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

Abstract

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.

Structural and functional identification of two distinct inspiratory neuronal populations at the level of the phrenic nucleus in the rat cervical spinal cord

“…Inspiratory depolarizing signals observed by voltage imaging in both central and medial motor areas in the present study could be attributed to inspiratory activity of not only motoneurons, but interneurons. Our finding is compatible with the notion by Streeter et al ( 2017 ), and it is considered that the motoneurons and interneurons together form inspiratory neural output patterns in the phrenic and scalene microcircuits.…”

“…However, due to technical limitations, the presence of interneurons within the phrenic nucleus has not been thoroughly studied either physiologically or anatomically (Lane 2011 ). Recently, Streeter et al ( 2017 ) combined the techniques of multielectrode array recording and histochemistry, and mapped interneuron populations in the mid cervical spinal cord in adult rats. They suggested that a number of interneurons generate synchronous respiratory discharge with mono- and polysynaptic connections among neurons.…”

Disproportionate loss of excitatory inputs to smaller phrenic motor neurons following cervical spinal hemisection

“…In addition to excitatory inputs from the rVRG, a complex network of interneurons throughout the spinal cord send excitatory inputs to PhMNs and can modulate DIAm activity (see reviews (Jensen et al 2019;Sunshine et al 2020)). There exist anatomical projections from interneurons in upper cervical segments to PhMNs (Dobbins & Feldman, 1994;Lane et al 2008a,b), constituting a premotor interneuron population that does not mediate inspiratory drive but may affect PhMN excitability (Lu et al 2004;Gonzalez-Rothi et al 2017;Streeter et al 2017). Following spinal cord injury, an increase in the excitatory input from these spinal interneurons may be important in recovery (Jensen et al 2019).…”

Intraspinal microstimulation for respiratory muscle activation

“…The respiratory phase dependence of these long-latency activations may reflect more than just inspiratory-expiratory oscillations in phrenic motoneuron excitability (Berger, 1979). Respiratory-related bursting patterns are often reported in cervical interneurons (Duffin and Iscoe, 1996; Hayashi et al, 2003; Hilaire et al, 1986; Lane et al, 2009; Palisses and Viala, 1987; Sandhu et al, 2015; Streeter et al, 2017b) and at least a portion of these cells can have direct input from the ventral respiratory group in the medulla (Duffin and Iscoe, 1996). Thus, oscillations in the excitability of the cervical propriospinal network, and specifically increased excitability during the inspiratory phase, may enable polysynaptic activations during inspiratory ISMS.…”

“…These longer latencies are consistent with activation of polysynaptic excitatory pathways (Borst et al, 1995; Borst and Sakmann, 1996; Meinrenken et al, 2003). Polysynaptic spinal activation of phrenic motoneurons is plausible since both neurophysiological (Marchenko et al, 2015; Sandhu et al, 2015; Streeter et al, 2017b) and anatomical data (Dobbins and Feldman, 1994; Lane et al, 2008; Lois et al, 2009) have shown mid cervical interneurons which are synaptically coupled with the phrenic motor pool. There is currently relatively little evidence, however, that cervical interneurons convey brainstem “respiratory drive” to phrenic motoneurons during spontaneous breathing (Duffin and Iscoe, 1996).…”