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

Abstract

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.

Keywords: Dopamine; Parkinson’s disease; calbindin-D28K; dopamine transporter; fast-scan cyclic voltammetry.

Figure 1

Calb1 knockdown leads to elevated [DA]_oin NAc but not CPu. (A) Fluorescence images of midbrain showing immunoreactivity to calb1 (green), TH-positive (red), and merged (yellow). Left: Note DA cells lacking calb1 in ventral SNc, and TH- and calb1-positive cells in lateral VTA in CalbWT (upper) and to a lesser degree in CalbKD (lower); scale bar 100 μm. Right: Note decreased calb1 fluorescence in TH-positive neurons in medial VTA of CalbKD relative to CalbWT mice. (B) Mean mouse weight ± SEM vs age. Two-way ANOVA, age × genotype interaction, F_3,40 = 1.4, P = 0.26; effect of genotype: F_1,40 = 16.2, P = 0.0002, n = 5 male mice per genotype tracked from 6 to 16 weeks, n = 11 male mice at time point of culling in weeks 22–36. (C) Mean [DA]_o ± SEM (shaded) vs time evoked by single pulses (arrow) in CPu (purple) and NAc (blue) in CalbWT (left) and CalbKD (right). (D) Peak [DA]_o ± SEM in CPu (circles) and NAc (squares) and mean [DA]_o ± SEM (black lines) for CalbWT (filled) and CalbKD (unfilled). Two-way ANOVA region × genotype, interaction F_1,52 = 5.0, P = 0.03; region: F_1,52 = 13.5, P = 0.001; genotype, F_1,52 = 5.11, P = 0.03; NAc, Sidak’s posthoc t test, t₅₂ = 3.18, P < 0.05; CPu, Sidak’s posthoc t test, t₅₂ = 0.02, P > 0.05 n = 14 sites from 5 pairs of mice. (E) DA content (pmol/mm³) measured by HPLC-ECD from CPu (circles) and NAc (squares) of CalbWT (filled) and CalbKD (unfilled), and mean data ± SEM (black lines). Two-way ANOVA, genotype effect: F_1,20 = 0.02, P = 0.88, n = 6 samples, 3 pairs of mice. FCV data were collected in the presence of nAChR blockade (DHβE 1 μM). *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 2

Modified relationship between Ca²⁺and [DA]_o in NAc after calb1 knockdown. (A,B) Variable slope sigmoidal curve fit of log–log transformed data of mean peak [DA]_o ± SEM evoked by 1p versus [Ca²⁺]_o in CPu: (A) one curve fits both genotypes, F_4,32 = 0.04, P = 0.99, n =. 4 Hill slope: 3.4. R² CPu: 0.81. NAc: (B) curves significantly different between genotypes, F_4,24 = 6.2, P = 0.0014. R² NAc 0.93 (calbWT) and 0.82 (calbKD). No change in Hill slope: CalbKD 2.9 vs CalbWT 2.5,F_1,24 = 0.4 P = 0.84, n = 4 experiments/animals. CalbWT (filled) and CalbKD (unfilled). (C) Peak evoked [DA]_o in CalbKD expressed normalized to value in CalbWT at each [Ca²⁺]_o used, linear regression not different from zero slope, P = 0.83. N = 4 pairs of animals. (D,G) Peak [DA]_o (% of control) ± SEM in the presence of BAPTA-AM (100 μM) (red) or EGTA-AM (100 μM) (blue) in CPu (D) or NAC (G). In CPu: two-way ANOVA: genotype × chelator interaction, F_1,51 = 1 × 10^–4, P = 0.99; genotype effect, F_1,51 = 1.60, P = 0.21. In NAc: two-way ANOVA: genotype × chelator interaction, F_1,48 = 18.9, P < 0.0001. (F) Ratio of DA release in BAPTA-AM:EGTA-AM (±SEM) in CPu and NAc in CalbWT (filled) and CalbKD (unfilled). Two-way ANOVA: genotype × region interaction, F_1,50 = 9.2, P = 0.004. (E,H) [DA]_o ± SEM (shaded) vs time in response to single pulses (1p, arrow) in CalbWT (left) and CalbKD (right) in control conditions (black) and BAPTA-AM (red) or EGTA-AM (blue) conditions, in CPu(E) and NAc (H). DHβE (1 μM) present throughout. *P < 0.05, **P < 0.01, ***P < 0.001 Sidak’s post-test following two-way ANOVA. N = 4 pairs of animals, 14 sites/genotype/region.

