Nicotine Exposure in a Phencyclidine-Induced Mice Model of Schizophrenia: Sex-Selective Medial Prefrontal Cortex Protein Markers of the Combined Insults in Adolescent Mice.

Tobacco misuse as a comorbidity of schizophrenia is frequently established during adolescence. However, comorbidity markers are still missing. Here, the method of label-free proteomics was used to identify deregulated proteins in the medial prefrontal cortex (prelimbic and infralimbic) of male and female mice modelled to schizophrenia with a history of nicotine exposure during adolescence. Phencyclidine (PCP), used to model schizophrenia (SCHZ), was combined with an established model of nicotine minipump infusions (NIC). The combined insults led to worse outcomes than each insult separately when considering the absolute number of deregulated proteins and that of exclusively deregulated ones. Partially shared Reactome pathways between sexes and between PCP, NIC and PCPNIC groups indicate functional overlaps. Distinctively, proteins differentially expressed exclusively in PCPNIC mice reveal unique effects associated with the comorbidity model. Interactome maps of these proteins identified sex-selective subnetworks, within which some proteins stood out: for females, peptidyl-prolyl cis-trans isomerase (Fkbp1a) and heat shock 70 kDa protein 1B (Hspa1b), both components of the oxidative stress subnetwork, and gamma-enolase (Eno2), a component of the energy metabolism subnetwork; and for males, amphiphysin (Amph), a component of the synaptic transmission subnetwork. These are proposed to be further investigated and validated as markers of the combined insult during adolescence.

Neonatal phencyclidine as a model of sex-biased schizophrenia symptomatology in adolescent mice.

Sex-biased differences in schizophrenia are evident in several features of the disease, including symptomatology and response to pharmacological treatments. As a neurodevelopmental disorder, these differences might originate early in life and emerge later during adolescence. Considering that the disruption of the glutamatergic system during development is known to contribute to schizophrenia, we hypothesized that the neonatal phencyclidine model could induce sex-dependent behavioral and neurochemical changes associated with this disorder during adolescence. C57BL/6 mice received either saline or phencyclidine (5, 10, or 20 mg/kg) on postnatal days (PN) 7, 9, and 11. Behavioral assessment occurred in late adolescence (PN48-50), when mice were submitted to the open field, social interaction, and prepulse inhibition tests. Either olanzapine or saline was administered before each test. The NMDAR obligatory GluN1 subunit and the postsynaptic density protein 95 (PSD-95) were evaluated in the frontal cortex and hippocampus at early (PN30) and late (PN50) adolescence. Neonatal phencyclidine evoked dose-dependent deficits in all analyzed behaviors and males were more susceptible. Males also had reduced GluN1 expression in the frontal cortex at PN30. There were late-emergent effects at PN50. Cortical GluN1 was increased in both sexes, while phencyclidine increased cortical and decreased hippocampal PSD-95 in females. Olanzapine failed to mitigate most phencyclidine-evoked alterations. In some instances, this antipsychotic aggravated the deficits or potentiated subthreshold effects. These results lend support to the use of neonatal phencyclidine as a sex-biased neurodevelopmental preclinical model of schizophrenia. Olanzapine null effects and deleterious outcomes suggest that its use during adolescence should be further evaluated.

Maternal protein restriction during the lactation period disrupts the ontogenetic development of behavioral traits in male Wistar rat offspring.

Neonatal undernutrition in rats results in short- and long-term behavioral and hormonal alterations in the offspring. It is not clear, however, whether these effects are present since the original insult or if they develop at some specific age later in life. Here, we assessed the ontogenetic profile of behavioral parameters associated with anxiety, exploration and memory/learning of Wistar rat offspring that were subjected to protein malnutrition during lactation. Dams and respective litters were separated into two groups: (1) protein-restricted (PR), which received a hypoproteic chow (8% protein) from birth to weaning [postnatal day (PN) 21]; (2) control (C), which received normoproteic chow. Offspring's behaviors, corticosterone, catecholamines, T3 and T4 levels were assessed at PN21 (weaning), PN45 (adolescence), PN90 (young adulthood) or PN180 (adulthood). PR offspring showed an age-independent reduction in the levels of anxiety-like behaviors in the Elevated Plus Maze and better memory performance in the Radial Arm Water Maze. PR offspring showed peak exploratory activity in the Open Field earlier in life, at PN45, than C, which showed theirs at PN90. Corticosterone was reduced in PR offspring, particularly at young adulthood, while catecholamines were increased at weaning and adulthood. The current study shows that considerable age-dependent variations in the expression of the observed behaviors and hormonal levels exist from weaning to adulthood in rats, and that protein restriction during lactation has complex variable-dependent effects on the ontogenesis of the assessed parameters.

