Neuropeptidergic regulation of compulsive ethanol seeking in C. elegans

Despite the catastrophic consequences of alcohol abuse, alcohol use disorders (AUD) and comorbidities continue to strain the healthcare system, largely due to the effects of alcohol-seeking behavior. An improved understanding of the molecular basis of alcohol seeking will lead to enriched treatments for these disorders. Compulsive alcohol seeking is characterized by an imbalance between the superior drive to consume alcohol and the disruption or erosion in control of alcohol use. To model the development of compulsive engagement in alcohol seeking, we simultaneously exploited two distinct and conflicting Caenorhabditis elegans behavioral programs, ethanol preference and avoidance of aversive stimulus. We demonstrate that the C. elegans model recapitulated the pivotal features of compulsive alcohol seeking in mammals, specifically repeated attempts, endurance, and finally aversion-resistant alcohol seeking. We found that neuropeptide signaling via SEB-3, a CRF receptor-like GPCR, facilitates the development of ethanol preference and compels animals to seek ethanol compulsively. Furthermore, our functional genomic approach and behavioral elucidation suggest that the SEB-3 regulates another neuropeptidergic signaling, the neurokinin receptor orthologue TKR-1, to facilitate compulsive ethanol-seeking behavior.


Introduction 1
Alcohol Use Disorder (AUD) is a chronic neurobehavioral disorder. A chronic exposure 2 leads to the development of tolerance contributing to increased consumption ( 1 , 2 , 3 ). Subsequently, 3 during the withdrawal or abstinence, alcohol craving and seeking are reinstated ( 4 , 5 , 6 ). Since these 4 three phases model has provided a inferred understanding of the development of AUD ( 7 ), 5 development of preference and compulsive seeking, known to be involved in progress of alcohol 6 dependence, have been identified as a pivotal and defining characteristic of AUD and other 7 substance use disorders ( 8 , 9 , 10 ). Furthermore, recent Human genome-wide association study 8 (GWAS) demonstrate that heavy drinking and increased consumption are not sufficient causes of 9 AUD, although binge drinking and increased consumption are key risk factors for AUD ( 11 ). 10 Hence, the neural substrates underlying compulsive seeking despite catastrophic consequences are 11 crucial components of AUD and comorbidities, but the molecular mechanism remains largely 12 elusive. The compulsive seeking in AUD is characterized by an imbalance between superior drive 13 to alcohol and disruption in control of alcohol use ( 12 , 13 ). In order to model this highly complex 14 neuromodulation, we addressed sophisticated behavioral paradigms in the simplest and most 15 completely defined connectome with the advantage of the straightforward genetic, behavioral, and 16 neurophysiological investigation, C. elegans ( 14 , 15 , 16 ). In a comparative proteomics study, 83% 17 of the worm proteome was found to have human homologous genes and recent meta-analysis of 18 orthology-prediction methods, about 52.6% of human protein-coding genome has recognizable 19 worm orthologues ( 17 , 18 ), allow the worm a suitable model organism for functional validation of 20 human genes. 21 C. elegans also has been shown to be a powerful and a deployable genetic tool to study 22 AUD ( 19 , 20 , 21 , 22 , 23 , 24 , 25 ). Worms showed comparable physiological effects in similar human 23 animals used for the experiment were the Bristol N2 strain. The strains tkr-1(ok2886), tkr-2 1 (ok1620) and tkr-3(ok381) were obtained from Caenorhabditis Genetics Center (CGC, 2 Minneapolis, MN, USA), which is supported by the National Institutes of Health -Office of 3 Research Infrastructure Programs (P40 OD010440). seb-3(eg696)gf previously isolated from a 4 genetic screening and the seb-3(tm1848)lf strain was obtained from S. Mitani (Tokyo Women's 5 Medical University, Tokyo, Japan) and backcrossed twice with N2 ( 22 ). 6 Trajectory Analysis of C.elegans locomotion in the development of ethanol preference 7 NGM was added to each well of 4 well tissue culture dish (1.9 cm2) to fill all the wells 8 equally to the top. Outside of well also filled up with NGM and the boundary were slightly covered 9 by disruption of surface tension when NGM was not solidified, which contained a gradient of 10 ethanol but an allowance of free moving of worms on the surface. Out of the 4 wells, one well was 11 selected and the glass Pasteur pipette (2ml volume) punctured to make a hole for adding ethanol 12 up to 300Mm. The plate was sealed with parafilm after adding ethanol then 1 hour later used. 1day 13 adult animals were washed twice in S-basal (100mM NaCl, 50mM potassium phosphate (pH 6.0),

