Anaplastic lymphoma kinase regulates binge-like drinking and dopamine receptor sensitivity in the ventral tegmental area

Abstract

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase associated with alcohol dependence in humans and behavioral responses to ethanol in mice. To characterize the ability of ALK to control ethanol consumption, we treated mice with the ALK inhibitors TAE684 or alectinib before testing them for binge-like drinking using the drinking in the dark protocol. Mice treated with ALK inhibitors drank less ethanol than controls. In addition, TAE684 treatment abolished ethanol conditioned place preference, indicating that ALK regulates the rewarding properties of ethanol. Because the ventral tegmental area (VTA) is a key brain region involved in the rewarding effects of ethanol, we determined if Alk expression in the VTA is important for binge-like ethanol consumption. Mice expressing a short hairpin ribonucleic acid targeting Alk in the VTA drank less ethanol compared with controls. ALK is expressed on dopamine (DA) neurons in the VTA, suggesting that ALK might regulate their firing properties. Extracellular recordings of putative DA neurons in VTA slices demonstrated that ALK inhibition did not affect the ability of ethanol to stimulate, or DA to inhibit, the firing of DA neurons. However, inhibiting ALK attenuated the time-dependent reversal of inhibition produced by moderate concentrations of DA, suggesting that ALK affects DA D2 autoreceptor (D2R) desensitization. Altered desensitization of the D2R changes the firing of DA neurons and is predicted to affect DA levels and alcohol drinking. These data support the possibility that ALK might be a novel target of pharmacotherapy for reducing excessive alcohol consumption.

Keywords: ALK; Alcohol addiction; binge-drinking; dopamine receptor; ventral tegmental area.

Figure 1

ALK inhibition attenuates binge-like ethanol consumption. (a) Ethanol consumed (g/kg) during 2 h of drinking on days 1–4 in mice treated with vehicle (n=12) or 10 mg/kg TAE684 (n=12). TAE684 treatment led to an average 37% decrease in ethanol consumption compared to vehicle treatment over 4 days. Inset bar graph shows the amount of ethanol consumed during 4 h on day 4. (b) Volume of 2% sucrose solution (ml/kg) consumed during 2 h of drinking on days 1–3 and 4 h on day 4 in mice treated with vehicle (n=10) or TAE684 (n=10). (c) Ethanol consumed during 2 h of drinking on days 1–4 in mice treated with vehicle (n=10) or 60 mg/kg alectinib (n=10). Alectinib treatment led to an average 61% decrease in ethanol consumption over 4 days. Inset bar graph shows the amount of ethanol consumed during 4 h on day 4. (d) Volume of 2% sucrose consumed during 2 h of drinking on days 1–3 and 4 h on day 4 in mice treated with vehicle (n=5) or alectinib (n=5). *p < 0.05, ***p < 0.001, ****p < 0.0001 by 2 way ANOVA or t-test.

Figure 2

ALK inhibition attenuates ethanol reward. (a) Schematic showing treatment conditions for the ethanol CPP test. (b) Preference score after ethanol conditioning in mice treated with vehicle (n=16) or 10 mg/kg TAE684 (n=13), showing that TAE684 significantly decreased ethanol CPP by 76%, *p = 0.05 by t-test. (c) Distance traveled (cm) during the 30 min preference test. (d) Distance traveled in 5 min immediately after a saline injection on conditioning days 3, 5, 7 & 9. (e) Distance traveled in 5 min after a 2 g/kg ethanol injection on conditioning days 2, 4, 6 & 8. Note that the mice sensitized to the ethanol injections, but there was no effect of TAE684 treatment on ethanol sensitization. (f) Schematic showing the treatment conditions for the TAE684 CPP test (in the absence of ethanol). (g) Time (s) spent on the TAE684-paired side before conditioning (pre-conditioning) and after conditioning (post-conditioning) in mice conditioned with 10 mg/kg TAE684 (n=11) instead of ethanol. Time spent on the TAE684-paired side did not change after conditioning.

