Structure-function relationships between aldolase C/zebrin II expression and complex spike synchrony in the cerebellum - PubMed (original) (raw)
Structure-function relationships between aldolase C/zebrin II expression and complex spike synchrony in the cerebellum
Shinichiro Tsutsumi et al. J Neurosci. 2015.
Abstract
Simple and regular anatomical structure is a hallmark of the cerebellar cortex. Parasagittally arrayed alternate expression of aldolase C/zebrin II in Purkinje cells (PCs) has been extensively studied, but surprisingly little is known about its functional significance. Here we found a precise structure-function relationship between aldolase C expression and synchrony of PC complex spike activities that reflect climbing fiber inputs to PCs. We performed two-photon calcium imaging in transgenic mice in which aldolase C compartments can be visualized in vivo, and identified highly synchronous complex spike activities among aldolase C-positive or aldolase C-negative PCs, but not across these populations. The boundary of aldolase C compartments corresponded to that of complex spike synchrony at single-cell resolution. Sensory stimulation evoked aldolase C compartment-specific complex spike responses and synchrony. This result further revealed the structure-function segregation. In awake animals, complex spike synchrony both within and between PC populations across the aldolase C boundary were enhanced in response to sensory stimuli, in a way that two functionally distinct PC ensembles are coactivated. These results suggest that PC populations characterized by aldolase C expression precisely represent distinct functional units of the cerebellar cortex, and these functional units can cooperate to process sensory information in awake animals.
Keywords: Purkinje cell; climbing fiber; microzone; zone.
Copyright © 2015 the authors 0270-6474/15/350843-10$15.00/0.
Figures
Figure 1.
Aldolase C compartments are visualized in vivo. A, Knock-in construct of Aldoc-tdTomato mouse line. Gray boxes indicate exon 1–9. Met, initial methionine; Tomato, tdTomato coding sequence followed by a polyA addition signal sequence; Neo, pgk-gb2 neomycin resistance cassette; E, EcoR V; S, SacI. B, Two-photon imaging of aldolase C compartments in vivo. A single horizontal plane in the molecular layer (left) and XZ (right top) and XY (right bottom) projection of the image stack. R, rostral; C, caudal; L, lateral; M, medial; D, dorsal; V, ventral. Scale bars: 40 μm. C, A representative coronal section of the cerebellum taken from an Aldoc-tdTomato mouse immunostained with anti-calbindin (blue) and anti-aldolase C (green) antibodies. Red fluorescence of tdTomato was overlaid. Scale bars: Ca, 1 mm; Cb, 200 μm; Cc, 50 μm.
Figure 2.
Aldolase C compartment boundaries correspond to those determined by complex spike synchrony at single-cell resolution. A, Left, Example waveforms of simple spike and complex spike. Right, Simultaneous cell-attached recording (top) and dendritic calcium imaging (bottom) from the same PC. Blue asterisks represent complex spike peaks. SS, simple spike; CS, complex spike. Ba, An example image from the raw movie of OGB-1 AM calcium imaging. Bb, Detected angle of PC dendrites. Bc, Color-coded correlation coefficient between a certain pixel and all the pixels in the strip. Bd, Color-coded correlation coefficient between seed pixels and all the pixels in the imaging field. Be, Adaptive threshold filtered map from Bd. Only one ROI around the seed pixels was selected. Bf, Pseudocolored map of the ROIs. C, Calcium imaging was performed at a representative aldolase C compartment boundary (5−/5+). Pseudocolored ROIs (PC dendrites) are overlaid to the corresponding RFP image. D, Representative Δ_F_/F waves taken from C. Red and blue traces are from aldolase C-positive and -negative PCs, respectively. E, Correlation matrix calculated by using Δ_F_/F waves from all the dendrite pairs in C. Red and blue bars denote aldolase C-positive and -negative PCs, respectively. R, rostral; C, caudal; L, lateral; M, medial. Scale bars: 40 μm.
Figure 3.
