Coupling multielectrode array recordings with silver labeling of recording sites to study cervical spinal network connectivity

Midcervical spinal interneurons form a complex and diffuse network and may be involved in modulating phrenic motor output. The intent of the current work was to enable a better understanding of midcervical "network-level" connectivity by pairing the neurophysiological multielectrode array (MEA) data with histological verification of the recording locations. We first developed a method to deliver 100-nA currents to electroplate silver onto and subsequently deposit silver from electrode tips after obtaining midcervical (C3-C5) recordings using an MEA in anesthetized and ventilated adult rats. Spinal tissue was then fixed, harvested, and histologically processed to "develop" the deposited silver. Histological studies verified that the silver deposition method discretely labeled (50-μm resolution) spinal recording locations between laminae IV and X in cervical segments C3-C5. Using correlative techniques, we next tested the hypothesis that midcervical neuronal discharge patterns are temporally linked. Cross-correlation histograms produced few positive peaks (5.3%) in the range of 0-0.4 ms, but 21.4% of neuronal pairs had correlogram peaks with a lag of ≥0.6 ms. These results are consistent with synchronous discharge involving mono- and polysynaptic connections among midcervical neurons. We conclude that there is a high degree of synaptic connectivity in the midcervical spinal cord and that the silver-labeling method can reliably mark metal electrode recording sites and "map" interneuron populations, thereby providing a low-cost and effective tool for use in MEA experiments. We suggest that this method will be useful for further exploration of midcervical network connectivity. We describe a method that reliably identifies the locations of multielectrode array (MEA) recording sites while preserving the surrounding tissue for immunohistochemistry. To our knowledge, this is the first cost-effective method to identify the anatomic locations of neuronal ensembles recorded with a MEA during acute preparations without the requirement of specialized array electrodes. In addition, evaluation of activity recorded from silver-labeled sites revealed a previously unappreciated degree of connectivity between midcervical interneurons.