The contribution of gap junctions (GJ’s) and connexin (Cx) proteins to pain facilitation may be more prominent than once thought. For example, the non-specific GJ uncoupler carbenoxolone (CBX) reduces mechanical allodynia in inflammatory and neuropathic pain (Spataro et al., 2004). Similarly, in formalin-injected rats CBX prolongs the paw thermal withdrawal latency compared to control (Qin et al., 2006). Despite growing knowledge about GJs in the CNS, there is still relatively little known about GJs in the spinal dorsal horn (DH) although CBX disrupts network-based activity within substantia gelatinosa (SG) in vitro (Chapman et al., 2009). In these studies, pharmacological tools that target GJs and electrophysiological single and multi-electrode array (MEA) recordings were used to further investigate the involvement of GJs in DH network activity. In addition, quantitative PCR (Q-PCR) was used to determine expression levels of selected Cx proteins in age-matched rat lumbar spinal cord. For single and MEA electrophysiological recordings, transverse spinal slices were obtained from terminally anaesthetized (Urethane, 2g/kg i.p.) Wistar rats aged 10-14 days old. Animal procedures accorded with current UK legislation. Perfusion of 4-Aminopyridine (4-AP, 25 µM) induced excitation within the DH that was characterized in terms of a) 4-12 Hz rhythmicity, b) population spike frequency and c) spatio-temporal characteristics. The effects of the GJ opener trimethylamine (TMA, 100 µM), the non-specific GJ uncoupler CBX (100 µM) and the GAP26 mimetic peptide (100 µM) which targets Cx43 were assessed. Lumbar spinal cord tissue was removed from terminally anaesthetized (Urethane, 2g/kg i.p.) Wistar rats for Q-PCR analysis of relative expression levels of glial and neuronal Cx subtypes. In single electrode recordings in SG, 4-AP induced population spikes and 4-12 Hz rhythmic activity. During co-application of TMA plus 4-AP, the spike frequency and 4-12 Hz rhythmic activity were significantly increased. Conversely, CBX and GAP26 reduced these quantified parameters. In MEA recordings, 4-AP-induced excitation was observed across the DH and characterized for its spatio-temporal characteristics and rhythmicity in superficial and deep DH laminae. GJ targeting compounds were assessed for their ability to differentially alter characteristics of 4-AP-induced activity across the DH. Q-PCR revealed spinal expression of glial Cx29, Cx32, Cx43 and neuronal Cx36 and Cx45. These data indicate that modulation of GJ connectivity can enhance or diminish excitation across DH, effects which are presumptively mediated via GJs in neurones and/or glia that express Cxs.