ABC Efflux Transporters and Solute Carriers in the Early Developing Brain of a Marsupial Monodelphis domestica (South American Gray Short-Tailed Opossum).

This study used a marsupial Monodelphis domestica, which is born very immature and most of its development is postnatal without placental protection. RNA-sequencing (RNA-Seq) was used to identify the expression of influx and efflux transporters (ATP-binding cassettes [ABCs] and solute carriers [SLCs]) and metabolizing enzymes in brains of newborn to juvenile Monodelphis. Results were compared to published data in the developing eutherian rat. To test the functionality of these transporters at similar ages, the entry of paracetamol (acetaminophen) into the brain and cerebrospinal fluid (CSF) was measured using liquid scintillation counting following a single administration of the drug along with its radiolabelled tracer [3H]. Drug permeability studies found that in Monodelphis, brain entry of paracetamol was already restricted at P5; it decreased further in the first week of life and then remained stable until the oldest age group tested (P110). Transcriptomic analysis of Monodelphis brain showed that expression of transporters and their metabolizing enzymes in early postnatal (P) pups (P0, P5, and P8) was relatively similar, but by P109, many more transcripts were identified. When transcriptomes of newborn Monodelphis brain and E19 rat brain and placenta were compared, several transporters present in the rat placenta were also found in the newborn Monodelphis brain. These were absent from E19 rat brain but were present in the adult rat brain. These data indicate that despite its extreme immaturity, the newborn Monodelphis brain may compensate for the lack of placental protection during early brain development by upregulating protective mechanisms, which in eutherian animals are instead present in the placenta.

Comparative analysis of novel decynium-22 analogs to inhibit transport by the low-affinity, high-capacity monoamine transporters, organic cation transporters 2 and 3, and plasma membrane monoamine transporter.

Growing evidence supports involvement of low-affinity/high-capacity organic cation transporters (OCTs) and plasma membrane monoamine transporter (PMAT) in regulating clearance of monoamines. Currently decynium-22 (D22) is the best pharmacological tool to study these transporters, however it does not readily discriminate among them, underscoring a need to develop compounds with greater selectivity for each of these transporters. We developed seven D22 analogs, and previously reported that some have lower affinity for α1-adrenoceptors than D22 and showed antidepressant-like activity in mice. Here, we extend these findings to determine the affinity of these analogs for OCT2, OCT3 and PMAT, as well as serotonin, norepinephrine and dopamine transporters (SERT, NET and DAT) using a combination of uptake competition with [3H]methyl-4-phenylpyridinium acetate in overexpressed HEK cells and [3H]citalopram, [3H]nisoxetine and [3H]WIN 35428 displacement binding in mouse hippocampal and striatal preparations. Like D22, all analogs showed greater binding affinities for OCT3 than OCT2 and PMAT. However, unlike D22, some analogs also showed modest affinity for SERT and DAT. Dual OCT3/SERT and/or OCT3/DAT actions of certain analogs may help explain their ability to produce antidepressant-like effects in mice and help account for our previous findings that D22 lacks antidepressant-like effects unless SERT function is either genetically or pharmacologically compromised. Though these analogs are not superior than D22 in discriminating among OCTs/PMAT, our findings point to development of compounds with combined ability to inhibit both low-affinity/high-capacity transporters, such as OCT3, and high-affinity/low-capacity transporters, such as SERT, as therapeutics with potentially improved efficacy for treatment of psychiatric disorders.

Evaluation of the antidepressant therapeutic potential of isocyanine and pseudoisocyanine analogues of the organic cation decynium-22.

