A critical area of inquiry in the neurobiology of alcohol abuse is the mechanism by which cues gain the ability to elicit alcohol use. Previously, we found that cue‐evoked activity in rat ventral pallidum robustly encodes the value of sucrose cues trained under both Pavlovian and instrumental contingencies, despite a stronger relationship between cue‐evoked activity and behavioral latency after instrumental training (Richard et al., 2018, Elife, 7, e33107). Here, we assessed: (a) ventral pallidal representations of Pavlovian versus instrumental cues trained with alcohol reward, and (b) the impact of non‐associative alcohol exposure on ventral pallidal representations of sucrose cues. Decoding of cue identity based on ventral pallidum firing was blunted for the Pavlovian alcohol cue in comparison to both the instrumental cue trained with alcohol and either cue type trained with sucrose. Further, non‐associative alcohol exposure had opposing effects on ventral pallidal encoding of sucrose cues trained on instrumental versus Pavlovian associations, enhancing decoding accuracy for an instrumental discriminative stimulus and reducing decoding accuracy for a Pavlovian conditioned stimulus. These findings suggest that alcohol exposure can drive biased engagement of specific reward‐related signals in the ventral pallidum.

Per- and polyfluoroalkyl substances (PFASs) are ubiquitous in the environment and are tied to myriad health effects. Despite the phasing out of the manufacturing of two types of PFASs (perfluorosulfonic acid (PFOS) and perfluorooctanoic acid (PFOA)), chemical composition renders them effectively indestructible by ambient environmental processes, where they thus remain in water. Exposure via water can affect both human and aquatic wildlife. PFASs easily cross the placenta, exposing the fetus at critical windows of development. Little is known about the effects of low-level exposure during this period; even less is known about the potential for multi- and transgenerational effects. We examined the effects of ultra-low, very low, and low-level PFAS exposure (7, 70, and 700 ng/L PFOA; 24, 240, 2400 ng/L PFOS; and stepwise mixtures) from 0–5 days post-fertilization (dpf) on larval zebrafish (Danio rerio) mortality, morphology, behavior and gene expression and fecundity in adult F0 and F1 fish. As expected, environmentally relevant PFAS levels did not affect survival. Morphological abnormalities were not observed until the F1 and F2 generations. Behavior was affected differentially by each chemical and generation. Gene expression was increasingly perturbed in each generation but consistently showed lipid pathway disruption across all generations. Dysregulation of behavior and gene expression is heritable, even in larvae with no direct or indirect exposure. This is the first report of the transgenerational effects of PFOA, PFOS, and their mixture in terms of zebrafish behavior and untargeted gene expression.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a potent and environmentally persistent endocrine disrupting chemical. Our previous work demonstrated the latent reproductive maladies of early-life TCDD exposure in zebrafish. Zebrafish acutely exposed to low, environmentally relevant levels of TCDD (50 pg/mL) during two windows of sexual differentiation in development (1 hour of exposure at 3 and 7 weeks postfertilization) were later infertile, showed a reduction in sperm, and exhibited gene expression consistent with an altered microenvironment, even months after exposure. Due to the highly heterogeneous cell- type and -stage landscape of the testes, we hypothesized various cell types contribute markedly different profiles toward the pathology of TCDD exposure. To investigate the contributions of the diverse cell types in the adult zebrafish testes to TCDD-induced pathology, we utilized single-cell RNA-seq and the 10x Genomics platform. The method successfully captured every stage of testicular germ cell development. Testes of adult fish exposed during sexual differentiation to TCDD contained sharply decreased populations of late spermatocytes, spermatids, and spermatozoa. Spermatogonia and early spermatocyte populations were, in contrast, enriched following exposure. Pathway analysis of differentially expressed genes supported previous findings that TCDD exposure resulted in male infertility, and suggested this outcome is due to apoptosis of spermatids and spermatozoa, even years after exposure cessation. Increased germ cell apoptosis was confirmed histologically. These results provide support for an environmental exposure explanation of idiopathic male infertility.

