2+ Calcium sensor regulation of the CaV2.1 Ca channel contributes to long-term potentiation and spatial learning

Evanthia Nanoua, Todd Scheuera, and William A. Catteralla,1

aDepartment of Pharmacology, University of Washington, Seattle, WA 98195-7280

Contributed by William A. Catterall, September 29, 2016 (sent for review September 2, 2016; reviewed by Diane Lipscombe and Gerald W. Zamponi)

Many forms of short-term synaptic plasticity rely on regulation of Presynaptic signals may also contribute to this series of events by 2+ presynaptic voltage-gated Ca type 2.1 (CaV2.1) channels. How- strengthening transmission at hippocampal syn- ever, the contribution of regulation of CaV2.1 channels to other apses (20); however, at the outset of these experiments, we did forms of neuroplasticity and to learning and memory are not not expect that changes in short-term synaptic plasticity would known. Here we have studied mice with a mutation (IM-AA) that have major effects on LTP. To further explore the role of Ca 2.1 channel regulation by disrupts regulation of CaV2.1 channels by calmodulin and related V calcium sensor proteins. Surprisingly, we find that long-term po- CaS proteins in spatial learning and memory, we used knockin tentiation (LTP) of synaptic transmission at the Schaffer collateral- mice with paired substitutions for the isoleucine and CA1 synapse in the hippocampus is substantially weakened, even methionine residues in the IQ-like motif (IM-AA) in their though this form of synaptic plasticity is thought to be primarily C-terminal domain (21), and we studied LTP and spatial learning and memory in IM-AA mice. In addition to the previously docu- generated postsynaptically. LTP in response to θ-burst stimulation mented alterations in short-term facilitation in Schaffer collateral and to 100-Hz tetanic stimulation is much reduced. However, a (SC)-CA1 synapses in acute hippocampal slices (21), we found normal level of LTP can be generated by repetitive 100-Hz stimu- major deficits in LTP and in spatial learning and memory, which lation or by depolarization of the postsynaptic cell to prevent 2+ reveal unexpected connections among presynaptic neuroplasticity, block of NMDA-specific glutamate receptors by Mg . The ratio postsynaptic LTP, and spatial learning and memory. NEUROSCIENCE of postsynaptic responses of NMDA-specific glutamate receptors to those of AMPA-specific glutamate receptors is decreased, but Results the postsynaptic current from activation of NMDA-specific gluta- IM-AA Mutation in CaV2.1 Channels Impairs LTP of SC-CA1 Pyramidal mate receptors is progressively increased during trains of stimuli Cell Synapses. In the acute hippocampal slice preparation, re- and exceeds WT by the end of 1-s trains. Strikingly, these impair- petitive bursts of stimulation at the frequency of θ-waves elicit ments in long-term synaptic plasticity and the previously documented LTP in response to a natural stimulus pattern (22, 23). We ex- impairments in short-term synaptic plasticity in IM-AA mice are asso- amined whether IM-AA synapses might have changes in long- ciated with pronounced deficits in spatial learning and memory in term synaptic modulation induced by θ-burst stimulation (TBS) context-dependent fear conditioning and in the Barnes circular maze. (Materials and Methods). We found that LTP, induced by TBS of Thus, regulation of CaV2.1 channels by calcium sensor proteins is re- SC fibers while holding the membrane potential of postsynaptic quired for normal short-term synaptic plasticity, LTP, and spatial CA1 neurons at −40 mV, was significantly reduced in IM-AA learning and memory in mice. synapses compared with WT (Fig. 1A). These results indicate that regulation of CaV2.1 channels by CaS proteins contributes calcium channel | calmodulin | synaptic plasticity | calcium sensor to long-term synaptic plasticity as well as short-term plasticity. proteins | hippocampus Significance ctivity-dependent modification of synaptic strength in syn- Aapses in the central nervous system is important for hippocampal- Learning and memory are caused by changes in strength of dependent information processing and for spatial learning and communication between neurons at synapses. Both brief changes memory (1). Short-term and long-term modifications in synaptic (short-term plasticity) and long-lasting changes (long-term plas- strength are regulated by the frequency and pattern of presynaptic ticity) are important. Synaptic transmission is initiated by calcium + spiking (2–5). Regulation of voltage-gated Ca2 channel type 2.1 channels, which are regulated by calcium-sensor proteins that 2+ (CaV2.1) by calmodulin (CaM) and related Ca sensor (CaS) induce short-term synaptic plasticity. We studied genetically + proteins causes Ca2 -dependent facilitation and inactivation of P/Q- modified mice with a mutation in the binding site for calcium- + type Ca2 currents (6–12) that results in short-term facilitation and sensor proteins on calcium channels, which alters short-term rapid depression of synaptic transmission (9, 12–14). Deletion synaptic plasticity. Surprisingly, we found that synapses in the of the gene encoding CaV2.1 channels (15) or mutation of their hippocampus of these mice also have impaired long-term po- CaS protein binding domain (12–14) impairs short-term synaptic tentiation. In addition, these mutant mice have impaired spa- plasticity. Although regulation of CaV2.1 channels contributes to tial learning and memory. Our results show that disruption of short-term synaptic plasticity in multiple types of synapses, the calcium-channel regulation by calcium-sensor proteins alters functional role of this form of synaptic regulation in learning both short-term and long-term plasticity, and these changes and memory is unknown. impair spatial learning and memory. Long-term potentiation (LTP) of synaptic transmission in the hippocampus is thought to be important for spatial learning and Author contributions: E.N., T.S., and W.A.C. designed research; E.N. performed research; memory (16). High-frequency stimuli induce LTP, which depends E.N., T.S., and W.A.C. analyzed data; and E.N. and W.A.C. wrote the paper. + on postsynaptic Ca2 entry via NMDA-specific glutamate recep- Reviewers: D.L., Brown University; and G.W.Z., University of Calgary. tors and activation of calcium/calmodulin-dependent protein The authors declare no conflict of interest. kinase II (17, 18). These events cause increased activity of AMPA- 1To whom correspondence should be addressed. Email: [email protected]. specific glutamate receptors via both direct phosphorylation This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. and enhanced trafficking to the postsynaptic membrane (19). 1073/pnas.1616206113/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1616206113 PNAS Early Edition | 1of6 Downloaded by guest on September 27, 2021 The response of NMDA receptors to glutamate is controlled A + 400 * by voltage-dependent Mg2 block of the pore. To determine TBS 400 + whether relief of Mg2 blockade of NMDA receptors would en- 300 300 hance LTP at IM-AA synapses, we induced LTP while depolarizing postsynaptic CA1 pyramidal neurons to 0 mV during the tetanus. 200 200 Under these conditions, there was no significant difference be- tween the potentiated EPSCs of WT and IM-AA synapses (Fig.

