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. 2016 May 17;6(5):e809.
doi: 10.1038/tp.2016.86.

Glucagon-like Peptide 1 Receptor Activation Regulates Cocaine Actions and Dopamine Homeostasis in the Lateral Septum by Decreasing Arachidonic Acid Levels

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Free PMC article

Glucagon-like Peptide 1 Receptor Activation Regulates Cocaine Actions and Dopamine Homeostasis in the Lateral Septum by Decreasing Arachidonic Acid Levels

I A Reddy et al. Transl Psychiatry. .
Free PMC article

Abstract

Agonism of the glucagon-like peptide 1 (GLP-1) receptor (GLP-1R) has been effective at treating aspects of addictive behavior for a number of abused substances, including cocaine. However, the molecular mechanisms and brain circuits underlying the therapeutic effects of GLP-1R signaling on cocaine actions remain elusive. Recent evidence has revealed that endogenous signaling at the GLP-1R within the forebrain lateral septum (LS) acts to reduce cocaine-induced locomotion and cocaine conditioned place preference, both considered dopamine (DA)-associated behaviors. DA terminals project from the ventral tegmental area to the LS and express the DA transporter (DAT). Cocaine acts by altering DA bioavailability by targeting the DAT. Therefore, GLP-1R signaling might exert effects on DAT to account for its regulation of cocaine-induced behaviors. We show that the GLP-1R is highly expressed within the LS. GLP-1, in LS slices, significantly enhances DAT surface expression and DAT function. Exenatide (Ex-4), a long-lasting synthetic analog of GLP-1 abolished cocaine-induced elevation of DA. Interestingly, acute administration of Ex-4 reduces septal expression of the retrograde messenger 2-arachidonylglycerol (2-AG), as well as a product of its presynaptic degradation, arachidonic acid (AA). Notably, AA reduces septal DAT function pointing to AA as a novel regulator of central DA homeostasis. We further show that AA oxidation product γ-ketoaldehyde (γ-KA) forms adducts with the DAT and reduces DAT plasma membrane expression and function. These results support a mechanism in which postsynaptic septal GLP-1R activation regulates 2-AG levels to alter presynaptic DA homeostasis and cocaine actions through AA.

Conflict of interest statement

SP has an active research contract with Lundbeck Pharmaceuticals; however, the work presented in the manuscript was supported entirely by the grants named above and Vanderbilt University. The remaining authors declare no conflict of interest.

