Optical mapping of neural network activity in chick spinal cord at an intermediate stage of embryonic development

We have applied multiple-site optical recording of transmembrane potential changes to recording of neuronal pathway/network activity from embryonic chick spinal cord slice preparations. Spinal cord preparations were dissected from 8-day-old chick embryos at Hamburger-Hamilton stage 33, and transverse slice preparations were prepared with the 13th cervical spinal nerve or with the 2nd or 5th lumbosacral spinal nerve intact. The slice preparations were stained with a voltage-sensitive merocyanine-rhodanine dye (NK2761). Transmembrane voltage-related optical (dye-absorbance) changes evoked by spinal nerve stimulation with positive square-current pulses using a suction electrode were recorded simultaneously from many loci in the preparation, using a 128- or 1,020-element photodiode array. Optical responses were detected from dorsal and ventral regions corresponding to the posterior (dorsal) and anterior (ventral) gray horns. The optical signals were composed of two components, fast spike-like and slow signals. In the dorsal region, the fast spike-like signal was identified as the presynaptic action potential in the sensory nerve and the slow signal as the postsynaptic potential. In the ventral region, the fast spike-like signal reflects the antidromic action potential in motoneurons, and the slow signal is related to the postsynaptic potential evoked in the motoneuron. In preparations in which the ventral root was cut microsurgically, the antidromic action potential-related optical signals were eliminated. The areas of the maximal amplitude of the evoked signals in the dorsal and ventral regions were located near the dorsal root entry zone and the ventral root outlet zone, respectively. Quasiconcentric contour-line maps were obtained in the dorsal and ventral regions, suggesting the functional arrangement of the dorsal and ventral synaptic connections. Synaptic fatigue induced by repetitive stimuli in the ventral synapses was more rapid than in the dorsal synapses. The distribution patterns of the signals were essentially similar among C13, LS2, and LS5 preparations, suggesting that there is no difference in the spatiotemporal pattern of the neural responses along the rostrocaudal axis of the spinal cord at this developmental stage. In the ventral root-cut preparations, comparing the delay times between the ventral slow optical signals, we have been able to demonstrate that neural network-related synaptic connections are generated functionally in the embryonic spinal cord at Hamburger-Hamilton stage 33.