eoe sub articles

EoE Sub Articles

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Presurgical preparations
<ol>
	<li>Electrode solution preparation
	<ol>
		<li>To make the electrode backfill solution, prepare a solution of 4 M potassium acetate with 140 mM potassium chloride.</li>
	</ol>
	</li>
	<li>Electrode preparation
	<ol>
		<li>Using vacuum suction, insert a T-650 carbon fiber (7 &#181;m in diameter) into a borosilicate glass capillary (length = 100 mm, diameter = 1.0 mm, inside diameter = 0.5 mm).</li>
		<li>Once the carbon fiber has been placed inside the glass capillary, place the glass capillary into a vertical electrode puller, with the heating element roughly in the middle of the capillary. Set the heater to 55 with the magnet turned off.</li>
		<li>After the capillary is pulled, carefully raise the upper capillary holder so that the tip of the electrode is not surrounded by the heating element.</li>
		<li>Using sharp scissors, cut the carbon fiber that is still connecting the two pieces of the capillary. This will result in two separate carbon fiber microelectrodes.</li>
		<li>Under a light microscope, carefully cut the exposed carbon fiber with a sharp scalpel so that it extends approximately 75-100 &#181;m beyond the end of the glass.</li>
		<li>Using a light microscope, ensure that the electrode is free of cracks along the capillary and that the seal where the carbon fiber exits the capillary is difficult to notice and free from cracks. 
		NOTE: A good seal will help reduce noise during recordings.</li>
	</ol>
	</li>
	<li>Reference electrode fabrication
	<ol>
		<li>Solder a gold pin to a 5 cm silver wire.</li>
		<li>Attach the anode to a metal paper clip or other conductor, the cathode to a pin, and apply a voltage (~2 V) while the paper clip and silver wire is submerged in 0.1 M HCl.</li>
		<li>Cease the voltage once a white coating (AgCl) appears on the silver wire.</li>
	</ol>
	</li>
	<li>Preparing electrodes for implantation
	<ol>
		<li>Solder a gold pin to a thin insulated wire (~10 cm in length, &#60;0.50 mm diameter).</li>
		<li>Remove ~5 cm of insulation from the wire opposite to the gold pin.</li>
		<li>Fill the electrode approximately halfway with the electrode solution.</li>
		<li>Insert insulated wire into the electrode. 
		NOTE: The wire should make contact with the carbon fiber inside the electrode.</li>
	</ol>
	</li>
</ol>
2. Electrode implantations
<ol>
	<li>Give adult, male, Sprague Dawley rats (250&#8722;450 g) an intraperitoneal injection (1.5 g/kg or 1 mL/kg volume) of 0.5 g/mL urethane dissolved in sterile saline. Start with an initial urethane dose of 1.0&#8722;1.2 g/kg. If the animal is still responsive to the noxious stimulus test (tail pinch) 20 min after urethane administration, administer an additional 0.3&#8722;0.5 g/kg urethane for a 1.5 g/kg total dose. 
	NOTE: To prepare the 0.5 g/mL urethane solution, add 10 g of urethane to 10 g (~10 mL) of saline. Urethane is a carcinogen and must be handled with care. However, it is an important anesthetic, as it does not alter levels of dopamine, as do other anesthetics such as ketamine/ xylazine and chloral hydrate.</li>
	<li>Once the animal is deeply anesthetized and is not responsive to noxious stimuli (e.g., toe pinch), place it in the stereotaxic frame. Apply ophthalmic lubricant to each eye of the rat. 
	NOTE: This is a non-survival surgery, but a good aseptic technique is encouraged.</li>
	<li>Clean the rat&#39;s scalp using a two-stage scrub (i.e., an iodopovidone scrub followed by a 70% ethanol scrub; perform with a 3-cycle repetition).</li>
	<li>Cut away the scalp tissue using sterilized needle nose tweezers and surgical scissors. Remove a significant amount of tissue to make room for the various implantations outlined below.</li>
	<li>Gently clean the skull surface using sterilized cotton tip applicators. Then apply 2&#8722;3 drops of 3% hydrogen peroxide to help identify the lambda and bregma.</li>
