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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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Cre-dependent,+anterograde+transsynaptic+viral+tracer+for+mapping+output+pathways+of+genetically+marked+neurons.">A Cre-dependent, anterograde transsynaptic viral tracer for mapping output pathways of genetically marked neurons.</a> Neuron. 72 (6), 938-950 (2011).</li><li>Li, J., Liu, T., Dong, Y., Kondoh, K., Lu, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trans-synaptic+Neural+Circuit-Tracing+with+Neurotropic+Viruses.">Trans-synaptic Neural Circuit-Tracing with Neurotropic Viruses.</a> Neuroscience bulletin. , 1-12 (2019).</li><li>Kuypers, H., Ugolini, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viruses+as+transneuronal+tracers.">Viruses as transneuronal tracers.</a> Trends in neurosciences. 13 (2), 71-75 (1990).</li><li>Atasoy, D., Aponte, Y., Su, H. 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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recombinant+adeno-associated+virus+vector:+use+for+transgene+expression+and+anterograde+tract+tracing+in+the+CNS.">Recombinant adeno-associated virus vector: use for transgene expression and anterograde tract tracing in the CNS.</a> Brain Research. 793 (1-2), 169-175 (1998).</li><li>Zingg, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AAV-mediated+anterograde+transsynaptic+tagging:+mapping+corticocollicular+input-defined+neural+pathways+for+defense+behaviors.">AAV-mediated anterograde transsynaptic tagging: mapping corticocollicular input-defined neural pathways for defense behaviors.</a> Neuron. 93 (1), 33-47 (2017).</li><li>Wang, C., Wang, C., Clark, K., Sferra, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recombinant+AAV+serotype+1+transduction+efficiency+and+tropism+in+the+murine+brain.">Recombinant AAV serotype 1 transduction efficiency and tropism in the murine brain.</a> Gene therapy. 10 (17), 1528 (2003).</li><li>Ahlgrim, N. 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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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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 ...

