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Behavioural Brain Research

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ole of inhibitory autophosphorylation ofalcium/calmodulin-dependent kinase II (�CAMKII) in persistent>24 h) hippocampal LTP and in LTD facilitated by novel object-placeearning and recognition in mice

inzhong Jeremy Goh, Denise Manahan-Vaughan ∗

epartment of Neurophysiology, Medical Faculty, Ruhr University Bochum, Universitaetsstr. 150, MA 4/150, 44780 Bochum, Germany

i g h l i g h t s

First study to address role of CAMKII in plasticity in freely behaving rodents.LTP induction threshold is increased and the LTD threshold is reduced.Object-place learning and object-place recognition learning is intact.LTD that is expressed as a consequence of object-place learning is enhanced.Object-place learning is encoded by LTD.

r t i c l e i n f o

rticle history:eceived 22 November 2013eceived in revised form 7 January 2014ccepted 17 January 2014vailable online xxx

eywords:ouseurine

ynaptic plasticityTDTPn vivo

a b s t r a c t

Experience-dependent synaptic plasticity is widely expressed in the mammalian brain and is believed tounderlie memory formation. Persistent forms of synaptic plasticity in the hippocampus, such as long-termpotentiation (LTP) and long-term depression (LTD) are particularly of interest, as evidence is accumulatingthat they are expressed as a consequence of, or at the very least in association with, hippocampus-dependent novel learning events. Learning-facilitated plasticity describes the property of hippocampalsynapses to express persistent synaptic plasticity when novel spatial learning is combined with affer-ent stimulation that is subthreshold for induction of changes in synaptic strength. In mice it occursfollowing novel object recognition and novel object-place recognition. Calmodulin-dependent kinase II(CAMKII) is strongly expressed in synapses and has been shown to be required for hippocampal LTPin vitro and for spatial learning in the water maze. Here, we show that in mice that undergo persistentinhibitory autophosphorylation of �CAMKII, object-place learning is intact. Furthermore, these animalsdemonstrate a higher threshold for induction of persistent (>24 h) hippocampal LTP in the hippocampalCA1 region during unrestrained behaviour. The transgenic mice also express short-term depression inresponse to afferent stimulation frequencies that are ineffective in controls. Furthermore, they express
stronger LTD in response to novel learning of spatial configurations compared to controls. These findingssupport that modulation of �CAMKII activity via autophosphorylation at the Thr305/306 site comprisesa key mechanism for the maintenance of synaptic plasticity within a dynamic range. They also indicatethat a functional differentiation occurs in the way spatial information is encoded: whereas LTP is likelyto be critically involved in the encoding of space per se, LTD appears to play a special role in the encodingof the content or features of space.
. Introduction

Please cite this article in press as: Goh JJ, Manahan-Vaughan D. Role of inkinase II (�CAMKII) in persistent (>24 h) hippocampal LTP and in LTD fBehav Brain Res (2014), http://dx.doi.org/10.1016/j.bbr.2014.01.022

The efficiency of signal transmission between two neurons isighly modifiable. This variability in transmission across a synapse

∗ Corresponding author. Tel.: +49 234 32 22042; fax: +49 234 32 14192.E-mail address: [email protected] (D. Manahan-Vaughan).

166-4328/$ – see front matter © 2014 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.bbr.2014.01.022

© 2014 Elsevier B.V. All rights reserved.

is termed synaptic plasticity. Depending on the parameters ofthe patterned electrical stimulation administered to the affer-ent fibers, synapses are capable of exhibiting changes in theirdegree of transmission that persists for periods of up to weeks

hibitory autophosphorylation of calcium/calmodulin-dependentacilitated by novel object-place learning and recognition in mice.

and months [1,2,76,77]. Such plastic changes at the synapses areknown to be bidirectional, meaning that the signal transmissioncan either be enhanced or weakened. A persistent enhancement ofsynaptic efficacy is referred to as long-term potentiation (LTP),

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dx.doi.org/10.1016/j.bbr.2014.01.022

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hereas long-term depression (LTD) describes an experience-ependent persistent decrease in synaptic efficacy. In freelyehaving mice and in hippocampal slices, high-frequency stimu-

ation (HFS), results in robust persistent synaptic potentiation atchaffer collateral (Sc)–CA1 synapses [3–6]. On the other hand, low-requency stimulation (LFS) results in LTD in isolated hippocampallices in vitro [7–9]. In freely behaving mice in vivo, LFS only induceshort-term depression (STD) of naïve CA1 synapses [3,10], whereasn rats it results in long-lasting LTD [76]. In contrast, robust and veryong-lasting LTD is enabled in freely behaving mice if LFS is coupled

ith a novel spatial learning event [11,12].Bidirectional synaptic plasticity is believed to comprise a cellu-

ar prerequisite for learning and memory formation [13–15]. In lineith this, it has been shown that specific aspects of spatial learning

acilitate hippocampal LTP [16,17], whereas other aspects facili-ate hippocampal LTD [17,18,77]. This phenomenon is referred tos learning-facilitated synaptic plasticity [18]. For instance, in freelyehaving rats, the learning of subtle contextual spatial featuresacilitates LTD at the hippocampal commissural associational-CA3nd Sc–CA1 synapses, whereas spatial learning of overt landmarkeatures of an environment facilitates LTD in the perforant pathway

dentate gyrus and mossy fiber – CA3 synapses [17–20]. Strikingly,TP is facilitated by novel empty space or generalised changes inovel space, in an input-specific manner, across all synapses thatarticipate in hippocampal circuitry i.e. a functional differentiation,uch as that seen for LTD has not yet been identified [17–20]. Thesendings suggest that a very tight relationship exists between thencoding of novel space and hippocampal LTP. LTD, on the otherand, may be associated with learning about the precise features,r content, of a spatial environment [18]. This phenomenon is notestricted to rats: more recently, it was demonstrated in freelyehaving mice that LTD is facilitated in the CA1 through the acqui-ition of novel object-place information [11,12]. This phenomenons very robust and even occurs in the absence of patterned afferenttimulation in the form of LFS [10]. In addition to its putative rolen enabling learning of spatial content [18], hippocampal LTD haseen implicated in reversal learning [21], in social memory [22] and

n cognitive flexibility [23], whereas a role for hippocampal LTP inear memory has also been described [24,25].