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The ethanol pre-exposure plates were prepared as previously described ( 19 , 21 ). Preexposure 1 plates were 6-cm NGM plates that had been seeded with bacteria on half of the plate and were 2 dried for 2 h. Ice-cold ethanol was added up to final concentration of 300 mM ethanol (from our 3 previous publication; 21 ) and worms were introduced to ethanol plates for 4 hours pre-exposure 4 with parafilm sealing. Briefly, well-fed naïve or ethanol pretreated animals (300mM) were washed 5 twice with S-buffer [100mM NaCl, 50mM potassium phosphate (pH 6.0)] and once in distilled 6 water. Then animals were placed on mark above B (arrow in fig. 2) at the chemotaxis assay plate 7 ( 36 , 37 ). Animals were allowed to move freely on an assay plate with different concentrations of 8 CuSO4 barrier to see the compulsive alcohol seeking behavior. The concentration of copper barrier 9 was taken from the previous publication of Hilliard et al, 2004 ( 38 ), starting from a lower 10 concentration of 2 mM to 20 mM. Before making the chemical barrier, allow the chemotaxis plates 11 to dry for 1 hr. To make a chemical barrier, pre-cut (3mm thickness) Whatman filter paper (cat Ethanol-pretreated animals were placed on the aversion-resistant assay plate with 5mM CuSO4 2 barrier then the locomotion was recorded for 30min. Time to respond to the copper barrier, which 3 was stopping forward locomotion and backward then turn to avoid it, was measured in each 4 animal's first encounter with the copper barrier. To assess with different types of aversive stimuli 5 than copper, 5mM or 10mM Denatonium benzoate was applied to the assay plate in the same way 6 to create an aversive barrier. SI in each trial was obtained from the population assay of 100 to 150 7 animals. Avoidance assay with drop test (0.1mM, 1mM, and 5mM CuSO4) was conducted as 8 shown in previous study ( 39 , 38 ), to measure the sensitivity to nociceptive stimuli of WT and seb-9 3(eg696) gf animals. Single L4 worms were transferred to each NGM plate with abundant OP50,     Approximately 10 one day adult animals where allowed to lay eggs per 15 NGM plates seeded 8 with OP 50 for 8 to 10 hours. After 10 hours, the adult animals were removed and embryo where 9 allowed to grow at 20 degree. After 72 hours, one-day adult animals from each plate were washed 10 out with M9 buffer and washed the worms three times before collected in 1.5ul nuclease free sterile 11 Eppendorf tubes and stored at -80 with RNA later until use. Isolation of nucleic acid (total RNA) 12 was done in Qi cube unit using Qiagen RNeasy mini kit according to manufacturer's instruction  Text files were retrieved from UTHSC Molecular Resource Center after normalization 6 performed by Affymetrix Expression Condole. Quality assurance was checked against reference 7 probes to ensure quality of data. Gene names, accession numbers, and expression were mined from 8 each text file for each sample. All non-annotated information was removed from the file leaving 9 only annotated gene expression. A student t test was run for pairwise interactions in order to obtain 10 pvalues for significance. Only genes with a pvalue < 0.05 were considered significant. The mean, 11 variance, standard deviation, and fold change were calculated for each pairwise comparison.

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Benjamini Hochberg false discovery rate method was applied in order to obtain the adjusted pvalue  shown that the internal concentration of ethanol for WT animals, which were exposed and forced 11 to be in 400mM ethanol plate, were comparable with 0.1% blood alcohol in human, corresponds 0% appearance in the ethanol area of the assay plate for some of the naïve animals such as #4, #5).

