Amphetamine Exposure Research Articles

An N-Terminal Threonine Mutation Produces an Efflux-Favorable, Sodium-Primed Conformation of the Human Dopamine Transporter

The dopamine transporter (DAT) reversibly transports dopamine (DA) through a series of conformational transitions. Alanine (T62A) or aspartate (T62D) mutagenesis of Thr62 revealed T62D-human (h)DAT partitions in a predominately efflux-preferring conformation. Compared with wild-type (WT), T62D-hDAT exhibits reduced [(3)H]DA uptake and enhanced baseline DA efflux, whereas T62A-hDAT and WT-hDAT function in an influx-preferring conformation. We now interrogate the basis of the mutants' altered function with respect to membrane conductance and Na(+) sensitivity. The hDAT constructs were expressed in Xenopus oocytes to investigate if heightened membrane potential would explain the efflux characteristics of T62D-hDAT. In the absence of substrate, all constructs displayed identical resting membrane potentials. Substrate-induced inward currents were present in oocytes expressing WT- and T62A-hDAT but not T62D-hDAT, suggesting equal bidirectional ion flow through T62D-hDAT. Utilization of the fluorescent DAT substrate ASP(+) [4-(4-(dimethylamino)styryl)-N-methylpyridinium] revealed that T62D-hDAT accumulates substrate in human embryonic kidney (HEK)-293 cells when the substrate is not subject to efflux. Extracellular sodium (Na(+) e) replacement was used to evaluate sodium gradient requirements for DAT transport functions. The EC50 for Na(+) e stimulation of [(3)H]DA uptake was identical in all constructs expressed in HEK-293 cells. As expected, decreasing [Na(+)]e stimulated [(3)H]DA efflux in WT- and T62A-hDAT cells. Conversely, the elevated [(3)H]DA efflux in T62D-hDAT cells was independent of Na(+) e and commensurate with [(3)H]DA efflux attained in WT-hDAT cells, either by removal of Na(+) e or by application of amphetamine. We conclude that T62D-hDAT represents an efflux-willing, Na(+)-primed orientation-possibly representing an experimental model of the conformational impact of amphetamine exposure to hDAT.

Amphetamine exposure during development causes epigenetic trans‐generational changes in drug sensitivity in Caenorhabditis elegans (803.8)

It’s well known that psychostimulants like amphetamine (AMPH) increase the amount of dopamine in the synaptic cleft. In C. elegans, it has been shown that a dopamine increase in the synapses causes a unique behavior named swimming induced paralysis (SWIP). Previous studies demonstrated that SWIP is a valid tool to investigate the effect of addictive drugs. Here, we explored the response to AMPH in C. elegans adult animals following previous exposure to the drug during development, as well as the second generation to see if a change in response was passed to progeny. Our results show that adult animals (F0) exposed to AMPH during development had a higher AMPH‐induced SWIP with respect to control‐exposed animals. Further, we found an elevated SWIP response in the second generation (F1) of animals originated from maternal lines that were exposed to AMPH during development. Epigenetic mechanisms were investigated to explain the inherited change in response to AMPH. Histone3 Lys4 trimethylation (H3K4me3) and histone3 Lys27 (H3K27me3) are promoter‐specific histone modifications associated with active transcription and gene repression, respectively. To test if H3K4 or H3K27 methylation were a contributing factor in the increased SWIP response seen after AMPH exposure, we performed western blot assays against H3K4me3 and H3K27me3 in protein samples collected from F0 and F1 animals. Our data shows that AMPH exposure during development decreases H3K4me3 expression in the F1 generation, while no change is seen in H3K27. These results suggest that epigenetic mechanisms underlie the trans‐generational effects induced by AMPH. Other epigenetic markers are currently under investigation.Grant Funding Source: Supported by P20 GM104360‐1 NIH Grant

The mechanism of amphetamine‐mediated trafficking of the dopamine transporter (1145.1)

