Article Figures & Data
Figures
- Figure 1.
Photoexcitability of A1 neurons remains unchanged during aging. A, Schematic of thalamocortical slicing. Slices were cut ∼15° from the horizontal plane to maintain the tonotopic organization in the slice. B, Left, Schematic of LSPS for cell-attached recording. Solid pink triangle represents recorded neuron. Middle, Infrared image of brain slice with patch pipette on L2/3 neuron. Stimulation grid is indicated by blue dots. Layer boundaries are indicated by white bars on the right. The scale bar at the bottom left is 200 μm. Right, Magnified infrared image with pipette patching a L2/3 pyramidal neuron. The scale bar at the bottom right is 15 μm. C, Relative position of recorded neurons within L2/3 relative to the borders of L4 and L1 (100%). Cells were sampled from a similar area in the middle of L2/3 in young adult and aged animals. D, E, Number of evoked action potentials (C) and effective stimulation distance (D) from cell-attached recordings of L2/3, L4 and L5/6 neurons. The numbers of evoked action potentials of neurons in all layers were similar and most spikes were evoked within 150 μm.
- Figure 2.
Sex-specific changes in excitatory connections to A1 L2/3 neurons in aged male mice. A, Schematic of LSPS when recording evoked EPSCs. Whole-cell voltage-clamp recordings were obtained from pink cell at a holding potential of −70 mV (EPSCs). If the presynaptic excitatory neuron (e.g., yellow cells) synapses on the recorded neuron, an EPSC will be observed. Shown on the right are examplar patch-clamp recordings of direct response (pink) and EPSCs (yellow), acquired at the holding potential of −70 mV. Black vertical line indicates time of photostimulation; shaded gray box labels the time window of evoked responses. B, Left, Schematic illustration of how the connection probability map is calculated. Connection maps from recorded neurons are aligned to the soma location and averaged to create the connection probability map. Right, Maps of connection probability for excitatory connections in young adult (left) and aged (right) animals. Soma location is indicated by the white circle. Connection probability is encoded according to the pseudocolor scale. White horizontal lines indicate averaged laminar borders and are 100 μm long. C, Left, Distributions of the area of excitatory input originating from L2/3 (top), L4 (middle), and L5/6 (bottom) of young adult (light green) or aged (dark green) animals. Middle, Distributions of the area of excitatory input originating from L2/3 (top), L4 (middle), and L5/6 (bottom) of adult female (light pink) or aged female (dark pink) animals. Right, Distributions of the area of excitatory input originating from L2/3 (top), L4 (middle), and L5/6 (bottom) of young adult male (light blue) or aged male (dark blue) animals. *pKS < 0.05; #pKruskal–Wallis < 0.05. D, Left, Integration distance to each L2/3 cell originating from L2/3 (top), L4 (middle), and L5/6 (bottom) of young adult or aged animals. Middle, Integration distance to each L2/3 cell originating from L2/3 (top), L4 (middle), and L5/6 (bottom) of adult female or aged female animals. Right, Integration distance to each L2/3 cell originating from L2/3 (top), L4 (middle), and L5/6 (bottom) of adult male or aged male animals.
- Figure 3.
Sex-specific changes of connection strength and percentage inputs to A1 L2/3 neurons in aged mice. A, Left, Distributions of mean EPSC charge of inputs originating from L2/3 (top), L4 (middle), and L5/6 (bottom) of young adult (light green) or aged (dark green) animals. $pRank-sum < 0.05. Middle, Distribution of mean charge of inputs to each L2/3 cell originating from L2/3 (top), L4 (middle), and L5/6 (bottom) of adult female (light pink) or aged female (dark pink) animals. **pKS < 0.01; #pKruskal–Wallis < 0.05. Right, Distribution of mean charge of inputs to each L2/3 cell originating from L2/3 (top), L4 (middle), and L5/6 (bottom) of adult male (light blue) or aged male (dark blue) animals. *pKS < 0.05; #pKruskal–Wallis < 0.05. B, Left, Distributions of mean EPSC amplitude of inputs originating from L2/3 (top), L4 (middle), and L5/6 (bottom) of young adult (light green) or aged (dark green) animals. Middle, Distribution of mean amplitude of inputs to each L2/3 cell originating from L2/3 (top), L4 (middle), and L5/6 (bottom) of adult female (light pink) or aged female (dark pink) animals. Right, Distribution of mean amplitude of inputs to each L2/3 cell originating from L2/3 (top), L4 (middle), and L5/6 (bottom) of adult male (light blue) or aged male (dark blue) animals. C, Left, Distributions of fraction of total excitatory input originating from L2/3 (top), L4 (middle), and L5/6 (bottom) of young adult or aged animals. *pKS < 0.05. Middle, Distributions of fraction of total excitatory input to each L2/3 cell originating from L2/3 (top), L4 (middle), and L5/6 (bottom) of adult female or aged female animals. *pKS < 0.05. Right, Distributions of fraction of total excitatory input to each L2/3 cell originating from L2/3 (top), L4 (middle), and L5/6 (bottom) of adult male or aged male animals.