Herein we describe the synthesis and evaluation of antidepressant properties of seven analogues (1-7) of the low affinity/high capacity transporter blocker decynium-22 (D-22). All analogues (1-7) were synthesized via base promoted coupling reactions between N-alkylated-2-methylquinolinium iodides or N-alkylated-4-methylquinolinium iodides and electrophilic N-alkylated-2-iodoquinolinium iodides. All final compounds were purified by re-crystallization or preparative HPLC and initial evaluation studies included; 1) screening for in vitro α1-adrenoceptor activity (a property that can lead to unwanted side-effects), 2) measuring antidepressant-like activity in a mouse tail suspension test (TST), and 3) measuring effects upon mouse locomotion. The results showed some analogues have lower affinities at α1-adrenoceptors compared to D-22 and showed antidepressant-like activity without the need for co-administration of SSRIs. Additionally, many analogues did not affect mouse locomotion to the same extent as D-22. Plans for additional evaluations of these promising analogues, including measurement of antidepressant-like activity with co-administration of selective serotonin re-uptake inhibitors (SSRIs), are outlined.

Bioaccumulation and Biodistribution of Selenium in Metamorphosing Tadpoles.

Selenium is an important macronutrient with a very narrow margin between essentiality and toxicity. Amphibians are hypothesized to be particularly sensitive due to the potential for metamorphosis-driven mobilization, which could transfer or concentrate contaminant burdens within specific organs. We explored the potential role of tissue degeneration and remodeling during anuran metamorphosis as a mechanism for altering tissue-specific Se burdens. Limnodynastes peronii tadpoles were exposed to dissolved 75Se (as selenite) for 7 days and depurated until completion of metamorphosis. Bioaccumulation and retention kinetics were assessed in whole tadpoles and excised tissues using gamma spectroscopy, and temporal changes in biodistribution were assessed using autoradiography. Tadpoles retained Se throughout metamorphosis, and partitioned the element predominantly within digestive and excretory tissues, including livers > mesonephros > guts > gallbladder. Importantly, our results demonstrate that Se biodistribution varies significantly throughout development. This is indicative of tissue transference, and particularly in tissues developing de novo after depuration. To the best of our knowledge, this is the first study demonstrating Se transference during metamorphic tissue remodelling. Further research is warranted to explore the fate and metabolism of Se (and other metal and metalloids) during anuran development and the implications of transference for influencing toxicity.

The Differential Binding of Antipsychotic Drugs to the ABC Transporter P-Glycoprotein Predicts Cannabinoid-Antipsychotic Drug Interactions.

Cannabis use increases rates of psychotic relapse and treatment failure in schizophrenia patients. Clinical studies suggest that cannabis use reduces the efficacy of antipsychotic drugs, but there has been no direct demonstration of this in a controlled study. The present study demonstrates that exposure to the principal phytocannabinoid, Δ9-tetrahydrocannabinol (THC), reverses the neurobehavioral effects of the antipsychotic drug risperidone in mice. THC exposure did not influence D2 and 5-HT2A receptor binding, the major targets of antipsychotic action, but it lowered the brain concentrations of risperidone and its active metabolite, 9-hydroxy risperidone. As risperidone and its active metabolite are excellent substrates of the ABC transporter P-glycoprotein (P-gp), we hypothesized that THC might increase P-gp expression at the blood-brain barrier (BBB) and thus enhance efflux of risperidone and its metabolite from brain tissue. We confirmed that the brain disposition of risperidone and 9-hydroxy risperidone is strongly influenced by P-gp, as P-gp knockout mice displayed greater brain concentrations of these drugs than wild-type mice. Furthermore, we demonstrated that THC exposure increased P-gp expression in various brain regions important to risperidone's antipsychotic action. We then showed that THC exposure did not influence the neurobehavioral effects of clozapine. Clozapine shares a very similar antipsychotic mode of action to risperidone, but unlike risperidone is not a P-gp substrate. Our results imply that clozapine or non-P-gp substrate antipsychotic drugs may be better first-line treatments for schizophrenia patients with a history of cannabis use.

Metal Transfer among Organs Following Short- and Long-Term Exposures Using Autoradiography: Cadmium Bioaccumulation by the Freshwater Prawn Macrobrachium australiense.

