Compulsive drug use is associated with imbalance of orbitofrontal- and prelimbic-striatal circuits in punishment-resistant individuals.

Substance use disorders (SUDs) impose severe negative impacts upon individuals, their families, and society. Clinical studies demonstrate that some chronic stimulant users are able to curtail their drug use when faced with adverse consequences while others continue to compulsively use drugs. The mechanisms underlying this dichotomy are poorly understood, which hampers the development of effective individualized treatments of a disorder that currently has no Food and Drug Administration-approved pharmacological treatments. In the present study, using a rat model of methamphetamine self-administration (SA) in the presence of concomitant foot shocks, thought to parallel compulsive drug taking by humans, we found that SA behavior correlated with alterations in the balance between an increased orbitofrontal cortex-dorsomedial striatal "go" circuit and a decreased prelimbic cortex-ventrolateral striatal "stop" circuit. Critically, this correlation was seen only in rats who continued to self-administer at a relatively high rate despite receiving foot shocks of increasing intensity. While the stop circuit functional connectivity became negative after repeated SA in all rats, "shock-resistant" rats showed strengthening of this negative connectivity after shock exposure. In contrast, "shock-sensitive" rats showed a return toward their baseline levels after shock exposure. These results may help guide novel noninvasive brain stimulation therapies aimed at restoring the physiological balance between stop and go circuits in SUDs.

Dopaminylation in Psychostimulant Use Disorder Protects Against Psychostimulant Seeking Behavior by Normalizing Nucleus Accumbens (NAc) Dopamine Expression.

Background: Repeated cocaine administration changes histone acetylation and methylation on Lys residues and Deoxyribonucleic acid (DNA) within the nucleus accumbens (NAc). Recently Nestler's group explored histone Arg (R) methylation in reward processing models. Damez-Werno et al. (2016) reported that during human investigations and animal self-administration experiments, the histone mark protein-R-methyltransferase-6 (PRMT6) and asymmetric dimethylation of R2 on histone H3 (H3R2me2a) decreased in the rodent and cocaine-dependent human NAc. Overexpression of PRMT6 in D2-MSNs in all NAc neurons increased cocaine seeking, whereas PRMT6 overexpression in D1-MSNs protects against cocaine-seeking. Hypothesis: The hypothesis is that dopaminylation (H3R2me2a binding) occurs in psychostimulant use disorder (PSU), and the binding inhibitor Srcin1, like the major DRD2 A2 allelic polymorphism, protects against psychostimulant seeking behavior by normalizing nucleus accumbens (NAc) dopamine expression. Discussion: Numerous publications confirmed the association between the DRD2 Taq A1 allele (30-40 lower D2 receptor numbers) and severe cocaine dependence. Lepack et al. (2020) found that acute cocaine increases dopamine in NAc synapses, and results in histone H3 glutamine 5 dopaminylation (H3Q5dop) and consequent inhibition of D2 expression. The inhibition increases with chronic cocaine use and accompanies cocaine withdrawal. They also found that the Src kinase signaling inhibitor 1 (Srcin1 or p140CAP) during cocaine withdrawal reduced H3R2me2a binding. Consequently, this inhibited dopaminylation induced a "homeostatic brake." Conclusion: The decrease in Src signaling in NAc D2-MSNs, (like the DRD2 Taq A2 allele, a well-known genetic mechanism protective against SUD) normalizes the NAc dopamine expression and decreases cocaine reward and motivation to self-administer cocaine. The Srcin1 may be an important therapeutic target.

Chemogenetic Inhibition of Dopamine D1-expressing Neurons in the Dorsal Striatum does not alter Methamphetamine Intake in either Male or Female Long Evans Rats.

