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The volatile compound 2,4,5-trimethylthiazoline (TMT, a synthetic predator scent) triggers fear, anxiety, and defensive responses in rodents that can outlast the encounter. The receptor systems underlying the development and persistence of TMT-induced behavioral changes remain poorly characterized, especially in females. Kappa opioid receptors regulate threat generalization and fear conditioning and alter basal anxiety, but their role in unconditioned fear responses in females has not been examined. Here, we investigated the effects of the long-lasting kappa opioid receptor antagonist, nor-binalthorphinmine dihydrochloride (nor-BNI; 10 mg/kg), on TMT-induced freezing and conditioned place aversion in female mice. We also measured anxiety-like behavior in the elevated plus maze three days after TMT and freezing behavior when returned to the TMT-paired context ten days after the single exposure. We found that 35μl of 10 % TMT elicited a robust freezing response during a five-minute exposure in female mice. TMT evoked persistent fear as measured by conditioned place aversion, reduced entries into the open arm of the elevated plus maze, and increased general freezing behavior long after TMT exposure. In line with the known role of kappa-opioid receptors in threat generalization, we found that kappa-opioid receptor antagonism increased basal freezing but reduced freezing during TMT presentation. Together, these findings indicate that a single exposure to TMT causes long-lasting changes in fear-related behavioral responses in female mice and highlights the modulatory role of kappa-opioid receptor signaling on fear-related behavioral patterns in females., Competing Interests: Declarations of interest None., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Adolescence is a critical period for social experience-dependent oligodendrocyte maturation and myelination. Adolescent stress predisposes to cause irreversible changes in brain structure and function with lasting effects on adulthood or beyond. However, the molecular mechanisms linking adolescent social isolation stress with emotional and social competence remain largely unknown. In our study, we found that social isolation during adolescence leads to anxiety-like behaviors, depression-like behaviors, impaired social memory and altered patterns of social ultrasonic vocalizations in mice. In addition, adolescent social isolation stress induces demyelination in the prefrontal cortex and hippocampus of mice, with decreased myelin-related gene expression and disrupted myelin structure. More importantly, clemastine was sufficient to rescue the impairment of emotional and social memory by promoting remyelination. These findings reveal the demyelination mechanism of emotional and social deficits caused by social isolation stress in adolescence, and provides potential therapeutic targets for treating stress-related mental disorders., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background: Postweaning is a pivotal period for brain development and individual growth. As an important chemical used in medicines, foods and beverages, sodium citrate (SC) is commonly available. Although some effects of SC exposure on individual physiology have been demonstrated, the potential long-lasting effects of postweaning dietary SC exposure on social behaviours are still elusive., Methods: Both postweaning male and female C57BL/6 mice were exposed to SC through drinking water for a total of 3 weeks. A series of behavioural tests, including social dominance test (SDT), social interaction test (SIT), bedding preference test (BPT) and sexual preference test (SPT), were performed in adolescence and adulthood. After these tests, serum oxytocin (OT) levels and gut microbiota were detected., Results: The behavioural results revealed that postweaning SC exposure decreased the social dominance of male mice in adulthood and female mice in both adolescence and adulthood. SC exposure also reduced the sexual preference rates of both males and females, while it had no effect on social interaction behaviour. ELISA results indicated that SC exposure decreased the serum OT levels of females but not males. 16S rRNA sequencing analysis revealed a significant difference in β-diversity after SC exposure in both males and females. The correlation coefficient indicated the correlation between social behaviours, OT levels and dominant genera of gut microbiota., Conclusion: Our findings suggest that postweaning SC exposure may have enduring and sex-dependent effects on social behaviours, which may be correlated with altered serum OT levels and gut microbiota composition., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

This report evaluates the effects of chlordiazepoxide, a benzodiazepine commonly prescribed to manage anxiety-related disorders in adolescent/pediatric populations, on elevated plus maze (EPM) performance in juvenile mice. This approach was taken because chlordiazepoxide produces anxiolytic-like effects in multiple models in adult rodents, however, less is known about the behavioral effects of this benzodiazepine in juveniles. Thus, we administered a single intraperitoneal injection of chlordiazepoxide (0, 5, or 10 mg/kg) to postnatal day 35 male C57BL/6 mice. Thirty minutes later, mice were allowed to explore the EPM for 5-min. We found that chlordiazepoxide-treated mice (5 and 10 mg/kg) spent more time exploring the open arms of the EPM. No differences in velocity (cm/s) or distance traveled (cm) were observed between the groups. These results indicate that chlordiazepoxide induces anxiolytic-related behavior in adolescent male mice., Competing Interests: Declaration of competing interest The authors declare no financial, or non-financial, conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Formosan wood mice (Apodemus semotus) are endemic rodents in Taiwan. Recently Formosan wood mice exhibit similar locomotor behaviors in the laboratory environment as in the field environment has shown. Contemporaneously, Formosan wood mice have higher moving distances of and central dopaminergic (DAergic) activities than C57BL/6 mice in behavioral test. This study tried to compare the behavioral responses between male Formosan wood mice and male C57BL/6 mice in the light-dark exploration tests. We also measured the levels of DA and 3,4-dihydroxyphenylacetic acid (DOPAC), the primary metabolite of DA, to assess the dopaminergic activity of the medial prefrontal cortex, striatum, and nucleus accumbens. Our data show that Formosan wood mice revealed higher exploration and central DAergic activities than did C57BL/6 mice in the light-dark exploration tests, and diazepam (an anxiolytics) treatment reduced the exploratory activity and central dopaminergic activities in Formosan wood mice, but not in C57BL/6 mice. After repeated exposure to light-dark exploration tests, the latency to dark zone was increased, and the duration in light zone as well as the central DAergic activity were decreased in C57BL/6 mice. This study provides comparative findings; Formosan wood mice showed the higher exploratory activities than C57BL/6 mice did, and their central DAergic activities were related to the behavioral responses in these two mice. This could potentially shed light on the reasons behind the prevalence of higher exploration and central dopaminergic activities. Using Formosan wood mice as a model to study human diseases related to hyperactivity adds significant value to the potential research., Competing Interests: Declaration of competing interest The authors have declared that no competing interests exist., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Psychological stress affects the neuroendocrine regulation, which modulates mental status and behaviors. Melatonin, a hormone synthesized primarily by the pineal gland, regulates many brain functions, including circadian rhythms, pain, sleep, and mood. Selective pharmacological melatonin agonist ramelteon has been clinically used to treat mood and sleep disorders. Posttraumatic stress disorder (PTSD) is a psychiatric condition associated with severe trauma; it is generally triggered by traumatic events, which lead to severe anxiety and uncontrollable trauma recall. We recently reported that repeated social defeat stress (RSDS) may induce robust anxiety-like behaviors and social avoidance in mice. In the present study, we investigated whether melatonin receptor activation by melatonin and ramelteon regulates RSDS-induced behavioral changes. Melatonin treatment improved social avoidance and anxiety-like behaviors in RSDS mice. Moreover, treatment of the non-selective MT 1 /MT 2 receptor agonist, ramelteon, markedly ameliorated RSDS-induced social avoidance and anxiety-like behaviors. Moreover, activating melatonin receptors also balanced the expression of monoamine oxidases, glucocorticoid receptors, and endogenous antioxidants in the hippocampus. Taken together, our findings indicate that the activation of both melatonin and ramelteon regulates RSDS-induced anxiety-like behaviors and PTSD symptoms. The current study also showed that the regulatory effects of neuroendocrine mechanisms and cognitive behaviors on melatonin receptor activation in repeated social defeat stress., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2024. Published by Elsevier Inc.)

