A technique for stereotaxic recordings of neuronal activity in awake, head-restrained mice

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Abstract

Neurophysiological recordings of brain activity during behavior in awake animals have traditionally been performed in primates because of their evolutionary close relationship to humans and comparable behavioral skills. However, with properly designed behavioral tasks, many fundamental questions about how the brain controls behavior can also be addressed in small rodents. Today, the rapid progress in mouse neurogenetics, including the development of mouse models of human brain disorders, provides unique and unparalleled opportunities for the investigation of normal and pathological brain function. The development of experimental procedures for the recording of neuronal activity in awake and behaving mice is an important and necessary step towards neurophysiological investigation of normal and pathological mouse brain function. Here we describe a method for stereotaxic recordings of neuronal activity from head-restrained mice during fluid licking. Fluid licking is a natural and spontaneous behavior in rodents, which mice readily perform under head-restrained conditions. Using a head-restrained preparation allows recordings of well-isolated single units at multiple sites during repeated experimental sessions. Thus, a large number of neurons can be tested for their relationship with behavior and detailed spatial maps of behavior related neuronal activity can be generated as exemplified here with recordings from lick-related Purkinje cells in the cerebellum.

Introduction

A central goal of brain research is to understand how the brain controls behavior and how the brain's ability to generate normal behavior is affected by brain disorders. Studies of behavior related neuronal activity in awake and behaving animals yield the most promising method of accurately assessing the complex interactions of multiple cell types and networks during the generation of behavior. Although traditionally a domain of primate research, many fundamental questions about normal and pathological brain function can be successfully studied in small rodents. In fact, the development of genetic mouse models of human brain disorders now offers unparalleled opportunities to study the neuronal and behavioral defects associated with brain disorders. While the use of anesthesia alters the brain's mode of operation, sometimes producing results irrelevant for our understanding of the function of the conscious brain (Schonewille et al., 2006, Bengtsson and Jorntell, 2007), recordings in awake, behaving mice avoid this issue.

Recordings of neuronal activity in awake mice have been successfully performed with permanently implanted electrodes to study hippocampal long term potentiation (LTP) based on local field potentials (LFPs) and multi-unit signals (Errington et al., 1997, Davis et al., 1997, Jones et al., 2001, Koranda et al., 2008). Advantages of this established technique are that it allows users to make long term observations of neuronal activity at one location and animals can behave more naturally as they move around freely. Limitations include that implanted electrode recordings are not suitable for mapping studies and that stable single unit recordings are more reliably and easily obtained in a head-restrained paradigm because of reduced tissue movements and because micromanipulators can be used for micrometer precise electrode placement. Furthermore, not all brain areas are as amenable to recordings with implanted electrodes as the neocortex and particularly the hippocampus. We attempted to use implanted electrodes in the cerebellum and failed to get stable single unit recordings. Anatomical examination of the brains revealed large lesions around the implanted electrodes, which may have resulted from movement of the cerebellar tissue relative to the stationary electrodes (unpublished observations). Using the head-restrained preparation described here overcomes these problems and additionally allows the use of optical imaging methods and intracellular recording techniques in the awake mouse. Optical and physiological recordings from head-restrained mice have been performed by a small number of labs (Cheron et al., 2004, Goossens et al., 2004, Schonewille et al., 2006, Ferezou et al., 2007) and so far no comprehensive description of the experimental procedures involved is available in the literature. The experimental technique described here was initially developed to perform cerebellar recordings in awake behaving rats (Heck et al., 2002, Heck et al., 2007) and has been adapted for the performance of stereotaxic recordings from behaving mice during head fixation. The behavioral spectrum of head-restrained mice is limited, but the animals will perform spontaneous orofacial behaviors such as whisker movements (Ferezou et al., 2007) and rhythmic fluid licking (Hayar et al., 2006). These behaviors also yield a large number of repetitions, which is an important prerequisite for electrophysiological investigations of the underlying neuronal processes. Here, we investigated the neuronal representation of rhythmic fluid licking behavior in the simple and complex spike activity of Purkinje cells in the mouse cerebellar cortex. Fluid licking has been studied in a variety of contexts, including studies of taste preference (Lewis et al., 2005), chronic or acute drug treatment (Hsiao and Spencer, 1983, Genn et al., 2003), and to phenotype mouse models of autism spectrum disorders such as Angelman syndrome (Heck et al., 2008).

The rhythm or inter-lick-interval (ILI) of fluid licking in mice is strain-specific (Horowitz et al., 1977, Boughter et al., 2007). The experimental paradigm described here can therefore be useful for the investigation of strain differences in the neuronal mechanisms controlling the licking rhythm. This approach can be readily adapted for recordings from other brain areas of the mouse while monitoring either licking or other behaviors, like movements of the mystacial whiskers.

Section snippets

Surgical procedures and head-fixation assembly

All experiments adhered to procedural guidelines approved by the University of Tennessee Health Science Center Animal Care and Use Committee. Principles of laboratory animal care (NIH publication No. 86–23, rev. 1996) were followed.

The following procedures were successfully performed with adult mice from four different strains (C57BL/6J, DBA/2J, Cbln1−/− and Lurcher mice). Here we present data examples obtained from C57BL/6J mice. Prior to surgery, mice were weighed and then anesthetized with

Results

We recorded Purkinje cell simple and complex spike activity in the cerebellar cortex of awake mice during licking behavior. Licking behavior was registered in the form of positive junction potentials, which lasted for the duration of each tongue-to-waterspout contact (Fig. 3A). Fig. 3B shows an example of single unit Purkinje cell activity recorded during licking. Stable extracellular recordings of single unit Purkinje cell activity were readily obtained and remained stable during licking

Discussion

We have described a method for acute stereotaxic recordings of neuronal activity from awake mice in a head-restrained paradigm. We demonstrated the ability to maintain stable single unit recordings of neuronal activity of Purkinje cells in the mouse cerebellum during rhythmic fluid licking behavior. Fluid licking is a natural and spontaneous behavior in rodents and is for several reasons ideally suited for neurophysiological investigations. It requires no training, can be readily quantified and

Acknowledgements

We would like to thank Bob Gallik and Michael Nguyen from the UTHSC Department of Biomedical Instrumentation for outstanding technical support and creative suggestions on the design of the head-fixation assembly. This work was supported in part by a grant from the National Institute of child health and human development (1R03HD057244-01), a grant from the National Institute of Mental Health (5R01MH068433-02) and an award from the American Psychological Association's Diversity Program in

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