Figure 3

Calb1 knockdown modifies DA uptake kinetics and DAT regulation of DA release in NAc. (A,B) One-phase exponential decay curve fits (±95% confidence intervals, shaded) for falling phases of mean [DA]_o transients versus time that were concentration-matched, evoked by single pulses in CalbWT (dark blue) and CalbKD (light blue) in CPu (A) and NAc (B). CPu, comparison of k, F_1,422 = 0.1, P = 0.76. NAc, comparison of k, F_1,350 = 64.9, P < 0.0001, n = 9 from 3 mice. Dashed lines show t_1/2. Insets show typical full [DA]_o profiles and indicate points in NAc of sampling decay rates shown in C. (C) Maximum decay rates seen for each transient versus [DA]_o at that rate for CalbWT (dark blue) and CalbKD (light blue). Unconstrained Michaelis–Menten curve fits (solid lines), V_max and K_m are indicated in dashed horizontal and vertical lines respectively ± SEM (shaded). Comparison of fits, P = 0.0032, n = 73 transients per genotype. (D,G) [DA]_o ± SEM (shaded) vs time in response to single pulses (1p, arrow) in CalbWT (left) and CalbKD (right) in control conditions (black), and cocaine (5 μM, red), in CPu (D) and NAc (G) of CalbWT and CalbKD mice. Data are normalized to predrug controls conditions, scale bar indicates 50% of control. (E,H) Area under the curve (AUC) ± SEM of DA transients seen in control (black) and cocaine (red) in CalbWT (filled) and CalbKD (unfilled) for CPu (E) of NAc (H). CPu: Two-way ANOVA: no drug × genotype interaction, F_1,8 = 0.04, P = 0.84; effect of genotype F_1,8 = 7 × 10^–6, P = 1.0. NAc: Two-way ANOVA: drug × genotype interaction, F_1,8 = 13.3, P = 0.007; effect of genotype F_1,8 = 10.7, P = 0.01. (F) Peak evoked [DA]_o ± SEM in the presence of cocaine expressed as % of control conditions. Two-way ANOVA: genotype × region interaction, F_1,8 = 9.1, P = 0.017; Sidak’s post-test: CalbWT vs CalbKD, CPU, t₈ = 0.56, P > 0.05, NAc, t₈ = 3.7, P < 0.05. (I) Peak evoked [DA]_o (μM) paired before and after cocaine ± SEM for individual experiments in CPu and NAc of CalbWT and CalbKD. Two-way ANOVA with repeated measures: cocaine × genotype interaction, F_1,4 = 124.6, P = 0.0004, Sidak’s post-tests: control vs cocaine, CalbWT, t₄ = 18.8, P < 0.001, CalbKD, t₄ = 3.0, P > 0.05, n = 3 experiments, 3 animals. (J,K) Paired-pulse ratios (PPR) for [DA]_o detected in response to a second pulse expressed as a fraction of [DA]_o detected by a single pulse, versus interpulse interval (IPI) in NAc in control conditions (black) and in cocaine (red) for CalbWT (J) and CalbKD (K). In CalbWT: Two-way ANOVA effect of cocaine: F_1,20 = 25.0, P < 0.0001. In CalbKD: F_1,20 = 0.05, P = 0.82, n = 3 sites from 3 animals. DHβE (1 μM) present throughout. *P < 0.05, **P < 0.01, ***P < 0.001, main effect of drug or Sidak’s post-tests following two-way ANOVA.