Potential Benefit of Add-on Δ9-Tetrahydrocannabinol in Pediatric Drug-Resistant Epilepsy: A Case Series.

We present five cases of pediatric drug-resistant epilepsy (DRE) that failed management using high cannabidiol (CBD) doses, but had significant reduction in seizure frequency with reintroduction or increasing doses of tetrahydrocannabinol (THC). There is growing evidence supporting the use of whole-plant CBD-rich extracts (containing THC and other cannabinoids) in the treatment of pediatric DRE. Based on our experiences and reports in the literature, we propose that, in patients who fail management with an initial trial of high-dose CBD-focused therapy, there may be a role for add-on THC-focused formulations.

Agitated behavior scale in pediatric traumatic brain injury.

Agitation following TBI commonly occurs during the acute recovery phase. The ABS is a valid measure of agitation in adults following TBI. The objective of the study was to determine if ABS scores accurately measure acute agitation in pediatric patients recovering from TBI. The ABS was completed twice daily for 4 days and mean ABS scores were calculated. Physicians assessed patients' agitation daily using a VAS. In addition, interventions for agitation were recorded. The association between ABS and VAS scores was assessed using Spearman's correlation. The relationship between the number of medication classes taken for agitation (0, 1-2, or ≥3) and ABS scores was assessed using one-way analysis of variance. Finally, the association between the use of hand restraints and ABS scores was examined using an unpaired two-sample t-test. Twenty-six pediatric patients with acute TBI were included. ABS scores significantly associated with VAS scores. Patients that required interventions for agitation (hand restraints or ≥3 medication classes) had higher ABS scores than patients that did not receive any intervention. The study supports the use of ABS scoring to measure agitation in pediatric patients with TBI. However, additional studies are warranted to further support the validity of this scale. Abbreviations: TBI: Traumatic brain injury; ABS: Agitated Behaviour Scale; VAS: visual analog scale; PCCU: Pediatric Critical Care Unit.

Time-dependent interacting effects of caffeine, diazepam, and ethanol on zebrafish behaviour.

Zebrafish have become a popular animal model for behavioural pharmacology due to their small size, rapid development, and amenability to high throughput behavioural drug screens. Furthermore, water-soluble compounds can be administered via immersion of the fish in the drug solution, which provides a non-invasive drug delivery method. Numerous studies have demonstrated stimulant effects of alcohol. Diazepam and caffeine, on the other hand have been found to have inhibitory effect on locomotor activity in zebrafish. However, the time-dependent changes induced by these psychoactive drugs are rarely reported, and potential drug interactions have not been examined in zebrafish, despite the translational relevance of this question. In the current study, we examine time- and dose-dependent changes in zebrafish following exposure to caffeine, diazepam, and ethanol quantifying four different behavioural parameters over a 30min recording session. We subsequently analyze potential drug-drug interactions by co-administering the three drugs in different combinations. Our time-course and dose-response analyses for each of the three drugs represent so far the most detailed studies available serving as a foundation for future psychopharmacology experiments with zebrafish. Furthermore, we report significant interactions between the three drugs corroborating findings obtained with rodent models as well as in humans, providing translational relevance for the zebrafish model.

Acute alcohol exposure increases tyrosine hydroxylase protein expression and dopamine synthesis in zebrafish.

Zebrafish have become a popular animal model for investigating the effects of alcohol on the brain and behaviour. Acute exposure to alcohol has been shown to alter dopaminergic signalling in zebrafish, but the underlying mechanisms have not been well defined. In the current study, we characterize the effects of alcohol on the zebrafish dopaminergic system by focusing on tyrosine hydroxylase, the rate-limiting enzyme in dopamine synthesis. Using western blot analysis, we demonstrate that a 60min exposure to 1% alcohol increases tyrosine hydroxylase protein expression in the zebrafish brain. Enzymatic activity assays confirmed that alcohol also increases tyrosine hydroxylase enzymatic activity, whereas HPLC analysis demonstrated increased levels of whole-brain dopamine and its metabolite DOPAC. In addition to activation of the dopaminergic system, behavioural analysis revealed accompanying increase of distance traveled following 1% alcohol exposure. These findings suggest that acute alcohol exposure elevates dopamine synthesis via increased tyrosine hydroxylase protein expression. Our results support the hypothesis that alcohol alters dopaminergic signalling in the zebrafish brain in a similar manner as compared to mammals.

