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Involvement of cAMP-guanine nucleotide exchange factor II in hippocampal long-term depression and behavioral flexibility.

Lee K, Kobayashi Y, Seo H, Kwak JH, Masuda A, Lim CS, Lee HR, Kang SJ, Park P, Sim SE, Kogo N, Kawasaki H, Kaang BK, Itohara S - Mol Brain (2015)

Bottom Line: Although cAMP-GEF II is expressed abundantly in several brain areas including the cortex, striatum, and hippocampus, its specific function and possible role in hippocampal synaptic plasticity and cognitive processes remain elusive.We found that deletion of cAMP-GEF II induced moderate decrease in long-term potentiation, although this decrease was not statistically significant.We concluded that cAMP-GEF II plays a key role in hippocampal functions including behavioral flexibility in reversal learning and in mechanisms underlying induction of long-term depression.

View Article: PubMed Central - PubMed

Affiliation: Behavioral Neural Circuitry and Physiology Laboratory, Department of Anatomy, Brain Science & Engineering Institute, Kyungpook National University Graduate School of Medicine, 2-101, Dongin-dong, Jung-gu, Daegu, 700-842, Korea. irislkm@knu.ac.kr.

ABSTRACT

Background: Guanine nucleotide exchange factors (GEFs) activate small GTPases that are involved in several cellular functions. cAMP-guanine nucleotide exchange factor II (cAMP-GEF II) acts as a target for cAMP independently of protein kinase A (PKA) and functions as a GEF for Rap1 and Rap2. Although cAMP-GEF II is expressed abundantly in several brain areas including the cortex, striatum, and hippocampus, its specific function and possible role in hippocampal synaptic plasticity and cognitive processes remain elusive. Here, we investigated how cAMP-GEF II affects synaptic function and animal behavior using cAMP-GEF II knockout mice.

Results: We found that deletion of cAMP-GEF II induced moderate decrease in long-term potentiation, although this decrease was not statistically significant. On the other hand, it produced a significant and clear impairment in NMDA receptor-dependent long-term depression at the Schaffer collateral-CA1 synapses of hippocampus, while microscopic morphology, basal synaptic transmission, and depotentiation were normal. Behavioral testing using the Morris water maze and automated IntelliCage system showed that cAMP-GEF II deficient mice had moderately reduced behavioral flexibility in spatial learning and memory.

Conclusions: We concluded that cAMP-GEF II plays a key role in hippocampal functions including behavioral flexibility in reversal learning and in mechanisms underlying induction of long-term depression.

No MeSH data available.


