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Journal of Neurophysiology logoLink to Journal of Neurophysiology
. 2016 Aug 17;116(5):2140–2151. doi: 10.1152/jn.00518.2016

Developmental restoration of LTP deficits in heterozygous CaMKIIα KO mice

Dayton J Goodell 1, Tim A Benke 1,2, K Ulrich Bayer 1,✉
PMCID: PMC5102309  PMID: 27535377

CaMKIIα+/− mice were described previously to have a schizophrenia-related phenotype. While the long-term potentiation (LTP) deficits seen here in young CaMKIIα+/− mice are restored during development, the initially normal basal synaptic transmission in the hippocampus becomes impaired.

Keywords: LTP, LTD, schizophrenia, CaMKII, synaptic transmission

Abstract

The Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a major mediator of long-term potentiation (LTP) and depression (LTD), two opposing forms of synaptic plasticity underlying learning, memory and cognition. The heterozygous CaMKIIα isoform KO (CaMKIIα+/−) mice have a schizophrenia-related phenotype, including impaired working memory. Here, we examined synaptic strength and plasticity in two brain areas implicated in working memory, hippocampus CA1 and medial prefrontal cortex (mPFC). Young CaMKIIα+/− mice (postnatal days 12–16; corresponding to a developmental stage well before schizophrenia manifestation in humans) showed impaired hippocampal CA1 LTP. However, this LTP impairment normalized over development and was no longer detected in older CaMKIIα+/− mice (postnatal weeks 9–11; corresponding to young adults). By contrast, the CaMKIIα+/− mice failed to show the developmental increase of basal synaptic transmission in the CA1 seen in wild-type (WT) mice, resulting in impaired basal synaptic transmission in the older CaMKIIα+/− mice. Other electrophysiological parameters were normal, including mPFC basal transmission, LTP, and paired-pulse facilitation, as well as CA1 LTD, depotentiation, and paired-pulse facilitation at either age tested. Hippocampal CaMKIIα levels were ∼60% of WT in both the older CaMKIIα+/− mice and in the younger WT mice, resulting in ∼30% of adult WT expression in the younger CaMKIIα+/− mice; levels in frontal cortex were the same as in hippocampus. Thus, in young mice, ∼30% of adult CaMKIIα expression is sufficient for normal LTD and depotentiation, while normal LTP requires higher levels, with ∼60% of CaMKIIα expression sufficient for normal LTP in adult mice.

NEW & NOTEWORTHY

CaMKIIα+/− mice were described previously to have a schizophrenia-related phenotype. While the long-term potentiation (LTP) deficits seen here in young CaMKIIα+/− mice are restored during development, the initially normal basal synaptic transmission in the hippocampus becomes impaired.

cognitive dysfunction and specific deficits in working and episodic memory are a hallmark of schizophrenia that are predictive of disease outcome severity (Tandon et al. 2009). Large genome screens of schizophrenia patients have found significant mutations in postsynaptic signaling complexes related to glutamatergic neuronal transmission and NMDA type glutamate receptor (NMDAR) signaling pathways, including in the calcium/calmodulin (CaM)-dependent protein kinase IIα (CaMKIIα) (Harrison and Weinberger 2005; Fromer et al. 2014; Purcell et al. 2014). The NMDAR is a Ca2+ permeable glutamate receptor important in the long-term potentiation (LTP) and long-term depression (LTD) of synaptic strength (Shipton and Paulsen 2013). These opposing forms of synaptic plasticity are thought to underlie cognition, learning, and memory (Martin et al. 2000; Kandel et al. 2014) and are impaired in schizophrenia patients (Fitzgerald et al. 2004; Daskalakis et al. 2008; Frantseva et al. 2008; Hasan et al. 2012). CaMKIIα plays a central role in decoding and transducing postsynaptic Ca2+ influx through NMDARs in response to glutamate stimuli (Coultrap and Bayer 2012; Lisman et al. 2012; Hell 2014). Full genetic knockout (KO) of CaMKIIα (CaMKII−/−) results in impaired LTP in 1- to 4-mo-old mice and impaired LTD in 2- to 3-wk-old mice at the hippocampal CA3 to CA1 synapse, as well as in impairments in several hippocampus-dependent behavioral tasks (Silva et al. 1992a; 1992b; Hinds et al. 1998; Coultrap et al. 2014).

The de novo CaMKII mutations that have been found in schizophrenia patients are heterozygous and, interestingly, the heterozygous CaMKIIα (CaMKIIα+/−) KO mice were described to show a schizophrenia-related phenotype (Yamasaki et al. 2008). The CaMKIIα+/− mice share several behavioral deficits with the full CaMKIIα KO, including deficient learning and memory in the Morris water maze and deficient retention of contextual fear conditioning in adult mice (Silva et al. 1996; Frankland et al. 2001). Additionally, adult CaMKIIα+/− mice have working memory deficits noted by repeat entry errors in the radial arms version of the Morris water maze and the T-maze (Yamasaki et al. 2008; Matsuo et al. 2009). Other correlates to schizophrenia described for the CaMKIIα+/− mice include abnormal social behaviors characterized by increased aggression toward their cagemates, hyperactivity, disrupted circadian activity (Yamasaki et al. 2008), and an increase in dopamine (D2High) receptors (a major target of antipsychotic medications) (Novak and Seeman 2010).

Although the onset of schizophrenia normally occurs in late adolescence to early adulthood (Tandon et al. 2009), many schizophrenia patients have preonset cognitive dysfunction at much younger ages (Nuechterlein et al. 2014). This indicates that early network malformation may play a role in the pathogenesis of schizophrenia. Thus the deficits in forms of LTP and LTD measured in schizophrenia patients by transcranial stimulation (Fitzgerald et al. 2004; Daskalakis et al. 2008; Frantseva et al. 2008; Hasan et al. 2012) may also precede the onset of the disease.

