National Institute on Drug Abuse
Director's Report to the National Advisory Council on Drug
Abuse
May, 1997
Research Findings
Basic Research
9-THC Stimulates Dopamine Neurons Via Their Afferent Monoaminergic
Neurons
9-Tetrahydrocannabinol (9-THC), the major active constituent of marijuana,
is believed to stimulate the brain's dopamine systems, as do other drugs
of abuse. Dr. Edward D. French's group at the University of Arizona College
of Medicine is determining the mechanisms of actions of 9-THC on dopamine
neurons using electrophysiological techniques. First, these investigators
confirmed that acute iv administration of 9-THC increases the firing rate
of substantia nigra and ventral tegmental area dopamine neurons (both about
a 50% maximum increase over baseline) (E.D. French, X. Wu & K. Dillon,
1997, NeuroReport 8, in press). Pretreatment with the selective cannabinoid
CB1 receptor antagonist SR141716A blocked this effect. Last year, the availability
of active and inactive enantiomers of the synthetic cannabinoid CB1 receptor
agonist WIN-55,212 provided an additional approach. The active enantiomer
(WIN-55,212-2) increased dopamine neuron firing rates in these brain areas,
while the inactive isomer (WIN-55,212-3) was ineffective. Thus, they confirmed
that the dopamine neuronal changes induced by 9-THC are mediated through
the CB1 cannabinoid receptor. Also last year, they demonstrated that reserpine
or alpha-methyl-p-tyrosine pretreatment in vivo each reduced the response
of the dopamine neurons to 9-THC. Thus, it would appear that catecholamine
or serotonin afferents to the dopamine neurons are needed for the cannabinoid
stimulation. This was confirmed by further experiments on dopamine neurons
in midbrain slice preparations, where the afferents are cut; neither 9-THC
nor WIN-55,212-2 stimulated cell firing in the slices.
Nitric Oxide Synthase Inhibitor Blocks Sensitization to Cocaine
The development of sensitization to cocaine and methamphetamine is thought
to underlie the processes of
1) addiction, 2) neurotoxicity and 3) psychopathology. In rodents, sensitization
is manifested by increased responsiveness to the locomotor-stimulating,
stereotypy- and seizure-inducing (kindling) effects of cocaine. It has been
established by the groups of Karler, White, and Wolf that glutamatergic
neurotransmission and the NMDA subtype of glutamate receptor are the primary
mediators of the induction of sensitization. Dr. Yossef Itzhak from the
University of Miami School of Medicine found an increase in the number of
NMDA receptors in the cortex of cocaine kindled mice, and further demonstrated
that activation of the NMDA receptor's intracellular messengers, Ca2+ and
nitric oxide (NO), mediates the development of sensitization to the convulsive
and lethal effects of cocaine. Recently, he reported that neuronal selective
NO synthase (NOS) inhibitors prevent the induction and expression of sensitization
(Itzhak, Neuropharmacology 35, pp. 1065-1073, 1996). Further, a neuronal
NOS inhibitor protects against methamphetamine neurotoxicity in mice (Itzhak
and S. Ali, J. Neurochem. 67, pp.1770 1773, 1996). Elucidation of these
mechanisms may advance the development of medications for the management
of psychostimulant abuse.
Cannabinoids and Excitotoxicity
Because many drugs of abuse are known to affect glutamatergic synaptic
transmission, Dr. Stanley Thayer and coworkers are testing the hypothesis
that such drugs might influence the progression of excitotoxicity. These
researchers found that cannabinoids significantly inhibit glutamatergic
synaptic transmission. Their report identifying a synaptic mechanism of
the cannabinoids is of considerable importance to understanding the cellular
effects of marijuana. Thus, focus was directed toward the study of cannabinoids
on glutamatergic synaptic transmission and excitotoxicity. In hippocampal
neurons, activation of cannabinoid receptors inhibits the N and P/Q subtypes
of Ca++ channels suggesting a likely molecular mechanism for the synaptic
inhibition produced by cannabinoids. Cannabinoids were found to protect
from excitotoxicity in vitro, suggesting that if compounds could be developed
in which the abuse properties were separated from this potentially therapeutic
effect, certain cannabimimetic drugs might be useful neuroprotective agents.