Acute amphetamine (AMPH) exposure elevates extracellular dopamine by a variety of mechanisms including increasing the rate of internalization of the plasma membrane dopamine transporter (DAT). We report here that AMPH also activates the small GTPases, Rho and Rac. In recombinant expression systems, primary cultures and midbrain slices, activation of Rho and the downstream Rho‐kinase (ROCK) triggers endocytosis of DAT. Inhibition of RhoA activity blocks AMPH‐induced DAT internalization, while Rac1 inhibition has no significant effect on DAT trafficking. ROCK inhibitors also prevent AMPH‐induced DAT internalization. Intriguingly, we found that AMPH must enter the cell to initiate this cascade. Further, intracellular AMPH also increases cAMP, leading to the phosphorylation of Rho by protein kinase A (PKA) that inactivates Rho and serves as a break on DAT internalization, thus demonstrating an important interaction between PKA‐ and RhoA‐dependent signaling in mediating the actions of AMPH. In agreement with our observations in cell lines and brain slices, we have also shown that the activation of receptors that couple to PKA in dopamine neurons within the brain can antagonize the behavioral effects of AMPH in mice. These observations imply the existence of a novel intracellular target that mediates the effects of AMPH on Rho and cAMP signaling and suggest new pathways to target in order to disrupt AMPH action.Grant Funding Source: Supported by the National Institute of Mental Health Intramural Program.

Label-Free Dopamine Imaging in Live Rat Brain Slices

Dopaminergic neurotransmission has been investigated extensively, yet direct optical probing of dopamine has not been possible in live cells. Here we image intracellular dopamine with sub-micrometer three-dimensional resolution by harnessing its intrinsic mid-ultraviolet (UV) autofluorescence. Two-photon excitation with visible light (540 nm) in conjunction with a non-epifluorescent detection scheme is used to circumvent the UV toxicity and the UV transmission problems. The method is established by imaging dopamine in a dopaminergic cell line and in control cells (glia), and is validated by mass spectrometry. We further show that individual dopamine vesicles/vesicular clusters can be imaged in cultured rat brain slices, thereby providing a direct visualization of the intracellular events preceding dopamine release induced by depolarization or amphetamine exposure. Our technique opens up a previously inaccessible mid-ultraviolet spectral regime (excitation ~270 nm, emission < 320 nm) for label-free imaging of native molecules in live tissue.

Amphetamine sensitisation and memory in healthy human volunteers: A functional magnetic resonance imaging study

Amphetamine sensitisation (AS) is an established animal model of the hypersensitivity to psychostimulants seen in patients with schizophrenia. AS also models the dysregulation of mesolimbic dopamine signalling which has been implicated in the development of psychotic symptoms. Recent data suggest that the enhanced excitability of mesolimbic dopamine neurons in AS is driven by a hyperactivity of hippocampal (subiculum) neurons, consistent with a strong association between hippocampal dysfunction and schizophrenia. While AS can be modelled in human volunteers, its functional consequences on dopaminoceptive brain regions (i.e. striatum and hippocampus) remains unclear. Here we describe the effects of a sensitising dosage pattern of dextroamphetamine on the neural correlates of motor sequence learning in healthy volunteers, within a randomised, double-blind, parallel-groups design. Behaviourally, sensitisation was characterised by enhanced subjective responses to amphetamine but did not change performance (i.e. learning rate) during an explicit sequence learning task. In contrast, functional magnetic resonance imaging (fMRI) measurements showed that repeated intermittent amphetamine exposure was associated with increased blood-oxygen-level dependent (BOLD) signal within the medial temporal lobe (MTL) (subiculum/entorhinal cortex) and midbrain, in the vicinity of the substantia nigra/ventral tegmental area (SN/VTA) during sequence encoding. Importantly, MTL hyperactivity correlated with the sensitisation of amphetamine-induced attentiveness. The MTL-midbrain hyperactivity reported here mirrors observations in sensitised rodents and is consistent with contemporary models of schizophrenia and behavioural sensitisation. These findings of meso-hippocampal hyperactivity during AS thus link pathophysiological concepts of dopamine dysregulation to cognitive models of psychosis.

