Evaluating the daily modulation of FADD and related molecular markers in different brain regions in male rats.

Fas-Associated protein with Death Domain (FADD), a key molecule controlling cell fate by balancing apoptotic versus non-apoptotic functions, is dysregulated in post-mortem brains of subjects with psychopathologies, in animal models capturing certain aspects of these disorders, and by several pharmacological agents. Since persistent disruptions in normal functioning of daily rhythms are linked with these conditions, oscillations over time of key biomarkers, such as FADD, could play a crucial role in balancing the clinical outcome. Therefore, we characterized the 24-h regulation of FADD (and linked molecular partners: p-ERK/t-ERK ratio, Cdk-5, p35/p25, cell proliferation) in key brain regions for FADD regulation (prefrontal cortex, striatum, hippocampus). Samples were collected during Zeitgeber time (ZT) 2, ZT5, ZT8, ZT11, ZT14, ZT17, ZT20, and ZT23 (ZT0, lights-on or inactive period; ZT12, lights-off or active period). FADD showed similar daily fluctuations in all regions analyzed, with higher values during lights off, and opposite to p-ERK/t-ERK ratios regulation. Both Cdk-5 and p35 remained stable and did not change across ZT. However, p25 increased during lights off, but exclusively in striatum. Finally, no 24-h modulation was observed for hippocampal cell proliferation, although higher values were present during lights off. These results demonstrated a clear daily modulation of FADD in several key brain regions, with a more prominent regulation during the active time of rats, and suggested a key role for FADD, and molecular partners, in the normal physiological functioning of the brain's daily rhythmicity, which if disrupted might participate in the development of certain pathologies.

Evaluating signs of hippocampal neurotoxicity induced by a revisited paradigm of voluntary ethanol consumption in adult male and female Sprague-Dawley rats.

BACKGROUND: Binge alcohol drinking is considered a prominent risk factor for the development of alcohol-use disorders, and could be model in rodents through the standard two-bottle preference choice test. The goal was to recreate an intermittent use of alcohol during 3 consecutive days each week to ascertain its potential impact on hippocampal neurotoxicity (neurogenesis and other neuroplasticity markers), and including sex as a biological variable, given the well-known sex differences in alcohol consumption. METHODS: Ethanol access was granted to adult Sprague-Dawley rats for 3 consecutive days per week, followed by 4 days of withdrawal, during 6 weeks, mimicking the most common pattern of intake in people, drinking over the weekends in an intensive manner. Hippocampal samples were collected to evaluate signs of neurotoxicity. RESULTS: Female rats consumed significantly more ethanol than males, although intake did not escalate over time. Ethanol preference levels remained below 40% over time and did not differ between sexes. Moderate signs of ethanol neurotoxicity were observed in hippocampus at the level of decreased neuronal progenitors (NeuroD + cells), and these effects were independent of sex. No other signs of neurotoxicity were induced by ethanol voluntary consumption when measured through several key cell fate markers (i.e., FADD, Cyt c, Cdk5, NF-L) by western blot analysis. CONCLUSIONS: Overall, the present results suggest that even though we modeled a situation where no escalation in ethanol intake occurred across time, mild signs of neurotoxicity emerged, suggesting that even the use of ethanol during adulthood in a recreational way could lead to certain brain harm.

Dose-dependent opposite effects of nortriptyline on affective-like behavior in adolescent rats: Comparison with adult rats.