Ca2+–calmodulin-dependent kinase II, or CAMKII, is a proteinith a distinct serine/threonine kinase enzymatic function that

s highly expressed in the brain, where it comprises a majoronstituent of the postsynaptic density [26]. CAMKII is of partic-lar importance, as it is known to be critical for the expressionf synaptic plasticity, especially in persistent synaptic potentiat-on, where it phosphorylates plasticity-related proteins, such ashe AMPA and ryanodine receptors, and modulates their chan-el conductance properties [27,28]. The activity of CAMKII itself

s modulated by the level of intracellular Ca2+. Increases in Ca2+

ontent result in the activation of the enzyme and, when thea2+ content is significantly elevated, this enables the enzyme tondergo autophosphorylation at its threonine-286 residue, whichesults in persistent activation of CAMKII even after Ca2+ lev-ls begin to decline [26]. CAMKII undergoes a second phase ofutophosphorylation at its threonine-305/306 sites, which serveso inactivate the enzyme (i.e. inhibitory autophosphorylation) afterhe dissociation of the Ca2+–calmodulin complex from the acti-ated enzyme [29–31,73]. Transgenic mice that are incapable ofndergoing inhibitory autophosphorylation express enhanced LTP,hilst those that undergo constitutive inhibitory autophospho-

ylation fail to express LTP in response to HFS in vitro [32]. Inddition, in transgenic mice that undergo constitutive inhibitory


utophosphorylation, the frequency-dependency of LTD changesn vitro [32]. In line with this, inhibitory autophosphorylationf CAMKII is needed for hippocampal metaplasticity [78], sug-esting that under normal conditions CaMKII is required for the

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maintenance of synaptic plasticity within dynamic and functionalranges. All of the abovementioned studies have been conducted inthe isolated hippocampus, however. As yet, an examination of therole of CAMKII, and in particular of inhibitory autophosphorylationof CAMKII in the intact hippocampus of freely behaving animals hasnot been conducted.

In the present study, we examined the importance of inhibitoryautophosphorylation of �CAMKII for synaptic plasticity in freelybehaving mice, and for learning-facilitated synaptic plasticity.The transgenic strain used, CAMKII-T305D, possesses an aspar-tate rather than threonine at residue 305, which mimics persistentinhibitory phosphorylation of the enzyme [32]. We observedthat learning-facilitated LTD is enhanced, and the frequency-dependency of both LTP and synaptic depression is altered inthese mice, such that in comparison to controls higher frequenciesare needed to induce LTP and with lower frequencies than usu-ally, synaptic depression is expressed. These findings suggest thatinhibitory autophosphorylation of CAMKII is required to maintainsynaptic plasticity within a workable dynamic range that enablesboth functional LTD and LTP. This, in turn, is likely to comprise anessential feature of effective synaptic information storage in asso-ciation with spatial learning.

2. Methods

2.1. Animals

The experiments in this study were carried out in accordancewith the European Communities Council Directive of September22nd, 2010 (2010/63/EU) for care of laboratory animals and afterapproval of the local ethics committee (Bezirksamt Arnsberg). Allefforts were made to minimize animal suffering and to reduce thenumber of animals. Male C57/BL6N mice (7–8 weeks at the timeof surgery; acquired from Charles River, Germany) and homozy-gote CAMKII-T305D transgenic mice and their wild-type (WT)littermates (7–8 weeks at time of surgery; kindly supplied by Dr.Ype Elgersma, Erasmus University Rotterdam, Netherlands) wereused in the experiments. In the CAMKII-T305D mice, Thr305 issubstituted by the negatively charged Asp residue which blocksCa2+–CAM binding, thus mimicking persistent inhibitory autophos-phorylation [32]. All mice attained the minimum weight of 22 gbefore being subjected to surgical electrode implantation. Themice were housed in individual cages in a temperature- (22 ± 2 ◦C)and humidity- (55 ± 5%) controlled vivarium (Scantainer VentilatedCabinets, Scanbur A/S, Denmark) with a constant 12-h light–darkcycle (lights on from 8 a.m. to 8 p.m.) where they had access to foodand water ad libitum. Prior to surgery, the animals were housed ingroups of maximally 6 animals in a single cage.

2.2. Surgery

All surgical procedures and experiments were conducted dur-ing the day. The mice were anaesthetized (sodium pentobarbital60 mg/kg, i.p.) and underwent stereotaxic chronic implantationof bipolar stimulating electrodes in the right Schaffer collat-eral pathway of the dorsal hippocampus (anterioposterior (AP):−2.0 mm; mediolateral (ML): 2.0 mm from bregma; dorsoventral(DV): ∼1.4 mm from brain surface) and monopolar recording elec-trodes in the right ipsilateral CA1 stratum radiatum (AP: −1.9; ML:1.4; DV: ∼1.2) to monitor the evoked potentials at the SchafferCollateral–CA1 synapses. The stimulating and recording electrodes


were made of polyurethane-coated stainless steel wire (100 �mdiameter; Gündel, BioMedical Intruments, Germany) and werelowered into the brain through a hole drilled on the skull. On thecontralateral side, 2 holes were drilled on the skull into which

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nchor screws were inserted. The anchor screws were attachedo stainless steel wires (A-M Systems, U.S.A.) that served as ref-rence and ground electrodes. The 5 wires were secured on a 6-pinocket (Conrad Electronic SE, Germany) and the whole assemblyas stabilized on the skull using dental cement. Test-pulse recor-ings during surgery aided depth-adjustment of the electrodes,hich was later verified by postmortem histology. After surgery,ice were housed individually and given at least 10 days recovery

ime before experiments began. Electrophysiological recordingsnd behavioral paradigms were performed in 20 (L) × 20 (W) × 30H) cm lidless recording chambers, wherein the mice could movereely and had access to food and water ad libitum. Animals wereransferred in their cages into the experiment room 1 d beforehe start of experiments to ensure adequate acclimatization to thexperimental environment.

