17β-estradiol induces Ca2+ influx, dendritic and nuclear Ca2+ rise and subsequent cyclic AMP response element-binding protein activation in hippocampal neurons: A potential initiation mechanism for estrogen neurotrophism

doi:10.1016/j.neuroscience.2004.11.054

Neuroscience

Volume 132, Issue 2, 2005, Pages 299-311

https://doi.org/10.1016/j.neuroscience.2004.11.054 Get rights and content

Abstract

Previous studies from our laboratory have shown that 17β-estradiol (E2) promotes neurite outgrowth in hippocampal and cortical neurons. The neurotrophic effect of E2 seen in vitro has also been observed in vivo by other investigators who found that E2 enhances the density of dendritic spines involved in neuronal synaptic connection. To investigate the rapid upstream mechanisms initiating the E2 neurotrophic effect, we tested the hypothesis that E2 would directly activate Ca²⁺ influx in primary hippocampal neurons, which would result in activation of the transcription factor, cyclic AMP response element-binding protein (CREB), and regulate E2 enhancement of neurite outgrowth. Using fura-2 ratiometric and fluo-3 Ca²⁺ imaging, we demonstrated that E2 induced a significant rise in intracellular Ca²⁺ concentration ([Ca²⁺]_i) through E2-induced Ca²⁺ influx. Interestingly, the rise in [Ca²⁺]_i occurred not only in the cytoplasm, but also in the nucleus and dendrites of hippocampal neurons. Since CREB is activated by Ca²⁺-dependent kinases and is required for certain aspects of synaptic plasticity, we investigated whether E2 would lead to activation of CREB. Western immunoblotting and immunocytochemical analyses revealed that E2 induced rapid CREB activation consistent with rapid intracellular Ca²⁺ signaling, which was dependent on the influx of extracellular Ca²⁺. E2-induced increase in dendritic spine marker protein spinophilin was abolished following treatment with a small interfering RNA against CREB, indicating that E2-induced neurotrophic effect requires the upstream CREB activation. Results of these analyses indicate that E2-induced neurotrophic responses are mediated by a Ca²⁺ signaling cascade that is dependent upon extracellular Ca²⁺ and CREB activation. These data provide insights into the initiating mechanisms required to activate the estrogen neurotrophic response and provide a mechanistic framework for determining the neurotrophic efficacy of existing and emerging estrogen therapies for the brain.

Section snippets

Cell culture preparation

Use of animals has been approved by the Institutional Animal Care and Use Committee at the University of Southern California (Los Angeles, CA, USA; protocol no. 10256), and all experiments conformed to University of Southern California guidelines on the ethical use of animals. Appropriate procedures were taken to minimize the number of animals used and their suffering. Primary cultures of hippocampal neurons were prepared as described in Zhao and Brinton (2002b). Briefly, hippocampi were

E2-induced [Ca²⁺]_i rise in hippocampal neurons and its dependence on extracellular Ca²⁺

To explore the initiation mechanism underlying E2 inducible neurotrophism, E2-induced rise in [Ca²⁺]_i in hippocampal neurons was determined by fura-2 ratiometric [Ca²⁺]_i imaging. Cultured hippocampal neurons were loaded with fura-2, and time-lapse fura-2 Ca²⁺ images were acquired prior to and following E2 perfusion (Fig. 1A). Perfusion of E2 (10 ng/ml) markedly increased [Ca²⁺]_i in the hippocampal neurons, while perfusion of buffer alone did not increase [Ca²⁺]_i above baseline (Fig. 1A). To

Discussion

The current study aimed to determine the initiation mechanism underlying estrogen-induced neurotrophism by investigating the direct effect of E2 on the rise of [Ca²⁺]_i and rapid CREB activation in cultured hippocampal neurons. Results from this study indicated that estrogen directly induced a dramatic increase in [Ca²⁺]_i concentration, which occurred not only in the cytoplasm, but interestingly also in the nuclei and the dendrites of hippocampal neurons. Following the rise in [Ca²⁺]_i, CREB was

Acknowledgments

This work was supported by the National Institutes of Aging, the Kenneth T. and Eileen L. Norris Foundation and the L. K. Whittier Foundation to R.D.B.

References (52)

R.D. Brinton
17 Beta-estradiol induction of filopodial growth in cultured hippocampal neurons within minutes of exposure
Mol Cell Neurosci
(1993)
M. Hagiwara et al.
Transcriptional attenuation following cAMP induction requires pp-1-mediated dephosphorylation of CREB
Cell
(1992)
M. Marino et al.
Estrogens cause rapid activation of IP3-PKC-alpha signal transduction pathway in hepg2 cells
Biochem Biophys Res Commun
(1998)
J. Nilsen et al.
Dual action of estrogen on glutamate-induced calcium signalingmechanisms requiring interaction between estrogen receptors and Src/mitogen activated protein kinase pathway
Brain Res
(2002)
L. Redmond et al.
Calcium regulation of dendritic growth via cam kinase IV and CREB-mediated transcription
Neuron
(2002)
D. Thomas et al.
A comparison of fluorescent Ca²⁺ indicator properties and their use in measuring elementary and global Ca²⁺ signals
Cell Calcium
(2000)
C.S. Woolley
Estrogen-mediated structural and functional synaptic plasticity in the female rat hippocampus
Horm Behav
(1998)
H. Yasuda et al.
Long-term depression in rat visual cortex is associated with a lower rise of postsynaptic calcium than long-term potentiation
Neurosci Res
(1996)
J.C. Yin et al.
CREB as a memory modulatorinduced expression of a dCREB2 activator isoform enhances long-term memory in Drosophila
Cell
(1995)
J.C. Yin et al.
Induction of a dominant negative CREB transgene specifically blocks long-term memory in Drosophila
Cell
(1994)

L. Zhao et al.

Vasopressin-induced cytoplasmic and nuclear calcium signaling in cultured cortical astrocytes

Brain Res

(2002)

A.S. Alberts et al.