Food consumption and weight gain after cessation of chronic amphetamine administration

Cessation of drug use often coincides with increased food consumption and weight gain in recovering addicts. However, it is not known whether this phenomenon (particularly the weight gain) is uniquely human, or whether it represents a consequence of drug cessation common across species. To address this issue, rats (n = 10/group) were given systemic injections of D-amphetamine (3 mg/kg) or an equal volume of saline vehicle for 9 consecutive days. Beginning 2 days after the final injection, rats were given free access to a highly palatable food mixture (consisting of sugar and butter) along with their standard chow diet, and food consumption and body weight were measured every 48 h for 30 days. Consistent with clinical observations, amphetamine-treated rats showed a greater increase in body weight over the course of the 30 days relative to vehicle-treated rats. Surprisingly, there was no difference in highly palatable food consumption between amphetamine- and vehicle-treated groups, but the amphetamine-treated group consumed significantly more standard chow than the control group. The finding that a history of chronic amphetamine exposure increases food consumption is consistent with previous work in humans showing that withdrawal from drugs of abuse is associated with overeating and weight gain. The current findings may reflect amphetamine-induced sensitization of mechanisms involved in reward motivation, suggesting that weight gain following drug cessation in humans could be due to similar mechanisms.

Systemic Administration of Fluoro-Gold for the Histological Assessment of Vascular Structure, Integrity and Damage

Fluoro-Gold (F-G) has been used extensively as a fluorescent retrograde neuronal-track tracer in the past. We now report that intraperitoneal administration of 10 to 30 mg/ kg of F-G from 30 min to 7 days prior to sacrifice labels vascular endothelial cells of the brain, choroid plexus and meninges and can be used to assess vascular integrity and damage. F-G vascular labeling co-localized with rat endothelial cell antigen (RECA-1) in the membrane. F-G also intensely labeled the nuclei of the endothelial cells, and co-localized with propidium iodide staining of these nuclei. As well, the administration of F-G during neurotoxic insults produced by amphetamine, kainic acid or "penetrating" wound to the brain can detect where vascular leakage/ hemorrhage has occurred. Histological methods to detect F-G labeled brain vasculature were performed in the same manner as that used for fluorescent visualization of neuronal elements labeled with F-G after perfusion fixation and coronal sectioning (15 to 40 µm) of the brain. This in vivo F-G labeling of endothelial cells and their nuclei yields a clear picture of the integrity of the vasculature and can be used to detect changes in structure. Vascular leaks after "penetrating" wounds through the cortex and striatum, hyperthermic amphetamine exposure or excitotoxic kainate exposure were detected by F-G in the extracellular space and via parenchymal F-G subsequently labeling the terminals and neurons adjacent to the lesioned or damaged vasculature. Further studies are necessary to determine the extent of the leakage necessary to detect vasculature damage. Visualization of the F-G labeling of vasculature structure and leakage is compatible with standard fluorescent immuno-labeling methods used to detect the presence and distribution of a protein in histological sections. This method should be directly applicable to studying brain vascular damage that occurs in the progression of Alzheimer's disease, diabetes and for monitoring the brain vascular changes during development.

