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. 2024 Jan 11;25(2):911.
doi: 10.3390/ijms25020911.

Cortical and Striatal Astrocytes of Neonatal Rats Display Distinct Molecular and Pharmacological Characteristics of Dopamine Uptake

Vesna Sočan  1 , Klemen Dolinar  2 , Mojca Kržan  1
Affiliations

Cortical and Striatal Astrocytes of Neonatal Rats Display Distinct Molecular and Pharmacological Characteristics of Dopamine Uptake

Vesna Sočan et al. Int J Mol Sci. .

Abstract

Astrocytes are crucial in the regulation of neurotransmitter homeostasis, and while their involvement in the dopamine (DA) tripartite synapse is acknowledged, it necessitates a more comprehensive investigation. In the present study, experiments were conducted on primary astrocyte cultures from the striatum and cortex of neonatal rats. The pharmacological intricacies of DA uptake, including dependence on time, temperature, and concentration, were investigated using radiolabelled [3H]-DA. The mRNA expression of transporters DAT, NET, PMAT, and OCTs was evaluated by qPCR. Notably, astrocytes from both brain regions exhibited prominent mRNA expression of NET and PMAT, with comparatively lower expression of DAT and OCTs. The inhibition of DA uptake by the DAT inhibitor, GBR12909, and NET inhibitors, desipramine and nortriptyline, impeded DA uptake in striatal astrocytes more than in cortical astrocytes. The mRNA expression of NET and PMAT was significantly upregulated in cortical astrocytes in response to the DA receptor agonist apomorphine, while only the mRNA expression of NET exhibited changes in striatal astrocytes. Haloperidol, a DA receptor antagonist, and L-DOPA, a DA precursor, did not induce significant alterations in transporter mRNA expression. These findings underscore the intricate and region-specific mechanisms governing DA uptake in astrocytes, emphasizing the need for continued exploration to unravel the nuanced dynamics of astrocytic involvement in the DA tripartite synapse.

Keywords: NET; PMAT; astrocyte; dopamine uptake; neonatal rat.

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Conflict of interest statement

The authors declare no conflicts of interest. The funders had no role in the design of the study, in the collection, analysis, or interpretation of data, in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
Dependence of [3H]-DA uptake on time, temperature, and concentration in cortical and striatal astrocytes of neonatal rats. Time dependence of total (37 °C) and nonspecific (4 °C) [3H]-DA uptake in cortical (a) and striatal (b) astrocytes, concentration dependence of [3H]-DA uptake (total, specific, and nonspecific) in cortical (c) and striatal (d) astrocytes and [3H]-DA uptake velocity in cortical (e) and striatal (f) astrocytes. Data are presented as mean ± SEM (n = 9) from three separate experiments.
Figure 2
Figure 2
Inhibition of (a) 30 nM concentration of [3H]-DA uptake by 0.1 mM and 1 mM concentration of ouabain and (b) 30 nM concentration of [3H]-DA uptake in uptake buffer (UB) without Na+ in cortical and striatal astrocytes from neonatal rats. Results are presented as percent of the control; each bar expressed as mean ± SEM from three or more separate experiments ((a): n = 9, (b): n = 22). Statistical significance of reduction of DA uptake in comparison to the control was determined by One-Way ANOVA: * p < 0.05, ** p < 0.01, **** p < 0.0001. Difference between cortical and striatal DA uptake was nonsignificant (Student t-test, p > 0.05).
Figure 3
Figure 3
mRNA expression of transporters NET, PMAT, DAT, OCT1, OCT2, and OCT3 in striatal and cortical astrocytes cultured from neonatal rats (Astrocytes) as well as in striatal and cortical tissue samples of neonatal rats (Tissue). mRNA expression is normalized to the mRNA expression of the endogenous control, β-actin. Data are presented as mean ± SEM (astrocytes: n ≥ 6, from at least two separate experiments, tissue: n ≥ 4 from 4–6 animal tissue samples). Statistical analysis of comparative mRNA expression of transporters between striatal and cortical astrocytes (Astrocytes), was performed by multiple unpaired Mann–Whitney U tests with multiple testing correction two-stage step-up (Benjamin, Kriger, and Yekutieli) to control the false discovery rate (FDR) *** p = 0.001, ns—nonsignificant.
Figure 4
Figure 4
Inhibition of [3H]-DA uptake by GBR12909, desipramine, nortriptyline, amitriptyline, corticosterone, and D22. Astrocytes were preincubated with various compounds for 20 min, which was followed by a 20 min incubation with 30 nM [3H]-DA at 37 °C. Results are presented as percent (mean ± SEM) of the total [3H]-DA uptake of the control from three to four separate experiments carried out in triplicates (n ≥ 9). IC50 and pIC50 (calculated as the negative log of the corresponding IC50 values) of compounds inhibiting the total [3H]-DA uptake into cultured adult rat astrocytes were calculated from the corresponding inhibition curves. GBR12909: cortex: IC50 = 2.97 ± 2.46 µM, pIC50 = 5.5, striatum: IC50 = 0.00956 ± 0.00571 µM, pIC50 = 8.0, desipramine: cortex IC50 = 0.0125 ± 0.0137 µM, pIC50 = 7.9, striatum: IC50 = 0.000157 ± 0.000223 µM, pIC50 = 9.8; nortriptyline: cortex ns, striatum: IC50 = 3.5 ± 32.1 µM, pIC50 = 5.5, amitriptyline: ns, corticosterone: cortex and striatum: ns, D22: cortex—IC50 = 30 ± 17 µM, pIC50 = 4.5, striatum—IC50 = 81 ± 235 µM, pIC50 = 4.1. Statistical analysis of the difference in DA uptake in comparison to the control was carried out using an unpaired t-test with Welch control, * p < 0.05.
Figure 5
Figure 5
Changes in mRNA expression of transporters NET (a,c) and PMAT (b,d) after 24 h treatment with apomorphine, L-DOPA, and haloperidol. Data are presented relative to the expression of endogenous control, β-actin, as mean ± SEM of at least two separate experiments ((a,b): n = 9; (c,d): n = 7). Statistical analysis was performed using One-Way ANOVA with post-hoc Dunnett’s test (Cortex: NET: F(3,30) = 6.701, p = 0.0014, PMAT: F(3,30) = 13.88, p < 0.0001; striatum: NET: F(3,24) = 10.98, p < 0.0001, PMAT: F(3,24) = 1.392, p = 0.2695); ** p = 0.0013, *** p < 0.001. Cell viability of cortical (e) and striatal (f) astrocytes after 24 h treatment with various concentrations (1, 10, 100, 150 µM) of apomorphine, L-DOPA, and haloperidol presented as percent of the control, mean ± SEM ((e): n = 16, 4 separate experiments, (f): n = 12, 3 separate experiments). Differences in cell viability between treatments and control were determined as nonsignificant by One-Way ANOVA with post-hoc Dunnett’s test, p > 0.05.
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