Name: measuring in vivo changes in extracellular neurotransmitters during naturally rewarding behaviors in female syrian hamsters
Uploaded: 2017-09-12T14:00:00.000+00:00
Duration: 10 min 23 s
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 behavior it is important to obtain sexually mature animals that are all approximately the same age.</li>
	<li>House animals in a controlled temperature (22 &#176;C) and lighting (14 h light followed by 10 h dark with lights out at 13:00 h) environment, with food and water available ad libitum except during periods of experimental testing. 
	NOTE: Since hamsters seasonally reproduce, this 14:10 light/dark cycle mimics their natural breeding conditions.</li>
	<li>Following a 1-week acclimation to the laboratory, perform aseptic bilateral ovariectomies and intracranial stereotaxic cannulations under general anesthesia (e.g., pentobarbital), and administer appropriate institutionally-approved post-operative analgesic and antibiotic treatment as previously described14,15. 
	NOTE: For the study of sexual behavior, ovariectomies are necessary to remove the major endogenous source of sexual hormones so that the animals can be uniformly induced to sexual receptivity with exogenous hormone in accordance with the experimental timeline.
	<ol>
		<li>For both dopaminergic and glutamatergic probe implantation, stereotaxically implant guide cannulae (0.7 mm diameter; Figure 3) into the area of interest (e.g., this lab targeted the right nucleus accumbens (NAc)) as described in stepwise detail elsewhere15. 
		NOTE: Although the data presented are from single sensor recordings of one glutamate or dopamine probe per animal, this system allows for simultaneous recording of up to 4 sensors in a single animal; thus, 1 - 4 cannulae could be implanted depending on desired experimental design and outcomes.
		<ol>
			<li>Determine the implant coordinates of the region of interest using a stereotaxic atlas (e.g., The Golden Hamster Brain by Morin &#38; Wood), keeping in mind that the sensor extends 1 mm below the cannula into brain tissue.</li>
		</ol>
		</li>
		<li>After a cannula is stereotaxically lowered to the desired dorsal-ventral location, affix it to the skull using a cap constructed from dental acrylic, secured with stainless steel bone screws and a plastic head mount (Figure 3; see15 for more details).</li>
		<li>For carbon fiber testing, insert a reference electrode (350 &#956;m diameter, 7.5 mm total length) into the contralateral hemisphere. 
		NOTE: A specific location or distance from the cannula is not required; reference electrodes may be placed anywhere in the contralateral hemisphere that is convenient.</li>
	</ol>
	</li>
</ol>
2. Biosensor and Carbon Fiber Testing
<ol>
	<li>Following 1-week of recovery from surgery, administer the hormone priming regimen to induce sexual receptivity.
	<ol>
		<li>Inject 10 &#181;g of estradiol benzoate in 0.1 mL of cottonseed oil subcutaneously (s.c.), approximately 48 h and 24 h prior to the sexual behavior testing, to induce sexual receptivity towards the male11.</li>
		<li>Inject 500 &#181;g of progesterone in 0.1 mL of cottonseed oil, s.c., 4 h prior to the introduction of the male to induce sexual receptivity11.</li>
	</ol>
	</li>
	<li>Test all animals 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 conditions16,17,18.</li>
	<li>For enzymatic biosensor glutamate testing, calibrate the sensors in vitro (Figure 4) before use as described previously15,19.
	<ol>
		<li>Make the calibration solutions fresh on the day of testing in 20 mL glass centrifuge tubes. For the analyte solution, dissolve 7.4 mg of L-glutamic acid into 10 mL of ultra-pure H2O by gently inverting the tube. Make the interferent solution by dissolving 176.1 mg AA in 10 mL of ultra-pure H2O.</li>
		<li>Set up for the calibration by placing a magnetic stirrer on the base of a basic lab ring stand. Place a 20-mL jacketed beaker on top of the magnetic stirrer, and clamp in place using a medium 2-prong clamp.
		<ol>
			<li>Add a magnetic stir bar and 20 mL of 100 mM phosphate buffered saline (PBS) into the jacketed beaker. Connect the jacketed beaker to a circulating water bath to heat the buffer solution to 37 &#176;C. 
			NOTE: The enzymatic biosensor calibrations must be performed at body temperature since enzymatic activity is influenced by temperature.</li>
		</ol>
		</li>
		<li>Place the sensor calibration holder on top of the jacketed beaker, and set a 4-channel calibration preamplifier on top, securing in place using a right-angle clamp. Connect the preamplifier to a data conditioning and acquisition device to record calibration data.</li>
		<li>Test the sensitivity of each enzymatic biosensor to glutamate (or other analytes of interest for other commercially available biosensor probes e.g., glucose) 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 (AgCl) reference wire in the buffer solution.
		<ol>
			<li>Before connecting each sensor (up to 4) being tested to a port on a 4-channel calibration preamplifier, initiate a new recording on the computer interface utilizing free downloadable acquisition software. Allow the sensors to reach a stable baseline.</li>
		</ol>
		</li>
		<li>Begin adding the analyte injections when the sensors have reached a stable baseline. Use the hole in the calibration holder to introduce a pipette tip to the buffer solution. Use the quick key annotation tool in the recording software to annotate when an injection is made.
		<ol>
