Determination of stereotaxic coordinates for the hippocampus in the domestic pig

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Abstract

In this study a stereotaxic instrument and a stereotaxic procedure based on external skull structures, to be used in prepubertal male Landrace pigs weighing less than 30 kg, is described. The instrument represents an adaptation of the apparatus designed by Marcilloux et al., Brain Res Bull 1989;22:591–597, but we have modified the instrument for stereotaxic procedures based on external skull structures, instead of intracerebral structures necessitating ventriculography (Marcilloux et al., Brain Res Bull 1989;22:591–597). For this reason the U-shaped frame and the ear-bar supports have been changed allowing the three-dimensional placement of the ear-bars into the oblique auditory canals. Firm fixation of the skulls of pigs weighing less than 30 kg, was furthermore secured with modified infraorbital ridges and hard palate pieces. Measurements of distances between external skull structures in animals of the same sex, age and weight showed a negligible variation, thus enabling definition of the horizontal, frontal and sagittal zero planes using external skull structures alone. Stereotaxic coordinates for the hippocampal region of male Landrace pigs weighing 10 kg were then provided and the coordinates from two different levels of the hippocampal region are presented in the text. The reliability of the stereotaxic instrument was finally secured by intrahippocampal injections of ink at predetermined coordinates.

Introduction

Domestic pigs have been used in biological studies and now extensively in biomedical research because of the anatomical and physiological resemblance of a number of different organ systems to those of humans (Douglas, 1972, Dodds, 1982, Tumbleson, 1986). The pig possesses a relatively large, gyrencephalic brain which facilitates surgical intervention, production of lesions, installation of electrodes and the placement of injections. A number of neuroanatomical studies have been performed on pig brains (Solnitzky, 1938, Woolsey and Fairman, 1946, Stephan, 1951, Breazile, 1967, Hereć, 1967, Kruska, 1970, Palmieri et al., 1987, Freeman et al., 1988, Niwa et al., 1988, Huffaker et al., 1989, Seeger, 1990, Van Eerdenburg et al., 1990, Van Eerdenburg et al., 1992Østergaard et al., 1992) including a comprehensive description of the hippocampal region (Holm and Geneser, 1989, Holm and Geneser, 1991a, Holm and Geneser, 1991b, Holm et al., 1990, Holm et al., 1992, Holm et al., 1993, Holm and West, 1994). Although behavioural studies have been reported in pigs, including the classical and operant conditioning tests (Mount and Ingram, 1971), very few reports on the integrated functions of the central nervous system of this species exist.

To study the neurophysiological characteristics of hippocampal functions in the pig, it is essential to have a stereotaxic instrument and a map of the hippocampal region with stereotaxic coordinates. Several stereotaxic instruments have been developed for pig brains (Steven-Poceta et al., 1981, Salinas-Zeballos et al., 1986, Marcilloux et al., 1989), whereas to our knowledge, no stereotaxic map of the hippocampal region in pigs has been published. All instruments are based on fixation of the skull by the auditory canals, the infraorbital ridges and the hard palate (Horsley and Clarke, 1908). During postnatal development, the orientation of the auditory canals changes, being perpendicular to the sagittal plane in the new-born piglet but oblique in the weaned pig (Steven-Poceta et al., 1981, Salinas-Zeballos et al., 1986, Marcilloux et al., 1989). For this reason, separate stereotaxic instruments were developed for piglets (Salinas-Zeballos et al., 1986) and weaned pigs weighing about 40 kg (Steven-Poceta et al., 1981). With the development of ear-bars carried by an orientable system it was possible to make an apparatus suitable for pigs from weaning to puberty (5–120 kg bodyweight) (Marcilloux et al., 1989).

The horizontal zero plane is classically defined as the plane through the center of the external auditory meati and the infraorbital ridges (Jasper and Ajmone-Marsan, 1954DeLucchi et al., 1965Kusama and Mabuchi, 1970; Berman and Jones, 1982Paxinos and Watson, 1986Mori et al., 1990). However, Marcilloux et al. (1989)found a considerable variation in skull morphology and therefore defined the horizontal zero plane, based on intracerebral structures using ventriculography, as the plane through the anterior border of the commissura posterior and the anterior border of the recessus preopticus.

We have found the recessus preopticus difficult to identify and the ventriculography technically demanding. For this reason, we have measured distances between various skull structures to reconsider the possibility of defining the horizontal zero plane by external skull structures alone. The aim of the present study was thus to secure stereotaxic coordinates of the hippocampal region in prepubertal pigs weighing 10 kg, using an adaptation of the stereotaxic apparatus designed by Marcilloux et al. (1989)and a technique based on external skull structures such as bregma, sutura coronalis and sagittalis.

Section snippets

Animal preparation

A total number of eighteen male Landrace pigs (Sus scrofa domesticus) were used. Weighing between 7.4 and 29.8 kg, nine of the pigs were used for skull measurements in connection with development of the stereotaxic instrument. The pigs fasted overnight before sacrifice by a rapid intravenous injection of sodium pentobarbital (50 mg/kg bodyweight, Abbott Laboratories, North Chicago, IL) followed by decapitation. The heads were then boiled and sonicated in a 1% (W/V) papain solution and finally

Skull measurements

The data from the skull measurements are shown in Table 1. The distance between bregma and lambda is 19.4±0.2 mm (n=3, Mean±S.E.M.) in the smallest group, but this value remains remarkably constant with increasing body weight, in contrast to the distance between bregma and nasion and between nasion and AEN, which increases with enlarged body weight. The distances a, b and c likewise increase with enlarged body weight. The angles A and B are 48.0°±2.6° and 136.0°±2.1° (n=3, Mean±S.E.M.) in the

Discussion

The stereotaxic instrument and methodology described in the present study provide a necessary tool for experimental studies of the hippocampal region of the domestic pig. The hippocampus of the pig is characterized by a general cytoarchitecture resembling that of other mammals. However, the dentate hilus is highly laminated, resembling the dentate hilus of primates, and occupies a larger percentage of the total volume of the hippocampus than it does in other species (Holm and West, 1994). We

Acknowledgements

We acknowledge with gratitude the valuable communications and advices for the stereotaxic instrument of Mr Jean-Claude Marcilloux, Drs Marie-Bernadette Felix and Denise Albe-Fessard, I.N.R.A., 79350 Jouy en Josas, France. The authors are grateful for the skillful technical assistance of Ms D. Jensen, Ms K. Møller, Ms K. Wiedemann, Mr A. Meier, Dr J.C. Sørensen, Dr F.A. Geneser and Dr I.E. Holm, Department of Neurobiology, University of Aarhus. The staff of the swine management section at the

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