Probes for INS

Cannabis can be rewarding or aversive. Cannabis reward is believed to be mediated by activation of cannabinoid CB1 receptors (CB1Rs) on GABAergic neurons that disinhibit dopaminergic neurons in the ventral tegmental area (VTA). However, little is known about the mechanisms underlying cannabis aversion in rodents. In the present study, CB1Rs are found not only on VTA GABAergic neurons, but also on VTA glutamatergic neurons that express vesicular glutamate transporter 2 (VgluT2). We then used Cre-Loxp transgenic technology to selectively delete CB1Rs in VgluT2-expressing glutamatergic neurons (VgluT2-CB1 -/-) and Cre-dependent viral vector to express light-sensitive channelrhodopsin-2 into VTA glutamatergic neurons. We found that photoactivation of VTA glutamatergic neurons produced robust intracranial self-stimulation (ICSS) behavior, which was dose-dependently blocked by DA receptor antagonists, but enhanced by cocaine. In contrast, Δ9-tetrahydrocannabinol (Δ9-THC), the major psychoactive component of cannabis, produced dose-dependent conditioned place aversion and a reduction in the above optical ICSS in VgluT2-cre control mice, but not in VgluT2-CB1 -/- mice. These findings suggest that activation of CB1Rs in VgluT2-expressing glutamate neurons produces aversive effects that might explain why cannabinoid is not rewarding in rodents and might also account for individual differences in the hedonic effects of cannabis in humans.

Tyrosine hydroxylase (TH) is the enzyme that catalyzes the rate-limiting step in the biosynthesis of the catecholamine neurotransmitters. In a previous communication, evidence was provided that TH mRNA is trafficked to the axon, where it is locally translated. In addition, a 50bp sequence element in the 3′untranslated region (3’UTR) of TH mRNA was identified that directs TH mRNA to distal axons (i.e. zip-code). In the present study, the hypothesis was tested that local translation of TH plays an important role in the biosynthesis of the catecholamine neurotransmitters in the axon and/or presynaptic nerve terminal. Toward this end, a targeted deletion of the axonal transport sequence element was developed, using the lentiviral delivery of the CRISPR/Cas9 system, and two guide RNA sequences flanking the 50bp cis-acting regulatory element in rat superior cervical ganglion (SCG) neurons. Deletion of the axonal transport element reduced TH mRNA levels in the distal axons and reduced the axonal protein levels of TH and TH activity as measured by phosphorylation of SER40 in SCG neurons. Moreover, deletion of the zip-code diminished the axonal levels of dopamine and norepinephrine. Conversely, the local translation of exogenous TH mRNA in the distal axon enhanced TH levels and activity, and elevated axonal norepinephrine levels. Taken together, these results provide direct evidence to support the hypothesis that TH mRNA trafficking and local synthesis of TH plays an important role in the synthesis of catecholamines in the axon and presynaptic terminal.

Significance Statement Tyrosine hydroxylase (TH) is the rate-limiting enzyme in the biosynthesis of the catecholamine neurotransmitters. Previous results suggested that TH mRNA is trafficked to the distal axons of primary sympathetic neurons and is locally translated. In the present study a gene editing strategy was employed to delete the axonal TH mRNA trafficking regulatory element (i.e. zip-code). Deletion of the zip-code reduced TH mRNA levels in the distal axons, reduced axonal protein levels of TH and diminished the axonal levels, and release of norepinephrine. Collectively, these studies demonstrate that the local synthesis of TH plays an important role in catecholamine synthesis and may facilitate the maintenance of catecholamine levels in response to long-term alteration in the need for neurotransmitters.

Genetic variants of Neuregulin 1 (NRG1) and its neuronal tyrosine kinase receptor ErbB4 are associated with risk for schizophrenia, a neurodevelopmental disorder characterized by excitatory/inhibitory imbalance and dopamine (DA) dysfunction. To date, most ErbB4 studies have focused on GABAergic interneurons in the hippocampus and neocortex, particularly fast-spiking parvalbumin-positive (PV+) basket cells. However, NRG has also been shown to modulate DA levels, suggesting a role for ErbB4 signaling in dopaminergic neuron function. Here we report that ErbB4 in midbrain DAergic axonal projections regulates extracellular DA levels and relevant behaviors. Mice lacking ErbB4 in tyrosine hydroxylase-positive (TH+) neurons, but not in PV+ GABAergic interneurons, exhibit different regional imbalances of basal DA levels and fail to increase DA in response to local NRG1 infusion into the dorsal hippocampus, medial prefrontal cortex and dorsal striatum measured by reverse microdialysis. Using Lund Human Mesencephalic (LUHMES) cells, we show that NRG/ErbB signaling increases extracellular DA levels, at least in part, by reducing DA transporter (DAT)-dependent uptake. Interestingly, TH-Cre;ErbB4f/f mice manifest deficits in learning, spatial and working memory-related behaviors, but not in numerous other behaviors altered in PV-Cre;ErbB4f/fmice. Importantly, microinjection of a Cre-inducible ErbB4 virus (AAV-ErbB4.DIO) into the mesencephalon of TH-Cre;ErbB4f/f mice, which selectively restores ErbB4 expression in DAergic neurons, rescues DA dysfunction and ameliorates behavioral deficits. Our results indicate that direct NRG/ErbB4 signaling in DAergic axonal projections modulates DA homeostasis, and that NRG/ErbB4 signaling in both GABAergic interneurons and DA neurons contribute to the modulation of behaviors relevant to psychiatric disorders.

Cannabinoid CB2 receptors (CB2Rs) are expressed in mouse brain dopamine (DA) neurons and are involved in several DA-related disorders. However, the cell type-specific mechanisms are unclear since the CB2R gene knockout mice are constitutive gene knockout. Therefore, we generated Cnr2-floxed mice that were crossed with DAT-Cre mice, in which Cre- recombinase expression is under dopamine transporter gene (DAT) promoter control to ablate Cnr2 gene in midbrain DA neurons of DAT-Cnr2 conditional knockout (cKO) mice. Using a novel sensitive RNAscope in situ hybridization, we detected CB2R mRNA expression in VTA DA neurons in wildtype and DAT-Cnr2 cKO heterozygous but not in the homozygous DAT-Cnr2 cKO mice. Here we report that the deletion of CB2Rs in dopamine neurons enhances motor activities, modulates anxiety and depression-like behaviors and reduces the rewarding properties of alcohol. Our data reveals that CB2Rs are involved in the tetrad assay induced by cannabinoids which had been associated with CB1R agonism. GWAS studies indicates that the CNR2 gene is associated with Parkinson's disease and substance use disorders. These results suggest that CB2Rs in dopaminergic neurons may play important roles in the modulation of psychomotor behaviors, anxiety, depression, and pain sensation and in the rewarding effects of alcohol and cocaine.