Striatal serotonin transporter gain-of-function in L-DOPA-treated, hemi-parkinsonian rats

Abstract

L-DOPA is the standard treatment for Parkinson’s disease (PD), but chronic treatment typically leads to L-DOPA-induced dyskinesia (LID). LID involves a complex interaction between the remaining dopamine (DA) system and the semi-homologous serotonin (5-HT) system. Since serotonin transporters (SERT) have some affinity for DA uptake, they may serve as a functional compensatory mechanism when DA transporters (DAT) are scant. DAT and SERT’s functional contributions in the dyskinetic brain have not been well delineated. The current investigation sought to determine how DA depletion and L-DOPA treatment affect DAT and SERT transcriptional processes, translational processes, and functional DA uptake in the 6-hydroxydopamine-lesioned hemi-parkinsonian rat. Rats were counterbalanced for motor impairment into equally lesioned treatment groups then given daily L-DOPA (0 or 6 mg/kg) for 2 weeks. At the end of treatment, the substantia nigra was processed for tyrosine hydroxylase (TH) and DAT gene expression and dorsal raphe was processed for SERT gene expression. The striatum was processed for synaptosomal DAT and SERT protein expression and ex vivo DA uptake. Nigrostriatal DA loss severely reduced DAT mRNA and protein expression in the striatum with minimal changes in SERT. L-DOPA treatment, while not significantly affecting DAT or SERT alone, did increase striatal SERT:DAT protein ratios. Using ex vivo microdialysis, L-DOPA treatment increased DA uptake via SERT when DAT was depleted. Overall, these results suggest that DA loss and L-DOPA treatment uniquely alter DAT and SERT, revealing implications for monoamine transporters as potential biomarkers and therapeutic targets in the hemi-parkinsonian model and dyskinetic PD patients.

1. Introduction

Parkinson’s disease (PD) is the second most prevalent neurodegenerative disorder and is typically characterized by a progressive deterioration of movement (Guttmacher et al., 2003; Kowal et al., 2013). Cardinal motor symptoms, including akinesia, rigidity, and resting tremor, have been primarily linked with nigrostriatal dopamine (DA) degeneration that causes imbalanced activity between the direct and indirect pathways of the basal ganglia (Albin, Young, & Penney, 1989). L-3,4-dihydroxyphenyl-L-alanine (L-DOPA) is the gold standard DA replacement therapy for PD and is highly effective at elevating synaptic DA and improving motor symptoms in early to moderate disease stages (Smith et al., 2012; Armstrong and Okun, 2020). However, chronic treatment typically results in the development of motor fluctuations in the majority of PD patients which are disabling abnormal involuntary movements (AIMs), referred to as L-DOPA-induced dyskinesia (LID; Ahlskog & Muenter, 2001; Eskow Jaunarajs et al., 2012; Cenci et al., 2020). As novel treatment strategies are still needed to optimize L-DOPA therapy, understanding the mechanisms contributing to L-DOPA’s efficacy and dyskinetic liability is critical.

To compensate for the low DA levels in the PD brain, several mechanisms appear to be engaged. For example, residual DA signaling can be maximized through reduced DA reuptake in the striatum and substantia nigra pars compacta (SNc). The DA transporter (DAT) is a primary auto-regulatory mechanism for DA signaling and is responsible for extracellular DA reuptake at dendrites, along the axon, and at axon terminals of DA neurons (Nirenberg et al., 1996). Attenuated DA uptake likely occurs in early PD with down-regulation of DAT and later by reduced striatal and SNc DAT resulting from DA neuronal degeneration (Cai et al., 2012; Kerenyi et al., 2003; Kraemmer et al., 2014; Li et al., 2009). Both may serve to prolong reduced synaptic DA effects (Adams et al., 2005; Lee et al., 2000; Sossi et al., 2009). This may provide relief from PD motor deficit and restore overall striatal DA neurotransmission but may be deleterious in the presence of larger amounts of extracellular DA, like that resulting from L-DOPA (Palermo and Ceravolo, 2019; Troiano et al., 2009).

Though no difference has been found in striatal DA markers, such as DAT, between dyskinetic and non-dyskinetic PD patients, higher extracellular DA levels have been found in those experiencing LID (Cheshire et al., 2015; Politis et al., 2014; Suwijn et al., 2013). This suggests that although remaining nigrostriatal DA terminals are structurally similar between PD patients suffering from LID and those that are not, the kinetics of DA release and uptake may differ between these patient populations. Furthermore, the increased release of L-DOPA-derived DA is negatively correlated with DAT binding, revealing that the DAT down-regulation or loss resulting from DA depletion is contributing to the large swings in extracellular DA that underlie the hyperkinetic side effects of L-DOPA treatment (Lee et al., 2008; Sossi et al., 2009; Troiano et al., 2009).

In recent years there has been a growing interest in the serotonin (5-HT) system in PD once DA cell loss is severe; it appears that 5-HT neurons may be both structurally and functionally altered (Beaudoin-Gobert et al., 2018; Cheshire et al., 2015; Gagnon et al., 2016; Li et al., 2009. For instance, variable loss of striatal 5-HT markers including tryptophan hydroxylase (TPH), which catalyzes 5-HT synthesis, and 5-hydroxyindo-leacetic acid, a 5-HT metabolite, is found in postmortem tissue of PD patients (Kish et al., 2008). One of the markers used to determine 5-HT cell loss is the 5-HT transporter (SERT) which can be found throughout the basal ganglia, likely residing on presynaptic raphe varicosities, axons, and cell bodies suggesting SERT’s role in synaptic and volumetric neurotransmission (Hahn et al., 2014; Zhou et al., 1998). How SERT expression and function is affected in PD is variable where many report no difference (Beaudoin-Gobert et al., 2018; Cheshire et al., 2015; Politis et al., 2010; Strecker et al., 2011; Suwijn et al., 2013; Walker et al., 2020) or reduced SERT binding in PD (Li et al., 2009; Roussakis et al., 2016; Rylander et al., 2010; Wang et al., 2010). Indeed, progressive decreases in striatal SERT have been shown with preferential loss in the caudate nucleus and disease duration negatively correlated with putamen SERT expression while raphe SERT loss does not occur until advanced PD stages (Kerenyi et al., 2003; Pagano et al., 2017; Politis et al., 2010). Still, negative correlations between SERT and DAT binding and increased striatal SERT:DAT in PD patients indicate a functional shift from DAT to SERT with escalating DA cell loss (Roussakis et al., 2016; Strecker et al., 2011).

