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Ablation of Ca(V)2.1 voltage-gated Ca²⁺ channels in mouse forebrain generates multiple cognitive impairments.

Mallmann RT, Elgueta C, Sleman F, Castonguay J, Wilmes T, van den Maagdenberg A, Klugbauer N - PLoS ONE (2013)

Bottom Line: Voltage-gated Ca(V)2.1 (P/Q-type) Ca²⁺ channels located at the presynaptic membrane are known to control a multitude of Ca²⁺-dependent cellular processes such as neurotransmitter release and synaptic plasticity.At the behavioural level, the forebrain-specific Ca(V)2.1 knock-out resulted in deficits in spatial learning and reference memory, reduced recognition memory, increased exploratory behaviour and a strong attenuation of circadian rhythmicity.In summary, we present a novel conditional Ca(V)2.1 knock-out model that is most suitable for analysing the in vivo functions of Ca(V)2.1 in the adult murine forebrain.

View Article: PubMed Central - PubMed

Affiliation: Institut für Experimentelle und Klinische Pharmakologie und Toxikologie, Albert-Ludwigs-Universität, Freiburg, Germany ; Fakultät für Biologie, Albert-Ludwigs-Universität, Freiburg, Germany.

ABSTRACT
Voltage-gated Ca(V)2.1 (P/Q-type) Ca²⁺ channels located at the presynaptic membrane are known to control a multitude of Ca²⁺-dependent cellular processes such as neurotransmitter release and synaptic plasticity. Our knowledge about their contributions to complex cognitive functions, however, is restricted by the limited adequacy of existing transgenic Ca(V)2.1 mouse models. Global Ca(V)2.1 knock-out mice lacking the α1 subunit Cacna1a gene product exhibit early postnatal lethality which makes them unsuitable to analyse the relevance of Ca(V)2.1 Ca²⁺ channels for complex behaviour in adult mice. Consequently we established a forebrain specific Ca(V)2.1 knock-out model by crossing mice with a floxed Cacna1a gene with mice expressing Cre-recombinase under the control of the NEX promoter. This novel mouse model enabled us to investigate the contribution of Ca(V)2.1 to complex cognitive functions, particularly learning and memory. Electrophysiological analysis allowed us to test the specificity of our conditional knock-out model and revealed an impaired synaptic transmission at hippocampal glutamatergic synapses. At the behavioural level, the forebrain-specific Ca(V)2.1 knock-out resulted in deficits in spatial learning and reference memory, reduced recognition memory, increased exploratory behaviour and a strong attenuation of circadian rhythmicity. In summary, we present a novel conditional Ca(V)2.1 knock-out model that is most suitable for analysing the in vivo functions of Ca(V)2.1 in the adult murine forebrain.

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Expression of functional CaV2.1 channels is strongly reduced in the hippocampus of cKO mice.(A and B) Representative EPSCs recorded in CA1 PCs evoked by extracellular stimulation of the stratum radiatum before (gray) and after (black) bath-application of ω-conotoxin GVIA (left) or ω -agatoxin IVA (right). PC recordings were obtained from CTR (A) and cKOs (B). (C) Bar graph summarizes the residual peak amplitude of EPSCs after toxin application for CTR (each 6 experiments) and cKO (5 vs. 6 experiments) mice. Means + SEM are shown. *** P≤0.001 (Two-tailed Student’s t-test)
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pone-0078598-g003: Expression of functional CaV2.1 channels is strongly reduced in the hippocampus of cKO mice.(A and B) Representative EPSCs recorded in CA1 PCs evoked by extracellular stimulation of the stratum radiatum before (gray) and after (black) bath-application of ω-conotoxin GVIA (left) or ω -agatoxin IVA (right). PC recordings were obtained from CTR (A) and cKOs (B). (C) Bar graph summarizes the residual peak amplitude of EPSCs after toxin application for CTR (each 6 experiments) and cKO (5 vs. 6 experiments) mice. Means + SEM are shown. *** P≤0.001 (Two-tailed Student’s t-test)

