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 ([125I]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. © 2012 PLoS On

3,4-Methylenedioxymethamphetamine (MDMA) neurotoxicity in rats: a reappraisal of past and present findings

RATIONALE: 3,4-Methylenedioxymethamphetamine (MDMA) is a widely abused illicit drug. In animals, high-dose administration of MDMA produces deficits in serotonin (5-HT) neurons (e.g., depletion of forebrain 5-HT) that have been interpreted as neurotoxicity. Whether such 5-HT deficits reflect neuronal damage is a matter of ongoing debate. OBJECTIVE: The present paper reviews four specific issues related to the hypothesis of MDMA neurotoxicity in rats: (1) the effects of MDMA on monoamine neurons, (2) the use of “interspecies scaling” to adjust MDMA doses across species, (3) the effects of MDMA on established markers of neuronal damage, and (4) functional impairments associated with MDMA-induced 5-HT depletions. RESULTS: MDMA is a substrate for monoamine transporters, and stimulated release of 5-HT, NE, and DA mediates effects of the drug. MDMA produces neurochemical, endocrine, and behavioral actions in rats and humans at equivalent doses (e.g., 1–2 mg/kg), suggesting that there is no reason to adjust doses between these species. Typical doses of MDMA causing long-term 5-HT depletions in rats (e.g., 10–20 mg/kg) do not reliably increase markers of neurotoxic damage such as cell death, silver staining, or reactive gliosis. MDMA-induced 5-HT depletions are accompanied by a number of functional consequences including reductions in evoked 5-HT release and changes in hormone secretion. Perhaps more importantly, administration of MDMA to rats induces persistent anxiety-like behaviors in the absence of measurable 5-HT deficits. CONCLUSIONS: MDMA-induced 5-HT depletions are not necessarily synonymous with neurotoxic damage. However, doses of MDMA which do not cause long-term 5-HT depletions can have protracted effects on behavior, suggesting even moderate doses of the drug may pose risks

Temporally distinct impairments in cognitive function following a sensitizing regimen of methamphetamine

Indiana University-Purdue University Indianapolis (IUPUI)Methamphetamine (MA) is a widely abused psychostimulant that has been shown to evoke an array of neurobiological abnormalities and cognitive deficits in humans and in rodent models (Marshall & O'Dell, 2012). Alterations in cognitive function after repeated drug use may lead to impaired decision-making, a lack of behavioral control, and ultimately the inability to abstain from drug use. Human studies have shown that alterations in neurobiology resulting from prolonged MA use may lead to a number of cognitive deficits, including impairments in executive function, learning, memory, and impulsivity. These impairments, specifically those that engage the prefrontal cortex (PFC) or hippocampus (HC), may persist or recover based on the duration of abstinence. In rodents, repeated intermittent injections of MA yield protracted changes in neurobiology and behavior, which have been shown to effectively model a number of the biological and cognitive abnormalities observed in addiction. In order to assess the temporal evolution of impaired cognitive function throughout abstinence, sensitization was first induced in rats (7 x 5.0 mg/kg MA over 14 days). MA-treated rats initially exhibited a robust increase in locomotion that transitioned to stereotypy as the induction phase progressed. Then, the effects of MA sensitization on social interaction (SI), temporal order recognition (TOR) and novel object recognition (NOR) was assessed at one-day and 30-days post induction. No differences were observed in SI in either group or after a single injection of MA. However, an acute injection of 5.0 mg/kg of MA 30-minutes prior to testing dramatically reduced SI time. Impairments in TOR and NOR were observed in MA-treated rats after one day of abstinence, and impairments in TOR, but not NOR, were observed on day 30 of abstinence. No differences in TOR and NOR after a single injection of MA or saline were observed. These data establish that after 30 days of abstinence from a sensitizing regimen of MA, the ability to recall the temporal sequence that two stimuli were encountered was impaired and that was not attributable to impaired novelty detection. These data also suggest that at least some of the neurocognitive abnormalities caused by chronic MA administration may normalize after prolonged abstinence, since the ability to detect novelty recovered after 30 days of abstinence. These data provide compelling support that, since MA-sensitization caused temporal deficits in memory, PFC and HC function may be differentially impaired throughout the time course of abstinence