Milk varieties and specific proteins exhibit anxiolytic-like actions in mice and rats exposed to several tests, the most prominent being the elevated plus-maze. Administrations of α s1 -casein, its 91-100 (α-casozepine), 91-97, 91-93, and 91-92 fragments, the 60-69 fragment of β-casein, lactoferrin, β-lactotensin, wheylin-1, wheylin-2, and α-lactalbumin have been reported to increase open arm exploration relative to enclosed arm exploration. Anxiolytic-like actions have also been described for 91-93 and 91-92 fragments of α s1 -casein, wheylin-1, α-lactalbumin, and lactoferrin in the open-field. Some effects appear to be mediated by the GABA A receptor complex, since antagonists mitigated the anxiolytic-like actions of α s1 -casein, the 91-92 fragment of α s1 -casein, and wheylin-1. Other neurotransmitters purported to affect such behaviors include 5HT, dopamine, and neurotensin. Further research is needed to identify their neuropharmacological actions., Competing Interests: Declaration of competing interest The author reports no conflict of interest for this review., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Research using zebrafish (Danio rerio) has begun to provide novel information in many fields, including the behavioral pharmacology of drug use and misuse. There have been limited studies on the effects of methamphetamine in adult zebrafish and the parameters of exposure (dose, test session length) have not been well-documented. Behavior following drug exposure is generally measured during relatively short sessions (6-10 min is common) in a novel tank environment. Many procedural variables (isolation, netting, novel tank) elicit anxiety-like behavior that is most apparent during the initial portion of a test session. This anxiety-like behavior might mask the initial effects of methamphetamine. During longer test sessions, these anxiety-like responses would be expected to habituate and drug effects should become more apparent. To test this idea, we measured several locomotor activity responses for 50-min following a range of methamphetamine doses (0.1-3.0 mg/L via immersion in methamphetamine solution). Methamphetamine failed to alter swimming velocity, distance travelled, or freezing time. In contrast, methamphetamine produced a dose-dependent decrease in time spent in the bottom of the tank, an increase in the number of visits to the top of the tank, and an increase in the number of transitions along the sides of the tank. The effects of methamphetamine were apparent 10-20 min following exposure and generally persisted throughout the session. These findings indicate that longer test sessions are required to measure methamphetamine-induced changes in behavior in zebrafish, as has been shown in other laboratory animals. The results also suggest that anxiety-like responses associated with various procedural aspects (netting, isolation, novel test apparatus) likely interfere with the ability to observe many behavioral effects of methamphetamine in zebrafish. Based on the current results, habituation to testing procedures to reduce anxiety-like behaviors is recommended in determining the effects of methamphetamine in zebrafish., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Autism is a complex neurodevelopmental disorder with no effective treatment available currently. Repetitive transcranial magnetic stimulation (rTMS) is emerging as a promising neuromodulation technique to treat autism. However, the mechanism how rTMS works remains unclear, which restrict the clinical application of magnetic stimulation in the autism treatment. In this study, we investigated the effect of low-frequency rTMS on the autistic-like symptoms and explored if this neuroprotective effect was associated with synaptic plasticity and neuroinflammation in the hippocampus. A rat model of autism was established by intraperitoneal injection of valproic acid (VPA) in pregnant rats and male offspring were treated with 1 Hz rTMS daily for two weeks continuously. Behavior tests were performed to identify behavioral abnormality. Synaptic plasticity was measured by in vivo electrophysiological recording and Golgi-Cox staining. Synapse and inflammation associated proteins were detected by immunofluorescence and Western blot analyses. Results showed prenatal VPA-exposed rats exhibited autistic-like and anxiety-like behaviors, and cognitive impairment. Synaptic plasticity deficits and the abnormality expression of synapse-associated proteins were found in the hippocampus of prenatal VPA-exposed rats. Prenatal VPA exposure increased the level of inflammation cytokines and promoted the excessive activation of microglia. rTMS significantly alleviated the prenatal VPA-induced abnormalities including behavioral and synaptic plasticity deficits, and excessive neuroinflammation. TMS maybe a potential strategy for autism therapy via rescuing synaptic plasticity and inhibiting neuroinflammation., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Depression and anxiety disorders have their pathophysiologies linked to inflammation and oxidative stress. In this context, celecoxib (CLX) and etoricoxib (ETR) inhibit cyclooxygenase 2 (COX-2), an enzyme expressed by cells involved in the inflammatory process and found in the brain. Studies have been using CLX as a possible drug in the treatment of depression, although its mechanisms at the central nervous system level are not fully elucidated. In this study, the effects of CLX and ETR on behavioral, oxidative, and inflammatory changes induced by systemic exposure to Escherichia coli lipopolysaccharide (LPS) were evaluated in adult male swiss mice. For ten days, the animals received intraperitoneal injections of LPS at 0.5 mg/kg. From the sixth to the tenth day, one hour after LPS exposure, they were treated orally with CLX (15 mg/kg), ETR (10 mg/kg), or fluoxetine (FLU) (20 mg/kg). Twenty-four hours after the last oral administration, the animals underwent evaluation of locomotor activity (open field test), predictive tests for depressive-like behavior (forced swim and tail suspension tests), and anxiolytic-like effect (elevated plus maze and hole board tests). Subsequently, the hippocampus, prefrontal cortex and striatum were dissected for the measurement of oxidative and nitrosative parameters (malondialdehyde, nitrite, and glutathione) and quantification of pro-inflammatory cytokines (IL-1β and IL-6). LPS induced depressive and anxious-like behavior, and treatment with CLX or ETR was able to reverse most of the behavioral changes. It was evidenced that nitrosative stress and the degree of lipid peroxidation induced by LPS were reduced in different brain areas after treatment with the drugs, as well as the endogenous defense system against free radicals was strengthened. CLX and ETR also significantly reduced LPS-induced cytokine levels. These data are expected to expand information on the role of inflammation in depression and anxiety and provide insights into possible mechanisms of COX-2 inhibitors in psychiatric disorders with a neurobiological basis in inflammation and oxidative stress., Competing Interests: Declaration of competing interest None., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Ketamine is a rapid-acting antidepressant associated with various cognitive side effects. To mitigate these side effects while enhancing efficacy, it can be co-administered with other antidepressants. In our study, we adopted a similar strategy by combining ketamine with environmental enrichment, a potent sensory-motor paradigm, in adult male Wistar rats. We divided the animals into four groups based on a combination of housing conditions and ketamine versus vehicle injections. The groups included those housed in standard cages or an enriched environment for 50 days, which encompassed a 13-day-long behavioral testing period. Each group received either two doses of ketamine (20 mg/kg, IP) or saline as a vehicle. We tested the animals in the novel object recognition test (NORT), forced swim test (FST), open field test (OFT), elevated plus maze (EPM), and Morris water maze (MWM), which was followed by ex vivo c-Fos immunohistochemistry. We observed that combining environmental enrichment with ketamine led to a synergistic antidepressant effect. Environmental enrichment also ameliorated the spatial memory deficits caused by ketamine in the MWM. There was enhanced neuronal activity in the habenula of the enrichment only group following the probe trial of the MWM. In contrast, no differential activity was observed in enriched animals that received ketamine injections. The present study showed how environmental enrichment can enhance the antidepressant properties of ketamine while reducing some of its side effects, highlighting the potential of combining pharmacological and sensory-motor manipulations in the treatment of mood disorders., Competing Interests: Declaration of competing interest None., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The present experiment used the trapped rat model to explore whether pharmacological manipulation of distress affects the likelihood of helping behavior. 120 Sprague-Dawley rats (30 male pairs and 30 female pairs) completed 12 consecutive, daily trials assessing helping behavior. During an individual trial, a trapped rat was placed in a restrainer in the center of an open field, while its cagemate could move around freely and possibly open the restrainer by lifting a door. Trapped rats received an intraperitoneal injection of either 1) physiological saline, 2) the anxiolytic midazolam (1.5 mg/kg), or 3) the anxiogenic yohimbine (2.5 mg/kg) 30 min prior to the start of each trial. Dependent variables measured were: 1) door opening latency (sec), 2) percentage of trials in which a door opening occurred, and 3) the number of free rats classified as "openers." Based on emotional contagion theory, we predicted that 1) free rats paired with midazolam-subjects would show attenuated helping behavior (e.g., higher door opening latency) compared to controls, and conversely 2) free rats paired with yohimbine-subjects would show enhanced helping behavior. First, a significant sex-difference was observed, in that more females were classified as openers than males. This supports previous evidence that females express higher altruistic motivation and experience stronger emotional contagion than males. Second, midazolam-treatment significantly attenuated helping behavior. From trials 4-12, free rats paired with midazolam-subjects expressed slower door opening latencies compared to controls. Third, yohimbine-treatment significantly increased helping behavior (e.g., reduced door opening latencies) - but only on trials 1-3; by trials 9-12, this pattern was reversed. These results are consistent with emotional contagion theory and indicate that intensity of distress directly modulates altruistic motivation through vicarious state-matching., (Copyright © 2024 Elsevier Inc. All rights reserved.)

