Transporters involved in adult rat cortical astrocyte dopamine uptake: Kinetics, expression and pharmacological modulation

Astrocytes, glial cells in the central nervous system, perform a multitude of homeostatic functions and are in constant bidirectional communication with neuronal cells, a concept named the tripartite synapse; however, their role in the dopamine homeostasis remains unexplored. The aim of this study was to clarify the pharmacological and molecular characteristics of dopamine transport in cultured cortical astrocytes of adult rats. In addition, we were interested in the expression of mRNA of dopamine transporters as well as dopamine receptors D1 and D2 and in the effect of dopaminergic drugs on the expression of these transporters and receptors. We have found that astrocytes possess both Na+‐dependent and Na+‐independent transporters. Uptake of radiolabelled dopamine was time‐, temperature‐ and concentration‐dependent and was inhibited by decynium‐22, a plasma membrane monoamine transporter inhibitor, tricyclic antidepressants desipramine and nortriptyline, both inhibitors of the norepinephrine transporter. Results of transporter mRNA expression indicate that the main transporters involved in cortical astrocyte dopamine uptake are the norepinephrine transporter and plasma membrane monoamine transporter. Both dopamine receptor subtypes were identified in cortical astrocyte cultures. Twenty‐four‐hour treatment of astrocyte cultures with apomorphine, a D1/D2 agonist, induced upregulation of D1 receptor, norepinephrine transporter and plasma membrane monoamine transporter, whereas the latter was downregulated by haloperidol and L‐DOPA. Astrocytes take up dopamine by multiple transporters and express dopamine receptors, which are sensitive to dopaminergic drugs. The findings of this study could open a promising area of research for the fine‐tuning of existing therapeutic strategies.


| INTRODUCTION
Astrocytes, the most abundant cell type in the central nervous system (CNS) (Parpura et al., 2012;Semyanov & Verkhratsky, 2021;Verkhratsky & Nedergaard, 2018), play a vital role in maintaining CNS homeostasis and are actively involved in various functions.They are crucial for synapse formation and regulation, influencing synaptic connectivity, plasticity, and responding to pathological insults (Haydon et al., 2009;Papouin et al., 2017;Prebil et al., 2011;Verkhratsky et al., 2019).Despite lacking the ability to generate action potentials, astrocytes engage in constant bidirectional communication with neurons, forming what is known as the tripartite synapse (Farhy-Tselnicker & Allen, 2018;Lim et al., 2021;Perea et al., 2009;Verharen et al., 2020).Through fluctuations in intracellular second messengers such as Ca 2+ and the release of gliotransmitters, astrocytes respond to synaptic activity and contribute to the regulation of synaptic transmission and plasticity (Bazargani & Attwell, 2016;Perea et al., 2009).While their involvement in glutamate homeostasis is well studied (Mahmoud et al., 2019;Parpura & Verkhratsky, 2012), their specific role in the homeostasis of neurotransmitter dopamine (DA) remains less explored.
DA plays a vital role in various processes including spatial memory formation, motivation, arousal, reward, pleasure, lactation, sexual behaviour, nausea and motor function (Beaulieu & Gainetdinov, 2011).Clearance of DA from the extracellular space back into the presynaptic neuronal cells occurs via two types of transport mechanisms: high-affinity, low-capacity uptake 1 transport and slower, low-affinity, high-capacity uptake 2 transport (Iversen, 2000;Jennings et al., 2016;Nishijima & Tomiyama, 2016).Uptake 1 transport of DA primarily relies on the dopamine transporter (DAT) (Cheng & Bahar, 2015) or norepinephrine transporter (NET), depending on the brain region and transporter availability.In certain brain regions with sparse DAT distribution, such as the cortex, NET has been found to have equal affinity for DA and plays a prominent role in DA uptake, especially in conditions like Parkinson's disease (PD) (Mor on et al., 2002;Nishijima & Tomiyama, 2016).Both uptake 1 transporters share structural similarities, with similar amino acid sequences (Klein et al., 2019), and their functionality depends on the Na + /Cl À ion gradient provided by the Na + /K + ATPase (Furihata & Anzai, 2017;Iversen, 2000;Jennings et al., 2016;Nishijima & Tomiyama, 2016).These transporters are important targets for drugs used in the treatment of various neuropsychiatric disorders (Iversen, 2000).In addition to the uptake 1 transporters, DA transport is also mediated by uptake 2 transporters, capable of transporting multiple organic cations, namely organic cation transporters OCT1, OCT2 and OCT3 (Chemuturi & Donovan, 2007;Gasser, 2019Gasser, , 2021) ) and the plasma membrane monoamine transporter (PMAT) (Dahlin et al., 2007;Duan & Wang, 2010;Vieira & Wang, 2021;Zhou et al., 2007).The coexistence of both low-and highaffinity neurotransmitter transporters enables rapid adjustments of monoamine neurotransmitter concentrations based on location and function.In the presence of drugs inhibiting high-affinity transporters, the role of lowaffinity transporters becomes crucial for the reuptake of neurotransmitters (Koepsell, 2021).
DA exerts its effects by binding to two types of DA receptors (DR).D1-type receptors (DRD1 and DRD5) are excitatory, while D2-type receptors (DRD2, DRD3 and DRD4) are inhibitory (Cooper et al., 2004;Jennings et al., 2016).Astrocytes have been reported to express various DA receptors, but their expression pattern can vary across studies (Bal et al., 1994;Horvat & Vardjan, 2019;Miyazaki et al., 2004).Activation of DA receptors in astrocytes can lead to changes in intracellular Ca 2+ levels and other signalling molecules, such as cAMP, with effects that require further investigation (Horvat & Vardjan, 2019;Perea & Araque, 2005).Emerging evidence suggests that astrocyte DA receptors respond to drugs used in the treatment of neuropsychiatric disorders, such as PD and schizophrenia, by increased astrocyte DA clearance in PD, uptake and accumulation of L-DOPA in rat astrocytes, and changes in intracellular signalling pathways in rat astrocytes after exposure to DA agonists and antipsychotic medications (Asanuma et al., 2014;Jennings et al., 2016;Miyazaki et al., 2004).
According to our knowledge, this is the first study to use primary astrocyte cultures prepared from Wistar adult rats as a model system to study DA uptake.The aim of our study was to investigate DA uptake into cultured adult rat cortical astrocytes, elucidate the kinetic properties of DA transport and identify the specific transporters involved.In addition to that, we explored whether treatment with drugs used to combat diseases such as PD, schizophrenia and depression might induce changes in the expression of transporters and receptors involved in astrocyte DA homeostasis.Understanding the role of astrocytes in the dopaminergic synapse may provide valuable insights into the pathogenesis of CNS disorders and potentially uncover new therapeutic strategies.