.3. Measurement of evoked potentials

The field excitatory postsynaptic potential (fEPSP) wasmployed as a measure of excitatory synaptic transmission in theA1 region. To obtain these measurements, an evoked responseas generated in the stratum radiatum by stimulating the Schaf-

er collaterals at low frequency (0.025 Hz) with single biphasicquare waves of 0.2 ms duration per half-wave, generated by a con-tant current isolation unit (World Precision Instruments, U.S.A.).he fEPSP signal was amplified using a differential AC amplifierA-M Systems, U.S.A.) and digitalized through a data acquisitionnit (Cambridge Electronic Design, U.K.). Recordings were madeith a stimulus strength which evoked a fEPSP which was 40%

f the maximum observed during acquisition of an input–outputi/o) relationship (100–900 �A). An i/o curve was determinedefore commencing every separate experiment. For each time-oint measured during the experiments, 5 consecutively-evokedEPSP responses, at 40 s intervals, were averaged. The first 6 time-oints, that were recorded at 5 min intervals, were averaged and allime-points were expressed as a mean percentage (±standard errorf the mean) of this value. Plasticity-inducing electrical stimula-ion, together with the relevant behavioral task (when appropriate)as applied immediately after the sixth time-point and synaptic

ransmission was recorded for another 4 h (240 min). A further 1 hf recordings was performed the next day, roughly 24 h after thexperiment began, to determine the degree of persistency of anyhanges in synaptic transmission. fEPSP responses were quantifiedy measuring the slope obtained on the first negative deflection ofhe evoked potential. Electroencephalography (EEG) activity was

onitored throughout the course of the experiment to control forhe occurrence of seizure activity. No behavioral signs or EEG activ-ty indicating seizures were observed.

.4. Electrical induction of synaptic plasticity

All animals were first tested in a “baseline” experiment whereest-pulse stimulation was given in the absence of any exter-al behavioral stimuli to ensure that the recordings were stable.est-pulse stimulation comprised afferent stimulation applied at.025 Hz, comprising 2 trains of 5 stimuli and a 5 min inter-train

nterval at 40% stimulation intensity (as determined by the i/ourve). High-frequency stimulation (HFS) to induce LTP was givenither at 100 Hz or 200 Hz (2 trains of 50 pulses, 5 min inter-trainnterval, 40% intensity). Low-frequency stimulation (LFS) to inducehort-term depression (STD) was given at either 2 Hz or 3 Hz (1 trainf 200 pulses, 70% intensity). The terms “electrically-induced” plas-


icity and “learning-facilitated” plasticity are used to distinguishetween synaptic plasticity that is induced solely through electricaltimulation and plasticity that is strengthened by the concurrencyf the patterned electrical stimulation (LFS) and the behavioral task.

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In a control experiment we verified that C57/BL6 and the WT lit-termates of the transgenic animals exhibited equivalent LTP andSTD. The data from these animals were therefore pooled into onecontrol group.

2.5. Object-place learning task

In the object-place learning task the novelty of the object posi-tion in space was examined (Fig. 5a). The recording chambers (3walls and the base) were completely grey in color except for thefront wall, which was white. This served to polarize the envi-ronment and provide a gross contextual feature to the recordingchambers. The experiments were performed 4 days in a row withthe same animals. A baseline experiment without the behaviouraltask was run on day 1, which also served to habituate the mice.On day 2 (novel exploration), two objects (e.g. A and B) were pre-sented against the grey back wall (novel object-place learning). Themice were then re-exposed to the objects in the same locations onthe day 3 (re-exposure). On day 4, the objects were shifted in aparallel fashion to the white front wall (novel configuration) to testfor object-place recognition memory. The objects were always pre-sented for 10 min simultaneously with the test-pulse stimulation,and were removed from the recording chamber after the presenta-tion. The objects as well as their relative positions were randomlyassigned for each animal. The objects and the recording chamberswere cleaned thoroughly between task trials to ensure the absenceof olfactory cues. The objects were cleaned with ethanol after everytrial and also before the first presentation. The objects were dis-tinctly different from one another and heavy, so that they couldnot be moved by the mice (e.g. transparent glass bottle filled withmono-colored sand). Several copies of each object were available.

2.6. Data analysis

To analyze the electrophysiological data between groups, ananalysis of variance (ANOVA) with repeated measures was appliedusing Statistica (StatSoft Inc, USA). Post-hoc Fisher LSD test wasimplemented when required. The fEPSPs from the period after elec-trical stimulation with or without the behavioral paradigm to theend of the experiment was compared between groups in order toassess the statistical difference between treatments.

In the behavioral experiment Student’s t-tests were used toassess the following:

• Comparison of the exploration behaviour (of control or transgenicmice) of the novel versus the familiar object-place exposure.

• Comparison of the exploration behaviour (of control or transgenicmice) of the novel versus the reconfigured object-place exposure.

• Comparison of the exploration behaviour of the transgenicsversus the control mice during novel exposure to the object-placeconfiguration, during re-exposure to the object-place configura-tion, or during exposure to the object-place re-configuration.