Relationships between aldolase C compartments and complex spike synchrony. A, Correlation matrices between Δ_F_/F waves from 20 adjacent dendrites across aldolase C boundaries; 10 dendrites each from both sides of the boundaries in Crus IIa of the left hemisphere. Data are averaged across mice. Red and blue bars represent aldolase C-positive and -negative PCs, respectively; n = 6, 7, 8, 6, and 3 mice for the 7+/6−, 6−/6+, 6+/5−, 5−/5+, and 5+/5a− boundaries, respectively. B, Synchrony between Δ_F_/F waves from PC dendrite pairs whose mediolateral distance were <100 μm and located within and across the aldolase C compartments. Data are represented as mean ± SEM. P-P (red bars), synchrony of Δ_F_/F waves between the aldolase C-positive PCs; P-N (green bars), that across aldolase C-positive and -negative PCs; and N-N (blue bars), that between the aldolase C-negative PCs. ***p < 0.001, two-way ANOVA followed by post hoc Tukey's test. C, Calcium transient synchrony plotted against the mediolateral separation between PC dendrite pairs. Data from 7+/6−, 5−/5+, and 5+/5a− boundaries are averaged (4670 dendrite pairs, n = 12 mice). P-P (red trace), P-N (green trace), and N-N (blue trace) represent synchrony between aldolase C-positive to -positive, aldolase C-positive to -negative, and aldolase C-negative to -negative PC dendrite pairs, respectively. Data are averaged within 20 μm bin and represented as mean ± SEM. Red and blue asterisks denote significant difference between P-P and P-N, and between N-N and P-N, respectively. ***p < 0.001, two-way ANOVA followed by post hoc Tukey's test. D, Same as A, but only 5+/5− boundary in Crus IIa of right hemisphere; n = 6 mice. E, Left, Calcium imaging using Cal-520 was performed within a single compartment (5−) in Crus IIa of right hemisphere. Pseudocolored ROIs are overlaid to the corresponding RFP image. Right, Correlation matrix calculated by using Δ_F_/F waves from all the dendrite pairs in the corresponding images on the left. Blue bars represent aldolase C-negative PCs. R, rostral; C, caudal; L, lateral; and M, medial. Scale bar, 40 μm.
Figure 4.
Relationship between aldolase C compartments and complex spike synchrony is unchanged after application of carbenoxolone or harmaline. A, Averaged frequency of calcium transient in PCs within aldolase C-positive (red) and -negative (blue) compartments before and after carbenoxolone application (baseline: 49 and 44 cells, carbenoxolone: 52 and 41 cells for 5+ and 5− compartment, respectively; n = 3 mice). Data are represented as mean ± SEM. No significant difference was observed (Mann–Whitney U test). B, Correlation matrices averaged across mice (n = 3 mice) before and after carbenoxolone application. Red and blue bars denote aldolase C-positive and -negative PCs, respectively. C, Same as A, but for harmaline (baseline: 46 and 38 cells; harmaline: 50 and 43 cells for 5+ and 5− compartment, respectively; n = 3 mice). Data are represented as mean ± SEM, *p < 0.05, ***p < 0.001, Mann–Whitney U test. D, Same as B, but for harmaline (n = 3 mice). Note that the boundaries of synchrony are unaffected by injections of either carbenoxolone or harmaline.
Figure 5.
Boundaries of sensory response profiles and sensory-evoked microzone correspond to those of aldolase C compartments. A, Schematic experimental settings. Mice received air-puff stimulation during calcium imaging. B, Representative RFP image at 7+/6− boundary where sensory response was consistently observed. Pseudocolored PC dendrites are overlaid. Black line represents 7+/6− aldolase C boundary. R, rostral; C, caudal; L, lateral; M, medial. Scale bar, 40 μm. C, Sensory-evoked calcium transients around the aldolase C boundary in B. Thick lines represent the average of responses in one imaging session (13 trials). Black inverted triangles denote the onset of air-puff stimuli. D, Color-coded response probability of PCs near aldolase C boundaries in left Crus II. White broken lines represent aldolase C boundaries. The numbers of ROI proximal to the aldolase C boundaries are shown for clarity; n = 7, 7, 8, 5, and 3 mice at the 7+/6−, 6−/6+, 6+/5−, 5−/5+, and 5+/5a− compartments, respectively. E, Correlation matrices during air-puff trials (top row) compared with that of the spontaneous events during the same period (middle row). Matrices in the bottom row were generated by subtracting the spontaneous matrices from the evoked ones. Red and blue bars represent PC dendrites in aldolase C-positive and -negative compartments, respectively; n = 6, 7, 8, 5, and 3 mice for the 7+/6−, 6−/6+, 6+/5−, 5−/5+, and 5+/5a− compartments, respectively.
Figure 6.
Microzones are coactivated during sensory stimulation in awake mice. A, Left, Color-coded response probability of PCs around the 7+/6− aldolase C boundary in Crus II (same data as Fig. 5_D_, left). Right, Same as the left, but during awake state. White broken lines represent the aldolase C boundary; n = 6 and 3 mice for anesthetized and awake states, respectively. B, Correlation matrices of calcium transients in PCs at the 7+/6− boundary in awake mice during periods with (right) and without (left) air-puff stimulation. Red and blue bars represent dendrites in aldolase C-positive and -negative compartments, respectively; n = 3 mice. C, Right, Averaged complex spike synchrony calculated using matrices in B within areas depicted on the left (a–f), without (−) and with (+) air-puff stimulation. Data are represented as mean ± SEM; ***p < 0.001, two-way ANOVA followed by post hoc Tukey's test.
References
- Bell CC, Kawasaki T. Relations among climbing fiber responses of nearby Purkinje cells. J Neurophysiol. 1972;35:155–169. -PubMed
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