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.