Age- and hormone-regulation of opioid peptides and synaptic proteins in the rat dorsal hippocampal formation

Abstract

Circulating estrogen levels and hippocampal-dependent cognitive functions decline with aging. Moreover, the responses of hippocampal synaptic structure to estrogens differ between aged and young rats. We recently reported that estrogens increase levels of post-synaptic proteins, including PSD-95, and opioid peptides leu-enkephalin and dynorphin in the hippocampus of young animals. However, the influence of ovarian hormones on synaptic protein and opioid peptide levels in the aging hippocampus is understudied. Here, young (3- to 5-month-old), middle-aged (9- to 12-month-old), and aged (about 22-month-old) female rats were ovariectomized and then, 4 weeks later, subcutaneously implanted with a silastic capsule containing vehicle or 17β-estradiol. After 48 h, rats were subcutaneously injected with progesterone or vehicle and sacrificed 1 day later. Coronal sections through the dorsal hippocampus were processed for quantitative peroxidase immunohistochemistry of leu-enkephalin, dynorphin, synaptophysin, and PSD-95. With age, females showed opposing changes in leu-enkephalin and dynorphin levels in the mossy fiber pathway, particularly within the hilus, and regionally specific changes in synaptic protein levels. 17β-estradiol, with or without progesterone, altered leu-enkephalin levels in the dentate gyrus and synaptophysin levels in the CA1 of young but not middle-aged or aged females. Additionally, 17β-estradiol decreased synaptophysin levels in the CA3 of middle-aged females. Our results support and extend previous findings indicating 17β-estradiol modulation of hippocampal opioid peptides and synaptic proteins while demonstrating regional and age-specific effects. Moreover, they lend credence to the "window of opportunity" hypothesis during which hormone replacement can modulate hippocampal structure and circuitry to improve cognitive outcomes.

Fig. 1. Examination of opioid peptide and synaptic protein immunoreactivity (-ir) in the hippocampal formation

A: Representative light photomicrograph showing dynorphin (DYN)-ir in the hilus of the dentate gyrus (DG) and stratum lucidum (SLu) of CA3. Three different sub-regions of the hilus (corresponding to levels between 3.80 and 4.30 caudal to Bregma, level 32) (Swanson, 2000) were analyzed as demonstrated by the dotted lines: (1) the tip, (2) the body or central region, and (3) the dorsal blade. SLu of the CA3 region was also divided into three different sub-regions demonstrated by the dotted lines: (1) CA3a, (2) CA3b, and (3) CA3c based on the classical divisions of (Lorente de No, 1934). B: Representative light photomicrograph showing leu-enkephalin (LE)-ir in the hilus of the DG and SLu of the CA3 region. C: Both large and small processes with DYN labeling were visible in SLu of CA3b. D: Both diffuse and punctuate DYN-immunoreactive processes were present in the body of the hilus. E: Many large processes with LE labeling were visible in SLu of CA3a. F: Both diffuse and punctuate LE-immunoreactive processes were observed in the body of the hilus. G: Representative light photomicrograph showing synaptophysin (SYP)-ir in the hilus, SLu of CA3, and stratum oriens (SO) and stratum radiatum (SR) of the CA1 region. H: Representative light photomicrograph showing PSD-95-ir in the hilus, SLu of CA3, and SO and SR of CA1. Dotted lines represent regions examined by densitometry in the CA1. I: Many large processes with SYP labeling were visible in SLu of CA3a. J: SYP-ir was diffusely distributed within SO and SR of CA1. K: Diffuse PSD-95 labeling was observed in SO of CA1 and distinct PSD95-immunoreactive processes were observed in SR of CA1. L: Representative light photomicrograph of a tissue section processed with omission of primary antisera showing lack of SYP-ir or PSD-95-ir in SO and SR of the CA1 region. GCL: Granule cell layer. SP: Stratum pyramidale. Scale bars = 300 μm (A, B, G, H); 100 μm (C – F, I – L). All photomicrographs were taken from young females.

