Beyond guidance: netrin-1 functions as a critical regulator of glutamatergic connectivity in the adult Ventral Tegmental Area

Abstract

The axonal guidance cue netrin-1 serves a critical role in neural circuit development by promoting growth cone motility, axonal branching, and synaptogenesis. Within the adult mouse brain, Ntn1 expression is highly enriched in the ventral midbrain where it is expressed in both GABAergic and dopaminergic neurons, but its function in this context remains largely obscured. To address this, we performed viral-mediated, cell-type specific CRISPR-Cas9 mutagenesis of Ntn1 in the ventral tegmental area (VTA) of adult mice. Ntn1 loss-of-function in either cell type resulted in a significant reduction in excitatory postsynaptic connectivity. In dopamine neurons, reduced excitatory tone had a minimal phenotypic outcome; however, reduced glutamatergic tone on VTA GABA neurons induced a hyperdopaminergic phenotype. Loss of Ntn1 function in both cell types simultaneously largely rescued the phenotype observed in the GABA-only mutagenesis. These findings demonstrate an important role for netrin-1 in maintaining excitatory connectivity in the adult midbrain and that a balance in this connectivity within two of the major cell types of the VTA is critical for the proper functioning of the mesolimbic system.Intriguingly, netrin-1 has not one but two receptors that have been identified as highly expressed in the adult VTA; Dcc and Unc5 homologue C (Unc5c). As the signaling cascades produced by these two receptors are in direct opposition to each other, it is possible that they are able to control differing aspects of netrin-1 circuitry regulation. To explore this, I used the same viral-mediated, cell-type specific CRISPR-Cas9 mutagenesis method to induce Dcc and Unc5c loss of function in dopamine neurons of the adult VTA. While similar to Ntn1 cKO in dopamine only VTA neurons, loss of Dcc or Unc5c produced distinct, phenotypically dissimilar, alterations in dopamine-mediated behaviors. Further research is needed to determine the individual roles of these axonal guidance receptors, and how they aid netrin-1 in modulating glutamatergic connectivity in the adult brain.