Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 27 (24), 6521-30

Segmental, Synaptic Actions of Commissural Interneurons in the Mouse Spinal Cord

Affiliations

Segmental, Synaptic Actions of Commissural Interneurons in the Mouse Spinal Cord

Katharina A Quinlan et al. J Neurosci.

Abstract

Left-right alternation depends on activity in commissural interneurons (CINs) that have axons crossing in the midline. In this study, we investigate the CIN connectivity to local motor neurons using a newly developed preparation of the in vitro neonatal mouse spinal cord that allows us to identify all classes of CINs. Nineteen of 29 short-range CINs with axonal projections <1.5 segments (sCINs) directly excited, directly inhibited, or indirectly inhibited contralateral motor neurons in the quiescent spinal cord. Excitation was glutamatergic and inhibition was mixed glycinergic and/or GABAergic. Long-range CINs were also found to have input to local, contralateral motor neurons. Thirteen of 29 descending CINs had similar synaptic connectivity to contralateral motor neurons as the sCINs, including direct excitation and direct and indirect inhibition. Some (9 of 23) rostrally projecting ascending CINs, and a few (2 of 10) CINs with bifurcating axons that both ascend and descend, indirectly inhibited local, contralateral motor neurons. Rhythmic firing during locomotor-like activity was observed in a number of CINs with segmental synaptic effects on contralateral motor neurons. This study outlines the basic connectivity pattern of CINs in the mouse spinal cord on a segmental level. Our study suggests that, based on observed synaptic connectivity, both short- and long-range CINs are likely involved in segmental left-right coordination and that the CIN system is organized into a dual-inhibitory and single-excitatory system. These systems are organized in a way that they could provide appropriate coordination during locomotion.

Figures

Figure 1.
Figure 1.
Experimental setup. A, Shown schematically, the stimulating electrodes (S) were positioned on rostral and caudal hemicords contralateral from the site of the patch electrode (IC), as well as on the ventral surface of the midline. Extracellular recording electrodes (R) on both left and right ventral roots were used in both AC and DC modes. B, A photograph of the preparation. A total of three intact segments remained in the spinal cords used. Scale bar, (red) 0.5 mm.
Figure 2.
Figure 2.
Differential antidromic activation from each of the CIN categories in the presence of glutamate antagonists. A, Antidromic spikes in dCINs were elicited from the midline (2) and the caudal, contralateral stimulating electrode (3). B, aCINs were identified from spikes with stimulation of rostral hemicord (1) and midline (2). C, Bifurcating adCINs responded to stimulation from all points with an antidromic spike. D, sCINs spiked only with midline stimulation, indicating that the contralateral projections extended <1.5 segments.
Figure 3.
Figure 3.
Examples of neurons recorded in this study. All are pictured in the transverse plane, with the ventral side to the bottom. A, An ipsilaterally projecting neuron with processes close to midline. B–D, Examples of sCINs with no postsynaptic effect on motor neurons on the contralateral side. The cell in B is filled with fluorescent tracer Alexa 568. The arrows mark axonal trajectory and the dotted line indicates the midline. Cells in A and C–E are filled with neurobiotin. E, sCIN with postsynaptic effects on motor neurons on the contralateral side.
Figure 4.
Figure 4.
Three examples of synaptic responses in contralateral motor neurons to sCIN firing. Traces shown are averages of DC ventral root recordings of between 300 and 1000 responses to sCIN spikes. Vertical line indicates the time of the sCIN spike, or time 0 in the spike-triggered average. Traces are the contralateral responses to one sCIN and are shown in chronological order from top to bottom. A, Depolarizing excitatory response (top trace) abolished with CNQX (20 μm) and AP5 (30 μm) that recovers and persists in AP5 and mephenesin (1 mm), indicating that the response is monosynaptic. B, Hyperpolarizing inhibitory response nearly completely blocked with bicuculline that recovers and persists in CNQX and AP5. C, Indirect inhibitory response, which was blocked by CNQX and AP5.
Figure 5.
Figure 5.
Three examples of synaptic responses in contralateral motor neurons to dCIN firing. Same setup as in Figure 4. A, Hyperpolarizing inhibitory response (top) that persists in CNQX and AP5 is partially blocked by strychnine and completely blocked by both strychnine and bicuculline. The short-latency “depolarizing response” that remained after blocking the glutamatergic transmission is part of the stimulus artifact. B, Inhibitory response that is attenuated by CNQX and AP5, recovers, and then is partially blocked by strychnine and completely blocked with bicuculline and strychnine. C, Excitatory response blocked with CNQX and AP5.
Figure 6.
Figure 6.
Rhythmic activity in sCIN during locomotion. A, Raw traces of activity in the ventral root (top) contralateral to the patched sCIN (bottom). Spikes in the sCIN are firing out of phase with the contralateral ventral root activity. B, Circular plot of the activity of the cell in A. The preferred firing phase is 241°.
Figure 7.
Figure 7.
Locations of the CINs recorded in the ventromedial spinal cord. All neurons were patched in lumbar segments L2 and L3. A, The location of recorded CINs grouped by axonal projection. B, Location of CINs grouped by neurotransmitter phenotype. CINs were distributed throughout the ventromedial area without any apparent grouping by neurotransmitter phenotype or axonal projection.
Figure 8.
Figure 8.
Typical sCIN responses to current pulses. The CINs were stimulated by two second pulses current pulses given in 20 pA steps. Traces from two representative sCINs are shown. A, sCIN displaying “sag” in response to hyperpolarizing pulses and PIR (top, intracellular recording; bottom, current monitor) (RP, −50 mV). B, sCIN displaying only postinhibitory rebound (RP, −52 mV). Arrows indicate PIR. Calibration applies to both A and B.
Figure 9.
Figure 9.
Summary diagram of local CIN connections. Inhibitory synapses are represented by closed circles whereas excitatory synapses are represented by flat lines. MN, Motor neuron; RC, Renshaw cells; IIN, ipsilateral inhibitory interneuron. The figure is modified from Kiehn (2006).

Similar articles

See all similar articles

Cited by 56 articles

See all "Cited by" articles

Publication types

MeSH terms

Substances

LinkOut - more resources

Feedback