Kevin A. Sevarino

Excitatory glycine receptors containing the NR3 family of NMDA receptor subunits

The N-methyl-d-aspartate subtype of glutamate receptor (NMDAR) serves critical functions in physiological and pathological processes in the central nervous system, including neuronal development, plasticity and neurodegeneration. Conventional heteromeric NMDARs composed of NR1 and NR2A–D subunits require dual agonists, glutamate and glycine, for activation. They are also highly permeable to Ca2+, and exhibit voltage-dependent inhibition by Mg2+. Coexpression of NR3A with NR1 and NR2 subunits modulates NMDAR activity. Here we report the cloning and characterization of the final member of the NMDAR family, NR3B, which shares high sequence homology with NR3A. From in situ and immunocytochemical analyses, NR3B is expressed predominantly in motor neurons, whereas NR3A is more widely distributed. Remarkably, when co-expressed in Xenopus oocytes, NR3A or NR3B co-assembles with NR1 to form excitatory glycine receptors that are unaffected by glutamate or NMDA, and inhibited by d-serine, a co-activator of conventional NMDARs. Moreover, NR1/NR3A or -3B receptors form relatively Ca2+-impermeable cation channels that are resistant to Mg2+, MK-801, memantine and competitive antagonists. In cerebrocortical neurons containing NR3 family members, glycine triggers a burst of firing, and membrane patches manifest glycine-responsive single channels that are suppressible by d-serine. By itself, glycine is normally thought of as an inhibitory neurotransmitter. In contrast, these NR1/NR3A or -3B ‘NMDARs’ constitute a type of excitatory glycine receptor.

Chronic Cocaine Treatment Decreases Levels of the G Protein Subunits Giα and Goα in Discrete Regions of Rat Brain

Abstract: A possible role for G proteins in contributing to the chronic actions of cocaine was investigated in three rat brain regions known to exhibit electrophysiological responses to chronic cocaine: the ventral tegmental area, nucleus accumbens, and locus coeruleus. It was found that chronic, but not acute, treatment of rats with cocaine produced a small (∼ 15%), but statistically significant, decrease in levels of pertussis toxin‐mediated ADP‐ribosylation of Giα and Goα in each of these three brain regions. The decreased ADP‐ribosylation levels of the G protein subunits were shown to be associated with 20–30% decreases in levels of their immunoreactivity. In contrast, chronic cocaine had no effect on levels of G protein ADP‐ribosylation or immunoreactivity in other brain regions studied for comparison. Chronic cocaine also had no effect on levels of GSα or Gβ immunoreactivity in the ventral tegmental area and nucleus accumbens. Specific decreases in Giα and Goα levels observed in response to chronic cocaine in the ventral tegmental area, nucleus accumbens, and locus coeruleus are consistent with the known electrophysiological actions of chronic cocaine on these neurons, raising the possibility that regulation of G proteins represents part of the biochemical changes that underlie chronic cocaine action in these brain regions.

Desipramine and contingency management for cocaine and opiate dependence in buprenorphine maintained patients

Co-dependence on opiates and cocaine occurs in about 60% of patients entering methadone treatment and has a poor prognosis. However, we recently found that desipramine (DMI) could be combined with buprenorphine to significantly reduce combined opiate and cocaine use among these dually dependent patients. Furthermore, contingency management (CM) has been quite potent in reducing cocaine abuse during methadone maintenance. To test the efficacy of combining CM with these medications we designed a 12-week, randomized, double blind, four cell trial evaluating DMI (150 mg/day) or placebo plus CM or a non-contingent voucher control in 160 cocaine abusers maintained on buprenorphine (median 16 mg daily). Cocaine-free and combined opiate and cocaine-free urines increased more rapidly over time in those treated with either DMI or CM, and those receiving both interventions had more drug-free urines (50%) than the other three treatment groups (25-29%). Self reported opiate and cocaine use and depressive and opioid withdrawal symptoms showed no differences among the groups and symptom levels did not correlate with urine toxicology results. Lower DMI plasma levels (average 125 ng/ml) were associated with greater cocaine-free urines. DMI and CM had independent and additive effects in facilitating cocaine-free urines in buprenorphine maintained patients. The antidepressant appeared to enhance responsiveness to CM reinforcement.