Memory impairment and alterations in prefrontal cortex gamma band activity following methamphetamine sensitization

RATIONALE: Repeated methamphetamine (MA) use leads to increases in the incentive motivational properties of the drug as well as cognitive impairments. These behavioral alterations persist for some time following abstinence, and neuroadaptations in the structure and function of the prefrontal cortex (PFC) are particularly important for their expression. However, there is a weak understanding of the changes in neural firing and oscillatory activity in the PFC evoked by repeated drug use, thus complicating the development of novel treatment strategies for addiction. OBJECTIVES: The purpose of the current study was to assess changes in cognitive and brain function following MA sensitization. METHODS: Sensitization was induced in rats, then temporal and recognition memory were assessed after 1 or 30 days of abstinence. Electrophysiological recordings from the medial PFC were also acquired from rats whereupon simultaneous measures of oscillatory and spiking activity were examined. RESULTS: Impaired temporal memory was observed after 1 and 30 days of abstinence. However, recognition memory was only impaired after 1 day of abstinence. An injection of MA profoundly decreased neuronal firing rate and the anesthesia-induced slow oscillation (SO) in both sensitized (SENS) and control (CTRL) rats. Strong correlations were observed between the SO and gamma band power, which was altered in SENS animals. A decrease in the number of neurons phase-locked to the gamma oscillation was also observed in SENS animals. CONCLUSIONS: The changes observed in PFC function may play an integral role in the expression of the altered behavioral phenotype evoked by MA sensitization

Role of Perirhinal to Prefrontal Cortex Reciprocal Connection in Methamphetamine-Induced Memory Deficits

Chronic use of methamphetamine (meth) disrupts cortical processing across multiple cognitive domains including impulsivity, decision making and memory. Our laboratory has consistently shown that extended access to contingent meth self-administration reliably produces memory deficits in novel object recognition (NOR) tasks designed to test the “what” component of episodic memory in a rodent model. This type of memory is dependent on an intact function of the perirhinal cortex (PRH). However, the ongoing role of the prefrontal cortex (PFC) in this task and the directionality of communication between the PRH and the PFC is not entirely understood. A set of four experiments were designed to characterize the bi-directional connection between the PFC and the PRH during the exploration of novel vs. familiar objects and whether the manipulation of the circuit will restore recognition memory following chronic meth self-administration (SA). Male and female rats were infused with retrograde GFP-tagged adeno-associated virus (AAV) in the PRH (Experiment 1) and the PFC (Experiment 2). Three weeks later rats were tested for NOR with half the animals exploring two familiar objects and half exploring a novel object. Brain tissue was processed for co-labeled cells containing both GFP and c-Fos, an indicator of neuronal activation. Rats spent more time exploring novel vs. familiar objects. During novel object exploration, animals that explored novel objects had more co-labeled cells that project from the PRH to the PFC, but not in cells that project from PFC to the PRH. A dual viral approach was utilized in the second set of experiments to activate the PRH to PFC pathway following meth SA (Experiment 3) or inhibit the PRH to PFC pathway in meth naïve animals (Experiment 4). AAV viral vectors containing CRE-dependent Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) were infused into the PRH while retrograde AAV encoding CRE recombinase was infused into the PFC. A recognition memory deficit was established in meth SA rats and restored through activation of excitatory DREADDS (Experiment 3). In meth naïve animals a NOR memory deficit was also induced by activation of inhibitory DREADDS within the same circuit (Experiment 4). In conclusion, this data set suggests communication from the PRH to the PFC directs novel object recognition

Voluntary Oral Methamphetamine Reveals Susceptibilities to Spatial Memory Deficits, Decreased Dopamine Marker Expression and Increased Neuroinflammation in the Hippocampus of Male and Female Mice

