Article Figures & Data
Figures
- Figure 1.
Individual identified cholinergic interneurons can respond to cortical and thalamic stimulation and receive synaptic input from the cortex and the thalamus. A, Recording of the spontaneous spike firing of an individual cholinergic interneuron (#AJS044) in an anesthetized rat; vertical scale bars, 1 mV; horizontal scale bar, 1 s. B, Raster plot (top) and PSTH (bottom) showing the response of interneuron in A to single-pulse stimulation of ipsilateral motor cortex. A single-trial example of an evoked spike waveform after cortical stimulation (arrow) is inset; horizontal scale bar, 5 ms; vertical scale bar, 1 mV. C, Raster plot (top) and corresponding PSTH (bottom) showing the response of the same interneuron to single-pulse stimulation (arrow) of the ipsilateral thalamus (targeted to the parafascicular nucleus). An example of an evoked spike waveform after thalamic stimulation is inset; horizontal scale bar, 5 ms; vertical scale bar, 1 mV. Note the short-latency excitations (<20 ms) and multiphasic responses evoked by cortical and thalamic stimuli. D, E, After recording, the same interneuron was juxtacellularly labeled with NB and tested positive for immunoreactivity against ChAT, thus confirming its cholinergic identity. Scale bars, 25 μm. F, Somata and dendrites of the identified cholinergic neuron digitally reconstructed in 3D. Scale bar, 25 μm. G, H, The same interneuron was then examined using electron microscopy. In G, a dendrite (d) is shown forming an asymmetric synapse (arrowhead) with an axon terminal (white asterisk) that is positive for VGluT1, a marker of cortical terminals. Note the crystalline deposits in the dendrite formed by the TMB. Scale bar, 0.25 μm. In H, another dendrite (d) is shown forming an asymmetric synapse (arrowhead) with an axon terminal (white asterisk) that is positive for VGluT2, a marker of thalamic terminals.
- Figure 2.
Terminals forming synapses with the dendrites of cholinergic interneurons. A, Example of a dendrite of a cholinergic interneuron (d) forming an asymmetric synapse (arrowhead) with a terminal negative for VGluT1 (n). Note that there is a terminal positive for VGluT1 (DAB product; white asterisk), forming an asymmetric synapse (arrowhead) with a MSN spine (sp). B, Cholinergic interneuron dendrite (d) forms two asymmetric synapses (arrowheads) with a VGluT2 positive terminal (asterisk) and terminal negative for VGluT2 (n). Note that within the same frame there is another positive terminal (asterisk) forming a synapse with an MSN spine (sp). C, A dendrite (d) forms an asymmetric synapse (arrowhead) with a terminal negative for VGluT2 (n). Note that the same terminal is also forming a synapse with a spine of an MSN (sp) and that within the same frame there is a VGluT2 positive terminal (asterisk). There is also a negative terminal (n) forming a synapse with a spine (sp). D, The dendrite of a cholinergic interneuron (d) forms a symmetric synapse (small arrows) with an unlabeled terminal (n), see inset. Note that within the frame there is a terminal positive for VGluT1. Scale bars, 0.25 μm. E, Percentages of terminals forming symmetric (blue) or asymmetric (green) synapses with the dendrites examined. Of the terminals that formed asymmetric synapses (green), some were positive for VGluT1 (red) or VGluT2 (violet). The remaining terminals were DAB negative in tissue labeled for either VGluT1 or VGluT2 (inset). F, Average number of asymmetric (green) and symmetric (blue) synapses normalized for every 10 μm of dendrite, within each compartment. G, Estimations of the total number of symmetric (blue) and asymmetric (green) synapses formed with the dendrites of cholinergic interneurons based on data collected.
- Figure 3.
