α-Synuclein-induced dysregulation of neuronal activity contributes to murine dopamine neuron vulnerability

Pathophysiological damages and loss of function of dopamine neurons precede their demise and contribute to the early phases of Parkinson’s disease. The presence of aberrant intracellular pathological inclusions of the protein α-synuclein within ventral midbrain dopaminergic neurons is one of the cardinal features of Parkinson’s disease. We employed molecular biology, electrophysiology, and live-cell imaging to investigate how excessive α-synuclein expression alters multiple characteristics of dopaminergic neuronal dynamics and dopamine transmission in cultured dopamine neurons conditionally expressing GCaMP6f. We found that overexpression of α-synuclein in mouse (male and female) dopaminergic neurons altered neuronal firing properties, calcium dynamics, dopamine release, protein expression, and morphology. Moreover, prolonged exposure to the D2 receptor agonist, quinpirole, rescues many of the alterations induced by α-synuclein overexpression. These studies demonstrate that α-synuclein dysregulation of neuronal activity contributes to the vulnerability of dopaminergic neurons and that modulation of D2 receptor activity can ameliorate the pathophysiology. These findings provide mechanistic insights into the insidious changes in dopaminergic neuronal activity and neuronal loss that characterize Parkinson’s disease progression with significant therapeutic implications.


INTRODUCTION
Volitional movement is a fundamental behavior of everyday life that is often taken for granted until control deteriorates. Dopaminergic neurons within the ventral midbrain play a critical role in the initiation and control of volitional movement 1,2 and the progressive demise of these neurons is a defining hallmark of Parkinson's disease (PD) 3 . Pathophysiological damages and loss of function of these neurons precedes their demise and contribute to the early phases of the movement impairments 4 . No current therapies reverse or slow the progression of PD or the many related neurodegenerative diseases associated with the demise of dopaminergic neurons due to the incomplete understanding of the etiology of PD.
The presence of aberrant intracellular inclusions comprised of the protein α-synuclein (α-syn) in the form of Lewy bodies and Lewy neurites within ventral midbrain dopaminergic neurons is another cardinal feature of PD [5][6][7] . Several missense mutations in the α-syn gene (SNCA), as well as duplication 8 or triplication 9 of SNCA, are sufficient to cause familial PD and the related disease Lewy body dementia. Thus, only a 50% increase in the expression of wild-type α-syn as in the multiplication of the SNCA gene is sufficient for a detrimental outcome on dopaminergic neurons resulting in disease. Furthermore, some studies indicate that elevated α-syn level also occurs in idiopathic PD, but the pathophysiological mechanisms associated with increased levels of α-syn remain poorly understood.
The location of midbrain dopamine neurons in deep neural structures creates a significant barrier of not only investigation but also control over the experimental milieu. For these reasons, we incorporated a primary culture model system of dopaminergic neurons, which provides unparalleled access and control over the experimental procedure to investigate potential mechanisms of how excessive α-syn level alters dopaminergic neuronal dynamics and dopamine transmission prior to neuronal demise. Importantly, using whole-cell current-clamp recording, we and others have shown that postnatally derived dopaminergic cultures exhibit spontaneous firing properties 10-14 similar to those found in ex vivo [15][16][17] and in vivo [18][19][20] . Thereby, the experimental model system used in this study provides mechanistic insights not achievable of those found in ex vivo and in vivo models.
The present study utilized complementary approaches in molecular biology, electrophysiology, and live-cell imaging to investigate the hypothesis that elevated α-syn expression in dopaminergic neurons perturbs intracellular calcium signaling, protein homeostasis, and dopamine transmission prior to neuronal demise. We demonstrated that D2 receptor (D2R) autoinhibition contributes to alterations in neuronal homeostatic properties and that modulation thereof can ameliorate the pathophysiology resulting from excessive α-syn levels. These results provide mechanistic insights into the pathobiological impact of α-syn on dopaminergic neuron function and their demise characteristic of PD.

