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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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Body weight and food consumption analyses of forebrain selective β3 knockouts. Growth curves of (A) male and (B) female mice (n = 4–12 mice of each gender per genotype at each age). Body weight of knockout mice was greater than controls (repeated measures ANOVA: p < 0.01 for males; p < 0.05 for females). Plotted are means ± SEM. For data points without error bars, the bars are obscured by the symbol. Body weight of individual (C) male and (D) female mice at 14 weeks of age. The horizontal line indicates the group mean. Male and female knockouts were heavier than controls (p ≤ 0.05). Note the great variability in the body weight of knockout mice, including several obviously obese male animals that are ~1.75× heavier than controls. (E) Obese forebrain selective male knockout mice consumed more food than controls (*, p < 0.05). Shown is the average daily consumption per mouse ± SEM.
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Figure 5: Body weight and food consumption analyses of forebrain selective β3 knockouts. Growth curves of (A) male and (B) female mice (n = 4–12 mice of each gender per genotype at each age). Body weight of knockout mice was greater than controls (repeated measures ANOVA: p < 0.01 for males; p < 0.05 for females). Plotted are means ± SEM. For data points without error bars, the bars are obscured by the symbol. Body weight of individual (C) male and (D) female mice at 14 weeks of age. The horizontal line indicates the group mean. Male and female knockouts were heavier than controls (p ≤ 0.05). Note the great variability in the body weight of knockout mice, including several obviously obese male animals that are ~1.75× heavier than controls. (E) Obese forebrain selective male knockout mice consumed more food than controls (*, p < 0.05). Shown is the average daily consumption per mouse ± SEM.

Mentions: During the course of these studies, we also noticed that some, but not all, β3F/F, cre+ became obese in adulthood. To quantify this, the body weights of mice from 4–16 weeks of age were measured. As shown in Figure 5A, B, cre+ mice attained significantly greater body weights by ~8–10 weeks of age compared to cre- controls. Furthermore, analysis of the distribution of body weights revealed that there was considerable variability in the body weights of the cre+ mice. For example, at 14 weeks of age, while some cre+ animals had body weights that overlaped with controls, others had body weights that were 1.75× average control values (Figure 5C, D).


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)

Body weight and food consumption analyses of forebrain selective β3 knockouts. Growth curves of (A) male and (B) female mice (n = 4–12 mice of each gender per genotype at each age). Body weight of knockout mice was greater than controls (repeated measures ANOVA: p < 0.01 for males; p < 0.05 for females). Plotted are means ± SEM. For data points without error bars, the bars are obscured by the symbol. Body weight of individual (C) male and (D) female mice at 14 weeks of age. The horizontal line indicates the group mean. Male and female knockouts were heavier than controls (p ≤ 0.05). Note the great variability in the body weight of knockout mice, including several obviously obese male animals that are ~1.75× heavier than controls. (E) Obese forebrain selective male knockout mice consumed more food than controls (*, p < 0.05). Shown is the average daily consumption per mouse ± SEM.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Body weight and food consumption analyses of forebrain selective β3 knockouts. Growth curves of (A) male and (B) female mice (n = 4–12 mice of each gender per genotype at each age). Body weight of knockout mice was greater than controls (repeated measures ANOVA: p < 0.01 for males; p < 0.05 for females). Plotted are means ± SEM. For data points without error bars, the bars are obscured by the symbol. Body weight of individual (C) male and (D) female mice at 14 weeks of age. The horizontal line indicates the group mean. Male and female knockouts were heavier than controls (p ≤ 0.05). Note the great variability in the body weight of knockout mice, including several obviously obese male animals that are ~1.75× heavier than controls. (E) Obese forebrain selective male knockout mice consumed more food than controls (*, p < 0.05). Shown is the average daily consumption per mouse ± SEM.
Mentions: During the course of these studies, we also noticed that some, but not all, β3F/F, cre+ became obese in adulthood. To quantify this, the body weights of mice from 4–16 weeks of age were measured. As shown in Figure 5A, B, cre+ mice attained significantly greater body weights by ~8–10 weeks of age compared to cre- controls. Furthermore, analysis of the distribution of body weights revealed that there was considerable variability in the body weights of the cre+ mice. For example, at 14 weeks of age, while some cre+ animals had body weights that overlaped with controls, others had body weights that were 1.75× average control values (Figure 5C, D).

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