Persisting neurobehavioral consequences of daily or intermittent paternal cannabis administration in F1 and F2 Rats.

Repeated paternal preconception exposure to Δ9-tetrahydrocannabinol (Δ9-THC) alone or together with the other constituents in a cannabis extract has been shown in our earlier studies in rats to cause significant neurobehavioral impairment in their offspring. In the current study, we compared the effects of daily cannabis extract (CE) exposure to cannabis on two consecutive days per week, modeling weekend cannabis use in human. The CE contained Δ9-THC as well as cannabidiol and cannabinol. We also extended the investigation of the study to cross-generational effects of grand-paternal cannabis exposure on the F2 generation and included testing the effects of paternal cannabis exposure on responding for opiate self-administration in F1 and F2 generation offspring. We replicated the findings of neurobehavioral impairment in F1 offspring of male rats exposed to cannabis extract containing 4 mg/kg/day of Δ9-THC daily for four weeks prior to mating with drug naïve females. The 4-week cannabis extract exposure caused a significant decrease in weight gain in the male rats exposed daily. In contrast, their offspring showed significantly greater body weights and anogenital distances (AGD) in the third to fourth weeks after birth. The behavioral effects seen in the F1 generation were increased habituation of locomotor activity in the figure-8 maze in female offspring and increased lever pressing for the opiate drug remifentanil in male offspring. The F2 generation showed significantly impaired negative geotaxis and an elimination of the typical sex-difference in locomotor activity, with effects not seen in the F1 generation. This study shows that daily paternal cannabis exposure for four weeks prior to mating causes significant neurobehavioral impairment in the F1 and F2 offspring. Intermittent exposure on two consecutive days per week for four weeks caused comparable neurobehavioral impairment. In sum, there should be concern about paternal as well as maternal exposure to cannabis concerning neurobehavioral development of their offspring.

Embryonic exposures to cadmium and PAHs cause long-term and interacting neurobehavioral effects in zebrafish.

Developmental exposure to either polycyclic aromatic hydrocarbons (PAHs) or heavy metals has been shown to cause persisting and overlapping neurobehavioral effects in animal models. However, interactions between these compounds have not been well characterized, despite their co-occurrence in a variety of environmental media. In two companion studies, we examined the effects of developmental exposure to cadmium (Cd) with or without co-exposure to prototypic PAHs benzo[a]pyrene (BaP, Exp. 1) or fluoranthene (FA, Exp. 2) using a developing zebrafish model. Zebrafish embryos were exposed to Cd (0-0.3 µM), BaP (0-3 µM), FA (0-1.0 µM), or binary Cd-PAH mixtures from 5 to 122 h post fertilization (hpf). In Exp. 1, Cd and BaP produced independent effects on an array of outcomes and interacting effects on specific outcomes. Notably, Cd-induced deficits in dark-induced locomotor stimulation were attenuated by BaP co-exposure in the larval motility test and BaP-induced hyperactivity was attenuated by Cd co-exposure in the adolescent novel tank test. Likewise, in Exp. 2, Cd and FA produced both independent and interacting effects. FA-induced increases on adult post-tap activity in the tap startle test were attenuated by co-exposure with Cd. On the predator avoidance test, FA- and 0.3 µM Cd-induced hyperactivity effects were attenuated by their co-exposure. Taken together, these data indicate that while the effects of Cd and these representative PAHs on zebrafish behavior were largely independent of one another, binary mixtures can produce sub-additive effects for some neurobehavioral outcomes and at certain ages. This research emphasizes the need for detailed risk assessments of mixtures containing contaminants of differing classes, and for clarity on the mechanisms which allow cross-class toxicant interactions to occur.

Prenatal and perinatal exposure to Per- and polyfluoroalkyl substances (PFAS)-contaminated drinking water impacts offspring neurobehavior and development.

