Article Figures & Data
Figures
- Figure 1.
A, Top, Schematic of brain regions associated with the expression of learned responses in trace eyelid conditioning. The boxes show schematic representations of the neural activity that has been observed in that region during expression of conditioned responses. The temporal relationship between the TONE CS (gray bar) and the US (black arrowhead) is shown to the left and above/below each brain region, along with the timing of the conditioned eyelid closure driven by the DCN via the red nucleus (RN, right). Single neurons in the mPFC show three patterns of tone-evoked responses: phasic (upper spike train), persistent (middle), and those restricted to the trace interval (bottom). Heavier arrows show anatomical flow of inputs between the CS and cerebellum. Lighter arrows point to brain regions previously shown to receive cerebellar output, which is reflected in the single-unit activity observed in those regions (e.g., bottom spike train in pons). Bottom, Schematic representation of the hypothesis tested in the current study: that persistent activity in the mPFC is upstream of the cerebellum whereas trace interval activity reflects downstream feedback from the output pathway of the cerebellum. To test this hypothesis, the DCN was inactivated with muscimol during trace eyelid conditioning and mPFC persistent and trace interval cells examined for changes in activity in the absence of cerebellar output and behavioral responses. B, Top, Autofluorescent coronal sections with plots of the estimated infusion sites in the cerebellar deep nuclei for all rabbits in this study (cannula was targeted to the anterior interpositus nucleus). Each marker represents a single site, with one or two sites per rabbit (if an infusion site was or became ineffective a deeper infusion was attempted in 3 of the rabbits). Effective sites are shown in red and ineffective in black, with the amount of muscimol infused represented by the size of the marker (range, 2–5 μl). The open marker represents one site in which six successful infusions were done followed by a 1 μl infusion of Alexa-conjugated dextran to estimate diffusion through the tissue. Bottom, Autofluorescent and dextran fluorescent images overlaid. Red fluorescence shows the spread of 1 μl infusion of dextran. Scale bars, 1 mm. C, Conditioned response likelihood during ineffective DCN infusions (light gray), noninfusion sessions (dark gray), and effective DCN infusions (red bars). CRs were abolished after effective DCN infusions (POST). The likelihood of CRs observed for ineffective infusion and noninfusion sessions were not different (t(11.26) = 1.08, p = 0.30), and so neural data from both session types were used as controls for comparison with successful DCN inactivation sessions.
- Figure 2.
Estimated sites of single-unit activity for cells recorded during each condition (Effective, Previous Day, and Ineffective) and average waveform and activity parameters from each single unit used to distinguish between putative pyramidal cells and interneurons. Colored markers indicate single units that showed a given response type (red represents persistent; green represents phasic CS; blue represents trace interval; black represents US/post-trial/no response), and whether the cell increased (filled marker) or decreased (open marker) spike activity in response to conditioning stimuli. Left, Representative Nissl-stained coronal sections at 3 locations along the rostrocaudal plane of the mPFC, and plots of the estimated recording location of single-unit activity for each condition. All cells were recorded within the medial agranular or caudal anterior cingulate regions of the mPFC. Scale bar, 1 mm. Right, Scatterplots of average spike width × average firing rate for each single unit, with representative examples of waveforms recorded from the four channels of a tetrode for 3 of the cell types analyzed, for each condition (color-coded as described above, with the single-unit for each example also highlighted with a larger marker with black outline in the scatterplots and coronal sections). Less than 5% of cells in each condition would be designated as putative interneurons based on spike width.
- Figure 3.
