Different Forms of AMPA Receptor Mediated LTP and Their Correlation to the Spatial Working Memory Formation

Spatial working memory (SWM) and the classical, tetanus-induced long-term potentiation (LTP) at hippocampal CA3/CA1 synapses are dependent on L-α-amino-3-hydroxy-5-methylisoxazole-4-propionate receptors (AMPARs) containing GluA1 subunits as demonstrated by knockout mice lacking GluA1. In GluA1 knockout mice LTP and SWM deficits could be partially recovered by transgenic re-installation of full-length GluA1 in principle forebrain neurons. Here we partially restored hippocampal LTP in GluA1-deficient mice by forebrain-specific depletion of the GluA2 gene, by the activation of a hypomorphic GluA2(Q) allele and by transgenic expression of PDZ-site truncated GFP-GluA1(TG). In none of these three mouse lines, the partial LTP recovery improved the SWM performance of GluA1-deficient mice suggesting a specific function of intact GluA1/2 receptors and the GluA1 intracellular carboxyl-terminus in SWM and its associated behavior.

Subsequent intensive research, analyzing the subunit composition of AMPARs in detail, led to a widely accepted model for the role of AMPAR subtypes in synaptic transmission and synaptic plasticity (for reviews see Derkach et al., 2007;Henley and Wilkinson, 2016). According to this model the Q/R site editing of the GluA2 subunit is essential for the formation of Ca 2+ -impermeable AMPAR assemblies (Sommer et al., 1991). The GluA2/3 AMPARs maintain basal synaptic transmission. In contrast, extra-synaptic GluA1/2-containing AMPARs are actively translocated into potentiated synapses upon LTP induction Shi et al., 2001). Immediately after LTP induction, Ca 2+ -permeable AMPARs are incorporated into the synapses (Plant et al., 2006;Rozov et al., 2012), (but see Adesnik and Nicoll, 2007) which might facilitate LTP expression. Due to their somatic and intracellular accumulation, GluA2 homomeric receptors contribute only poorly to AMPAR mediated signaling (Greger et al., 2002).
Despite the normal SRM of Gria1 −/− mice a robust impairment in the rewarded alternation task on the elevated T-maze-the standard behavioral test for the spatial working memory (SWM) performance in rodents (Rawlins and Olton, 1982;Deacon et al., 2002)-was detected in Gria1 −/− mice (Reisel et al., 2002). This SWM deficit was directly correlated to the LTP impairment, as shown by the partial restoration of SWM and LTP in Gria1 −/− mice that express GFP-tagged-GluA1 in principal forebrain neurons (Mack et al., 2001;Schmitt et al., 2005).
A recent study showed that AMPAR-mediated CA3-to-CA1 LTP is not strictly GluA1 dependent but requires a reserve pool of extra-synaptic ionotropic glutamate receptors (iGluRs; Granger et al., 2013). An increased surface expression of Ca 2+permeable iGluRs provided e.g., by the Q/R site unedited, trafficking competent GluA2(Q), a kainate receptor GluK1 or C-terminally truncated GluA1, was sufficient to restore LTP at mature CA1 synapses in absence of the endogenous AMPAR subunits (GluA1-3; Granger et al., 2013). Similarly, PDZ-site truncated GluA1 was sufficient for CA1 LTP as reported for gene targeted mice .
We noticed in previous studies that CA3-to-CA1 LTP is not necessarily linked to the SWM performance. The forebrain-specific depletion of GluA2 in Gria2 Fb mice was associated with SWM impairment although CA3-to-CA1 LTP was well-developed (Shimshek et al., 2006). Similarly, the transgenic expression of PDZ-site truncated GFP-GluA1(TG) was comparable to the GFP-GluA1 expression, but the GFP-GluA1(TG) expression could not rescue the SWM impairment in GluA1 deficient mice (Freudenberg et al., 2013a,b). To further dissect AMPAR functions in LTP and SWM, we genetically activated AMPARs containing homomeric GluA3, heteromeric GluA2(Q)/3 or PDZ-site truncated GFP-GluA1(TG) in principal forebrain neurons of Gria1 −/− mice and analyzed AMPAR subunit expression, pairing-induced and field-LTP and the SWM of the three different mouse lines.