Per- and polyfluoroalkyl substances (PFAS) are persistent organic pollutants ubiquitous in the environment and humans. In-utero PFAS exposure is associated with numerous adverse health impacts. However, little is known about how prenatal PFAS mixture exposure affects offspring's neurobehavioral function. This study aims to determine the causal relationship between in-utero PFAS mixture exposure and neurobehavioral changes in Sprague-Dawley rat offspring. Dams were exposed via drinking water to the vehicle (control), an environmentally relevant PFAS mixture, or a high-dose PFAS mixture. The environmentally relevant mixture was formulated to resemble measured tap water levels in Pittsboro, NC, USA (10 PFAS compounds; sum PFAS =758.6 ng/L). The high-dose PFAS load was 3.8 mg/L (5000×), within the range of exposures in the experimental literature. Exposure occurred seven days before mating until birth. Following exposure to PFAS-laden water or the vehicle during fetal development, neurobehavioral toxicity was assessed in male and female offspring with a battery of motor, cognitive, and affective function tests as juveniles, adolescents, and adults. Just before weaning, the environmentally relevant exposure group had smaller anogenital distances compared to the vehicle and high-dose groups on day 17, and males in the environmentally relevant exposure group demonstrated lower weights than the high-dose group on day 21 (p < 0.05). Reflex development delays were seen in negative geotaxis acquisition for both exposure groups compared to vehicle-exposed controls (p = 0.009). Our post-weaning behavioral measures of anxiety, depression, and memory were not found to be affected by maternal PFAS exposure. In adolescence (week five) and adulthood (week eight), the high PFAS dose significantly attenuated typical sex differences in locomotor activity. Maternal exposure to an environmentally relevant PFAS mixture produced developmental delays in the domains of pup weight, anogenital distance, and reflex acquisition for rat offspring. The high-dose PFAS exposure significantly decreased typical sex differences in locomotor activity.

Developmental exposure to pesticides that disrupt retinoic acid signaling causes persistent retinoid and behavioral dysfunction in zebrafish.

Early developmental exposure to environmental toxicants may play a role in the risk for developing autism. A variety of pesticides have direct effects on retinoic acid (RA) signaling and as RA signaling has important roles in neurodevelopment, such compounds may cause developmental neurotoxicity through an overlapping adverse outcome pathway. It is hypothesized that a pesticide's embryonic effects on retinoid function may correspond with neurobehavioral disruption later in development. In the current studies, we determined the effects of RA-acting pesticides on neurobehavioral development in zebrafish. Buprofezin and imazalil caused generalized hypoactivity in the larval motility test, whereas chlorothalonil and endosulfan I led to selective hypoactivity and hyperactivity, respectively. With buprofezin, chlorothalonil, and imazalil, hypoactivity and/or novel anxiety-like behaviors persisted in adulthood and buprofezin additionally decreased social attraction responses in adulthood. Endosulfan I did not produce significant adult behavioral effects. Using qPCR analyses of adult brain tissue, we observed treatment-induced alterations in RA synthesis or catabolic genes, indicating persistent changes in RA homeostasis. These changes were compound-specific, with respect to expression directionality, and potential patterns of homeostatic disruption. Results suggest the likely persistence of disruptions in RA signaling well into adulthood and may represent compensatory mechanisms following early life stage exposures. This study demonstrates that early developmental exposure to environmental toxicants that interfere with RA signaling causes short as well as long-term behavioral disruption in a well-established zebrafish behavioral model and expand upon the meaning of the RA adverse outcome pathway, indicating that observed effects likely correspond with the nature of underlying homeostatic effects.

Developmental exposure of zebrafish to neonicotinoid pesticides: Long-term effects on neurobehavioral function.

Neonicotinoid compounds are commonly used insecticides which have become increasingly used as replacements of older generations of insecticides, such as organophosphates. Given the established neurotoxicity of cholinergic toxicants, developmental neurotoxicity studies are needed to identify in vertebrate species the potential toxicity of these insecticides which act on nicotinic cholinergic receptors. Previously, developmental exposure to a neonicotinoid insecticide imidacloprid was shown to cause persisting neurobehavioral toxicity in zebrafish. The current study evaluated neurobehavioral effects of embryonic exposure to two other neonicotinoid insecticides, clothianidin (1-100 µM) and dinotefuran (1-100 µM) in zebrafish (5-120 h post-fertilization), concentrations below the threshold for increased lethality and overt dysmorphogenesis. Neurobehavioral tests were conducted at larval (6 days), adolescent (10 weeks) and adult (8 months) ages. Both compounds caused short-term behavioral effects on larval motility, although these effects were distinct from one another. At a lower concentration (1 µM) clothianidin increased dark-induced locomotor stimulation the second time the lights turned off, while a higher concentration (100 µM) reduced activity in the dark at its second presentation. By contrast, dinotefuran (10-100 µM) caused a general decrease in locomotion. Specific longer-term neurobehavioral toxicity after early developmental exposure was also seen. clothianidin (100 µM) reduced locomotor activity in the novel tank in adolescence and adulthood, as well as reduced baseline activity in the tap startle test (1-100 µM) and reduced activity early (1-10 µM) or throughout the predator avoidance test session (100 µM). In addition to locomotor effects, clothianidin altered the diving response in a dose-, age- and time-block-dependent manner (1 µM, 100 µM), causing fish to remain further away from a fast predator cue (100 µM) relative to controls. Dinotefuran produced comparatively fewer effects, increasing the diving response in adulthood (10 µM), but not adolescence, and suppressing initial locomotor activity in the predator avoidance test (1-10 µM). These data indicate that neonicotinoid insecticides may carry some of the same risks for vertebrates posed by other classes of insecticides, and that these adverse behavioral consequences of early developmental exposure are evident well into adulthood.

