Calcium-permeable AMPA receptors mediate timing-dependent LTP elicited by 6 coincident 1 action potentials at Schaffer collateral-CA1 synapses

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Long-term potentiation (LTP) and long-term depression (LTD) of synaptic transmission can be observed in 48 response to repetitive activation of synapses and are believed to represent cellular models of learning 49 and memory processes in the brain (see e.g., Bi and Poo, 1998;Bliss and Cooke, 2011;Malenka and Bear, 50 2004). While LTP leads to a stable enhancement of synaptic transmission between connected neurons, 51 LTD yields a long-lasting decrease in synaptic responses. Depending on the time frame that is investigated, 52 LTP as well as LTD can be divided into an early phase lasting roughly 1h and a late phase that starts 2-3h 53 after induction of the synaptic change. While early LTP is mediated by posttranslational modifications, 54 late LTP was found to depend on synthesis of new proteins (Lynch, 2004; but see Wang et al., 2016). LTP 55 was initially discovered using long-lasting high frequency stimulation of glutamatergic synapses in the 56 mammalian hippocampus (Bliss and Lomo, 1973), a brain region essential for encoding episodic memory 57 (Tonegawa et al., 2018). While in these pioneering studies, LTP was recorded in vivo using extracellular 58 field potential recordings (Bliss and Lomo, 1973), LTP is also observed in acutely isolated brain slices ex

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Like memory formation in vivo, t-LTP in acute ex vivo brain slices is strongly controlled by 79 neuromodulatory inputs, which can regulate the efficacy of induction paradigms to elicit plasticity 80 Lessmann, 2011, 2013 Seol et al., 2007). Such kind of neuromodulation can also bridge the temporal gap 82 between synaptic plasticity and behavioral time scales for learning processes (Gerstner et al., 2018;3 Our present study demonstrates, that Schaffer collateral (SC)-CA1 t-LTP can be induced robustly by only 96 three to six repeats of coincident pre-and postsynaptic spiking at 0.5 Hz. Moreover, our study reveals 97 that, depending on specific STDP paradigms (i.e. 1:1 vs. 1:4) the low repeat protocols recruit distinct 98 sources for postsynaptic Ca 2+ elevation during induction of t-LTP, are mediated by distinct pre-and 99 postsynaptic expression mechanisms, and are differentially regulated by dopamine receptor signaling.

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Together these data suggest that hippocampal SC-CA1 synapses can recruit multiple types of synaptic 101 plasticity in response to low repeat STDP protocols for processing of information and memory storage in 102 the hippocampus. Altogether, these distinct cellular STDP pathways might form the basis for the 103 pluripotency of hippocampal functions in learning and memory.

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Timing-dependent LTP at Schaffer collateral-CA1 synapses requires 3-6 spike pairings 108 Using whole cell patch clamp recordings, we investigated timing-dependent (t-)LTP at Schaffer collateral 109 (SC) -CA1 synapses in acute hippocampal slices obtained from juvenile (i.e. P28-35) male C57BL/6J mice. neurons that were not subjected to STDP stimulation (negative controls (0:0): 105.0% ± 6.5%; Fig 1A). The 125 time course of changes in synaptic strength in an individual cell that was potentiated with the low repeat 126 1:1 protocol is depicted at the right side in blue. These data indicate that t-LTP can be induced at SC-CA1 127 synapses with low repeat t-LTP paradigms (i.e. 6 x 1:1), that might more closely resemble the natural 128 pattern of pre-and postsynaptic activity that can be observed during memory formation in CA1 in vivo 129 than any high frequency or high repeat LTP protocol.

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In general, the magnitude of t-LTP induced with the 6 x 1:4 stimulation (159.6% ± 8.0%) was comparable 6 transmission without any decline in magnitude. Moreover, the overall time course of the potentiation was 198 indistinguishable for both types of low repeat t-LTP (Fig 2D). Together, these findings indicate that the 199 low repeat STDP paradigms identified in this study induce Hebbian plasticity selectively at short positive 200 spike timings with similar properties as have been described in earlier studies using high repeat canonical 201 and burst type STDP protocols (e.g., Bi and Poo, 1998;Edelmann et al., 2015;Froemke et al., 2006).

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In the next series of experiments, we determined the mechanisms of induction and expression as well as 203 the intracellular signaling cascades involved in modulation of both types of low repeat t-LTP.

