Target-dependent expression of the netrin-1 receptor, UNC5C, in projection neurons of the ventral tegmental area
Introduction
Netrin-1 is a bi-functional guidance cue critically implicated in the formation of neural networks during development (Manitt and Kennedy, 2002, Manitt et al., 2010, Manitt et al., 2011). Our group has demonstrated a role for the netrin-1 receptor, DCC (deleted in colorectal cancer), in the selective development and organization of mesocortical dopamine (DA) circuitry (Flores et al., 2005, Grant et al., 2007, Yetnikoff et al., 2007, Yetnikoff et al., 2010, Flores, 2011, Manitt et al., 2011). Briefly, we have found that mice with a heterozygous loss-of-function mutation in the dcc gene (dcc+/−) display elevated baseline levels of extracellular DA in the medial prefrontal cortex (mPFC; “mesocortical”), but not in the nucleus accumbens (NAcc; “mesolimbic”), when compared to wild-type controls (Grant et al., 2007). Furthermore, adult dcc+/− mice have a significant increase in the span of DA synapses within the cingulate 1 and prelimbic subregions of the mPFC, but not in the NAcc, relative to wild-type controls (Manitt et al., 2011). These changes are associated with reduced spine density and dendritic arbor length of mPFC layer V pyramidal neurons selectively, which receive robust DA innervation (Benes et al., 2000, Grant et al., 2007, Manitt et al., 2011). However, no structural modifications are observed in NAcc medium spiny neurons.
The phenotypic traits of dcc+/− mice only become evident after adolescence (Grant et al., 2009, Manitt et al., 2011, Yetnikoff et al., 2011). This finding is intriguing because the DA innervation of the mPFC, which continues to develop until early adulthood, undergoes dramatic refinement during adolescence (Kalsbeek et al., 1988, Voorn et al., 1988, Rosenberg and Lewis, 1995, Benes et al., 2000, O’Donnell, 2010). In contrast, the DA innervation of the NAcc achieves an adult level of density soon after birth (Voorn et al., 1988).
Recent findings from our group suggest that the UNC5 homologue netrin-1 receptors, may also contribute to the adolescent development of ventral tegmental area (VTA) projections to the mPFC (Manitt et al., 2010, Auger et al., 2013). Both DCC and UNC5 receptors are highly expressed in the VTA by DA and non-DA neurons in rodents. However, whereas DCC is highly expressed in this region throughout life, UNC5 expression increases dramatically during adolescence (Manitt et al., 2010). In fact, VTA DA neurons only begin to express UNC5 receptors from adolescence onwards (Manitt et al., 2010). We now have demonstrated that DA neurons express the unc-5 homologue C (UNC5C) homologue specifically (Manitt et al., 2010, Auger et al., 2013). Importantly, we have shown that (a) single VTA DA neurons co-express DCC and UNC5C in adulthood (Manitt et al., 2010); (b) dcc+/− and unc5c+/- mice exhibit almost identical behavioral responsiveness to stimulant drugs of abuse; and (c) the behavioral phenotypes of both dcc+/− and unc5c+/- mice become evident only after adolescence (Flores et al., 2005, Grant et al., 2007, Manitt et al., 2011, Auger et al., 2013). Together, these findings suggest that the emergence of UNC5C receptors by VTA DA neurons during adolescence may be a contributing factor to the effects of DCC on the development of the DA projection to the mPFC. We therefore hypothesized that UNC5C receptors may be expressed predominantly by VTA DA neurons that project to the mPFC. To address this idea, we assessed whether UNC5 receptors in the VTA are expressed differentially by neurons that target the mPFC versus the NAcc. We combined retrograde transport of fluorescent tracer beads injected into the mPFC or NAcc with immunofluorescence against TH and UNC5C in the VTA of adult male wild-type mice.
Section snippets
Animals
All experiments were conducted according to the guidelines of the Canadian Council of Animal Care and all animal procedures were approved by the McGill University/Douglas Mental Health University Institute Animal Care Committee. Animals were kept on a 12-h light/dark cycle with food access given ad libitum. In all studies, we used adult male wild-type C57/Bl6 mice (post-natal day 75 ± 15; average body weight of 30 ± 5 g) bred from our in-house colony.
Primary antibodies
A monoclonal tyrosine hydroxylase (TH) antibody
Results
The mean total count of TH-positive VTA cells (±SEM), from all subjects, was 10,796 ± 1008, which corresponds with data from previous stereological analyses in the VTA (data not shown, Flores et al., 2005, Liss et al., 2005, Martinez de Lagran et al., 2007, Vadasz et al., 2007, Nair-Roberts et al., 2008, Baquet et al., 2009, Mouton et al., 2012). Consistent with our previous findings in adult mice, UNC5C immunolabeling was observed in cells within all subregions of the VTA (parabrachial pigmented
Expression of UNC5C by DA neurons in the VTA
In the VTA, DA neurons only begin to express UNC5C during adolescence (Manitt et al., 2010, Auger et al., 2013). This change coincides with the critical period of the effects of DCC, the other netrin-1 receptor, on the selective organization of the mesocortical DA circuitry (Grant et al., 2007, Grant et al., 2009, Manitt et al., 2011). Here we assessed, in adult wild-type mice, whether UNC5C receptors are expressed primarily, or exclusively, by DA neurons projecting to the mPFC. Contrary to our
Acknowledgements
This work was supported by grants from the National Science and Engineering Research Council of Canada (NSERC), the Canadian Institute for Health Research (CIHR), the Fonds de la Recherche en Santé du Québec (FRSQ), and the McGill University Faculty of Medicine Internal Studentship Program. The authors would also like to thank Dr. Tony Pawson for kindly donating the UNC5H antiserum to us, and Drs. Pablo Henny and Leora Yetnikoff for their advice on stereological procedures and critical feedback
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DCC Receptors Drive Prefrontal Cortex Maturation by Determining Dopamine Axon Targeting in Adolescence
2018, Biological PsychiatryCitation Excerpt :In fact, VTA dopamine neurons begin to express another netrin-1 receptor, UNC5C, during adolescence, and the ratio of DCC:UNC5C expression inverts (17). This developmental switch may contribute to mesolimbic dopamine axon targeting and to determining which axons pause in the NAcc before continuing to the mPFC (27,65). Cognitive control relies on PFC function and matures in tandem with its circuitry (66–69).