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stage. The TARP γ-4 subunit, on the other hand, showed high expression until P7, which decreased at P14 through P21, which was the latest time point tested in this study. Age- dependent changes in TARP expression beyond P60 were unclear. As such, our study is the first to investigate the temporal expression profile of AMPAR regulatory proteins, stargazin, CNIH2, and GSG1L, over a significant fraction of adult life. From this analysis we suggest that GSG1L exhibits a unique age dependent increase. The findings reported here will serve as a valuable resource for future functional studies of GSG1L in various brain regions throughout development
Previous investigations on the synaptic phenotypes of GSG1L KO rats in CA1 and DG neurons (Gu et al., 2016; Mao et al., 2017) were both conducted at time points when the expression of the lacZ reporter for GSG1L promoter activity is low (Figures 3.3).
Specifically, GSG1L KO rat acute slices at postnatal day (P) 13–P19 were used. Although GSG1L protein is detectable in a subset of dendritic spines in older neurons (Schwenk et al., 2012; Shanks et al., 2012; Willems et al., 2020), the overall expression of GSG1L may be low in the hippocampus compared with the anterior thalamus at young age (i.e., P13–
P19). In agreement with such a hypothesis, a previous study on GSG1L KO rat reported a substantial contribution of another auxiliary subunit, CNIH2, superimposed on the functional effect of GSG1L (Gu et al., 2016). However, because the lacZ reporter is an indirect indicator of GSG1L protein expression, future studies using more direct methods are necessary to determine whether its expression is low in young hippocampus.
The AD/AV nuclei, where GSG1L is abundantly expressed, are at the core of the extended hippocampal-diencephalic network with crucial roles in memory (Aggleton &
Brown, 1999), and thus their damage is associated with severe anterograde amnesia
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(Ghika-Schmid & Bogousslavsky, 2000; Gold & Squire, 2006). AD/AV nuclei also contain neurons that sense head direction during spatial navigation (Clark & Taube, 2012), and have been implicated in seizure initiation and/or propagation (Mirski & Ferrendelli, 1984;
Kerrigan et al., 2004). In contrast to the extensively studied LGN, molecular and functional characterization of the synapses of AD/AV nuclei is largely lacking.
Our studies, delineated in this chapter, show that GSG1L is functionally expressed at AD/AV nuclei, where it regulates AMPAR activity in a subset of synapses that receive inputs from the cortex, but not mammillary bodies or subiculum. In corticothalamic synapses, GSG1L reduces the amplitude and slows the kinetics of AMPAR mediated qEPSCs and further suppresses short-term facilitation by slowing the recovery from desensitization. Previous investigations have shown that GSG1L may play a role in trafficking of AMPARs, where its presence is associated with a reduction in surface expression of AMPARs in hippocampal neurons (Gu et al., 2016), and such mechanism may partially contribute to the properties of corticothalamic synapses. However, we suggest that the effect of GSG1L on gating modulation plays a dominant role in regulating short-term plasticity given the CTZ sensitivity of these synapses in the wild type but not in GSG1L KO (Figure 3.8).
In contrast to GSG1L, stargazin plays a major role in regulating AMPAR activity in mammillothalamic or subiculum-thalamic synapses. The mechanism that restricts the functions of GSG1L and stargazin to specific afferent synapses remains to be determined.
We speculate that presynaptic terminals may provide some trans-synaptic molecular cues, considering that GSG1L lacks a PDZ (PSD-95, dlg, ZO-1) domain binding motif in the cytoplasmic C terminus as it would normally facilitate synaptic anchoring. However,
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future studies will be needed to more definitively determine the mechanisms underlying the segregations of AMPAR complexes.
Coexpression of GSG1L and stargazin in AD/AV nuclei is particularly interesting. In Xenopus oocytes, stargazin outcompetes GSG1L in AMPAR modulation (Schwenk et al., 2012), which agrees with GSG1L and stargazin sharing identical binding sites on AMPARs (Twomey et al., 2016, 2017b). These observations would suggest that the two auxiliary subunits would likely compete for AMPAR regulation in neuronal populations where they are co-expressed. Indeed, in AD/AV nuclei, we also found that under basal conditions, stargazin outcompetes GSG1L, functionally, in most synapses (Figures 3.15).
The effect of GSG1L is unmasked in GSG1L/γ-2dKO mice, with changes in AMPAR- mediated mEPSCs amplitude and frequency, as well as changes in the kinetics of mEPSCs. These observations also indicate that in AD/AV, the corticothalamic synapses are the smaller population compared with the combined sets of mammillothalamic and subiculum-thalamic synapses.
Similar to GSG1L, CKAMP44 also slows the recovery from desensitization and has been previously shown to modulate short-term plasticity in hippocampal and retinogeniculate synapses (von Engelhardt et al., 2010; Chen et al., 2018). Based on the in situ hybridization data, CKAMP44 is highly expressed in AD/AV nuclei. We find that mammillothalamic synapses in the AD/AV nuclei undergo pronounced short-term depression (Figure 3.6C-E, Figure 3.7A-C), which is highly sensitive to CTZ (data not shown). Given that GSG1L does not modulate AMPAR function at mammillothalamic synapses, we speculate that CKAMP44 could be postsynaptically functional in these
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synapses, which is yet to be investigated. Again, input specific functional expression of an AMPAR auxiliary subunit in the thalamus may extend to the case of CKAMP44.
AMPAR desensitization is critical for normal brain function (Christie et al., 2010).
Introduction of a mutation that abolishes AMPAR desensitization in knock-in, GluA2L483Y mutant mice results in lethality of homozygous mice. Heterozygous mice carrying this mutation exhibit severe neurological phenotype, including seizures and early mortality (Christie et al., 2010). These investigations highlight the critical importance of AMPAR desensitization in vivo.
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Circuit-level and behavioral changes in GSG1L knockout mice Adapted from Kamalova et al., “AMPA receptor auxiliary subunit GSG1L suppresses short-term facilitation in corticothalamic synapses and determines seizure susceptibility”