The hippocampus and medial prefrontal cortex (mPFC), as well as their reciprocal connectivity, are thought to be important for learning, proper memory formation, and working memory (Cohen 2011; Gordon 2011; van Kesteren et al. 2012; Kandel et al. 2014). Here, we tested and compared bidirectional synaptic plasticity and basal synaptic function in area CA1 of the hippocampus in young [postnatal day (p)12–16] and young adult (9–11 wk) CaMKIIα+/− mice, ages corresponding to preonset or disease manifestation in human schizophrenia patients, respectively. We found a specific impairment in CA1 LTP in p12–16 young mice that was reversed by 9–11 wk. By contrast, over the same time period, a deficit in CA1 basal synaptic transmission developed. Additionally, we examined synaptic functions in the mPFC at 9–11 wk and found both LTP and basal transmission to be normal (along with any other synaptic functions tested in either mPFC or CA1). Thus the only synaptic deficit found here in either hippocampus or mPFC of the CaMKIIα+/− mice at older ages (at which the impaired working memory is manifested) was basal transmission in the CA1.

MATERIALS AND METHODS

Materials

All reagents were purchased from Sigma-Aldrich unless otherwise noted.

Mice

All procedures were approved by the University of Colorado Institutional Animal Care and Use Committee and carried out in accordance with the National Institutes of Health best practices for animal use. The University of Colorado Anschutz Medical Campus is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care, International (AAALAC). The day of birth was defined as p1. Male and female p12–16 and male 9–11 wk CaMKIIα+/− mice on C57/Bl6 background were taken from het/het or het/wt breeding within our CaMKIIα−/− line described elsewhere (Coultrap et al. 2014). Primarily littermates, but also WT from WT/WT breeders derived from the CaMKIIα−/− line, were used as controls. A minimum of four animals (from at least two separate litters) for each genotype were used for electrophysiology, and the number of mice (N) and the number of slices (n) are indicated for each experiment. No more than three slices were used per mouse for any plasticity protocol. Three different animals per genotype were used for the protein expression experiments.

Electrophysiological Field Recordings

Hippocampal preparation.

Slice electrophysiology was performed essentially as described previously (Buard et al. 2010; Corser-Jensen et al. 2014; Coultrap et al. 2014; Barcomb et al. 2016) with some modification. Mice were briefly anesthetized with isoflourane (until the loss of foot pinch response) and rapidly decapitated. Brains were dissected into ice-cold high sucrose dissection solution containing the following (in mM): 220 sucrose, 12 MgSO4, 10 glucose, 0.2 CaCl2, 0.5 KCl, 0.65 NaH2PO4, 13 NaHCO3, and 1.8 ascorbate. Hippocampi were removed and transverse slices (400 μm) were made using a tissue chopper (McIlwain). Slices were transferred into 32°C artificial cerebral spinal fluid (ACSF) containing the following (in mM): 124 NaCl, 2 KCl, 1.3 NaH2PO4, 26 NaHCO3, 10 glucose, 2 CaCl2, 1 MgSO4, and 1.8 ascorbate. All solutions were constantly infused with 95% O2/5% CO2. Slices were recovered for a minimum of 1.5 h before experimentation. During recordings slices were continually perfused with 30.5 ± 0.5°C ACSF at a rate of 3.5 ± 0.5 ml/min in a recirculation volume of 50 ml. Recordings of field excitatory postsynaptic potentials (fEPSPs) were made in the CA1 dendritic layer using a glass micropipette (typical resistance 0.4–0.8 MΩ when filled with ACSF) in response to 0.1-ms constant-current stimulation with a tungsten bipolar electrode placed in the Schaffer collaterals at the CA2 to CA1 interface. Stimuli were delivered every 20 s and three responses (1 min) were averaged for analysis. After a stable response was achieved, an input/output (I/O) curve was generated by increasing the amplitude of stimulus intensity at a constant interval until a maximal response or population spike was noted. Stimulus intensity was set to 40 or 70% of the maximum slope for LTP and LTD experiments, respectively. Paired-pulse recordings were done post-I/O with a 50-ms interpulse interval from 40% of maximal response before baseline acquisition. A stable baseline was maintained for a minimum of 20 min before induction protocols. LTP was induced with two trains of 100 pulses delivered at 100 Hz separated by 20 s (HFS). LTD and depotentiation were induced with 900 pulses at 1 Hz (LFS). Paired-pulse recordings were again taken at the end of each experiment, and stimulus intensity was reduced if necessary to avoid contamination with population spiking in the second pulse. Responses were recorded and analyzed using WIN LTP software (Anderson and Collingridge 2001). Slope (mV/ms) was defined as the initial rise from 10 to 60% of response peak and excluded the fiber volley. Slices with a maintained population spike 30 min post-LTP induction were eliminated from all analysis (leading to the following exclusions: 4 of 20 slices from 9–11 wk WT; 3 of 15 slices from 9–11 wk CaMKIIα+/−; none from p12–16 of either genotype). Similarly, data containing population spikes in the I/O and paired-pulse responses were eliminated.

Cortical preparation.

Euthanasia and brain dissection were conducted the same as for the hippocampal preparation. Coronal whole brain slices containing the mPFC were made using a vibratome (Leika) in ice-cold cutting solution and recovered for a minimum of 1.5 h in ACSF. Recordings were made in mPFC layer 5 using a glass micropipette (typical resistance of 0.4–0.8 MΩ when filled with ACSF) in response to 0.1-ms constant-current stimulation with a tungsten bipolar electrode placed parallel to the recording electrode in layer 2–3. Stimuli were delivered every 20 s and three responses (1 min) were averaged for analysis. I/O, paired-pulse, and LTP induction were conducted using the same protocols as for the hippocampal preparation. Due to slope contamination, amplitude of response peak was calculated for comparisons in mPFC recordings.