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In contrast, ethanol-pretreated WT animals headed straight to the ethanol zone and stayed in the 23 ethanol zone ( Fig. 1b and Fig. 1d). The naïve animals stayed longer in the non-ethanol area (66% 1 of time spent) whereas ethanol pretreated animals spent 89% of the time in the ethanol area ( Fig.   2 1d). First, we addressed the question of whether the change of locomotion behavior is due to an 3 inability to keep moving. Ethanol pretreated (300mM) animals were placed on the non-ethanol 4 NGM and their locomotion was analyzed. The ability to move was not defective after ethanol 5 pretreatment. While animals exposed to 300mM exogenous ethanol for the pretreatment (Fig.1), 6 the speed of locomotion was reduced initially but recovered. Thus, no difficulty was observed in 7 the movement of pretreated animals (Fig. 2a, 2b). Ethanol preexposure produces a reversible barrier. The sensitivity of ethanol-pretreated animals to aversive stimuli was not altered (Fig. 3b). (data not shown). Then we also introduced another aversive stimulus, denatonium benzoate as a 23 barrier. Without pretreatment of ethanol, all the tested concentrations of denatonium barrier 1 successfully block to interfere the naïve animals' chemotaxis to ethanol. However, pretreated 2 animals showed endurance against the denatonium barrier to reach the ethanol area and crossed 3 the low concentration of obstacle (Fig. 3d). Since the compulsivity is defined as the urge to perform 4 and persist to take a substance that escapes control despite serious negative consequences ( 51 , 52 , 5 53 ), these results suggest that aversion-resistant ethanol seeking approach can be used as a proxy 6 measure for the compulsive ethanol seeking behavior. also be able to exhibit enhanced alcohol seeking against the higher concentration of Cu 2+ barrier.

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A seb-3(eg696), dominant mutation, was suggested and functionally evaluated as a gain of 17 function mutation due to its identity of mutation, which was single amino acid change in a 18 conserved residue at the third intracellular loop, the binding region for G proteins ( 22 , 54 , 55 , 56 ).

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Significant ethanol preference was observed in animals pretreated with ethanol for 2 hours whereas 4 WT animals did not (Fig. 4c). Additionally, greater development of preference was observed in  The pretreated seb-3(eg696) gf animals exhibited significantly high SI values not only in lower 14 but also in the higher concentration of Cu 2+ (Fig. 5a). Nevertheless, sensory perception of aversive 15 Cu 2+ stimuli was not defective in seb-3(eg696) gf animals (Fig. 5b, 5c, 5d, and 5e) represented 16 enhanced ethanol preference, which could override the interference of noxious stimuli. Although shows 51% similarity with human NK1R in overall 406 amino acids and 52% similarity with 21 human NK3R. TKR-1 represents the putative ligand-binding pocket defined according to the NMR 22 evidence of Substance P (SP) docking to the NK1R pocket (Fig.7a , 60 ), The crystal structure of 23 NK1R in complex with clinically used antagonist also endorse the important role of this region, 1 that is a ligand bind deeper within the receptor core ( 61 ). Tachykinin/Neurokinin system is 2 functionally pleiotropic and has been reported to be involved in the stress responses, anxiety, pain, 3 inflammation, immune response, and sensory processing ( 59 , 62 , 63 , 64 , 65 , 66 , 67 ). For the functional 4 evaluation of tkr-1 in association with the ethanol preference and compulsive ethanol seeking, we 5 tested the KO mutant of tkr-1 (ok2886) in the assay for aversion-resistant ethanol seeking. The C. 6 elegans genome includes 3 genes predicted to encode Neurokinin receptor family proteins, tkr-1, 7 tkr-2, and tkr-3 as shown in the phylogenetic analysis of the Tachykinin/Neurokinin Receptor 8 family (Fig. 7b). The phylogenetic tree was constructed by COBALT (constraint-based alignment 9 tool for multiple protein sequences) using minimum evolution method ( 25 ). We also tested KO 10 mutant animals of tkr-2 (ok1620) and tkr-3(ok381) in the neurokinin receptor family. The tested 11 are putative KO strains due to the deletion of genomic loci (Fig. 7c, 7d, and 7e). The evident Compulsivity is defined as repetitive attempts despite facing adverse consequences and 2 compel individuals to perform to be relieved from stress or anxiety ( 68 ). The repetitive or obsessive 3 aspects and compulsive drinking scale has been adapted and help to evaluate the severity of AUD 4 in human genetic studies for reliable assessing alcohol craving ( 6 , 26 , 27 ), that has been described 5 as a compelling urge to intake alcohol and considered crucial for the maintenance of AUD ( 5 ). The 6 compulsive alcohol seeking is characterized by an imbalance between superior drive to alcohol 7 and disruption in control of alcohol use ( 12 , 13 ). To model the development of compulsive 8 engagement of alcohol seeking in C. elegans, we showed following: i) enhanced preference of 9 ethanol, ii) repetitive attempts to seek, and iii) enhanced aversion-resistant ethanol seeking.

