Introduction
Activity-regulated cytoskeleton associated protein (Arc/Arg3.1) is one of the major molecular players in cognition, as it is required for protein-synthesis dependent synaptic plasticity and learning and memory (Park et al. , 2008; Waung et al. , 2008; Bramham et al. , 2010; Shepherd & Bear, 2011). Arc plays a key role in determining synaptic strength through promoting endocytosis of AMPA-type glutamate receptors (AMPARs) during mGluR-LTD which has been most studied in area CA1 of the hippocampus (Chowdhury et al. , 2006; Park et al. , 2008; DaSilva et al. , 2016; Wall & Correa, 2018).
One key feature of Arc protein expression in area CA1 of the hippocampus, is its transient nature. Following increased network activity or exposure to a novel environment Arc expression increases and then rapidly declines (Guzowski et al. , 2000; Ramirez-Amaya et al. , 2005; Miyashita et al. , 2009). The regulation of Arc protein induction occurs at the level of mRNA transcription, mRNA trafficking, and protein translation (Bramham et al. , 2008; Korb & Finkbeiner, 2011). Arc protein expression is then reduced by rapid proteasomal degradation (Rao et al. , 2006; Greer et al. , 2010) following ubiquitination by the RING domain ubiquitin ligase Triad3A/RNF216 (Mabb et al. , 2014). However, not all of Arc expression is transient, as a basal level of Arc appears to be retained at synapses, where Arc interacts with several proteins in the post-synaptic density (PSD) scaffold (Fiuza et al. , 2017) and reviewed in (Zhang & Bramham, 2020).
To determine the importance of the Arc removal process in spatial learning behaviour and synaptic plasticity, a mutant mouse line (ArcKR) was generated in which mutations were introduced within Exon 1 of the Arc gene. When encoded, the introduction of the two point mutations replace lysine to arginine at positions 268 and at 269 (Wall et al. , 2018). These sites have been previously shown to be ubiquitinated by Triad3A (Greer et al. , 2010; Mabb et al. , 2014). Hippocampal neurons isolated from mice bearing the mutation of the Triad3A-dependent sites (ArcKR) showed markedly increased Arc protein 30 minutes after activation of Group I metabotropic glutamate receptors (GI-mGluR), with the agonist DHPG, consistent with diminished degradation of Arc protein (Wall et al. , 2018). In addition to these effects on Arc expression, ArcKR mice displayed impaired cognitive flexibility, which was coupled with elevated levels of Arc protein expression in the hippocampus, a reduced threshold to induce GI-mGluR-mediated long-term depression (GI-mGluR-LTD) induced by the GI agonist DHPG (DHPG-LTD), and enhanced DHPG-LTD amplitude (Mabb & Ehlers, 2018; Wall et al. , 2018).
Although the role of Arc in mGluR-LTD is consistent with its action in facilitating the internalisation of synaptic AMPARs (Waung et al. , 2008; Wall & Correa, 2018; Wilkerson et al. , 2018), the mechanism by which Arc regulates potentiation, particularly long term potentiation (LTP) is less clear. For example, an LTP-inducing stimulus in the hippocampus increased Arc mRNA levels, transportingArc mRNA to activated synapses (Lyford et al. , 1995; Steward et al. , 1998; Steward & Worley, 2001a; b). Arc has also been implicated in F-actin stability, which is responsible for dendritic spine maintenance and plasticity. Knocking down Arc expression blocks LTP in the dentate gyrus by reducing F-actin formation and cofilin phosphorylation (Messaoudi et al. , 2007) . The genetic knockout of Arc was also shown to be associated with impaired LTP at SC-CA1 synapses in vitro and in the perforant pathway in vivo (Plath et al. , 2006). Arc is also involved in inverse synaptic tagging during LTP induction, where Arc mediates endocytosis of AMPARs at inactive synapses that recently experienced strong stimulation (Okunoet al. , 2012; Okuno et al. , 2018).
A recent study has directly addressed the role of Arc in LTP at SC-CA1 synapses using two different Arc knock-out (Arc KO) mouse lines and a conditional KO floxed line (Arc cKO), for both in vitro andin vivo LTP analysis (Kyrke-Smithet al. , 2021). It was found that Arc was not required for the maintenance of high frequency stimulation (HFS)-induced LTP. In contrast, theta burst stimulation (TBS)-induced LTP had an enhanced amplitude in Arc KO mice. This effect on LTP was not observed in the conditional Arc KO mouse line. It was therefore suggested that deletion of Arc may have developmental compensatory effects, which leads to the indirect enhancement of LTP, rather than being a direct effect of Arc removal (Kyrke-Smith et al. , 2021). Kyrke-Smith et al (2021) also demonstrated that the deletion of Arc had no effect on the threshold for LTP induction at SC-CA1 synapses. Here we investigated whether expression of ArcKR leads to an LTP phenotype in area CA1 of the hippocampus.