This study investigated the effects of anxiogenic and anxiolytic drugs on zebrafish (Danio rerio) behaviour using a modified novel tank dive test with higher walls and a narrower depth. Zebrafish were administered chondroitin sulfate, beta-carboline, delta-9-tetrahydrocannabinol (THC), ethanol, and beta-caryophyllene, and their behaviours were evaluated for geotaxis, swimming velocity, and immobility. Both anxiogenic and anxiolytic compounds generally increased bottom-dwelling behaviour, suggesting that the tank's modified dimensions significantly influence zebrafish responses. EC 50 values for ethanol showed a lower threshold for velocity reduction compared to zone preference. Chondroitin sulfate uniquely caused a sex-specific increase in male swimming velocity, whereas no other sex-differences were observed with any compound. Interestingly, the presence of drug-treated fish did not alter the behaviour of observer fish, suggesting limited social buffering effects. The findings underscore the complexity of zebrafish behavioural phenotypes and highlight the need for considering tank dimensions and multiple behavioural parameters to accurately assess the effects of anxiety-modulating drugs. This study demonstrates the utility of the modified novel tank dive test in providing nuanced insights into the behavioural effects of different pharmacological agents in zebrafish., Competing Interests: Declaration of competing interest The authors declare they have no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Nitric oxide (NO) is a molecule involved in plasticity across levels and systems. The role of NOergic pathways in stress-induced sensitization (SIS) of behavioral responses, in which a particular stressor triggers a state of hyper-responsiveness to other stressors after an incubation period, was assessed in adult zebrafish. In this model, adult zebrafish acutely exposed to a fear-inducing conspecific alarm substance (CAS) and left undisturbed for an incubation period show increased anxiety-like behavior 24 h after exposure. CAS increased forebrain glutamate immediately after stress and 30 min after stress, an effect that was accompanied by increased nitrite levels immediately after stress, 30 min after stress, 90 min after stress, and 24 h after stress. CAS also increased nitrite levels in the head kidney, where cortisol is produced in zebrafish. CAS-elicited nitrite responses in the forebrain 90 min (but not 30 min) after stress were prevented by a NOS-2 blocker. Blocking NOS-1 30 min after stress prevents SIS; blocking NOS-2 90 min after stress also prevents stress-induced sensitization, as does blocking calcium-activated potassium channels in this latter time window. Stress-induced sensitization is also prevented by blocking guanylate cyclase activation in both time windows, and cGMP-dependent channel activation in the second time window. These results suggest that different NO-related pathways converge at different time windows of the incubation period to induce stress-induced sensitization., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Alcohol use disorder (AUD) is a chronic relapsing disease that is deleterious at individual, familial, and societal levels. Although AUD is one of the highest preventable causes of death in the USA, therapies for the treatment of AUD are not sufficient given the heterogeneity of the disorder and the limited number of approved medications. To provide better pharmacological strategies, it is important to understand the neurological underpinnings of AUD. Evidence implicates the endogenous dynorphin (DYN)/κ-opioid receptor (KOR) system recruitment in dysphoric and negative emotional states in AUD to promote maladaptive behavioral regulation. The nucleus accumbens shell (AcbSh), mediating motivational and emotional processes that is a component of the mesolimbic dopamine system and the extended amygdala, is an important site related to alcohol's reinforcing actions (both positive and negative) and neuroadaptations in the AcbSh DYN/KOR system have been documented to induce maladaptive symptoms in AUD. We have previously shown that in other nodes of the extended amygdala, site-specific KOR antagonism can distinguish different symptoms of alcohol dependence and withdrawal. In the current study, we examined the role of the KOR signaling in the AcbSh of male Wistar rats in operant alcohol self-administration, measures of negative affective-like behavior, and physiological symptoms during acute alcohol withdrawal in alcohol-dependence. To induce alcohol dependence, rats were exposed to chronic intermittent ethanol vapor for 14 h/day for three months, during which stable escalation of alcohol self-administration was achieved and pharmacological AcbSh KOR antagonism ensued. The results showed that AcbSh KOR antagonism significantly reduced escalated alcohol intake and negative affective-like states but did not alter somatic symptoms of withdrawal. Understanding the relative contribution of these different drivers is important to understand and inform therapeutic efficacy approaches in alcohol dependence and further emphasis the importance of the KOR/DYN system as a target for AUD therapeutics., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The population of most countries in the world is increasing and understanding risk factors that can influence the health of the older population is critical. Older adults consume alcohol often in a risky, binge manner. Previous work has demonstrated that aged rats are more sensitive to many of the effects of acute ethanol. In the current project aged, adult, and adolescent female and male rats were tested on the elevated plus maze and open field following either a 1.0 g/kg alcohol injection or a saline injection. We report sex- and age-dependent effects whereas aged female rats, but not aged male rats, showed an increased anxiolytic effect of alcohol in the elevated plus maze while aged male rats, but not aged female rats, showed increased stimulatory movement in the open field. In addition, significant age effects were found for both female and male rats. It is proposed that the sex- and age-dependent effects reported in the current studies may be due to differential levels of alcohol-induced allopregnanolone for the anxiolytic effects and differential levels of alcohol-induced dopamine for the stimulatory effects. The current work provides insights into factors influencing alcohol consumption in older adults., (Copyright © 2024 Elsevier Inc. All rights reserved.)

In the search for anxiolytic drugs with fewer adverse effects, calcium blockers were proposed as a benzodiazepines (BZDs) alternative. In this context, the anxiolytic effect of nimodipine has been demonstrated. However, its low bioavailability and solubility could be improved by using nanostructured drug delivery systems such as liposomes. In this way, liposomal formulation containing nimodipine (NMD-Lipo) was developed. The NMD-lipo is a formulation capable of improving the kinetic characteristics of the drug, as well as the anxiolytic effect of nimodipine. In this work, the serotonergic system participation in the anxiolytic mechanism of the liposomal formulation containing nimodipine (NMD-Lipo) was investigated. A possible 5-HT1A receptor mediation on the NMD-Lipo anxiolytic effect was demonstrated by using WAY 100635 (5-HT1A receptor antagonist) since the antagonist reversed the NMD-Lipo anxiolytic effect in the light/dark test and elevated plus maze test. The results demonstrated that the NMD-Lipo administration had anxiolytic activity through 5-HT1A receptors without causing sedation or compromising the motor coordination of the tested animals., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Obsessive-compulsive disorder (OCD) is a disabling mental condition that poses recurring bothersome intrusive thoughts, obsessions, and compulsions. Considering the positive impact of probiotics on neuropsychiatric disorders, herein, we investigated the effect of multistrain probiotic (Bifidobacterium lactis UBBLa-70, Bacillus coagulans Unique IS-2, Lactobacillus rhamnosus UBLR-58, Lactobacillus plantarum UBLP-40, Bifidobacterium infantis UBBI-01, Bifidobacterium breve UBBr-01, and glutamine) in the management of OCD-like phenotype in rats. Rats injected with quinpirole for 5 weeks showed an increased number of marble burying and self-grooming episodes. Quinpirole-injected animals also did less head dipping in the hole board test and avoided exploration of open spaces in the elevated-plus maze. These repetitive, compulsive, self-directed, and anxiety-like phenotypes were abolished after 8-week of multistrain probiotic treatment. The probiotic formulation also prevented the elevated mRNA expression of interleukin-6, tumor-necrosis factor-α, and C-reactive protein in the amygdala and dysregulated levels of 5-hydroxytryptamine, dopamine, and noradrenaline in the frontal cortex of quinpirole-injected rats. The level of brain-derived neurotrophic factor in the frontal cortex remained unaffected across the groups. The altered levels of goblet cells and crypt-to-villi ratio in quinpirole rats were prevented by multistrain probiotic treatment. The results of 16S-rRNA gene-sequencing of gut microbiota from feces contents revealed an elevation in the abundance of Allobaculum and Bifidobacterium species (specifically Bifidobacterium animalis), while the presence of Lactobacillus species (including Lactobacillus reuteri and Lactobacillus vaginalis) exhibited a decline in quinpirole-induced rats. These results imply that modifying the gut-brain axis may be a possible mechanism by which selective multistrain probiotic therapy prevents OCD-like behaviors., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2023. Published by Elsevier Inc.)