The uptake, depuration, and organ distribution of the radioisotope 109Cd were used to explore the internal kinetics of this nonessential metal following accumulation from waterborne cadmium by the freshwater decapod crustacean Macrobrachium australiense. Short- (6 h) and long-term (7 to 14 days) exposures to the radioisotope in solutions of 0.56 µg Cd/L were followed by depuration in metal- and isotope-free water for up to 21 days. The anatomical distribution of the radionuclide was visualized using autoradiography at predefined time points. The gills did not become saturated with cadmium after 14 days of exposure and demonstrated a greater rate of cadmium uptake relative to the hepatopancreas. Cadmium concentrations decreased rapidly during depuration from both gills and hepatopancreas after short exposures but slowly following long-term exposures. This suggests that the duration of cadmium exposure influences the depuration rate for this organism. The study demonstrates the complex behavior of cadmium accumulated by M. australiense and improves our understanding of how exposure duration will influence the internal location and potential toxicity of metals.

Bioaccumulation kinetics and organ distribution of cadmium and zinc in the freshwater decapod crustacean Macrobrachium australiense.

This study used the radioisotopes (109)Cd and (65)Zn to explore the uptake, retention and organ distribution of these nonessential and essential metals from solution by the freshwater decapod crustacean Macrobrachium australiense. Three treatments consisting of cadmium alone, zinc alone, and a mixture of cadmium and zinc were used to determine the differences in uptake and efflux rates of each metal individually and in the metal mixture over a three-week period, followed by depuration for 2 weeks in metal-free water using live-animal gamma-spectrometry. Following exposure, prawns were cryosectioned and the spatial distribution of radionuclides visualized using autoradiography. Metal uptake and efflux rates were the same in the individual and mixed-metal exposures, and efflux rates were close to zero. The majority of cadmium uptake was localized within the gills and hepatopancreas, while zinc accumulated in the antennal gland at concentrations orders of magnitude greater than in other organs. This suggested that M. australiense may process zinc much faster than cadmium by internally transporting the accumulated zinc to the antennal gland. The combination of uptake studies and autoradiography greatly increases our understanding of how metal transport kinetics and internal processing may influence the toxicity of essential and nonessential metals in the environment.

High levels of intravenous mephedrone (4-methylmethcathinone) self-administration in rats: neural consequences and comparison with methamphetamine.

Mephedrone (MMC) is a relatively new recreational drug that has rapidly increased in popularity in recent years. This study explored the characteristics of intravenous MMC self-administration in the rat, with methamphetamine (METH) used as a comparator drug. Male Sprague-Dawley rats were trained to nose poke for intravenous MMC or METH in daily 2 h sessions over a 10 d acquisition period. Dose-response functions were then established under fixed- and progressive-ratio (FR and PR) schedules over three subsequent weeks of testing. Brains were analyzed ex vivo for striatal serotonin (5-HT) and dopamine (DA) levels and metabolites, while autoradiography assessed changes in the regional density of 5-HT and serotonin transporter (SERT) and DA transporter (DAT) and induction of the inflammation marker translocator protein (TSPO). Results showed that MMC was readily and vigorously self-administered via the intravenous route. Under a FR1 schedule, peak responding for MMC was obtained at 0.1 mg/kg/infusion, versus 0.01 mg/kg/infusion for METH. Break points under a PR schedule peaked at 1 mg/kg/infusion MMC versus 0.3 mg/kg/infusion for METH. Final intakes of MMC were 31.3 mg/kg/d compared to 4 mg/kg/d for METH. Rats self-administering MMC, but not METH, gained weight at a slower rate than control rats. METH, but not MMC, self-administration elevated TSPO receptor density in the nucleus accumbens and hippocampus, while MMC, but not METH, self-administration decreased striatal 5-hydroxyindolacetic acid (5-HIAA) concentrations. In summary, MMC supported high levels of self-administration, matching or exceeding those previously reported with other drugs of abuse.

PET imaging of brain inflammation during early epileptogenesis in a rat model of temporal lobe epilepsy.