Embryonic exposure to PFAS causes long-term, compound-specific behavioral alterations in zebrafish.

Per- and polyfluoroalkyl substances (PFAS) are commonly used as surfactants and coatings for industrial processes and consumer products. These compounds have been increasingly detected in drinking water and human tissue, and concern over their potential effects on health and development is growing. However, relatively little data are available for their potential impacts on neurodevelopment and the degree to which different compounds within this class may differ from one another in their neurotoxicity. The present study examined the neurobehavioral toxicology of two representative compounds in a zebrafish model. Zebrafish embryos were exposed to 0.1-100uM perfluorooctanoic acid (PFOA) or 0.01-1.0uM perfluorooctanesulfonic acid (PFOS) from 5 to 122 h post-fertilization. These concentrations were below threshold for producing increased lethality or overt dysmorphologies, and PFOA was tolerated at a concentration 100× higher than PFOS. Fish were maintained to adulthood, with behavioral assessments at 6 days, 3 months (adolescence) and 8 months of age (adulthood). Both PFOA and PFOS caused behavioral changes in zebrafish, but PFOS and PFOS produced strikingly different phenotypes. PFOA was associated with increased larval motility in the dark (100uM), and enhanced diving responses in adolescence (100uM) but not adulthood. PFOS was associated with a reversed light-dark response in the larval motility test (0.1-1uM), whereby the fish were more active in the light than the dark. PFOS also caused time-dependent changes in locomotor activity in the novel tank test during adolescence (0.1-1.0uM) and an overall pattern of hypoactivity in adulthood at the lowest concentration (0.01uM). Additionally, the lowest concentration of PFOS (0.01uM) reduced acoustic startle magnitude in adolescence, but not adulthood. These data suggest that PFOS and PFOA both produce neurobehavioral toxicity, but these effects are quite distinct from one another.

Developmental exposure to the flame retardant, triphenyl phosphate, causes long-lasting neurobehavioral and neurochemical dysfunction.

BACKGROUND: Human exposures to organophosphate flame retardants result from their use as additives in numerous consumer products. These agents are replacements for brominated flame retardants but have not yet faced similar scrutiny for developmental neurotoxicity. We examined a representative organophosphate flame retardant, triphenyl phosphate (TPP) and its potential effects on behavioral development and dopaminergic function. METHODS: Female Sprague-Dawley rats were given low doses of TPP (16 or 32 mg kg-1  day-1 ) via subcutaneous osmotic minipumps, begun preconception and continued into the early postnatal period. Offspring were administered a battery of behavioral tests from adolescence into adulthood, and littermates were used to evaluate dopaminergic synaptic function. RESULTS: Offspring with TPP exposures showed increased latency to begin eating in the novelty-suppressed feeding test, impaired object recognition memory, impaired choice accuracy in the visual signal detection test, and sex-selective effects on locomotor activity in adolescence (males) but not adulthood. Male, but not female, offspring showed marked increases in dopamine utilization in the striatum, evidenced by an increase in the ratio of the primary dopamine metabolite (3,4-dihydroxyphenylacetic acid) relative to dopamine levels. CONCLUSIONS: These results indicate that TPP has adverse effects that are similar in some respects to those of organophosphate pesticides, which were restricted because of their developmental neurotoxicity.

Sperm DNA methylation alterations from cannabis extract exposure are evident in offspring.