A key feature of many abused drugs is the development of tolerance upon
repeated exposure. Experimenters have found that the effects of full cannabinoid
agonists on synaptic transmission desensitize during prolonged exposure.
In contrast, the effects of one of the synthetic compounds CP55940, a compound
that acts as a partial agonist, does not desensitize, suggesting that this
drug may be effective in preventing the neurodegeneration that results from
glutamate release. Thayer, S.A., and Shen, M. Cannabinoid Receptor Agonists
Inhibit Glutamatergic Synaptic Transmission in Rat Hippocampal Cultures.
Symposium on Cannabis and the Cannabinoids (Int. Cannabinoid Res. Soc.)
p. 45, 1996. Shen, M. and Thayer, S.A. Desensitization of Cannabinoid-Mediated
Inhibition of Glutamatergic Synaptic Transmission between Cultured Rat Hippocampal
Neurons. Soc. Neurosci. Abst. 22: 82, 1996.
Tropane Derivative Holds Promise as a Methadone-Like Approach for Treating
Cocaine Addiction As reported in the February 1997 issue of the Journal
of Pharmacology and Experimental Therapeutics, Dr. Michael Nader and his
co-workers from the Drug Abuse Center Neuroscience Program at the Bowman
Gray School of Medicine in Winston Salem, NC tested PTT, a tropane derivative,
in nonhuman primates. These researchers found that the drug produces internal
cues similar to cocaine (that is, it is perceived as having properties like
cocaine), but it will not substitute for cocaine in monkeys trained to self-administer
cocaine. Also, PTT itself is not self-administered. Further, a single pretreatment
dose of PTT to monkeys trained to lever press for intravenous cocaine prevented
cocaine self-administration for 4 hours or more. The findings that the compound
is not self-administered, prevents cocaine self-administration, and has
subjective effects similar to cocaine, suggest that this tropane derivative,
or a drug like it, would be useful in preventing cocaine relapse without
compliance problems.
Biosensors
A recent analytical report describes a fiber optic system, known as an
immunobiosensor, capable of determining the cocaine content of coca leaf
samples in the field. It requires optical fibers which have been coated
with a monoclonal antibody, in this case, one generated against benzoyl
ecgonine-fluorescein as a sensor, and a portable fluorometer. Since cocaine
competes with benzoyl ecgonine-fluorescein in binding to the antibody, its
concentration is proportional to the decrease in fluorescence of the sensor
when both are present in a sample. The method is rapid, of reasonable precision,
and can be performed directly on acid extracts of coca leaves. Mohyee Eldefrawi,
Charles Helling et al., Biosensors and Bioelectronics, 12(2), pp.113 124,
1997.
Blood Barrier and Narcotic Peptides
The tritiated mu-selective antagonist CTAP has been shown in a rat brain
perfusion study to cross the blood brain barrier and the cerebral spinal
fluid barrier in amounts approximating those of tritiated morphine. CTAP
was bound to albumin in the perfusion medium and to rat serum protein, and
remained 63% "intact" in the brain after a 20 minute perfusion,
based on radioactive counts for the major HPLC peak in the detection system
used. It is suggested that the blood brain barrier transport is based on
passive diffusion rather than saturation transport, since the degree of
transport is unaffected by the presence of unlabeled CTAP. The compound
may have a potential role in narcotic addiction treatment. T. Abbruscato,
S. Thomas, V. Hruby, T. Davis, J. Pharmacology and Experimental Therapeutics,
280, pp. 402 409, 1997.
