JNK Signaling Positively Regulates Acute Ethanol Tolerance in C. elegans.

Alcohol use disorder (AUD) is a chronic neurobehavioral condition characterized by a cycle of tolerance development, increased consumption, and reinstated craving and seeking behaviors during withdrawal. Understanding the intricate mechanisms of AUD necessitates reliable animal models reflecting its key features. Caenorhabditis elegans (C. elegans), with its conserved nervous system and genetic tractability, has emerged as a valuable model organism to study AUD. Here, we employ an ethanol vapor exposure model in Caenorhabditis elegans, recapitulating AUD features while maintaining high-throughput scalability. We demonstrate that ethanol vapor exposure induces intoxication-like behaviors, acute tolerance, and ethanol preference, akin to mammalian AUD traits. Leveraging this model, we elucidate the conserved role of c-jun N-terminal kinase (JNK) signaling in mediating acute ethanol tolerance. Mutants lacking JNK signaling components exhibit impaired tolerance development, highlighting JNK's positive regulation. Furthermore, we detect ethanol-induced JNK activation in C. elegans. Our findings underscore the utility of C. elegans with ethanol vapor exposure for studying AUD and offer novel insights into the molecular mechanisms underlying acute ethanol tolerance through JNK signaling.

Nicotine Motivated Behavior in C. elegans.

To maximize the advantages offered by Caenorhabditis elegans as a high-throughput (HTP) model for nicotine dependence studies, utilizing its well-defined neuroconnectome as a robust platform, and to unravel the genetic basis of nicotine-motivated behaviors, we established the nicotine conditioned cue preference (CCP) paradigm. Nicotine CCP enables the assessment of nicotine preference and seeking, revealing a parallel to fundamental aspects of nicotine-dependent behaviors observed in mammals. We demonstrated that nicotine-elicited cue preference in worms is mediated by nicotinic acetylcholine receptors and requires dopamine for CCP development. Subsequently, we pinpointed nAChR subunits associated with nicotine preference and validated human GWAS candidates linked to nicotine dependence involved in nAChRs. Functional validation involves assessing the loss-of-function strain of the CACNA2D3 ortholog and the knock-out (KO) strain of the CACNA2D2 ortholog, closely related to CACNA2D3 and sharing human smoking phenotypes. Our orthogonal approach substantiates the functional conservation of the α2δ subunit of the calcium channel in nicotine-motivated behavior. Nicotine CCP in C. elegans serves as a potent affirmation of the cross-species functional relevance of GWAS candidate genes involved in nicotine seeking associated with tobacco abuse, providing a streamlined yet comprehensive system for investigating intricate behavioral paradigms within a simplified and reliable framework.

Intermittent-access operant alcohol self-administration promotes binge-like drinking and drinking despite negative consequences in male and female heterogeneous stock rats.

The study of Alcohol Use Disorders (AUD) in preclinical models is hampered by difficulty in training rodents to voluntarily consume high levels of alcohol. The intermittency of alcohol access/exposure is well known to modulate alcohol consumption (e.g., alcohol deprivation effect, intermittent-access two-bottle-choice) and recently, intermittent access operant self-administration procedures have been used to produce more intense and binge-like self-administration of intravenous psychostimulant and opioid drugs. In the present study, we sought to systematically manipulate the intermittency of operant self-administered alcohol access to determine the feasibility of promoting more intensified, binge-like alcohol consumption. To this end, 24 male and 23 female NIH Heterogeneous Stock rats were trained to self-administer 10% w/v ethanol, before being split into three different-access groups. Short Access (ShA) rats continued receiving 30-min training sessions, Long Access (LgA) rats received 16-h sessions, and Intermittent Access (IntA) rats received 16-h sessions, wherein the hourly alcohol-access periods were shortened over sessions, down to 2 min. IntA rats demonstrated an increasingly binge-like pattern of alcohol drinking in response to restriction of alcohol access, while ShA and LgA rats maintained stable intake. All groups were tested on orthogonal measures of alcohol-seeking and quinine-punished alcohol drinking. The IntA rats displayed the most punishment-resistant drinking. In a separate experiment, we replicated our main finding, that intermittent access promotes a more binge-like pattern of alcohol self-administration using 8 male and 8 female Wistar rats. In conclusion, intermittent access to self-administered alcohol promotes more intensified self-administration. This approach may be useful in developing preclinical models of binge-like alcohol consumption in AUD.

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.

Neurobiological Basis of Aversion-Resistant Ethanol Seeking in C. elegans.

