National Institute on Drug Abuse IRP

Based on animal data, there is speculation that (؎)-3,4-methylenedioxymethamphetamine (MDMA) is neurotoxic to humans. Extrapolation of MDMA findings from animals to humans requires assessment of pharmacokinetics in various species, and low-dose administration data from rats are lacking. In this study, we examine MDMA pharmacokinetics in rats given low (2 mg/kg) and high (10 mg/kg) doses of racemic MDMA via intraperitoneal, subcutaneous, and oral routes. Repeated blood specimens were collected from venous catheters, and plasma was assayed for MDMA and its metabolites, 4-hydroxy-3-methoxymethamphetamine (HMMA) and 3,4-methylenedioxyamphetamine (MDA), by gas chromatographymass spectrometry. After 2 mg/kg, maximum MDMA concentrations (C max ) were ϳ200 ng/ml for intraperitoneal and subcutaneous routes, but less for the oral route. MDMA plasma half-lives were <1 h for low-dose groups, whereas HMMA and MDA half-lives were >2 h. After 10 mg/kg, MDMA areas under the curve (AUCs) were 21-fold (intraperitoneal), 10-fold (subcutaneous), and 36-fold (oral) greater than those at 2 mg/kg. In contrast, HMMA AUC values in high-dose groups were <3-fold above those at 2 mg/kg. Several new findings emerge from this report of low-dose MDMA pharmacokinetics in rats. First, 2 mg/kg MDMA in rats can produce MDMA C max values similar to those in humans, perhaps explaining why both species discriminate 1.5 mg/kg MDMA in laboratory paradigms. Second, our data provide additional support for nonlinear kinetics of MDMA in rats, and, analogous to humans, this phenomenon appears to involve impaired drug metabolism. Finally, given key similarities between MDMA pharmacokinetics in rats and humans, data from rats may be clinically relevant when appropriate dosing conditions are used.

Previous studies suggest prenatal methamphetamine (MA) exposure inhibits fetal growth. We examined neonatal growth effects of prenatal MA exposure in a prospective cohort study. After adjusting for covariates, exposed neonates had a higher incidence of being small for gestational age (SGA) than unexposed neonates.

A fully validated, highly sensitive and specific method for the extraction and quantification of 9 -tetrahydrocannabinol (THC), 11-hydroxy-9 -tetrahydrocannabinol (11-OH-THC) and 11-nor-9-carboxy-9 -tetrahydrocannabinol (THCCOOH) in plasma is presented. This method incorporates Escherichia coli ␤-glucuronidase hydrolysis to cleave glucuronic acid moieties to capture total analyte concentrations, and simultaneous solid phase extraction (SPE) of the three analytes in a single eluant with separation and quantification on a bench-top positive chemical ionization (PCI) gas chromatography-mass spectrometry (GC-MS) in the selected ion monitoring (SIM) mode. Quantitation was achieved by the addition of deuterated analogues for each analyte as internal standards (IS). Limits of quantitation (LOQ) were 0.5, 0.5 and 1.0 for THC, 11-OH-THC and THCCOOH, respectively, with linearity ranging up to 50 ng/ml for THC and 11-OH-THC, and 100 ng/ml for THCCOOH. Absolute recoveries ranged from 67.3 to 83.5% for all three analytes. Intra-assay accuracy and precision ranged from 1.2 to 12.2 and 1.4 to 4.7%, respectively. Inter-assay accuracy and precision ranged from 1.4 to 12.2 and 3.1 to 7.3%, respectively. This method was used to analyze plasma samples collected from individuals participating in a controlled oral THC administration study. Statistically significant (P ≤ 0.05) increases of 40% for 11-OH-THC and 42% for THCCOOH concentrations were found between hydrolyzed and non-hydrolyzed results. This method will be utilized in ongoing controlled cannabinoid administration studies and may be a useful analytical procedure for the fields of forensic toxicology and cannabinoid pharmacology.

Addiction is characterized by a lack of insight into the likely outcomes of one&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s behavior. Insight, or the ability to imagine outcomes, is evident when outcomes have not been directly experienced. Using this concept, work in both rats and humans has recently identified neural correlates of insight in the medial and orbital prefrontal cortices. We found that these correlates were selectively abolished in rats by cocaine self-administration. Their abolition was associated with behavioral deficits and reduced synaptic efficacy in orbitofrontal cortex, the reversal of which by optogenetic activation restored normal behavior. These results provide a link between cocaine use and problems with insight. Deficits in these functions are likely to be particularly important for problems such as drug relapse, in which behavior fails to account for likely adverse outcomes. As such, our data provide a neural target for therapeutic approaches to address these defining long-term effects of drug use.