Western Blotting and Protein Expression Levels

Euthanasia and brain dissection were conducted as in the electrophysiology experiments for p12–16 and 9–11 wk WT and CaMKIIα+/− mice to examine hippocampal expression (N = 3 each age and genotype) and 9–11 wk WT and CaMKIIα+/− mice (N = 3) to examine hippocampal and cortex expression. A 1-mm section of cortex containing the mPFC ranging from approximately bregma 1 to 2 mm was used for cortical comparisons and here called “frontal cortex.” Hippocampi and frontal cortex were dissected and sonicated in 1% SDS, 1 mM EDTA, and 10 mM Tris, pH 8. Protein content was determined using the BCA method (Bradford). Ten micrograms of total protein were subjected to SDS-PAGE on a 10% polyacrylamide gel and transferred to a PVDF membrane for Western blotting essentially as previously described (Goodell et al. 2014). Blots were probed for the α- and β-isoforms of CaMKII (BD Biosciences) 1:2,000 in 1% milk dissolved in Tris buffered saline containing 0.1% Tween-20, pH 7.55 (TBST) followed by horseradish peroxidase-conjugated sheep anti-mouse secondary (GE Healthcare) 1:5,000 in 1% milk in TBST. Blots were stripped and reprobed for β-actin loading control (Cell Signaling) 1:2,500 in 1% BSA in TBST followed by horseradish peroxidase-conjugated donkey anti-rabbit secondary (GE Healthcare) 1:5,000 in 1% BSA in TBST. Blots were developed using Western Lighting Plus ECL (Perkins Elmer). Image acquisition and densitometry were performed as previously described (Goodell et al. 2014; Barcomb et al. 2015).

Statistical Analysis

Statistical analysis was performed in SPSS 22 (IBM) or GraphPad Prism. Specific tests are indicated in the text and figure legends. Data are expressed as mean ± SE using slice number (n) for electrophysiological experiments. Any statistical significance determined using slice number (n) was confirmed using the number of animals from which the slices were derived (N) to determine experimental subject number and standard error. Analysis of variance is abbreviated as ANOVA.

RESULTS

Hippocampal CA1 LTP but Not Depotentiation or LTD Is Impaired in p12–16 CaMKIIα+/− Mice

As schizophrenia patients often have preonset developmental deficits including early cognitive dysfunction (Nuechterlein et al. 2014), synaptic plasticity in hippocampal CA1 was here first examined in p12–6 young CaMKIIα+/− mice. Synaptic responses were recorded in CA1 in response to stimulation of CA1 Shaffer collaterals, and differences were assessed as differences between the last 20 min of baseline and the last 10 min of either LTP, depotentiation, or LTD. While both WT and CaMKIIα+/− mice showed significant HFS (2 × 100 Hz) induced potentiation, LTP in slices from CaMKIIα+/− mice was significantly impaired (Fig. 1A) [WT (N = 5, n = 6) = 173.33 ± 4.73%, CaMKIIα+/− (N = 5, n = 7) = 149.45 ± 4.6%, F(5) = 59.215, P < 0.001, one-way ANOVA, P < 0.001 difference from baseline in both genotypes, P = 0.004 difference between genotypes, Tukey's honestly significant difference (HSD)]. Depotentiation induced with LFS (900 × 1 Hz) 30 min post-LTP induction did not differ between groups, neither without (Fig. 1A; P = 0.154) nor with renormalization to the last 10 min of LTP [Fig. 1B; WT = 63.48 ± 4.02%, CaMKIIα+/− = 62.77 ± 3.02%, F(3) = 83.97, P < 0.001, one-way ANOVA, P < 0.001 difference from baseline in both genotypes, P = 1.00 no difference between genotypes, Tukey's HSD]. As NMDAR-dependent LTD is abolished in the full CaMKIIα KO mice at this age (Coultrap et al. 2014), LTD was also tested here for the heterozygous CaMKIIα+/− mice. Slices from both genotypes had significant de novo LFS induced LTD and did not differ between genotypes indicating that LTD is normal in young CaMKIIα+/− mice [Fig. 1C; WT (N = 5, n = 6) = 65.71 ± 6.95%, CaMKIIα+/− (N = 4, n = 6) = 68.98 ± 5.65%, F(3) = 17.79, P < 0.001, one-way ANOVA, P < 0.001, difference from baseline in both genotypes, P = 0.95, no difference between genotypes, Tukey's HSD].

Fig. 1.

Fig. 1.

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Hippocampal CA1 LTP but not depotentiation or long-term depression (LTD) is impaired in the young p12–16 CaMKIIα+/− mice. Field excitatory postsynaptic potentials (fEPSPs) were recorded in the CA1 dendritic layer in response to CA1 Schaffer collateral stimulation. Long-term potentiation (LTP) was induced by high frequency stimulation (HFS; two 1 strains of 100-Hz stimulation separated by 20 s). LTD and depotentiation were induced by low -frequency stimulation (LFS; 900 pulses at 1 Hz). A: LTP and depotentiation plots and quantification from young p12–16 mice. Quantifications are from time points A, B, and C as indicated, representing a 20-min baseline, and last 10 min of LTP and depotentiation respectively. Right: example traces (average of 3 responses, representing 1 min) pre (black)- or post (grey)-HFS stimulation from time points A and B. B: depotentiation plot and quantification after renormalization to the last 10 min of LTP. Quantifications are from time points A and B as indicated, representing the renormalized last 10 min of LTP and the last 10 min of depotentiation. Right: example traces (average of 3 responses, representing 1 min) pre (black)- or post (grey)-LFS stimulation from time points A and B. LTP traces are the same as time point B traces in A. C: LTD plot and quantification from young postnatal day (p) 12–16 mice. Quantifications are from time points A and B as indicated, representing a 20-min baseline and last 10 min of LTD, respectively. Right: example traces (average of 3 responses, representing 1 min) pre (black)- or post (grey)-LFS from time points A and B. WT, wild type. **P < 0.01, ***P < 0.001 significantly different from prestimulus value indicating LTP, depotentiation, or LTD or as indicated between groups, one-way ANOVA followed by Tukey's honestly significant difference (HSD); NS, not significant.