RESULTS AND DISCUSSION
Tyrosine hydroxylase (TH) promoter-driven adeno-associated virus (AAV) efficiently transduces cultures of midbrain dopamine neurons In order to investigate the pathophysiological changes associated with α-syn overexpression, we first developed a cell model with high-fidelity AAV-mediated α-syn expression in midbrain dopaminergic neuronal culture. The ventral midbrain neuronal culture contains the dopaminergic nuclei substantia nigra (SNc) and ventral tegmental area (VTA) that have been frequently used to study dopamine transmission [10][11][12][13][21][22][23][24][25] . Notably, SNc dopaminergic neurons are more sensitive than VTA dopaminergic neurons 22,[26][27][28][29][30][31] . Therefore, midbrain neuronal culture is likely to contain more VTA dopaminergic neurons than SNc dopaminergic neurons 22,[26][27][28][29][30][31] (see limitations of this model system in the "Methods" section). We utilized a TH promoter-driven AAV to specifically express wild-type human α-syn in cultured dopaminergic neurons. First, to demonstrate the specificity of the TH promoter vector, cultures were transduced with AAV-TH-GFP for visual confirmation of expression and quantification of dopaminergic specificity. As demonstrated in Fig. 1a, b, 91 ± 3% of neurons expressing green fluorescent protein (GFP) are TH positive, indicating high specificity. The same pAAV1-TH backbone, but with the human α-syn complementary DNA (cDNA) only, was utilized to overexpress human α-syn in dopamine neurons. The transduction of pAAV1-TH-human-αsyn in cultured midbrain dopamine neurons was confirmed via immunocytochemistry (ICC) and western blot analyses, demonstrating elevated expression of α-syn in these neurons (Fig. 1c, e and Supplementary Fig. 1, p = 0.005, two-tailed t test, n = 3 independent experiments). It should be noted that the midbrain neuronal culture contains many cell types, i.e., neuronal and non-neuronal cells. While our model provided 91% transduction specificity of αsyn overexpression in TH-positive neurons, it is likely that α-syn is also expressed in 9% of TH-negative cells-a mix of neuronal and glial cells-which is a limitation of AAV transduction in general. As shown in Fig. 1d, α-syn overexpression decreases neuronal survival as identified by TH-positive neurons expressing GFP (Fig. 1d, p < 0.05). It should be noted that functional analyses are conducted on surviving neurons. Since SNc neurons exhibit increased sensitivity to degeneration, it is possible that the relative proportion of VTA neurons to SNc neurons in cultures is altered by α-syn overexpression. No pre or post hoc techniques were carried out to investigate this ratio or selectively record from a subset of DA cells. Thus, these data may be over-representative of the response of surviving VTA cells; to investigate this question in realtime experiments, future studies with genetic targeting of SNc or VTA subtypes of dopamine neurons will be required (neurod6, pit3x, calbindin, aldh1, and GIRK3). Immunolabeling of TH confirmed 91 ± 3% of TH-positive neurons co-express GFP, suggesting a high fidelity for pAAV1-TH-GFP viral transduction in the TH-positive neurons (n = 3 independent experiments). c, e The transduction specificity was confirmed via immunocytochemistry analysis and western blot (n = 3 independent experiments). Scale bars: 50 μm. d Dopaminergic neuron counts revealed that α-syn overexpression decreases neuronal survival (naive = 223 ± 33.52, α-syn = 113.7 ± 33.52, two-tailed unpaired t test, naive vs. α-syn, p = 0.03), *p < 0.05.
Overexpression of α-synuclein disrupts calcium dynamics and firing activity of dopamine neurons Increased α-syn burden in dopamine neurons is correlated with neuronal loss in neurodegenerative diseases such as PD 32,33 .
Although extensively studied in cortical neurons, yeast, and heterologous expression systems [34][35][36][37][38][39] , α-syn regulation of intracellular calcium and firing activity in dopaminergic neurons prior to cell death remains less clear. The maintenance of calcium homeostasis is a vital process in neurons [40][41][42] . Calcium is a ubiquitous second messenger that helps to transmit depolarization status and synaptic activity to the biochemical machinery of a neuron 43 . Extensive calcium signaling requires high ATP consumption to restore basal (low) intracellular calcium levels.
Increased intracellular calcium may also lead to increased generation of mitochondrial reactive oxygen species 44,45 . Failure to maintain cellular energy levels and to suppress oxygen species may impact calcium signaling during aging and in neurodegeneration 5,6 . To investigate if α-syn overexpression regulates dopaminergic neuronal activity prior to neuronal demise, we employed live-cell calcium imaging in dopamine neurons conditionally expressing GCaMP6f under the control of the dopamine transporter (DAT) promoter (DAT-GCaMP6f) containing either endogenous levels of α-syn (naive) or overexpressing α-syn. Compared to calcium events in naive dopaminergic neurons, both width and amplitude of calcium peaks were increased in the presence of α-syn overexpression, creating repeated burdens on the neuron (Fig. 2a-c, width-p = 0.0152, unpaired two-tailed t test, n = 33 wild-type neurons, n = 40 α-syn-overexpressing neurons, amplitude-p = 0.0000198, unpaired two-tailed t test, n = 33 wild-type neurons, n = 40 α-syn-overexpressing neurons). These data suggest that increased levels of α-syn in dopamine neurons lead to disturbances in calcium homeostasis that can alter biophysical properties of neurons, neuronal activity, neurotransmission 42,43,[46][47][48][49][50] , and neuronal death, all of which are shared hallmarks in neurodegenerative diseases 22,46,51,52 . While measurement of calcium activity provides inferential information about the neuronal firing, we did not observe a change in calcium event rates (Fig. 2c, left). The firing rate of dopamine neurons ranges from 0.5 to 20 spike/s 10-21 . We and others have shown that electrophysiological properties of cultured dopamine neurons are consistent with those of dopamine neurons in vivo or in vitro 10,11,14,21 . Therefore, next, we investigated whether α-syn overexpression modulates the firing activity of dopaminergic neurons. We utilized whole-cell current-clamp recordings to measure the spontaneous firing activity of cultured DAT-GCaMP6f neurons with endogenous levels or overexpression of α-syn, which were identified by GCAMP6f fluorescence. While dopaminergic neurons containing endogenous levels of α-syn exhibited characteristic pace-making activity 53,54 , the spontaneous firing activity of α-syn-overexpressing dopamine neurons showed an irregular and clustered firing pattern with increased burst firing activity within the clusters (Fig. 2d, e, data are expressed as the percent of control from eight independent experiments, firing frequency-100 ± 21.21 for naive neurons vs. 281.7 ± 61.30 for α-syn-overexpressing neurons, two-tailed unpaired t test, naive vs. α-syn, p = 0.0142, raw ISI distribution, Kolmogorov-Smirnov test, D = 0.26529, p < 0.001). Thus far, our data suggest that increased α-syn levels in dopaminergic neurons lead to altered calcium dynamics and increased firing activity. Both firing activity and calcium dynamics in dopamine neurons are tightly regulated by the activity of the D2 autoinhibitory receptors [53][54][55][56][57] . Therefore, we asked whether α-syn-induced dysregulation of dopamine neuronal activity and calcium dynamics is due to reduced D2R autoinhibition.