BACKGROUND: Cannabis legalization is expanding and men are the predominant users. We have limited knowledge about how cannabis impacts sperm and whether the effects are heritable. RESULTS: Whole genome bisulfite sequencing (WGBS) data were generated for sperm of rats exposed to: (1) cannabis extract (CE) for 28 days, then 56 days of vehicle only (~ one spermatogenic cycle); (2) vehicle for 56 days, then 28 days of CE; or (3) vehicle only. Males were then mated with drug-naïve females to produce F1 offspring from which heart, brain, and sperm tissues underwent analyses. There were 3321 nominally significant differentially methylated CpGs in F0 sperm identified via WGBS with select methylation changes validated via bisulfite pyrosequencing. Significant methylation changes validated in F0 sperm of the exposed males at the gene 2-Phosphoxylose Phosphatase 1 (Pxylp1) were also detectable in their F1 sperm but not in controls. Changes validated in exposed F0 sperm at Metastasis Suppressor 1-Like Protein (Mtss1l) were also present in F1 hippocampal and nucleus accumbens (NAc) of the exposed group compared to controls. For Mtss1l, a significant sex-specific relationship between DNA methylation and gene expression was demonstrated in the F1 NAc. Phenotypically, rats born to CSE-exposed fathers exhibited significant cardiomegaly relative to those born to control fathers. CONCLUSIONS: This is the first characterization of the effect of cannabis exposure on the entirety of the rat sperm methylome. We identified CE-associated methylation changes across the sperm methylome, some of which persisted despite a "washout" period. Select methylation changes validated via bisulfite pyrosequencing, and genes associated with methylation changes were involved in early developmental processes. Preconception CE exposure is associated with detectable changes in offspring DNA methylation that are functionally related to changes in gene expression and cardiomegaly. These results support that paternal preconception exposure to cannabis can influence offspring outcomes.

Embryonic exposure to benzo[a]pyrene causes age-dependent behavioral alterations and long-term metabolic dysfunction in zebrafish.

Polycyclic aromatic hydrocarbons (PAH) are products of incomplete combustion which are ubiquitous pollutants and constituents of harmful mixtures such as tobacco smoke, petroleum and creosote. Animal studies have shown that these compounds exert developmental toxicity in multiple organ systems, including the nervous system. The relative persistence of or recovery from these effects across the lifespan remain poorly characterized. These studies tested for persistence of neurobehavioral effects in AB* zebrafish exposed 5-120 h post-fertilization to a typical PAH, benzo[a]pyrene (BAP). Study 1 evaluated the neurobehavioral effects of a wide concentration range of BAP (0.02-10 µM) exposures from 5 to 120 hpf during larval (6 days) and adult (6 months) stages of development, while study 2 evaluated neurobehavioral effects of BAP (0.3-3 µM) from 5 to 120 hpf across four stages of development: larval (6 days), adolescence (2.5 months), adulthood (8 months) and late adulthood (14 months). Embryonic BAP exposure caused minimal effects on larval motility, but did cause neurobehavioral changes at later points in life. Embryonic BAP exposure led to nonmonotonic effects on adolescent activity (0.3 µM hyperactive, Study 2), which attenuated with age, as well as startle responses (0.2 µM enhanced, Study 1) at 6 months of age. Similar startle changes were also detected in Study 2 (1.0 µM), though it was observed that the phenotype shifted from reduced pretap activity to enhanced posttap activity from 8 to 14 months of age. Changes in the avoidance (0.02-10 µM, Study 1) and approach (reduced, 0.3 µM, Study 2) of aversive/social cues were also detected, with the latter attenuating from 8 to 14 months of age. Fish from study 2 were maintained into aging (18 months) and evaluated for overall and tissue-specific oxygen consumption to determine whether metabolic processes in the brain and other target organs show altered function in late life based on embryonic PAH toxicity. BAP reduced whole animal oxygen consumption, and overall reductions in total basal, mitochondrial basal, and mitochondrial maximum respiration in target organs, including the brain, liver and heart. The present data show that embryonic BAP exposure can lead to neurobehavioral impairment across the life-span, but that these long-term risks differentially emerge or attenuate as development progresses.

Persistent neurobehavioral and neurochemical anomalies in middle-aged rats after maternal diazinon exposure.