			<li>Make 10 &#181;M additions of glutamate by pipetting 40 &#956;L of the analyte solution into the buffer solution. Wait for the sensor to stabilize between additions. Add 3 injections of analyte solution before adding a single injection from interferent AA solution (50 &#956;L injection volume for 250 &#956;M change in concentration). 
			NOTE: The calibration allows for conversion of the changes in the amperometric signal (seen during the enzymatic experimental recording in step 2.12) to changes in glutamate concentration, if desired. See reference15 for stepwise instruction. The dopaminergic probes used by this lab are calibrated during the manufacturing process by the commercial provider. Glutamate probes must be calibrated at the time of use as their sensitivity can decrease over time as the enzymatic components degrade19. Because carbon fiber electrodes do not undergo degradation, the calibration performed by the company prior to shipment is sufficient along as there is an increased likelihood of damaging the thin carbon fiber (350 &#956;m diameter) through additional handling.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Line the test chamber with bedding taken from the animal&#39;s home cage. 
	NOTE: This increases the female&#39;s familiarity with the testing chamber, as well as exposes the male to sexually stimulating olfactory mating cues that were distributed by the female in response to the estradiol treatment.</li>
	<li>Lightly anesthetize the animals prior to insertion of the probe by either isoflurane or another volatile anesthetic in an induction chamber.</li>
	<li>Remove the occlusive obdurator from the guide cannula, and insert either the carbon fiber electrode or enzymatic probe through the guide shaft.</li>
	<li>After the probe insertion, place the animal in the testing chamber. 
	NOTE: This lab uses a 10-gal glass aquarium (24.5 x 48.9 x 29.4 cm), but round chambers of equal area may also be employed.</li>
	<li>Begin recording the video and time-locked amperometric signal in a commercially available software program, described in detail elsewhere15.</li>
	<li>Connect the sensor to the recording system: the potentiostat via an electrically shielded cable and an electrical swivel. Stabilize the sensor connection by screwing a pin attachment onto the head mount. Reinforce the connection using lab film if necessary. 
	NOTE: A customized connector is needed to attach both the carbon fiber electrode and reference electrode to the potentiostat, while the enzymatic glutamate sensors can be directly connected to the potentiostat.</li>
	<li>Allow the sensor to equilibrate in the brain before experimental testing. 
	NOTE: Equilibration times differ depending on the recording sensor (e.g., enzymatic sensors require 2 - 4 h equilibration, while carbon-fiber electrodes only require about 30 min). If both types of sensors are implanted, equilibrate for the longer enzymatic sensor time.</li>
	<li>After sensor equilibration, introduce a stimulus male into the testing chamber.</li>
	<li>Following the first mount with penile insertion (termed intromission) by the stimulus male, continue recording for an additional 10 - 30 min, depending on the experimental goals.</li>
	<li>Following behavioral testing, disconnect the female&#39;s sensor.</li>
	<li>Remove both hamsters from the testing chamber. Remove the bedding and clean the chamber using 70% ethanol. 
	NOTE: Due to the brief equilibration required for carbon fiber recordings, steps 2.3 - 2.14 can be repeated to test multiple animals in the same day. In contrast, because the enzymatic sensors require about 4 h of equilibration, test only one animal per day to limit possible inter-subject variation due to differences in the animals&#39; circadian time at testing.</li>
</ol>
3. Sacrifice and Perfusion
<ol>
	<li>Following the conclusion of the testing period, deeply anesthetize the female test subject with a euthanizing agent (e.g., this lab uses an intraperitoneal injection of 0.2 mL phenytoin and pentobarbital solution).</li>
	<li>Transcardially perfuse the animal with 25 mM PBS, pH 7.6, to remove all circulating blood, followed by a 20 min fixation using 4% paraformaldehyde-PBS.</li>
	<li>Remove the brain and postfix in ~ 30 mL of the 4% paraformaldehyde solution in a 50-mL conical tube overnight.
	<ol>
		<li>Store the brain in a 10% sucrose-PBS solution until ready to serial section. 
		NOTE: Brains may be stored up to 2-weeks, with weekly sucrose solution changes to avoid potential growth of contaminants.</li>
	</ol>
	</li>
	<li>Serial section the brain in 40 &#181;m slices through the implanted region with either a freezing microtome or cryostat as previously described19.</li>
	<li>Mount slices serially on commercially available adhesive-coated slides (or see20 to make). Allow to dry, at least overnight.</li>
	<li>Stain the slides with cresyl violet dye, clear, and coverslip as previously described20.</li>
	<li>Image the slides using bright field microscopy to confirm the anatomical placement of the sensor.</li>
</ol>
4. Behavioral Coding
<ol>
	<li>View and annotate the videos using free commercially downloadable software (see Table of Materials) in slow motion to precisely code behaviors time-locked to the amperometric signal. 
	NOTE: To use the MATLAB code in the analysis (see below), annotate the start and end of each of the female&#39;s and male&#39;s behaviors.
	<ol>
		<li>Annotate startLordosis in the frame when the female initiates a dorsoflexion of her back and defects her tail upwards. Annotate endLordosis when the female terminates this posture, which often occurs when the female readjusts to another location in the testing chamber.</li>