5-HT markers and function are not only altered from parkinsonian DA depletion, but also with L-DOPA DA replacement therapy. Chronic L-DOPA has been reported to influence the 5-HT system in various ways by promoting 5-HT sprouting (Gagnon et al., 2016; Rylander et al., 2010), while also reducing 5-HT neuronal content and raphe TPH (Eskow Jaunarajs et al., 2011; Stansley & Yamamoto, 2014), or has no effect (Eskow Jaunarajs et al., 2012). LID expression in hemi-parkinsonian rats is shown to be positively and negatively correlated with striatal DA and 5-HT levels, respectively (Gil et al., 2011; Lindgren et al., 2010). Indeed, lesioning over 50% of or inhibiting 5-HTergic raphe neurons reduces LID expression and development implicates the role 5-HT neurons play in L-DOPA’s side effects (Eskow et al., 2009). Some of these lesion and L-DOPA-induced changes appear compensatory in nature. A significant body of literature has shown that 5-HT neurons are able to take up L-DOPA, convert it to DA, and release it in the DA lesioned striatum (Carta et al., 2007; Navailles et al., 2010). SERT has been shown to transport not only 5-HT but also DA back up into the presynaptic 5-HT neuron for re-packaging and release (Berger, 1978; Kaminska et al., 2018; Kannari et al., 2006). LID severity is shown to be positively correlated with increased SERT binding in dyskinetic PD rats, primates, and patients (Beaudoin-Gobert et al., 2018; Politis et al., 2014; Roussakis et al., 2016; Rylander et al., 2010; Walker et al., 2020) while others show no difference in striatal SERT binding in dyskinetic PD patients (Pagano et al., 2017). Interestingly, dyskinetic PD patients and hemi-parkinsonian rats show significantly higher striatal SERT:DAT than those without LID further implying functional plasticity between these monoamine transporters (Conti et al., 2016a; Roussakis et al., 2016). In order to further validate SERT as a potential therapeutic target for regulating DA, determining the relative contribution of SERT and DAT in DA reuptake is paramount.

Of late there has been renewed interest in targeting monoamine transporters for parkinsonian motor symptoms and LID (Chotibut, Fields, & Salvatore, 2014; Conti et al., 2016a, b; Devos et al., 2008; Müller, 2021; Nishijima and Tomiyama, 2016; Paumier et al., 2012; Wile et al., 2017). However, there is a lack of a direct comparison of how DA depletion and L-DOPA treatment affects monoamine transporter expression and function. Therefore, in the present series of experiments, we determined transcriptional, translational, and functional monoamine transporter modifications in the L-DOPA-treated hemi-parkinsonian rat model. First, we investigated the effects of DA loss and L-DOPA treatment on DAT and SERT gene expression in the SNc and dorsal raphe nucleus (DR), respectively, and striatal synaptosomal protein expression. Second, we determined the effects of DA loss and L-DOPA treatment on striatal DAT and SERT function in taking up DA using ex vivo microdialysis techniques. We hypothesized the treatment with L-DOPA in lesioned animals would initiate a gain-of-function in SERT therefore adding to the unregulated DA activity originating at serotoninergic terminals characteristic of the LID phenotype.

2. Materials and methods

Animals

Adult male Sprague-Dawley rats were used (N = 55; approximately 2 months old and 225–250 g upon arrival; Harlan Farms, USA). Rats were housed in plastic cages (22 cm high, 45 cm deep, and 23 cm wide) and given free access to standard lab chow (Rodent Diet 5001; Lab Diet, Brentwood, MO, USA) and water. The colony room was kept on a 12 h light/dark cycle (light on at 0700 h) and maintained at 22–23 °C. Rats were maintained in accordance with the guidelines of the Institutional Animal Care and Use Committee of Binghamton University and the “Guide for the Care and Use of Laboratory Animals” (Institute for Laboratory Animal Research, National Academies Press 2011).

Medial forebrain bundle 6-hydroxydopamine surgery

One week after arrival, rats received active (n = 47) or sham (n = 8) unilateral 6-hydroxydopamine (6-OHDA) infusions of the left medial forebrain bundle (MFB) to lesion DA neurons. All rats received injections of Buprenex (buprenorphine HCl; 0.03 mg/kg, i.p.; Reckitt Benckiser Pharmaceuticals Inc., Richmond, VA) as analgesic treatment 5 min pre-surgery. In order to isolate cell loss in the DA system, desipramine HCl (25 mg/kg, i.p.; Sigma, St. Louis, MO, USA) was given to each rat 30 min prior to 6-OHDA injection to protect norepinephrine neurons. Rats were anesthetized with inhalant isoflurane (2–3%; Sigma) in oxygen (1000 cc/min) and placed in a stereotaxic apparatus (David Kopf Instruments, Tujunga, CA, USA). The coordinates for vehicle or 6-OHDA injections were AP: −1.8 mm, ML: +2.0 mm, DV: −8.6 mm relative to bregma, with the incisor bar positioned 5.0 mm below the interaural line (Paxinos & Watson, 1998). After drilling a small hole in the skull above the injection site, a 10 μl Hamilton syringe attached to a 26-gauge needle was used to deliver 4 μl of vehicle or 6-OHDA (3μ g/ μL; Sigma; Conti et al., 2014) dissolved in 0.9% NaCl + 0.1% ascorbic acid at a rate of 2 μl/min. The needle was withdrawn 5 min later to allow the drug to disperse. Stainless steel wound clips were used to close the surgical site. Post-surgery, rats were pair-housed in clean cages and provided with soft chow, fruit, and saline as needed to facilitate recovery during the following 10 days. Wound clips were removed 1-week post-surgery.

Abnormal involuntary movements (AIMs)

Rats were monitored for rodent dyskinesia using a procedure previously described (Bishop et al., 2012; Lundblad et al., 2003). During testing (0900–1700 h), rats were placed in clear plastic cylinders (22.2 cm diameter, 25.4 cm height; Thermo Fisher Scientific, Rochester, NY) immediately after L-DOPA injection. After injections, a trained observer blind to treatment conditions recorded AIMs involving axial, limb, and orolingual (ALO) regions. “Axial” AIMs include dystonic twisting of the neck and torso directed toward the side of the body that is contralateral to lesion. “Limb” AIMs refer to excessive and purposeless movements of the forelimb contralateral to lesion. “Orolingual” AIMs are repetitive openings and closings of the jaw as well as multiple tongue protrusions that are not associated with eating or grooming. ALO AIMs were rated and given a severity score (0–4) for 1 min every 10 min for 3 h: 0, not present; 1, present for < 30 s or half the time; 2, present for 30–59 s or majority of the time; 3, present for the whole 60 s but interrupted by stimulus (tap on the cylinder); or 4, present for the whole 60 s and was not interrupted by stimulus. Additionally, contralateral rotations, defined as complete 360° turns away from lesion, were recorded. Axial, limb, and orolingual scores were summed to create a single ALO AIMs score for data analysis.

Forepaw adjustment steps test (FAS)

The FAS test was used to measure rodent forelimb akinesia since motor deficit due to DA loss and therapeutic reversal has been demonstrated in hemi-parkinsonian rats (Chang et al., 1999; Conti et al., 2014; Olsson et al., 1995). An experimenter blind to treatment held the rat’s hindlimbs and one forepaw so that weight would be imposed on the other forepaw. Rats were moved laterally across a table at a rate of 90 cm/10 s. Each test consisted of 6 trials for each forepaw for both forehand (adjusting for movement toward the body) and backhand (adjusting for movement away from the body) directions. Data are presented as “percent intact”, where the sum of steps taken by the lesioned forepaw is divided by the sum of steps taken by the intact forelimb and multiplied by 100 thus indicating the degree of disability seen in the lesioned paw. Lower percent intact scores indicate greater parkinsonian motor impairment.

