DZF-Project: Mario Raggenbass

Cellular electrophysiology of mammalian motoneurones: an in vitro approach using brainstem slices of the rat

Mario Raggenbass, PhD

Department of Basic Neurosciences, University Medical Center, 1211 Geneva 4, Switzerland


Keywords: brain slices, motoneurons, whole-cell patch clamp recording

Begin and End of the Project: 1994-1996

Background and Aim

Background: In the motor system, brainstem and spinal cord motoneurons play a pivotal role. They receive and integrate movement-related information coming from the brain and control the contraction of the muscle fibers they innervate. Knowledge of the cellular electrophysiological properties of motoneurons is thus essential for understanding the physiology of movement.

For ethical reasons, living motoneurons cannot be directly studied in man. Experiments have thus been carried out in animals, in particular in living cats. This species is widely used because cat motoneurons are relatively large and recording microelectrodes can be easily inserted into these cells. Although useful information has been gathered using this approach, much remains to be discovered in the field of motoneuron physiology. In order to reduce the number of animals used as well as the degree of pain and distress created in these in vivo approaches, alternative biological preparations are needed.

Aim: Our main objective was to develop an in vitro preparation of brainstem and spinal motoneurons of the rat. In a preliminary series of experiments, we have prepared brainstem slices containing vagal preganglionic motoneurons and have studies their responsiveness to the neuropeptide oxytocin. The results obtained have been published in a scientific journal (Alberi et al., 1997).

Methods and Results

After the animal is sacrificed, the brain is rapidly removed and coronal brainstem slices, 300-400-mm-thick, cut with a vibrating microtome. They are incubated in a recording chamber and continuously perfused with a physiological solution. Electrophysiological recordings are performed using the whole-cell patch clamp technique.

We have found that oxytocin excites vagal motoneurons by activating two distinct intracellular pathways. One is cAMP-dependent and one, unidentified, is phospholipase C- and cAMP-independent. Each pathway account for about half of the peptide effect and both involve G-protein activation.

Conclusions and Relevance for 3R

Our results show that survival of motoneurons in brainstem slices is excellent. Stable, long-term electrophysiological recordings can be easily obtained and pharmacological experiment can be performed under strictly controlled conditions. Since then, this technique has been successfully extended in our laboratory to other classes of brainstem motoneurons as well as to spinal motoneurons (see, for example, Ogier et al., 2004). It should be regarded as a valuable alternative to animal experiments aimed at characterizing the physiological and pharmacological properties of mammalian motoneurons.

Reference cited

Alberi S, Dreifuss JJ, Raggenbass M (1997) The oxytocin-induced inward current in vagal neurons of the rat is mediated by G protein activation but not by an increase in the intracellular calcium concentration. Eur J Neurosci 9:2605-2612.

Ogier R, Liu X, Tribollet E, Bertrand D, Raggenbass M (2004) Identified spinal motoneurons of young rats possess nicotinic acetylcholine receptors of the heteromeric family. Eur J Neurosci 20:2591-2597.