Lifetime caffeine and adolescent nicotine exposure in mice: effects on anxiety-like behavior and reward.

Caffeine consumption occurs throughout life, while nicotine use typically begins during adolescence, the period when caffeine-nicotine epidemiological association begins in earnest. Despite that, few studies in animal models parallel the pattern of coexposure that occurs in humans. Therefore, the neurobehavioral consequences of the association between these drugs remain unclear. Here, we exposed Swiss mice to lifetime caffeine. Caffeine solutions of 0.1 g/L (CAF0.1), 0.3 g/L (CAF0.3), or water (CTRL) were used as the sole liquid source, being offered to progenitors until weaning and, after that, directly to the offspring until the last day of adolescent behavioral evaluation. The open field test was used to evaluate acute effects of nicotine, of lifetime caffeine and of their interaction on locomotion and anxiety-like behavior, while the conditioned place preference test was used to assess the impact of caffeine on nicotine (0.5 mg/Kg, i.p.) reward. Frontal cerebral cortex dopamine content, dopamine turnover, and norepinephrine levels, as well as hippocampal serotonin 1A receptor expression were assessed. CAF0.3 mice exhibited an increase in anxiety-like behavior when compared to CAF0.1 and CTRL ones, but nicotine coexposure mitigated the anxiogenic-like caffeine-induced effect. Distinctively, caffeine had no effect on locomotion and failed to interfere with both nicotine-induced hyperactivity and place preference. There were no significant effects on dopaminergic and serotonergic markers. In conclusion, although caffeine did not affect nicotine reward, considering the strong association between anxiety disorders and tobacco consumption, caffeine-induced anxiety-like behavior advises limiting its consumption during development, including adolescence, as caffeine could be a risk factor to nicotine use.

Nicotine exposure during breastfeeding alters the expression of endocannabinoid system biomarkers in female but not in male offspring at adulthood.

Early nicotine exposure compromises offspring's phenotype at long-term in both sexes. We hypothesize that offspring exposed to nicotine during breastfeeding show deregulated central and peripheral endocannabinoid system (ECS), compromising several aspects of their metabolism. Lactating rats received nicotine (NIC, 6 mg/Kg/day) or saline from postnatal day (PND) 2 to 16 through implanted osmotic minipumps. Offspring were analyzed at PND180. We evaluated protein expression of N-acylphosphatidylethanolamide-phospholipase D (NAPE-PLD), fatty acid amide hydrolase (FAAH), diacylglycerol lipase (DAGL), monoacylglycerol lipase (MAGL) and cannabinoid receptors (CB1 and/or CB2) in lateral hypothalamus, paraventricular nucleus of the hypothalamus, liver, visceral adipose tissue (VAT), adrenal and thyroid. NIC offspring from both sexes did not show differences in hypothalamic ECS markers. Peripheral ECS markers showed no alterations in NIC males. In contrast, NIC females had lower liver DAGL and CB1, higher VAT DAGL, higher adrenal NAPE-PLD and higher thyroid FAAH. Endocannabinoids biosynthesis was affected by nicotine exposure during breastfeeding only in females; alterations in peripheral tissues suggest lower action in liver and higher action in VAT, adrenal and thyroid. Effects of nicotine exposure during lactation on ECS markers are sex- and tissue-dependent. This characterization helps understanding the phenotype of the adult offspring in this model and may contribute to the development of new pharmacological targets for the treatment of several metabolic diseases that originate during development.

The Implementation of a Geriatrics Co-Management Model of Care Reduces Hospital Length of Stay.