Persistent alcohol seeking despite the risk of aversive consequences is a crucial characteristic of alcohol use disorders (AUDs). Therefore, an improved understanding of the molecular basis of alcohol seeking despite aversive stimuli or punishment in animal models is an important strategy to understand the mechanism that underpins the pathology of AUDs. Aversion-resistant seeking (ARS) is characterized by disruption in control of alcohol use featured by an imbalance between the urge for alcohol and the mediation of aversive stimuli. We exploited C. elegans, a genetically tractable invertebrate, as a model to elucidate genetic components related to this behavior. We assessed the seb-3 neuropeptide system and its transcriptional regulation to progress aversion-resistant ethanol seeking at the system level. Our functional genomic approach preferentially selected molecular components thought to be involved in cholesterol metabolism, and an orthogonal test defined functional roles in ARS through behavioral elucidation. Our findings suggest that fmo-2 (flavin-containing monooxygenase-2) plays a role in the progression of aversion-resistant ethanol seeking in C. elegans.

CRF-like receptor SEB-3 in sex-common interneurons potentiates stress handling and reproductive drive in C. elegans.

Environmental conditions can modulate innate behaviours. Although male Caenorhabditis elegans copulation can be perturbed in the presence of stress, the mechanisms underlying its decision to sustain copulation are unclear. Here we describe a mating interference assay, which quantifies the persistence of male C. elegans copulation in noxious blue light. We show that between copulations, the male escapes from blue light illumination at intensities over 370 µW mm(-2). This response is attenuated in mutants with constitutive activation of the corticotropin-releasing factor receptor family homologue SEB-3. We show that activation of this receptor causes sex-common glutamatergic lumbar ganglion interneurons (LUA) to potentiate downstream male-specific reproduction circuits, allowing copulatory behaviours to partially override the light-induced escape responses in the male. SEB-3 activation in LUA also potentiates copulation during mild starvation. We suggest that SEB-3 activation allows C. elegans to acclimate to the environment and thus continue to execute innate behaviours even under non-optimal conditions.

TMC-1 attenuates C. elegans development and sexual behaviour in a chemically defined food environment.

Although diet affects growth and behaviour, the adaptive mechanisms that coordinate these processes in non-optimal food sources are unclear. Here we show that the C. elegans tmc-1 channel, which is homologous to the mammalian tmc deafness genes, attenuates development and inhibits sexual behaviour in non-optimal food, the synthetic CeMM medium. In CeMM medium, signalling from the pharyngeal MC neurons and body wall muscles slows larval development. However, in the non-standard diet, mutation in tmc-1 accelerates development, by impairing the excitability of these cells. The tmc-1 larva can immediately generate ATP when fed CeMM, and their fast development requires insulin signalling. Our findings suggest that the tmc-1 channel indirectly affects metabolism in wild-type animals. In addition to regulating the development, we show that mutating tmc-1 can relax diet-induced inhibition of male sexual behaviour, thus indicating that a single regulator can be genetically modified to promote growth rate and reproductive success in new environments.

Caenorhabditis elegans male sensory-motor neurons and dopaminergic support cells couple ejaculation and post-ejaculatory behaviors.

The circuit structure and function underlying post-coital male behaviors remain poorly understood. Using mutant analysis, laser ablation, optogenetics, and Ca2+ imaging, we observed that following C. elegans male copulation, the duration of post-coital lethargy is coupled to cellular events involved in ejaculation. We show that the SPV and SPD spicule-associated sensory neurons and the spicule socket neuronal support cells function with intromission circuit components, including the cholinergic SPC and PCB and the glutamatergic PCA sensory-motor neurons, to coordinate sex muscle contractions with initiation and continuation of sperm movement. Our observations suggest that the SPV and SPD and their associated dopamine-containing socket cells sense the intrauterine environment through cellular endings exposed at the spicule tips and regulate both sperm release into the hermaphrodite and the recovery from post-coital lethargy.

CNP-1 (ARRD-17), a novel substrate of calcineurin, is critical for modulation of egg-laying and locomotion in response to food and lysine sensation in Caenorhabditis elegans.

Calcineurin is a Ca(2+)/calmodulin-dependent protein phosphatase involved in calcium signaling pathways. In Caenorhabditis elegans, the loss of calcineurin activity causes pleiotropic defects including hyperadaptation of sensory neurons, hypersensation to thermal difference and hyper-egg-laying when worms are refed after starvation. In this study, we report on arrd-17 as calcineurin-interacting protein-1 (cnp-1), which is a novel molecular target of calcineurin. CNP-1 interacts with the catalytic domain of the C. elegans calcineurin A subunit, TAX-6, in a yeast two-hybrid assay and is dephosphorylated by TAX-6 in vitro. cnp-1 is expressed in ASK, ADL, ASH and ASJ sensory neurons as TAX-6. It acts downstream of tax-6 in regulation of locomotion and egg-laying after starvation, ASH sensory neuron adaptation and lysine chemotaxis, that is known to be mediated by ASK neurons. Altogether, our biochemical and genetic evidence indicates that CNP-1 is a direct target of calcineurin and required in stimulated egg-laying and locomotion after starvation, adaptation to hyperosmolarity and attraction to lysine, which is modulated by calcineurin. We suggest that the phosphorylation status of CNP-1 plays an important role in regulation of refed stimulating behaviors after starvation and attraction to amino acid, which provides valuable nutritious information.

