Deficits in dopaminergic transmission precede neuron loss and dysfunction in a new Parkinson model

Abstract

The pathological end-state of Parkinson disease is well described from postmortem tissue, but there remains a pressing need to define early functional changes to susceptible neurons and circuits. In particular, mechanisms underlying the vulnerability of the dopamine neurons of the substantia nigra pars compacta (SNc) and the importance of protein aggregation in driving the disease process remain to be determined. To better understand the sequence of events occurring in familial and sporadic Parkinson disease, we generated bacterial artificial chromosome transgenic mice (SNCA-OVX) that express wild-type α-synuclein from the complete human SNCA locus at disease-relevant levels and display a transgene expression profile that recapitulates that of endogenous α-synuclein. SNCA-OVX mice display age-dependent loss of nigrostriatal dopamine neurons and motor impairments characteristic of Parkinson disease. This phenotype is preceded by early deficits in dopamine release from terminals in the dorsal, but not ventral, striatum. Such neurotransmission deficits are not seen at either noradrenergic or serotoninergic terminals. Dopamine release deficits are associated with an altered distribution of vesicles in dopaminergic axons in the dorsal striatum. Aged SNCA-OVX mice exhibit reduced firing of SNc dopamine neurons in vivo measured by juxtacellular recording of neurochemically identified neurons. These progressive changes in vulnerable SNc neurons were observed independently of overt protein aggregation, suggesting neurophysiological changes precede, and are not driven by, aggregate formation. This longitudinal phenotyping strategy in SNCA-OVX mice thus provides insights into the region-specific neuronal disturbances preceding and accompanying Parkinson disease.

Keywords: behavioral phenotyping; dopamine transmission; in vivo electrophysiology; neurodegeneration; voltammetry.

Fig. 1.

Characterization of α-syn transgenic mice. (A and B) Double immunofluorescence labeling for α-syn and TH confirms human α-syn transgene expression in TH-immunoreactive dopamine neurons of the (A) SNc and (B) VTA in 3-mo-old SNCA-OVX and hα-syn animals. (Scale bars, 200 µm.) (C) Quantitative Western analysis of striatal α-syn expression in 3-mo-old SNCA-OVX, hα-syn, Snca^−/−, and C57/Bl6 animals reveals that SNCA-OVX animals express the human wild-type SNCA transgene at 1.9-fold higher levels compared with endogenous mouse α-syn protein in C57/Bl6 animals. No α-syn expression was observed in Snca^−/− control animals. One-way ANOVA with Bonferroni post hoc analysis; ****P < 0.0001, ***P < 0.001, n = 2–3. (D) Immunohistochemical analysis revealed somatic cytoplasmic α-syn immunostaining in cells of the SNc in 18-mo-old SNCA-OVX mice using the Syn-1 antibody with formic acid (FA) pretreatment. Similar structures were weakly labeled in 18-mo-old SNCA-OVX mice using the LB509 antibody autoclaved (AC) in citric buffer. This staining was abolished when proteinase K (PK) antigen retrieval was applied instead. All of these immunohistochemical stainings were carried out together with control tissue (entorhinal cortex from a PD patient with dementia that shows prominent Lewy body and neuritic pathology). No “amyloid” pathology was detected with thioflavine S in either 18-mo-old SNCA-OVX or Snca^−/−, whereas several Lewy bodies and dystrophic neurites were detected in the positive PD control. (Scale bars, 20 μm; magnification: all pictures, 400×.) (E and G) At 3 mo of age, stereological cell counting revealed no differences in the number of TH-immunoreactive neurons in the SNc of SNCA-OVX mice compared with hα-syn and Snca^−/− mice. One-way ANOVA: no main effect of genotype: F < 1, P > 0.05, n = 5 per genotype. Data are expressed as the mean ± SEM. Representative images of TH-immunoreactivity in the SNc are shown. (Scale bar, 200 µm.) (F and H) Analysis of 18-mo-old animals revealed a 30% loss of TH-immunoreactive neurons in the SNc of SNCA-OVX mice compared with hα-syn mice. One-way ANOVA with Bonferroni post hoc analysis: main effect of genotype: F_(2,12) = 6.3, *P < 0.05, n = 5 per genotype. Data are expressed as the mean ± SEM. Representative images of TH immunoreactivity in the SNc are shown. (Scale bar, 200 µm.)

Fig. 2.