Vibrissae-evoked forelimb placing (VEFP)

Due to the VEFP test’s sensitivity to 6-OHDA-induced DA loss (Anstrom et al., 2007; Woodlee et al., 2005), it was used along with the FAS test to characterize motor deficit and therapeutic improvement. As previously described by Woodlee and colleagues (2005), an experimenter blind to treatment held the animal by the torso, restrained a forelimb, and brushed its vibrissae against the edge of a table to elicit a forelimb placement from the ipsilateral limb, or same-side placing. Animals were then turned sideways so that vibrissae were perpendicular to the table and the downwardly oriented limb was restrained. The vibrissae were then brushed against the tabletop to evoke forelimb placement from the contralateral limb, or cross-midline placing. Animals underwent extensive handling to minimize struggle. Trials where the animal struggled were not counted. Successful placements out of 10 trials were recorded for each limb. Data are presented as “percent intact” for ipsilateral placements, where the sum of the placements taken by the lesioned paw is divided by the sum of the placements taken by the intact by and multiplied by 100. Lower percent intact scores refer to more severe motor deficit.

Sample preparation for western blot analysis

Using the procedure described by Santerre et al. 2014, striatal samples underwent synaptosome (P2) fractionation to determine synaptosomal expression of DAT and SERT. Following dissection, P2 fractions were homogenized in 0.32 M sucrose/PBS solution, and spun at 1,000 g followed by spinning the resulting supernatant at 12,000 g for 20 min. The pellet (P2 fraction) was re-suspended in a modified Krebs Ringer’s solution (2 mM KCl, 1 mM KH2PO4, 1.2 mM Ca2Cl, 6 Na2HPO4, 136 mM NaCl) and 5 mM glucose. Aliquots of 300 μl were used for ex vivo microdialysis in experiment 2. The remaining sample was used for calculating protein concentrations using a bicinchoninic acid method and stored at − 80 °C until western blot analysis in experiment 1.

Western blot analysis

DAT and SERT protein expression was analyzed by western blotting using P2 fractions (Santerre et al., 2014; Werner et al., 2011). Briefly, protein samples underwent sodium dodecyl sulfate-polyacrylamide gel electrophoresis using Novex Tris-Glycine gels (8–16%) and were transferred to polyvinylidene difluoride membranes (Invitrogen, Carlsbad, CA, USA). Membranes were probed with antibodies for the DAT (rabbit polyclonal raised against rat DAT, Millipore, Lake Temecula, CA, USA) and SERT (mouse monoclonal raised against rat SERT, Santa Cruz Biotechnology Inc., Dallas, TX, USA) proteins. Blots were subsequently exposed to an antibody directed against β-actin (Millipore) to verify equivalent protein loading and transfer. Secondary antibodies were obtained from Thermo Scientific (Waltham, MA). All bands were detected by enhanced chemiluminescence under nonsaturating conditions (GE Healthcare, Piscataway, NJ, USA) and exposed to x-ray films and analyzed using NIH Image J.

3. Experiment 1. Effects of DA lesion and L-DOPA treatment on monoamine transporter gene and protein expression

As described in Fig. 1A, 3 weeks post-surgery, sham- (n = 8) and 6-OHDA-lesioned (n = 18) rats were habituated to the FAS and VEFP tasks. FAS motor performance was used prior to testing to counterbalance treatment groups. For the following 2 weeks, sham rats were treated with daily vehicle while lesioned rats with equivalent parkinsonian motor deficit were given either daily vehicle (n = 9) or L-DOPA (n = 9; 6 mg/kg, s.c.; Sigma) + DL-serine 2-(2,3,4-trihydroxybenzyl) hydrazine hydrochloride (benserazide; 15 mg/kg, s.c.; Sigma) dissolved in 0.9% NaCl + 0.1% ascorbic acid to prime for maximal LID (Dupre et al., 2011). During this time, rats were rated on ALO AIMs development on days 1, 8, and 14 while FAS and VEFP (off treatment) were assayed on days 4 and 11. Approximately 3 days after the last treatment day, rats were euthanized via rapid decapitation and bilateral striatal tissue was dissected and P2 fractioned for western blot analysis of membrane bound DAT and SERT expression and ex vivo microdialysis for experiment 2. Posterior brains were flash frozen in methyl butane and stored at − 20 °C for later tissue dissection of SN and DR for several transcripts (see below).

Fig. 1. Experimental timeline and design.

In Experiments 1 and 2, rats were either given unilateral Sham or 6-hydroxydopamine (6-OHDA) lesions. Then, after 3 weeks, rats were tested for motor performance using the Vibrissae Evoked Forepaw Placement (VEFP) and Forepaw Adjustment Steps (FAS) tests to counterbalance 6-OHDA-lesioned animals into treatment groups. Rats were then given vehicle (Veh) or L-DOPA (6 mg/kg) daily for 14 days, Sham rats in Experiment 1 were treated with vehicle for 14 days. All rats were rated for abnormal involuntary movements (AIMs) on day 1, 8, and 14, then tested off treatment on FAS and VEFP on day 4 and 11. A) Approximately 3 days after the last treatment, rats in Experiment 1 were euthanized and bilateral striatal tissue was dissected for synaptosomal fractions for western blot analyses of dopamine transporter (DAT) and serotonin transporter (SERT). Posterior brains were flash frozen for later RT-PCR analysis of the substantia nigra (SN) and dorsal raphe nucleus (DR). B) In Experiment 2, 3 days after the last treatment, rats meeting behavioral criteria (n = 29/43) were sacrificed and bilateral striatal tissue dissected for synaptosomal fractions for ex vivo microdialysis. Samples were aliquoted and incubated in Veh and either selective DAT or SERT blockers GBR182020 (GBR) or Citalopram (CIT), respectively; 10 μM) followed by dopamine (DA) at 50 nM. Six dialysate samples (50 μl every 2.5 min) were collected and stored at − 80 °C for high performance liquid chromatography (HPLC) analysis of remaining synaptic DA concentrations. Created with BioRender.com.

Real-time reverse transcription polymerase chain reaction (RT-PCR)

SN (AP: −5.7; ML: ±2.5; DV: −7.0) and DR (AP: −8.0; ML: 0.0; DV: −6.0) tissue was processed for changes in DAT and SERT gene expression, respectively, using the housekeeper gene β-actin through RT-PCR techniques previously described (Dupre et al., 2013; Lindenbach et al., 2011). For SN tissue, changes in DAT and tyrosine hydroxylase (TH) mRNA was determined (Hurd et al., 1994). For DR tissue, SERT and TPH2 mRNA was quantified (Eskow Jaunarajs et al., 2011; Huot, Johnston, Koprich, et al., 2012). Tissue samples were homogenized into Trizol using TissueLayer 2 (Qiagen), extracted with RNeasy mini protocol (Qiagen), normalized, and converted into cDNA using the QuantiTect Reverse Transcription Kit (Qiagen), which includes a DNase treatment step. cDNA was amplified with the IQ SYBR Green Supermix kit (BioRad, Hercules, CA, USA). A reaction master mix of volume 10 μl consisted of 5 μl SYBR Green, 4.4 μl RNase-free water, 0.5 μl cDNA template, and 0.1 μl of primer. The mix ran in triplicate in a 384 well plate (BioRad) and captured in real-time using the CFX Real-Time PCR detection system (BioRad). Relative gene expression was quantified using the 2^−ΔΔCT method, with expression values normalized to 100% of ultimate control values. Gene sequences were obtained from GenBank at the National Center for Biotechnology Information and primer specificity was verified by the Basic Local Alignment Search Tool (https://www.ncbi.nlm.nih.gov/).