The significance level was set at p < 0.05.Behavioural data for the experiments were recorded from

cameras positioned above the chambers, and digitally stored.Exploration of the objects was then analyzed post-hoc using thewithin-object area scoring system, which was defined as sniffingof the object (with nose contact or head directed to the object)within ∼2 cm radius of the object [10,33]. Standing, sitting or lean-


ing on the object was not scored as object exploration. The datawere expressed as a percentage of the total exploration time dur-ing novel (first-time) exploration of the objects [10,34]. The resultsacross animals were expressed in terms of mean ± s.e.m. The data

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Fig. 1. (a) Application of 100 Hz HFS to Schaffer collateral–CA1 stratum radiatumsynapses of freely moving control mice (WT) results in long-term potentiation (LTP,grey triangles) that lasts for over 25 h. The same HFS protocol, when applied totransgenic animals that cannot undergo inhibitory autophosphorylation of CAMKII(CAMKII-T305D), fails to induce LTP (open circles). The evoked potentials do notdiffer from transgenic animals that received test-pulses only (black circles). (b)Analog traces represent field potentials pre-HFS (left), 5 min (middle) and 24 h(1440 min) after HFS (right) for control mice (WT, grey triangles) or CAMKII trans-genics (CAMKII-T305D, open circles) that received HFS, or for equivalent time-pointsic

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Fig. 2. (a) Application of 200 Hz HFS to Schaffer collateral–CA1 stratum radiatumsynapses of freely moving control mice (WT) fails to induce LTP (black circles).The same HFS protocol, when applied to transgenic animals that cannot undergoinhibitory autophosphorylation of CAMKII (CAMKII-T305D), results in robust LTPthat lasts for over 25 h (grey triangles). (b) Analog traces represent field potentials

we confirmed this effect in control mice (n = 8): LFS at 2 Hz (200

n transgenic animals that received test-pulses only (black circles). Vertical scale barorresponds to 2 mV and horizontal scale bar corresponds to 5 ms.

ere then statistically assessed using the Student’s t-test with theignificance level set at p < 0.05.

. Results

.1. In CAMKII transgenic mice 100 Hz stimulation does not elicitTP

Baseline, test-pulse only, recordings of evoked potentials inreely behaving CAMKII-T305D mice were stable for at least 25 huring the entire recording period (Fig. 1a, b). In a previoustudy, we showed that 100 Hz high-frequency stimulation (HFS)2 trains of 50 pulses, 5 min ITI) applied to the Schaffer col-aterals results in robust LTP in freely behaving mice [3]. Here,

e confirmed that in control mice (n = 10), 100 Hz stimulationnduced robust LTP that persisted for at least 25 h (Fig. 1a). Inontrast, freely behaving CAMKII-T305D transgenic mice (n = 7)ailed to exhibit LTP following 100 Hz HFS (Fig. 1a). The profilef synaptic transmission following 100 Hz HFS in CAMKII-T305Dice was not significantly different from recordings of basal

ynaptic transmission (evoked test-pulses only) when no HFSas applied (ANOVA, F1,12 = 0.23435, p = 0.63703), but significantlyifferent from the LTP that was induced in control mice follow-

ng 100 Hz HFS (ANOVA, F1,13 = 25.089, p < 0.001) (Fig. 1a, b). Themall initial potentiation observed in the CAMKII-T305D micefter HFS was not significant compared to potentials evoked byest-pulses alone (t = 0–30 min: ANOVA, F1,12 = 1.1863, p = 0.29746;

= 0–60 min: ANOVA, F1,12 = 1.4173, p = 0.25687) (Fig. 1a). Stable


asal synaptic transmission (in response to test-pulses only) waslso exhibited in control animals throughout the recording periodsee Fig. 5).

pre-HFS (left), 5 min (middle) and 24 h after HFS (right) for control mice (black cir-cles) or CAMKII transgenics (grey triangles). Vertical scale bar corresponds to 2 mVand horizontal scale bar corresponds to 5 ms.

3.2. High-frequency stimulation at 200 Hz elicits persistent LTP inCAMKII-T305D but not in control mice

When freely behaving control mice (n = 8) were stimulated with200 Hz HFS (2 trains of 50 pulses, 5 min ITI), a modest initial synapticpotentiation occurred that was not persistent and rapidly returnedto basal values after approximately 90 min (Fig. 2a). In contrast,200 Hz HFS resulted in robust LTP in the CA1 of freely behav-ing CAMKII-T305D mice (n = 6) (Fig. 2a, b). The initial potentiationevoked was 138.79 ± 17.27% and the synaptic potentiation per-sisted for at least 25 h. The synaptic potentiation that was inducedvia 200 Hz HFS was significantly larger in the CAMKII-T305D micein comparison to control mice (ANOVA, F1,6 = 10.181, p < 0.05) andin comparison to potentials evoked in CAMKII-T305D animals bytest-pulses alone (ANOVA, F1,8 = 12.483, p < 0.01). This suggests thatin the absence of inhibitory autophosphorylation of CAMKII it isharder to induce LTP by HFS and that the frequency-dependency ofLTP becomes shifted to the right.

3.3. In CAMKII-T305D mice the range of frequencies at whichshort-term depression is induced becomes expanded

We previously reported that 3 Hz LFS low frequency stimulation(LFS) (200 pulses) induces strong STD in the CA1 of freely behavingmice [3,10]. Here, we also observed that LFS at 3 Hz induces STDin control mice (n = 8) (Fig. 3a, b). Freely behaving CAMKII-T305Dmice (n = 9) expressed an equivalent STD to that of the control mice(ANOVA, F1,13 = 0.44036, p = 0.51854) (Fig. 3a, b).