Diazinon is an organophosphate pesticide that has a history of wide use. Developmental exposures to organophosphates lead to neurobehavioral changes that emerge early in life and can persist into adulthood. However, preclinical studies have generally evaluated changes through young adulthood, whereas the persistence or progression of deficits into middle age remain poorly understood. The current study evaluated the effects of maternal diazinon exposure on behavior and neurochemistry in middle age, at 1 year postpartum, comparing the results to our previous studies of outcomes at adolescence and in young adulthood (4 months of age) (Hawkey 2020). Female rats received 0, 0.5 or 1.0 mg/kg/day of diazinon via osmotic minipump throughout gestation and into the postpartum period. The offspring were tested on a battery of locomotor, affective, and cognitive tests at young adulthood and during middle age. Some of the neurobehavioral consequences of developmental DZN seen during adolescence and young adulthood faded with continued aging, whereas other neurobehavioral effects emerged with aging. At middle age, the rats showed few locomotor effects, in contrast to the locomotor hyperactivity that had been observed in adolescence. Notably, though, DZN exposure during development impaired reference memory performance in middle-aged males, an effect that had not been seen in the younger animals. Likewise, middle-aged females exposed to DZN showed deficient attentional accuracy, an effect not seen in young adults. Across adulthood, the continued potential for behavioral defects was associated with altered dopaminergic function, characterized by enhanced dopamine utilization that was regionally-selective (striatum but not frontal/parietal cortex). This study shows that the neurobehavioral impairments from maternal low dose exposure to diazinon not only persist, but may continue to evolve as animals enter middle age.

The organophosphate insecticide diazinon and aging: Neurobehavioral and mitochondrial effects in zebrafish exposed as embryos or during aging.

Organophosphate (OP) compounds comprise one of the most widely used classes of insecticides worldwide. OPs have been shown to have negative human health impacts, particularly developmental neurotoxicity. However, neurotoxic impacts in later adulthood and during the aging process are relatively uncharacterized. The present study examined diazinon (DZN), an OP, to determine the neurobehavioral consequences, in addition to mitochondrial dysfunction on a macroscale (whole organism basal respiration) and on a microscale (whole organ mitochondrial respiration), using zebrafish (ZF) as a model. One group of 14-month-old adult ZF were exposed acutely as adults (0.4, 1.25, and 4.0 µM) for five days and tested as adults, and another group was exposed developmentally 5-120 h post-fertilization (70, 210, and 700 nM) and tested at larval, adolescent, adult, and aging life stages. ZF exposed acutely as adults did not display many significant neurobehavioral impacts or mitochondrial dysfunction. Conversely, the embryonically exposed ZF showed altered behavioral functions at each stage of life which emerged and attenuated as fish transitioned from each developmental stage to the next. Mitochondrial oxygen consumptions measurement results for developmentally DZN exposed ZF showed significant increases in the low and middle dose groups in organs such as the brain and testes. Overall, there is an indication that early developmental exposure to DZN had continuing adverse neurobehavioral and cellular consequences throughout their lives well into adulthood and aging periods.

Subchronic effects of plant alkaloids on anxiety-like behavior in zebrafish.

Zebrafish provide a valuable emerging complementary model for neurobehavioral research. They offer a powerful way to screen for the potential therapeutic effects of neuroactive drugs. A variety of behavioral tests for zebrafish have been developed and validated for assessing neurobehavioral function. The novel tank diving test is a straightforward, reproducible way of measuring anxiety-like behavior in zebrafish. When introduced into a novel tank, zebrafish normally dive to the bottom of the tank and then gradually explore the higher levels of the water column as time progresses. Buspirone is an effective anxiolytic drug in humans, which has been found, with acute administration, to reduce this anxiety-like response in zebrafish. The current study used the zebrafish model to evaluate the potential anxiolytic effects of alkaloids, commonly found in Solanaceae plants, with known neuropharmacology relevant to mood regulation. In line with previous findings, acute treatment with anxiolytic positive controls buspirone and the plant alkaloid nicotine reduced the anxiety-like diving response in the zebrafish novel tank diving test. Further, both buspirone and nicotine continued to produce anxiolytic-like effects in zebrafish after 5 days of exposure. In the same treatment paradigm, the effects of five other alkaloids-cotinine, anatabine, anabasine, harmane, and norharmane-were investigated. Cotinine, the major metabolite of nicotine, also caused anxiolytic-like effects, albeit at a dose higher than the effective dose of nicotine. Nicotine's anxiolytic-like effect was not shared by the other nicotinic alkaloids, anabasine and anatabine, or by the naturally present monoamine oxidase inhibitors harmane and norharmane. We conclude that nicotine uniquely induces anxiolytic-like effects after acute and subchronic treatment in zebrafish. The zebrafish model with the novel tank diving test could be a useful complement to rodent models for screening candidate compounds for anxiolytic effects in nonclinical studies.

