Calbindin-D28K Limits Dopamine Release in Ventral but Not Dorsal Striatum by Regulating Ca2+ Availability and Dopamine Transporter Function

The calcium-binding protein calbindin-D28K, or calb1, is expressed at higher levels by dopamine (DA) neurons originating in the ventral tegmental area (VTA) than in the adjacent substantia nigra pars compacta (SNc). Calb1 has received attention for a potential role in neuroprotection in Parkinson’s disease. The underlying physiological roles for calb1 are incompletely understood. We used cre-loxP technology to knock down calb1 in mouse DA neurons to test whether calb1 governs axonal release of DA in the striatum, detected using fast-scan cyclic voltammetry ex vivo. In the ventral but not dorsal striatum, calb1 knockdown elevated DA release and modified the spatiotemporal coupling of Ca2+ entry to DA release. Furthermore, calb1 knockdown enhanced DA uptake but attenuated the impact of DA transporter (DAT) inhibition by cocaine on underlying DA release. These data reveal that calb1 acts through a range of mechanisms underpinning both DA release and uptake to limit DA transmission in the ventral but not dorsal striatum.

DAT-Cre mice have prolonged uptake kinetics compared to WT mice but unaltered peak [DA] o Heterozygous DAT-Cre (DATcre +/-) mice used here as controls for wild-type calbindin (CalbWT), have ~17% lower DAT levels relative to wild-type mice 2 , and correspondingly, have slightly prolonged timecourses for decay of [DA] o transients relative to background wild-type (C57Bl6/J) mice, in both CPu and NAc (Supplementary Figure S2A,B) Figure S2C; Two-way ANOVA effect of genotype, F 1,73 =1.9, P=0.17).

DA release is enhanced in NAc of CalbKD relative to CalbWT mice in the absence of nAChR inhibition
Electrically evoked striatal DA release in the absence of nAChR inhibition results from two release events 3 . The first DA release event is due to rapid direct depolarisation of dopaminergic axons by the electrical stimulation while the second DA release event is driven by ACh released from local striatal cholinergic interneurons which activates nAChRs on DA axons with a short latency (~10 ms). These two events cannot be temporally resolved using FCV but appear combined as a summed [DA] o signal. The nAChR-driven release event strongly governs the output of DA including dictating short-term plasticity and frequencydependence 4 , and can mask control by other underlying Ca 2+ -dependent events (Brimblecombe and Cragg, unpublished observations). In our study we therefore inhibited nAChRs to enable us to probe the roles of calb1 without the confounding effects of nAChR activation or variable ACh release. We nonetheless assessed here whether evoked [DA] o was modified in the absence of nAChR inhibition. We confirmed that [DA] o evoked in NAc by single pulses were greater in calbKD than CalbWT mice in the absence of nAChR antagonists (Supplementary Figure S3; t-test, t 50 =2.3, P=0.025).

Response to stimulation frequency does not differ between CalbWT and CalbKD mice
At many synapse types, there is an inverse relationship between the amount of neurotransmitter released by initial stimuli compared to consecutive stimulations over short inter-pulse intervals, typically resulting from summation of residual intracellular Ca 2+ at high frequencies. This relationship has been observed only weakly for DA release 5,6 , and only when nAChRs are inhibited 4 2), there was however no difference in the relative release in response to frequency of stimulation between CalbKD and CalbKD mice for trains of 5 pulses at frequencies spanning 5-100 Hz (in either the presence or absence of nAChR inhibition, DHβE, 1 µM, which is well documented to govern frequency responsiveness 4,7,9-11 ) (Supplementary Figure S4A, . These data in turn also support previous conclusions that short-term plasticity of DA release is only weakly associated with initial release probability.

D2-receptor regulation of DA release does not differ between CalbWT and CalbKD mice
D 2 -type DA receptors (D 2 R) are strong negative regulators of striatal DA release 12 and are known to be regulated by Ca 2+ dependent processes 13 , and therefore we assessed whether elevated DA release in NAc of CalbKD mice might be underpinned by lower levels of D 2 R function compared to CalbWT controls. We first identified an IC 50 for D 2

Cocaine (0.5-5 µM) promotes peak [DA] o to a lesser extent in CalbKD than CalbWT mice
Low concentrations of cocaine (nM) have been shown to increase peak [DA] o without affecting uptake kinetics, which is thought to be due to cocaine at low concentrations liberating a DAT-limited release pool at lower cocaine concentrations than required to prevent DAT from translocating DA across the plasma membrane 14 . We therefore sought to expand upon our findings with 5 µM cocaine in NAc, using 0.