The preclinical pharmacology of mephedrone; not just MDMA by another name.

The substituted ß-keto amphetamine mephedrone (4-methylmethcathinone) was banned in the UK in April 2010 but continues to be used recreationally in the UK and elsewhere. Users have compared its psychoactive effects to those of 3,4-methylenedioxymethamphetamine (MDMA, 'ecstasy'). This review critically examines the preclinical data on mephedrone that have appeared over the last 2-3 years and, where relevant, compares the pharmacological effects of mephedrone in experimental animals with those obtained following MDMA administration. Both mephedrone and MDMA enhance locomotor activity and change rectal temperature in rodents. However, both of these responses are of short duration following mephedrone compared with MDMA probably because mephedrone has a short plasma half-life and rapid metabolism. Mephedrone appears to have no pharmacologically active metabolites, unlike MDMA. There is also little evidence that mephedrone induces a neurotoxic decrease in monoamine concentration in rat or mouse brain, again in contrast to MDMA. Mephedrone and MDMA both induce release of dopamine and 5-HT in the brain as shown by in vivo and in vitro studies. The effect on 5-HT release in vivo is more marked with mephedrone even though both drugs have similar affinity for the dopamine and 5-HT transporters in vitro. The profile of action of mephedrone on monoamine receptors and transporters suggests it could have a high abuse liability and several studies have found that mephedrone supports self-administration at a higher rate than MDMA. Overall, current data suggest that mephedrone not only differs from MDMA in its pharmacological profile, behavioural and neurotoxic effects, but also differs from other cathinones.

Cathinone increases body temperature, enhances locomotor activity, and induces striatal c-fos expression in the Siberian hamster.

Cathinone is a ß-keto alkaloid that is the major active constituent of khat, the leaf of the Catha edulis plant that is chewed recreationally in East Africa and the Middle East. Related compounds, such as methcathinone and mephedrone have been increasing in popularity as recreational drugs, resulting in the recent proposal to classify khat as a Class C drug in the UK. There is still limited knowledge of the pharmacological effects of cathinone. This study examined the acute effects of cathinone on core body temperature, locomotor and other behaviors, and neuronal activity in Siberian hamsters. Adult male hamsters, previously implanted with radio telemetry devices, were treated with cathinone (2 or 5mg/kg i.p.), the behavioral profile scored and core body temperature and locomotor activity recorded by radio telemetry. At the end of the study, hamsters received vehicle or cathinone (5mg/kg) and neuronal activation in the brain was determined using immunohistochemical evaluation of c-fos expression. Cathinone dose-dependently induced significant (p<0.0001) increases in both temperature and locomotor activity lasting 60-90min. Cathinone (2mg/kg) increased rearing (p<0.02), and 5mg/kg increased both rearing (p<0.001) and lateral head twitches (p<0.02). Both cathinone doses decreased the time spent at rest (p<0.001). The number of c-fos immunopositive cells were significantly increased in the striatum (p<0.0001) and suprachiasmatic nucleus (p<0.05) following cathinone, indicating increased neuronal activity. There was no effect of cathinone on food intake or body weight. It is concluded that systemic administration of cathinone induces significant behavioral changes and CNS activation in the hamster.

Differential effects of cathinone compounds and MDMA on body temperature in the rat, and pharmacological characterization of mephedrone-induced hypothermia.

BACKGROUND AND PURPOSE: Recreational users report that mephedrone has similar psychoactive effects to 3,4-methylenedioxymethamphetamine (MDMA). MDMA induces well-characterized changes in body temperature due to complex monoaminergic effects on central thermoregulation, peripheral blood flow and thermogenesis, but there are little preclinical data on the acute effects of mephedrone or other synthetic cathinones. EXPERIMENTAL APPROACH: The acute effects of cathinone, methcathinone and mephedrone on rectal and tail temperature were examined in individually housed rats, with MDMA included for comparison. Rats were killed 2 h post-injection and brain regions were collected for quantification of 5-HT, dopamine and major metabolites. Further studies examined the impact of selected α-adrenoceptor and dopamine receptor antagonists on mephedrone-induced changes in rectal temperature and plasma catecholamines. KEY RESULTS: At normal room temperature, MDMA caused sustained decreases in rectal and tail temperature. Mephedrone caused a transient decrease in rectal temperature, which was enhanced by α(1) -adrenoceptor and dopamine D(1) receptor blockade, and a prolonged decrease in tail temperature. Cathinone and methcathinone caused sustained increases in rectal temperature. MDMA decreased 5-HT and/or 5-hydroxyindoleacetic acid (5-HIAA) content in several brain regions and reduced striatal homovanillic acid (HVA) levels, whereas cathinone and methcathinone increased striatal HVA and 5-HIAA. Cathinone elevated striatal and hypothalamic 5-HT. Mephedrone elevated plasma noradrenaline levels, an effect prevented by α-adrenoceptor and dopamine receptor antagonists. CONCLUSIONS AND IMPLICATIONS: MDMA and cathinones have different effects on thermoregulation, and their acute effects on brain monoamines also differ. These findings suggest that the adverse effects of cathinones in humans cannot be extrapolated from previous observations on MDMA.

