Dopamine Dysregulation and Altered Responses to Drugs Affecting Dopaminergic Transmission in a New Dopamine Transporter Knockout (DAT-KO) Rat Model.

Under normal conditions, dopamine (DA) clearance after release largely depends on uptake by the DA transporter (DAT). DAT expression/activity is reduced in some neuropsychiatric and neurological disorders. Our aim was to characterize the behavioral, neurochemical and electrophysiological effects of eliminating DAT in a novel knockout rat model we generated using CRISPR/Cas9. Consistent with existing DAT-KO models, our DAT-KO rats displayed increased locomotion, paradoxical calming by amphetamine, and reduced kinetics of DA clearance after stimulated release. Reduced DA kinetics were demonstrated using fast-scan cyclic voltammetry in brain slices containing the striatum or substantia nigra pars compacta (SNc) and in the dorsal striatum in vivo. Cocaine enhanced DA release in wild-type (WT) but not DAT-KO rats. Basal extracellular DA concentration measured with fast-scan controlled-adsorption voltammetry was higher in DAT-KO rats both in the striatum and SNc and was enhanced by L-DOPA (particularly after pharmacological block of monoamine oxidase), confirming that DA release after L-DOPA is not due to DAT reversal. The baseline firing frequency of SNc neurons was similar in both genotypes. However, D2 receptor-mediated inhibition of firing (by quinpirole or L-DOPA) was blunted in DAT-KO rats, while GABAB-mediated inhibition was preserved. We have also provided new data for the DAT-KO rat regarding the effects of slowing DA diffusion with dextran and blocking organic cation transporter 3 with corticosterone. Together, our results validate our DAT-KO rat and provide new insights into the mechanisms of chronic dysregulation of the DA system by addressing several unresolved issues in previous studies with other DAT-KO models.

Electrophysiological Characterization of Novel Effects of the Uptake-2 Blocker Decynium-22 (D-22) on Dopaminergic Neurons in the Substantia Nigra Pars Compacta.

Extracellular levels of dopamine (DA) and other monoamines in the brain depend not only on the classic transporters encoded by SLC6A gene family such as DAT, NET and SERT, but also a more recently identified group of low-affinity/high-capacity 'Uptake-2' transporters, mainly OCT3 and PMAT. The most frequently used pharmacological tool in functional studies of Uptake-2 is decynium-22 (D-22) known to block these transporters. However, the effectiveness of this drug in enhancing extracellular DA remains uncertain. Our aim was to test the hypothesis that D-22 increases extracellular levels of DA released from the somatodendritic region of dopaminergic neurons in the substantia nigra pars compacta (SNc) by reducing the OCT3/PMAT-dependent component of DA uptake. Extracellular DA was assessed indirectly, by evoking D2-IPSCs in SNc neurons following stimulated release of this neurotransmitter in midbrain slices obtained from mice. Recordings were conducted after partial inhibition of DAT with nomifensine, and after application of L-DOPA which increased the releasable DA pool. Contrary to our expectations, D-22 reduced, rather than increased, the amplitude of D2-IPSCs. Other effects included inhibition of GABAB-IPSCs and Ih current, and a reduction in firing frequency of nigral neurons. These results show that in addition to the previously known non-specific inhibitory action on α1 adrenoceptors, D-22 exerts additional off-target effects by inhibiting dopaminergic and GABAergic synaptic transmission in the SNc and the spontaneous (pacemaker) activity of nigral neurons. It remains to be established if these novel effects contribute to a reduction in spontaneous locomotor activity reported in previous studies after systemic drug administration.