Hippocampal CA1 LTP and Depotentiation Are Normal in 9–11 wk CaMKIIα+/− Mice

Due to our above finding that CA1 LTP is impaired in young mice, we next tested LTP also in young adult (9–11 wk) mice, the age at which working memory deficits were described in the CaMKIIα+/− mice (Yamasaki et al. 2008). We found that both WT and CaMKIIα+/− mice had significant LTP induced by HFS (2 × 100 Hz) that did not differ between genotypes, indicating that the LTP deficit seen in young mice was completely normalized already by 9–11 wk in CaMKIIα+/− mice [Fig. 2A; WT (N = 9, n = 16) = 149.39 ± 6.59%, CaMKIIα+/− (N = 8, n = 12) = 145.42 ± 7.83%, F(3) = 29.89, P < 0.001, one-way ANOVA, P < 0.001 difference from baseline in both genotypes, P = 0.95, no difference between genotypes, Tukey's HSD]. Consistent with these results, prior studies using theta burst stimulation have also indicated normal hippocampal CA1 LTP in older CaMKIIα+/− mice (Silva et al. 1996; Frankland et al. 2001).

Fig. 2.

Fig. 2.

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Hippocampal CA1 LTP and depotentiation are normal in the older 9–11 wk CaMKIIα+/− mice. fEPSPs were recorded in the CA1 dendritic layer in response to CA1 Schaffer collateral stimulation. LTP was induced by HFS (two 1 strains of 100-Hz stimulation separated by 20 s). Depotentiation was induced by LFS (900 pulses at 1 Hz). A: LTP plot and quantification from 9–11 wk mice. Quantifications are from time points A and B as indicated, representing 20 min baseline, and last 10 min of LTP, respectively. Right: example traces (average of 3 responses, representing 1 min) pre (black)- or post (grey)-HFS from time points A and B. B: Depotentiation plot and quantification after renormalization to the last 10 min of LTP. Quantifications are from time points A and B as indicated, representing the renormalized last 10 min of LTP and the last ten min of depotentiation. Right: example traces (average of 3 responses, representing 1 min) pre (black)- or post (grey)-LFS from time points A and B. LTP traces are the same as time point B traces in A. ***P < 0.001, significantly different from pre stimulus value indicating LTP or depotentiation; NS, not significant, one-way ANOVA followed by Tukey's HSD.

A subset of slices subjected to LTP were additionally depotentiated 60 min post-LTP with LFS (900 × 1 Hz). Slices from both genotypes had significant depotentiation (from last 10 min of LTP) that did not differ between genotypes, indicating that depotentiation is normal in CaMKIIα+/− mice also at this age [Fig. 2B; WT (N = 8, n = 11) = 86.16 ± 2.87%, CaMKIIα+/− (N = 7, n = 11) = 85.81 ± 3.08%, F(3) = 14.81, P < 0.001, one-way ANOVA, P < 0.001 difference from baseline in both genotypes, P = 1.00, no difference between genotypes, Tukey's HSD].

While there was no effect of the genotype on depotentiation, there was a significant main effect of age when all depotentiation data was compared, with p12–16 young mice of both genotypes having significantly more depotentiation (with a reduction in slope of ∼35%; see Fig. 1B) compared with 9–11 wk mice [with a reduction in slope of ∼15%; see Fig. 2B; F(1,34) = 49.66, P < 0.001, two-way ANOVA, age × genotype, P < 0.001 WT difference between ages, P < 0.001 CaMKIIα+/− difference between ages, Tukey's HSD]. Thus, as described for NMDAR-dependent de novo LTD in naïve slices (Dudek and Bear 1993), depotentiation of previously potentiated slices decreases with developmental age.

Hippocampal CA1 Paired-Pulse Ratio Is Normal in Both Young and Older CaMKIIα+/− Mice

To assess presynaptic function in the two ages of CaMKIIα+/− mice, paired-pulse ratios were conducted at the beginning of each experiment (after slice stabilization and the generation of the I/O curve but before baseline acquisition) and also at the end of each experiment (post a 30- to 60-min run out after plasticity inducing stimuli). We found no differences in paired-pulse ratios (50-ms interpulse interval) either before or after the delivery of plasticity inducing stimuli within genotype or between genotypes in p12–16 mice [Fig. 3A; WT n = 8 pre, 8 post, CaMKIIα+/− n = 11 pre, 8 post, F(3) = 0.096, P = 0.962, one-way ANOVA, no post hoc]. This was also true for 9–11 wk mice [Fig. 3B; WT n = 16 pre, 9 post, CaMKIIα+/− n = 12 pre, 9 post, F(3) = 0.504, P = 0.682, one-way ANOVA, no post hoc].

Fig. 3.

Fig. 3.

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Hippocampal CA1 paired-pulse ratio is normal in both young and older CaMKIIα+/− mice. Paired pulses (50-ms interpulse interval) were conducted at the beginning (after slice stabilization) and end of each experiment. A: quantification of paired-pulse ratio (first pulse/second pulse) from p12–16 young mice recorded at the beginning and end of each experiment. Right: example paired-pulse traces (average of 3 responses, representing 1 min) (pulse 1 black, pulse 2 grey) recorded at the beginning and end of each experiment. B: quantification of paired-pulse ratio (first pulse/second pulse) from 9–11 wk mice. Right: example traces (average of 3 responses, representing 1 min) (pulse 1 black, pulse 2 grey) recorded at the beginning and end of each experiment. C: percent amplitude of facilitation and decay during the first 50 pulses of 100-Hz stimuli used to induce LTP from young p12–16 mice. D: percent amplitude of facilitation and decay during the first 50 pulses of 100-Hz stimuli used to induce LTP from 9–11 wk mice. NS, not significant, one-way ANOVA for A and B, and for C, not significant at any pulse number by nonpaired, two-tailed t-test.

