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. 2015 Jul 1;87(1):111-23.
doi: 10.1016/j.neuron.2015.05.045. Epub 2015 Jun 18.

Activity Regulates the Incidence of Heteronymous Sensory-Motor Connections

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Free PMC article

Activity Regulates the Incidence of Heteronymous Sensory-Motor Connections

Alana I Mendelsohn et al. Neuron. .
Free PMC article

Abstract

The construction of spinal sensory-motor circuits involves the selection of appropriate synaptic partners and the allocation of precise synaptic input densities. Many aspects of spinal sensory-motor selectivity appear to be preserved when peripheral sensory activation is blocked, which has led to a view that sensory-motor circuits are assembled in an activity-independent manner. Yet it remains unclear whether activity-dependent refinement has a role in the establishment of connections between sensory afferents and those motor pools that have synergistic biomechanical functions. We show here that genetically abolishing central sensory-motor neurotransmission leads to a selective enhancement in the number and density of such "heteronymous" connections, whereas other aspects of sensory-motor connectivity are preserved. Spike-timing-dependent synaptic refinement represents one possible mechanism for the changes in connectivity observed after activity blockade. Our findings therefore reveal that sensory activity does have a limited and selective role in the establishment of patterned monosynaptic sensory-motor connections.

Figures

Figure 1
Figure 1. Patterns of Heteronymous Sensory-Motor Connections in the Anterolateral Crural Synergy Group
(A) Schematic of anterolateral crural muscle anatomy. TA = tibialis anterior. EDL = extensor digitorum longus. PL = peroneus longus. Image generated using The Mouse Limb Anatomy Atlas (Delaurier et al., 2008). (B) Lines of action of muscles in cat. Axes represent torques (in newton-meters) evoked by individual muscle nerve stimulation. Posterior crural muscles shown in grey. MG = medial gastrocnemius. LG = lateral gastrocnemius. Adapted from (Nichols, 1994). (C) Number of self and synergist motor neurons innervated by sensory afferents from a given muscle in p21 wild-type mice (n = 14-30 MNs; 3-7 mice per pair). (D) Density of sensory synaptic connections with self and synergist motor neurons in p21 wild-type mice. Each point represents one motor neuron (n as in (C)). Red lines indicate mean ± SEM for motor neurons receiving input. (E) Number of self and synergist motor neurons innervated by TA sensory afferents in p7 wild-type mice (n = 11-20 MNs; 2-3 mice per pair). (F) Density of TA sensory synaptic connections with self and synergist motor neurons in p7 wild-type mice (n as in (E)). (G) Schematic depicting organization of sensory-motor connectivity within the anterolateral crural synergy group. All data reported as mean ± SEM. For related data, see also Figure S1.
Figure 2
Figure 2. Functional Validation of Heteronymous Connections
(A) Schematic of lumbar spinal-hindlimb preparation. Stimulating electrodes were placed in TA, EDL, and PL muscles to activate proprioceptive fibers. Ventral roots were cut and placed into suction electrodes for either stimulation or recording. Motor neurons (green) were visually identified following muscle-specific labeling by CTB-488 and recorded intracellularly using whole-cell patch clamp. (B) Image of PL motor neurons retrogradely labeled at p0 with CTB-488 and showing three cells filled with intracellular dye after whole-cell recording. (C) Intracellularly recorded EPSPs in a single retrogradely labeled TA motor neuron upon stimulation of TA or EDL muscle. Traces averaged across 5 trials at 0.1 Hz. Black arrow indicates stimulus artifact. First dashed line indicates onset of EPSP response. Second dashed line indicates the maximum amplitude of the monosynaptic response, as determined at 3 ms after EPSP onset (Mears and Frank, 1997; Shneider et al., 2009). (D) Average EPSP amplitudes induced in TA motor neurons upon TA or EDL muscle stimulation (n = 4 MNs). Inset represents corresponding relationship within each recorded motor neuron. (E) Average latency of EPSP onset upon TA or EDL stimulation, as defined in relation to stimulus artifact. (F) Average ratio of the EPSP amplitude induced in each TA motor neuron by EDL stimulation to the EPSP amplitude induced by TA stimulation. (G) Intracellularly recorded EPSPs in a single retrogradely labeled PL motor neuron upon stimulation of PL or TA muscle. Single trials shown. The longer latency of response is due to the younger age at the time of recording. Conduction velocity increases with age due to a developmental increase in myelination (Li and Burke, 2002). (H) Average EPSP amplitudes induced in PL motor neurons upon PL or TA muscle stimulation (n = 3 MNs). (I) Average latency of EPSP onset upon PL or TA stimulation. Differences are significant at p = 0.02, indicating the lack of monosynaptic response from TA stimulation (Paired t-test). (J) Average ratio of the EPSP amplitude induced in each PL motor neuron by TA stimulation to the EPSP amplitude induced by PL stimulation. Scale bar represents 50 µm in (B). All data reported as mean ± SEM. For related data, see also Figure S2. For detailed methodology, see Supplemental Experimental Procedures.
Figure 3
Figure 3. Spatial Organization within the Anterolateral Crural Synergy Group
