Calcium-stimulated adenylyl cyclases modulate ethanol-induced neurodegeneration in the neonatal brain

Abstract

Fetal alcohol exposure results in cognitive and neurobehavioral deficits, but the effects of modifying genetic loci on the severity of these sequelas have not been well characterized. Although the cAMP signaling pathway has been shown to be an important modulator of ethanol sensitivity in adult mice, its potential role in modulating ethanol-induced neurodegeneration has not been examined. Adenylyl cyclases (ACs) 1 and 8 produce cAMP in response to intracellular calcium elevation and modulate several aspects of neuronal function, including ethanol sensitivity. AC1 and AC8 are expressed widely throughout the brain of neonatal mice, and genetic deletion of both AC1 and AC8 in double-knock-out (DKO) mice enhances ethanol-induced neurodegeneration in the brains of neonatal mice. In addition, ethanol treatment induces significantly greater levels of caspase-3 activation in the brains of DKO mice compared with wild-type (WT) mice, reflecting higher numbers of apoptotic neurons. Administration of the NMDA receptor antagonist MK801 [(+)-5-methyl-10,11-dihydro-5H-dibenzo [a,d] cyclohepten-5,10-imine hydrogen maleate] or the GABA(A) receptor potentiator phenobarbital, which mimics components of the effects of ethanol on neurons, results in significantly greater neurodegeneration in the brains of neonatal DKO mice than WT mice. Furthermore, loss of a single calcium-stimulated AC isoform potentiates neurodegeneration after administration of ethanol, MK801, or phenobarbital. In contrast, the levels of physiological cell death, death after hypoxia/ischemia, and excitotoxic cell death are not increased in the brains of DKO mice. Thus, AC1 and AC8 are critical modulators of neurodegeneration induced by activity blockade in the neonatal brain and represent genetic loci that may potentially modify the severity of fetal alcohol syndrome.

Figure 1.

AC1 and AC8 are expressed widely in the brain at P7 and do not modulate ethanol metabolism. A, In situ hybridization for mRNAs for AC1 and AC8 in coronal (left) and sagittal (right) brain sections at P7. AC1 and AC8 are both present in cingulate cortex (Cing), parietal cortex, retrosplenial cortex (RSC), cerebellum (Cbl), area CA1 of the hippocampus (CA1), and thalamus (Thal). In addition, AC1 is present in dentate gyrus (DG), and AC8 is present in olfactory bulb (OB). B, Plasma ethanol concentration at various times after ethanol administration in P7 mice. We administered 2.2 g/kg ethanol subcutaneously at 0 and 2 h (indicated by arrows). n = 5 mice for all points except t = 3 h, where n = 14 mice.

Figure 2.

Ethanol-induced neurodegeneration is enhanced in the brains of neonatal DKO mice. A, Representative sections depicting the median amount of silver deposition for each genotype in the anterodorsal (AD) and anteroventral (AV) thalamic nuclei of WT and DKO brains 24 h after administration of two doses of 2.2 g/kg ethanol. B, Representative sections depicting the median amount of silver deposition for each genotype in the hippocampus, cingulate cortex (Cing), and parietal cortex (P Ctx) of WT and DKO brains 24 h after administration of two doses of 2.2 g/kg ethanol. C, Representative sections depicting the median amount of silver deposition for each genotype in the retrosplenial cortex (RSC) and subiculum (Sub) of WT and DKO brains 24 h after administration of two doses of 2.2 g/kg ethanol. D, Quantitation of neurodegeneration in various brain regions of neonatal WT and DKO mice 24 h after administration of two doses of 2.2 g/kg ethanol (^*p < 0.05 vs WT; ^**p < 0.01 vs WT; n = 9-10 mice per genotype). E, Quantitation of neurodegeneration in various brain regions of neonatal WT and DKO mice 24 h after administration of two doses of 2.5 g/kg ethanol (^*p < 0.05 vs WT; ^**p < 0.01 vs WT; n = 9 mice per genotype). F, Survival of hippocampal neurons from WT and DKO mice cultured in the presence of 50 mm ethanol or 1 μm tetrodotoxin (TTX) (^*p < 0.05 vs WT; n = 5-6 per genotype).

Figure 3.