α-Syn overexpression increases intracellular and extracellular dopamine levels and TH expression
Our findings so far suggest that α-syn may induce a feedforward adaptive mechanism that decreases the ability of inhibitory D2 autoreceptors to act as a brake on neuronal excitability and increase extracellular dopamine levels 53,56 .
To test this hypothesis, we used two complementary approaches of high-performance liquid chromatography (HPLC) analysis and an engineered dopamine sensor to measure intracellular and extracellular dopamine levels, at baseline. First, we used GRABDA 2M (G-protein-coupled receptor activationbased DA-expressing HEK293 cells) to measure extracellular dopamine levels. GRABDA 2M is a genetically encoded fluorescent dopamine sensor that is engineered by coupling a conformationally sensitive circular-permutated enhanced GFP (cpEGFP) to D2R. In GRABDA 2M -expressing HEK293 cells, dopamine binding to the sensor induces a conformational change that results in a robust increase in fluorescence signal in a concentration-dependent manner (Fig. 4a, b). Constitutive GRABDA 2M fluorescence signal in the absence of dopamine neurons was obtained at the beginning of each experiment, where GRABDA 2M -expressing HEK293 cells were plated in similar conditions, but sans neurons (F c , Fig. 4c). To compare baseline dopamine release amongst the experimental groups, the average ratio of the fluorescence signal of cells adjacent to the soma and neuronal processes to the average ratio of the fluorescence signal of GRABDA 2M cells (only) was calculated To confirm that the increase in GRABDA 2M fluorescence signal is due to dopamine release, we ran a positive control experiment ( Supplementary Fig. 2), where GRABDA 2M fluorescent signal around the soma and neuronal processes was measured following KCl (90 mM) stimulation of dopamine release 72 . The average fluorescence signal of cells adjacent to the soma and neuronal processes before and after KCl was calculated Fig. 2A, B). KCl-induced neuronal depolarization 73,74 produced a robust fluorescence increase in both experimental groups (Supplementary Fig. 2B, n = 10 from three independent replicates; the data are mean ± SEM, two-tailed t test, p = 0.8991). The KCl-evoked dopamine release was similar in both experimental groups.
After confirming the ability of GRABDA 2M cells to detect evoked dopamine release, we measured spontaneous (i.e., baseline, unstimulated) dopamine release in naive and α-syn-overexpressing neurons. The neurons were cocultured with GRABDA 2M cells 20-24 h prior to live-cell confocal imaging. This experimental design enables real-time detection of endogenous dopamine released at baseline, i.e., spontaneous dopamine release (Fig. 4d, e). Using a blinded experimental design, we found a significantly higher spontaneous dopamine level, as measured by a higher GRABDA 2M fluorescence signal around the soma and neuronal processes of α-syn-overexpressing neurons (Fig. 4f, n = 33 naive, 89 α-syn-overexpressing from three independent replicates; the data are mean ± SEM, two-tailed t test, p = 0.0013). These data support the interpretation that α-syn overexpression increases spontaneous neuronal activity, leading to increased extracellular dopamine levels. We previously reported that α-syn overexpression decreases dopamine uptake via the DAT 75 and we have also reported that α-syn overexpression increases the DAT-mediated dopamine efflux 24 . These data are consistent with the hypothesis that α-syn overexpression can increase extracellular dopamine levels. Collectively, our previous reports 72, 73 , combined with the data shown in Figs. 2-4, provide a reasonable cellular mechanism for the puzzling observation by Lam et al. 76 that in mice overexpressing α-syn there is an initial increase in extracellular dopamine levels in the striatum prior to neuronal death.
Because GRAB DA readouts report only the relative difference in dopamine release between α-syn and control neurons, we used HPLC to measure absolute dopamine levels in the external milieu of α-syn-transduced and naive DAT-GCaMP6f cultures (i.e., spontaneous dopamine release) via a blinded experimental design. HPLC analysis showed significantly higher extracellular dopamine levels in α-syn-overexpressing neurons compared to naive neurons (Fig. 4g, h; n = 8 each, from eight independent replicates; two-tailed t test, p = 0.0139). Collectively, these data, combined with live-cell detection of extracellular dopamine levels (at baseline), support the notion that α-syn modulation of dopaminergic neuronal activity leads to increased extracellular dopamine levels. An increase in extracellular dopamine could be due to increased neuronal activity, increased dopamine synthesis, or both possibilities. Since we have already examined the former (Figs. 2 and 3) to test the latter possibility, we used HPLC to measure intracellular dopamine levels. The measurement of dopamine in the cell lysate of naive and α-syn-overexpressing neurons revealed significantly higher intracellular dopamine levels ( Fig. 4h, n = 8 each, from eight independent replicates, two-tailed t test, p = 0.0071). These data suggest that the decreased autoinhibition of dopamine neurons following α-syn overexpression not only increases neuronal excitability but also dysregulates dopamine synthesis and secretion. Furthermore, in Fig. 2, we showed that increased neuronal α-syn increases the magnitude and duration of intracellular calcium burden, which would promote increased basal dopamine release.
Multiple mechanisms likely contribute to the increased intracellular dopamine following α-syn overexpression. For example, increased dopamine uptake via the DAT, decreased DAT-mediated dopamine efflux, increased expression of TH (a key enzyme involved in dopamine synthesis), or a combination of these mechanisms would possibly contribute to a higher intracellular dopamine level. Previously, we and others have shown that α-syn overexpression reduces dopamine recycling by reducing dopamine uptake 75,77,78 . In addition, we have shown that α-syn overexpression increases reverse transport of dopamine, i.e., dopamine efflux 24 , without changing surface DAT levels. Therefore, α-syn regulation of dopamine uptake or dopamine efflux would decrease intracellular dopamine and not increase it.
increased TH levels that can contribute to increased intracellular dopamine 80,[82][83][84][85][86][87] . Since the frequently used approaches of western blotting or ICC do not provide purely quantitative data of protein expression to test this hypothesis, we developed an enzyme-linked immunosorbent assay (ELISA) 88 to quantify TH levels in α-syn-overexpressing neurons (Fig. 4i). For these experiments, we used HEK293 cells as a negative control group and PC12 cells as a positive control group, and a purified full-length recombinant TH protein was used to generate a standard curve (Fig. 4j, k). α-Syn-overexpressing neurons show significantly higher TH levels compared to naive (Fig. 4l, n = 8-10, two-tailed t test, p = 0.0289). While ELISA provides quantitative data for total TH level across these experimental groups, a limitation of this assay is that it cannot discriminate TH phosphorylation that is associated with TH activity and thus dopamine synthesis 80,89,90 . Nevertheless, these data support the interpretation that increased intracellular dopamine in α-synoverexpressing neurons, at least in part, is due to increased TH protein levels.

