Modulation of Rhythmic Activity in Mammalian Spinal Networks Is Dependent on Excitability State

Dopamine evokes multirhythmic patterns of motor activity. A, Neurograms recorded from the left and right ventral roots of the L2 and L5 isolated thoracolumbar spinal cord. B, Dopamine evokes a multirhythmic pattern of motor activity that is composed of two rhythms: a slow, synchronous rhythm with a superimposed fast rhythm. C, These fast and slow rhythms (red boxes) are apparent when the frequency power is represented over time as a spectrogram with frequency on the left y-axis, time on the x-axis, and warmer colors representing higher frequency power. Di, Dii, Div, The slow rhythm features including cycle period (Di), episode duration (Dii), and power (Div) did not differ between L2 (red bars) and L5 (black bars) segments. Diii, Div, The frequency of the fast rhythm occurring within bouts (Diii) decreases over the course of the bout (Div) with no difference in power between L2 and L5 (Div). Data are displayed as the mean ± SD. Ei, Example phase plots from a single bout of rhythmic activity recorded from the L2 segment illustrate that the bouts are synchronous, with the fast superimposed rhythm exhibiting a biphasic phase distribution. Eii, Separating the fast rhythm into two bins illustrates that the fast bursts start off synchronous and end alternating. Eiii, The pie charts represent the distribution of the possible patterns over the course of a bout recorded between the left and right L2 and ipsilateral L2–L5 ventral roots. Fi, Reflects the mean phase of all bouts. Fii, The predominantly synchronous bursting pattern in ipsilateral ventral root pairs is represented in the mean phase plot. The phases in circular plots are reported in degrees with the length of arrows representing mean vector length (r) and angle.

High concentrations of dopamine evoke rhythmicity. Ai–Aiii, Neurograms from single L2 ventral roots from individual experiments with dopamine (DA) bath applied (red arrows) at 30 µm (Ai), 100 µm (Aii), and 300 µm (Aiii) to naive preparations evoke rhythmic motor activity. Bi–Biii, The fast and slow rhythms are illustrated in the autowavelet frequency power spectrograms over time with frequency on the left y-axis, time on the x-axis, and warmer colors representing higher power. Rhythm frequency and power of the fast and slow rhythms were analyzed by selecting regions of interest selected within bouts of the fast rhythm and within the range of the slow rhythm. Ci, Cii, The frequency of the fast rhythm slowed down (Ci) and power increased (Cii) with dopamine concentration. Ciii, Civ, No differences were found in frequency (Ciii) or power (Civ) of the slow rhythm between dopamine concentrations. Data are presented as the mean ± SD, with asterisks denoting significance (*p < 0.05, **p < 0.01, ***p < 0.001) with Tukey post hoc test following a two-way ANOVA between time (5 min bins) and dopamine concentration.

Sequentially boosting network excitation with KCl modulates dopamine-evoked rhythmicity. A, Neurograms recorded from the left and right L2 and right L5 ventral roots show raw data and the effect on rhythmicity. Bath application of 50 µm dopamine evokes a multirhythmic pattern of motor activity that is modulated as network excitation is boosted by sequentially increasing the concentration of KCl in the bath (red arrows). When KCl concentration is increased to 10 mm, the pattern switches from multirhythmic to a single, continuous locomotor-like rhythm. B, The frequency power distribution following dopamine application and subsequent excitability manipulation is illustrated in the cross-wavelet frequency power spectrogram over time with frequency on the left y-axis, time on the x-axis, and increasing power represented as warmer colors. At 10 mm KCl, the pattern switches from multiple rhythms to a single continuous rhythm. Ci–Ciii, Representative neurograms showing rhythm at baseline (Ci), dopamine with 8 mm KCl (Cii), and a continuous locomotor-like rhythm expressed with dopamine and 10 mm KCl (Ciii). D, Regions of interest were selected around the fast and slow rhythms, and the respective frequency and power for left and right L2 and L5 neurograms were analyzed over time. Di, Dii, Increasing network excitation increased the power of the fast rhythm (Di) and decreased the power of the slow rhythm (Dii). Data are presented as the mean ± SD, with asterisks denoting a significant difference between the respective point and the rhythm at 20 min following dopamine application (*p < 0.05, **p < 0.01, ***p < 0.001) obtained from Tukey post hoc analysis when significant main effects on a repeated-measures ANOVA were found. Nonparametric statistical analysis was performed when assumptions of normality failed, and significance was denoted as follows: #p < 0.05, ##p<0.01, ###p < 0.001. Ei, Eii, The phase between L2 neurograms for the fast rhythm is presented in the circular plots in Ei and Eii and illustrate the switch to a locomotor-like pattern at 10 mm KCl as the vector length increases and phase moves toward 180° (alternating) in both the left and right L2s and ipsilateral L2–L5. Each dot represents the average phase for an individual preparation for each respective experimental condition. The length of the arrows represents the mean vector length (r) or the robustness of the pattern.

