Gender-Specific Effects of Estrogen in the Developing Hippocampus – Detailed Outline
Background:
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Estrogen in the brain
o Estradiol (E2) – main form of estrogen; not only reproductive hormone, but also
has many widespread effects in brain
o Acts via nuclear estrogen receptors alpha and beta; both must be present; form a
dimer to regulate transcription
BDNF and the hippocampus
o Hippocampus – center of learning and memory in the brain; region most affected
by Alzheimer’s disease
o BDNF = Brain Derived Neurotrophic Factor – member of neurotrophin family of
growth factors, vital to learning and memory; expression of BDNF and its
receptor TrkB is reduced in hippocampi of Alzheimer’s disease patients
Past studies (Dreyfus lab)
o Astrocytes (neural support cells) from female postnatal day 1 (P1) mice respond
to E2 administration with a twofold increase of BDNF
o Male P1 astrocytes are unaffected by E2
o This effect is mediated by ER’s alpha and beta
The present study
o In vivo examination of the hippocampus as a whole (includes both glia and
neurons)
o Examines changes in E2 dependence from P1 to adulthood
o Compares ERα knockout (ERαKO) mice to wild-type mice
Hypothesis
o There are gender differences in E2 regulation of BDNF levels only during early
development
o Female ERα knockouts will have less BDNF than wild-type controls
o Male ERα knockouts will have as much BDNF as wild-type controls
Methods
o Western blot – purpose = to quantify BDNF in wild-type versus ER alpha
knockout mice; works with primary antibody binding to target protein, secondary
antibody binding to primary antibody, and chemiluminescent detection signal
allows visualization and quantification of BDNF present
o Immunocytochemistry – purpose = to visualize the areas in which E2 receptors
are located; same principle w/ antibodies as Western Blot, but difference is that
this happens on a section of the brain; coimmunostain for NeuN detects neurons,
stain for GFAP detects astrocytes;
Ages examined
o Early postnatal development (days P1 and P7);
Intermediate development (adolescence/sexual maturity – 1 month and 3 months)
Adulthood/late adulthood (6 months and 12 months – pre and post menopausal)
Results – Early postnatal development
o [Slide shows original western blots and quantification in charts – P1 and P7
females, ERAKO vs. control at each age; P1 and P7 males, ERAKO vs. control
at each age]
o Main finding = during postnatal development, E2 regulates BDNF production in
females but not in males
o Follow-up slide – compares (combined P1 and P7) males and females in one
graph to more prominently show that female KO’s produce a significantly
smaller percentage of control BDNF than males [graph]
Results – Intermediate development
o [Slide shows original western blots and quantification in charts – 1 month and 3
month females, ERAKO vs. control at each age; 1 month and 3 month males,
ERAKO vs. control at each age]
o Main finding = during intermediate development, E2 regulates BDNF production
in both females and males
Results – Late adulthood
o [Slide shows original western blots and quantification in charts – 6 month and 12
month females, ERAKO vs. control at each age; 6 month and 12 month males,
ERAKO vs. control at each age]
o Main finding = during late adulthood, E2 does not regulate BDNF production in
either males or females
Other results –
o BDNF levels higher in wild-type males than wild-type females at P1 [Western
blot and graph]
o ERA levels slightly higher in wild-type males than wild-type females at P1
[Western blot and graph]
o Immunohistochemistry slides (4) – show that at 6 months, males and females
express ERA in both neurons and astrocytes; at also, during early development,
males and females express ERA in both neurons and astrocytes
Summary slide – summarize all findings, and conclude that It is probable that BDNF
levels in males are regulated by a mechanism independent of E2/ERα interactions during
postnatal development
Further research
o Quantify ERα/NeuN and ERα/GFAP costaining in the entire hippocampus for a
more accurate determination of where the ERα receptors are located
o Observe density of ERα receptors in the hippocampus
o Evaluate whether knocking out ERβ has a different effect from knocking out ERα
Acknowledgements slide
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