E2 Deficiency Involves in Age-dependent Cognitive Decline in Female Aging Mice
Female C57BL/6 mice at age of 18-month-old are close to the average age of menopause in women, 22-month-old approximately correspond to the human at age of 70 years old [26]. Therefore, female 18- and 22-month-old mice were selected to investigate the effects of short-term and long-term E2 deficiency on cognitive function and neuronal dendrite plasticity. It has been reported that the reduction of serum E2 involved in the process of cognitive decline [27]. However, the relation between serum E2 level and risk of AD in women is still controversial in clinic [2, 28]. To ascertain the association between E2 and cognitive function of female aging mice, we detected the E2 level in serum and brain by ELISA and found that the serum E2 level was age-dependently decreased in female aging mice (Fig. 1a). Compared to the serum E2 level, the content of E2 in brain is much higher that is consistent with the clinical findings [2]. Interestingly, although the hippocampal E2 level was also age-dependent reduced but this phenomenon is not happened in the cortex (Fig. 1b). Then, we investigated the short-term memory and context-dependent memory by passive avoidance task and active avoidance task, separately. Compared to the young mice, the latency of female 18- and 22-month-old mice was shorter with age in the passive avoidance task (Fig. 1c). In the active avoidance task, female 18- and 22-month-old mice decreased the percentage of active avoidance task response (Fig. 1d) and prolonged the latency (Fig. 1e) during the training period. In the test phase, the latency of active avoidance was age-dependently increased (Fig. 1g). However, although female 22-month-old mice reduced the successful active avoidance response, female 18-month-old mice did not change that compared with 6-month-old female mice (Fig. 1f), suggesting the age-dependent decline of the context-dependent learning and memory.
Considering sex difference, male mice are not the good choice to clarify the contribution of E2 in cognition during female aging, so we chose female mice at the age of 3-month-old underwent OVX surgery and raised for 5 months to mimic the time of E2 deficiency of female 22-month-old mice. Surprisingly, OVX successfully induced the decline of E2 level in serum, hippocampus and cortex, and E2 treatment could powerfully reverse that (Fig. 1h and i). Accordingly, the latency of OVX mice significantly reduced in the passive avoidance task (Fig. 1j). Similarly, in the active avoidance test, OVX decreased the percentage of successful avoidance (Fig. 1k, m) and prolonged the latency (Fig. 1l and n). Importantly, the administration of E2 reversed the decline of cognitive function in OVX mice (Fig. 1k-n).
E2 Deficiency Contributes to Female Aging-induced Dendritic Degeneration of Hippocampal CA1 Pyramidal Neurons and DG Granular Cells
To investigate how E2 affects hippocampal dendritic remodeling during female aging, we quantitatively compared dendritic number and length of hippocampal CA1 pyramidal and DG granular neurons by Golgi staining. We found that, compared with female 6-month-old mice, 18- and 22-month-old mice dramatically reduced the total length and number of dendrites of hippocampal CA1 pyramidal neurons and that did not show age-dependent reduction (Fig. 2a-c). The same phenomenon was displayed in the primary, secondary and tertiary dendrites (Fig. 2a-c). Neuronal dendritic complexity reflects the function of the neural network. Next, we analyzed the dendritic complexity by Sholl analysis and found that the intersection number of dendrites in hippocampal CA1 pyramidal neurons was dramatically reduced in 18- and 22-month-old mice and that did not exacerbate in 22-month-old mice (Fig. 2d).
Apical and basal dendrites raising from different sides of the cone-shaped soma of pyramidal neurons ensure that pyramidal neurons could receive synaptic inputs from different afferent sources [29]. Therefore, we evaluated apical and basal dendrites separately. Compared with the young group, female 18- and 22-month-old mice decreased the number and length of all basal dendrites and reduced the dendritic intersection number (Fig. 2e-g). For the hippocampal CA1 apical dendrites, the number and length of the total, secondary and tertiary dendrites and the dendritic complexity were significantly decreased, however, the primary apical dendrites did not changed in female 18- and 22-month-old mice (Fig. 2h-j). Like the total dendrites, the degeneration of apical and basal dendrites did not show age-dependent decline in female aging mice (Fig. 2e-i). These results suggested that dendrites of hippocampal CA1 pyramidal neurons degenerated at onset of menopause, and that would not further aggravate with age.
In the hippocampal DG region, axons of granular cells form mossy fibers play a critical role in spatial memory [13]. In the present study, the number and length accompanied by dendritic complexity of total dendrites of hippocampal DG granular cells were significantly reduced in both female 18- and 22-month-old mice (Fig. 2k-n). However, although the number and length of primary and secondary dendrites dramatically reduced in 22-month-old group that did not changed in 18-month-old mice (Fig. 2l, m). The intriguing results implied that age deteriorates the dendrites degeneration of hippocampal DG granular cells post menopause.
