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Director's Report to the National Advisory Council on Drug Abuse
May, 1995

Research Findings

Basic Research

Cocaine and Amphetamine Sensitization and NMDA Receptors

Sensitization, defined as the progressive enhancement of the locomotor stimulatory effects of drugs such as amphetamine or cocaine during their repeated administration, is often considered to model processes contributing to (1) drug addiction, and (2) certain drug-induced and spontaneous psychotic symptoms. Dr. Marina Wolf and others have previously shown that the development of sensitization in rats to effects of amphetamine and cocaine is prevented by co-administration of antagonists of the Nmethyl-d-aspartate (NMDA) receptor complex, a result which is consistent with the demonstrated role of NMDA receptors in other forms of neuronal and behavioral plasticity. Several experiments recently performed by this group have contributed to our understanding of sensitization. (1) Lesion studies indicate that sensitization requires a neuronal circuit involving prefrontal cortex, amygdala, and ventral tegmental area. (2) The response of ventral tegmental area dopaminergic neurons to glutamate was increased in amphetamine and cocaine-sensitized rats, while nucleus accumbens neurons were less sensitive to glutamate. Thus, sensitization appears to be associated with changes in glutamate transmission at both the origin and termination of the mesoaccumbens dopaminergic pathway (White FJ, Hu X-T, Zhang X-F & Wolf ME: Repeated Administration of Cocaine or Amphetamine Alters Neuronal Responses to Glutamate in the Mesoaccumbens Dopamine System. J. Pharmacol. Exp. Ther., in press).

Neurobiology Of Sensitization

NIDA has several grantees studying various aspects of stimulant-induced behavioral sensitization in animals. Among them are Dr. Terry Robinson from the University of Michigan (Ann Arbor) and Dr. James Steketee from LSU (Shreveport). Sensitization is a common effect whereby the repeated administration of a psychomotor stimulant results in an increased, rather than a decreased, drug effect over time. The clinical relevance of sensitization is that repeated exposure of the stimulant often results in a drug-associated paranoid psychosis that is clinically similar to paranoid schizophrenia. This increased effect is thought to be due to a neural adaptation produced by repeated exposure.

Of particular significance are the findings by Dr. Robinson that there is stimulus control of amphetamine sensitization (both development and expression). These studies are of particular relevance to drug craving induced by environmental cues (eg, an addict sees an old drug supplier, which makes him return to his drug-seeking ways).

Dr. Steketee is a young investigator who is studying the role of the ventral tegmental area in the development and expression of behavioral sensitization of cocaine in the rat. He is examining the potential role that changes in intracellular signal transduction play in the development of behavioral sensitization to cocaine. In his initial studies, Dr. Steketee determined the role of GABAB receptors in the A10 region of the rat brain by the direct injection of the antagonist 2hydroxysaclofen alone or in combination with cocaine, with measurement of locomotor activity as the endpoint. The antagonist did not alter the spontaneous locomotor activity or the cocaineinduced hyperactivity. These data suggest that blocking the activation of the GABAB receptor may not be important in the development of sensitization.

D1 Receptorless Mice Do Not Show Motor Stimulation In Response To Cocaine

The brain mesoaccumbens DA system is involved in the psychomotor stimulation activities of cocaine. However, the extent to which different DA receptors mediate these effects is not clear. This study used a DA D1 receptor mutant mice produced by gene targeting to investigate the role of this receptor in the effects induced by cocaine. Wild-type mice, showed a dose-dependent increase in locomotion, D1 mutant mice exhibited a dose-dependent decrease. Electrophysiological studies of DA sensitive nucleus accumbens neurons demonstrated a marked reduction in the inhibitory effects of cocaine on the generation of action potentials. Also, the inhibitory effects of DA as well as D1 and D2 agonists were nearly completely abolished, whereas those of 5HT were unaffected. D2-like DA receptor binding was also normal. The results indicate the essential role of the D1 receptor in the locomotor stimulant effects of cocaine and in DA-mediated neurophysiological effects within the nucleus accumbens. Xu, Ming, Hu, Siu-Ti, Cooper, D., Moratalla, Rosaria, Graybiel, A.,White, F., and Tonegawa, S. Elimination of Cocaine-Induced Hyperactivity and Dopamine-Mediated Neurophysiological Effects in Dopamine D2 Receptor Mutant Mice. Cell, 79: 945-955, 1994.

An Opioid-like Brain Derived Chemotactic Factor

During normal brain development macrophages are targeted to areas of degeneration, including sites where cell death naturally occurs, and glial scaffolds are eliminated. Similarly, macrophages also are targeted to sites of neuronal injury. What molecular mechanisms control this specific migration? Some investigators have proposed release of a chemotactic signal by degenerating neuronal elements, and, indeed, numerous chemotactic factors (including growth factors and cytokines) have been identified outside the central nervous system. Now, NIDA grantee Dr. Pat Levitt of the Robert Wood Johnson Medical School in New Jersey and coworkers report identifying an injury-induced brain derived chemotactic factor (BDCF) whose activity appears to be opioid-like. The factor attracts both brain and resident peritoneal macrophages. In tests with the latter, delta receptor antagonists naltrindole and ICI174,864 blocked chemotaxis, but mu and kappa antagonists failed to show an effect; however tests with five opioid ligands failed to induce chemotaxis, indicating that the factor is not one of those conventionally known. The investigators report that their results "...suggest that opioids are released as a response to neuronal injury and have critical roles in recruiting phagocytic cells and subsequent immune modulation." Carolanne E. Milligan, Linda Webster, Elmer T. Piros, Christopher J. Evans, Timothy J. Cunningham, and Pat Levitt. 1995. "Induction of Opioid Receptor-Mediated Macrophage Chemotactic Activity After Neonatal Brain Injury. The Journal of Immunology, in press.

Transgenic Mice Model of Endogenous Opioid Gene Regulation by Exogenous Opioid

Stress strongly induces proenkephalin gene expression in the hypothalamus. Using a transgenic mice model, NIDA grantees Steven E. Hyman, David Borsook and their coworkers at Harvard Medical School observed that acute or subacute morphine administration prior to stress produced marked superinduction of transgene expression compared with stress alone. In contrast, chronic morphine administration decreased basal expression of the transgene, and inhibited stress-induced expression of the transgene. The endogenous proenkephalin mRNA and c-fos were induced in parallel with the transgene. These data suggest that acute or subacute morphine administration sensitizes proenkephalin neurons within the hypothalamus to stress and that chronic morphine administration desensitizes this response. With special reference to mechanisms of opioid dependence, this model appears to be a useful tool to investigate the mechanistic aspects of the regulation of endogenous opioid genes by exogenous opioids.

Effects of Prenatal Cocaine Exposure

Clinical case reports have described cerebral vascular insults in fetuses born to cocaine abusing mothers. Fetal hypoxemia is thought to be one of the mechanisms involved in the patho-physiology of abnormal cerebral growth and neurodevelopment in newborns. Findings from in vivo fetal sheep studies reveal that cocaine causes fetal hypoxemia but does not affect fetal brain oxygen delivery probably due to enhanced compensatory blood flow and oxygen delivery to the heart. Increases in cerebral vascular resistance were also observed following cocaine and its metabolites, benzoylecgonine and cocaethylene; however, the magnitude of change in vascular resistance seen with cocaine metabolites was different from that of the parent drug.

Data from in vitro studies indicate that cocaine affects fetal cerebral vasculature probably through its direct inhibitory effect on neurotransmitter uptake and sodium channels as well as through its major metabolites. Thus, the development of fetal cerebral vasculature and its response to physiological or pathophysiological stimuli may be altered by prenatal cocaine exposure. (Covert, Schreiber et al., J Pharmacol. Exp. Therap. 270:1-9, 1994; Schreiber et al., J. Dev. Physiol. 20:141-147, 1994; Schreiber et al., J. Applied Physiology 77:834-839, 1994; Pena, Burchfield and Abrams, Pediatric Research 35:248A, 1994)

Effects of Prenatal Morphine Exposure

Dr. Vathy and her associates have reported recently that prenatal morphine exposure produces long-lasting, sexually dimorphic alterations in norepinephrine (NE) content and turnover rate in specific brain regions. This may indicate altered firing of NE cell bodies of origin or modification in local mechanisms regulating NE utilization at the level of terminals. Morphine also induced postsynaptic alterations in the brains of female rat offspring as mu opioid receptor binding was reduced in females but not in males. Like morphine, prenatal cocaine exposure in modest doses also produced long-lasting, sexually dimorphic alterations in adult sexual behavior and brain catecholamines in rat offspring; however, the alterations in the magnitude of behavioral changes were different than after morphine exposure. (Vathy et al., Dev. Brain Res. 73:1115-1122, 1993; Vathy et al., Brain Res. in press, 1995; Rimanoczy and Vathy, Society for Neurosci. 20:504A, 1994).

Opioid Regulation of Calcium Channels

Using specific antibodies directed at the G protein alpha subunits ( alphao, alphai1, alphai2 and alphai3) to block G protein coupling of µ receptors to calcium channels, Dr. Robert L. Macdonald and his colleagues at The University of Michigan Medical School have demonstrated that µ opioid induced inhibition of calcium current in acutely dissociated rat primary afferent neurons occurs through activation of a Gi or Go-type G protein and that the reduction is independent of changes in adenylate cyclase activity. Intracellular dialysis with an antiserum specific for Go attenuated calcium current inhibition by a µ agonist in 5 of 6 neurons but no alteration of responses to the µ agonist were produced when neurons were dialyzed with an anti-Gi1alpha/Gi2alpha antiserum or antibody specific for the a subunits of Gi proteins. Thus, in rat DOG neurons, µ opioid receptors coupled to calcium channels via the pertussis toxin sensitive Go subclass of GTP binding proteins

Furthermore, studies were conducted to examine the regulations of voltage dependent calcium channels, using whole cell patch clamp recordings, on acutely isolated rat dorsal ganglia neurons by µ and K opioid agonists. The results indicated that inhibitory response to one agonist was occluded when tested in the presence of the other, suggesting that µ and K opioid receptors are co-expressed on at least some primary afferent neurons and appear to be functionally coupled to a common pool of calcium channels. The effect of µ agonists was studied on high threshold calcium currents recorded from these neurons. Using selective blockade (a combination of omega-conotoxin, omega-agatoxin IV A and nifedipine), they demonstrated the presence of N type, L type and possibly P type calcium currents. These data demonstrated that µ agonists reduced N type and possibly P type calcium currents but did not reduce currents mediated by L calcium channel currents. Thus, µ opioid receptors were negatively coupled to several pharmacological distinct types of high threshold calcium channels in rat primary afferent neurons, probably N type and P type calcium current channels.

Studies were also conducted to investigate the effects of direct intracellular application of a constitutively active form of protein kinase (PKC), PKM, on whole cell calcium currents recorded from acutely dissociated rat primary afferent neurons to determine which high threshold calcium currents are regulated by phosphorylation by protein kinase A (PKA) or PKC. PKM enhanced high threshold voltage activated calcium currents. This enhancement was blocked by the synthetic PKC inhibitor peptide (PKC-I) confirming that this was a specific PKC mediated effect. The enhancement of high threshold calcium current appeared to involve both N and L calcium current components.

Additionally, Dr. Macdonald and his colleagues have succeeded in co-expressing mouse brain ß subunit isoforms with a neuronal class C alpha1 subunit in HEK 293 cells. Whole cell voltage clamp recording demonstrated that addition of mouse brain as ß subunit isoforms with the alpha1 subunit produces a marked enhancement of calcium current amplitude.

Finally, the effect of PKM on K opioid receptor agonist mediated inhibition of N type current in primary afferent neurons was investigated. In the presence of intracellular PKM, the dynorphin A, a K opioid agonist, was less effective in decreasing N type calcium current. This reduction in efficacy was blocked by co-introduction of the inhibitory peptide PKC-I. These data demonstrate that PKC mediated phosphorylation decreases the coupling between opioid receptor activation and N type calcium current reduction. The target for this phosphorylation remains uncertain and could represent phosphorylation of either K opioid receptor, G protein or calcium channel.

These studies have demonstrated the feasibility of determining the type of G protein which couples µ and K opioid receptors to calcium currents and of identifying the calcium current target. They show the coupling of µ and k opioid receptors to the same calcium channel target via the same G protein and reveal a role for protein kinase C mediated phosphorylation in regulating this coupling. Moreover, with the ability to express recombinant calcium channels and opioid receptors in an heterologous expression system, it will become possible to determine the sites of phosphorylation mediated regulation of this coupling and to examine the signal transduction mechanism occurring between G proteins and calcium channels in these cells. ( Moises, Russin and Macdonald, J. Neurosci. 14: 3842, 1994; Moises, Russin and Macdonald, J. Neurosci. 14: 5903, 1994; Hall, Browning, Dudek and Macdonald, J. Neurosci, in press; Massa, Kelly, Yule, MA, Macdonald, and Uhler, in press; Kelly, Esmaeil, and Macdonald, Soc Neurosci Abstr, 20:899, 1994; Hall, Browning, Dudek, and Macdonald, Soc Neurosci Abstr, 20: 902, 1994.

NMDA Elevation of Extracellular Dopamine and Serotonin in the Nucleus Accumbens

A property common to addicting drugs, including opiates, ethanol, nicotine, amphetamine and cocaine, is the ability to increase extracellular concentrations of dopamine in the nucleus accumbens. Many of these drugs also elevate extracellular serotonin in the n. accumbens. Although MDMA increases in extracellular dopamine measured by in vivo voltammetry in the accumbens have been demonstrated, it was not clear whether MDMA also increases serotonin in vivo in this nucleus. Susan White and colleagues at Washington State University used microiontophoresis combined with extracellular recording to determine the effects of MDMA on glutamate-evoked firing of neurons in the n. accumbens in vivo. Then they used in vivo microdialysis to determine whether local infusion of MDMA into the accumbens alters extracellular levels of DA and/or serotonin.

These investigators found that local application of MDMA inhibited glutamate-evoked firing of n. accumbens neurons, and that this effect was mediated partially by dopamine and partially by serotonin. Extracellular levels of both of these monoamines were elevated in the n. accumbens following local application of MDMA. These results permit MDMA to be added to the list of abused drugs that have been demonstrated to elevate extracellular levels of dopamine and serotonin in the nucleus accumbens (White et al., Neuroscience 62: 41-50, 1994).

In even more recent (unpublished) experiments, they found that repeated exposure to MDMA decreased inhibitory effects of dopamine on accumbens cells that were tested 2 weeks after the last MDMA injection. This observation suggests that chronic MDMA, like chronic cocaine, causes long-term changes in dopamine neurotransmission in the accumbens, an effect which may underlie the addictive property of these drugs.

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