Major depression disorder is a debilitating psychiatric disease affecting millions of people worldwide. This disorder is the leading cause of morbidity and mortality in high-income countries. Selective serotonin reuptake inhibitors such as fluoxetine are first-line drugs for treating depression-related disorders, but not all patients respond well to these antidepressants. This study aimed to evaluate whether fluoxetine combined with aerobic exercise can affect lipopolysaccharide (LPS)-induced depressive-like behavior, hypothalamic-pituitary-adrenal (HPA) axis dysregulation, and brain inflammation in mice. Male mice were exposed to fluoxetine, swimming exercise, or a combination of both and finally treated with LPS. We measured depression-related symptoms such as anhedonia, behavioral despair, weight gain, and food intake. Hormones (corticosterone and testosterone) and cytokines (IL-1β, IL-6, TNF-α, IL-10) were also measured in serum and brain (hippocampus and prefrontal cortex), respectively. The findings indicated that LPS induced anhedonia and behavioral despair and increased corticosterone, hippocampal IL-1β, TNF-α, and decreased testosterone and hippocampal IL-10 in mice. Fluoxetine and exercise separately reduced LPS-induced depressive-like behavior, while their combination synergistically reduced these symptoms in LPS-treated mice. We found fluoxetine alone increased food intake and body weight in LPS-treated mice. Fluoxetine and exercise combination reduced corticosterone, hippocampal TNF-α, and prefrontal IL-6 and TNF-α levels and increased testosterone and hippocampal and prefrontal IL-10 levels more effectively than fluoxetine alone in LPS-treated mice. This study suggests that swimming exercise combined with fluoxetine can affect depression-related behavior, HPA axis, and brain inflammation more effectively than when they are used separately., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Despite considerable evidence suggesting that sweet foods are a substitute for nicotine in humans, no formal behavioral economic analysis of this interrelationship has been conducted in nonhumans. The purpose of the present study was to examine this phenomenon in rats using concurrent schedules of sucrose pellet, chow pellet, and nicotine reinforcer delivery. Rats responded on separate levers that delivered sucrose pellets, chow pellets, or nicotine infusions under concurrent fixed-ratio (FR) 1 schedules for each commodity within a closed economy. Following stable food and nicotine intake, the unit price of either sucrose or nicotine (the primary commodity) was increased while the two alternative commodities remained unchanged. Substitution was quantified using a behavioral economic cross-price model, as well as a novel commodity relation index that normalizes consumption of dissimilar commodities. Asymmetrical partial substitution was observed, wherein sucrose served as a partial substitute for nicotine, but nicotine failed to substitute for sucrose. Moreover, sucrose was a stronger partial substitute for nicotine than chow in most rats. These findings indicate that substitution of food for nicotine depends on the type of food. These findings mirror the selective increase in carbohydrate intake that can occur during smoking cessation and demonstrate a behavioral economic mechanism that may mediate it., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Anxiety disorders, characterized by high prevalence rates, cause psychiatric disabilities and are related to impairments in serotoninergic system function. Frequent anxiety recurrence, resistance, and drug adverse effects have driven searches for new therapies. We initially evaluated the anxiolytic-like activity of 3-selanyl-benzo[b]furan compounds (SeBZF1-5) (50 mg/kg, i.g.) in male Swiss mice using the light-dark test (LDT). The compound 3-((4-methoxyphenyl)selanyl)-2-phenylbenzofuran (SeBZF3) exhibited anxiolytic-like activity. SeBZF3 anxiolytic-like effects were also observed in the novelty-suppressed feeding test (NSFT) (50 mg/kg) and elevated plus-maze test (EPMT) (25 and 50 mg/kg). In the EPMT, anxiolytic-like effects of SeBZF3 (50 mg/kg) were abolished by pretreatment with p-chlorophenylalanine, a selective tryptophan hydroxylase inhibitor (100 mg/kg, i.p. for 4 days), suggesting the involvement of serotonergic mechanisms. Furthermore, we conducted experiments to investigate the synergistic effects of SeBZF3 subeffective doses (5 mg/kg, i.g.) in combination with fluoxetine (a selective serotonin reuptake inhibitor, 5 mg/kg, i.p.) or buspirone (a partial agonist of the 5-HT 1A receptor, 2 mg/kg, i.p.). This coadministration resulted in pronounced synergistic effects. We also examined the effects of repeated oral treatment with SeBZF3 at doses of 1 and 5 mg/kg over 14 days and both reduced anxiety signals. In vitro and ex vivo findings revealed that SeBZF3 inhibited cerebral MAO-A activity. These findings collectively imply the potential involvement of serotonergic mechanisms in the anxiolytic-like activity of SeBZF3 in mice. These data offer contributions to the research field of organoselenium compounds and anxiolytics, encouraging the broadening of the search for new effective drugs while offering improved side effect profiles., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2023. Published by Elsevier Inc.)

Adolescent alcohol exposure is associated with lasting behavioral changes in humans and in mice. Prior work from our laboratory and others have demonstrated that C57BL/6J and DBA/2J mice differ in sensitivity to some effects of acute alcohol exposure during adolescence and adulthood. However, it is unknown if these strains differ in cognitive, anxiety-related, and addiction-related long-term consequences of adolescent intermittent alcohol exposure. This study examined the impact of a previously validated adolescent alcohol exposure paradigm (2-3 g/kg, i.p., every other day PND 30-44) in C57BL/6J and DBA/2J male and female mice on adult fear conditioning, anxiety-related behavior (elevated plus maze), and addiction-related phenotypes including nicotine sensitivity (hypothermia and locomotor depression) and alcohol sensitivity (loss of righting reflex; LORR). Both shared and strain-specific long-term consequences of adolescent alcohol exposure were found. Most notably, we found a strain-specific alcohol-induced increase in sensitivity to nicotine's hypothermic effects during adulthood in the DBA/2J strain but not in the C57BL/6J strain. Conversely, both strains demonstrated a robust increased latency to LORR during adulthood after adolescent alcohol exposure. Thus, we observed strain-dependent cross-sensitization to nicotine and strain-independent tolerance to alcohol due to adolescent alcohol exposure. Several strain and sex differences independent of adolescent alcohol treatment were also observed. These include increased sensitivity to nicotine-induced hypothermia in the C57BL/6J strain relative to the DBA/2J strain, in addition to DBA/2J mice showing more anxiety-like behaviors in the elevated plus maze relative to the C57BL/6J strain. Overall, these results suggest that adolescent alcohol exposure results in altered adult sensitivity to nicotine and alcohol with some phenotypes mediated by genetic background., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Cannabinoid use has increased among aging individuals. However, little information on age-related differences in the behavioral effects of these agents is available. To explore potential differences in the behavioral effects of cannabinoids, we determined effects of Δ 9 -tetrahydrocannabinol (THC, 1-10 mg/kg) or rimonabant (0.3-3.2 mg/kg) on operant fixed-ratio responding (FR10) for food in young adult (6 months) and aged (29 months) rats. THC dose-dependently decreased responding for food. Rimonabant alone had little or no effect on responding up to 1.0 mg/kg, but disrupted responding following a 3.2 mg/kg dose. Rimonabant (1.0 mg/kg) partially antagonized response disruption by THC. These effects were similar in young adult and aged rats. However, aging has been reported to change the neurobiology of cannabinoid CB1 receptors. To confirm our rats exhibited such differences, we assessed CB1 receptor binding sites and function in six subcortical (caudate, nucleus accumbens CA1, and CA2/CA3), and three cortical regions (medial prefrontal, temporal, entorhinal) in young adult (6 months) or aged (26 months) male Lewis rats using quantitative autoradiography. CB1 receptor binding sites were reduced in cortical, but not subcortical brain regions of aged rats. CB1 receptor function, at the level of receptor-G protein interaction, was not different in any region studied. Results indicate that down-regulation of CB1 receptor binding sites observed in cortical regions of aged rats was not accompanied by a commensurate decrease in CB1 receptor-stimulated [ 35 S]GTPγS binding, suggesting a compensatory increase in receptor function in cortical areas. Together, our results provide additional evidence of age-related changes in central CB1 receptor populations. However, the functional compensation for decreased CB1 receptor binding may mitigate changes in behavioral effects of cannabinoids. With the rising use of cannabinoid-based therapeutics among aging populations, further evaluation of age-related changes in the cannabinoid system and the impact of these changes on effects of this class of drugs is warranted., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Rationale: We define behavioral sensitization as an augmented response to subsequent dosing after chronic intermittent administration of a drug. However, the biphasic effects of ethanol (EtOH), first stimulatory followed by depressive, make animal models of behavioral sensitization rare., Objectives: This study aimed to determine a dose of EtOH that did not depress wheel-running (WR) in CD1 mice and then to develop a model of EtOH-induced behavioral sensitization., Methods: For the first part of this study, male CD1 mice (n = 24, 6/group) were administered either phosphate buffer saline (PBS), 0.5 g/kg, 1 g/kg, or 2 g/kg EtOH at a volume of 3 ml/kg, intraperitoneally (IP). Mice were divided into equal groups and received the weight-based dose once daily on Days 1, 2, 3, 4, and 5. All mice received a challenge dose of 0.5 g/kg on Day 10. In both parts of the study, mice were habituated to the running wheel for 5 min prior to dosing and wheel running was measured for 10 min after each dose., Results: The acute dose-response of EtOH effects on wheel running determined a significant difference between doses in wheel running (p < 0.05), with a post-hoc analysis establishing that 0.5 g/kg EtOH resulted in significantly more WR compared to 2 g/kg EtOH (p < 0.05). The chronic study demonstrated a significant main effect of Day (1 vs. 5 vs. Challenge, p < 0.001) and an interaction between Day and Treatment, with post-hoc analysis determining the effect to be between PBS and EtOH WR on Day 5 (p < 0.05). In addition, Bonferroni post-hoc analysis determined no differences between Days in the PBS condition, but a significant difference in the EtOH condition between Day 1 and Day 5 (p < 0.001) and that difference from Day 1 persisted when comparing to the Challenge Day (p < 0.01)., Conclusion: After chronic, intermittent, low dose administration of EtOH, male mice showed an increase in activity as measured by wheel running. Therefore, we laid the groundwork for a potentially useful rodent model for EtOH-induced behavioral sensitization., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Adverse early life experiences influence behavioral and physiological functions and increase vulnerability to neuropsychiatric disorders. Maternal separation (MS) is an established animal model that reproduces the features of chronic stress or adverse experiences during early life. Previous studies have been shown that MS may lead to impairments of social behaviors. Here, we investigated the effects of MS on mutual reward preferences in a double T-maze prosocial choice task. Since enriched environment (EE) and intranasal oxytocin (OT) administration have beneficial effects on cognition and social behaviors, in the present study we tested whether these treatments, alone or in combination, would affect prosocial behavior of rats which underwent MS during infancy. Rat pups underwent MS paradigm for 180 min/day from postnatal day (PND) 1-21. From PND 22-34, rats were exposed to an EE and/or received intranasal OT (2 μg/μl, 7 days). Hence, the 8 groups consisted of control (CTRL), MS, CTRL+EE, CTRL+OT and the saline groups. Assessment of prosocial choice behavior was started in adolescence. MS impaired prosocial choice behavior and reduced mutual reward preferences. Getting exposed to EE and intranasal OT administration could overcome MS-induced deficits and promoted mutual reward preferences of MS rats. Combination of short-term EE and OT strengthened prosocial behavior. Obtained results showed that EE and OT may be considered as profitable therapeutic approaches for promoting some aspects of social behavior., (Copyright © 2021. Published by Elsevier Inc.)