Effects of amphetamine exposure in adolescence or young adulthood on inhibitory control in adult male and female rats

Heightened impulsivity is a feature of some psychiatric disorders, including addiction, that also have sex-specific patterns of expression. The relationship between addiction and impulsivity may be driven by drug-induced changes in behavior caused by long term adaptations in signaling within the medial prefrontal cortex (mPFC). Here, we used a response inhibition task that is sensitive to changes in mPFC function to examine the effects of sex and exposure to amphetamine (AMPH) on impulsive action and vigilance. We also examined drug-induced alterations in glutamatergic and dopaminergic signaling through challenge injections with the NMDA receptor antagonist MK-801 (dizocilpine) and AMPH. Male and female Sprague Dawley rats were injected (i.p.) with saline or 3mg/kg AMPH every other day during adolescence (postnatal day (P) 27–45) or adulthood (P85–103). Starting on P125–135, rats were tested for their ability to lever press for a food reward during periods of signaled availability and withhold responding during a “premature response” phase. In experiment 1, rats received challenge injections (i.p.) of MK-801 and AMPH followed by tests of task performance and locomotor activity. In experiment 2, rats received intra-mPFC infusion of MK-801. We found that females had better inhibitory control and poorer vigilance than males and that AMPH exposure had both sex- and age-of-exposure dependent effects on impulsivity. Systemic drug challenges disrupted task performance, particularly in females, and increased impulsivity while intra-mPFC infusions had modest effects. AMPH exposure did not affect responses to drug challenges. Together, these results suggest that sex mediates both trait and drug-induced impulsivity.

Intracellular gene transcription factor protein‐guided MRI by DNA aptamers in vivo

The mechanisms by which transcription factor (TF) protein AP-1 modulates amphetamine's effects on gene transcription in living brains are unclear. We describe here the first part of our studies to investigate these mechanisms, specifically, our efforts to develop and validate aptamers containing the binding sequence of TF AP-1 (5ECdsAP1), in order to elucidate its mechanism of action in living brains. This AP-1-targeting aptamer, as well as a random sequence aptamer with no target (5ECdsRan) as a control, was partially phosphorothioate modified and tagged with superparamagnetic iron oxide nanoparticles (SPIONs), gold, or fluorescein isothiothianate contrast agent for imaging. Optical and transmission electron microscopy studies revealed that 5ECdsAP1 is taken up by endocytosis and is localized in the neuronal endoplasmic reticulum. The results of magnetic resonance imaging (MRI) with SPION-5ECdsAP1 revealed that neuronal AP-1 TF protein levels were elevated in neurons of live male C57black6 mice after amphetamine exposure; however, pretreatment with SCH23390, a dopaminergic receptor antagonist, suppressed this elevation. As studies in transgenic mice with neuronal dominant-negative A-FOS mutant protein, which has no binding affinity for the AP-1 sequence, showed a completely null MRI signal in the striatum, we can conclude that the MR signal reflects specific binding between the 5ECdsAP1 aptamer and endogenous AP-1 protein. Together, these data lend support to the application of 5ECdsAP1 aptamer for intracellular protein-guided imaging and modulation of gene transcription, which will thus allow investigation of the mechanisms of signal transduction in living brains.

Persistent gene expression changes in NAc, mPFC, and OFC associated with previous nicotine or amphetamine exposure

Highly addictive drugs like nicotine and amphetamine not only change an individual's behaviour in the short and long-term, they also induce persistent changes in neuronal excitability and morphology. Although research has started to examine the epigenetic changes that occur immediately after drug exposure, there has been little investigation into the persistent modifications to the epigenome that likely moderate the stable maintenance of the neurological changes. Male Long-Evans rats were administered amphetamine, nicotine, or saline for 14 consecutive days, given a 14 day withdrawal period, and then sacrificed. DNA from the mPFC, OFC, and nucleus accumbens (NAc) was used for global DNA methylation analysis and RNA from the same brain regions was used for gene expression analysis. Following the two-week withdrawal period, exposure to amphetamine or nicotine was associated with a decrease in global DNA methylation in each brain region examined. Previous exposure to nicotine was associated with changes in expression of 16 genes (NAc:6, mPFC:5, OFC:5) whereas exposure to amphetamine was associated with changes in expression of 25 genes (NAc:13, OFC:8, mPFC:4). The persistent epigenetic changes associated with exposure to amphetamine and nicotine were region and drug dependent, and differ from the latent epigenetic changes that occur immediately after drug exposure. The changes in DNA methylation are consistent with the gene expression results and provide further support to the notion that DNA methylation is the key regulatory mechanism for experience dependent changes.