	<li>Using a stereotaxic or hand drill (1.0 mm, ~20,000 rpm), drill a 1.5 mm diameter hole 2.5 mm anterior to the bregma and 3.5 mm lateral to the bregma. Partially (about halfway, until it is firmly in place) implant a screw (1.59 mm O.D., 3.2 mm long) in this hole. It is recommended to use sterile saline to irrigate while drilling to prevent thermal injury.</li>
	<li>For the reference electrode, drill a 1.0 mm diameter hole 1.5 mm anterior and 3.5 mm lateral to the bregma, in the left hemisphere.</li>
	<li>By hand, insert ~2 mm of reference wire into this hole while wrapping the reference wire around and under the head of the implanted screw.</li>
	<li>Fully implant the screw, pinning down the reference electrode in place.</li>
	<li>In the right hemisphere, drill a 1.5 mm diameter hole 1.2 mm anterior and 1.4 mm lateral to the bregma.</li>
	<li>Gently remove the dura using tweezers.</li>
	<li>For the stimulating electrode, drill a square hole (2 mm anterior-posterior, 5 mm medial-lateral) centered at 5.2 mm posterior and 1.0 mm lateral to the bregma.</li>
	<li>Using the stereotactic arm bars, lower the bipolar stimulating electrode/guide cannula 5 mm below the dura. In case of bleeding during the implantation of the electrode, use sterile cotton swabs and gauze to minimize bleeding. 
	NOTE: The bipolar stimulating electrode used in this method is prefitted with a guide cannula (Table of Materials). The internal cannula used with this item should be flushed with the prongs on the bipolar stimulating electrode when fully inserted into the guide cannula. This will allow the internal cannula to sit directly in between the two prongs of the stimulator, which sit about 1 mm apart. A similar protocol is described elsewhere.</li>
	<li>Using the stereotactic arm bars, lower the carbon fiber microelectrode 4 mm below the dura. This location is at the most dorsal portion of the striatum.</li>
	<li>Connect the reference wire and carbon fiber to a potentiostat.</li>
	<li>Apply a triangular waveform (-0.4&#8722;1.3 V, 400 V/s) for 15 min at 60 Hz, and again for 10 min at 10 Hz. 
	NOTE: Typically, when applying waveforms to carbon fiber microelectrodes in the brain, oxide groups are added to the surface of the carbon fiber. Equilibrium of this reaction must be reached prior to recording; otherwise, significant drift will occur19. Cycling the electrode at higher frequencies (60 Hz) allows the carbon fiber to achieve equilibrium faster.</li>
</ol>
3. Optimizing carbon fiber and stimulating electrode/guide cannula locations
<ol>
	<li>Set the stimulator to produce a bipolar electrical waveform, with a frequency of 60 Hz, 24 pulses, 300 &#181;A current, and a pulse width of 2 ms/phase.</li>
	<li>Gently lower the stimulator in increments of 0.2 mm from 5 mm to 7.8 mm below the dura. At each increment, stimulate the VTA. 
	NOTE: At more dorsal depths (5&#8722;6 mm), stimulation of the brain will typically (~80% of the time) cause the whiskers of the rat to twitch. At further depths, the whiskers will cease twitching, which occurs between 7.5&#8722;8.2 mm below the dura. When the whiskers cease twitching, the stimulating electrode will be near or at the VTA. This will not occur in every rat, and lack of whisker twitching should not be taken as a sign that the bipolar stimulating electrode/infusion cannula is misplaced. Whisker twitching may not occur for all anesthetics (e.g., isoflurane).</li>
	<li>Continue to lower the bipolar stimulating electrode/guide cannula until a stimulation produces phasic DA release at the carbon fiber microelectrode (currently in the dorsal striatum). 
	NOTE: DA release in the dorsal striatum will not always occur if the bipolar electrode is implanted in the VTA, but observation of DA release in the dorsal striatum upon VTA stimulation is usually a good sign that a good signal will be observed in the NAc core.</li>