BACKGROUND: Recently, inflammatory cascades have been suggested as a target for epilepsy therapy. Positron emission tomography (PET) imaging offers the unique possibility to evaluate brain inflammation longitudinally in a non-invasive translational manner. This study investigated brain inflammation during early epileptogenesis in the post-kainic acid-induced status epilepticus (KASE) model with post-mortem histology and in vivo with [18F]-PBR111 PET. METHODS: Status epilepticus (SE) was induced (N = 13) by low-dose injections of KA, while controls (N = 9) received saline. Translocator protein (TSPO) expression and microglia activation were assessed with [125I]-CLINDE autoradiography and OX-42 immunohistochemistry, respectively, 7 days post-SE. In a subgroup of rats, [18F]-PBR111 PET imaging with metabolite-corrected input function was performed before post-mortem evaluation. [18F]-PBR111 volume of distribution (Vt) in volume of interests (VOIs) was quantified by means of kinetic modelling and a VOI/metabolite-corrected plasma activity ratio. RESULTS: Animals with substantial SE showed huge overexpression of TSPO in vitro in relevant brain regions such as the hippocampus and amygdala (P < 0.001), while animals with mild symptoms displayed a smaller increase in TSPO in amygdala only (P < 0.001). TSPO expression was associated with OX-42 signal but without obvious cell loss. Similar in vivo [18F]-PBR111 increases in Vt and the simplified ratio were found in key regions such as the hippocampus (P < 0.05) and amygdala (P < 0.01). CONCLUSION: Both post-mortem and in vivo methods substantiate that the brain regions important in seizure generation display significant brain inflammation during epileptogenesis in the KASE model. This work enables future longitudinal investigation of the role of brain inflammation during epileptogenesis and evaluation of anti-inflammatory treatments.

Mephedrone in adolescent rats: residual memory impairment and acute but not lasting 5-HT depletion.

Mephedrone (4-methylmethcathinone, MMC) is a popular recreational drug, yet its potential harms are yet to be fully established. The current study examined the impact of single or repeated MMC exposure on various neurochemical and behavioral measures in rats. In Experiment 1 male adolescent Wistar rats received single or repeated (once a day for 10 days) injections of MMC (30 mg/kg) or the comparator drug methamphetamine (METH, 2.5 mg/kg). Both MMC and METH caused robust hyperactivity in the 1 h following injection although this effect did not tend to sensitize with repeated treatment. Striatal dopamine (DA) levels were increased 1 h following either METH or MMC while striatal and hippocampal serotonin (5-HT) levels were decreased 1 h following MMC but not METH. MMC caused greater increases in 5-HT metabolism and greater reductions in DA metabolism in rats that had been previously exposed to MMC. Autoradiographic analysis showed no signs of neuroinflammation ([(125)I]CLINDE ligand used as a marker for translocator protein (TSPO) expression) with repeated exposure to either MMC or METH. In Experiment 2, rats received repeated MMC (7.5, 15 or 30 mg/kg once a day for 10 days) and were examined for residual behavioral effects following treatment. Repeated high (30 mg/kg) dose MMC produced impaired novel object recognition 5 weeks after drug treatment. However, no residual changes in 5-HT or DA tissue levels were observed at 7 weeks post-treatment. Overall these results show that MMC causes acute but not lasting changes in DA and 5-HT tissue concentrations. MMC can also cause long-term memory impairment. Future studies of cognitive function in MMC users are clearly warranted.

Aggregation in quads but not pairs of rats exposed to cat odor or bright light.

In many prey species aggregation of individuals is a defensive strategy commonly employed in response to predators and predator-related cues. However, very little work has explored this adaptive response in laboratory rats. It is known that individual rats show characteristic defensive responses to predator odors, such as hiding, avoidance, inhibition of foraging, feeding and reproduction, and risk assessment directed toward the odor source. However, whether these species-typical responses in individuals are altered in the presence of other conspecifics is yet to be characterized. The present study therefore examined the defensive response of groups of two rats (dyads) or four rats (quads) to two unconditioned stressors: bright ambient light and cat odor (a 2g ball of cat fur). The dyads and quads were formed from familiar cage mates and test sessions (20 min) occurred in a large open arena (1200 mm(2)) to which the rats had been extensively habituated under dark conditions. The results showed that when quads of rats were exposed to either cat odor or bright light in this arena, they showed characteristic increases in close social proximity, termed "huddling". A tight grouping of 3 (triplet) or 4 (quad) rats was commonly seen in response to cat fur, while triplets were more commonly seen in response to bright light. Interestingly there was no evidence for increased social proximity in dyads exposed to either stressor, only in quads. However, cat odor caused other signs of fear (such as decreased locomotor activity and increased defecation) in both quads and dyads. It is concluded that huddling is a rodent defensive strategy in rats when anxiogenic stimuli are encountered by larger groups of rats.