Smoking Cessation during Alcohol Treatment: A Randomized Trial of Combination Nicotine Patch plus Nicotine Gum

AIMS The primary aim was to compare the efficacy of smoking cessation treatment using a combination of active nicotine patch plus active nicotine gum versus therapy consisting of active nicotine patch plus placebo gum in a sample of alcohol-dependent tobacco smokers in an early phase of out-patient alcohol treatment. A secondary aim was to determine whether or not there were any carry-over effects of combination nicotine replacement on drinking outcomes. DESIGN Small-scale randomized double-blind placebo-controlled clinical trial with 1-year smoking and drinking outcome assessment. SETTING Two out-patient substance abuse clinics provided a treatment platform of behavioral alcohol and smoking treatment delivered in 3 months of weekly sessions followed by three monthly booster sessions. PARTICIPANTS Participants were 96 men and women with a diagnosis of alcohol abuse or dependence and smoking 15 or more cigarettes per day. INTERVENTION All participants received open-label transdermal nicotine patches and were randomized to receive either 2 mg nicotine gum or placebo gum under double-blind conditions. FINDINGS Analysis of 1-year follow-up data revealed that patients receiving nicotine patch plus active gum had better smoking outcomes than those receiving patch plus placebo gum on measures of time to smoking relapse and prolonged abstinence at 12 months. Alcohol outcomes were not significantly different across medication conditions. CONCLUSIONS Results of this study were consistent with results of larger trials of smokers without alcohol problems, showing that combination therapy (nicotine patch plus gum) is more effective than monotherapy (nicotine patch) for smoking cessation.

Ketoconazole increases cocaine and opioid use in methadone maintained patients

Stress plays an important role in substance abuse problems. For example, in studies with rodents stress induces reinstatement of opioid and cocaine self-administration. In addition, attenuation of the stress response by pharmacological adrenalectomy using ketoconazole, a cortisol synthesis inhibitor, reduces cocaine self-administration in rodents. In contrast, studies in primates and humans have produced conflicting results using cortisol synthesis inhibitors for attenuating cocaine-related behaviors and subjective effects. To explore the treatment implications of these findings, ketoconazoles (600-900 mg daily) ability to reduce heroin and cocaine use was compared with placebo in 39 methadone maintained patients with a history of cocaine abuse or dependence during a 12-week double blind trial. Contrary to the predicted effects, both heroin and cocaine use increased after patients were stabilized on methadone and ketoconazole. Depressive and withdrawal symptoms improved no more with ketoconazole than with placebo treatment, and side effects were greater on ketoconazole than placebo. As reported before with methadone treatment, morning cortisol levels were significantly lower than normal values throughout the clinical trial, but were not lower with ketoconazole than placebo treatment. Thus, in agreement with the negative results from acute dosing studies in primates and humans, chronic ketoconazole treatment does not appear to reduce cocaine or opioid use in humans maintained on methadone.

Concurrent Alcohol and Tobacco Treatment: Effect on Daily Process Measures of Alcohol Relapse Risk

OBJECTIVE The aim of this study was to compare the effects of alcohol treatment along with concurrent smoking treatment or delayed smoking treatment on process measures related to alcohol relapse risk. METHOD Alcohol dependent smokers (N = 151) who were enrolled in an intensive outpatient alcohol treatment program and were interested in smoking cessation were randomized to a concurrent smoking cessation (CSC) intervention or to a waiting list for delayed smoking cessation (DSC) intervention scheduled to begin 3 months later. Daily assessments of relapse process measures were obtained using an Interactive Voice Response (IVR) system for 12 weeks after the onset of smoking treatment in the CSC condition, and before beginning smoking treatment in the DSC condition. Smoking outcomes were assessed at 2 and 13 weeks after starting treatment. RESULTS Seven-day carbon monoxide (CO) verified smoking abstinence in the CSC condition was 50.5% at 2 weeks and 19.0% at 13 weeks compared with 2.2% abstinence at 2 weeks and 0% abstinence at 13 weeks for those in the DSC condition. Drinking outcomes were not significantly different for CSC versus DSC treatment conditions. On daily IVR assessments, CSC participants had significantly lower positive alcohol outcome expectancies relative to DSC participants. Multilevel modeling (MLM) analyses of within-person effects across the 12 weeks of daily monitoring showed that daily smoking abstinence was significantly associated with same day reports of lower alcohol consumption, lower urge to drink, lower negative affect, lower positive alcohol outcome expectancies, greater alcohol abstinence self-efficacy, greater alcohol abstinence readiness to change, and greater perceived self-control demands. CONCLUSIONS Analyses of process measures provide support for recommending smoking intervention concurrent with intensive outpatient alcohol treatment. (PsycINFO Database Record

Neurobiological adaptations to psychostimulants and opiates as a basis of treatment development.