Lost in translation: preclinical studies on 3,4-methylenedioxymethamphetamine provide information on mechanisms of action, but do not allow accurate prediction of adverse events in humans.

3,4-Methylenedioxymethamphetamine (MDMA) induces both acute adverse effects and long-term neurotoxic loss of brain 5-HT neurones in laboratory animals. However, when choosing doses, most preclinical studies have paid little attention to the pharmacokinetics of the drug in humans or animals. The recreational use of MDMA and current clinical investigations of the drug for therapeutic purposes demand better translational pharmacology to allow accurate risk assessment of its ability to induce adverse events. Recent pharmacokinetic studies on MDMA in animals and humans are reviewed and indicate that the risks following MDMA ingestion should be re-evaluated. Acute behavioural and body temperature changes result from rapid MDMA-induced monoamine release, whereas long-term neurotoxicity is primarily caused by metabolites of the drug. Therefore acute physiological changes in humans are fairly accurately mimicked in animals by appropriate dosing, although allometric dosing calculations have little value. Long-term changes require MDMA to be metabolized in a similar manner in experimental animals and humans. However, the rate of metabolism of MDMA and its major metabolites is slower in humans than rats or monkeys, potentially allowing endogenous neuroprotective mechanisms to function in a species specific manner. Furthermore acute hyperthermia in humans probably limits the chance of recreational users ingesting sufficient MDMA to produce neurotoxicity, unlike in the rat. MDMA also inhibits the major enzyme responsible for its metabolism in humans thereby also assisting in preventing neurotoxicity. These observations question whether MDMA alone produces long-term 5-HT neurotoxicity in human brain, although when taken in combination with other recreational drugs it may induce neurotoxicity.

Impact of regional 5-HT depletion on the cognitive enhancing effects of a typical 5-ht(6) receptor antagonist, Ro 04-6790, in the Novel Object Discrimination task.

RATIONALE: Selective 5-ht(6) receptor antagonists like Ro 04-6790 prolong memory in many rodent preclinical paradigms, possibly by blocking tonic 5-HT-evoked GABA release and allowing disinhibition of cortico-limbic glutamatergic and cholinergic neurones. If this is the case, behavioural responses to Ro 04-6790 should be abolished by depletion of endogenous 5-HT, and selective lesions of dorsal raphé (DR) or median raphé (MR) 5-HT pathways would allow the neuroanatomical substrates underlying the cognitive effects of 5-ht(6) receptor antagonists to be elucidated. OBJECTIVES: This study compared the effect of Ro 04-6790 on novel object discrimination (NOD) before and after sham or 5,7-dihydroxytryptamine (5,7-DHT)-induced lesions produced by injection into the lateral ventricles (LV), DR or MR. MATERIALS AND METHODS: NOD tests used a 4 h inter-trial interval (ITI) and Ro 04-6790 (10 mg kg(-1) i.p.) was administered 20 min before the familiarization trial. Brain region-specific 5-HT depletion was assessed by high performance liquid chromatography with electrochemical detection (HPLC-ED). RESULTS: Widespread LV or selective MR, but not DR lesions, abolished the ability of Ro 04-6790 to delay natural forgetting. Successful performance of all lesioned rats in subsequent 'drug-free' NOD tests using a 1 h ITI excluded the possibility of any confounding effects on visual acuity or motivation. CONCLUSIONS: The ability of Ro 04-6790 to prolong object recognition memory requires blockade of MR 5-HT function. Because DR lesions did not produce the expected depletion of striatal 5-HT an additional contribution of DR inputs to this region cannot be completely excluded.

5-HT6 receptor antagonists reverse delay-dependent deficits in novel object discrimination by enhancing consolidation--an effect sensitive to NMDA receptor antagonism.