The biochemical and molecular substrates of methamphetamine (METH) use disorder remain to be elucidated. In rodents, increased METH intake is associated with increased expression of dopamine D1 receptors (D1R) in the dorsal striatum. The present study assessed potential effects of inhibiting striatal D1R activity on METH self-administration (SA) by rats. We microinjected Cre-activated adeno-associated viruses to deliver the inhibitory DREADD construct, hM4D (Gi) - mCherry, into neurons that expressed Cre-recombinase (D1-expressing neurons) in the dorsal striatum of male and female transgenic Long Evans rats (Drd1a-iCre#3). Two weeks later, we trained rats to self-administer METH. Once this behavior was acquired, intraperitoneal injections of clozapine-N-Oxide (CNO) or its vehicle (sterile water) were given to rats before each METH SA session to determine the effect of DREADD-mediated inhibition on METH intake. After the end of the experiments, histology was performed to confirm DREADD delivery into the dorsal striatum. There were no significant effects of the inhibitory DREADD on METH SA by male or female rats. Post-mortem histological assessment revealed DREADD expression in the dorsal striatum. Our results suggest that inhibition of D1R in the dorsal striatum does not suppress METH SA. It remains to be determined if activating D1R-expressing neurons might have differential behavioral effects. Future studies will also assess if impacting D1R activity in other brain regions might influence METH SA.

Methamphetamine causes microglial activation in the brains of human abusers.

Methamphetamine is a popular addictive drug whose use is associated with multiple neuropsychiatric adverse events and toxic to the dopaminergic and serotonergic systems of the brain. Methamphetamine-induced neuropathology is associated with increased expression of microglial cells that are thought to participate in either pro-toxic or protective mechanisms in the brain. Although reactive microgliosis has been observed in animal models of methamphetamine neurotoxicity, no study has reported on the status of microglial activation in human methamphetamine abusers. The present study reports on 12 abstinent methamphetamine abusers and 12 age-, gender-, and education-matched control subjects who underwent positron emission tomography using a radiotracer for activated microglia, [(11)C](R)-(1-[2-chlorophenyl]-N-methyl-N-[1-methylpropyl]-3-isoquinoline carboxamide) ([(11)C](R)-PK11195). Compartment analysis was used to estimate quantitative levels of binding potentials of [(11)C](R)-PK11195 in brain regions with dopaminergic and/or serotonergic innervation. The mean levels of [(11)C](R)-PK11195 binding were higher in methamphetamine abusers than those in control subjects in all brain regions (>250% higher; p < 0.01 for all). In addition, the binding levels in the midbrain, striatum, thalamus, and orbitofrontal and insular cortices (p < 0.05) correlated inversely with the duration of methamphetamine abstinence. These results suggest that chronic self-administration of methamphetamine can cause reactive microgliosis in the brains of human methamphetamine abusers, a level of activation that appears to subside over longer periods of abstinence.

EEG of chronic marijuana users during abstinence: relationship to years of marijuana use, cerebral blood flow and thyroid function.

OBJECTIVE: Marijuana abuse is associated with neurological changes including increases in frontal EEG alpha during abstinence. Research is needed to assess to what extent these EEG patterns are indicative of cerebral perfusion deficits. METHODS: We recorded the resting eyes closed EEG of 75 abstinent marijuana users and 33 control subjects. Fifty-six marijuana users used marijuana for less than eight years and 19 used for eight years or more. The EEG evaluation occurred within 72h of admission to an inpatient unit. Fifty-nine marijuana users remained abstinent for a month and were tested twice. Supplemental psychological and physiological data were also collected. RESULTS: Log alpha2 and beta2 power at posterior sites were significantly lower for the marijuana abusers that used eight years or more than the other marijuana abusers and the control subjects. These EEG changes continued for the month of abstinence. The marijuana users who used marijuana for more than eight years, also, had lower heart rates and thyroid function (T4) compared to the other marijuana users and the control subjects. CONCLUSIONS: Chronic marijuana use was also associated with reduced EEG power in alpha and beta bands at posterior sites. These reductions in EEG power appear to be related to cerebral perfusion deficits and/or thyroid function in marijuana abusers. SIGNIFICANCE: Our results suggest EEG, cerebral blood flow velocity, cardiovascular and thyroid function alterations in marijuana abuser with an extended period of use. These alterations reflect under arousal in these systems.

Postmortem diagnosis and toxicological validation of illicit substance use.