Mentions: To test whether knock-out of CaV2.1 channels may result in alterations of synaptic transmission, we performed whole-cell recordings from CA1 pyramidal cells (PCs) in hippocampal slice preparations of CTR and cKO mice (Fig. 2A) and examined EPSCs evoked by extracellular stimulation of the Schaffer collaterals at different intensities. Input-output curves clearly showed that evoked EPSCs were significantly lower in cKO animals at all stimulation intensities that reliably evoked EPSCs (Fig. 2B and C), suggesting that synaptic transmission at the Schaffer collaterals-CA1 synapse is strongly impaired in cKO animals. To further analyse glutamate release at these synapses, we applied repetitive stimulation at 50 Hz (100 pulses). The synaptic charge transferred during trains of stimulation was by a factor ∼10 smaller in cKOs than CTR mice (Fig. 2D). Facilitation of EPSCs at the onset of the train (∫5th to 10th EPSC/∫1st to 5th EPSC) was similar in CTR and cKO animals but significantly higher at the end of the train (∫90th to 100th EPSC/∫1st to 10th) for cKO animals (Fig. 2D). To examine whether the observed reduction in glutamate release from Schaffer collaterals was mediated by a functional loss of P/Q-type Ca2+ channels, we bath-applied selective Ca2+ channel blockers (Fig. 3A and B) [27]. The N-type Ca2+ channel blocker ω-conotoxin GVIA (1 µM) reduced the amplitude of EPSCs in CTR animals to 56.4±5.6%, whereas EPSCs recorded in CA1 PCs of cKO mice were almost entirely blocked by the toxin to a residual signal of 7.6±1.7% (Fig. 3C). Application of the P/Q-type Ca2+ channel blocker ω-agatoxin IVA (500 nM) reduced EPSC peak amplitudes in PCs of CTR animals to 31.2±7.3% (5 cells), but had no significant effect on EPSCs recorded in cKO mice (110.5±11.4%; n = 6; Fig. 3C). Thus NEX/Cre-mediated loss of CaV2.1 channels resulted in a marked reduction in transmitter release at Schaffer collateral-PC synapses. In contrast, application of ω-agatoxin IVA (500 nM) strongly reduced EPSCs recorded in Purkinje cells after stimulation of parallel fibers to 30.6±6.8% of the control response in cKO animals, further demonstrating the regional specificity of our CaV2.1 KO model.


Ablation of Ca(V)2.1 voltage-gated Ca²⁺ channels in mouse forebrain generates multiple cognitive impairments.

Mallmann RT, Elgueta C, Sleman F, Castonguay J, Wilmes T, van den Maagdenberg A, Klugbauer N - PLoS ONE (2013)

Expression of functional CaV2.1 channels is strongly reduced in the hippocampus of cKO mice.(A and B) Representative EPSCs recorded in CA1 PCs evoked by extracellular stimulation of the stratum radiatum before (gray) and after (black) bath-application of ω-conotoxin GVIA (left) or ω -agatoxin IVA (right). PC recordings were obtained from CTR (A) and cKOs (B). (C) Bar graph summarizes the residual peak amplitude of EPSCs after toxin application for CTR (each 6 experiments) and cKO (5 vs. 6 experiments) mice. Means + SEM are shown. *** P≤0.001 (Two-tailed Student’s t-test)
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Related In: Results  -  Collection

