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¹ MRC National Institute for Medical Research, Mill Hill, London NW7 1AA, UK
² Department of Physiology, Trinity College Dublin, Dublin 2, Ireland
³ Wolfson Institute for Biomedical Research, University College London, London WC1E 1BT, UK

	Abstract

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Autophosphorylation of -Ca²⁺/calmodulin kinase II (CaMKII) at Thr²⁸⁶ is thought to be a general effector mechanism for sustaining transcription-independent long-term potentiation (LTP) at pathways where LTP is NMDA receptor-dependent. We have compared LTP at two such hippocampal pathways in mutant mice with a disabling point mutation at the Thr²⁸⁶ autophosphorylation site. We find that autophosphorylation of CaMKII is essential for induction of LTP at Schaffer commissural–CA1 synapses in vivo, but is not required for LTP that can be sustained over days at medial perforant path–granule cell synapses in awake mice. At these latter synapses LTP is supported by cyclic AMP-dependent signalling in the absence of CaMKII signalling. Thus, the autophosphorylation of CaMKII is not a general requirement for NMDA receptor-dependent LTP in the adult mouse.

(Received 11 May 2006; accepted after revision 18 May 2006; first published online 25 May 2006)
Corresponding authors S. F. Cooke: National Institute for Medical Research, Mill Hill, London NW7 1AA, UK. Email: scooke{at}nimr.mrc.ac.uk; K. P. Giese: Wolfson Institute for Biomedical Research, University College London, London WC1E 1BT, UK. Email: p.giese{at}ucl.ac.uk

	Introduction

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Induction of long-term potentiation (LTP) at many types of synapse relies upon the voltage dependency of the NMDA receptor to detect coincident pre- and postsynaptic activity (Collingridge et al. 1983; Nowak et al. 1984; Bliss & Collingridge, 1993), with a resultant calcium influx that activates postsynaptic effector mechanisms (Lynch et al. 1983). Although NMDA receptor-dependent LTP has been most widely investigated in area CA1 of the hippocampus, it occurs at many synapses throughout the nervous system, including those between medial perforant path (MPP) fibres and granule cells in the dentate gyrus (Morris et al. 1986; Errington et al. 1987), where LTP was first identified (Bliss & Lømo, 1973).

Models of synapse-specific LTP mechanisms insensitive to protein turnover (Crick, 1984) posit a central role for an autophosphorylating kinase (Lisman, 1985; Lisman & Goldring, 1988) that responds to Ca²⁺ influx and maintains local changes in synaptic efficacy through a variety of postsynaptic LTP expression mechanisms (Barria et al. 1997; Hayashi et al. 2000; Lee et al. 2000; Bayer et al. 2001; see Lisman et al. 2002). Work focusing predominantly on the CA1 subfield of the hippocampus has demonstrated that autophosphorylation of -Ca²⁺/calmodulin-dependent kinase II (CaMKII) at threonine 286 could fulfil this role in the adult animal (Fukunaga et al. 1995; Barria et al. 1997; Ouyang et al. 1997; Giese et al. 1998) by switching the kinase to an autonomously active state (for review, see Hanson & Schulman, 1992). The autonomous kinase can phosphorylate AMPA receptors that contain the GluR1 subunit, leading to enhanced conductance of the receptor channel (Barria et al. 1997; Benke et al. 1998; Lee et al. 2000), thus providing a mechanism by which autonomously active CaMKII may mediate enhanced synaptic efficacy (but see Lee et al. 2003; Lengyel et al. 2004). A targeted point mutation at residue 286 in the mouse CaMKII gene, in which threonine is replaced by alanine (T286A), prevents autophosphorylation at that site, resulting in the block of NMDA receptor-dependent LTP both in area CA1 in vitro (Giese et al. 1998) and in neocortical slices (Hardingham et al. 2003). Interestingly, LTP at CA1 synapses is not supported by CaMKII autophosphorylation in juvenile mice, but instead requires the cAMP-dependent protein kinase A (PKA) (Yasuda et al. 2003). Similarly, alternative enzymes to CaMKII, such as PKA, have been shown to support LTP at Schaffer collateral–CA1 synapses in juvenile rats (Wikstrom et al. 2003). Also, application of pharmacological inhibitors of CaMKII activity does not block LTP in the adult animal at another set of hippocampal synapses, the MPP-granule cell synapses in the dentate gyrus (Wu et al. 2004; Cooke et al. 2004; Zhang et al. 2005).

In this study we have analysed LTP in the dentate gyrus of adult CaMKII^T286A mice to test whether the autophosphorylation of CaMKII is required. We find that LTP can be induced at MPP–granule cell synapses in CaMKII^T286A mice. LTP at these synapses is supported by the cAMP-dependent signalling pathway, a cascade involving sequential activity of PKA and the mitogen activated protein kinase (MAPK) (for review see Waltereit & Weller, 2003). Thus, LTP in the adult dentate gyrus is mechanistically similar to LTP at Schaffer commissural–CA1 synapses in the juvenile animal, and we conclude that the autophosphorylation of CaMKII is not a general requirement for NMDA receptor-dependent LTP in the adult mouse.

	Methods

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Knock-in mice

Heterozygous CaMKII^T286A mutants backcrossed into the C57BL/6 genetic background were interbred to obtain homozygous and wild-type (WT) littermates. Genotyping was carried out by PCR analysis, as previously described (Giese et al. 1998), with DNA obtained from tail biopsies on postnatal day 21, the day of weaning. Adult male mice homozygous for CaMKII^T286A and WT littermates aged 3–6 months were used in all experiments and recordings were made blind to the genotype. All experiments were undertaken in accordance with the UK Animals (Scientific Procedures) Act 1986.