EPSC (%) A 100 100 2 ). Furthermore, LTP comparable to WT could be elicited

LTP (% baseline) LTP using a stronger, three-train stimulus paradigm in IM-AA syn- 0 apses held at −60 mV (Fig. 2B). Taken together, these results 20 40 60 80 demonstrate that the IM-AA mutation causes weakened LTP, Time (min) which can be restored by stronger activation of NMDA receptors by + B using depolarization to reverse Mg2 block (Fig. 2A)orbyapplying 400 400 *** TET additional tetanic stimuli (Fig. 2B). To better understand the basis for altered LTP induction, we 300 300 directly recorded the EPSC during the trains of stimuli used to 200 elicit LTP. Mean peak amplitudes of EPSCs during 100-Hz trains 200 in IM-AA synapses were slightly increased relative to WT synapses,

EPSC (%) 100 and the integral of EPSCs was greater (Fig. S1). These EPSCs re- 100 flect primarily the contribution of AMPA receptors, which are

LTP (% baseline) LTP 0 rapidly activated by released glutamate. To estimate the efficacy of postsynaptic depolarization to induce LTP more accurately, we 20 40 60 80 Time (min) C 400 400 D-AP5 A 400 TET+DEP 400 300 TET 300 300 300 200 200

(% baseline) EPSC (%) 200 200 100 100

LTP EPSC (%) 100 0 100

LTP (% baseline) LTP 20 40 60 80 0 Time (min) 20 40 60 80 Fig. 1. IM-AA mutation reduces LTP in SC-CA1 synapses. (A) LTP induced at Time (min) − θ B 40 mV by eight trains of -burst stimulation (10 bursts delivered at 5 Hz, 400 TET 400 each burst consisted of 10 pulses at 100 Hz) from WT (black, n = 6) and IM- = AA (red, n 7) (Left). LTP values from individual cells (open symbols) and 300 300 average LTP values (solid symbols) from WT (black) and IM-AA (red) (Right). (B) LTP induced by two 100-Hz trains at −60 mV in the presence of 1 μM 200 ω-Ctx, 50 μM picrotoxin, and 10 μM CGP55845 hydrochloride from WT (black, 200 = =

n 8) and IM-AA (red, n 8) (Left). (Right) Summarized LTP values as in A. (% baseline)

EPSC (%) (C) LTP induced by two 100-Hz trains at 0 mV in the presence of 1 μM ω-Ctx, 100 100