| Animals
For the purpose of our study, we have obtained the permission of the Administration of the Republic of Slovenia for Food Safety, Veterinary and Plant Protection issue U34401-20/2017/2.This study was approved by the National Veterinary Administration (approval numbers U34401-23/2022/6, approval date 20.06.2017 and U34401-23/2022/6, approval date 23.12.2022).We used brain from redundant (10 in total) sexually mature animals, weighing 180-200 g of both genders, and all procedures complied with the relevant Slovenian and European legislation.Animals sacrificed for the purpose of removing organs (brains) were euthanized by decapitation after anaesthesia with CO 2 , which was assessed as the most suitable method.All necessary measures were used for the prevention of unnecessary suffering and discomfort of the used laboratory animals.

| Primary astrocyte cultures
We obtained cortical and kidney tissue from adult rats, species Rattus norvegicus, breed Wistar.Cultured rat cortical astrocytes were prepared from adult rat cortices by a well-established protocol, routinely used in our laboratory (Krzan & Schwartz, 2006).Astrocyte cultures were grown and maintained in T-75 flasks (Falcon, Corning, USA) and 12-well plates (Falcon).Briefly, cells were grown in high-glucose Dulbecco's modified Eagle's medium (DMEM), containing 10% FBS, 1 mM pyruvate, 2 mM glutamine and 25 μg/mL gentamycin/streptomycin in humidified 95% air-5% CO 2 atmosphere.Confluent cultures were shaken at 225 RPM overnight, the medium was changed the next morning and this process was repeated a total of three times.After the third overnight shaking, the cells were trypsinized, washed in DMEM and subcultured into 12-well plates; the medium was changed each week from then on.After 3 weeks, the cultures contained 93%-100% type 1 astrocytes.