Short-latency responses of cholinergic interneurons to cortical and thalamic stimulation. Ai, Unit-activity recording (single representative sweep of stimulation artifacts and evoked responses, arrow indicates stimulation onset) of a cholinergic interneuron that fired at short latency (∼10 ms) in response to single-pulse electrical stimulation of the cortex. The coincident ECoG is shown below; note evoked potential. Aii, Expanded view of the same response showing that, after the short-latency spike, there is a cessation of firing (∼250 ms) followed by a period of renewed spiking. B, PSTHs (2 ms bins) of the same interneuron's response to cortical stimulation (single pulses delivered at 0 ms, with artifact removed). Inset, Response during the first 50 ms after stimulation. C, Mean PSTH of all cholinergic interneurons that showed a significant response to cortical stimulation (n = 16). Inset, Responses of each interneuron (thin lines) during the first 50 ms after stimulation. Di, The same interneuron (as in A) also fired at short latency (∼10 ms) in response to single-pulse electrical stimulation of the thalamus. A second spike follows after ∼40 ms. Dii, Expanded view highlighting a later pause in firing that is followed by renewed spiking. E, PSTHs (2 ms bins) of the same interneuron's response to thalamic stimulation (single pulses delivered at 0 ms). Inset, Response during the first 50 ms after stimulation. F, Mean PSTH for all interneurons that responded significantly to thalamic stimulation (n = 9). Note the evoked increase in firing between 25 and 50 ms (arrowhead) that is not present after cortical stimulation (see C). G, Latencies of the first spikes evoked at short latency (lag of 1.5–20 ms) of all interneurons significantly responding to either cortical or thalamic stimulation. Box plots show the medians (white line), the interquartile ranges (box), and extremes of the range (whiskers, within 99% of the distribution). Dots show the mean latency of each individual interneuron. H, Latencies of the first spikes evoked at short latency in all interneurons significantly responding to both cortical and thalamic stimulation. Lines join data from individual interneurons.
- Figure 4.
Paired-pulse stimulation of cortex, but not thalamus, leads to a decrease in the evoked firing probability of cholinergic interneurons. A, Mean peristimulus histogram of all cholinergic interneurons that showed a significant short-latency response to paired cortical stimulation (n = 10). Inset, Magnification of the first 50 ms after stimulation. B, Mean peristimulus histogram of all cholinergic interneurons that showed a significant short-latency response to paired thalamic stimulation (n = 9). Inset, Magnification of the first 50 ms after stimulation. C, Short-latency firing probabilities for first and second pulses of paired cortical stimuli. Dots show the mean firing probability of each individual interneuron. On average, firing probability to the second pulse was significantly lower (asterisk, Wilcoxon signed-rank, p < 0.005). D, Short-latency firing probabilities of each interneuron to the first and second pulses of paired thalamic stimuli. On average, the firing probabilities to the first and second pulses were similar (Wilcoxon signed-rank test, p > 0.05).
- Figure 5.
Differential responses of cholinergic interneurons to high-frequency stimulation of cortex and thalamus. A, B, Unit-activity recording (single representative sweep of stimulation artifacts and evoked responses, with arrows indicating stimulation onset) of a cholinergic interneuron that fired at short latency (∼10 ms) in response to 40 Hz trains(5 pulses) of electrical stimuli delivered to the cortex (A) or thalamus (B). Ci, Mean PSTH of all cholinergic interneurons (n = 9) that showed significant short-latency responses during 40 Hz cortical stimulation. Cii, Magnified view of Ci during the first 150 ms after the start of train stimulation. Di, Mean PSTH of all cholinergic interneurons (n = 9) that showed significant short-latency responses during 40 Hz thalamic stimulation. Dii, Magnified view of Di during the first 150 ms after the start of train stimulation. E, Mean firing probability of spikes fired at short latency on each pulse for cortical (red) and thalamic (blue) stimulation. The firing probability for the first thalamic pulse was significantly lower than the mean of the following four pulses (Wilcoxon signed-rank test, p = 0.004). F, Mean percentage of spikes fired on each pulse for cortical (red) and thalamic (blue) stimulation. The percentage of spikes fired after first thalamic pulse was significantly higher than the mean of the following four pulses (Wilcoxon signed-rank test, p = 0.027), whereas for cortical stimulation, it was significantly lower (Wilcoxon signed-rank test, p = 0.027). G, Mean cumulative sums (cusum) of firing of all interneurons in response to cortical (red) and thalamic (blue) stimulation over each pulse. H, Slopes of the cusum for each interneuron (dots) to cortical and thalamic stimulation trains. The slope was significantly steeper for responses to thalamic stimulation (Mann–Whitney U test, p = 0.0078).
