of the carbon fiber recording, due to the small width of fiber, breakage can occur, and the probe must be inserted with deliberate care. In ...

The ability to measure neurotransmitter release in awake behaving animals allows researchers to link specific behaviors with spatial and temporal patterns of neurotransmission-a powerful tool to investigate mechanisms and circuitry underlying both natural and operant behaviors in real-time. Historically, microdialysis has been employed to measure both electrically reactive and nonreactive substances in the extracellular milieu of the brain1. This technique uses a continuous flow of an aqueous solution of similar ionic composition to the extracellular fluid, through a microdialysis probe composed of a small shaft with a tip made of a semipermeable hollow fiber membrane2. After insertion of the probe, neurotransmitters or other analytes of interest can cross the semipermeable membrane by passive diffusion before being collected at intervals for subsequent analysis by high-performance liquid chromatography (HPLC), an analytical chemistry technique commonly utilized to separate, identify, and quantify components in a heterogeneous mixture3.
Although microdialysis is a sensitive technique that can be used to measure virtually any analyte of interest, the temporal resolution is low, with maximum sampling rates on the order of minutes to tens of minutes1,2. The invention of fast scan cyclic voltammetry (FSCV), a technique that relies on the redox potential of electroactive species, can elucidate near instantaneous concentrations of the analyte of interest in the extracellular fluid. In brief (see Robinson et al.4 for an extensive review), an electrode is applied to raise and lower the voltage in a triangular wave fashion on a fast time scale4. When the voltage is in the correct range, the compound of interest is repeatedly oxidized and reduced. This oxidization and reduction results in a movement of electrons that creates a small alternating current. Scan rates take place on the sub-second scale with oxidization and reduction of compounds occurring in microseconds. By subtracting the background current created by the probe from the resulting current, one can generate a voltage vs. current plot unique to each compound. Since the time scale of the voltage oscillations is known, these data can be used to calculate a plot of the current as a function of time. Thus, the relative concentrations of the compound may be determined as long as the number of electrons transferred in each oxidation and reduction reaction is known4.
This chemical specificity and high temporal resolution make FSCV a powerful technique for detecting changing chemical concentrations in vivo. However, despite these manifold advantages, this technique requires extensive technical expertise and expensive equipment and setup. Further, nonelectroactive neurotransmitters (e.g., glutamate) cannot be measured using this technique. Fortunately, technological advancements in the field of electrochemistry5, as well as commercialization of these inventions, has introduced a relatively simple approach to measure non-electroactive neurotransmitters in awake behaving animals without compromising temporal precision-a technique known as enzymatic biosensor technology. This technique uses enzymatic conversion of the nonelectroactive neurotransmitter of interest into two substrates, one of which is electroactive hydrogen peroxide that is detected as an amperometric oxidation current generated by an applied potential5. Commercially available biosensor probes (see Figure 1) selectively measure analytes of interest by competitively reducing the contribution of endogenous interferents. In the case of glutamate, the contribution of the common interferent ascorbic acid (AA) is competitively reduced to the measured current by co-localizing AA oxidase onto the active enzymatic surface of the sensor, converting AA to non-electroactive dihydroascorbate and water. In addition, a negatively charged Nafion polymer layer present under the enzyme layer excludes endogenous anionic compounds.
This same biosensor experimental setup can measure electroactive neurotransmitters as in FSCV, but instead it employs a fixed-potential recording6. In contrast to the oscillating voltage applied in FSCV, in a fixed-potential recording the voltage is kept at the redox potential for the analyte of interest. Although it is less chemically selective than FSCV as multiple neurotransmitters may have the same redox potential, in brain areas that overwhelmingly skew towards one neurotransmitter, the turn-key nature of this approach outweighs the lack of chemical specificity.
The ability to measure both electroactive and nonelectroactive neurotransmitter release in near real-time and link it to specific behavioral events provides an opportunity to examine converging neurotransmitter release. This manuscript details the use of this system to interrogate both dopamine and glutamate neurotransmission in response to natural reward in awake behaving hamsters. The aim of this paper is to detail the process of measuring this neurotransmitter release during sexual behavior in female hamsters, with the goal of demonstrating its feasibility for examining other behaviors and experimental paradigms.
Hamsters are an ideal model for use in electrochemical recordings 
Historically, rat and mice models have been employed in the study of sexual behavior. These rodent species engage in a dynamic copulatory sequence, involving numerous female solicitation behaviors that include hopping, darting, and ear wiggling to entice the male to chase and ultimately mount the female7. The mounting by the male (with or without vaginal penetration) lasts only a few seconds, during which the female engages in her sexual behavior posture (termed lordosis) also only for a few seconds before resuming active solicitation behaviors. This pattern of behavior, composed of high levels of activity interspersed with brief periods of immobility, is problematic for measuring neurotransmission in behaving animals. First, there can be movement artifacts in the amperometric recordings that are unrelated to neural activity. Second, the locomotion is associated with the release of particular neurotransmitters in certain brain regions. For example, dopamine release has been coupled to locomotor activity in the dorsal and ventral striatum8,9, a finding that formed the basis for microdialysis measurements of dopamine following psychostimulant administration10. Because the female-typical solicitation behaviors in most rodents involve high levels of locomotor activity, and are represented by the bulk of a 10 minute sexual behavior test, this makes it difficult to ascribe changes in neurotransmission to the explicit components of sexual behavior that collectively last only minutes.
To analyze the neurochemical profile of female sexual behavior, this lab sought out a species in which there is minimal locomotor activity accompanying sexual behavior. The copulatory sequence in Syrian hamsters (Mesocricetus auratus) is ideal for neurochemical recordings due to the lack of solicitation behaviors typically seen in rats and mice11. As a consequence, female hamsters will enter and maintain the lordosis posture for upwards of 9 minutes out of a 10 minute testing session12. With the lack of extraneous locomotor movements by the female, in vivo electrochemical recordings that can be associated with components of sexual interactions with the male can be obtained.
Copulatory bouts in hamsters 
After the introduction of a male stimulus animal into the testing chamber, the male will initially engage in anogenital investigation (AI) of the female before mounting her (Figure 2A). In order for the male to mount, the female must assume a receptive sexual posture known as lordosis, in which she arches her back and deflects her tail so that the mounting male can gain penile access to her vagina. The male will mount the female, clasping her hindquarters with both paws (Figure 2B), and begin thrusting in an attempt to gain penile intromission (Figure 2C). The male will mount the female (without insertion) as well as intromit a number of times before eventually achieving ejaculation. This sequence of mounts and intromissions leading to ejaculation is termed a &#34;copulatory bout&#34;. Males will have several copulatory bouts within a single session.