- Figure 4.
Sex-specific changes in inhibitory connections to A1 L2/3 neurons in aged male mice. A, Schematic of LSPS when recording evoked IPSCs. Whole-cell voltage-clamp recordings were obtained from pink cell at holding potentials of 0 mV (IPSCs). If the presynaptic inhibitory neuron (e.g., green cells) synapses on the recorded neuron, an IPSC will be observed. Shown on the right are examplar patch-clamp recordings of IPSCs (green), acquired at the holding potential of 0 mV. Black vertical line indicates time of photostimulation; shaded gray box labels the time window of evoked responses. B, Average maps of connection probability for inhibitory connections in young adult (left) and aged (right) animals. Maps are aligned to the soma location (white circle). Connection probability is encoded according to the pseudocolor scale. White horizontal lines indicate averaged laminar borders and are 100 μm long. C, Left, Distributions of the area of inhibitory input originating from L2/3 (top), L4 (middle), and L5/6 (bottom) of young adult (light green) or aged (dark green) animals. *pKS < 0.05; $$pRank-sum < 0.01. Middle, Distributions of the area of inhibitory input originating from L2/3 (top), L4 (middle), and L5/6 (bottom) of adult female (light pink) or aged female (dark pink) animals. Right, Distributions of the area of inhibitory input originating from L2/3 (top), L4 (middle), and L5/6 (bottom) of young adult male (light blue) or aged male (dark blue) animals. *pKS < 0.05; **pKS < 0.01; #pKruskal–Wallis < 0.05; ##pKruskal–Wallis < 0.01. D, Left, Integration distance to each L2/3 cell originating from L2/3 (top), L4 (middle), and L5/6 (bottom) of young adult or aged animals. *pKS < 0.05. Middle, Integration distance to each L2/3 cell originating from L2/3 (top), L4 (middle), and L5/6 (bottom) of adult female or aged female animals. Right, Integration distance to each L2/3 cell originating from L2/3 (top), L4 (middle), and L5/6 (bottom) of adult male or aged male animals. *pKS < 0.05.
- Figure 5.
Inhibitory connection strength and percentage of inhibitory inputs originating from each layer remain unchanged between young and aged animals. A, Left, Distributions of mean IPSC charge of inputs originating from L2/3 (top), L4 (middle), and L5/6 (bottom) of young adult (light green) or aged (dark green) animals. **pKS < 0.01; ###pKruskal–Wallis < 0.001. Middle, Distribution of mean charge of inputs to each L2/3 cell originating from L2/3 (top), L4 (middle), and L5/6 (bottom) of adult female (light pink) or aged female (dark pink) animals. #pKruskal–Wallis < 0.05. Right, Distribution of mean charge of inputs to each L2/3 cell originating from L2/3 (top), L4 (middle), and L5/6 (bottom) of adult male (light blue) or aged male (dark blue) animals. **pKS < 0.01; #pKruskal–Wallis < 0.05. B, Left, Distributions of mean IPSC amplitude of inputs originating from L2/3 (top), L4 (middle), and L5/6 (bottom) of young adult or aged animals. $pRank-sum < 0.05. Middle, Distributions of mean amplitude of inputs to each L2/3 cell originating from L2/3 (top), L4 (middle), and L5/6 (bottom) of adult female or aged female animals. *pKS < 0.05. Right, Distributions of fraction of mean amplitude of inputs to each L2/3 cell originating from L2/3 (top), L4 (middle), and L5/6 (bottom) of adult male or aged male animals. *pKS < 0.05; **pKS < 0.01; #pKruskal–Wallis < 0.05. C, Left, Distributions of fraction of total inhibitory input originating from L2/3 (top), L4 (middle), and L5/6 (bottom) of young adult or aged animals. Middle, Distributions of fraction of total inhibitory input to each L2/3 cell originating from L2/3 (top), L4 (middle), and L5/6 (bottom) of adult female or aged female animals. Right, Distributions of fraction of total inhibitory input to each L2/3 cell originating from L2/3 (top), L4 (middle), and L5/6 (bottom) of adult male or aged male animals.