Figure 3

ALK is expressed in DA neurons and in non-DA cells in the VTA. Confocal microscope images showing (a) nuclear (DAPI) staining in blue, (b) immunofluorescence to tyrosine hydroxylase (TH) in green, (c) immunofluorescence to ALK in red, and (d) a merged image of DAPI, TH and ALK immunofluorescence. Yellow arrows point to TH neurons expressing ALK and white arrowheads indicate expression of ALK in non-TH expressing cells. Scalebar, 50 µm.

Figure 4

ALK expression in the VTA regulates binge-like ethanol consumption. (a) Ethanol consumed (g/kg) during 2 h drinking sessions on days 1–4 in mice infected with lentivirus expressing shScr (control, n=11) or shAlk (n=11) in the VTA. Inset bar graph shows the amount of ethanol consumed during the 4 h drinking session on day 4. Mice expressing shAlk consumed approximately 22% less ethanol than controls on average over 4 days. *p < 0.05 and **p < 0.01 by 2 way ANOVA. (b) Fluorescent image demonstrating lentiviral infection in the VTA. Mouse brain sections containing infected VTA were incubated with fluorescent antibodies to tyrosine hydroxylase (TH, red) to indicate DA neurons and green fluorescent protein (GFP, green), expressed by the lentivirus. Abbreviations: IPN, interpeduncular nucleus; ml, medial lemniscus; SNC, substantia nigra pars compacta. Scalebar, 1 mm. (c) Fluorescent images showing ALK expression in VTA sections infected with shScr (top panel) or shAlk lentiviruses (bottom panel). Confocal images were taken with identical laser settings. Note the reduced intensity of ALK immunostaining in VTA infected with shAlk compared to shScr. Scalebar, 50 µm. (d) Ethanol consumed during 2 h drinking sessions on days 1–4 in mice infected with lentivirus expressing shScr (n=12) or shAlk (n=12) in the nucleus accumbens (Acb). Inset bar graph shows the amount of ethanol consumed during the 4 h drinking session on day 4. (e) Lentiviral infection in the Acb. GFP staining is shown in brown. Sections were counterstained with Cresyl violet. ac, anterior commissure.

Figure 5

ALK inhibitors attenuate DIR but not the response of DA neurons to acutely administered ethanol or DA. Extracellular recordings of VTA DA neurons were performed in the presence or absence of ALK inhibitors or the DMSO control. (a) Ethanol dose-response curves, showing that ethanol-induced stimulation of DA neuron firing is unaffected by incubation of slices with 100 nM TAE684 (n=10) compared to DMSO controls (n=10). (b) Ethanol dose-response curves, showing that ethanol-induced stimulation of DA neuron firing is unaffected by incubation of slices with 100 nM alectinib (n=11) compared to DMSO controls (n=11). (c) DA dose-response curves, showing that inhibition of DA neurons by DA is unaffected by incubation of slices with TAE684 (open squares, n=9) or alectinib (open triangles, n=8) compared to DMSO controls (solid circles, n=9). (d) Percent change in firing rate over 40 min of exposure to DA in cells treated with DMSO (filled circles, n=9), TAE684 (open squares, n=10), or alectinib (open triangles, n=6). Change in firing rate at 5 min intervals is plotted as a function of time after the initiation of DA administration. DIR was delayed in TAE684-treated and abolished in alectinib-treated slices compared to DMSO-treated controls. (e–g) Representative ratemeter graphs from single neurons showing the effect of DMSO (e), TAE684 (f), or alectinib (g) treatment on DA inhibition. Vertical bars indicate the firing rate over 5 second intervals. Horizontal bars indicate the duration of drug application.

Figure 6

Model for regulation of D2R desensitization by ALK and predicted effect on ethanol consumption. Prolonged DA exposure leads to desensitization of the D2R through transactivation of ALK signaling, presumably resulting in endocytosis and degradation of ALK and D2R. This allows for the recovery of DA neuron firing. The prediction is increased DA release in target regions, leading to an increase in ethanol consumption. ALK inhibitors such as TAE684 and alectinib are predicted to block the process of ALK and D2R endocytosis and degradation, resulting in attenuated DA release and ethanol consumption. ALK ligands such as midkine (MDK) or pleiotrophin (PTN) may participate in this process.