Visual patch clamp recording of neurons in thick portions of the adult spinal cord

The study of visually identified neurons in slice preparations from the central nervous system offers considerable advantages over in vivo preparations including high mechanical stability in the absence of anaesthesia and full control of the extracellular medium. However, because of their relative thinness, slices are not appropriate for investigating how individual neurons integrate synaptic inputs generated by large numbers of neurons. Here we took advantage of the exceptional resistance of the turtle to anoxia to make slices of increasing thicknesses (from 300 to 3000 μm) from the lumbar enlargement of the spinal cord. With a conventional upright microscope in which the light condenser was carefully adjusted, we could visualize neurons present at the surface of the slice and record them with the whole-cell patch clamp technique. We show that neurons present in the middle of the preparation remain alive and capable of generating action potentials. By stimulating the lateral funiculus we can evoke intense synaptic activity associated with large increases in conductance of the recorded neurons. The conductance increases substantially more in neurons recorded in thick slices suggesting that the size of the network recruited with the stimulation increases with the thickness of the slices. We also find that that the number of spontaneous excitatory postsynaptic currents (EPSCs) is higher in thick slices compared with thin slices while the number of spontaneous inhibitory postsynaptic currents (IPSCs) remains constant. These preliminary data suggest that inhibitory and excitatory synaptic connections are balanced locally while excitation dominates long-range connections in the spinal cord.