| [ 3 H]-dopamine uptake experiments
i. Temperature, time and concentration dependence of [ 3 H]-DA uptake Monolayer cultures in 12-well plates were washed twice in the uptake buffer containing CaCl 2 (25 mM HEPES, 125 mM NaCl, 4.8 mM KCl, 1.2 mM KH 2 PO 4 , 1.2 mM MgSO 4 , 1.4 mM CaCl 2 , and 5.6 mM glucose, pH 7.4) and MAO (tropolone, 0.1 mM) and COMT (Ldeprenyl, 0.1 mM) inhibitors at 37 C (total uptake) and at 4 C (non-specific uptake).The uptake buffer was added to each well in addition to [ 3 H]-DA, resulting in a total volume of 500 μL.For the time course (0-60 min) curve, transport was initialized by the addition of radiolabelled DA and stopped by placing the plates into an icewater bath at different time points.The buffer was removed, and the plates were washed four times with icecold uptake buffer without Ca 2+ .The cells were lysed in 300 μL of 0.5 M NaOH.From each well, 250 μL of the sample was transferred to a scintillation vial, and the radioactivity was measured.From 4 μL of each sample, we measured the protein concentration using the Bradford method using the Bio-Rad Protein Assay (Hercules, CA, USA).The design of the experiments was the same for the concentration dependence curve, where the experiments were performed with the [ 3 H]-DA concentration ranging from 0.03 to 750 μM and the reaction was stopped after 15 min for each tested concentration.
ii. Inhibition by antidepressants and decynium-22 and ouabain sensitivity of [ 3 H]-DA uptake For the inhibition studies, monolayer cultures in 12-well plates were prepared in the same manner as described previously and preincubated in different test compounds at various concentrations (10 À8 -10 À3 M) for 20 min and then incubated with the [ 3 H]-DA concentration of 30 nM for 15 min.The chosen test compounds were desipramine, nortriptyline and amitriptyline and non-specific uptake transporter inhibitor decynium-22 (D22).The reaction was stopped by placing the plates on ice and washing with ice-cold uptake buffer without Ca 2+ in the same manner as described above.The cells were lysed by the addition of 0.5 M NaOH, samples collected and DA uptake determined in the same manner as described above.Ouabain sensitivity was tested by a 20-min preincubation of cell plates.

| Quantitative polymerase chain reaction (qPCR)
Total RNA was extracted from adult rat cortical astrocyte cell cultures, as well as cortical and kidney tissue samples using the E.Z.N.A. ® Total RNA Kit I.One microgram of RNA was used to synthesize cDNA utilizing High-Capacity cDNA Reverse Transcription Kit, according to manufacturer's instructions.qPCR was performed using TaqMan™ Universal PCR Master Mix and TaqMan Assays: , SLC22A3 (Rn00570264_m1), SLC29A4 (Rn01453824_m1), DRD1 (Rn03062203_s1), DRD2 (Rn00561126_m1) and ß-actin (Rn00667869_m1), according to the manufacturer's instructions, in QuantStudio™ 3 System.Expression of target genes was normalized to expression of ß-actin according to the equation [target/reference] = [EFF refer- ence ^Cq reference ]/[EFF target ^Cq target ], where Cq is quantification cycle and EFF is amplification efficiency (expressed as a value between 1 and 2).EFF was determined with LinRegPCR software (Ruijter et al., 2009).

| Cell treatments
Confluent 3-week-old adult rat cortical astrocyte cell cultures plated in 12-well plates were treated with three different compounds, haloperidol, apomorphine and L-DOPA, for 24 h.The concentration of drugs used for the cell treatment was set at 100 μM, based on cell viability experiments.The viability of astrocytes, plated in 96-well plates, was examined after 24-h treatment with the drugs at concentrations up to 150 μM, using CellTiter 96 ® AQueous One Solution Cell Proliferation Assay (MTS).

| Data analysis
The uptake experiments were routinely carried out in triplicates, and each experiment was repeated at least twice.All data are indicated as arithmetic means ± SEM.From the cell cultures on the 12-well plates, three wells were used as a triplicate in each experiment, which was then repeated at least twice (n = 6) or three times (n = 9), using different cell cultures, cultivated from different animals.Data and statistical analysis were performed in consideration to each experiment with GraphPad Prism 9.5 software (San Diego, USA).Comparison between means of two samples was carried out using the t-test (Student's ttest, Welch's t-test, Holm-Šidak method t-test vs. control).Comparison between multiple groups of data was performed using ANOVA.Ordinary one-way ANOVA with post hoc Dunnett's correction or Brown-Forsythe ANOVA test with Dunnett's T3 multiple comparisons test was used to analyse the difference between the means of more than two groups.Two-way ANOVA with post hoc test Bonferroni correction for multiple comparisons was used for analysis of the cell culture treatments.Differences were considered significant at p < 0.05.