Representative examples of behavioral and simultaneously recorded single-unit responses before (PRE) and after (POST) DCN inactivation (A), and examples from the control session recorded on the previous day from the same animal (B). A, Eyelid responses (waterfall plot) for individual trials before (PRE) and after (POST) DCN inactivation with muscimol; upward deflection indicates eyelid closure, gray rectangles represent onset and duration of the CS (500 ms); gray line (50 ms) indicates timing of the US. The average preinfusion eyelid response is shown above the individual trial examples, and the average postinfusion eyelid response is shown below. The percentage conditioned responses before and after infusion are also indicated. There are anticipatory eyelid closures during the trace interval before US presentation during the preinfusion epoch, with few observed during the postinfusion epoch. The rabbit in this and the following figures still shows full amplitude reflexive responses during DCN inactivation in the absence of CRs. Raster plots represent the spike activity of three isolated single units (type of categorical response and cluster identification given above, e.g., the first example was recorded from tetrode 9 as cluster number 1). Stimulus markers are the same as the behavioral data. Each row of a raster plot represents one trial and includes a 1 s baseline before and after the trial, with dots indicating the occurrence of a single spike. Histograms show cumulative number of spikes for 100 ms bins for preinfusion trials (top row) and for postinfusion trials (bottom row). White markers in the histograms indicate time bins with significant increases in activity relative to baseline. *Significant changes in activity for postinfusion trials. This infusion abolished conditioned responses (left) and trace interval activity (c16.1 and c10.2) but did not change persistent tone-evoked response patterns (c9.1). c16.1 displayed a significant phasic tone response that was still observed after DCN infusion while the trace interval response was abolished along with the learned behavior. B, Changes were not observed for persistent (left raster/histograms) or trace interval (right) single-unit responses from cells recorded on the same tetrodes during a noninfusion control session taken the previous day.
- Figure 4.
Additional examples of persistent mPFC cell activity during DCN inactivation and during control sessions, with figure layout as described for Figure 2. Examples of a single-unit response that did not change (left) and one that did change (right) are given for both inactivation (A) and control sessions (B). A similar proportion of mPFC cells showed a change in activity pattern during both inactivation and control sessions. For both infusion and control sessions, changes in persistent responses were often one of consistency during the last half of the session that did not meet statistical criteria relative to baseline, and did not reflect a complete absence of the activity pattern.
- Figure 5.
Additional examples of trace interval activity during DCN inactivation and during control sessions, with figure layout as described for Figure 2. Trace interval responses were significantly impacted by DCN inactivation (e.g., A), whereas only a small proportion showed significant changes during control sessions (e.g., B). In most cases, trace interval activity that changed during control sessions showed significant increases in activity early in the trace interval, before the onset of behavioral responses, which was not typical for that response type (B, c25.1). A, The data are examples in which analysis has been corrected for the delayed effects of DCN inactivation. Arrowheads in waterfall plots indicate the actual time of muscimol infusion.
- Figure 6.
Proportions of mPFC cell types that maintained the same response pattern for two control conditions (A), and between DCN inactivation and pooled control sessions (B). A, Cells were categorized as persistent, trace interval, or phasic based on response patterns during the first half of control sessions, and then categorized again during the second half of control sessions. Similar proportions of mPFC single-unit response types continued to meet the same categorical criteria for both control session types (light gray represents ineffective DCN infusions; dark gray represents noninfusion sessions from the previous day; persistent increase: ineffective infusion vs previous day, n = 3,11, χ2 = 0.01, p = 0.56; trace increase: n = 5,12, χ2 = 0.004, p = 0.63; phasic increase: n = 5,19, χ2 = 1.70, p = 0.42; persistent decrease: n = 6,12, χ2 = 0.12, p = 0.86; trace decrease: n = 14,39, χ2 = 0.29, p = 0.59; phasic decrease: n = 14,28, χ2 = 0.05, p = 0.83). Therefore, mPFC cells recorded during both control types were pooled for control comparisons with cells recorded during DCN inactivation. B, Compared with controls, DCN inactivation significantly affected the response pattern of trace interval cells but did not significantly affect the activity patterns of persistent or phasic tone cells (DCN vs controls, persistent increase: n = 8,14, χ2 = 0.43, p = 0.51; trace increase: n = 16,17, χ2 = 13.50, p = 0.0002; phasic increase: n = 15,24, χ2 = 0.13, p = 0.72; persistent decrease: n = 14,18, χ2 = 0.40, p = 0.53; trace decrease: n = 24,53, χ2 = 6.17, p = 0.01; persistent decrease: n = 19,42, χ2 = 0.55, p = 0.46). *Bonferroni-corrected, p < 0.017.