- Figure 6.
Aged female animals show altered E/I ratio. A, Left, Distributions of E/I based on input numbers (density) from L2/3 (top), L4 (middle), and L5/6 (bottom) of young adult (light green) or aged (dark green) animals. *pKS < 0.05. Middle, Distributions of E/I based on input numbers (density) to each L2/3 cell originating from L2/3 (top), L4 (middle), and L5/6 (bottom) of adult female (light pink) or aged female (dark pink) animals. *pKS < 0.05. Right, Distributions of E/I based on input numbers (density) from L2/3 (top), L4 (middle), and L5/6 (bottom) of adult male (light blue) or aged male (dark blue) animals. B, Left, Distributions of E/I based on transferred charge originating from L2/3 (top), L4 (middle), and L5/6 (bottom) of young adult or aged animals. Middle, Distributions of E/I based on transferred charge to each L2/3 cell originating from L2/3 (top), L4 (middle), and L5/6 (bottom) of adult female or aged female animals. Right, Distributions of E/I based on transferred charge to each L2/3 cell originating from L2/3 (top), L4 (middle), and L5/6 (bottom) of adult male or aged male animals.
- Figure 7.
Circuit similarity decreased in aged mice. Fano factors (variance/mean) for excitatory connection maps of adult (left), aged (middle), and the differences between adult and aged (right). The Fano factor is represented in pseudocolor. The smaller the Fano factor, the greater the similarity in the population. In the maps, white represents areas where the probability of the connections was <5% and a Fano factor was not calculated. A, Fano factors for the excitatory connection maps. B, Fano factors for the inhibitory connection maps.
- Figure 8.
Decreased similarity of input maps in aged mice. A, Schematic illustration of how pairwise correlation between two maps is calculated. Filled white circle represents recorded neuron. A yellow square represents a stimulus location that has monosynaptic connection to the recorded cell; a blue square represents a location with no connection to the recorded cell. For the pairwise correlation calculation, yellow and blue squares are assigned values of 1 and 0, respectively. B, Correlation of excitatory and inhibitory circuit patterns across all layers. Left, Correlation of circuit patterns for all adult (light green) and aged (dark green) animals. $$$pRank-sum < 0.001. Middle, Correlation of circuit patterns for adult female (light pink) and aged female (dark pink) animals. Right, Correlation of circuit patterns for adult male (light blue) and aged male (dark blue) animals. #pKruskal–Wallis < 0.05; ###pKruskal–Wallis < 0.001. C, Correlation of circuit patterns within L2/3 (left), L4 (middle), and L5/6 (right). Left, Circuit similarity from different layers in adult and aged animals. $$$pRank-sum < 0.001. Middle, Circuit similarity from different layers in adult and aged female animals. #pKruskal–Wallis < 0.05. Right, Circuit similarity from different layers in adult and aged male animals. #pKruskal–Wallis < 0.05; ##pKruskal–Wallis < 0.01; ###pKruskal–Wallis < 0.001.
