In vitro Brainstem-spinal Cord Preparation from Newborn Rat

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Original research article

A brief version of this protocol appeared in:
The Journal of Neuroscience
Jan 2016


The brainstem-spinal cord preparation of newborn rat contains neural networks able to produce motor output in absence of sensory feedback. These neural structures, commonly called central pattern generators (CPGs), are involved in many vital functions such as respiration (Morin and Viala, 2002; Giraudin et al., 2008) or locomotion (Juvin et al., 2005). Here we describe a procedure for the isolation of the brainstem-spinal cord tissue of neonatal rat (0-2 days old). A surgical method under binocular microscope allows the brainstem and the spinal cord to be isolated in vitro and the motor outputs to be recorded. This preparation can then be used for diverse experimental approaches, such as electrophysiology, pharmacology or anatomical studies, and constitutes a useful model to study the interaction between CPGs (Juvin et al., 2007; 2012; Giraudin et al., 2012; Le Gal et al., 2014; 2016).


Historically, the in vitro spinal cord of neonatal rodent was developed to study the spinal reflexes (Otsuka and Konishi, 1974). In 1984, Suzue was the first to develop the in vitro brainstem-spinal cord preparation of newborn rat. Thus, it was possible to demonstrate that an isolated central nervous system was able to generate spontaneously what is referred as fictive respiratory activity. Later, it was then possible to determine the location of the CPGs underlying the locomotor rhythm generation (Cazalets et al., 1995; Kjaerulff and Kiehn, 1996; Ballion et al., 2001) and those engaged in respiratory rhythm generation (Smith et al., 1991; Onimaru and Homma, 2003). In our research team, this preparation has been mainly used to study the neural mechanisms underlying the interaction between CPGs. For instance, in a context of interaction between CPGs involved in the same function, our results have contributed to characterize the role played by the sensory afferents and the spinal thoracic segments in the coordination between the cervical and the lumbar locomotor CPGs (Juvin et al., 2005; 2012). Similarly, this preparation allows studies on the neural mechanisms involved in coordination between CPGs engaged in different functions. Based on electrical stimulation of dorsal roots, it was shown that the proprioceptive inputs originating from both hindlimb and forelimb are involved in the respiratory rhythm entrainment observed during locomotion (Morin and Viala, 2002; Giraudin et al., 2012). These ascending entraining signals from the cervical and lumbar afferents are conveyed to the brainstem respiratory centers via a brainstem pontine relay located in the parabrachial/Kölliker-Fuse complex (Giraudin et al., 2012). Using pharmacological and intracellular (patch-clamp recording) approaches on the same preparation, recent results have demonstrated for the first time the existence of an ascending pathway from the lumbar locomotor CPGs to the respiratory CPGs. This central neurogenic mechanism, involving a substance P-dependent modulating mechanism, could play a crucial role in the increased respiratory frequency observed during locomotion (Le Gal et al., 2014). In addition, it was also demonstrated that the locomotor related signal from the lumbar locomotor CPGs selectively modulates the intracellular activity of spinal expiratory neurons (Le Gal et al., 2016). Altogether, our results obtained on the in vitro brainstem spinal cord preparation of new born rat have contributed to increase our understanding of the cellular bases engaged in the coordination of rhythmic neural circuitry responsible for different functions.

How to cite:  Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Le Gal, J., Nicolosi, A., Juvin, L. and Morin, D. (2016). In vitro Brainstem-spinal Cord Preparation from Newborn Rat. Bio-protocol 6(22): e2003. DOI: 10.21769/BioProtoc.2003.
  2. Le Gal, J. P., Juvin, L., Cardoit, L. and Morin, D. (2016). Bimodal respiratory-locomotor neurons in the neonatal rat spinal cord. J Neurosci 36(3): 926-937.

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