Antidepressant drugs elicit different behavioral and neurochemical responses with age. In fact, the use of antidepressants during adolescence is associated with an increased risk of suicidal thinking, being the best pharmacological treatment during this critical period a matter of constant debate in terms of its risk-benefit outcome. In this regard, the present study compared the effects of nortriptyline (3-10 mg/kg, 7 days) on regulating different aspects of affective-like behavior by screening adolescent and adult Sprague-Dawley rats through several consecutive tests (forced-swim, open field, sucrose preference). Brains were later collected to evaluate hippocampal neurogenesis and mBDNF protein content as potential molecular correlates of the observed behavioral responses. The main results in adolescent rats showed that nortriptyline induced dose-dependent opposite effects: while 3 mg/kg decreased immobility and increased mBDNF (indicative of an antidepressant-like response), 10 mg/kg decreased exploratory time in the open field and mBDNF (suggestive of an anxiogenic-like response). These effects were not associated with changes in neurogenesis regulation. In adult rats, nortriptyline failed to modulate affective-like behavior or the neuroplasticity markers evaluated at the doses tested. In conclusion, clear behavioral and neurochemical differences were observed between adolescent and adult rats in response to nortriptyline treatment. Interestingly, while nortriptyline displayed an antidepressant-like potential at the lowest dose examined in adolescence, a higher dose shifted these results towards a negative outcome, thus reinforcing the need to extreme caution when considering this treatment for our younger population.

Electroconvulsive seizures protect against methamphetamine-induced inhibition of neurogenesis in the rat hippocampus.

Following methamphetamine consumption and during abstinence many behavioral consequences emerge (i.e., cognitive deficits, ongoing episodes of psychosis, depression, severe cravings, brain neurotoxicity), which are likely linked to propensity to relapse. In this line of thought, we recently showed that binge methamphetamine administration enhanced negative affect and voluntary drug consumption in rats, while it induced persistent neurotoxic effects (i.e., impaired hippocampal neurogenesis), effects that emerged long after drug removal. To date, no pharmacological strategies have been proven to be effective for the treatment of methamphetamine toxicity. A few studies have evaluated the impact of combining methamphetamine pretreatment with electroconvulsive seizures (ECS) post-treatment, an alternative non-pharmacological option used in psychiatry for resistant depression that offers a safe and really potent therapeutic response. Against this background, the present study aimed at testing whether repeated ECS treatment could ameliorate some of the long-term neurotoxicity effects induced by adolescent methamphetamine exposure in rats and emerging after drug removal. At the behavioral level, the main results showed that methamphetamine administration did not alter negative affect immediate during adolescence or later on in adulthood. Interestingly, repeated ECS improved the negative impact of methamphetamine administration on reducing hippocampal neurogenesis, demonstrating that ECS can attenuate certain degree of methamphetamine-induced neurotoxicity in rats, and suggesting ECS as a good therapeutical candidate that deserves further studies.

Adolescent cocaine induced persistent negative affect in female rats exposed to early-life stress.

RATIONALE: The combination of several risk factors (sex, a prior underlying psychiatric condition, or early drug initiation) could induce the emergence of negative affect during cocaine abstinence and increase the risk of developing addiction. However, most prior preclinical studies have been centered in male rodents, traditionally excluding females from these analyses. OBJECTIVES: To ascertain the behavioral and neurochemical consequences of adolescent cocaine exposure when the combination of several risk factors is present (female, early-life stress). METHODS: Whole litters of Sprague-Dawley rats were exposed to maternal deprivation for 24 h on postnatal day (PND) 9. Cocaine was administered in adolescence (15 mg/kg/day, i.p., PND 33-39). Negative affect was assessed by several behavioral tests (forced swim, open field, novelty-suppressed feeding, sucrose preference). Hippocampal cell fate markers were evaluated by western blot (FADD, Bax, cytochrome c) or immunohistochemistry (Ki-67; cell proliferation). RESULTS: Maternal deprivation is a suitable model of psychiatric vulnerability in which to study the impact of adolescent cocaine in female rats. While adolescent cocaine did not alter affective-like behavior during adolescence, a pro-depressive-like state emerged during adulthood, exclusively in rats re-exposed to cocaine during abstinence. FADD regulation by cocaine in early-life stressed female rats might contribute to certain hippocampal neuroadaptations with some significance to the observed induced negative affect. CONCLUSIONS: Adolescent cocaine induced persistent negative affect in female rats exposed to early-life stress, highlighting the risk of early drug initiation during adolescence for the emergence of negative reinforcement during abstinence likely driving cocaine addiction vulnerability, also in female rats.

Antidepressant-like effects of cannabidiol in a rat model of early-life stress with or without adolescent cocaine exposure.

