Colleen Manitt

Netrin Participates in the Development of Retinotectal Synaptic Connectivity by Modulating Axon Arborization and Synapse Formation in the Developing Brain

Netrin has been implicated in retinal ganglion cell (RGC) axon pathfinding in a number of species. In Xenopus laevis, RGC axons reaching their target in the optic tectum can be repelled by a netrin-1 gradient in vitro, suggesting that netrin may also function in wiring events that follow successful axon pathfinding. Here, we examined the contribution of netrin to RGC axon arborization and synapse formation at the target. Time-lapse confocal microscopy imaging of individual RGC axons coexpressing GFP-synaptobrevin and DsRed in the intact Xenopus brain demonstrated a role for deleted in colorectal cancer (DCC)-mediated netrin signaling. Microinjection of netrin-1 into the tectum induced a rapid and transient increase in presynaptic site addition that resulted in higher presynaptic site density over a 24 h observation period. Moreover, netrin induced dynamic axon branching, increasing branch addition and retraction; a behavior that ultimately increased total branch number. In contrast, microinjection of DCC function-blocking antibodies prevented the increase in presynaptic site number normally observed in control axons as well as the associated increase in branch number and axon arbor growth. Dynamic analysis of axon arbors demonstrated that the effects of anti-DCC on axon morphology and presynaptic connectivity were attributable to a specific decrease in new synapse and branch additions, without affecting the stability of existing synapses and branches. Together, these results indicate that, in the absence of DCC signaling, RGC axons fail to branch and differentiate, and support a novel role for netrin in later phases of retinotectal development.

The Netrin Receptor DCC Is Required in the Pubertal Organization of Mesocortical Dopamine Circuitry

Netrins are guidance cues involved in neural connectivity. We have shown that the netrin-1 receptor DCC (deleted in colorectal cancer) is involved in the functional organization of the mesocorticolimbic dopamine (DA) system. Adult mice with a heterozygous loss-of-function mutation in dcc exhibit changes in indexes of DA function, including DA-related behaviors. These phenotypes are only observed after puberty, a critical period in the maturation of the mesocortical DA projection. Here, we examined whether dcc heterozygous mice exhibit structural changes in medial prefrontal cortex (mPFC) DA synaptic connectivity, before and after puberty. Stereological counts of tyrosine-hydroxylase (TH)-positive varicosities were increased in the cingulate 1 and prelimbic regions of the pregenual mPFC. dcc heterozygous mice also exhibited alterations in the size, complexity, and dendritic spine density of mPFC layer V pyramidal neuron basilar dendritic arbors. Remarkably, these presynaptic and postsynaptic partner phenotypes were not observed in juvenile mice, suggesting that DCC selectively influences the extensive branching and synaptic differentiation that occurs in the maturing mPFC DA circuit at puberty. Immunolabeling experiments in wild-type mice demonstrated that DCC is segregated to TH-positive fibers innervating the nucleus accumbens, with only scarce DCC labeling in mPFC TH-positive fibers. Netrin had an inverted target expression pattern. Thus, DCC-mediated netrin-1 signaling may influence the formation/maintenance of mesocorticolimbic DA topography. In support of this, we report that dcc heterozygous mice exhibit a twofold increase in the density of mPFC DCC/TH-positive varicosities. Our results implicate DCC-mediated netrin-1 signaling in the establishment of mPFC DA circuitry during puberty.

Peri-Pubertal Emergence of UNC-5 Homologue Expression by Dopamine Neurons in Rodents