4. Experiment 2. Monoamine transporter uptake of DA in the vehicle- or L-DOPA-treated intact or DA-lesioned striatum

According to Fig. 1B, 3 weeks post-surgery, 6-OHDA-lesioned rats (n = 29) were habituated to FAS and VEFP tasks. FAS motor performance was then used to counterbalance treatment groups. For the following 2 weeks, rats with equivalent parkinsonian motor deficit were primed with either daily vehicle (n = 15) or L-DOPA 6 mg/kg (n = 14). During this time, rats were rated on ALO AIMs development on days 1, 8, and 14 while FAS and VEFP (off treatment) were measured on days 4 and 11. Approximately 3 days after the last treatment day, rats were euthanized by rapid decapitation and bilateral striatal tissue was dissected and P2 fractioned for ex vivo microdialysis. Intact and lesioned synaptosomes from either vehicle- or L-DOPA-treated rats counterbalanced based on averaged FAS motor performance from behavioral days 4 and 11 were incubated in either vehicle (20% dimethyl sulfoxide + 80% distilled water) or the respective DAT or SERT blocker: GBR12909, 1-(2-(bis-(4-fluorophenyl)-methoxy)ethyl)-4-(3-phenylpropyl)piperazine)dihydrochloride (GBR; 10 μM; Sigma) or Citalopram (CIT; 10 μM; LKT Laboratories Inc, St. Paul, MN). Afterwards, custom microdialysis probes (see below) were submerged and DA (50 nM) was added. Six samples (50 μl every 2.5 min) were collected and DA concentrations were determined with high performance liquid chromatography (HPLC).

Synaptosomal uptake assay

Using an ex vivo microdialysis technique previously described (Huff et al., 2013), an aliquot (150 μl) of the isolated P2 fraction was incubated in either vehicle, GBR, or CIT (10 μM; Hansard et al., 2002; Thompson et al., 2011) for 10 min at 37 °C. Looped microdialysis probes were constructed from microbore PTFE tubing, PE-20 tubing, stainless steel wire, and Hospal AN69 HF membrane with an active membrane length of 16 mm. The probes were connected to an infusion pump which delivered modified Krebs Ringer’s solution (2 mM KCl, 1 mM KH2PO4, 1.2 mM Ca2Cl, 6 Na2HPO4, 136 mM NaCl) and 5 mM glucose at a constant rate of 20 μl/min. Probes were submerged into the sample upon the addition of DA (50 nM). Then 6 dialysate samples (50 μl each) were collected and immediately stored at − 80 °C for HPLC analysis of DA concentrations. Mean DA pM concentrations were adjusted for probe recovery (~10%) and protein. The synaptosomal uptake of DA was equated to the loss of DA from the medium. Data is presented as mean “percent vehicle” which was calculated by dividing the amount of remaining DA after transporter inhibitor treatment by the amount of DA remaining after vehicle treatment multiplied by 100.

High-performance liquid chromatography coupled to electrochemical detection (HPLC-ED) analysis

Previously described in (Nakano et al., 1994), ex vivo dialysate samples were measured for remaining DA via HPLC-ED (Eicom USA, San Diego, CA). DA peaks were quantified using peak heights of standard solutions and corrected for in vitro probe recovery. The system detection limit is reliably 26.4 pM of DA. Chromatographs obtained every 10 min/sample were analyzed using the software program Envision (provided by Eicom, USA).

Data analysis

All AIMs data (expressed as medians + median absolute difference; M.A.D.) were analyzed by the non-parametric Kruskal-Wallis ANOVA with Mann-Whitney U post-hoc test used to determine significant differences. The remaining behavioral data and post-mortem analyses (presented as mean + standard mean error; S.E.M.) were measured by ANOVAs with Tukey HSD post-hoc analyses. Pearson correlations were used for comparing SERT:DAT ratios from the lesioned striatum with day 14 AIMs data in experiment 1. Significant differences in ex vivo data in experiment 2 (expressed as mean + S.E.M.) were analyzed using ANOVAs with Fisher LSD post-hoc analyses or when a priori hypotheses were postulated, planned comparisons. Outliers in ex vivo data were removed from analyses based on specific exclusion criteria. For extracellular DA concentrations that were normalized to vehicle treatment: values over 2 standard deviations above the mean were considered outliers and replaced with mean substitution (~5% of total data points). Additionally, low values ≤ 0.03 were removed leaving final treatment groups with n = 5 – 8 (~7% of total data points). Analyses for all experiments were performed with Statistica software’98 (StatSoft Inc., Tulsa, OK, USA) and alpha was set at p < 0.05.

5. Results

5.1. Experiment 1. Effects of DA lesion and L-DOPA treatment on monoamine transporter gene and protein expression

To examine transcriptional and translational effects of DA lesion and L-DOPA, vehicle-treated sham rats or unilateral DA-lesioned rats received either daily vehicle or L-DOPA (6 mg/kg) for 2 weeks. On day 4 and 11, motor performance was measured via FAS and VEFP off treatment. Since results did not differ between test days 4 and 11, according to t-test analysis, motor performance scores were averaged. DA-lesioned rats exhibited ~ 70% motor deficit compared with sham rats on FAS (F_2,23 = 192.21, p < 0.05; Fig. 2A). Similarly, rats showed ~ 60–70% movement reduction compared with sham rats during VEFP (F_2,23 = 16.61, p < 0.05; Fig. 2B). Importantly, lesioned rats exhibited similar degrees of deficit between treatment groups on both motor performance measures. DA-lesioned rats that received daily L-DOPA displayed substantive AIMs (χ² (2) = 17.95, p < 0.05; Fig. 2C) and rotations (F_2,23 = 7.07, p < 0.05; Fig. 2D) by day 14 compared to rats that received daily vehicle. Vehicle-treated DA-lesioned rats expressed scant but detectable AIMs compared to sham rats (p < 0.05).

Fig. 2. Motor performance on A) Forepaw Adjustment Steps (FAS), B) Vibrissae Evoked Forepaw Placement (VEFP), C) L-DOPA-induced abnormal involuntary movements (AIMs) and D) rotations in sham or 6-hydroxydopamine (6-OHDA)-lesioned rats.

Rats with unilateral sham lesions were treated with vehicle (Veh; n = 8) while rats with unilateral 6-OHDA lesions were treated with either vehicle or L-DOPA (LD; 6 mg/kg) daily for 14 days (n = 9/group). On days 1, 8, and 14, axial, limb, and orolingual (ALO) AIMs and contralateral rotations were recorded for 3 h following LD. Total AIMs values are expressed as medians (ALO AIMs + median absolute difference; M.A.D.). Total rotation values are expressed as means + standard mean error (S.E.M.). On days 4 and 11 prior to treatment, VEFP and FAS were used to evaluate motor deficit. Values (as means + S.E.M.) are expressed as ipsilateral (ipsi) percent intact and percent intact for VEFP and FAS, respectively. Significant group differences in ALO AIMs were determined by non-parametric Kruskal-Wallis ANOVAs and Mann-Whitney U post-hoc tests. The remaining data were analyzed by one-way ANOVAs. When appropriate, motor performance differences were analyzed with Tukey HSD post-hocs. * p < 0.05 vs Sham-Veh.