Unlike freely behaving rats and hippocampal slices derived fromrats or mice, freely behaving mice fail to exhibit synaptic depressionin the CA1 region in response to 1 Hz or 2 Hz LFS [3,10]. In this study,


pulses) did not induce any changes in the efficacy of synaptic trans-mission (Fig. 4a, b). In contrast, LFS at 2 Hz in CAMKII-T305D mice(n = 7) resulted in potent STD (Fig. 4a, b). Post-hoc tests revealed

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Fig. 3. (a) Application of 3 Hz low frequency stimulation (LFS) to Schaffercollateral–CA1 stratum radiatum synapses of freely moving control mice (WT)induces equivalent short-term depression in both control mice (black circles) andtransgenic mice that cannot undergo inhibitory autophosphorylation of CAMKII(CAMKII-T305D, grey triangles). (b) Analog traces represent field potentials pre-Loh

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FS (left), 5 min (middle) and 24 h after HFS (right) for control mice (black circles)r CAMKII transgenics (grey triangles). Vertical scale bar corresponds to 2 mV andorizontal scale bar corresponds to 5 ms.

hat the initial synaptic depression in the CAMKII-T305D mice was


ignificantly different from the profile exhibited by control miceor up to 30 min after the LFS was applied (t = 0–30 min, ANOVA,1,13 = 28.683, p < 0.001; t = 0–1500 min: ANOVA, F1,13 = 0.15882,

= 0.69672). This finding suggests that in the absence of inhibitory

ig. 4. (a) Application of 2 Hz low frequency stimulation (LFS) to Schafferollateral–CA1 stratum radiatum synapses fails to induce STD in freely moving con-rol mice (WT, black circles). In contrast, 2 Hz LFS results in STD in transgenic micehat cannot undergo inhibitory autophosphorylation of CAMKII (CAMKII-T305D,rey triangles). (b) Analog traces represent field potentials pre-LFS (left), 5 min (mid-le) and 24 h after HFS (right) for control mice (black circles) or CAMKII transgenicsgrey triangles). Vertical scale bar corresponds to 2 mV and horizontal scale barorresponds to 5 ms.


autophosphorylation of CAMKII, the frequency-dependency of STDbecomes expanded to the left and the frequency-range at whichSTD can be induced becomes larger.

3.4. Learning facilitated LTD that occurs in association with novelobject-place learning is enhanced in CAMKII-T305D mice

In Schaffer collateral–CA1 synapses of freely behaving controlmice, exposure to novel object-place information (Fig. 5a) resultsin the expression of persistent synaptic depression [11,12,35]. Thecontrol animals in the current study behaved in the same way:mice (n = 11) expressed robust LTD in response to novel object-space learning (Fig. 5b, c). When no objects were presented andonly test-pulses were applied, the animals exhibited stable basalsynaptic transmission for the entire recording period (Fig. 5b, c).During novel exposure, two unfamiliar objects were presented. Thisresulted in persistent LTD compared to basal synaptic transmis-sion evoked by test-pulses alone (ANOVA, F1,13 = 55.058, p < 0.001)(Fig. 5b, c). Upon re-exposure to the objects in their familiar loca-tions 1 day later, a facilitation of LTD did not occur and the profileof evoked potentials was no different from that seen when test-pulses were given in the absence of object exposure (Fig. 5b, c)(ANOVA, F1,17 = 0.29317, p = 0.59522). On the next day, spatial re-configuration of the familiar objects resulted anew in robust LTDexpression compared to potentials evoked by test-pulses alone(ANOVA, F1,16 = 36.986, p < 0.001) (Fig. 5b, c).

When CAMKII-T305D mice (n = 8) participated in the object-space learning task, they demonstrated behavioral learning andelectrophysiological responses that were qualitatively similar tothe control mice (Fig. 6a, b). For example CAMKII-T305D miceexpressed LTD in association with novel object-space learning(ANOVA, F1,12 = 40.931, p < 0.001) and following novel configura-tion of the familiar objects (ANOVA, F1,10 = 35.314, p < 0.001), butthey did not express LTD during re-exposure to objects in theirfamiliar locations (Fig. 6a, b) (ANOVA, F1,19 = 0.15161, p = 0.70606).The transgenic animals also exhibited stable fEPSP responses totest-pulse stimulation for the entire recording session (Fig. 6a, b).

The behavioural data obtained from the object-place taskshowed that both control and CAMKII-T305D mice acquired thetask equally successfully. The levels of object exploration dur-ing the object-place recognition test were significantly higher inboth groups when the positions of the objects were reconfiguredcompared to re-exposure (Fig. 7a). Thus, during object-place re-exposure, control mice (n = 11) exhibited a significant reduction inexploratory activity directed towards the familiar objects in theirfamiliar locations (63.72 ± 4.69% of novel exploration; p < 0.001).In contrast, during novel configuration of the objects (object-place recognition test), the exploratory activity was stronglyelevated (214.22 ± 14.15% of novel exploration; p < 0.001). TheCAMKII-T305D mice (n = 8) showed a similar prominent lackof interest to the objects in their familiar locations during re-exposure (53.36 ± 6.40% of novel exploration; p < 0.001) and amarked increase in exploration of spatially re-configured objectsduring novel configuration (182.07 ± 17.75% of novel exploration;p < 0.001). It was also found that there was no significant differ-ence between the amount of exploratory activity of the controland CAMKII-T305D mice during both re-exposure (p = 0.19829) andnovel configuration (p = 0.17009).

