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, 17 (10), e3000477
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NF1-cAMP Signaling Dissociates Cell Type-Specific Contributions of Striatal Medium Spiny Neurons to Reward Valuation and Motor Control

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NF1-cAMP Signaling Dissociates Cell Type-Specific Contributions of Striatal Medium Spiny Neurons to Reward Valuation and Motor Control

Laurie P Sutton et al. PLoS Biol.

Abstract

The striatum plays a fundamental role in motor learning and reward-related behaviors that are synergistically shaped by populations of D1 dopamine receptor (D1R)- and D2 dopamine receptor (D2R)-expressing medium spiny neurons (MSNs). How various neurotransmitter inputs converging on common intracellular pathways are parsed out to regulate distinct behavioral outcomes in a neuron-specific manner is poorly understood. Here, we reveal that distinct contributions of D1R-MSNs and D2R-MSNs towards reward and motor behaviors are delineated by the multifaceted signaling protein neurofibromin 1 (NF1). Using genetic mouse models, we show that NF1 in D1R-MSN modulates opioid reward, whereas loss of NF1 in D2R-MSNs delays motor learning by impeding the formation and consolidation of repetitive motor sequences. We found that motor learning deficits upon NF1 loss were associated with the disruption in dopamine signaling to cAMP in D2R-MSN. Restoration of cAMP levels pharmacologically or chemogenetically rescued the motor learning deficits seen upon NF1 loss in D2R-MSN. Our findings illustrate that multiplex signaling capabilities of MSNs are deployed at the level of intracellular pathways to achieve cell-specific control over behavioral outcomes.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Lack of NF1 in D1R-MSNs but not in D2R-MSNs reduces opioid reward.
Scheme of NF1 deletion from (A) D1R-MSNs or (F) D2R-MSNs (mouse graphic was adapted from [44]). Effects of morphine-induced CPP (10 mg/kg) for (B) Nf1flx/flxD1Cre mice (n = 6–7 mice/group) or (G) Nf1flx/flxD2Cre mice (n = 7 mice/group). Place preference scores are calculated as the difference between time spent in the drug-paired side during post-conditioning versus preconditioning tests. *P < 0.05, Student t test. Rewards earned during morphine self-administration at 0.3 mg/kg/infusion for (C) Nf1flx/flxD1Cre (n = 5–8 mice/group) and (H) Nf1flx/flxD2Cre mice (n = 6–10 mice/group). ***P < 0.001, two-way RM ANOVA. Self-administration criteria were set under a fixed-ratio 5 with a time-out 20-second schedule of reinforcement. Number of infusions earned during morphine self-administration at varying doses (mg/kg/infusions) for (D) Nf1flx/flxD1Cre mice and (I) Nf1flx/flxD2Cre mice. ***P < 0.001, ****P < 0.0001 compared with Nf1flx/flx mice, two-way RM ANOVA. Intake of morphine self-administration calculated from the last three stable sessions for (E) Nf1flx/flxD1Cre mice and (J) Nf1flx/flxD2Cre mice. ****P < 0.0001, two-way RM ANOVA. Data are represented as mean ± SEM. Underlying data for this figure can be found in S1 Data. CPP, conditioned place preference; D1R, D1 dopamine receptor; D2R, D2 dopamine receptor; MSN, medium spiny neuron; NF1, neurofibromin 1; RM-ANOVA, repeated measures analysis of variance.
Fig 2
Fig 2. MSNs lacking NF1 are resistant to morphine-induced reduction in excitability.
