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Functional roles of astroglial connexins in the generation of sharp wave ripples

Final Report Summary - ASTRORIPPLES (Functional roles of astroglial connexins in the generation of sharp wave ripples)

Sleep has been found to be essential for memory consolidation, the conversion of unstable memory traces into a stable form [1]. In the hippocampus, different types of memory including episodic and spatial memory are formed [2, 3]. It was discovered that some hippocampal pyramidal neurons called “place cells” selectively discharge when the animal is in a specific location in space [4]. In subsequent sleep, the entire sequences of activities in these cell assemblies are spontaneously replayed during a time scale optimal for induction of long-term potentiation, the principal candidate molecular mechanism of synaptic plasticity underlying memory [5, 6]. It was proposed that spontaneous reactivations during sharp wave-ripples (SPW-R) complexes could induce long-lasting synaptic changes in the hippocampus and target structures underlying some form of memory consolidation [7]. Findings by the team of Michael Zugaro at host institute revealed that hippocampal ripples mediate memory consolidation during sleep [8].

Astrocytes sense neuronal inputs with their ion channels, neurotransmitter receptors and transporters and can respond by complex Ca2+ signalling and in turn modulate neighbouring pre- and postsynaptic elements [9, 10]. They abundantly express gap junction (GJ) proteins, connexins (Cxs). GJ channels are made up of two connexons, each composed of six Cx subunits that align between adjacent cells to form intercellular channels. They mediate direct cell-to-cell diffusion of ions and small signaling molecules (<1.5 kDa), providing electrical and metabolic coupling between connected cells [11]. Importantly, Cxs also function as hemichannels (single connexons), mediating exchange with the extracellular space, as well as channel-independent functions involving protein interactions, cell adhesion and intracellular signalling [11-14]. The two main Cxs in mature astrocytes include Cx43, present from embryonic to adult stages, and Cx30, which is expressed later in development (from P10). Indeed, many astroglial factors (e.g. glutamate and ATP) and properties (e.g. glutamate and K+ clearance) are thought to mediate neurotransmission and plasticity. Recent evidence shows that astrocytes can even alter neocortical excitability, modulate ongoing slow oscillations [15, 16], and sleep [17, 18], in part through modulations of excitatory synaptic activity by gliotransmitters. Furthermore, the host team has demonstrated significant roles of astroglial Cxs in synaptic and network activities [9, 19-24]. Cx30 is also up-regulated in mice raised in enriched environments [25], known to promote structural changes in the brain and to enhance learning and memory performance. In addition, Cx30 elimination alters the reactivity of mice to novel environments and impairs behaviour [26]. Similarly, Cx43 has also been found to mediate fear memory consolidation [27]. These interesting roles of Cxs in behavioural, cognitive processes, and physiological network activity, raise the intriguing possibility that astroglial Cxs may also modify hippocampal excitability and regulate SPW-R activities. However, this regulation as well as the cellular and molecular mechanisms involved remains to be characterised.

This project was designed to first establish whether astroglial Cxs regulate ripples physiology as well as the associated learning and memory processes. Then, we investigated the physiological and molecular mechanisms underlying this regulation. Using transgenic mice lacking the expression of astroglial Cx30, we have demonstrated that Cx30 plays a role in SPW-R. Specifically, we have performed ex vivo recordings on acute brain slices using multielectrode arrays. With behavioural test, Cx30 was also found to be important for spatial learning and memory. As for the mechanisms involved, we have demonstrated that Cx30 specific channel functions are important for its functional roles in ripples. At the same time, it also contributes to excitatory and inhibitory balance in the CA1 region of the hippocampus important for ripples physiology. In addition, we also tested whether Cx43 plays a role in ripples. Interestingly, we have discovered that similar to Cx30, Cx43 also modulates ripples ex vivo. Given that Cx43 has also been shown to modulate fear memory consolidation [27], this prompted us to investigate the contributions of Cx43 (in addition to Cx30) to ripples activities and to determine if they have similar or complementary roles. Using electrophysiological studies, we observed that Cx43 modulates hippocampal excitatory synaptic transmission and short term plasticity.

In all, this project has taken on a multidisciplinary experimental approach to demonstrate the roles of astroglial Cxs in hippocampal high frequency oscillations and associated learning and memory in mice. We have demonstrated that two major Cxs found in astrocytes contribute to hippocampal sharp wave ripples physiology and that differential mechanisms mediated synaptic activity might be involved. Specifically, we found that Cx30 channel functions modulate hippocampal excitatory and inhibitory balance whereas Cx43 supports short term plasticity via astroglial glutamine supply to neuronal networks. Our results are novel and important to the fields of hippocampal ripples, neuroglial interactions and Cx functions. Furthermore, the understanding of the roles of astrocytes in ripples in healthy brain may shed lights on potential mechanisms underlying pathologically synchronous activity such as seizures and epilepsy. These findings offer insights into astroglial Cxs as an alternative target for future drug development to regulate memory consolidation and pathological network activities.

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