Expression of a peptide inhibitor of protein phosphatase 1 increases phosphorylation and activity of CREB in NIH 3t3 fibroblasts

Mol Cell Biol

(1994)

S.M. Aronica et al.

Estrogen action via the camp signaling pathwaystimulation of adenylate cyclase and camp-regulated gene transcription

Proc Natl Acad Sci USA

(1994)

R. Bi et al.

The tyrosine kinase and mitogen-activated protein kinase pathways mediate multiple effects of estrogen in hippocampus

Proc Natl Acad Sci USA

(2000)

R. Bi et al.

Cyclic changes in estradiol regulate synaptic plasticity through the map kinase pathway

Proc Natl Acad Sci USA

(2001)

R.D. Brinton

Cellular and molecular mechanisms of estrogen regulation of memory function and neuroprotection against Alzheimer’s diseaserecent insights and remaining challenges

Learn Mem

(2001)

R.D. Brinton et al.

The Women’s Health Initiative estrogen replacement therapy is neurotrophic and neuroprotective

Neurobiol Aging

(2000)

R.D. Brinton et al.

17 Beta-estradiol enhances the outgrowth and survival of neocortical neurons in culture

Neurochem Res

(1997)

D.B. Dubal et al.

Estrogen receptor alpha, not beta, is a critical link in estradiol-mediated protection against brain injury

Proc Natl Acad Sci USA

(2001)

M.R. Foy et al.

17Beta-estradiol enhances NMDA receptor-mediated EPSPs and long-term potentiation

J Neurophysiol

(1999)

A. Ghosh et al.

Calcium signaling in neuronsmolecular mechanisms and cellular consequences

Science

(1995)

G.A. Gonzalez et al.

A cluster of phosphorylation sites on the cyclic AMP-regulated nuclear factor CREB predicted by its sequence

Nature

(1989)

G. Gu et al.

Hormonal regulation of CREB phosphorylation in the anteroventral periventricular nucleus

J Neurosci

(1996)

J.F. Guzowski et al.

Antisense oligodeoxynucleotide-mediated disruption of hippocampal camp response element binding protein levels impairs consolidation of memory for water maze training

Proc Natl Acad Sci USA

(1997)

C. Hansel et al.

Relation between dendritic Ca²⁺ levels and the polarity of synaptic long-term modifications in rat visual cortex neurons

Eur J Neurosci

(1997)

J. Hao et al.

Estrogen increases the number of spinophilin-immunoreactive spines in the hippocampus of young and aged female rhesus monkeys

J Comp Neurol

(2003)

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17β-estradiol induces Ca2+ influx, dendritic and nuclear Ca2+ rise and subsequent cyclic AMP response element-binding protein activation in hippocampal neurons: A potential initiation mechanism for estrogen neurotrophism

Abstract

Section snippets

Cell culture preparation

E2-induced [Ca2+]i rise in hippocampal neurons and its dependence on extracellular Ca2+

Discussion

Acknowledgments

Mol Cell Neurosci

Cell

Biochem Biophys Res Commun

Brain Res

Neuron

Cell Calcium

Horm Behav

Neurosci Res

Cell

Cell

Brain Res

Expression of a peptide inhibitor of protein phosphatase 1 increases phosphorylation and activity of CREB in NIH 3t3 fibroblasts

Mol Cell Biol

Estrogen action via the camp signaling pathwaystimulation of adenylate cyclase and camp-regulated gene transcription

Proc Natl Acad Sci USA

The tyrosine kinase and mitogen-activated protein kinase pathways mediate multiple effects of estrogen in hippocampus

Proc Natl Acad Sci USA

Cyclic changes in estradiol regulate synaptic plasticity through the map kinase pathway

Proc Natl Acad Sci USA

Cellular and molecular mechanisms of estrogen regulation of memory function and neuroprotection against Alzheimer’s diseaserecent insights and remaining challenges

Learn Mem

The Women’s Health Initiative estrogen replacement therapy is neurotrophic and neuroprotective

Neurobiol Aging

17 Beta-estradiol enhances the outgrowth and survival of neocortical neurons in culture

Neurochem Res

Estrogen receptor alpha, not beta, is a critical link in estradiol-mediated protection against brain injury

Proc Natl Acad Sci USA

17Beta-estradiol enhances NMDA receptor-mediated EPSPs and long-term potentiation

J Neurophysiol

Calcium signaling in neuronsmolecular mechanisms and cellular consequences

Science

A cluster of phosphorylation sites on the cyclic AMP-regulated nuclear factor CREB predicted by its sequence

Nature

Hormonal regulation of CREB phosphorylation in the anteroventral periventricular nucleus

J Neurosci

Antisense oligodeoxynucleotide-mediated disruption of hippocampal camp response element binding protein levels impairs consolidation of memory for water maze training

Proc Natl Acad Sci USA

Relation between dendritic Ca2+ levels and the polarity of synaptic long-term modifications in rat visual cortex neurons

Eur J Neurosci

Estrogen increases the number of spinophilin-immunoreactive spines in the hippocampus of young and aged female rhesus monkeys

J Comp Neurol

17β-estradiol induces Ca²⁺ influx, dendritic and nuclear Ca²⁺ rise and subsequent cyclic AMP response element-binding protein activation in hippocampal neurons: A potential initiation mechanism for estrogen neurotrophism

E2-induced [Ca²⁺]_i rise in hippocampal neurons and its dependence on extracellular Ca²⁺

Relation between dendritic Ca²⁺ levels and the polarity of synaptic long-term modifications in rat visual cortex neurons