Effects of withdrawal from repeated amphetamine exposure in peri-puberty on neuroplasticity-related genes in mice

Although extensive evidence demonstrates that repeated administration of amphetamine (AMPH) induces behavioral and neurochemical sensitization, the influence of the developmental timing of AMPH administration is unknown. This is an important issue to address because it could help clarify the influence of early drug exposure on neuronal plasticity and the involvement of dopaminergic sensitization in the etiopathology of neuropsychiatric disorders.Thus, we decided to investigate the molecular alterations induced by the administration of AMPH during adolescence, when repeated exposure to the psychostimulant may interfere with developmental neuroplasticity. We investigated the expression of the neurotrophin brain-derived neurotrophic factor (BDNF) and of two inducible-early genes (arc and cfos) that bridge neuronal activity with long-lasting functional alterations. We found that peri-pubertal treatment with AMPH induces long-lasting changes in the expression of bdnf and of activity-regulated genes in the hippocampus and in the prefrontal/frontal cortex, and leads to alterations of their short-term modulation in response to a subsequent acute AMPH challenge. These data suggest that AMPH exposure in peri-puberty may negatively affect the maturation of brain structures, such as the prefrontal cortex, which facilitate the development of dopamine sensitization and may contribute to dopamine-dependent behavioral dysfunctions and molecular alterations in adulthood.

Acetylcholine Encodes Long-Lasting Presynaptic Plasticity at Glutamatergic Synapses in the Dorsal Striatum after Repeated Amphetamine Exposure

Locomotion and cue-dependent behaviors are modified through corticostriatal signaling whereby short-term increases in dopamine availability can provoke persistent changes in glutamate release that contribute to neuropsychiatric disorders, including Parkinson's disease and drug dependence. We found that withdrawal of mice from repeated amphetamine treatment caused a chronic presynaptic depression (CPD) in glutamate release that was most pronounced in corticostriatal terminals with a low probability of release and lasted >50 d in treated mice. An amphetamine challenge reversed CPD via a dopamine D1-receptor-dependent paradoxical presynaptic potentiation (PPP) that increased corticostriatal activity in direct pathway medium spiny neurons. This PPP was correlated with locomotor responses after a drug challenge, suggesting that it may underlie the sensitization process. Experiments in brain slices and in vivo indicated that dopamine regulation of acetylcholine release from tonically active interneurons contributes to CPD, PPP, locomotor sensitization, and cognitive ability. Therefore, a chronic decrease in corticostriatal activity during withdrawal is regulated around a new physiological range by tonically active interneurons and returns to normal upon reexposure to amphetamine, suggesting that this paradoxical return of striatal activity to a more stable, normalized state may represent an additional source of drug motivation during abstinence.

Amphetamine makes caudate tissue more susceptible to oxygen and glucose deprivation

Amphetamine is being investigated to reduce morbidity following stroke. However, the medicinal use of amphetamine is complicated because this drug is addictive, cardiotoxic, and can be neurotoxic. Thus, further research into the safety of giving amphetamine to stroke patients is required. Here, we examine whether prior treatment with amphetamine has any effect on oxygen–glucose deprivation (OGD)-evoked transmitter efflux and mitochondrial function. To circumvent the well-documented cardiovascular effects of amphetamine we have used rat brain slices.Brain slices were exposed to 30μM of amphetamine for 10min 1h before being exposed to OGD. Using fast cyclic voltammetry in rat caudate, dopamine efflux induced by OGD was measured. The effect of amphetamine on mitochondrial function was examined using triphenyltetrazolium chloride (TTC) staining.Prior amphetamine exposure decreased the time to onset of OGD-evoked dopamine efflux (from 460 to 220s), suggesting that the caudate was more sensitive to OGD. This increased sensitivity to OGD was attenuated by pre-treatment with the dopamine transporter blocker GBR12909 (1μM). Pre-treatment with the dopamine D2 receptor antagonist metoclopramide (1μM) had no effect on the amphetamine-evoked sensitisation to OGD. Amphetamine decreased TTC staining in the caudate suggesting that amphetamine compromised the dopamine system by disrupting mitochondrial function.Amphetamine treatment may be harmful in stroke recovery by making the brain more vulnerable to ischaemia. These data also suggest that amphetamine abusers might be more susceptible to cerebral ischaemia.

