A new method to study sensory modulation of locomotor networks by activation of thermosensitive cutaneous afferents using a hindlimb attached spinal cord preparation

Abstract

The use of isolated in vitro spinal cord preparations to examine the underlying networks that control locomotion has become popular. It is also well known that afferent feedback can excite and modulate these networks. However, it is often difficult to selectively activate classes of afferents that subserve specific modalities using in vitro preparations. Here, we describe a technique where afferent receptors that detect temperature were selectively activated. To accomplish this we used an in vitro preparation of the mouse where the spinal cord was isolated (T5-cauda equina) with one hind limb left attached. We designed a special chamber allowing the hind paw to be placed in such a way that it remained attached to the spinal cord but received a separate supply of artificial cerebrospinal fluid (aCSF). This allowed us to alter the temperature of the hind limb compartment without affecting the temperature of the central compartment containing the spinal cord. We also demonstrate using this approach that agonists which activate receptors which detect noxious heat could be intradermally injected into the hind limb without it diffusing into the central compartment.

Introduction

Sensory information from receptors in the hind limb is well known to control spinal circuits and a wealth of data has been generated using cat and rodent preparations (Pearson, 2004). Most of this work has focused on proprioceptors from muscle partly for historical and technical reasons. Part of the difficulty with activating cutaneous afferents is that they are heterogeneous and transduce sensation ranging from touch to pain (Lumpkin and Caterina, 2007). Consequently, relatively few tools have been available to selectively activate these classes of afferents. This has changed over the last 5 years with the cloning of ion channels which transduce temperature (Mandadi and Roufogalis, 2008). These are classes of transient receptor potential (TRP) ion channels which are present in skin receptors and can be activated by agonists or by changes in skin temperature. We focus here on two subclasses within the group of ThermoTRPs that have been well characterized for their role in somatosensation and nociception. These receptors have been found on both peripheral and central projections of C and Aδ cutaneous afferents (Caterina et al., 1997, McKemy et al., 2002, Bautista et al., 2007). For example, transient receptor potential vanilloid 1 (TRPV1) is an ion channel that is activated by noxious heat (>43 °C and, more specifically, by capsaicin) (Caterina et al., 1997). In contrast, transient receptor potential melastatin 8 (TRPM8) is an ion channel present primarily in a segregated group of primary afferents and is activated by cool temperatures (15–27 °C) (McKemy et al., 2002). Therefore the prospect of examining the effects of modulating TRPs either by using agonists or by changing skin temperature and examining the effects on spinal motor networks is now possible.

An important model for examining sensorimotor activation of spinal circuits is the isolated spinal cord preparation. This preparation has proven useful but lacks sensory input from the hind limb. To counter this issue, hind limb-spinal cord preparations have been developed with hind limb afferent input left intact. However, studies that have selectively activated receptors to examine locomotor function using this preparation have been limited. In this short communication, we outline the development of a technique to selectively activate cutaneous receptors in the mouse hind paw that transduce temperature sensation. We developed a hind limb attached to spinal cord preparation where we isolated the paw from the rest of the limb and the spinal cord. By doing so the paw could be superfused with different temperatures of artificial cerebrospinal fluid (aCSF) to selectively activate cutaneous afferents which in turn are known to activate specific classes of ThermoTRPs. In addition, intradermal skin injections of capsaicin activating cutaneous TRPV1 expressing afferents could be completed without any issues of diffusion into the aCSF superfusing the spinal cord. Part of the data used in this manuscript has been published (Mandadi et al., 2009).