Aging affects GABAergic function and calcium homeostasis in the mammalian central clock

Abstract

Introduction: Aging impairs the function of the central circadian clock in mammals, the suprachiasmatic nucleus (SCN), leading to a reduction in the output signal. The weaker timing signal from the SCN results in a decline in rhythm strength in many physiological functions, including sleep-wake patterns. Accumulating evidence suggests that the reduced amplitude of the SCN signal is caused by a decreased synchrony among the SCN neurons. The present study was aimed to investigate the hypothesis that the excitation/inhibition (E/I) balance plays a role in synchronization within the network.

Methods: Using calcium (Ca²⁺) imaging, the polarity of Ca²⁺ transients in response to GABA stimulation in SCN slices of old mice (20-24 months) and young controls was studied.

Results: We found that the amount of GABAergic excitation was increased, and that concordantly the E/I balance was higher in SCN slices of old mice when compared to young controls. Moreover, we showed an effect of aging on the baseline intracellular Ca²⁺ concentration, with higher Ca²⁺ levels in SCN neurons of old mice, indicating an alteration in Ca²⁺ homeostasis in the aged SCN. We conclude that the change in GABAergic function, and possibly the Ca²⁺ homeostasis, in SCN neurons may contribute to the altered synchrony within the aged SCN network.

Keywords: calcium imaging; chloride transporters; circadian; excitatory/inhibitory balance; old mice; suprachiasmatic nucleus.

Figure 1

More GABAergic excitation in SCN slices from old mice. (A) Upper panels: examples of fura-2-AM loaded SCN neurons in slices from young (left) and old (right) mice. Color scale indicates fluorescence at 380 nm excitation in arbitrary units (Scale bar, 20 μm). Lower panels: example traces of Ca²⁺ transients in response to two GABA administrations recorded from one SCN slice from a young (left) and old (right) mouse. Excitatory responses are shown in blue, inhibitory responses in orange and non-responding cells in black (Scale bars, 50 nM, 20 s). (B) The percentages of inhibitory, excitatory, non-responding, and biphasic cells. Each dot represents the mean percentage of responses per response type per SCN. Every single dot in one response type category adds up to 100% together with the corresponding dots in the other categories. (C) E/I ratio’s in young and old mice, determined by dividing the number of excitatory responses by the number of inhibitory responses measured from each SCN. Open dots represent values from young mice (n = 11) and filled, green dots represent values from old (n = 9) mice. Bars indicate mean ± SEM. *p < 0.05, **p < 0.01; distribution of GABAergic response types: GEE with Bonferroni correction (B), E/I ratio: unpaired t-test with Welch’s correction (C).

Figure 2

Spatial differences in GABAergic responses along the anteroposterior axis. (A–C) The percentages of inhibitory, excitatory, non-responding, and biphasic cells in the anterior (A), central (B), and posterior (C) part of the young and old SCN. Each dot represents the mean percentage of responses per response type per SCN. Every single dot in one response type category adds up to 100% together with the corresponding dots in the other categories. (D) Distribution of GABAergic response types for the anterior, central, and posterior part of the young (left) and the old (right) SCN. Orange represents the percentage of inhibitory responses, dark blue represents excitatory responses, white represents non-responding cells, and light blue represents biphasic responses. The value on top of the bar shows the total number of cells measured. (E) E/I ratios per SCN region in young and old mice, determined by dividing the number of excitatory responses by the number of inhibitory responses measured from different parts of the SCN along the anteroposterior axis. Open dots represent values from young mice and filled, green dots represent values from old mice. Bars indicate mean ± SEM. **p < 0.01; distribution of GABAergic response types: GEE with Bonferroni correction (A,B,C,E).

Figure 3

GABAergic responses along the dorsoventral axis. (A) Distribution of GABAergic response types for the ventral and dorsal part of the young (left) and the old (right) SCN. Orange represents the percentage of inhibitory responses, dark blue represents excitatory responses, white represents non-responding cells, and light blue represents biphasic responses. The value on top of the bar represents the total number of cells measured. (B,C) The percentages of inhibitory, excitatory, non-responding, and biphasic cells in the dorsal and ventral part of the young (B) and old (C) SCN. Each dot represents the mean percentage of responses per response type per SCN sub-region. Every single dot in one response type category adds up to 100% together with the corresponding dots in the other categories. Filled triangles represent values from the ventral part and open triangles represent values from the dorsal part of the SCN. Bars indicate mean ± SEM. GEE for each category with Bonferroni correction (B,C) n. s.

Figure 4

Baseline [Ca²⁺]_i is higher in old SCN neurons. (A) Violin plots show baseline [Ca²⁺]_i levels (nM) from all SCN neurons measured in slices from young (n = 1,204) and old (n = 924) mice. (B) Violin plots show baseline [Ca²⁺]_i levels (nM) from all SCN neurons measured categorized per GABAergic response type. White violins represent data from young mice and grey violins represent data from old mice. Violin plots show median and quartiles, **p < 0.01, ****p < 0.0001, unpaired t-test with Welch’s correction (A), GEE with Bonferroni correction (B).

Figure 5

Age-dependent changes in Ca²⁺ homeostasis. (A) Example of depolarization-induced Ca²⁺ transient to explain the parameters analyzed and plotted in (B–G). (B) Violin plot showing the amplitude of the Ca²⁺ response to depolarization caused by elevated extracellular K⁺ for all cells recorded. (C) Rise time of the depolarization-induced Ca²⁺ transient is significantly increased in old SCN neurons compared to young controls. (D,E) Analysis of subregions show significant higher amplitude of High K⁺ response in old neurons of posterior and dorsal SCN compared to young controls. (F,G) Time constant of rise in [Ca²⁺]_i is significantly increased in ventral neurons of SCN neurons from old mice compared to young controls. *p < 0.05, **p < 0.01, independent samples t-test (B,C), GEE with Bonferroni correction (D–G).