Electrophysiology in transverse slices

Transverse hippocampal slices were taken from young adult mice (males, 3–4 months of age; weight, 25–30 g). Mice were culled by cervical dislocation and then decapitated using scissors. The brain was rapidly removed and placed in cold oxygenated (95% O₂, 5% CO₂) medium containing (mM): 120 NaCl, 2.5 KCl, 1.25 NaH₂PO₄, 26 NaHCO₃, 2.0 MgSO₄, 2.0 CaCl₂, and 10 D-glucose. Slices were cut at a thickness of 350 µm using an vibratome (Intracell Plus 1000, Intracell, Herts, UK) and placed in a storage container with oxygenated medium at room temperature (20–22°C) for 1 h. The slices were then transferred to a submerged recording chamber and continuously superfused at a rate of 5–6 ml min^–1 at 30–32°C. Solutions for experiments in the dentate gyrus contained 100 µM picrotoxin (Sigma, St Louis, MO, USA) to block GABA_A-mediated activity. The following drugs were used at the indicated concentrations: KN-62 (10 µM, Calbiochem), KT5720 (5 µM, Calbiochem) and U0126 (10 µM, Tocris Cookson), all dissolved in DMSO to give a maximal final concentration of 0.1%. Control experiments were carried out using the same vehicle.

Standard electrophysiological techniques were used to record field EPSPs (fEPSPs). Test stimuli were applied at a frequency of 0.033 Hz to the MPP in the dentate gyrus using a bipolar insulated tungsten wire electrode, and fEPSPs were recorded similarly in the middle third of the molecular layer of the dentate gyrus with a glass microelectrode. The inner blade of the dentate gyrus was used in all studies. In each experiment, an input–output curve (stimulus intensity versus fEPSP amplitude) was plotted at the test frequency, and the amplitude of the test fEPSP was adjusted to one-quarter to one-third of the maximum. LTP was evoked by high-frequency stimulation (HFS) consisting of eight trains, each of eight stimuli at 200 Hz, and an intertrain interval of 2 s, with the stimulation voltage increased during the HFS to elicit an initial fEPSP double the normal test fEPSP amplitude. In experiments involving kinase inhibitors, the agents were preperfused over the slices for 1 h before HFS. Recordings were analysed using pCLAMP software (Axon Instruments, Union City, CA, USA).

Electrophysiology in the anaesthetized mouse

Animals were anaesthetized with an intraperitoneal (I.P.) injection of urethane (1.8 g kg^–1). Rectal temperature was maintained at 37°C using a heat lamp. Holes were made in the skull of the anaesthetized animal using a dental drill and electrodes were placed according to established stereotactic coordinates on the left-hand side of the brain. For experiments in area CA1, the recording electrode was placed in stratum radiatum, 2 mm posterior to bregma, 1.6 mm lateral to the midline at a depth of approximately 1 mm from the brain surface. The stimulating electrode was positioned near the midline in the contralateral ventral hippocampal commissure approximately 1.8 mm posterior to bregma and 1.5 mm below the brain surface. In the dentate gyrus, a concentric bipolar stimulating electrode was positioned in the MPP, 3 mm lateral to lambda and at a depth of approximately 1.5 mm from brain surface, and a glass micropipette was lowered into the hilus of the ipsilateral dentate gyrus, 2 mm posterior to bregma, 1.6 mm lateral to the mid-line and at a depth of approximately 1.5 mm, to record evoked field responses. Correct placement of the stimulating electrode in the MPP was confirmed by the characteristic relatively short latency to onset of the evoked field response (2–2.5 ms) and the early onset of the population spike (4 ms approx.). Lateral perforant path (LPP) responses could be distinguished from MPP responses by the longer latency to onset (3–3.5 ms) and a characteristic late onset of the population spike (6–7 ms approx.). MPP responses also have a faster rise time than LPP responses (for a discussion of these issues see McNaughton & Barnes, 1977). We are confident that, taken together, these features allow us to distinguish MPP responses from LPP responses. In the rare event when a response with LPP characteristics was observed, the stimulating electrode was repositioned to obtain a MPP response. However, it is not possible, at least in the mouse, to restrict stimulation exclusively to a single component of the PP. For the in vivo experiments described here, therefore, stimulation was limited predominantly to the MPP, but we cannot discount the possibility that there was also some activation of the LPP.

For all experiments input–output relationships were assessed using a range of stimulus intensities from 0 µA through to a stimulus strength that evoked a maximal response. Three responses were collected at each intensity and averaged. Pairs of pulses (interpulse intervals, 10–100 ms) were used to study paired-pulse interactions at two intensities, one to evoke a pure EPSP and the other a population spike of approximately 1 mV (to the first of the pair of stimuli). Again, three responses were collected at each intensity and averaged. Test responses for LTP experiments were evoked by monophasic stimuli set at an intensity to evoke a response approximately 50% of maximum fEPSP slope (0.033 Hz; 70–300 µA, 60 µs). In the CA1 subfield such stimulation was below threshold for generation of a population spike. In the dentate gyrus baseline stimulus intensity was set to evoke a population spike of approximately 1 mV before the tetanus. In CA1 experiments the tetanus consisted of two trains of 50 pulses at 100 Hz, with an intertrain interval of 30 s. In the dentate gyrus experiments the tetanus comprised six series of six trains of six stimuli at 400 Hz, 200 ms between trains, 20 s between series. Pulse width was doubled during the tetanus. Only the very early component of the EPSP slope was measured for analysis in order to ensure that there was no contamination by the population spike in the dentate gyrus experiments. The slope of the fEPSP was expressed as a percentage change relative to the mean response in the 10 min prior to tetanic stimulation. In some experiments mice received an injection of CPP (10 µg g^–1, I.P., Tocris, UK) in saline 1 h before the tetanus. At the end of each experiment the anaesthetized mouse was killed by cervical dislocation.

