Romeo D. Ando

Damage of serotonergic axons and immunolocalization of Hsp27, Hsp72, and Hsp90 molecular chaperones after a single dose of MDMA administration in dark agouti rat: Temporal, spatial, and cellular patterns

3,4‐Methylenedioxymethamphetamine (MDMA, “ecstasy”) causes long‐term disturbance of the serotonergic system. We examined the temporal, spatial, and cellular distribution of three molecular chaperones, Hsp27, Hsp72, and Hsp90, 3 and 7 days after treatment with 7.5, 15, and 30 mg/kg single intraperitoneal (i.p.) doses of MDMA in Dark Agouti rat brains. Furthermore, we compared the immunostaining patterns of molecular chaperones with serotonergic axonal‐vulnerability evaluated by tryptophan‐hydroxylase (TryOH) immunoreactivity and with astroglial‐activation detected by GFAP‐immunostaining. There was a marked reduction in TryOH‐immunoreactive axon density after MDMA treatment in all examined areas at both time points. Three days after treatment, a significant dose‐dependent increase in Hsp27‐immunoreactive protoplasmic astrocytes was found in the cingulate, frontal, occipital, and pyriform cortex, and in the hippocampus CA1. However, there was no increase in astroglial Hsp27‐immunoreactivity in the caudate putamen, lateral septal nucleus, or anterior hypothalamus. A significant increase in the GFAP immunostaining density of protoplasmic astrocytes was found only in the hippocampus CA1. In addition, numerous strong Hsp72‐immunopositive neurons were found in some brain areas only 3 days after treatment with 30 mg/kg MDMA. Increased Hsp27‐immunoreactivity exclusively in the examined cortical areas reveals that Hsp27 is a sensitive marker of astroglial response to the effects of MDMA in these regions of Dark Agouti rat brain and suggests differential responses in astroglial Hsp27‐expression between distinct brain areas. The co‐occurrence of Hsp27 and GFAP response exclusively in the hippocampus CA1 may suggest the particular vulnerability of this region. The presence of strong Hsp72‐immunopositive neurons in certain brain areas may reflect additional effects of MDMA on nonserotonergic neurons. J. Comp. Neurol. 497:251–269, 2006.

Partial lesion of the serotonergic system by a single dose of MDMA results in behavioural disinhibition and enhances acute MDMA-induced social behaviour on the social interaction test

The acute effects of 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) on anxiety-related behaviours were studied using indices of social interaction in Dark Agouti (DA) both drug naive rats and those pretreated with MDMA (15 mg/kg i.p.) 3 weeks earlier. The functional neuroanatomy of these MDMA effects was visualised using 2-deoxyglucose imaging of local cerebral glucose use (LCMRglu), whilst MDMA-induced serotonergic neurotoxicity was measured by radioligand binding with [3H]paroxetine. Acute MDMA alone markedly decreased most typical elements of social interaction but increased adjacent lying, a behaviour that also contains social elements. In animals pre-exposed to MDMA, decreased [3H]paroxetine binding indicated serotonergic terminal depletion, and in these animals significant increases in locomotor activity, exploratory behaviour and aggressive behaviour were found. Both behavioural effects and also the metabolic activation induced by acute MDMA were potentiated in rats previously exposed to the drug. In conclusion, a single dose of MDMA caused marked changes in social behaviour acutely that might be interpreted either as a decrease or increase in anxiety. Three weeks after MDMA a behavioural disinhibition similar to psychomotor agitation, a symptom connected to depression or mania, and a sensitization to the acute effects of MDMA are apparent in both the behavioural and brain metabolic effects of the drug.

Synthesis of some diazino-fused tricyclic systems via Suzuki cross-coupling and regioselective nitrene insertion reactions

Suzuki coupling of 5-chloro-2-methyl-6-phenylpyridazin-3(2H)-one, 6-chloro-1,3-dimethyluracil and 2-chloropyrazine with protected aminoaryl boronic acids resulted in the corresponding pivaloylaminophenyl diazines which were transformed to diazino-fused indole and cinnoline derivatives. Suzuki coupling of 5-amino-6-chloro-1,3-dimethyluracil with 2-formylphenyl boronic acid afforded a novel pyrimidoisoquinoline ring system in a one-pot reaction.

Single dose of MDMA causes extensive decrement of serotoninergic fibre density without blockage of the fast axonal transport in Dark Agouti rat brain and spinal cord.