Puberty is a critical period in mesocorticolimbic dopamine (DA) system development, particularly for the medial prefrontal cortex (mPFC) projection which achieves maturity in early adulthood. The guidance cue netrin-1 organizes neuronal networks by attracting or repelling cellular processes through DCC (deleted in colorectal cancer) and UNC-5 homologue (UNC5H) receptors, respectively. We have shown that variations in netrin-1 receptor levels lead to selective reorganization of mPFC DA circuitry, and changes in DA-related behaviors, in transgenic mice and in rats. Significantly, these effects are only observed after puberty, suggesting that netrin-1 mediated effects on DA systems vary across development. Here we report on the normal expression of DCC and UNC5H in the ventral tegmental area (VTA) by DA neurons from embryonic life to adulthood, in both mice and rats. We show a dramatic and enduring pubertal change in the ratio of DCC:UNC5H receptors, reflecting a shift toward predominant UNC5H function. This shift in DCC:UNC5H ratio coincides with the pubertal emergence of UNC5H expression by VTA DA neurons. Although the distribution of DCC and UNC5H by VTA DA neurons changes during puberty, the pattern of netrin-1 immunoreactivity in these cells does not. Together, our findings suggest that DCC:UNC5H ratios in DA neurons at critical periods may have important consequences for the organization and function of mesocorticolimbic DA systems.

dcc orchestrates the development of the prefrontal cortex during adolescence and is altered in psychiatric patients

Adolescence is a period of heightened susceptibility to psychiatric disorders of medial prefrontal cortex (mPFC) dysfunction and cognitive impairment. mPFC dopamine (DA) projections reach maturity only in early adulthood, when their control over cognition becomes fully functional. The mechanisms governing this protracted and unique development are unknown. Here we identify dcc as the first DA neuron gene to regulate mPFC connectivity during adolescence and dissect the mechanisms involved. Reduction or loss of dcc from DA neurons by Cre-lox recombination increased mPFC DA innervation. Underlying this was the presence of ectopic DA fibers that normally innervate non-cortical targets. Altered DA input changed the anatomy and electrophysiology of mPFC circuits, leading to enhanced cognitive flexibility. All phenotypes only emerged in adulthood. Using viral Cre, we demonstrated that dcc organizes mPFC wiring specifically during adolescence. Variations in DCC may determine differential predisposition to mPFC disorders in humans. Indeed, DCC expression is elevated in brains of antidepressant-free subjects who committed suicide.

DCC Confers Susceptibility to Depression-like Behaviors in Humans and Mice and Is Regulated by miR-218

BACKGROUD Variations in the expression of the Netrin-1 guidance cue receptor DCC (deleted in colorectal cancer) appear to confer resilience or susceptibility to psychopathologies involving prefrontal cortex (PFC) dysfunction. METHODS With the use of postmortem brain tissue, mouse models of defeat stress, and in vitro analysis, we assessed microRNA (miRNA) regulation of DCC and whether changes in DCC levels in the PFC lead to vulnerability to depression-like behaviors. RESULTS We identified miR-218 as a posttranscriptional repressor of DCC and detected coexpression of DCC and miR-218 in pyramidal neurons of human and mouse PFC. We found that exaggerated expression of DCC and reduced levels of miR-218 in the PFC are consistent traits of mice susceptible to chronic stress and of major depressive disorder in humans. Remarkably, upregulation of Dcc in mouse PFC pyramidal neurons causes vulnerability to stress-induced social avoidance and anhedonia. CONCLUSIONS These data are the first demonstration of microRNA regulation of DCC and suggest that, by regulating DCC, miR-218 may be a switch of susceptibility versus resilience to stress-related disorders.

DCC Receptors Drive Prefrontal Cortex Maturation by Determining Dopamine Axon Targeting in Adolescence

