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New insight into the role of the beta3 subunit of the GABAA-R in development, behavior, body weight regulation, and anesthesia revealed by conditional gene knockout.

Ferguson C, Hardy SL, Werner DF, Hileman SM, Delorey TM, Homanics GE - BMC Neurosci (2007)

Bottom Line: However, developmental abnormalities, compensation, reduced viability, and numerous behavioral abnormalities limited the usefulness of that murine model.Survivors had reduced reproductive fitness, reduced sensitivity to etomidate, were hyperactive, and some became obese.Conditional inactivation of the beta3 gene revealed novel insight into the function of this GABAA-R subunit.

View Article: PubMed Central - HTML - PubMed

Affiliation: Departments of Anesthesiology and Pharmacology, University of Pittsburgh, Pittsburgh, PA 15261, USA. fergusonc@anes.upmc.edu

ABSTRACT

Background: The beta3 subunit of the gamma-aminobutyric acid type A receptor (GABAA-R) has been reported to be important for palate formation, anesthetic action, and normal nervous system function. This subunit has also been implicated in the pathogenesis of Angelman syndrome and autism spectrum disorder. To further investigate involvement of this subunit, we previously produced mice with a global knockout of beta3. However, developmental abnormalities, compensation, reduced viability, and numerous behavioral abnormalities limited the usefulness of that murine model. To overcome many of these limitations, a mouse line with a conditionally inactivated beta3 gene was engineered.

Results: Gene targeting and embryonic stem cell technologies were used to create mice in which exon 3 of the beta3 subunit was flanked by loxP sites (i.e., floxed). Crossing the floxed beta3 mice to a cre general deleter mouse line reproduced the phenotype of the previously described global knockout. Pan-neuronal knockout of beta3 was achieved by crossing floxed beta3 mice to Synapsin I-cre transgenic mice. Palate development was normal in pan-neuronal beta3 knockouts but ~61% died as neonates. Survivors were overtly normal, fertile, and were less sensitive to etomidate. Forebrain selective knockout of beta3 was achieved using alpha CamKII-cre transgenic mice. Palate development was normal in forebrain selective beta3 knockout mice. These knockouts survived the neonatal period, but ~30% died between 15-25 days of age. Survivors had reduced reproductive fitness, reduced sensitivity to etomidate, were hyperactive, and some became obese.

Conclusion: Conditional inactivation of the beta3 gene revealed novel insight into the function of this GABAA-R subunit. The floxed beta3 knockout mice described here will be very useful for conditional knockout studies to further investigate the role of the beta3 subunit in development, ethanol and anesthetic action, normal physiology, and pathophysiologic processes.

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Western blot analysis of forebrain selective neuronal β3 knockouts. Western blot analysis of the β3 subunit of the GABAA-R from individual 13–16 week old mice revealed dramatically reduced amounts of β3 in hippocampus (A) and cortex (B) of α CamKII-cre positive samples compared to cre negative control samples. The amount of β3 in cerebellum (C) did not differ between genotypes. Analysis of cortex for the GABAA-R α1 subunit revealed a ~95% reduction in knockout samples compared to cre negative controls. All blots were reprobed for β-actin as a loading control. Shown are representative western blots. Four mice of each genotype were analyzed and all samples were analyzed on at least two different blots. D. Summary graph of western blot analysis demonstrating a significant (*, p ≤ 0.01; **, p < 0.001) reduction in β3 protein in hippocampus and cortex, but not in cerebellum. Data are expressed as mean ± SEM of percent change in band intensity relative to cre negative controls following normalization to actin. (E) Western blot analysis of β3 in cortex from individual 4 week old mice revealed dramatically reduced amounts of β3 in cre positive samples compared to cre negative control samples.
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Figure 4: Western blot analysis of forebrain selective neuronal β3 knockouts. Western blot analysis of the β3 subunit of the GABAA-R from individual 13–16 week old mice revealed dramatically reduced amounts of β3 in hippocampus (A) and cortex (B) of α CamKII-cre positive samples compared to cre negative control samples. The amount of β3 in cerebellum (C) did not differ between genotypes. Analysis of cortex for the GABAA-R α1 subunit revealed a ~95% reduction in knockout samples compared to cre negative controls. All blots were reprobed for β-actin as a loading control. Shown are representative western blots. Four mice of each genotype were analyzed and all samples were analyzed on at least two different blots. D. Summary graph of western blot analysis demonstrating a significant (*, p ≤ 0.01; **, p < 0.001) reduction in β3 protein in hippocampus and cortex, but not in cerebellum. Data are expressed as mean ± SEM of percent change in band intensity relative to cre negative controls following normalization to actin. (E) Western blot analysis of β3 in cortex from individual 4 week old mice revealed dramatically reduced amounts of β3 in cre positive samples compared to cre negative control samples.

Mentions: To examine the impact of α CamKII-cre mediated recombination on the β3 gene product, western blot analysis was conducted on hippocampus, cortex, cerebellum, and hypothalamus using a β3 specific antibody. As shown in Figure 4, β3 protein was dramatically reduced in hippocampus and was nearly completely ablated in cortex of cre+ mice compared to cre- controls. In contrast, the amount of β3 in cerebellum was not significantly reduced by the α CamKII-cre transgene. Cortex from 4 week old mice was also examined for the amount of β3. This analysis revealed a ~50% reduction in β3 in forebrain selective knockout mice compared to controls (Figure 4E).