Because paired-pulse deficits have been noted in CaMKIIα+/− mice previously (Chapman et al. 1995; Silva et al. 1996), we further explored presynaptic function by quantifying the facilitation and decay during the first 50 pulses of the high-frequency stimuli used to induce LTP (100 Hz; Fig. 3, C and D). No difference was noted between genotypes at any pulse number (nonpaired two-way Student's t-test of each pulse) further indicating a similarity in presynaptic function between genotypes. When paired-pulse facilitation between young and older mice for each genotype was compared, p-12–16 young WT mice showed a significant enhancement [t(22) = −4.02, P = 0.001, nonpaired two-tailed t-test] and a similar trend that narrowly missed significance was also seen in CaMKIIα+/− mice [t(21) = −2.06, P = 0.052; see Fig. 3, A and B]. Consistent with the increase in paired-pulse facilitation, an enhancement in the facilitation of response during HFS (100 Hz) was also noted at the younger age for both genotypes during the initial HFS pulses. Within ∼20 pulses, the increased facilitation in young mice returned to the facilitation levels seen in the older mice and the facilitation had a similar decay in both age groups (nonpaired, two-tailed t-test of each pulse; see Fig. 3, C and D).

Additive Effects of Age and Genotype on CaMKIIα Expression in the Hippocampus Result in Four Times Lower Expression in the Young CaMKIIα+/− Mice

Expression of CaMKIIα in rats and mice begins around p5 and does not reach full expression levels in the hippocampus until early adulthood, while the CaMKIIβ-isoform is expressed already at birth and plateaus at approximately p8 (Kelly and Vernon 1985; Kelly et al. 1987; Burgin et al. 1990; Bayer et al. 1999). Thus the ratio of CaMKIIα to CaMKIIβ in the hippocampus changes during development from ∼1:4 at p5 to ∼3:1 at p90 due mostly to the increases in CaMKIIα expression, with little change in CaMKIIβ expression during development (Kelly et al. 1987). Here, we directly compared CaMKIIα and -β expression in the two ages of mice used in this study with Western blot analysis of hippocampal extracts taken from p12–16 and 9–11 wk WT and CaMKIIα+/− mice (N = 3 each age and genotype). As expected, CaMKIIα expression levels in WT were slightly more than double that in age matched CaMKIIα+/− mice [Fig. 4, A and B; main effect of genotype, F(1,11) = 38.46, P < 0.001, genotype × age two-way ANOVA, P = 0.004 difference between genotype 9–11 wk mice, P = 0.027 difference by genotype in p12–16 young mice, Tukey's HSD]. Additionally, there was also an approximately twofold difference between CaMKIIα expression at the different ages with young mice having ∼60% of 9–11 wk expression [Fig. 4, A and B; main effect of age, F(1,11) = 34.58, P < 0.001, P = 0.005, WT difference between ages, P = 0.037, CaMKIIα+/− difference between ages, Tukey's HSD]. CaMKIIα expression levels in the young CaMKIIα+/− mice were therefore ∼30% of the total WT expression in 9–11 wk WT mice (P < 0.001, p12–16 CaMKIIα+/− difference from 9–11 wk WT, Tukey's HSD).

Fig. 4.

Fig. 4.

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Additive effects of age and genotype on CaMKIIα expression in the hippocampus results in 4 times lower expression in the young CaMKIIα+/− mice. Hippocampal homogenates were made from p12–16 and 9–11 wk WT and CaMKIIα+/− mice (N = 3 for each age and genotype) and compared with SDS-PAGE and Western blot analysis for CaMKIIα and β, normalized to β-actin loading control. Densitometry is quantified as immunodetection value (IDV) normalized to values from 9–11 wk WT mice. A: representative blots. B: quantification of CaMKIIα expression. C: quantification of CaMKIIβ expression. *P < 0.05, **P < 0.01, ***P < 0.001, significantly different from 9–11 wk WT mice or between indicated groups; NS, not significant, two-way ANOVA (age × genotype) followed by Tukey's HSD; #main effect of age and different by t-test between respective ages but not significant by Tukey's HSD multiple comparison.

Also as expected, CaMKIIβ expression levels in the hippocampus did not differ between genotypes [Fig. 4, A and C; no main effect of genotype, F(1,11) = 0.11, P = 0.749 genotype × age two-way ANOVA]. There was a significant main effect of age on CaMKIIβ expression [F(1,11) = 13.39, P = 0.006]; however, differences did not reach significance in multiple comparison post hoc analysis indicating that the increase in CaMKIIβ expression between p12–16 young mice and 9–11 wk mice over development was minimal.

mPFC LTP and Paired-Pulse Facilitation Is Normal in 9–11 wk CaMKIIα+/− Mice

CaMKIIα+/− mice have deficits in cortical LTP in the parietal and visual cortex (Frankland et al. 2001), but the mPFC, an area important in working memory and implicated in schizophrenia (Eisenberg and Berman 2010; Cohen 2011; Lewis 2012; Li et al. 2015), has not been previously examined. Synaptic responses were recorded in layer 5 in response to stimulation in layers 2–3, and amplitude of response was used for comparison. Both WT and CaMKIIα+/− mice showed significant HFS (2 × 100 Hz)-induced potentiation, and did not differ between genotype indicating normal LTP in the mPFC of CaMKIIα+/− mice [Fig. 5A; WT (N = 4, n = 12) = 138.61 ± 7.87%, CaMKIIα+/− (N = 4, n = 10) = 133.20 ± 7.35%, F(3) = 14.85, P < 0.001, one-way ANOVA, WT, P < 0.001, CaMKIIα+/−, P = 0.001, difference from baseline; P = 0.897, no difference between genotypes, Tukey's HSD]. Similar to hippocampus, no differences in paired-pulse facilitation were noted in mPFC [Fig. 5B; WT N = 4, n = 12 CaMKIIα+/− N = 4, N = 10, F(1,3) = 0.943, P = 0.429, one-way ANOVA, no post hoc].

Fig. 5.

Fig. 5.