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			<td style="width:183px;">Oak Pharmaceuticals Inc.</td>
			<td style="width:323px;">76478-501-50</td>
			<td style="width:504px;">Pentobarbital sodium injection, USP. This lab uses 8.5mg/100g body weight, injected intraperotineally.&#160;</td>
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			<td height="42" style="height:42px;width:329px;">Loxicom analgesic&#160;</td>
			<td style="width:183px;">Norbrook Laboratories&#160;</td>
			<td style="width:323px;">6451603670</td>
			<td style="width:504px;">NSAID antinflammatory and analgesic used for post-operative pain control. Generic: meloxicam.&#160;</td>
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			<td height="42" style="height:42px;width:329px;">Enroflox antibiotic&#160;</td>
			<td style="width:183px;">Norbrook Laboratories&#160;</td>
			<td style="width:323px;">5552915411</td>
			<td style="width:504px;">Fluoroquinolone antibiotic for post-operative infection prevention. Generic: Enrofloxacin.</td>
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			<td height="21" style="height:21px;width:329px;">Beuthanasia-D&#160;</td>
			<td style="width:183px;">Merck Animal Health</td>
			<td style="width:323px;">00061047305</td>
			<td style="width:504px;">Pentobarbital Sodium, Phenytoin Sodium euthanasia agent.</td>
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			<td height="21" style="height:21px;">Bone screws</td>
			<td>Pinnacle Technologies, Inc.</td>
			<td>8111-16</td>
			<td style="width:504px;">1/8&#34; bone screw (Pkg. of 16) used to affix skull cap to skull.&#160;</td>
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			<td height="42" style="height:42px;">Dental acrylic (Bosworth Duz-All)</td>
			<td>Bosworth&#160;</td>
			<td>166261C&#160;</td>
			<td style="width:504px;">Self curing dental acrylic is used in construction of a skull cap to affix cannula and head mount to skull.</td>
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			<td height="63" style="height:63px;">Hardware biosensor setup&#160;</td>
			<td>Pinnacle Technologies, Inc.</td>
			<td>8400-K2</td>
			<td style="width:504px;">Pinnacle offers complete hardware kits for new users of our tethered biosensor system for rats. Kits include a commutator, preamplifier, and data conditioning and acquisition system</td>
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			<td height="60" style="height:60px;">Base video computer package</td>
			<td>Pinnacle Technologies, Inc.</td>
			<td>9000-K1</td>
			<td style="width:504px;">The base computer package includes a preconfigured computer with ample hard disk storage, a high-definition monitor, a keyboard and mouse, an uninterruptible power supply, and all necessary cables.&#160;</td>
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			<td height="60" style="height:60px;">Video EQ700 EverFocus camera&#160; package</td>
			<td>Pinnacle Technologies, Inc.</td>
			<td>&#160;9000-K10&#160;</td>
			<td style="width:504px;">EQ700 night vision capable box camera with independent IR source was obtained as part of Pinnacle video computer package. Dome camera (9000-K9) and HD camera (9000-K11) options are also available.&#160;</td>
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			<td height="126" style="height:126px;">Sirenia Acquisition software</td>
			<td>Pinnacle Technologies, Inc.</td>
			<td>Free--available to download from pinnaclet.com</td>
			<td style="width:504px;">Sirenia&#160;Acquisition provides a single platform for recording data from any Pinnacle hardware system. The software features synchronization of all data streams, user-configurable settings, data consolidation, and multiple export options. In addition, the software includes basic review and analysis modules for biosensor recordings. Sirenia&#160;delivers free ll-in-one software that is ideal for data acquisition and review.</td>
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			<td height="147" style="height:147px;">Tethered rat&#160;in vitro&#160;calibration kit</td>
			<td>Pinnacle Technologies, Inc.</td>
			<td>7000-K2-T-BAS&#160;</td>
			<td style="width:504px;">In order to relate the current changes measured by a biosensor to actual changes in analyte concentration, it is necessary to calibrate the biosensor prior to implantation into the animal. The process also confirms the integrity and selectivity of the sensors.&#160;Calibration kit includes 20 mL jacketed beaker (#7058), 1/2&#34; by 1/8&#34; magnetic stir bar (#7059), right angle clamp (#7056), 2 prong single-adjustment clamp (#7055), 4-channel calibration preamplifer (#7053), and calibration holder (#7051).&#160;</td>
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			<td height="63" style="height:63px;">Stir plate&#160;</td>
			<td>Corning</td>
			<td>6795-410D</td>
			<td style="width:504px;">Corning digital Stirrer, 5&#34; x 7&#34;, 120 VAC used to spin magnetic stirrer in jacketed beaker during in vitro calibration of glutamate biosensors.</td>
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			<td height="63" style="height:63px;">Water bath capable of closed loop circulation</td>
			<td>PolyScience</td>
			<td>8006A11B</td>
			<td style="width:504px;">PolyScience 8006A11B 6L Standard Digital Heated Circulating Bath, 120VAC water bath was used with plastic tubing to heat jacketed beaker to physiological temperature.&#160;</td>
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			<td height="21" style="height:21px;">Carbon fiber sensor with BASi rat cannulae</td>
			<td>Pinnacle Technology, Inc.</td>
			<td>7002-CFS</td>
			<td style="width:504px;">Carbon fiber electrode used for recording dopamine neurotransmission.</td>
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			<td height="21" style="height:21px;">Ag/AgCl reference electrode</td>
			<td>Pinnacle Technology, Inc.</td>
			<td>7065</td>
			<td style="width:504px;">Necessary for carbon fiber recordings.</td>
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			<td height="42" style="height:42px;">Glutamate biosensors&#160;</td>
			<td>Pinnacle Technology, Inc.</td>
			<td>7001&#160;</td>
			<td style="width:504px;">Enzymatic biosensor probe used for recording glutamatergic neurotransmission.</td>
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			<td height="21" style="height:21px;">BASi guide cannulae</td>
			<td>Pinnacle Technologies, Inc.</td>
			<td>7030&#160;</td>
			<td style="width:504px;">Guide cannulae implanted into brain region of interest to guide probe.</td>
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			<td height="21" style="height:21px;">BASi cannula plastic headpiece for rats&#160;</td>
			<td>Pinnacle Technologies, Inc.</td>
			<td>7011&#160;</td>
			<td style="width:504px;">Headmount stabilizes probe and attaches to potentiostat.</td>
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measuring in vivo changes in extracellular neurotransmitters during naturally rewarding behaviors in female syrian hamsters

