Matthew P. Galloway, Ph.D. - US grants

cocaine; psychotropic drugs; neurotransmitter metabolism; amphetamines; brain mapping; dopamine; neurochemistry; dosage; synapses; antiadrenergic agents; neural transmission; tissue /cell culture; high performance liquid chromatography;

In monoamine neurons, the potential importance of autoregulation of neuronal activity has received varied attention. For example, although the pharmacological concept of dopamine autoreceptors is slightly more than 10 years old, clinical trials are actively attempting to exploit this important regulatory step of chemical neurotransmission in diseases associated with hyperdopaminergic activity. In contrast, clinical studies utilizing direct, selective pre- or postsynaptic serotonin (5-HT) receptor modulation are relatively rare. On reason for this deficit is the lack of basic, in vivo neuropharmacological studies specifically directed at the question of 5-HT agonist-induced modulation of presynaptic 5-HT function. The objective of the studies proposed herein is a further understanding of autoregulation in central 5-HT neurons and the functional consequences of such mechanisms in rats. Proposed studies to be conducted in vivo are designed to examine the mechanism by which 5-HT agonists attenuate 5-HT synthesis, the role of impulse flow in controlling 5-HT synthesis, and the potential role of nerve-terminal 5-HT autoreceptors modulating transmitter synthesis. Moreover, parallel issues concerning 5-HT synthesis and release will be addressed in vitro using brain slices as a simplified system to study isolated nerve terminals. In addition to further defining the neurobiology of 5-HT autoregulation, these studies are designed to elucidate the functional pharmacology of novel 5-HT agonists such as 8-hydroxy-2-(di-n-propylamino)tetralin (8-OHDPAT). After studies on the mechanism of 5-HT autoregulation are in progress, the longer-term objectives of the proposal concerning changes in autoreceptor responsiveness will be initiated. Specifically, experiments will test the hypothesis that repeated exposure of terminal 5-HT autoreceptors to elevated levels of 5-HT (subsequent to chronic treatment with reuptake blockers) will lead to an alteration in autoreceptor modulation of neuronal activity. Thus, these studies will refine and extend concepts of 1) 5-HT autoregulation, and 2) the possible mechanisms by which chronic antidepressants modify functional presynaptic 5-HT activity.

The ability of either cocaine or amphetamine to enhance monoaminergic neurotransmission via inhibition of neuronal uptake is thought to be one mechanism that contributes to their psychotropic properties. The proposed experimental plan seeks to understand potential changes in synaptic neurobiology as a consequence of increased transmitter levels. Specifically, studies are proposed to 1) define the interaction of serotonin (5HT) and dopamine (DA) in areas of the brain relevant to drug self-administration such as the medial prefrontal cortex and nucleus accumbens, 2) determine the consequence of repeated cocaine or amphetamine on presynaptic autoreceptor function, postsynaptic DA sensitive adenylate cyclase, and the relative balance of D1/D2 influence at each site, 3) define further the mechanisms by which amphetamine and cocaine alter DA or 5HT synthesis in intact nerve terminals. Objectives will be realized with a combination of neurochemical techniques, including DA and 5HT synthesis in vivo, synthesis and release in striatal brain slices in vitro, brain microdialysis to measure transmitter release in vivo, and adenylate cyclase determinations. Advances in understanding the neurobiology of cocaine and amphetamine have not kept pace with the exponential increase in drug abuse. Nonetheless, successful, pharmacological-based withdrawal programs for drug abstinence require knowledge of the molecular mechanisms associated with drug self- administration and craving. By examining the effects of both acute and chronic exposure to these drugs of abuse, the proposed experiments will yield insight into both the mechanism of action and the functional consequences of stimulant abuse.