Neurobehavioral anomalies in zebrafish after sequential exposures to DDT and chlorpyrifos in adulthood: Do multiple exposures interact?

A sequence of different classes of synthetic insecticides have been used over the past 70 years. Over this period, the widely-used organochlorines were eventually replaced by organophosphates, with dichlorodiphenyltrichloroethane (DDT) and chlorpyrifos (CPF) as the principal prototypes. Considerable research has characterized the risks of DDT and CPF individually, but little is known about the toxicology of transitioning from one class of insecticides to another, as has been commonplace for agricultural and pest control workers. This study used adult zebrafish to investigate neurobehavioral toxicity following 5-week chronic exposure to either DDT or CPF, to or their sequential exposure (DDT for 5 weeks followed by CPF for 5 weeks). At the end of the exposure period, a subset of fish were analyzed for brain cholinesterase activity. Behavioral effects were initially assessed one week following the end of the CPF exposure and again at 14 months of age using a behavioral test battery covering sensorimotor responses, anxiety-like functions, predator avoidance and social attraction. Adult insecticide exposures, individually or sequentially, were found to modulate multiple behavioral features, including startle responsivity, social approach, predator avoidance, locomotor activity and novel location recognition and avoidance. Locomotor activity and startle responsivity were each impacted to a greater degree by the sequential exposures than by individual compounds, with the latter being pronounced at the early (1-week post exposure) time point, but not 3-4 months later in aging. Social approach responses were similarly impaired by the sequential exposure as by CPF-alone at the aging time point. Fleeing responses in the predator test showed flee-enhancing effects of both compounds individually versus controls, and no additive impact of the two following sequential exposure. Each compound was also associated with changes in recognition or avoidance patterns in a novel place recognition task in late adulthood, but sequential exposures did not enhance these phenotypes. The potential for chemical x chemical interactions did not appear related to changes in CPF metabolism to the active oxon, as prior DDT exposure did not affect the cholinesterase inhibition resulting from CPF. This study shows that the effects of chronic adult insecticide exposures may be relevant to behavioral health initially and much later in life, and that the effects of sequential exposures may be unpredictable based on their constituent exposures.

Translating Neurobehavioral Toxicity Across Species From Zebrafish to Rats to Humans: Implications for Risk Assessment.

There is a spectrum of approaches to neurotoxicological science from high-throughput in vitro cell-based assays, through a variety of experimental animal models to human epidemiological and clinical studies. Each level of analysis has its own advantages and limitations. Experimental animal models give essential information for neurobehavioral toxicology, providing cause-and-effect information regarding risks of neurobehavioral dysfunction caused by toxicant exposure. Human epidemiological and clinical studies give the closest information to characterizing human risk, but without randomized treatment of subjects to different toxicant doses can only give information about association between toxicant exposure and neurobehavioral impairment. In vitro methods give much needed high throughput for many chemicals and mixtures but cannot provide information about toxicant impacts on behavioral function. Crucial to the utility of experimental animal model studies is cross-species translation. This is vital for both risk assessment and mechanistic determination. Interspecies extrapolation is important to characterize from experimental animal models to humans and between different experimental animal models. This article reviews the literature concerning extrapolation of neurobehavioral toxicology from established rat models to humans and from zebrafish a newer experimental model to rats. The functions covered include locomotor activity, emotion, and cognition and the neurotoxicants covered include pesticides, metals, drugs of abuse, flame retardants and polycyclic aromatic hydrocarbons. With more complete understanding of the strengths and limitations of interspecies translation, we can better use animal models to protect humans from neurobehavioral toxicity.

Paternal cannabis extract exposure in rats: Preconception timing effects on neurodevelopmental behavior in offspring.

Maternal toxicant exposure during gestation can have deleterious effects on neurobehavioral development of the offspring. The potential risks engendered by paternal toxicant exposure prior to conception have been largely understudied. Recently, we found that chronic THC exposure prior to conception in male rats causes long-lasting behavioral impairment in their offspring. The current study examined the effects of chronic preconception exposure to cannabis smoke extract in Sprague-Dawley rats at two different phases in sperm development. One group received daily subcutaneous (sc) injections of THC in cannabis extract at 4 mg/kg/day for 28 days until three days prior to mating with untreated females (late exposure group). Another group received the same regimen except they underwent 56 days of drug abstinence prior to mating (early exposure group). These were compared with a control group treated with vehicle. The offspring underwent a battery of tests for behavioral function to assess motor, emotional and cognitive function. On the elevated plus maze test, the offspring of both paternal cannabis smoke extract (CSE) exposure groups had significantly more time on the open arms than control offspring, indicative of greater risk-taking behavior. No significant main effects of CSE exposure were seen on adolescent or adult locomotor activity in the figure-8 apparatus. In the novel object recognition test, there was a significantly greater drop-off in novel object preference across the session in the male, but not female offspring of the late exposure group. There was also a sex-selective effect of paternal CSE treatment in the 16-arm radial maze test of memory function. Female offspring of the late exposure group had significantly more working memory errors than control females in the first half of the 12-session training sequence. No significant effects were seen in the operant visual signal sustained detection test of attention. This study shows that there are long-lasting behavioral consequences of preconception CSE exposure through the paternal lineage in rats.