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To investigate whether the low repeat STDP paradigms introduced here, rely on pre-or postsynaptic 220 expression mechanisms of synaptic plasticity, we determined changes in the paired pulse ratio (PPR) 221 before and 30 min after t-LTP induction that was obtained when two successively evoked EPSPs were 222 elicited at 50 ms inter-stimulus interval (Fig 3A). Commonly, a decrease in PPR after induction of LTP is 7 presynaptically expressed synaptic plasticity. When t-LTP was induced with the 6 x 1:1 paradigm we found 225 on average in fact a significant decrease in PPR (paired Student's t-test, t(34) =2.3471; p= 0.0249, whereas 226 the PPR remained unchanged after inducing 6 x 1:4 t-LTP (paired Student's t-test, t(20) = 1.0146; p = 0.3224; 227 Fig 3A). This decreased PPR after induction of 6 x 1:1 t-LTP hints at a presynaptic expression mechanism.

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In contrast, the PPR analysis clearly speaks against any presynaptic contribution in the expression of 6 x 229 1:4 t-LTP. Interestingly, the initial PPR before inducing t-LTP was not significantly different between the 230 tested groups (negative control (0:0): 1.77 ± 0.09; 6 x 1:1: 1.78 ± 0.08; 6 x 1:4: 1.74 ± 0.06; Kruskal-Wallis 231 test, H (2) = 0.059; p = 0.9710), indicating that the initial release probability was similar and a stable basal 232 parameter in our slices. As additional measures for a presynaptic expression mechanism, we determined 233 miniature EPSCs and coefficient of variation (CV) analysis before and after successful induction of our 6x are consistent with presynaptic expression of 6 x 1:1 t-LTP, as the overall mean for CV after LTP was 236 decreased, and miniature EPSC frequencies (determined as shorter inter event intervals (IEI)) were 237 increased after 6 x 1:1 t-LTP induction (see Fig S1).

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To test for involvement of ER-resident ryanodine receptors (RyR) in the low repeat burst protocol, we  (Nyberg et al., 2016). A classical role for D1 receptor signaling was also described for 371 for high repeat (70 x) canonical t-LTP in rat hippocampal slices Lessmann, 2011, 2013). To clarify whether repeat number or species matter for the contribution of D1 and D2 receptors in t-LTP, we 373 examined DA dependence of high repeat 70 x 1:1 t-LTP in mouse hippocampal slices. We found that also 374 in mouse slices 70 x 1:1 t-LTP was fully blocked by bath application of the D1 antagonist SCH23390 375 (unpaired Student's t-test, t (22) = 3.028; p= 0.0062; Fig 6C). These data reveal that high repeat number 376 induced t-LTP is regulated by D1 signaling whereas D2 signaling is selectively involved in low repeat t-LTP.

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Further, the extent of D2 receptor involvement in low repeat t-LTP is regulated by the postsynaptic spike 378 pattern used for t-LTP induction (compare Fig 6A and B).   Fig 8A, B). Surprisingly, these results indicate 434 that the influx of Ca 2+ via GluA2-lacking, cp-AMPARs is mandatory to elicit low-repeat t-LTP induction.

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To rule out off-target effects of NASPM, we verified cp-AMPAR contribution in low repeat t-LTP with a 436 second inhibitor of cp-AMPARs (IEM-1460, 100 µM). As shown in Figure 8C 1A). As for the canonical protocol, we also 524 determined the threshold for successful t-LTP induction also for the burst protocol (compare Fig 1B). The

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Interestingly, our data show that both low repeat t-LTP variants tested are blocked when signaling of 553 endogenously released DA is inhibited (Fig 6). Our results are in line with the previously described effects  Regarding the magnitude of t-LTP induced by low repeat canonical and burst protocols we, found that 561 both, 6 x 1:1 and 6 x 1:4 t-LTP, were equally successful to induce t-LTP at positive spike timings (Fig 2C).

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Because it is reasonable to assume that 1:4 burst protocols induce longer lasting and stronger Ca 2+ 563 elevations than 1:1 pairings, it might be expected that the time course of synaptic potentiation could differ 564 between the two protocols. However, both protocols induced t-LTP with comparable onsets and rise times 565 of potentiation and also resulted in similar magnitudes of t-LTP after 1 h of recording (compare Fig 2D).

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Thus, except for the lower threshold number of repeats to elicit t-LTP (see last paragraph), the burst 567 protocol does not seem to be more effective in inducing t-LTP at SC-CA1 synapses than the canonical 568 protocol.

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We also compared different spike timings (with negative and positive delays), to compare the full capacity 570 to induce bidirectional plasticity with low repeat protocols (Fig 2A, B). For positive pairings with Δt: +20 571 ms we observed a similar decline (compared to Δt: +10 ms) in t-LTP magnitude as described previously for 572 higher numbers of repeats (compare Bi and Poo, 1998;Edelmann et al., 2015). When applying negative 573 pairings (i.e. post before pre pairings) t-LTP was absent, but we did not observe robust t-LTD for either of 574 the two protocols. While these results stress that successful induction of t-LTP is critically dependent on 575 the sequence of presynaptic and postsynaptic spiking and on the pairing interval, future studies should 576 address under which conditions low repeat t-LTD can be induced by anti-causal synaptic activation.

