Spike-timing dependent plasticity in primate corticospinal connections induced during free behavior

Strengthening of corticospinal connections through spike-triggered stimulation

Figure 2A illustrates the output effect in SpTAs of three muscles from a representative CM cell that had an excitatory effect on two flexor muscles and an inhibitory linkage to an extensor muscle. The magnitude and shape of SpREs from this CM cell were stable for two days prior to conditioning (cf. Day-1 and Day0). After the first 22 hours of spike-triggered stimulation, there was a statistically significant increase in all the SpREs, both facilitation and suppression (cf. Day1 vs Day0). The facilitation increased further after a second day of conditioning (Day2). The day after the end of conditioning (Day3) the SpREs in one muscle (FCU) remained above preconditioning levels.

The SpREs of CM cells could include “synchrony effects”, whose onsets were too early relative to the triggering spike to be mediated by the triggering cell, as seen in FCU and PL muscles of Fig. 2A. These early effects are mediated by other CM cells that are synchronized with the trigger cell. Cross-correlograms of synchronized CM cells typically show central peaks straddling the reference cell (Jackson et al., 2003; Smith and Fetz, 2009 and Supplementary Fig. S1B), so the average time of the synchronized spikes would be the same as those of the recorded CM cell. Thus, the conditioning paradigm would also strengthen terminals of the synchronized cells and lead to increases in the synchrony effects (see supplementary material for further discussion). Additional examples of increases in both PSpF and synchrony effects are shown in Supplementary Fig. S2.

To confirm that the terminals of the triggering CM cell alone could be strengthened, we also measured the effects of conditioning on a pure serial PSpF, shown in Fig. 2C. In this case the PSpF was re-measured after a relatively brief conditioning period of 3.5 hours, which proved sufficient to enhance the PSpF (cf. Day0’ with Day0). Moreover, in this case the increase in PSpF lasted over 24 hours following conditioning (cf. Day1 with Day0). These changes were confirmed a second time on the subsequent days after further conditioning (Day1 and Day2).

We investigated the effects of this conditioning paradigm in 19 different sessions, using 15 different pairs of CM cells and spinal sites in two monkeys. Conditioning produced significant changes in the 16 sessions that had appropriate spike-stimulus delays (for details of all sessions see Supplementary Table S1). The amplitudes of spike-related facilitation and suppression were both found to increase after overnight conditioning with spike-stimulus delays between 12 and 25 ms. The increased output effects were sustained through multiple successive days of conditioning (Fig. 2A and Supplementary Fig. S2). Fig. 3 plots the sizes of the SpREs before and after conditioning in all the sessions, obtained with spike-stimulus delays between 0 and 500 ms. For delays between 12 and 25 ms the points are distributed above the unity line, indicating increases in the SpRE, for both facilitation (Fig. 3Aa) and suppression (Fig. 3Ab). These changes tended to be sustained 24 hours after the termination of conditioning (Fig. 3B), despite the resumption of normal activity. The SpREs remained larger than their original amplitudes 24 hours after the end of conditioning in 71.4 % of the cases tested (Facilitation: 16 of 22; Suppression: 4 of 6) (Fig. 2A and ​and3B).3B). The SpREs for those cells tested after a second post-conditioning day are plotted individually in Fig. 3C. The SpREs remained larger than their original amplitudes 48 hours after the end of conditioning in 44.4 % of the cases tested (Facilitation: 2 of 7; Suppression: 2 of 2).

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Figure 3

Summary of conditioning effect on output

A: MPI of spike-related effects in SpTAs compiled before and after conditioning (pre MPI and post MPI). Facilitation in a; Suppression in b. Data points are color coded for different spike-stimulus delays. Data points above the dashed line indicate SpREs that increased after conditioning. The red arrow identifies session in Fig. 2A. For sessions involving multiple days of conditioning, the post-conditioning values were obtained after the last conditioning period. B: Average MPI on pre-and post-conditioning day, and one and two days after the end of conditioning, for facilitation (a) and suppression (b). Error bars show standard deviation. Significance of comparisons is given above horizontal bars. C: Individual MPI values in successive days for cell-muscle pairs that were documented for two post-conditioning days. Open circles for subsequent days indicate significant changes (p<0.05) relative to Day 0. A & b: spike-related facilitation and suppression.