Ethical Statement
Experiments were performed according to the institutional guidelines of the Max Planck Society and of the animal core facility (IBF) of the Heidelberg University. These guidelines adhere to the German Animal Welfare Act: Regulation for the Protection of Animals Used for Experimental or Other Scientific Purposes (Animal Welfare Regulation Governing Experimental Animals (TierSchVersV). Animal numbers for molecular and histological experiments were recorded under the protocol MPI/T-6/06; 15/08; 20/; 28/11. Genetic manipulations and behavioral experiments were licensed by the Regional Council in Karlsruhe, Germany (35-9185.81/G-4/02 and 35-9185.81/G-71/10). Efforts were made to minimize the number of animals used.

Genotyping
Mice were genotyped by tail-PCR with specific primers. Indicated below are the names of primers, primer sequences, and the approximate lengths of the amplified gene fragments.

Low Frequency Induced LTP in Whole-Cell Recordings
Pairing-induced LTP was induced by pairing low frequency stimulation (120 pulses, 0.67 Hz) with postsynaptic depolarization to 0 mV for 3 min as published in Chen et al. (1999). Monopolar stimulation electrodes were placed in the str. radiatum and in the str. oriens. The former was used to induce LTP, whereas the latter activated the control pathway. Excitatory postsynaptic currents (EPSCs) were elicited by activation of the two pathways (0.2 Hz) and were recorded for 20 min at -70 mV after the LTP-induction. The following intra-and extra-cellular solutions were used: Intracellular (in mM): 120 CsGluconate, 10 CsCl, 8 NaCl, 10 HEPES, 10 phosphocreatine, 0.2 EGTA, 4 MgATP, 0.3 NaGTP. The pH was set to 7.24 with CsOH and osmolarity was analyzed (295-310 mOsm). Extracellular (in mM): 124 NaCl, 26 NaHCO 3 , 2.5 KCl, 1.25 NaH 2 PO 4 , 4 MgSO 4 , 4 CaCl 2 , 10 glucose. All chemicals were obtained from Sigma. Statistical analysis was done by a two-tailed paired Student's t-test.

Tetanus Induced LTP in Hippocampal Field Recordings
Potentiation of hippocampal field excitatory postsynaptic potentials (EPSPs) was induced by tetanic stimulation as previously published. In all these studies Vidar Jensen and Øivind Hvalby performed the experiments under the same conditions and at the same E-Phys. setups (Feldmeyer et al., 1999;Zamanillo et al., 1999;Mack et al., 2001;Jensen et al., 2003;Shimshek et al., 2006). To standardize tetanization strength in different experiments, the tetanic stimulation strength was set in response to a single shock at intensity just above the threshold for generating a population spike. Synaptic efficacy was assessed measuring the slope of the fEPSP in the middle third of its rising phase. Six consecutive responses (1 min) were averaged and normalized to the mean value recorded 4-7 min prior to tetanic stimulation. In some experiments D-AP5 (50 µM, Sigma) was present during the recordings. Statistical significance of LTP levels between tetanized and non-tetanized pathways were calculated by Student's paired two-tailed t-test. LTP levels between genotypes were evaluated by linear mixed model statistical analysis (SAS 9.2, RRID:SCR_008567).

