Positive autoregulation of GDNF levels in the ventral tegmental area mediates long-lasting inhibition of excessive alcohol consumption

Abstract

Glial cell line-derived neurotrophic factor (GDNF) is an essential growth factor for the survival and maintenance of the midbrain dopaminergic (DA-ergic) neurons. Activation of the GDNF pathway in the ventral tegmental area (VTA), where the GDNF receptors are expressed, produces a long-lasting suppression of excessive alcohol consumption in rats. Previous studies conducted in the DA-ergic-like cells, SHSY5Y, revealed that GDNF positively regulates its own expression, leading to a long-lasting activation of the GDNF signaling pathway. Here we determined whether GDNF activates a positive autoregulatory feedback loop in vivo within the VTA, and if so, whether this mechanism underlies the long-lasting suppressive effects of the growth factor on excessive alcohol consumption. We found that a single infusion of recombinant GDNF (rGDNF; 10 μg) into the VTA induces a long-lasting local increase in GDNF mRNA and protein levels, which depends upon de novo transcription and translation of the polypeptide. Importantly, we report that the GDNF-mediated positive autoregulatory feedback loop accounts for the long-lasting inhibitory actions of GDNF in the VTA on excessive alcohol consumption. Specifically, the long-lasting suppressive effects of a single rGDNF infusion into the VTA on excessive alcohol consumption were prevented when protein synthesis was inhibited, as well as when the upregulation of GDNF expression was prevented using short hairpin RNA to focally knock down GDNF mRNA in the VTA. Our results could have implications for the development of long-lasting treatments for disorders in which GDNF has a beneficial role, including drug addiction, chronic stress and Parkinson's disease.

Keywords: GDNF; addiction; alcohol; growth factors; ventral tegmental area.

Figure 1

A single infusion of recombinant glial cell line-derived neurotrophic factor (rGDNF) into the ventral tegmental area (VTA) produces a sustained induction of GDNF expression and phosphorylation of extracellular-regulated protein kinase 1/2 (ERK1/2). rGDNF (10 μg, unilateral) or vehicle was infused into the VTA of rats 12 h (a), 24 h (b) or 48 h (c) before the VTA dissection. GDNF and GAPDH mRNA levels were determined using semi-quantitative reverse transcription PCR. GAPDH levels were measured to standardize sample loading. Data are expressed as mean±s.e.m. of GDNF/GAPDH expression (t's>3.50, ^*P<0.05; n=3–5). (d) Shown is a dual-channel immunofluorescence for phospho-ERK1/2 (p-ERK1/2, red), tyrosine hydroxylase (TH, a marker for dopaminergic neurons, green), and overlay (yellow). Left panel: images depict ERK1/2 phosphorylation in the midbrain 24 h after rGDNF (left brain side) or PBS (right side) infusion into the VTA. Images are representative of results from four rats (six sections per rat), scale bar: 500 μm. Right panel: magnified image of the VTA infused with rGDNF, as marked in a white square in Figure 2a. The arrowheads point to cells immunostained for both p-ERK1/2 and TH, scale bar: 50 μm.

Figure 2

Recombinant glial cell line-derived neurotrophic factor (rGDNF)-mediated upregulation of GDNF mRNA and protein requires de novo protein translation. rGDNF (10 μg, unilateral) or vehicle was infused into the ventral tegmental area (VTA) of rats 48 h (a) or 24 h (b) before VTA dissection. Cycloheximide (CHX; 2 mg kg⁻¹, i.p.) was administered 50 min before and 3 h after rGDNF infusion. CHX was administered twice at these intervals to provide efficient inhibition of protein synthesis for at least 6–8 h. (a) GDNF and GAPDH mRNA levels were determined using semi-quantitative reverse transcription PCR. Data are expressed as mean±s.e.m. of GDNF/GAPDH levels (main effects of CHX pretreatment (F (1,6)=67.03; P<0.0002) and rGDNF infusion (F (1,6)=156.88; P<0.0001), and a significant interaction (F (1,6)=101.09; P<0.0001). Post-hoc comparison: a significant difference between the rGDNF- and the vehicle-infused sides in rats that were pretreated with vehicle, but not in CHX-pretreated rats; ^**P<0.001; n=4). (b) GDNF and GAPDH protein levels were determined using western blot analysis. Data are expressed as mean±s.e.m. of GDNF/GAPDH levels (main effects of CHX pretreatment (F (1,6)=171.01; P<0.0001) and rGDNF infusion (F (1,6)= 281.47; P<0.0001), and a significant interaction (F (1,6)=279.17; P<0.0001). Post-hoc comparison: a significant difference between the rGDNF- and the vehicle-infused sides in rats that were pretreated with vehicle, but not in CHX-pretreated rats; ^**P<0.001; n=4). (c) Adenovirus shGDNF (AdV-shGDNF) or AdV-shSCR (1.2 μl per side, 1.3 × 10⁸ TU ml⁻¹) was infused into the VTA of rats. rGDNF was infused into the VTA 14 days after virus infection. The VTA was dissected 24 h later and GDNF mRNA levels were quantified by real-time reverse transcription PCR. Data are expressed as mean±s.e.m. of GDNF/GAPDH expression (main effects of viral infection (F (1,12)=61.19, P<0.0001) and rGDNF infusion (F (1,12)=16.57, P<0.002), and a significant interaction (F (1,12)=18.37, P<0.002); post-hoc comparisons: a significant difference between rGDNF- and vehicle-infused VTA in the AdV-shSCR-infected rats (^**P<0.001), but not in the AdV-shGDNF-infected rats; n=3–4).