Dynamic downregulation of Nogo receptor expression in the rat forebrain by amphetamine

Nogo receptors (NgRs) are a family of cell surface receptors that are broadly expressed in the mammalian brain. These receptors could serve as an inhibitory element in the regulation of activity-dependent axonal growth and spine and synaptic formation in the adult animal brain. Thus, through balancing the structural response to changing cellular and synaptic inputs, NgRs participate in constructing activity-dependent morphological plasticity. Psychostimulants have been well documented to induce morphological plasticity critical for addictive properties of stimulants, although underlying molecular mechanisms are poorly understood. In this study, we initiated a study to investigate the response of NgRs to a stimulant. We tested the effect of acute administration of amphetamine on protein expression of two principal NgR subtypes (NgR1 and NgR2) in the rat striatum, medial prefrontal cortex (mPFC) and hippocampus. We found that a single injection of amphetamine induced a rapid and time-dependent decrease in NgR1 and NgR2 expression in the striatum and mPFC. A relatively delayed and time-dependent decrease in expression of the two receptors was seen in the hippocampus. The drug-induced decrease in NgR1 and NgR2 expression in the three forebrain regions was dose-dependent. A behaviorally active dose of the drug was required to trigger a significant reduction in NgR1 and NgR2 expression. These data indicate that NgRs are subject to the regulation by the stimulant. Amphetamine exposure exerts the inhibitory modulation of basal NgR1 and NgR2 expression in the key structures of reward circuits in vivo.

Age of exposure-dependent effects of amphetamine on behavioral flexibility

Drug use typically begins during adolescence, which is a period of ongoing neurobiological development that may confer heightened vulnerability to develop drug dependence. Previously, our lab has shown that amphetamine (AMPH)-induced deficits in a medial prefrontal cortex (mPFC)-sensitive working memory task are greater in rats exposed to the drug during adolescence compared to adulthood. Here, we examine potential age-dependent effects of AMPH exposure on behavioral flexibility tasks that are sensitive to disruptions in mPFC and orbitofrontal cortex (OFC) function. Male Sprague-Dawley rats were injected (i.p.) with saline or 3mg/kg AMPH every other day between postnatal days (PNDs) 27–45 and PNDs 85–103. Starting around PND 125, rats were tested in an attentional set-shifting task and a subset of those was then tested in an operant strategy shifting task. Following completion of the operant task, rats were challenged with 3mg/kg AMPH and monitored in open field chambers. Our results demonstrate that AMPH-exposed rats were faster to acquire simple and compound discriminations, but were impaired during the first stimulus-reward reversal when compared to controls. In the operant strategy shifting task, adolescent-exposed rats shifted more rapidly between strategies and completed reversals faster than adult-exposed and control rats, respectively. The final AMPH challenge revealed evidence for sensitization in drug pre-exposed rats, with adult-exposed animals exhibiting the most significant effects. Together, these results suggest that AMPH induces long-lasting changes in behavioral flexibility that are at least partially dependent on age of exposure and may be due to adaptations in OFC function.