The present study examines the diagnostic challenges of identifying ante-mortem illicit substance use in human postmortem cases. Substance use, assessed by clinical case history reviews, structured next-of-kin interviews, by general toxicology of blood, urine and/or brain, and by scalp hair testing, identified 33 cocaine, 29 cannabis, 10 phencyclidine and nine opioid cases. Case history identified 42% cocaine, 76% cannabis, 10% phencyclidine and 33% opioid cases. Next-of-kin interviews identified almost twice as many cocaine and cannabis cases as Medical Examiner (ME) case histories, and were crucial in establishing a detailed lifetime substance use history. Toxicology identified 91% cocaine, 68% cannabis, 80% phencyclidine and 100% opioid cases, with hair testing increasing detection for all drug classes. A cocaine or cannabis use history was corroborated by general toxicology with 50% and 32% sensitivity, respectively, and with 82% and 64% sensitivity by hair testing. Hair testing corroborated a positive general toxicology for cocaine and cannabis with 91% and 100% sensitivity, respectively. Case history corroborated hair toxicology with 38% sensitivity for cocaine and 79% sensitivity for cannabis, suggesting that both case history and general toxicology underestimated cocaine use. Identifying ante-mortem substance use in human postmortem cases are key considerations in case diagnosis and for characterization of disorder-specific changes in neurobiology. The sensitivity and specificity of substance use assessments increased when ME case history was supplemented with structured next-of-kin interviews to establish a detailed lifetime substance use history, while comprehensive toxicology, and hair testing in particular, increased detection of recent illicit substance use.

Cocaine and Caffeine Effects on the Conditioned Place Preference Test: Concomitant Changes on Early Genes within the Mouse Prefrontal Cortex and Nucleus Accumbens.

Caffeine is the world's most popular psychostimulant and is frequently used as an active adulterant in many illicit drugs including cocaine. Previous studies have shown that caffeine can potentiate the stimulant effects of cocaine and cocaine-induced drug seeking behavior. However, little is known about the effects of this drug combination on reward-related learning, a key process in the maintenance of addiction and vulnerability to relapse. The goal of the present study was thus to determine caffeine and cocaine combined effects on the Conditioned Place Preference (CPP) test and to determine potential differential mRNA expression in the Nucleus Accumbens (NAc) and medial prefrontal cortex (mPFC) of immediate-early genes (IEGs) as well as dopamine and adenosine receptor subunits. Mice were treated with caffeine (5 mg/kg, CAF), cocaine (10 mg/kg, COC), or their combination (caffeine 5 mg/kg + cocaine 10 mg/kg, CAF-COC) and trained in the CPP test or treated with repeated injections inside the home cage. NAc and mPFC tissues were dissected immediately after the CPP test, after a single conditioning session or following psychostimulant injection in the home cage for mRNA expression analysis. CAF-COC induced a marked change of preference to the drug conditioned side of the CPP and a significant increase in locomotion compared to COC. Gene expression analysis after CPP test revealed specific up-regulation in the CAF-COC group of Drd1a, cFos, and FosB in the NAc, and cFos, Egr1, and Npas4 in the mPFC. Importantly, none of these changes were observed when animals received same treatments in their home cage. With a single conditioning session, we found similar effects in both CAF and CAF-COC groups: increased Drd1a and decreased cFos in the NAc, and increased expression of Drd1a and Drd2, in the mPFC. Interestingly, we found that cFos and Npas4 gene expression were increased only in the mPFC of the CAF-COC. Our study provides evidence that caffeine acting as an adulterant could potentiate reward-associated memories elicited by cocaine. This is associated with specific changes in IEGs expression that were observed almost exclusively in mice that received the combination of both psychostimulants in the context of CPP memory encoding and retrieval. Our results highlight the potential relevance of caffeine in the maintenance of cocaine addiction which might be mediated by modifying neural plasticity mechanisms that strengthen learning of the association between drug and environment.

Methamphetamine causes alterations in the MAP kinase-related pathways in the brains of mice that display increased aggressiveness.

Aggressive behaviors have been reported in patients who suffer from some psychiatric disorders, and are common in methamphetamine (METH) abusers. Herein, we report that multiple (but not single) injections of METH significantly increased aggressiveness in male CD-1 mice. This increase in aggressiveness was not secondary to METH-induced hyperactivity. Analysis of protein expression using antibody microarrays and Western blotting revealed differential changes in MAP kinase-related pathways after multiple and single METH injections. There were statistically significant (p<0.05) decreases in MEK1, Erk2p, GSK3alpha, 14-3-3e, and MEK7 in the striata of mice after multiple injections of METH. MEK1 was significantly decreased also after a single injection of METH, but to a much lesser degree than after multiple injections of METH. In the frontal cortex, there was a statistically significant decrease in GSK3alpha after multiple (but not single) injections of METH. These findings suggest that alterations in MAP kinase-related pathways in the prefronto-striatal circuitries might be involved in the manifestation of aggressive behaviors in mice.

