Preparation of an Awake Mouse for Recording Neural Responses and Injecting Tracers

Summary

Electrophysiological characterization of neuronal responses is important for understanding brain function and for guiding the placement of dyes for pathway tracing. However, many studies are performed in anesthetized animals. To understand brain function without anesthetics, we developed a method to record neuronal response properties and inject dyes in awake mouse.

Abstract

It is well known that anesthesia alters neural response properties in various regions of the brain.¹³. In the auditory system, fundamental response properties of brainstem neurons including threshold, frequency specificity, and inhibitory sidebands are altered in significant ways under anesthesia^1-2. These observations prompted physiologists to seek ways to record from single neurons without the contaminating effects of anesthesia. One result was a decerebrate preparation, where the brainstem was completely transected at the level of the midbrain⁴. The drawbacks of this preparation are a formidable surgery, the elimination of descending projections from the forebrain, and an inability to use sensory stimulation to examine structures above the midbrain. A different strategy has been to implant electrode arrays chronically to record from single neurons and multiunit clusters while the animal is awake and/or behaving^5,6. These techniques however are not compatible with injecting tracer dyes after first electrophysiologically characterizing a brain structure. To avoid altering neural response properties with anesthetics while recording electrophysiological response properties from single neurons, we have adapted a head restraint technique long used in bats^7-9 to mouse^10-12. Using this method, we are able to conduct electrophysiological recordings over several days in the unanesthetized mouse. At the end of the recording sessions, we can then inject a dye to reconstruct electrode positions and recording sites or inject a tracer so that pathways to and from the recording loci can be determined. This method allows for well isolated single neuron recordings over multiple days without the use anesthetics.

Protocol

1. Head-restraint Overview

1. To assemble a custom-built head-post, insert a 1/16" stainless steel roll-pin perpendicularly into a hole drilled in a 3/32" stainless steel rod to form a cross. The vertical piece of the head-post should be about 20 mm and the horizontal cross piece about 15 mm. Tap one end of the vertical rod to accept a #1-72 screw. (Fig. 1).
2. During surgery and recording, the head-post is secured in a custom-built brass or aluminum mounting bar with a #1-72 screw (Fig. 2). The long piece of the mounting bar should be about 90 mm long and 5-10 mm wide. The head-post f....

Discussion

The advantage of the head-post restraint system for electrophysiological and neuroanatomical experiments in mouse is that experiments can be conducted with unanesthetized, awake mice, eliminating potential response contamination due to anesthetic drugs. In addition, the set up can be used over multiple days to allow for more efficient animal use.

Most components for the head-post are available off-the-shelf. Only the head-post and mounting apparatus are custom-made, both of which are relativ.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Etch gel for cleaning skull prior to dental cement	Henry Schein	101-5396	12/pk with 1.2 mL syringes
Primer for dental cement	Kerr Optibond	25881	8 mL bottle
Adhesive for dental cement	Kerr Optibond	25882	8 mL bottle
Applicators for dental cement	Kerr Optibond	24680	200/pk
Dental Cement	Charisma, Heraeus Kulzer	66000085	4gm Syringe Refill
Tungsten Rod (Ground Pin)	A-M Systems	717200	0.010" diameter
Economy UV Curing Light	Henry Schein	CU-80
Head-post	Built in-house
Mounting bar	Built in-house
Mounting block	Built in-house
Stereotaxic Frame	David Kopf Instruments	902
Mouse and Neonatal Rat Adaptor	Stoelting Co.	51625
Deltaphase Isothermal Pad	Braintree Scientific, Inc.	39DP