What was striking, however, is that a closer examination of theLTD data revealed that the LTD expressed in the CAMKII transgenicmice in response to object-place learning and object-place recogni-tion was stronger than their wild-type counterparts (Fig. 7b). After


novel exploration of the objects, freely behaving control mice expe-rienced an average CA1 synaptic depression of 82.97 ± 0.56% whichwas significantly higher (Student’s t-test, p < 0.001) than the synap-tic depression exhibited by CAMKII-T305D mice (78.33 ± 0.87%).

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Fig. 5. (a) In the object-place task, on day 1, animals were given either test-pulsestimulation in the absence of any objects in the recording chamber to obtain thesynaptic transmission profile of electrically induced plasticity without the tasks.After day 1, the tasks were presented simultaneously with test-pulse stimulation.Animals were exposed to 2 novel objects on day 2 (training phase) and then re-exposed to the same objects at the same positions on day 3 (re-exposure). Onday 4 (novel configuration) the same objects were repositioned to the other sideof the chamber to test for spatial object-place recognition of the objects. Thearrows represent the delay period between the individual phases of the tasksand the additional line indicates the side of the recording chamber with the dis-tinct wall. (b) In control animals, stable fEPSP responses were obtained whenonly test-pulses were applied to Schaffer collateral–CA1 stratum radiatum synapses(black circles) (n = 14). When test-pulse stimulation was paired with the expo-sure to novel objects (grey triangles), a progressive decrease in synaptic strengthoccurred that stabilized within 30 min and lasted for at least 24 h (1440 min). Nodecrease in synaptic strength was observed when the mice were re-exposed to thesame objects at the same locations the next day (open circles). When a novel spatialconfiguration of the same objects was presented (object-place recognition test), thesynaptic depression re-emerged (black squares). (c) Analog traces represent fieldpotentials pre-task, and 5 min and 24 h after commencement of exposure to thetask (left to right). Black circles: test pulse only, grey triangles: novel object expo-sure, open circles: re-exposure, black squares object-place recognition test. Verticals

S(prdt

Fig. 6. (a) In transgenic (CAMKII-T305D), animals, stable fEPSP responses wereevoked when only test-pulses were applied to Schaffer collateral–CA1 stratum radia-tum synapses (black circles) (n = 14). When test-pulse stimulation was paired withthe exposure to novel objects (grey triangles), a progressive decrease in synap-tic strength occurred that stabilized within 30 min and lasted for at least 24 h(1440 min). No decrease in synaptic strength was observed when the mice were re-exposed to the same objects at the same locations the next day (open circles). Whena novel spatial configuration of the same objects was presented (object-place recog-nition test), the synaptic depression re-emerged (black squares). (b) Analog tracesrepresent field potentials pre-task, and 5 min and 24 h after commencement of expo-sure to the task (left to right). Black circles: test pulse only, grey triangles: novel

the frequency-dependency of short-term depression (STD) was

cale bar corresponds to 2 mV and horizontal scale bar corresponds to 5 ms.

imilarly, the synaptic depression shown by CAMKII-T305D mice80.37 ± 0.78%) was also significantly greater (Student’s t-test,

< 0.01) than control mice (82.98 ± 0.63%) when the objects were


elocated in space during novel configuration. Taken together, theseata suggest that in animals that lack inhibitory autophosphoryla-ion of CAMKII, object-place learning is unaffected, but the degree

object exposure, open circles: re-exposure, black squares object-place recognitiontest. Vertical scale bar corresponds to 2 mV and horizontal scale bar corresponds to5 ms.

of synaptic information storage through LTD that occurs during theobject-place learning event is greater.

4. Discussion

The data of this study represent the first examination of the roleof inhibitory phosphorylation at Thr305/306 of �CAMKII for synap-tic plasticity and learning-facilitated plasticity in freely behavingmice. All previous studies were conducted in the hippocampal slicepreparation or in separate studies of spatial learning. Althoughthe data are in line with previous in vitro findings that suggestthat inhibitory autophosphorylation of CAMKII is likely to serveto optimize the degree of experience-dependent synaptic plas-ticity expressed, they also offer new insights into the role ofautophosphorylation of CAMKII in synaptic plasticity and learning.In contrast to in vitro data, where it was reported that STP but notLTP could not be induced during persistent inhibitory autophos-phorylation of CAMKII [32], we found that LTP was expressedwhereby the frequency-dependency for induction of LTP wasshifted to the right. Thus, LTP could be induced but the induc-tion threshold had become higher. In addition we observed thatalthough synaptic depression that was induced by low frequencyafferent stimulation (LFS) was equivalent in control mice and thosethat undergo persistent inhibitory autophosphorylation of CAMKII,


expanded to the left, thereby indicating that under these conditionsSTD can be induced with lower afferent frequencies. Strikingly,LTD that was enabled by the coupling of test-pulse afferent

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Fig. 7. (a) The bar charts represent the total time spent by the animals exploring theobjects during re-exposure to the now familiar objects in their familiar locations, andduring the object-place recognition test, when the familiar objects were placed in anew spatial location. The data are expressed as a percentage of the total explorationtime spent during novel (first-time) exploration of the objects (% NE). No differencein exploration behaviour of control and transgenic animals was observed. Both con-trol (WT) and transgenic (CAMKII-T305D) animal groups displayed significantly lessinterest in the familiar objects in familiar locations (re-exposure test) compared tothe novel configuration of the familiar objects (object-place recognition test). (b) Thebar charts represent the average fEPSP, expressed as a percentage of the baselineobtained prior to the commencement of the behavioural test for a given experiment.During novel object exposure (left bars) the transgenic (CAMKII-T305D), animalsexpressed significantly greater LTD compared to the control animals (WT). Simi-lap

sdserihr

ta

arly, during the object-place recognition test (right bars) the transgenic animalslso expressed significantly greater LTD compared to the control mice (* signifies

< 0.001).