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mPFC LTP and paired-pulse facilitation is normal in 9–11 wk CaMKIIα+/− mice. fEPSPs were recorded in mPFC layer 5 in response to layer 2–3 stimulation. LTP was induced by HFS (two 1 strains of 100-Hz stimulation separated by 20 s). Paired pulses (50-ms interpulse interval) were conducted at the beginning (after slice stabilization) and end of each experiment. A: LTP plots and quantification from young p12–16 mice. Quantifications are from time points A and B as indicated, representing 15 min baseline, and last 10 min of LTP respectively. Right: example traces (average of 3 responses, representing 1 min) pre (black)- or post (grey)-HFS stimulation from time points A and B. B: quantification of paired-pulse ratio (first pulse/second pulse). Right: example traces (average of 3 responses, representing 1 min) (pulse 1, black, pulse 2 grey) recorded at the beginning and end of each experiment. **P < 0.01, ***P < 0.001 significantly different from prestimulus value indicating LTP, one-way ANOVA followed by Tukey's HSD; NS, not significant.

Frontal Cortex CaMKIIα Expression Does Not Differ From Hippocampus in 9–11 wk Mice

A past study has indicated that CaMKIIα expression in the whole cortex is ∼60% of hippocampal levels (Erondu and Kennedy 1985), which would result in cortex CaMKIIα levels in older CaMKIIα+/− mice similar to what we noted in the hippocampus of young CaMKIIα+/− mice (∼30%). As we found that ∼30% of adult CaMKIIα expression was insufficient to allow the expression of normal LTP in the hippocampus of young CaMKIIα+/− mice, we next directly compared CaMKIIα and -β expression in the frontal cortex of 9–11 wk WT and CaMKIIα+/− mice (N = 3, frontal cortex and hippocampus taken from the same mouse). As we saw before in hippocampus, in this set of animals CaMKIIα+/− mice had roughly half of WT expression and this held true for frontal cortex also [Fig. 6, A and B, main effect of genotype, F(1,11) = 33.63, P < 0.001, brain area × genotype two-way ANOVA, P = 0.032 difference between genotype in hippocampus, P = 0.007 difference by genotype in cortex, Tukey's HSD between the genotypes in both brain areas]. In contrast to the previous reports in whole cortex, we found no differences in CaMKIIα expression between hippocampus and frontal cortex for either genotype [no main effect of brain area, F(1,11) = 0.599, P = 0.461], consistent with the notion that ∼60% of adult WT hippocampal levels is sufficient for normal LTP induction also in mPFC. No differences were noted in CaMKIIβ expression between brain areas or between genotypes [Fig. 6, A and C; no main effect of brain area, F(1,11) = 0.002, P = 0.963, no main effect of genotype, F(1,11) = 0.614, P = 0.456].

Fig. 6.

Fig. 6.

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Frontal cortex CaMKIIα expression does not differ from hippocampus in 9–11 wk mice. Hippocampal and frontal cortex homogenates were made from 9–11 wk WT and CaMKIIα+/− mice (N = 3 hippocampus and cortex were taken from same mouse) and compared with SDS-PAGE and Western blot analysis for CaMKIIα and β, normalized to β-actin loading control. Densitometry is quantified as immunodetection value (IDV) and normalized to values from WT hippocampus. A: representative blots. B: quantification of CaMKIIα expression. C: quantification of CaMKIIβ expression. *P < 0.05, **P < 0.01, two-way ANOVA (brain area × genotype) followed by Tukey's HSD; NS, not significant.

CaMKIIα+/− Mice Show a Selective Deficit in Synaptic Efficacy at the Hippocampal CA1 Synapse That Develops with Age

Full genetic knockout of CaMKIIα results in a decreased basal synaptic transmission as measured by a decrease in the slope of input/output (I/O) curve at the hippocampal CA1 synapse (Hinds et al. 1998). Thus input/output (I/O) curves were here generated by increasing the stimulus intensity by 5 μA in 1-min intervals. The slope (mV/ms) for hippocampus or amplitude (mV) for mPFC was plotted against stimulus intensity and regression analysis to generate best-fit lines was conducted. We found that despite the deficits seen in LTP, p12–16 CaMKIIα+/− mice had no difference in I/O response [Fig. 7A; WT, N = 4, n = 10, r2 = 0.569, CaMKIIα+/−, N = 4, n = 10, r2 = 0.633, F(3,94) = 0.544, P = 0.653, one curve fits both genotypes, sum of squares F-test]. Thus basal synaptic strength is not a main contributor for the reduction in LTP seen in p12–16 young CaMKIIα+/− mice. Interestingly, although LTP was no longer impaired at 9–11 wk, 9–11 wk CaMKIIα+/− mice had developed a significant reduction in their I/O response [Fig. 7B; WT, N = 4, n = 9, r2 = 0.720; CaMKIIα+/−, N = 4, n = 9, r2 = 0.533, F(3,80) = 7.134, P < 0.001, different curves are needed for each genotype, sum of squares F-test]. Basal synaptic strength increased significantly during development from p12–16 to 9–11 wk in WT mice [see Fig. 7, A and B; WT young, N = 4, n = 10, r2 = 0.569; WT old, N = 4, n = 9, r2 = 0.720, F(3,86) = 5.531, P = 0.002, different curves are needed for each age, sum of squares F-test] but not in the CaMKIIα+/− mice [CaMKIIα+/− young, N = 4, n = 10, r2 = 0.626; CaMKIIα+/− old, N = 4, n = 9, r2 = 0.524, F(3,88) = 0.554, P = 0.647, one curve fits both ages, sum of squares F-test]. Thus the reduced basal synaptic strength in the older CaMKIIα+/− mice is because of the lack of the developmental increase in synaptic strength seen in WT mice.

Fig. 7.

Fig. 7.

