Working memory impairment in calcineurin knock-out mice is associated with alterations in synaptic vesicle cycling and disruption of high-frequency synaptic and network activity in prefrontal cortex

Abstract

Working memory is an essential component of higher cognitive function, and its impairment is a core symptom of multiple CNS disorders, including schizophrenia. Neuronal mechanisms supporting working memory under normal conditions have been described and include persistent, high-frequency activity of prefrontal cortical neurons. However, little is known about the molecular and cellular basis of working memory dysfunction in the context of neuropsychiatric disorders. To elucidate synaptic and neuronal mechanisms of working memory dysfunction, we have performed a comprehensive analysis of a mouse model of schizophrenia, the forebrain-specific calcineurin knock-out mouse. Biochemical analyses of cortical tissue from these mice revealed a pronounced hyperphosphorylation of synaptic vesicle cycling proteins known to be necessary for high-frequency synaptic transmission. Examination of the synaptic vesicle cycle in calcineurin-deficient neurons demonstrated an impairment of vesicle release enhancement during periods of intense stimulation. Moreover, brain slice and in vivo electrophysiological analyses showed that loss of calcineurin leads to a gene dose-dependent disruption of high-frequency synaptic transmission and network activity in the PFC, correlating with selective working memory impairment. Finally, we showed that levels of dynamin I, a key presynaptic protein and calcineurin substrate, are significantly reduced in prefrontal cortical samples from schizophrenia patients, extending the disease relevance of our findings. Our data provide support for a model in which impaired synaptic vesicle cycling represents a critical node for disease pathologies underlying the cognitive deficits in schizophrenia.

Figure 1.

Synaptic vesicle cycling proteins are hyperphosphorylated in CN-KO cortex. A, Western blotting was performed on cerebral cortex protein extracts from three control and three CN-KO mice. Protein was probed with antibodies against total and phosphorylated dynamin I (Dnm1), synapsin I (Syn1), and Pip5k1γ (Pip5k1c) at the indicated residues. Densitometry analysis was performed. B, Mean (±SEM) phospho:total band densitometry ratios, normalized to mean ratio of control bands. *p < 0.05. **p < 0.01. ***p < 0.001. ****p < 0.0001.

Figure 2.

Synaptic vesicle cycling is impaired after knockdown of calcineurin levels. A–C, Neurons transfected with either (1) vGlut1-pHluorin alone (Cont), (2) vGlut1-pHluorin and an shRNA targeting CNB1 (CNB1-KD), or (3) vGlut1-pHluorin, the CNB1-shRNA, and an shRNA-resistant version of CNB1 (CNB1-Res) were subjected to stimulus trains of 300 pulses delivered at (1) 10 Hz, (2) 50 Hz, and (3) 10 Hz. A, vGlut1-pHluorin fluorescence responses from single neurons to the first 10 Hz and the 50 Hz pulse trains from control neurons and CNB1-shRNA transfected neurons. B, Mean (±SEM) response amplitudes normalized to the response of the first 10 Hz stimulus train from control (n = 5), CNB1-shRNA (n = 5), and CNB1-shRNA rescued (n = 4) neurons. C, The vGlut1-pHluorin waveform decays were fit with a single-exponential decay curve. Shown are the mean (±SEM) recovery time constants from control (n = 5), CNB1-shRNA (n = 5), and CNB1-shRNA rescued (n = 4) neurons. D–F, Primary rat cortical neurons expressing the synaptophysin-pHluorin reporter delivered by AAV transduction were treated with vehicle (Veh; n = 5) or cyclosporin A (CsA, 20 μm; n = 5) and analyzed as described above (A–C). There was a significant reduction in the amplitude of responses to the 50 Hz stimulus (t₍₈₎ = 8.63, p < 0.0001) and a significant increase in the recovery time constant for the 10 Hz stimulus 1 (t₍₈₎ = 4.39, p < 0.01), the 50 Hz stimulus (t₍₈₎ = 4.13, p < 0.01), and the 10 Hz stimulus 2 (t₍₈₎ = 4.17, p < 0.01) in cyclosporin A-treated versus vehicle-treated neurons. *p < 0.05. **p < 0.01. ****p < 0.0001.

