Frequency-dependent recruitment of V2a interneurons during fictive locomotion in the mouse spinal cord

Abstract

The principles governing the recruitment of interneurons during acceleration in vertebrate locomotion are unknown. In the mouse, the V2a spinal interneurons are dispensable for left-right coordination at low locomotor frequencies, but their function is essential for maintaining left-right coordination at high frequencies. Here we explore the mechanisms driving this frequency-dependent role using four methods to determine how V2a interneurons are recruited at different locomotor frequencies. We show that half of the V2a interneurons receive rhythmic locomotor synaptic drive, which increases with cycle frequency, recruiting more of the neurons to fire at higher frequencies. The other V2a interneurons do not receive locomotion-related synaptic drive and are not recruited into the locomotor network at any frequency. The increased role of V2a interneurons at higher locomotor frequencies arises from increased synaptic drive to recruit subthreshold oscillating V2a neurons, and not from recruitment of a second set of silent V2a interneurons.

Figure 1

Firing patterns of V2a interneurons during tonic BES-induced fictive locomotion. (a) Recording of a rhythmically firing V2a interneuron and its associated L2 ventral root activity during tonic BES induced fictive locomotion. The smoothed neuronal trace (S-V2a) is shown below the real recording. The ventral root activity is integrated and smoothed and shown under the VR recordings (∫iL2). (b) Recording of a subthreshold rhythmic V2a interneuron and the associated L2 ventral root activity during tonic BES induced fictive locomotion. (c) Recording of a non-rhythmic tonically firing and L2 ventral root activity during tonic BES induced fictive locomotion. (d) Recording of a silent V2a interneuron and L2 ventral root activity during tonic BES induced fictive locomotion. (e) Circular plot of activity of theV2a interneuron from a. The gray area indicates the ipsilateral L2 activity phase in the cycle. (f) Cross-correlogram of the smoothed membrane potential oscillations of the subthreshold V2a interneurons with the rectified and smoothed ventral root output from b. (g) Circular plot of activity of the V2a interneuron from c. The gray area indicates the ipsilateral L2 activity phase in the cycle. (h) Cross-correlogram of the smoothed membrane potential oscillations of the silent V2a interneurons and the rectified and smoothed ventral root output from d. (i) Summary of the firing phase and rhythmicity of all V2a interneurons that fired action potentials during BES induced fictive locomotion. The dashed line shows the r value for statistically significant rhythmicity. Filled circles: significantly rhythmic (as in a); open circles, not rhythmic (as in c). (j) Relation between locomotor cycle frequency and circular plot r-values of 45 V2a interneurons during tonic BES induced fictive locomotion. The dashed line indicates the r value for statistically significant rhythmicity. (k) Subthreshold V2a interneurons have lower input resistance than those which fire action potentials during low frequency BES induced fictive locomotion.

Figure 2

Subthreshold V2a interneurons during low frequency fictive locomotion are recruited at higher frequencies. (a) Activity of a subthreshold V2a interneuron and the associated ipsilateral ventrol root activity during fictive locomotion induced by 8μM NMDA and 8μM 5-HT. (b) The cross-correlogram between the smoothed membrane potential oscillations of the V2a interneurons and the rectified and smoothed ventral root output from a. (c) Recording of the same V2a interneuron and ventral root at a higher locomotor frequency after raising the NMDA/5-HT concentrations to 10μM each. (d) The cross-correlogram between the smoothed membrane potential oscillations of the V2a interneurons and the rectified and smoothed ventral root output from c. (e) Current traces of the same V2a interneuron using voltage clamp recording at lower fictive locomotor frequencies. The is shown beneath the voltage clamp trace. (f) Current traces of the same V2a interneuron using voltage clamp recording at higher fictive locomotor frequencies, as shown by the smoothed and rectified ventral root activity below the trace. (g) Averaged current oscillation of the V2a interneuron at the lower cycle frequency. (h) Averaged current oscillation of the V2a interneuron during a single cycle (shown by integrated ventral root activity beneath it) at the higher cycle frequency. (i) Relationship between the action potential number per cycle and the locomotor cycle frequency for this interneuron.

Figure 3

Enhanced activity of rhythmically firing V2a interneurons with increased locomotor frequency. (a) Recording of a rhythmically firing V2a interneuron during fictive locomotion induced by 8μM NMDA/5-HT, with ipsilateral ventral root trace shown beneath it. (b) This V2a interneuron increases its spike frequency when the NMDA/5-HT concentrations are raised to 10 μM. (c) In 6 cords, the locomotor cycle frequency increased as the concentration of NMDA/5-HT was increased (n=6; P=0.0013: ^**, paired t-test). Larger circles show mean values at lower and higher drug concentrations. (d) V2a action potential number per cycle is increased in 5 of 6 neurons after increasing the NMDA/5-HT concentration (n=6; P=0.007: ^*). Data are expressed as mean ± SD.

