Genistein partly eases aging and estropause-induced primary cortical neuronal changes in rats

Abstract

Gonadal hormones can modulate brain morphology and behavior. Recent studies have shown that hypogonadism could result in cortical function deficits. To this end, hormone therapy has been used to ease associated symptoms but the risk may outweigh the benefits. Here we explored whether genistein, a phytoestrogen, is effective in restoring the cognitive and central neuronal changes in late middle age and surgically estropause female rats. Both animal groups showed poorer spatial learning than young adults. The dendritic arbors and spines of the somatosensory cortical and CA1 hippocampal pyramidal neurons were revealed with intracellular dye injection and analyzed. The results showed that dendritic spines on these neurons were significantly decreased. Remarkably, genistein treatment rescued spatial learning deficits and restored the spine density on all neurons in the surgically estropause young females. In late middle age females, genistein was as effective as estradiol in restoring spines; however, the recovery was less thorough than on young OHE rats. Neither genistein nor estradiol rectified the shortened dendritic arbors of the aging cortical pyramidal neurons suggesting that dendritic arbors and spines are differently modulated. Thus, genistein could work at central level to restore excitatory connectivity and appears to be potent alternative to estradiol for easing aging and menopausal syndromes.

Figure 1. The effects of 5-day genistein treatment on spatial learning memory evaluated with Morris water maze task.

The swimming distance (A and B) and escape latency (C and D) of the experimental animals to find the platform during the 3 consecutive days of Morris water maze task was plotted. The average swimming speed of each animal was shown in E. Young adult: young adult female rats; OHE-2W: OHE and survived for 2 weeks and vehicle-treated; OHE-2W+GS: OHE-2W females treated with genistein; OHE-8W: OHE for 8 weeks and vehicle-treated; OHE-8W+GS: OHE-8W females treated with genistein; late middle age: late middle age females without vehicle treatment; late middle age+V: late middle age females treated with vehicle; late middle age+GS: late middle age females treated with genistein; late middle age+E2: late middle age females treated with estradiol. *, p<0.05 between the marked and young adult, two-way ANOVA followed by S-N-K test. Vertical bar represents standard error of the mean.

Figure 2. A representative layer III pyramidal neuron of the primary somatosensory cortex (control animal) revealed with intracellular dye injection.

A is a low-power picture of the neuron from one of the 60-µm-thick sections of the brain slice in which the cell was filled. A’ was the dendritic arbor of the neuron reconstructed 3-dimensionally from all serial sections of the injected brain slice with computer software. The dendritic segments (B1–B4) indicated in A were illustrated at higher magnification at the bottom. Bar = 50 µm in A and A’ and 5 µm for B1∼B4.

Figure 3. Changes in the dendritic spines of somatosensory cortical layer III pyramidal neurons in OHE and late middle age females and following treatments with gensitein (GS) and estradiol (E2).

A shows the micrograph of a representative segment of the proximal apical dendrite of a neuron of the young adult, OHE-2W, OHE-2W+GS, OHE-8W, OHE-8W+GS, late middle age, late middle age+GS and late middle age+E2 group, respectively. Spine densities of these animal groups were plotted in B. Please see legend of figure 1 for abbreviations. *, p<0.05 between the marked and young adult; #, p<0.05 between the marked (GS or E2-treated) and its corresponding control, one-way ANOVA followed by Newman-Keuls test. Bar = 10 µm for all.

Figure 4. Changes in the dendritic spines of somatosensory cortical layer V pyramidal neurons.

A shows the micrograph of a representative segment of the proximal apical dendrite of neuron of the young adult, OHE-2W, OHE-2W+GS, OHE-8W, OHE-8W+GS, late middle age, late middle age+GS and late middle age+E2 groups, respectively. Densities of dendritic spines in these groups were analyzed in B. *, p<0.05 between the marked and young adult; #, p<0.05 between the genistein or estradiol-treated and its corresponding control, one-way ANOVA followed by Newman-Keuls test. Bar = 10 µm for all.

Figure 5. Changes in the dendritic spines of hippocampal CA1 pyramidal neurons.

A shows micrographs of a representative proximal apical dendritic segment of the hippocampal CA1 pyramidal neuron of young adult, OHE-2W, OHE-2W+GS, OHE-8W, OHE-8W+GS, late middle age, late middle age+GS and late middle age+E2 groups, respectively. Please see figure 1 legend for abbreviations of experimental groups. Analyses of the spine density of these neurons were plotted in B. *, p<0.05 between the marked and young adult; #, p<0.05 between genistein or estradiol-treated and its corresponding control, one-way ANOVA followed by Newman-Keuls test. Bar = 10 µm for all.

Figure 6. The dendritic arbors of somatosensory cortical layer III pyramidal neurons of normal and experimentally treated late middle age female rats.

Representative 3-dimensionally reconstructed dendritic arbors of neurons from the young adult (A), late middle age (B), late middle age+GS (C) and late middle age+E2 (D) animals are illustrated. Roman numerals and bars to the left of each panel mark cortical laminae. The dendrogram of each neuron was shown below its dendritic arbor plot. Branches of the same dendritic trunk are shown with the same color. Bar = 100 µm for A–D.

Figure 7. The dendritic arbors of the somatosensory cortical layer V pyramidal neuron in the somatosensory cortex of normal and experimentally treated late middle age females rats.

Representative 3-dimensionally reconstructed dendritic arbor of the layer V pyramidal neurons of the young adult, late middle age, late middle age+E2 and late middle age+GS animals were illustrated in A–D respectively. Roman numerals and bars to the left of each panel mark cortical laminae. The dendrogram of each neuron was shown below. Branches of the same dendritic trunk are shown in the same color. Bar = 100 µm for A–D.

Figure 8. Analyses of the dendritic arbors of somatosensory cortical layer III and V pyramidal neurons of late middle age females.

The total, apical and basal dendritic lengths (A and B), numbers of terminal ends of the basal and apical dendrites (C and D) and lengths of the terminal segment of the basal and apical dendrites (E and F) of the two categories of neurons were analyzed based on the 3-dimensionally reconstructed dendritic arbors with Neurolucida.