Effect of rate of administration on subjective and physiological effects of intravenous cocaine in humans.

The rate hypothesis of psychoactive drug action holds that the faster a drug reaches the brain and starts to act, the greater its reinforcing effects and abuse liability. A previous human study using a single cocaine dose confirmed the rate hypothesis for subjective responses, but found no rate effect on cardiovascular responses. We evaluated the rate hypothesis in 17 experienced cocaine users (7 [all men] provided complete data; 6 participated in only 1-2 sessions) by administering IV cocaine at each of three doses (10, 25, 50 mg) and injection durations (10, 30, 60 s) in a double-blind, placebo-controlled, escalating dose design. Heart rate, blood pressure, and positive (e.g., rush, high) and negative (e.g., feel bad, anxious) subjective effects (100-mm visual analogue scales) were measured for 1h after dosing. Peak change from baseline, time to peak, and area under the time-response curve were evaluated with repeated measures mixed linear regression analyses, allowing use of data from all sessions for all subjects, including non-completers. Both dose (mg) and infusion rate (mg/s) significantly influenced most subjective and cardiovascular variables. Analysis of the interaction suggested that dose had a stronger impact than rate. Rate had a stronger influence on positive subjective effects than on negative subjective effects or cardiovascular variables. These findings provide support for the rate hypothesis as it applies to both subjective and cardiovascular effects of IV cocaine administration in humans.

Protective effects of Delta(9)-tetrahydrocannabinol against N-methyl-d-aspartate-induced AF5 cell death.

The neuroprotective effects of Delta(9)-tetrahydrocannabinol (THC) were examined using an in vitro model in which the AF5 CNS cell line was exposed to toxic levels of N-methyl-d-aspartate (NMDA), an agonist of the NMDA glutamate receptor. NMDA toxicity was reduced by THC, but not by the more specific cannabinoid receptor agonist, WIN55,212-2. Addition of dibutyryl cAMP (dbcAMP) to the culture medium did not alter the neuroprotective effect of THC and did not unmask a neuroprotective effect of WIN55,212-2. The cannabinoid antagonist SR141716A did not inhibit the neuroprotection induced by THC or alter the response to WIN55,212-2, even in the presence of dbcAMP, indicating that the neuroprotective effect of THC was cannabinoid receptor-independent. On the other hand, both THC and WIN55,212-2 produced cellular toxicology at higher dosages, an effect which was blocked in part by SR141716A. Capsaicin, an antioxidant and vanilloid receptor agonist, also produced a protective effect against NMDA toxicology. The protective effect of capsaicin was blocked by co-application of ruthenium red, but was not blocked by the specific vanilloid receptor antagonist capsazepine, and the transient receptor potential vanilloid type 1 (TRPV1) and ANKTM1 transcripts were not detected in AF5 cells. Thus, the neuroprotective effects of THC and capsaicin did not appear to be mediated by TRP ion channel family receptors. The antioxidant alpha-tocopherol prevented neurotoxicity in a dose-dependent manner. Therefore, THC may function as an antioxidant to increase cell survival in NMDA-induced neurotoxicity in the AF5 cell model, while higher dosages produce toxicity mediated by CB1 receptor stimulation.

Neuropsychiatric alterations in MDMA users: preliminary findings.

