PERISLEEP · Imaging dendrites across wake and sleep: fiberoptic measurements of calcium activity in freely behaving animals

Imaging dendrites across wake and sleep: fiberoptic measurements of calcium activity in freely behaving animals

Despite the lack of consensus on the core function of sleep, there is converging evidence that sleep is important to process and integrate our every day’s experience into existing brain networks throughout life, and thereby is key to brain plasticity. This function is supported by accumulating evidence that sleep is important for memory formation. But how sleep achieves this function, and in particular how it modulates brain plasticity at the cellular and network level, is not well understood. Experience and learning can modulate important hallmarks of brain activity during sleep, measured by electroencephalogram (EEG) recordings in the neocortex (i.e., EEG slow-waves [0.5-4Hz] and spindles [9-16Hz]) and hippocampus (e.g., ripples [200Hz]), two important brain regions for memory formation. Enhancing or disrupting those oscillations in humans and animals can in turn improve or impair memory performances, respectively. These observations are central to the view that there is a functional relationship between network oscillations (EEG), brain plasticity, and memory formation during sleep. Yet how those oscillations impact cortical plasticity is not known. Dendrites are elaborate processes that branch out from cell bodies of neurons and receive most inputs from other neurons. Those communication sites on dendrites (i.e., synapses) are the locus of synaptic plasticity processes. Dendrites are therefore the main site for the integration and processing of information, and most probably the physical substrate of our memories. Dendrites also constitute individual functional units both at the electrical and molecular levels, allowing localised dendritic plasticity to occur. Recent studies suggest that sleep actively contributes to structural plasticity in localised dendritic regions (i.e., spines) during development and adulthood, but if and how sleep regulates dendritic activity is currently unknown. In this context, the main goal of this project was to study changes in dendritic activity and plasticity during natural sleep states. A well-recognised index of activity is the changes in intracellular calcium (Ca2+) which, in turn, is critical to induce plasticity processes. In the first 2 years of this project, we successfully developed a method for combined Ca2+ imaging and polysomnography (EEG) recordings in freely behaving rats. This method uses state-of-the art fiber-optic imaging technique combined with specific labelling of neocortical dendrites of layer 5 (L5) neurons with a Ca2+ indicator. Using this method, we could, for the first time, monitor Ca2+ activity in dendrites across the 24hrs sleep-wake cycle. Comparison of amounts of Ca2+ activity during four different brain states (active wake, quiet wake, slow-wave sleep [SWS], and rapid-eye-movement [REM] sleep, also known as paradoxical sleep), revealed that activity in large populations of dendrites was highest during periods of SWS particularly rich in spindles oscillations. In the last 2 years, we completed this project by performing advanced analysis on those data. We found that both spindle and dendritic activities also gradually increased during individual SWS episodes and that both physiological measures showed strong and specific correlations, compared with other brain oscillations during sleep. Finally, we confirmed this result by measuring Ca2+ activity in individual dendrites using two-photon microscopy combined with EEG recordings in naturally sleeping mice. Two photon imaging further demonstrated that EEG spindles not only correlated with increased Ca2+ activity in single dendrites but also reflect synchronization of ensembles of dendrites. Importantly, those results were specific to dendrites as Ca2+ activity recorded from cell bodies in L2/3 and L5 failed to reproduce such relationship with sleep spindles. Taken together, by measuring Ca2+ activity in population and single dendrites in naturally sleeping rodents (i.e., mice and rats) using two independent methods, we showed that changes in dendritic activity are tightly correlated with changes of a specific sleep oscillation in the spindle frequency range (9-16Hz). Those results have implications at the fundamental and clinical levels. First, although it is known that sleep spindles are linked to memory in humans and animals, we provide here, for the first time, a cellular mechanism for this function. In the context of memory, repeated occurrence of spindles during episodes of SWS would provide reactivation of dendrites, thus enabling repeated Ca2+ increase necessary for plastic changes. Current experiments looking at the effect of learning and experience on this relationship will further contribute to a better understanding of the role of spindles in memory and cognitive function in humans. Secondly, our data also provide the first physiological correlate of EEG spindles. In humans, it is known that spindles have significant inter- and intra-individual variability and that this variability also correlates with fluid intelligence (e.g., IQ). Our data therefore suggest that this differences in spindle activity across population might reflect a difference in dendritic processing. Further studies will reveal if EEG spindles can be used as a biomarker for dendritic function and brain plasticity in humans. Finally, cognitive impairment associated with ageing and neurodegenerative diseases is correlated with decrease in spindle activity. Our results therefore advocate for spindle enhancement (e.g., transcranial magnetic stimulation) as a potential therapeutic approach in conditions associated with natural and pathological cognitive decline.

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