Homer, a PDZ-domain Protein Selectively Binds Metabotropic Glutamate
Receptors
Using differential cloning strategies, NIDA Grantee Paul Worley of Johns
Hopkins University discovered Homer, a novel, brain-specific, small protein
of 186 amino acids that differs from the conventional immediate early genes
in that it can directly modify cellular function.
Recently a new protein motif called PDZ domain has been shown to be important
in the targeting of a variety of membrane proteins to cell-cell junctions
including synapses. The most widely recognized member of this family of
proteins containing a PDZ domain is PSD95 which was originally identified
as a component of the postsynaptic density and recently shown to interact
with the C-terminus of the ionotropic glutamate receptor, N-methyl-D-aspartate
receptor (NMDA receptor). By virtue of the physical interaction between
PSD95 and the NMDA receptor, it is hypothesized that PSD95 functions to
restrict the spatial distribution of the NMDA receptor.
While sequence comparisons fail to demonstrate a significant homology
between Homer and PSD95, there were a number of functional similarities
that suggested they may possess a similar domain for interaction. First,
Dr. Worley and his coworkers demonstrated that Homer interacts with the
C-terminal 4 amino acids of metabotropic glutamate receptor, mGluR5. This
is the same C-terminal dependency for the interaction between PSD95 and
NMDA receptor. Second, Homer possesses a sequence GLGF that is present in
all PDZ family members and deletion of this region destroys the interaction
between Homer and mGluR5. Third, the crystal structure of PSD95 demonstrated
that the GLGF sequence forms the critical interaction site. Researchers
also demonstrated that Homer is remarkably selective for mGluR receptors
that mediate turnover of the phosphoinositide pathway (mGluR1 and mGluR5).
Immuno- localization of Homer showed that it is concentrated at synaptic
spines; this selective expression at neuronal spines suggests that Homer
may be targeted to the spines.
In an accompanying paper in the same issue of Nature, another team at
Johns Hopkins University led by Richard Huganir described GRIP, a larger
synaptic protein possessing seven PDZ domains that interacts with another
ionotropic glutamate receptor, a-amino-3-hydroxy- 5-methyl-4-isoxazole propionic
acid receptor (AMPA receptor). Because glutamate is the major excitatory
neurotransmitter in the mammalian brain, the identification of these two
proteins, Homer and GRIP, will undoubtedly advance our understanding of
the molecular mechanism of synaptic clustering of receptors.
Cocaine produces long lasting changes in the responsiveness of neurons
in the basal ganglia and neuronal circuits in the frontal cortex and midbrain.
These changes may play a role in the addictive potential of cocaine. Dr.
Worley and his coworkers discovered that Homer is markedly induced in neurons
of the basal ganglia in response to acute administration of cocaine. Homer
is also persistently upregulated in the frontal cortex following chronic
administration of cocaine. Metabotropic receptors have recently been demonstrated
to play an important role in motor and behavioral responses of the nucleus
accumbens and striatum and to be regulated by dopamine signaling from the
midbrain. Homer may be critical in regulating this interaction. Brakeman,
P.R., Lanahan, A.A., O'Brien, R., Roche, K., Barnes, C.A., Huganir, R.L.
and Worley, P.F., Nature, p. 284, March 20, 1997.
Opioid Receptor, Hematopoiesis, and Reproduction
Using a knockout mouse deficient in mu opioid receptor, NIDA grantee
Dr. Lei Yu and his colleagues at the Indiana University School of Medicine
recently have discovered that the mu receptor gene disruption affected a
number of aspects of the mouse physiology. Of particular interest is the
observation that a lack of the functional mu receptor resulted in changes
in both the host defense system and the reproductive system. They found
increased proliferation of hematopoietic progenitor cells in both bone marrow
and spleen, indicating a link between hematopoiesis and the opioid system,
both of which are stress-responsive systems. They also detected changes
in sexual function in male homozygous mice, including reduced mating activity,
decrease in sperm count and motility, and smaller offspring litter size.