		<li>Annotate startAI when the female is in the lordosis posture and the male moves his snout towards the female&#39;s anogenital region, where sniffing, licking, or nuzzling of her perineal region may occur. Annotate endAI when the male removes his snout from close proximity to the female&#39;s anogenital region.</li>
		<li>Annotate startMount when the male approaches and places his forepaws on the female in a mounting posture, regardless of the orientation of the mount attempt (e.g., side, rump).</li>
		<li>Annotate startIntromission when successful thrusting gains the mounted male penile access to the female&#39;s vagina. 
		NOTE: An intromission is behaviorally distinguishable from the mounted thrusting that occurs prior to intromission, as the male visibly pulls his upper limbs towards his body, thrusts his pelvis forward, and curls his tail upwards, indicating penetration (see Figure 4C).
		<ol>
			<li>When applicable, annotate ejaculation. 
			NOTE: At the end of a mating bout and in conjunction with a successful intromission, the male will ejaculate. Although one is unable to visualize the actual emission, male hamsters have a characteristic treading motion with their hind foot during an intromission21, thus annotate the ejaculation at the occurrence of the foot movement.</li>
		</ol>
		</li>
		<li>Annotate endIntromission and endMount 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, simultaneous code these two behaviors to allow for consistency and reliability across mating bouts. 
		Note: A mating bout either follows a successful ejaculation or is when the female breaks the lordosis posture following at least one mount.</li>
	</ol>
	</li>
</ol>
5. Example Data Analysis
Note: The use of fixed-potential carbon fiber and enzymatic recordings for dopamine and glutamate, respectively, allows for the ability to measure both tonic and phasic patterns of electrochemical release. This lab uses a free, commercial acquisition system and software to record and convert recorded signals from analog to digital (bias 0.600 V, sampling rate = 1 Hz).
<ol>
	<li>Description of example data analysis protocol
	<ol>
		<li>Although other programs can be used, quantify the tonic and phasic (transient) changes in the recorded electrochemical signal using MATLAB.
		<ol>
			<li>For the presented data, calculate the tonic signal using a 50-point moving average filter. A transient-only normalized representation of the signal was calculated by subtracting the tonic signal from the raw data. When identifying phasic peaks, there are various algorithmic options (e.g., labeling peaks as changes in the signal more than 1 SD above the mean).</li>
			<li>Identify the locations of these peaks, i.e., local maxima, from the normalized signal using the MATLAB function peakfinder with the minimum peak amplitude set to the root mean square of the signal. The location of peaks detected from the normalized signal was used to extract the amplitude from each peak. The ability to detect peaks allows for the identification of any time-locked behaviors that may be coinciding with, or driving the relative peak in the neurochemical under investigation.</li>
		</ol>
		</li>
		<li>Segment the processed data in accordance with the behavioral annotations (see step 3); a Student&#39;s t-test was used to evaluate differences in the tonic neurotransmitter level, the occurrence of transients, and the amplitude of transients during the pre-mating bout lordosis versus lordosis during mating bouts. To measure changes to tonic patterns of electrochemical release, the data points occurring during the Lordosis position directly before a mating bout (i.e., pre-mating bout lordosis) were compared to the data points during a mating bout session using a t-test.</li>
	</ol>
	</li>
	<li>Preparation of raw data for described data analysis protocol 
	Note: As stated, although other programs or analyses may be equally viable options, in order to use the MATLAB code described above, the raw data must be prepared in the following way:
	<ol>
		<li>Export the annotation and voltage files. To do this, under the &#39;File&#39; tab, select the &#39;Export&#39; function and select the &#39;Annotations&#39; tab to save the annotations. Under the &#39;File&#39; tab, select the &#39;Export&#39; function and choose the &#34;TSV&#39; tab to save the voltage measurements as a TSV extension file.</li>
		<li>Open each file with a spreadsheet program and save as &#39;xls/xlsx&#39; to make the files compatible with MATLAB. 
		NOTE: For the &#39;Annotation&#39; file, there are additional annotations that must manually added post-export in a spreadsheet program to dictate the start and end of both the pre-mating bout lordosis (annotated as PreMBlordorsis) and the mating bout so that these time points can be directly compared as mentioned in step 5.1.2.</li>
		<li>Annotate the start of the pre-mating bout lordosis (startPreMBlordosis) at the same time as a lordosis behavior begins (startLordosis) that also includes a mount behavior, and also at the start of a new mating bout if the female remains in the lordosis posture.</li>
		<li>Annotate the end of the pre-mating bout lordosis (endPreMBlordosis) when the first mount begins.</li>
		<li>Annotate the start of a mating bout (startMB) at the same time of the endPreMBlordosis, as this signifies the start of a mating bout.</li>
		<li>Annotate the end of a mating bout (endMB) at either the end of a mount that includes an ejaculation (see step 4.1.4.1), or following the female&#39;s termination of the lordosis posture (endLordosis).</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

