Research Question: To establish new methods for monitoring the activity of astrocyte networks in vivo and use this approach to define their role in learning-induced changes in the brain.
Interdisciplinary Approach: This project uses a multidisciplinary approach to explore astrocyte function, combining in vivo, selective genetic manipulation of astrocytes, fiber optic-based imaging of fluorescent calcium indicators, and behavioral training of mice in reward-based learning paradigms.
Potential Implications of Research: Our studies indicate that astrocytes in cortical networks are reliably engaged by stimuli that increase arousal/attention, such as sleep-wake transitions, by appetitive stimuli and during exploration of novel objects, raising the possibility that astrocytes participate in the modification of neural circuits during learning.
Project Description: The ability to predict the timing of salient events from limited sensory experience is a fundamental aspect of learning accomplished by the brain. Previous studies suggest that acetylcholine (ACh) is necessary and sufficient to invoke reinforcement learning of reward-timing; however, the sites of action of ACh crucial for this plasticity have not been defined. Astrocytes are a prominent target of cholinergic efferents to the cortex, express G-protein coupled acetylcholine receptors (mAChRs) and in vivo cholinergic activity evoked by sensory or electrical stimulation causes widespread, yet transient elevation of cytosolic Ca2+ in astrocytes, events that have been correlated with the release of neurotransmitters and modulation of neural activity. As a single astrocyte has direct access to tens of thousands of synapses and neighboring astrocytes are extensively coupled through gap junctions, the astrocyte network could serve as a conduit through which sparse neuromodulatory inputs convey signaling of behavior outcome. However, it has not yet been possible to monitor astrocyte activity in freely moving animals to define their activity patterns.
To define the activity of cortical astrocytes in area V1 in relation to behavior, we developed a novel fiber optic imaging instrument in which an optical fiber with 30,000 individually isolated fiber optic cores is used to monitor fluorescence changes. We optimized the coupling of this fiber to an achromatic lens pair for optimum resolution and built a platform to allow excitation and efficient collection of fluorescence from brain tissue in living mice. We designed a lightweight mount to allow this probe to be attached to the mouse head and developed custom acquisition and analysis software to enable simultaneous recording of fluorescence changes and animal behavior (see Figure). We monitored calcium levels in astrocytes using transgenic mice that we developed (GLAST-CreER;R26-lsl-GCaMP3 mice), in which astrocytes express the genetically encoded calcium indicator GCaMP3. Our studies indicate that astrocyte networks in this region are robustly activated during periods of enhanced vigilance, pointing to a possible role for these glial cells in promoting the enhanced responsiveness of cortical networks during arousal. In addition, we discovered that this modulation of astrocytes markedly enhanced their sensitivity to visual input. These studies were published in Neuron (82:1263) as a collaboration between the Kang and Bergles laboratories. Moreover, our results suggest that astrocyte networks are activated in a variety of behavioral contexts, including sleep-wake transitions, exposure to appetitive stimuli such as water and food, and during the exploration of novel environments or objects. Although we hypothesized that astrocyte networks are modulated by ACh through direct activation of muscarinic receptors, our studies suggest that cholinergic modulation of astrocytes is indirect, at least for the majority of astrocytes in V1. Based on these findings, we conclude that ACh does not have a direct excitatory effect on astrocytes, but that it may work indirectly by activating the noradrenergic system. Our studies point to an important role of astrocytes in the modulation of neural networks in the cerebral cortex during periods when learning occurs.