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Despite baseline aversive response to ethanol in acute exposure, C. elegans exhibit state-11 dependent ethanol seeking behavior, which is a significantly potentiated preference for ethanol 12 after the prolonged experience of ethanol ( 21 ). In addition, C. elegans also recapitulates the 13 behavioral traits of alcohol-dependent animals, known as studies in mammals. Ethanol pretreated 14 worms showed distinct orientation while seeking for ethanol and a pronounced tendency to stay in 15 the limited region where ethanol is, even though their locomotion and exploratory behavior were 16 not damaged by the pretreatment of ethanol ( Fig. 1 and 2). The increase in exploratory behavior Ethanol pretreated animals also represented repetitive attempts and endurance to cross the 3 chemical barrier to move to the ethanol area and consequently, crossed the barrier (Fig. 3). as an obstacle to interfere with the animals' ethanol seeking behavior. Since ethanol pretreatment 11 does not change the worm's sensitivity to the aversive stimulus (Fig.3b), ethanol seeking against 12 the aversive chemical barrier promise as an endophenotype for compulsive ethanol seeking. and must monitor its environment to determine whether stressful conditions warrant the expression 23 of their innate urges. The CRF (corticotropin-releasing factor) system plays a pivotal role in 1 mediating stress responses in the brain from amphibians to primates ( 95 , 96 , 97 , 98 ). A CRF 2 (corticotropin-releasing factor) receptor has been implicated in the pathophysiology of compulsive 3 behavior such as anxiety and AUD ( 99 ; 100 ; 101 ). Stress pathways have been studied as key 4 fundamentals of neural systems that drive alcohol and drug dependence. The brain stress system 5 is activated and sensitized during repeated withdrawal and lead to a negative emotional state that 6 promotes dependence ( 102 ). Indeed, the seb-3(eg696) gf animals, originally isolated as a genetic and Antagonism of NK1R decreases alcohol self-administration in alcohol-preferring rats ( 114 ).

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Furthermore, adeno-associated virus-mediated overexpression of NK1R in the central amygdala 1 increased alcohol self-administration ( 115 ), which is consistent with our finding. A Family-based 2 association study in human genetics have also reported NK3R is associated with alcohol and 3 cocaine dependence ( 116 ). 4 We report that SEB-3 facilitates animal drive to seek ethanol over noxious stimuli 5 representing enhanced compulsive seeking. Our functional genomics study revealed that TKR-1 6 is upregulated in seb-3 gf strain, which is defined as compulsive ethanol seeking animals, and 7 functions in the development of compulsive ethanol seeking. Interestingly, sodh-1, which showed 8 a significant alcohol intoxication phenotype in the orthogonal test for functional validation of ADH 9 (alcohol dehydrogenase) as a human GWAS candidate related to heavy alcohol consumption ( 25 ), 10 is also upregulated in seb-3 gf animals (Sup.1). These results also demonstrate the potential of our 11 investigation as a scalable model to accelerate the functional validation of alcohol dependence         between no barrier versus 2mM, 5mM, or 10mM in each genotype (p<0.001, ***; p<0.0001, ****).