Electrophysiology in awake, freely moving mice

Animals were habituated to the handler for 3 days prior to implantation of recording and stimulating electrodes. Surgery was performed under pentobarbital anaesthesia (6 µg g^–1, I.P.) and carprofen analgesia (5 µg g^–1, S.C.). Conditions were kept aseptic throughout. Electrodes were made from 50 µm diameter formvar-insulated nickel-chrome wire. A mechanical encased microdrive was attached to the recording electrode. Stimulating and recording electrodes were placed according to the stereotactic coordinates described above for dentate gyrus experiments in anaesthetized animals. Electrode depths were adjusted to maximize the amplitude of evoked responses. Electrodes were fixed in place with dental cement, leaving the connectors and the microdrive screwcap exposed on the surface of the headplug. Aureomycin (chlortetracycline hydrochloride) antibiotic powder was administered topically to the edge of the incision to prevent the development of infections postoperatively. A single suture was applied to draw the incision closed around the headplug. Post-operatively the mice were fed on a wet diet for a period of 2 days immediately postoperatively and then allowed to recover fully for 6 further days before 2 days of habituation to the recording chamber. Throughout this recovery period mice were monitored for signs of discomfort or illness. Finally, cables were connected to the headcap, via a junction field effect transistor (JFET) to condition the signal before amplification, and evoked responses sampled. Low frequency baseline stimuli were given for 20 min per day until stable responses were obtained over two successive days. The tetanus consisted of six series of six trains of six stimuli at 400 Hz, 200 ms between trains, 20 s between series. The slope of the fEPSP was expressed as a percentage change relative to the mean response over 20 min prior to tetanic stimulation. Each data point presented is an average of four successive sampled responses over 2 min.

Attempts to record LTP from area CA1 in the freely moving mouse were frustrated by the sensitivity of areas CA3/CA1 to tetanic stimulation of the commissural input, leading to a high probability of seizures. Experiments in vivo in area CA1 were therefore confined to the anaesthetized animal. All animals used for the recordings were killed at the end of the experiment by cervical dislocation.

Biochemistry

LTP was induced in anaesthetized adult male mice as described above. The animals were killed by cervical dislocation 1 h after LTP induction in area CA1 or the dentate gyrus, and the relevant brain areas were microdissected immediately. For the dentate gyrus LTP experiments the contralateral dentate gyrus was used as unstimulated control tissue. For CA1 LTP experiments, control tissue was obtained from another unstimulated mouse. The brain regions were dissected in ice-cold lysate buffer (10 mM Tris-HCl pH 7.6, 320 mM sucrose, 1 mM EDTA, 1 mM EGTA and 0.025% NaN₃) containing protease and phosphatase inhibitors (complete protease inhibitor tablets (Roche), 0.1 mM ammonium molybdate, 0.2 mM phenylarsenine oxide, 50 mM sodium fluoride, 10 mM sodium pyrophosphate and 2 mM sodium vanadate) to preserve the integrity and phosphorylation state of hippocampal proteins. Subfields were dissected under a magnifying lens using cold steel implements and homogenized in lysate buffer with 1% Triton X-100 and 1% CHAPS using a glass pestle and mortar. Samples were stored at –80°C. Protein concentration was determined with the BCA protein assay (Pierce Biotechnology, Inc., Rockford, IL, USA). Proteins were separated on 4–16% gel by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes using standard protocols. Immunoblots were probed with primary antibodies (CaMKII (Chemicon, Temecula, CA, US), phospho-CaMKII pThr²⁸⁶ (Affinity BioReagents, Golden, CO, USA), phospho-CaMKII /ß (Thr^286/287) (Upstate Ltd., Dundee, UK), phospho-CREB (Ser¹³³) (Cell Signalling Technology, Beverly, MA, USA), phospho-Synapsin I (Ser⁶⁰³), phospho-NR2B (Tyr¹⁴⁷²) and ßCaMKII (all from Zymed Laboratories Inc., South San Francisco, CA, USA)) and signals visualized with horseradish peroxidase-conjugated secondary antibodies and an enhanced chemiluminescent system (Pierce). Signals from the X-ray film were quantified with Densitometer Quantity One (Bio-Rad) in the linear range. Blots were stripped with stripping buffer (Pierce) and reprobed with anti-ß-actin and antisynaptotagmin antibody (Sigma) to normalize for protein content.

Synaptosomal lysate

Crude synaptosomal fractionation of hippocampus was performed as described in Gray & Whittaker (1962). The hippocampi were dissected and Dounce homogenized in ice-cold lysate buffer as described above. The homogenate was centrifuged at 1000 g. The resulting supernatant was re-centrifuged at 30 000 g and the pellet fraction (P2) was resuspended in 100 µl lysate buffer.