50 μM picrotoxin, and 10 μM CGP55845 hydrochloride from WT (black, n = 7) LTP and IM-AA (red, n = 6) (Left). (Right) Summarized LTP values as in A.*P < 0 0.05; ***P < 0.001. 20 40 60 80 Time (min) C We investigated whether reduced LTP in IM-AA SC-CA1 250 synapses was specific to the mode of induction of LTP by com- paring LTP in WT and IM-AA mice induced by tetanic stimu- 200 lation. Application of two 100-Hz trains of stimuli to the SC

fibers resulted in robust LTP in WT, but not in IM-AA SC-CA1 LTP % 150 B synapses (Fig. 1 ). Potentiation immediately following the tet- 100 anus was similar for WT and IM-AA (WT, 265 ± 36%; IM-AA, 295 ± 33%), which is expected because posttetanic potentiation 0 100 200 300 400 500 600 does not involve calcium channel regulation (3, 13). On the other Postsynaptic charge (pC) hand, excitatory postsynaptic current (EPSC) amplitude was Fig. 2. Induction of LTP in IM-AA SC-CA1 synapses by stronger stimulation sustained in WT but decreased rapidly in IM-AA to only 122 ± and postsynaptic depolarization. (A) LTP induced by two 100-Hz trains at 18% of the pretetanus value by 60 min after the end of the te- 0 mV in the presence of 1 μM ω-Ctx, 50 μM picrotoxin, and 10 μM CGP55845 tanic stimulus (Fig. 1B). Because LTP in SC-CA1 synapses de- hydrochloride from WT (black, n = 7) and IM-AA (red, n = 6) (Left). LTP values + pends on postsynaptic Ca2 influx through NMDA-specific from individual cells (open symbols) and average LTP values (solid symbols) glutamate receptors (17, 18), we examined whether application from WT (black) and IM-AA (red) are shown at Right.(B) LTP induced at −60 mV by two 100-Hz trains from WT (black, n = 8) and by three 100-Hz of the NMDA receptor blocker D-AP5 would affect potentiation trains from IM-AA (red, n = 8) in the presence of 1 μM ω-Ctx, 50 μM picro- of the EPSC. We found that D-AP5 completely abolished LTP in μ C toxin, and 10 M CGP55845 hydrochloride (Left). (Right) Summarized LTP both WT and IM-AA synapses (Fig. 1 ), consistent with a nor- values as in A.(C) Relationship between postsynaptic charge during 1x, 2x, mal mechanism of induction of LTP dependent on NMDA re- 3x 100-Hz trains used to induce LTP and the normalized percentage of LTP ceptors in IM-AA synapses, which is less effective than in WT. from WT (black, n = 4–7) and IM-AA (red, n = 4–6).

