The in vivo physiological properties of these neurons have traditionally been studied using extracellular recording techniques.
While approach is useful for characterizing neuron responsiveness it provides little information about the intrinsic or synaptic properties of DH neurons. Accordingly, we developed a mouse preparation, which allows in vivo patch clamp analysis of intrinsic and synaptic properties in DH neurons that Ku-0059436 solubility dmso receive input from the colon. Methods: Male mice (C57Bl/6J, 6–7 weeks) were anesthetized (isoflurane) and mounted in a stereotaxic frame. An incision was made to expose the T13-L2 vertebral bodies, which were clamped, before a laminectomy exposed the L6-S1 spinal segments2. Colonic inputs are thought to synapse, via the pelvic nerve, with DH neurons in these segments. The dura and pia mater were removed
GS-1101 cell line and a recording pipette (5–7 MΩ) was lowered until it touched the surface of the cord. The electrode was advanced 100 μm to reach the grey matter, and then advanced in 3 μm steps until a DH neuron was encountered. The whole-cell recording configuration was established and we then tested whether the neuron received colonic inputs by distending the colon at both innocuous and noxious pressures. A series of protocols were also run to assess the intrinsic and additional synaptic properties of the recorded DH neuron. Results: Of the 48 neurons obtained so far, three responded to colonic distension. Responses were observed at noxious pressures (80 mmHg) in 3/3 cells. Two of these neurons also responded to gentle brushing of the tail. Two of three neurons responded to depolarizing current injection with a tonic firing pattern, while one responded with an initial bursting pattern. One neuron displayed the Ih current during hyperpolarization. These three neurons did not display spontaneous action potentials, but exhibited excitatory and CYTH4 inhibitory post-synaptic currents (EPSCs and IPSCs). Based
on what we know about DH neurons our preliminary data suggest these DH neurons we were inhibitory interneurons. Considerable heterogeneity in firing patterns and spontaneous activity was observed in other DH neurons. Conclusions: In vivo patch clamp can be used to study the properties of DH neurons that receive input from the colon. Importantly, the patch clamp technique has the power to putatively classify neurons as excitatory or inhibitory and study their intrinsic and synaptic properties. This preparation will allow future detailed analysis of the mechanisms that determine DH neuron excitability in mice with normal and inflamed colons. 1. Farrell KE, Keely S, Graham BA, Callister R, Callister RJ: A systematic review of the evidence for central nervous system plasticity in animal models of inflammatory-mediated gastrointestinal pain. Inflamm Bowel Dis 2014; 20, 176–195. 2.