Hippocampal Gαq/₁₁ but not Gαo-coupled receptors are altered in aging

Abstract

Normal aging may limit the signaling efficacy of certain GPCRs by disturbing the function of specific Gα-subunits and leading to deficient modulation of intracellular functions that subserve synaptic plasticity, learning and memory. Evidence suggests that Gαq/₁₁ is more sensitive to the effects of aging relative to other Gα-subunits, including Gαo. To test this hypothesis, the functionality of Gαq/₁₁ and Gαo were compared in the hippocampus of young (6 months) and aged (24 months) F344 × BNF₁ hybrid rats assessed for spatial learning ability. Basal GTPγS-binding to Gαq/₁₁ was significantly elevated in aged rats relative to young and but not reliably associated with spatial learning. mAChR stimulation of Gαq/₁₁ with oxotremorine-M produced equivocal GTPγS-binding between age groups although values tended to be lower in the aged hippocampus and were inversely related to basal activity. Downstream Gαq/₁₁ function was measured in hippocampal subregion CA₁ by determining changes in [Ca(2+)]i after mAChR and mGluR (DHPG) stimulation. mAChR-stimulated peak change in [Ca(2+)]i was lower in aged CA₁ relative to young while mGluR-mediated integrated [Ca(2+)]i responses tended to be larger in aged. GPCR modulation of [Ca(2+)]i was observed to depend on intracellular stores to a greater degree in aged than young. In contrast, measures of Gαo-mediated GTPγS-binding were stable across age, including basal, mAChR-, GABABR (baclofen)-stimulated levels. Overall, the data indicate that aging selectively modulates the activity of Gαq/₁₁ within the hippocampus leading to deficient modulation of [Ca(2+)]i following stimulation of mAChRs but these changes are not related to spatial learning.

Fig 1. Performance of young and aged rats in the Morris water maze

Young (6 months; n=21) and aged (24 months; n=47) rats were trained on a hidden-platform/place-learning version of the Morris water maze organized into 4 blocks that each included 5 training trials (A) and a single probe trial (B) administered at the end of each training block. After the end of place training, rats received a single block of six visible-platform/cue-training trials (C). Probe trial measures were summed to compute “proximity scores” to characterize the range of individual performance differences within this study population (D; horizontal line denotes mean of each group). Proximity scores of each cohort used for GTPγS-binding (n=10 young, n=22 aged; E), oxotremorine-M-stimulated [Ca²⁺]_i (n=6 young, n=15 aged; F) or DHPG-stimulated [Ca²⁺]_i (n=5 young, n=10 aged; G). **p<0.01 and ***p<0.001 vs. young according to Bonferroni post hoctest (A&B). *** p<0.001 vs. young by independent samples t-test (D–G).

Fig 2. GTPγS-binding to Gα_q/11 in the hippocampus of young and aged rats

[³⁵S]GTPγS-binding assay was combined with an antibody-capture scintillation proximity counting approach to measure Gα_q/11-specific GTPγS-binding in the 3 major hippocampal subregions (n=10 young, n=22 aged; A and D). Basal (agonist-free) GTPγS-binding to Gα_q/11 (A) was averaged for all 3 subregions (B; *p<0.05 vs. young by independent samples t-test) and subsequently tested for association with proximity scores (C; solid line denotes significant trend line; INSET: r and p-values for aged group). GTPγS-binding to Gα_q/11 stimulated by 100 μM oxotremorine-M (D) was similarly averaged across all 3 subregions (E) and tested for association with proximity scores (F; dashed line denotes non-significant trend line; INSET: r and p-values for aged group).

Fig. 3. Basal and oxotremorine-M stimulated GTPγS-binding to Gα_q/11 are inversely correlated in the hippocampus of young and aged rats

Basal and oxotremorine-M [³⁵S]GTPγS-binding were measured for correlation in the average of 3 hippocampal subregions (A), DG (B), CA3 (C) and CA1 (D). Solid lines denote significant trend lines and dashed lines denote non-significant trend line; INSET: r and p-values for young (n=10) and aged (n=22) group tested together.

Fig. 4. GTPγS-binding to Gα_o in the hippocampus of young and aged rats

[³⁵S]GTPγS-binding assay was combined with an antibody-capture scintillation proximity counting approach to measure basal activity (A) as well as GTPγS-binding to Gα_o stimulated by 100 μM oxotremorine-M (B) and 300 μM baclofen (C) in the 3 major hippocampal subregions of young and aged rats (n=10 young, n=22 aged; A–C).

Fig. 5. Oxotremorine-M-stimulated changes to intracellular Ca²⁺ concentration in CA1 of young and aged rats

Representative results of Ca²⁺ imaging in hippocampal slices showing time-course of changes to intracellular Ca²⁺ concentration stimulated by 50 μM oxotremorine-M (Oxo-M) in young (A) and aged CA1 cells (B); INSET: number of cells and mean peak and area under curve (AUC), range of values is given in parentheses. Peak change in intracellular Ca²⁺ concentration of young and aged cells (C; n=72 cells from young, n=136 cells from aged), averaged by rat (D; n=6 young, n=15 aged) and tested for association with proximity scores (E; dashed line denotes non-significant trend line; INSET: r and p-values for aged group). Area under curve of intracellular Ca²⁺ response of young and aged cells (F) and averaged by rat (G). Black bar in A and B denotes time period of agonist application. *p<0.05 vs. young according to independent-samples t-test; ###p<0.001 vs. oxotremorine-M control according to paired-samples t-test.

Fig 6. DHPG-stimulated changes to intracellular Ca²⁺ concentration in CA1 of young and aged rats

Representative results of Ca²⁺ imaging showing time-course of changes to intracellular Ca²⁺ concentration in hippocampal slices stimulated by 50 μM DHPG in young (A) and aged CA1 cells (B); INSET: number of cells and mean peak and area under curve (AUC), range of values is given in parentheses. Peak change in intracellular Ca²⁺ concentration of young and aged cells (C; n=35 cells from aged, n=67 cells from young) and averaged by rat (D; n=5 young, n=10 aged). Area under curve of intracellular Ca²⁺ response of young and aged cells (E) and averaged by rat (F). Black bar in A and B denotes time period of agonist application. *p<0.05 vs. young according to independent-samples t-test; #p<0.05, ###p<0.001 vs. DHPG control according to paired-samples t-test.