| Time and temperature dependence of [ 3 H]-DA uptake into cultured adult rat astrocytes
Astrocyte cultures were prepared from cortices of adult Wistar rats as previously described (Krzan & Schwartz, 2006;Schwartz & Wilson, 1992).Basic characteristics of DA transport (time and concentration dependence) were determined first, since phenotypic properties of cultured astrocytes can be affected by the method of preparation, culture conditions and animal species used (Codeluppi et al., 2011).Astrocytes were incubated with 30 nM [ 3 H]-DA, and the characteristics of time and temperature dependence were assessed at 37 C, determined as total DA uptake, and at 4 C, determined as nonspecific uptake, based on previous studies on cultured rat astrocytes (Perdan-Pirkmajer et al., 2012).Cortical astrocytes were exposed to 30 nM concentration of [ 3 H]-DA for a maximal time span of 60 min.[ 3 H]-DA uptake reached a plateau at 20 min (Figure 1).Based on these experiments, the incubation time of DA uptake was set to 15 min.

| Ouabain sensitivity of [ 3 H]-DA uptake into adult rat astrocytes
Cell cultures were preincubated at 37 C with 0.1 and 1 mM concentration of ouabain for 20 min (Inazu, Takeda, et al., 1999).Ouabain is a cardiac glycoside that specifically targets and blocks the Na + /K + ATPase, which is responsible for maintaining the electrochemical gradient of sodium and potassium ions across the cell membrane and is therefore crucial for transport mediated by uptake 1 transporters such as NET (Ogawa et al., 2009).DA uptake was measured after 15 min incubation with 30 nM [ 3 H]-DA at 37 C. DA uptake was significantly reduced in comparison to the control by 1 mM concentration of ouabain (44 ± 8%), whereas 0.1 mM concentration of ouabain had no significant effect of DA uptake, which was reduced to 69 ± 13% in comparison to the control 100 ± 23% (Figure 2).

| Concentration dependence of [ 3 H]-DA uptake in cultured adult rat astrocytes
Astrocytes were exposed to different concentrations of [ 3 H]-DA (0.03-750 μM) for a time span of 15 min, in line with the time course experiments, and the total (37 C) and the non-specific (4 C) DA uptake were measured.Specific uptake, from which the uptake kinetic characteristics were determined, was calculated as the difference between the total (37 C) and non-specific (4 C) uptake.DA uptake into cultured astrocytes, presented in Figure 3a, was clearly dependent on DA concentration; however, it does not appear fully saturable at even the highest concentration of 750 μM.DA uptake velocity (Figure 3b) was calculated from the specific uptake and the time span of [ 3 H]-DA incubation (15 min).The kinetic parameters of uptake velocity were calculated using Michaelis-Menten equation.The apparent maximal uptake rate, V max , of DA was calculated as 75 pmol/ mg/min, and the apparent Michaelis-Menten constant, K m , was calculated as 1134 μM.
Astrocytes are capable of DA uptake, which is time-, temperature-and concentration-dependent.We presumed DA uptake is mediated by multiple transporters; therefore, we examined the mRNA expression of transporters involved in DA uptake, such as uptake F I G U R E 1 Time dependence of [ 3 H]-DA uptake into adult rat cortical astrocytes.Concentration (30 nM) of [ 3 H]-DA was added at different time stamps (60, 45, 30, 20, 15, 10 and 5 min).Data from three separate experiments are represented as mean ± SEM (n = 9).The non-specific uptake was lower compared to the total uptake at each time stamp (Student's t-test, p < 0.05).
F I G U R E 2 Ouabain sensitivity of the total [ 3 H]-DA uptake into adult rat astrocytes.Astrocytes were incubated with 30 nM [ 3 H]-DA for 15 min at 37 C under normal conditions and in the presence of 0.1 mM and 1 mM concentration of ouabain.Data are presented as mean of [ 3 H]-DA uptake ± SEM (n = 5), and statistical significance was determined by ordinary one-way ANOVA with Dunnett's correction, ns = non-significant, **p = 0.004. 1 transporters DA, DAT and NET and uptake 2 transporters OCT1, OCT2, OCT3 and PMAT.