Ethanol-Induced ADH Activity in Zebrafish: Differential Concentration-Dependent Effects on High- Versus Low-Affinity ADH Enzymes.

Zebrafish express enzymes that metabolize ethanol in a manner comparable to that of mammals, including humans. We previously demonstrated that acute ethanol exposure increases alcohol dehydrogenase (ADH) activity in an inverted U-shaped dose-dependent manner. It was hypothesized that the biphasic dose-response was due to the increased activity of a high-affinity ADH isoform following exposure to low concentrations of ethanol and increased activity of a low-affinity ADH isoform following exposure to higher concentrations of ethanol. To test this hypothesis, we exposed zebrafish to different concentrations of ethanol (0%, 0.25%, 0.5%, and 1.0% v/v) for 30 min and measured the total ADH activity in the zebrafish liver. However, we also repeated this enzyme activity assay using a low concentration of the substrate (ethanol) to determine the activity of high-affinity ADH isoforms. We found that total ADH activity in response to ethanol induces an inverted U-shaped dose-response similar to our previous study. Using a lower substrate level in our enzyme assay targeting high-affinity isozymes, we found a similar dose-response. However, the difference in activity between the high and low substrate assays (high substrate activity - low substrate activity), which provide an index of activity for low-affinity ADH isoforms, revealed no significant effect of ethanol exposure. Our results suggest that the inverted U-shaped dose-response for total ADH activity in response to ethanol is driven primarily by high-affinity isoforms of ADH.

Neurochemical factors underlying individual differences in locomotor activity and anxiety-like behavioral responses in zebrafish.

Variation among individuals may arise for several reasons, and may have diverse underlying mechanisms. Individual differences have been studied in a variety of species, but recently a new model organism has emerged in this field that offers both sophistication in phenotypical characterization and powerful mechanistic analysis. Recently, zebrafish, one of the favorites of geneticists, have been shown to exhibit consistent individual differences in baseline locomotor activity. In the current study, we further explore this finding and examine whether individual differences in locomotor activity correlate with anxiety-like behavioral measures and with levels of dopamine, serotonin and the metabolites of these neurotransmitters. In addition, we examine whether individual differences in locomotor activity are also associated with reactivity to the locomotor stimulant effects of and neurochemical responses to acute ethanol exposure (30min long, 1% v/v ethanol bath application). Principal component analyses revealed a strong association among anxiety-like responses, locomotor activity, serotonin and dopamine levels. Furthermore, ethanol exposure was found to abolish the locomotion-dependent anxiety-like behavioral and serotonergic responses suggesting that this drug also engages a common underlying pathway. Overall, our results provide support for an important role of the serotonergic system in mediating individual differences in anxiety-like responses and locomotor activity in zebrafish and for a minor modulatory role of the dopaminergic system.

Interaction between handling induced stress and anxiolytic effects of ethanol in zebrafish: A behavioral and neurochemical analysis.

Stress is often considered an important factor in the development of alcohol addiction. In rodents, various types of stressors have been shown to potentiate the effects of alcohol on behavioral responses, and to increase consumption of this substance. However, few have investigated the interaction between stress and alcohol in zebrafish. In the current study we present a repeated handling stress paradigm we developed for zebrafish, and examine whether stress alters alcohol induced behavioral and neurochemical responses. Our results show that repeated handling of zebrafish conducted for 2 consecutive days is sufficient to increase anxiety-like behavioral responses quantified 24h post-stressor. Repeatedly handled zebrafish also exhibited a reduction in the levels of serotonin's metabolite, 5-hydroxyindole acetic acid (quantified by high precision liquid chromatography) compared to unhandled controls. A 60-min acute exposure to 1% ethanol was found to significantly increase locomotor activity and decrease anxiety-like behavioral responses in stressed zebrafish but not in controls. Furthermore, unhandled control zebrafish exhibited a significant increase in whole-brain dopamine levels following exposure to ethanol but the increase was not observed in repeatedly handled fish. Our findings suggest that ethanol induced locomotor activity and anxiolysis is potentiated by handling stress and may be partially mediated by changes in dopaminergic and serotonergic activity. Overall, we demonstrate the validity of our repeated handling stressor paradigm for zebrafish, which can be used to investigate the interaction between stress and ethanol.