MDMA-induced c-Fos expression in oxytocin-containing neurons is blocked by pretreatment with the 5-HT-1A receptor antagonist WAY 100635.

The popular party drug MDMA (3,4-methylenedioxymethamphetamine, "Ecstasy") increases sociability in both humans and laboratory animals. Recent research suggests that these prosocial effects may involve serotonin (5-HT)-stimulated hypothalamic release of the neuropeptide oxytocin. WAY 100635, a 5-HT(1A) receptor antagonist, prevents MDMA-induced increases in plasma oxytocin and also reduces MDMA-mediated increases in social interaction in rats. The present study used c-Fos immunohistochemistry to determine the possible role of 5-HT(1A) receptors in MDMA-mediated activation of oxytocin synthesizing neurons. Male Wistar rats (n=8/group) were administered MDMA (10 mg/kg, i.p.) with or without WAY 100635 (1 mg/kg, i.p.) pre-treatment and c-Fos expression was then assessed throughout the brain. MDMA significantly increased locomotor activity and this effect was partly prevented by WAY 100635, in agreement with previous studies. WAY 100635 significantly reduced MDMA-induced c-Fos expression in a subset of brain regions examined. A particularly prominent reduction was seen in the oxytocin-positive neurons of the supraoptic nucleus and paraventricular hypothalamus, with more modest reductions in the Islands of Calleja, median preoptic nucleus, somatosensory cortex and nucleus of the solitary tract. WAY 100635 did not alter MDMA-induced c-Fos expression in the striatum, thalamus, or central amygdala. These results indicate that MDMA's action on oxytocin producing cells in the hypothalamus is mediated through 5-HT(1A) receptors and that certain specific cortical, limbic and brainstem sites are also activated by MDMA via these receptors.

Differential behavioural and neurochemical outcomes from chronic paroxetine treatment in adolescent and adult rats: a model of adverse antidepressant effects in human adolescents?

Selective serotonin reuptake inhibitor use is associated with increased risk of suicidal ideation in adolescent humans, yet the neuropharmacological basis of this phenomenon is unknown. Consequently, we examined the behavioural and neurochemical effects of chronic paroxetine (PRX) treatment in adult and adolescent rats. Rats received PRX in their drinking water (target dose 10 mg/kg) for 22 d, during which time they were assessed for depression- and anxiety-like behaviours. Subsequent ex-vivo analyses examined serum PRX concentrations, striatal neurotransmitter content, and regional serotonin and dopamine transporter (SERT, DAT) binding density. After 11-12 d treatment, PRX-treated adolescent rats showed a significant inhibition of social interaction while adults were unaffected. After 19-20 d treatment, adolescents failed to show an antidepressant-like effect of PRX treatment on the forced swim test (FST), while PRX-treated adults showed a typical decrease in immobility and increase in swimming. Two PRX-treated adolescents died unexpectedly after the FST suggesting a compromised response to physical stress. Despite their greater apparent adverse reaction to the drug, adolescents had significantly lower plasma PRX than adults at day 22 of treatment. Chronic PRX treatment had similar effects in adults and adolescents on striatal 5-HT (unchanged relative to controls) and 5-HIAA levels (decreased), while markers of dopaminergic function (DOPAC, HVA, DA turnover) were increased in adults only. SERT density was up-regulated in the amygdala in PRX-treated adolescents only while DAT density in the nucleus accumbens was down-regulated only in PRX-treated adults. These data suggest that the immature rat brain responds differently to PRX and that this might be of use in modelling the atypical response of human adolescents to antidepressants. The age-specific PRX-induced changes in dopaminergic markers and SERT and DAT binding provide clues as to the neural mechanisms underlying adverse PRX effects in adolescent humans.

