A decerebrate, artificially-perfused in situ preparation of rat: Utility for the study of autonomic and nociceptive processing

Abstract

By extending established technology, we have developed a relatively simple in situ preparation from juvenile rat that overcomes some technical restrictions present in vivo (e.g. need for anaesthesia, mechanical instability for intracellular recording and control over the extracellular milieu). The in situ preparation is decerebrate and artificially perfused via the left ventricle with a colloid containing solution. It exhibits an eupneic pattern of respiratory motor activity and demonstrates numerous somatic and visceral reflexes including those evoked by stimulation of the tail, hindlimbs, bladder, baroreceptors and peripheral chemoreceptors. We have employed this preparation to allow recordings from multiple sympathetic motor outflows such as thoracic and lumbar chain, inferior cardiac, splanchnic, renal and adrenal nerves. We show that the sympathetic motor discharge shows strong respiratory modulation and exhibits pronounced reflex modulation indicating intact communication between the periphery, the brainstem and the spinal cord. Further, we have made extracellular and whole cell recordings from neurones in the spinal cord, demonstrating good mechanical stability. The decerebrate, artificially-perfused rat (DAPR) provides a powerful methodology with which to study peripheral and central control of the autonomic nervous system with many of the benefits of an in vitro environment.

Introduction

The desire to probe neuronal mechanisms at the cellular and sub-cellular levels has driven the widespread adoption of reduced, in vitro preparations. Over the last 30 years there has been a strong trend for in vivo approaches to be supplanted by cellular neurophysiological studies in vitro. In particular the use of brain slice preparations has allowed the precise study of neuronal integration and synaptic mechanisms (Dingledine, 1989, Edwards et al., 1989, Llinas, 1988). Such studies have been facilitated by the ability to control the extracellular milieu, good access for drug application, the ability to visualise cells and better mechanical stability for recording without the requirement for anaesthesia. Most of these studies have used relatively thin sections of tissue to allow oxygen and other nutrients to diffuse from the bathing medium into the tissue to maintain viability. However, in order to relate cellular properties to integrated functions it has been necessary to study larger blocks of brain tissue with better connectivity, therefore a number of investigators have developed arterially perfused in vitro preparations of hypothalamus (Bourque and Renaud, 1983), brainstem-cerebellum (Llinas and Muhlethaler, 1988), brainstem (Morin-Surun and Denavit-Saubie, 1989) and subsequently of the whole brain (Muhlethaler et al., 1993).

This approach has been further extended with a number of in situ artificially perfused preparations that leave the nervous tissue accessible within the body of the animal (Chizh et al., 1997, Hayashi et al., 1991, Paton, 1996, Richerson and Getting, 1987). These preparations were mainly developed initially to allow improved mechanical stability of the brain, for intracellular studies and good pharmacological access—a situation that is analagous to in vitro. Importantly, these arterially perfused preparations were advantaged by the preservation of part or all of a functional system, often containing the afferent pathway of interest, so allowing peripheral inputs to be physiologically driven. These preparations also contained the motor outflow pathways and some target tissues allowing simultaneous monitoring of motor outputs (Chizh et al., 1997, Hayashi et al., 1991, Paton, 1996, Richerson and Getting, 1987).

Our laboratory has described several artificially perfused preparations for performing integrative physiological experiments such as the working heart-brainstem preparation (WHBP; Paton, 1996) and the perfused hindlimb and trunk preparation (HLTP; Chizh et al., 1997). We have used these in situ preparations for studies at a molecular level (Wong et al., 2002), intra- and extra-cellular recording studies (Dutschmann and Paton, 2003, Paton et al., 2001, Paton and St-John, 2005) and systems level analysis including motor nerve recording (Pickering et al., 2002, St-John and Paton, 2000) as well as kinesiological studies (Dutschmann and Paton, 2005, Potts et al., 2003). However, as the WHBP is bisected above the level of the diaphragm, it is without hindlimbs, tail and abdominal organs. Furthermore, only the proximal spinal cord and a limited number of sympathetic motor outflows remain intact (upper thoracic chain plus branches and cervical sympathetic trunk). Indeed, other in situ preparations have also either removed the hindquarters (Richerson and Getting, 1987) or left them without direct perfusion (Hayashi et al., 1991). Similarly, there are limitations of the isolated spinal cord-hindlimb preparation (Fulton and Walton, 1986) and the HLTP (Chizh et al., 1997), in that only spinal processes can be studied in the absence of crucial brainstem control, and there is no equivalent to phrenic nerve activity that has proved to be very useful for assessment of preparation viability (Hayashi et al., 1991, Paton, 1996, Richerson and Getting, 1987).

To further extend the utility of arterially perfused preparations, we report a new experimental model. This is an extension of the approach that we developed for the WHBP and HLTP. We describe a decerebrate, artificially perfused preparation of the whole rat (DAPR, recently demonstrated at the Physiological Society (Simms et al., 2005)). We show its physiological viability and durability to allow rigorous investigations at both the cellular and systems levels. We also demonstrate that the preparation has utility for studying multiple somatic and visceral responses as well as the central respiratory modulation of cardiac vagal and sympathetic motor outflows.