Related in: MedlinePlus

Characterization of cAMP-GEF II−/− mice. a. Schematic diagram for wild-type, floxed, and knockout (KO) alleles of cAMP-GEF II. Floxed mice were generated by gene targeting using MS12 ES cells derived from the B6 strain, and KO mice were generated by expressing Cre recombinase in the germ cells of the floxed mice (arrow, locus of primer (P1, P2, and P3) for genomic PCR). b, Genomic PCR analysis of cAMP-GEF II gene deletion in cAMP-GEF II+/− (HT, heterozygous), cAMP-GEF II+/+ (WT, wild-type), and cAMP-GEF II−/− (KO, knockout) mice. c, Western blot analysis of cAMP-GEF II protein expression in fractionated brain lysates. cAMP-GEF II protein expression was compared among S1 (postnuclear), P2 (crude membrane), and SPM (synaptic plasma membrane) fractions. cAMP-GEF II protein was highly expressed in SPM fractions, which also presented high expression of PSD95. Note that cAMP-GEFs protein expression was completely abolished in the brain of cAMP-GEF II−/− mice. d, Immunohistochemical analysis of cAMP-GEF II expression in brain tissue sections. Strong immunolabeling was observed in the cortex and hippocampus of WT mice, but was absent in KO mice. In the hippocampus, immunoreactivity for cAMP-GEF II was relatively low in the stratum pyramidale (sp) of the Cornu Ammonis (CA) as well as in the granular cell layer (gcl) of the dentate gyrus; while the stratum oriens (so), radiatum (sr), and lacunosum moleculare (sl-m), as well as the molecular layer (ml) of the dentate gyrus showed strong immunoreactivity for cAMP-GEF II. e, Immunofluorescence for NeuN showed that there was no difference in morphology of the hippocampus between the two genotypes. Scale bars = 500 μm in D, E. Abbreviations: SM, size marker
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Fig1: Characterization of cAMP-GEF II−/− mice. a. Schematic diagram for wild-type, floxed, and knockout (KO) alleles of cAMP-GEF II. Floxed mice were generated by gene targeting using MS12 ES cells derived from the B6 strain, and KO mice were generated by expressing Cre recombinase in the germ cells of the floxed mice (arrow, locus of primer (P1, P2, and P3) for genomic PCR). b, Genomic PCR analysis of cAMP-GEF II gene deletion in cAMP-GEF II+/− (HT, heterozygous), cAMP-GEF II+/+ (WT, wild-type), and cAMP-GEF II−/− (KO, knockout) mice. c, Western blot analysis of cAMP-GEF II protein expression in fractionated brain lysates. cAMP-GEF II protein expression was compared among S1 (postnuclear), P2 (crude membrane), and SPM (synaptic plasma membrane) fractions. cAMP-GEF II protein was highly expressed in SPM fractions, which also presented high expression of PSD95. Note that cAMP-GEFs protein expression was completely abolished in the brain of cAMP-GEF II−/− mice. d, Immunohistochemical analysis of cAMP-GEF II expression in brain tissue sections. Strong immunolabeling was observed in the cortex and hippocampus of WT mice, but was absent in KO mice. In the hippocampus, immunoreactivity for cAMP-GEF II was relatively low in the stratum pyramidale (sp) of the Cornu Ammonis (CA) as well as in the granular cell layer (gcl) of the dentate gyrus; while the stratum oriens (so), radiatum (sr), and lacunosum moleculare (sl-m), as well as the molecular layer (ml) of the dentate gyrus showed strong immunoreactivity for cAMP-GEF II. e, Immunofluorescence for NeuN showed that there was no difference in morphology of the hippocampus between the two genotypes. Scale bars = 500 μm in D, E. Abbreviations: SM, size marker

Mentions: We confirmed first the disruption of the cAMP-GEF II gene in cAMP-GEF II−/− mice by genomic PCR using tail tissues (Fig. 1a and b), and assessed expression of cAMP-GEF II protein by western blotting using fractionated brain tissues of wild-type and cAMP-GEF II−/− mice, before assessing the physiological functions of cAMP-GEF II in the hippocampus. We observed that cAMP-GEF II protein was expressed in wild-type mice, but abolished in cAMP-GEF II−/− mice (Fig. 1c). Moreover, western blot analysis revealed prominent expression or reduction of cAMP-GEF II in the synaptic plasma membrane (SPM) fraction of wild-type and cAMP-GEF II−/− mice, respectively (Fig. 1c), suggesting that cAMP-GEF II protein is mainly expressed at the postsynaptic membrane and postsynaptic density (PSD). In addition, we confirmed that cAMP-GEF II was highly expressed in dendritic processes (i.e., the stratum oriens, radiatum, lacunosum moleculare, and lucidum of the CA, as well as in the molecular layer of the dentate gyrus), rather than in cellular layers of hippocampus (i.e., stratum pyramidale of the CA and granular layer of the dentate gyrus) of wild-type mice, while its expression was completely abolished in the hippocampus of cAMP-GEF II−/− mice (Fig. 1d). Finally, there were no morphological anomalies in the hippocampus (Fig. 1e), or other brain areas (data not shown) of cAMP-GEF II−/− mice compared to wild-type mice.Fig. 1


Involvement of cAMP-guanine nucleotide exchange factor II in hippocampal long-term depression and behavioral flexibility.