	<li>Lower the carbon fiber microelectrode until it is at least 6.0 mm below the dura. This is the most dorsal part of the NAc core.</li>
	<li>Stimulate the VTA and record the peak amplitude of the DA peak.</li>
	<li>Lower or raise the carbon fiber microelectrode at the site that produces the greatest DA release.</li>
	<li>Ensure that the DA response peak is a clear oxidation peak at 0.6 V and a reduction peak at -0.2 V. These peaks indicate DA.</li>
</ol>
4. Combination infusion and stimulation FSCV recording
NOTE: Figure 1 shows the timeline for recording before and after VTA microinfusion.
<ol>
	<li>Once the carbon fiber and stimulating electrode/guide cannula location has been optimized, stimulate for ~20&#8722;30 min. 
	NOTE: Under the current stimulation parameters, do not stimulate any more than once every 3 min, to allow for vesicular reloading22.</li>
	<li>After achieving a stable baseline (&#60;20% variation over five stimulations), gently lower the internal cannula by hand into the guide cannula that is prefitted into the bipolar stimulator.</li>
	<li>Take an additional 2&#8722;3 baseline recordings to ensure that the cannula insertion itself did not change the evoked signal. In some cases, the insertion and removal of the internal cannula can damage the VTA. If the signal drastically changes over this baseline period (&#62;20%), then take an additional 3&#8722;4 recordings until the baseline restabilizes.</li>
	<li>Using a syringe pump and micro syringe, infuse 0.5 &#181;L of solution (e.g., 0.9% saline, N-methyl-D-aspartate [NMDA], (2R)-amino-5-phosphonovaleric acid [AP5]) into the VTA over a 2 min period.</li>
	<li>Post infusion, leave the internal cannula for at least 1 min prior to removal. 
	NOTE: Some drugs may require leaving the internal cannula for a longer time based on the drug kinetics, and removal of the internal cannula may cause the drug to travel back up through the internal cannula. If there is concern, one could leave the internal cannula in the guide cannula during the entirety of the recording. Otherwise, recording can begin after this 1 min interval.</li>
	<li>Continue recording every 3 min to measure post-infusion effects. 
	NOTE: If infusing a control solution, and no effect is observed, it is possible to infuse a second time10. If there is altered DA release caused by inserting the internal cannula or saline infusion, the signal typically recovers to baseline within 30 min.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Electrode Filling Solution/Supplies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette</td>
			<td>World Precision Instruments</td>
			<td>MF286-5 (28 gauge)</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Acetate</td>
			<td>Sigma</td>
			<td>236497-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Sigma</td>
			<td>P3911-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrode Supplies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Carbon fiber</td>
			<td>Thornel</td>
			<td>T650</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrode puller</td>
			<td>Narishige International</td>
			<td>PE-22</td>
			<td>Note: horizontal pullers can be used as well</td>
		</tr>
		<tr>
			<td>Glass capillary</td>
			<td>A-M systems</td>
			<td>626000</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulated wires for electrodes</td>
			<td>Weico Wire and Cable Incorporated</td>
			<td>UL 1423</td>
			<td>Length; 10 cm; diameter,0.4mm; must get custom made; insulated material should cover 5 cm of the wire</td>
		</tr>
		<tr>
			<td>Light Microscope (for viewing and cutting electrode)</td>
			<td>Fischer Scientific</td>
			<td>M3700</td>
			<td></td>
		</tr>
		<tr>
			<td>Pin</td>
			<td>Phoenix Enterprises</td>
			<td>HWS1646</td>
			<td>To be soldered onto the insuled electrode wire and reference electrode; connects to headstage</td>
		</tr>
		<tr>
			<td>Putty</td>