The use of marijuana is rampant among 3,4-methylenedioxymethamphetamine (MDMA) users. The co-occurrence of abuse of these two drugs has made it difficult to assess the specific residual effects of MDMA alone. As a first step toward identifying the effects of long-term MDMA use, we studied 8 MDMA abusers, 8 marijuana/MDMA abusers, 15 marijuana abusers (matched in marijuana use without MDMA use), and 17 control subjects. EEG, cerebral blood velocity by pulsed transcranial Doppler (TCD), and psychological measures were collected. Three-minute resting eyes-closed EEG recordings were obtained from 16 electrodes. The EEG was converted to 6 frequency bands (delta, theta, alpha-1, alpha-2, beta-1, and beta-2) using a fast Fourier transformation. Blood flow velocity was determined using a temporal window for the right and left middle cerebral arteries using TCD. Absolute log delta power in the EEG of MDMA abusers at central electrode sites was significantly higher than that of the MDMA/marijuana, marijuana abusers, and control subjects. There were also increases in alpha-2 EEG power observed only in marijuana abusers. The blood flow measure, diastolic velocity, was increased in MDMA abusers whether they used marijuana or not. Because increases in delta power and perfusion deficits are associated with some chronic disorders, our findings in these ecstasy abusers suggest that MDMA use may be associated with a drug-induced neuropathological state. More research is necessary to test these ideas.

Stress hormone responses to corticotropin-releasing hormone in substance abusers without severe comorbid psychiatric disease.

BACKGROUND: Preclinical data indicate a crucial role of stress in the acute effects of drugs of abuse, maintenance of self-administration, and susceptibility to relapse. Stress system activation may serve as a marker for a neurochemical dysfunction with prognostic significance in patients with addiction. METHODS: We tested pituitary adrenocorticotrophin (ACTH) and adrenal cortisol response to ovine corticotropin-releasing hormone (oCRH) to assess the reactivity of the hypothalamic-pituitary-adrenal (HPA) axis in seven nonsubstance-abusing subjects, 31 polysubstance-abusing subjects without depressive symptoms, and seven subjects with substance abuse and depressive symptoms. No subject met diagnostic criteria for depression or other severe psychiatric disease. RESULTS: Compared with normal control subjects, substance abusers showed significantly lower ACTH and cortisol responses over the course of oCRH stimulation (p <.0001). Substance abusers with depressive symptoms showed similarly blunted responses. CONCLUSIONS: Polysubstance abusers with no past or current diagnosis of other Axis I disorders show blunted ACTH and cortisol responses to oCRH administration. The finding of an activated HPA axis in this population suggests an overlapping role of central CRH and HPA axis activation in affective disorders and substance abuse, which is likely to constitute an endocrine milieu necessary for the maintenance of addictive behavior. These data support the role of future therapeutic trials with nonpeptide CRH receptor 1 antagonists in these patients.

Methadone treatment induces attenuation of cerebrovascular deficits associated with the prolonged abuse of cocaine and heroin.

Opiate replacement therapy has been useful in reducing heroin use and in keeping patients in treatment programs. However, neuropsychological and neurophysiological effects of this treatment regimen have not been evaluated systematically. To determine whether methadone treatment reduces the magnitude of cerebral blood flow alternations in polysubstance (heroin and cocaine) abusers, we compared blood flow parameters in control subjects (n=26), polysubstance abusers (n=28) maintained on methadone for 24 weeks, and polysubstance abusers (n=22) who were not seeking treatment. Blood flow velocity was recorded from the anterior and middle cerebral arteries using transcranial Doppler sonography on an outpatient visit. The pulsatility index, a measure of cerebrovascular resistance, was significantly (p&<0.05) increased in both groups of polysubstance abusers compared to control subjects. Increased pulsatility in the two groups of substance abusers suggests constriction of the small cortical arteries. Nevertheless, the methadone-maintained polysubstance abusers had significantly lower pulsatility values than the nontreatment substance-abusing group. These findings suggest that maintenance on methadone might have significant beneficial neurovascular effects on this population of patients.

EEG deficits in chronic marijuana abusers during monitored abstinence: preliminary findings.

