Mid-cervical interneuron networks following high cervical spinal cord injury

Abstract

Spinal interneuron (IN) networks can facilitate respiratory motor recovery after spinal cord injury (SCI). We hypothesized that excitatory synaptic connectivity between INs located immediately caudal to unilateral cervical SCI would be most prevalent in a contra- to ipsilateral direction. Adult rats were studied following chronic C2 spinal cord hemisection (C2Hx) injury. Rats were anesthetized and ventilated and a multi-electrode array was used to simultaneously record INs on both sides of the C4-5 spinal cord. The temporal firing relationship between IN pairs was evaluated using cross-correlation with directionality of synaptic connections inferred based on electrode location. During baseline recordings, the majority of detectable excitatory IN connections occurred in a contra- to- ipsilateral direction. However, acute respiratory stimulation with hypoxia abolished this directionality, while simultaneously increasing the detectable inhibitory connections within the ipsilateral cord. We conclude that propriospinal networks caudal to SCI can display a contralateral-to-ipsilateral directionality of synaptic connections and that these connections are modulated by acute exposure to hypoxia.

Keywords: Cervical interneurons; Connectivity; Plasticity; Spinal cord injury.

Fig. 1.

Spinal segments C1–C5 illustrating a unilateral C2 hemisection (C2Hx) injury, C3, C4, C5 cervical rootlets, phrenic nerve output and multi-electrode array recordings at C4/C5 (center panel). Representative ipsilateral and contralateral recordings from 13 mid-cervical spinal INs and their corresponding locations within the cervical spinal cord.

Fig. 2.

A. Schematic illustrating bilateral phrenic nerve recordings following C2Hx. B. Representative compressed raw and integrated phrenic neurograms and instantaneous burst frequency before, during, and after hypoxia. C. Average phrenic burst frequency during the center 50 breaths of each experimental time point. D, E. Ipsilateral and contralateral phrenic traces showing the average (center line) and standard error of the mean (SEM; shaded region) and individual data points for phasic inspiratory nerve activity during baseline, hypoxia (shaded yellow) and post-hypoxia. * Significantly different from ipsilateral; ^ significantly different from baseline; # significantly different from hypoxia. All symbols denote P < 0.05.

Fig. 3.

A. A schematic illustrating the method for multi-electrode array recordings of spinal INs following C2Hx. B. Average firing rate (pulses per second) of all ipsilateral and contralateral spinal INs before, during (shaded yellow), and after hypoxia. C. Representative compressed firing rate traces, from one experiment illustrating hypoxia-activated neurons (i.e. those which increase firing during hypoxia) and hypoxia-inhibited INs (i.e. those which decrease firing during hypoxia). D. Averaged firing rate of activated and E. inhibited INs before, during (shaded yellow), and after hypoxia. Post-hoc analysis indicated ipsilateral INs had a lower burst rate during the post-hypoxic period as compared to baseline (P = 0.0195, indicated by *).

Fig. 4.

A. Schematic illustrating cross-correlation analysis of bilateral multi-electrode array recordings of spinal INs following C2Hx. For each pair of neurons, the trigger cell (open circle) was used to form the correlation histogram, and the target cell (filled circle) fired in synchrony or after the trigger cell. Based on the recording location of the cell soma, a directionality (ex: ipsi to contra) of the connection was assigned. B, C. The proportion of excitatory, and D, E. inhibitory connections (as a % of possible connections) during baseline, hypoxia (shaded yellow) and post-hypoxia across the hemicord (ipsi-contra; contra-ipsi) and within each hemicord (ipsi-ipsi; contra-contra). The number of excitatory/inhibitory connections present at each time is noted in each bar. Note: the total number of possible connections varies within each group at each time point. * Denotes P < 0.05.