Individual differences in the conditioned and unconditioned rat 50-kHz ultrasonic vocalizations elicited by repeated amphetamine exposure

Adult rats often produce 50-kHz ultrasonic vocalizations (USVs), particularly the frequency-modulated varieties, in appetitive situations. These calls are thought by some to reflect positive affective states and the reinforcing value of drugs such as amphetamine and cocaine. The objective of this study was to determine whether the number of unconditioned 50-kHz USVs elicited by amphetamine predicts the development and/or magnitude of drug-conditioned motivation. In three experiments, we recorded USVs before and after injections of 1mg/kg amphetamine (i.v. or i.p.) administered once per session. Rats were categorized as "high callers" or "low callers" according to individual differences in the number of 50-kHz USVs elicited by their first amphetamine injection. We examined the conditioned appetitive behavior and conditioned place preference (CPP) that emerged in high and low callers after repeated pairings of amphetamine with specific contexts. We also examined whether amphetamine-induced calling was affected by treatment within an unfamiliar (test chamber) versus familiar (home cage) context. Within an unfamiliar environment, the high callers consistently produced more amphetamine-induced 50-kHz USVs than the low callers. Compared to the low callers, high callers showed significantly greater amphetamine CPP as well as enhanced conditioned 50-kHz USVs and locomotor activity during anticipation of amphetamine. Individual differences were stable when amphetamine was administered in test chambers, but when it was administered in home cages, low callers showed an increase in 50-kHz calling that matched the high callers. These findings suggest that individual differences in drug-induced USVs can reveal environment-sensitive traits involved in drug-related appetitive motivation.

Differential targeting of the dopamine transporter to recycling or degradative pathways during amphetamine‐ or PKC‐regulated endocytosis in dopamine neurons

The dopamine transporter (DAT) clears the extracellular dopamine released during neurotransmission and is a major target for both therapeutic and addictive psychostimulant amphetamines. Amphetamine exposure or activation of protein kinase C (PKC) by the phorbol ester PMA has been shown to down-regulate cell surface DAT. However, in dopamine neurons, the trafficking itinerary and fate of internalized DAT has not been elucidated. By monitoring surface-labeled DAT in transfected dopamine neurons from embryonic rat mesencephalic cultures, we find distinct sorting and fates of internalized DAT after amphetamine or PMA treatment. Although both drugs promote DAT internalization above constitutive endocytosis in dopamine neurons, PMA induces ubiquitination of DAT and leads to accumulation of DAT on LAMP1-positive endosomes. In contrast, after amphetamine exposure DAT is sorted to recycling endosomes positive for Rab11 and the transferrin receptor. Furthermore, quantitative assessment of DAT recycling using an antibody-feeding assay reveals that significantly less DAT returns to the surface of dopamine neurons after internalization by PMA, compared with vehicle or amphetamine treatment. These results demonstrate that, in neurons, the DAT is sorted differentially to recycling and degradative pathways after psychostimulant exposure or PKC activation, which may allow for either the transient or sustained inhibition of DAT during dopamine neurotransmission.

Serum myoglobin, but not lipopolysaccharides, is predictive of AMPH-induced striatal neurotoxicity