New insight into the role of the beta3 subunit of the GABAA-R in development, behavior, body weight regulation, and anesthesia revealed by conditional gene knockout.

Ferguson C, Hardy SL, Werner DF, Hileman SM, Delorey TM, Homanics GE - BMC Neurosci (2007)

Western blot analysis of forebrain selective neuronal β3 knockouts. Western blot analysis of the β3 subunit of the GABAA-R from individual 13–16 week old mice revealed dramatically reduced amounts of β3 in hippocampus (A) and cortex (B) of α CamKII-cre positive samples compared to cre negative control samples. The amount of β3 in cerebellum (C) did not differ between genotypes. Analysis of cortex for the GABAA-R α1 subunit revealed a ~95% reduction in knockout samples compared to cre negative controls. All blots were reprobed for β-actin as a loading control. Shown are representative western blots. Four mice of each genotype were analyzed and all samples were analyzed on at least two different blots. D. Summary graph of western blot analysis demonstrating a significant (*, p ≤ 0.01; **, p < 0.001) reduction in β3 protein in hippocampus and cortex, but not in cerebellum. Data are expressed as mean ± SEM of percent change in band intensity relative to cre negative controls following normalization to actin. (E) Western blot analysis of β3 in cortex from individual 4 week old mice revealed dramatically reduced amounts of β3 in cre positive samples compared to cre negative control samples.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2100059&req=5

Figure 4: Western blot analysis of forebrain selective neuronal β3 knockouts. Western blot analysis of the β3 subunit of the GABAA-R from individual 13–16 week old mice revealed dramatically reduced amounts of β3 in hippocampus (A) and cortex (B) of α CamKII-cre positive samples compared to cre negative control samples. The amount of β3 in cerebellum (C) did not differ between genotypes. Analysis of cortex for the GABAA-R α1 subunit revealed a ~95% reduction in knockout samples compared to cre negative controls. All blots were reprobed for β-actin as a loading control. Shown are representative western blots. Four mice of each genotype were analyzed and all samples were analyzed on at least two different blots. D. Summary graph of western blot analysis demonstrating a significant (*, p ≤ 0.01; **, p < 0.001) reduction in β3 protein in hippocampus and cortex, but not in cerebellum. Data are expressed as mean ± SEM of percent change in band intensity relative to cre negative controls following normalization to actin. (E) Western blot analysis of β3 in cortex from individual 4 week old mice revealed dramatically reduced amounts of β3 in cre positive samples compared to cre negative control samples.
Mentions: To examine the impact of α CamKII-cre mediated recombination on the β3 gene product, western blot analysis was conducted on hippocampus, cortex, cerebellum, and hypothalamus using a β3 specific antibody. As shown in Figure 4, β3 protein was dramatically reduced in hippocampus and was nearly completely ablated in cortex of cre+ mice compared to cre- controls. In contrast, the amount of β3 in cerebellum was not significantly reduced by the α CamKII-cre transgene. Cortex from 4 week old mice was also examined for the amount of β3. This analysis revealed a ~50% reduction in β3 in forebrain selective knockout mice compared to controls (Figure 4E).

Bottom Line: However, developmental abnormalities, compensation, reduced viability, and numerous behavioral abnormalities limited the usefulness of that murine model.Survivors had reduced reproductive fitness, reduced sensitivity to etomidate, were hyperactive, and some became obese.Conditional inactivation of the beta3 gene revealed novel insight into the function of this GABAA-R subunit.

View Article: PubMed Central - HTML - PubMed

Affiliation: Departments of Anesthesiology and Pharmacology, University of Pittsburgh, Pittsburgh, PA 15261, USA. fergusonc@anes.upmc.edu

ABSTRACT

Background: The beta3 subunit of the gamma-aminobutyric acid type A receptor (GABAA-R) has been reported to be important for palate formation, anesthetic action, and normal nervous system function. This subunit has also been implicated in the pathogenesis of Angelman syndrome and autism spectrum disorder. To further investigate involvement of this subunit, we previously produced mice with a global knockout of beta3. However, developmental abnormalities, compensation, reduced viability, and numerous behavioral abnormalities limited the usefulness of that murine model. To overcome many of these limitations, a mouse line with a conditionally inactivated beta3 gene was engineered.

Results: Gene targeting and embryonic stem cell technologies were used to create mice in which exon 3 of the beta3 subunit was flanked by loxP sites (i.e., floxed). Crossing the floxed beta3 mice to a cre general deleter mouse line reproduced the phenotype of the previously described global knockout. Pan-neuronal knockout of beta3 was achieved by crossing floxed beta3 mice to Synapsin I-cre transgenic mice. Palate development was normal in pan-neuronal beta3 knockouts but ~61% died as neonates. Survivors were overtly normal, fertile, and were less sensitive to etomidate. Forebrain selective knockout of beta3 was achieved using alpha CamKII-cre transgenic mice. Palate development was normal in forebrain selective beta3 knockout mice. These knockouts survived the neonatal period, but ~30% died between 15-25 days of age. Survivors had reduced reproductive fitness, reduced sensitivity to etomidate, were hyperactive, and some became obese.

Conclusion: Conditional inactivation of the beta3 gene revealed novel insight into the function of this GABAA-R subunit. The floxed beta3 knockout mice described here will be very useful for conditional knockout studies to further investigate the role of the beta3 subunit in development, ethanol and anesthetic action, normal physiology, and pathophysiologic processes.

Show MeSH
Related in: MedlinePlus