Approximately 3 days after the final treatment day, rats were killed and posterior brains were flash frozen for later dissection of bilateral SN to determine 6-OHDA lesions and L-DOPA treatment induced changes in DAT and TH mRNA, and midline DR to determine effects on SERT and TPH2 mRNA, using RT-PCR. As shown in Fig. 3, β-actin was similar across groups in the SN (Fig. 3C) and DR (Fig. 3F) and was therefore used to normalize the genes of interest. Main effects of side (F_1,22 = 62.43, p < 0.05) and treatment (F_2,22 = 18.06, p < 0.05) were shown in SN DAT revealing the impact of the 6-OHDA lesion against the intact SN and against the sham-lesioned group, respectively (Fig. 3A). Similar results occurred with TH in the SN where main effects of side (F_1,21 = 21.64, p < 0.05) and treatment (F_2,21 = 9.92, p < 0.05) revealed the effect of DA depletion rather than L-DOPA treatment (Fig. 3B). In the DR, no statistically significant changes were found in either SERT (Fig. 3D.) or TPH2 (Fig. 3E) mRNA.

Fig. 3. Changes in monoamine transporter and rate-limiting enzyme gene expression in in substantia nigra (SN) and dorsal raphe nucleus (DR) from vehicle-treated sham, and vehicle- or L-DOPA-treated 6-hydroxydopamine (6-OHDA)-lesioned rats.

Rats with unilateral sham lesions were treated with vehicle (Sham-Veh, n = 8) while rats with unilateral 6-OHDA lesions were treated with either vehicle (Les-Veh, n = 9) or L-DOPA (Les-LD; 6 mg/kg, n = 9) daily for 14 days. Rats were then euthanized and bilateral SN and DR were dissected and analyzed with reverse transcript polymerase chain reaction (RT-PCR) for mRNA changes induced by lesion and L-DOPA treatment. Using Sham-Veh as the control, changes in A) dopamine transporter (DAT) and B) tyrosine hydroxylase (TH) in SN and D) serotonin transporter (SERT) and E) tryptophan hydroxylase 2 (TPH2) in the DR, were normalized to the housekeeper β-actin (C & F). One-way ANOVAs were used for the DR while 3 × 2 (Treatment × Side) mixed ANOVAs with Tukey post-hoc analyses were used to determine significant differences for the SN targets. * Main effect of side, p < 0.05 vs Intact. + Main effect of treatment, p < 0.05 vs Sham-Veh.

From the same rats, striatal tissue was dissected for P2-fractioned SERT and DAT protein levels via western blot. β-actin levels were equivalent across groups within blots, therefore it was used as a housekeeper for SERT and DAT levels. To determine SERT:DAT, optical density values for SERT were divided by optical density values for DAT. All data was normalized to the intact striatum of the sham and vehicle-treated group which served as the control. Fig. 4A and 4B show representative blots and values for striatal SERT and DAT, respectively, across all conditions. A main effect of side was seen for DAT expression where the lesioned striatum exhibited significantly less DAT than the intact striatum (F^1,22 = 23.31, p < 0.05; Fig. 4B). A significant side by treatment interaction was also revealed for DAT (F_2,22 = 3.65, p < 0.05). DAT in the DA-lesioned striatum of vehicle-treated rats was ~ 80% lower than DAT of the intact striatum of sham- and DA-lesioned, vehicle-treated rats. A similar reduction was seen in DAT of DA-lesioned, L-DOPA-treated animals compared with the intact striatum of all treatment groups (all p < 0.05). While no effect of DA lesion or L-DOPA treatment was seen for measures of striatal SERT, a main effect of side revealed that membrane-bound SERT: DAT ratios were elevated in the lesioned compared to the intact striatum (F_1,22 = 10.05, p < 0.05; Fig. 4C). A trending main effect of treatment (F_2,22 = 3.13, p = 0.06) suggested an increase in SERT:DAT in the L-DOPA-treated striatum compared with sham (p = 0.058). Interestingly, a side by treatment interaction (F_2,22 = 3.35, p = 0.05) revealed the significant rise in SERT: DAT dependent upon L-DOPA treatment and DA depletion (p < 0.05). Although higher SERT: DAT were found in the L-DOPA-treated, lesioned striata, there was no correlation with ALO AIMs severity (Fig. 4D).

Fig. 4. Synaptosomal monoamine transporter expression in intact and 6-hydroxydopamine (6-OHDA)-lesioned striatum of vehicle (Veh)-treated sham rats and Veh- or L-DOPA (LD)-treated 6-OHDA-lesioned rats.

Rats with unilateral sham lesions (n = 8) were treated with Veh while rats with unilateral 6-OHDA lesions were treated with either Veh or L-DOPA (LD; 6 mg/kg) daily for 14 days (n = 9/group). Rats were then euthanized, and bilateral striatal tissue dissected for P2 A) serotonin transporter (SERT) B) dopamine transporter (DAT). Representative blots are shown for SERT and DAT (A, B, respectively). C) SERT:DAT ratios were calculated, and D) were examined for correlations with axial, limb, and orolingual (ALO) AIMs scores from day 14 of priming (see Fig. 2C). Values (as means + standard mean error; S.E.M.) are expressed as percent (%) control. Significant differences were determined by 3 × 2 (Treatment × Lesion) mixed ANOVAs within each transporter. When appropriate, expression differences were analyzed with Tukey HSD post-hocs. Main effect of lesion: * p < 0.05 vs Intact; + p < 0.05 vs Sham-Veh-Intact; ^ p < 0.05 vs Sham-Veh-Lesion; # p < 0.05 vs Les-Veh-Intact; % p < 0.05 vs Les-LD-Intact.

6. Experiment 2. Monoamine transporter uptake of DA in the vehicle- or L-DOPA-treated intact or DA-lesioned striatum

To determine functional changes in DAT and SERT uptake of DA, ex vivo microdialysis techniques were employed on P2 fractioned striatal tissue from unilateral DA-lesioned rats that received either daily vehicle or L-DOPA (6 mg/kg) for 2 weeks. On day 4 and 11, rats were examined for motor deficit using FAS and VEFP. Like data from experiment 1, since results did not differ between test days, motor performance scores were averaged across days. Indeed, all DA-lesioned rats showed equivalent parkinsonian motor deficit across treatment groups in both FAS (Fig. 5A) and VEFP (Fig. 5B). Rats that received daily L-DOPA showed substantially more LID (χ² (5) = 43.0, p < 0.05; Fig. 5C) by day 14 compared to rats that received daily vehicle. Additionally, a main effect of L-DOPA treatment was found with contralateral rotations where L-DOPA-treated rats rotated more than vehicle-treated rats (F_1,37 = 15.51, p < 0.05; Fig. 5D).

Fig. 5. Motor performance and L-DOPA-induced dyskinesia on A) Forepaw Adjustment Steps (FAS), B) Vibrissae Evoked Forepaw Placement (VEFP), C) L-DOPA-induced abnormal involuntary movements (AIMs) and D) rotations in sham or 6-hydroxydopamine (6-OHDA)-lesioned rats.