(1) Background: Older adults comprise a large proportion of hospitalized patients. Many are frail and require complex care. Geriatrics has developed models of care specific to this inpatient population. Our objective was to demonstrate the effect of a geriatric co-management team on clinical administrative indicators of care in Clinical Teaching Units (CTUs) that have adopted the Age-friendly Hospital (AFH) principles in Brazilian hospitals. (2) Methods: Following 3 months of implementation of the AFH principles in CTUs, two periods of the same 6 months of two consecutive years were compared. (3) Results: The total number of participants in the study was 641 and 743 in 2015 and 2016, respectively. Average length of patient-stay (length of stay: 8.7 ± 2.7 vs. 5.4 ± 1.7 days) and number of monthly complaints (44.2 ± 6.5 vs. 13.5 ± 2.2) were significantly lower with the co-management model. Number of homecare service referrals/month was also significantly higher (2.5 ± 1 vs. 38.3 ± 6.3). The 30-day readmission rates and total hospital costs per patient remained unchanged. (4) Conclusion: The presence of a geriatric co-management team in CTUs is of added benefit to increase the efficiency of the AFH for vulnerable older inpatients with reduced LOS and increased referrals to homecare services without increasing hospital costs.

Adolescent nicotine potentiates the inhibitory effect of raclopride, a D2R antagonist, on phencyclidine-sensitized psychotic-like behavior in mice.

The association between schizophrenia and nicotine addiction becomes evident during adolescence. Here, to investigate interactive events that might underlie the early establishment of this comorbidity, we used phencyclidine-evoked locomotor sensitization, a proxy model of psychotic behavior, and nicotine minipump infusions in adolescent mice. Considering the involvement of dopamine D2 receptors in both schizophrenia and addiction, we further tested their role by exposing mice to raclopride. Adolescent mice that were either exposed to nicotine (24 mg/Kg/day) or not, received single daily raclopride (0.5 mg/kg, s.c.) or saline followed by phencyclidine injections (10 mg/Kg, s.c.) during open field testing for 6 consecutive days (Acquisition phase, ACQ). Phencyclidine and nicotine challenges (Sensitization Test, ST) were carried out after a 5-day withdrawal. Ambulation escalated in response to repeated phencyclidine exposure during ACQ and was increased after phencyclidine challenge, evidencing development and expression of locomotor sensitization. Raclopride prevented phencyclidine-evoked development of sensitization. However, raclopride pre-exposure during ACQ only shortened its expression in phencyclidine-challenged mice. Nicotine failed to interfere with phencyclidine stimulatory effects during ACQ but potentiated raclopride inhibition during the first ACQ days. During ST, nicotine history shortened the expression of phencyclidine-evoked sensitization. Nicotine challenge had no impact on locomotion, which is consistent with a lack of nicotine/phencyclidine cross-sensitization. In conclusion, our results show that nicotine does not worsen, and may even ameliorate phencyclidine-sensitized psychotic-like behavior in adolescent mice. The potentiation of raclopride-mediated inhibition further suggests that nicotine transiently improves the therapeutic efficacy of medication on psychotic symptoms through mechanisms that converge on D2 receptors.

Neonatal nicotine exposure changes insulin status in fat depots: sex-related differences.

Nicotine is the main psychoactive substance present in cigarette smoke that is transferred to the baby by breast milk. In rats, maternal nicotine exposure during breastfeeding induces obesogenesis and hormone dysfunctions in adult male offspring. As glucocorticoid (GC), insulin, and vitamin D change both adipogenesis and lipogenesis processes, we assessed parameters related to metabolism and action of these hormones in visceral and subcutaneous adipose tissues (VAT and SAT) of adult male and female rats in a model of neonatal nicotine exposure. At postnatal (PN) day 2, dams were kept with six pups (three per sex) and divided into nicotine and control groups for implantation of osmotic minipumps that released 6 mg/kg nicotine or saline, respectively. At PN180, fat mass, hormone levels, and protein contents of biomarkers of the GC activation and receptor (11beta-hydroxysteroid dehydrogenase type 1 and glucocorticoid receptor alpha), insulin signaling pathway [insulin receptor beta (IRß), phosphorylated insulin receptor substrate 1, insulin receptor substrate 1 (IRS1), phosphorylated serine/threonine kinase (pAKT), serine/threonine kinase, glucose transporter type 4 (GLUT4)], and vitamin D activation and receptor (1α-hydroxylase and vitamin D receptor) were evaluated. While nicotine-exposed males showed increased fat mass, hypercorticosteronemia, hyperinsulinemia, and higher 25-hydroxyvitamin D, these alterations were not observed in nicotine-exposed females. Nicotine-exposed males only showed lower IRS1 in VAT, while the females had hyperglycemia, higher pAKT in VAT, while lower IRß, IRS1, and GLUT4 in SAT. Parameters related to metabolism and action of GC and vitamin D were unaltered in both sexes. We evidence that exposure exclusively to nicotine during breastfeeding affects the hormone status and fat depots of the adult progeny in a sex-dependent manner.