| mRNA expression of transporters involved in DA uptake in adult rat cortical astrocytes
We examined the mRNA expression of high-affinity and low-affinity transporters involved in DA uptake in adult rat astrocytes as well as adult rat cerebral cortex and kidney tissue serving as positive control.qPCR was performed after extraction of the total RNA from 3-week-old confluent cultures and tissue samples; the latter were used as a positive control to validate our gene expression assays.
The mRNA expression profile of the transporters in adult rat cortical and kidney tissue (Figure 4b) was in line with our expectations.In the cortical tissue, among the low-affinity transporters, PMAT expression was most prominent, followed by OCT3 expression, which is among the three subtypes of OCTs considered the primary uptake 2 transporter in the CNS.In the kidney tissue, as expected, OCT1 and OCT2 mRNA expression exceeded OCT3 and PMAT mRNA expression.DAT and NET were present in both rat tissue samples; however, NET expression was more prominent in rat cortical tissue than in kidney tissue.We observed a distinct mRNA expression profile in astrocyte cultures, as tissue samples include neurons, where mRNA of the examined transporters, particularly DAT, is greater.Astrocyte cultures lack DAT mRNA expression, which may be either due to their inherent characteristic or due to the long-term cultivation under non-physiological conditions, lacking DA stimulation.Transporter expression was statistically significantly different (Brown-Forsythe ANOVA test: F* [DFn, DFd] = 117.0[5.000, 4.785], p < 0.0001).The mRNA expression of all three OCTs in adult rat cortical astrocytes appears to be relatively low.Interestingly, among the three examined OCTs, OCT1 exhibits the highest mRNA expression, followed by OCT2 expression, while comparatively, OCT3 expression is nearly 100 times lower (Figure 4a).In astrocytes, mRNA expression of PMAT statistically significantly F I G U R E 3 Concentration dependence of [ 3 H]-DA into cultured adult rat cortical astrocytes.DA uptake into astrocytes is concentration-and temperature-dependent.Experiments were repeated three times (n = 9), and results are expressed as mean ± SEM.Specific uptake was calculated as the difference between total uptake (37 C) and non-specific uptake (4 C) (a).Uptake velocity was calculated from the Michaelis-Menten equation, apparent maximal uptake rate V max = 75 ± 48 pmol/mg/min and apparent Michaelis-Menten constant K m = 1134 ± 1051 μM (b).
F I G U R E 4 mRNA expression of transporters DAT, NET, PMAT, OCT1, OCT2 and OCT3 in adult rat cortical astrocytes (a), adult rat cortical tissue (b) and adult rat kidney tissue (c).Results are expressed relative to the expression of OCT3.Data are represented as mean ± SEM (n = 6).mRNA expression is normalized to the mRNA expression of the endogenous control ß-actin.
exceeded mRNA expression of all three OCTs; however, it was lower than NET mRNA expression, contrarily to the rat cortical tissue, where PMAT mRNA expression was the most prominent.We have not been able to confirm DAT mRNA expression in adult rat cortical astrocytes, suggesting NET and PMAT might serve as the primary transporters involved in DA uptake into cultured adult rat cortical astrocytes.The statistical analysis of pairwise comparisons between mRNA expression of the examined transporters is displayed in Table 1.

| The effect of inhibitor decynium-22 and antidepressants on [ 3 H]-dopamine uptake
After establishing that the main transporters that could transport DA into cultured rat astrocytes are NET and PMAT, we examined the effect of norepinephrine uptake inhibitors (Gillman, 2007) nortriptyline and desipramine (Figure 5a), non-selective reuptake inhibitor amitriptyline (data not shown) and D22 (Figure 5b), a PMAT and OCT inhibitor (Fraser-Spears et al., 2019;Wang, 2016).Threeweek-old cultures were preincubated with the transporter inhibitors for 20 min in the uptake buffer at 37 C prior to the addition of 30 nM [ 3 H]-DA.The uptake was measured after 15 min.
Antidepressants desipramine and nortriptyline (0.01-100 μM) produced a significant inhibition of [ 3 H]-DA uptake from 0.1 μM onwards for desipramine and from 1 μM onwards for nortriptyline (Figure 4), whereas amitriptyline had no significant effect on DA uptake.Nevertheless, these results suggest NET may have a significant role in DA uptake into cultured astrocytes.D22 inhibited DA uptake at 0.01 μM already, and uptake was reduced to approximately 80% (78.318 ± 5.153%) in comparison to the control.The maximal total DA uptake inhibition by all three compounds reached only approximately 50%.

| Dopamine receptor expression in adult rat cortical astrocytes
After establishing the mRNA transporter expression in our cell cultures of adult rat cortical astrocytes, we wanted to examine whether astrocytes express DA receptor mRNA.Results presented in Figure 6 show a fairly low mRNA expression of both receptor subtypes (Welch's t-test, p > 0.05).
The mRNA expression signal of DRD2 was at the limit of detection; therefore, we proceeded to perform only qPCR analysis of DRD1 in further experiments.