Spatial Working Memory in Rewarded
Alternation on a T-Maze (Non-matching-to-Place Paradigm) SWM was administrated in the rewarded alternation task on an elevated T-maze Reisel et al., 2002). The Tmaze consisted of a start arm (47 × 10 cm) and two identical goal arms (35 × 10 cm) with 10 cm high walls made out of black-painted wood. Mice were kept on diet at 85-90% of the starting body weight and were habituated to the investigator and the T-maze 2 days before testing. For the test, each trial consisted of a sample run followed by a choice run; the two separated by 15 s. During each run a food reward (30 µl sweetened, condensed milk; 4% fat, 10% fat-free dry milk, 27% sugar) was available in a food vial at the end of both arms. On the sample run the choice arms was blocked and the mouse picked up the reward in the sample arm. For the choice run, both arms of the T-maze were open and mice were rewarded for choosing the choice arm and unrewarded when choosing the previously visited sample arm. Correct choices in the choice runs of eight trials per day (four trials in the morning and four trials in the afternoon) were pooled and monitored as daily "block" performance. Behavior was statistically evaluated by analysis of variance (ANOVA) measurements followed by Holm-Sidak's multiple comparison and Bonferroni post-hoc tests (Prism 6, RRID:SCR_002798; IGOR Pro, RRID:SCR_000325).
Spatial Reference Memory on an Elevated Y-Maze (Non-matching-to-Place Paradigm) Acquisition of SRM was performed with mice kept on a strict food diet (remain to 85-90% of the starting body weight) on an elevated Y-shaped maze with prominent extra-maze cues as previously described (Shimshek et al., 2006). In brief, the Ymaze consisted of three identical arms without walls (arms: 50 × 10 × 0.5 cm; angle: 120 • ; height: 110 cm) made of black painted wood. Mice were trained in 10 sessions per day (intertrial interval of 10-15 min; 10 sessions in total) to find a milk reward (30 µl sweetened milk) at the end of a designated target arm (marked by a checkerboard pattern as extra-maze cue). The other two arms were assigned as starting position in a pseudorandom order (no more than three successive starts from the same arm with equal numbers of starting positions per day). On a given trial, the mouse was placed at the distal end of the starting arm and the initial entering of one of the other two arms was evaluated as correct (target arm) or incorrect (other start arm) trial. During the initial two sessions, exploring the maze and consuming the bait in the target arm (including entering and re-entering of all arms) was allowed to habituate to the spatial reward location. From session three on, the mouse was removed from the Y-maze when entering the wrong arm. To avoid any olfactory, visible or tactile cue inside the setup directed to a particular arm, the Y-maze was rotated by 120 • in random direction between each trial. Mice were trained in two daily blocks of five trials (one in the morning, the other in the afternoon) for 10 days (100 trials in total). Successful trials were recorded and pooled as daily performance. Data represent mean ± SEM. Behavior was statistically evaluated analysis of variance (ANOVA) measurements followed by Holm-Sidak's multiple comparison and Bonferroni post-hoc tests (Prism 6, RRID:SCR_002798; IGOR Pro, RRID:SCR_000325).