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CaMKIIα+/− mice show a selective deficit in synaptic efficacy at the hippocampal CA1 synapse that develops with age. Input/output (I/O) curves were generated by increasing stimulus strength by 5 μA until responses leveled off or in hippocampus showed signs of population spiking. Regression analysis of the slope (mV/ms) of response vs. the corresponding stimulus intensity (μA) are compared; A–C include best fit lines for each genotype (left) and representative traces (right). A: I/O plot from hippocampus CA1 in p12–16 mice. B: I/O plot from hippocampus CA1 in 9–11 wk mice. C: I/O plot for mPFC layer 5 from 9–11 wk mice. ***P < 0.001, different curves are needed for each genotype; NS, one curve fits both genotypes, sum of squares F-test. For clarity, best-fit lines are shown for each genotype rather than one fit in A and C.

Although I/O responses were impaired in hippocampus in CaMKIIα+/− mice at 9 to 11 wk old, we found no such change in the I/O in mPFC [Fig. 7C; WT, N = 4, n = 12, r2 = 0.402, CaMKIIα+/−, N = 4, n = 10, r2 = 0.294, F(3,153) = 0.105, P = 0.957, one curve fits both genotypes, sum of squares F-test]. Thus the only impairments we noted in CaMKIIα+/− mice at the age when working memory impairments are apparent was the reduction in hippocampal CA1 synaptic efficacy.

DISCUSSION

CaMKIIα mediates NMDAR-dependent LTP and LTD, two opposing forms of synaptic plasticity thought to underlie learning, memory, and cognition (Coultrap and Bayer 2012; Lisman et al. 2012; Coultrap et al. 2014; Hell 2014). Furthermore, the CaMKIIα+/− mice have been described to have a schizophrenia-related phenotype, specifically including impaired working memory (Yamasaki et al. 2008). Thus we here examined CaMKIIα+/− mice for synaptic functions in hippocampus and prefrontal cortex, two brain regions implicated in both working memory and schizophrenia (Eisenberg and Berman 2010; Cohen 2011; Lewis 2012; Li et al. 2015). Young CaMKIIα+/− mice (p12–16) had deficits in hippocampal CA1 LTP, while LTD, depotentiation, and paired-pulse ratio were normal. Interestingly, the LTP deficits normalized over development by 9–11 wk. LTP in prefrontal cortex was also normal in the adult CaMKIIα+/− mice. The only detected impairment that could explain the working memory deficit in the CaMKIIα+/− mice was basal synaptic transmission in the adult hippocampus (but not prefrontal cortex). Interestingly, basal transmission was normal in the young mice; however, the CaMKIIα+/− mice failed to show the developmental increase seen in the WT mice. Thus, while development restored the LTP deficits in the CaMKIIα+/− mice, basal synaptic transmission in the hippocampus became impaired.

Comparing the effects on plasticity at different ages and in different brain regions with the corresponding CaMKIIα expression levels suggests an interesting correlation: ∼30% of the CaMKIIα levels in adult hippocampus appear sufficient for normal LTD and depotentiation, while normal LTP appears to require higher (∼60%) levels. Of course direct comparison of different developmental stages and brain regions has limitations. However, our results clearly show that the ∼30% of adult WT level in the hippocampus of the young CaMKIIα+/− mice is sufficient to support normal LTD and depotentiation but not LTP and that ∼60% expression is sufficient for normal LTP in both hippocampus and prefrontal cortex of the older CaMKIIα+/− mice. Additionally, previous data indicate that other cortical areas should have only ∼30% expression (compared to adult WT hippocampus) even in the older CaMKIIα+/− mice (Erondu and Kennedy 1985) and that these expression levels are not sufficient to support normal LTP in neither visual nor temporal cortex (Frankland et al. 2001). Furthermore, consistent with LTP requiring higher levels of CaMKIIα expression than LTD, the full induction of LTP requires the additional spine accumulation of CaMKIIα via binding to GluN2B, while NMDAR-dependent LTD does not (Zhou et al. 2007; Halt et al. 2012). Thus the requirement of higher CaMKII levels for normal LTP may be the result of the direct acute requirement of higher levels during the induction process; however, alternatively, it could potentially arise from indirect effects on other molecules or synaptic maturation.

A connection of CaMKIIα expression level also to basal synaptic strength is indicated both by the results here and by previous studies. In dissociated hippocampal cultures, basal synaptic transmission as measured by miniature EPSC amplitude is bidirectionally affected by manipulating CaMKIIα expression levels: synaptic strength decreases after knockdown and increases after overexpression (Barcomb et al. 2014). However, in native hippocampal tissue expression of CaMKIIα is 10-fold higher than in dissociated culture (Barcomb et al. 2014), and in organotypic hippocampal slice cultures additional overexpression of exogenous CaMKIIα does not further increase synaptic strength (Pi et al. 2010a,b). Likewise, acute slices from mice that overexpress CaMKIIα (CaMKIIα F89G Tg) do not have changes in hippocampal or mPFC I/O response (Wang et al. 2003; Ma et al. 2015), which suggests that a maximum effect of CaMKIIα expression can be reached. In acute hippocampal slices, full genetic knockout of CaMKIIα results in decreased basal synaptic transmission as measured by a decrease in the slope of I/O response (Hinds et al. 1998), and we found a similar reduction also for the heterozygous CaMKIIα+/− mice at 9–11 wk. Additionally, in WT mice, we found an increase in basal hippocampal synaptic strength over development, coinciding with the increased CaMKIIα expression. In fact, young WT mice and older CaMKIIα+/− mice have similar levels of expression in the hippocampus, and their synaptic strength in this brain region was also similar. However, in young hippocampus, further decreasing expression by heterozygous knockout did not significantly further reduce synaptic strength at this developmental stage. Overall, this lead to an interesting reciprocal change of LTP vs. basal synaptic transmission over development: while the LTP impairment seen in the young CaMKIIα+/− mice normalized over development, the initially normal basal transmission became impaired during the same time period.

