Towards trans-diagnostic mechanisms in psychiatry: neurobehavioral profile of rats with a loss-of-function point mutation in the dopamine transporter gene

Abstract

The research domain criteria (RDoC) matrix has been developed to reorient psychiatric research towards measurable behavioral dimensions and underlying mechanisms. Here, we used a new genetic rat model with a loss-of-function point mutation in the dopamine transporter (DAT) gene (Slc6a3_N157K) to systematically study the RDoC matrix. First, we examined the impact of the Slc6a3_N157K mutation on monoaminergic signaling. We then performed behavioral tests representing each of the five RDoC domains: negative and positive valence systems, cognitive, social and arousal/regulatory systems. The use of RDoC may be particularly helpful for drug development. We studied the effects of a novel pharmacological approach metabotropic glutamate receptor mGluR2/3 antagonism, in DAT mutants in a comparative way with standard medications. Loss of DAT functionality in mutant rats not only elevated subcortical extracellular dopamine concentration but also altered the balance of monoaminergic transmission. DAT mutant rats showed deficits in all five RDoC domains. Thus, mutant rats failed to show conditioned fear responses, were anhedonic, were unable to learn stimulus-reward associations, showed impaired cognition and social behavior, and were hyperactive. Hyperactivity in mutant rats was reduced by amphetamine and atomoxetine, which are well-established medications to reduce hyperactivity in humans. The mGluR2/3 antagonist LY341495 also normalized hyperactivity in DAT mutant rats without affecting extracellular dopamine levels. We systematically characterized an altered dopamine system within the context of the RDoC matrix and studied mGluR2/3 antagonism as a new pharmacological strategy to treat mental disorders with underlying subcortical dopaminergic hyperactivity.

Keywords: In vivo microdialysis; Molecular modeling; RDoC matrix; Rat mutagenesis; mGluR2/3 antagonist LY341495.

Fig. 1.

Molecular characterization of a novel mutant rat model with a subcortical hyperdopaminergic state. (A) Secondary structures of the wild-type (gray) and N157K-mutant (red) dopamine transporter (DAT) following energy minimization (1 ns) within an environment resembling the intracellular space. (B-D) Subconfluent HEK293 cells transiently transfected with rat DAT-wt (WT) or rat DAT-N157K (N157K). (B) Immunofluorescence and confocal analysis of DAT protein using an antibody targeted against an extracellular epitope of DAT in the absence (top row) and presence (bottom row) of detergent saponine (sap). A weak cell surface labeling is seen for DAT-N157K in the absence of detergent (top right) and a globular distribution of protein is seen for both DAT-wt and DAT-N157K (bottom row). Shown are example z-projections of single cells. (C) [³H]DA uptake in HEK293 cells expressing DAT. Specific [³H]DA uptake is defined as the difference between monoamine transporter mediated uptake minus control uptake in HEK293 cells that have been transiently transfected with empty vector pcDNA3.1(pcDNA). (D) Total (-CFT) and nonspecific (+CFT) binding of [³H]WIN35,428 to DAT. Nonspecific binding was determined in the presence of 50 µM β-CFT (WIN35,428). (E) Total [³H]DA uptake in synaptosomes obtained from the dorsal striatum (caudate putamen, CPu) from wild-type (WT, n=3) and DAT mutant (N157K, n=3) rats. (F) Basal extracellular DA levels in the CPu and medial prefrontal cortex (PFC) of WT (n=12) and N157K (n=9) rats. (G) Quantitative analysis of DAT ([³H]-mazindol) binding levels in the CPu, nucleus accumbens core (AcbC), nucleus accumbens shell (AcbS) and ventral tegmental area (VTA) of WT (n=10) and N157K (n=9) rats. (H) Representative dark-field images showing [³H]-mazindol binding on a coronal brain section from WT and N157K rats at the striatal level. (I) Tissue concentration of dopamine in homogenates of CPu, AcbC, AcbS, VTA and PFC of WT (n=5) and N157K (n=5) rats. All data are expressed as means±s.e.m. *P<0.05, significant difference from DAT-wt cells/WT rats; ⁺P<0.05, significant difference from the total binding.

Fig. 2.

Systematic analysis of the DAT-N157K mutant rats according to the RDoC matrix. (A-C) Negative valence systems are represented as time spent freezing (calculated as percentage of the total exposure time) during footshock-conditioned (A) context and (B) cue in wild-type (WT, n=6) and DAT mutant (N157K, n=5) rats in fear-conditioning paradigm. (C) Time (s) spent in open arms by WT (n=9) and N157K (n=15) rats during a 5 min plus-maze test. (D,E) Positive valence systems are represented as (D) preference for 0.5% sucrose solution over water during the 24-h free-choice sucrose preference test in WT (n=16) and N157K (n=25) rats and as (E) number of conditioned responses during the autoshaping task in WT (n=15) and N157K (n=11) rats. In this task, the first three daily sessions consisted of 20 trials and the fourth session consisted of 40 trials. (F,G) Cognitive systems are represented as (F) time spent exploring a novel object (calculated as percentage of the total exploration time) during the novel object recognition test in WT (n=22) and N157K (n=23) rats and as (G) percentage of correct choices during the T-maze spontaneous alternation test in WT (n=5) and N157K (n=5) rats. (H,I) Systems for social processes are represented as number of (H) anogenital and non-anogenital exploration and (I) approach/following events in WT (n=12) and N157K (n=11) rats during 5 min interaction with an unknown social partner in the social interaction test. (J-L) Arousal and regulatory systems are represented as (J) circadian activity recordings in WT (n=11) and N157K (n=9) rats measured as the number of movements during five consecutive days in the home cage [black horizontal bars mark the dark (active) phases of the circadian cycle] and as locomotor vigilance measured as (K) distance travelled (m) every 10 min and as (L) total number of rearings in the 60 min open-field test in WT (n=20) and N157K (n=24) rats. All data are expressed as means±s.e.m. *P<0.05, significant difference from WT rats.

Fig. 3.

Pharmacological reversal of phenotypic alterations in DAT-N157K rats. (A,B,D,E) Distance traveled (cm) during 60 min testing in the open field in wild-type (WT, n=7-15) and DAT mutant (N157K, n=6-18) rats. Thirty minutes before the test, all rats were administered vehicle, 2 mg/kg amphetamine (A,B) or 10 mg/kg LY-341495 (D,E). The data are shown as total distance traveled (A,D) and as distance traveled every 10 min (B,E). (C,F) Extracellular DA levels in the caudate putamen of WT (n=6-7) and N157K (n=5-7) rats. At the time point 0 min, all rats were administered with the vehicle; 60 min later animals were given either 2 mg/kg amphetamine (C) or 10 mg/kg LY341495 (F). Microdialysis samples were collected every 20 min. All data are expressed as means±s.e.m. *P<0.05, significant difference from WT rats.