Increasing [DA] increases GRAB
Altered neural dynamics mediated by α-syn may emerge from altered D2 activity and expression patterns Our data, thus far, support the interpretation that the canonical D2R-mediated autoinhibition, such as inhibitory modulation of spontaneous firing activity, is reduced in α-syn-overexpressing neurons. While D2R agonist quinpirole silenced naive dopamine neurons, the response to quinpirole in α-syn-overexpressing neurons is significantly reduced, possibly due to desensitization or reduced activity of the D2R (Fig. 3). Therefore, next, we tested the hypothesis that blockade of D2Rs in naive dopaminergic neurons simulates the firing activity observed in α-syn-overexpressing neurons. We performed whole-cell current-clamp recordings to measure spontaneous firing activity of dopaminergic neurons before and during bath application of sulpiride (D2 antagonist, 5 µM). In naive dopaminergic neurons, bath application of sulpiride produced burst firing patterns with intermediated periods of quiescence and firing frequencies similar to α-synoverexpressing dopaminergic neurons in the presence of sulpiride ( Fig. 5a-e, n = 8 from three independent biological replicates, distribution of raw ISIs in naive and α-syn-overexpressing dopaminergic neurons, Kolmogorov-Smirnov test, D = 0.13114, p < 0.001, two-tailed unpaired t test, firing frequency: 100 ± 22.94 naive vs. 158.8 ± 30.37 α-syn-overexpressing neurons, p = 0.148; ISI: 100 ± 14.51 naive vs. 68.15 ± 11.84 α-syn-overexpressing neurons, p = 0.1147; CV of ISI: 100 ± 17.02 naive vs. 85.94 ± 6.599 α-syn-overexpressing neurons, p = 0.456). These data support the hypothesis that α-syn-overexpressing dopamine neurons exhibit reduced functional availability of D2-mediated response, which could be due to receptor desensitization 89,90 , decreased membrane expression of D2Rs, or a combination of these possibilities.
To investigate if α-syn overexpression in dopaminergic neurons alters D2R expression, we performed cell surface biotinylation of D2R via a blinded experimental design with striatal lysate as a positive control group and CHO cells as the negative control group. When total D2R was normalized to HSP60 loading control, there were no significant differences between naive and α-synoverexpressing neurons ( Supplementary Fig. 3, from three independent biological replicates, two-tailed unpaired t test, p = 0.3417). We also found that the ratio of cytoplasmic D2R to total D2R in α-syn-overexpressing neurons was not significantly different from naive neurons ( Supplementary Fig. 3, from three independent biological replicates, two-tailed unpaired t test, p = 0.9426). However, when comparing the ratio of membrane-tocytoplasmic D2R, we found that α-syn-overexpressing neurons have a significantly lower ratio of membrane-to-cytoplasmic D2R than naive neurons ( Supplementary Fig. 3B, from three independent biological replicates, two-tailed unpaired t test, p = 0.0039). It should be noted that biotinylation assay detects total (both functional and desensitized receptors). The double ICC of fixed, but not permeabilized, dopamine neurons stained for both D2R and an integral membrane protein such as Na + /K + -ATPase, or GM1-CTxB would have been a suitable complementary approach to examine membrane-localized D2R across the experimental groups in this study. However, the frequently used D2R antibodies in the field 91-94 are raised against the intracellular N-terminal domain of the receptor. This limitation decreases the confidence in the identification of membrane vs. intracellular protein levels. A similar technical limitation applies to the single-cell qPCR assay, where total transcript levels do not necessarily reflect functional D2Rs at the membrane. The latter limitation somewhat applies to the biotinylation assay used in this study. Unless an antibody is raised against the active or inactive form of the receptor, a biotinylation assay detects both functional and desensitized receptors. Therefore, although our data suggest that membrane D2Rs are decreased in α-syn-overexpressing neurons, it is possible that the detected membrane D2Rs are desensitized, i.e., a lesser receptor-effector coupling [95][96][97][98] . Therefore, live-cell functional assays, such as electrophysiology and calcium imaging, combined with pharmacological manipulations are more reliable strategies to assess the mechanism of α-syn regulation of neuronal activity.
α-Syn overexpression reduces arborization of dopamine neurons and pretreatment with a D2R agonist partially rescues the detrimental impact of α-syn Dopaminergic neurons have extensive axonal arborizations and large terminal fields 99-101 , where one dopamine neuron is estimated to have~245,000 release sites 102,103 . Studies in animal models of PD and postmortem data in human PD 104 show that decreased axonal complexity and dendritic arborization, reduction of the number of axon terminals, and global neuronal size precede neuronal death 77,102,105 . Our data suggest that prior to cell death, via a D2R mechanism, α-syn overexpression can induce neuronal disinhibition, leading to increased intracellular and extracellular dopamine levels that are implicated in increased neuronal vulnerability 35,106,107 . Therefore, we investigated the potential link between α-syn-mediated dopamine neuronal dysfunction and neuronal complexity. Fig. 4 Overexpression of α-synuclein increases intracellular and extracellular dopamine levels with concurrent increased tyrosine hydroxylase expression. a Schematic and representative baseline-subtracted images of GRABDA 2M -HEK cells exposed to increasing concentration of dopamine. Scale bar = 20 μm. b A standard curve of GRABDA 2M -HEK cells against known extracellular dopamine concentrations (R 2 = 0.98). c Constitutive GRABDA 2M -HEK cell fluorescence signal in the absence of dopamine neurons (in culture). Scale bar = 10 μm. d Schematic of GRABDA 2M -HEK cells seeded into dopaminergic cultures. In the presence of dopamine, GRABDA 2M -HEK cells rapidly increase in fluorescence intensity. e Baseline fluorescence levels denote unstimulated and spontaneous dopamine release from the neurons. The average ratio of the fluorescence signal of the cells adjacent to neuron soma and neuronal processes to the average ratio of GRABDA 2M -HEK cells (only) were calculated (relative fluorescence = (FGRABDA2M-HEK cells grown with neurons − F c )/F c ). GRABDA 2M -HEK cells cocultured with naive and α-syn-overexpressing neurons. Scale bar = 50 μm. f GRABDA 2M -HEK cells cocultured with α-syn-overexpressing neurons show higher basal fluorescence, indicating higher baseline dopamine release (relative fluorescence) compared to naive neurons (n = 10 from three independent replicates; the data are means ± SEM, two-tailed t test, p = 0.0013). g, h HPLC analysis complements the GRABDA 2M -HEK results. Extracellular milieu (g) and cell lysate intracellular milieu (h) revealed increased intracellular and extracellular dopamine levels in α-synoverexpressing neurons compared to naive neurons (n = 8 each, from eight independent replicates, two-tailed t test; intracellular: p = 0.0071; extracellular: p = 0.0139). i Schematic diagram of quantitative ELISA experimental design for TH in dopaminergic neurons. j Standard curve for TH sandwich ELISA shows average absorbance values for each purified TH protein concentration from multiple consecutive experiments (R 2 = 0.99). k TH protein levels were detected and quantified in positive control groups, PC12 cells, whereas no protein was detected in the negative control group, HEK293 cells. l α-Syn-overexpressing neurons exhibited increased levels of TH compared to naive (n = 8-10, two-tailed t test, p = 0.0289). These experiments were performed through a double-blinded experimental design. *p < 0.05, **p < 0.01.