Statistical analysis

Statistical comparisons of electrophysiological data were made using a two-way Student's t test unless otherwise stated. In cases where variance was inhomogeneous between groups, the non-parametric Mann–Whitney U test was applied. For each experiment, the slope of the fEPSP was normalized to the mean value in the 5 min preceding the tetanus (unless otherwise stated), and LTP was calculated as the percentage increase measured over the last 5 min of the experiment. Biochemical data were analysed with one-way ANOVA. Group values are reported as means ± S.E.M.

	Results

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LTP at MPP–granule cell synapses in the dentate gyrus does not require CaMKII autophosphorylation

We first examined LTP at MPP–granule cell synapses in the dentate gyrus of adult, anaesthetized CaMKII^T286A mutant mice and WT littermates. In contrast to previously presented in vitro findings in the CA1 subfield (Giese et al. 1998) we found that the loss of CaMKII autophosphorylation did not block LTP at MPP–dentate gyrus synapses in vivo (Fig. 1A). One hour after the tetanus, the increase in the field EPSP remained significant in both WT (17.6 ± 4.0%, n = 6; P < 0.005, Mann–Whitney U test) and mutant mice (7.9 ± 3.7%, n = 7; P < 0.01, Mann–Whitney U test), although there was a non-significant trend towards less LTP in the mutants relative to WTs (P = 0.09). Induction of LTP at MPP–granule cell synapses does not therefore require the autophosphorylation of CaMKII.

View larger version (27K): [in this window] [in a new window]   Figure 1.  LTP in anaesthetized CaMKIIT286A mice A, LTP is induced at PP–granule cell synapses in urethane-anaesthetized CaMKIIT286A (• in all panels; n = 7) and WT mice (; n = 6), and sustained for at least 1 h in both. Sample responses are shown on the right. Dentate gyrus responses are sampled from cell bodies (hilus). Response onset is approx. 2 ms and pretetanus spike onset is approx. 3.5–4 ms after the stimulus, indicative of stimulation of predominantly MPP fibres. B, LTP is blocked at PP–granule cell synapses in urethane-anaesthetized CaMKIIT286A mutant mice by I.P. injection of the NMDA receptor antagonist CPP (10 µg g–1) 1 h prior to tetanus (grey circles; n = 5). LTP is induced at PP–granule cell synapses in interleaved control CaMKIIT286A mutant mice receiving saline vehicle alone (n = 5) and sustained for at least 1 h. Sample responses on the right again have latencies and shapes characteristic of predominantly MPP stimulation. C, LTP is not induced at Schaffer commissural–CA1 pyramidal cells of urethane-anaesthetized CaMKIIT286A mice (n = 4) but is induced and sustained for at least 1 h in WT littermates (n = 4). Sample responses are on the right. CA1 responses are recorded from the synaptic layer in apical dendrites (stratum radiatum). Calibrations: 5 mV, 5 ms.

LTP at MPP-dentate gyrus synapses of CaMKII^T286A mutant mice is NMDA receptor-dependent

In order to determine if LTP at MPP–granule cell synapses is dependent upon the NMDA receptor in the CaMKII^T286A mutant mice, we injected the competitive NMDA receptor antagonist CPP (10 µg g^–1, I.P.) 1 h before delivering the high frequency tetanus. LTP induction was blocked in animals receiving CPP (EPSP slope 1.0 ± 3.5%, n = 5). Interleaved control mutant animals that received injections of vehicle alone showed significant LTP of the fEPSP 1 h after the tetanus (13.3 ± 5.5%, n = 5; P < 0.001) in comparison (Fig. 1B). This finding confirms that LTP induced in the CaMKII^T286A mutant mice at MPP–granule cell synapses is dependent upon the NMDA receptor.

LTP in area CA1 requires CaMKII autophosphorylation in vivo

We next examined LTP at Schaffer commissural–CA1 pyramidal cell synapses in anaesthetized CaMKII^T286A mutant mice and WT littermates. Consistent with a previous in vitro study (Giese et al. 1998) and our own slice experiments (data not shown), we found that the loss of CaMKII autophosphorylation fully blocked CA1 LTP in vivo (Fig. 1C). In WT mice, LTP of the fEPSP remained significantly above baseline 1 h after the tetanus (18.6 ± 6.4%, n = 4; P < 0.005, Mann–Whitney U test) whereas, in CaMKII^T286A mutants, synapses were not significantly potentiated (1.3 ± 1.7%, n = 4). This result confirms that autophosphorylation of CaMKII is necessary for the induction of LTP at Schaffer commissural–CA1 pyramidal cell synapses in vivo. Basal synaptic transmission and paired-pulse facilitation were not affected in CaMKII^T286A mutants in either subfield (see online Supplemental material, Supplementary Fig. 1).

LTP persists over days in the dentate gyrus of awake, freely moving CaMKII^T286A mutant mice

We next investigated LTP at MPP–granule cell synapses in the dentate gyrus of awake, freely moving adult CaMKII^T286A mice and WT littermates by chronically implanting stimulation electrodes in the MPP and recording electrodes in the hilus of the dentate gyrus. Following two days of baseline recording, tetanic stimulation of the MPP induced significant potentiation of synaptic transmission that persisted for over 2 days in CaMKII^T286A mutant mice (the mean increase at 48 h was 43.7 ± 15.7%, n = 4, P < 0.05, Mann–Whitney U test). The degree of potentiation seen at 48 h in mutant animals was not significantly different from that observed in WT mice (52.4 ± 22.3%, n = 3; Fig. 2A and B). Induction and persistence of LTP over days at MPP–granule cell synapses in awake, freely moving mice therefore does not require the autophosphorylation of CaMKII.

