Name: combined in vivo anatomical and functional tracing of ventral tegmental area glutamate terminals in the hippocampus
Uploaded: 2020-09-09T12:30:00
Description: Watch this Scientific Journal Video about Combined In Vivo Anatomical and Functional Tracing of Ventral Tegmental Area Glutamate Terminals in the Hippocampus at JoVE.com

This work is funded by CBS Bridging Grant awarded to OOM. OOM, PAA, and AS designed the study and performed the experiments. AS and PAA analyzed the results. OOM and PAA prepared the manuscript.&#160;We thank Dr. Karl Disseroth (Stanford University) for making the AAV available for our use.

All experimental and animal handling procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of the Louisiana State University School of Veterinary Medicine.
1. Experimental animal
<ol>
	<li>Use 5&#8211;6 weeks old mice.</li>
	<li>House 3&#8211;5 animals per cage under standard conditions of 12 h alternating light and dark cycle. Food and water should be provided ad libitum.</li>
</ol>
2. Craniotomy and animal preparation
<p class="jove_c...

<ol>
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In the past decade, the design of AAV constructs has advanced significantly. As such, more neuron-specific promoters have been incorporated into an array of AAV serotypes for improved transfection specificity14. By combining genes for fluorescence proteins, transporters, receptors, and ion channels, libraries of AAV now exists for imaging, neuromodulation, and synaptic activity detection. In commercially available AAV-constructs, a combination of a genetically encoded fluorophore and ion channels ...

In the past decade, an array of genetic tools was developed to increase the specificity of neuron-type modulation, and the mapping of complex neural networks1. Notably, neurotropic viruses with an inherent ability to infect and replicate in neuronal cells have been deployed to express or ablate specific proteins in neuron sub-types. When harboring fluorescence proteins or genetically encoded synaptic activity indicators, transfected AAV vectors label and delineate neural networks across brain regions2,3. The choice of a promoter in the AAV construct directs the expression of the vector in neuron types with some level of specificity (promoter-dependent expression). However, through Cre-lox recombination, AAV constructs are deployed with greater specificity for&#160;neuron labeling4,5,6,7. Of note, photoactivated microbial opsins and fluorescence proteins packaged in AAV vectors can be expressed in various neuron subtypes8, and are ideal for imaging, neuron-type circuit tracing, and photomodulation9,10.
AAVs constructs stereotactically injected into a brain region (or nucleus) drives the expression of the reporter protein in the soma, dendrite, and axons terminals. Neural expression of AAV harboring a reporter gene (eYFP) facilitates the labeling of neuron cell bodies and anatomical tracing of projections to and from other brain regions11,12,13,14. AAV-eYFP constructs carrying light-controlled opsin (e.g., hChR2), can be deployed as a tool for imaging6,15 and stimulation-based physiological tracing of neural projections to target brain areas in vivo16. Depending on the AAV serotype, the direction of neuron labeling may be anterograde or retrograde11,12. Previous studies have established that AAV5 travels anterogradely in neurons12. Thus, photostimulation&#160;of cell bodies expressing hChR2 produces presynaptic effects elsewhere in the brain (target)17.
Here, AAV (serotype 5) with a CaMKII&#945; promoter was used to express eYFP (reporter) and hChR2 (opsin) in VTA glutamate neurons and axonal projections. Results from this study demonstrate the layer-dependent distribution of VTA-glutamate presynaptic terminals in the CA1, CA3, and DG hippocampal regions. Also, photostimulation of VTA glutamate neurons increased CA1 multi-unit and single-unit firing rates in vivo when compared with baseline values. This protocol utilizes affordable tools and commercially available software that can increase the quality of data obtained from neural circuit tracing experiments.