| Changes in mRNA expression of NET, PMAT and DRD1 in adult rat cortical astrocytes
In previous in vitro studies, concentrations of drugs such as (atypical) antipsychotics varied within the range of 1-100 μM (Bai et al., 2006).Therefore, in this study, we chose the concentrations of apomorphine, haloperidol and L-DOPA in this range and, based on preliminary experiments, decided on the largest concentration, which did not cause a reduction in cell viability.Adult rat cortical astrocytes were treated with 100 μM concentration of apomorphine, haloperidol and L-DOPA for 24 h.The viability of the cells at concentrations from 1 to 150 μM of the drugs was tested in a separate experiment using the MTS assay (Figure 7).Astrocyte viability was significantly reduced (two-way ANOVA, Bonferroni correction for multiple comparisons, ****p < 0.0001), by apomorphine at 150 μM concentration, whereas haloperidol and L-DOPA had no significant effect on astrocyte viability even at the highest concentration of 150 μM (Figure 7).
After establishing the effect the compounds on cell viability, we proceeded to test whether these drugs affect mRNA expression of transporters NET and PMAT, which have been shown to have the most prominent expression in our astrocyte cultures, as well as of receptor DRD1.We found a significant interaction between mRNA expression (NET [F = 7.014 (3.000, 12.19); p = 0.0021], PMAT [F = 96.25 (3.000,7.238);p < 0.0001], DRD1 [F = 10.07 (3.000, 12.12); p = 0.0013]) and dopaminergic treatments (Figure 8).This interaction was subsequently evaluated with pairwise comparison for each specific treatment.