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.

<table><tbody><tr><td>Nembutal</td><td>Oak Pharmaceuticals Inc.</td><td>76478-501-50</td><td>Pentobarbital sodium injection, USP. This lab uses 8.5 mg/100 g body weight, injected intraperotineally.&amp;nbsp;</td></tr><tr><td>Loxicom analgesic&amp;nbsp;</td><td>Norbrook Laboratories&amp;nbsp;</td><td>6451603670</td><td>NSAID antinflammatory and analgesic used for post-operative pain control. Generic: meloxicam.&amp;nbsp;</td></tr><tr><td>Enroflox antibiotic&amp;nbsp;</td><td>Norbrook Laboratories&amp;nbsp;</td><td>5552915411</td><td>Fluoroquinolone antibiotic for post-operative infection prevention. Generic: Enrofloxacin.</td></tr><tr><td>Beuthanasia-D&amp;nbsp;</td><td>Merck Animal Health</td><td>00061047305</td><td>Pentobarbital Sodium, Phenytoin Sodium euthanasia agent.</td></tr><tr><td>Bone screws</td><td>Pinnacle Technologies, Inc.</td><td>8111-16</td><td>1/8&quot; bone screw (Pkg. of 16) used to affix skull cap to skull.&amp;nbsp;</td></tr><tr><td>Dental acrylic (Bosworth Duz-All)</td><td>Bosworth&amp;nbsp;</td><td>166261C&amp;nbsp;</td><td>Self curing dental acrylic is used in construction of a skull cap to affix cannula and head mount to skull.</td></tr><tr><td>Hardware biosensor setup&amp;nbsp;</td><td>Pinnacle Technologies, Inc.</td><td>8400-K2</td><td>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></tr><tr><td>Base video computer package</td><td>Pinnacle Technologies, Inc.</td><td>9000-K1</td><td>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.&amp;nbsp;</td></tr><tr><td>Video EQ700 EverFocus camera&amp;nbsp; package</td><td>Pinnacle Technologies, Inc.</td><td>&amp;nbsp;9000-K10&amp;nbsp;</td><td>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.&amp;nbsp;</td></tr><tr><td>Sirenia Acquisition software</td><td>Pinnacle Technologies, Inc.</td><td>Free--available to download from pinnaclet.com</td><td>Sirenia&amp;nbsp;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&amp;nbsp;delivers free ll-in-one software that is ideal for data acquisition and review.</td></tr><tr><td>Tethered rat&amp;nbsp;in vitro&amp;nbsp;calibration kit</td><td>Pinnacle Technologies, Inc.</td><td>7000-K2-T-BAS&amp;nbsp;</td><td>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.&amp;nbsp;Calibration kit includes 20 mL jacketed beaker (#7058), 1/2&quot; by 1/8&quot; magnetic stir bar (#7059), right angle clamp (#7056), 2 prong single-adjustment clamp (#7055), 4-channel calibration preamplifer (#7053), and calibration holder (#7051).&amp;nbsp;</td></tr><tr><td>Stir plate&amp;nbsp;</td><td>Corning</td><td>6795-410D</td><td>Corning digital Stirrer, 5&quot; x 7&quot;, 120 VAC used to spin magnetic stirrer in jacketed beaker during &lt;em&gt;in vitro&lt;/em&gt; calibration of glutamate biosensors.</td></tr><tr><td>Water bath capable of closed loop circulation</td><td>PolyScience</td><td>8006A11B</td><td>PolyScience 8006A11B 6L Standard Digital Heated Circulating Bath, 120VAC water bath was used with plastic tubing to heat jacketed beaker to physiological temperature.&amp;nbsp;</td></tr><tr><td>Carbon fiber sensor with BASi rat cannulae</td><td>Pinnacle Technology, Inc.</td><td>7002-CFS</td><td>Carbon fiber electrode used for recording dopamine neurotransmission.</td></tr><tr><td>Ag/AgCl reference electrode</td><td>Pinnacle Technology, Inc.</td><td>7065</td><td>Necessary for carbon fiber recordings.</td></tr><tr><td>Glutamate biosensors&amp;nbsp;</td><td>Pinnacle Technology, Inc.</td><td>7001&amp;nbsp;</td><td>Enzymatic biosensor probe used for recording glutamatergic neurotransmission.</td></tr><tr><td>BASi guide cannulae</td><td>Pinnacle Technologies, Inc.</td><td>7030&amp;nbsp;</td><td>Guide cannulae implanted into brain region of interest to guide probe.</td></tr><tr><td>BASi cannula plastic headpiece for rats&amp;nbsp;</td><td>Pinnacle Technologies, Inc.</td><td>7011&amp;nbsp;</td><td>Headmount stabilizes probe and attaches to potentiostat.</td></tr></tbody></table>