			<td>Alcolin</td>
			<td>23922-1003</td>
			<td>Used to place electrode on while cutting the carbon fiber</td>
		</tr>
		<tr>
			<td>Scalpal Blade</td>
			<td>World Precision Instruments</td>
			<td>500239</td>
			<td>For cutting carbon fiber to the apprpriate length</td>
		</tr>
		<tr>
			<td>Silver Wire</td>
			<td>Sigma</td>
			<td>327026-4G</td>
			<td></td>
		</tr>
		<tr>
			<td>FSCV Hardware/Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Faraday Cage</td>
			<td>U-Line</td>
			<td>H-3618 (36&#34; x 24&#34; x 42&#34;)</td>
			<td></td>
		</tr>
		<tr>
			<td>Potentiostat</td>
			<td>Univ. of N. Carolina, Electronics Facility</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stimulating electrode</td>
			<td>PlasticsOne</td>
			<td>MS303/2-A/SPC</td>
			<td>when ordering, request a 22 mm cut below pedestal</td>
		</tr>
		<tr>
			<td>TarHeel HDCV Software</td>
			<td>University of North Carolina-Chapel Hill</td>
			<td>-</td>
			<td>https://chem.unc.edu/critcl-main/criticl-electronics/criticl-electronics-hardware/ for ordering information</td>
		</tr>
		<tr>
			<td>UEI breakout box</td>
			<td>Univ. of N. Carolina, Electronics Facility</td>
			<td></td>
			<td>https://chem.unc.edu/critcl-main/criticl-electronics/criticl-electronics-hardware/ for ordering information</td>
		</tr>
		<tr>
			<td>UEI power supply</td>
			<td>Univ. of N. Carolina, Electronics Facility</td>
			<td></td>
			<td>https://chem.unc.edu/critcl-main/criticl-electronics/criticl-electronics-hardware/ for ordering information</td>
		</tr>
		<tr>
			<td>Stimulator Hardware</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Neurolog stimulus isolator</td>
			<td>Digitimer Ltd.</td>
			<td>DS4</td>
			<td>Neurolog 800A</td>
		</tr>
		<tr>
			<td>Infusion/Stimulation Supplies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Infusion Pump</td>
			<td>New Era Syringe Pump</td>
			<td>NE-300</td>
			<td></td>
		</tr>
		<tr>
			<td>Internal Cannula</td>
			<td>PlasticsOne</td>
			<td>C315I/SPC INTERNAL 33GA</td>
			<td></td>
		</tr>
		<tr>
			<td>Microliter Syringe</td>
			<td>Hamilton</td>
			<td>80308</td>
			<td></td>
		</tr>
		<tr>
			<td>Tubing</td>
			<td>PlasticsOne</td>
			<td>C313CT/ PKG TUBING 023 X 050 PE50</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Supplies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cannula Holder</td>
			<td>Kopf Instruments</td>
			<td>1776 P-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton Tip Applicators</td>
			<td>Vitality Medical</td>
			<td>806</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrode Holder</td>
			<td>Kopf Instruments</td>
			<td>1770</td>
			<td></td>
		</tr>
		<tr>
			<td>Heating Pad</td>
			<td>Kent Scientific</td>
			<td>RT-0501</td>
			<td></td>
		</tr>
		<tr>
			<td>Povidone Iodine</td>
			<td>Vitality Medical</td>
			<td>29906-004</td>
			<td></td>
		</tr>
		<tr>
			<td>Screws</td>
			<td>Stoelting</td>
			<td>Bone Anchor Screws/Pkg.of 100</td>
			<td>1.59 mm O.D., 3.2 mm long</td>
		</tr>
		<tr>
			<td>Silver wire reference with AgCl</td>
			<td>InVivo Metric</td>
			<td>E255A</td>
			<td></td>
		</tr>
		<tr>
			<td>Square Gauze</td>
			<td>Vitality Medical</td>
			<td>441408</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereotax</td>
			<td>Kopf Instruments</td>
			<td>Model 902 (Dual Arm Bar)</td>
			<td></td>
		</tr>
		<tr>
			<td>Drugs for infusions</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>((2R)-amino-5-phosphonovaleric acid</td>
			<td>Sigma Aldrich</td>
			<td>A5282</td>
			<td></td>
		</tr>
		<tr>
			<td>N-methyl-D-aspartate</td>
			<td>Sigma Aldrich</td>
			<td>M3262</td>
			<td></td>
		</tr>
		<tr>
			<td>Mecamylamine hydrochloride (M9020-5mg)</td>
			<td>Sigma Aldrich</td>
			<td>M9020</td>
			<td></td>
		</tr>
		<tr>
			<td>Scopolamine hydrobromide (S0929-1g)</td>
			<td>Sigma Aldrich</td>
			<td>S0929</td>
			<td></td>
		</tr>
	</tbody>
</table>