Reduced sensitivity to MDMA-induced facilitation of social behaviour in MDMA pre-exposed rats.

The acute effects of the party drug 3,4-methylenedioxymethamphetamine (MDMA, "Ecstasy") in humans include feelings of love, closeness towards other people and an increased acceptance of others views and feelings. Some evidence suggests that regular MDMA users develop a subsensitivity to the positive effects of the drug and escalate their intake of the drug over time as a result. The current study investigated whether brief exposure to relatively high doses of MDMA in rats produces a subsequent attenuation in the ability of MDMA to enhance social interaction. Male Wistar rats were exposed to either MDMA (4 x 5 mg/kg over 4 h) or vehicle on two consecutive days. Twelve weeks later, MDMA pre-exposed rats displayed a significantly shorter period of time spent in social interaction than controls when tested in the drug-free state. MDMA pre-exposed rats also showed a blunted prosocial response to MDMA (2.5 mg/kg) relative to controls. This difference was overcome by increasing the MDMA dose to 5 mg/kg. The 5-HT(1A) agonist 8-OH-DPAT (250 microg/kg but not 125 microg/kg) increased social interaction and this effect did not differ in MDMA and vehicle pre-exposed rats. HPLC analysis showed a small but significant depletion of prefrontal 5-HT and 5-HIAA in MDMA pre-exposed rats. Prefrontal 5-HIAA concentrations were also reduced in the subset of vehicle and MDMA pre-exposed rats that received additional testing with MDMA. These results indicate that treatment with MDMA not only causes lasting reductions in social interaction in rats but causes an attenuation of the prosocial effects of subsequent MDMA administration. The lack of a differential response to 8-OH-DPAT agrees with other findings that the 5-HT(1A) receptor system remains functionally intact following MDMA pre-exposure and suggests that other neuroadaptations may underlie the lasting social deficits caused by MDMA.

High ambient temperature increases intravenous methamphetamine self-administration on fixed and progressive ratio schedules in rats.

Methamphetamine is a drug that is often consumed at dance parties or nightclubs where the ambient temperature is high. The present study determined whether such high ambient temperatures alter intravenous methamphetamine self-administration in the rat. Male Hooded Wistar rats were trained to self-administer intravenous methamphetamine (0.1 mg/kg/infusion) under a fixed ratio 1 (FR1) or progressive ratio (PR) schedule of reinforcement at an ambient temperature of 23 +/- 1 degrees C. They were then given their daily self-administration session at a raised ambient temperature of 30 +/- 1 degrees C. Methamphetamine self-administration was increased at 30 degrees C under both FR1 and PR reinforcement schedules, with the latter effect indicating that heat enhances the motivation to obtain methamphetamine. High temperatures did not alter self-administration of the D1 receptor agonist SKF 82958 in methamphetamine-experienced rats suggesting some specificity in the methamphetamine effect. When rats were given access to drink isotonic saline solution during methamphetamine self-administration sessions they drank much more solution at 30 degrees C than 23 degrees C. However, availability of isotonic saline to drink did not alter the heat-induced facilitation of methamphetamine self-administration (PR schedule) indicating that the heat effect does not simply reflect increased motivation for intravenous fluids. Hyperthermia was evident in rats self-administering methamphetamine at high ambient temperatures and fluid consumption did not prevent this effect. Heat did not affect blood levels of methamphetamine, or its principal metabolite amphetamine indicating that the facilitatory effect of heat did not reflect altered methamphetamine pharmacokinetics. Overall, these results show that high ambient temperatures increase the reinforcing efficacy of methamphetamine and encourage higher levels of drug intake.

Adolescent rats find repeated Delta(9)-THC less aversive than adult rats but display greater residual cognitive deficits and changes in hippocampal protein expression following exposure.