- Figure 6.
Multiphasic responses of cholinergic interneurons to stimulation of cortical and thalamic afferents. A, B, Classification of types of multiphasic responses to cortical stimulation. Ai, Percentage of different response types evoked by single-pulse cortical stimulation. Aii, PSTHs of all interneurons with the pause/rebound type of response (top) and the initial excitation/pause/rebound type of response (bottom). Single stimuli were delivered at 0 ms. Bi, Percentage of different response types evoked by a high-frequency cortical stimulation (train of 5 pulses at 40 Hz). Bii, PSTHs of the one interneuron that displayed a pause/rebound response (top) and all the interneurons with initial excitation/pause/rebound responses (bottom). Start of train of high-frequency stimuli at 0 ms. C, D, Classification of types of multiphasic responses to thalamic stimulation. Ci, Percentage of different responses types to single-pulse thalamic stimulation. Cii, PSTHs of the only interneuron with a pause/rebound response (top) and all the interneurons that displayed initial excitation/pause/rebound responses (bottom). Di, Percentage of different responses types evoked by a high-frequency thalamic stimulation. Dii, PSTHs of all interneurons with initial excitation/pause responses (top) and two neurons that displayed initial excitation/pause/rebound responses (bottom).
- Figure 7.
Initial excitation predicts the temporal profile of the multiphase response in cholinergic interneurons. A, Raster plot with trials sorted by pause duration (top) and PSTH (bottom) for a cholinergic interneuron responding to single-pulse cortical stimulation. B, As in A, for the same neuron responding to single-pulse thalamic stimulation. C, Mean PSTHs across all responses (cortical and thalamic) sorted for the numbers of spikes in the initial excitation phase. Data from single-pulse stimulation of cortex and thalamus are combined. Responses were only included for a given neuron if there were ≥3 trials with the requisite number of spikes in the initial excitation. Number of spikes in the initial excitation phase is shown using color code and the numbers of responses that were averaged are shown in parentheses. D, Scatter plot of the mean number of spikes fired in the initial excitation phase and the mean normalized pause length for all interneurons (dots) after single-pulse electrical stimulation of cortex (red) and thalamus (blue). The mean number of spikes in the initial excitation phase was positively correlated with the duration of the pause phase. Dotted line shows the best linear fit between the two variables. E, F, Change in firing rate in the rebound phase was significantly negatively correlated with the mean number of spikes in the initial excitation phase (E), but not significantly correlated with the pause duration (F). G, H, Identical plots as A and B but for a different interneuron responding to high-frequency stimulation of cortex (E) and thalamus (F). I, Mean PSTHs across all trials with the same numbers of spikes in the intitial excitation phase for high-frequency stimulation of cortex and thalamus combined. Format as in C. J, Scatter plot of the mean number of spikes fired in the initial excitation phase and the mean normalized pause length for all neurons after cortical (red) and thalamic (blue) high-frequency electrical stimulation. The mean number of spikes in the initial excitation phase was positively correlated with the length of the pause phase across cortical and thalamic responses to stimulation. Dotted line shows the best linear fit between the two variables. K, L, The change in firing rate in the rebound phase was significantly negatively correlated with the mean number of spikes in the initial excitation phase (K) and with the pause duration (L; format as in J). Spearman correlation coefficients were used in all cases. NS, Not significant.
- Figure 8.
Multiphasic responses of primate TANs to motivationally salient stimuli. A, Mean response (Ai) and the percentage of significant responses per bin (Aii) across the population of recorded TANs after the reward-predicting stimulus (given at 0 ms). B, Classification of the types of multiphasic responses of all the TANs recorded in this first behavioral condition. C, PSTHs of all TANs with pause/rebound responses (Ci) and initial excitation/pause/rebound responses (Cii) to the reward-predicting stimulus. D, Mean response (Di) and the percentage of significant responses (Dii) across the population of recorded TANs after reward-only. E, Classification of multiphasic responses of all of the TANs recorded in this second behavioral condition. F, PSTHs of all TANs with pause/rebound responses (Fi) and initial excitation/pause/rebound responses (Fii) to reward-only.