A novel calcineurin-interacting protein, CNP-3, modulates calcineurin deficient phenotypes in Caenorhabditis elegans.

Calcineurin (Cn) is a calcium/calmodulin-dependent serine/threonine protein phosphatase that has diverse functions in different cell types and organisms. We screened proteins interacting with the C. elegans CnA homolog, TAX-6, by the yeast two-hybrid system. CNP-3 (Calcineurin interacting protein-3) is a novel protein that physically interacts with the catalytic domain of TAX-6. It is strongly expressed in the nuclei of intestine, hypodermis, dorsal uterine regions and spermatheca. Expression begins around the 60-cell stage and proceeds during all larval stages and the adult. To elucidate the biological function of cnp-3 we isolated a cnp-3 deletion mutant. Since CNP-3 binds CnA, we looked at factors associated with calcineurin loss-of-function mutants, such as brood size, body size, serotonin- and levamisole-mediated egg-laying behavior. The cnp-3(jh145) single mutant had no gross defects compared to wild-type animal. However, the phenotypes of the double mutants, tax-6(p675);cnp-3(jh145) and cnb-1(jh103);cnp-3(jh145), were more severe in terms of brood size, body size and serotonin-mediated egg-laying defects than tax-6(p675) and cnb-1(jh103), respectively. These results suggest that dysfunction of cnp-3 enhances certain calcineurin loss-of-function phenotypes in C. elegans.

Calcineurin interacts with KIN-29, a Ser/Thr kinase, in Caenorhabditis elegans.

Calcineurin is a Ca2+/Calmodulin activated Ser/Thr phosphatase that is well conserved from yeast to human. In Caenorhabditis elegans, tax-6 and cnb-1 encode catalytic and regulatory subunits of calcineurin, respectively. We performed yeast two-hybrid screening using TAX-6 as a bait to identify calcineurin interacting proteins. KIN-29 is one of proteins that specifically interacted with TAX-6. KIN-29 is a Ser/Thr kinase previously shown to be involved in regulating gene expression of a subset of chemoreceptors in specific neurons. Both TAX-6 and KIN-29 are expressed in hypodermis, muscles, and neurons. Moreover, both calcineurin and kin-29 mutants exhibit similar phenotypes, namely small body size, small brood size, and slow growth. Here we describe specific genetic interaction between tax-6 and kin-29 in regulating body size, serotonin mediated egg laying, and chemoreceptor expression.

Thioredoxin is related to life span regulation and oxidative stress response in Caenorhabditis elegans.

Thioredoxin, an oxidoreductase, is a multifunction protein. The thioredoxin system is composed of NADPH, thioredoxin reductase and thioredoxin. This enzyme is highly conserved from bacteria to humans. We have characterized TRX-1, a thioredoxin homolog in C. elegans, which has about 36% identity in amino acid sequence with human thioredoxin. By gfp reporter system, trx-1 has been shown to be restrictedly expressed in ASI and ASJ neurons and in intestine. Immunostaining confirmed the intestinal expression. Full-length cDNA of trx-1 has been isolated by cDNA library PCR and subsequently cloned and sequenced. We have shown that the encoded protein functions as a reductase in the insulin reducing assay. Moreover, we have isolated a deletion mutant by PCR-based TMP-UV mutagenesis method. Mutant animals have reduced life span and are sensitive to oxidative stress. Reintroduction of trx-1 into mutant worms fully restored the wild-type phenotype. Our results suggest that trx-1 has important functions in life span regulation and oxidative stress response in C. elegans.

Calcineurin regulates enteric muscle contraction through EXP-1, excitatory GABA-gated channel, in C. elegans.

The enteric muscle contraction (EMC) is the last step of the defecation behavior which occurs every 50 s in Caenorhabditis elegans. This EMC is regulated by intestinal and anal depressor muscles, which are innervated by GABA motor neurons. Our data show that calcineurin (tax-6) is expressed in intestinal muscle and anal depressor muscle, and the gain-of-function mutant of calcineurin, tax-6(jh107), shows defects in enteric muscle contractions. In addition, the intracellular region of EXP-1, an excitatory GABA receptor, specifically binds to calcineurin A. This interaction between TAX-6 and EXP-1 appears to be independent of both calcium and CNB, which is the calcium-binding regulatory subunit. Genetic evidence of epistasis between cnb-1(jh103) and exp-1(sa6) suggests that calcineurin functions as a negative regulator of excitatory GABA receptor in GABA signaling in C.elegans.