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Despite the similarities described above, both low repeat protocols recruited different expression 580 mechanisms. Synaptic potentiation induced with the 6 x 1:1 protocol is most likely expressed by 581 presynaptic alterations (see below), whereas the 6 x 1:4 protocol relies on postsynaptic insertion of AMPA 582 receptors (Fig 3). Commonly, LTP at SC-CA1 synapses that is induced by high-frequency stimulation and is 583 also thought to be expressed by a postsynaptic increase in AMPA receptor mediated currents (Granger 584 and Nicoll, 2014;Nicoll, 2003). For STDP, however, different mechanisms of expression have been 585 described that varied between brain regions and depending on experimental conditions (see e.g., Costa  3).

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For the 6 x 1:1 t-LTP, the absence of an increase in AMPAR mediated currents (Fig 5) and the observed 599 decrease in paired pulse ratio (PPR) after successful LTP induction and the increased mEPSC frequency 600 (Fig S1), are consistent with presynaptic enhancement of glutamate release probability. AMPARs for the expression of 6 x 1:4 t-LTP (Fig 3C). with these previous studies, we found that 6 x 1:1 t-LTP can in addition to NMDARs also be induced by 630 Ca 2+ entry through L-type VGCCs (Fig 4A, C).

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In contrast to these conventional Ca 2+ sources for the canonical low repeat t-LTP, the situation is much 632 different for 6 x 1:4 burst t-LTP. Although a requirement for postsynaptic Ca 2+ elevation is clearly evident 633 from the BAPTA experiments (Fig 5A), Ca 2+ entry via NMDARs or VGCCs was not involved (compare Fig 4B, 649   5D). The resulting calcium rise and additional Ca 2+ influx via cp-AMPARs might than be strengthened by 650 additional IP3 and RyR mediated calcium induced Ca 2+ release to successfully boost low repeat induced 651 burst t-LTP (compare Fig 9).

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The accurate timing of pre-and postsynaptic activity is necessary for hebbian plasticity. In addition,

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Moreover, while the D1 receptor inhibitor SCH23390 alone did not show any signs of 6 x 1:1 t-LTP 676 inhibition, it was nevertheless able to impair the slightly reduced t-LTP in the presence of Sulpiride down 677 to control levels, when both antagonists were co-applied (Fig 6A). The interpretation of this 678 pharmacological profile of 6 x 1:1 t-LTP needs to take into consideration that D1-like and D2-like receptors

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To interpret the combined regulation of the 6 x 1:1 t-LTP by D1-and D2-like receptors it also needs to be 688 taken into consideration that D2-like receptors are generally believed to display a higher affinity for DA 689 compared to D1-like receptors (Beaulieu and Gainetdinov, 2011). Therefore, the complex D1-and D2 690 receptor-dependent regulation of 6 x 1:1 t-LTP might assure that this type of t-LTP is on the one hand 691 regulated by the presence of DA, but on the other hand remains intact at high and low DA concentrations.

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Interestingly, both variants of low repeat t-LTP were strictly dependent on activation of GluA2-lacking 731 calcium-permeable (cp-)AMPA receptors (Fig 8). In the respective experiments, NASPM or IEM were 732 present in the ACSF from the start of the recording to assure complete inhibition of cp-AMPARs during t-733 LTP induction. The respective solvent controls were treated in the same way. In CA1 neurons, cp-AMPARs

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Spike timing intervals (i.e. Δt, ms) were measured as the time between onset of the evoked EPSP and the 867 peak of the first action potential. Cells were only included for analysis if the initial resting membrane 868 potential (RMP) was between -55 and -70 mV. Cells were excluded when the input resistance varied more 869 than 25% over the entire experiment. Furthermore, traces showing visible "run-up" or "run-down" during 870 baseline recording were excluded. Data were binned at 1 min intervals.

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For analysis of presynaptic short-term plasticity before and after t-LTP induction, paired pulse facilitation 878 was recorded in voltage clamp mode at a holding potential of -70 mV, and the paired pulse ratio (PPR) 879 was determined by dividing the peak current amplitudes of the second EPSC by the first EPSC at an inter-880 stimulus interval of 50 ms.

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To further check for a presynaptic LTP expression locus of the 6x 1:1 t-LTP, coefficient of variance (CV) 882 analysis was performed. CV is expressed as standard deviation/mean (Faber and Korn, 1991). The ratio of 883 CV² before and after the pairing (20-30 min after induction) was plotted against the respective ratio of 884 mean EPSP slopes (EPSP after/EPSP baseline, Malinow and Tsien, 1990;Manabe et al., 1993). CV² ratio 885 was calculated by dividing 1/CV² after LTP with 1/CV² of baseline. Presynaptic LTP is supposed to influence 924 925