The current study examined whether adolescent rats are more vulnerable than adult rats to the lasting adverse effects of cannabinoid exposure on brain and behavior. Male Wistar rats were repeatedly exposed to Delta-9-tetrahydrocannabinol (Delta(9)-THC, 5 mg/kg i.p.) in a place-conditioning paradigm during either the adolescent (post-natal day 28+) or adult (post-natal day 60+) developmental stages. Adult rats avoided a Delta(9)-THC-paired environment after either four or eight pairings and this avoidance persisted for at least 16 days following the final Delta(9)-THC injection. In contrast, adolescent rats showed no significant place aversion. Adult Delta(9)-THC-treated rats produced more vocalizations than adolescent rats when handled during the intoxicated state, also suggesting greater drug-induced aversion. After a 10-15 day washout, both adult and adolescent Delta(9)-THC pretreated rats showed decreased social interaction, while only Delta(9)-THC pretreated adolescent rats showed significantly impaired object recognition memory. Seventeen days following their last Delta(9)-THC injection, rats were euthanased and hippocampal tissue processed using two-dimensional gel electrophoresis proteomics. There was no evidence of residual Delta(9)-THC being present in blood at this time. Proteomic analysis uncovered 27 proteins, many involved in regulating oxidative stress/mitochondrial functioning and cytoarchitecture, which were differentially expressed in adolescent Delta(9)-THC pretreated rats relative to adolescent controls. In adults, only 10 hippocampal proteins were differentially expressed in Delta(9)-THC compared to vehicle-pretreated controls. Overall these findings suggest that adolescent rats find repeated Delta(9)-THC exposure less aversive than adults, but that cannabinoid exposure causes greater lasting memory deficits and hippocampal alterations in adolescent than adult rats.

In vivo analysis of serotonin clearance in rat hippocampus reveals that repeated administration of p-methoxyamphetamine (PMA), but not 3,4-methylenedioxymethamphetamine (MDMA), leads to long-lasting deficits in serotonin transporter function.

p-Methoxyamphetamine (PMA) has been implicated in fatalities as a result of 'ecstasy' (MDMA) overdose worldwide. Like MDMA, acute effects are associated with marked changes in serotonergic neurotransmission, but the long-term effects of PMA are poorly understood. The aim of this study was to determine the effect of repeated PMA administration on in vitro measures of neurodegeneration: serotonin (5-HT) uptake, 5-HT transporter (SERT) density and 5-HT content in the hippocampus, and compare with effects on in vivo 5-HT clearance. Male rats received PMA, MDMA (4 or 15 mg/kg s.c., twice daily) or vehicle for 4 days and 2 weeks later indices of SERT function were measured. [(3)H]5-HT uptake into synaptosomes and [(3)H]cyanoimipramine binding to the SERT were significantly reduced by both PMA and MDMA treatments. 5-HT content was reduced in MDMA-, but not PMA-treatment. In contrast, clearance of locally applied 5-HT measured in vivo by chronoamperometry was only reduced in rats treated with 15 mg/kg PMA. The finding that 5-HT clearance in vivo was unaltered by MDMA treatment suggests that in vitro measures of 5-HT axonal degeneration do not necessarily predict potential compensatory mechanisms that maintain SERT function under basal conditions.

Repeated administration of the substituted amphetamine p-methoxyamphetamine produces reductions in cortical 5-HT transporter binding but not 5-HT content, unlike 3,4-methylenedioxyamethamphetamine.

Worldwide growth in p-methoxyamphetamine (PMA) usage amongst 'ecstasy' users indicates a proportionally greater incidence of acute toxicity compared to 3,4-methylenedioxymethamphetamine (MDMA). While longer-term use of MDMA appears to produce degeneration of 5-hydroxytryptamine (5-HT, serotonin) neurons, PMA effects are poorly understood. The aim of this study was to determine the effect of repeated PMA administration on two indices of 5-HT axonal degeneration, cortical brain 5-HT transporter (SERT) density and 5-HT/5-hydroxyindolacetic acid (5-HIAA) content. Treatment of male rats once daily for 4 days (10 or 20 mg/kg) with PMA or MDMA resulted in significant reductions (20 mg/kg: 53% and 23% of vehicle treatment respectively) in [(3)H]-paroxetine binding (SERT density) one week after final drug administration. When rats were housed at a higher ambient temperature (28 degrees C vs. 22 degrees C) for 6 h after dosing, no additive effect was seen for either drug. A more intensive dosing regimen (10 or 20 mg/kg twice daily for 4 days) was used to examine PMA/MDMA effects on cortical 5-HT content. Two weeks after MDMA treatment, significant reductions in cortical 5-HT content (20 mg/kg: 39% of vehicle treatment) were seen. However, PMA did not alter cortical 5-HT content, yet reduced cortical 5-HIAA content (20 mg/kg: 72% of vehicle treatment). These data suggest PMA has severe long-term implications clinically for alteration of 5-HT neurotransmission that may differ from MDMA, but may not necessarily be interpreted as a degeneration of 5-HT fibres.