Figure 4

Response of a subthreshold V2a interneuron as the locomotor cycle frequency increases during slow wash-in of 10 μM NMDA/5-HT. (a) Initiation and acceleration of bursting in a V2a interneuron as fictive locomotion accelerates during 10μM NMDA/5-HT application. In this preparation, the fictive locomotion starts slowly and gradually increases with time. S-V2a: Smoothed and filtered V2a activity. iL2: Activity of the ipsilateral ventral root. ∫iL2: integrated and rectified iL2 activity. (b) Activity of this V2a interneuron at the locomotor frequency indicated by the gray area 1 in a. (c) Activity of this V2a interneuron at the locomotor frequency indicated by the gray area 2 in a. (d) Activity of this V2a interneuron at the locomotor frequency indicated by the gray area 3 in a. (e) Cross-correlogram between the smoothed V2a membrane potential oscillations and the rectified and smoothed ventral root output from b. (f) Cross-correlograms between the smoothed V2a membrane potential oscillations and the rectified and smoothed ventral root output from c. (g) Cross-correlograms between the smoothed V2a membrane potential oscillations and the rectified and smoothed ventral root output from d. (h) Averaged membrane potential oscillations during one locomotor cycle from b-d. (i) Summary of the relationship of the V2a membrane oscillation amplitude and cycle frequencies from 4 different cords. (j) Relationship between the action potential number per cycle of the V2 interneuron from a and locomotor cycle frequency.

Figure 5

V2a interneurons fire action potentials at the initial higher frequencies during brief BES induced locomotor bouts. (a) Response of a rhythmic V2a interneuron during brief high-frequency BES-induced locomotor bouts. BES stimulation (10Hz, 4ms, 1.5mA, 1s duration) was applied before the start of the recording. Below the V2a recording are the iL2 and integrated iL2 recordings. The initial cycle frequency is high as 1Hz, and gradually decreases until the bout ceases. The interneuron is more active at the higher initial cycle frequencies. (b) Response of another rhythmic V2a interneuron during brief high-frequency BES-induced locomotor bouts. BES stimulation was applied before the start of the recording. The initial higher cycle frequency is around 0.5Hz and gradually decreases until the bout ceases. This interneuron is also more active at the higher initial cycle frequencies. (c) Summary of relationship between action potential number per cycle and cycle frequency from 8 preparations. Each shape indicates a different V2a interneuron.

Figure 6

V2a interneurons increase their firing frequency with spontaneous frequency increases during tonic BES-induced fictive locomotion. (a) Recording of a rhythmic V2a interneuron during tonic BES induced fictive locomotion when the frequency spontaneously increased as seen in the shaded area. S-V2a: Smoothed and filtered V2a activity. iL2: Activity of the ipsilateral ventral root. ∫iL2: integrated and rectified iL2 activity. (b) Change in V2a action potential number per cycle and cycle frequency during each cycle of the spontaneous increase, from a. (c) Increase in action potential number per cycle as frequency spontaneously increased.

Figure 7

V2a interneurons that do not receive locomotor-related synaptic drive at lower locomotor frequencies are not recruited at higher frequencies. (a) Activity of a tonically firing V2a interneuron during 8μM NMDA/5-HT induced fictive locomotion. (b) Increase in tonic activity but not rhythmicity after the cycle frequency is accelerated by 10μM NMDA/5-HT. (c) Voltage clamp recording of this V2a interneuron during fictive locomotion induced by 10μM NMDA/5-HT. (d) Circular plot of the V2a interneuron from a, showing no rhythmicity in firing. (e) Circular plot of the V2a interneuron from b, showing no rhythmicity in firing at the elevated cycle frequency. (f) Recording from a silent V2a interneuron during fictive locomotion induced by lower concentrations of NMDA and 5-HT (7 μM). (g) Recording from the same V2a interneuron during faster fictive locomotion induced by higher concentrations of NMDA and 5-HT (9 μM). (h) Voltage clamp recording from this neuron during fictive locomotion induced by 9μM NMDA/5-HT. (i) Response of a tonic V2a interneuron during a brief high frequency BES-induced locomotor bout. (j) Response of a silent V2a interneuron during a brief high frequency BES-induced locomotor bout. Neither neuron becomes rhythmically active.