The role of Arc/Arg3.1 during early development in the wiring of hippocampal networks

Abstract

Critical periods are time windows in early development when genetical programs, and environmental and experiential factors confluence to tune functional properties of brain circuits towards their maturation. Concurrently, neural activity, synaptogenesis, and plasticity complete the wiring of hippocampal circuits giving rise to rhythmic activity. Work from our group has previously identified a critical period for hippocampal learning, wherein spontaneous upregulation of Arc/Arg3.1 in the hippocampus during the first postnatal month, permanently influences adult learning and hippocampal oscillations. Here, I propose that Arc/Arg3.1 shapes hippocampal circuits’ wiring and functional maturation during the critical period. This thesis aimed to delve into the impact of Arc/Arg3.1 on the maturation of synaptic transmission in the hippocampus. My approach was to investigate the molecular, structural, and functional properties of hippocampal circuits in conditional Arc/Arg3.1 knockout (cKO) mice engineered in earlier studies, featuring deletions of the gene at various time points during the first postnatal month. These included a germline KO line, in which the deletion occurs during embryogenesis; an “early-cKO” line, where Arc/Arg3.1 is present during the first postnatal week and the deletion is completed between postnatal days 7-14 (P7-P14); and a “late-cKO” line, where the deletion is completed between P21-P36. I employed a broad range of methods encompassing: in-vitro field recordings, patch-clamp techniques, 3D dendritic reconstructions, immunohistochemistry and quantitative confocal microscopy, electron-microscopy, mass spectrometry-based proteomics, and subcellular fractionation with Western blotting. The first part of this thesis evaluated the adult KO and cKO mice, at a time point where Arc/Arg3.1 had been fully deleted in the hippocampus and deficits in oscillatory activity and and learning had been observed. The findings presented here revealed an essential role of Arc/Arg3.1 in regulating the temporal dynamics of excitatory synapses in a development-dependent manner, with the most pronounced effects observed upon the earliest deletion. These effects were associated with changes in critical components of the postsynaptic density, including the transmembrane AMPA receptor regulatory proteins (TARPs) and PSD-95. Remarkably, my findings also showed alterations in the inhibitory synaptic transmission, hitherto believed to be independent of Arc/Arg3.1 plasticity, providing a first mechanistic understanding of the oscillatory deficits. Part II of this thesis described the proteomic profile of the hippocampus in adult WT and Arc/Arg3.1 KO mice, focusing on differences between the CA1, CA3, and DG subfields. The results proved the efficacy of a nanosecond infrared laser (NIRL) ablation method to reliably isolate distinct regions of the mouse brain for subsequent proteomic analysis. Furthermore, I demonstrated that Arc/Arg3.1 regulates the proteomic hippocampal profile in a subfield-specific manner and identified novel exciting candidate proteins regulated by Arc/Arg3.1 under low activity levels. Additionally, enrichment analyses highlighted Arc/Arg3.1’s role in protein transport. Finally, part III evaluated the role of Arc/Arg3.1 in the development of hippocampal function by examining mice of two and four weeks of age. My findings demonstrated that the kinetics of excitatory synaptic transmission are not altered in any of the KO lines at this stage of development, indicating that the alterations seen in the adult hippocampus develop at a later stage. In contrast to its effects in the adult brain, Arc/Arg3.1’s effects in the developing brain are more pronounced in response to early postnatal deletion, resulting in the active elimination of functional excitatory synapses and possibly the elimination of non-functional inhibitory synapses. Collectively, this work provides further evidence of the existence of a critical period for the development of hippocampal function and demonstrates that Arc/Arg3.1 plays a vital role in the modulation of this critical period by shaping the wiring of hippocampal circuitry, not only by modulating excitatory synapses but, notably, also inhibitory transmission. My findings open new directions for investigating Arc/Arg3.1-dependent molecular pathways and cellular processes involved in brain wiring and plasticity.