Loss of Cdk5 function in the nucleus accumbens decreases wheel running and may mediate age-related declines in voluntary physical activity

Abstract

Key points: Physical inactivity, which drastically increases with advancing age, is associated with numerous chronic diseases. The nucleus accumbens (the pleasure and reward 'hub' in the brain) influences wheel running behaviour in rodents. RNA-sequencing and subsequent bioinformatics analysis led us to hypothesize a potential relationship between the regulation of dendritic spine density, the molecules involved in synaptic transmission, and age-related reductions in wheel running. Upon completion of follow-up studies, we developed the working model that synaptic plasticity in the nucleus accumbens is central to age-related changes in voluntary running. Testing this hypothesis, inhibition of Cdk5 (comprising a molecule central to the processes described above) in the nucleus accumbens reduced wheel running. The results of the present study show that reductions in synaptic transmission and Cdk5 function are related to decreases in voluntary running behaviour and provide guidance for understanding the neural mechanisms that underlie age-dependent reductions in the motivation to be physically active.

Abstract: Increases in age are often associated with reduced levels of physical activity, which, in turn, associates with the development of numerous chronic diseases. We aimed to assess molecular differences in the nucleus accumbens (NAc) (a specific brain nucleus postulated to influence rewarding behaviour) with respect to wheel running and sedentary female Wistar rats at 8 and 14 weeks of age. RNA-sequencing was used to interrogate transcriptomic changes between 8- and 14-week-old wheel running rats, and select transcripts were later analysed by quantitative RT-PCR in age-matched sedentary rats. Voluntary wheel running was greatest at 8 weeks and had significantly decreased by 12 weeks. From 619 differentially expressed mRNAs, bioinformatics suggested that cAMP-mediated signalling, dopamine- and cAMP-regulated neuronal phosphoprotein of 32 kDa feedback, and synaptic plasticity were greater in 8- vs. 14-week-old rats. In depth analysis of these networks showed significant (∼20-30%; P < 0.05) decreases in cell adhesion molecule (Cadm)4 and p39 mRNAs, as well as their proteins from 8 to 14 weeks of age in running and sedentary rats. Furthermore, Cadm4, cyclin-dependent kinase 5 (Cdk5) and p39 mRNAs were significantly correlated with voluntary running distance. Analysis of dendritic spine density in the NAc showed that wheel access increased spine density (P < 0.001), whereas spine density was lower in 14- vs. 8-week-old sedentary rats (P = 0.03). Intriguingly, intra-NAc injection of the Cdk5 inhibitor roscovitine, dose-dependently decreased wheel running. Collectively, these experiments suggest that an age-dependent loss in synaptic function and Cdk5/p39 activity in the NAc may be partially responsible for age-related declines in voluntary running behaviour.

Keywords: Cdk5; nucleus accumbens; physical activity; reward; synapse; wheel running.

Figure 1. Characteristics of RNA‐seq dataset and transcript filtering

A, overview of the filtering process used to generate differentially expressed transcripts between 8‐ and 14‐week‐old wheel running rats. For each group, a transcript was omitted if it was not a known transcript. B, RNA‐seq data were highly reliable as demonstrated by the high correlation (r = 0.995) of RPKM values from a single rat compared to RPKM average values from all rats. Both 8‐ and 14‐week‐old groups expressed high amounts of Grd1 (C) and Grd2 (D), which is indicative of NAc MSNs. FDR, false discovery rate; DET, differentially expressed transcripts.

Figure 2. NAc roscovitine injection

A, overview of the study design for intra‐NAc vehicle or roscovitine injection. The schematic illustration of ascending distances of voluntary running to the peak fourth night mimics the 4‐day running cycles of female rats employed in the experiment. Rats were introduced to running wheels at 10 weeks of age, had cannulae surgically implanted bilaterally in the NAc core at 13 weeks of age, and began the injection protocol at ∼14 weeks of age (wheel running was monitored for 8 days post‐surgery to ensure that surgery did not disrupt running pattern). Baseline vehicle (VEH) injection was performed in all rats. Rats were then randomly administered VEH, 40 nmol roscovitine or 80 nmol roscovitine for 5 continuous days ∼10–15 min before the beginning of the dark cycle. Running distance was monitored for 120‐min post‐injection for the baseline injection and on the first and fifth night of VEH or roscovitine injection. B, coronal section of rat brain, in accordance with a rat brain atlas (Paxinos & Watson, 1998), which shows the cannulae location as determined by cresyl violet staining (with the position of each section given in mm relative to Bregma). Black dots indicate the location of injector tips.

