Nucleus accumbens neuronal maturation differences in young rats bred for low versus high voluntary running behaviour

Abstract

We compared the nucleus accumbens (NAc) transcriptomes of generation 8 (G8), 34-day-old rats selectively bred for low (LVR) versus high voluntary running (HVR) behaviours in rats that never ran (LVR(non-run) and HVR(non-run)), as well as in rats after 6 days of voluntary wheel running (LVR(run) and HVR(run)). In addition, the NAc transcriptome of wild-type Wistar rats was compared. The purpose of this transcriptomics approach was to generate testable hypotheses as to possible NAc features that may be contributing to running motivation differences between lines. Ingenuity Pathway Analysis and Gene Ontology analyses suggested that 'cell cycle'-related transcripts and the running-induced plasticity of dopamine-related transcripts were lower in LVR versus HVR rats. From these data, a hypothesis was generated that LVR rats might have less NAc neuron maturation than HVR rats. Follow-up immunohistochemistry in G9-10 LVR(non-run) rats suggested that the LVR line inherently possessed fewer mature medium spiny (Darpp-32-positive) neurons (P < 0.001) and fewer immature (Dcx-positive) neurons (P < 0.001) than their G9-10 HVR counterparts. However, voluntary running wheel access in our G9-10 LVRs uniquely increased their Darpp-32-positive and Dcx-positive neuron densities. In summary, NAc cellularity differences and/or the lack of running-induced plasticity in dopamine signalling-related transcripts may contribute to low voluntary running motivation in LVR rats.

Figure 1. Experimental strategy to assess influential NAc mRNAs differentially expressed between G8 HVR and LVR rats

A, no inherent difference, but acquired difference with voluntary running. B, inherent difference is maintained after 6 days of voluntary running. C, inherent difference is lost after 6 days of voluntary running. NAc transcripts from HVR and LVR sitters (HVR_non-run and LVR_non-run) were also compared to age-matched control (CTL) Wistar rats to provide further evidence for line type differences.

Figure 2. Western blotting confirmation of NAc Cadm4 as a line type-specific gene

All animals were female and were 35 days of age, and HVR_run and LVR_run animals spent 6 days in a voluntary running wheel (n = 6–7 per bar). *P < 0.05.

Figure 3. Enrichment of an NAc MSN-specific mRNA marker in all groups as well as the reliability of RNA-seq measurements

All groups presented high amounts of Grd1 (A) and Grd2 (B), which is indicative of NAc MSNs as previously shown by Chen et al. (2011). C, high correlation of RPKM values from a single rat compared to RPKM average values from all rats, demonstrating high reliability from the RNA-seq data.

Figure 4. Differentially expressed genes (DEGs) in HVR_run and LVR_run and similar or inherently different between HVR_non-run and LVR_non-run rats as well as CTL rats in RNA-seq analysis

Figure 5. Differences in NAc immature neurons (Dcx⁺) and MSNs (Darpp-32⁺) between G9–10 LVR and HVR rats

Line-type differences in NAc Dcx-positive (A) and Darpp-32-positive neurons (C), which are indicative of immature neurons and mature/differentiated MSNs, respectively. B and D, representative NAc micrographs of Dcx-positive and Darpp-32-positive cell staining, respectively. All animals were female and were 39–40 days of age, and HVR_run and LVR_run animals spent ∼10–12 days in a voluntary running wheel (n = 6–7 per bar). **P < 0.01, ***P < 0.001.

Figure 6. Differences in NAc immature neurons (Dcx⁺) and MSNs (Darpp-32⁺) between G9–10 LVR and HVR rats Correlations between NAc IHC measures and 10-day running distance in G9–10 LVR and HVR runner rats

Negative associations exist between 10-day running distances and NAc MSN density in HVR (A) and LVR runners (B). Note that one HVR runner was excluded from analysis due to it being an outlier (* in A). However, r values are presented for both the inclusion and the exclusion of this animal.

Figure 7. NAc tissue dopamine content between G10–11 LVR and HVR lines

No significant differences (NS) existed between LVR_non-run and HVR_non-run rats 1–2 h prior to the running/dark cycle (P = 0.28), LVR_run and HVR_run rats 1–2 h prior to the running/dark cycle (P = 0.28), or LVR_run and HVR_run rats 2–3 h into the running/dark cycle (P = 0.72). All animals were female and were 35 days of age, and HVR_run and LVR_run animals spent 6 days in a voluntary running wheel (n = 6–7 per bar).

Figure 8. Summary figure of new hypotheses developed from current observations between the LVR and HVR lines

Hypotheses on neuron development between lines: the inherent up-regulation of ‘cell-cycle’-related transcripts in the HVR and LVR line and the higher densities of Dcx- and Darpp-32- positive neurons indicates that MSN development is inherently greater in the HVR line. However, voluntary running reverses these trends (MSNs: HVR = LVR; Dcx-positive neurons: LVR > HVR). Methamphetamine administration to rodents, which increases striatal dopamine levels, has been shown to decrease striatal neurogenesis. Hence, high pulsatile dopamine secretions into the NAc on a nightly basis due to high running may be the mechanism whereby HVRs experience a decrease in MSN density. Conversely, glutamate–NMDA receptor signalling is linked to an increase in striatal neurogenesis. Hence, this may be the mechanism whereby voluntary running in LVRs increases NAc Dcx- and Darpp-32-positive neurons. Hypotheses on MSN function between lines: initially, we hypothesized that lower inherent MSN density in LVR versus HVR non-runners may lead to the lack of voluntary running reward. However, there were negative associations between running distance and Darpp-32-positive neurons in the HVR and LVR runners (Fig.4A and B). Hence, we speculate that a greater MSN density may inhibit efferent targets that lead to a decreased motivation for voluntary running. Abbreviations: DA, dopamine; MSN, medium spiny neuron.