A critical area of inquiry in the neurobiology of alcohol abuse is the neural mechanisms by which cues gain the ability to elicit alcohol use. We previously showed that cue-evoked activity in rat ventral pallidum (VP) robustly encodes the value of cues trained under both Pavlovian and instrumental contingencies, despite a stronger relationship between cue-evoked responses and behavioral latency after instrumental training. Here, we assessed VP neural representations of cue value in rats trained with a Pavlovian conditioned stimulus (CS+) that predicted alcohol delivery, and in rats trained with an instrumental discriminative stimulus (DS) that predicted alcohol availability if the rat entered the reward port during the cue. We also examined the impact of alcohol exposure itself on the integrity of this type of signaling in rats trained with sucrose. Decoding of cue value based on VP firing was blunted for an alcohol CS+ versus an alcohol DS, as well as in comparison to a sucrose DS or CS+. Further, homecage alcohol exposure had opposing effects on VP encoding of cue value for a sucrose DS versus a sucrose CS+, enhancing decoding accuracy for the DS and reducing decoding accuracy for the CS+. These findings suggest that problem alcohol seeking may result from biased engagement of specific reward-related processes via changes in VP signaling.

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In this report, we compare the outcomes and limitations of two methods of transcriptomic inquiry on adult zebrafish testes exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) during sexual differentiation: conventional or bulk RNA-seq (bulk-seq) and single cell RNA sequencing (scRNA-seq) data. scRNA-seq has emerged as a valuable tool for uncovering cell type-specific transcriptome dynamics which exist in heterogeneous tissue. Our lab previously showed the toxicological value of the scRNA-seq pipeline to characterize the sequelae of TCDD exposure in testes, demonstrating that loss of spermatids and spermatozoa, but not other cell types, contributed to the pathology of infertility in adult male zebrafish exposed during sexual differentiation. To investigate the potential for technical artifacts in scRNA-seq such as cell dissociation effects and reduced transcriptome coverage, we compared bulk-sequenced and scRNA-seq-paired samples from control and TCDD-exposed samples to understand what is gained and lost in scRNA-seq vs bulk-seq, both transcriptomically and toxicologically. We hypothesized that the testes may be sensitive to tissue disruption as they contain multiple cell types under constant division and/or maturation, and that TCDD exposure may mediate the extent of sensitivity. Thus, we sought to understand the extent to which this dissociation impacts the toxicological value of data returned from scRNA-seq. We confirm that the required dissociation of individual cells from intact tissue has a significant impact on gene expression, affecting gene pathways with the potential to confound toxicogenomics studies on exposures if findings are not well-controlled and well-situated in context. Additionally, a common scRNA-seq method using cDNA amplified from the 3’ end of mRNA under-detects low-expressing transcripts including transcription factors. We confirm this, and show TCDD-related genes may be overlooked by scRNA-seq, however, this under-detection effect is not mediated by TCDD exposure. Even so, scRNA-seq generally extracted toxicologically relevant information better than the bulk-seq method in the present study. This report aims to inform future experimental design for transcriptomic investigation in the growing field of toxicogenomics by demonstrating the differential information extracted from sequencing cells—despite being from the same tissue and exposure scheme—is influenced by the specific protocol used, with implications for the interpretation of exposure-induced risk.

Triclosan, triclocarban and 4-nonylphenol are all chemicals of emerging concern found in a wide variety of consumer products that have exhibited a wide range of endocrine-disrupting effects and are present in increasing amounts in groundwater worldwide. Results of the present study indicate that exposure to these chemicals at critical developmental periods, whether long-term or short-term in duration, leads to significant mortality, morphologic, behavioral and transcriptomic effects in zebrafish (Danio rerio). These effects range from total mortality with either long- or short-term exposure at 100 and 1000 nM of triclosan, to abnormalities in uninflated swim bladder seen with long-term exposure to triclocarban and short-term exposure to 4-nonylphenol, and cardiac edema seen with short-term 4-nonylphenol exposure. Additionally, a significant number of genes involved in neurological and cardiovascular development were differentially expressed after the exposures, as well as lipid metabolism genes and metabolic pathways after exposure to each chemical. Such changes in behavior, gene expression, and pathway abnormalities caused by these three known endocrine disruptors have the potential to impact not only the local ecosystem, but human health as well.