Opposite Control of Excitatory and Inhibitory Synapse Formation by Slitrk2 and Slitrk5 on Dopamine Neurons Modulates Hyperactivity Behavior

Abstract

Graphical Abstract Highlights d Lmx1a/b control Slitrk2 and Slitrk5 expression in midbrain dopamine neurons d Slitrk2 and Slitrk5 regulate excitatory and inhibitory synapse formation, respectively d Chronic inhibition of VTA neurons during development results in hyperactivity d Developmental changes in E/I balance on dopamine neurons lead to hyperlocomotion SUMMARY The neurodevelopmental origin of hyperactivity disorder has been suggested to involve the dopaminergic system, but the underlying mechanisms are still unknown. Here, transcription factors Lmx1a and Lmx1b are shown to be essential for midbrain dopaminergic (mDA) neuron excitatory synaptic inputs and dendritic development. Strikingly, conditional knockout (cKO) of Lmx1a/b in postmitotic mDA neurons results in marked hyperactivity. In seeking Lmx1a/b target genes, we identify positively regulated Slitrk2 and negatively regulated Slitrk5. These two synaptic adhesion proteins promote excitatory and inhibitory synapses on mDA neurons, respectively. Knocking down Slitrk2 reproduces some of the Lmx1a/b cKO cellular and behavioral phenotypes, whereas Slitrk5 knockdown has opposite effects. The hyperactivity caused by this imbalance in excitatory/inhibitory synaptic inputs on dopamine neurons is reproduced by chronically inhibiting the ventral tegmental area during development using pharmacogenetics. Our study shows that alterations in developing dopaminergic circuits strongly impact locomotor activity, shedding light on mechanisms causing hyperactivity behaviors.

Inhibitory control in neuronal networks relies on the extracellular matrix integrity

“…Neuronal activity is regulated through a dynamic balance of excitation and inhibition (E-I balance) [35] that requires coordinated plasticity of excitatory and inhibitory synapses [6,69]. In the brain, excitatory and inhibitory synapses are regulated cooperatively [25,60], and their modulation can be synergistic, reciprocal, and antagonistic. Over several years, experimental studies have gathered solid evidence on the plasticity of individual excitatory synapses, establishing the modulation of presynaptic neurotransmitter release and postsynaptic responsiveness to glutamate as key neural correlates for memory and learning [39].…”

SLITRK5 is a negative regulator of hedgehog signaling in osteoblasts

“…Supporting this, Slitrk5 expression is upregulated in tumors marked by active Hh signaling, such as Hh dependent medulloblastoma 16 . Similarly, missense mutations in SHH and Slitrk5 have been implicated in the attention deficit hyperactivity disorder 34 , 35 , raising the possibility that SLITRK5-regulation of Hh signaling contributes to disease processes outside of bone.…”

RIT2 reduces LRRK2 kinase activity and protects against alpha-synuclein neuropathology

“…Visualization was performed using a Leica SP8-X confocal laser scanning microscope system equipped with an oil immersion 63X objective and images were analysed using CellProfiler to quantify the number and intensity of investigated objects (object size in pixel units: pS129-aSyn 20-250; LC3B 5-90; pipelines available upon request). Immunohistochemistry (IHC) was performed as described in (Salesse et al, 2020). For DA neuron survival quantification, sections of midbrain (4 sections interval) were stained with NeuN (Millipore, MAB377, 1:500) and TH (Pel-Freez Biologicals, P60101, 1:1000) antibodies, followed by incubation with Cy3 (Jackson Immuno, 715-165-150, 1:200) and Alexa Fluor 647 (Life Technologies, A-21448, 1:400) respectively.…”

“…RNAscope probes for mouse TH and RIT2 (TH – 317621, RIT2 – 58904) were designed by Advanced Cell Diagnostics (Newark, CA, USA). The in situ hybridization was carried out following the manufacturers protocol for fixed and frozen tissue and as described in (Salesse et al, 2020).…”

“…Immunohistochemistry (IHC) was performed as described in (Salesse et al, 2020). For DA neuron survival quantification, sections of midbrain (4 sections interval) were stained with NeuN (Millipore, MAB377, 1:500 ) and TH (Pel-Freez Biologicals, P60101, 1:1000)…”