View larger version (21K): [in this window] [in a new window]   Figure 2.  LTP in awake CaMKIIT286A mice A, individual experiments on CaMKIIT286A and WT mice. Sample responses shown on the right from 24 h pretetanus are compared with 48 h post-tetanus. Again these responses have latencies and shapes characteristic of predominantly MPP stimulation. B, LTP is induced at PP–granule cell synapses in awake freely moving CaMKIIT286A (• in both panels; n = 4) and WT (; n = 3) mice, and sustained over days. Calibrations: 5 mV, 5 ms.

Normal levels of ßCaMKII in hippocampal synapses of CaMKII^T286A mice

The ß isoform of CaMKII is expressed at high levels in the brain (for review see Hanson & Schulman, 1992). ßCaMKII, like CaMKII, can also be switched into an autonomously active state, in this case through autophosphorylation of threonine 287. It has been suggested that ßCaMKII can compensate for LTP deficits in area CA1 of CaMKII null mutant mice (Hinds et al. 1998; Elgersma et al. 2002), via retargeting of ßCaMKII into postsynaptic densities (Elgersma et al. 2002). We asked if there was an up-regulation of ßCaMKII or autophosphorylated ßCaMKII in synaptosomes from CaMKII^T286A mutant mice (Fig. 3A and B). We observed no differences in levels of ßCaMKII or autophosphorylated ßCaMKII in synaptosomal lysates from WT and mutant mice (n = 3 in both cases), suggesting that retargeting of ßCaMKII does not occur in CaMKII^T286A mice.

View larger version (31K): [in this window] [in a new window]   Figure 3.  Autophosphorylation of - and ßCaMKII in unstimulated and potentiated dentate gyrus A, representative immunoblots from crude synaptosomal fractions of hippocampal lysates from WT mice and CaMKIIT286A mutant mice probed with CaMKII, ßCaMKII and phospho-/ßCaMKII (T286/287) antibodies. B, quantification of immunoblots in A. Left panel, ratio of phospho-ßCaMKII to total enzyme is the same for WT (open bars in all panels; n = 3) and mutant mice (filled bars; n = 3). Right panel, ratio of ßCaMKII to CaMKII is the same for WT (n = 3) and mutant mice (n = 3). C,representative immunoblots and quantitative analysis of lysates from the dentate gyrus of WT mice 1 h after LTP induction (tet) and unstimulated control tissue (cont; n = 5 for both) tested with phospho-/ßCaMKII (T286/287) antibody. D, quantification of phospho-ßCaMKII (T287) signal from immunoblots of lysates from the dentate gyrus of CaMKIIT286A mice 1 h after LTP induction (tet) and in control tissue (cont; n = 5 for both). ***P < 0.001, **P < 0.01.

Next we investigated the regulation of CaMKII autophosphorylation in the dentate gyrus after LTP induction using phospho-specific antibodies directed at lysed tissue (Fig. 3C and D and Supplementary Fig. 2). To ensure that our tissue extraction technique retained phospho-proteins at a physiological level, we compared phosphorylation of two synaptic proteins, NR2B (Tyr¹⁴⁷²) and synapsin I (Ser⁶⁰³), as well as a nuclear protein, CREB (Ser¹³³) in potentiated and control tissue (Supplementary Fig. 3). We confirmed previous findings that there is an increased phosphorylation of each of these proteins following induction of LTP (Nayak et al. 1996; Rostas et al. 1996; Schulz et al. 1999). Next, using a phospho-specific antibody to the autophosphorylation sites of both CaMKII isoforms, we found that autophosphorylation of and ßCaMKII is significantly increased in the dentate gyrus 15 m after LTP induction in anaesthetized C57BL/6 WT mice (CaMKII: 145%, n = 8, P < 0.01 and ßCaMKII: 153%, n = 8, P < 0.001; Supplementary Figs 3 and 2A). The increased autophosphorylation of both isoforms was sustained for at least 1 h post-induction (CaMKII: 162%, n = 5, P < 0.001 and ßCaMKII: 132%, n = 5, P < 0.01; Fig. 3C, Supplementary Fig. 2B). We also analysed the autophosphorylation of ßCaMKII in potentiated dentate gyrus tissue from CaMKII^T286A mutants 1 h after LTP induction. Again, we found that the autophosphorylation of ßCaMKII was significantly greater in potentiated than control tissue (ßCaMKII: 136%, n = 5, P < 0.001; Fig. 3D, Supplementary Fig. 2C). These results suggest that autonomous ßCaMKII activity may contribute to dentate gyrus LTP. However, this conclusion is not supported by the pharmacological experiments described below.

