Rapid and precise method to locate microdialysis probe implantation in the rodent brain

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Abstract

An important concern about microdialysis methodology is the histological validation of the dialysis probe implantation site in brain tissue of rodents (rat, mouse). Several methods have been described on standard histological staining (i.e., cresyl violet, formalin fixation, fast green perfusion, etc.). However, this methodology is time consuming. These requirements are not compatible with a histological validation prior to analysis of microdialysis samples. Here, we developed a new method to locate the track of the dialysis probe in the rodent brain. This method is based on a digital photomicrograph of a coronal section of the rodent frozen brain. The fitting of an appropriate coronal diagram of the rats’ and mice’ brain atlas with this photomicrograph, allowed us to locate precisely and quickly the track of the dialysis probe.

Introduction

The idea of using the principles of dialysis to sample extracellular fluid in brain is over 30 years old (Bito et al., 1966, Delgado et al., 1972). Microdialysis became a powerful and versatile neurochemical extraction technique for a routine use between 1985 and 1990 by Ungerstedt (1984). Microdialysis has then been rapidly adopted in the neuroscience research area as the in vivo neurochemistry reference method. Since 1996, 700 papers were published every year in this scope dealing with various components such as neurotransmitters and their major metabolites (for review, see Benveniste and Huttemeier, 1990, Westerink, 2000).

An important concern about microdialysis methodology is the histological validation of the dialysis probe implantation site in brain tissues of rodents. These requirements are now crucial in microdialysis experiments in mice where the structures explored are smaller than in rats. This point is especially important knowing the current access to numerous genetically modified transgenic or knockout mice. Several methods based on post-mortem standard histological staining (i.e., cresyl violet, formalin fixation) have been described. For a precise location of the dialysis probe, the in vivo perfusion of a dye (fast green or pontamine sky blue) (Ortemann et al., 1993, Ungerstedt, 1991), before brain removal, is necessary. However, these methodologies are time consuming, and thus not compatible with a histological validation prior to analysis of microdialysis samples.

In order to decrease the time needed to generate histological data, we developed a new method to locate precisely and quickly the track of the dialysis probe in the rat and mouse brain. This method is based on the fitting of a digital photomicrograph of a coronal section of the animal frozen brain with an appropriate coronal diagram of the corresponding brain atlas (Paxinos and Watson, 1998 for rats; Hof et al., 2003 for mice).

Section snippets

Animals

Male Wistar rats (Janvier, Le Genest sur l’Isle, France), weighing 280–300 g were used. They were kept at constant temperature (21 ± 1 °C) and relative humidity (50–60%) with 12 h light–dark cycle (light at 7:00 a.m.) and had free access to food and water. All the experiments were carried out according to the guidelines of the European Community's Council for Animal experiments (DL 116/92) with the permission of the local ethical committee at the Institut de Recherches Servier.

Male C57BL/6 mice

Rats

As shown in Fig. 1A, the track of the microdialysis probe is easily visible on the photomicrograph. In the Acb, the percentage of all tracks created by an implanted microdialysis probe that were visible is 91 ± 1% (n = 38) and 83 ± 4% (n = 38) using the photomicrograph and the cresyl violet technique, respectively. In the PFC (data not shown), we found 99 ± 1% (n = 48) and 81 ± 7% (n = 48) of visible tracks, respectively. The index scores for qualifying the probe tracks indicate that both in

Conclusion

The comparison of the two techniques shows that the photomicrograph technique is more precise and reliable than the cresyl violet staining in the rat brain. The photomicrograph technique can be used for either rats’ or mice’ brain combined with chronic and acute dialysis approaches and is ready for probe location in other species. This technique could also be used for the location of electrodes when their diameter is more than 100 μm, such as bipolar electrode. This low cost technique

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