- Figure 9.
Dependence of multiphase responses of primate TANs on their initial excitation is behaviorally specific. A, Raster plot with trials sorted by pause duration (top) and PSTH (bottom) for a TAN responding to the reward-predicting stimulus with an initial excitation/pause/rebound response. B, Plots for a different TAN responding to the same condition with a pause/rebound response. C, Mean PSTHs for all trials with the same number of spikes in the initial excitation phase, in response to the reward-predicting stimulus. Responses were only included for a given TAN if there were ≥3 trials with the requisite number of spikes in the initial excitation. Number of spikes in the initial excitation phase is shown using color code and the number of TANs that were averaged is shown in parenthesis. D, Mean number of spikes in the initial excitation was positively correlated with the length of the pause phase across all neurons after partial correlation with firing rate (parentheses). E, F, Increase in firing rate in the rebound phase was significantly negatively correlated and the mean number of spikes in the initial excitation phase (E) and with the length of the pause (F). Both correlations were significant after partialization with firing rate (parentheses). G, Raster plot with trials sorted by pause duration (top) and PSTH (bottom) for a TAN responding to reward-only with an initial excitation/pause/rebound response. H, Identical plots for a different TAN responding to the same condition with a pause/rebound response. I, Mean PSTH for all neurons for trials with the same numbers of spikes in the initial excitation phase, in response to the unpredicted reward. Format as in C. J, Mean number of spikes in the initial excitation phase was correlated with the length of the pause phase across all neurons, but was no longer significant after partial correlation with firing rate (parentheses). K, L, Increase in firing rate in the rebound phase was not significantly correlated with the mean number of spikes in the initial excitation phase (K) or with the length of the pause (L). Spearman correlation coefficients were used in all cases. NS, Not significant.
Tables
Properties examined Neurons Average NJX009 AJS044 NJX014 Total number of ultrathin sections examined (50 nm) 849 1039 789 892.33 Total number of dendritic fragments analyzed 20 24 20 21.33 In VGluT1-labeled tissue 11 14 11 12.00 In VGluT2-labeled tissue 9 10 9 9.33 Total number of unlabeled (DAB-negative) asymmetric synapses 23 32 46 33.67 In VGluT1-labeled tissue 11 22 27 20.00 In VGluT2-labeled tissue 12 10 19 13.67 Total number of VGluT1-positive synapses 0 2 0 0.67 Total number of VGluT2-positive synapses 6 5 3 4.67 Total number of (unlabeled) symmetric synapses 61 62 64 62.33 For each neuron examined, several properties were analyzed over a number of sections in either VGluT1- or VGluT2-labeled tissue.
Properties examined Proximal (0–19%) Distal (20–79%) Most distal (80–100%) Number of dendritic fragments analyzed 17 38 9 Average distance from soma (±SEM) (μm) 59.96 (±12.19) 276.53 (±34.22) 558.22 (±17.00) Average distance from soma as a percentage of total distance (±SEM) 9.31 (±1.90) 43.64 (±5.40) 85.48 (±2.17) Ultrathin sections analyzed (total) 730 1568 379 Average number of ultrathin sections analyzed in serial section (±SEM) 42.94 (±7.02) 41.26 (±6.26) 42.11 (±10.13) Total number of terminals forming asymmetric synapses 24 58 19 In VGluT1-labeled tissue 14 40 6 In VGlut2-labeled tissue 10 18 13 Total number of VGlut1-positive terminals forming synapses 1 1 0 Total number of VGlut2-positive terminals forming synapses 2 10 2 Total number of terminals forming symmetric synapses 55 103 29 The distribution of terminals forming synapses on dendrites at varying distances from the soma was analyzed for the three neurons examined.