timulation with novel object-place learning was more potenturing persistent inhibitory autophosphorylation of CAMKII. Thisuggests that CAMKII may serve to maintain synaptic informationncoding that is associated with spatial learning within a functionalange. This could, for example, subserve the function of ensur-ng that less salient or redundant information is not encoded byippocampal synapses or that more synapses than necessary are


ecruited into the encoding of an experience.In the hippocampus, most forms of synaptic plasticity depend on

he activation of the N-methyl-d-aspartate (NMDA) receptor. Whenctivated, this receptor is permeable to Ca2+ that in turn activates


CAMKII. If sufficient levels of Ca2+ are available, autophosphory-lation of CAMKII at the Thr286 site occurs [26] that enables theenzyme to remain active after Ca2+ levels decline. This autophos-phorylation state of CAMKII is believed to comprise a critical factorin the induction of LTP, for certain forms of spatial learning [36]and for place cell stability [37]. Here, whereas high frequency stim-ulation (HFS) that results in LTP is accompanied by elevations ofCa2+ such that maximal Thr286 phosphorylation occurs within15 min [75], this autophosphorylation state can persist for up to1 h after HFS was given [75]. The activation of calmodulin by Ca2+

is a crucial step in the subsequent activation of CAMKII. WhenCa2+ levels fall and Ca2+–calmodulin becomes dissociated fromactivated CAMKII, the Ca2+–calmodulin binding domain becomesexposed, thereby permitting a different phase of phosphorylationat the Thr305/306 sites of CAMKII [29–31]. The consequence of thisinhibitory autophosphorylation is a reduced affinity of the enzymefor Ca2+–calmodulin and thus, a reduced effectivity of �CAMKII[30,38–41]. This manifests itself, for example, in a reduced affin-ity of �CAMKII for the postsynaptic density (PSD) [32,42] and mayexplain why in in vitro studies transgenic animals that undergo per-sistent autophosphorylation at the Thr305/306 site do not expressLTP [32].

In the present study, we observed that transgenic animalsthat undergo persistent inhibitory autophosphorylation at theThr305/306 site of CAMKII exhibit a shift in their frequency-dependency of LTP to the right. In contrast to in vitro studies [32],where no LTP was observed in the hippocampal slice prepara-tion, we found that robust and very long-lasting LTP is expressedin freely behaving transgenic mice if higher tetanus frequencieswere applied. Whereas LTP could not be induced using HFS at100 Hz, in line with in vitro reports [32], we observed robustLTP using HFS at 200 Hz. Persistent inhibitory autophosphoryla-tion at the Thr305/306 reduces but does not abolish the affinityof �CAMKII for the PSD [32]. Thus, the higher afferent stimula-tion frequency of 200 Hz may have been sufficient to compensateto some extent for the lowered levels of �CAMKII, thus enablingLTP to be expressed. The metabotropic glutamate receptor 5(mGlu5) plays a decisive role in the induction and maintenanceof LTP [43,44]. This receptor potentiates NMDA receptor cur-rents [45] and mobilizes calcium from intracellular stores [46].Through the latter process, kinases such as protein kinase C becomeactivated that contribute to LTP-promoting phosphorylation pro-cesses [46]. In the absence of affective CAMKII function, it isthinkable that strong tetanisation resulted in potent activation ofmGlu5 that enabled LTP to be expressed in CAMKII transgenic ani-mals.

Interestingly, the tetanisation frequency that enabled LTP inthe CAMKII transgenic animals failed to induce anything morethan short-term potentiation in the control mice, in line withprevious reports [3]. There is evidence that �CAMKII translo-cates to inhibitory synapses in response to afferent stimulationthat promotes inhibitory transmission [47,48]. �CAMKII also phos-phorylates the GABAA receptor [49,74], can potentiate GABAAreceptor currents [50], increases GABAA receptor expression inhippocampal synaptosomes [51] and potentiates hippocampalinhibitory synapses [47]. We speculate that this serves not onlyto ensure that synaptic plasticity remains within a functionaldynamic range but also prevents “pathological” overexcitation thatresults in epileptiform activity. Kindling, as a means to experi-mentally induce epilepsy, comprises the prolonged repetition ofLTP-inducing tetani [52] suggesting that LTP and epileptiformactivity could occur as a continuum, especially when inhibitoryprocesses that guard against this fail. The higher frequency of


afferent stimulation in the control animals may thus have trig-gered inhibitory mechanisms that prevented long-term changesin synaptic strength. Therefore, the absence of robust LTP in the

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ontrol animals may reflect the effective increase of inhibitoryontrol by the GABAergic system mediated by CAMKII activa-ion.

Strikingly however, the assumed absence of this kind ofnhibitory regulation in the transgenic animals did not nega-ively affect short-term synaptic depression or LTD. In contrast,TD was expressed at an afferent frequency that was ineffec-ive in control animals (2 Hz) whereas STD was equivalent inontrol and transgenic animals following 3 Hz stimulation. LTD,hat was facilitated by learning, was improved in the transgenicnimals. Experience-dependent synaptic depression is supportedy changes in GABAergic transmission, but recent evidence sug-ests that hippocampal LTD is more dependent upon changesn GABAB receptors than GABAA receptors [53]. As it is theABAA receptor that appears to be regulated more tightly byAMKII [49,51,74], this may explain why LTD is not impairedy the persistent autophosphorylation of CAMKII. Hippocam-al LTD is strongly dependent upon the activation of mGlu543,44,54]. Thus, in the absence of GABAA receptor regulation andAMKII-mediated, LTP-promoting, phosphorylation mechanisms,his receptor may also be able to support STD induction at lowerfferent frequencies and enhanced LTD induction in associationith learning as seen in the transgenic animals in the present