Ethanol-induced caspase-3 activation is greater in the brains of neonatal DKO mice than WT mice. A, Western blot analysis for the levels of cleaved (activated) caspase-3 in the brains of WT and DKO mice at various times after administration of two doses of 2.2 g/kg ethanol. B, Western blot analysis for the levels of cleaved (activated) caspase-3 in the brains of WT and DKO mice 8 h after administration of two doses of 2.2 g/kg ethanol. C, Quantitation of cleaved caspase-3 levels in the brains of WT and DKO mice 8 h after administration of two doses of 2.2 g/kg ethanol (n = 3 mice per genotype; ^**p < 0.01 vs WT 8 h). D, Quantitation of cleaved caspase-3 levels in the brains of WT and DKO mice 12 h after administration of two doses of 2.2 g/kg ethanol (n = 5-7 mice per genotype; ^*p < 0.05 vs WT 12 h).

Figure 4.

Neurodegeneration after administration of the GABAergic modulator phenobarbital is enhanced in the brains of neonatal DKO mice compared with WT mice. A, Representative sections depicting the median amount of silver deposition for each genotype in the anterodorsal (AD) and anteroventral (AV) thalamic nuclei of WT and DKO brains 24 h after administration of 70 mg/kg phenobarbital. B, Representative sections depicting the median amount of silver deposition for each genotype in the hippocampus, cingulate cortex (Cing), and parietal cortex (P Ctx) of WT and DKO brains 24 h after administration of 70 mg/kg phenobarbital. C, Representative sections depicting the median amount of silver deposition for each genotype in the retrosplenial cortex (RSC) and subiculum (Sub) of WT and DKO brains 24 h after administration of 70 mg/kg phenobarbital. D, Quantitation of neurodegeneration in various brain regions of neonatal WT and DKO mice 24 h after administration of 70 mg/kg phenobarbital (^*p < 0.05 vs WT; ^**p < 0.01 vs WT; n = 6 mice per genotype).

Figure 5.

Neurodegeneration after administration of the NMDA receptor antagonist MK801 is enhanced in the brains of neonatal DKO mice compared with WT mice. A, Representative sections depicting the median amount of silver deposition for each genotype in the anterodorsal (AD) and anteroventral (AV) thalamic nuclei of WT and DKO brains 24 h after administration of three doses of 0.65 mg/kg MK801. B, Representative sections depicting the median amount of silver deposition for each genotype in the hippocampus, cingulate cortex (Cing), and parietal cortex (P Ctx) of WT and DKO brains 24 h after administration of three doses of 0.65 mg/kg MK801. C, Representative sections depicting the median amount of silver deposition for each genotype in the retrosplenial cortex (RSC) and subiculum (Sub) of WT and DKO brains 24 h after administration of three doses of 0.65 mg/kg MK801. D, Quantitation of neurodegeneration in various brain regions of neonatal WT and DKO mice 24 h after administration of three doses of 0.65 mg/kg MK801 (^*p < 0.05 vs WT; ^**p < 0.01 vs WT; n = 6 mice per genotype).

Figure 6.

Loss of a single calcium-stimulated AC isoform is sufficient to potentiate neurodegeneration after administration of ethanol, phenobarbital, or MK801. A, Quantitation of neurodegeneration in various brain regions of neonatal WT, AC1 KO, AC8 KO, and DKO mice 24 h after administration of two doses of 2.2 g/kg ethanol (^*p < 0.05 vs WT; n = 6-10 mice per genotype). B, Quantitation of neurodegeneration in various brain regions of neonatal WT, AC1 KO, AC8 KO, and DKO mice 24 h after administration of two doses of 2.5 g/kg ethanol (^*p < 0.05 vs WT; n = 6-10 mice per genotype). C, Quantitation of neurodegeneration in various brain regions of neonatal WT, AC1 KO, AC8 KO, and DKO mice 24 h after administration of 70 mg/kg phenobarbital (^*p < 0.05 vs WT; n = 6-7 mice per genotype). D, Quantitation of neurodegeneration in various brain regions of neonatal WT, AC1 KO, AC8 KO, and DKO mice 24 h after administration of three doses of 0.65 mg/kg MK801 (^*p < 0.05 vs WT; n = 6 mice per genotype).

Figure 7.

Loss of calcium-stimulated ACs does not sensitize neurons to excitotoxic or hypoxic/ischemic death. A, Representative section of excitotoxic cell death in brain of neonatal WT mouse after administration of 1.25 g/kg monosodium glutamate. B, Representative section of excitotoxic cell death in brain of neonatal DKO mouse after administration of 1.25 g/kg monosodium glutamate. C, Quantitation of excitotoxic cell death in arcuate nucleus of neonatal WT and DKO mice (n = 7 mice per genotype). D, Caspase-3 activity (as assessed by cleavage of substrate DEVD) in lysates of hippocampi ipsilateral and contralateral to the site of carotid artery ligation 24 h after hypoxia/ischemia.