These results suggest a novel role of the mu opioid receptor in hematopoiesis
and reproductive physiology, in addition to its known involvement in pain
relief. Journal of Experimental Medicine, April 21, 1997.
Gender-Related Differences
A recent article published by NIDA supported researchers demonstrates
pronounced gender-related differences in the antinociceptive effects of
morphine. These differences appear to reflect markedly enhanced CNS sensitivity
to morphine in males compared with females, as opposed to any intrinsic
differences in the bioavailability of morphine. Furthermore, these gender-related
differences appear to exist at both spinal and supraspinal levels. Their
findings also suggested that the acute effects of steroids play little role
in the gender-related differences observed; rather, it appears more probable
that the organizational effects of steroids, which occur in the late prenatal
and early postnatal stages and in large part determine gender-related distinctions
in males and females, may be more significant. Although the clinical significance
and the underlying mechanisms of these findings are unknown at this point,
these results may provide a means to begin examining gender-related differences
in abuse liability of psychoactive drugs. Cicero, T.J., Nock, B. and Meyer,
E.R. J Pharmacol. Exp. Therap. 279, pp. 767-773, 1996.
Gender-Related Differences
Dr. Marlene Wilson and her co-workers have examined gender related differences
and regional variations in the ability of neuroactive steroid derivatives
of progesterone, testosterone and glucocorticoids to alter physiological
gama-aminobutyric acid (GABA) responses in brain regions of male and female
rats. All four steroids examined increased GABA-activated chloride influx;
however, the maximal enhancement in GABA responses differed significantly
among brain regions. Limbic areas, such as hippocampus, amygdala and cortex,
displayed greater maximal responses to these steroids than hypothalamic
or cerebellar preparations. Since gender-related differences in neuroactive
steroid modulation of GABA responses were observed with the glucocorticoid
derivative, tetrahydrodeoxy-corticosterone (THDOC) but not with the progesterone
derivative, 3-alpha 5-alpha tetrahydro-progesterone (THP), it would further
suggest that the ability of THDOC and THP to potentiate GABA responses are
affected differentially by the hormonal milieu. Wilson, M.A. and Biscardi,
R. Life Sciences, In press.
Opiate Withdrawal Increases ProTRH Gene Expression in the Ventrolateral
Column of the Midbrain Periaqueductal Gray
The midbrain periaqueductal gray matter (PAG) has a critical role in
the modulation of behavioral and autonomic manifestations of the opiate
withdrawal syndrome. Utilizing multi disciplinary approaches including behavioral
studies, in situ hybridization histochemistry, immunohistochemistry and
radioimmunoassay, Dr. Ronald M. Lechan of the New England Medical Center
and his research team have demonstrated a nearly 5 fold increase in proTRH
gene expression in neurons of the ventrolateral column of the PAG following
naltrexone precipitated morphine withdrawal. The accumulation of immunoreactive
proTRH-derived peptides, but not the mature TRH tripeptide, was concomitantly
observed in these cells. These observations along with the fact that the
ventrolateral PAG mediates a hyporeactive pattern of behavioral and autonomic
reactions indicate that proTRH-derived peptides synthesized in neurons of
the ventro-lateral PAG may function as modifiers of opiate withdrawal responses.
Currently Dr. Lechan and his colleagues are attempting to elucidate the
anatomical connectivity of this unique population of opiate-responsive proTRH
neurons and to determine how these neurons are integrated into the control
system that responds to the hyperactive state of morphine withdrawal.
The observation that there is a nearly 5-fold increase in proTRH gene
expression in neurons of the ventrolateral column of the PAG following naltrexone
precipitated morphine withdrawal is a novel and exciting finding. It opens
up an entirely new area of opiate research and offers opportunities to design
new approaches for the treatment of opioid addiction and the withdrawal
syndrome. The data generated from this work could have significant clinical
relevance. Legradi,G., Rand, W.M., Hitz, S., Nillni, E.A., Jackson, I.M.D.,
and Lechan, R.M. Opiate Withdrawal Increases ProTRH Gene Expression in the
Ventrolateral Column of the Midbrain Periaqueductal Gray. Brain Research,
729, pp.10-19, 1996.