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1616206113 Nanou et al. Downloaded by guest on September 27, 2021 integrated the EPSCs to yield a measure of total postsynaptic charge postsynaptic membrane potential (−20mV)inboth(Fig.3D). To in response to one, two, or three 100-Hz trains, and we plotted the determine whether alterations in NMDA receptor subunit compo- amount of LTP produced vs. postsynaptic charge (Fig. 2C). The sition contribute to the decreased NMDA receptor/AMPA recep- IM-AA mutation dramatically weakened the coupling between tor ratio in IM-AA synapses, we examined the effect of a NR2B the production of LTP and the integrated EPSC charge during subunit-specific blocker () on NMDA receptor-mediated the induction protocol in IM-AA SC-CA1 synapses, as illustrated current (Fig. 3C). Interestingly, no difference in NR2B/NR2A ratio by the rightward shift of the LTP vs. postsynaptic charge rela- was found between WT and IM-AA synapses, suggesting that the tionship, but it had no effect on the maximal LTP response. ratio between NMDA receptor subunit types remains constant. We then measured current–voltage (I–V) curves for pharmacologically E IM-AA Mutation in CaV2.1 Channels Affects NMDA Receptor-Mediated isolated AMPA receptor-mediated currents (Fig. 3 ). AMPA Excitatory Synaptic Transmission in SC-CA1 Synapses. The ability of receptor-mediated inward currents were similar between WT E postsynaptic depolarization to rescue LTP induction suggested and IM-AA synapses (Fig. 3 ). Taken together, these results that a reduced contribution of NMDA receptors to the postsyn- show that the NMDA receptor-mediated postsynaptic response aptic current might be responsible for reduced LTP. Therefore, we is reduced in mutant IM-AA SC-CA1 synapses, whereas inward characterized the postsynaptic currents in hippocampal slices from current conducted by AMPA receptors was unchanged. These WT and IM-AA mice in more detail. We recorded evoked AMPA results are consistent with a selective reduction in the number of functional NMDA receptors in IM-AA synapses. receptor-mediated EPSCs at a holding potential, Vh,of−80 mV. We then added an AMPA receptor blocker (CNQX), recorded To probe the relationship between NMDA receptor currents and impaired LTP induction, we measured postsynaptic currents NMDA receptor-mediated EPSCs at V of +40 mV, and calcu- h before and after application of the NMDA receptor blocker lated the ratio of NMDA receptor-mediated to AMPA receptor- D-AP5 during 100-Hz trains of stimuli. The NMDA receptor mediated EPSCs. We found that IM-AA synapses had significantly current in response to a single stimulus does not contribute lower NMDA receptor/AMPA receptor ratio than WT in response − A B significantly to the total postsynaptic current recorded at 60 mV to a single presynaptic stimulus (Fig. 3 and ). The peak AMPA (Fig. 4A, example traces). However, the NMDA receptor current receptor-mediated amplitude was unchanged, indicating a selective increased during repetitive stimuli (Fig. 4A), and at the end of the reduction in the NMDA receptor-mediated component of the train it was actually larger in hippocampal slices from IM-AA mice postsynaptic response. (Fig. 4B). Thus, greater NMDA receptor current at the end of NMDA receptor-mediated inward current was significantly 100-Hz trains is coupled to less LTP in IM-AA synapses. Evi- smaller in IM-AA vs. WT synapses, although it peaked at the same dently, postsynaptic depolarization is less effective in activating the NEUROSCIENCE molecular pathways that induce LTP in the postsynaptic cell at IM-AA synapses, consistent with the rightward shift of the de- pendence of LTP on postsynaptic charge (Fig. 2C). A WT IM-AA BC* In hippocampal synapses, LTP and long-term depression 50 pA (LTD) balance each other in controlling synaptic strength (16). 2.5 1.0 100 ms Low-frequency stimulation induces LTD, whereas high-frequency 2.0 0.8 stimulation induces LTP via similar signaling pathways. Enhanced +40 mV LTD could potentially reduce the sensitivity for induction of LTP, -80 mV 1.5 0.6 as we have observed in IM-AA synapses. Therefore, we tested 0.4 whether low-frequency stimulation induces LTD similarly in WT 1.0 and IM-AA synapses. We found no significant difference between 0.5 0.2 WT and IM-AA synapses in LTD (Fig. S2A). Therefore, IM-AA 50 pA ratio NR2B:NR2A synapses have a primary defect in LTP, which causes reduced 50 ms ratio NMDAR:AMPAR 0.0 0.0 sensitivity to induction of LTP by postsynaptic depolarization. In neural circuits, the balance of excitation and inhibition is DE** ** 400 also important for information processing. To assess the effects 300 of the IM-AA mutation on the overall balance of excitation vs. * 300 * inhibition, we measured postsynaptic AMPA receptor EPSCs 200 and compared them to GABAA receptor inhibitory postsynaptic WT 200 B–D IM-AA currents (IPSCs) (Fig. S2 ). We found that the ratio of ex- 100 100 citatory to inhibitory neurotransmission in the hippocampal slice was unchanged in IM-AA mice. Therefore, the effects of the -80 -40 40 -80 -40 40 IM-AA mutation are primarily on short-term synaptic plasticity ** -100 -100 and LTP, with no effect on LTD and the overall ratio of exci- ** tation to inhibition. -200 -200 IM-AA Mice Display Impaired Spatial Learning and Memory. Because Fig. 3. IM-AA mutation alters NMDAR-to-AMPAR ratio in SC-CA1 synapses. hippocampal plasticity is important for spatial learning, we ex- (A) Example of AMPAR-mediated EPSCs recorded at −80 mV in control so- amined the performance of WT and IM-AA mice in the context- lution, and, after AMPAR block by 10 μM CNQX, NMDAR-mediated EPSCs dependent fear-conditioning test, a hippocampal-dependent recorded at +40 mV from WT (black) and IM-AA (red). Stimulus artifacts cognitive task in which spatial memory is assessed from the fear- were erased for clarity. (B) NMDAR-to-AMPAR ratio from individual cells related freezing behavior of mice when returned to the spatial (open symbols) and average values (solid symbols) from WT (black, n = 9) = μ ω μ context of a previous fearful event (24). WT and IM-AA mice and IM-AA (red, n 8) in the presence of 1 M -Ctx, 50 M picrotoxin, and explored a cage with an electrified floor grid similarly when first 10 μM CGP55845 hydrochloride. (C) NR2B:NR2A ratio calculated after block by 10 μM ifenprodil from individual cells (open symbols) and average values introduced into it (Fig. 5, control). During the training phase, (solid symbols) for WT (black, n = 9) and IM-AA (red, n = 11) as described in mice were subjected to a mild foot shock. WT and IM-AA mice Materials and Methods.(D) I–V relationship of NMDAR-mediated currents in exhibited comparable fear-related freezing behavior in response the presence of 20 μMCNQX,1μM ω-Ctx, 50 μM picrotoxin, and 10 μM to the shock (Fig. 5, training). The mice were returned to their CGP55845 hydrochloride from WT (black, n = 9) and IM-AA (red, n = 11). (E) I–V home cage. When they were brought back into the spatial con- relationship of AMPAR-mediated currents in the presence of 50 μMAPV,1μM text in which they had experienced the foot shock 10 min or 24 h ω-Ctx, and 50 μM picrotoxin from WT (black, n = 9) and IM-AA (red, n = 8). *P < earlier, IM-AA mice showed dramatically impaired freezing re- 0.05; **P < 0.01. sponses compared with WT (Fig. 5). These results reveal a nearly

