Estrogen levels regulate the subcellular distribution of phosphorylated Akt in hippocampal CA1 dendrites

Abstract

In addition to genomic pathways, estrogens may regulate gene expression by activating specific signal transduction pathways, such as that involving phosphatidylinositol 3-kinase (PI3-K) and the subsequent phosphorylation of Akt (protein kinase B). The Akt pathway regulates various cellular events, including the initiation of protein synthesis. Our previous studies showed that synaptogenesis in hippocampal CA1 pyramidal cell dendritic spines is highest when brain estrogen levels are highest. To address the role of Akt in this process, the subcellular distribution of phosphorylated Akt immunoreactivity (pAkt-I) in the hippocampus of female rats across the estrous cycle and male rats was analyzed by light microscopy (LM) and electron microscopy (EM). By LM, the density of pAkt-I in stratum radiatum of CA1 was significantly higher in proestrus rats (or in estrogen-supplemented ovariectomized females) compared with diestrus, estrus, or male rats. By EM, pAkt-I was found throughout the shafts and in select spines of stratum radiatum dendrites. Quantitative ultrastructural analysis identifying pAkt-I with immunogold particles revealed that proestrus rats compared with diestrus, estrus, and male rats contained significantly higher pAkt-I associated with (1) dendritic spines (both cytoplasm and plasmalemma), (2) spine apparati located within 0.1 microm of dendritic spine bases, (3) endoplasmic reticula and polyribosomes in the cytoplasm of dendritic shafts, and (4) the plasmalemma of dendritic shafts. These findings suggest that estrogens may regulate spine formation in CA1 pyramidal neurons via Akt-mediated signaling events.

Fig. 1.

By LM and EM, pAkt-I is prominent in dendritic processes within stratum radiatum of the hippocampal CA1 region.A, Low magnification LM photomicrograph shows the distribution of pAkt-I (as demonstrated by peroxidase) in a coronal section through the dorsal rat hippocampal formation [corresponds to level 34 of Swanson (1992)]. B, Higher magnification of the boxed region in A shows that in the CA1 region pAkt-I is concentrated around somata in the pyramidal cell layer (pcl) and in dendritic processes radiating through stratum radiatum (sr).C, By EM, pAkt-I is found throughout most large dendritic shafts (pAKT-D) and in some dendritic spines (arrowheads), contacted by unlabeled terminals (uT). D, Some dendritic spines do not contain pAkt-I (arrowhead). DG, Dentate gyrus; gcl, granule cell layer;h, hilus; ml, molecular layer;slm, stratum lacunosum-moleculare; so, stratum oriens. Scale bars: A, 1 mm; B, 100 μm; C, D, 50 μm.

Fig. 2.

The density of pAkt-I in CA1 stratum radiatum is highest in proestrus rats. Representative LM micrographs showing peroxidase pAkt labeling in the CA1 region of a coronal hippocampal section from a diestrus (A) and proestrus (B) rat. C, The density of pAkt-I (measured in pixel density units) in CA1 stratum radiatum differs between female rats across estrus and male rats.Asterisk indicates significant differences from proestrus (ANOVA; Fisher's post hoc).pcl, Pyramidal cell layer; so, stratum oriens; sr, stratum radiatum. N, Number of animals per condition. Scale bar: (in B)A, B, 100 μm.

Fig. 3.

By EM, pAkt-IG labeling associated with dendritic spines is significantly higher in proestrus rats.A–C, Representative electron micrographs showing the distribution of pAkt-IG particles (arrowheads) associated with the spine apparatus (A, B) or the plasma membrane (C) of dendritic spines. Unlabeled terminals (uT) contact both labeled spines. InB, the labeled spine arises from a pAkt-IG-labeled dendritic shaft (pAKT-D). D, The total number of pAkt-IG particles in dendritic spines (number of pAkt-IG particles associated with both cytoplasm and plasma membrane of spines per 100 μm² of tissue) is significantly higher in proestrus rats. E, The relative number of labeled spines is highest in proestrus. The relative number of labeled spines was calculated as the number of dendritic spines containing pAkt-IG particles (i.e., pAkt-labeled) divided by the total number of dendritic spines (i.e., labeled + unlabeled) as represented per 100 μm² of the total field of 9632 μm² for each rat. Asteriskindicates significant differences from proestrus (unpairedt test). N, Number of rats per condition. Scale bar: (in C) A, B,C, 50 μm.

Fig. 4.

In dendritic shafts, pAkt-IG labeling associated with endoplasmic reticula and polyribosomes is highest in proestrus rats. A, Representative electron micrograph showing pAkt-IG particles associated with endoplasmic reticula (arrowheads) of a pAkt-IG-containing dendritic shaft profile (pAKT-D). B, Variations in the number of pAkt-IG particles per 1 μm² of dendritic area that are associated with endoplasmic reticula and polyribosomes in dendritic shafts of female rats across estrous and male rats. Asterisk indicates significant differences (unpaired t test) from proestrus rats. n = number of dendrites analyzed from three rats per condition. Scale bar, 50 μm.

Fig. 5.

pAkt-IG labeling near the base of dendritic spines is highest in proestrus rats. A, Representative electron micrograph showing a pAkt-IG particle (arrowhead) affiliated with a portion of the spine apparatus located within 0.1 μm from the base of a dendritic spine. B, Variations in the number of pAkt-IG particles within 0.1 μm of the bases of dendritic spines in female rats across estrous and male rats, calculated per dendrite. Asterisk indicates significant differences from proestrus (unpaired t test).n = number of dendrites analyzed from three rats. Scale bar, 50 μm.

Fig. 6.

The number of pAkt-IG particles on the plasma membrane of dendritic shafts is highest in proestrus rats.A, Representative electron micrograph of a dendritic shaft profile showing pAkt-IG particles in the cytoplasm (arrows) and on the plasma membrane (arrowheads). B, The total number of pAkt-IG particles per 1 μm of dendritic plasma membrane of dendritic shafts is significantly different in proestrus rats compared with disestrus rats (unpaired t test; *p< 0.05). C, Cytoplasmic pAkt-IG particles in dendritic shafts (expressed per 1 μm²) are not significantly different between female rats across estrous and male rats.n = number of dendrites analyzed from three rats per condition. Scale bar, 50 μm.