The overall purpose of the research program is a further understanding of the neurochemical basis of drug abuse. In particular, the experimental approach consists of two objectives: 1) To gain insight into the regulatory functions of the neuronal systems that mediate drug reinforcement, and 2) to determine the effects of repeated exposure to either cocaine or amphetamine on these systems. It is anticipated that the convergence of these two strategies will generate knowledge required for the rationale development of clinically useful, therapeutic agents capable of decreasing the craving and or reward associated with drugs of abuse. The proposed studies constitute a program to examine the interaction of serotonin and dopamine with each other and their response to drugs of abuse such as cocaine, amphetamine, and MDMA. The studies involve protocols to examine transmitter release and synthesis utilizing brain slices (in vitro) and microdialysis (in vivo). The specific objectives set forth to investigate the above issues are: A) What is the pharmacological basis mediating the serotonin (5HT)- facilitation of dopamine (DA) release at the level of the nerve terminal. Additionally, experiments are designed to elucidate the second messenger system of the 5HT receptor and to determine potential involvement of either potassium or calcium fluxes. Subsequent experiments focus on the role of endogenous 5HT on DA release, and systemically administered 5HT agonists on both DA release and synthesis. Finally, does chronic cocaine alter the efficacy of 5HT to facilitate DA release. B) The second major goal of the proposal tests the possibility that DA autoreceptors are the functional counterpart of the recently discovered D3 receptor. In vitro (brain slices) and in vivo protocols are presented to characterize the pharmacological actions of selective DA agonists and antagonists at DA autoreceptors. Lastly, experiments are proposed to discover if repeated exposure to either cocaine or amphetamine modify the functional responsivity of nerve terminal DA autoreceptors.

DESCRIPTION (provided by applicant): Despite a wealth of preclinical information on stimulant-induced adaptations (plasticity or toxicity) in the CNS, the translation of that knowledge to clinical neuroscience has been hampered by the inability to accurately assess human brain function in vivo. The advent of magnetic resonance (MR) neuroimaging techniques, with increasingly high-resolution and sensitivity, has revolutionized research strategies in studies of human substance abusers. However the physiological significance of MR-derived measures in humans is not yet fully understood. In fact, preclinical MR studies of CNS function lag far behind the number of MR studies with humans, despite the fact that preclinical studies are amenable to controlled pharmacological manipulation and independent verification. With an emphasis on stimulant abuse, the present proposal addresses the physiological significance of several neurochemicals that can be measured with proton MR spectroscopy (1H-MRS). By applying state-of-the-art MR techniques (at 11.7 T) to proven paradigms of drug study, the results should provide 1) novel insight into the neurobiology and neurotoxicology of acute and repeated exposure to stimulants, and 2) interpretations and hypotheses that can be directly translated into clinical utility. In an effort to validate the physiological significance of MR-visible compounds, a major objective of the research plan is to use high-resolution magic angle spinning (HR-MAS) 1H-MRS to determine ex vivo the basic neuropharmacology of the MR-visible neurochemical profile in rat brain. Thus, in discrete monoamine-related areas of the rat brain, we plan to determine the effect of in vivo pretreatment with 1) specific neurotoxins, 2) three psychostimulants with distinct mechanisms of action (MDMA, cocaine, and methamphetamine), and 3) D1 and 5HT2 receptor ligands on the MR-visible neurochemical profile. In each case, direct comparisons will be made between the 1H-MRS observations and those determined with HPLC-EC. Neurochemicals that are readily measured by HR-MAS 1H-MRS reflect several key cellular functions including neuronal viability (Nacetylaspartate), neurotransmission (glutamate, glutamine, GABA), energy status (lactate, creatine), 2 nd-messenger pathways (myo-inositol), and membrane phospholipid metabolism (phosphocholine and glycerophosphocholine). After establishing the pharmacological and physiological parameters that modulate the MR-visible neurochemical profile, a series of experiments will use in vivo 1H-MRS and MRI (at 11.7 T) to determine the longitudinal effects of stimulant exposure. A critical component of validating the in vivo observations will be a direct comparison to the results obtained with HR-MAS 1H-MRS. MRI T2 images, water diffusion maps, and in vivo neurochemistry obtained in a longitudinal design in vivo, are expected to define regionally selective lesions following drug exposure. MDMA abuse has increased unabated in adolescents, despite convincing evidence that street doses are toxic to both 5HT and DA neurons in rodents and monkeys, up to 7 yrs after drug cessation. The potential loss of 5HT innervation subsequent to MDMA exposure may have profound effects on the emergence of mood disorders in the relatively young cohort of MDMA users. Increased use, combined with preclinical toxicity, speaks to the necessity to resolve the question of MDMA toxicity in humans. MR neuroimaging, especially 1H-MRS, provides a non-invasive window into human brain function. Development of a neurochemical fingerprint describing the effects of 3 stimulants will provide a solid basis for hypothesis-driven investigations of stimulant-induced plasticity and/or toxicity in the CNS.