Figure 3

Inhibition of protein synthesis prevents the long-lasting, but not the acute, effects of recombinant glial cell line-derived neurotrophic factor (rGDNF) on alcohol consumption. Rats consumed a solution of 20% alcohol for 7 weeks. Cycloheximide (CHX; 2 mg kg⁻¹, i.p.) was administered 1 h before and 3 h after the beginning of the alcohol-drinking session. rGDNF (10 μg per side) was infused into the ventral tegmental area (VTA) 10 min before the beginning of the drinking session. (a) Timeline of treatments and consumption measurements schedule. (a and b) Data are expressed as mean±s.e.m. of alcohol intake in g kg⁻¹ (^*P<0.01 vs vehicle–vehicle; n=12). (b) Alcohol intake during the first 30 min of the drinking session (two-way repeated measurements analysis of variance (ANOVA): a main effect of rGDNF infusion (F (1,13)=34.79, P<0.0001), no effect of CHX pretreatment (F (1,13)=1.28, P=0.28), and no interaction (F (1,13)=0.07, P=0.79)). (c) Alcohol intake during the 4–24-h time period after the beginning of the drinking session (two-way repeated measurements ANOVA: no effect of rGDNF infusion (F (1,13)=2.19, P=0.16), a significant main effect of CHX pretreatment (F (1,13)=10.46, P<0.007), and a significant interaction (F (1,13)=13.16, P<0.005). Post-hoc comparisons: a significant difference between rGDNF- and vehicle-infused rats in the vehicle control rats (P<0.01), but not in rats that were pretreated with CHX (P=0.086)).

Figure 4

Downregulation of glial cell line-derived neurotrophic factor (GDNF) expression in the ventral tegmental area (VTA) prevents the sustained, but not the rapid, effects of recombinant GDNF (rGDNF) on alcohol consumption. Rats consumed a solution of 20% alcohol for 7 weeks. After the establishment of a baseline level of alcohol intake, VTA were infected with adenovirus (AdV)-shGDNF or control AdV-shSCR (SCR), after which the basal levels of alcohol intake were reestablished. Ten to 15 days after the infection, rGDNF (10 μg per side) or vehicle was infused into the VTA 10 min before the drinking session onset. Data are expressed as mean±s.e.m. of alcohol intake in g kg⁻¹ (^*P<0.01 vs vehicle; n=7). (a) Alcohol intake during the first 30 min of the drinking session (main effects of rGDNF infusion (F (1,12)=40.55, P<0.0001) and of viral infection (F (1,12)=4.89, P<0.05), but no interaction (F (1,12)=0.10, P=0.75)). (b) Alcohol intake during the 4–24 h time period after the beginning of the drinking session (mixed-model analysis of variance (ANOVA): a main effect of rGDNF infusion (F (1,12)=6.67, P<0.025), no effect of viral infection (F (1,12)=2.53, P=0.13), and a marginally significant interaction (F (1,12)=3.88, P=0.072). Post-hoc comparisons: a difference between rGDNF- and vehicle-infused rats in the adenovirus (AdV)-shSCR infected control rats (P<0.01), but not in rats that were infected with AdV-shGDNF (P=0.67)).

Figure 5

A schematic illustration of the glial cell line-derived neurotrophic factor (GDNF) autoregulatory loop within the ventral tegmental area (VTA). (a) Recombinant GDNF (rGDNF) binds to the GFRα1-Ret receptor complex in the VTA (1), which leads to the activation of Ret. Activation of the GDNF-mediated signaling pathway leads to the upregulation of the transcription of the GDNF gene (2). GDNF mRNA is translated to GDNF protein, and secreted (3). Secreted GDNF binds to its receptor complex (4), and activates its signaling pathway to maintain this positive feedback cycle. (b and c) Inhibition of protein synthesis by cycloheximide (CHX) (b) or adenovirus (AdV)-shGDNF-mediated downregulation of GDNF in the VTA (c) disrupts the autoregulatory feedback cycle by reducing the level of endogenously-produced GDNF, thus preventing the subsequent activation of the loop.