The current protocol analyzes an affordable and straightforward method for combined neuroanatomical and electrophysiological tracing of neurocircuits in vivo. The technique permits mapping of adjunct terminals from specific neuron population to a target site. Likewise, the anatomical map for these terminals can be verified by optogenetic stimulation during extracellular recording at the target site.This protocol can be applied for studying multiple neural circuits associated with sensory, motor, and cognitive function. Performing this procedure requires some knowledge of rodent stereotaxic surgery and handling of delicate neural electrodes. The recording system connection to prevent noise in recordings.Visual demonstration highlights basic steps in setting up an affordable LED system for in vivo optogenetics. Also, it shows how to connect the amplifier with the LED driver for synchronized recording and stimulation in an anesthetized mouse. Place a heating pad on the stereotaxic frame so that the body of the mouse is lying on it which will help maintain its body temperature throughout the procedure, then gently fix the head on the stereotaxic apparatus.Prepare the surgical area by cleaning it first with iodine solution and then with alcohol to remove the iodine. Next, apply topical lidocaine to block sensation on the scalp. Then using a scalpel, make a midline incision extending from the frontal to the occipital region.For ventral tegmental area or VTA injection, position an ultra-fine blunt point needle syringe at negative 3.08 millimeter anterior posterior and 0.5 millimeter medial lateral coordinates relative to the bregma. Use a drilling tool to bore a one millimeter hole in the skull at the marked coordinate. Follow the manufacturer's instructions to fix the syringe holder on a micromanipulator and fill the syringe with double distilled water to clean and test the flow of fluid.Then dispense the water to test the flow of the syringe. Thaw aliquots of adeno-associated virus or AAV cocktail on ice. Fill the mounted syringe with 1, 000 nanoliters of AAV solution and dispense 10 nanoliters of the solution to confirm the flow of the liquid.Use the micromanipulator to lower the needle to the injection site and inject 600 to 800 nanoliters of AAV into the VTA, delivering the solution at 60 nanoliters per minute. Three weeks after the AAV injection, affix the head of the animal on a stereotactic frame as previously described and perform a craniotomy to expose the dura. Use a drilling tool to remove part of the parietal bone.Under a dissection microscope, use a bent 27 gauge needle tip to excise the exposed dura, taking care not to pull apart the delicate peel covering and cortical tissues in this area. Apply drops of artificial cerebrospinal fluid over the craniotomy area to prevent dryness. Bore a hole in the occipital bone to hold the ground screw and connect a stainless steel ground wire.Before lowering the cannula, connect the fiber optic cable to a fiber coupled LED source. Lower the 400 micrometer diameter optic fiber into the VTA at coordinates of AP negative 3.08 millimeters and ML 0.5 millimeters. Using a micromanipulator, position the electrode contact sites in the pyramidal cell layer of the CA1.To synchronize the light pulse with the neural recording, connect the LED driver and recording controller digital import to a transistor-transistor logic pulser with a BNC splitter. Adjust the knob to determine the effective intensity that can generate a response without producing photoelectric artifacts and connect the ground on the skull to the ground on the adapter. Connect the preamplifier head stage to the recording controller via a serial peripheral interface cable and check the LED color lights on the recording controller ports.Green and yellow LEDs indicate proper voltage on the connected amplifier board and the red led indicates a working software head stage control. After launching the software, select the data file format and change the filename. Switch to the port for the connected head stage and click on disable all on port if the electrode has fewer channels than the head stage.Select the appropriate number of channels to be displayed on the screen. To capture the TTL timestamp for synchronized neural recording and light pulse triggered timestamp, click on the digital imports and enable digital in zero one. This must correspond with the BNC connection on the amplifier digital imports.Adjust the timescale and voltage scale for appropriate waveform display and select the sampling rate, keeping in mind that a higher sampling rate and number of channels will increase the file size. Next, set the amplifier bandwidth for single unit recording. A 300 to 5, 000 Hertz cutoff frequency was used here.For spike sound, click on analog out audio and enable the analog port. Then adjust the gain and silencer for optimum sound. To synchronize the amplifier recording and light pulse train, click on the display tab and enable show marker.Switch to the appropriate channel that corresponds with the BNC connection and reinspect the ports to make sure that the recording channels and digital in channel are enabled to capture neural spikes and light pulse timestamps. Click on spike scope to view waveforms that constitute the continuously recorded spike train. Use the mouse to set the threshold for the waveforms to be displayed.Then inspect the RMS to determine the noise level in your recording. Adeno-associated virus expression was verified by immunofluorescence imaging of EYFP in the ventral tegmental area of mice 21 days post-injection. Fluorescence imaging was also used to detect pre-synaptic VTA glutamate projections in the hippocampus layers DG, CA3, and CA1.Once extracellular voltage activity was detected, the baseline activity was recorded for approximately 10 minutes before triggering the light pulse at the desired frequency. This made it possible to compare the firing or burst rates of CA1 putative neurons before and after VTA glutamate photostimulation. To support this outcome, statistical comparison of the CA1 network firing rates before and after photostimulation revealed a significant increase for the post-stimulation period.Subsequent analysis of the raster train to detect bursts also showed an increased burst rate for the CA1 putative pyramidal neurons after photostimulation. Response to photosimulation was identified by fluorescence of the AAV reporter expressions at the stimulus sites. Also, the description of should be correlated with the site of recording for responsive putative units.This method can also be performed in awake mice undertaking behavioral tasks. In this case, chronic implantable probes and fiber optic cannula should be used. This technique will allow for robust tracing of neurocircuits through a combination of presynaptic terminal distribution in the target site and modulation of presynaptic terminals to detect target site response in vivo.

combined-vivo-anatomical-functional-tracing-ventral-tegmental-area

Research

JoVE Journal

Neuroscience

3.7K ビュー.  Louisiana State University School of Veterinary Medicine. The current protocol analyzes an affordable and straightforward method for combined neuroanatomical and electrophysiological tracing of neurocircuits in vivo. The technique permits mapping of adjunct terminals from specific neuron population to a target site. Likewise, the anatomical map for these terminals can be verified by optogenetic stimulation during extracellular recording at the target site.This protocol can be applied for studying multiple neural circuits associated with senso...