Figures

Figure 1
Figure 1
The glucagon-like peptide 1 receptor (GLP-1R) is highly expressed in the lateral septum (LS) where GLP-1R agonists block cocaine-induced dopamine (DA) in vivo. (a-c) Cells expressing the GLP-1R (white in a, red in c) and terminals expressing dopamine transporter (DAT; white in b, green in c) in rostral LS (GLP-1R in a, DAT in b, merge in c). Confocal images in z stack through 13 planes taken with a × 10 objective. (d-f) Cells expressing the GLP-1R (white in d, red in f) and terminals expressing DAT (white in e, green in f) in caudal LS (GLP-1R in d, DAT in e, merge in f). Confocal images in z stack through 14 planes taken with a × 10 objective. (g) DA release induced by 30 mm KCl (representative of n=3) in section from the caudal LS. Approximate site of recording is demarcated by * in f. Ld, laterodorsal tegmental nucleus; LSd, dorsal LS; LSi, intermediate LS; LSv, ventral LS; MS, medial septum; SHi, septohippocampal nucleus. Dotted lines delineate region boundaries. Scale bars, 100 μm. (h) Representative c-fos expression in the LS 30 min following vehicle (top) or Ex-4 (30 μg kg−1, intraperitoneally, bottom) injection. Dotted box includes the LS. (i) Quantification of optical density analysis of c-fos autoradiographs in LS (t(14)=3.26; **P<0.01 by Student's t-test; n=8). (j) Cocaine (50 μm) greatly increased septal DA levels in mice pretreated with saline (intraperitoneally, solid circles). This cocaine-induced DA increase is significantly diminished in mice pretreated with Ex-4 (1 μg kg−1 intraperitoneally, solid triangles; 30 μg kg−1 intraperitoneally, solid squares). Extracellular concentrations of DA are expressed as the percentage of basal levels in two fractions collected before the intervention; significant analysis of variance. Open symbols indicate significance by post hoc test where P<0.001 at 150 min and P<0.05 at 120 min; n=5–6 per group.
Figure 2
Figure 2
Local activation of the glucagon-like peptide 1 receptor (GLP-1R) promotes septal dopamine transporter (DAT) membrane expression and [3H]DA uptake. (a) Representative immunoblot of surface and total DAT following slice treatment with vehicle (artificial cerebrospinal fluid, aCSF) or 1 nm GLP-1. (b) Ratio of DAT surface expression in GLP-1-treated ex vivo septal slices as percentage of average vehicle values (t(3)=10.12; **P<0.01 by Student's t-test; n=4). The slices were treated with 1 nm GLP-1 for 20 min. (c) [3H]DA uptake in the LS from ex vivo slice preparation following 20 min treatment with vehicle (aCSF) or 10 nm GLP-1. Uptake was significantly elevated (t(4)=2.35; *P<0.05 by Student's t-test; n=5). (d) Pretreatment with 100 nm Ex-9 for 10 min blocks the increase in [3H]DA uptake following treatment with 10 nm GLP-1 (t(3)=1.047; P=0.19 by Student's t-test; n=4).
Figure 3
Figure 3
Treatment of mice with Ex-4 reduces septal arachidonic acid (AA) levels and AA is a strong modulator of dopamine transporter (DAT) function in lateral septum (LS). (a) Ex-4 (2.4 μg kg−1, intraperitoneally) administered in vivo 30 min before killing resulted in lower levels of septal 2-AG (t(11)=2.62; *P<0.05 by Student's t-test; n=6–7). (b) Exenatide (Ex-4; 2.4 μg kg−1, intraperitoneally) administered 30 min before sacrifice reduces levels of septal AA (t(10)=5.018; ***P<0.001 by Student's t-test; n=6). (c) Eighty micromolar AA applied for 20 min to slices containing the LS reduces [3H]DA uptake to 42% of vehicle (dimethyl sulfoxide)-treated slices (t(3)=2.59; *P<0.05 by Student's t-test; n=4). (d) [3H]DA uptake kinetics in human DAT (hDAT) cells treated either with vehicle (open circles) or Fe+3/Asc (open triangles) or with 40 μm AA for 1 h at 37 °C in absence (gray squares) or presence of Fe+3/Asc (gray triangles). The kinetic parameters (e) Vmax and (f) Km were obtained from the Michaelis–Menten fit to the influx of [3H]DA. Each value represents mean±s.e.m. of ⩾ three independent experiments. *P<0.05, ****P<0.001, ##P<0.01 and &&P<0.01 indicate significant differences between vehicle (Veh) and AA, Veh and Fe+3/Asc+AA, AA and Fe+3/Asc+AA and Fe+3/Asc and Fe+3/Asc+AA, respectively. Significant analysis of variance followed by post hoc tests.
Figure 4
Figure 4
Arachidonic acid (AA) decreases dopamine transporter (DAT) cell surface expression in human DAT (hDAT) cells. (a) Cell surface biotinylation and immunoblots for DAT using anti-DAT in extracts from hDAT cells treated with AA (40 μm) for 1 h at 37 °C in the absence or presence of Fe+3/Asc. (b) Results expressed as the ratio of optical density between biotinylated (surface) and total DAT signals for each experimental condition. Each value represents mean ±s.e.m. of ⩾ three independent experiments. *P<0.05 indicates significant differences with respect to vehicle condition (0.1% ethanol in Krebs–Henseleit buffer). Significant analysis of variance followed by post hoc tests.
Figure 5
Figure 5
Synthetic isoketals form adducts with human dopamine transporter (hDAT) and decrease its function. (a) Immunoblots with anti-γ-KA protein adduct and anti-DAT antibodies using green fluorescent protein (GFP)-DAT immunoprecipitates from hDAT cells treated with synthetic isoketals (1 μm) for 1 h at 37 °C. (b) Quantification of immunoprecipitates as a ratio of γ-KA:DAT. Each value represents mean±s.e.m. of ⩾ four independent experiments (t(6)=3.94; **P<0.01 by Student's t-test; n=4). (c) Uptake of [3H]DA in hDAT cells treated with vehicle (open circles) or with 1 μm synthetic isoketals (gray triangles) for 1 h at 37 °C. The kinetic parameters (d) Vmax and (e) Km were obtained from the Michaelis–Menten fit to [3H]DA uptake. Each value represents mean±s.e.m. of three independent experiments (Vmax: t(4)=4.21; *P<0.05 by Student's t-test; n=3; Km: t(4)=0.88; *P=0.43 by Student's t-test). (f) Influx of [3H]DA in hDAT cells pretreated with salicylamine (0.5 mm) and then treated with vehicle (open circles, 0.1% ethanol in Krebs–Henseleit buffer), Fe+3/Asc (open triangles) or with 40 μm AA for 1 h at 37 °C in the absence (gray squares) or presence of Fe+3/Asc (gray triangles). The kinetic parameters (g) Vmax and (h) Km were obtained from the Michaelis–Menten fit to the influx of [3H]DA. Each value represents mean±s.e.m. of ⩾ three independent experiments. No significant differences were observed between the groups. The statistical analysis was performed with analysis of variance. AA, arachidonic acid; Veh, vehicle.

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