Nanou et al. PNAS Early Edition | 3of6 Downloaded by guest on September 27, 2021 A that the IM-AA mutation resulted in greatly impaired spatial learning and memory. Discussion Our previous studies of IM-AA mice showed that regulation of presynaptic CaV2.1 channels by CaS proteins contributes sub- stantially to short-term synaptic plasticity in hippocampal syn- apses (21). In those studies, we found that the IM-AA mutation does not alter the frequency or amplitude of miniature EPSCs caused by presynaptic release of single quanta of glutamate or the amplitudes of EPSCs recorded in response to increasing presynaptic stimulus intensities. We also found that the slow + Ca2 chelator EGTA had similar effects on evoked EPSCs in WT and IM-AA synapses. Together, these results indicated that the function of the presynaptic release machinery and its spatial rela- tionship to presynaptic CaV2.1 channels and postsynaptic glutamate receptors were unaltered in IM-AA synapses. Here we tested B whether this form of regulation of CaV2.1 channels also has a role in LTP and in spatial learning and memory. Our findings show that the IM-AA mutation impairs long-term synaptic plasticity as well as hippocampus-dependent spatial learning and memory in mice. These results forge a link between regulation of CaV2.1 channels, LTP, and spatial learning and memory.

Weakened LTP. A key synaptic outcome of bursts of presynaptic action potentials is NMDA receptor-dependent LTP of the postsynaptic response at excitatory glutamatergic synapses in the hippocampus. Extensive studies have shown that LTP arises 2+ Fig. 4. IM-AA mutation induces increases in NMDAR currents in response to postsynaptically via a pathway involving Ca entry via NMDA 2+ 100-Hz tetanus in SC-CA1 synapses. (A) Example EPSCs recorded at −60 mV in receptors, activation of Ca /calmodulin-dependent protein ki- response to a single stimulus in control solution from WT (Top, black) and nase II, phosphorylation of AMPA receptors, and increased IM-AA (Bottom, red) and after NMDAR block by 100 μM D-AP5 WT (Top, trafficking of AMPA receptors into the postsynaptic membrane gray) and IM-AA (Bottom, pink) (Left). Stimulus artifacts were erased for (16, 18, 19). Additional studies suggest involvement of presynaptic clarity. Average EPSCs were recorded at −60 mV in response to 100-Hz mechanisms as well (20). Surprisingly, we found that LTP was tetanus in control solution from WT (Top,black)andIM-AA(Bottom, weakened in IM-AA synapses and required stronger postsynaptic red) and after NMDAR block by 100 μM D-AP5 WT (Top,gray)andIM-AA depolarization to achieve the same level of LTP as WT synapses. (Bottom,pink)(Left). Example EPSCs were recorded at −60 mV in control Although the initial activation of NMDA receptors was reduced, solution from WT (Top, black) and IM-AA (Bottom, red) and after NMDAR the integral of NMDA receptor postsynaptic current during a μ block by 100 M D-AP5 WT (Top, gray) and IM-AA (Bottom, pink) (Right). train of stimuli was greater than WT. Our working hypothesis is (B) Average peak amplitude of evoked NMDAR-mediated EPSCs during a that the postsynaptic molecular pathways leading to stable in- 100-Hz train from WT (black, n = 4) and IM-AA (red, n = 4) in the presence of creases in synaptic strength downstream of the NMDA receptor 1 μM ω-Ctx, 50 μM picrotoxin, and 10 μM CGP55845 hydrochloride. + are altered in IM-AA synapses. It seems unlikely that Ca2 entry via CaV2.1 channels is directly involved in inducing LTP in our complete lack of spatial learning and memory for IM-AA mice in experiments because QX-314 present in our recording pipettes this behavioral test. to block postsynaptic action potential generation via direct block In context-dependent fear conditioning, mice must recognize of voltage-gated sodium channels also blocks CaV2.1 channels. fear as well as encode and remember the spatial context of the Further work will be required to define the postsynaptic mech- fearful event. Because WT and IM-AA mice exhibited similar anisms involved in these changes in the strength of LTP. freezing behavior in the training phase of the experiments, it is In addition to potential postsynaptic mechanisms acting di- likely that the IM-AA mice can recognize fear but fail to encode rectly on coupling of NMDA receptor activation to LTP, it is or remember the spatial context of that fear. To further test their possible that the alteration of presynaptic plasticity caused by the spatial learning and memory, we examined WT and IM-AA mice in the Barnes circular maze test, which does not involve fear (25). This test measures whether mice can learn and remember the 70 WT location of a single escape hole with a dark refuge among 20 IM-AA *** *** 60 *** ** identical holes at the perimeter of a brightly lighted circular field. *** WT mice learned to locate the escape hole by the third training 50 ** trial as indicated by the reduced distance they traveled to the 40 vicinity of the hole (Fig. 6A). The amount of time required be- fore entering the escape hole also decreased with each trial 30