Lee K, Kobayashi Y, Seo H, Kwak JH, Masuda A, Lim CS, Lee HR, Kang SJ, Park P, Sim SE, Kogo N, Kawasaki H, Kaang BK, Itohara S - Mol Brain (2015)

Characterization of cAMP-GEF II−/− mice. a. Schematic diagram for wild-type, floxed, and knockout (KO) alleles of cAMP-GEF II. Floxed mice were generated by gene targeting using MS12 ES cells derived from the B6 strain, and KO mice were generated by expressing Cre recombinase in the germ cells of the floxed mice (arrow, locus of primer (P1, P2, and P3) for genomic PCR). b, Genomic PCR analysis of cAMP-GEF II gene deletion in cAMP-GEF II+/− (HT, heterozygous), cAMP-GEF II+/+ (WT, wild-type), and cAMP-GEF II−/− (KO, knockout) mice. c, Western blot analysis of cAMP-GEF II protein expression in fractionated brain lysates. cAMP-GEF II protein expression was compared among S1 (postnuclear), P2 (crude membrane), and SPM (synaptic plasma membrane) fractions. cAMP-GEF II protein was highly expressed in SPM fractions, which also presented high expression of PSD95. Note that cAMP-GEFs protein expression was completely abolished in the brain of cAMP-GEF II−/− mice. d, Immunohistochemical analysis of cAMP-GEF II expression in brain tissue sections. Strong immunolabeling was observed in the cortex and hippocampus of WT mice, but was absent in KO mice. In the hippocampus, immunoreactivity for cAMP-GEF II was relatively low in the stratum pyramidale (sp) of the Cornu Ammonis (CA) as well as in the granular cell layer (gcl) of the dentate gyrus; while the stratum oriens (so), radiatum (sr), and lacunosum moleculare (sl-m), as well as the molecular layer (ml) of the dentate gyrus showed strong immunoreactivity for cAMP-GEF II. e, Immunofluorescence for NeuN showed that there was no difference in morphology of the hippocampus between the two genotypes. Scale bars = 500 μm in D, E. Abbreviations: SM, size marker
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Related In: Results  -  Collection