Fig. 2. Age and hormone replacement influence DYN-ir in the mossy fiber pathway

As a statistical interaction between the variables age and hormone treatment was observed for DYN-ir in the dorsal blade of the DG hilus and SLu of CA3a, graphs present data from all age and treatment groups separately (9 groups). A: In the dorsal blade of the DG hilus, middle-aged and aged females that did not receive hormone replacement had significantly less DYN-ir than young females that did not receive hormone replacement (**p<0.01, ***p < 0.001, compared to young). B: In all hilar subregions (DG-whole), middle-aged and aged females displayed significantly less DYN-ir than young females (***p < 0.001, compared to young). C: In SLu of CA3a, middle-aged and aged females that did not receive hormone replacement had significantly less DYN-ir than young females that did not receive hormone replacement (*p<0.05, ***p < 0.001, compared to young). In addition, in young females only, hormone replacement decreased DYN-ir (^p < 0.05, compared to control). D: In all CA3 sub-regions (CA3-whole), middle-aged and aged females displayed significantly less DYN-ir than young females (*p < 0.05, ***p < 0.001, compared to young). The numbers at the bottom of each bar represent the number of animals per group and error bars represent SEM.

Fig. 3. LE-ir increases with age but not hormone replacement in the mossy fiber pathway

As no statistical interaction between the variables age and hormone treatment was observed for LE-ir in any region, graphs demonstrating age effects pool data from all treatment groups and graphs demonstrating treatment effects pool data from all age groups. A: In all hilar sub-regions (DG-whole), young and middle-aged females displayed significantly less LE-ir than aged females (post-hoc test ***p < 0.001, compared to aged). B: In all CA3 sub-regions (CA3-whole), middle-aged females displayed significantly less LE-ir than aged females (***p < 0.001). C: LE-ir did not vary with steroid hormone replacement in the DG. D: LE-ir did not vary with steroid hormone replacement in the CA3. For this and all subsequent figures error bars represent SEM and numbers in parentheses represent number of animals per group.

Fig. 4. SYP-ir varies with age in the mossy fiber pathway and CA1

As no statistical interaction between the variables age and hormone treatment was observed for SYP-ir in any region, graphs demonstrating age effects pool data from all treatment groups. A: In all hilar sub-regions, aged females displayed significantly less SYP-ir than young females (**p < 0.01). B: In all CA3 sub-regions, young and middle-aged females displayed significantly less SYP-ir than aged females (**p < 0.01, ***p < 0.001, compared to aged). C: In SO of CA1, middle-aged and aged females displayed significantly less SYP-ir than young females (*p < 0.05, **p < 0.01, compared to young).

Fig. 5. PSD-95-ir decreases with age in the mossy fiber pathway and CA1

As no statistical interaction between the variables age and hormone treatment was observed for PSD-95-ir in any region, graphs demonstrating age effects pool data from all treatment groups. A: In all hilar sub-regions, aged females displayed significantly less PSD-95-ir than young females (**p < 0.01). B: In all CA3 sub-regions, middle-aged and aged females displayed significantly less PSD-95-ir than young females (***p < 0.001, compared to young). C: In SR of CA1, middle-aged females displayed significantly less PSD-95-ir than young females (**p < 0.01).

Fig. 6. Hormone replacement selectively alters LE-ir and SYP-ir in young and middle-aged females

A: In the DG-whole of young females, hormone replacement significantly decreased LE-ir (*p < 0.05, compared to control). B: In the CA1 SR of young females, hormone replacement significantly increased SYP-ir (*p < 0.05, **p < 0.01, compared to control). C: In the CA3-whole of middle-aged females, hormone replacement with 17β-estradiol alone significantly decreased SYP-ir (*p < 0.05, compared to control). The numbers at the bottom of each bar represent the number of animals per group.

Fig. 7. Summary of age and treatment effects on opioid peptide and synaptic protein levels in the dorsal hippocampal formation

A: With age, females showed opposing changes in leu-enkephalin and dynorphin levels in the mossy fiber pathway, particularly within the hilus, and regionally specific changes in levels of pre-synaptic protein synaptophysin and post-synaptic protein PSD-95. For each region, significant changes with age, if evident, were found in comparison to the young (Y) or middle-aged (M) group noted by the thick black outline. B: Hormone replacement (E or P) significantly altered leu-enkephalin and dynorphin peptide levels within sub-regions of the mossy fiber pathway and synaptophysin levels in the CA1 of young but not middle-aged or aged female rats. Additionally, 17β-estradiol (E) decreased synaptophysin levels in the CA3 of middle-aged females. For each region, significant changes with hormone treatment, if evident, were found in comparison to the control (C) group noted by the thick black outline.