- Figure 7.
Average difference scores of the number of spikes observed during the trace interval before and after DCN inactivation for mPFC persistent (A), trace (B), and phasic tone (C) cell response types, and difference scores for the expression of CRs within the sessions in which those cells were recorded. A, Difference scores derived from the average number of spikes observed during the stimulus-free trace interval between pre-DCN and post-DCN infusion epochs (red bars) were not significantly different from controls (gray bars) for persistent cells (left: n = 8 and 14 cells, t = 0.34, p = 0.74), even though a significant difference in CRs was observed for sessions in which those cells were recorded (right: n = 6 and 12 sessions, t = 15.77, p < 0.001). B, In contrast, trace interval cells showed a significant decrease in the post-pre difference in the average number of spikes observed between DCN infusion and control sessions (left: n = 16 and 16 cells, t = 2.51, p = 0.02), as was also observed for the change in behavioral responses from those sessions (right: n = 10 and 9 sessions, t = 13.58, p < 0.001). C, As a within manipulation control, phasic tone cells were analyzed for differences in post-pre average spike responses during the tone CS and showed no significant difference between control and DCN inactivation sessions (left: n = 15 and 25 cells, t = 0.62, p = 0.54) even though the behavioral responses were different during the corresponding sessions (right: n = 9 and 14 sessions, t = 14.07, p < 0.001). *Bonferroni-corrected, p < 0.017.
- Figure 8.
All three mPFC response types maintained significant increases in activity relative to baseline during the second half of control sessions (left bar graph: persistent, n = 14 cells, t = 3.29, p = 0.005; trace, n = 16, t = 3.40, p = 0.004; tone, n = 25, t = 4.83, p < 0.001), whereas only persistent and phasic tone response types maintained significant increases in activity during DCN inactivation (right bar graph: persistent, n = 8, t = 4.36, p = 0.002; trace, n = 16, t = 2.19, p = 0.024; tone, n = 15, t = 4.15, p < 0.001). The activity of trace interval cells was not different from baseline activity during DCN inactivation, indicating that this pattern of activity was abolished along with the expression of CRs. *Bonferroni-corrected, p < 0.008.
- Figure 9.
Pseudo-colored raster plots of average increases in activity (number of spikes/100 ms bin × trial) during sessions for persistent (A) and trace interval (B) cells recorded during control (left) and DCN infusion sessions (right). Black bars indicate CS and US presentation during trials. A, Trace interval cells appeared to show decreased activity within just a few trials after DCN inactivation. Right matrix, red box indicates inactivation epoch. The mean number of spikes observed during the trace interval was averaged for blocks of trials (right) and shows that trace cell activity was abolished to baseline by the second block (n = 16 cells, t = 0.16, p = 0.88 relative to baseline). *Significant difference between DCN and controls, n = 16 and 16 cells, t = 2.64, p = 0.018. B, Persistent cells showed gradually decreasing activity over the session, which was not affected by DCN inactivation (right: n = 8 and 14 cells, t = 0.49, p = 0.63). C, Temporal profile of mean spike response of trace cells over the entire trial (100 ms bins), averaged across preinfusion (top) or postinfusion (bottom) trials. Black bars indicate presentation of conditioning stimuli. The response profile of trace cells changed significantly during DCN inactivation (Kolmogorov–Smirnov test, D = 0.55, *p < 0.001), specifically during the trace interval, US, and post-trial time points, whereas CS responses were similar. D, Temporal profile of mean spike response of persistent cells over the entire trial during preinfusion (top) or postinfusion (bottom) trials. The profile during the postinfusion trials was not significantly different from controls (Kolmogorov–Smirnov test, D = 0.25, p = 0.14), suggesting that persistent responses were unaffected by DCN inactivation.