tudy.LTP is predominantly kinase-dependent, whereas LTD is pre-

ominantly phosphatase-dependent and it has been proposed thatt is the balance, or competition, between postsynaptic phos-hatases and kinases that determines whether LTP or LTD isxpressed [55]. At afferent frequencies such as 10 Hz, typicallyo change in synaptic strength is evident in the hippocam-us, although Ca2+-dependent activation of plasticity-relevanthosphatases and kinases occurs. In vitro, persistent inhibitoryutophosphorylation of �CAMKII results in a small synaptic depres-ion in CA1 synapses following 10 Hz stimulation [32], suggestinghat the frequency range at which LTD can be induced is shiftedo the right. In the dentate gyrus, priming of perforant path-entate gyrus synapses using 10 Hz reduced the subsequent abilityf the synapses to express LTP using 100 Hz HFS [78]. This type ofetaplasticity is absent in mice that express persistent inhibitory

utophosphorylation of CAMKII [78], in line with a role for CAMKIIn maintaining a functional dynamic range for the expression ofynaptic plasticity.

Our findings argue against a critical role for CAMKII in LTD mech-nisms but support the theory that by regulating the relative degreef LTP and LTD expressed, CAMKII plays an intrinsic role in main-aining the degree of synaptic plasticity expressed as a result ofxperience, within a functional dynamic range.

Along these lines, one would not necessarily expect that thedditional LTD that was expressed in association with object-placeearning reflects better learning or a better propensity to learnhis kind of information. Given its putative involvement in reversalearning [21] and in cognitive flexibility [23] one could speculatehat too much LTD could even hinder effective learning. In ourtudy, our behavioural data indicated that there was no differencen the behaviour of the control and transgenic animals towardshe objects. Both animal groups showed significantly higher explo-ation when exposed to the objects for the first time and whenhe object-place configuration was changed. In contrast, both ani-

al groups showed habituation to the objects when re-exposed tohem in their original locations. In association with this behaviour,TD was expressed when the animals saw the objects for the firstime and when the animals were exposed to a new spatial con-


guration of the same objects. No significant change in synaptictrength occurred when the animals were re-exposed to the famil-ar objects in familiar locations. Although the transgenic animalshowed significantly stronger LTD in response to novel spatial


learning the degree of learning was equivalent in transgenics andcontrols. Thus, we saw no evidence of improved learning ability inassociation with the more pronounced LTD in the transgenic ani-mals. In rats, differences in hippocampal synaptic plasticity abilityfailed to show clear correlations with differences in spatial learn-ing ability [56]. However in both this case and in the present studythe demands of the spatial task may simply be too low to reveal acorrelation.

An impairment of spatial learning has been reported in ani-mals that undergo persistent inhibitory autophosphorylation of�CAMKII [32]. Here, spatial learning in the Morris Water Maze wasstudied, that is believed to be associated with LTP [57]. Based onour data, we propose that the elevated threshold for LTP induc-tion we observed in animals that undergo persistent inhibitoryautophosphorylation of �CAMKII would necessitate a higher infor-mational saliency to pass the threshold for information encodingthrough LTP. Here, neuromodulators such as dopamine [58] andnoradrenalin [35,59] that are released in the hippocampus duringevents of high novelty or motivation could be expected to facilitatepostsynaptic activity leading to LTP induction. In contrast to theimpairment of spatial learning in the water maze [32], we saw noimpairment of object-place learning and object-place recognition.This can be explained by the likelihood that synaptic informationstorage that underlies this kind of learning depends on LTD and notLTP [11,12,35]. Although object recognition per se is widely believedto depend on information processing in extrahippocampal struc-tures [50–62], others have provided evidence for a significant roleof the hippocampus through lesion and pharmacological studies[63–67]. Recently it has become apparent that under circumstanceswhere spatial information processing is a component of objectrecognition, it is likely that the hippocampus mediates the encodingspatial and “temporal order” aspects of object recognition [68–72].This process may involve hippocampal LTD: in mice, preventionof the activation of neurotransmitter receptors that are essentialfor the induction of hippocampal LTD such as the NMDA receptor,the mGlu receptor or the beta-adrenergic receptor, prevent bothobject recognition memory and object-place recognition memory[12,35]. Most strikingly, even in the absence of LFS, the couplingof test-pulse stimulation of Schaffer collateral-CA1 synapses withnovel object or object-place learning results in long-lasting LTDin the hippocampus [11,12,35]. Thus, we would argue that thereason why we saw no impairment of object-place learning andobject-place recognition in the presence of persistent inhibitoryautophosphorylation of �CAMKII in the present study is becausethis form of learning does not depend on effective induction of LTP,rather it requires the expression of persistent LTD at hippocampalsynapses.

In summary, these data support that inhibitory autophospho-rylation of �CAMKII regulates the threshold for induction of bothLTP and LTD and that CAMKII plays a pivotal role in ensur-ing that experience-dependent synaptic plasticity is expressed ina dynamic range that promotes efficient and optimal informa-tion encoding. In addition, these data strongly indicate that adivision of labour occurs in terms of the forms or componentsof spatial memory that may be encoded by LTP or LTD. Thus,whereas LTP may be essential for the synaptic storage of spatialrepresentations, LTD may play a special role in the encoding ofinformation about spatial content and/or spatial features. Thesefindings also support that object-place recognition memory is pro-cessed by the hippocampus and enabled by means of hippocampalLTD.


Grant sponsor

German Research foundation (DFG, SFB874, B1).

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cknowledgments

We thank Jens Klausnitzer and Stefan Jansen for technical assis-ance and Nadine Kollosch for animal care. We are particularlyrateful to Prof. Ype Elgersma for his kind gift of the CAMKII-T305Dice used in this study.

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Role of inhibitory autophosphorylation of calcium/calmodulin-dependent kinase II (αCAMKII) in...

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