5-HT(6) receptors are expressed in brain regions associated with learning and memory, and blockade of their function increases central cholinergic and glutamatergic neurotransmission and enhances cognitive processes. This study examined the effects of acute systemic administration of two selective 5-HT(6) receptor antagonists Ro 04-6790 and SB-271046 (10 mg kg(-1) i.p.) on acquisition, consolidation, and retrieval in the novel object discrimination (NOD) task, a two-trial test of recognition memory in which rats exposed to two identical objects during a familiarisation trial can discriminate a novel from a familiar object during the subsequent choice trial, following inter-trial delays of up to 3 h. 5-HT(6) receptor antagonist administration 20 min prior to or immediately after the familiarisation trial, but not 20 min prior to the choice trial reversed the deficit in object discrimination produced by a 4 h inter-trial interval. The nootropic effects of the 5-HT(6) receptor antagonists in this task thus appear to involve enhanced consolidation. Pre-treatment with the non-competitive NMDA receptor antagonist MK-801 (0.05 mg kg(-1) i.p.) prevented the effect of Ro 04-6790 on delay-induced deficits in object discrimination. This suggests that the 5-HT(6) receptor antagonist-induced enhancement of consolidation involves increased central glutamatergic neurotransmission.

Dimensional changes in cells and tissues during specimen preparation for the electron microscope.

Studies on dimensional changes incurred during preparation of tissue specimens for the transmission and scanning electron microscopes are reviewed, with emphasis on quantitative measurements pertinent to morphometry and three-dimensional reconstruction. The scope of the review includes fixation, dehydration, plastic embedment, critical-point drying, and freeze-drying. Recommendations are presented for monitoring dimensional changes; a strategy for the choice of method of specimen preparation is outlined.

[The cross desensitization and modulation of Cl currents activated by gamma-aminobutyric acid and L-glutamate in the isolated neurons of Aplysia]. / Perekrestnaia desensitizatsiia i moduliatsiia Cl-tokov, aktivirovannykh gamma-aminomaslianoi kislotoi i L-glutamatom, u odinochnykh neironov aplizii.

Chlorine conductance gated by gamma-aminobutyric acid (GABA) and L-glutamate in the medial pleural neurons of aplysia was studied using voltage clamp technique and a continuous microperfusion system that allowed rapid agonist application. Both GABA and glutamate elicited current responses that rapidly activated and then decayed. Glutamate response could be blocked by perfusion of aspartate or taurine and the GABA current showed voltage dependence. Thus the currents exhibited cross desensitization. It has been found that very low concentrations of acetylcholine (10(-8) to 10(-14) M) which have no electrophysiologic responses of their own, modulate the response to a constant application of GABA. During cooling the preparation blocked this effect, it is possible to suggest that the small doses of acetylcholine effect the membrane chemosensitivity through the cell biochemical mechanism.

High-voltage electron microscopy and three-dimensional graphic study of R and T cells in head and neck carcinomas.

Individual head and neck carcinomas show extreme regional cellular differentiation. Some cells are rich in keratin filaments (T cells) and some have little keratin and a high density of free ribosomes (R or RT cells). We attempted to isolate these two cell types in order to test their relative invasiveness in an in vitro model. The high frequency of mitosis of hyperkeratinized cells showed that there was no constraint on the motility of cell division. High-voltage electron microscopy of serial thick sections and three-dimensional graphic reconstruction demonstrated that keratin cytoskeleton filaments were cross-linked into short, thick bundles. However, the keratin cytoskeleton was absent from some portions of the cytoplasm. In normal differentiated keratinized cells, a more uniform spanning of the whole cell by thin keratin intermediate-filament bundles was evident. The cytoplasm may be more mobile in the keratinized tumor cells. Even heavily keratinized T cells, like the less keratinized cell types, may have invasive motility.

Asbestos-contaminated drinking water: its impact on household air.

Asbestos contamination in excess of 10 billion fibers per liter was detected in a community's drinking water. To assess the possibility of waterborne asbestos becoming airborne, air samples were collected from impacted houses receiving contaminated water from three control houses. Collected within each house were three samples on 0.6-micrometer-pore Nuclepore filters and three samples on 0.8-micrometer-pore Millipore filters. In addition, bulk samples of suspect material and water samples were collected. Mean waterborne asbestos concentrations were 24 million fibers per liter (MFL) in the impacted houses versus only 1.1 MFL in the control houses. Transmission electron microscopy revealed that airborne asbestos concentrations were highest in impacted houses, with airborne asbestos concentrations positively correlated with waterborne concentrations. For fiber and mass measurements on both filter types, airborne asbestos concentrations were significantly higher in the impacted houses: mean concentrations in impacted houses were 0.12 fibers/cm3 and 1.7 ng/m3 on Nuclepore filters and 0.053 fibers/cm3 and 2.3 ng/m3 on Millipore filters versus only 0.037 fibers/cm3 and 0.31 ng/m3 on Nuclepore filters and 0.0077 fibers/cm3 and 0.14 ng/m3 on Millipore filters from control houses. Also detected in the air samples from impacted houses were clusters of chrysotile, often with several hundreds of fibers. When estimates of these individual fibers were added to the total fiber count, the difference between the impacted and control houses became even greater. The increased concentrations in impacted houses were due primarily to short (less than 1 micrometer) fibers. Bulk samples did not reveal likely materials within the impacted houses to account for these differences. Thus high levels of waterborne asbestos were apparently the source of increased concentrations of airborne asbestos within these houses.

