Sensory feedback synchronizes motor and sensory neuronal networks in the neonatal rat spinal cord

Abstract

Early stages of sensorimotor system development in mammals are characterized by the occurrence of spontaneous movements. Whether and how these movements support correlated activity in developing sensorimotor spinal cord circuits remains unknown. Here we show highly correlated activity in sensory and motor zones in the spinal cord of neonatal rats in vivo. Both during twitches and complex movements, movement-generating bursts in motor zones are followed by bursts in sensory zones. Deafferentation does not affect activity in motor zones and movements, but profoundly suppresses activity bursts in sensory laminae and results in sensorimotor uncoupling, implying a primary role of sensory feedback in sensorimotor synchronization. This is further supported by largely dissociated activity in sensory and motor zones observed in the isolated spinal cord in vitro. Thus, sensory feedback resulting from spontaneous movements is instrumental for coordination of activity in developing sensorimotor spinal cord circuits.

Figure 1. In vivo functional mapping of sensory-motor spinal cord zones.

(a) Schematic illustration of the experimental setup. We recorded spinal cord, translaminar network events, either evoked by mechanical stimulation or spontaneous (and in association with motor behaviour), following which we performed ISMS. (b) Silicone-based electrode array track visualized on a transverse spinal cord section (P6 rat). Left: ChAT⁺ cells—motoneurons (Alexa633, confocal image). Expression profiles of ChAT⁺ and NeuN⁺ cells were used to estimate borders of different spinal cord laminae (Supplementary Fig. 1). Right: Overlaid dark-field image and epi-fluorescence image (DiI, red) of the same slice and to-scale scheme of the electrode array (100 μm between recording sites), denoting the approximate anatomical location each recording site (dashed lines—Rexed laminae borders). Scale bar: 200 μm. (c) Mechanical stimulus (stim)-evoked MUA frequency represented as colour-coded dot overlaid on the corresponding stimulation point in a aged-matched hindlimb photograph (response map, single, anesthetized animal). Note that all firing frequencies correspond to the two recording sites showing the shortest response onsets, which were consistent across all stimulation points (depths of 100 and 200 μm). The response characterized by the highest MUA frequency (marked by #) was considered topographic and is described in more detail in d–e. (d) Stim-triggered mean LFP traces and CSD map (n=100 stimuli, single, non-anesthetized animal). (e) Corresponding normalized translaminar peri-stim time-histograms (PSTHs) of MUA. Note the duration of response across non-anesthetized animals (mean±s.d., n=10). A comparative description of sensory-evoked responses obtained in anesthetized animals is included in Supplementary Fig. 2. (f) Mean movement (mov) traces obtained for each depth of ISMS (n≈50 pulses per depth, under 1.5% isoflurane) overlaid on a respective normalized (colour-coded) amplitude map (single animal). Mov onsets and offsets were as graphed (mean±s.d., n=9 animals). (g) Occurrence, per depth, of mechanical stimulus-evoked MUA responses (light blue) and ISMS-evoked movements (black) per animal (n=9). (h) Left: Mean stim-evoked MUA frequency (light blue, n=15 animals) and ISMS evoked-mov amplitude (black, n=9 animals) per depth. Right: Corresponding mean stim-evoked response onsets and ISMS-evoked mov onsets per depth. *P<0.05 (Wilcoxon rank-sum test).

Figure 2. In vivo network correlates of spontaneous behaviour.

(a) Original wide-band LFP signal and MUA (filtered LFP, 300–4,000 Hz) corresponding to one sensory zone (sen) recording site (light-blue traces) and one motor zone (mot) recording site (black traces), and simultaneously recorded hindlimb movements (mov) signal (red: twitches, blue: complex mov, dark grey: short-lasting mov, light grey: baseline). (b) Spike waveforms for the two channels included in a (original wide-band LFP signal, mean±s.d., single animal). Left: sen spike-triggered sen and mot LFP. Right: mot spike-triggered mot and sen LFP. (c) Histogram of hindlimb movement durations (colour code as in a). (d) The twitch highlighted in a) is shown here in more detail (back vertical line: onset). (e) Mean LFP traces and CSD map triggered by twitch onset (n=330 twitches, single animal). (f) Corresponding normalized peri-event time-histograms (PETHs) of MUA, and mean twitch waveform (black and grey traces: mean±s.d.). (g) Top: normalized time-histograms of sen, intermediate zone (int), and mot MUA (black—mean; grey—individual animals; n=15 animals). Bottom: Sen, int and mot MUA peak frequency and return to baseline times in relation to twitch onset (mean±s.d., n=2217 twitches of 15 animals). (h) Top: Normalized cross-correlogram of mot and sen spikes referent to twitching epochs (black and grey—mean±s.e.m., n=15 animals); the mean peak lag was 54 ms. Bottom: Equivalent normalized cross-correlogram of mot and int spikes. (i) Exemplification of behavioural events classified as complex movements (highlighted a; black vertical lines—onsets); (j) Normalized time-histograms of MUA triggered by complex movement onset, and complex movement amplitude per bin (black and grey—mean±s.d., n=231 complex movements of a single animal). (k) Corresponding histograms of MUA for three main zones analyzed, for all animals (n=15). (l) Top: Normalized cross-correlogram of motor and sensory spikes occurring during complex movement epochs (black and grey traces: mean±s.e.m., n=15 animals); the mean peak lag was 41 ms. Bottom: Equivalent normalized cross-correlogram of mot and int spikes.