Neural mechanisms of conditioning effect on corticospinal connections

The synaptic efficacy of the CM cells’ corticospinal terminals was documented by the magnitude of SpREs seen in SpTAs of rectified muscle activity. The SpRE often include two distinguishable components that are superimposed (e.g., Figs. 1C, ​,2A2A and S2) and are produced by different mechanisms. A post-spike facilitation (PSpF) with an onset time consistent with serial conduction times from cortex to muscles (i.e., greater than 5 ms) is likely to be mediated by monosynaptic or disynaptic linkages from the trigger cell to motoneurons of the target muscle (Fetz and Cheney, 1980; Porter and Lemon, 1993). In addition, many SpREs show a “synchrony facilitation” evident in an increase above baseline that begins earlier and that would be produced by other CM cells that fire synchronously and have some spikes that occur prior to the trigger cell spikes (Jackson et al., 2003; Smith and Fetz, 2009; Davidson et al, 2007). Many of the SpREs show evidence of a clear PSpF superimposed on a broad synchrony peak (Figs. 2A, ​,5B,5B, S1C and S2). Sometimes these two features look distinct enough to invite separation, but reliable quantification of the separate components is problematic for several reasons. The separation would require an accurate estimate of the time course of the synchrony component beneath the PSpF, which is ultimately unknown. Even if it could be reliably estimated, subtraction of the synchrony component would assume linear summation, which is not the case for averages of rectified EMG (Baker and Lemon, 1995). Moreover, in many cases these two components are hard to distinguish because the rise of the serial PSpF merges with the synchrony peak, as in Fig. S2. For these reasons we have not attempted to separate and extract quantitative measures of these superimposed components.

We found that the conditioning paradigm changed the size of the synchrony component similarly to the changes in the pure PSpF. This would be predicted from the fact that spikes of synchronized cells are distributed around the trigger cell and therefore have the same average timing relative to the spinal stimuli. Thus spike-triggered stimulation would affect the spinal terminals of other corticospinal neurons firing synchronously with the trigger cell in the same way as terminals of the trigger cell. This was seen with the increase in the size of the early synchrony component, similar to the increase in the serial PSpF (e.g., Fig. 2A, FCU, Day2, and Fig. S2; see discussion in Supplementary Materials) as well as the decrease in the synchrony component for zero delays (Fig. 5A, B).

Fig. 6 illustrates the likely mechanism producing conditioned increases in the strength of corticospinal synapses and in the resultant post-spike effects for positive spike-stimulus delays. When the spinal neurons were activated 12–25 ms after arrival of the descending action potential, both excitatory and inhibitory SpREs were enhanced. Increases in the excitatory SpREs probably involve the strengthening of corticomotoneuronal terminals of the CM cells on target motoneurons, as illustrated. The mechanisms for enhancing the disynaptically mediated post-spike suppression could involve changes at two sites: in the terminals of the CM cell or terminals of the mediating inhibitory interneurons (IINs) or both. Previous studies found that the polarity of the bidirectional STDP rule is reversed for some synapses from excitatory to inhibitory neurons (Bell et al., 1997; Tzounopoulos et al., 2004). This would produce decreases in suppression, counter to our observation, but the rule for descending excitatory connections to spinal IINs remains to be determined. Given that post-spike suppression appeared in antagonists of the facilitated muscles, the underlying pathway probably involved Ia inhibitory interneurons (Jankowska et al., 1976), which are glycinergic (Fyffe, 1991). Other spinal IINs are GABAergic. In any case, assuming that stimuli activated the IINs, our evidence suggests the standard STDP rule for positive delays would apply to the corticospinal input to spinal IINs. Consistent with this, Iriki et al. found that tetanic stimulation of the pyramidal tract produced increases in corticospinal postsynaptic potentials in all of their unidentified spinal interneurons (Iriki et al., 1990). Our conditioning protocol could also strengthen the synaptic connection of the IINs to motoneurons, consistent with the STDP rule for GABAergic synapses (Haas et al., 2006; Woodin et al., 2003). Future experiments are needed to resolve the plasticity rules at these two synapses. Whatever the outcome, our results show that the net effect mediated via the disynaptic inhibitory linkage is enhanced by appropriate spike-triggered stimulation. Simultaneously strengthening both excitatory outputs to agonist muscles and inhibitory linkages to antagonists makes functional sense in maintaining the balance of effects that CM cells have on their reciprocal targets.

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Figure 6

Proposed mechanisms for conditioning effect

Spike-triggered stimulation with appropriate positive delay produces a strengthening of CM cell terminals, increasing direct excitatory post-spike effects. Changes in inhibitory effects could involve terminals of CM cell and/or inhibitory interneuron. The strengthened terminals are illustrated in red.

