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

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.

[1]  J. Paton,et al.  An arterially-perfused trunk-hindquarters preparation of adult mouse in vitro , 1997, Journal of Neuroscience Methods.

[2]  F. A. Edwards,et al.  A thin slice preparation for patch clamp recordings from neurones of the mammalian central nervous system , 1989, Pflügers Archiv.

[3]  F. Cerveró,et al.  Understanding the signaling and transmission of visceral nociceptive events. , 2004, Journal of neurobiology.

[4]  L. Renaud,et al.  A perfused in vitro preparation of hypothalamus for electrophysiological studies on neurosecretory neurons , 1983, Journal of Neuroscience Methods.

[5]  Jin Mo Chung,et al.  An experimental model for peripheral neuropathy produced by segmental spinal nerve ligation in the rat , 1992, PAIN.

[6]  M. Denavit-Saubié,et al.  Rhythmic discharges in the perfused isolated brainstem preparation of adult guinea pig , 1989, Neuroscience Letters.

[7]  R. Llinás,et al.  The Isolated and Perfused Brain of the Guinea‐pig In Vitro , 1993, The European journal of neuroscience.

[8]  R. Llinás The intrinsic electrophysiological properties of mammalian neurons: insights into central nervous system function. , 1988, Science.

[9]  K. Walton,et al.  Electrophysiological properties of neonatal rat motoneurones studied in vitro. , 1986, The Journal of physiology.

[10]  W. Jänig,et al.  Respiratory modulation in the activity of sympathetic neurones , 1994, Progress in Neurobiology.

[11]  J. Paton,et al.  Dynamic Changes in Glottal Resistance during Exposure to Severe Hypoxia in Neonatal Rats In Situ , 2005, Pediatric Research.

[12]  J. Paton,et al.  Investigation of Systemic Bupivacaine Toxicity using the In situ Perfused Working Heart-Brainstem Preparation of the Rat , 2002, Anesthesiology.

[13]  J. Schwaber,et al.  Response Properties of Baroreceptive NTS Neurons , 2001, Annals of the New York Academy of Sciences.

[14]  M. Dutschmann,et al.  Whole Cell Recordings From Respiratory Neurones in an Arterially Perfused in situ Neonatal Rat Preparation , 2003, Experimental physiology.

[15]  Julian F. R. Paton,et al.  A working heart-brainstem preparation of the mouse , 1996, Journal of Neuroscience Methods.

[16]  Peter A. Getting,et al.  Maintenance of complex neural function during perfusion of the mammalian brain , 1987, Brain Research.

[17]  J. Paton,et al.  Long-term intracellular recordings of respiratory neuronal activities in situ during eupnea, gasping and blockade of synaptic transmission , 2005, Journal of Neuroscience Methods.

[18]  R. Callister,et al.  An in vivo mouse spinal cord preparation for patch-clamp analysis of nociceptive processing , 2004, Journal of Neuroscience Methods.

[19]  J. Paton,et al.  Characterizations of eupnea, apneusis and gasping in a perfused rat preparation. , 2000, Respiration physiology.

[20]  E. Kumamoto,et al.  Responsiveness of rat substantia gelatinosa neurones to mechanical but not thermal stimuli revealed by in vivo patch‐clamp recording , 1999, The Journal of physiology.

[21]  D. Dubuisson,et al.  The formalin test: A quantitative study of the analgesic effects of morphine, meperidine, and brain stem stimulation in rats and cats , 1977, Pain.

[22]  S. Kasparov,et al.  Genetic and pharmacological dissection of pathways involved in the angiotensin II‐mediated depression of baroreflex function , 2002, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[23]  R. Llinás,et al.  An electrophysiological study of the in vitro, perfused brain stem‐cerebellum of adult guinea‐pig. , 1988, The Journal of physiology.

[24]  E D Adrian,et al.  Discharges in mammalian sympathetic nerves , 1932, The Journal of physiology.

[25]  A. Light,et al.  Spinal laminae I-II neurons in rat recorded in vivo in whole cell, tight seal configuration: properties and opioid responses. , 1999, Journal of neurophysiology.

[26]  Gary J. Bennett,et al.  A peripheral mononeuropathy in rat that produces disorders of pain sensation like those seen in man , 1988, Pain.

[27]  J. Mitchell,et al.  Contraction-sensitive skeletal muscle afferents inhibit arterial baroreceptor signalling in the nucleus of the solitary tract: role of intrinsic GABA interneurons , 2003, Neuroscience.

[28]  J. Lipski,et al.  Intracellular recording from respiratory neurones in the perfused ‘in situ’ rat brain , 1991, Journal of Neuroscience Methods.