Inhibition of CaMKII activity does not affect LTP at MPP–granule cell synapses in CaMKII^T286A mutant mice in vitro

We found that we could induce LTP at MPP–granule cell synapses in transverse hippocampal slices taken from CaMKII^T286A mutants (Fig. 4A). LTP evoked at these synapses was similar in WT and mutant animals, measuring 56.6 ± 6.5% (n = 10) in slices from WT mice and 42.6 ± 3.6% (n = 10, not significant, Mann–Whitney U test) in slices from mutant mice 1 h post-tetanus. This result matches our in vivo findings. Even in the absence of any compensatory increase in synaptosomal ßCaMKII in CaMKII^T286A mice, the increased autophosphorylation of the ß isoform (Fig. 3D) could, in principle, support LTP at MPP–granule cell synapses. In order to test this possibility we bath-applied the general CaMKII inhibitor KN-62 (10 µM). LTP induced at MPP–granule cell synapses in slices taken from mutant mice measured 33.7 ± 6.0% (n = 5) in KN-62 1 h post-tetanus (•, Fig. 4B), not significantly different from the value seen in hippocampal slices taken from WT mice treated with KN-62 (41.0 ± 4.9%; n = 5; , Fig. 4B) or the mutant group in control medium (42.6 ± 3.6%; n = 10, same data as in Fig. 4A). This finding demonstrates that LTP at MPP–granule cell synapses is not dependent on the activity of either isoform of CaMKII in CaMKII^T286A mice. The increase in ßCaMKII autophosphorylation observed in mutant mice following high-frequency stimulation of the MPP cannot therefore be causally related to the induction of LTP. The experiments with KN-62 demonstrate that alternative signalling pathway(s) to - and ßCaMKII are recruited to sustain MPP–granule cell synapses in CaMKII^T286A mutants.

View larger version (21K): [in this window] [in a new window]   Figure 4.  Effects of general CaMKII inhibitor KN-62 on LTP in vitro in CaMKIIT286A and WT mice A, tetanically induced LTP at MPP–dentate gyrus synapses in WT mice () and mutant mice (•; n = 10 for both) in hippocampal slices perfused with ACSF containing 100 µM picrotoxin. B, LTP at MPP–dentate gyrus synapses in hippocampal slices from WT mice () and mutant mice (•; n = 5 for both) is sustained for at least 1 h in the presence of the general CaMKII inhibitor KN-62 (10 µM), perfused throughout the experiment, beginning 1 h prior to the tetanus (ACSF also contains 100 µM picrotoxin). C, LTP at Schaffer collateral–CA1 pyramidal cell synapses in WT C57BL/6 mice is completely inhibited by KN-62 (10 µM), perfused throughout the experiment, beginning 1 h prior to tetanus (grey circles; n = 6) and is significantly impaired in comparison with LTP at the same synapses in hippocampal slices perfused with control medium only (; n = 5). Calibrations: 1 mV, 5 ms.

In order to confirm that KN-62 (10 µM) inhibits the activity of CaMKII sufficiently to prevent the induction of LTP we bath-applied the drug to slices taken from WT mice and monitored synaptic potentials at Schaffer collateral–CA1 synapses. LTP was completely blocked at these synapses by KN-62 (–0.6 ± 1.1% 1 h after the tetanus, n = 6). LTP was sustained 1 h after the tetanus in interleaved control experiments without KN-62 (54.0 ± 6.0%, n = 5; P < 0.005 compared to WT in KN-62, Fig. 4C). This result confirms the importance of CaMKII signalling at Schaffer collateral–CA1 synapses and also demonstrates that 10 µM KN-62 blocks the activity of CaMKII.

Inhibition of PKA reduces LTP in CaMKII^T286A mutant mice in vitro

Since PKA signalling can support LTP in area CA1 in juvenile CaMKII^T286A mutants (Yasuda et al. 2003), we tested whether PKA signalling supports LTP at MPP synapses in adult CaMKII^T286A mutants. We bath-applied KT5720 (5 µM), an inhibitor of PKA, to slices from WT and mutant mice, and monitored LTP for 1 h after the tetanus (Fig. 5A). KT5720 did not affect the level of LTP in slices from WT mice measured 1 h after induction (45.3 ± 4.1%, n = 5; not significantly different from WTs in control medium, Fig. 4A), but caused a reduction of LTP in interleaved slices from CaMKII^T286A mice (16.4 ± 5.9%, n = 6; P < 0.005, compared to mutants in control medium, Fig. 4A). This result demonstrates that PKA signalling contributes to LTP at adult MPP–granule cell synapses in the absence of autonomous CaMKII activity.

View larger version (28K): [in this window] [in a new window]   Figure 5.  Effects of PKA and MAPK inhibitors on MPP–dentate gyrus LTP in vitro in CaMKIIT286A and WT mice A, the induction of LTP is inhibited by the PKA inhibitor KT5720 in mutant mice but not in WT mice. The graph shows the effect of KT5720 (5 µM), perfused throughout the experiment beginning 1 h prior to the tetanus, on the induction of LTP in WT mice (; n = 5), and in CaMKIIT286A mutant mice (•; n = 6). B, the MAPK inhibitor UO126 suppresses LTP in mutant mice and weakly inhibits LTP in WT mice. The graph shows the effect of UO126 (10 µM), perfused throughout the experiment beginning 1 h prior to the tetanus, on the induction of LTP in WT mice (), and in CaMKIIT286A mutant mice (•; n = 6 for both). Calibrations: 1 mV, 5 ms.

Inhibition of MAPK blocks LTP in CaMKII^T286A mutant mice in vitro

MAPK is thought to act downstream from PKA signalling (for review see Waltereit & Weller, 2003) so we assessed the effects of the MAPK inhibitor UO126 (10 µM) on LTP in the dentate gyrus of CaMKII^T286A mice and WT littermates. Bath-applied UO126 completely blocked LTP at 1 h in the dentate gyrus of CaMKII^T286A mutant mice (Fig. 5B). In the presence of UO126, LTP in slices from WT mice was slightly reduced relative to the value in control medium (38.0 ± 3.3%, n = 6, P < 0.05, Mann–Whitney U test), and severely reduced in mutant mice (4.6 ± 7.3%, n = 6; P < 0.005 compared to WT slices in UO126, and P < 0.001 compared to mutants in control medium). This result establishes a role for MAPK in the induction of LTP at MPP–granule cell synapses in adult mice.