Does nicotine exposure during adolescence modify the course of schizophrenia-like symptoms? Behavioral analysis in a phencyclidine-induced mice model.

The first symptoms of schizophrenia (SCHZ) are usually observed during adolescence, a developmental period during which first exposure to psychoactive drugs also occurs. These epidemiological findings point to adolescence as critical for nicotine addiction and SCHZ comorbidity, however it is not clear whether exposure to nicotine during this period has a detrimental impact on the development of SCHZ symptoms since there is a lack of studies that investigate the interactions between these conditions during this period of development. To elucidate the impact of a short course of nicotine exposure across the spectrum of SCHZ-like symptoms, we used a phencyclidine-induced adolescent mice model of SCHZ (2.5mg/Kg, s.c., daily, postnatal day (PN) 38-PN52; 10mg/Kg on PN53), combined with an established model of nicotine minipump infusions (24mg/Kg/day, PN37-44). Behavioral assessment began 4 days after the end of nicotine exposure (PN48) using the following tests: open field to assess the hyperlocomotion phenotype; novel object recognition, a declarative memory task; three-chamber sociability, to verify social interaction and prepulse inhibition, a measure of sensorimotor gating. Phencyclidine exposure evoked deficits in all analyzed behaviors. Nicotine history reduced the magnitude of phencyclidine-evoked hyperlocomotion and impeded the development of locomotor sensitization. It also mitigated the deficient sociability elicited by phencyclidine. In contrast, memory and sensorimotor gating deficits evoked by phencyclidine were neither improved nor worsened by nicotine history. In conclusion, our results show for the first time that nicotine history, restricted to a short period during adolescence, does not worsen SCHZ-like symptoms evoked by a phencyclidine-induced mice model.

Forced swimming stress increases natatory activity of lead-exposed mice.

Recent evidence points to the relationship between lead toxicity and the function of the hypothalamic-pituitary-adrenal axis, which suggests that lead exposure could influence how an individual cope with stress. Here we test this hypothesis by investigating the behavioral effects of lead exposure in mice during the forced swimming test (FST), a parading in which animals are exposed to a stressful situation and environment. Swiss mice received either 180 ppm or 540 ppm of lead acetate (Pb) in their ad-lib water supply for 60-90 days, starting at postnatal day 30. Control (Ctrl) mice drank tap water. At the end of the exposure period, mice were submitted to a 5-min session of FST or to an open-field session of the same duration. Data from naïve animals showed that corticosterone levels were higher for animals tested in the FST compared to animals tested in the open-field. Blood-lead levels (BLL) in Pb-exposed mice ranged from 14.3 to 106.9 µg/dL. No differences were observed in spontaneous locomotion between Ctrl and Pb-exposed groups in the open-field. However, in the FST, Pb-treated mice displayed higher swimming activity than Ctrl ones and this effect was observed even for animals with BLL higher than 20 µg/dL. Furthermore, significant differences in brain glutathione levels, used as an indicator of led toxicity, were only observed for BLL higher than 40 µg/dL. Overall, these findings suggest that swimming activity in the FST is a good indicator of lead toxicity and confirm our prediction that lead toxicity influences behavioral responses associated to stress.

Sex- and age-dependent differences in nicotine susceptibility evoked by developmental exposure to tobacco smoke and/or ethanol in mice.