Cognitive, cerebrovascular, and psychiatric impairments have been documented with chronic marijuana users. To better understand the nature and duration of these neurocognitive changes in marijuana abusers, we recorded the resting EEG of 29 abstinent chronic marijuana abusers and 21 control subjects. The marijuana abusers were tested twice: the first evaluation occurred within 72 hours of admission to the inpatient research unit; the second evaluation occurred after 28 to 30 days of monitored abstinence. A three-minute period of EEG was recorded during resting eyes-closed conditions from eight electrodes (F(3), C(3), P(3), O(1), F(4), C(4), P(4), and O(2)). The artifacted EEG was converted to six frequency bands (delta, theta, alpha(1), alpha(2), beta(1), and beta(2)) using a fast Fourier transform. During early abstinence, absolute power was significantly lower (p < 0.05) for the marijuana abusers than for the control subjects for the theta and alpha(1) bands. These reductions in theta and alpha(1) power persisted for 28 days of monitored abstinence. These EEG changes, together with cerebral blood flow deficits, might underlie the cognitive alterations observed in marijuana abusers. Additional research is needed to determine how long these deficits persist during abstinence and if treatment with neuroprotective agents may reverse them.

Substituted amphetamines that produce long-term serotonin depletion in rat brain ("neurotoxicity") do not decrease serotonin transporter protein expression.

Administration of high-dose D-fenfluramine (D-FEN) or parachloroamphetamine (PCA) produces long-lasting decreases in serotonin transporter (SERT) binding and tissue levels of serotonin (5-HT) in rat forebrain. These changes have been viewed as evidence for 5-HT neurotoxicity, but few studies have measured SERT protein levels. Thus, in the present study we determined the effect of high-dose D-FEN or PCA, administered according to a "neurotoxic" dosing regimen, on the density of SERT sites using ligand binding methods and on SERT protein levels using Western blots. Rats were sacrificed 2 days and 2 weeks after administration of drug or saline. The density of SERT was determined in homogenates of caudate and whole brain minus caudate. d-FEN and PCA decreased SERT binding by 30 to 60% in both tissues and at both time points. Similarly, D-FEN and PCA administration profoundly decreased tissue 5-HT and 5-HIAA in frontal cortex. Despite the large decreases in SERT binding and depletion of tissue 5-HT that occurred with d-FEN administration, SERT protein expression, as determined by Western blot analysis, did not change in either tissue or time point. PCA administration decreased SERT protein by about 20% only at the 2-day point in the caudate. Drug treatments did not change expression of glial fibrillary acidic protein (GFAP), a hallmark indicator of neuronal damage, in whole brain minus caudate in the 2-week group. These results support the hypothesis that D-FEN- and PCA-induced decreases in tissue 5-HT and SERT binding sites reflect neuroadaptive changes rather than neurotoxic effects.

Lack of persistent changes in the dopaminergic system of rats withdrawn from methamphetamine self-administration.

A continuing challenge for studies in the neurobiology of drug abuse is to identify and characterize long-lived neuroadaptations that can trigger craving and relapse. We previously reported that rats that had actively self-administered methamphetamine for 5 weeks and were then withdrawn from methamphetamine for 24 h showed marked decreases in somatodendritic dopamine D(2) autoreceptor levels in the ventral tegmental area and median and dorsal part of the substantia nigra zona compacta with a corresponding down-regulation of dopamine D(1) receptors in the shell of the nucleus accumbens. The purpose of the present study was to determine whether neuroadaptive changes in dopamine receptors or transporters in the brains of rats withdrawn for 24 h from chronic methamphetamine self-administration are persistent changes that can be demonstrated long after withdrawal. A "yoked" procedure was used in which rats were tested simultaneously in groups of three, with only one rat actively self-administering methamphetamine while the other two received yoked injections of either methamphetamine or saline. In vitro quantitative autoradiography was used to determine densities of dopamine uptake sites and dopamine D(1) and D(2) receptors in different brain regions following 7- and 30-day periods of withdrawal from chronic methamphetamine self-administration. No changes in dopamine transporter and dopamine receptor numbers were detected in any brain region examined in rats self-administering methamphetamine compared with littermates receiving yoked infusions of either methamphetamine or saline. Thus, neuroadaptive changes in densities of dopamine receptors or transporters in certain brain areas may contribute to the reinforcing effects of methamphetamine during the acquisition and maintenance phases of self-administration, but do not appear to contribute to the long-lasting neuroadaptive effects of chronic methamphetamine self-administration, which may trigger craving and relapse.

Incubation of methamphetamine and palatable food craving after punishment-induced abstinence.