Memory deficits produced by marijuana arise partly via interaction of the psychoactive component, ⌬ 9 -tetrahydrocannabinol (⌬ 9 -THC), with cannabinoid receptors in the hippocampus. Although cannabinoids acutely reduce glutamate release and block hippocampal long-term potentiation (LTP), a potential substrate for learning and memory, the consequences of prolonged exposure to ⌬ 9 -THC for hippocampal function are poorly understood. Rats were injected with ⌬ 9 -THC (10 mg/kg, i.p., q.d.) for 1, 3, or 7 d, and electrophysiological recordings were performed in hippocampal slices 1d after the final injection. At this time, ⌬ 9 -THC was undetectable in hippocampus using liquid chromatography-mass spectrometry (LC-MS). Hippocampal LTP generated using high-frequency (HFS) or theta burst stimulation was not observed in brain slices from the 7-d ⌬ 9 -THC-treated animals. ⌬ 9 -THC also blocked HFS-LTP after 3 d, but not 1 d of treatment. The complete blockade of LTP persisted for 3 d after the last ⌬ 9 -THC injection, and full reversal of the LTP deficit was not observed up to 14 d following ⌬ 9 -THC withdrawal. The cannabinoid antagonist AM251 (2 mg/kg), administered before each ⌬ 9 -THC injection prevented the blockade of LTP, and 7-d treatment with AM251 alone significantly increased the level of LTP. Chronic ⌬ 9 -THC also produced tolerance to the inhibition of synaptic GABA, but not glutamate release by the agonist WIN55,212-2. These data define consequences of repeated ⌬ 9 -THC exposure for synaptic plasticity in the hippocampus that may help explain memory impairments in humans following chronic marijuana use.

Background. We initiated differentiation of human embryonic stem cells (hESCs) into dopamine neurons, obtained a purified population of neuronal precursor cells by cell sorting, and determined patterns of gene transcription. Methodology. Dopaminergic differentiation of hESCs was initiated by culturing hESCs with a feeder layer of PA6 cells. Differentiating cells were then sorted to obtain a pure population of PSA-NCAM-expressing neuronal precursors, which were then analyzed for gene expression using Massive Parallel Signature Sequencing (MPSS). Individual genes as well as regions of the genome which were activated were determined. Principal Findings. A number of genes known to be involved in the specification of dopaminergic neurons, including MSX1, CDKN1C, Pitx1 and Pitx2, as well as several novel genes not previously associated with dopaminergic differentiation, were expressed. Notably, we found that a specific region of the genome located on chromosome 11p15.5 was highly activated. This region contains several genes which have previously been associated with the function of dopaminergic neurons, including the gene for tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis, IGF2, and CDKN1C, which cooperates with Nurr1 in directing the differentiation of dopaminergic neurons. Other genes in this region not previously recognized as being involved in the functions of dopaminergic neurons were also activated, including H19, TSSC4, and HBG2. IGF2 and CDKN1C were also found to be highly expressed in mature human TH-positive dopamine neurons isolated from human brain samples by laser capture. Conclusions. The present data suggest that the H19-IGF2 imprinting region on chromosome 11p15.5 is involved in the process through which undifferentiated cells are specified to become neuronal precursors and/or dopaminergic neurons.

Background: Stromal-Derived Inducing Activity (SDIA) is one of the most efficient methods of generating dopaminergic (DA) neurons from embryonic stem cells (ESC). DA neuron induction can be achieved by co-culturing ESC with the mouse stromal cell lines PA6 or MS5. The molecular nature of this effect, which has been termed ''SDIA'' is so far unknown. Recently, we found that factors secreted by PA6 cells provided lineage-specific instructions to induce DA differentiation of human ESC (hESC).

Formation of lasting memories is believed to rely on structural alterations at the synaptic level. We had found that increased neuronal activity down-regulates Nogo receptor-1 (NgR1) in brain regions linked to memory formation and storage, and postulated this to be required for formation of lasting memories. We now show that mice with inducible overexpression of NgR1 in forebrain neurons have normal long-term potentiation and normal 24-h memory, but severely impaired month-long memory in both passive avoidance and swim maze tests. Blocking transgene expression normalizes these memory impairments. Nogo, Lingo-1, Troy, endogenous NgR1, and BDNF mRNA expression levels were not altered by transgene expression, suggesting that the impaired ability to form lasting memories is directly coupled to inability to down-regulate NgR1. Regulation of NgR1 may therefore serve as a key regulator of memory consolidation. Understanding the molecular underpinnings of synaptic rearrangements that carry lasting memories may facilitate development of treatments for memory dysfunction.

This study determined the consequences of dopamine denervation of the striatum on synaptic plasticity and prevention of these changes with gene therapy using an adeno-associated viral vector (AAV) expressing glial cell line-derived neurotrophic factor (GDNF). C57BL6/J mice were injected with the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP); long-term depression (LTD) or potentiation (LTP) were measured in vitro. Fast-scan cyclic voltammetry measured electrically released dopamine from a functionally relevant pool in these same striatal slices. After MPTP, dopamine release and uptake were greatly diminished, and LTP and LTD were blocked in the striatal slices. The loss of plasticity resulted directly from the loss of dopamine since its application rescued synaptic plasticity. Striatal GDNF expression via AAV, before MPTP, significantly protected against the loss of dopamine and prevented the blockade of corticostriatal LTP. These data demonstrate that dopamine plays a role in supporting several forms of striatal plasticity and that GDNF expression via AAV prevents the loss of dopamine and striatal plasticity caused by MPTP. We propose that impairment of striatal plasticity after dopamine denervation plays a role in the symptomology of Parkinson's disease and that AAV expression of neurotrophic factors represents a tenable approach to protecting against or slowing these neurobiological deficitsLupica, C. R. MPTP-induced deficits in striatal synaptic plasticity are prevented by glial cell line-derived neurotrophic factor expressed via an adeno-associated viral vector. FASEB J. 22, 000 -000 (2008)