BACKGROUND: Further studies are needed to better understand the effects of potential novel antidepressants, such as cannabidiol, for the treatment of psychiatric disorders during adolescence. In this context, we evaluated in a rodent model of early-life stress (a single 24-h episode of maternal deprivation, PND 9), the antidepressant-like effects of adolescent cannabidiol alone and/or in combination with adolescent cocaine exposure (given the described beneficial effects of cannabidiol on reducing cocaine effects). METHODS: Maternally deprived Sprague-Dawley male rats were treated in adolescence with cannabidiol (with or without concomitant cocaine) and exposed to a battery of behavioral tests (forced-swim, novelty-suppressed feeding, open field, sucrose preference) across time. Putative enduring molecular correlates (CB receptors, BDNF) were evaluated in the hippocampus by western blot. RESULTS: Cannabidiol exerted antidepressant- and anxiolytic-like effects in rats exposed to early-life stress. Cocaine did not alter affective-like behavior during adolescence in rats exposed to early-life stress; however, a depressive- and anxiogenic-like phenotype emerged during adulthood, and cannabidiol exerted some behavioral improvements, along with the growing literature supporting its beneficial role for reducing cocaine intake and/or reinstatement in rodents. Finally, cannabidiol did not modulate hippocampal CB receptors or BDNF proteins, and although the data raised interesting questions about the possible role of CB1 receptors on modulating the long-term effects of cocaine, future research is needed to expand these findings. CONCLUSION: Cannabidiol showed a promising therapeutic response in terms of ameliorating affect in a rat model of early-life stress during adolescence and up to adulthood.

Increased negative affect when combining early-life maternal deprivation with adolescent, but not adult, cocaine exposure in male rats: regulation of hippocampal FADD.

RATIONALE: Besides early drug initiation during adolescence, another vulnerability factor associated with increased risk for substance abuse later in life is early-life stress. One way of assessing such combined risk is by evaluating the emergence of increased negative affect during withdrawal (i.e., linked to persistence in drug seeking). OBJECTIVES: To compare the impact of maternal deprivation with cocaine exposure at different ages on affective-like behavior and hippocampal neuroplasticity regulation. METHODS: Maternal deprivation was performed in whole-litters of Sprague-Dawley rats (24 h, PND 9-10). Cocaine (15 mg/kg, 7 days, i.p.) was administered in adolescence (PND 33-39) or adulthood (PND 64-70). Changes in affective-like behavior were assessed by diverse tests across time (forced-swim, open field, novelty-suppressed feeding, sucrose preference). Hippocampal multifunctional FADD protein (balance between cell death and plasticity) was evaluated by Western blot. RESULTS: Exposing rats to either maternal deprivation or adolescent cocaine did not modulate affective-like behavior immediately during adolescence, but increased negative affect in adulthood. Maternal deprivation combined with adolescent cocaine advanced the negative impact to adolescence. Adult cocaine exposure alone and/or in combination with maternal deprivation did not induce any behavioral changes at the time-points analyzed. FADD regulation might participate in the neural adaptations taking place in the hippocampus in relation to the observed behavioral changes. CONCLUSIONS: Adolescence is a more vulnerable period, as compared to adulthood, to the combined impact of cocaine and early maternal deprivation, thus suggesting that the accumulation of stress early in life can anticipate the negative behavioral outcome associated with drug consumption.

Sex differences in the antidepressant-like potential of repeated electroconvulsive seizures in adolescent and adult rats: Regulation of the early stages of hippocampal neurogenesis.