(A) Representative traces at 250-pA injection level and the mean number of APs generated for a given level of current injection in D1R-MSNs and D2R-MSNs following morphine administration. n = 8–9 neurons/group. Comparison of the (B) first spike latency (ms) (C) firing threshold (rheobase), (D) RMP, and (E) input resistance (Rin) in D1R-MSNs and D2R-MSNs of drug-naïve and morphine-treated mice. n = 8–9 neurons/group. (F) Image of the NAc in Nf1flx/flxD2Cre mouse stereotaxically injected with AAV9-Flex-EGFP. (G) Representative traces and the mean number of APs generated for a given level of current injection in Nf1flx/flxD1Cre and Nf1flx/flxD2Cre mice following morphine administration. n = 8–16 neurons/group. Comparison of the (H) first spike latency (ms), (I) firing threshold (rheobase), (J) RMP, and (K) input resistance (Rin). n = 5–16 neurons/group. *P < 0.05, ***P < 0.001, two-way RM ANOVA. Data are represented as mean ± SEM. Underlying data for this figure can be found in S1 Data. AP, action potential; D1R, D1 dopamine receptor; D2R, D2 dopamine receptor; MSN, medium spiny neuron; NAc, nucleus accumbens; NF1, neurofibromin 1; RM-ANOVA, repeated measures analysis of variance; RMP, resting membrane potential.
Fig 3
Fig 3. Elimination of NF1 in D2R-MSNs blunts morphine-induced locomotion.
(A) Locomotor activity for naïve Nf1flx/flxD1Cre mice. n = 5–10 mice/group. (B) Cumulative distance traveled in the open-field chamber for varying concentrations of morphine (1, 5, 10, and 20 mg/kg) and (C) time course for 10 mg/kg morphine for Nf1flx/flxD1Cre mice. n = 9–10 mice/group. (D) Locomotor activity for naïve Nf1flx/flxD2Cre mice. (E) Cumulative distance traveled in the open-field chamber for varying concentrations of morphine (1, 5, 10, and 20 mg/kg) and (F) time course for 10 mg/kg morphine for Nf1flx/flxD2Cre mice. n = 5–11 mice/group. Data are represented as mean ± SEM. **P < 0.01, ****P < 0.0001, two-way RM ANOVA. Underlying data for this figure can be found in S1 Data. D2R, D2 dopamine receptor; MSN, medium spiny neuron; NF1, neurofibromin 1; RM-ANOVA, repeated measures analysis of variance.
Fig 4
Fig 4. Key role of striatal NF1 in motor learning.
(A) Scheme of NF1 deletion from MSNs and accelerating rotarod paradigm. (B) Performance of Nf1flx/flx and Nf1flx/flxRgs9cre mice on the accelerating rotarod. n = 14 mice/group, two-way RM ANOVA. (C) Percentage of mice able to reach maximal speed. (D) Learning rate of Nf1flx/flx and Nf1flx/flxRgs9cre mice on the accelerating rotarod. Student t test. (E) Grip strength and (F) wire hang for Nf1flx/flx and Nf1flx/flxRgs9cre mice. n = 9 mice/group. (G) Time course of the terminal speed (trial 4) over 10 days of consecutive training on the accelerating rotarod for Nf1flx/flx and Nf1flx/flxRgs9cre mice. n = 8–13 mice/group, two-way RM ANOVA. (H) Probability of reaching maximal speed over the 10 days of training on the accelerating rotarod. Data are represented as mean ± SEM. *P < 0.05, ***P < 0.001. Underlying data for this figure can be found in S1 Data. MSN, medium spiny neuron; NF1, neurofibromin 1; RM-ANOVA, repeated measures analysis of variance.
Fig 5
Fig 5. Action of NF1 in D2R-MSNs but not in D1R-MSNs is required for motor learning.