(A and C) Organization of motor pool positions from L3-L4 in p21 (A) and p1-p7 (C) wild-type mice after CTB and Rh-Dex injection into specific muscles. Standard spinal cord dimensions shown in µm. (B and D) Contour density plots showing the distribution of individual cell body positions at p21 (B) and p1-p7 (D). Position coordinates were determined as distance in micrometers with respect to the central canal and normalized to standard spinal cord dimensions. At both ages, X coordinates are significant between TA and PL, and EDL and PL at p < 0.001 (Student’s t-test). At both ages, Y coordinates are significant between TA and EDL, and TA and PL at p < 0.001 (Student’s t-test) (p21: TA: n = 99 MNs; 5 mice. EDL: n = 33 MNs; 4 mice. PL: n = 65 MNs; 4 mice. p1-p7: TA: n = 78 MNs; 5 mice. EDL: n = 32 MNs; 3 mice. PL: n = 30 MNs; 2 mice). Scale bars represent 30 µm in (A) and (C). For related data, see also Figure S3.
Figure 4
Figure 4. Sensory-Motor Synapses Maintained Following Tetanus Toxin Expression in Proprioceptors
(A) Axon trajectories of proprioceptor afferents in p8 wild-type and PvTeNT mice. (B) Pv+/vGluT1+ boutons in contact with ChAT+ motor neurons in p18 PvTeNT mice. (C) Number of vGluT1+ boutons on the soma and proximal ~100 µm dendrites in p21 wild-type and p18 PvTeNT mice (TA: WT n = 48 MNs, PvTeNT n = 40. EDL: WT n = 43, PvTeNT n = 25. PL: WT n = 52, PvTeNT n = 24). (D) Density of vGluT1+ boutons on motor neuron surface in p18 wild-type and PvTeNT mice (both WT and PvTeNT: n = 9 MNs; 3 mice). (E) vGluT1+ boutons in contact with ChAT+ motor neurons no longer express VAMP 1 and VAMP 2 in p18 PvTeNT mice. Scale bars represent 100 µm in (A), 5 µm in (B) and 2 µm in (E). All data reported as mean ± SEM. For related data, see also Figure S4.
Figure 5
Figure 5. Tetanus Toxin Expression in Proprioceptors Blocks Sensory-Motor Transmission
(A and B) Extracellular recordings from ventral root L5 following dorsal root L5 stimulation in p8 wild-type and PvTeNT mice. Trace averaged across 5 trials at 0.1 Hz. Black arrow indicates stimulus artifact. Traces are shown in a time expanded scale in (B). (C) Reflex amplitude is reduced by 92% in p8 PvTeNT mice. Differences are significant at p < 0.001 (Student’s t-test, n = 3 mice). (D and E) Intracellular recordings from L4 motor neurons following dorsal root L4 stimulation in p4 wild-type and PvTeNT mice. Single trials shown. Traces are shown in a time expanded scale in (E). First dashed line indicates onset of EPSP response. Second dashed line indicates the maximum amplitude of the monosynaptic response, as determined at 3 ms after EPSP onset. (F) Monosynaptic EPSP amplitude is reduced by 96% in p4 PvTeNT mice. Differences are significant at p < 0.001 (Student’s t-test; n = 4 MNs). All data reported as mean ± SEM. For related data, see also Figure S5.
Figure 6
Figure 6. Increased Incidence of Heteronymous Connections Following Transmission Blockade
(A) Percentage of motor neurons contacted by TA sensory afferents in p21 WT and p18 PvTeNT mice (WT: data as in Figure 1C. PvTeNT: n = 3-5 mice). Difference in percentage of EDL motor neurons receiving TA input is significant at p = 0.003 (Student’s t-test). (B) Percentage of motor neurons contacted by TA sensory afferents in p7 WT and PvTeNTmice (WT: data as in Figure 1E. PvTeNT: 3-4 mice). Difference in EDL motor neurons receiving TA input is significant at p = 0.008 (Student’s t-test). (C) Density of TA sensory input to TA motor neurons in p7 wild-type and PvTeNTmice. Each point represents one motor neuron (WT: n = 11 MNs, as in Figure 1F. PvTeNT: n = 14 MNs). Red lines indicate mean ± SEM for motor neurons receiving TA input. (D) Density of TA sensory input to EDL motor neurons in p7 wild-type and PvTeNT mice (WT: n = 19 MNs, as in Figure 1F. PvTeNT: n = 20 MNs). For EDL motor neurons contacted by TA sensory afferents, the density of contacts increases ~2-fold (p = 0.05; Student’s t-test). (E) In the absence of neurotransmission, sensory afferents contact a greater proportion of heteronymous motor neurons and initially contact each heteronymous neuron with increased density. All data reported as mean ± SEM. For related data, see also Figure S6.
Figure 7
Figure 7. A Spike-Timing Model of Sensory-Motor Refinement
(A) Schematic of the spike-timing dependent plasticity model. A motor neuron (grey) receives sensory input from homonymous (blue) and heteronymous (red) sensory afferent populations. The strength of the sensory-motor synapses is subject to STDP, resulting in a dependence on the phase relationship between the firing patterns of the sensory afferent and the motor neuron. (B) Long-tailed STDP model. The horizontal axis is the difference tpre-tpost in ms between the pre- and postsynaptic spike times. The vertical axis is the change in synaptic strength relative to the maximal strength produced by a single spike pair. (C) Simulated motor neuron spike train. The neuron fires a burst of ~6 spikes twice per second. (D) Distribution of sensory afferent firing phases relative to the phase of the simulated motor neuron activity. On each cycle, homonymous and heteronymous afferent activity was phase shifted relative to the motor oscillation by a random amount chosen from a zero-mean Gaussian distribution with a standard deviation of 10 or 15 degrees, respectively (n = 100 each). The distributions shown have been normalized to a peak value of 1. (E) Sensory-motor synapse strengths shortly after application of STDP model (left), and following application of model over 200 minutes of simulated muscle contraction (right). Synaptic strengths are reported in units of the maximum allowed synaptic strength (1 nS), and all synapses were set to a relative strength of 1.0 at the beginning of the simulation. (F) Percentage of sensory-motor synapses refined during a representative application of the STDP model.

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