Isozyme-Specific Opioid-Induced Adenylyl Cyclase Supersensitization Acute
stimulation of opiate receptors inhibits adenylyl cyclase (AC) and reduces
cAMP in the cell, while chronic activation has been shown to lead to a progressive
increase in AC activity. This phenomenon, particularly manifest upon withdrawal
of the opiate agonist, is referred to as AC superactivation. The mechanism
of AC superactivation is not clear, although it seems to play an important
role in opiate addiction.
Zvi Vogel of The Weizman Institute of Science, Rohovot, Israel has transfected
AC of types I-VIII (currently ten AC isozymes are known) into COS cells
and studied the regulations of these AC isozymes by acute and chronic opiate
exposures. The results show that the various AC isozymes are differently
regulated by opiates. AC types I, V, VI and VIII are inhibited by acute
opiate exposure and super-activated following chronic exposure. AC II, IV
and VII are activated by acute opiate exposure and do not show the superactivation.
AC type III is not affected by the presence of opiates. AC-V yielded the
largest superactivation. This information will enable identification of
the particular AC isozymes which participate in opiate signaling , and the
reward system, in vivo and hence may facilitate the development of effective
treatment strategies for opioid abuse. Avidor-Reiss, T., Nevo, I., Saya,
D., Bayewitch, M. and Vogel, Z. Opioid-Induced Adenylyl Cyclase Supersensitization
is Isozyme-Specific. J. Biol. Chem. 272, pp. 5040-5047, 1997.
Evaluation of Discriminative Stimulus Anandamide was shown to produce
behavioral effects in mice characteristic of psychoactive cannabinoids;
however, differences have also been found between anandamide and delta-9
THC. Drs. Billy Martin and Raj Razdan, and their colleagues designed a study
to examine the discriminative stimulus effects of anandamide in rhesus monkeys
trained to discriminate delta-9 THC from vehicle. While anandamide failed
to produce reliable substitution for delta-9 THC and did not reduce response
rates, 2-methylarachidonyl-2 prime-fluoroethylamide (methylated fluoroanandamide,
a stable analog of anandamide), produced full dose-dependent substitution
for delta-9 THC at doses that caused no significant changes in response
rates. The results suggest that systematically-administered anandamide may
be metabolized in monkeys before behaviorally active concentrations could
reach the brain and further suggest that the metabolically stable analog
of anandamide, methylated fluoroanandamide, may aid in the discovery of
functional properties of the endogenous cannabinoid system. Wiley, J.L.,
Golden, K.M., Ryan, W.J., Balster, R.L., Razdan, R.K., and Martin, B.R.
Discriminative Stimulus Effects of Anandamide and Methylated Fluoroanadamide
in delta-9-THC-Trained Rhesus Monkeys. Pharmacol. Biochem. Behav., In press.
Hippocampus and GIRK1 Regulation of potassium channels by receptors coupled
to heterotrimeric G proteins such as the different types of opioid receptors
can have a profound effect on neuronal excitability by changing the duration
of an electrical impulse or action potential, altering the membrane potential
of neurons, and the number of action potentials fired by neurons. These
changes can indirectly alter the amount of neurotransmitter released by
a neuron which in turn can affect the excitability of neighboring neurons.
One family of potassium channels modulated by opioid receptors, dopamine
receptors, and other receptors coupled to G proteins that have been recently
cloned is the G coupled inwardly rectifying potassium channels (GIRKs).