DISCUSSION
In our study we used genetically modified Gria1 and Gria2 genes to modulate hippocampal AMPAR expression in GluA1deficient mice. The cell-type specific modulation of AMPARs was achieved by inactivating a floxed Gria2 gene, by activating a hypomorphic Gria2 neo gene and by expressing a transgenic GFP-tagged-GluA1(TG) in principal forebrain neurons of GluA1 knockout mice. In the three different mouse lines-Gria1 −/− /2 Fb , Gria1 −/− /2 QFb , and Gria1 −/− /Tg 8.1 -the remaining AMPAR levels and the ratios of Ca 2+ -permeable and Ca 2+ -impermeable AMPARs is very different in principal neurons of the hippocampus.
In hippocampal neurons of Gria1 −/− /2 Fb mice, the GluA3 level, was about 25% lower compared to GluA3 levels of wildtype mice, where GluR3 subunits already represent only 10% of the AMPAR subunits (Wenthold et al., 1996;Lu et al., 2009). In Gria1 −/− /2 QFb , which express both GluA2 and GluA2(Q), the fall in GluA3 expression was less pronounced than in Gria1 −/− /2 Fb mice even though the difference reached no statistical difference (p = 0.22). This might suggest that AMPARs containing only Glutamine (Q) in the pore-forming segment (Sprengel et al., 2001) are less stable and might be faster degraded than Ca 2+ -impermeable channel assemblies containing GluA2 with an Arginine (R) at homologous position. Similarly the twofold reduction of GluA2 levels in Gria1 −/− /2 QFb mice is less pronounced when GluA1 is present in Gria2 QFb (also called Gria2 ECS ) mice, as demonstrated in an earlier study (Feldmeyer et al., 1999). On the other hand, we cannot exclude changes in Gria2 and Gria3 gene expression in response to GluA1 depletion.
The immunohistological analysis of coronal brain slices confirmed the absence and reduced GluA2 expression in Gria1 −/− /2 Fb and Gria1 −/− /2 QFb mice, respectively. In addition, the somatic accumulation of GluA2 immunosignals in the str. pyramidale of Gria1 −/− /2 QFb mice showed that a substantial fraction of GluA2 is trapped in the cell somata. Despite the loss of synaptic AMPARs in Gria1 −/− /2 Fb and Gria1 −/− /2 QFb mice, the recorded I/V curves of CA1 pyramidal cells documented the contribution of the remaining AMPAR subunits in fast synaptic signal transmission. As expected from the expression analysis, the AMPAR currents in CA1 cells were strongly reduced when GluA1 and GluA2 were not expressed in Gria1 −/− /2 Fb . The remaining GluA3-containing AMPAR in CA1 cells of Gria1 −/− /2 Fb mice could be identified by a high rectification index (RI)-the hallmark of Ca 2+ -permeable AMPARs (Burnashev et al., 1992). In CA1 pyramidal neurons of Gria1 −/− /2 QFb mice the presence of GluA2(Q) in AMPAR assemblies could also be monitored by the formation of synaptic Ca 2+ -permeable AMPARs, as shown by the small but significant shift of the RI compared to the RI monitored in wild-type mice; the AMPAR-mediated current amplitude was similar to wild type.
The expression of endogenous encoded AMPARs in Gria1 −/− /2 Fb and Gria1 −/− /2 QFb mice was sufficient for the induction and expression of pairing-induced and field-LTP in GluA1-deficient mice. However, the different amount of AMPARs affected the potentiation level. The GluA3-containing AMPARs of Gria1 −/− /2 Fb mice showed slightly lower LTP levels compared to the partial LTP rescue of Gria1 −/− /2 QFb mice. A partial recovery of field-LTP in Gria1 −/− mice was also achieved by the transgenic GFP-GluA1(TG) subunit in Gria1 −/− /Tg 8.1 mice confirming that the GluA1-PDZ domain is dispensable for LTP . Thus, for the pairinginduced and field-LTP, there is no strict requirement for functional GluA1 subunits, but the pool of extracellular iGluRs affects the level of potentiation as described earlier (Granger et al., 2013).
Despite the partially restored hippocampal LTP in our three mouse lines, the SWM performance of all three lines remained at the chance level in the T-maze task. The lower amplitudes of LTP are unlikely to be the main reason for the failure to rescue the SWM impairment of GluA1 knockout mice. As we described earlier a partial LTP rescue with similar amplitudes obtained by the transgenic GFP-tagged-GluA1 expression was sufficient to improve the SWM performance in Gria1 −/− mice (Mack et al., 2001;Schmitt et al., 2005) whereas a fully developed LTP in forebrain-specific GluA2 knockout mice (Gria2 Fb ) was associated with strong SWM impairment (Shimshek et al., 2006). Therefore, we conclude that the hippocampal LTP cannot be used to predict the behavioral performance of mice. Their SWM performance might be influenced by many factors modulating the excitatory and inhibitory systems, which might be more important than experimentally induced synaptic plasticity.

AUTHOR CONTRIBUTIONS
DS, TB, LL, and RS designed, generated, and molecularly analyzed the mouse lines. VJ, BS, and GK performed and analyzed the electrophysiological experiments. VM and DS performed the behavioral experiments. RS, DS, and TB wrote the manuscript.