In a rat model of drug craving and relapse, cue-induced drug seeking progressively increases after withdrawal from methamphetamine and other drugs, a phenomenon termed 'incubation of drug craving'. However, current experimental procedures used to study incubation of drug craving do not incorporate negative consequences of drug use, which is a common factor promoting abstinence in humans. Here, we studied whether incubation of methamphetamine craving is observed after suppression of drug seeking by adverse consequences (punishment). We trained rats to self-administer methamphetamine or palatable food for 9 h per day for 14 days; reward delivery was paired with a tone-light cue. Subsequently, for one group within each reward type, 50% of the lever-presses were punished by mild footshock for 9-10 days, whereas for the other group lever-presses were not punished. Shock intensity was gradually increased over time. Next, we assessed cue-induced reward seeking in 1-h extinction sessions on withdrawal days 2 and 21. Response-contingent punishment suppressed extended-access methamphetamine or food self-administration; surprisingly, food-trained rats showed greater resistance to punishment than methamphetamine-trained rats. During the relapse tests, both punished and unpunished methamphetamine- and food-trained rats showed significantly higher cue-induced reward seeking on withdrawal day 21 than on day 2. These results demonstrate that incubation of both methamphetamine and food craving occur after punishment-induced suppression of methamphetamine or palatable food self-administration. Our procedure can be used to investigate mechanisms of relapse to drug and palatable food seeking under conditions that more closely approximate the human condition.

Altered gene expression in pulmonary tissue of tryptophan hydroxylase-1 knockout mice: implications for pulmonary arterial hypertension.

The use of fenfluramines can increase the risk of developing pulmonary arterial hypertension (PAH) in humans, but the mechanisms responsible are unresolved. A recent study reported that female mice lacking the gene for tryptophan hydroxylase-1 (Tph1(-/-) mice) were protected from PAH caused by chronic dexfenfluramine, suggesting a pivotal role for peripheral serotonin (5-HT) in the disease process. Here we tested two alternative hypotheses which might explain the lack of dexfenfluramine-induced PAH in Tph1(-/-) mice. We postulated that: 1) Tph1(-/-) mice express lower levels of pulmonary 5-HT transporter (SERT) when compared to wild-type controls, and 2) Tph1(-/-) mice display adaptive changes in the expression of non-serotonergic pulmonary genes which are implicated in PAH. SERT was measured using radioligand binding methods, whereas gene expression was measured using microarrays followed by quantitative real time PCR (qRT-PCR). Contrary to our first hypothesis, the number of pulmonary SERT sites was modestly up-regulated in female Tph1(-/-) mice. The expression of 51 distinct genes was significantly altered in the lungs of female Tph1(-/-) mice. Consistent with our second hypothesis, qRT-PCR confirmed that at least three genes implicated in the pathogenesis of PAH were markedly up-regulated: Has2, Hapln3 and Retlna. The finding that female Tph1(-/-) mice are protected from dexfenfluramine-induced PAH could be related to compensatory changes in pulmonary gene expression, in addition to reductions in peripheral 5-HT. These observations emphasize the intrinsic limitation of interpreting data from studies conducted in transgenic mice that are not fully characterized.

PKCδ inhibition enhances tyrosine hydroxylase phosphorylation in mice after methamphetamine treatment.

The present study was designed to evaluate the specific role of protein kinase C (PKC) δ in methamphetamine (MA)-induced dopaminergic toxicity. A multiple-dose administration regimen of MA significantly increases PKCδ expression, while rottlerin, a PKCδ inhibitor, significantly attenuates MA-induced hyperthermia and behavioral deficits. These behavioral effects were not significantly observed in PKCδ antisense oligonucleotide (ASO)-treated- or PKCδ knockout (-/-)-mice. There were no MA-induced significant decreases of dopamine (DA) content or tyrosine hydroxylase (TH) expression in the striatum in rottlerin-treated-, ASO-treated- or PKCδ (-/-)-mice. The administration of MA also results in a significant decrease of TH phosphorylation at ser 40, but not ser 31, while the inhibition of PKCδ consistently and significantly attenuates MA-induced reduction in the phosphorylation of TH at ser 40. Therefore, these results suggest that the MA-induced enhancement of PKCδ expression is a critical factor in the impairment of TH phosphorylation at ser 40 and that pharmacological or genetic inhibition of PKCδ may be protective against MA-induced dopaminergic neurotoxicity in vivo.