<table>
	<tbody>
		<tr>
			<td>3% Hydrogen peroxide</td>
			<td>Fisher chemical</td>
			<td>H312</td>
			<td></td>
		</tr>
		<tr>
			<td>AAV-CaMKII&#945;-ChR2-eGYP</td>
			<td>Addgene</td>
			<td>Plasmid #26969</td>
			<td></td>
		</tr>
		<tr>
			<td>BNC cable</td>
			<td>Amazon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BNC Splitter</td>
			<td>Amazon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ceramic Split Mating Sleeve for &#216;1.25mm Ferrules.</td>
			<td>Thorlabs</td>
			<td>ADAL1-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Drill</td>
			<td>Dremel</td>
			<td>LR 39098</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin coated slides</td>
			<td>Fisher scientific</td>
			<td>OBSLD01CS</td>
			<td></td>
		</tr>
		<tr>
			<td>Hamilton&#39;s syringe (Neuros)</td>
			<td>WPI Inc.</td>
			<td>06H</td>
			<td></td>
		</tr>
		<tr>
			<td>Head stage adapter</td>
			<td>Neuronexus</td>
			<td>Adpt-Q4-OM32</td>
			<td></td>
		</tr>
		<tr>
			<td>High impedance silicon probe</td>
			<td>Neuronexus</td>
			<td>Q1x1-tet-5mm-121-CQ4</td>
			<td></td>
		</tr>
		<tr>
			<td>INTAN 512ch Recording Controller</td>
			<td>INTAN</td>
			<td>RHD2000</td>
			<td></td>
		</tr>
		<tr>
			<td>Iodine solution</td>
			<td>Dynarex</td>
			<td>1425</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Piramal</td>
			<td>NDC 66794-017-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>Spectrum</td>
			<td>K1068</td>
			<td></td>
		</tr>
		<tr>
			<td>LED Driver</td>
			<td>Thorlabs</td>
			<td>LEDD1B</td>
			<td></td>
		</tr>
		<tr>
			<td>LED light source (470 nm)-blue light</td>
			<td>Thorlabs</td>
			<td>M470F3</td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>Narishige</td>
			<td>M0-203</td>
			<td></td>
		</tr>
		<tr>
			<td>Optic fiber</td>
			<td>Thorlabs</td>
			<td>CFMLC14L05</td>
			<td></td>
		</tr>
		<tr>
			<td>Pan head philips screw (M0.6 X 2mm)</td>
			<td>Amazon</td>
			<td>M0.6 X 2mm</td>
			<td></td>
		</tr>
		<tr>
			<td>Pre-amplifier headstage (32 Channel)</td>
			<td>INTAN</td>
			<td>C3314</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereotaxic frame</td>
			<td>Kopf</td>
			<td>1530</td>
			<td></td>
		</tr>
		<tr>
			<td>TTL pulser</td>
			<td>Prizmatix</td>
			<td>4031</td>
			<td></td>
		</tr>
		<tr>
			<td>Urethane</td>
			<td>Sigma</td>
			<td>U2500</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Alfa Aesar</td>
			<td>J61430</td>
			<td></td>
		</tr>
		<tr>
			<td>Software</td>
			<td>Company</td>
			<td>Version</td>
			<td></td>
		</tr>
		<tr>
			<td>Graphpad Prism</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Intan Recording Controller</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Neuroexplorer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plexon Offline Spike Sorter</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ACSF Composition:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>oxygenated ACSF with 95% Oxygen/5%CO2 constantly being bubbled through the ACSF (ACSF; in mM 125 NaCl, 25 NaHCO3, 3 KCl, 1.25 NaH2PO4, 1 MgCl2, 2 CaCl2 and 25 Glucose).</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Anterograde tracing
AAV expression was verified by immunofluorescence imaging of reporter protein (eYFP) in the VTA of C57BL/6 mice 21 days post-injection (Figure 2). Successful anterograde labeling of presynaptic VTA glutamate projections in the hippocampus was also verified by eYFP detection in the layers of the DG, CA3, and CA1 (Figure 6a&#8211;d; Movie 2 and 3).
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Watch this Scientific Journal Video about Combined In Vivo Anatomical and Functional Tracing of Ventral Tegmental Area Glutamate Terminals in the Hippocampus at JoVE.com

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

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.

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

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