D2R activation partially restores neuronal activity in α-synoverexpressing dopamine neurons
The energy homeostasis principle suggests that the balance between energy income, expenditure, and availability is the key parameter in determining neuronal endurance 125 . APs impose the highest energy demands on neurons 102,125,126 . In addition, dopamine metabolism is strongly linked to oxidative stress, as its degradation generates reactive oxygen species 35,127,128 that have shown to increase the vulnerability of dopamine neurons to oxidative stress 101,[128][129][130][131][132][133][134] . So far, we have identified multiple interrelated mechanisms that can potentially lead to the vulnerability of α-syn-overexpressing dopamine neurons. We identified an increase in AP frequency, increased intracellular and extracellular dopamine levels, and elevated intraneuronal calcium dynamics in α-syn-overexpressing neurons that are directly or indirectly related to decreased D2R activity. The unexpected observation that protracted (48 h) application of D2R agonist increased neuronal survival and nearly restored neuronal complexity of α-syn-overexpressing neurons to the levels measured in naive dopaminergic neurons at baseline suggests that the pharmacological activation of D2Rs might be a possible target to alleviate the untoward consequences of α-syn overexpression on neuronal activity prior to neuronal death. To test this hypothesis, we treated α-syn-overexpressing neurons with 0.5 µM quinpirole for 48 h before assessing calcium dynamics, spontaneous firing activity, and dopamine release and synthesis in these neurons (Fig. 7). We compared the results of these experiments to our previous data obtained in α-syn-overexpressing neurons. To reduce the impact of type 1 statistical errors, naive primary midbrain cultures were produced alongside each experimental group. While it may be intriguing to study the effects of pretreatment of quinpirole on naive neurons, we found that this was not feasible for spontaneous calcium activity and firing as these neurons were silent in the recording chamber (zero values for firing activity that cannot be included in statistical analyses of the data in Fig. 7). Furthermore, D2 autoreceptor activity has been shown to be a potent regulator of neuronal activity 13 100,141 , and protein expression 55,90,106,138,140,[142][143][144][145] . Specifically, the purpose of quinpirole pretreatment was to examine whether modulation of D2R Event rate, width, and amplitude after quinpirole pretreatment, respectively (n = 28 quinpirole-treated α-syn-overexpressing neurons, two-tailed unpaired t test, α-syn vs. α-syn + quinpirole p = 0.2024 event rate, p = 0.0277 event widths, p = 0.6204 event height, untreated α-syn data presented in Fig. 2). Box plot whiskers represent the 95% confidence interval, the upper and lower bounds of the box represent the 75th and 25th percentiles, respectively; the middle line indicates the median value of the sample. Representative firing activity of an untreated naive (g) and quinpirolepretreated α-syn-overexpressing neuron (h). i-k Firing frequency (i), interspike interval (j), and firing regularity (k) in quinpirole-pretreated αsyn-overexpressing neuron (n = 7, 1.325 ± 0.2735 Hz for quinpirole-treated α-syn-overexpressing neurons, two-tailed unpaired t test, α-syn vs. α-syn + quinpirole p = 0.0342 for firing frequency, p = 0.1053 for ISI, p = 0.4778 for CV of ISI, untreated α-syn data are presented in Fig. 2). l GRABDA 2M -HEKs seeded with untreated naive (left), untreated α-syn-overexpressing (middle), and quinpirole-pretreated α-synoverexpressing neurons (right). m Quinpirole rescued extracellular dopamine level in α-syn-overexpressing neurons (n = 6, one-way ANOVA, naive vs. α-syn + quinpirole p = 0.9948, α-syn vs. α-syn + quinpirole p = 0.0003, untreated α-syn and naive data presented in Fig. 4). n, o HPLC quantification of dopamine confirm that quinpirole pretreatment of α-syn-overexpressing neurons reduces extracellular (n) and intracellular (o) dopamine levels vs. untreated α-syn-overexpressing neurons (n = 3 each, one-way ANOVA, intracellular: α-syn vs. α-syn + quinpirole p = 0.0325 and naive vs. α-syn + quinpirole p = 0.9959; extracellular: α-syn vs. α-syn + quinpirole p = 0.0449 and naive vs. αsyn + quinpirole p = 0.6197, untreated α-syn and naive data presented in Fig. 4). p Quantitative TH ELISA (n = 3, one-way ANOVA, naive vs. αsyn + quinpirole p = 0.4288 and α-syn vs. α-syn + quinpirole p = 0.6809, untreated α-syn and naive data are presented in Fig. 4). Data are presented as mean ± SEM, from at least three independent biological replicates. n.s. not significant. *p < 0.05, ****p < 0.0001.
activity would restore or augment the properties of neurons with increased α-syn burdens to those of naive neurons, which has previously been shown to be true for MPP + exposure 117 , and not whether quinpirole pretreatment alters naive neurons. The black dotted line represents the average values measured for untreated naive neurons. The pink dotted line represents the average values for untreated α-syn-overexpressing neurons. The shaded region indicates the respective SEM for each measurement. We found that a prolonged D2R activation partially restores calcium dynamics in these neurons, approximating calcium dynamics measured in untreated naive neurons (Fig. 7a-f, n = 28 quinpirole-treated αsyn-overexpressing neurons, two-tailed unpaired t test, α-syn vs. α-syn pretreated with quinpirole p = 0.2024 event rate, p = 0.0277 event widths, p = 0.6204 event height, untreated α-syn, and naive data are presented in Fig. 2), suggesting a restoration of calcium homeostasis in these neurons that might be causal or a consequence of a shift in neuronal activity. To test this hypothesis, we employed whole-cell current-clamp recordings to measure the spontaneous firing activity of α-syn-overexpressing neurons after treatment with quinpirole (0.5 µM for 48 h). Quinpirole pretreatment on α-syn-overexpressing neurons decreased the burst firing frequency, shortened the intermediate periods of quiescence, and restored firing regularity near to the values measured in untreated naive neurons (Fig. 7g-k, n = 7 from three independent biological replicates, 117.1 ± 21.17 for quinpirole-treated α-syn-overexpressing neurons, two-tailed unpaired t test, α-syn vs. α-syn pretreated with quinpirole p = 0.0342 for firing frequency, p = 0.1053 for ISI, p = 0.4778 for CV of ISI, untreated α-syn, and naive data are presented in Fig. 2). These results suggest that dysregulation of D2R in α-syn-overexpressing dopamine neurons can be partially rescued with prolonged activation of the remaining functional D2Rs on the cell surface.
The observed changes in neuronal responses and calcium activity following extended D2R activation could be predictive of downstream changes in dopamine synthesis in α-syn-overexpressing neurons. To test the hypothesis that D2R activation decreases α-syn modulation of dopamine release, we measured extracellular dopamine levels via two complementary approaches: live-cell imaging utilizing an engineered dopamine sensor and HPLC. To measure D2R-mediated modulation of baseline extracellular dopamine levels (0.5 µM quinpirole 48 h) in α-syn-overexpressing neurons, we cocultured GRABDA 2M -expressing cells with the quinpirole-treated, α-syn-overexpressing dopamine neurons for 20-24 h prior to imaging. Quinpirole pretreatment of α-synoverexpressing neurons decreased basal GRABDA 2M fluorescence (used as a proxy to measure basal dopamine release) around the soma and dendritic fields (Fig. 7l, m), comparable to values measured in naive untreated neurons shown in Fig. 4 (represented as a black dotted line in Fig. 7).
As a complementary approach, we used HPLC, as described in the "Methods" section and in Fig. 4, to measure extracellular dopamine level in the external milieu of neurons after quinpirole pretreatment (0.5 µM for 48 h) via a blinded experimental design. HPLC analysis revealed a reduction in basal dopamine release in all quinpirole-treated experimental groups, with the largest fold decrease in α-syn-overexpressing neurons (Fig. 7n, n = 3 from independent biological replicates, one-way ANOVA followed by Tukey's HSD, α-syn vs. α-syn treated with quinpirole p = 0.0325 and naive vs. α-syn treated with quinpirole p = 0.9959). Therefore, through pharmacological manipulation of D2Rs, the α-syn dysregulation of dopamine transmission is potentially reversible (n = 3 each, from three independent biological replicates, one-way ANOVA followed by Tukey's HSD, α-syn vs. α-syn treated with quinpirole p = 0.0325 and naive vs. α-syn treated with quinpirole p = 0.9959). The restoration of extracellular dopamine could be due to decreased neuronal activity, decreased dopamine synthesis, or both. Since we have already examined the former (Fig. 7a-k), to test the possibility of decreased dopamine synthesis, we used HPLC to measure intracellular dopamine levels via a blinded experimental design (described in the "Methods" section). Intracellular dopamine levels in quinpirole-treated α-syn-overexpressing neurons were significantly reduced compared to untreated α-syn-overexpressing neurons (Fig. 7o, n = 3 independent biological replicates, one-way ANOVA followed by Tukey's HSD, α-syn vs. α-syn treated with quinpirole p = 0.0449 and naive vs. α-syn treated with quinpirole p = 0.6197) shown in Fig. 4 (n = 3 each, from 3 independent replicates, one-way ANOVA followed by Tukey's HSD, α-syn vs. αsyn treated with quinpirole p = 0.0325 and naive vs. α-syn treated with quinpirole p = 0.9959). Since activation of D2R negatively regulates TH 55,56,80,89,90,[146][147][148] and neuronal activity 16,[53][54][55][56]58 , we then tested the hypothesis that reduced intracellular and extracellular dopamine are, in part, due to decreased TH protein levels. Via a blinded experimental design, we utilized quantitative ELISA, as described in Fig. 4, to measure TH levels. As shown in Fig. 7p, TH protein level is similar in quinpirole-treated, α-synoverexpressing neurons compared to untreated (0.5 µM for 48 h; n = 3, one-way ANOVA followed by Tukey's HSD, naive vs. α-syn treated with quinpirole p = 0.4288 and α-syn vs. α-syn treated with quinpirole p = 0.6809). The partial rescue of α-syn-induced neuronal dysregulation after D2R activation is consistent with neuroprotective properties of D2Rs described previously 117,[149][150][151][152] . It has been shown that D2 autoreceptors suppress dopamine synthesis through a negative feedback mechanism, and thus reduce oxidative stress caused by a high level of cytoplasmic dopamine [149][150][151] . In addition, consistent with our data, activation of D2 autoreceptors mediates neuroprotection by reducing neuronal excitability, cytoplasmic dopamine, and calcium levels 117,152 that can restore the balance between energy income, expenditure, and its availability 125 . The data presented in this study provide a potential druggable target that may revert or prevent the untoward consequences of α-syn burden on dopamine neuronal activity and viability.
To summarize (Fig. 8), we found that α-syn overexpression dysregulates the structural and functional properties of dopaminergic neurons. The untoward consequences of increased α-syn likely cascade across the neuron, protracting the neuronal processes, increasing calcium burdens, and biophysical properties of dopamine neurons as measured by increased burst firing activity. We found that the endogenous self-regulation of dopaminergic neurons fails to restrain the exacerbation of these phenotypes. Thereby, the signaling of these neurons in their networks becomes erratic, potentially creating avalanching neuronal dysfunction. The dysregulation of dopamine signaling within the brain therefore precedes neuronal demise. However, we show that these progressive dysregulations can be reversed through pharmacological manipulation.
The D2 autoreceptor-mediated feedback mechanism is one of the main autoinhibitory mechanisms regulating dopamine neuronal activity 16,56,153 . We found that D2 autoreceptor activity is diminished in α-syn-overexpressing dopamine neurons, and prolonged incubation with a D2R agonist, quinpirole (48 h, 0.5 μM), nearly restored the firing activity to its canonical levels, reinstated intracellular and extracellular dopamine levels, and prevented neuronal loss and structural neural complexity. Notably, D2R agonists (full and partial) have attained Food and Drug Administration approval and have made their way into the clinic; however, these are often tested in late-stage PD. Our results suggest that the current treatment timeline may occur too late and that the efficacy of this strategy requires early intervention to reduce the rate of neuronal demise. Most crucially, our results suggest that neuronal loss might be preventable, and future exploration across other mechanistic pathways will reveal intersectional treatments that may have the capacity to ameliorate PD.