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Fig1: Characterization of cAMP-GEF II−/− mice. a. Schematic diagram for wild-type, floxed, and knockout (KO) alleles of cAMP-GEF II. Floxed mice were generated by gene targeting using MS12 ES cells derived from the B6 strain, and KO mice were generated by expressing Cre recombinase in the germ cells of the floxed mice (arrow, locus of primer (P1, P2, and P3) for genomic PCR). b, Genomic PCR analysis of cAMP-GEF II gene deletion in cAMP-GEF II+/− (HT, heterozygous), cAMP-GEF II+/+ (WT, wild-type), and cAMP-GEF II−/− (KO, knockout) mice. c, Western blot analysis of cAMP-GEF II protein expression in fractionated brain lysates. cAMP-GEF II protein expression was compared among S1 (postnuclear), P2 (crude membrane), and SPM (synaptic plasma membrane) fractions. cAMP-GEF II protein was highly expressed in SPM fractions, which also presented high expression of PSD95. Note that cAMP-GEFs protein expression was completely abolished in the brain of cAMP-GEF II−/− mice. d, Immunohistochemical analysis of cAMP-GEF II expression in brain tissue sections. Strong immunolabeling was observed in the cortex and hippocampus of WT mice, but was absent in KO mice. In the hippocampus, immunoreactivity for cAMP-GEF II was relatively low in the stratum pyramidale (sp) of the Cornu Ammonis (CA) as well as in the granular cell layer (gcl) of the dentate gyrus; while the stratum oriens (so), radiatum (sr), and lacunosum moleculare (sl-m), as well as the molecular layer (ml) of the dentate gyrus showed strong immunoreactivity for cAMP-GEF II. e, Immunofluorescence for NeuN showed that there was no difference in morphology of the hippocampus between the two genotypes. Scale bars = 500 μm in D, E. Abbreviations: SM, size marker
Mentions: We confirmed first the disruption of the cAMP-GEF II gene in cAMP-GEF II−/− mice by genomic PCR using tail tissues (Fig. 1a and b), and assessed expression of cAMP-GEF II protein by western blotting using fractionated brain tissues of wild-type and cAMP-GEF II−/− mice, before assessing the physiological functions of cAMP-GEF II in the hippocampus. We observed that cAMP-GEF II protein was expressed in wild-type mice, but abolished in cAMP-GEF II−/− mice (Fig. 1c). Moreover, western blot analysis revealed prominent expression or reduction of cAMP-GEF II in the synaptic plasma membrane (SPM) fraction of wild-type and cAMP-GEF II−/− mice, respectively (Fig. 1c), suggesting that cAMP-GEF II protein is mainly expressed at the postsynaptic membrane and postsynaptic density (PSD). In addition, we confirmed that cAMP-GEF II was highly expressed in dendritic processes (i.e., the stratum oriens, radiatum, lacunosum moleculare, and lucidum of the CA, as well as in the molecular layer of the dentate gyrus), rather than in cellular layers of hippocampus (i.e., stratum pyramidale of the CA and granular layer of the dentate gyrus) of wild-type mice, while its expression was completely abolished in the hippocampus of cAMP-GEF II−/− mice (Fig. 1d). Finally, there were no morphological anomalies in the hippocampus (Fig. 1e), or other brain areas (data not shown) of cAMP-GEF II−/− mice compared to wild-type mice.Fig. 1

Bottom Line: Although cAMP-GEF II is expressed abundantly in several brain areas including the cortex, striatum, and hippocampus, its specific function and possible role in hippocampal synaptic plasticity and cognitive processes remain elusive.We found that deletion of cAMP-GEF II induced moderate decrease in long-term potentiation, although this decrease was not statistically significant.We concluded that cAMP-GEF II plays a key role in hippocampal functions including behavioral flexibility in reversal learning and in mechanisms underlying induction of long-term depression.

View Article: PubMed Central - PubMed

Affiliation: Behavioral Neural Circuitry and Physiology Laboratory, Department of Anatomy, Brain Science & Engineering Institute, Kyungpook National University Graduate School of Medicine, 2-101, Dongin-dong, Jung-gu, Daegu, 700-842, Korea. irislkm@knu.ac.kr.

ABSTRACT

Background: Guanine nucleotide exchange factors (GEFs) activate small GTPases that are involved in several cellular functions. cAMP-guanine nucleotide exchange factor II (cAMP-GEF II) acts as a target for cAMP independently of protein kinase A (PKA) and functions as a GEF for Rap1 and Rap2. Although cAMP-GEF II is expressed abundantly in several brain areas including the cortex, striatum, and hippocampus, its specific function and possible role in hippocampal synaptic plasticity and cognitive processes remain elusive. Here, we investigated how cAMP-GEF II affects synaptic function and animal behavior using cAMP-GEF II knockout mice.

Results: We found that deletion of cAMP-GEF II induced moderate decrease in long-term potentiation, although this decrease was not statistically significant. On the other hand, it produced a significant and clear impairment in NMDA receptor-dependent long-term depression at the Schaffer collateral-CA1 synapses of hippocampus, while microscopic morphology, basal synaptic transmission, and depotentiation were normal. Behavioral testing using the Morris water maze and automated IntelliCage system showed that cAMP-GEF II deficient mice had moderately reduced behavioral flexibility in spatial learning and memory.

Conclusions: We concluded that cAMP-GEF II plays a key role in hippocampal functions including behavioral flexibility in reversal learning and in mechanisms underlying induction of long-term depression.

No MeSH data available.


Related in: MedlinePlus