Pamela Roque

Project title: Modulation of the Astrocytic Influence on Synapse Structure and Function by Ethanol and Cholinergic Stimulation

Degree: PhD | Program: Environmental Toxicology (Tox) | Project type: Thesis/Dissertation
Completed in: 2013 | Faculty advisor: Lucio G. Costa


It is increasingly becoming clear that astrocytes release numerous factors that contribute to the process of synaptogenesis. The fact that astrocytes express a wide range of neurotransmitter receptors suggest that they are capable of responding to environmental clues from surrounding neurons, yet knowledge about the signals controlling the release of factors from astrocytes is relatively sparse. Understanding how astrocytes influence typical synapse formation may provide insight as to their role in mediating disease. Using an in vitro co-culture system, the present study investigates how an important physiological neurotransmitter, acetylcholine, and a known developmental neurotoxicant, ethanol, each independently and differently act on astrocytes to modulate synapse formation and function. Here we report that primary rat hippocampal neurons (E21) grown in culture for 13 days and co-cultured for 24 hours with astrocytes pre-treated with either carbachol, an acetylcholine receptor agonist (0.010, 0.100, 1 mM) or ethanol (25, 50, 75 mM) for 24 hours, show increased expression of the pre- and post-synaptic proteins, synaptophysin and PSD-95, with carbachol pre-treatment inducing a greater effect than ethanol. Immunocytochemical labeling of the same proteins, followed by confocal imaging and 3-dimensional object analysis shows that carbachol (1 mM) pre-treatment of astrocytes resulted in a 3.2-fold potentiation of synaptic structure formation, an effect mediated by cholinergic actions on the M3 muscarinic and nicotinic ACh receptors. Ethanol pre-treatment of astrocytes induced a bi-phasic increase in the number of synapses, with 50 mM ethanol inducing a 4.5-fold increase, an effect greater than 75 mM pre-treatment (2.6-fold). To corroborate that the observed increase in synaptic structures is reflected in an increase in functionality, whole cell patch clamp techniques were used to measure spontaneous miniature excitatory post-synaptic currents in neurons after co-culture with pre-treated astrocytes. A higher frequency of events was observed in neurons of both treatment groups, suggesting more functional synapses, however, a second population of neurons in the ethanol (50 mM) pre-treated group showed a decreased frequency of events. Both astrocyte-released thrombospondin (TSP) and cholesterol have been shown to potentiate synapse formation in retinal ganglion cells. Our laboratory has previously shown that treatment of astrocytes with carbachol induces increased TSP1 release, while ethanol causes increased efflux of cholesterol-containing lipoproteins from astrocytes. Investigations of these two factors as potential candidates in the observed effects showed that both TSP1 and cholesterol-containing lipoproteins (CCL) are sufficient to induce synapse formation in hippocampal neurons. Pharmacologically blocking the neuronal receptors which these factors act upon showed that the release of TSP1 from carbachol-treated astrocytes and the release of cholesterol-containing lipoproteins from ethanol pre-treated astrocytes are responsible, at least in part, for the observed potentiation of synaptic structures in the co-culture system. Together these data confirm the astrocytic role in synapse formation and development, and clearly shows that they may mediate portions of the important physiological process of cholinergic stimulation during brain development. Additionally, the effects observed after astrocyte pre-treatment with ethanol suggests that astrocytes also may play a role in influencing some of the cognitive and learning disabilities observed in children exposed in utero to alcohol. While both cholinergic stimulation of astrocytes and pre-treatment with ethanol show broadly similar effects, the differences suggest that cholinergic stimulation may move the process of synapse formation forward in a regulated manner, while ethanol may be acting in a way that dysregulates the typical process.