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Title: Estradiol Induced Changes In Neuronal Excitability And Neuron-Astrocyte Signaling In Mixed Hippocampal Cultures
Authors: Rao, Shilpa P
Advisors: Sikdar, Sujit K
Keywords: Brain Reception
Neural Network (Brain)
Signal Transduction
Hippocampal Neurons - Estradiol Effects
Neuronal Networks - Estradiol Effects
Estradiol
Astrocyte-Neuron Signaling - Estradiol Effects
Astrocyte-Astrocyte - Estradiol Effects
Hippocampal Cultures
17ß-estradiol
Submitted Date: Aug-2007
Series/Report no.: G21643
Abstract: One of the defining characteristics of the brain is its plasticity, which is the ability to alter and reorganize neuronal circuits. The brain is constantly being shaped and moulded by the external world through endogenous factors like neurotransmitters, growth factors and circulating hormones. 17β-estradiol, which is the most potent estrogen among the group of ovarian steroid hormones, has widespread effects throughout the central nervous system. Apart from its actions on regions of the brain concerned with reproduction, estradiol has profound effects on brain areas not classically associated with reproductive function like cerebral cortex, midbrain, brainstem, hippocampus and spinal cord. This enables the hormone to influence learning and memory, emotions, affective state, cognition, motor coordination and pain sensitivity. Estradiol exerts these effects by regulating gene expression via intracellular estrogen receptors. In addition to this, the hormone interacts with receptors at the cell membrane to rapidly alter the electrical activity of neurons and astrocytes, and regulate second messenger systems. The aim of this study was to investigate the cellular and functional effects of estradiol on neuronal networks and on signaling between neurons and astrocytes in primary mixed hippocampal cultures. Estradiol is proconvulsant; it increases neuronal excitability and decreases the threshold for seizures. This property of estradiol is instrumental in precipitating catamenial seizures in women with epilepsy. These are epileptic seizures influenced by cyclical hormone changes and occur in over one-third to half of women with epilepsy. In the first part of the work, the effects of 24-hour estradiol treatment on hippocampal neurons were investigated using fluorescence imaging and electrophysiological techniques. Further, the ability of gabapentin, an antiepileptic drug sometimes used to treat hormone sensitive seizures, to counteract the effects of estradiol was studied. Synaptic vesicles were labeled by uptake of FM 1-43, and high K+- triggered exocytotic release was monitored by fluorescence imaging. The reduction in intensity of FM 1-43 fluorescence, which is a measure of vesicular release, was enhanced by estradiol, suggesting that estradiol upregulates the exocytotic machinery. The high K+-evoked intracellular Ca2+ rise in neurons, studied by loading the neurons with the Ca2+ indicator dye fluo-3 AM, was potentiated following estradiol treatment. Electrophysiological recordings from neurons following estradiol treatment showed an increase in the frequency of miniature excitatory postsynaptic currents (mEPSCs) and a larger number of mEPSC events with a predominant NMDA component. Many of the estradiol-induced excitatory effects on the neuronal network were abolished by incubating the cultures with a combination of estradiol and gabapentin suggesting a mechanism of action for the drug in the treatment of hormone sensitive seizures. Glial cells were once regarded as passive, supportive elements in the nervous system. This view of glial cells has drastically changed over the past decade and it is now known that glial cells are dynamic signaling elements in the brain. In view of the emerging importance of glia in the physiology of the nervous system and accumulating evidence of direct effects of steroid hormones on these cells, the subsequent part of the work delves into the consequences of 24-hour estradiol treatment on astrocytes and neuron-to-astrocyte signaling. Estrogen receptors have been described on both neurons and astrocytes in the hippocampus suggesting a complex interplay between the two cell types in mediating the effects of the hormone. Astrocytes sense and respond to neuronal activity with a rise in intracellular calcium concentration, ([Ca2+]i). Astrocyte ([Ca2+]i) transients can modulate neuronal activity, indicating a bi-directional form of communication between neurons and astrocytes. Using simultaneous electrophysiology and calcium imaging techniques, neuronal activity-evoked ([Ca2+]i) changes in fluo-3 AM loaded astrocytes were monitored. Action potential firing in neurons, elicited by injecting depolarizing current pulses, was associated with ([Ca2+]i) elevations in adjacent astrocytes which could be blocked by 200 µM MCPG and also 1 µM TTX. Comparison of astrocytic ([Ca2+]i) transients in control and estradiol treated cultures revealed that the amplitude of the ([Ca2+]i) transient, the number of responsive astrocytes and the ([Ca2+]i) wave velocity were all significantly reduced in estradiol treated cultures. ([Ca2+]i) rise in astrocytes in response to local application of the metabotropic glutamate receptor agonist t-ACPD was attenuated in estradiol treated cultures suggesting functional changes in the astrocyte metabotropic glutamate receptor following 24-hour treatment with estradiol. Since astrocytes can modulate synaptic transmission by release of glutamate, the attenuated ([Ca2+]i) response seen following estradiol treatment could have functional consequences on astrocyte-neuron signaling. The acute effects of estradiol on astrocyte-to-astrocyte and astrocyte-to-neuron signaling have been addressed in the next part of the study. Bidirectional communication between neurons and astrocytes involves integration of neuronal inputs by astrocytes, and release of gliotransmitters that modulate neuronal excitability and synaptic transmission. In addition to its rapid actions on neuronal electrical activity, estradiol can rapidly alter astrocyte ([Ca2+]i) levels through a plasma membrane-associated estrogen receptor. The functional consequences of acute estradiol treatment (5 min) on astrocyte-astrocyte and astrocyte-neuron communication were investigated using calcium imaging and electrophysiological techniques. Mechanical stimulation of an astrocyte evoked a ([Ca2+]i) rise in the stimulated astrocyte, which propagated to the surrounding astrocytes as a ([Ca2+]i) wave. Following acute treatment with estradiol, the amplitude of the ([Ca2+]i) elevation in astrocytes around the stimulated astrocyte was attenuated. Further, estradiol inhibited the ([Ca2+]i) rise in individual astrocytes in response to the metabotropic glutamate receptor agonist, t-ACPD. Mechanical stimulation of astrocytes induced ([Ca2+]i) elevations and electrophysiological responses in adjacent neurons. Estradiol rapidly attenuated the astrocyte-evoked glutamate-mediated ([Ca2+]i) rise and slow inward current in neurons. Also, the incidence of astrocyte-induced increase in spontaneous postsynaptic current frequency was reduced in presence of estradiol. The effects of estradiol were stereo-specific and reversible following washout. These findings indicate that the regulation of neuronal excitability and synaptic transmission by astrocytes is sensitive to rapid estradiol mediated hormonal control.
URI: http://hdl.handle.net/2005/645
Appears in Collections:Molecular Biophysics Unit (mbu)

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