| DISCUSSION
We examined the pharmacological and molecular characteristics of DA uptake into cultured cortical astrocytes of adult rats in the presence of MAO and COMT inhibitors, while earlier studies focused on glial cell cultures prepared from neonatal rats.Although, in the rodent forebrain, the majority of astrocytes are F I G U R E 5 Effect of antidepressants (a) nortriptyline and desipramine and D22 (b) on DA uptake into cultured adult rat astrocytes.Astrocytes were preincubated with these compounds for 20 min prior to the 15 min incubation with 30 nM concentration of [ 3 H]-DA at 37 C (n = 9).Results are represented as percent (mean ± SEM) of the control.IC 50 and pIC 50 (calculated as the negative log of the corresponding IC 50 values) of compounds inhibiting the total [ 3 H]-DA uptake into cultured adult rat astrocytes were calculated from the corresponding inhibition curves.Nortriptyline: IC 50 = 0.225 ± 0.188 μM, pIC 50 = 6.647, maximal inhibition in comparison to the control: 64%; desipramine: IC 50 = 0.0254 ± 0.0140 μM, pIC 50 = 7.595, maximal inhibition in comparison to the control: 54%; D22: IC 50 = 0.813 ± 0.956 μM, pIC 50 = 6.090, maximal inhibition in comparison to the control: 46%.Statistical significance was determined by Holm-Šidak method t-test versus control, *p < 0.05.F G U R E 6 mRNA expression of DA receptors DRD1 and DRD2 in adult rat cortical astrocytes.Data are normalized to the endogenous control ß-actin and expressed as mean ± SEM (n = 10 for DRD1, n = 8 for DRD2, three separate experiments).Statistical significance of the difference between the expressions of the two receptor subtypes was determined with Welch's t-test, p >0.05, ns = non-significant.generated during the early postnatal phase, many of the plasma membrane proteins develop during later stages of animal development (Felix et al., 2021); therefore, adult rat cultures might be a more suitable in vitro model for the study of DA transporter systems in astrocytes.
In the present study, we provide evidence that adult rat cortical astrocytes are capable of time-, temperatureand concentration-dependent DA uptake, which is significantly reduced by the lack of presence of sodium ions, indicating involvement of both Na + -dependent and Na +independent uptake mechanisms.Astrocyte cultures F I G U R E 7 Adult rat astrocyte viability after 24-h treatment with apomorphine, haloperidol and L-DOPA was tested using the MTS assay and is presented as percent of the control ± SEM from three separate experiments (n = 12).Apomorphine induced a significant reduction in cell viability at the concentration of 150 μM; statistical significance was tested using two-way ANOVA with Bonferroni correction for multiple comparisons, ****p = 0.0002.
F I G U R E 8 Changes in mRNA expression of (a) DRD1, (b) NET and (c) PMAT after 24-h treatment with apomorphine, haloperidol and L-DOPA.Data are expressed as mean ± SEM (n = 6) from two separate experiments and normalized to the mRNA expression of the endogenous control, ß-actin.Changes in mRNA expression expressed relative to the control: DRD1: control 1.000 ± 0.119, apomorphine 4.733 ± 0.835, haloperidol 2.115 ± 0.668, L-DOPA 1.596 ± 0.442; NET: control 1.000 ± 0.360, apomorphine 3.452 ± 1.061, haloperidol 1.573 ± 0.674, L-DOPA 1.161 ± 0.317; PMAT: control 1.000 ± 0.080, apomorphine 2.575 ± 0.225, haloperidol 0.649 ± 0.058, L-DOPA 0.604 ± 0.048.Statistical significance was determined by Brown-Forsythe ANOVA test with Dunnett's T3 multiple comparisons test: ns = nonsignificant, *p < 0.05, **p = 0.0025, ***p < 0.0005.exhibited characteristics of saturation, which was however not reached even at the highest tested concentration of 750 μM, emphasizing the importance of a highcapacity carrier system in DA uptake as well as highlighting astrocytes' role in the cortical synapse, as they are capable of continuous DA clearance, even at high concentrations of DA.The kinetic characteristics of DA uptake presented in the present study are in line with previous work on neonatal rat astrocytes.Inazu et al. examined DA uptake into cortical astrocytes of neonatal rats and found DA uptake is time-, temperature-and concentration-dependent (Inazu, Takeda, et al., 1999), which however not saturable at even the highest concentration used (1 mM).Pelton et al.'s and Semenoff et al.'s works on primary astrocyte cultures from the cerebral hemispheres of neonatal rats also reached a similar conclusion, and astrocyte DA uptake was concentration dependent and metabolized in by COMT and MAO.Additionally, these studies reported a significant reduction of DA uptake in the presence of Na + /K + -ATPase inhibitor ouabain as well as presence of Na + -dependent uptake at lower concentrations of DA (Kimelberg, 1986;Pelton et al., 1981;Semenoff & Kimelberg, 1985), indicating the existence of an active transporter in neonatal rat astrocytes.Nevertheless, it is worth noting that Hansson et al. (Hansson, 1985;Hansson et al., 1985) and Hösli and Hösli (1997) reported neonatal astrocytes do not show saturable kinetics for DA, which they found to be Na + -independent leading them to question the existence of a high-affinity carrier system in astrocytes.Differences in Na + -dependency of DA uptake can be explained by culture conditions and heterogeneity among astrocytes.
Inhibition of DA uptake by antidepressants desipramine and nortriptyline, both NET selective inhibitors as well as an OCT and PMAT inhibitor D22, in combination with results of mRNA expression indicate a presence of a single high-affinity, low-capacity uptake site, involving NET, and low-affinity, high-capacity uptake site involving PMAT in adult rat cortical astrocytes.We have not been able to confirm the presence of DAT mRNA in our astrocyte cultures; on the other hand, mRNA expression of NET was the most prominent among the studied transporters and closely followed by PMAT, a high-capacity, low-affinity transporter.Inazu et al. reached a similar conclusion concerning the role of NET in cortical neonatal rat astrocyte DA uptake as they were unable to confirm DAT mRNA expression; however, they confirmed NET mRNA expression (Takeda et al., 2002).Additionally, Takeda et al. reported significant inhibition of astrocyte Na + -dependent DA uptake by nisoxetine, a NET selective inhibitor, and not GBR1235, a potent DAT inhibitor, highlighting the importance of NET in astrocyte active DA transport (Inazu, Kubota, et al., 1999).Selective inhibitors for the high-capacity, low-affinity DA transporters, such as the OCTs and PMAT, are lacking; however, due to the low mRNA expression of the OCTs, which is in line with previous studies on both adult and neonatal cortical rat astrocyte cultures in our laboratory (Perdan-Pirkmajer et al., 2012;Perdan-Pirkmajer et al., 2013), we may assume that their role in astrocyte DA uptake is negligible in comparison to the role of PMAT.