Figure 3. Voluntary wheel running characteristics

Average nightly running (A) distance (km), (B) time (min) and (C) velocity (m min^–1) over the course of the study. ^* P < 0.05, ^** P < 0.01, ^*** P < 0.001.

Figure 4. Top IPA generated networks up‐regulated in 8‐ vs. 14‐week old wheel running rats

Top associated networks as defined by IPA for NAc mRNAs differently expressed in 8‐ and 14‐week‐old wheel running rats included (A) ‘nervous system and function, organ morphology, and organismal development’ (Focus molecules: 24/35; Score: 35) and (B) ‘cellular assembly and organization, nervous system development and function, and tissue morphology’ (Focus molecules: 23/35; Score: 35). Nodes represent genes/molecules. Shading is proportional to fold change size (red, up‐regulated; green, down‐regulated). Direct and indirect relationships are denoted with solid and dashed lines, respectively. White nodes denote network members that were not altered in the network. Lines ending in an arrow or blunt end indicate the known direction of molecular activation or inhibition, respectively.

Figure 5. Cadm4 mRNA and protein are up‐regulated at 8‐ vs. 14‐weeks of age independent of wheel running

Cadm4 mRNA is up‐regulated at 8 vs. 14 weeks of age in wheel running (A) and sedentary (B) rats. Note the units in (A) and (B) differ as a result of (A) being determined by RNA‐seq and (B) by qRT‐PCR. Cadm4 mRNA is strongly correlated with running distance during the final week of the study (C). Similarly, Cadm4 protein decreases from 8 to 14 weeks of age independent of wheel running (data normalized to the 8‐week‐old wheel running group) (D). ^* P < 0.05, ^** P < 0.01, ^*** P < 0.001.

Figure 6. Cdk5‐associated mRNA and protein differences in 8‐ vs. 14‐week old wheel running and sedentary rats

Cdk5r2 (p39) mRNA is higher at 8 vs. 14 weeks of age in wheel running (A) and sedentary (B) rats. Note the units in (A) and (B) differ as a result of (A) being determined by RNA‐seq and (B) by qRT‐PCR. p39 mRNA is strongly correlated with running distance during the final week of the study (C). Similarly, p39 protein decreases from 8 to 14 weeks of age independent of wheel running (data normalized to the 8‐week‐old wheel running group) (D). Wheel running and age had no effect on Cdk5r1 (p35) protein level (E); however, wheel running increased protein expression of the p35 cleavage product p25 (F). Cdk5 mRNA was modestly but significantly greater in 8‐ vs. 14‐week‐old wheel running rats (G) but was not different between 8‐ and 14‐week‐old sedentary rats (H). Cdk5 mRNA was significantly correlated with running distance during the final week of the study (I). ^* P < 0.05, ^** P < 0.01, ^*** P < 0.001.

Figure 7. Dendritic spine density is increased in 8‐ vs. 14‐week old wheel running and sedentary rats

A, Darrp‐32 protein was not different between any of the four experimental groups assessed. B, analysis of dendritic spine density showed that wheel running increased spine density, whereas increases in age from 8 to 14 weeks were associated with a decrease in dendritic spine density. C, analysis of spine type showed that wheel running decreased the percentage of stubby spines and tended to increase the percentage of thin spins. D, representative images for the four experimental groups assessed. Scale bar = 5 μm. ^* P < 0.05, ^** P < 0.01, ^*** P < 0.001.

Figure 8. Intra‐NAc roscovitine infusion decreases voluntary wheel running

A, running distance (km) shown in 30 min increments for baseline injection and on the first and fifth nights of vehicle (black) or 40 nmol (grey) or 80 nmol (white) roscovitine (ROS) injection. B, percentage running distance on the first and fifth nights of vehicle or drug injection compared to baseline injection values. Note: two outlier points (±2 SD) were removed from this analysis (not shown). C, total running distance over the 120 min test session for the baseline injection and for the first and fifth night of vehicle or drug injection. All data were analysed with repeated‐measures ANOVA. ^* P < 0.05, ^** P < 0.01, ^*** P < 0.001.