Either tobacco smoking or alcohol consumption during pregnancy sex-selectively increases susceptibility to drugs of abuse later in life. Considering that pregnant smoking women are frequently intermittent consumers of alcoholic beverages, here, we investigated whether a short-term ethanol exposure restricted to the brain growth spurt period when combined with chronic developmental exposure to tobacco smoke aggravates susceptibility to nicotine in adolescent and adult mice. Swiss male and female mice were exposed to tobacco smoke (SMK; research cigarettes 3R4F, whole-body exposure, 8 h/daily) or ambient air during the gestational period and until the tenth postnatal day (PN). Ethanol (ETOH, 2 g/Kg, 25%, i.p.) or saline was injected in the pups every other day from PN2 to PN10. There were no significant differences in cotinine (nicotine metabolite) and ethanol serum levels among SMK, ETOH and SMK + ETOH groups. During adolescence (PN30) and adulthood (PN90), nicotine (NIC, 0.5 mg/Kg) susceptibility was evaluated in the conditioned place preference and open field tests. NIC impact was more evident in females: SMK, ETOH and SMK + ETOH adolescent females were equally more susceptible to nicotine-induced place preference than control animals. At adulthood, SMK and SMK + ETOH adult females exhibited a nicotine-evoked hyperlocomotor profile in the open field, with a stronger effect in the SMK + ETOH group. Our results indicate that ethanol exposure during the brain growth spurt, when combined to developmental exposure to tobacco smoke, increases nicotine susceptibility with stronger effects in adult females. This result represents a worsened outcome from the early developmental dual exposure and may predispose nicotine use/abuse later in life.

Mood-related behavioral and neurochemical alterations in mice exposed to low chlorpyrifos levels during the brain growth spurt.

Organophosphates are among the most used pesticides. Particularly, chlorpyrifos (CPF) is responsible for a number of deleterious effects on brain development, which may program behavioral changes later in life. Here, we investigated whether a regimen of early low level CPF exposure that did not result in a significant inhibition of acetylcholinesterase (AChE) had deleterious effects on mood-related behaviors, as well as on cholinergic and serotonergic biomarkers in the mice brain. From the 3rd to 9th postnatal day (PN), male and female Swiss mice were subcutaneously injected with CPF. Mice were submitted to a battery of behavioral tests from PN60 to PN63: open field, elevated plus maze and forced swimming tests. The cholinergic and serotonergic biomarkers were assessed at PN10 and PN63. Our data indicated that early CPF exposure increased anxiety-like behavior in females and altered decision-making behavior in both sexes. Most biochemical alterations were sex-dependent and restricted to females. At PN10, CPF female mice showed increased serotonin and choline transporter binding in cerebral cortex. Distinctively, in adult females, the effects indicated a hypoactive state: CPF exposure reduced 5-HT1a receptor binding in cerebral cortex, as well as serotonin transporter binding and choline acetyltransferase activity in brainstem. Our results indicate that CPF exposure during the brain growth spurt deregulates serotonergic and cholinergic biomarkers. The effects are consistent with impaired synaptic function, may be related to long-term mood disorders and point out to higher female susceptibility.

Ethanol exposure during the brain growth spurt period increases ethanol-induced aggressive behavior in adolescent male mice.

Ethanol exposure during development is associated with deficient social behavior, such as aggressive behavior, and ethanol consumption is associated with violent crimes, thus raising the possibility that individuals with fetal alcohol spectrum disorder may exhibit exacerbated social deficits in response to ethanol exposure. The present study evaluated the effects of ethanol exposure during the brain growth spurt period (i.e., a critical time period during which ethanol's effects are augmented) on aggressive behavior and ethanol-induced aggression during adolescence. From postnatal Day 2 (PD2) to PD8, Swiss mice received either ethanol (5 g/kg, i.p.) or saline on alternate days. On PD39, aggressive behavior was assessed using the resident-intruder paradigm in male mice, and social dominance was investigated using the tube dominance test in both males and females. Testis structure and testosterone levels were evaluated in male mice. Early ethanol exposure increased the gonadosomatic index and the number of Leydig cells. The thickness of the seminiferous tube decreased. No difference in testosterone levels was found. The ethanol-exposed resident mice exhibited increased number and duration of aggressive episodes only when challenged with a low ethanol dose (1 g/kg) before confrontation. Female mice early-exposed to ethanol won more confrontations in the tube dominance test. The present findings suggest a critical brain growth spurt period that is susceptible to ethanol-induced alterations of social dominance behavior in females. Although basal levels of aggression were unaffected, early ethanol exposure resulted in greater susceptibility to ethanol-induced aggression in adolescent male mice.