Paternal factors in neurodevelopmental toxicology: THC exposure of male rats causes long-lasting neurobehavioral effects in their offspring.

The potential health risks of cannabis are of growing concern, including effects on reproduction and development. Extensive research has investigated risks associated with maternal exposure to THC during gestation and its impacts on the development of offspring, but little research has been done regarding paternal THC exposure effects prior to conception. We have previously found that paternal THC exposure in rats causes changes in sperm methylation. In an initial study we also showed that a 12-day paternal THC exposure prior to conception alters locomotor activity and impairs cognitive function of their offspring. This study investigated the cross-generational effects of chronic paternal THC exposure in rats (0, 2, or 4 mg/kg/day SC for 28 days) prior to mating with drug naïve females. The offspring of THC-exposed male rats had significant alterations in locomotor activity and cognitive function. Specifically, during adolescence there was significant locomotor hyperactivity in the offspring of males exposed to 2 mg/kg/day of THC. During the novel object recognition task, the controls maintained their relative preference for the novel object across the duration of the ten-min session while the rats whose fathers received THC (2 mg/kg/day) showed a significantly greater drop-off in interest in the novel object during the second half of the session. Learning in the radial-arm maze was significantly delayed in the offspring of males exposed to 4 mg/kg/day of THC. This study shows that premating chronic paternal THC exposure at multiple dose regimens can cause long-lasting detrimental behavioral effects in their offspring, including abnormal locomotor activity and impaired cognitive function. Future studies should investigate the underlying mechanisms driving these aberrant developmental outcomes and seek to identify possible treatments of alleviation in the presence of paternal THC exposure.

Adult exposure to insecticides causes persistent behavioral and neurochemical alterations in zebrafish.

Farmers are often chronically exposed to insecticides, which may present health risks including increased risk of neurobehavioral impairment during adulthood and across aging. Experimental animal studies complement epidemiological studies to help determine the cause-and-effect relationship between chronic adult insecticide exposure and behavioral dysfunction. With the zebrafish model, we examined short and long-term neurobehavioral effects of exposure to either an organochlorine insecticide, dichlorodiphenyltrichloroethane (DDT) or an organophosphate insecticide chlorpyrifos (CPF). Adult fish were exposed continuously for either two or 5 weeks (10-30 nM DDT, 0.3-3 µM CPF), with short- and long-term effects assessed at 1-week post-exposure and at 14 months of age respectively. The behavioral test battery included tests of locomotor activity, tap startle, social behavior, anxiety, predator avoidance and learning. Long-term effects on neurochemical indices of cholinergic function were also assessed. Two weeks of DDT exposure had only slight effects on locomotor activity, while a longer five-week exposure led to hypoactivity and increased anxiety-like diving responses and predator avoidance at 1-week post-exposure. When tested at 14 months of age, these fish showed hypoactivity and increased startle responses. Cholinergic function was not found to be significantly altered by DDT. The two-week CPF exposure led to reductions in anxiety-like diving and increases in shoaling responses at the 1-week time point, but these effects did not persist through 14 months of age. Nevertheless, there were persistent decrements in cholinergic presynaptic activity. A five-week CPF exposure led to long-term effects including locomotor hyperactivity and impaired predator avoidance at 14 months of age, although no effects were apparent at the 1-week time point. These studies documented neurobehavioral effects of adult exposure to chronic doses of either organochlorine or organophosphate pesticides that can be characterized in zebrafish. Zebrafish provide a low-cost model that has a variety of advantages for mechanistic studies and may be used to expand our understanding of neurobehavioral toxicity in adulthood, including the potential for such toxicity to influence behavior and development during aging.