To better understand the neuronal function of these GIRKS Dr. Charles Chavkin
and coworkers at the University of Washington used immunohistochemistry
and high resolution electron microscopy to define the subcellular localization
and cell type that expresses GIRK1 in the hippocampus. Chavkin reports that
GIRK1 immunoreactivity is regionalized within stratum lacunosum molecular
and the superficial striatum radiatum in the hippocampus. At the cellular
level electron microscopy revealed that GIRK immunoreactivity is found immediately
adjacent to asymmetric (excitatory type) post synaptic densities along dendritic
spines and shafts of pyramidal cells. Post synaptic densities are areas
where receptors are located at synapses, the junctions where neurons communicate.
Chavkin suggests that the localization of GIRK1 in dendritic shafts and
spines could play a significant role in modulating the post synaptic responses
at excitatory synapses by decreasing the likelihood of propagation of synaptic
currents from distal dendrites. Drake, C.T., Bausch, S.B., Milner, T.A.,
Chavkin, C. GIRK1 Immunoreactivity is Present Predominantly in Dendrites,
Dendritic Spines, and Somata in the CA1 Region of the Hippocampus. Proc.
Natl. Acad. Sci. USA. 94, pp. 1007-1012, 1997.
µ3 opiate receptor and Morphine Morphine causes human monocytes,
granulocytes, and endothelial cells as well as molluscan immunocytes and
microglia to change from a flattened to a rounded shape. This change in
morphology produced by morphine may play an important role in the ability
of morphine to reduce inflammation by altering cell adhesion and cell migration
through endothelial cells. Dr. Harold Magazine and his colleagues examined
the role of nitric oxide in morphine-induced rounding of monocytes, granulocytes,
and endothelial cells. Dr. Magazine and his colleagues showed that morphine
induces the synthesis of nitric oxide. The synthesis of nitric oxide appears
to play an important role in morphine-induced rounding because exposure
to morphine elicits nitric oxide production, nitric oxide agonists induce
cell rounding, and nitric oxide antagonists block morphine induced rounding.
The synthesis of nitric oxide by morphine was blocked by naloxone; however,
opioid peptides failed to elicit the production of nitric oxide. Magazine
suggests that the morphine-induced NO release may be mediated by the activation
of the opiate alkaloid-selective, opioid peptide insensitive m3 receptor
and that the coupling of nitric oxide to the m3 opiate receptor has been
conserved throughout evolution. Magazine, H.I., Liu, Y., Bilfinger, T.V.,
Fricchione, G.L., and Stefano, G.B. Morphine-Induced Conformational Changes
in Human Monocytes, Granulocytes, and Endothelial Cells and in Invertebrate
Immunocytes and Microglia are Mediated by Nitric Oxide. Journal of Immunology,
156, pp. 4845-4850, 1996.
Separation and Detection of Neurotransmitters and Neurochemicals in
Ultra-Small Volumes
Richard N. Zare, Ph.D., Professor of Chemistry at Stanford University
who co-authored a paper last summer suggesting possible fossil evidence
of life on Mars is also a NIDA grantee who is devising methods to separate
and detect chemical compounds in single synaptic vesicles that normally
contain ultra small volumes of neurotransmitters. This is important because
biological systems are partitioned into small compartments from single cells
(pico-to femtoliters) to single mitochondria and vesicles (attoliters).
Analysis of the composition of individual synaptic vesicles will provide
insight into the basic mechanisms by which synaptic transmission is modified.
In a paper published in Science he reports significant progress in separating
neurotransmitters by capillary electrophoresis whose biological activity
and presence are detected by patch-clamp electrophysiology. Capillary electrophoresis
can be used to separate compounds dissolved in volumes as small as the low
femtoliter range and patch clamp electrophysiology can detect the presence
of a small number of molecules of agonist that can open a single ion channel.
Orwar, O., Jardemark, K., Jacobson, I., Moscho, A., Fishman, H.A., Scheller,
R.H., and Zare, R.N. Patch-Clamp Detection of Neurotransmitters in Capillary
Electrophoresis. Science, 272, pp. 1779-1782, 1996.
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