Inhibition of phosphorylated tyrosine hydroxylase attenuates ethanol-induced hyperactivity in adult zebrafish (Danio rerio).

Zebrafish have been successfully employed in the study of the behavioural and biological effects of ethanol. Like in mammals, low to moderate doses of ethanol induce motor hyperactivity in zebrafish, an effect that has been attributed to the activation of the dopaminergic system. Acute ethanol exposure increases dopamine (DA) in the zebrafish brain, and it has been suggested that tyrosine hydroxylase, the rate-limiting enzyme of DA synthesis, may be activated in response to ethanol via phosphorylation. The current study employed tetrahydropapaveroline (THP), a selective inhibitor of phosphorylated tyrosine hydroxylase, for the first time, in zebrafish. We treated zebrafish with a THP dose that did not alter baseline motor responses to examine whether it can attenuate or abolish the effects of acute exposure to alcohol (ethanol) on motor activity, on levels of DA, and on levels of dopamine's metabolite 3,4-dihydroxyphenylacetic acid (DOPAC). We found that 60-minute exposure to 1% alcohol induced motor hyperactivity and an increase in brain DA. Both of these effects were attenuated by pre-treatment with THP. However, no differences in DOPAC levels were found among the treatment groups. These findings suggest that tyrosine hydroxylase is activated via phosphorylation to increase DA synthesis during alcohol exposure in zebrafish, and this partially mediates alcohol's locomotor stimulant effects. Future studies will investigate other potential candidates in the molecular pathway to further decipher the neurobiological mechanism that underlies the stimulatory properties of this popular psychoactive drug.

Differential effects of acute administration of SCH-23390, a D1 receptor antagonist, and of ethanol on swimming activity, anxiety-related responses, and neurochemistry of zebrafish.

RATIONALE: The zebrafish has become an increasingly popular animal model for investigating ethanol's actions in the brain and its effects on behavior. Acute exposure to ethanol in zebrafish has been shown to induce a dose-dependent increase of locomotor activity, to reduce fear- and anxiety-related behavioral responses, and to increase the levels of dopamine and its metabolite 3,4-dihydroxyphenylacetic acid (DOPAC). OBJECTIVES: The objective of the present study was to investigate the role of dopamine D1 receptors (D1-R) in ethanol-induced locomotor activity in zebrafish. METHODS: Zebrafish were pre-treated with SCH-23390 (0 or 1 mg/L bath concentration), a D1-R antagonist, and subsequently exposed to ethanol (0, 0.25, 0.5, 1.0 % v/v). To explore potential underlying mechanisms, we quantified levels of dopamine, DOPAC, serotonin, and 5-HIAA from whole-brain tissue using high-precision liquid chromatography. RESULTS: We found pre-treatment with the D1-R antagonist to attenuate locomotor activity independent of ethanol concentration. Furthermore, unlike ethanol, D1-R antagonism did not alter behavioral responses associated with fear and anxiety. Pre-treatment with SCH-23390 decreased levels of dopamine and DOPAC, but this effect was also independent of ethanol concentration. The D1-R antagonist also reduced serotonin and 5-hydroxyindole acetic acid (5-HIAA) levels. CONCLUSION: These results suggest a multifaceted and at least partially independent role of dopamine D1 receptors in ethanol-induced locomotor activity and anxiety-related responses as well as in the functioning of the dopaminergic and serotoninergic neurotransmitter systems in zebrafish.

Differential effects of dopamine D1 and D 2/3 receptor antagonism on motor responses.

RATIONALE: The zebrafish dopaminergic system is thought to be evolutionarily conserved and may be amenable to pharmacological manipulation using drugs developed for mammalian receptors. However, only few studies have examined the role of specific receptor subtypes in behaviour of adult zebrafish. OBJECTIVES: The objectives of this study are to determine the translational relevance of the zebrafish and examine the psychopharmacology of specific dopamine receptors in this species. METHODS: Using a behavioural pharmacological approach, we examine the effect of D1 and D2/3 receptor antagonisms on motor patterns of adult zebrafish during acute drug exposure and withdrawal. RESULTS: Acute exposure to SCH-23390 (D1 receptor antagonist) decreased total distance travelled in a dose-dependent manner. Exposure to amisulpride (D2/3 receptor antagonist) induced a biphasic dose-response in total distance travelled and in angular velocity. The results provide support for the existence of structurally and functionally conserved postsynaptic D1 and D2 receptors, as well as presynaptic D2 autoreceptors in the zebrafish brain. The behavioural effects of the employed antagonists did not persist following 30 min of withdrawal. CONCLUSION: The results suggest that zebrafish, a cheaper and simpler model organism compared to the rat and the mouse, may be an efficient translationally relevant tool for the analysis of the psychopharmacology of receptors of the vertebrate dopaminergic system.