Boosting network excitation with KCl prior to the application of dopamine evokes locomotor-like rhythmicity. A, Neurograms recorded from left and right L2 and right L5 ventral roots illustrate the experimental paradigm and the resultant effect on rhythmicity. KCl concentration was increased to 10 mm to boost network excitation 20 min prior to application of 50 µm dopamine (DA). Subsequent application of DA resulted in the direct expression of a continuous locomotor-like rhythm that returned to a multirhythm when washed out with regular (4 mm KCl) aCSF and 50 µm DA. B, Frequency power spectrogram with frequency on the left y-axis, time on the x-axis, and increasing power represented as warmer colors. Ci, Cii, Raw data showing zoomed regions represented in B of 50 µm dopamine plus 10 mm KCl at a longer time point (Ci) and following wash in of 4 mm KCl (Cii). D, Region-of-interest analysis of fast and slow rhythms within L2 root pairs illustrate significantly higher power of the fast rhythm with 10 mm KCl compared with the expected rhythm power at 4 mm KCl. Di, The slow rhythm showed significantly lower power compared with the expected multirhythm evoked in the 4 mm KCl condition. Dii, The expected power values returned to the same level as the expected condition following a washout with 4 mm KCl (Blue lines). Ei, Eii, Circular plots in Ei and Eii illustrate a locomotor-like pattern with vector length increases accompanied by phase angles moving toward 180° (alternating) in both the left and right L2s and ipsilateral L2–L5 at higher KCl concentrations. The length of arrows represents mean vector length (r) and angle or robustness of the pattern. Red lines represent mean data (n = 20 preparations) when 50 µm DA (aCSF, 4 mm KCl) was applied, and 20 min of baseline data were analyzed. Black lines represent the rhythm evoked by 50 µm DA under enhanced network excitation (aCSF, 10 mm KCl), and blue lines represent the washout condition of DA (aCSF, 4 mm KCl). Each dot in the phase plots represents the average phase for an individual preparation for each respective experimental condition. Data are presented as the mean ± SD. A two-way ANOVA between each excitability condition (DA-evoked rhythm in 4 mm KCl, 10 mm KCl, and washing with 4 mm KCl) and time to examine the effects of manipulating network excitation prior to DA application. When significant main effects of interactions were detected Tukey post hoc analysis between time-matched points following DA application were conducted. Asterisks denote significance, as follows: *p < 0.05, **p < 0.01, ***p < 0.001.

Decreasing network excitation disrupts the ability of dopamine to evoke rhythmicity. A–D, Neurograms from single ventral roots within the L5 illustrate the overall effect of reducing network excitation via several pharmacological approaches on dopamine-evoked rhythmicity. A, In the first experiment, network excitability was reduced by washing in aCSF containing 1 mm KCl after evoking a rhythm. B, In a second experiment, preparations were recovered in low KCl (1 mm) aCSF for 1 h prior to dopamine application. C, In a third experiment, excitability was reduced by sequentially increasing bath MgSO₄ concentration (1.0–2.5 mm) after evoking a rhythm with dopamine. D, In a final experiment, AP5 was bath applied to antagonize NMDA receptors (5 µm) 20 min prior to the addition of dopamine. E, An example cross-wavelet frequency power spectrogram illustrates the degradation of rhythmic activity when aCSF with dopamine and low KCl washed into the bath. F, Regions of interest were analyzed for cross-wavelet spectrograms between left and right L2 and L5 neurograms around fast and slow rhythm frequency bands. Fi, Fii, The power of the fast rhythm was reduced under all conditions in the L2 (Fi) and L5 (Fii) neurograms. Fiv, Slow rhythm power was reduced in the L2s but was reduced only in the L5s by MgSO₄ and washing in low KCl. Data are presented as the mean ± SD, with asterisks denoting a significant difference between the respective point and the rhythm at 20 min following dopamine application. Nonparametric one-way ANOVAs were performed for fast or slow rhythms in L2 and L5 power. Significant differences on post hoc analyses between each respective condition compared to rhythm power from 50 µm dopamine alone (#p < 0.05, ##p < 0.01, ###p < 0.001).