Rats with unilateral 6-OHDA lesions were sorted into treatment groups (n = 7–8/group) with equivalent motor deficit based on FAS motor performance prior to commencing daily treatment (data not shown). The 4 treatment groups received: either 14 consecutive days of vehicle (Veh) or L-DOPA (LD: 6 mg/kg, s.c.) and then ex vivo striatal treatment with either the dopamine transporter (DAT) inhibitor GBR 12909 (GBR), or the serotonin transporter (SERT) inhibitor citalopram (CIT). Therefore, data is separated into the following groups: GBR-Veh, GBR-LD, CIT-Veh, CIT-LD. Rats were treated with either vehicle or L-DOPA (6 mg/kg) daily for 14 days. On days 1, 8, and 14, axial, limb, and orolingual (ALO) AIMs and contralateral rotations were recorded for 3 h following LD. Total ALO values are expressed as medians (ALO AIMs + median absolute difference; M.A.D.). Total rotation values are expressed as means + standard mean error (S.E.M.). On days 4 and 11, VEFP and FAS were used to evaluate motor deficit. Motor performance tasks were performed prior to treatment and therefore values show off treatment effects. Values (as means + S.E.M.) are expressed as ipsilateral (Ipsi) percent intact and percent intact for VEFP and FAS, respectively. ALO AIMs significant differences were determined by non-parametric Kruskal-Wallis ANOVAs and Mann-Whitney U post-hoc tests. The remaining data were analyzed by 2 × 2 (LD treatment X Transporter blocker) ANOVAs. When appropriate, motor performance differences were analyzed with Tukey HSD post-hocs. * p < 0.05 vs GBR-Veh. ^ p < 0.05 vs CIT-Veh. # Main effect of LD treatment, p < 0.05 vs Veh.

Approximately 3 days after the last treatment day, striatal tissue was dissected and processed for ex vivo microdialysis. Extracellular DA concentrations from selective monoamine transporter blockade were normalized to vehicle treatment. As shown in Fig. 6A, pharmacological DAT or SERT inhibition with GBR or CIT, respectively was applied to ostensibly prevent uptake of extracellular DA, therefore providing a measure of DA uptake via each monoamine transporter. Thus, results in Fig. 6 reveal how vehicle or L-DOPA treatment in intact or DA-lesioned rats (Int-Veh, Int-LD, Les-Veh, Les-LD) affected ex vivo striatal monoamine transporter function.

Fig. 6. Effect of 6-hydroxydopamine (6-OHDA) lesion and L-DOPA treatment on ex vivo DA uptake via striatal synaptic dopamine transporter (DAT) and serotonin transporter (SERT).

Unilateral 6-OHDA-lesioned rats (n = 7–8/group) were treated with either vehicle (Veh) or L-DOPA (LD; 6 mg/kg, s.c.) daily for 2 weeks. Three days after the last treatment, rats were euthanized and bilateral striatal tissue was prepared for synaptosomal (P2) fraction sample analyses. A) Striatal P2 samples from Veh- and LD-treated intact (Int-Veh and Int-LD, respectively) and lesioned (Les-Veh and Les-LD, respectively) rats were aliquoted for a 10 min incubation in vehicle and B) GBR12909 (GBR; 10 μM) or C) Citalopram (CIT; 10 μM). Thereafter, DA (50 nM) was injected into the P2 sample and the dialysis loop probe inserted. For the next 30 min, 6 dialysate samples (50 μl across 2.5 mins) were collected every 5 mins. Samples were immediately frozen and later analyzed for DA concentrations. As shown in A) data are presented as mean percent (%) vehicle per time point and averaged across the timepoints. Data which was calculated by dividing the DA in the aqueous analyte after transporter inhibitor treatment by DA remaining after vehicle treatment and multiplying the value by 100. Effects were determined by 2 × 2 × 6 (Lesion × Treatment × Time) mixed ANOVAs. When appropriate, Fisher LSD post-hocs or planned comparisons were used. In B) # Timepoints (T) T10-T30 min Veh- vs. L-DOPA-treated groups; C) + p < 0.05 T10 Les-LD vs. Int-LD; * p < 0.05 Les-LD-T10 vs. all other Les-LD timepoints; ^ p < 0.05 vs. Int-LD, Les-Veh. Created with BioRender.com.

For GBR-treated striata (Fig. 6B), a significant interaction between L-DOPA treatment X time was found (F_5,120 = 2.45, p < 0.05). Post-hoc analyses indicated that DA uptake from vehicle-treated rat striata was occluded by DAT blockade as compared to DA uptake in L-DOPA-treated striata (p < 0.05). This supports that DA uptake via DAT as the primary mechanism of peri-synaptic DA removal in the striatum, and by extension that L-DOPA treatment may compromise DAT-mediated DA update. For CIT-treated striata (Fig. 6C), a 3-way interaction of lesion X L-DOPA treatment X time was observed (F_5,115 = 2.40, p < 0.05). Post hoc analyses indicated that CIT blockade of SERT significantly modified DA uptake in L-DOPA-treated, lesioned rat striata within 10 min after DA application compared with the remaining other Int-LD and all other Les-LD samples (all p < 0.05). When examining average uptake per condition, planned comparisons demonstrated increased overall DA uptake via SERT in the Les-LD condition vs. Int-LD and Les-Veh. These data support DA uptake via SERT as an emergent mechanism of DA removal in the dyskinetic striatum.

7. Discussion

Targeting monoamine transporters in PD and LID has received recent interest given their burgeoning role in DA neurotransmission. However, without a clearer understanding of DAT and SERT expression and function in parkinsonian and dyskinetic brain, therapeutic effects or lack thereof may be misleading in a preclinical or clinical setting. The current work characterized how DA depletion and L-DOPA treatment affect monoamine transporters at the structural and functional levels in a hemi-parkinsonian rat model. Our findings were threefold: 1) not surprisingly DA lesion profoundly reduced DAT mRNA and P2 protein in the SN and striatum, 2) L-DOPA treatment increased SERT:DAT expression in the DA-depleted striatum and 3) SERT exhibited a gain-in-function, as a surrogate to DA uptake when DAT function was reduced in the DA-depleted, L-DOPA-treated striatum.

DAT is expressed along DA projections and terminals throughout the striatal patch-matrix to facilitate synaptic and diffused DA transmission (Nirenberg et al., 1996; Salinas et al., 2016). Although DAT expression naturally declines in aging, it has been used as a pathophysiological marker in PD rodents, primates, and patients (Bang et al., 2016; Buddhala et al., 2015; Kanazawa et al., 2016; Simuni et al., 2020). In the current study, unilateral 6-OHDA MFB lesion reduced DAT both on the transcriptional and translational levels as shown by RT-PCR (Fig. 3) and western blot analysis (Fig. 4) which corresponded with motor impairment severity (Fig. 2) suggesting that striatal DAT expression negatively correlates with motor symptom severity (Liu et al., 2015; Niccoli Asabella et al., 2015).