Prolonged neurotoxicity of the recreational drug, MDMA (3,4‐methylenedioxymethamphetamine) on serotoninergic axon terminals has been suggested. The effect of a single (15 mg/kg) dose of intraperitoneally administered MDMA on serotoninergic fibre density, defined by tryptophan hydroxylase (TpH) and serotonin transporter (5‐HTT) immunoreactivity, has been evaluated in the spinal cord and brain areas in Dark Agouti rats, 7 and 180 days after MDMA applications. Immunostaining for amyloid precursor protein (APP) has been performed to examine possible defects of the fast axonal transport, and 5‐HTT mRNA expressions were quantified in neurones of medullary raphe nuclei. Seven days after MDMA treatment, a substantial decrease in the density of TpH‐immunoreactive fibres was detectable in the frontal cortex, the caudate‐putamen, the CA1 region of the hippocampus, and marked decreases were found in the spinal cord. These changes in TpH density showed a high correlation with 5‐HTT densities. In contrast, APP‐immunoreactive axonal bulbs were not detected in any of the brain regions studied. Seven days after MDMA administrations, significantly elevated 5‐HTT mRNA expressions were found in the raphe pallidus and obscurus. Our results suggest that a single dose of MDMA elicits widespread depletion of TpH and 5‐HTT immunoreactivity in serotoninergic axons without morphological sign of the blockage of the fast anterograde axonal transport. Our results do not support the notion of MDMA‐induced axotomy of serotoninergic neurones. The up‐regulation of 5‐HTT mRNA expressions 1 week after MDMA injections might indicate the potential recovery of the serotonin system.

Effect of two noncompetitive AMPA receptor antagonists GYKI 52466 and GYKI 53405 on vigilance, behavior and spike-wave discharges in a genetic rat model of absence epilepsy

The present study was conducted to investigate the effects of two noncompetitive alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonists, GYKI 52466 and GYKI 53405 (the racemate of talampanel) on the generation of spike-wave discharges (SWD) parallel with the vigilance and behavioral changes in the genetic absence epilepsy model of WAG/Rij rats. Intraperitoneal (i.p.) administration of GYKI 52466 (1-[4-aminophenyl]-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine; 3, 10 and 30 mg/kg, i.p.), the prototypic compound of the 2,3-benzodiazepine family, caused a fast dose-dependent increase in the number and cumulative duration of SWD. These changes were accompanied by dose-dependent increase in duration of light slow wave sleep (SWS1) and passive awake, vigilance states associated with the presence of SWD. In addition a short, transient behavioral activation occurred that was followed by strong ataxia and immobility, decrease of active wakefulness and increase in deep slow wave sleep. GYKI 53405 (7-acetyl-5-(4-aminophenyl)-8-methyl-8,9-dihydro-7H-1,3-dioxolo[4,5-b][2,3]benzodiazepine, the racemate of talampanel, 16 mg/kg, i.p.) failed to affect any measure of SWD and vigilance. When used as a pretreatment, GYKI 52466 (10 mg/kg) slightly attenuated SWD-promoting effects of the 5-HT1A receptor agonist 8-OH-DPAT, it decreased cumulative duration and average time of paroxysms. In conclusion, AMPA receptors play moderate role in regulation of epileptic activity, and some of these effects are connected to their effects on vigilance in this model.

Acute and long-term effects of a single dose of MDMA on aggression in Dark Agouti rats.

MDMA causes selective depletion of serotonergic terminals in experimental animals and the consequent decrease in synaptic 5-HT may, inter alia, increase impulsivity. To study the effects of MDMA upon brain function, the behaviour of male Dark Agouti rats exposed to MDMA (15 mg/kg i.p.), two 5-HT1B agonists (CGS-12066A and CP-94,253, both 5 mg/kg i.p.) or saline were investigated in the resident-intruder test. Studies were performed in drug-naive rats and also in rats exposed to MDMA (15 mg/kg i.p.) 21 d earlier. In parallel experiments the functional neuroanatomy of MDMA effects were assessed using 2-deoxyglucose imaging of local cerebral metabolic rate of glucose utilization (LCMRGlu) and neurotoxicity was assessed by measuring [3H]paroxetine binding. There was no significant difference in aggressive behaviour (biting, boxing, wrestling and their latencies) between drug-naive rats and rats previously exposed to MDMA 21 d earlier, despite reduced social behaviour, decreased LCMRGlu in several brain areas involved in aggression, and reductions in paroxetine binding by 30-60% in the forebrain. CGS-12066A, CP-94,253 and acute MDMA produced marked decreases in aggressive behaviours, especially in biting, boxing and kicking found in drug-naive rats. In animals previously exposed to the drug, acute anti-aggressive effects of MDMA were, in general, preserved as were MDMA-induced increases in LCMRGlu. Our studies provide evidence that in the resident-intruder test, where social isolation is a requirement, aggressive behaviour and acute anti-aggressive effects of MDMA and 5-HT1B receptor agonists remain intact 3 wk after a single dose of the drug despite significant damage to the serotonergic system.

Transcriptional Evidence for the Role of Chronic Venlafaxine Treatment in Neurotrophic Signaling and Neuroplasticity Including also Glutatmatergic- and Insulin-Mediated Neuronal Processes.

