Cre recombinase-mediated restoration of nigrostriatal dopamine in dopamine-deficient mice reverses hypophagia and bradykinesia

Abstract

A line of dopamine-deficient (DD) mice was generated to allow selective restoration of normal dopamine signaling to specific brain regions. These DD floxed stop (DDfs) mice have a nonfunctional Tyrosine hydroxylase (Th) gene because of insertion of a NeoR gene flanked by lox P sites targeted to the first intron of the Th gene. DDfs mice have trace brain dopamine content, severe hypoactivity, and aphagia, and they die without intervention. However, they can be maintained by daily treatment with l-3,4-dihydroxyphenylalanine (L-dopa). Injection of a canine adenovirus (CAV-2) engineered to express Cre recombinase into the central caudate putamen restores normal Th gene expression to the midbrain dopamine neurons that project there because CAV-2 efficiently transduces axon terminals and is retrogradely transported to neuronal cell bodies. Bilateral injection of Cre recombinase into the central caudate putamen restores feeding and normalizes locomotion in DDfs mice. Analysis of feeding behavior by using lickometer cages revealed that virally rescued DDfs mice are hyperphagic and have modified meal structures compared with control mice. The virally rescued DDfs mice are also hyperactive at night, have reduced motor coordination, and are thigmotactic compared with controls. These results highlight the critical role for dopamine signaling in the dorsal striatum for most dopamine-dependent behaviors but suggest that dopamine signaling in other brain regions is important to fine-tune these behaviors. This approach offers numerous advantages compared with previous models aimed at examining dopamine signaling in discrete dopaminergic circuits.

Conflict of interest statement

Conflict of interest statement: No conflicts declared.

Figures

Fig. 1

Illustration of viral rescue strategy to restore Th production to dopamine neurons in DDfs mice (Thfs/fs; DbhTh/+) (see Fig. 6 for more details). (A) Before viral rescue, DDfs mice have a Pgk-NeoR cassette flanked by lox P sites disrupting expression of the Th gene. (B) 1, CAVCre is injected into the central CPu (dashed circle approximates extent of viral spread); 2, CAVCre transduces local axon terminals near the site of injection, including those of midbrain dopamine neurons; 3, CAVCre is retrogradely transported to the dopamine cell bodies where it drives expression of Cre recombinase leading to the excision of the stop cassette allowing expression of the endogenous Th gene; 4, Th is then transported to the axon terminals of the infected dopaminergic neurons where it synthesizes l-dopa. CAVCre is retrogradely transported to cortical glutamatergic neurons as well, but recombination in those neurons is inconsequential because the Th gene is not expressed in those cells. (C) After Cre recombinase-mediated excison of the Pgk-NeoR cassette, Th expression and dopamine production are restored.

Fig. 2

Th (green) and dopamine transporter (red) immunostaining in coronal sections of brains from DDfs, control, and vrDDfs mice. (A–F) Sections through the SNc and VTA were subjected to immunohistochemistry with antisera to Th and DAT. There was no Th signal in DDfs mice, but DAT staining indicates the presence of intact dopamine neurons. Th+ neurons are abundant in the SNc of control and vrDDfs mice. There are a few Th+ cell bodies in the dorsal VTA of vrDDfs mice. (G–L) Sections through the striatum, showing projections of the midbrain dopamine neurons. Th staining was present throughout the CPu and ventrolateral striatum of vrDDfs mice. The medial shell and core of the NAc were devoid of Th signal in vrDDfs mice.

Fig. 3

Feeding behavior in lickometer cages by control (n = 8; open symbols) and vrDDfs (n = 7; filled symbols) mice on a novel, highly palatable liquid diet. (A) Lick time course in 2-h bins over 11 days (white bars) and nights (dark bars). The experiment is separated into four phases as shown. vrDDfs mice required more time to achieve a steady-state baseline feeding pattern (rm-ANOVA on acclimation days 1 to 3, genotype × time interaction, P < 0.001; rm-ANOVA on days 4–8, genotype effect P < 0.01). (B) Average baseline licking was significantly greater for vrDDfs mice over the 24-h feeding cycle (rm-ANOVA, genotype effect P < 0.05, genotype × time interaction, P < 0.001). (C) Food intake (by weight) was also greater in vrDDfs mice, demonstrating that the increased licking does not simply reflect decreased lick efficiency (rm-ANOVA on days 4–8, genotype effect P < 0.01). (D) The distribution of meal sizes during the baseline phase is different in vrDDfs mice compared with controls (rm-ANOVA, genotype × size interaction, P < 0.05) with the largest difference being the increase in very large meals (>1,000 licks) in vrDDfs mice. (E) The distribution of lick rates during baseline phase meals is shifted in vrDDfs mice, indicating that they spend more time licking at faster rates (rm-ANOVA, genotype × rate interaction P < 0.001). All data expressed as means ± SEM.

Fig. 4

Lickometer analysis of fed control (n = 8) and vrDDfs (n = 8) mice with 2 h of access to highly palatable liquid diet for 10 consecutive days. Both genotypes increased consumption of liquid diet over the course of 10 days, but the vrDDfs mice had more licks per day and a steeper rate of increase; most of this increase occurred during the first 20 min (shown here) of access to the diet (rm-ANOVA, genotype effect P < 0.05, day effect P < 0.001, genotype × day interaction P < 0.05). All data expressed as means ± SEM.

Fig. 5

Locomotor activity of control (n = 8) and vrDDfs (n = 8) mice. (A) Representative 3-day locomotor time course. vrDDfs mice are hyperactive relative to control mice during the nocturnal phase (dark bars). (B) l-dopa induces a dose-dependent locomotor response in vrDDfs but not control mice (rm-ANOVA, treatment effect P < 0.05, genotype × treatment interaction P < 0.05). (C) Rotarod performance improved over ten trials in both genotypes but was impaired in the vrDDfs mice compared with control mice (rm-ANOVA, genotype effect P < 0.05, trial effect P < 0.001). All data expressed as means ± SEM.