Some have suggested that lower DAT levels independent of DA cell loss may reflect beneficial compensatory processes that prolong synaptic DA signaling (Hansard et al., 2002; Lee et al., 2000; Madras et al., 2006; Sossi et al., 2009). Indeed, although not significant, reduced DA uptake via DAT was observed in the lesioned compared to the intact striatum (Fig. 6). However, it is unclear whether this was due to a specific loss of function or reduced DAT expression. Downregulation of DAT to elevate synaptic DA may be a more relevant compensatory mechanism in early PD stages when there are remaining striatal DA terminals versus the negligible levels that occur in late-stage PD (Bezard et al., 2000; Sossi et al., 2009). Instead, reduced DA uptake in a DAT-depleted state may have adverse effects on DA release as evidenced by reduced DA terminal TH levels in DAT knock out models (Bergstrom & Garris, 2003; Salvatore, Calipari, & Jones, 2016). While an overall deficit due to lesion was seen in DAT mRNA, P2 protein, and DA uptake, differences in the extent of these deficits indicate independent roles for DAT expression and function in PD motor symptoms and, potentially, L-DOPA-induced motor effects (Hong et al., 2016).

DAT has been associated with L-DOPA-related motor activity and efficacy. For instance, genetic variants of DAT differentially impact PD patients’ therapeutic response to L-DOPA, suggesting that genotyping may offer insight into clinical treatment regiments (Michałowska et al., 2020; Moreau et al., 2015; Schumacher-Schuh et al., 2013). The extent of DAT binding, even in a healthy state, is inversely associated with L-DOPA-induced hyperactivity where low and high DA uptake is linked to high and low locomotion, respectively (Nikolaus et al., 2016). Large swings in synaptic DA are the driving mechanism for LID development and expression; however, reports on L-DOPA’s effect on striatal DA markers, including DAT, are mixed, where no difference or even less DAT is found in the dyskinetic state (Cai et al., 2012; Cheshire et al., 2015; Hong et al., 2014; Politis et al., 2014; Purcaro et al., 2019; Suwijn et al., 2013). In such cases, DAT loss may lead to reduced L-DOPA-derived DA uptake contributing to LID development and even non-motor complications in PD patients (Kiferle et al., 2014; Löhle et al., 2016; Santangelo et al., 2015). While the severity of DA lesion may have caused no significant effect of L-DOPA on either DAT mRNA (Fig. 3) or P2 expression (Fig. 4), downward trends may indicate a potential detriment from L-DOPA thereby affecting extracellular DA kinetics.

Extracellular DA predominantly diffuses and targets distal receptors until it is taken back up by DAT, located primarily in non-synaptic locations (Hersch et al., 1997; Pickel, Nirenberg, & Milner, 1996). Depending on L-DOPA treatment duration and species, L-DOPA can reduce DAT binding, suggesting competitive uptake, in the DA-intact system, leaving DA clearance to diffusion or other monoamine transporters present (Bang et al., 2016; Borgkvist et al., 2012; Nikolaus et al., 2013; Sossi et al., 2010). L-DOPA has been shown to enhance oxidative stress by increasing cytosolic DA, resulting in increased vulnerability in DAT-expressing neurons (Chen et al., 2008; Stednitz et al., 2015). Interestingly, Cai and colleagues (2012) reported that rats with sub-chronic L-DOPA treatment, that did not develop dyskinesia, exhibited increased DAT expression, implicating increased DA clearance in the restoration of a steady state of equivalent DA uptake and release. This effect may be concentration dependent since lower amounts of L-DOPA have been shown to increase DA uptake while higher amounts have inhibited DA uptake via DAT (Hashimoto et al., 2005). L-DOPA-induced increase in DAT may be mediated by L-DOPA-derived DA overactivation of D2 receptors which have been shown to upregulate DAT activity and membrane expression through intracellular pathways, including extracellular signal-regulated kinases and protein kinase A (PKA; Batchelor & Schenk, 1998; Bolan et al., 2007). However, DAT binding has also been unaffected by L-DOPA treatment which potentially reflects how L-DOPA can also be taken up by the amino acid transport system (Audus and Borchardt, 1986; Diesel et al., 1998; Schillaci et al., 2005). Presently, L-DOPA treatment dampened the increase extracellular DA seen in the vehicle-treated striatum suggesting L-DOPA-induced interference of DAT function (Fig. 6). The present impact of L-DOPA on DAT appears to be specific to function rather than expression, likely contributing to the irregularities in DA transmission that underlie LID expression. Reduced DAT uptake and altered DA release following L-DOPA may serve to promote healthy movement early in PD, but may eventually lead to LID development (Hong et al., 2014; Lee et al., 2008; Sossi et al., 2009; Troiano et al., 2009).

SERT levels are often cited as a proxy for 5-HT neuronal integrity given their location on raphestriatal projections and soma (Hahn et al., 2014; Nicastro et al., 2020; Zhou et al., 1998). Although changes in striatal SERT expression in parkinsonian pathophysiology are variable, SERT appears to play a role in PD manifestation. Not only have SERT genetic polymorphisms been associated with elevated risk for PD (Zhang et al., 2014), but reduced striatal SERT expression negatively correlates with clinical tremor, anxiety, and fatigue as well as with 6-OHDA lesion severity in PD rodents (Joling et al., 2018; Loane et al., 2013; Pavese et al., 2010; Weng et al., 2013). Indeed, progressive SERT loss in the DA-depleted striatum may be indicative of raphestriatal 5-HT neuronal damage in PD patients and preclinical models (Buddhala et al., 2015; Kerenyi et al., 2003; Pagano et al., 2017; Politis et al., 2010; Roussakis et al., 2016). Conversely, reports of no change or even increased striatal SERT expression in PD suggest compensatory measures when DAT expression is limited (Bedard et al., 2011; Cheshire et al., 2015; Gagnon et al., 2016; Lanza and Bishop, 2018; Strecker et al., 2011;). The current neurochemical analysis indicated no effect of unilateral DA loss on DR SERT mRNA (Fig. 3) or P2 striatal SERT protein (Fig. 4). Reports of striatal SERT loss in preclinical models have typically required bilateral DA deficit (Berghauzen-Maciejewska et al., 2016; Li et al., 2009) or found large variations in the degree of SERT loss in a unilateral DA lesion (Wang et al., 2010). The present lack of effect on striatal SERT expression may reflect the nature of the unilateral DA lesion or individual differences across 6-OHDA-lesioned rats. Most reports show that striatal SERT positively correlates with DAT in the parkinsonian state, indicating related down-regulation to the 5-HT system (Berghauzen-Maciejewska et al., 2016; Li et al., 2009; Wang et al., 2010). Interestingly, in the progressive MPTP toxin non-human primate model that exhibits motor symptom recovery, SERT in the putamen was elevated in the full symptomatic state and inversely correlated with DA cell death and maximal parkinsonian score (Ballanger et al., 2016). This may reflect the transient nature of the symptoms in this model or an increase in SERT to compensate for the loss of DA uptake via DAT (Kannari et al., 2006).

