Conserved role of unc-79 in ethanol responses in lightweight mutant mice.

An ENU-induced QTL for locomotor activity and body weight maps to distal mouse chromosome 12.(A) A mutagenized (B6:D2*) male 28C4 was backcrossed to C57BL/6J wild-type females and spontaneous locomotor activity of male progeny was monitored for a period of two hours followed by QTL analysis (see reference [23] for details). Significant (P<0.01) levels of linkage (dashed black line) were determined by permutation testing [39]. (B) Male B6:D2*×B6 progeny were weighed between the age of 8 to 10 weeks and QTL analysis was performed. (C) Haplotype analysis of the mapping population indicates that Mutant/+ mice, which are unambiguous carriers of mutagenized chromosome 12 across the 95% confidence interval (n = 50), were modestly hyperactive relative to +/+ animals (n = 43) across the same confidence interval. Inset: Cumulative distance traveled (CDT) over two hours (in centimeters) is significantly elevated in Mutant/+ males (white bar) versus +/+ animals (black bar) (***P≪0.0001, Student's t-test). (D) Scatterplot of body weight haplotypes of unambiguous +/+ males (•, n = 43) and Mutant/+ males (○, n = 49) across the 95% confidence interval on chromosome 12. Black line indicates average weight of population. +/+ males weigh significantly more than Mutant/+ males (***P≪0.0001, Student's t-test). See Figure 2 for haplotype structure of Mutant animals.

Chromosome 12 haplotype structure of animals used for behavioral testing.B6:D2*×B6 N2 (Mutant) mice harbor the mutagenized D2 chromosome 12 across the 95% confidence interval (between D12Mit158 and D12Mit263). The rest of the genome is a mixed B6 and D2 genetic background. Lwt and Control (see [30]) mice are congenic strains on a pure B6 genetic background. Lwt/+ and Control/+ males were backcrossed to either B6 or D2 females to produce populations for behavioral testing. B6 chromosomal DNA is represented in black, D2 chromosomal DNA represented in red, and mutagenized D2 chromosomal DNA is represented by hatched red.

Positional cloning of the unc-79 Lightweight mutation.(A) Haplotype analysis of surviving animals from Mutant/+ intercross. Mutant/+ animals were intercrossed (Left plot) and animals that survived were genotyped across the distal portion of mouse chromosome 12. Black boxes indicate homozygosity for the mutagenized D2 strain and grey boxes indicate either homozygosity for the B6 strain or heterozygosity for B6 and D2. Numbers of surviving animals with a particular haplotype are indicated at the bottom. Three different male carriers (Right plot) with the indicated haplotypes were crossed to Mutant/+ females, and the ratio of surviving progeny/total number born was monitored. Both crosses indicate that the lethality maps to a region between D12Mit180 and D112Mit195. Note that survival of animals born to Mutant/+ females is lower than expected for unknown reasons. (B) Sequence analysis of +/+ and Lwt/Lwt DNA. Note the G→T transversion in Lwt/Lwt which changes a glutamic acid residue to a premature stop codon. (C) Exon structure of unc-79 gene (AB257853) with position of Lwt point mutation indicated by (*). Genome coordinates were determined using NCBI Build 37. The C-terminal portion of the protein used to generate the antibody is indicated by a rectangle. (D) Western analysis of +/+ and Lwt/Lwt P0 whole brain lysates. Note the absence of unc-79 protein in Lwt/Lwt homozygotes at ∼300 kDa.

B6.D2-Unc-79Lwt (Lwt) congenic strain captures locomotor and body weight phenotypes.(A) Spontaneous locomotor activity was assayed in +/+ (n = 34) and Lwt/+ (n = 26) male littermates. Inset: Cumulative distance traveled (CDT) over two hours (in centimeters) in Lwt/+ males (white bar) was significantly higher than in +/+ (black bar) littermates (**P<0.01, Student's t-test). (B) Congenic strain B6.D2.12D heterozygotes (denoted here and elsewhere as Control/+) was used to control for polymorphic differences between the B6 and D2 strains on mouse distal chromosome 12. There was no significant difference between +/+ (n = 34) and Control/+ (n = 34) male littermates in locomotor activity. Inset: Cumulative distance traveled (CDT) over two hours (in centimeters) did not differ significantly between Control/+ (white bar) and +/+ (black bar) male littermates (Student's t-test). (C) At all ages tested Lwt/+ (n = 13) male animals weighed less than their +/+ (n = 15) littermates (***P<0.001, post hoc Tukey test). (D) Control/+ (n = 15) and +/+ (n = 15) male littermates did not differ significantly in body weight.