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 ...

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10.1K Views.  University of Minnesota. 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.

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

Comprehensive Analysis of Transcription Dynamics from Brain Samples Following Behavioral Experience

The encoding of experiences in the brain and the consolidation of long-term memories depend on gene transcription. Identifying the function of specific genes in encoding experience is one of the main objectives of molecular neuroscience. Furthermore, the functional association of defined genes with specific behaviors has implications for understanding the basis of neuropsychiatric disorders. Induction of robust transcription programs has been observed in the brains of mice following various behavioral manipulations. While some genetic elements are utilized recurrently following different behavioral manipulations and in different brain nuclei, transcriptional programs are overall unique to the inducing stimuli and the structure in which they are studied1,2.
In this publication, a protocol is described for robust and comprehensive transcriptional profiling from brain nuclei of mice in response to behavioral manipulation. The protocol is demonstrated in the context of analysis of gene expression dynamics in the nucleus accumbens following acute cocaine experience. Subsequent to a defined in vivo experience, the target neural tissue is dissected; followed by RNA purification, reverse transcription and utilization of microfluidic arrays for comprehensive qPCR analysis of multiple target genes. This protocol is geared towards comprehensive analysis (addressing 50-500 genes) of limiting quantities of starting material, such as small brain samples or even single cells.
The protocol is most advantageous for parallel analysis of multiple samples (e.g. single cells, dynamic analysis following pharmaceutical, viral or behavioral perturbations). However, the protocol could also serve for the characterization and quality assurance of samples prior to whole-genome studies by microarrays or RNAseq, as well as validation of data obtained from whole-genome studies.

This manuscript describes a protocol that applies comprehensive profiling for analysis of transcriptional programs induced in specific brain nuclei of rodents following behavioral paradigms. Herein, this approach is illustrated in the context of profiling genes induced in the nucleus accumbens (NAc) of mice following acute cocaine exposure, utilizing microfluidic qPCR arrays.

The encoding of experiences in the brain and the consolidation of long-term memories depend on gene transcription. Identifying the function of specific ...

Behavior

The Modified Hole Board - Measuring Behavior, Cognition and Social Interaction in Mice and Rats

This protocol describes the modified hole board (mHB), which combines features from a traditional hole board and open field and is designed to measure multiple dimensions of unconditioned behavior in small laboratory mammals (e.g., mice, rats, tree shrews and small primates). This paradigm is a valuable alternative for the use of a behavioral test battery, since a broad behavioral spectrum of an animal&#8217;s behavioral profile can be investigated in one single test.
The apparatus consists of a box, representing the &#8216;protected&#8217; area, separated from a group compartment. A board, on which small cylinders are staggered in three lines, is placed in the center of the box, representing the &#8216;unprotected&#8217; area of the set-up. The cognitive abilities of the animals can be measured by baiting some cylinders on the board and measuring the working and reference memory. Other unconditioned behavior, such as activity-related-, anxiety-related- and social behavior, can be observed using this paradigm. Behavioral flexibility and the ability to habituate to a novel environment can additionally be observed by subjecting the animals to multiple trials in the mHB, revealing insight into the animals&#8217; adaptive capacities.
Due to testing order effects in a behavioral test battery, na&#239;ve animals should be used for each individual experiment. By testing multiple behavioral dimensions in a single paradigm and thereby circumventing this issue, the number of experimental animals used is reduced. Furthermore, by avoiding social isolation during testing and without the need to food deprive the animals, the mHB represents a behavioral test system, inducing if any, very low amount of stress.