Age and sex are critical factors for the diagnosis and treatment of major depression, since there is a well-known age-by-sex difference in the prevalence of major depression (being females the most vulnerable ones) and in antidepressant efficacy (being adolescence a less responsive period than adulthood). Although the induction of electroconvulsive seizures (ECS) is a very old technique in humans, there is not much evidence reporting sex- and age-specific aspects of this treatment. The present study evaluated the antidepressant- and neurogenic-like potential of repeated ECS across time in adolescent and adult rats (naïve or in a model of early life stress capable of mimicking a pro-depressive phenotype), while including a sex perspective. The main results demonstrated age- and sex-specific differences in the antidepressant-like potential of repeated ECS, since it worked when administered during adolescence or adulthood in male rats (although with a shorter length in adolescence), while in females rendered deleterious during adolescence and ineffective in adulthood. Yet, repeated ECS increased cell proliferation and vastly boosted young neuronal survival in a time-dependent manner for both sexes and independently of age. Moreover, pharmacological inhibition of basal cell proliferation prevented the antidepressant-like effect induced by repeated ECS in male rats, but only partially blocked the very robust increase in the initial cell markers of hippocampal neurogenesis. Overall, the present results suggest that the induction of the early phases of neurogenesis by ECS, besides having a role in mediating its antidepressant-like effect, might participate in some other neuroplastic actions, opening the path for future studies.

Decreased sensitivity in adolescent versus adult rats to the antidepressant-like effects of cannabidiol.

RATIONALE: Cannabidiol is a non-psychoactive phytocannabinoid with great therapeutic potential in diverse psychiatric disorders; however, its antidepressant potential has been mainly ascertained in adult rats. OBJECTIVES: To compare the antidepressant-like response induced by cannabidiol in adolescent and adult rats and the possible parallel modulation of hippocampal neurogenesis. METHODS: Male Sprague-Dawley rats were repeatedly treated with cannabidiol (3, 10, and 30 mg/kg) or vehicle (1 mL/kg) during adolescence (postnatal days, PND 27-33) or adulthood (PND 141-147) and exposed to 3 consecutive tests (forced swim, open field, two-bottle choice) that quantified behavioral despair, anxiety, and sucrose intake respectively. RESULTS: Cannabidiol induced differential effects depending on the age and dose administered, with a decreased sensitivity observed in adolescent rats: (1) cannabidiol (30 mg/kg) decreased body weight only in adult rats; (2) cannabidiol ameliorated behavioral despair in adolescent and adult rats, but with a different dose sensitivity (10 vs. 30 mg/kg), and with a different extent (2 vs. 21 days post-treatment); (3) cannabidiol did not modulate anxiety-like behavior at any dose tested in adolescent or adult rats; and (4) cannabidiol increased sucrose intake in adult rats. CONCLUSIONS: Our findings support the notion that cannabidiol exerts antidepressant- and anorexigenic-like effects in adult rats and demonstrate a decreased potential when administered in adolescent rats. Moreover, since cannabidiol did not modulate hippocampal neurogenesis (cell proliferation and early neuronal survival) in adolescent or adult rats, the results revealed potential antidepressant-like effects induced by cannabidiol without the need of regulating hippocampal neurogenesis.

Adolescent morphine induces emotional signs of withdrawal paired with neurotoxicity selectively in male rats: Female resilience.

This study evaluated the immediate and persistent behavioral and molecular consequences (i.e., emotional signs emerging during withdrawal and signs of neurotoxicity) following adolescent morphine exposure with a sex perspective. Basally, and prior to drug treatment, adolescent female rats showed smaller body weight and lower pain threshold than their male counterparts. Adolescent morphine treatment induced some sex-specific differences, while morphine impaired normal weight gain in male rats, no effects were observed for female rats. Plus, morphine produced an attenuated antinociceptive response in female rats. Moreover, following adolescent morphine treatment some emotional signs of withdrawal emerged exclusively in male rats in conjunction with signs of neurotoxicity, while female rats were not affected. In particular, an anxiolytic-like effect in adolescence during early withdrawal was followed by the development of a depressive-like phenotype (i.e., behavioral despair, anxiety-like behavior and anhedonia) in adulthood during prolonged withdrawal and paired with decreased contents of neurofilaments proteins in the prefrontal cortex. In conclusion, morphine administration during adolescence induced persistent changes in negative affect and brain toxicity selectively in male rats, suggesting female rats were resilient to these harmful effects. Given the widespread availability and use of opiate-based painkillers, the interplay between addiction, analgesia and emotional behaviors, and since adolescents and young adult humans are the age group with the highest abuse potential, these results add to the current literature by reporting distinct sex-specific opioid actions when administered in adolescence.