Early nonmotor and late motor phenotypes in SCNA-OVX mice. (A) Male SNCA-OVX animals displayed an increased dry stool weight independent of age. Three-way ANOVA; main effect of age: F_(1,84) = 20.66, ****P < 0.0001; genotype/sex interaction: F_(2,83) = 3.19, *P < 0.05; no interaction between genotype and age: F_(2,83) = 1.44, P > 0.05; separate ANOVA for males: main effect of genotype: F_(2,45) = 7.76, ***P < 0.001 (Tukey post hoc test *P < 0.05, ***P < 0.001), n = 4–11; females: main effect of genotype: F < 1, P > 0.05, n = 3–8. Data are expressed as mean ± SEM. (B) Rotarod performance was impaired in 18- but not in 3-mo-old SNCA-OVX mice. Two-way ANOVA of square-root transformed data: main effect of age: F_(1,28) = 25.95, ***P < 0.001; main effect of genotype: F_(2,27) = 3.49, *P < 0.05; genotype/age interaction: F_(2,27) = 6.14, **P < 0.01; separate ANOVAs revealed no main effect of genotype at 3 mo (F < 1, P > 0.05), but a significant main effect of genotype at 18 mo (F_(2,15) = 10.04, **P < 0.01) (Tukey post hoc test *P < 0.05, **P < 0.01), n = 3–8), which remained significant when body weight was included as a covariate (ANCOVA: main effect of genotype: F_(2,14) = 4.93, *P < 0.05). Data are expressed as mean ± SEM. (C–E) SNCA-OVX animals (18 mo old) were impaired on the multiple static rods test compared with hα-syn and Snca^−/− animals. (C) Orientation: ANOVA of square-root transformed data collapsed across both rods; main effect of genotype: F_(2,24) = 3.35, P = 0.05, n = 7–10. Data are expressed as mean ± SEM. (D) Traversing: Kruskal–Wallis one-way ANOVA on ranked data collapsed across both rods. Effect of genotype: H₂ = 12.37; **P < 0.01 (Dunn’s post hoc pairwise comparison *P < 0.05), n = 7–10. Data are expressed as median ± IQ range. (E) SNCA-OVX animals displayed increased foot slips when traversing rod 2 (**P < 0.01, Welch t test: t = 3.79, df = 6, n = 4 per group). Rods had a diameter of 22 mm (rod 1) and 9 mm (rod 2). (F) SNCA-OVX animals displayed reduced stride length compared with Snca^−/− mice (Kruskal–Wallis one-way ANOVA on ranked data, comprising data from both ages and sexes: main effect of genotype: H₍₂₎ = 10.79, **P < 0.01 (Dunn’s post hoc pairwise comparison *P < 0.05), n = 15–30). Data are expressed as median ± IQ range.

Fig. 3.

α-syn overexpression limits dopamine release from an early age in CPu, but not NAc. (A–C) Mean extracellular dopamine concentration ([dopamine]_o) profiles vs. time (mean ± SEM) following single pulse stimulation (↑200 µs, 0.6 mA) in CPu (A–C, left traces) or NAc (A–C, right traces) of Snca^−/− vs. SNCA-OVX at increasing ages. Mean peak evoked [dopamine]_o was significantly lower in SNCA-OVX compared with Snca^−/− in CPu (**P < 0.01, ***P < 0.001; unpaired t test; n = 60–72) but not NAc (P > 0.05; unpaired t test; n = 19–23) at 3–18 mo. (D–F) Dopamine content (expressed as a percentage of Snca^−/−) in CPu and NAc dissected from striatal slices used for FCV recordings. Mean dopamine content was not significantly different between SNCA-OVX and Snca^−/− in CPu or NAc (P > 0.05, unpaired t test, n = 10) between ages 3 and 12 mo. However, at 18 mo mean dopamine content was significantly greater in CPu of SNCA-OVX compared with Snca^−/− (F; **P < 0.01, unpaired t test, n = 12) but not NAc. (G) Typical FCV recording sites include six CPu and two NAc recordings. Mean peak [dopamine]_o from individual sites from 3- to 4-mo-old Snca^−/− and SNCA-OVX highlights more prominent [dopamine]_o deficit in dorsal striatum (ventrolateral striatum **P < 0.01; dorsomedial and dorso-mid striatum *P < 0.05; all other regions P > 0.05; unpaired t test; n = 10–14 per region). (H and I) The hα-syn mice show no difference compared with Snca^−/− mice in mean peak evoked [dopamine]_o (P > 0.05; unpaired t test; H: 3 mo, n = 18 NAc, n = 60 CPu; I: 18 mo, n = 15 NAc, n = 47 CPu) or dopamine content (P > 0.05; unpaired t test; J: 3 mo, n = 10; K: 18 mo, n = 7) in CPu or NAc. Absolute values for dopamine content ranged from 97 to 240 pmol/mm³ tissue in CPu to 47–104 pmol/mm³ tissue in NAc.