A % Freeze (Fig. 6 ). The performance of IM-AA mice did improve during 20 the training period, indicating that they were learning, but the decreases in distance traveled to the escape hole and in latency 10 before entering the escape hole were markedly greater for WT 0 than for IM-AA at each trial (Fig. 6 A and B). On the day fol- Control Training 10 min 24 hrs lowing the last training trials, a probe test was conducted in Fig. 5. IM-AA mice have impaired spatial learning and memory in context- which the refuge was removed and the latency before moving to dependent fear conditioning. Mean freezing time (percent of total) in the the position of the former refuge for the first time was measured. conditioning chamber for WT (black, n = 10) and IM-AA mice (red, n = 10) In this probe test, the average latency was far higher for IMAA before (control), immediately after (training), and at 10 min and 24 h after mice than for WT (Fig. 6C). Taken together, these data suggest delivery of foot shock. **P < 0.01; ***P < 0.001.

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1616206113 Nanou et al. Downloaded by guest on September 27, 2021 ABChippocampus-dependent spatial learning and memory. These results *** open unexplored avenues of inquiry into the relationship be- Day Day 1 2 3 4 1 2 3 4 tween synaptic plasticity at the molecular and cellular levels 1000 200 30 * ** and spatial learning and memory at the levels of neural circuits 800 * * * 150 * and behavior. ** 20 600 * * * * * ** * 100 * * Possible Relationship Among Short-Term Synaptic Plasticity, LTP, and 400 ** ** * * 10 Spatial Learning. Our results do not yet reveal the mechanistic 200 50 connections among short-term synaptic plasticity, LTP, and

0 Latency to goal box (s) 0 spatial learning and memory. Exploratory behavior in rodents

2 4 6 8 10 12 Latency to enter goal box (s) 2 4 6 8 10 12 generates θ-rhythm in the hippocampus (33). Points of demarcation

Distance to enter goal box (cm) Training trials Training trials or reward in the navigation pathway are denoted by formation of Fig. 6. IM-AA mice have impaired spatial learning and memory in the sharp-wave ripples superimposed on θ-rhythm, which are replayed Barnes circular maze. (A) Average distance traveled to locate and enter the to consolidate spatial memory retention (32–34). Formation of goal box in the Barnes maze test plotted against number of training trials sharp-wave ripples depends on coordinated firing of CA3 neurons, for WT (black, n = 6) and IM-AA (red, n = 8) mice. (B) Average time spent to orchestrated by parvalbumin-expressing fast-spiking interneurons enter the goal box in the Barnes maze test plotted against number of training (35). Place cells fire when an animal reaches a specific position in trials for WT (black, n = 6) and IM-AA (red, n = 8) mice. (C)Timerequiredto space, and firing of place cells in turn can induce LTP in vitro first reach the former location of the goal box (goal box removed) 24 h after (32, 36, 37). How might the changes in short-term and long-term the last training session in the Barnes maze test from individual mice (open synaptic plasticity that we observe in IM-AA mice alter sharp- symbols) and average values (solid symbols) for WT (black, n = 6) and IM-AA wave ripples, place cell formation and stability, and ultimately (red, n = 8) mice. *P < 0.05; **P < 0.01; ***P < 0.001. spatial learning and memory? One possibility is that short-term synaptic plasticity on the millisecond time scale may contribute to formation of sharp-wave ripples, which are generated on a similar impaired regulation of CaV2.1 channels by CaS proteins that we time scale. On the other hand, LTP on the time scale of hundreds have observed could reduce the strength of LTP by homeostatic of seconds and longer may contribute to place cell formation mechanisms. In IM-AA synapses, the initial facilitation of EPSCs and stability. Thus, the deficits in short-term synaptic plasticity in response to a train of action potentials is reduced in ampli- and LTP caused by loss of regulation of CaV2.1 channels in IM- tude, but facilitation eventually reaches a comparable level to