Alterations in acute responses to ethanol in Lwt/+ congenic mice.(A) Duration of loss of righting reflex in response to the indicated dose of ethanol (g/kg, i.p.) was determined for both Lwt/+ (n = 13–18) and +/+ male littermates (n = 12–23). There was a highly significant increase in time to recovery at all doses tested in Lwt/+ animals (***P<0.001, post hoc Tukey test). (B) Male Lwt/+ (n = 9) and +/+ littermates (n = 9) were tested for ethanol clearance after a 3.6 g/kg injection of ethanol (i.p.). These animals were sampled repeatedly at the times indicated. There were no significant differences between the genotypes at any timepoint. In addition, three additional animals of each genotype were sampled only at the 180 minute timepoint to control for repeated sampling. No significant differences were found between the 180 minute values from the repeatedly and acutely sampled groups. (C) Mean locomotor activity of +/+ male mice (n = 12–16) for the first five minutes after treatment with either saline (solid lines) or ethanol (dotted lines). (D) Mean locomotor activity of Lwt/+ male mice (n = 12–15) for the first five minutes after treatment with either saline (solid lines) or ethanol (dotted lines). Note that there is significantly higher locomotor activity (P = 0.038, post hoc Tukey test) in response to saline injection in Lwt/+ mice relative to +/+ mice in the locomotor activation experiment shown in (C) and (D). (**P<0.01; ***P<0.001, post hoc Tukey test).

Lwt/+ congenic mice are resistant isoflurane anesthesia.(A) The minimum alveolar concentration (MAC) required to suppress movement in response to a tail pinch was determined for several anesthetic agents in both Lwt/+ (white bars) (n = 9) and +/+ littermates (black bars) (n = 9). There was no significant difference in MAC for halothane, cyclopropane, or sevoflurane; however, there was a significant increase in the MAC in Lwt/+ animals in response to isoflurane (***P<0.0001, Student's t-test). (B) MAC was determined for halothane and isoflurane in Control/+ (white bar) (n = 13) and +/+ (black bar) (n = 13) male littermates. There was no significant difference between genotypes in MAC.

Lwt/+ congenic mice exhibit a higher preference for and consumption of ethanol.(A) Lwt/+ (n = 13) animals have a higher preference for ethanol than +/+ littermates (n = 14). (B) There was no difference in ethanol preference in Control/+ (n = 15) relative to +/+ (n = 15) littermates. (C) Lwt/+ (n = 13) animals consume more ethanol than +/+ littermates (n = 14). (D) There was no difference in ethanol consumption in Control/+ (n = 15) relative to +/+ (n = 15) littermates. (*P<0.05; **P<0.01; ***P<0.001, post hoc Tukey test).

Taste preference is not altered in Lwt/+ congenic mice.Relative to +/+ littermates (n = 14) (black bars), Lwt/+ (n = 13) (white bars) animals do not show differences in preference for (A) or consumption of (B) saccharin (%, w/v), or in preference for (C) or consumption of (D) quinine (units in mM). The same is true for the control strain (data not shown).

Lwt/+ congenic mice develop a conditioned place preference to ethanol.Wild-type (n = 11) and Lwt/+ (n = 11) littermates developed a conditioned place preference to a 1.75 g/kg dose of ethanol. (*P<0.05; ***P<0.001 compared with saline).

Swimming behavior is hypersensitive to ethanol in C. elegans unc-79, unc-80, and nca-1;nca-2 double mutants.Swimming sensitivity to ethanol (400mM). The mean relative frequency of body bends during swimming of wild-type (N2) and various mutants is shown. Error bars indicate SEM. Asterisk indicates a statistical difference as tested by Student's t-test (*P<0.05, **P<0.005, ***P<0.0001).