BACKGROUND Dopaminergic input to the prefrontal cortex (PFC) increases throughout adolescence and, by establishing precisely localized synapses, calibrates cognitive function. However, why and how mesocortical dopamine axon density increases across adolescence remains unknown. METHODS We used a developmental application of axon-initiated recombination to label and track the growth of dopamine axons across adolescence in mice. We then paired this recombination with cell-specific knockdown of the netrin-1 receptor DCC to determine its role in adolescent dopamine axon growth. We then assessed how altering adolescent PFC dopamine axon growth changes the structural and functional development of the PFC by quantifying pyramidal neuron morphology and cognitive performance. RESULTS We show, for the first time, that dopamine axons continue to grow from the striatum to the PFC during adolescence. Importantly, we discover that DCC, a guidance cue receptor, controls the extent of this protracted growth by determining where and when dopamine axons recognize their final target. When DCC-dependent adolescent targeting events are disrupted, dopamine axons continue to grow ectopically from the nucleus accumbens to the PFC and profoundly change PFC structural and functional development. This leads to alterations in cognitive processes known to be impaired across psychiatric conditions. CONCLUSIONS The prolonged growth of dopamine axons represents an extraordinary period for experience to influence their adolescent trajectory and predispose to or protect against psychopathology. DCC receptor signaling in dopamine neurons is a molecular link where genetic and environmental factors may interact in adolescence to influence the development and function of the prefrontal cortex.

unc5c haploinsufficient phenotype: striking similarities with the dcc haploinsufficiency model

DCC and UNC5 homologs (UNC5H) are guidance cue receptors highly expressed by mesocorticolimbic dopamine neurons. We have shown that dcc heterozygous mice exhibit increased dopamine, but not norepinephrine, innervation and function in medial prefrontal cortex. Concomitantly, dcc heterozygotes show blunted mesolimbic dopamine release and behavioral responses to stimulant drugs. These changes appear only in adulthood. Recently, we found an adolescent emergence of UNC5H expression by dopamine neurons and co‐expression of DCC and UNC5H by single dopamine cells. Here, we demonstrate selective expression of unc5 homolog c mRNA by dopamine neurons in adulthood. We show that unc5c haploinsufficiency results in diminished amphetamine‐induced locomotion in male and female mice. This phenotype is identical to that produced by dcc haploinsufficiency and is observed after adolescence. Notably, and similar to dcc haploinsufficiency, unc5c haploinsufficiency leads to dramatic increases in tyrosine hydroxylase expression in the medial prefrontal cortex, but not in the nucleus accumbens. In contrast, medial prefrontal cortex dopamine‐β‐hydroxylase expression is not altered. We confirmed that UNC5C protein is reduced in the ventral tegmental area of unc5c heterozygous mice, but that DCC expression in this region remains unchanged. UNC5C receptors may also play a role in dopamine function and influence sensitivity to behavioral effects of stimulant drugs of abuse, at least upon first exposure. The striking similarities between the dcc and the unc5c haploinsufficient phenotypes raise the possibility that functions mediated by DCC/UNC5C complexes may be at play.

Dynamic responses of Xenopus retinal ganglion cell axon growth cones to netrin-1 as they innervate their in vivo target

Netrin‐1 influences retinal ganglion cell (RGC) axon pathfinding and also participates in the branching and synaptic differentiation of mature RGC axons at their target. To investigate whether netrin also serves as an early target recognition signal in the brain, we examined the dynamic behavior of Xenopus RGC axons soon after they innervate the optic tectum. Time‐lapse confocal microscopy imaging of RGC axons expressing enhanced yellow fluorescent protein demonstrated that netrin‐1 is involved in early axon branching, as recombinant netrin‐1 halted further advancement of growth cones into the tectum and induced back branching. RGC growth cones exhibited differential responses to netrin‐1 that depended on the degree of differentiation of the axon and the developmental stage of the tadpole. Netrin‐1 decreased the total number of branches on newly arrived RGC growth cones at the target, but increased the dynamic branching of more mature arbors at the later developmental stage. To further explore the response of axonal growth cones to netrin, Xenopus RGC axons were followed in culture by time‐lapse imaging. Exposure to netrin‐1 rapidly increased the forward advancement of the axon and decreased the size and expanse of the growth cone, while also inducing back branching. Taken together, the differential in vivo and in vitro responses to netrin‐1 suggest that netrin alone is not sufficient to induce the cessation of growth cone advancement in the absence of a target but can independently modulate axon branching. Collectively, our findings reveal a novel role for netrin on RGC axon branch initiation as growth cones innervate their target.