This protocol describes the modified hole board, which is a behavioral test set-up that comprises the characteristics of an open field and a traditional hole board. This set-up enables the differential analysis of unconditioned behavior of small laboratory mammals as well as the analysis of cognitive abilities.

This protocol describes the modified hole board (mHB), which combines features from a traditional hole board and open field and is designed to measure ...

Quantifying Social Motivation in Mice Using Operant Conditioning

In this protocol, social motivation is measured in mice through a pair of operant conditioning paradigms. To conduct the experiments, two-chambered shuttle boxes were equipped with two operant levers (left and right) and a food receptacle in one chamber, which was then divided from the second chamber by an automated guillotine door covered by a wire grid. Different stimulus mice, rotated across testing days, served as a social stimulus behind the wire grid, and were only visible following the opening of the guillotine door. Test mice were trained to lever press in order to open the door and gain access to the stimulus partner for 15 sec. The number of lever presses required to obtain the social reward progressively increased on a fixed schedule of 3. Testing sessions ended after test mice stopped lever pressing for 5 consecutive minutes. The last reinforced ratio or breakpoint can be used as a quantitative measure of social motivation. For the second paradigm, test mice were trained to discriminate between left and right lever presses in order to obtain either a food reward or the social reward. Mice were rewarded for every 3 presses of each respective lever. The number of food and social rewards can be compared as a measurement of the value placed upon each reward. The ratio of each reward type can also be compared between mouse strains and the change in this ratio can be monitored within testing sessions to measure satiation with a given reward type. Both of these operant conditioning paradigms are highly useful for the quantification of social motivation in mouse models of autism and other disorders of social behavior.

This article describes a new protocol for the quantitative measurement of social motivation in mice using operant conditioning for a social reward. The protocol is useful for the measurement of social motivation in mouse models of autism and other disorders of social behavior.

In this protocol, social motivation is measured in mice through a pair of operant conditioning paradigms. To conduct the experiments, two-chambered ...

Survivable Stereotaxic Surgery in Rodents 

The ability to measure extracellular basal levels of neurotransmitters in the brain of awake animals allows for the determination of effects of different systemic challenges (pharmacological or physiological) to the CNS. For example, one can directly measure how the animal's midbrain dopamine projections respond to dopamine-releasing drugs like d-amphetamine or natural stimuli like food. In this video, we show you how to implant guide cannulas targeting specific sites in the rat brain, how to insert and implant a microdialysis probe and how to use high performance liquid chromatography coupled with electrochemical detection (HPLC-EC) to measure extracellular levels of oxidizable neurotransmitters and metabolites. Local precise introduction of drugs through the microdialysis probe allows for refined work on site specificity in a compound s mechanism of action. This technique has excellent anatomical and chemical resolution but only modest time resolution as microdialysis samples are usually processed every 20-30 minutes to ensure detectable neurotransmitter levels. Complementary ex vivo tools (i.e., slice and cell culture electrophysiology) can assist with monitoring real-time neurotransmission.

Survivable Stereotaxic Surgery in Rodents

The monitoring of extracellular neurotransmitter levels in distinct brain regions of freely moving animals offers insights on the link between neurotransmitter release and behavior. In vivo microdialysis coupled with electrochemical detection provides excellent anatomical and chemical resolution; and information on how basal neurotransmission is altered by pharmacological or physiological manipulations.

The ability to measure extracellular basal levels of neurotransmitters in the brain of awake animals allows for the determination of effects of different ...

Biology

A Conflict Model of Reward-seeking Behavior in Male Rats

The present protocol describes a novel conflict task as a model of inhibitory control in rats. In this model, a natural rewarding stimulus (sexual stimulus) that represents a high-value reward, and the aversive stimuli (pins), are concurrently presented. The male rats have to climb or jump over the obstacle full of pins to approach and investigate the sexual partner. If the animal persists in their approaching behavior regardless of the aversive stimuli, it is considered as a maladaptive or risky reward-seeking behavior. The conflict task permits the evaluation of deficit in inhibitory control resulting from exposure to abused drug, such as morphine, or a stressful event. 
The main advantage of this model is that it provides a simple and quick way to discover the deficit in inhibitory control after exposure to opiate drugs or other stressful events. In addition to opiates, this behavioral model would also be useful for quickly discovering the inhibitory control deficits induced by other addictive drugs. However, the limitation is that the male rats' performance may be subject to exercising effects with repeated testing under this conflict task. In the future, one can hope that the individuals with the compulsive phenotype of reward-seeking behavior after exposure to opiates will be identified based on modifying this conflict model.

This conflict model is used to measure the impairment of inhibitory control after exposure to addictive drugs, or other factors that may influence inhibitory control. A sexual stimulus and an aversive obstacle are concurrently presented, thus male rats have to conquer the obstacle to approach the sexual reward.