Methamphetamine is an addictive illicit psychostimulant that produces lasting neurochemical and behavioral changes. The mechanisms underlying these deficits have been characterized in animal models using extremely high doses. Currently, better translational models are needed to understand the onset and progression of these deficits that more accurately reflect the gradual and voluntary dosing parameters as chosen by an abuser. To that end, a new model of methamphetamine administration, labeled Voluntary Oral Methamphetamine Administration (VOMA), offers a means to examine the progression of neurotoxicity, behavioral deficits, and the addiction process through a voluntary consumption framework. Female populations show consistent vulnerabilities to methamphetamine, including greater severity of abuse, and greater shifts in psychological health. Females also show increased neurochemical susceptibilities during abstinence from methamphetamine, exhibiting increased grey-matter loss compared to controls. The mechanisms underlying these female-specific susceptibilities to methamphetamine are unknown. The overall GOAL of these studies was to establish a voluntary methamphetamine administration model in mice that recapitulates the cognitive and neurochemical deficits shown with previous models to further our understanding of the long-term susceptibilities of at-risk populations, including adolescent and female subjects. Rodents can exhibit drug-preference and drug-seeking behaviors, and methamphetamine challenges can reproduce the behavioral and neurochemical deficits that human addicts show. However, it is unclear if mice would voluntarily consume methamphetamine, and what effects this administration design would produce in this mice. Thus, the studies presented in this dissertation characterized the utility of the VOMA model to produce 1. naturalistic methamphetamine consumption 2. behavioral deficits and 3. neurochemical changes. This was done in an effort to understand the 4. mechanisms underlying adolescent- and female-specific vulnerabilities to the drug. To achieve our GOAL, we carried out three (3) specific aims: Specific Aim 1: Determine the utility of Voluntary Oral Methamphetamine Administration (VOMA) to produce behavioral and neurochemical deficits as seen in previous models [Chapter 2] In modeling MA abuse with rodents, researchers have developed paradigms that inject neurotoxic or binge doses in mice that are 10 times higher than the lower limits we have characterized in our models 1 (reviewed in2). Previous work in this line of research has found that two (2) 30mg/kg MA doses can acutely increase spatial working-memory performance but also increase spatial working-memory errors in the long-term. These effects were correlated to decreased DA and synaptic plasticity maker expression in the hippocampus 1. These prior studies have contributed significantly to our understanding of the mechanisms underlying MA toxicity 2, but offered very little in understanding the voluntary nature of methamphetamine administration. In order to characterize the utility of VOMA in producing MA consumption that produced cognitive and neurochemical deficits, we combined a spatial cognition design with a drug administration design as follows: mice were randomly assigned to either a water/control group or MA for 28 consecutive days. To carry out voluntary oral MA administration (VOMA) in mice, MA was mixed into a palatable sweetened oatmeal flake that mice were drawn to and ingested orally. Mice were allowed to consume MA throughout a 3-hour administration period, within 15 min intervals. Following 28-days of VOMA, mice were sacrificed, and tissues were collected. We found that VOMA: (1) Decreased working-memory and reference-memory performance of male mice on the radial arm maze. These behavioral deficits were observed after the 28-day drug administration period over a 2-week abstinence period. (2) Increased neurotoxicity in the hippocampus, as observed through lower dopamine marker expression, increased neuroinflammation, and lower synaptic-plasticity marker expression. Overall, these results indicate that VOMA can produce the behavioral and the neurochemical deficits observed in previous animal models as well as in human methamphetamine addicts. Additionally, that the molecular deficits were observed in the hippocampus highlights the utility of the model to easily characterize correlative cognitive behaviors and the underlying molecular shifts. Specific Aim 2: Characterize the role of abstinence in perpetuating methamphetamine-induced deficits using VOMA [Chapter 3] MA abuse can produce long-term cognitive impairments in abstinent individuals3-5 but other studies have shown that long periods of abstinence can improve the recovery of decision making-skills and emotional symptoms6. This may help individuals to avert relapse and yet, other reports indicate that relapse can occur after abstinence7, as a result