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pone-0078598-g003: Expression of functional CaV2.1 channels is strongly reduced in the hippocampus of cKO mice.(A and B) Representative EPSCs recorded in CA1 PCs evoked by extracellular stimulation of the stratum radiatum before (gray) and after (black) bath-application of ω-conotoxin GVIA (left) or ω -agatoxin IVA (right). PC recordings were obtained from CTR (A) and cKOs (B). (C) Bar graph summarizes the residual peak amplitude of EPSCs after toxin application for CTR (each 6 experiments) and cKO (5 vs. 6 experiments) mice. Means + SEM are shown. *** P≤0.001 (Two-tailed Student’s t-test)
Mentions: To test whether knock-out of CaV2.1 channels may result in alterations of synaptic transmission, we performed whole-cell recordings from CA1 pyramidal cells (PCs) in hippocampal slice preparations of CTR and cKO mice (Fig. 2A) and examined EPSCs evoked by extracellular stimulation of the Schaffer collaterals at different intensities. Input-output curves clearly showed that evoked EPSCs were significantly lower in cKO animals at all stimulation intensities that reliably evoked EPSCs (Fig. 2B and C), suggesting that synaptic transmission at the Schaffer collaterals-CA1 synapse is strongly impaired in cKO animals. To further analyse glutamate release at these synapses, we applied repetitive stimulation at 50 Hz (100 pulses). The synaptic charge transferred during trains of stimulation was by a factor ∼10 smaller in cKOs than CTR mice (Fig. 2D). Facilitation of EPSCs at the onset of the train (∫5th to 10th EPSC/∫1st to 5th EPSC) was similar in CTR and cKO animals but significantly higher at the end of the train (∫90th to 100th EPSC/∫1st to 10th) for cKO animals (Fig. 2D). To examine whether the observed reduction in glutamate release from Schaffer collaterals was mediated by a functional loss of P/Q-type Ca2+ channels, we bath-applied selective Ca2+ channel blockers (Fig. 3A and B) [27]. The N-type Ca2+ channel blocker ω-conotoxin GVIA (1 µM) reduced the amplitude of EPSCs in CTR animals to 56.4±5.6%, whereas EPSCs recorded in CA1 PCs of cKO mice were almost entirely blocked by the toxin to a residual signal of 7.6±1.7% (Fig. 3C). Application of the P/Q-type Ca2+ channel blocker ω-agatoxin IVA (500 nM) reduced EPSC peak amplitudes in PCs of CTR animals to 31.2±7.3% (5 cells), but had no significant effect on EPSCs recorded in cKO mice (110.5±11.4%; n = 6; Fig. 3C). Thus NEX/Cre-mediated loss of CaV2.1 channels resulted in a marked reduction in transmitter release at Schaffer collateral-PC synapses. In contrast, application of ω-agatoxin IVA (500 nM) strongly reduced EPSCs recorded in Purkinje cells after stimulation of parallel fibers to 30.6±6.8% of the control response in cKO animals, further demonstrating the regional specificity of our CaV2.1 KO model.

Bottom Line: Voltage-gated Ca(V)2.1 (P/Q-type) Ca²⁺ channels located at the presynaptic membrane are known to control a multitude of Ca²⁺-dependent cellular processes such as neurotransmitter release and synaptic plasticity.At the behavioural level, the forebrain-specific Ca(V)2.1 knock-out resulted in deficits in spatial learning and reference memory, reduced recognition memory, increased exploratory behaviour and a strong attenuation of circadian rhythmicity.In summary, we present a novel conditional Ca(V)2.1 knock-out model that is most suitable for analysing the in vivo functions of Ca(V)2.1 in the adult murine forebrain.

View Article: PubMed Central - PubMed

Affiliation: Institut für Experimentelle und Klinische Pharmakologie und Toxikologie, Albert-Ludwigs-Universität, Freiburg, Germany ; Fakultät für Biologie, Albert-Ludwigs-Universität, Freiburg, Germany.

ABSTRACT
Voltage-gated Ca(V)2.1 (P/Q-type) Ca²⁺ channels located at the presynaptic membrane are known to control a multitude of Ca²⁺-dependent cellular processes such as neurotransmitter release and synaptic plasticity. Our knowledge about their contributions to complex cognitive functions, however, is restricted by the limited adequacy of existing transgenic Ca(V)2.1 mouse models. Global Ca(V)2.1 knock-out mice lacking the α1 subunit Cacna1a gene product exhibit early postnatal lethality which makes them unsuitable to analyse the relevance of Ca(V)2.1 Ca²⁺ channels for complex behaviour in adult mice. Consequently we established a forebrain specific Ca(V)2.1 knock-out model by crossing mice with a floxed Cacna1a gene with mice expressing Cre-recombinase under the control of the NEX promoter. This novel mouse model enabled us to investigate the contribution of Ca(V)2.1 to complex cognitive functions, particularly learning and memory. Electrophysiological analysis allowed us to test the specificity of our conditional knock-out model and revealed an impaired synaptic transmission at hippocampal glutamatergic synapses. At the behavioural level, the forebrain-specific Ca(V)2.1 knock-out resulted in deficits in spatial learning and reference memory, reduced recognition memory, increased exploratory behaviour and a strong attenuation of circadian rhythmicity. In summary, we present a novel conditional Ca(V)2.1 knock-out model that is most suitable for analysing the in vivo functions of Ca(V)2.1 in the adult murine forebrain.

Show MeSH
Related in: MedlinePlus