Reagents and chemicals
The source, catalog number, and concentration of reagents, antibodies, and chemicals used in this study are outlined in Table 1. All viral vectors utilized in this study are listed in Table 2.

Animals
All experiments were approved by the Institutional Animal Care and Use Committee at the University of Florida. Mice were housed in the animal care facility at the University of Florida, 2-4 per cage with food and water available ad libitum in the home cage. The room was maintained under 12 h light/dark cycle. Wild-type C57BL/6J mice, or DAT IREScre and Ai95(RCL-GCaMP6f)-D (Ai95D) knock-in mice were obtained from The Jackson Laboratory (stock number: 006660 (DAT IREScre ), 024105 (Ai95D), Bar Harbor, ME, USA). C57BL/6J pups or pups expressing GCaMP6f in dopamine neurons were used for this study. Mice of both sexes were used.

Primary neuronal culture
Primary culture was prepared as previously described, with small distinctions 23 . Briefly, acutely dissociated mouse midbrains from 0-to 2day-old male and female pups were isolated and incubated in a dissociation medium at 35-37°C under continuous oxygenation for 60-90 min. Dissociated cells were triturated with pipettes of decreasing bore size (including a punctured fire-polished pipette), then pelleted by centrifugation at 1500 r.p.m. for 3-5 min, and finally resuspended and plated in glial medium (Table 1). Cells were plated at a density of 100,000 cells/coverslip on a 12 mm coverslip coated with 0.1 mg/ml poly-D-lysine and 5 μg/ml laminin and maintained in neuronal media. After 2 h, cells were supplemented with neuronal media (days in vitro 0 (DIV0) composition). Every 4 days, 1/3 of the media was replaced with fresh media. On DIV5, cultures were transduced with the desired AAV1 (see Table 2). The experiments described in this study were performed on DIV9-11. Reagents and chemicals utilized for midbrain neuronal culture are listed in Table 1.

Electrophysiology
Spontaneous firing activity of midbrain dopamine neurons was examined via whole-cell current-clamp recordings as previously described 10,11,21 . The neurons were continuously perfused with artificial cerebral spinal fluid (ACSF) (composition is described in Table 1) equilibrated with 95% O 2 /5% CO 2 ; pH was adjusted to 7.4 at 37°C. Patch electrodes were fabricated from borosilicate glass (Cat. No. 1B150F-4, 1.5 mm outer diameter; World Precision Instruments, Sarasota, FL) with the P-2000 puller (Sutter Instruments, Novato, CA). The tip resistance was in the range 3-5 MΩ. The electrodes were filled with a pipette solution containing (in mM): 120 potassium gluconate, 20 KCl, 2 MgCl 2 , 10 HEPES, 0.1 EGTA, 2 ATP, and 0.25 GTP, with pH adjusted to 7.25 with KOH. All experiments were performed at 37°C. To standardize AP recordings, neurons were held at their resting membrane potential (see below) by DC application through the recording electrode. AP was recorded if the following criteria were met: a resting membrane potential more polarized than −35 mV and an AP peak amplitude >60 mV. AP half-width was measured as the spike width at the half-maximal voltage using Clampfit 10 software (Molecular Devices LLC, San Jose, CA). The steady-state basal activity was recorded for 2-3 min before bath application of the drug. Each coverslip was used for only one recording; this is specifically important for experiments involving drug application. The spontaneous spike activity of midbrain dopamine neurons was obtained by averaging 1 min interval activities at baseline and after 3-5 min of drug exposure.