of exposure to specific environmental cues8. The neurochemical pathways affected by abstinence from MA are also unclear. An understanding of the progression of behavioral and neurochemical changes produced by MA abuse and by abstinence would provide pharmacological targets to aid in recovery from addiction. In order to characterize the role of abstinence in producing MA-induced behavioral and molecular deficits, we used an acute VOMA design to do the following: put adolescent mice through cognitive assessments directly after 14 days of MA exposure and after a prolonged abstinence period. Tissue was collected directly after VOMA and after a 4-week abstinence period. We found that VOMA: (1) Produced a transient sensitization to MA as seen through behavior analyses in the drug-context (2) Produced an acute spatial working-memory deficit on the RAM directly after VOMA We found that abstinence: (1) Did not produce long-term working-memory, reference learning, nor retention deficits on the RAM (2) Increased expression of neuroinflammatory markers in the hippocampus (3) Modulated expression of monoamine proteins in the hippocampus Overall, these results indicate that abstinence from VOMA acutely affects behavioral performance that is ameliorated by abstinence. Further, abstinence produces neurochemical changes not observed directly following VOMA. This demonstrates that even acute MA exposure is sufficient to produce molecular changes in the brain despite not manifesting in behavior. This highlights the susceptibility of the adolescent brain to acute MA exposure and provides new avenues for pharmacological studies aimed at preventing MA-induced deficits. Specific Aim 3: Characterize the sex-differences in behavior and neurochemistry following escalating dose of methamphetamine using VOMA [Chapter 4] Previous work has shown that females exhibit susceptibilities to psychostimulant-addiction 9-12. It has been reported that methamphetamine use in female populations can be attributed to factors including weight loss, desire for increased energy and work output, and decreased exhaustion 13-14. Data on drug-rehabilitation admissions show that young female participants are admitted at higher rates compared to males 15. One hypothesis for sex differences in psychostimulant addiction highlights estrogen’s facilitation of neuronal and behavioral development of drug addiction processes in the brain. 16-18. However, this idea remains untested. Additionally, previous work has shown that escalating doses of methamphetamine have intrinsic neuroprotective qualities that protect the brain and behavior from a chronic administration of the drug. The nature and source of this neuroprotection is unclear, but previous work has shown that this phenomenon occurs in binge injection models, 19-20, associated with decreases in methamphetamine binding to DAT and decreases in the resulting DA availability in the synapse. However, the underlying mechanisms that produce this change in DA sensitivity remain unknown. To test how female subjects respond differentially to VOMA, we modified our administration design to include a 10-day escalation period, wherein mice would receive gradually escalating doses of and access to methamphetamine prior to receiving the maximum access and dose. We hypothesized that that this administration design would produce the maximal voluntary consumption over a shorter (18-day) period and produce the maximal behavioral and molecular deficits that could be observed. We found that Escalation-VOMA: (1) Decreased working-memory of female mice on the radial arm maze shortly after the conclusion of VOMA. This deficit was not observed in male mice that were placed into VOMA. (2) Discrete shifts in neuroprotective signaling (D1, ERα and Akt/GSK3β pathway) in the hippocampus and nucleus accumbens of female mice after a 2-week abstinence period. Male mice that underwent VOMA did not exhibit any molecular changes. Overall, this study showed that through VOMA, and with the aid of an escalating-dose design, we are able to observe sex-differences in the behavioral and neurochemical changes that methamphetamine can produce. Interestingly, we also observed a previously reported neuroprotective effect of escalating doses in male subjects, but not in females. Our molecular investigations will provide future direction in basic research and clinical endeavors to target methamphetamine addiction. Conclusion: The novel VOMA design has provided a more naturalistic approach to investigating the behavioral and molecular deficits produce by methamphetamine and abstinence during adolescence. With this model, we have successfully characterized longer-term susceptibilities in mice than previous studies have shown. Furthermore, it has provided new insights into the sex-differences surrounding meth-abuse, including the role of neuroprotective pathway signaling as markers for susceptibilities/resilience to methamphetamine abus