The present protocol describes a novel conflict task as a model of inhibitory control in rats. In this model, a natural rewarding stimulus (sexual ...

Simultaneous Monitoring of Wireless Electrophysiology and Memory Behavioral Test as a Tool to Study Hippocampal Neurogenesis

Brainwaves amplitude obtained from electroencephalography (EEG) has been well-recognized as a basis for cognitive capacity, memory, and learning on animals and humans. Adult neurogenesis mechanism is also linked to memory and learning improvement. Traditionally, researchers used to assess learning and memory parameters in rodent models by behavioral tasks. Therefore, the simultaneous monitoring of behavioral changes and EEG is particularly interesting in correlating data between brain activity and task-related behaviors. However, most of the equipment required to perform both studies are either complex, expensive, or uses a wired setup network that hinders the natural animals&#8217; movement. In this study, EEG was recorded with a wireless electrophysiology device along with the execution of a novel object recognition task (NORT). The animal&#8217;s behavior was monitored simultaneously by a video tracking system. Both recordings were analyzed offline by their timestamps which were synched to link EEG signals with the animal&#8217;s actions. Subjects consist of adult Wistar rats after medium-term environmental enrichment treatment. Six skull screw electrodes were fixed in pairs on both hemispheres over frontal, central, and parietal regions and were referenced to an electrode located posterior of the nasal bone. NORT protocol consists of exposing the animal to two identical objects for 10 min. After 2 h and 24 h, one of the objects was replaced with a novel one. Exploration time for each object was monitored by a behavioral tracking software (BTS) and EEG data recording. The analysis of the EEG synced with behavioral data consists of estimations of alpha and beta relative band power and comparisons between novel object recognition versus familiar object exploration, between three experimental stages. In this manuscript, we have discussed electrodes manufacturing process, epidural electrodes implantation surgery, environmental enrichment protocol, NORT protocol, BTS setup, EEG &#8211; BTS coupling for simultaneous monitoring in real-time, and EEG data analysis based on automatic events detection.

The protocol presented here provides information on the simultaneous electroencephalography (EEG) and behavioral assessment in real-time. We have discussed all steps involved in this protocol as an attractive solution for researchers in many fields of neuroscience, particularly in learning and memory areas.

Brainwaves amplitude obtained from electroencephalography (EEG) has been well-recognized as a basis for cognitive capacity, memory, and learning on ...

Environmental Modulations of the Number of Midbrain Dopamine Neurons in Adult Mice

Long-lasting changes in the brain or &#8216;brain plasticity&#8217; underlie adaptive behavior and brain repair following disease or injury. Furthermore, interactions with our environment can induce brain plasticity. Increasingly, research is trying to identify which environments stimulate brain plasticity beneficial for treating brain and behavioral disorders. Two environmental manipulations are described which increase or decrease the number of tyrosine hydroxylase immunopositive (TH+, the rate-limiting enzyme in dopamine (DA) synthesis) neurons in the adult mouse midbrain. The first comprises pairing male and female mice together continuously for 1 week, which increases midbrain TH+ neurons by approximately 12% in males, but decreases midbrain TH+ neurons by approximately 12% in females. The second comprises housing mice continuously for 2 weeks in &#8216;enriched environments&#8217; (EE) containing running wheels, toys, ropes, nesting material, etc., which increases midbrain TH+ neurons by approximately 14% in males. Additionally, a protocol is described for concurrently infusing drugs directly into the midbrain during these environmental manipulations to help identify mechanisms underlying environmentally-induced brain plasticity. For example, EE-induction of more midbrain TH+ neurons is abolished by concurrent blockade of synaptic input onto midbrain neurons. Together, these data indicate that information about the environment is relayed via synaptic input to midbrain neurons to switch on or off expression of &#8216;DA&#8217; genes. Thus, appropriate environmental stimulation, or drug targeting of the underlying mechanisms, might be helpful for treating brain and behavioral disorders associated with imbalances in midbrain DA (e.g. Parkinson&#8217;s disease, attention deficit and hyperactivity disorder, schizophrenia, and drug addiction).

This protocol describes two different environmental manipulations and a concurrent brain infusion protocol to study environmentally-induced brain changes underlying adaptive behavior and brain repair in adult mice.

Long-lasting changes in the brain or &#8216;brain plasticity&#8217; underlie adaptive behavior and brain repair following disease or injury. Furthermore, ...