Objectives Venlafaxine (VLX), a serotonine-noradrenaline reuptake inhibitor, is one of the most commonly used antidepressant drugs in clinical practice for the treatment of major depressive disorder (MDD). Despite being more potent than its predecessors, similarly to them, the therapeutical effect of VLX is visible only 3–4 weeks after the beginning of treatment. Furthermore, recent papers show that antidepressants, including also VLX, enhance the motor recovery after stroke even in non depressed persons. In the present, transcriptomic-based study we looked for changes in gene expressions after a long-term VLX administration. Methods Osmotic minipumps were implanted subcutaneously into Dark Agouti rats providing a continuous (40 mg/kg/day) VLX delivery for three weeks. Frontal regions of the cerebral cortex were isolated and analyzed using Illumina bead arrays to detect genes showing significant chances in expression. Gene set enrichment analysis was performed to identify specific regulatory networks significantly affected by long term VLX treatment. Results Chronic VLX administration may have an effect on neurotransmitter release via the regulation of genes involved in vesicular exocytosis and receptor endocytosis (such as Kif proteins, Myo5a, Sv2b, Syn2 or Synj2). Simultaneously, VLX activated the expression of genes involved in neurotrophic signaling (Ntrk2, Ntrk3), glutamatergic transmission (Gria3, Grin2b and Grin2a), neuroplasticity (Camk2g/b, Cd47), synaptogenesis (Epha5a, Gad2) and cognitive processes (Clstn2). Interestingly, VLX increased the expression of genes involved in mitochondrial antioxidant activity (Bcl2 and Prdx1). Additionally, VLX administration also modulated genes related to insulin signaling pathway (Negr1, Ppp3r1, Slc2a4 and Enpp1), a mechanism that has recently been linked to neuroprotection, learning and memory. Conclusions Our results strongly suggest that chronic VLX treatment improves functional reorganization and brain plasticity by influencing gene expression in regulatory networks of motor cortical areas. These results are consonant with the synaptic (network) hypothesis of depression and antidepressant-induced motor recovery after stroke.

Acute SSRI-induced anxiogenic and brain metabolic effects are attenuated 6 months after initial MDMA-induced depletion

To assess the functional state of the serotonergic system, the acute behavioural and brain metabolic effect of SSRI antidepressants were studied during the recovery period after MDMA-induced neuronal damage. The effects of the SSRI fluoxetine and the serotonin receptor agonist meta-chloro-phenylpiperazine (m-CPP) were investigated in the social interaction test in Dark Agouti rats, 6 months after treatment with a single dose of MDMA (15 or 30 mg kg(-1), i.p.). At earlier time points these doses of MDMA have been shown to cause 30-60% loss in axonal densities in several brain regions. Densities of the serotonergic axons were assessed using serotonin-transporter and tryptophan-hydroxylase immunohistochemistry. In a parallel group of animals, brain function was examined following an acute challenge with either fluoxetine or citalopram, using 2-deoxyglucose autoradiographic imaging. Six months after MDMA treatment the densities of serotonergic axons were decreased in only a few brain areas including hippocampus and thalamus. Basal anxiety was unaltered in MDMA-treated animals. However, the acute anxiogenic effects of fluoxetine, but not m-CPP, were attenuated in animals pretreated with MDMA. The metabolic response to both citalopram and fluoxetine was normal in most of the brain areas examined with the exception of ventromedial thalamus and hippocampal sub-fields where the response was attenuated. These data provide evidence that 6 months after MDMA-induced damage serotonergic axons show recovery in most brain areas, but serotonergic functions to challenges with SSRIs including anxiety and aggression remain altered.

Recovery and aging of serotonergic fibers after single and intermittent MDMA treatment in dark agouti rat

3,4‐Methylenedioxymethamphetamine (MDMA; ecstasy) is a popular party drug known to cause selective serotonergic damage. Here we examined the long‐term recovery and aging of serotonergic fibers and levels of brain‐derived neurotrophic factor (BDNF) after intermittent MDMA administration (15 mg kg−1 i.p. every 7th day for 4 weeks, MDMA ×4) and a single‐dose treatment (15 mg kg−1 i.p., MDMA ×1) in adolescent/young adult male Dark Agouti rats. After MDMA treatment, tryptophan hydroxylase‐immunoreactive fiber density decreased and then recovered in all brain regions. Recovery was more pronounced in the MDMA ×4 group compared with the MDMA ×1 group, but similar long‐term BDNF responses were found after both treatments. Twenty‐two months after treatment, there were fewer clusters of aberrant serotonergic fibers in the parietal cortex in the MDMA ×4 group compared with the MDMA ×1 group. There was no difference in the density of microglial cells or astrocytes in treated groups versus the control 22 months after the treatments. These results indicate that recovery of serotonergic fibers is faster after intermittent MDMA treatment than after single‐dose administration, and differences in BDNF levels per se are unlikely to account for this difference. Moreover, it seems that intermittent MDMA treatment attenuates the morphological signs of aging in serotonergic fibers. In addition, neither intermittent nor single‐dose MDMA exposition of young animals induces accelerated aging processes or neurodegeneration in senescence, as indicated by the unaltered densities of microglial cells and astrocytes in the treated groups compared with the control. J. Comp. Neurol. 519:2353–2378, 2011.