While we found differences in paired-pulse facilitation between ages, we found no such differences between the genotypes. Additionally, paired-pulse ratios after induction of synaptic plasticity did not differ from basal preplasticity ratios, indicating that the plasticity induced here most likely did not involve changes in presynaptic glutamate release probability. It should be noted that our results differ from prior reports that described deficits in paired-pulse facilitation in the CaMKIIα+/− mice in CA1 at several stimulus intervals (Chapman et al. 1995; Silva et al. 1996). Notably, the mice used in previous reports included mice much older than those used here (2.5 to 7 mo), and it is possible that further presynaptic deficits could develop in CaMKIIα+/− mice as they age. Another possibility for the difference to previous findings is the separate derivation of the CaMKIIα KO line used here (on a C57Bl/6 background; described in Coultrap et al. 2014). The latter is of note as differences in synaptic plasticity were noted between earlier and later generations of the original CaMKIIα KO mice (on mixed 129SvOla/SvJ;BALB/c vs. C57Bl/6 backgrounds) (Silva et al. 1992a; Hinds et al. 1998).

By reducing CaMKIIα expression, the heterozygous knockout also reduces the ratio of CaMKIIα to CaMKIIβ. CaMKIIα enzymatic activity and targeting are regulated in part by intersubunit autophosphorylation events made possible by the 12-meric holoenzyme structure formed by CaMKII, and this regulation is modulated by the addition of CaMKIIβ subunits within some holoenzymes (Coultrap and Bayer 2012). Notably, CaMKIIβ binds to and regulates F-actin dynamics (Shen et al. 1998; Fink et al. 2003; O'Leary et al. 2006; Okamoto et al. 2007; Sanabria et al. 2009). The regulation of F-actin by CaMKIIβ is important for synapse remodeling (Kim et al. 2015) and the CaMKIIβ interaction with F-actin is important for the basal spine targeting of CaMKIIα subunits (Borgesius et al. 2011). Therefore, the altered CaMKIIα-to-CaMKIIβ ratio in CaMKIIα+/− mice could have consequences on enzymatic function and intracellular targeting of CaMKIIα in parallel with the overall reduction in CaMKIIα expression.

The amount of CA1 LTP during normal development is tightly regulated, such that LTP is first observed at P5, peaks at P15, and then settles to adult levels (Harris and Teyler 1984; Teyler et al. 1989; Harris et al. 1992). This developmental specificity in the magnitude of LTP at P15 is thought to correlate with a critical developmental period specifically related to network organizational changes at this time (Harris et al. 1992). Perturbations in LTP at this time are likely to critically impact how the future network will function (Hensch 2004) and could thus permanently impact mature CA1 function as well as hippocampal connections to other brain areas. However, despite the continued impairments in working memory noted in older CaMKIIα+/− mice (Yamasaki et al. 2008; Matsuo et al. 2009), we show here that hippocampal LTP normalized during development in these mice. Therefore, we additionally tested plasticity in the prefrontal cortex, as this brain region receives direct input from the CA1 of the hippocampus and dysfunction in this circuit has been implicated in working memory deficits including those seen in schizophrenia patients (Eisenberg and Berman 2010; Cohen 2011; Lewis 2012; Li et al. 2015). Based on the LTP deficits in the parietal and visual cortex of CaMKIIα+/− mice (Frankland et al. 2001), it was initially surprising that LTP in prefrontal cortex was normal. The previous study in these other cortical areas used slightly younger adult mice than our study (6–8 instead of 9–11 wk old), and thus we cannot rule out that LTP in these cortical areas normalizes with further development. However, there is another explanation for the apparent differences among the cortical areas: while CaMKIIα expression levels in overall cortex are ∼60% compared with adult hippocampus (Erondu and Kennedy 1985) and thus similar to young hippocampus (where further decreased expression by heterozygous knockout impaired LTP), we found that expression in frontal cortex is instead similar to adult hippocampus (where heterozygous knockout also had no effect on LTP).

Altered processing of glutamatergic signaling has been implicated in the pathophysiology of schizophrenia (Moghaddam and Javitt 2012; Howes et al. 2015; Volk et al. 2015), and schizophrenia patients have several altered synaptic functions including decreases in postsynaptic spine density in several brain areas (Moyer et al. 2015), abnormal neural oscillations (Uhlhaas and Singer 2010), and impairments in forms of LTP and LTD (Fitzgerald et al. 2004; Daskalakis et al. 2008; Frantseva et al. 2008; Hasan et al. 2012). The CaMKIIα+/− mice have been highlighted as a potential genetic model to study working-memory processing in schizophrenia based on cellular, behavioral, and electrophysiological phenotypes similar to those seen in schizophrenia patients (Frankland et al. 2001; Yamasaki et al. 2008; Matsuo et al. 2009; Novak and Seeman 2010). Our finding here that hippocampal LTP is impaired in young CaMKIIα+/− mice provides an additional correlate within this genetic model to human schizophrenia, which is often accompanied by preonset hippocampal-dependent cognitive deficits (Nuechterlein et al. 2014). Thus our results indicate that early LTP deficits could provide a potential biomarker for the early detection and diagnosis of preonset schizophrenia in at risk populations. However, the only synaptic deficit found here in either hippocampus CA1 or mPFC of older CaMKIIα+/− mice was hippocampal basal transmission.

GRANTS

This research was supported by National Institutes of Health Grants T32-HD-041697 (neuroscience training grant), F31-NS-092265 (to D. J. Goodell), R01-NS-076577 (to T. A. Benke), and R01-NS-081248 (to K. U. Bayer).

DISCLOSURES

K. U. Bayer is owner of Neurexus Therapeutics, LLC.

AUTHOR CONTRIBUTIONS

D.J.G., T.A.B., and K.U.B. conception and design of research; D.J.G. performed experiments; D.J.G. analyzed data; D.J.G., T.A.B., and K.U.B. interpreted results of experiments; D.J.G. prepared figures; D.J.G. and K.U.B. drafted manuscript; D.J.G., T.A.B., and K.U.B. edited and revised manuscript; D.J.G., T.A.B., and K.U.B. approved final version of manuscript.

ACKNOWLEDGMENTS

We thank Dr. Vincent Zaegel and Janna Mize-Berge for mouse colony maintenance and genotyping, and Dr. Steven Coultrap for helpful discussions.

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