Sequential enhancement of network excitation with NMDA modulates dopamine-evoked rhythmicity. A, Neurograms recorded from left and right L2 and right L5 ventral roots illustrate the experimental paradigm and resultant effect on rhythmicity. Bath application of 50 µm dopamine (DA) evoked a multirhythmic pattern of motor activity that is modulated as network excitation is boosted by sequentially increasing the concentration of NMDA in the bath (red arrows). When NMDA is increased to 6 µm, the pattern switches from multirhythmic to a single, continuous locomotor-like rhythm. B, The frequency power distribution following dopamine application and subsequent excitability manipulation is illustrated in the cross-wavelet frequency power spectrogram over time with frequency on the left y-axis, time on the x-axis, and increasing power represented as warmer colors. C, At 6 µm NMDA, the pattern switches from multiple rhythms to a single continuous rhythm. D, Regions of interest were selected around the fast and slow rhythms, and respective frequency and power for left and right L2 and L5 neurograms were analyzed over time. Increasing network excitation increased the power of the fast rhythm and decreased the power of the slow rhythm. Ei, Eii, The phase and regularity of the fast rhythm is presented in the circular plots in Ei and Eii, and illustrate the switch to a locomotor-like pattern at 6 µm NMDA as the vector length increases and phase moves closer to 180° in both the left and right L2s and ipsilateral L2–L5. The phase in circular plots is reported in degrees, with 180° indicating an alternating pattern and 0° being synchronous. The length of the arrows represents the mean vector length (r) or robustness of the pattern. Each dot represents the average phase for an individual preparation for each respective experimental condition. Data are presented as the mean ± SD, with asterisks denoting a significant difference between the respective point and the rhythm at 20 min following dopamine application (#p < 0.05, ##p < 0.01, ###p < 0.001) using pairwise multiple-comparisons Tukey post hoc analysis when significant main effects on a repeated-measures ANOVA were found.

5-HT evokes multirhythmic patterns of motor activity that become locomotor like as network excitation is enhanced with KCl. Ai–Aiii, Neurograms recorded in separate experiments from single L2 ventral roots illustrate that bath application of 10 µm 5-HT evokes a single slow rhythm (Ai), 15 µm evokes a slow and fast rhythm (Aii), and 20 µm evokes a single fast rhythm (Aiii). B, Autowavelet spectrograms depicted in Bi–Biii illustrate these rhythms. C, Boosting network excitation following the generation of multiple rhythms with 15 µm 5-HT caused a transition from a multirhythm to a single locomotor-like rhythm. Network manipulations are represented and highlighted as a red downward arrow in the spectrogram. D, Regions of interest were selected within cross-wavelet spectrograms around the fast and slow rhythms, and the respective frequency and power for left and right L2 and L5 were analyzed over time. Di, Div, Boosting network excitation increased the power of the fast rhythm (Di) and decreased the power of the slow rhythm (Div). Ei, Eii, The bursting pattern of the fast rhythm is presented in the circular plots in Ei and Eii, and illustrates an increase in the vector length at 10 mm KCl in the left and right L2s and ipsilateral L2–L5 at 8 mm KCl, but at 10 mm the length declined as activity became tonic. The phases in circular plots are reported in degrees, with 180° indicating an alternating pattern and 0° indicating a synchronous pattern. The lengths of arrows represent the mean vector length (r) and angle or robustness of the pattern. Each dot represents the average phase for an individual preparation for each respective experimental condition. Data are presented as the mean ± SD, with asterisks denoting a significant difference between the respective point and the rhythm at 20 min following dopamine application (*p < 0.05) from Tukey post hoc analysis when significant main effects on a repeated-measures ANOVA were found.

Neuromodulators evoke rhythmicity by moving the network through an excitation parameter space with the end point pattern being locomotor like. State 1 (basal state) in isolated rodent spinal cords is characterized by spontaneous network activity. Depolarization of the network moves the network into a higher excitation state (state 2), which is characterized by tonic activity with no rhythmicity. State 3 is characterized by multirhythmic patterns of motor activity where modulator-specific patterns of rhythmic motor activity may exist. Finally, in state 4, the locomotor state is characterized by continuous rhythmic activity with an alternating locomotor-like pattern expressed as a network-emergent property at the highest level of network excitation. Neurograms depicted in the schematic are from ventral root recordings in the right L5 and left and right L2 spinal segments. Curved lines in the schematic represent transition zones between network states, where dopamine would be expected to have the greatest effects on network output.