5-HT neurons play an active role in steady state DA release and uptake as they have the machinery to synthesize DA from exogenous L-DOPA and release DA into the synapse for subsequent reuptake via SERT (Berger, 1978; Carta et al., 2007; Kannari et al., 2006; Miguelez et al., 2016; Navailles et al., 2010). L-DOPA has been shown to upregulate striatal SERT expression and 5-HT terminals implicating the enhanced role for 5-HT neurons in DA neurotransmission (Rylander et al., 2010; Walker et al., 2020). However, 5-HT neurons lack high-affinity DA auto-regulatory mechanisms, like D2 and DAT, which lead to large surges in synaptic DA resulting in LID (Carta et al., 2007; Lindren et al., 2014; Politis et al., 2014; Sellnow et al., 2019). While reductions in striatal DAT binding corresponded with L-DOPA-induced hyperlocomotion, increases in striatal SERT binding have been shown to positively correlate with LID severity in dyskinetic rat and primate models as well as in PD patients implicating that SERT is in a position to clear aberrant DA transmission (Beaudoin-Gobert et al., 2018; Conti et al., 2016a; Nikolaus et al., 2016; Politis et al., 2014; Rylander et al., 2010; Smith et al., 2015). However, Pagano and colleagues (2017) report no difference in SERT binding in putamen or caudate nucleus of non-dyskinetic and dyskinetic PD patients. In the current findings, L-DOPA had no significant impact on DR SERT mRNA or P2 striatal SERT expression (Figs. 3 & 4). Even though L-DOPA treatment did not alter striatal SERT expression, a treatment-induced increase in the P2 striatal SERT: DAT ratio was found. This increase was not simply due to a lesion-induced DAT reduction but also depended upon DA depletion and L-DOPA treatment together. Such a shift suggests compensatory mechanisms due to severe DAergic cell loss that are contingent upon L-DOPA-induced elevations in DA signaling. While the current data reflects post-mortem SERT:DAT ratios, elevated striatal SERT:DAT expression quantified by in vivo imaging studies have been shown in dyskinetic versus non-dyskinetic PD patients (Lee et al., 2015; Politis et al., 2011; Roussakis et al., 2016). In previous work utilizing such ratios, PD patients who experienced dyskinesias had greater SERT:DAT in the putamen compared to those who did not experience dyskinesia (Roussakis et al., 2016). As such, SERT:DAT may serve as a potential biomarker for dyskinesia or indicate the efficacy of 5-Htergic treatments. An important caveat to P2 preparation is that the entire synaptosomal space is quantified, including some cytosolic transporter expression and not sole membrane surface expression. Therefore, other techniques, such as measuring binding with a membrane-specific biotin analog or uptake of radioactive tracers, are necessary for determining changes in functionally expressed, surface monoamine transporters in the DA-lesioned and L-DOPA-treated striatum (Cai et al., 2012; Salvatore et al., 2003).

A functional change in SERT plasticity was further supported by preferential DA uptake via SERT in the dyskinetic striatum (Fig. 6). This resonates well with increased SERT:DAT ratios in the dyskinetic striatum, further suggesting SERT’s prominent role in LID expression and how SERT:DAT may serve as a marker for the L-DOPA response. Increased uptake may be an adaptation whereby 5-HT neurons clear the synapse of dysregulated extracellular DA. Indeed, even with SERT’s high affinity for 5-HT uptake, DA uptake via SERT engages a separate uptake mechanism which leads to non-competitive 5-HT and DA transport (Kaminska et al., 2018; Larsen et al., 2011; Tatsumi et al., 1997). However, when other modes of DA regulation, such as enzymatic breakdown or other reuptake transporters, are present, SERT’s role in DA uptake can be limited (Larsen et al., 2011). Therefore, optimizing SERT function in the raphe-striatal circuit may temper L-DOPA-derived DA levels to alleviate dyskinesia (Meadows et al., 2018; Smith et al., 2022). A limitation of the ex vivo microdialysis technique is that residual DA release from remaining TH and catabolism of extracellular DA were not accounted for when determining changes in DA uptake (Salvatore et al., 2016; Snyder et al., 1990). This may be especially relevant considering the potential differences in basal DA levels across striatal conditions: intact versus DA-lesioned and vehicle versus L-DOPA treatment (Jeon et al., 1995; Omiatek et al., 2013). However, each sample served as its own control and DA uptake data was normalized to monoamine transporter vehicle treatment thus accounting for endogenous DA levels.

Because DA deficit was necessary for L-DOPA’s effects, there may be a threshold of DA depletion that is necessary for L-DOPA-induced SERT gain-in-function similar to a threshold for LID development and expression (Bez et al., 2016; Shan et al., 2015). Additionally, understanding the status of other mechanisms that are capable of DA uptake across PD stages would allow for a more complete view of DA transmission influenced by both DA loss and L-DOPA treatment. The norepinephrine transporter has previously been shown to have the capacity for DA uptake (Chotibut et al., 2014). Glial expression of DAT, SERT, and organic cation transporters, also capable of DA uptake, implicates not only neuronal, but also glial regulation of DA signaling (Asanuma et al., 2014; Cui et al., 2009; Inazu et al., 2001; Takeda et al., 2002).

Although the present data supports the 5-HT hypothesis of LID, there are several limitations of the current study worth mentioning for future studies. Firstly, the male only subject pool leaves the question of how the serotonergic circuit may reorganize following 6-OHDA lesion and chronic L-DOPA treatment in the female rodent brain. While MFB lesions like those used in the present model that induce > 80% dopaminergic loss are not sensitive to biological sex (Gillies et al., 2014 Aug), it remains unknown if there are significant functional differences in SERT between male and female rodent models. Studies evaluating sex differences and selective serotonin reuptake inhibitor (SSRIs) treatments have hinted that females respond better to SSRIs than to men, suggesting a more responsive serotonergic system to neuroplasticity in females not only in depressive states, but also neurodegenerative states (Sramek et al., 2016). In human PD condition, there is a strong prevalence in men over women, which further supports the need for future studies evaluating the differences with LID treatments.

Despite debilitating side effects, L-DOPA remains the gold-standard symptomatic treatment in PD. In the present work we revealed a compensatory shift and gain-in-function of SERT in the dyskinetic, parkinsonian striatum. Such mechanisms must be directly tested in more clinically relevant designs since compounds that selectively block DAT and SERT are readily available. Monoamine transporters are shown to be vital players in tempering dysregulated DA, working with such endogenous compensatory mechanisms may prove to be an ideal therapeutic strategy for dyskinetic PD patients.

Abbreviations:

6-OHDA

6-hydroxydopamine

AIMs

Abnormal involuntary movements

ALO

Axial, limb, orolingual

Benserazide

DL-serine 2-(2,3,4-trihydroxybenzyl) hydrazine hydrochloride

CIT

Citalopram

DA

Dopamine

DAT

Dopamine transporter

DR

Dorsal raphe nucleus

FAS

Forepaw adjustment steps

GBR12909

1-(2-(bis-(4-fluorophenyl)-methoxy)ethyl)-4-(3-phenylpropyl) piperazine) dihydrochloride

HPLC

High-performance liquid chromatography

L-DOPA

L-3,-4-dihydroxyphenylalanine

LID

L-DOPA-induced dyskinesia

MFB

Medial forebrain bundle

M.A.D

Median absolute deviation

P2

Synaptosome

PD

Parkinson’s disease

RT-PCR

Real-time reverse transcription polymerase chain reaction

SSRI

Selective serotonin reuptake inhibitor

5-HT

Serotonin

SERT

Serotonin transporter

S.E.M

Standard error of the mean

SNc

Substantia nigra pars compacta

TH

Tyrosine hydroxylase

TPH

Tryptophan hydroxylase

VEFP

Vibrissae-evoked forelimb placing

Data availability

Data will be made available on request.