Multiple signalling pathways can support MPP LTP

WT and CaMKII^T286A mutant mice show an equivalent magnitude of LTP (Fig. 4A), suggesting that autonomously active CaMKII plays little part in supporting LTP at MPP–dentate gyrus synapses. However, other evidence suggests that this may be an oversimplification of the situation. Inhibition of either PKA or MAPK signalling has a much greater effect on LTP in mutant than in WT mice (Fig. 5A and B), indicating that MPP LTP can be supported at near-normal levels by autonomously active CaMKII in the absence of either of these two other kinases. The converse also holds: in the absence of all CaMKII signalling, LTP can still be expressed at near-normal levels (Fig. 4B), supported by cAMP-dependent signalling pathways (Fig. 5A and B). Thus, our evidence suggests that MPP LTP can be mediated by parallel signalling pathways, such that the loss of one alone does not lead to a significant reduction in LTP (Fig. 6). This contrasts with the situation in area CA1 (Figs 1C and 4C, Giese et al. 1998), where the absence of autonomously active CaMKII leads to the complete loss of LTP.

View larger version (19K): [in this window] [in a new window]   Figure 6.  Schematic illustrating parallel signalling pathways mediating early LTP Schematic illustrating parallel signalling pathways mediating early LTP (E-LTP) at MPP–granule cell synapses in the dentate gyrus, as compared to the single pathway at LPP–granule cell synapses (Zhang et al. 2005) and Schaffer collateral–CA1 synapses (Giese et al. 1998) in the adult mouse. Blockade of CaMKII activity has little effect on E-LTP at MPP–granule cell synapses because PKA/MAPK signalling is unhindered. The reverse situation also holds: blockade of PKA/MAPK has little effect on E-LTP at these synapses unless CaMKII signalling is also disabled. At present it is not possible to conclude which of these signalling pathways, if any, predominates under normal circumstances.

	Discussion

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In CaMKII null mutants, LTP can be induced at Schaffer collateral–CA1 pyramidal cell synapses (Silva et al. 1992; Hinds et al. 1998). Moreover, experience-dependent plasticity in the neocortex is not abolished in these knockout animals, suggesting again the engagement of a compensatory mechanism when expression of CaMKII is absent during development (Glazewski et al. 2000). Recent work has demonstrated that developmental compensation in these mutants is likely to be mediated by the ß-isoform of CaMKII because this enzyme is found at unusually high concentrations in the postsynaptic density of the CaMKII null mutants (Elgersma et al. 2002). However, we did not observe an increase of ßCaMKII in synaptosomes isolated from CaMKII^T286A mice, nor were we able to induce LTP at Schaffer commissural–CA1 pyramidal cell synapses, either in vitro (data not shown) or in vivo. These findings are consistent with the observation that experience-dependent neocortical plasticity is abolished in CaMKII^T286A mice (Glazewski et al. 2000). Our data reveal that, despite lack of compensation, LTP can be induced at MPP–granule cell synapses in the dentate gyrus. This observation is consistent with recent findings in the adult rat (Wu et al. 2004; Zhang et al. 2005) where inhibitors of CaMKII signalling that block LTP in the CA1 subfield fail to do so at MPP–granule cell synapses. Here we have extended these findings in two ways. First, we show that LTP at MPP–granule cell synapses in mice can survive a complete disabling of the autophosphorylation mechanism of CaMKII, in contrast to the complete block seen at Schaffer commissural–CA1 synapses; second, we provide an initial analysis of the signalling pathways that mediate LTP at these synapses in the absence of CaMKII signalling. We have shown that PKA and MAPK pathways contribute to LTP in the dentate gyrus of the WT animal when CaMKII signalling is blocked with a pharmacological inhibitor and that these two pathways assume a greater importance in supporting MPP LTP in the CaMKII^T286A mutant mice, where autophosphorylation of CaMKII is completely prevented. Our observations present an intriguing dissociation between the molecular mechanisms for LTP in the two hippocampal subfields.

It has previously been shown that LTP at Schaffer collateral–CA1 synapses shifts from a PKA-dependent mechanism in juvenile animals to a CaMKII-dependent mechanism in the adult animal (Yasuda et al. 2003). Our findings therefore suggest that juvenile features of synaptic function are retained in the adult dentate gyrus. The continuous addition of newly generated neurons into the dentate gyrus (Song et al. 2002; van Praag et al. 2002) makes it possible that at least some neurons in this subfield retain a juvenile phenotype. Alternatively, the requirement for PKA/MAPK signalling in LTP at MPP–granule cell synapses may not relate to the developmental status of neurons, but may instead reflect some other physiological difference between CA1 and the dentate gyrus. Interestingly, it has recently been demonstrated that, in contrast to the MPP, LTP at lateral perforant path (LPP)–granule cell synapses is wholly dependent on CaMKII signalling (Zhang et al. 2005), suggesting that heterogeneous synaptic signalling mechanisms support plasticity at distinct inputs to individual granule cells. We can speculate that newly introduced granule cells in the adult dentate gyrus mature slowly even after they have developed electrical properties (Song et al. 2002) and are incorporated into the neural network of the dentate gyrus. It is possible that their dendrites invade the middle molecular layer, which is innervated by the MPP, for a substantial period before extending to the outer molecular layer, innervated by the LPP. The outer molecular layer is a greater distance from the cell bodies of the granule cells than the middle molecular layer. It may be that the heterogeneous character of signalling at MPP–granule cell synapses reflects two classes of dendrites, one class originating from newborn cells utilizing juvenile signalling mechanisms and the other from mature cells utilizing CaMKII; in contrast the LPP–granule cell population may contain only those cells that are sufficiently mature to have extended dendrites into the outer molecular layer, by which stage CaMKII has become the predominant signalling pathway.