Live-cell calcium imaging
Live-cell calcium imaging and analysis are described previously 23 . Briefly, naive (non-transduced) and α-syn-overexpressing (transduced with AAV1-TH-α-syn) midbrain neuronal cultures were imaged with a Nikon Eclipse FN1 upright microscope (Nikon Instruments, Melville, NY). A Spectra X (Lumencor, Inc., Beaverton, OR) was used to stimulate   GCaMP6f (λ ex = 470 nm) fluorescence through a custom quad-pass filter (Chroma Technologies, Battleboro, VT), and emission was filtered through visible spectra bandpass filter. Experiments were performed under gravity perfusion of ACSF (Table 1). The average fluorescence of the first 60 s recording is defined as the baseline. After baseline imaging, vehicle (ACSF), 1 μM DA, or 10 μM quinpirole was administered via a perfusion system (flow rate of 2 ml/min) and recorded for an additional 2 min. Background fluorescence was subtracted from each frame. Fold fluorescence change from baseline was calculated and plotted against time. Each coverslip was used for only one recording; this is specifically important for experiments involving drug application. All resources and reagents used for live-cell calcium imaging experiments are listed in Table 1.
Live-cell confocal imaging using GRABDA 2M -expressing HEK293 cells to measure extracellular dopamine These experiments were performed via a blinded experimental design. GRABDA 2M is a genetically encoded fluorescent dopamine sensor that is engineered by coupling a conformationally sensitive cpEGFP to D2R. In GRABDA 2M -expressing HEK293 cells, dopamine binding to the sensor induces a conformational change that results in a robust increase in fluorescence signal in a concentration-dependent manner 156

Generation of a standard curve
To generate a standard curve, the baseline fluorescence signal (F c ), which is the constitutive fluorescent signal in the absence of extracellular dopamine, was recorded. Changes in fluorescence signal after adding various dopamine concentrations (1-20 nM) were plotted against dopamine concentration.

Measurement of basal dopamine release
GRABDA 2M cells are cocultured with tdTomato-expressing dopamine neurons (DAT IREScre -LoxP-tdTomato) containing endogenous α-syn or its overexpression 20-24 h prior to live-cell confocal imaging. At the beginning of each experiment, the constitutive GRABDA 2M fluorescence signal (F c ) of the cells that are plated in similar conditions sans neurons was obtained. To compare baseline dopamine release amongst the experimental groups, the average fluorescence signal of cells adjacent to the soma and neuronal processes to the average fluorescence signal of GRABDA 2M cells (only) were calculated in Eq. (1): Visualization and quantification of real-time dopamine release following KCl stimulation Twenty to twenty-four hours prior to live-cell confocal imaging, GRABDA 2M cells are cocultured with tdTomato-expressing dopamine neurons containing endogenous α-syn or its overexpression. GRABDA 2M fluorescent signal around the soma and neuronal processes were measured before (F baseline ) and following KCl stimulation (90 mM) of dopamine release 69 . The average fluorescence signal of cells adjacent to the soma and neuronal processes before and after KCl were calculated in Eq. (2):

Immunocytochemistry
These experiments were performed via a blinded experimental design. On DIV9-11, naive (non-transduced) and α-syn-overexpressing neuronal cultures were fixed with 4% paraformaldehyde (PFA) in PBS for 30 min at room temperature (RT), followed by blocking, permeabilizations, and  A. Dagra et al.
overnight incubation (at 4°C) with primary antibodies diluted in blocking buffer, followed by three 20 min phosphate-buffered solution (PBS) washes. Then, a 1 h incubation in blocking buffer with Alexa Fluorconjugated secondary antibodies at RT, followed by three 20 min washes and an overnight PBS wash at RT. Coverslips were mounted on slides using Fluoromount-G. Images were captured on a Nikon A1 laser-scanning confocal microscope (×20 or ×40 oil-immersion objective). Reagents and chemicals utilized for ICC are listed in Table 1.

Western blot analysis
These experiments were performed via a blinded experimental design. For detection of endogenous and human α-syn, total cell lysates of neurons transduced with AAV1-TH-α-syn (n = 4) or naive (non-transduced) neurons (n = 4) were used, as described previously 157 . Briefly, the cells were harvested in 200 µl of 2% sodium dodecyl sulfate (SDS) buffer, protein concentrations were determined using the bicinchoninic acid assay (Pierce), and further diluted in sample buffer (10 mM Tris, pH 6.8, 1 mM EDTA, 40 mM dithiothreitol, 0.005% bromophenol blue, 0.0025% pyronin yellow, 1% SDS, 10% sucrose). Following harvest of total cell lysate, samples were heated to 100°C for 10 min prior to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (13% polyacrylamide gels, 10 µg lysate per well) followed by electrophoretic transfer onto 0.2 µm nitrocellulose membranes as previously described 145 Table 1). Fluorescent images were analyzed using ImageJ (NIH) to measure band optical density. Values were normalized to total protein per lane. Beta-tubulin (Aves, TUJ) was probed to demonstrate membrane fraction isolation during biotinylation.

ELISA quantification of TH
These experiments were performed via a blinded experimental design.

Cell lysis
For total protein quantification via ELISA, neuronal cultures were washed three times with cold PBS, then lysed in BufferD lysis buffer (10% glycerol, 125 mM NaCl, 1 mM EDTA, 1 mM EGTA, pH 7.6) containing 1% Triton X-100 and protease inhibitor cocktail (Millipore, 539131) for 1 h at 4°C with rocking, followed by centrifugation for 15 min at 12,000 × g. Samples were denatured in Laemmli Sample Buffer 4× (containing 10% beta-mercaptoethanol) at 37°C for 30 min and separated by 10% SDS-PAGE, transferred to 0.45 μm nitrocellulose, and probed with antibodies against proteins of interest. Values were normalized to total protein per lane.