In vitro studies have elucidated the properties of the bidirectional STDP rules in a variety of supraspinal synapses (Bi and Poo, 2001; Caporale and Dan, 2008; Markram et al., 2011). The long corticospinal conduction time allowed demonstration that stimulation of postsynaptic cells prior to the arrival of the presynaptic impulses produced decreases in synaptic efficacy, consistent with bidirectional STDP documented in in vitro experiments. Evidence for such decreases in synaptic efficacy when postsynaptic responses precede presynaptic input has been found in sensory systems of other in vivo preparations (Jacob et al., 2007; Meliza and Dan, 2006; Mu and Poo, 2006). In most previous studies the conditioning protocol employed paired electrical stimulation. In contrast, we triggered stimuli from action potentials generated during normal behavior, which may produce more effective conditioning (Jackson et al., 2006a; Rebesco et al., 2010), perhaps due to the enhanced efficacy of natural activity patterns, known to affect plasticity (Caporale and Dan, 2008; Sjostrom et al., 2001) and possible activity-dependent delivery of facilitating neuromodulators.

Duration of conditioned effects

In most in vitro experiments the duration of so-called “long-term” potentiation and depression has typically been tested for up to several hours, and found to last that long in the absence of neural activity. In most in vivo studies the conditioned effects decay over periods of hours following the end of conditioning (Lucas and Fetz, 2013; Rebesco et al., 2010; Wolters et al., 2003). An exceptionally long-lasting effect of many days was seen with spike-triggered stimulation of nearby motor cortical sites (Jackson et al., 2006a). We found that conditioned changes in the strength of corticospinal connections lasted for variable times, in some cases up to at least two days post-conditioning. The conditioned changes of connections to different motoneuron pools from the same CM cell could survive for different durations (e.g., Fig. 2A). Thus, after the end of spike-triggered stimulation the natural activity patterns of the pre- and post-synaptic neurons tend to eventually drive synaptic strengths back to pre-conditioning levels. The synaptic strengths can be influenced by two opposing factors. Under purely random pre- and postsynaptic timings the strength of synapses would tend to decrease because the two components of the bidirectional STDP function are usually asymmetric, with greater net suppression. However, this effect is countered when presynaptic inputs tend to occur more often before the postsynaptic activity. Thus under normal behavioral conditions the balance of these and possibly other factors would determine the strength of the “steady-state” synaptic connections. Differences in the decay rate of the conditioned changes would reflect differences in the relative efficacy of these factors.

Recording

Activity of single CM cells in the precentral gyrus was recorded over successive days using chronically implanted, hand-moveable tungsten microwire electrodes (Jackson and Fetz, 2007) (diameter 50 µm; impedance 0.5MΩ; inter-electrode spacing 500 µm). The electrodes could be positioned along the bank of the precentral gyrus, where many CM cells reside (Rathelot and Strick, 2009; Smith and Fetz, 2009b). Each session began by recording the cell’s responses during an isometric, 2D eight-target wrist torque tracking task. Action potentials of cells were initially identified by a spike-sorting device based on a template-matching algorithm (MSD, Alpha Omega Engineering). The output effects of CM cells on forearm muscles were identified by peri-spike features in SpTAs of rectified EMG. Between these sessions of task performance, lasting about 3–5 hours, the monkey was returned to the home cage where movements were unrestricted.

Stimulation

Spinal stimuli were delivered through polyurethane-coated platinum iridium wire electrodes (diameter 30 µm; impedance 200–600kΩ) implanted chronically in the cervical spinal cord at the levels of C6-T1 (Mushahwar and Horch, 1998). Sites from which trains of stimuli evoked responses in one or more target muscles of the CM cell were chosen for conditioning. During conditioning stimulation intensities were subthreshold for generating antidromic or orthodromic responses in the recorded cells, but activated the cells’ target motoneurons.

Conditioning

The Neurochip was programmed to generate acceptance pulses when the waveform of the CM cell exceeded a threshold above the noise level and passed through two time-amplitude windows (Jackson et al., 2006; Mavoori et al., 2005). Following a specified delay after the action potential the Neurochip delivered a biphasic, constant-current stimulus pulse (0.2 ms/phase) to a wire electrode in the cervical spinal cord. To confirm unit isolation and reliable triggering during free behavior, short sections of raw recording (sampled at 11.7 kHz) were stored to on-board memory, interspersed with stimulation rate in 1 second bins during the conditioning period (Jackson et al., 2006; Mavoori et al., 2005).

We thank A. Price and R. Robinson for technical help and L. Shupe for programming. The work was supported by grants from the National Institutes of Health NS 12542, Christopher & Dana Reeve Foundation, Life Sciences Discovery Fund, the W.M. Keck Foundation for E.F., the National Institutes of Health NS 40867 for S.I.P and Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency for Y.N.