A procedure for making phosphor viewing screens for the transmission electron microscope.

A technique is described for preparing uniform, durable phosphor layers for viewing screens suitable for the transmission electron microscope; a settling procedure is used. The example described here is for a high-voltage instrument, but with adjustment of the coating density, the technique should be equally suitable for screen preparation for transmission electron microscopes that operate at lower acceleration voltages.

Ultrastructural characterization of isolated human head and neck squamous carcinoma cells. Assessment of isopycnic centrifugation.

In an experiment to evaluate the merit of isopycnic centrifugation as a method of separating cell types in human head and neck squamous cell carcinomas, cells have been isolated from four specimens of these tumors and subjected to isopycnic centrifugation in continuous Percoll gradients. Cell types were identified by electron microscopy. The R- (ribosome-rich), T- (tonofilament-rich), and RT- (intermediate) cell types yielded broad bands overlapping extensively with one another, and partially with the bands of leukocytes. The pattern differed for each tumor studied, so that universal density levels separating given cell types could not be found. Isopycnic centrifugation proves less suitable in analyzing cells dispersed from solid tumors than for cells in suspended culture, blood, effusions, etc., probably because of heterogeneous growth conditions of cells in solid tumors.

Detection and characterization of circulating rat mammary tumor cells in buffy coat and correlation with metastasis.

A new method for detecting bloodborne TMT-081 rat mammary tumor cells in buffy coat has revealed dose-dependent variations in the latency period after inoculation of tumor cells, the concentration of circulating tumor cells, and the incidence of metastases. Cells isolated from buffy coat of right ventricular blood were more tumorigenic than tryptically dispersed cells from solid tumors. With the new method circulating tumor cells can be detected at concentrations as low as 3 cells/microliter of buffy coat, or approximately 60 cells/ml of whole blood. The morphologic and ultrastructural features of the primary tumor were generally retained in both the circulating and tryptically dispersed cells, as shown by light and electron microscopy. A sparse distribution of intermediate filaments was revealed by high-voltage electron microscopy, although the filaments were not evident in conventional transmission electron micrographs. They were identified as keratin by immunofluorescence studies.

A quantitative sampling device for use at hazardous waste sites.

A device to draw air through up to 10 direct reading indicator tubes simultaneously has been developed. The qualitative results obtained can be used to obtain a rapid preliminary classification of the unknown chemical substances at spill scenes or at hazardous waste sites, and to devise protocols for laboratory sample analysis.

Protecting workers who clean up hazardous waste sites.

On April 21, 1980, an explosion and fire among the 40 000 drums of unlabeled chemical wastes at the former site of Chemical Control Corporation in Elizabeth, New Jersey, attracted national attention and raised serious concern about the health and safety of workers who would be called upon to clean up the disaster site. Evaluation of workers' exposure during clean up operation and an assessment of the level of respiratory protection needed were necessary. NIOSH conducted 8 days of air monitoring. The chemicals of concern were organic vapors, metals, pesticides, polychlorinated biphenyls, cyanide compounds, and dioxins (TCDD). No excessive exposure to chemical substances by inhalation was found. However, continuous use of respiratory protective equipment and other personal protective devices was necessary because of frequent drum ruptures.

Use of fast x-ray film for low-influence biological electron microscopy at 100 kV and 1000 kV.

A user evaluation has been made by electron microscopists of an X-ray film for routine electron microscopy. The recent improvements in mammographic X-ray films, with the attempt to reduce the patient dose required to produce a high-resolution mammogram, have resulted in some useful films for medium- and high-voltage electron microscopy. They can yield essential cytological information with a reduction of the electron fluence (exposure) applied to the specimen of more than an order of magnitude compared with conventional electron-microscope films. Their use is indicated in situations where beam damage is severe.

A perspicuous technique for directly visualizing radiation-damage artefacts in biological electron microscopy.

Levels of impairment of electron-microscopic images of biological specimens stemming from radiation damage are assessed in a rapid visual procedure that involves taking a pair of low-fluence micrographs of a specimen area before and after a fraction of the picture area has been more seriously damaged by applying a measured electron fluence. The pair of micrographs is treated as a mock-stereo pair and is given contrasting colours. Lateral displacements of specimen details appear as false relief and changes in electron lucency as false colour.