Our results also demonstrate that LTP at MPP synapses can be supported by parallel signalling pathways. This finding is reminiscent of observations at Schaffer collateral–CA1 synapses in 2-week-old rats (Wikstrom et al. 2003). LTP in the MPP can be expressed in adult WT animals in the presence of inhibitors of PKA and MAPK, but in adult CaMKII^T286A mice LTP is severely attenuated by both classes of inhibitor. These results imply that in WT animals autonomous CaMKII activity has the potential to support LTP in the dentate gyrus when alternative signalling pathways are compromised. Only in circumstances where more than one pathway is disabled, as when MAPK or PKA inhibitors are applied to slices from mutant mice lacking autonomously active CaMKII, is there a significant reduction in LTP. Note that our results do not allow us to determine the relative contributions of the CaMKII and cAMP-dependent signalling pathways to the expression of LTP in the dentate gyrus under normal conditions, since it is clear that when one pathway is inhibited by drugs or genetic modification, the other is able to compensate (Fig. 6). Moreover, it is not clear why the dentate gyrus should be endowed with parallel signalling mechanisms when early LTP in the CA1 subfield appears to rely exclusively on the CaMKII pathway.

Autophosphorylation of CaMKII is an attractive candidate mechanism for ensuring that only synapses undergoing coincident pre- and postsynaptic activity undergo LTP, because it could potentially produce changes local to the microenvironment of the synapse without the requirement for whole-cell mechanisms, such as de novo gene expression (Lisman & Goldring, 1988). Our results suggest that cAMP-dependent signalling can somehow mediate a similar process. PKA is capable of autonomous activity after calcium/calmodulin-mediated increase in adenylyl cyclase activity has waned and cAMP concentration has fallen (Greenberg et al. 1987). This independence is due to a persistent reduction in the number of regulatory subunits that normally prevent activity of the catalytic subunit (Hegde et al. 1993). Autonomous PKA activity by this means is, however, dependent on protein synthesis and changes in regulatory subunit turnover, and contributes only to the later phase of synaptic plasticity in Aplysia (Chain et al. 1999), so it does not provide a suitable mechanism for rapidly induced local changes. A possible alternative mechanism could rely on enhanced PKA activity resulting from autophosphorylation of the regulatory subunit and a resultant reduction in its affinity for the catalytic subunit (First et al. 1988). However, this or any other continued post-translational modification of PKA independent of cAMP levels, would be more susceptible to molecular turnover than CaMKII autophosphorylation, because there are only two catalytic subunits in any one holoenzyme of PKA (Taylor et al. 1990), in contrast to the 12 that normally comprise CaMKII (Lisman et al. 2002). Furthermore, in contrast to CaMKII, these subunits do not remain associated with each other once the kinase is activated (Gibbs et al. 1992). Nevertheless, a cAMP-dependent mechanism of this kind must maintain localized change at MPP-granule cell synapses for at least as long as it takes for more permanent alterations, such as structural change, to take place. This may involve other, as yet unidentified signalling mechanisms.

Our observations are relevant to the finding that mice that do not express the endogenous inhibitor (I-1) of protein phosphatase 1 (PP1) exhibit normal LTP at Schaffer commissural–CA1 pyramidal cell synapses, but deficient LTP at perforant path–granule cell synapses (Allen et al. 2000). This dissociation is the converse of our observations in the CaMKII^T286A mutant mice and may well also relate to the differential involvement of PKA at the two populations of synapses. The kinases PKA and CaMKII are thought to respond to calcium influx during the induction of LTP, but this process is regulated by the activity of phosphatases, such as PP1, that dephosphorylate activated kinases, such as CaMKII, and kinase substrates such as glutamate receptors (Blitzer et al. 1998; Malleret et al. 2001). I-1 is activated by PKA (Blitzer et al. 1998) and can then inhibit the activity of PP1 (Hemmings et al. 1984). The greater importance of PKA in the induction of LTP at perforant path–granule cell synapses, as demonstrated here, may explain why the removal of I-1 has a more significant impact at these synapses than at Schaffer commissural–CA1 pyramidal synapses.

In summary, while both CaMKII and cAMP-dependent signalling pathways can support early LTP at MPP–granule cell synapses, neither pathway is obligatory. The cAMP-dependent pathway mediates LTP at Schaffer commissural–pyramidal cell synapses in juvenile animals, and the continuing availability of cAMP-dependent signalling during adulthood suggests that plasticity in the dentate gyrus retains juvenile characteristics. Finally, our results make clear that autophosphorylation of CaMKII is not a universal requirement for NMDA receptor-dependent LTP.

	Footnotes

 
S. F. Cooke, J. Wu and F. Plattner contributed equally to this work.

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	Acknowledgements

 
This work was supported in part by predoctoral fellowships from the Schering Stiftung (to F.P.) and the Boehringer Ingelheim Fonds (to M.P.) as well as by an MRC Career Establishment grant (to K.P.G.). We would also like to thank Noah Russell and Derek Brewer for technical assistance.

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