Article Figures & Data
Figures
- Figure 1.
Hearing status of the different groups. A, Examples of averaged ABR traces in response to tone pips at 4 kHz, played back at levels ranging from 40 to 80 dB. The dashed line indicates the onset of a 10-ms tone pip. The asterisks mark the level at which the threshold was set. Left, Individual from the agedB6 group. Right, Animal from the agedB6.CAST group without AHL, both animals were measured at an age of 14 months. B, Mean threshold for all four groups at different sound frequencies. Circles depict B6 animals, squares display animals from the B6.CAST line. Open symbols represent young and filled symbols aged animals (mean ± SEM).
- Figure 2.
Analysis of histology. A, Identification of the pAC. The pAC was identified by using the hippocampal fissure (HF; DG, dentate gyrus) as a reference structure. Three selected regions along the rostro-caudal axis are shown (from left to right, y coordinate relative to bregma: −3.68, −3.005, −2.48 mm). The landmark was chosen according to the mouse brain reference atlas and centered under the microscope. By following a straight line, the camera section was moved to the cortex (CTX) and the center of pAC was photographed. The magenta rectangle symbolizes the ROI that was used for the counting procedure. For the confirmation of the cortex position, see Extended Data Figure 2-1 and Tsukano et al. (2016). TH: thalamus; CP: caudoputamen. B, Example image with ROI for the counting of PV+ neurons. C, Example image with ROI for the counting of Nissl-stained neurons. Detailed image, distinguishing between neurons and other cell types (arrow, neuron; asterisk, non-neuronal cell type).
- Figure 3.
Total number of neurons and PV+ immunoreactivity of the different groups. Circles depict B6 animals, squares display animals from the B6.CAST line. Open symbols represent young and filled symbols aged animals (mean ± SEM), significance values are displayed by number of asterisks: *p < 0.01; **p < 0.001. A, Mean total number of neurons/ROI. No significant difference could be revealed for the total number of neurons. B, Mean number of PV+ neurons/ROI. When compared with youngB6/B6.CAST, both agedB6 and agedB6.CAST show a significant decrease in number of positive neurons. C, Mean PV+ density. Young animals show a significant higher density of PV+ neurons compared to aged animals of both groups.
- Figure 4.
Distribution of PV+ neurons across layers and along the rostro-caudal axis. A, Illustration of the relative laminar mapping of the cell positions within the single slices. The pia was set to depth = 0 and the border to white matter was set to depth = 1. B, Population overview. Relative density of PV+ neurons resolved by the rostro-caudal position of slices and cortical depth of cell body is shown. Here, density was averaged over all slices from all animals to reveal an age-independent overview about the general PV pattern in the pAC. Density was normalized to the maximum of the distribution. C, Cell density resolved by rostro-caudal position of the slices (relative to bregma). Each bar depicts mean density for all slices within the respective bin along the rostro-caudal axis. Left, Young animals of both lines. Middle, agedB6 group. Right, agedB6.CAST group. D, Laminar distribution of PV+ neurons in the three groups (mean ± SEM). Positions are relative to the pia (see A). E, Relative density of PV+ neurons in the slices resolved by rostro-caudal position and cortical depth in the three groups. Color-coded densities are normalized to the maximum mean density in the young group (left, youngB6/B6.CAST; middle, agedB6; right, agedB6.CAST). Plots (B–E) were generated using MATLAB (version R2018b; The MathWorks Inc.).
Tables
Group Age Histology ABR Double Total YoungB6 10–12 weeks 6 6 1 11 YoungB6.CAST 10–12 weeks 2 9 1 7 AgedB6 12–15 months 8 7 2 13 AgedB6.CAST 12–15 months 6 9 3 12 Sum 22 28 7 43 Ref # Data structure Parameters tested Type of test Power (1-β err prob) Figure A Three independent samples:
youngB6/B6.CAST (n = 8),
agedB6 (n = 8),
agedB6.CAST (n = 6)Total number of neurons/ROI ANOVA 0.3739166 3A B Three independent samples:
youngB6/B6.CAST (n = 8),
agedB6 (n = 8),
agedB6.CAST (n = 6)Number of PV+ interneurons/ROI ANOVA 0.9993094 3B C Three independent samples:
youngB6/B6.CAST (n = 8),
agedB6 (n = 8),
agedB6.CAST (n = 6)Density of PV+ interneurons, number of PV+/mm2 ANOVA 0.8839026 3C
Extended Data Figure 2-1
Confirmation of the position of auditory cortex in B6.cast mice. A, A movable electrode array was implanted, stereotactically positioned at 2.6 mm anterior to bregma. The path of the probe was angled at 24° in order to follow the path indicated by the black line. Arrowheads indicate the positions of the starting point after surgery and the last position that was recorded from in the subject with furthest travel of the probe. Final positions varied from subject to subject. Recordings were obtained from an array of eight tetrodes at each position in awake, unrestrained animals, typically yielding 20–30 units per position. The orange area depicts cortical fields labeled as “auditory areas” according to the Allen mouse brain reference atlas (version 2012), which is based on tracing data. The thick yellow line at the top of the cortex indicates auditory areas in B6/C57 mice as determined by a study using physiological imaging (Tsukano et al., 2016). The red box is our window for counting PV+ cells, aligned by anatomical landmarks at 2.6 anterior to bregma. B, Proportion of auditory and non-auditory units recorded at each position along the probe path. Each horizontal sequence of pie charts represents data from a single animal (n = 6), the horizontal position of the pie center marks the position of the probe along the path. The grey area depicts the medial-lateral position of the window used for counting PV+ cells at 2.6 anterior to bregma. Each pie chart displays the proportion of non-auditory units (white), determined by a stimulus set containing both simple tone and complex naturalistic stimuli. Auditory units were classified as primary (blue) and non-primary (red) based on response latency (<20 ms) to tone stimuli, responsiveness to repeated simple stimuli, and the shape of the frequency tuning curve. Data was collected in the context of another study, for further methodological details see Gothner et al. (2019). Download Figure 2-1, TIF file.
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