Abstract: Abuse of illicit substances, in particular psychostimulants and opiates, is a worldwide public health issue. Chronic use of cocaine and amphetamine causes common neurobiological adaptations that may guide new treatment development. These include perturbations in dopamine and serotonin neurotransmission, leading to trials of antidepressants, and serotonin and dopamine augmentation strategies. The detection of cerebral perfusion abnormalities caused by psychostimulants has led to examination of antiplatelet and excitatory amino acid (EAA) antagonist therapies. Further, development of cocaine vaccines allows for testing of peripheral blockade approaches to cocaine addiction. New approaches to behavioral treatments for cocaine dependence are also reviewed. For opiate dependence, understanding of heroins effects on μ and κ opiate receptors has led to investigations of the partial μ agonist buprenorphine in opiate maintenance. Evidence for hyper‐excitability of locus coeruleus (LC) noradrenergic neurons and EEA inputs to the LC guides trials of new α2‐adrenergic agonists and EEA antagonists to alleviate opiate withdrawal. Finally, clinical experience with withdrawal from methadone and LAAM has led to trials of antagonist‐accelerated opiate withdrawal. Improved treatment of psychostimulant and opiate addiction is critically needed, and likely to have wide‐reaching impact in health care and society.

In organello mitochondrial protein and RNA synthesis systems from Saccharomyces cerevisiae.

Publisher Summary This chapter describes yeast in organello systems that have been optimized for mitichondrial protein and RNA synthesis. The mitochondrial genome of Saccharomyces cerevisiae encodes eight polypeptide subunits of respiratory proteins (cytochrome-c oxidase subunits I, II, and III, cytochrome b, and ATP synthase subunits 6, 8, and 9), var1 (a ribosomal protein), several proteins involved in RNA splicing, large and small rRNAs, 24 tRNAs, and an RNA component of an RNase P enzyme involved in tRNA processing. In contrast, isolated yeast mitochondrial (in organello) translation and transcription systems have been developed for studying the various aspects of mitochondrial gene expression in vitro . These systems are used to determine the stoichiometry of transcription and translation of the major mitochondrial gene products to determine that mitochondrial transcription and translation are not obligately coupled, to establish that mitochondrial translation products are inserted into the inner mitochondrial membrane cotranslationally, and to study proteolytic turnover of mitochondrial translation products.

An anti-χ-1 antibody recognizes a heavily glycosylated protein in rat brain

Abstract The χ-1 subunit is a recently identified member of a new class of the ionotropic glutamate receptor family that attenuates NMDA receptor current. We have generated a polyclonal C-terminal antibody to the χ-1 subunit which recognizes a 135-kDa protein in membranes prepared from χ-1 transfected HEK-293 cells and in rat brain. In the post-natal day 7 (P7) rat brain, Western blot analysis revealed a 135-kDa band in the thalamus and cortex but not the striatum, cerebellum or peripheral tissues. De-glycosylation of the χ-1 subunit in both transfected cell lines and in the brain reduced the 135-kDa band to 110 kDa, near the predicted molecular weight of the χ-1 subunit. These studies demonstrate the χ-1 subunit is expressed as a glycosylated protein subunit in a distribution that parallels that observed for χ-1 mRNA by in situ hybridization.

Naltrexone for Initiation and Maintenance of Opiate Abstinence

Pharmacotherapies for opiate dependence first involve detoxification from physical dependence on opiates and then maintenance of that abstinent state. There are now three agents of distinct pharmacological classes available to achieve both phases: the opiate agonist methadone, the partial opioid agonist buprenorphine, and the opioid antagonist naltrexone. This chapter reviews the role of naltrexone in current opiate detoxification strategies and its use in long-term maintenance of sobriety from opiates.