Reduced striatal dopamine D1–D2 receptor heteromer expression and behavioural subsensitivity in juvenile rats
Highlights
► Dopamine D1 and D2 receptors are coexpressed with dynorphin and enkephalin. ► Striatal coexpression of D1 and D2 receptors is age-dependent. ► Juvenile rats have lower striatal D1–D2 receptor heteromer expression than adults. ► Juvenile rats are subsensitive to D1–D2 receptor heteromer-induced grooming.
Introduction
Dopaminergic signaling within the basal ganglia has classically been thought to occur within two distinct neuronal pathways; the direct striatonigral pathway which contains the dopamine D1 receptor (D1R) and the neuropeptides dynorphin (DYN) and substance P, and the indirect striatopallidal pathway which expresses the dopamine D2 receptor (D2R) and enkephalin (ENK). A number of studies have also shown, however, that D1R and D2R can co-exist within a certain fraction of medium spiny neurons (MSN) (Bertran-Gonzalez et al., 2008, Valjent et al., 2009, Perreault et al., 2010, Lim et al., 2012). Emerging evidence additionally indicates that these D1R/D2R-coexpressing neurons, which also express DYN and ENK (Perreault et al., 2010) as well as the neurotransmitters GABA and glutamate (Perreault et al., 2012), may comprise a distinct neuronal network, as evidenced by a recent report showing D1R and D2R coexpression in the cell bodies and presynaptic terminals of regions of both the striatonigral and striatopallidal projections of the basal ganglia (Perreault et al., 2010). Furthermore, within these coexpressing neurons it has been shown that the D1R and D2R can form a novel and pharmacologically distinct receptor complex, the dopamine D1–D2 receptor heteromer. The D1–D2 heteromer has been shown to exhibit cell signaling properties distinct from its constituent receptors (Lee et al., 2004, Rashid et al., 2007, Hasbi et al., 2009, So et al., 2009, Verma et al., 2010), emphasizing the functionally unique role for these coexpressing MSNs in the brain.
Evidence suggests that striatal coexpression of the D1R and D2R, and the expression of the D1–D2 receptor heteromer, may be age-dependent thus implicating age as a critical factor in the contribution of these neurons to the regulation of striatal neurotransmission. For example, while it has been shown that the majority of rat neonatal striatal neurons may coexpress D1R and D2R (Aizman et al., 2000, Falk et al., 2006, Iwatsubo et al., 2007, Hasbi et al., 2009) and form D1–D2 receptor heteromers (Hasbi et al., 2009), receptor coexpression is greatly reduced and anatomically circumscribed in adults with approximately 17–35% of nucleus accumbens (NAc) MSNs and 5–6% of caudate putamen (CP) MSNs expressing both receptors (Bertran-Gonzalez et al., 2008, Valjent et al., 2009, Perreault et al., 2010). In addition, increased D1–D2 receptor heteromer-induced signaling was more robust in mid-life compared to younger adults (Rashid et al., 2007), an effect that may correspond to increased D1R and D2R coexpression and/or increased D1–D2 receptor heteromer densities.
There have been many reports detailing the differences in behavioral responding to dopamine drugs in immature versus mature rats, such as enhanced responses to pychostimulant-induced reward (Shahbazi et al., 2008, Zakharova et al., 2009a, Zakharova et al., 2009b, Anker and Carroll, 2010), but reduced responsiveness to the locomotor activating effects of the drugs (Bolanos et al., 1998, Banerjee et al., 2009, Zakharova et al., 2009a), findings indicative of age-dependent differences in dopamine neurotransmission. Thus, given the evidence of time-dependent changes in striatal D1–D2 receptor heteromer expression, in the present study we sought to compare the expression levels of the D1–D2 heteromer in NAc and CP in juvenile and adult rats, and additionally, to evaluate their grooming responses, a behavior previously shown to be mediated by the D1–D2 heteromer (Perreault et al., 2012), following activation of the receptor complex. We showed that juvenile animals exhibited reduced coexpression of D1R and D2R in NAc shell and CP, but not in NAc core, suggestive of lower D1–D2 receptor heteromer densities and region-specific decreases in D1–D2 heteromer-induced neurotransmission. Accordingly, these changes in heteromer expression were associated with reduced responsiveness to the induction of grooming induced by activation of the receptor complex.
Section snippets
Neuronal cultures
Neonatal rat striata (1 day of age) were trypsinized in Hanks’ balanced salt solution (HBSS) with 0.25% trypsin and 0.05% DNase (Sigma) at 37 °C, and cells were washed three times in HBSS with 12 mM MgSO4. Cells were dissociated in DMEM with 2 mM glutamine and 10% FBS and plated at 2 × 105 cells per poly-l-lysine-coated well (Sigma; 50 g/mL). The next day, media were changed to Neurobasal medium with 50X B27 Supplement and 2 mM glutamine (Invitrogen). On day 3 of culture, 5 μM of cytosine arabinoside was
Neuronal coexpression of the D1R and D2R is linked with DYN and ENK
It has been reported that in adult rat striatum coexpression of the D1R and D2R occurs in MSNs also expressing both DYN and ENK, (Perreault et al., 2010). To determine whether the D1R/D2R coexpression was consistently linked to DYN and ENK expression, we first examined the phenotype of D1R/D2R-coexpressing cultured striatal neurons from 1-day-old rat pups, and individual and coexpression of D1R and D2R with DYN, ENK or both neuropeptides was assessed (Fig. 1). There was a high degree of
Discussion
The present study showed that in juvenile striatum the D1R and D2R were coexpressed solely in a unique subset of neurons that also contained the neuropeptides DYN and ENK. Similarly in cultured striatal neurons, the majority of which coexpress the D1R and D2R, an identical phenotype was observed. Furthermore, we showed that the number of MSNs that exhibited D1R/D2R coexpression, and therefore the ability to express the D1–D2 receptor heteromer, showed an age-dependent variation, with reduced
Acknowledgements
This work was supported by a grant from the National Institute on Drug Abuse (to S.R.G. and B.F.O.), and a Canadian Institute of Health Research Postdoctoral Fellowship (to M.L.P.). S.R.G. holds a Canada Research Chair in Molecular Neuroscience.
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2016, European NeuropsychopharmacologyCitation Excerpt :In the present study adolescent rats (PND 37) showed a marked decrease in expression levels of the D1–D2 heteromer in NAc compared with their adult counterparts. This, together with the higher D1–D2 heteromer expression in cultured neonatal (Hasbi et al., 2009) or intact (Biezonski et al., 2015) postnatal striatal neurons is indicative of a maturation-dependent decline of these coexpressing MSNs and, indeed, the present findings are consistent with our previous studies that showed reduced D1–D2 heteromer expression in the NAc shell of juvenile rats (PND 26-28), subsensitivity to the behavioral effects of D1–D2 heteromer activation in these animals, and an absence of NAc BDNF induction by the receptor complex (Perreault et al., 2013a, 2012b). A recent study also showed supra-additive locomotor and stereotypic responses in adult rats following coadministration of the D1–D2 heteromer partial agonist SKF 83959 and the D2 receptor agonist quinpirole, a phenomenon completely lacking in adolescent animals and speculated to be the result, as least in part, from reduced D1–D2 heteromer expression in adolescent animals (Dwyer and Leslie, 2016).
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These authors contributed equally to the preparation of this manuscript.