ArcKR expression may regulate LTP magnitude through indirect mechanisms such as mGluR priming.
The magnitude of LTP can be regulated by many different mechanisms which could be classified as direct: such as modulating Ca2+signalling, NMDA receptor opening, AMPA receptor trafficking etc, or indirect for example through metaplastic processes such as mGluR priming. mGluR priming is the low-level activation of G1-mGluRs prior to the induction of LTP leading to a significant enhancement in LTP amplitude (Cohen et al. , 1998; Mellentin et al. , 2007; Privitera et al. , 2019). We therefore investigated whether the priming of the mGluR signalling pathway prior to the induction of LTP is altered in ArcKR mice. To investigate this, we used the same protocol as in (Privitera et al. , 2019): HFS (100 Hz for 1s) to evoke LTP and 20 µM of the GI mGluR agonist DHPG to produce low-level mGluR activation. Using this LTP induction protocol, there was no significant difference in the amplitude of LTP between the genotypes after 1 hour (Figure 3A, B). To investigate mGluR priming, interleaved slices (WT and ArcKR) were exposed to DHPG (20 µM) for 10 min to prime intracellular mGluR-dependent signalling (Cohen et al. , 1998; Privitera et al. , 2019). After the DHPG was washed out for 20 min, LTP was then induced with HFS (1s burst of stimuli at 100Hz). The application of 20 µM DHPG produced a peak reduction of ~ 40% in fEPSP slope, which was not significantly different between WT and ArcKR hippocampal slices (Figure 3C). We observed that the amplitude of LTP in WT mice was significantly enhanced compared to the LTP in ArcKR mice following application of DHPG (20 µM Fig 3C, D). Strikingly, there was no enhancement in LTP amplitude in slices obtained from ArcKR mice that had previously been exposed to DHPG (20 µM) compared to control slices (Figure 3C, D).
Although not directly comparable (as recordings were not performed using interleaved slices), the increased magnitude of LTP in control conditions was approx. 60 % for WT mice and ~ 74% (Fig 3A, B) for ArcKR when normalized to the baseline. After priming with DHPG, in WT slices LTP magnitude had increased to ~ 80 % but in ArcKR slices it had fallen to approx. 40% (Figure 3B, D). This apparent decrease in LTP amplitude could potentially be produced by an mGluR-mediated increase in Arc/Arg3.1 protein expression and facilitation of AMPA receptor endocytosis. To investigate this, we first examined the DHPG-induced inhibition in more detail. Although there was no difference in the peak inhibition produced by 20 µM DHPG, there did appear to be more inhibition after DHPG wash in ArcKR compared to WT slices (Figure 3E, F). Although this difference did not reach significance, there were some ArcKR slices that appeared to exhibit more DHPG-induced inhibition of the fEPSP than in WT slices (Figure 3F). In these slices, DHPG may have induced a low level of LTD via an increased accumulation of Arc expression (as the threshold for LTD induction is reduced in ArcKR mice (Wall et al. , 2018). These slices showed the largest fall in LTP amplitude.
To investigate this further, we incubated hippocampal slices with either vehicle (control), DHPG (100 µM) or the DHPG priming dose (20 µM) and examined levels of Arc protein 30 min after DHPG washout. Previous findings have demonstrated there is a significant increase in Arc expression in hippocampal cultures obtained from ArcKR compared to WT mice 30 min after DHPG washout (Wallet al. , 2018). As expected, there was a significant increase in Arc protein expression in hippocampal slices obtained from WT mice exposed to DHPG (100 µM) when compared vehicle (*p=0.01; Figure 3G, H) but no significant changes in Arc protein expression was observed between control (vehicle) and DHPG (20 µM) in both genotypes (p = 0.26). Similarly, exposure of DHPG (100 µM) caused a significant increase in expression of Arc protein levels in ArcKR when compared to control (**p=0.008; Figure 3G, H), but exposure to 20 µM DHPG had no detectable effect on Arc expression when compared to control (p=0.75; Figure 3G, H). Thus, the mechanism underlying the loss of mGluR priming in ArcKR mice remains unclear, but it may involve alterations in Arc localisation or posttranslational modifications.