Simultaneous Detection of c-Fos Activation from Mesolimbic and Mesocortical Dopamine Reward Sites Following Naive Sugar and Fat Ingestion in Rats

This study uses cellular c-fos activation to assess effects of novel ingestion of fat and sugar on brain dopamine (DA) pathways in rats. Intakes of sugars and fats are mediated by their innate attractions as well as learned preferences. Brain dopamine, especially meso-limbic and meso-cortical projections from the ventral tegmental area (VTA), has been implicated in both of these unlearned and learned responses. The concept of distributed brain networks, wherein several sites and transmitter/peptide systems interact, has been proposed to mediate palatable food intake, but there is limited evidence empirically demonstrating such actions. Thus, sugar intake elicits DA release and increases c-fos-like immunoreactivity (FLI) from individual VTA DA projection zones including the nucleus accumbens (NAC), amygdala (AMY) and medial prefrontal cortex (mPFC) as well as the dorsal striatum. Further, central administration of selective DA receptor antagonists into these sites differentially reduce acquisition and expression of conditioned flavor preferences elicited by sugars or fats. One approach by which to determine whether these sites interacted as a distributed brain network in response to sugar or fat intake would be to simultaneous evaluate whether the VTA and its major mesotelencephalic DA projection zones (prelimbic and infralimbic mPFC, core and shell of the NAc, basolateral and central-cortico-medial AMY) as well as the dorsal striatum would display coordinated and simultaneous FLI activation after oral, unconditioned intake of corn oil (3.5%), glucose (8%), fructose (8%) and saccharin (0.2%) solutions. This approach is a successful first step in identifying the feasibility of using cellular c-fos activation simultaneously across relevant brain sites to study reward-related learning in ingestion of palatable food in rodents.

The goal of this study is to identify reward-related distributed brain networks by delineating a reliable immunohistological technique using cellular c-fos activation to measure simultaneous changes in dopamine pathways and terminal sites after novel ingestion of fat and sugar in rats.

This study uses cellular c-fos activation to assess effects of novel ingestion of fat and sugar on brain dopamine (DA) pathways in rats. Intakes of sugars ...

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We demonstrate a simple way to tap into the fly reward system, modify experiences related to natural reward, and use voluntary ethanol consumption as a measure for changes in reward states. The approach serves as a relevant tool to study the neurons and genes that play a role in experience-mediated changes of internal state. The protocol is composed of two discrete parts: exposing the flies to rewarding and nonrewarding experiences, and assaying voluntary ethanol consumption as a measure of the motivation to obtain a drug reward. The two parts can be used independently to induce the modulation of experience as an initial step for further downstream assays or as an independent two-choice feeding assay, respectively. The protocol does not require a complicated setup and can therefore be applied in any laboratory with basic fly culture tools.

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for inducing rewarding and nonrewarding experiences in fruit flies (Drosophila melanogaster) using voluntary ethanol consumption as a measure for changes in reward states.

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We ...

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

In the central nervous system, the D1-alpha subtype receptor (Drd1&#945;) is the most abundant dopamine (DA) receptor, which plays a vital role in regulating neuronal growth and development. However, the mechanisms underlying Drd1&#945; receptor abnormalities mediating behavioral responses and modulating working memory function are still unclear. Using a novel RNA in situ hybridization assay, the current study identified dopamine Drd1&#945; receptor and tyrosine hydroxylase (TH) RNA expression from DA-related circuitry in the nucleus accumbens (NAc) area and substantia nigra region (SNR), respectively. Drd1&#945; expression in the NAc shows a &#34;discrete dot&#34; staining pattern. Clear sex differences in Drd1&#945; expression were observed. In contrast, TH shows a &#34;clustered&#34; staining pattern. Regarding TH expression, female rats displayed a higher signal expression per cell relative to male animals. The methods presented here provide a novel in situ hybridization technique for investigating changes in dopamine system dysfunction during the progression of central nervous system diseases.

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

Identification of dopamine D1-alpha receptor in the nucleus accumbens is critical for clarifying D1 receptor dysfunction during a central nervous system disease. We performed a novel RNA in situ hybridization assay to visualize single RNA molecules in a specific brain area.

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

Summary

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Chapters in this video

Comprehensive Analysis of Transcription Dynamics from Brain Samples Following Behavioral Experience

The Modified Hole Board - Measuring Behavior, Cognition and Social Interaction in Mice and Rats

Quantifying Social Motivation in Mice Using Operant Conditioning

Survivable Stereotaxic Surgery in Rodents

A Conflict Model of Reward-seeking Behavior in Male Rats

Simultaneous Monitoring of Wireless Electrophysiology and Memory Behavioral Test as a Tool to Study Hippocampal Neurogenesis

Environmental Modulations of the Number of Midbrain Dopamine Neurons in Adult Mice

Simultaneous Detection of c-Fos Activation from Mesolimbic and Mesocortical Dopamine Reward Sites Following Naive Sugar and Fat Ingestion in Rats

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology