Bruce T. Hope

Neuronal NADPH diaphorase is a nitric oxide synthase.

NADPH diaphorase histochemistry selectively labels a number of discrete populations of neurons throughout the nervous system. This simple and robust technique has been used in a great many experimental and neuropathological studies; however, the function of this enzyme has remained a matter of speculation. We, therefore, undertook to characterize this enzyme biochemically. With biochemical and immunochemical assays, NADPH diaphorase was purified to apparent homogeneity from rat brain by affinity chromatography and anion-exchange HPLC. Western (immunoblot) transfer and immunostaining with an antibody specific for NADPH diaphorase labeled a single protein of 150 kDa. Nitric oxide synthase was recently shown to be a 150-kDa, NADPH-dependent enzyme in brain. It is responsible for the calcium/calmodulin-dependent synthesis of the guanylyl cyclase activator nitric oxide from L-arginine. We have found that nitric oxide synthase activity and NADPH diaphorase copurify to homogeneity and that both activities could be immunoprecipitated with an antibody recognizing neuronal NADPH diaphorase. Furthermore, nitric oxide synthase was competitively inhibited by the NADPH diaphorase substrate, nitro blue tetrazolium. Thus, neuronal NADPH diaphorase is a nitric oxide synthase, and NADPH diaphorase histochemistry, therefore, provides a specific histochemical marker for neurons producing nitric oxide.

Neuroadaptation: Incubation of cocaine craving after withdrawal

Relapse to cocaine addiction is frequently associated with subjective reports of craving, a poorly understood state that precedes and accompanies cocaine-seeking behaviours. It has been suggested that over the first few weeks of withdrawal from cocaine, human addicts become sensitized to drug-associated environmental cues that act as external stimuli for craving, although the evidence for this is inconsistent. Here we provide behavioural evidence from laboratory animals suggesting that the onset of craving is delayed and that craving does not decay, but rather increases progressively, over a two-month withdrawal period.

Drug addiction: a model for the molecular basis of neural plasticity.

Although some aspects of drug addiction can occur relatively rapidly in response to acute administration of a drug of abuse, most changes in brain function associated with addiction occur gradually over time in response to prolonged drug exposure. These gradually developing changes can persist for a long time after cessation of chronic drug administration. Such adaptive changes are described by the terms tolerance, sensitization, dependence, and withdrawal. Tolerance represents a reduced effect upon repeated exposure to a constant drug dose, or the need for an increased dose to maintain the same effect. Sensitization, or “reverse-tolerance,” describes the opposite situation, in which a constant drug dose elicits increasing effects. Dependence is defined as the need for continued drug exposure to avoid a withdrawal syndrome, characterized by physical or psychological disturbances, when the drug is withdrawn. The traditional distinction between physical and psychological dependence is artificial, because both are mediated by the brain, possibly even by similar neural mechanisms, as will be seen below. Addiction can be defined as the accumulated effects of tolerance, sensitization, and dependence. However, the most useful clinical definition of addiction is the compulsive use of a drug despite adverse consequences, which is presumably due to certain adaptive changes that arise in specific brain areas. The prominence of delayed, progressively develop ing, and persistent adaptations in brain function during drug addiction suggests that long-term changes in the brain are important in mediating addictive phenomena. Drug addiction can thus be viewed as druginduced neural plasticity and as such can serve as a model system to investigate the types of neurobiological mechanisms involved in plasticity. Drug addiction can also serve as a model system to establish the biological basis of a complex and clinically relevant behavioral abnormality. This is because prominent features of drug addiction can be reproduced accurately in laboratory animals, enabling detailed investigations of the molecular and cellular mechanisms underlying addictive phenomena. An improved understanding of the molecular neurobiology of drug addiction must include two types of information: mechanisms of pathophysiology-identification of the changes that drugs of abuse produce in the brain that lead to addiction, and mechanisms of individual risk-identification of specific genetic and environmental factors that predispose certain individuals to addiction. In this review, weoutline an experimental approach bywhich the neurobiological mechanisms underlying the neural plasticity responsible for drug addiction can be investigated. This approach focuses on two anatomically well-defined brain regions known from behavioral and pharmacological studies to mediate aspects of drug addiction: the locus ceruleus, which plays an important role in physical opiate dependence and withdrawal (Aghajanian, 1978; Redmond and Krystal, 1984; Koob et al., 1992; Nestler, 1992), and the mesolimbic dopamine system, which plays an important role in psychological dependence to opiates and many other drugs of abuse (Bozarth, 1989; Wise, 1990; Kuhar et al., 1991; Fibiger et al., 1992; Koob, 1992; Dworkin and Smith, 1993). These brain regions have served as useful models for biochemical studies of drug addiction, based on their prior electrophysiological and behavioral characterizatian. The approach we outline here has three aims: to identify molecular and cellular adaptations that are induced in these regions by drugs of abuse; to relate the altered biochemical phenotype of cells in those regions to the altered electrophysiological phenotype of the cells and to the altered behavioral phenotype of the organism; and to elaborate the detailed molecular mechanisms by which the drugs produce the altered biochemical and electrophysiological phenotypes. Information concerning the molecular basis of drug action can then be used to identify genetic and environmental factors that determine individual differences in responsiveness to the drugs, including predilection for drug addiction.

Induction of a long-lasting AP-1 complex composed of altered Fos-like proteins in brain by chronic cocaine and other chronic treatments

Following chronic cocaine treatment, we have found a long-lasting increase in AP-1 binding in the rat nucleus accumbens and striatum, two important targets of the behavioral effects of cocaine. This increase develops gradually over several days and remains at 50% of maximal levels 7 days after the last cocaine exposure. Supershift experiments, along with one- and two-dimensional Western blots, indicate that this chronic AP-1 complex contains at least four Fos-related antigens (FRAs), some of which display delta FosB-like immunoreactivity, that are induced selectively by chronic, but not acute, cocaine treatment. The same chronic FRAs were also induced by several different types of chronic treatments in a region-specific manner in the brain. Thus, the chronic FRAs and associated chronic AP-1 complex could mediate some of the long-term changes in gene expression unique to the chronic-treated state as opposed to the acute-treated and normal states.

Discovery of the Presence and Functional Expression of Cannabinoid CB2 Receptors in Brain

Abstract:  Two well‐characterized cannabinoid receptors (CBrs), CB1 and CB2, mediate the effects of cannabinoids and marijuana use, with functional evidence for other CBrs. CB1 receptors are expressed primarily in brain and peripheral tissues. For over a decade several laboratories were unable to detect CB2 receptors in brain and were known to be intensely expressed in peripheral and immune tissues and have traditionally been referred to as peripheral CB2 CBrs. We have reported the discovery and functional presence of CB2 cannabinoid receptors in mammalian brain that may be involved in depression and drug abuse and this was supported by reports of identification of neuronal CB2 receptors that are involved in emesis. We used RT‐PCR, immunoblotting, hippocampal cultures, immunohistochemistry, transmission electron microscopy, and stereotaxic techniques with behavioral assays to determine the functional expression of CB2 CBrs in rat brain and mice brain exposed to chronic mild stress (CMS) or those treated with abused drugs. RT‐PCR analyses supported the expression of brain CB2 receptor transcripts at levels much lower than those of CB1 receptors. In situ hybridization revealed CB2 mRNA in cerebellar neurons of wild‐type but not of CB2 knockout mice. Abundant CB2 receptor immunoreactivity (iCB2) in neuronal and glial processes was detected in brain and CB2 expression was detected in neuron‐specific enolase (NSE) positive hippocampal cell cultures. The effect of direct CB2 antisense oligonucleotide injection into the brain and treatment with JWH015 in motor function and plus‐maze tests also demonstrated the functional presence of CB2 cannabinoid receptors in the central nervous system (CNS). Thus, contrary to the prevailing view that CB2 CBrs are restricted to peripheral tissues and predominantly in immune cells, we demonstrated that CB2 CBrs and their gene transcripts are widely distributed in the brain. This multifocal expression of CB2 immunoreactivity in brain suggests that CB2 receptors may play broader roles in the brain than previously anticipated and may be exploited as new targets in the treatment of depression and substance abuse.

Central amygdala ERK signaling pathway is critical to incubation of cocaine craving.

Using a rat model of craving and relapse, we have previously found time-dependent increases in cue-induced cocaine seeking over the first months of withdrawal from cocaine, suggesting that drug craving incubates over time. Here, we explored the role of the amygdala extracellular signal–regulated kinase (ERK) signaling pathway in this incubation. Cocaine seeking induced by exposure to cocaine cues was substantially higher after 30 withdrawal days than after 1 withdrawal day. Exposure to these cues increased ERK phosphorylation in the central, but not the basolateral, amygdala after 30 d, but not 1 d, of withdrawal. After 30 d of withdrawal from cocaine, inhibition of central, but not basolateral, amygdala ERK phosphorylation decreased cocaine seeking. After 1 d of withdrawal, stimulation of central amygdala ERK phosphorylation increased cocaine seeking. Results suggest that the incubation of cocaine craving is mediated by time-dependent increases in the responsiveness of the central amygdala ERK pathway to cocaine cues.

Incubation of cocaine craving after withdrawal: a review of preclinical data.

Using a rat model of drug craving and relapse, we recently found that cocaine seeking induced by re-exposure to drug-associated cues progressively increases over the first 2 months after withdrawal from cocaine self-administration, suggesting that drug craving incubates over time [Nature 412 (2001) 141]. Here, we summarize data from studies that further characterized this incubation phenomenon and briefly discuss its implications for drug addiction. The main findings of our ongoing research are: 1. Incubation of cocaine craving is long-lasting, but not permanent: cocaine seeking induced by exposure to cocaine cues remains elevated for up to 3 months of withdrawal, but decreases after 6 months. 2. Incubation of reward craving is not drug specific: sucrose seeking induced by re-exposure to the reward cues also increases after withdrawal, but for a time period that is shorter than that of cocaine. 3. Incubation of cocaine craving is not evident after acute re-exposure to cocaine itself: cocaine seeking induced by cocaine priming injections remains essentially unchanged over the first 6 months of withdrawal. 4. Incubation of cocaine craving after withdrawal is associated with increases in the levels of brain-derived neurotrophic factor (BDNF) in mesolimbic dopamine areas.

Synergistic interaction between adenosine A2A and glutamate mGlu5 receptors: Implications for striatal neuronal function

The physiological meaning of the coexpression of adenosine A2A receptors and group I metabotropic glutamate receptors in γ- aminobutyric acid (GABA)ergic striatal neurons is intriguing. Here we provide in vitro and in vivo evidence for a synergism between adenosine and glutamate based on subtype 5 metabotropic glutamate (mGluR5) and adenosine A2A (A2AR) receptor/receptor interactions. Colocalization of A2AR and mGluR5 at the membrane level was demonstrated in nonpermeabilized human embryonic kidney (HEK)-293 cells transiently cotransfected with both receptors by confocal laser microscopy. Complexes containing A2AR and mGluR5 were demonstrated by Western blotting of immunoprecipitates of either Flag-A2AR or hemagglutinin-mGluR5 in membrane preparations from cotransfected HEK-293 cells and of native A2AR and mGluR5 in rat striatal membrane preparations. In cotransfected HEK-293 cells a synergistic effect on extracellular signal-regulated kinase 1/2 phosphorylation and c-fos expression was demonstrated upon A2AR/mGluR5 costimulation. No synergistic effect was observed at the second messenger level (cAMP accumulation and intracellular calcium mobilization). Accordingly, a synergistic effect on c-fos expression in striatal sections and on counteracting phencyclidine-induced motor activation was also demonstrated after the central coadministration of A2AR and mGluR5 agonists to rats with intact dopaminergic innervation. The results suggest that a functional mGluR5/A2AR interaction is required to overcome the well-known strong tonic inhibitory effect of dopamine on striatal adenosine A2AR function.

Neurobiology of the incubation of drug craving

It was suggested in 1986 that cue-induced drug craving in cocaine addicts progressively increases over the first several weeks of abstinence and remains high for extended periods. During the past decade, investigators have identified an analogous incubation phenomenon in rodents, in which time-dependent increases in cue-induced drug seeking are observed after withdrawal from intravenous cocaine self-administration. Such an incubation of drug craving is not specific to cocaine, as similar findings have been observed after self-administration of heroin, nicotine, methamphetamine and alcohol in rats. In this review, we discuss recent results that have identified important brain regions involved in the incubation of drug craving, as well as evidence for the underlying cellular mechanisms. Understanding the neurobiology of the incubation of drug craving in rodents is likely to have significant implications for furthering understanding of brain mechanisms and circuits that underlie craving and relapse in human addicts.

Histochemical characterization of neuronal NADPH-diaphorase.

We examined the properties of neuronal NADPH-diaphorase in sections of rat striatum, using histochemical procedures. NADPH-diaphorase histochemistry stained discrete populations of central neurons and provided a Golgi-like image of the neurons exhibiting this activity. The NADPH-diaphorase reaction appeared to be enzyme catalyzed, since it was abolished by pre-treatment with proteases, heat, and acid or alkaline denaturation. Under anaerobic conditions, any tetrazolium salt with a redox potential more positive than NADPH could be reduced by the enzyme. NADPH-diaphorase activity was sensitive to inhibition by sulfhydryl reagents but was unaffected by metal chelators, superoxide dismutase, and catalase. Therefore, the enzyme is unlikely to be a metalloenzyme or to reduce tetrazoliums by producing superoxide anions or hydrogen peroxide. Various analogues of beta-NADPH could be used by the enzyme; however, beta-NADH, which can be used by DT-diaphorase, was ineffective. The enzyme was also resistant to dicumarol, an inhibitor of DT-diaphorase activity. Electron microscopy indicated that the NADPH-diaphorase reaction resulted in staining of various membranous organelles. We conclude that neuronal NADPH-diaphorase is a membrane-bound enzyme distinct from DT-diaphorase and other known enzymes with diaphorase activity. The histochemical characteristics presented here should now enable meaningful biochemical studies of neuronal NADPH-diaphorase to be undertaken.