Determinants of amphetamine (AMPH)-induced neurotoxicity are poorly understood. The role of lipopolysaccharides (LPS) and organ injury in AMPH-induced neurotoxicity was examined in adult male Sprague-Dawley rats that were give AMPH and became hyperthermic during the exposure. Environmentally-induced hyperthermia (EIH) in the rat was compared to AMPH to determine whether AMPH-induced increases in LPS and peripheral toxicities were solely attributable to hyperthermia. Muscle, liver, and kidney function were determined biochemically at 3h or 1 day after AMPH or EIH exposure and histopathology at 1 day after treatment. Circulating levels of LPS were monitored (via limulus amoebocyte coagulation assay) during AMPH or EIH exposure. Blood LPS levels were detected in 40–50% of the AMPH and EIH rats, but the presence of LPS in the serum had no effect on organ damage or striatal dopamine depletions (neurotoxicity). In both CR and NCTR rats, serum bound urea nitrogen and creatinine levels increased at 3h after EIH or AMPH (2- to 3-fold above control) but subsided by 1 day. Alanine transaminase was increased (indicating liver dysfunction) by both AMPH and EIH at 3h (2- to 10-fold above control) in CR rats, but the levels were not significantly different between the control and AMPH groups in NCTR animals. Mild liver necrosis was detected in 1 of 7 rats examined in the AMPH group and in 1 of 5 rats examined in the EIH group (only NCTR rats were examined). Serum myoglobin increased (indicating muscle damage) in both CR and NCTR rats at 3h and was more pronounced with AMPH (≈5-fold above control) than EIH. Our results indicate that: (1) “free” blood borne LPS often increases with EIH and AMPH but may not be necessary for striatal neurotoxicity and CNS immune responses; (2) liver or kidney dysfunction may result from muscle damage; however, it is not sufficient nor necessary to produce, but may exacerbate, neurotoxicity; (3) AMPH-induced serum myoglobin release is a potential biomarker and possibly a factor in AMPH-induced toxicity processes.

The atypical dopamine transport inhibitor, JHW 007, prevents amphetamine-induced sensitization and synaptic reorganization within the nucleus accumbens

Benztropine (BZT) analogs, a family of agents with high affinity for the dopamine transporter have been postulated as potential treatments in stimulant abuse due to their ability to attenuate a wide range of effects evoked by psychomotor stimulants such as cocaine and amphetamine (AMPH). Repeating administration of drugs, including stimulants, can result in behavioral sensitization, a progressive increase in their psychomotor activating effects. We examined in mice the sensitizing effects and the neuroplasticity changes elicited by chronic AMPH exposure, and the modulation of these effects by the BZT derivative and atypical dopamine uptake inhibitor, JHW007, a candidate medication for stimulant abuse. The results indicated that JHW007 did not produce sensitized locomotor activity when given alone but prevented the sensitized motor behavior induced by chronic AMPH administration. Morphological analysis of medium spiny neurons of the nucleus accumbens revealed that JHW 007 prevented the neuroadaptations induced by chronic AMPH exposure, including increments in dendritic arborization, lengthening of dendritic processes and increases in spine density. Furthermore, data revealed that AMPH produced an increase in the density of asymmetric, possibly glutamatergic synapses in the nucleus accumbens, an effect that was also blocked by JHW007 pretreatment. The present observations demonstrate that JHW007 is able to prevent not only AMPH-induced behavioral sensitization but also the long-term structural changes induced by chronic AMPH in the nucleus accumbens. Such findings support the development and evaluation of BZT derivatives as possible leads for treatment in stimulant addiction.

Withdrawal effect of chronic amphetamine exposure during adolescence on complex maze performance

National survey data suggest a steady increase in the diagnosis and treatment of mental disorders in children, particularly Attention Deficit/Hyperactivity Disorder (ADHD). As nearly all children diagnosed with ADHD are prescribed stimulant drugs, rationale exists to quantitatively characterize behavioral responses following withdrawal from chronic stimulant dosing. These rodent experiments involved chronic administration of 7.5 mg/kg, s.c. amphetamine to subjects throughout adolescence followed by cognitive tests to gauge learning and performance during the withdrawal stage 7 to 14 days past withdrawal. Tests used a complex Stone 14-unit multiple T-maze, which is a robust paradigm for demonstrating age-related differences in rodent models when behavioral cognitive endpoints are used. Results reveal that amphetamine-treated subjects committed fewer major and retracing errors with increased minor errors and a significantly lower mean completion time. These findings suggest that pharmacotherapy aimed at adolescent-phase treatment of ADHD does not provoke spatial memory deficits at times proximal to drug withdrawal and lends support to amphetamine use in the treatment of ADHD children.