Age- and sex-dependent alterations in primary somatosensory cortex neuronal calcium network dynamics during locomotion

Abstract

Over the past 30 years, the calcium (Ca²⁺ ) hypothesis of brain aging has provided clear evidence that hippocampal neuronal Ca²⁺ dysregulation is a key biomarker of aging. Age-dependent Ca²⁺ -mediated changes in intrinsic excitability, synaptic plasticity, and activity have helped identify some of the mechanisms engaged in memory and cognitive decline based on work done mostly at the single-cell level and in the slice preparation. Recently, our lab identified age- and Ca²⁺ -related neuronal network dysregulation in the cortex of the anesthetized animal. Still, investigations in the awake animal are needed to test the generalizability of the Ca²⁺ hypothesis of brain aging. Here, we used in vigilo two-photon imaging in ambulating mice, to image GCaMP8f in the primary somatosensory cortex (S1), during ambulation and at rest. We investigated aging- and sex-related changes in neuronal networks in the C56BL/6J mouse. Following imaging, gait behavior was characterized to test for changes in locomotor stability. During ambulation, in both young adult and aged mice, an increase in network connectivity and synchronicity was noted. An age-dependent increase in synchronicity was seen in ambulating aged males only. Additionally, females displayed increases in the number of active neurons, Ca²⁺ transients, and neuronal activity compared to males, particularly during ambulation. These results suggest S1 Ca²⁺ dynamics and network synchronicity are likely contributors of locomotor stability. We believe this work raises awareness of age- and sex-dependent alterations in S1 neuronal networks, perhaps underlying the increase in falls with age.

Keywords: Ca2+; behavior; locomotor stability; mice; neuronal network; neuroscience; somatosensation; two-photon imaging.

FIGURE 1

Grip strength in young and aged mice. Measures of grip strength for forelimb, hind limb, and all limbs combined were obtained in young male (n = 8), aged male (n = 9), young female (n = 11), and aged female (n = 8) C57BL/6J mice. While a limb effect (i.e., forelimb vs. hind limb) was noted, no sex‐ or age‐associated changes were present. Data are expressed in Newtons (N). Main effect of limb was identified with p < 0.0001.

FIGURE 2

S1 neuronal network dynamics in young and aged mice on flat surface. (a) Individual GCaMP8f‐positive neurons are distinguishable in S1 in an aged male during ambulation and rest across a flat surface. Dark circular areas reflect penetrating blood vessels. (b) Representative GCaMP8f intensity traces from ROIs identified in panel (a) during periods of rest and ambulation. Scale bar represents 30 secs. (c) Individual raw GCaMP8f signals during rest and ambulation underwent a continuous wavelet transform (d) and were then thresholded (red line) and binarized (yellow vertical lines) (e) to extract events across multiple frequencies. (f) Corresponding animal's ambulation velocity trace across time. Measures of network communication (g–j) were obtained in young male (n = 7), aged male (n = 5), young female (n = 6), and aged female (n = 5) C57BL/6J mice and here, we report measures in the 0.1–2 Hz frequency domain. A main effect of sex was detected on measures of active neurons (g), connectivity (i), and synchronicity (j). A significant locomotion effect (ambulating vs. resting) was noted for measures of connectivity (i) and synchronicity (j). Additionally, a main effect of age was reported for measures of synchronicity (j) with age males having a significantly greater synchronicity compared to young males. No significant changes were detected on measures of connection length (p > 0.05). Main effects with p < 0.05, p < 0.001, and p < 0.0001 are represented with *, ***, and ****, respectively. Main effects of age, locomotion, and sex are represented with A, L, and S, respectively.

FIGURE 3

Neuronal network dynamics across frequency on flat surface. Measures of network communication were obtained in young male (n = 7), aged male (n = 5), young female (n = 6), and aged female (n = 5) C57BL/6J mice across multiple frequency domains during ambulation and rest. Main effects of locomotion status and frequency were detected on measures of connectivity (a, b) and synchronicity (e, f) in both males and females. A main effect of frequency was noted for measures of synchronicity. Interestingly, we note a significant effect of age on measures of connection length (c) and synchronicity (e) in males only, where aged males had greater lengths of connections and synchronicity than young males. No significant changes were detected on measures of connection length in female mice (d; p > 0.05). Main effects with p < 0.05, p < 0.01, p < 0.001, and p < 0.0001 are represented with *, **, ***, and ****, respectively. Main effects of age, locomotion, and frequency are represented with A, L, and F, respectively.

FIGURE 4

Neuronal network dynamics in young and aged mice across multiple surfaces. Measures of network communication were obtained in young male (n = 7), aged male (n = 5), young female (n = 6), and aged female (n = 5) C57BL/6J mice during ambulation and rest across multiple surfaces (a; 3‐mm, 4‐mm, and 5‐mm plastic mesh), and here, we report measures in the 0.1–2 Hz frequency domain. A main effect of locomotion status was detected on measures of connectivity (c, d), connection length (e, f), and synchronicity (g, h) in both males and females. Additionally, we note a significant effect of age on measures of synchronicity in both male and female mice (g, h), where aged males had greater synchronicity than young males, while aged females had decreased synchronicity compared to young females. No significant changes were detected on measures of active neurons (b) across sex, age, or surface (p > 0.05). Main effects with p < 0.05, p < 0.01, p < 0.001, and p < 0.0001 are represented with *, **, ***, and ****, respectively. Main effects of age and locomotion are represented with A and L, respectively.

FIGURE 5

Single‐cell (ΔF/F) neuronal Ca²⁺ dynamics on flat surface. Measures of single‐cell (ΔF/F) neuronal Ca²⁺ dynamics were obtained in young male (n = 7), aged male (n = 5), young female (n = 5), and aged female (n = 5) C57BL/6J mice during ambulation and rest across a flat surface. We report a main effect of locomotion status for measures of neuronal Ca²⁺ transients (a), area‐under‐curve (b), rise time (c), and decay time (d). Additionally, we report a main effect of sex for measures of neuronal activity (a) and area‐under‐curve (b). Main effects with p < 0.01, p < 0.001, and p < 0.0001 are represented with **, ***, and ****, respectively. Main effects of locomotion and sex are represented with L and S, respectively.

FIGURE 6

Gait behavior in young and aged mice across multiple surfaces. Measures of gait behavior were obtained in young male (n = 8), aged male (n = 6), young female (n = 10), and aged female (n = 7) C57BL/6J mice during ambulation across a flat (control), 3 mm, 4 mm, and 5 mm plastic mesh surface. A main effect of age was noted for measure of velocity (a) and stride time deviance (c), highlighting that aged animals ambulate slower and perhaps exhibit impaired gait rhythm or cadence. We report a main effect of sex for measures of stride time deviance (c) and stride length (e). Interestingly, we report a main effect of surface for measures of velocity (a). No main effects were seen for measures of deviance from center (b), stride length deviance (d), and paw precision (f). Main effects with p < 0.05, p < 0.01, and p < 0.001 are represented with *, **, and ***, respectively. Main effects of age, sex, and surface are represented with A, S, and Sur, respectively.