Fig. 4.

Overexpression of α-syn alters vesicle clustering in dopaminergic synaptic boutons in the dorsal striatum. (A) Frequency distribution of intervesicle distance in TH-immunoreactive profiles in the striatum of SNCA-OVX and Snca^−/− mice. The two-sample Kolmogorov–Smirnov test was performed to test for differences between the two distributions and revealed a significant difference between the groups (****P < 0.0001). (B) Cumulative distribution functions of the two data groups. Application of an index of dispersion, D = σ²/μ, to each raw dataset (D_SNCA-OVX, D_Snca^−/−) shows that the Snca^−/− vesicles are three times more dispersed than the SNCA-OVX (D_SNCA-OVX/D_Snca^−/− = 0.34). (C) Electron micrograph of a TH immunogold-labeled axon terminal in the dorsal striatum identified by the accumulation of electron dense silver-intensified immunogold particles. The bouton forms symmetrical synaptic contact (arrowhead) with a dendritic shaft. Note the accumulation of vesicles within the bouton. (Scale bar, 0.2 μm.)

Fig. 5.

α-syn overexpression does not alter nondopamine amine signaling in bed nucleus of the stria terminalis and substantia nigra pars reticulata. (A) Typical cyclic voltammograms from electrically evoked recordings of NE in BNST (stimulated) and following calibration in 2 µM NE (calibration). Note single oxidation and reduction current peaks at +560–580 and –200 mV, respectively. (B) Typical recording site in ventromedial portion of BNST (vmBNST) ∼0.26 mm anterior of bregma. Mean profiles of extracellular NE concentration ([NE]_o) vs. time (mean ± SEM) following train stimulation (30 pulses, 50 Hz) in vmBNST in young adult (C) (3 mo, n = 33–35) or aged adult (E) (18 mo, n = 70–74) Snca^−/− vs. SNCA-OVX mice. Mean peak [NE]_o vs. frequency during 30 pulse trains (10–100 Hz) in vmBNST in young adult (D) (n = 33–35) or aged adult (F) (n = 70–74). (G) Typical cyclic voltammograms from electrically evoked recordings of 5-HT in SNr (stimulated) and following calibration in 0.5 µM 5-HT (calibration). Note single oxidation and dual reduction current peaks at approximately +600, −20, and −670 mV, respectively. (H) Typical recording sites in the SNr ∼3.2 mm posterior of bregma. Note recording sites located on ventral side of SNr to minimize any possible contribution of dendritic dopamine in recordings. Mean profiles of [5-HT]_o vs. time following 50-Hz train stimulation (I: 20 pulses or J: 40 pulses; n = 31–44) in the SNr in young adult (3 mo) and aged adult (18 mo) (K: 20 pulses or L: 40 pulses; n = 26–32) Snca^−/− vs. SNCA-OVX mice.

Fig. 6.

Spontaneous firing of identified dopaminergic neurons in the substantia nigra in vivo. Unit activity of individual dopamine neurons recorded in (A) 3- to 4-mo-old and (B) 18- to 22-mo-old Snca^−/− and SNCA-OVX mice during robust slow-wave activity in electrocorticograms (ECoG). Neurons were juxtacellularly labeled with Neurobiotin, verified as dopaminergic by TH expression, and tested for expression of α-syn. (C and D) Locations of identified dopamine neurons in (C) 3- to 4-mo-old and (D) 18- to 22-mo-old Snca^−/− (red) and SNCA-OVX (green) mice (dorsal top, lateral right) grouped at three rostro-caudal levels (distances from bregma shown left). PBP, parabrachial pigmented nucleus of the VTA; SNc, substantia nigra pars compacta; SNr, substantia nigra pars reticulata; VTA, ventral tegmental area. [Adapted from ref. .] (E and F) Mean firing rates and action potential durations of SNc dopamine neurons in (E) 3- to 4-mo-old (n = 17, Snca^−/− and 22 neurons, SNCA-OVX) and (F) 18- to 22-mo-old mice (n = 11 and 21; *P < 0.05). (Inset) Average wideband-filtered action potential from an SNCA-OVX mouse indicating duration measurement (scale 0.5 mV, 1 ms). (G and H) Mean interspike interval (ISI) coefficients of variation and the proportions of SNc neurons exhibiting each firing mode in (G) 3- to 4-mo-old mice (n = 15 and 20 neurons analyzed for mode in Snca^−/− and SNCA-OVX) and (H) 18- to 22-mo-old mice (n = 10 and 14 neurons). The firing pattern did not differ significantly with genotype. In E–H, group means ± SEM are shown in black.