AA mice may both contribute significantly to the impairment of NEUROSCIENCE WT and is sustained longer because the rapid phase of synaptic spatial learning and memory. Future experiments may reveal depression is slowed (21). Overall, the integral of EPSCs is ac- the functional relationships among impairments of short-term tually increased during a train of stimuli (21). This persistent neuroplasticity, LTP, and spatial learning and memory in IM- increase in EPSC amplitude during trains of stimuli in vivo may AA mice through correlation of synaptic deficits with changes in engage homeostatic regulatory mechanisms that weaken LTP, as neural circuit functions assessed in recordings of sharp-wave ripples we have reported here. Such homeostatic regulation might in- and place cells. volve inhibition of NMDA receptors in the postsynaptic membrane or internalization of NMDA receptors in intracellular vesicles under Materials and Methods

basal conditions. It will be interesting to determine the time course Animals. IM-AA mice with a point mutation in the IQ-like motif of CaV2.1 of weakening of LTP during neural development to determine (IM>>AA), (ATCATG to GCCGCT) were generated by Ingenious Targeting whether homeostatic compensatory mechanisms are involved and at Laboratory. The mutation (within exon 40) was generated by PCR mutagen- what age they are engaged. It will also be important to probe the esis and confirmed by sequencing. Traditional blastocyst injection of ES cells downstream signaling mechanisms that connect NMDA receptor expressing the targeting vector resulted in chimeric mice. These chimeric mice activation to LTP to find the step(s) in that process that is subject to were mated first to generate heterozygotes, which were then backcrossed for homeostatic regulation by short-term synaptic plasticity. 10 generations with C57BL/6J to generate homozygous IM-AA mutant mice in Importantly, weakened LTP was observed during stimulations a pure genetic background. All experiments were performed according to the at θ-frequency, which is thought to represent a natural pattern of guidelines for the care and use of animals approved by the Animal Care and stimulation during spatial exploration in vivo (22, 23, 26). Increased Use Committee at the University of Washington. firing of action potentials by CA3 neurons is thought to induce – LTP in CA1 neurons, which contributes to formation and stability of Electrophysiology in Hippocampal Slices. WT and IM-AA mice 16 21 d old were – anesthetized with . Brains were rapidly removed and placed in ice-cold, place cells whose firing denotes location during movement (27 31). high sucrose cutting solution containing (in mM): 75 sucrose, 25 NaHCO ,25 Because LTP would contribute to increased excitability of hippo- 3 – glucose, 2.5 KCl, 1.25 NaH2PO4,87NaCl,7MgCl2,and0.5CaCl2.Acute campal place cells during spatial learning (27 31), these deficits in transverse hippocampal slices (400 μm) were cut on a 1000 Plus Vibratome in the LTP we have observed would be expected to cause deficits in spatial high sucrose cutting solution, and transferred immediately to an incubation learning and memory in IM-AA mice. chamber containing artificial cerebrospinal fluid (ACSF) (in mM): 125 NaCl, 3 KCl,

2CaCl2,2MgCl2, 125 NaH2PO4,26NaHCO3, and 10 glucose, saturated with 95% Impaired Spatial Learning and Memory. In IM-AA mice, impaired (vol/vol) O2 and 5% (vol/vol) CO2. The slices were allowed to recover at 37 °C for short-term facilitation and weakened hippocampal LTP were ac- 45 min and then maintained at room temperature for at least 30 min before companied by deficits in two well-established tests of hippocampal- recording. dependent spatial learning and memory. IM-AA mice failed to learn Slices were transferred to a submerged recording chamber mounted on a the spatial context of a fear-provoking foot shock or remember the Nikon microscope (E600FN) equipped for infrared differential interference location of a dark refuge in the Barnes circular maze. Together, contrast microscopy and were perfused with ACSF at a rate of 1.5 mL/min these results document a major loss of spatial learning and memory at room temperature. All experiments unless specified otherwise were performed in the presence of the GABAA blocker picrotoxin (50 μM), GABAB in IM-AA mice. As both synaptic facilitation and LTP are thought to μ ω be required for development of place cells that encode spatial blocker CGP55845 hydrochloride (10 M), and CaV2.2 blocker -conotoxin (ω-Ctx) GVIA. In addition, a cut was made between CA1 and CA3 to prevent learning and memory (32), it is not surprising that these changes in the propagation of epileptiform activity. Evoked postsynaptic responses synaptic plasticity are accompanied by failure of spatial learning and were recorded from CA1 pyramidal cells, which were visualized by infrared memory. In all, our data show that activity-dependent regulation of differential interference contrast. EPSCs were induced by stimulating presynaptic CaV2.1 channels by CaS proteins contributes significantly Schaffer collaterals in stratum radiatum by a concentric bipolar stimulat- to short-term synaptic plasticity in presynaptic terminals in vivo and ing electrode (FHC). Whole-cell recording pipettes (4–6MΩ) were filled