Doctor of Philosophy

dissertationMethamphetamine (METH) causes partial dopamine (DA) loss in the caudate/putamen and has long-term detrimental effects on cognitive function. We have previously shown that the positive correlation between expression of the immediate-early gene Arc in dorsomedial (DM) striatum and learning on a motor response reversal task is lost in rats with METH-induced striatal DA loss, despite normal behavioral performance. This discrepancy suggests that METH-pretreated rats no longer use DM striatum in this task. When function of or Arc expression in DM striatum of saline (SAL)-pretreated rats is disrupted, reversal learning and retention of learning, respectively, are impaired. However, METH-pretreated rats are unaffected by either treatment, suggesting that METH-pretreated rats no longer use DM striatum to perform this task. In situ hybridization histochemical staining for Arc mRNA expression in various brain regions of rats revealed a correlation between Arc and response reversal learning in nucleus accumbens (NAc) shell of METH-pretreated rats that did not exist in SAL-pretreated rats. When Arc was knocked down in the NAc shell, memory consolidation on the reversal task in METH-pretreated rats was impaired, whereas it was unaffected in SAL-pretreated rats, suggesting that METH-pretreated rats are relying on the NAc shell instead of DM striatum to consolidate reversal memories. Since the above evidence strongly suggests that METH-induced damage to the striatum forces rats to rely on a different brain region to complete this reversal task, we attempted to restore striatal function in METH-pretreated rats by manipulating extracellular DA levels. METH-pretreated rats are selectively deficient in phasic DA signaling, which generates transient DA changes in response to rewards and their cues. We stimulated the brains of METH- and SAL-pretreated rats in a phasic-like manner and found that the reduced striatal preprotachykinin gene expression in METH-pretreated rats was restored to control levels. Furthermore, we found that L-DOPA, the biochemical precursor to DA, restored phasic DA signals in METH-pretreated rats back to the baseline levels in SAL-pretreated rats. These results suggest that METH-induced neurotoxicity results in altered circuitry used in the brain during a reversal learning task, but that restoration of phasic DA signaling may be able to rescue striatal function

Effects of methamphetamine on sexual behavior

Methamphetamine (Meth) is a highly addictive psychostimulant associated with enhanced sexual desire, arousal, and sexual pleasure. Moreover, Meth abuse is frequently linked with the practice of sexual risk behavior and increased prevalence of Human Immunodeficiency Virus (HIV). Currently, the neurobiological basis for this drug-sex nexus is unknown. Moreover, there is a lack of studies investigating the effects of Meth on sexual behavior and more importantly, compulsive sex-seeking behavior, under controlled experimental settings in animal models. First, using immuhistochemistry for mating- and Meth-induced neural activation it was demonstrated that Meth administration in male rats activates neurons in brain regions of the limbic system that are involved in the regulation of sexual behavior. Specifically, Meth and mating co-activated neurons in the nucleus accumbens (NAc) core and shell, basolateral amygdala (BLA), anterior cingulate (ACA) and orbitofrontal (OFC) cortices. Second, the effects of acute or chronic administration of Meth on different aspects of sexual behavior were tested including motivation and performance, compulsive behavior, and reward. Results showed that high doses of Meth inhibited sexual motivation and performance. Next, to investigate Meth effects on compulsive sexual behavior a paradigm was established in which visceral illness induced by lithium chloride (LiCl) was paired with sexual reward. A low Meth dose (1mg/kg; s.c.) that does not impair sexual function had long-term effects on compulsive sexual behavior. Specifically, two weeks following the last Meth administration, Meth-pretreated males displayed sex-seeking behavior despite having learned the adverse consequences of mating. This effect was dependent on Meth administration being concurrent with sexual experience. Finally, using a conditioned place preference (CPP) paradigm, it was shown that concurrent Meth and sex experience was required for enhanced CPP for mating with Meth and for Meth alone. In contrast, reward for mating alone was decreased. Together, these findings illustrate that Meth can activate the same neurons as sexual behavior and in turn may alter this natural reward behavior. Moreover, these data indicate that the association between drug use and mating may be required for expression of compulsive sex behavior reported by Meth users and is correlated with increased reward seeking for concurrent Meth exposure and mating