Thyroid redox imbalance in adult Wistar rats that were exposed to nicotine during breastfeeding.

Maternal nicotine exposure causes several consequences in offspring phenotype, such as obesity and thyroid dysfunctions. Nicotine exposure can increase oxidative stress levels, which could lead to thyroid dysfunction. However, the mechanism by which nicotine exposure during breastfeeding leads to thyroid gland dysfunction remains elusive. We aimed to investigate the long-term effects of maternal nicotine exposure on redox homeostasis in thyroid gland, besides other essential steps for thyroid hormone synthesis in rats from both sexes. Lactating Wistar rats were implanted with osmotic minipumps releasing nicotine (NIC, 6 mg/kg/day) or saline (control) from postnatal day 2 to 16. Offspring were analyzed at 180-day-old. NIC males showed lower plasma TSH, T3 and T4 while NIC females had higher T3 and T4. In thyroid, NIC males had higher sodium-iodide symporter protein expression, whereas NIC females had higher thyroid-stimulating hormone receptor (TSHr) and thyroperoxidase (TPO) protein expression. TPO activity was lower in NIC males. Hydrogen peroxide generation was decreased in NIC males. Activities of superoxide dismutase, catalase and glutathione peroxidase were compromised in NIC animals from both sexes. 4-Hydroxynonenal was higher only in NIC females, while thiol was not affected in NIC animals from both sexes. NIC offspring also had altered expression of sex steroid receptors in thyroid gland. Both sexes showed similar thyroid morphology, with lower follicle and colloid size. Thyroid from female offspring exposed to nicotine during breastfeeding developed oxidative stress, while the male gland seemed to be protected from redox damage. Thyroid dysfunctions seem to be associated with redox imbalance in a sex-dependent manner.

Malnourishment during early lactation disrupts the ontogenetic distribution of the CART and α-MSH anorexigenic molecules in the arcuate/paraventricular pathway and lateral hypothalamus in male rats.

Developmental malnourishment impacts the energetic metabolism control throughout life. In rat offspring, a 0% protein diet during the first 10 days of lactation results in leptin resistance and in alterations in: feeding behavior, serum leptin and neuropeptide Y (NPY) levels in the hypothalamic arcuate nucleus (ARC)/paraventricular (PVN) pathway. Here, the distributions of alpha-melanocyte stimulating hormone (α-MSH) and cocaine and amphetamine regulated transcript (CART), anorexigenic molecules, were immunohistochemically assessed in the ARC, PVN and lateral hypothalamus (LH) nuclei. Rat dams were subjected to one of the following diet protocols from postnatal day (P) 1-10: 1) Protein-free (PFG, 0% protein chow); 2) Pair-fed (UFG, normoprotein chow); 3) Control group (CG, normoprotein chow). PFG, UFG and CG male offspring were analyzed at different time points, from P5 to P180. In the ARC, PFG α-MSH and CART were increased from P10 to P45 when compared to CG and UFG. In the PVN, α-MSH and CART peaks in PFG animals were delayed from P20 to P30 when compared to CG. In the LH, CART was more intense in PFG animals than in UFG and CG ones by P20, and, by P30, UFG immunostaining became less intense than in CG. In conclusion, aproteic diet altered the ontogenetic distribution of both anorexigenic molecules. In the PVN, the peak was delayed to P30, which coincides with the leptin peak and follows the previously described NPY (orexigenic) peak in this model. The permanent LH CART and α-MSH increase may be associated with the previously observed PFG hypophagia.

Ethanol exposure during the brain growth spurt affects social behavior and increases susceptibility to acute ethanol effects during adolescence in male mice.