Dissociative-based new psychoactive substances (NPSs) are increasingly available through the Internet, and public health problems related to the recreational use of these substances have been increasing globally. Two such NPSs are deschloroketamine and diphenidine, which are primarily used recreationally as ketamine substitutes. However, there is little scientific evidence to describe the dependence liability of NPSs. This study aimed to evaluate the dependence liability of deschloroketamine and diphenidine via animal behavioral experiments. We evaluated the rewarding and reinforcing effects of these NPSs using the conditioned place preference (CPP) and the self-administration (SA) paradigms in mice. Psychomotor effects and behavioral features of these compounds were assessed by quantifying locomotor activity, stereotypic movements, and dopaminergic neurotransmission. Both deschloroketamine (10 mg/kg) and diphenidine (10-60 mg/kg) produced increased locomotor activation and stereotypy that were similar to the effects of ketamine (10 mg/kg). Both deschloroketamine (10 mg/kg) and diphenidine (10, 20 mg/kg) increased the animals' preference for the drug-paired compartment in the CPP testing. In the SA testing, deschloroketamine (1 mg/kg/infusion) increased the number of active lever presses and the number of infusions received, whereas diphenidine administration (1, 2 mg/kg/infusion) did not alter either of these. Furthermore, both deschloroketamine and diphenidine increased dopamine levels in PC-12 cells. Collectively, the data suggest that deschloroketamine may have both rewarding and reinforcing effects, whereas diphenidine only induced rewarding effect., (Copyright © 2022. Published by Elsevier Inc.)

Background: Autism spectrum disorders (ASDs) are a group of neurodevelopmental conditions defined by behavioral deficits in social communication and interactions, mental inflexibility and repetitive behaviors. Converging evidence from observational and preclinical studies suggest that excessive repetitive behaviors in people with ASD may be due to elevated histaminergic H3 receptor signaling in the striatum. We hypothesized that systemic administration of pharmacological histamine H3 receptor antagonists would attenuate the expression of repetitive behaviors in the BTBR T+Itpr3tf/J (BTBR) mouse inbred strain, an established mouse model presenting autism-like repetitive behaviors and novelty-induced hyperactivity. We further aimed to investigate whether agonism of the histamine H3 receptor would be sufficient to induce repetitive behaviors in the C57BL/6J control mouse strain., Methods: Different doses of H3 receptor agonists (i.e., (R)-α-methylhistamine and immethridine) and H3 receptor antagonists/inverse agonists (i.e., ciproxifan and pitolisant) were administered via intraperitoneal (i.p.) injection in male mice to characterize the acute effects of these compounds on ASD-related behavioral readouts., Results: The highly selective H3 receptor agonist immethridine significantly increased the time spent in stereotypic patterns in C57BL/6J mice, but this effect appeared to be driven by general sedative properties of the compound. High doses of pitolisant significantly decreased locomotor hyperactivity in novel environments in BTBR mice, without significant effects on repetitive behaviors., Conclusions: Based on our findings, we conclude that acute H3 receptor manipulation mainly affected general motor activity levels in novel environments. Small changes in stereotyped behaviors were observed but appeared to be driven by altered general activity levels., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The metabotropic glutamate 2/3 (mGlu2/3) receptor antagonists are reported to produce ketamine-like rapid-acting and sustained antidepressant-like effects in rodents. In this study, we compared the effects of single administration of the new mGlu2/3 receptor antagonist TP0178894 and the selective serotonin reuptake inhibitor (SSRI) escitalopram in the chronic social defeat stress (CSDS) model of depression, a model which has been shown to be resistant to treatment with a single dose of SSRI. In the tail suspension test and forced swimming test, high dose (3.0 mg/kg) of TP0178894 significantly attenuated the increased immobility time of these tests in CSDS susceptible mice, compared with vehicle-treated mice. In contrast, low doses (0.3 and 1.0 mg/kg) of TP0178894 and escitalopram (10 mg/kg) did not alter the increased immobility time of these two tests. In the sucrose preference test, TP0178894 (3.0 mg/kg) significantly improved the reduced sucrose preference of CSDS susceptible mice, three and seven days after a single dose. In addition, Western blot analyses showed that TP0178894 (3.0 mg/kg), but not low doses of TP0178894 and escitalopram, significantly attenuated the reduced expression of synaptic proteins [α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (GluA1) and postsynaptic density protein 95 (PSD-95)] in the prefrontal cortex from CSDS susceptible mice. This study suggests that TP0178894 shows rapid-acting and sustained antidepressant-like effects in CSDS model, as ketamine does., (Copyright © 2021 Elsevier Inc. All rights reserved.)

We previously identified in laboratory mice an inactive state [being awake with eyes open motionless within the home cage; inactive but awake, 'IBA'] sharing etiological factors and symptoms with human clinical depression. We further test the hypothesis that greater time spent displaying IBA indicates a depression-like state in mice by investigating whether the antidepressant Venlafaxine, environmental enrichment, and their combination, alleviate IBA. Seventy-two C57BL/6J and 72 DBA/2J female mice were pseudo-randomly housed post-weaning in mixed strain-pairs in non-enriched (NE; 48 pairs) or in environmentally enriched (EE; 24 pairs) cages. After 34 days, half of the mice housed in NE cages were either relocated to EE cages or left in NE cages. For each of these conditions, half of the mice drank either a placebo or the antidepressant Venlafaxine (10 mg/kg). The 48 mice housed in EE cages were all relocated to NE cages and allocated to either the placebo (n = 24) or Venlafaxine (n = 24). IBA data were collected prior to and after environmental adjustment by trained observers blind to the pharmacological and environmental adjustment treatments. Data were analyzed using GLM models. NE cages triggered more IBA than EE cages (Likelihood-Ratio-Test Chi 2 3  = 53.501, p < 0.0001). Venlafaxine and environmental enrichment appeared equally effective at reducing IBA (LRT Chi 2 3  = 18.262, p < 0.001), and combining these approaches did not magnify their effects. Enrichment removal triggered IBA increase (LRT Chi 2 1  = 23.050, p < 0.001), but Venlafaxine did not overcome the increase in IBA resulting from enrichment loss (LTR Chi 2 1  = 0.081, p = 0.775). Theoretical implications for putative depression-like states in mice, and further research directions, are discussed., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Alcohol abuse is a widespread cause of aggressive and impulsive behaviors that impact the users as well as their entourage. However, only a few medications are effective. Recently, cannabidiol has been reported to improve mood disorders and recovery from substance abuse, yet the psychopharmacologic effects of cannabidiol in ethanol-induced drug reward and aggressivity remain unexplored. In the present study, we investigated the effects of cannabidiol on ethanol-induced place preference and aggressivity in individually and group-housed male rats using the conditioned place preference test, and intruder evoc aggression test, respectively. The obtained results showed that ethanol significantly increased locomotor activity, induced conditioned place preference in all animals, and, specifically, increased aggressivity in individually housed rats. These behavioural impairments induced by ethanol were associated with decreased glucocorticoid and mineralocorticoid receptors transcription in the prefrontal cortex. Notwithstanding, cannabidiol at a dose of 10 mg/kg significantly inhibited Et-OH-induced place preference in group-housed, but not in individually housed rats, and markedly inhibited the aggressive behaviour. These findings suggest that ethanol-induced behavioural impairments are dependent on the housing condition that may affect corticosterone receptors expression and subsequently the animal responsivity to cannabidiol treatment., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Evidence demonstrates that psychiatric disorders during pregnancy are detrimental to the offspring. Many disorders are treated with SSRIs and increasing numbers of pregnant women now receive these drugs during gestation. The long-term neurobehavioral consequences of prenatal SSRI exposure require further evaluation. This study examined the effects of prenatal fluoxetine exposure in mice in an extensive battery of behaviors related to neurodevelopment, mood, social, and repetitive behaviors. C57BL/6J dams were administered fluoxetine at a low (0.6 mg/kg/day) or high (6 mg/kg/day) dose or saline from embryonic days 8 to 18. Juvenile mice were tested for changes in ultrasonic vocalizations and neuromotor development. In adulthood, offspring were tested for changes in behaviors related to anxiety, depression, social, and repetitive behaviors. Prenatal exposure to fluoxetine impaired surface righting reflex at P5, and sex-dependently reduced the frequency of ultrasonic vocalizations in juvenile males but not females. In adulthood, both males and females prenatally exposed to high, but not low, doses of fluoxetine exhibited an increase in repetitive behaviors in the marble burying task and a decrease in sucrose preference. Males, but not females, exposed to fluoxetine exhibited increased anxiety-related behaviors in the elevated plus maze. Prenatal fluoxetine exposure did not affect other adult behaviors including social preference, self-grooming, passive avoidance and open field activity. These findings suggest males are more sensitive than females to disruptions in serotonin balance during prenatal development and highlight the need for additional systematic and mechanistic studies to evaluate the impact of fluoxetine exposure during other periods of gestation., (Copyright © 2021 Elsevier Inc. All rights reserved.)

