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 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 a dual imbalance 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 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/f mice. 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 NRG/ErbB4 signaling directly in DAergic axonal projections contributes to the modulation of DA homeostasis, and that NRG/ErbB4 signaling in both GABAergic interneurons and DA neurons contribute to the modulation of behaviors with relevance to psychiatric disorders.

High voltage-gated calcium channels consist of a pore-forming subunit (␣ 1 ) and three nonhomologous subunits (␣ 2 /␦, ␤, and ␥). Although it is well established that the ␤-subunit promotes traffic of channels to the plasma membrane and modifies their activity, the reversible nature of the interaction with the ␣ 1 -subunit remains controversial. Here, we address this issue by examining the effect of purified ␤ 2a protein on Ca V 1.2 and Ca V 2.3 channels expressed in Xenopus oocytes. The ␤ 2a -subunit binds to the ␣ 1 -interaction domain (AID) in vitro, and when injected into oocytes, it shifts the voltage dependence of activation and increases charge movement to ionic current coupling of Ca V 1.2 channels. This increase depended on the integrity of AID but was not abolished by bafilomycin, demonstrating that the ␣ 1 -␤ interaction through the AID site can take place at the plasma membrane. Furthermore, injection of ␤ 2a protein inhibited inactivation of Ca V 2.3 channels and converted fast inactivating Ca V 2.3/␤ 1b channels to slow inactivating channels. Inhibition of inactivation required larger concentration of ␤ 2a in oocytes expressing Ca V 2.3/␤ 1b channels than expressing Ca V 2.3 alone but reached the same maximal level as expected for a competitive interaction through a single binding site. Together, our data show that the ␣ 1 -␤ interaction is reversible in intact cells and defines calcium channels ␤-subunits as regulatory proteins rather than stoichiometric subunits.High voltage-gated calcium channels are multi-subunit proteins complexes where a pore-forming subunit combines with one or more nonhomologous auxiliary subunits (1). One of these auxiliary subunit, the ␤-subunit, is crucial for channel function, because in addition to stimulating channel activity it appears to be required for surface expression of the channel protein (2). These two effects combined result in a severalfold increase in the ionic current density in heterologous expression systems, but the relative contribution, biological relevance, and extent to which both processes are independent from each other remain elusive. Early studies show that in Xenopus oocytes, coexpression of ␤ 2a with the pore-forming ␣ 1 subunit from cardiac cells (Ca V 1.2) augments ionic currents mostly by increasing ionic current to charge movement ratio (3). Later on, it was shown that the addition of the ␤-subunit as purified protein is capable of modulating channel activity of the ␣ 1 subunit expressed in Xenopus oocytes (4, 5) and also on isolated membranes from skeletal muscle (6). These results suggest that modulation of function is separated from the effect on channel expression and predicts that binding sites remain available on the mature channel. However, the ␣ 1 -␤-subunit association depends primarily on the so-called ␣-interaction domain (AID), 2 located within the intracellular loop joining the first and second repeats of the ␣ 1 -subunit. Secondary binding sites have been identified, but they appear to be specific to certain ␣ 1 -␤ pairs, an...

Background: Viloxazine was historically described as a norepinephrine reuptake inhibitor (NRI). Since NRIs have previously demonstrated efficacy in attention deficit/hyperactivity disorder (ADHD), viloxazine underwent contemporary investigation in the treatment of ADHD. Its clinical and safety profile, however, was found to be distinct from other ADHD medications targeting norepinephrine reuptake. Considering the complexity of neuropsychiatric disorders, understanding the mechanism of action (MoA) is an important differentiating point between viloxazine and other ADHD medications and provides pharmacology-based rationale for physicians prescribing appropriate therapy. Methods: Viloxazine was evaluated in a series of in vitro binding and functional assays. Its effect on neurotransmitter levels in the brain was evaluated using microdialysis in freely moving rats. Results: We report the effects of viloxazine on serotoninergic (5-HT) system. In vitro, viloxazine demonstrated antagonistic activity at 5-HT 2B and agonistic activity at 5-HT 2C receptors, along with predicted high receptor occupancy at clinical doses. In vivo, viloxazine increased extracellular 5-HT levels in the prefrontal cortex (PFC), a brain area implicated in ADHD. Viloxazine also exhibited moderate inhibitory effects on the norepinephrine transporter (NET) in vitro and in vivo, and elicited moderate activity at noradrenergic and dopaminergic systems. Conclusion: Viloxazine's ability to increase 5-HT levels in the PFC and its agonistic and antagonistic effects on certain 5-HT receptor subtypes, which were previously shown to suppress hyperlocomotion in animals, indicate that 5-HT modulating activity of viloxazine is an important (if not the predominant) component of its MoA, complemented by moderate NET inhibition. Supported by clinical data, these findings suggest the updated psychopharmacological profile of viloxazine can be best explained by its action as a serotonin norepinephrine modulating agent (SNMA).