Proceedings of The Physiological Society
University of Manchester (2010) Proc Physiol Soc 19, PC239
Investigating the involvement of gap junctions in patterned activity within sensory networks of the spinal dorsal horn: a study using a multi-electrode array technology
C. W. Kay1, R. J. Chapman1, D. Ursu2, E. Sher2, A. King1
1. Institute for Membrane & Systems Biology, Leeds, United Kingdom. 2. Eli Lilly UK, Windlesham, Surrey, United Kingdom.
In the CNS, neuronal networks may provide a platform for the emergence of complex high order functions linked to sensory and motor outputs. In forebrain network connectivity is co-ordinated via both chemical and electrical synapses. The spinal dorsal horn (DH) is important for integration of somatosensory inputs. Previously, we used a single microelectrode technique to characterize within the substantia gelatinosa (SG) synchronized network behaviour that is co-reliant on chemical- and gap junction (GJ)-mediated synapses (Chapman et al., 2009, J. Physiol. 587.11: 2499-2510). However, with this technique we were unable to extract information about the spatio-temporal spread of network-based activity across other DH laminae. Here, we have used multi-electrode array (MEA) technology to analyse patterns of activity emerging across deep and superficial DH laminae in vitro and have further characterised the contribution of GJs to such activity. Transverse lumbar spinal slices (250 µm) were prepared from terminally anaesthetized (Urethane, 2g/kg i.p.) Wistar rats. All animal procedures were performed in accordance with UK legislation. Spinal cord slices were mounted onto 8 x 8 arrays of 3-D microelectrodes (40 µm diameter; 35-45 µm height; 100 or 200 µm spaced; Ayanda Biosystems). 4-aminopyridine (4-AP, 25 µM) induced widespread excitation across the DH that manifested as large amplitude extracellular population field potentials (amplitude range 20-150 µV; peak frequency ~ 0.8 Hz). Auto- and cross-correlation analysis respectively revealed a) a rhythmic characteristic to 4-AP-induced activity, as indicated by a narrow range of inter-spike intervals (ISI) (1.5 - 4 s) and b) a high degree of unilateral synchrony between superficial and deep DH laminae. Pseudo-heat maps were constructed to profile bilateral and dorso-ventral spatio-temporal patterns of the emergent activity. Application of GJ uncouplers such as Carbenoxolone (CBX) (200 μm) or Quinine modified quantified parameters of activity including the ISI range, periodicity of population field potentials and total duration of spiking activity. In a preliminary study with age-matched rat spinal cords, we used qPCR to profile the expression of neuronal and glial GJ connexin (Cx) proteins (Cx36, Cx43, Cx45 and Cx29). These data indicate the utility of MEA technology to characterize more complex patterns of multi-cell activity in DH networks and indicate that GJs expressing specific Cx subtypes may shape network output.
Where applicable, experiments conform with Society ethical requirements