This study analyzed whether the positive allosteric modulator of metabotropic glutamate receptor type 5 (mGlu5) 3-Cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl) benzamide (CDPPB) would alleviate deficits in prepulse inhibition (PPI) and affect dopamine (DA) D 2 signaling in the dorsal striatum and prefrontal cortex (PFC) in the neonatal quinpirole (NQ) model of schizophrenia (SZ). Male and female Sprague-Dawley rats were neonatally treated with either saline (NS) or quinpirole HCL (1 mg/kg; NQ), a DAD 2 receptor agonist, from postnatal days (P) 1-21. Rats were raised to P44 and behaviorally tested on PPI from P44-P48. Before each trial, rats were subcutaneous (sc) administered saline or CDPPB (10 mg/kg or 30 mg/kg). On P50, rats were given a spontaneous locomotor activity test after CDPPB or saline administration. On P51, the dorsal striatum and PFC were evaluated for both arrestin-2 (βA-2) and phospho-AKT protein levels. NQ-treated rats demonstrated a significant deficit in PPI, which was alleviated to control levels by the 30 mg/kg dose of CDPPB. There were no significant effects of CDPPB on locomotor activity. NQ treatment increased βA-2 and decreased phospho-AKT in both the dorsal striatum and PFC, consistent with an increase DAD 2 signaling. The 30 mg/kg dose of CDPPB significantly reversed changes in βA-2 in the dorsal striatum and PFC and phospho-AKT in the PFC equivalent to controls. Both doses of CDPPB produced a decrease of phospho-AKT in the PFC compared to controls. This study revealed that a mGlu5 positive allosteric modulator was effective to alleviate PPI deficits and striatal DAD 2 signaling in the NQ model of SZ., (Copyright © 2021. Published by Elsevier Inc.)

Animal models are important tools for studying neuropsychological disorders. Considering their limitations, a more extensive translational research must encompass data that are generated from several models. Therefore, a comprehensive characterization of these models is needed in terms of behavior and neurophysiology. The present study evaluated the behavioral responses of Carioca Low-conditioned Freezing (CLF) rats to haloperidol and methylphenidate. The CLF breeding line is characterized by low freezing defensive responses to contextual cues that are associated with aversive stimuli. CLF rats exhibited a delayed response to haloperidol at lower doses, needing higher doses to reach similar levels of catatonia as control randomly bred animals. Methylphenidate increased freezing responses to conditioned fear and induced motor effects in the open field. Thus, CLF rats differ from controls in their responses to both haloperidol and methylphenidate. Because of the dopamine-related molecular targets of these drugs, we hypothesize that dopaminergic alterations related to those of animal models of hyperactivity and attention disorders might underlie the observed phenotypes of the CLF line of rats., (Copyright © 2021. Published by Elsevier Inc.)