To ascertain how much effect of E2 deficiency contributes to the dendrite degeneration of hippocampal CA1 pyramidal neurons and DG granular cells, OVX mice were used to evaluate the same indexes in female aging groups. Surprisingly, similar to female 22-month-old mice, the number and length of all dendrites, as well as the dendrite intersection number of hippocampal CA1 pyramidal neurons were dramatically decreased in OVX mice (Fig. 3a-d). Apart from apical primary dendrites, OVX mice decreased all the number, length, and intersection number of apical and basal dendrites (Fig. 3e-j). The data indicated that E2 deficiency plays an important role in the dendritic remodeling of hippocampal CA1 pyramidal neurons in female mice during aging. Interestingly, in the hippocampal DG granular cells, E2 deprivation induced by OVX failed to mimic the degeneration of primary dendrite that observed in female 22-month-old mice (Fig. 3l-m). As predicted, the E2 application reversed all the above-mentioned changes (Fig. 3a-n). These results suggested that long-term E2 deficiency leads to severe degeneration in hippocampal CA1 pyramidal neurons relative to DG granular neurons.
E2 Deficiency Involves in Dendrite Degeneration of Cortical Pyramidal and Nonpyramidal Neurons in Female Aging Mice
The cortical pyramidal and nonpyramidal neurons play essential roles in emotion control and receiving, modulating sensory information to memory formation [14, 15]. Next, we quantified the dendritic number and length by tracing pyramidal and non-pyramidal neurons in cortex. Different from hippocampal CA1 pyramidal neurons, although female 18- and 22-month-old mice reduced the overall number and length of total, secondary and tertiary dendrites, as well as the dendritic intersection number of cortical pyramidal neurons, whereas the number and length of primary dendrites did not change (Fig. 4a-d). Similar results were also observed in apical dendrites (Fig. 4e-g). For the basal dendrites, we found that female 18-month-old mice decreased the number and length of total and tertiary dendrites excepting primary and secondary dendrites, while, female 22-month-old mice reduced that of total, secondary and tertiary dendrites but not primary dendrites (Fig. 4h- j). Intriguingly, the length and number of nonpyramidal neuronal dendrites and the dendrite intersection number were significantly decreased in female 18- and 22-month-old mice (Fig. 4k-n). These results demonstrated that female aging from menopause induced dendritic degeneration of cortical pyramidal and nonpyramidal neurons in an age-independent manner.
Accordingly, we quantified dendritic number and length in the cortex of OVX mice. Surprisingly, OVX perfectly reproduced the result of age female mice at the age of 22-month-old (Fig. 5a-n). As shown in Fig. 5a-d, the number and length of the total, secondary and tertiary dendrites, as well as the dendrite intersection number of cortical pyramidal neurons in OVX group were significantly decreased without changing that of primary dendrites. Similar results were observed in apical dendrites (Fig. 5e-g) and basal dendrites (Fig. 5h-j). As expected, E2 treatment could powerfully reverse these changes (Fig. 5a-j). Same to the female 22-month-old mice, OVX group reduced the number and length of all dendrite, as well as the dendrite intersection number of cortical nonpyramidal neurons, and application of E2 could rescue that (Fig. 5k-n). These results demonstrated that long-term E2 deficiency could induce dendritic degeneration of cortical pyramidal and nonpyramidal neurons.
E2 Deficiency-induced Similar Decrease of ERα and ERβ in Female Aging Mice.
Most function mediating by estrogen works by ERα and ERβ [30]. It has been reported that the expression of both ERα and ERβ significantly decreased in the hippocampal CA1 region of female aging rats [31, 32]. However, whether long-term E2 deficiency alone could mimic the changes of ERα and ERβ in hippocampus and cortex of female aging mice is obscure. Using the immunofluorescence staining, we found that the number of ERα and ERβ positive cells was reduced in the hippocampal CA1 (Fig. 6a-c), CA3 (Fig. 6d-f) and DG (Fig. 6g-i) as well as in the cortex (Fig. 6j-l) of female 18- and 22-month-old mice. These results suggested that the level of ERα and ERβ in the hippocampus and cortex decreased from the early stage of menopause. Similar to the female 22-month-old mice, OVX mice decreased the number of ERα and ERβ positive cells in hippocampal subfields and cortex and that was effectively prevented by E2 treatment (Fig. 7). These results suggested that E2 deficiency-induced decreased expression of ERα and ERβ in the hippocampus and cortex may be involved in the cognitive deficits in female aging mice.