TH ELISA
Antibodies and concentrations used are given in Table 1. In brief, Immulon 4 HBX High-Binding 96-well plates were coated with 100 μl per well of 1:1000 dilution of mouse anti-TH (EnCor, MCA-4H2) in coating buffer (28.3 mM Na 2 CO 3 , 71.42 mM NaHCO 3 , pH 9.6) for 20 h at 4°C. Edge lanes 1 and 12 were left empty. Wells were blocked with 5% fat-free milk in 1× TBS (pH 7.4) for 1 h at RT on an orbital shaker set to 90 r.p.m.

Generation of a standard curve
To produce a standard curve, two standard curve lanes were generated, with six serial dilutions, beginning at 10 ng/ml and 1 ng/ml in TBS-T containing 1% fat-free milk (with the last well in each standard curve lane left with incubation buffer only as a blank). The remaining wells were incubated in duplicate with lysates from cells of interest. Incubation was completed for 20 h at 4°C on an ELISA shaker set to 475 r.p.m. After each well was washed and aspirated six times with TBS-T, anti-TH rabbit (EnCor, RPCA-TH) conjugated to biotin was diluted 1:6000 from a stock concentration of 1.65 mg/ml in TBS-T with 1% fat-free milk and incubated for 1 h at RT by centrifugation at 425 r.p.m. One hundred microliters of Avidin-HRP (Vector labs, A-2004), diluted 1:2500 in TBS-T with 1% fat-free milk, was added to each well following washing, and then incubated for 1 h at RT by centrifugation at 425 r.p.m. Following final washes, 150 μl TMB-ELISA reagent (Thermo Fisher, 34028) was added to each well at RT. The reaction was allowed to continue for 20 min, protected from light, and stopped by the addition of 50 μl 2 N H 2 SO 4 . The plate was immediately read at 450 nm. Duplicate standard and sample wells were averaged and background subtracted based on blank wells. TH concentration was calculated using a quadratic curve equation calculated in GraphPad Prism 8, and then normalized to total protein concentration per sample as calculated using Lowry assay. Final TH values shown are presented as pg TH/mg total protein after the multiplication of the nanogram TH value by 1000.

HPLC
These experiments were performed via a blinded experimental design. Midbrain primary cultures were incubated in KH buffer (Table 1), at 37°C, for 1 h before collecting intracellular and extracellular milieu for HPLC analysis 72,160 . For extracellular milieu, KH buffer incubated with neurons was collected, treated with 1 M perchloric acid, and snap frozen for analysis. For intracellular milieu, coverslips were washed with KH buffer, scraped, and treated with 1 M perchloric acid, before sonicating. Then, the sample was centrifuged at 12,000 r.p.m. at 4°C for 10 min, and the supernatant was snap frozen in liquid nitrogen for analysis. The pellet was resuspended using 0.2 NaOH and RIPA buffer 161 for protein quantification via Lowry assay. Samples were centrifuged at 16,000 × g for 15 min (4°C) and the supernatant was filtered through a 0.2 mm pore membrane (Nanosep with 0.2 mm bioinsert, Pall Life Sciences) and 15 μl of the supernatant was injected directly into an HPLC-ECD (HTEC-510; Eicom). Dopamine was separated on a CAX column (EICOMPAK 2.0 i.d. × 200 mm) maintained at 35°C. The mobile phase consisted of 70% 0.1 M ammonium acetate buffer (pH 6.0) containing sodium sulfate (0.025 M), EDTA-2Na (50 mg/l), and 30% methanol at a flow rate of 250 μl/min. An electrochemical detector that used a glassy working electrode (+450 mV) against a silver-silver chloride reference electrode (WE-3G; Eicom) was used to quantify dopamine in the samples. A dopamine standard was used to identify and quantify the dopamine concentration in the samples.

Morphometric analysis
These experiments were performed via a blinded experimental design. Control (DAT IREScre ) neurons and α-syn-overexpressing neurons (DAT IREScre / α-syn) were transduced with AAV1-LoxP-tdTomato (Addgene) on DIV5. Neurons were fixed with 4% PFA for 30 min at room temperature on DIV10, and coverslips were mounted using Fluoromount-G and allowed to dry. Alternatively, neurons were fixed, co-immunolabeled with TH and GFP antibodies, and mounted for imaging, as described above. Images were captured on a Nikon A1 laser-scanning confocal microscope (visualized through a ×20 oil-immersion objective). Images of neurons with minimal interference from neighboring neurons were analyzed in ImageJ (FIJI) and converted to 8-bit binary images after threshold adjustment. Sholl analysis plugin was used to draw concentric circles starting from 15 μm, followed by 5 μm successive shells in order to identify the number of intersections along the radii 108,162,163 . Sholl analysis was performed, and the number of intersections was plotted (Fig. 6). Cell area measurements were attained by manually drawing ROI around cell soma using the free polygon selection tool in ImageJ. ROIs were drawn to encompass the complete projection area of the cell and selection was finalized by a convex hull to attain final projection area measurement. Results obtained were plotted to analyze the complexity of morphology.

Statistical analysis
Data analysis was performed using GraphPad Prism version 8.02 and MATLAB version 2020a. Student's t test, linear regression, one-way, twoway, or repeated-measures ANOVA were used where appropriate and corrected for multiple comparisons. The significance of P < 0.05 was considered statistically significant. Data are presented with a mean and standard error, unless otherwise stated.
Limitations to the methodology and model system used in this study The primary neuronal culture used in this study is derived from the ventral midbrain, which contains the dopaminergic nuclei SNc and VTA. Notably, it has been shown that SNc dopaminergic neurons are more sensitive than VTA dopaminergic neurons 22,23,[26][27][28][29][30][31]164 . Therefore, this model is likely to contain more VTA dopaminergic neurons than SNc dopaminergic neurons 22,[26][27][28][29][30][31] . In addition, α-syn-mediated neuronal loss can also lead to a higher loss of SNc dopaminergic neurons than VTA dopamine neurons. Since functional analyses are conducted on surviving neurons, our data may overrepresent effects in the surviving VTA midbrain dopamine neurons.
In this study, we utilized TH promoter-driven expression of GFP and α-syn to confirm the specificity of TH promoter-dependent viral expression. We also performed calcium imaging, TH expression, and neuronal cell count in dopaminergic neurons expressing control vector (AAV-TH-GFP). We found that, compared to naive neurons, the AAV-TH-GFP transduction did not change neuronal activity and calcium dynamics in response to dopamine and quinpirole exposure, TH levels, and cell count (data not shown); therefore, the control AAV was not used for the rest of the experiments.
In regards to investigating if α-syn overexpression in dopaminergic neurons alters D2R expression, we utilized the biotinylation assay to assess the differences in membrane vs. cytoplasmic D2 levels [91][92][93][94] . Whether or not the detected membrane D2Rs are functional or desensitized remains unclear.

Reporting summary
Further information on research design is available in the Nature Research Reporting Summary linked to this article.