Serotonin (5-HT) receptors have been implicated in responses to aversive stimuli in mammals and fish, but its precise role is still unknown. Moreover, since at least seven families of 5-HT receptors exist in vertebrates, the role of specific receptors is still debated. Aversive stimuli can be classified as indicators of proximal, distal, or potential threat, initiating responses that are appropriate for each of these threat levels. Responses to potential threat usually involve cautious exploration and increased alertness, while responses to distal and proximal threat involve a fight-flight-freeze reaction. We exposed adult zebrafish to a conspecific alarm substance (CAS) and observed behavior during (distal threat) and after (potential threat) exposure, and treated with the 5-HT 2C receptor agonists MK-212 or WAY-161503 or with the antagonist RS-102221. The agonists blocked CAS-elicited defensive behavior (distal threat), but not post-exposure increases in defensive behavior (potential threat), suggesting inhibition of responses to distal threat. MK-212 blocked changes in freezing elicited by acute restraint stress, a model of proximal threat, while RS-102221 blocked changes in geotaxis elicited this stressor. We also found that RS-102221, a 5-HT 2C receptor antagonist, produced small effect on behavior during and after exposure to CAS. Preprint: https://www.biorxiv.org/content/10.1101/2020.10.04.324202; Data and scripts: https://github.com/lanec-unifesspa/5-HT-CAS/tree/master/data/5HT2C., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Opioids such as morphine are the most effective treatment for pain, but termination of opioid use can produce severe withdrawal symptoms. The present study models this process by using home cage wheel running to assess well-being as a result of pain, morphine analgesia, and opioid withdrawal. Injection of CFA into the right hindpaw caused a dramatic decrease in wheel running and body weight. Implantation of two morphine pellets (75 mg each) resulted in an increase in body weight on Day 1 of administration and a more gradual restoration of wheel running that was only evident during the dark phase of the circadian cycle on Days 3 and 4 of morphine administration. Continuous morphine administration decreased wheel running during the relatively inactive light phase. These findings are consistent with the clinical goal of pain therapeutics to restore normal activity during the day and facilitate sleep at night. Administration of naloxone (1 mg/kg) on Day 5 of morphine administration depressed wheel running for approximately 4 h and caused an increase in wet dog shakes. Naloxone-precipitated changes were no longer evident 6 h after administration. These findings demonstrate that the use of morphine to treat pain does not protect against opioid withdrawal. Moreover, this study provides additional support for the use of home cage wheel running as a method to assess changes in well-being as a result of pain, analgesia, and opioid withdrawal., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The development of sensitization is one of the hallmarks of addictive drugs such as morphine. We administered morphine (10 mg/kg; MOR) to induce locomotor sensitization and ERK activation in the VTA and NAc. In the first experiment, four groups of rats received five daily 30 min sessions in an open-field, and locomotion was measured. For the first four sessions, one group received MOR pre-test (MOR-P); a second group received vehicle pre-test (MOR-UP) and MOR 30 min post-test; the remaining 2 groups received vehicle (VEH) pre-test. On the fifth session, the MOR-P, MOR-UP, and one VEH group received MOR pre-test and the remaining VEH group received VEH. Sensitization emerged in the first 5 min and progressed over to the second and third 5 min blocks only in the MOR-P group. For the second experiment, 4 groups received MOR and 4 groups VEH, and were then returned to their home cage and after 5, 15, 30 or 60 min post-injection, were euthanized for ERK measurements in VTA and NAc. ERK activation increased and peaked at 5 min post injection in the MOR group and then declined to VEH levels by 30 min. Another two groups received either MOR or VEH immediately before a 5 min arena test and ERK was measured immediately post-test. MOR had no effect on locomotion but increased ERK in the VTA and NAc. The peak ERK activation in VTA reflected activation of reward systems by morphine that reinforced locomotor behavior and with repeated treatments, induced a sensitization effect., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Neuronal adaptations involving dopaminergic, glutamatergic, and serotonergic neurotransmitter systems are responsible for behavioral sensitization. Because of common underlying mechanisms, cross-sensitization between compounds of different drug classes can be observed. The purpose of the present study was to determine whether a one- or four-day pretreatment regimen of RU 24969 (a 5-HT 1A/1B receptor agonist) would reciprocally cross-sensitize with cocaine or methamphetamine in male and female preweanling rats. Rats were pretreated with RU 24969 (0 or 5 mg/kg) for 4 days (PD 17-20) and then challenged with cocaine (10 or 20 mg/kg) or methamphetamine (1 or 2 mg/kg) on PD 22. Reciprocal cross-sensitization was also assessed (i.e., rats were pretreated with psychostimulants and tested with RU 24969). In a follow-up experiment, the ability of RU 24969 and cocaine to reciprocally cross-sensitize was assessed using a one-day pretreatment regimen. Reciprocal cross-sensitization between cocaine and RU 24969 was evident in preweanling rats, whereas methamphetamine and RU 24969 did not cross-sensitize. When a one-trial pretreatment regimen was used, cross-sensitization was only detected when rats were pretreated with RU 24969 and tested with cocaine, but not the reverse. In sum, the present results show that the nonselective 5-HT 1A/1B receptor agonist RU 24969 cross-sensitizes with cocaine, but not methamphetamine, in preweanling rats. This dichotomy may be a function of cocaine having a greater affinity for the serotonin transporter than methamphetamine., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Prolonged alcohol consumption has been considered as an important risk factor for various diseases. Chronic ethanol (EtOH) intake is associated with deleterious effects on brain functions culminating in robust behavioral changes. Notably, drugs available to treat the effects of EtOH have low therapeutic efficacy so far. Taurine (TAU) appears as a promising neuroprotective molecule due to its pleiotropic action in the brain. Here, we investigated whether TAU plays a beneficial role in different behavioral domains of zebrafish submitted to an intermittent EtOH exposure model, specially focusing on social behavior, anxiety-like responses, and memory. Moreover, since monoamines play a role in EtOH-mediated responses, we also evaluated the influence of both TAU and EtOH exposures on brain monoamine oxidase (Z-MAO) activity. Fish were exposed to non-chlorinated water or 1% EtOH for 8 consecutive days (20 min per day). From the 5th day until the end of the experimental period (8th day), animals were kept in the absence or presence of TAU (42, 150, or 400 mg/L) 1 h per day immediately after EtOH exposure. Behavioral measurements started 24 h after the last EtOH exposure. We observed that TAU showed modest attenuating effects on shoaling behavior and anxiety-like responses, while 42 and 150 mg/L TAU abolished the memory acquisition deficit in the inhibitory avoidance task. Biochemical analysis revealed that TAU did not modulate EtOH-induced increase on brain Z-MAO activity. Collectively, our novel data show a potential beneficial effect of TAU in an intermittent EtOH exposure model in zebrafish. Moreover, these findings foster the growing utility of this aquatic species to investigate the neurobehavioral basis of EtOH- and TAU-mediated responses in vertebrates., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Metoprine increases the content of histamine in brain by inhibiting histamine N-methyltransferase (HMT), a centrally acting histamine degrading enzyme. We present data demonstrating that pretreatment with metoprine attenuates the hyperlocomotive effects of METH in mice using a multi-configuration behavior apparatus designed to monitor four behavioral outcomes [horizontal locomotion, appetitive behavior (food access), and food and water intake]. Metoprine pretreatment itself induced hyperlocomotion in mice challenged with saline during the large part of light phase. The trend was also observed during the following dark phase. This is the first report that metoprine has a long-lasting locomotor stimulating property. Similarly, in a tail suspension test, a single injection of metoprine significantly reduced total time of immobility in mice, consistent with the idea that metoprine possesses motor stimulating properties. Metoprine pretreatment did not affect other aspects of behavior. Metoprine did not affect the appetitive and drinking behavior while exerted an effect on stereotypy. No stereotyped behavior was observed in mice pretreated with vehicle followed by METH, while stereotyped sniffing was observed in mice pretreated with metoprine followed by METH. The metoprine pretreatment attenuated METH-induced hyperlocomotion during the first 2 h of light phase, suggesting that metoprine-induced locomotor stimulating property might be different from that of METH. The hypothalamic content of histamine (but not its brain metabolite) was increased after metoprine or METH administration. Both METH and metoprine reduced dopamine and histamine turnover in the striatum and the nucleus accumbens and the hypothalamus, respectively, and there is a significant metoprine pretreatment x METH challenge interaction in the histamine turnover. It is likely that metoprine may attenuate METH-induced hyperlocomotion via activation of histaminergic neurotransmission. Metoprine also might induce a long-lasting locomotor stimulating effect via a putative mechanism different from that whereby METH induces the locomotor stimulating effect., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The increasingly popular combination of "energy drinks" containing high amounts of caffeine and alcohol has been shown to induce a stimulated, rather than sedated, state which may result in increased binge drinking and increased risk for alcohol-attributable accidents. We sought to examine consumption patterns of and withdrawal from alcohol and caffeine using a voluntary co-consumption animal model. Male and female adult C57BL/6J mice were given access to increasing doses of caffeine (0.01-0.05%) and/or alcohol (3-20%) in a two-bottle choice, intermittent access voluntary paradigm with fluid consumption recorded daily. Anxiety-like behavior during withdrawal was assessed via elevated plus maze or open field test in experiment 2. Increasing both alcohol and caffeine simultaneously in Experiment 1 resulted in no significant changes in co-consumption compared to mice given access to only alcohol or caffeine. Experiment 2 held caffeine concentration steady while slowly increasing alcohol content and resulted in mice consuming more alcohol when it was consumed in tandem with low dose caffeine. Both male and female mice consumed more caffeine when it was paired with alcohol; however, no significant differences were observed during withdrawal behavior. These results suggest that caffeine may dose-dependently positively influence alcohol consumption in mice and echo clinical literature suggesting that caffeine and alcohol together may result in a heightened state of stimulation and lead to further binge drinking. The intermittent access paradigm affords increased translational validity regarding investigations of alcohol and caffeine co-consumption and may be useful in identifying the neurobiological mechanisms concerning co-consumption of such substances., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The present studies investigated the effects of withdrawal from a single binge-like dose of ethanol (hangover) on fear conditioning in male and female Sprague Dawley rats. In Experiment 1, males and females were given 0 or 3.5 g/kg ethanol intraperitoneally (i.p.) and then conditioned to contextual fear 24 h post injection. Withdrawal from acute ethanol enhanced expression of the conditioned freezing response in males, but not in females. Experiment 2 demonstrated that in males, withdrawal from acute ethanol administered 24 h prior to conditioning enhanced contextual fear conditioning, but not auditory-cued fear conditioning. In Experiment 3, male and female rats were given 3.5 g/kg ethanol, and blood ethanol concentrations (BECs) were assessed at various time points for determination of ethanol clearance. Female rats cleared ethanol at a higher rate than males, with 10 h required for females and 14 for males to eliminate ethanol from their systems. Because females cleared ethanol faster than males, in Experiment 4, females were conditioned 18 h after ethanol administration to keep the interval between ethanol clearance and fear conditioning similar to that of males. Withdrawal from acute ethanol given 18 h prior to conditioning did not affect both contextual and auditory-cued fear conditioning in females. In summary, these results highlight sex differences in the impact of withdrawal from acute ethanol (hangover) on fear learning; suggesting that males are more sensitive to hangover-associated enhancement of negative affect than females., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Several studies have reported that mirtazapine attenuated the induction and expression of cocaine-induced locomotor sensitization. Animals placed in enriched housing environments have shown a decrease in cocaine-induced locomotor activity and sensitization. In addition, it has been suggested that a pharmacological treatment combined with a behavioral intervention increases the efficacy of the former. Thus, the objective of this study was to determine if dosing of mirtazapine in an enriched housing environment enhanced the mirtazapine-induced decrease on the induction and expression of cocaine-induced locomotor sensitization. Wistar male rats were dosed with cocaine (10 mg/kg, i.p.). During the drug-withdrawal phase, mirtazapine (30 mg/kg, i.p.) was administered under standard and enriched housing environmental conditions. The environmental enrichment consisted of housing the animals in enclosures with plastic toys, tunnels, and running wheels. After each administration, locomotor activity for each animal was recorded for 30 min. The study found that treatment with mirtazapine in an enriched housing environment produced an enhanced and persistent attenuation of the induction and expression of cocaine-induced locomotor sensitization. Additionally, it reduced the duration of cocaine-induced locomotor activity in the expression phase of locomotor sensitization. Dosing of mirtazapine in an enriched housing environment enhanced the effectiveness of mirtazapine to decrease cocaine-induced locomotor sensitization. This suggests the potential use of enriched environments to enhance the effect of mirtazapine., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Anxiety is the most prevalent brain disorder and a common cause of human disability. Animal models are critical for understanding anxiety pathogenesis and its pharmacotherapy. The zebrafish (Danio rerio) is increasingly utilized as a powerful model organism in anxiety research and anxiolytic drug screening. High similarity between human, rodent and zebrafish molecular targets implies shared signaling pathways involved in anxiety pathogenesis. However, mounting evidence shows that zebrafish behavior can be modulated by drugs beyond conventional anxiolytics or anxiogenics. Furthermore, these effects may differ from human and/or rodent responses, as such 'unconventional' drugs may affect zebrafish behavior despite having no such profiles (or exerting opposite effects) in humans or rodents. Here, we discuss the effects of several putative unconventional anxiotropic drugs (aspirin, lysergic acid diethylamide (LSD), nicotine, naloxone and naltrexone) and their potential mechanisms of action in zebrafish. Emphasizing the growing utility of zebrafish models in CNS drug discovery, such unconventional anxiety pharmacology may provide important, evolutionarily relevant insights into complex regulation of anxiety in biological systems. Albeit seemingly complicating direct translation from zebrafish into clinical phenotypes, this knowledge may instead foster the development of novel CNS drugs, eventually facilitating innovative treatment of patients based on novel 'unconventional' targets identified in fish models., (Copyright © 2021 Elsevier Inc. All rights reserved.)