Figure 3.

Synaptic fatigue is augmented in the PFC of CN-KO and CN-Het mice. A, Paired pulse ratios were measured in sagittal slices containing PFC from CN-KO (n = 24), CN-Het (n = 5), and littermate controls (n = 16). B, Synaptic fatigue was induced in PFC slices from control (n = 8), CN-Het (n = 5), and CN-KO (n = 6) mice by stimulating at 40 Hz and monitoring the left slope of evoked fEPSPs. C, Synaptic fatigue data from PFC slices stimulated at 20 Hz or 40 Hz were fit with single exponential curves, and the magnitude of fatigue was determined as the plateau of the fit; control (n = 8), CN-Het (n = 5), CN-KO (n = 6). D, Basal synaptic transmission was assessed in PFC by relating fEPSP slope to fiber volley amplitudes in response to a series of electrical stimuli. PFC slices isolated from CN-KO (n = 24) animals exhibited lower I/O relationships relative to either CN-Het (n = 5) or control (n = 16) animals. E, Synaptic fatigue in response to a 40 Hz stimulus train was measured at the mossy fiber synapse in which calcineurin is intact in postsynaptic neurons but deficient in presynaptic terminals of CN-KO mice; CN-KO (n = 4), control (n = 4). F, Summary data for fatigue experiments in E. ****p < 0.0001. Data shown in A, C, D, and F are mean (±SEM).

Figure 4.

High-frequency oscillatory network activity is impaired in PFC of CN-KO and CN-Het mice. A, Extracellular potentials were recorded in PFC from sagittal slices from CN-KO (n = 10), CN-Het (n = 12), and littermate control animals (n = 12) using a pMEA. Oscillations were evoked with carbachol (red bar, 20 μm). Representative traces above summary data indicate spontaneous activity in the γ band (30–90 Hz) before (Baseline) and after application of carbachol (Carbachol). Data shown are mean (±SEM). B, Neural activity was recorded from the PFC of freely behaving CN-KO (n = 4), CN-Het (n = 20), and littermate control mice (n = 16) during the first 15 min of exposure to a novel environment. Representative traces show Gamma_Hi (65–90 Hz) bandpass filtered signals. Summary histogram illustrates the relative number of times a particular level of Gamma_Hi power was observed in each animal cohort.

Figure 5.

CN-KO and CN-Het mice have impaired performance in the DNMTP task. The performance of CN-KO and CN-Het mice was analyzed in a DNMTP working memory task. A, D, Mean (±SEM) number of trials for mice to reach criterion for basic performance of the task with no imposed delay. B, E, Mean (±SEM) number of completed trials per test session. C, F, Mean (±SEM) percentage correct above chance on the DNMTP task after imposition of delays within the task. ****p < 0.0001.

Figure 6.

Levels of the synaptic vesicle cycling protein dynamin I are reduced in schizophrenia PFC. Western blotting was performed for dynamin I and β-III-tubulin on protein extracts generated from slices of PFC (Brodmann's area 46) from control individuals (n = 14) and from patients with schizophrenia (n = 11) or bipolar disease (n = 12). (See Table 1 for cohort information.) A, Representative Western blot showing dynamin I and β-III-tubulin expression from normal (N), schizophrenia (S), and bipolar (B) brain samples; C indicates a normalization control consisting of a commercial human cerebral cortex sample. B, Mean (±SEM) dynamin I protein levels measured and calculated as described in Materials and Methods. **p < 0.01.

Figure 7.

A presynaptic model for working memory deficits. Calcineurin deficiency impairs the ability of neurons in PFC to efficiently mobilize and recycle synaptic vesicles during high-frequency activity, leading to a disruption of high-frequency neuronal and network activities in PFC that are required for working memory. Additional molecular alterations implicated in schizophrenia may converge on the synaptic vesicle cycle and thereby contribute to specific cognitive impairments associated with the disease.