Dcc haploinsufficiency regulates dopamine receptor expression across postnatal lifespan

Adolescence is a period during which the medial prefrontal cortex (mPFC) undergoes significant remodeling. The netrin-1 receptor, deleted in colorectal cancer (DCC), controls the extent and organization of mPFC dopamine connectivity during adolescence and in turn directs mPFC functional and structural maturation. Dcc haploinsufficiency leads to increased mPFC dopamine input, which causes improved cognitive processing and resilience to behavioral effects of stimulant drugs of abuse. Here we examine the effects of Dcc haploinsufficiency on the dynamic expression of dopamine receptors in forebrain targets of C57BL6 mice. We conducted quantitative receptor autoradiography experiments with [3H]SCH-23390 or [3H]raclopride to characterize D1 and D2 receptor expression in mPFC and striatal regions in male Dcc haploinsufficient and wild-type mice. We generated autoradiograms at early adolescence (PND21±1), mid-adolescence (PND35±2), and adulthood (PND75±15). C57BL6 mice exhibit overexpression and pruning of D1, but not D2, receptors in striatal regions, and a lack of dopamine receptor pruning in the mPFC. We observed age- and region-specific differences in D1 and D2 receptor density between Dcc haploinsufficient and wild-type mice. Notably, neither group shows the typical pattern of mPFC dopamine receptor pruning in adolescence, but adult haploinsufficient mice show increased D2 receptor density in the mPFC. These results show that DCC receptors contribute to the dynamic refinement of D1 and D2 receptor expression in striatal regions across adolescence. The age-dependent expression of dopamine receptor in C57BL6 mice shows marked differences from previous characterizations in rats.

Mesocorticolimbic Connectivity and Volumetric Alterations in DCC Mutation Carriers

The axon guidance cue receptor DCC (deleted in colorectal cancer) plays a critical role in the organization of mesocorticolimbic pathways in rodents. To investigate whether this occurs in humans, we measured (1) anatomical connectivity between the substantia nigra/ventral tegmental area (SN/VTA) and forebrain targets, (2) striatal and cortical volumes, and (3) putatively associated traits and behaviors. To assess translatability, morphometric data were also collected in Dcc-haploinsufficient mice. The human volunteers were 20 DCC+/− mutation carriers, 16 DCC+/+ relatives, and 20 DCC+/+ unrelated healthy volunteers (UHVs; 28 females). The mice were 11 Dcc+/− and 16 wild-type C57BL/6J animals assessed during adolescence and adulthood. Compared with both control groups, the human DCC+/− carriers exhibited the following: (1) reduced anatomical connectivity from the SN/VTA to the ventral striatum [DCC+/+: p = 0.0005, r(effect size) = 0.60; UHV: p = 0.0029, r = 0.48] and ventral medial prefrontal cortex (DCC+/+: p = 0.0031, r = 0.53; UHV: p = 0.034, r = 0.35); (2) lower novelty-seeking scores (DCC+/+: p = 0.034, d = 0.82; UHV: p = 0.019, d = 0.84); and (3) reduced striatal volume (DCC+/+: p = 0.0009, d = 1.37; UHV: p = 0.0054, d = 0.93). Striatal volumetric reductions were also present in Dcc+/− mice, and these were seen during adolescence (p = 0.0058, d = 1.09) and adulthood (p = 0.003, d = 1.26). Together these findings provide the first evidence in humans that an axon guidance gene is involved in the formation of mesocorticolimbic circuitry and related behavioral traits, providing mechanisms through which DCC mutations might affect susceptibility to diverse neuropsychiatric disorders. SIGNIFICANCE STATEMENT Opportunities to study the effects of axon guidance molecules on human brain development have been rare. Here, the identification of a large four-generational family that carries a mutation to the axon guidance molecule receptor gene, DCC, enabled us to demonstrate effects on mesocorticolimbic anatomical connectivity, striatal volumes, and personality traits. Reductions in striatal volumes were replicated in DCC-haploinsufficient mice. Together, these processes might influence mesocorticolimbic function and susceptibility to diverse neuropsychiatric disorders.