monitoring dopamine release in a rat model with vta dopaminergic neuron receptor manipulation

Source: Wickham, R. J., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/60886">Combined Infusion and Stimulation with Fast-Scan Cyclic Voltammetry (CIS-FSCV) to Assess Ventral Tegmental Area Receptor Regulation of Phasic Dopamine.</a> J. Vis. Exp. (2020).
This video demonstrates the implantation of electrodes and cannulas in an anesthetized rat to measure dopamine release in the nucleus accumbens using a potentiostat and the infusion of drugs to modulate dopaminergic activity in the ventral tegmental area.

<img alt="Figure 1" src="/files/ftp_upload/22744/22744_Figure1.jpg" /> 
Figure 1: Timeline for recording before and after VTA microinfusion.

Monitoring Dopamine Release in a Rat Model with VTA Dopaminergic Neuron Receptor Manipulation

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

Begin with an immobilized, anesthetized rat with an activated carbon microelectrode implanted in the nucleus accumbens or NAc region and a reference wire in the left hemisphere.
These electrodes are connected to a potentiostat, which measures dopamine-generated electrical signals.
A stimulating electrode with a guide cannula is implanted into the ventral tegmental area or VTA, targeting dopaminergic neurons that project into the NAc region.
Apply electrical pulses via the stimulating electrode that activate the VTA neurons, releasing dopamine, a chemical messenger, into the NAc region.
In the NAc region, dopamine interacts with the carbon electrode and undergoes oxidation and reduction, generating baseline electrical signals.
Insert an internal cannula into the guide cannula to infuse either an activator or an inhibitor drug.
The activator drug stimulates the VTA neurons, increasing dopamine release and elevating electrical signals.
In contrast, the inhibitory drug decreases the electrical signal by preventing dopamine release.

monitoring-dopamine-release-rat-model-with-vta-dopaminergic-neuron

Universidad de Cartagena UDEC

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neurophysiology

Stimulation and Modulation Techniques

58 ビュー. 出典:Wickham, R. J., et al. ファシックドーパミンの腹側被蓋領域受容体調節を評価するための高速スキャンサイクリックボルタンメトリー(CIS-FSCV)による注入と刺激の組み合わせ。J. Vis. Exp.(2020年)。 このビデオは、麻酔をかけたラットに電極とカニューレを埋め込んで、ポテンショスタットを使用して側坐核のドーパミン放出を測定し、腹側被蓋領域のドーパミン作動性活性?...

ビデオ: VTAドーパミン作動性ニューロン受容体操作によるラットモデルにおけるドーパミン放出のモニタリング - 概念

Examination of Rapid Dopamine Dynamics with Fast Scan Cyclic Voltammetry During Intra-oral Tastant Administration in Awake Rats

Rapid, phasic dopamine (DA) release in the mammalian brain plays a critical role in reward processing, reinforcement learning, and motivational control. Fast scan cyclic voltammetry (FSCV) is an electrochemical technique with high spatial and temporal (sub-second) resolution that has been utilized to examine phasic DA release in several types of preparations. In vitro experiments in single-cells and brain slices and in vivo experiments in anesthetized rodents have been used to identify mechanisms that mediate dopamine release and uptake under normal conditions and in disease models. Over the last 20 years, in vivo FSCV experiments in awake, freely moving rodents have also provided insight of dopaminergic mechanisms in reward processing and reward learning. One major advantage of the awake, freely moving preparation is the ability to examine rapid DA fluctuations that are time-locked to specific behavioral events or to reward or cue presentation. However, one limitation of combined behavior and voltammetry experiments is the difficulty of dissociating DA effects that are specific to primary rewarding or aversive stimuli from co-occurring DA fluctuations that mediate reward-directed or other motor behaviors. Here, we describe a combined method using in vivo FSCV and intra-oral infusion in an awake rat to directly investigate DA responses to oral tastants. In these experiments, oral tastants are infused directly to the palate of the rat &#8211; bypassing reward-directed behavior and voluntary drinking behavior &#8211; allowing for direct examination of DA responses to tastant stimuli.

Rapid fluctuations in extracellular dopamine (DA) mediate both reward processing and motivated behavior in mammals. This manuscript describes the combined use of fast scan cyclic voltammetry (FSCV) and intra-oral tastant administration to determine how tastants alter rapid dopamine release in awake, freely moving rats.

覚醒ラットにおける口腔内味物質投与の間に高速スキャンサイクリックボルタンメトリーと迅速ドーパミンダイナミクスの検討

Rapid, phasic dopamine (DA) release in the mammalian brain plays a critical role in reward processing, reinforcement learning, and motivational control. ...