Plasma drug concentrations and physiological measures in 'dance party' participants.

The increasing use of (+/-) 3,4-methylenedioxymethamphetamine (MDMA) in the setting of large dance parties ('raves') and clubs has been the source of some concern, because of potential acute adverse events, and because animal studies suggest that MDMA has the potential to damage brain serotonin (5-HT) neurons. However, it is not yet known whether MDMA, as used in the setting of dance parties, leads to plasma levels of MDMA that are associated with toxicity to 5-HT neurons in animals. The present study sought to address this question. Plasma MDMA concentrations, vital signs, and a variety of blood and urine measures were obtained prior to, and hours after, individuals attended a dance party. After the dance party, subjects were without clinical complaints, had measurable amounts of residual MDMA in plasma, and nearly half of the subjects also tested positive for methamphetamine, another amphetamine analog that has been shown to have 5-HT neurotoxic potential in animals. Plasma concentrations of MDMA did not correlate with self-reported use of 'ecstasy' and, in some subjects, overlapped with those that have been associated with 5-HT neurotoxicity in non-human primates. Additional subjects were likely to have had similar concentrations while at the dance party, when one considers the reported time of drug ingestion and the plasma half-life of MDMA in humans. Hematological and biochemical analyses were generally unremarkable. Moderate increases in blood pressure, heart rate and body temperature were observed in the subjects with the highest MDMA plasma concentrations. These findings are consistent with epidemiological findings that most people who use MDMA at dance parties do not develop serious clinical complications, and suggest that some of these individuals may be at risk for developing MDMA-induced toxicity to brain serotonin neurons.

Differences in the in vivo dynamics of neurotransmitter release and serotonin uptake after acute para-methoxyamphetamine and 3,4-methylenedioxymethamphetamine revealed by chronoamperometry.

Illicit use of p-methoxyamphetamine (PMA) is rapidly increasing. However, little is known about the acute effects of PMA on neurotransmission in vivo. High-speed chronoamperometry was used to monitor neurotransmitter release and clearance in anesthetized rats after local application of PMA or 3,4-methylenedioxymethamphetamine (MDMA). In striatum, PMA caused less neurotransmitter release than MDMA. PMA-evoked release could be partially blocked by pre-treatment with a serotonin (5-HT) reuptake inhibitor, suggesting that evoked 5-HT release contributed to the electrochemical signal and was mediated by the 5-HT transporter (SERT). MDMA-evoked release was not blocked by a SERT inhibitor, suggesting that primarily DA was released. To study the effect of these amphetamines on clearance of 5-HT mediated specifically by the SERT, clearance of exogenously applied 5-HT was measured in the CA3 region of the hippocampus. In contrast to the striatum where 5-HT is cleared by both the SERT and the dopamine transporter (DAT), 5-HT is cleared primarily by the SERT in the CA3 region. This is also a region where neither PMA nor MDMA evoked release of neurotransmitter. The maximal inhibition of 5-HT clearance was greater after PMA than MDMA. These data demonstrate in vivo (1) brain region variability in the ability of PMA and MDMA to evoke release of neurotransmitter; (2) that clearance of 5-HT in the striatum is mediated by both the SERT and the DAT; (3) distinct differences in the amount and nature of neurotransmitter released in the striatum after local application of PMA and MDMA and (4) that PMA is a more efficacious inhibitor of 5-HT clearance in the hippocampus than MDMA. These fundamental differences may account for the more severe adverse reactions seen clinically after PMA, compared to MDMA.