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

Summary

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.

Abstract

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 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.

Introduction

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 regions^2,3. The choice of a promoter in the AAV construct directs the expression of the vector ....

Protocol

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

1. Use 5–6 weeks old mice.
2. House 3–5 animals per cage under standard conditions of 12 h alternating light and dark cycle. Food and water should be provided ad libitum.

2. Craniotomy and animal preparation

NOTE: This section describes pre- and peri-operative procedures for mouse craniotomy. Use standard stereotactic apparatus and appropriate coor....

Results

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–d; Movie 2 and 3).

Discussion

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 .......

Materials

Name	Company	Catalog Number	Comments
3% Hydrogen peroxide	Fisher chemical	H312
AAV-CaMKIIα-ChR2-eGYP	Addgene	Plasmid #26969
BNC cable	Amazon
BNC Splitter	Amazon
Ceramic Split Mating Sleeve for Ø1.25mm Ferrules.	Thorlabs	ADAL1-5
Drill	Dremel	LR 39098
Gelatin coated slides	Fisher scientific	OBSLD01CS
Hamilton's syringe (Neuros)	WPI Inc.	06H
Head stage adapter	Neuronexus	Adpt-Q4-OM32
High impedance silicon probe	Neuronexus	Q1x1-tet-5mm-121-CQ4
INTAN 512ch Recording Controller	INTAN	RHD2000
Iodine solution	Dynarex	1425
Isoflurane	Piramal	NDC 66794-017-25
Ketamine	Spectrum	K1068
LED Driver	Thorlabs	LEDD1B
LED light source (470 nm)-blue light	Thorlabs	M470F3
Micromanipulator	Narishige	M0-203
Optic fiber	Thorlabs	CFMLC14L05
Pan head philips screw (M0.6 X 2mm)	Amazon	M0.6 X 2mm
Pre-amplifier headstage (32 Channel)	INTAN	C3314
Stereotaxic frame	Kopf	1530
TTL pulser	Prizmatix	4031
Urethane	Sigma	U2500
Xylazine	Alfa Aesar	J61430
Software	Company	Version
Graphpad Prism
Intan Recording Controller
Neuroexplorer
Plexon Offline Spike Sorter
ACSF Composition:
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).