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., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The adverse effects of mu opioid agonists have spurred a renewed interest in using kappa opioid receptor (KOR) agonists as analgesics. KOR agonists also have potential for development as diuretics for the treatment of edema and hypertension. Here, we evaluated the discriminative stimulus, antinociceptive, and diuretic effects of the kappa agonist (±)-trans-U-50488 and its stereoisomers (-)-(1S,2S)-U-50488 or (+)-(1R,2R)-U-50488) alone and in combination with the cannabinoid agonist (-)-CP 55,940. To establish (±)-U-50488 as a discriminative stimulus, rats (n = 12) were trained to discriminate intraperitoneal (i.p.) administration of 5.6 mg/kg of (±)-trans-U-50488 from saline under a fixed-ratio 20 (FR-20) schedule of food reinforcement. Then, antinociception was assessed using two procedures: warm water tail withdrawal and von Frey paw withdrawal. Diuretic effects were assessed in separate rats (n = 6/group). Doses of (±)-U-50488 and (-)-U-50488 that served as discriminative stimuli produced significant increases in urine output, but at lower doses than those that produced antinociception. In contrast, (+)-U-50488 alone had no discriminative stimulus or diuretic effects at the doses tested, but did produce antinociception in the von Frey assay. When three cannabinoids and morphine were tested in the (±)-U-50488 discrimination procedure to determine the similarity of these drugs' discriminative stimulus effects to those for (±)-U-50488, the rank order similarity was (-)-CP 55,940 > (-)-trans-THC > (+)-WIN 55,212-2 ≥ morphine. (-)-CP 55,940 alone (0.056 mg/kg) partially substituted for the discriminative stimulus effects of (±)-U-50488 and produced significant diuretic and antinociceptive effects. (-)-CP 55,940 in combination with (±)-U-50488 also produced a two-fold leftward shift in the discriminative stimulus curve for (±)-U-50488, and near-additive antinociception with (±)-U-50488 and (+)-U-50488. Further, the diuretic effect of (-)-CP 55,940 was enhanced by a dose of (+)-U50488, which itself did not alter urine output. These data together indicate that a combination of cannabinoid and kappa opioid agonists can enhance diuresis, but may have limited potential for serving as opioid-sparing pharmacotherapeutics for treatment of pain., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Very few people are able to quit smoking, and therefore it is essential to know which factors contribute to the development of compulsive nicotine use. These studies aimed to investigate if early-adolescent nicotine exposure causes locomotor sensitization and affects anxiety-like behavior and the spontaneous acquisition of intravenous nicotine self-administration. Early-adolescent male and female rats were treated with nicotine from postnatal (P) days 24 to 42, and anxiety-like behavior and locomotor activity were investigated one day after the cessation of nicotine treatment and in adulthood (>P75). The spontaneous acquisition of nicotine self-administration was also investigated in adulthood. The rats self-administered 0.03 mg/kg/infusion of nicotine for six days under a fixed-ratio (FR) 1 schedule and four days under an FR2 schedule (3-h sessions). Repeated nicotine administration increased locomotor activity, rearing, and stereotypies in a small open field in adolescent male and female rats. One day after the last nicotine injection, the percentage of open arm entries in the elevated plus-maze test was decreased in the males and increased in the females. However, locomotor activity in the small open field was unaffected. Adolescent nicotine treatment did not affect anxiety-like behavior and locomotor activity in adulthood. During the 10-day nicotine self-administration period, the females had a higher level of nicotine intake than the males. Adolescent nicotine treatment decreased nicotine intake in the females. In conclusion, these findings indicate that repeated nicotine administration during adolescence causes robust behavioral sensitization and leads to lower nicotine intake in females throughout the acquisition period in adulthood in rats., (Copyright © 2021. Published by Elsevier Inc.)

Amphetamine (AMP) and atomoxetine (ATX) represent two of the most widely studied drug treatments used in the investigation of impulsive behaviour. While both drugs have relatively well defined effects in tests designed to investigate impulsive action (e.g. 5-choice task; 5-CSRTT), the effects of both drugs in tests of impulsive choice (e.g. delay discounting) are less consistent. In the present study both AMP and ATX were tested in a rodent gambling task (rGT) and delay discounting in rats separately trained to either an ascending or descending delay schedule. Effects of both drugs were compared to measures of impulsive action (premature (PREM) responses) and perseverative (PSV) responses measured in the 5-choice and rGT tasks. Consistent with previous studies, AMP (0.1-1 mg/kg) increased both PREM and PSV responses, and ATX (0.5-2 mg/kg) reduced both measures in the 5-choice and rGT tasks. At equivalent doses ATX had no reliable effect on choice behaviour in either the rGT or delay discounting suggesting a null effect of this drug on impulsive choice and risky decision making. The effects of AMP were more complex, with a subtle shift in preference to a low risk (P1) choice in the rGT, and an effect on discounting that was unrelated to reinforcer value, but instead dependent on delay sequence and baseline choice preference. One aspect to these outcomes is to highlight the importance of multiple methodological factors when assessing drug effects on complex behaviours such as impulsive choice, and question what are the most appropriate test conditions under which to examine these drugs on discounting., (Copyright © 2021. Published by Elsevier Inc.)

Methylphenidate (MET) has a putative cognitive enhancer effect that has led adolescents and young adults to increase and indiscriminate its use aiming to ameliorate their productivity. However, the impacts of MET on addiction-related behaviors, emotional levels, and cognition are still not fully understood. To investigate the influence of chronic treatment with MET during adolescence on addiction-like behaviors, memory, and anxiety in adult mice. Thirty-day-old female mice received i.p. 10 mg/kg MET or Veh injections for 10 consecutive days. Forty days after the treatment (mice were 70-days-old), animals were submitted to the behavioral evaluation under the effects of MET, which included: MET-induced conditioned place preference (CPP), behavioral sensitization, and plus-maze discriminative avoidance task. Pre-exposure to MET during adolescence promoted an early expression of CPP and also facilitated the development of MET-induced behavioral sensitization during adulthood. These addictive-like behaviors were accompanied by anxiogenic effects of MET but not by any memory-enhancing effect. We demonstrated that exposure to MET during adolescence can increase the vulnerability to addiction-like behaviors and anxiety during adulthood. Our results reinforce the necessity of a more efficient system to control MET indiscriminate use, thus avoiding its potential tardive addictive effects., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Dopamine, orexin (hypocretin), and adenosine systems have dual roles in reward and sleep/arousal suggesting possible mechanisms whereby drugs of abuse may influence both reward and sleep/arousal. While considerable variability exists across studies, drugs of abuse such as cocaine induce an acute sleep loss followed by an immediate recovery pattern that is consistent with a normal response to loss of sleep. Under more chronic cocaine exposure conditions, an abnormal recovery pattern is expressed that includes a retention of sleep disturbance under withdrawal and into abstinence conditions. Conversely, experimentally induced sleep disturbance can increase cocaine seeking. Thus, complementary, sleep-related therapeutic approaches may deserve further consideration along with development of non-human models to better characterize sleep disturbance-reward seeking interactions across drug experience., (Published by Elsevier Inc.)

Electronic-cigarette's (ECIGs) popularity has grown over the last decade and changed the way individuals administer nicotine. Preclinical research is imperative for understanding the addictive properties and health-risks associated with ECIG use; however, there is not a standard dosing regimen used across research laboratories. The main objective was to determine how vapor puff durations, administration session length, and flavored e-liquid alter general and mood-disorder related behaviors while providing a foundation of vapor administration parameters. Adult male and female C57BL/6 mice were exposed to several nicotine-free unflavored vapor puff durations (1, 3, 6, or 10 s) and vapor administration session lengths (10 and 30 min) then measured on the following assays: locomotor activity (LMA), tail suspension test (TST), and light-dark test. The effects of mecamylamine and the time-course of vapor-induced depression of LMA also were assessed. Additionally, mice were exposed to flavored (strawberry and adventurers tobacco blend) vapor inhalation and measured on locomotor activity, tail suspension test, and light-dark test. Following both 10 and 30 min vapor administration session, there was a puff duration-dependent decrease in distance traveled, time in center, and rearing. The vapor-induced depression of LMA was not mediated by nicotine or nicotinic acetylcholine receptor (nAChR) activation and lasted 60-90 min. The 10 s puff duration produced an anxiogenic-like effect in the light-dark test by decreasing the time spent in the light side. Vapor inhalation did not significantly alter TST behavior. No significant effects of sex or flavor were found. The anxiogenic-like effects of nicotine-free vapor inhalation are concerning as many adolescents vape nicotine-free flavored e-liquid, and there is an association between ECIGs and mood disorders. Additionally, these studies demonstrate that vapor puff duration, but not vapor administration session length, is an important variable to consider during research design as it can become a confounding variable and alter baseline behaviors., (Copyright © 2021 Elsevier Inc. All rights reserved.)