In the brain, DRD1 and DRD2 expression has been shown to be prominent in the frontal cortex, whereas DRD3, DRD4 and DRD5 have a more limited expression pattern (Beaulieu & Gainetdinov, 2011).Multiple studies have reported mRNA expression of DA receptors in astrocytes (Corkrum & Araque, 2021;Miyazaki et al., 2004); however, reports of their expression vary between animal species and the studied brain region.Perhaps astrocytes in different brain regions may express different DA receptors, or different DA receptors serve distinct brainarea-specific signalling (Corkrum et al., 2020).In the present study, we examined the presence of the two most prominently expressed DA receptors in the frontal cortex, DRD1 and DRD2.Our results indicate a fairly low expression of both receptor subtypes in adult rat cortical astrocytes; additionally, mRNA expression of DRD2 was at the limit of detection of our qPCR analysis method.Activation or blockade of brain DA receptors has been embraced as a mechanism for the therapeutic efficacy of dopaminergic drugs.Although this concept is correct in principle, it may be too simplistic.Receptors are functionally linked with intracellular molecular networks that control membrane excitability, as well as neurotransmitter synthesis, release and metabolism, and by these mechanisms, neuronal cells can regulate their own activity.Thus, drugs targeting DRDs can impact neurotransmitter synthesis, release and metabolism (Amato et al., 2019).Astrocyte DRDs have been shown to induce fluctuations in intracellular signalling molecules in different brain areas.Some studies have connected these changes to activation of DRD1 (Requardt et al., 2010) and some to DRD2 (Cui et al., 2009;Jennings et al., 2017), by various dopaminergic compounds.These changes have yet to be thoroughly explored, not only how astrocyte DA receptors respond to different stimuli but also whether they lead to any possible changes in astrocyte DA transport.Therefore, we decided to examine whether treatment of our adult rat astrocyte cultures with high concentrations of apomorphine, haloperidol and L-DOPA might induce changes in the expression of DRD1, which has been studied to some extent previously, as well as their effect on transporters involved in astrocyte DA uptake, NET and PMAT.
The three used compounds, apomorphine, a potent DRD1/DRD2 agonist, currently used as an antiparkinsonian drug (Li et al., 2006), haloperidol, a competitive post-synaptic DRD2 antagonist with limited action on DRD1 (Lidow & Goldman-Rakic, 1994), and L-DOPA, a prodrug of DA that is administered to patients with Parkinson's due to its ability to cross the blood-brain barrier, produced varied results.Apomorphine induced changes of all examined samples, the greatest change was observed in DRD1 mRNA expression, followed lesser upregulation of NET and PMAT.Haloperidol has been found to change the expression of neuronal D1 and D2 receptors (Damask et al., 1996;D'Souza et al., 1997;Lidow & Goldman-Rakic, 1994), which however interestingly, in our study, had no effect on astrocyte DRD1 receptor mRNA expression.L-DOPA can interact with DRD1 or DRD2 receptors independent of its conversion to endogenous DA (Viaro et al., 2021) and has been found to upregulate DRD1 sensitivity and promote DA release through the reversal of DAT (Viaro et al., 2021), which however had no effect on either DRD1 or NET mRNA expression in adult rat cortical astrocytes.Although haloperidol and L-DOPA did not induce any significant change in the expression of NET mRNA, we observed a significant downregulation of PMAT mRNA expression.Haloperidol has been found to inhibit hPMAT at micromolar concentrations (Haenisch & Bönisch, 2010), whereas we have not found similar data on L-DOPA in regard to PMAT.Interestingly, all three compounds affected the expression of the high-capacity transporter PMAT, and only apomorphine induced upregulation of both NET and DRD1.
Our results concerning pharmacological and molecular characteristics of DA uptake suggest that adult rat cortical astrocyte DA uptake is mediated by multiple transporters, which however may not include DAT.The key transporter involved in DA reuptake, DAT, critically regulates the duration of the actions of DA and the extent to which DA diffuses.In the cortex, particularly prefrontal cortex, evidence for perisynaptic DAT is lacking, although a low density of DAT protein is observed along preterminal portions of DA axons, axonal varicosities; therefore, DA appears to undergo less regulation by DAT-mediated re-uptake, thus less extensive neuronal uptake and greater extracellular diffusion, in comparison to the striatum, where DA is released at synaptic sites.Although cortical astrocytes form the tripartite synapse in conjunction with cortical neurons and are crucial for cortical neuronal development (Farhy-Tselnicker & Allen, 2018), the release of DA from the varicosities of the dopaminergic axons, rather than axonal terminals in the cortex, poses an interesting challenge of defining the cortical dopaminergic tripartite synapse, as well as question the importance of DAT in cortical astrocyte reuptake of DA (Sesack et al., 1998).
The present study has some limitations.We conducted experiments on primary astrocyte cultures of adult rats.Primary cell cultures represent the in vivo state of cells more closely than immortalized cell lines, which however are due to their origin more difficult to cultivate and the variability of the obtained results is greater.Additionally, they may only serve as a model of the in vivo state due to cultivation under 'non-physiological' conditions lacking interaction with neuronal and other brain cells.

| CONCLUSION
Astrocytes contribute to DA homeostasis and are capable of DA clearance and metabolism.DA uptake into astrocytes is not mediated by the uptake 1 transporter DAT, but by NET and high-capacity, low-affinity transporter PMAT.Astrocytes are sensitive to treatment with dopaminergic drugs, which is reflected by the changes in mRNA expression of transporters NET and PMAT, as well as DRD1.The involvement of glial cells in DA homeostasis needs further research.

AUTHOR CONTRIBUTIONS
Vesna Sočan and Mojca Kržan designed the project, Vesna Sočan and Klemen Dolinar performed and analysed the measurements and Vesna Sočan and Mojca Kržan interpreted the data, wrote and edited the manuscript.
Pairwise comparison of transporter mRNA expression in adult rat cortical astrocytes.