(A) Performance of Nf1flx/flx and Nf1flx/flxD1cre mice on the accelerating rotarod. n = 9–13 mice/group. Two-way RM ANOVA. (B) Percentage of mice able to reach maximal speed and (C) learning rate on the accelerating rotarod for Nf1flx/flx and Nf1flx/flxD1cre mice. (D) Performance of Nf1flx/flx and Nf1flx/flxD2cre mice on the accelerating rotarod. n = 11–16 mice/group. Two-way RM ANOVA. (E) Percentage of mice able to reach maximal speed and (F) learning rate on the accelerating rotarod for Nf1flx/flx and Nf1flx/flxD2cre mice. (G) Time course of the terminal speed (trial 4) over 10 days of consecutive training on the accelerating rotarod for Nf1flx/flx and Nf1flx/flxD2cre mice. Two-way RM ANOVA. (H) Probability of reaching maximal speed over the 10 days of training on the accelerating rotarod. *P < 0.05, **P < 0.01. Data are represented as mean ± SEM. Underlying data for this figure can be found in S1 Data. D1R, D1 dopamine receptor; D2R, D2 dopamine receptor; MSN, medium spiny neuron; NF1, neurofibromin 1; RM-ANOVA, repeated measures analysis of variance.
Fig 6
Fig 6. Cell-specific effects of NF1 on cAMP signaling in the striatum.
(A) Quantification of total striatal cAMP in Nf1flx/flx and Nf1flx/flxRgs9Cre mice. n = 6 mice/group, Student t test. (B) Representative image of western blot analysis for AC5 expression in the striatum of Nf1flx/flx and Nf1flx/flxRgs9Cre mice. n = 6 mice/group. Student t test. (C) Comparison of mRNA for Nf1 and Adcy5 in striatum of Nf1flx/flx and Nf1flx/flxRgs9Cre mice. n = 6 mice/group. Student t test. (D) Scheme and Image of TEPACVV fluorescence in transfected primary MSNs. Mouse graphic was adapted from [44]. Scale bar represents 20 μm. Quantification of baseline striatal cAMP levels for (E) Nf1flx/flxD1Cre and (K) Nf1flx/flxD2Cre compared with Nf1flx/flx mice. n ≥ 4 neurons/genotype. Change in cAMP following bath application of forskolin/IBMX to (F) D1R-MSNs and (L) D2R-MSNs. n ≥ 4 neurons/genotype. Quantification of maximum cAMP change following forskolin/IBMX treatment for (G) Nf1flx/flxD1Cre and (M) Nf1flx/flxD2Cre neurons. n ≥ 4 neurons per genotype. Representative traces of cAMP dynamics for (H) Nf1flx/flxD1Cre and (N) Nf1flx/flxD2Cre to a 1-second pulse of dopamine at the indicated micromolar concentration. Quantification of maximum change in cAMP response to phasic dopamine pulses for (I) Nf1flx/flxD1Cre and (O) Nf1flx/flxD2Cre neurons. Discrimination ratio (Vmax/Baseline) for cAMP response to dopamine in (J) Nf1flx/flxD1Cre and (P) Nf1flx/flxD2Cre. n ≥ 4 neurons per genotype, Kolmogorov-Smirnov test. *P < 0.05, **P < 0.01, ***P < 0.001. Data are represented as mean ± SEM. Underlying data for this figure can be found in S1 Data. AC5, adenylyl cyclase type 5 (protein); Adcy5, adenylyl cyclase type 5 (gene); D1R, D1 dopamine receptor; D2R, D2 dopamine receptor; IBMX, 3-isobutyl-1-methylxanthine; MSN, medium spiny neuron; NF1, neurofibromin 1.
Fig 7
Fig 7. Chemogenetic and pharmacological rescue of motor deficits associated with NF1 deficiency.
(A) Scheme and Image of Nf1flx/flxD2cre mice bilaterally injected in the striatum with AAV-DIO-rM3Gs. Mouse graphic was adapted from [44]. (B) Quantification of striatal cAMP levels of Nf1flx/flxD2cre bilaterally injected with AAV-DIO-rM3Gs or DIO-mcherry upon CNO administration. n = 6 mice/group. (C) Increase in performance and (D) learning rate on the accelerating rotarod of DIO-rM3Gs mice compared with DIO-mcherry mice following CNO injection in Nf1flx/flxD2cre mice. n = 6–7 mice/group. (E) Quantification of striatal cAMP levels in Nf1flx/flx and Nf1flx/flxRgs9cre treated with rolipram. (F) Performance and (G) learning rate on the rotarod task for Nf1flx/flx and Nf1flx/flxRgs9cre mice treated with rolipram. n = 7–10 mice/group. Two-way RM ANOVA. *P < 0.05, **P < 0.01. Data are represented as mean ± SEM. Underlying data for this figure can be found in S1 Data. DIO, double-floxed inverted open reading frame; NF1, neurofibromin 1; RM-ANOVA, repeated measures analysis of variance; Rol, rolipram; Veh, vehicle.

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