A new stabilizing craniotomy–duratomy technique for single-cell anatomo-electrophysiological exploration of living intact brain networks

Abstract

Standard large craniotomies induce undesirable brain motions during intracellular recordings in whole animal preparations. Practically all of the papers available in the literature outline a number of specific methodological approaches designed to avoid this inconvenience. Our study describes a new craniotomy–duratomy, which consists of the maintenance of a thin bone membrane and dura mater surrounding the small hole opened for lowering the recording micropipette. This new surgical preparation avoids brain movements by keeping the brain’s volume constant within the cranial cavity and does not require additional technical procedures. It is an all-purpose surgical technique, although it was developed in anaesthetized rats while studying spatio-temporal dynamics of cellular interactions associated with thalamocortical oscillations. It significantly improves both the precision of stereotaxic approaches and the success rate of single-cell recordings (e.g., current-clamp intracellular and paired recordings) compared to standard craniotomy/electrophysiology techniques.

Introduction

Understanding the properties of cells and neural networks underlying normal and pathological brain function has always been a tremendous challenge, requiring the continuous development of new equipment and techniques. The saline-filled sharp micropipette for intracellular recording was invented at the turn of the twentieth century, and represented a break-through in electrophysiology. Intracellular recordings in in vitro preparations are now a common means of studying the electrophysiological properties of single neurons in simplified networks. Although such investigations have increasingly improved our understanding of the integrative and oscillatory properties of single neurons and neural networks, it is still difficult to relate these in vitro data with global brain functions. Single-cell electrophysiology in whole animal preparations is the only means of verifying hypotheses based on in vitro investigations.

The success rate of in vivo single-cell electrophysiology experiments—especially intracellular recordings from the brain of whole animal preparations—depends primarily on mechanical stability at the submicron scale of the living brain (Britt and Rossi, 1982, Fee, 2000, Konopacki et al., 2003). Most papers describe the same specific methodological approaches which make it possible to stabilize the living brain of any anaesthetized animal held in a stereotaxic frame (Chiaia et al., 1991, Deschênes and Hu, 1990, Lipski et al., 1993, Lu et al., 1992, Purpura and Cohen, 1962, Steriade and Contreras, 1995, Yamamoto et al., 1991). Cisternal drainage is often used to prevent the development of edema. It is also tacitly accepted that large craniotomies and duratomies will be performed and, for recording sessions in particular, that the exposed brain will be covered with a saline-based agar gel. Furthermore, in an attempt to minimize cardiac and/or respiratory pulsations, a pneumothorax is performed, the hips are suspended, and the rectal temperature is usually maintained at least 1 or 2 °C below the physiological mean. Sometimes, it is even necessary to elevate the animal’s body to minimize the vascular pressure differential between the body and the brain (Konopacki et al., 2003).

The opening of the cisterna magna and the large craniotomy cause most if not all of the cerebrospinal fluid to flow out, making the brain slump down progressively into the cranium in the course of experiments. These conditions tend to reduce the precision with which single neurons can be reached stereotaxically in a target region, especially in deep brain structures. Under these conditions, undesirable and sometimes even attenuated, non-neuronal rhythms (particularly vascular and respiratory pulsations) may still be present in intracellular recordings.

Furthermore, standard large craniotomy–duratomy (CD) may be regarded as improper opening because of bleeding or inadequate maintenance of the brain surface. Bleeding, drying, or edema are potential causes of clogging or damage of the sharp tip of the recording glass micropipette.

Regarding the intracranial pressure, two different sorts of problems should be considered: (1) excess pressure producing edema, which may be dependent upon level of anaesthesia along with damage to the brain, and (2) inadequate pressure producing collapse of the brain, which is presumably due to artificial drainage of the cerebrospinal fluid. These conditions might result in pulsatile artifacts (due to heart or respiratory pulsations) in single-unit recordings (phasic changes in membrane potential and/or in action potential amplitude).

Consequently, in order to avoid these inconveniences we performed a new type of CD, which prevented significant outflow of the cerebrospinal fluid and made it possible to keep the living brain totally free of unwanted pulsations. This all-purpose surgical procedure was devised in anaesthetized adult rats and has been improved over the last 6 years thereby enhancing stereotaxic accuracy when it comes to reaching target structures and increasing the success rate and quality of intracellular recordings and paired recording-labelling of single neurons (Pinault et al., 2001, Pinault, 2003).