The ability to measure neurotransmitter release on a rapid time scale allows patterns of neurotransmission to be linked to specific behaviors or manipulations; a powerful tool in elucidating underlying mechanisms and circuitry. While the technique of microdialysis has been used for decades to measure nearly any analyte of interest in the brain, this technique is limited in temporal resolution. Alternatively, fast scan cyclic voltammetry is both temporally precise and extremely sensitive; however, because this technically difficult method relies on the electroactivity of the analyte of interest, the possibility to detect nonelectroactive substances (e.g., the neurotransmitter glutamate) is eliminated. This paper details the use of a turn-key system that combines fixed-potential amperometry and enzymatic biosensing to measure both electroactive and nonelectroactive neurotransmitters with temporal precision. The pairing of these two powerful techniques allows for the measurement of both tonic and phasic neurotransmission with relative ease, and permits recording of multiple neurotransmitters simultaneously. The aim of this manuscript is to demonstrate the process of measuring dopamine and glutamate neurotransmission in vivo using a naturally rewarding behavior (i.e., sexual behavior) in female hamsters, with the ultimate goal of displaying the technical feasibility of this assay for examining other behaviors and experimental paradigms.

Using the electrochemical and behavioral coding methodology described above, this lab has begun to characterize both tonic and phasic fluctuations in both dopamine and glutamate during in vivo recordings of sexual behavior. Due to the temporally-precise manner of this methodology, we can more accurately characterize neurotransmission during sexual behavior; as well as ascribe specific changes in release patterns to corresponding tonic changes during mating bouts, and correspondin...

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

This paper details the use of fixed-potential amperometric recordings using carbon fiber electrodes and enzymatic biosensor technology to measure the release of dopamine and glutamate with high temporal resolution during natural rewarding behavior in the female hamster.

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

The ability to measure neurotransmitter release on a rapid time scale allows patterns of neurotransmission to be linked to specific behaviors or ...

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'

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