Cannabis use is associated with potentially heritable widespread changes in autism candidate gene DLGAP2 DNA methylation in sperm.

Parental cannabis use has been associated with adverse neurodevelopmental outcomes in offspring, but how such phenotypes are transmitted is largely unknown. Using reduced representation bisulphite sequencing (RRBS), we recently demonstrated that cannabis use is associated with widespread DNA methylation changes in human and rat sperm. Discs-Large Associated Protein 2 (DLGAP2), involved in synapse organization, neuronal signaling, and strongly implicated in autism, exhibited significant hypomethylation (p < 0.05) at 17 CpG sites in human sperm. We successfully validated the differential methylation present in DLGAP2 for nine CpG sites located in intron seven (p < 0.05) using quantitative bisulphite pyrosequencing. Intron 7 DNA methylation and DLGAP2 expression in human conceptal brain tissue were inversely correlated (p < 0.01). Adult male rats exposed to delta-9-tetrahydrocannabinol (THC) showed differential DNA methylation at Dlgap2 in sperm (p < 0.03), as did the nucleus accumbens of rats whose fathers were exposed to THC prior to conception (p < 0.05). Altogether, these results warrant further investigation into the effects of preconception cannabis use in males and the potential effects on subsequent generations.

Chronic memantine decreases nicotine self-administration in rats.

Neurobehavioral bases of tobacco addiction and nicotine reinforcement are complex, involving more than only nicotinic cholinergic or dopaminergic systems. Memantine is an NMDA glutamate antagonist used to improve cognitive function in people with Alzheimer's disease. Glutamate may be an important component of the reinforcing effects of nicotine, so memantine was evaluated as a potential smoking cessation aid. Two studies were conducted with adult female rats, one testing acute effects of memantine over a range of doses for changing nicotine self-administration and the other testing the chronic effects of memantine to reduce nicotine self-administration. Acute memantine injections slightly, but significantly, increased nicotine self-administration in a dose-related manner. In contrast, chronic memantine treatment significantly reduced nicotine self-administration. During the first day of memantine administration in the chronic study, nicotine self-administration was significantly elevated replicating the acute study. Starting in the second week of treatment there was a significant reduction of nicotine self-administration relative to controls. This was seen because memantine treatment prevented the increase in nicotine self-administration shown by controls. There even continued to be a memantine-induced lowered nicotine self-administration during the week after the cessation of memantine treatment. Memantine or other drugs affecting NMDA glutamate receptors may be useful aids to smoking cessation. Full efficacy for reducing nicotine self-administration was seen as the NMDA drug treatment is given chronically. Importantly, the effect persisted even after treatment is ended, indicating the high potential for NMDA glutamate receptors to impact nicotine addiction.

Paternal nicotine exposure in rats produces long-lasting neurobehavioral effects in the offspring.

Studies of intergenerational effects of parental chemical exposure have principally focused on maternal exposure, particularly for studies of adverse neurobehavioral consequences on the offspring. Maternal nicotine exposure has long been known to cause adverse neurobehavioral effects on the offspring. However, paternal toxicant exposure has also been found to cause neurobehavioral toxicity in their offspring. Recent work suggests that paternal nicotine exposure can have epigenetic effects, although it remains unclear whether such changes lead to neurobehavioral effects. In the current study, we investigated the effects of paternal nicotine exposure on neurobehavioral development of their offspring. Male Sprague-Dawley rats were exposed to 0 or 2 mg/kg/day nicotine (sc) for 56 consecutive days with two consecutive 2ML4 osmotic minipumps. Following treatment, these males were mated with drug-naïve female rats. Offspring of both sexes were tested in a behavioral battery to assess locomotion, emotional function and cognition. Paternal nicotine exposure did not impact offspring viability, health or growth. However, behavioral function of the offspring was significantly altered by paternal nicotine exposure. Male offspring with paternal nicotine exposure exhibited locomotor hyperactivity in the Figure-8 apparatus when tested during adolescence. When retested in adulthood and regardless of sex, offspring of the nicotine exposed father showed significantly reduced habituation of locomotor activity over the course of the session. Compared to controls, female offspring of nicotine-exposed fathers showed significantly reduced response latency in the radial arm maze test. In addition to locomotor hyperactivity, the offspring of nicotine-exposed fathers also showed significantly diminished habituation in the novel object recognition test. These results indicate that chronic paternal nicotine exposure can impact the behavior of offspring, producing locomotor hyperactivity and impaired habituation.