reveal unexpectedly that this key regulatory process is also required with a solution containing (in mM): 145 Cs-gluconate, 2 MgCl2, 10 Hepes, for normal LTP in postsynaptic hippocampal neurons and for 0.5 EGTA, 2 Tris-ATP, 0.2 Na2GTP, and 5 QX-314. Data were collected with a

Nanou et al. PNAS Early Edition | 5of6 Downloaded by guest on September 27, 2021 MultiClamp 700A amplifier (Axon Instruments), filtered at 2 kHz and digi- procedure. The scoring of freezing behavior was quantified using a com- tized at 10 kHz. Multiple step depolarizations were given at the beginning puterized beam-grid system (TruScan, Coulbourn Instruments). of every experiment to induce block of Na2+ and Ca2+ currents in the CA1 pyramidal cells by QX-314 (21). Barnes Circular Maze. The Barnes circular maze test was conducted on a white LTP was induced in both WT and IM-AA in SC-CA1 synapses by eight trains circular surface, 92 cm in diameter, with 20 holes equally spaced around the of θ-burst stimulation (10 bursts delivered at 5 Hz, each burst consisting of 10 perimeter. A black Plexiglas escape box (15 × 7 × 7 cm) containing paper cage pulses at 100 Hz), while CA1 neurons were depolarized to −40 mV. LTP was bedding on its floor was located under one of the holes. The location of the also induced by two or three trains of high-frequency stimulation (100 Hz, target was consistent for a given mouse and two 500-lux (1 lux = 1 lumen per 1 s) separated by 20 s, while cells were held at −60 mV or depolarized to sq. m.) lights were turned on during trials. The platform and the escape box 0 mV. The induction protocols were applied within 5 min of achieving whole-cell were cleaned thoroughly with 70% ethanol and paper towels between each “ ” configuration, to prevent wash-out of LTP. The magnitude of LTP was trial to remove olfactory cues. Training lasted for 4 successive days and three calculated based on the EPSC values 30 min after the end of the induction trials 15 min apart were performed each day. At the beginning of each trial, protocol. Data were analyzed using Clampfit (Axon) and Igor Pro software a test mouse was placed in the center of the maze, held there for 10 s, and (Wavemetrics). Postsynaptic charge during the 100-Hz stimulation was cal- then given 3 min to locate the target hole. If the mouse failed to find the culated by integrating the area under the EPSCs. LTD was induced by low target hole it was gently guided to the escape box. When the mouse entered ± frequency (1 Hz, 15 min). All data are presented as means SEMs. Statistical the escape box the light was turned off and the mouse remained undis- ’ significance was calculated using a Student s t test. turbed for 1 min in the escape box. On the fifth day, a probe trial was conducted after the last training session without the escape box to confirm Context-Dependent Fear Conditioning. Two-month-old male mice were placed that this spatial task was performed based on navigation cues. Time latency in a conditioning chamber (Coulbourn Instruments) and allowed to explore. to reach the target hole and the distance traveled to reach the target hole After 2 min, a footshock of 0.7 mA was delivered for 2 s. Animals were then were recorded and analyzed by Noldus analysis software. allowed to recover for 1 min and then returned to their home cages. At increasing time intervals after training, animals were returned to the con- ACKNOWLEDGMENTS. We thank Dr. Jane Sullivan (Department of Physiol- ditioning chamber (10 min, 24 h). The formation of memory for fear con- ogy and Biophysics, University of Washington) for valuable discussions and ditioning was measured by testing freezing behavior upon return to the comments on the manuscript. This work was supported by NIH Grant R01 conditioning chamber. Freezing behavior was defined as cessation of all but NS022625 (to W.A.C.) and by a postdoctoral fellowship from the Swedish respiratory movement and was assessed by means of a time-sampling Society for Medical Research (to E.N.).

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