Tables
Excitation Adult Aged p values Input area (μm2) Mean Median Mean Median KS test Ranksum All L2/3 1.84 × 104 1.60 × 104 2.23 × 104 1.68 × 104 0.68 0.57 L4 7.96 × 103 3.20 × 103 6.37 × 103 2.40 × 103 0.96 0.97 L5/6 1.04 × 104 3.20 × 103 1.00 × 104 4.80 × 103 0.49 0.19 Mean Median Mean Median KS test Kruskal–Wallis Female L2/3 1.97 × 104 1.60 × 104 3.31 × 104 2.88 × 104 0.025 0.14 L4 7.92 × 103 1.60 × 103 1.09 × 104 8.00 × 103 0.21 0.74 L5/6 1.06 × 104 1.60 × 103 1.47 × 104 8.80 × 103 0.041 0.011 Male L2/3 1.73 × 104 1.44 × 104 1.30 × 104 1.20 × 104 0.44 1.0 L4 8.00 × 103 4.80 × 103 2.46 × 103 1.60 × 103 0.22 0.16 L5/6 1.02 × 104 4.80 × 103 5.94 × 103 3.20 × 103 0.25 1.0 Integration distance (μm) Mean Median Mean Median KS test Ranksum All L2/3 191.1 174.5 198.6 210.0 0.17 0.23 L4 113.0 100.0 124.5 98.0 0.96 0.53 L5/6 191.9 214.1 223.1 272.0 0.36 0.30 Mean Median Mean Median KS test Kruskal–Wallis Female L2/3 191.1 165.1 231.6 228.0 0.079 0.43 L4 111.0 96.5 180.7 200.0 0.098 0.30 L5/6 198.6 248.4 261.2 302.3 0.12 1.00 Male L2/3 191.1 189.9 172.9 173.0 0.36 1.00 L4 114.7 100.0 80.6 0.0 0.35 1.00 L5/6 186.2 196.7 193.3 234.1 0.87 1.00 Mean charge (pC) Mean Median Mean Median KS test Ranksum All L2/3 1.28 1.26 0.95 1.12 0.26 0.078 L4 0.54 0.95 0.42 0.59 0.074 0.023 L5/6 0.45 0.38 0.44 0.39 0.99 0.94 Mean Median Mean Median KS test Kruskal–Wallis Female L2/3 1.18 1.09 1.18 1.28 0.86 1.00 L4 0.70 1.11 0.65 0.64 0.004 0.021 L5/6 0.53 0.54 0.56 0.37 0.44 0.51 Male L2/3 1.35 1.28 0.77 0.86 0.028 0.025 L4 0.40 0.64 0.24 0.46 0.14 1.00 L5/6 0.38 0.35 0.35 0.40 0.63 1.00 Mean amplitude (pA) Mean Median Mean Median KS test Ranksum All L2/3 28.55 28.57 28.08 30.48 0.81 0.57 L4 17.13 30.38 19.94 28.95 0.56 0.78 L5/6 18.21 23.15 22.38 25.11 0.96 0.73 Mean Median Mean Median KS test Kruskal–Wallis Female L2/3 27.73 27.63 35.42 31.01 0.19 1.00 L4 18.65 33.59 27.50 27.82 0.30 1.00 L5/6 18.85 27.72 27.82 26.75 0.46 1.00 Male L2/3 29.25 29.48 22.34 29.54 0.76 1.00 L4 15.85 25.36 14.03 32.01 0.59 1.00 L5/6 17.98 23.09 18.13 21.61 0.70 1.00 Fractional input area (%) Mean Median Mean Median KS test Ranksum All L2/3 63.50 63.15 60.13 60.00 0.076 0.64 L4 16.80 10.80 12.00 12.50 0.22 0.53 L5/6 19.71 13.27 27.88 22.22 0.047 0.085 Mean Median Mean Median KS test Kruskal–Wallis Female L2/3 66.41 61.29 62.86 60.61 0.046 1.00 L4 18.09 12.50 15.98 16.78 0.21 1.00 L5/6 15.50 12.50 21.15 20.94 0.028 0.74 Male L2/3 60.84 65.00 57.70 56.76 0.83 1.00 L4 15.61 9.09 8.45 5.00 0.47 0.93 L5/6 23.55 21.43 33.85 30.00 0.89 1.00 Inhibition Adult Aged p values Input area (μm2) Mean Median Mean Median KS test Ranksum All L2/3 7.90 × 104 