The brain is particularly vulnerable to ethanol effects during its growth spurt. Outcomes of early ethanol exposure such as hyperactivity have been extensively investigated; however, persons with fetal alcohol spectrum disorder frequently have social impairments and are heavy drinkers. Despite that, scant information is available regarding the neurobiological basis of these latter behavioral issues. Here, Swiss mice exposed to ethanol (Etoh, 5 g/kg i.p., alternate days) or saline during the brain growth spurt [postnatal day (PN) 2 to 8] were used to assess social behavior after an ethanol challenging during adolescence. At PN39, animals were administered with a single ethanol dose (1 g/Kg) or water by gavage and were then evaluated in the three-chamber sociability test. We also evaluated corticosterone serum levels and the frontal cerebral cortex serotoninergic system. Etoh males showed reductions in sociability. Ethanol challenging reverted these alterations in social behavior, reduced corticosterone levels, and increased the 5-HT2 receptor binding of male Etoh mice. No alterations were observed in 5-HT and 5-HIAA contents. These data support the idea that ethanol exposure during the brain growth spurt impacts social abilities during adolescence, alters ethanol reexposure effects, and suggests that stress response and serotoninergic system play roles in this phenomenon.

Profiling of behavioral effects evoked by ketamine and the role of 5HT2 and D2 receptors in ketamine-induced locomotor sensitization in mice.

Ketamine has addictive potential, a troublesome fact due to its promising use as a therapeutic drug. An important phenomenon associated with drug addiction is behavioral sensitization, usually characterized as augmented locomotion. However, other behaviors may also be susceptible to sensitization, and/or interfere with locomotor activity. Thus, this study drew a comprehensive behavioral 'profiling' in an animal model of repeated administration of ketamine. Adult Swiss mice received single daily ketamine injections (30 or 50 mg/Kg, i.p.), which were followed by open field testing for 7 days (acquisition period, ACQ). A ketamine challenge (sensitization test, ST) was carried out after a 5-day withdrawal. Locomotion, rearing, grooming, rotation and falling were assessed during ACQ and ST. All behaviors were affected from the first ACQ day onwards, with no indication of competition between locomotion and the other behaviors. Only locomotion in response to 30 mg/Kg of ketamine both escalated during ACQ and expressed increased levels at ST, evidencing development and expression of locomotor sensitization. Considering the involvement of serotonin 5HT(2) and dopamine D(2) receptors on addiction mechanisms, we further tested the involvement of these receptors in ketamine-induced sensitization. Ketanserin (5HT2 antagonist, 3 mg/Kg, s.c.) prevented ketamine-evoked development of locomotor sensitization. However, ketanserin pretreatment during ACQ failed to inhibit its expression during ST. Raclopride (D2 antagonist, 0.5 mg/Kg, s.c.) evoked less robust reductions in locomotion but prevented the development of ketamine-evoked sensitization. Pretreatment during ACQ further inhibited the expression of sensitization during ST. These results indicate that a partial overlap in serotonergic and dopaminergic mechanisms underlies ketamine-induced locomotor sensitization.

Lifelong exposure to caffeine increases anxiety-like behavior in adult mice exposed to tobacco smoke during adolescence.

Caffeine and tobacco smoke are among the most frequently self-administered licit psychoactive drugs in the world. Both drugs affect anxiety levels, however, little is known on the impact of the dual exposure in the adolescent brain, the period during which smoking begins. Considering that anxiety is a relevant factor for smoking maintenance and relapse, we investigated the effects of lifelong exposure to caffeine on anxiety levels of Swiss mice exposed to tobacco smoke during adolescence. Caffeine was administrated during all prenatal and postnatal life (CAF, 0.1 g/l to drink). From postnatal day 30-45, animals were exposed to tobacco smoke (SMK, whole body exposure, 8 h/day) generated from research cigarettes type 3R4F (nicotine = 0.73 mg/per cigarette). Four groups were analyzed: (1) CAF + SMK exposure; (2) SMK exposure; (3) CAF exposure; (4) Control. Anxiety levels were assessed in the elevated plus maze at the end of smoke exposure (PN45), at short- (PN55) and long-term (PN75) withdrawal. Caffeine exposure reduced decision making time (time in center of maze) during adolescence (PN45 and PN55). In addition, caffeine increased anxiety-like behavior during long-term tobacco smoke withdrawal. The present study provides experimental evidence that caffeine and tobacco smoke during adolescence interact resulting in emotional dysregulation during tobacco smoke withdrawal. Particularly, increased anxiety-like behavior during long-term withdrawal in CAF + SMK animals demonstrates late-emergent effects. In this sense, our data suggest that lifelong caffeine exposure may be an important factor in tobacco relapse.