Excess amounts of 3-iodo-l-tyrosine induce Parkinson-like features in experimental approaches of Parkinsonism.

3-iodo-l-tyrosine might play a role in Parkinson's disease since this molecule is able, at high concentration, to inhibit tyrosine-hydroxylase activity, the rate-limiting enzyme in dopamine biosynthesis. The possible Parkinson-like effects of 3-iodo-l-tyrosine were tested on three experimental approaches in mice: cultured substantia nigra neurons, the enteric nervous system of the jejunum after intra-peritoneal infusions, and the nigrostriatal system following unilateral intrabrain injections. 3-iodo-l-tyrosine, a physiological molecule, was used at concentrations higher than its serum levels in humans. Parkinson-like signs were evaluated through abnormal aggregation of α-synuclein and tyrosine-hydroxylase, loss of tyrosine-hydroxylase-expressing and striatum-projecting neurons and fibers, reduced tyrosine-hydroxylase density, and Parkinson-like motor and non-motor deficits. The retrograde tracer FluoroGold was used in the brain model. The findings revealed that excess amounts of 3-iodo-l-tyrosine induce Parkinson-like effects in the three experimental approaches. Thus, culture neurons of substantia nigra show, after 3-iodo-l-tyrosine exposure, intracytoplasmic inclusions that express α-synuclein and tyrosine-hydroxylase. Intra-peritoneal infusions of 3-iodo-l-tyrosine cause, in the long-term, α-synuclein aggregation, thicker α-synuclein-positive fibers, and loss of tyrosine-hydroxylase-positive cells and fibers in intramural plexuses and ganglia of the jejunum. Infusion of 3-iodo-l-tyrosine into the left dorsal striata of mice damages the nigrostriatal system, as revealed through lower striatal tyrosine-hydroxylase density, reduced number of tyrosine-hydroxylase-expressing and striatum-projecting neurons in the left substantia nigra, as well as the emergence of Parkinson-like behavioral deficits such as akinesia, bradykinesia, motor disbalance, and locomotion directional bias. In conclusion, excess amounts of 3-iodo-l-tyrosine induce Parkinson-like features in cellular, enteric and brain approaches of Parkinsonism in mice.

Methamphetamine binge administration during late adolescence induced enduring hippocampal cell damage following prolonged withdrawal in rats.

A recent study from our laboratory demonstrated that binge methamphetamine induced hippocampal cell damage (i.e., impaired cell genesis) in rats when administered specifically during late adolescence (postnatal day, PND 54-57) and evaluated 24 h later (PND 58). The results also suggested a possible role for brain-derived neurotrophic factor (BDNF) regulating cell genesis and survival. This subsequent study evaluated whether these effects persisted in time as measured following prolonged withdrawal. Male Sprague-Dawley rats were treated (i.p.) with BrdU (2 × 50 mg/kg, 3 days, PND 48-50) followed by a binge paradigm (3 pulses/day, every 3 h, 4 days, PND 54-57) of methamphetamine (5 mg/kg, n = 14, M) or saline (0.9% NaCl, 1 ml/kg, n = 12, C). Following 34 days of forced withdrawal (PND 91), rats were killed 45 min after a challenge dose of saline (Sal: C-Sal, n = 6; M-Sal, n = 7) or methamphetamine (Meth: C-Meth, n = 6; M-Meth, n = 7). Neurogenesis markers (Ki-67: cell proliferation; NeuroD: early neuronal survival; BrdU: prolonged cell survival, 41-43 days old cells) were evaluated by immunohistochemistry while neuroplasticity markers (BDNF and Fos forms) were evaluated by Western blot. The main results showed that a history of methamphetamine administration (PND 54-57) induced enduring hippocampal cell damage (i.e., observed on PND 91) by decreasing cell survival (BrdU + cells) and mature-BDNF (m-BDNF) protein content, associated with neuronal survival, growth and differentiation. Interestingly, m-BDNF regulation paralleled hippocampal c-Fos protein content, indicating decreased neuronal activity, and thus reinforcing the persisting negative effects induced by methamphetamine in rat hippocampus following prolonged withdrawal.