Figure 3. In vivo deafferentation suppresses spontaneous dorsal horn bursting.

(a) Schematic representation of the local deafferentation procedure performed in a subset of animals (n=3 of 15). (b) Mechanical stimulus (stim)-triggered mean LFP traces and corresponding normalized PSTHs of MUA before (Ctrl) and after deafferentation (Deaffer), (n≈100 stimuli per condition, single animal). Note the lack of stim-evoked responses in the after condition. (c) Global effect of deafferentation on bursting and mean firing frequencies in sensory (sen, light blue) and motor (mot, black) zones, graphed as percentage of control (mean±s.d., n=3 animals). (d) Effect of deafferentation on spinal cord network dynamics during twitching (single animal). Top: twitch onset-triggered mean LFP traces and CSD maps before and after deafferentation (n_{BeforeDeaffer}=66 and n_AfterDeaffer=108 twitches). Bottom: Corresponding normalized PETHs of MUA, and mean twitch waveforms (black and grey traces: mean±s.d.). (e) Effect of deafferentation on network dynamics during complex movement epochs (single animal). Normalized time-histograms of MUA aligned to complex movement onset (n_{BeforeDeaffer}=66 and n_AfterDeaffer=108 complex movements). (f) Top: normalized twitch onset-triggered time-histograms of sen, int and mot MUA before and after deafferentation (black and grey traces: mean±s.d., n_{BeforeDeaffer}=273 twitches and n_AfterDeaffer=321 twitches). Bottom: normalized complex movement onset-triggered histograms of sen, int and mot MUA before and after deafferentation (black and grey traces: mean±s.d., n_{BeforeDeaffer}=273 twitches and n_AfterDeaffer=321 twitches). (g) Cross-correlograms of peri-twitching and peri-movement mot and sen spikes before and after deafferentation (black and grey traces: mean±s.e.m.). (h) Effect of deafferentation on peri-twitching ventral spiking activity (% of control, mean±s.d.). Graphs showing, for mot, the twitches-related within-bursts MUA peak frequency (spikes ms⁻¹) and respective time in relation to twitch onset (latency), as well as bursts offset (peak frequency to baseline return—duration). (i) Spontaneous behaviour. Twitches and complex movements frequency (min⁻¹), maximal amplitude, power (normalized to duration) and duration after versus before transection of afferent fibers (% of control). ***P<0.001 (a full description of the statistical test used is included in the ‘Materials and methods’ section).

Figure 4. In vitro spontaneous patterns of activity.

(a) Original wide-band LFP traces representing stereotypical sensory zone (sen) bursts and heterogeneous motor zone (mot) events (S: short-lasting mot event; L: long-lasting mot even). (b) Autocorrelograms of spikes in selected sen and mot recording sites. (c) Histogram of inter-sensory event intervals (with a peak at 3 s). (d) Left: original wide-band LFP traces and MUA (red) across all spinal depths; the long-lasting mot event indicated in (a) is shown here in more detail. Top right: filtered LFP (1–15 Hz) trace and coherent MUA at a recording depth of 900 μm. Bottom right: Mean power spectrum of bursts referent to a selected mot recording site (mean±s.d., n≈760 bursts of 5 animals). (e) Exemplification of short-lasting mot events. Original wide-band LFP traces and MUA (red). (f) Sensory events-triggered mean LFP and CSD maps (single animal). (g,h) Equivalent analysis, with long-lasting or short-lasting motor events as triggers, respectively.

Figure 5. Dissociation of ongoing dorsal and ventral activities in vitro.

(a) Top: Sensory (sen)-bursts triggered normalized histograms of MUA across all spinal depths (single animal). Middle: corresponding normalized PETHs of MUA of sensory (Sen), intermediate (Int) and motor (Mot) zones (Z-scores, grey traces: single animals; black trace: mean, n=5 animals). Bottom: percentage of detected surrounding motor events (mean±s.d., n=5 animals). (b,c) Equivalent graphics instead triggered by long- and short-lasting motor events, respectively. (d) Left: Representative histograms of inter-sensory event intervals during non-bursting and bursting motor zone periods (black and orange, respectively, single animal). Right: Mean inter-event interval values during non-bursting and bursting motor zone periods per animal (n=4). (e) Cross-correlograms of all mot and sen units recorded per animal in vitro (mean±s.e.m., n=5 animals), in vivo under control conditions (mean±s.e.m., n=15 animals) and in vivo after deafferentation (mean±s.e.m., n=3 animals). **P<0.01 (paired two-tailed Student’s t-test).