7.20 × 104 6.71 × 104 5.52 × 104 0.042 0.12 L4 1.60 × 104 1.44 × 104 1.15 × 104 8.00 × 103 0.046 0.007 L5/6 4.27 × 104 3.68 × 104 3.62 × 104 2.96 × 104 0.18 0.19 Mean Median Mean Median KS test Kruskal–Wallis Female L2/3 8.23 × 104 8.32 × 104 9.16 × 104 8.64 × 104 0.95 1.00 L4 1.65 × 104 1.44 × 104 1.68 × 104 1.28 × 104 0.69 1.00 L5/6 3.77 × 104 3.36 × 104 4.87 × 104 4.48 × 104 0.65 0.91 Male L2/3 7.61 × 104 6.72 × 104 4.77 × 104 4.48 × 104 0.003 0.031 L4 1.56 × 104 1.44 × 104 7.28 × 103 4.80 × 103 0.003 0.002 L5/6 4.71 × 104 4.88 × 104 2.64 × 104 1.76 × 104 0.029 0.030 Integration distance (μm) Mean Median Mean Median KS test Ranksum All L2/3 248.9 244.7 251.5 275.9 0.030 0.12 L4 270.4 282.0 233.3 264.2 0.38 0.40 L5/6 311.7 347.0 280.8 318.4 0.89 0.36 Mean Median Mean Median KS test Kruskal–Wallis Female L2/3 257.3 245.5 262.5 275.9 0.092 1.00 L4 286.0 299.2 263.1 298.0 0.94 1.00 L5/6 305.1 301.1 295.4 335.4 0.66 1.00 Male L2/3 241.7 243.2 242.9 274.7 0.32 1.00 L4 257.1 247.3 210.1 196.4 0.039 1.00 L5/6 317.3 357.1 269.4 317.3 0.26 0.94 Mean charge (pC) Mean Median Mean Median KS test Ranksum All L2/3 5.64 6.06 4.76 4.61 0.17 0.27 L4 2.45 1.97 1.32 0.93 0.003 0.001 L5/6 1.18 1.06 0.99 0.78 0.36 0.18 Mean Median Mean Median KS test Kruskal–Wallis Female L2/3 5.46 5.45 0.18 5.71 0.92 1.00 L4 2.97 2.20 1.47 1.69 0.15 0.56 L5/6 1.17 1.06 0.95 0.96 0.97 1.00 Male L2/3 5.79 6.53 4.03 3.85 0.007 0.21 L4 2.01 1.94 1.21 0.70 0.004 0.002 L5/6 1.20 1.05 1.03 0.72 0.22 0.87 Mean amplitude (pA) Mean Median Mean Median KS test Ranksum All L2/3 82.58 84.93 78.61 73.42 0.43 0.55 L4 46.64 38.92 31.12 31.97 0.11 0.034 L5/6 29.20 30.55 27.18 28.70 0.62 0.72 Mean Median Mean Median KS test Kruskal–Wallis Female L2/3 76.49 83.77 98.75 98.11 0.30 0.86 L4 52.17 36.84 37.48 39.91 0.80 1.00 L5/6 27.32 28.49 28.51 34.50 0.046 0.42 Male L2/3 87.72 90.28 62.88 62.88 0.080 0.055 L4 41.96 39.32 26.15 26.15 0.032 0.094 L5/6 30.79 30.70 26.14 26.14 0.009 0.047 Fractional percentage input area (%) Mean Median Mean Median KS test Ranksum All L2/3 56.68 58.14 60.72 57.51 0.36 0.33 L4 12.35 10.29 9.44 9.73 0.15 0.083 L5/6 30.98 30.56 29.84 32.36 0.55 0.77 Mean Median Mean Median KS test Kruskal–Wallis Female L2/3 58.73 58.14 62.37 58.23 0.13 1.00 L4 13.13 11.94 9.90 10.81 0.48 1.00 L5/6 28.15 29.93 27.74 33.63 0.28 1.00 Male L2/3 54.88 56.95 59.42 56.76 0.26 1.00 L4 11.66 9.18 9.08 7.94 0.078 1.00 L5/6 33.45 31.04 31.51 32.05 0.26 1.00 Area Distance Charge % input M F M F M F M F Excitation L2/3 o + o o − o o + L4 o o o o o − o o L5/6 o + o o o o o + Inhibition L2/3 − o o o o o o o L4 − o o o − o o o L5/6 − o o o o o o o
In this issue