Opposing functions of calcineurin and CaMKII regulate G-protein signaling in egg-laying behavior of C.elegans.

Ca(2+)/calmodulin-dependent calcineurin has been shown to have important roles in various Ca(2+) signaling pathways. We have previously reported that cnb-1(jh103) mutants, null mutants of a regulatory B subunit, displayed pleiotropic defects including uncoordinated movement and delayed egg laying in Caenorhabditis elegans. Interestingly, gain-of-function mutants of a catalytic A subunit showed exactly opposite phenotypes to those of cnb-1(null) mutants providing an excellent genetic model to define calcium-mediated signaling pathway at the organism level. Furthermore, calcineurin is also important for normal cuticle formation, which is required for maintenance of normal body size in C.elegans. Genetic interactions between tax-6 and several mutants including egl-30 and egl-10, which are known to be involved in G-protein signaling pathways suggest that calcineurin indeed regulates locomotion and serotonin-mediated egg laying through goa-1(Goalpha) and egl-30(Gqalpha). Our results indicate that, along with CaMKII, calcineurin regulates G-protein-coupled phosphorylation signaling pathways in C.elegans.

SHN-1, a Shank homologue in C. elegans, affects defecation rhythm via the inositol-1,4,5-trisphosphate receptor.

Protein localization in the postsynaptic density (PSD) of neurons is mediated by scaffolding proteins such as PSD-95 and Shank, which ensure proper function of receptors at the membrane. The Shank family of scaffolding proteins contain PDZ (PSD-95, Dlg, and ZO-1) domains and have been implicated in the localizations of many receptor proteins including glutamate receptors in mammals. We have identified and characterized shn-1, the only homologue of Shank in Caenorhabditis elegans. The shn-1 gene shows approximately 40% identity over 1000 amino acids to rat Shanks. SHN-1 protein is localized in various tissues including neurons, pharynx and intestine. RNAi suppression of SHN-1 did not cause lethality or developmental abnormality. However, suppression of SHN-1 in the itr-1 (sa73) mutant, which has a defective inositol-1,4,5-trisphosphate (IP(3)) receptor, resulted in animals with altered defecation rhythm. Our data suggest a possible role of SHN-1 in affecting function of IP(3) receptors in C. elegans.

Calcineurin, a calcium/calmodulin-dependent protein phosphatase, is involved in movement, fertility, egg laying, and growth in Caenorhabditis elegans.

Calcineurin is a Ca(2+)-calmodulin-dependent serine/threonine protein phosphatase that has been implicated in various signaling pathways. Here we report the identification and characterization of calcineurin genes in Caenorhabditis elegans (cna-1 and cnb-1), which share high homology with Drosophila and mammalian calcineurin genes. C. elegans calcineurin binds calcium and functions as a heterodimeric protein phosphatase establishing its biochemical conservation in the nematode. Calcineurin is expressed in hypodermal seam cells, body-wall muscle, vulva muscle, neuronal cells, and in sperm and the spermatheca. cnb-1 mutants showed pleiotropic defects including lethargic movement and delayed egg-laying. Interestingly, these characteristic defects resembled phenotypes observed in gain-of-function mutants of unc-43/Ca(2+)-calmodulin-dependent protein kinase II (CaMKII) and goa-1/G(o)-protein alpha-subunit. Double mutants of cnb-1 and unc-43(gf) displayed an apparent synergistic severity of movement and egg-laying defects, suggesting that calcineurin may have an antagonistic role in CaMKII-regulated phosphorylation signaling pathways in C. elegans.

Characterization of CeHDA-7, a class II histone deacetylase interacting with MEF-2 in Caenorhabditis elegans.

As a result of screen searching for proteins interacting with MEF-2 transcription factor, we have identified the hda-7 gene in Caenorhabditis elegans. The hda-7 locus encodes a class II histone deacetylase containing a highly conserved catalytic domain. C. elegans HDA-7 protein translated in vitro demonstrated a direct interaction with CeMEF-2, as shown in other organisms. CeHDA-7 is abundantly expressed in body-wall muscle cells, neurons, and hypodermal seam cells, similar to CeMEF-2 expression patterns. Consistent with previously known phenotypes observed in mef-2 deletion mutants [Dev. Biol. 223 (2000) 431], RNA interference targeted for hda-7 did not result in muscle function or developmental defects.