A Method for Remotely Silencing Neural Activity in Rodents During Discrete Phases of Learning

Summary

This protocol describes how to temporarily and remotely silence neuronal activity in discrete brain regions while rats are engaged in learning and memory tasks. The approach combines pharmacogenetics (Designer-Receptors-Exclusively-Activated-by-Designer-Drugs) with a behavioral paradigm (sensory preconditioning) that is designed to distinguish between different components of learning.

Abstract

This protocol describes how to temporarily and remotely silence neuronal activity in discrete brain regions while animals are engaged in learning and memory tasks. The approach combines pharmacogenetics (Designer-Receptors-Exclusively-Activated-by-Designer-Drugs) with a behavioral paradigm (sensory preconditioning) that is designed to distinguish between different forms of learning. Specifically, viral-mediated delivery is used to express a genetically modified inhibitory G-protein coupled receptor (the Designer Receptor) into a discrete brain region in the rodent. Three weeks later, when designer receptor expression levels are high, a pharmacological agent (the Designer Drug) is administered systemically 30 min prior to a specific behavioral session. The drug has affinity for the designer receptor and thus results in inhibition of neurons that express the designer receptor, but is otherwise biologically inert. The brain region remains silenced for 2-5 hr (depending on the dose and route of administration). Upon completion of the behavioral paradigm, brain tissue is assessed for correct placement and receptor expression. This approach is particularly useful for determining the contribution of individual brain regions to specific components of behavior and can be used across any number of behavioral paradigms.

Introduction

An exciting challenge within the field of behavioral neuroscience is to determine the neural substrates of complex behaviors. A number of techniques such as permanent lesions, temporary brain inactivation via cannulae implants and optogenetics have been employed to identify the contributions of discrete brain regions to subcomponents of complex behaviors. While these approaches inform our understanding of regional specificity during learning, each technique is not without limitations. Specifically, permanent lesions are typically conducted prior to behavioral testing, thus their effects are present throughout the duration of the paradigm. Cannulation studies that invo....

Protocol

The use of animals are approved by the Oberlin College Institutional Animal Care and Use committee and are in accordance with the Guide for the Care and Use of Laboratory Animals²¹.

1. Preparation for Viral Infusion

Note: This protocol uses BSL-1 precautions. When employing BSL-2 precautions, a disposable lab coat, gloves, shoe covers, eye protections and a particulate respirator (type N95) are required. All individuals handling BSL-2 compounds must be fit tested for a particulate respirator by a local public health agency. Refer to Lowery & Majewska (2010)²² for additional details on hand....

Results

Behavioral Results

Upon completion of the experiment, the effectiveness of the region-specific temporary inactivation should be quantitatively and qualitatively assessed. The present example involves a 3-phase behavioral paradigm (sensory preconditioning), in which CNO was administered to attenuate neural activity in the RSC during the Preconditioning sessions to test the hypothesis that the RSC is necessary for the formation of associations among neutral stimuli¹². Importantly, experi.......

Discussion

This protocol describes how to apply a pharmacogenetic approach (DREADD) to investigate how a specific brain region contributes to a multi-phase complex learning task. With the ability to temporarily and remotely silence neural activity in discrete brain regions across phases of learning, this combination of approaches provides a platform to investigate a wide range of behaviors, including more nuanced or masked forms of learning. In the example described in this protocol, control rats and rats that express the designer .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Male, Long Evans Rats, 55-60 days old	Hilltop Lab Animals Inc
rAAV8/hSyn-HA-hM4D(Gi)-IRES-mCitrine	Virus Vector Core		Caution: This is a BSL-1 compound
rAAV8/hSyn-GFP	Virus Vector Core		Caution: This is a BSL-1 compound
Clozapine-N-oxide	R&D Systems	4936-10	Designer drug
Rat Cage lid (polycarbonate)	Alternative Design	FT 8XL-PC	Used to cover animal cages 48-72 hr post infusion
Filter paper (replacement)	Alternative Design	FP-R-1018XAD	Filter paper that goes with cage lids
Table top vise	JETS	2201-265	For holding microscentrifuge tubes containing AAV in the hood
Biohazard trash bags	Staples	113444	Medline biohazard liners
Biohazard trash can	Amazon.com		United Solutions 34 gallon rectangular wheeled trashcan with hook and lock handle
Isoflurane, 100 ml	Patterson Veterinary Supply Inc.	07-890-8540	Anesthetic
Dual small animal stereotaxic with digital display readout console	David Kopf	Model 942	Surgical equipment
Non-rupture ear bars, set of 2 (rat)	David Kopf	Model 955	Surgical equipment
Anesthesia mask (rat)	David Kopf	Model 906	Surgical equipment
High speed stereotaxic drill includes table top motor controller, foot pedal, handpiece, stereotaxic handpiece holder	David Kopf	Model 1474	Surgical equipment
Microdrill burrs, 0.9 mm	Fine Science Tools Inc	19007-09	Surgical supply
Automated syringe pump with micro4 controller	David Kopf	Model UMP3-1	Surgical equipment
Pro-animal detachable ceramic blade clipper kit	Ahdis	21420	Surgical supply
Betadine skin cleanser	Perdue Products L.P	67618-149-04	Surgical supply
Triple antiobiotic ointment	Medline Supply	53329-087-01	Surgical supply
Puralube vet ointment	Only Veterinary Supply	17033-211-38	Surgical supply
Dino-lite	Microscope	AD7013MTL	An alternative to the traditional disection scope
Dino-lite rigid table-top boom stand	Microscope	MS36B	Surgical equipment
28 G 10 μl syringe	Hamilton	80308-701SN	Surgical equipment
Extra tall MDF sound attenuating cubicle	Med Associates, Inc	ENV-018MD	22' W x 22" H x 16" D
Extra tall modular test chamber	Med Associates, Inc	ENV-007	Behavioral equipment
Stainless steel grid floor	Med Associates, Inc	ENV-005	Behavioral equipment
House light	Med Associates, Inc	ENV-215M	Used as the house light and stimulus light
Modular pellet dispenser	Med Associates, Inc	ENV-203M-45	Behavioral equipment
Pellet receptacle, cup type	Med Associates, Inc	ENV-200R1M	Behavioral equipment
Head entry detector for rat	Med Associates, Inc	ENV-254-CB	Behavioral equipment
Dustless precision food pellets, 45 mg	Bio-Serv	F0165	Behavioral supply
Cage speaker for rat chamber	Med Associates, Inc	ENV-224AM	Behavioral equipment
Programmable audio generator	Med Associates, Inc	ANL-926	Behavioral equipment
Smart ctrl 8 input/16 output package	Med Associates, Inc	DIG-716P2	Behavioral equipment
Large table-top cabinet and power supply	Med Associates, Inc	SG-6510D	Behavioral equipment
PCI interface package	Med Associates, Inc	DIG-700P2-R2	Behavioral equipment
MED Intel core computer pkg with X Pro 19" monitor	Med Associates, Inc	COM-103V	Behavioral equipment
Paraformaldehyde (grannular), 1 kg	Electron Microsopy Sciences	19210	Hazard: carcinogen, weigh in hood
Rabbit monoclonal antibody (HA-Tag)	Cell Signaling Technologies	3724S	Histology reagent
XP Rabbit monoclonal antibody (GFP)	Cell Signaling Technologies	2956S	Histology reagent
Anti-rabbit IgG	Cell Signaling Technologies	4412S	Histology supplies
Superfrost plus slides	VWR international	483111-703	Histology supplies