JoVE Journal

行動学

Laser Capture Microdissection - A Demonstration of the Isolation of Individual Dopamine Neurons and the Entire Ventral Tegmental Area

Laser capture microdissection (LCM) is used to isolate a concentrated population of individual cells or precise anatomical regions of tissue from tissue sections on a microscope slide. When combined with immunohistochemistry, LCM can be used to isolate individual cells types based on a specific protein marker. Here, the LCM technique is described for collecting a specific population of dopamine neurons directly labeled with&#160;tyrosine hydroxylase immunohistochemistry and for isolation of the dopamine neuron containing region of the ventral tegmental area using indirect tyrosine hydroxylase immunohistochemistry on a section adjacent to those used for LCM. An infrared (IR) capture laser is used to both dissect individual neurons as well as the ventral tegmental area off glass slides and onto an LCM cap for analysis. Complete dehydration of the tissue with 100% ethanol and xylene is critical. The combination of the IR capture laser and the ultraviolet (UV) cutting laser is used to isolate individual dopamine neurons or the ventral tegmental area when using PEN membrane slides. A PEN membrane slide has significant advantages over a glass slide as it offers better consistency in capturing and collecting cells, is faster collecting large pieces of tissue, is less reliant on dehydration and results in complete removal of the tissue from the slide. Although removal of large areas of tissue from a glass slide is feasible, it is considerably more time consuming and frequently leaves some residual tissue behind. Data shown here demonstrate that RNA of sufficient quantity and quality can be obtained using these procedures for quantitative PCR measurements. Although RNA and DNA are the most commonly isolated molecules from tissue and cells collected with LCM, isolation and measurement of microRNA, protein and epigenetic changes in DNA can also benefit from the enhanced anatomical and cellular resolution obtained using LCM.

The isolation of individual dopamine neurons or the ventral tegmental area with direct or indirect immunohistochemistry is demonstrated using laser capture microdissection. Parameters for isolation of tissue from a glass slide using an infrared laser and from membrane slides using the combination of an infrared and ultraviolet laser are discussed.

レーザーキャプチャーマイクロダイセクション - 個々のドーパミンニューロンの単離と全体の腹側被蓋領域の実証

Laser capture microdissection (LCM) is used to isolate a concentrated population of individual cells or precise anatomical regions of tissue from tissue ...

神経科学

Combined In Vivo Anatomical and Functional Tracing of Ventral Tegmental Area Glutamate Terminals in the Hippocampus

Optogenetic modulation of neuron sub-populations in the brain has allowed researchers to dissect neural circuits in vivo and ex vivo. This provides a premise for determining the role of neuron types within a neural circuit, and their significance in information encoding relative to learning. Likewise, the method can be used to test the physiological significance of two or more connected brain regions in awake and anesthetized animals. The current study demonstrates how VTA glutamate neurons modulate the firing rate of putative pyramidal neurons in the CA1 (hippocampus) of anesthetized mice. This protocol employs adeno-associated virus (AAV)-dependent labeling of VTA glutamate neurons for the tracing of VTA presynaptic glutamate terminals in the layers of the hippocampus. Expression of light-controlled opsin (channelrhodopsin; hChR2) and fluorescence protein (eYFP) harbored by the AAV vector permitted anterograde tracing of VTA glutamate terminals, and photostimulation of VTA glutamate neuron cell bodies (in the VTA). High-impedance acute silicon electrodes were positioned in the CA1 to detect multi-unit and single-unit responses to VTA photostimulation in vivo. The results of this study demonstrate the&#160;layer-dependent distribution of presynaptic VTA glutamate terminals in the hippocampus (CA1, CA3, and DG). Also, the photostimulation of VTA glutamate neurons increased the firing and burst rate of putative CA1 pyramidal units in vivo.

The current protocol demonstrates a simple method for tracing of ventral tegmental area (VTA) glutamate projections to the hippocampus. Photostimulation of VTA glutamate neurons was combined with CA1 recording to demonstrate how VTA glutamate terminals modulate CA1 putative pyramidal firing rate in vivo.

海馬の腹側テグメンタル領域グルタミン酸末端のインビボ解剖学的および機能的なトレース

Optogenetic modulation of neuron sub-populations in the brain has allowed researchers to dissect neural circuits in vivo and ex vivo. This provides a ...

Monitoring Dopamine Release in a Rat Model with VTA Dopaminergic Neuron Receptor Manipulation

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Monitoring Dopamine Release in a Rat Model with VTA Dopaminergic Neuron Receptor Manipulation