Tobacco smoke and ethanol during adolescence: Both combined- and single-drug exposures lead to short- and long-term disruption of the serotonergic system in the mouse brain.

The impairment of the serotonergic system contributes to nicotine and ethanol effects on mood, suggesting that this system is targeted by each of these drugs and that co-exposure possibly worsens the disruption. Here, we tested this hypothesis in an adolescent mice model of tobacco smoke and/or ethanol exposure. From postnatal day (PN) 30-45, Swiss mice were exposed to one of the following: 1) tobacco smoke (SMK; research cigarettes 2R1F, whole-body exposure, 8 h/daily); 2) ethanol (ETOH; 2 g/kg i.p., every other day); 3) SMK + ETOH; 4) Control (VEH). At PN45 (end-of-exposure), hippocampal serotonin transporter (5 H TT) binding was increased in SMK and decreased in ETOH male mice. At PN50 (short-term deprivation), cortical 5 H TT was reduced in all drug-exposed mice. In the hippocampus, similar deficits were identified in females. In both brain regions, the effects of SMK + ETOH deprivation on 5 H TT were equivalent to the damage caused by either drug. At PN50, hippocampal 5 H T1A receptor binding was reduced in ETOH and SMK + ETOH mice. Similar results were observed in the male cortex. In females, deficits were identified in SMK mice. In both brain regions, SMK + ETOH 5 H T1A deficits reflected the summation of SMK and ETOH outcomes. At PN75 (long-term deprivation), there was a late-emergent increase in cortical 5 H T1A binding in SMK mice, while cortical 5 H T2 receptor binding was similarly increased in SMK and SMK + ETOH groups. Adolescent SMK and/or ETOH serotonergic impairment is sex-dependent and most evident during short-term deprivation. SMK + ETOH deprivation evokes serotonergic disruption that is at least equivalent to that caused by either drug alone.

Hyperactivity and memory/learning deficits evoked by developmental exposure to nicotine and/or ethanol are mitigated by cAMP and cGMP signaling cascades activation.

Pregnant smoking women are frequently episodic drinkers. Here, we investigated whether ethanol exposure restricted to the brain growth spurt period when combined with chronic developmental exposure to nicotine aggravates memory/learning deficits and hyperactivity, and associated cAMP and cGMP signaling disruption. To further investigate the role of these signaling cascades, we verified whether vinpocetine (a phosphodiesterase inhibitor) ameliorates the neurochemical and behavioral outcomes. Swiss mice had free access to nicotine (NIC, 50 µg/ml) or water to drink during gestation and until the 8th postnatal day (PN8). Ethanol (ETOH, 5 g/kg, i.p.) or saline were injected in the pups every other day from PN2 to PN8. At PN30, animals either received vinpocetine (20 mg/kg, i.p.) or vehicle before being tested in the step-down passive avoidance or open field. Memory/learning was impaired in NIC, ETOH and NIC + ETOH mice, and vinpocetine mitigated ETOH- and NIC + ETOH-induced deficits. Locomotor hyperactivity identified in ETOH and NIC + ETOH mice was ameliorated by vinpocetine. While cyclic nucleotides levels in cerebral cortex and hippocampus were reduced by NIC, ETOH and NIC + ETOH, this outcome was more consistent in the latter group. As observed for behavior, vinpocetine normalized NIC + ETOH nucleotides levels. pCREB levels were also increased in response to vinpocetine, with stronger effects in the NIC + ETOH group. Exposure to both drugs of abuse worsens behavioral and neurochemical disruption. These findings and the amelioration of deleterious effects by vinpocetine support the idea that cAMP and cGMP signaling contribute to nicotine- and ethanol-induced hyperactivity and memory/learning deficits.