Acute and chronic ethanol exposure differentially alters alcohol dehydrogenase and aldehyde dehydrogenase activity in the zebrafish liver.

Chronic ethanol exposure paradigms have been successfully used in the past to induce behavioral and central nervous system related changes in zebrafish. However, it is currently unknown whether chronic ethanol exposure alters ethanol metabolism in adult zebrafish. In the current study we examine the effect of acute ethanol exposure on adult zebrafish behavioral responses, as well as alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) activity in the liver. We then examine how two different chronic ethanol exposure paradigms (continuous and repeated ethanol exposure) alter behavioral responses and liver enzyme activity during a subsequent acute ethanol challenge. Acute ethanol exposure increased locomotor activity in a dose-dependent manner. ADH activity was shown to exhibit an inverted U-shaped curve and ALDH activity was decreased by ethanol exposure at all doses. During the acute ethanol challenge, animals that were continuously housed in ethanol exhibited a significantly reduced locomotor response and increased ADH activity, however, ALDH activity did not change. Zebrafish that were repeatedly exposed to ethanol demonstrated a small but significant attenuation of the locomotor response during the acute ethanol challenge but ADH and ALDH activity was similar to controls. Overall, we identified two different chronic ethanol exposure paradigms that differentially alter behavioral and physiological responses in zebrafish. We speculate that these two paradigms may allow dissociation of central nervous system-related and liver enzyme-dependent ethanol induced changes in zebrafish.

Serotonin antagonists induce anxiolytic and anxiogenic-like behavior in zebrafish in a receptor-subtype dependent manner.

Motor function and anxiety-like responses are easily quantifiable in zebrafish, a novel model organism for behavioral pharmacology. Activation of serotonin receptors through the use of selective agonists has been shown to alter anxiety-like behaviors in zebrafish. However, few studies have examined the effect of blockade of specific serotonin receptors. In the current study, we examine the effect of 4 serotonin receptor antagonists selective for 5-HT1A, 5-HT1B/D, 5-HT2, and 5-HT3 receptors on zebrafish motor and anxiety-like responses. Exposure to the receptor antagonists did not change baseline motor responses. However, when placed in a novel environment, zebrafish previously exposed to GR 55562 (5-HT1B/D antagonist) exhibited reduced anxiety-like behavior, whereas zebrafish previously exposed to p-MPPF (5-HT1A antagonist), Ketanserin (5-HT2 antagonist), or Ondasetron (5-HT3 antagonist) exhibited increased anxiety-like behaviors. These results show that drugs developed for mammalian serotonin receptors are efficacious in the zebrafish too, a finding that demonstrates evolutionary conservation of the serotoninergic system. The results also imply that zebrafish may be an appropriate animal model for examining the serotonergic neurotransmitter system in vertebrates.

Cross-modal signatures in maternal speech and singing.

We explored the possibility of a unique cross-modal signature in maternal speech and singing that enables adults and infants to link unfamiliar speaking or singing voices with subsequently viewed silent videos of the talkers or singers. In Experiment 1, adults listened to 30-s excerpts of speech followed by successively presented 7-s silent video clips, one from the previously heard speaker (different speech content) and the other from a different speaker. They successfully identified the previously heard speaker. In Experiment 2, adults heard comparable excerpts of singing followed by silent video clips from the previously heard singer (different song) and another singer. They failed to identify the previously heard singer. In Experiment 3, the videos of talkers and singers were blurred to obscure mouth movements. Adults successfully identified the talkers and they also identified the singers from videos of different portions of the song previously heard. In Experiment 4, 6- to 8-month-old infants listened to 30-s excerpts of the same maternal speech or singing followed by exposure to the silent videos on alternating trials. They looked longer at the silent videos of previously heard talkers and singers. The findings confirm the individuality of maternal speech and singing performance as well as adults' and infants' ability to discern the unique cross-modal signatures. The cues that enable cross-modal matching of talker and singer identity remain to be determined.