Edward D. French - US grants

The presence of specific high-affinity binding sites for phencyclidine (PCP) in brain and the fact that PCP can produce in man profound behavioral changes often resembling a schizophrenic psychosis requires that substantial efforts be made to determine the effects of PCP on central nervous system processes. The overall goal of the studies described in this proposal is to examine the action of phencyclidine and a recently isolated PCP-like brain peptide on normal neurobiological processes within the rat central nervous system. Specifically, dopamine cell bodies (A10) in the ventral tegmental area and the mesolimbic-mesocortical areas innervated by those DA neurons have been chosen for study. These pathways have been strongly implicated as anatomical and biochemical substrates likely to be involved in schizophrenia. Using electrophysiological methods of extracellular unit recording, microiontophoretic-micropressure administration, and electrical stimulation of selected pathways to identifiable target cells, PCP and the PCP-like peptide will be studied and compared for their selective actions on specific sites and chemical modes of synaptic transmission. Pharmacological manipulations and selective lesioning techniques will be used to discriminate between a preferential presynaptic or postsynaptic site of action as well as to characterize interactions of the test compounds with dopaminergic and noradrenergic systems. Finally, we will determine if PCP and PCP-like peptide neuronal effects differ between naive animals and those chronically treated with systemically administered PCP. The projects outlined here should enable us to characterize the sites, mechanisms and functional consequences of PCP and PCP-like peptide action on selected neuronal pathways. The resulting data will provide essential information concerning PCP's unique pharmacology. In turn, such findings may be useful for the understanding of specific chemical processes involved in the etiology of schizophrenia.

The fact that phencyclidine (PCP) continues to be an abused drug which can produce in man profound alterations in behavior, requires that substantial efforts are crucially necessary to understand its effects on central nervous system function. The overall goal of the studies described in this proposal is to determine the mechanism(s) of action of PCP on normal neurobiological processes within the rat central nervous system. Specifically, the effects of PCP on midbrain dopamine neurons (A10) within the ventral tegmental area (VTA) and the influence of VTA afferents on those cells have been selected for study. As with other drugs of abuse (e.g. cocaine), the A10- dopamine neurons and the mesolimbic-mesocortical structures they innervate have been implicated as anatomical and biochemical substrates likely to be involved in the reinforcing properties of PCP. Also, these same structures are considered underpinnings in the pathophysiology of schizophrenia and, as such, plausibly linked to the psychosis-like effects frequently elicited by PCP. In the present proposal, we will use electrophysiological methods of extracellular recording combined with selective lesions and pharmacological manipulations to determine the transmitter identity contribution to either the intensification or diminishment of PCP's unique excitatory/inhibitory effects on VTA neurons. In addition, intracellular recordings from neurons in the in vitro VTA brain slice preparation will be used to provide an indepth analysis of the effects of acute and chronic PCP on neuronal membrane properties, chemical synapses, and voltage sensitive ion conductances of the neuronal A10 dopamine cells. The projects outlined here should enable us to characterize the consequences of PCP on VTA A10 neurons directly, and the relative contributions made various by afferent inputs to these effects. The data derived from these experiments will provide essential information regarding PCP's unique pharmacological actions on this midbrain dopamine containing system. This in turn may help delineate the neurobiological consequence of phencyclidine abuse, and as such possibly lead to a more rational design of treatments for the various behavioral effects and psychopathologies related to PCP abuse.

Approximately 10 million persons a=use marijuana on a monthly or more frequent basis making it the most commonly used illicit drug. 2% of 12th graders use marijuana on a daily basis. Moreover, marijuana use at even younger ages (eight graders) increased a full percentage point in 1992. Healthy People 2000 refers to marijuana use as typifying the age-at-first- use phenomenon whereby its use prior to age 15 relates to its greater abuse and that of other drugs after 15. These facts coupled with the knowledge that psychoactive cannabinoids produce a myriad of behavioral effects and alterations in information processing within the CNS through actions at endogenous cannabinoid receptors requires that substantial efforts should be made to understand its effects on central nervous system function. The overall goal of these studies is to determine the mechanism(s) of action of the psychoactive cannabinoid, delta-9-tetrahydrocannabinol (delta-9-THC) on those dopamine containing systems thought to be pivotally involved in the reinforcing and psychotomimetic properties of drugs of abuse. Specifically, the effects of acute and chronic administration of delta-9- THC, and the endogenous brain constituent anandamide, on midbrain dopamine neurons (A9 & A10) have been selected for study. In the proposed experiments we will use single neuron extracellular recordings to determine the effects of various cannabinoids (psychoactive and non-psychoactive) on the rate and patterns of action potential generation, and importantly the effects of prolonged exposure to delta-9-THC on these parameters. In addition, intracellular recording from neurons in the in vitro midbrain slice preparation will be used to analyze the effects of acute and chronic delta-9-THC administration on neuronal membrane properties and excitability. These projects should enable us to characterize the consequences of delta-9-THC exposure on midbrain dopamine neurophysiology. The data derived from these experiments will provide information relevant to delta-9-THC's pharmacological effects on those dopamine neurons implicated in the emotional, cognitive and sensorimotor changes occurring with marijuana use.

Opioids are known to have important behavioral and physiological actions that are mediated by interactions with neurons in the central nervous system. However, while the behavioral actions of the opioids, including analgesia and euphoria, are well-known, the functional roles these molecules play as neuromodulators in the mammalian brain remain poorly understood. The following experiments will evaluate the actions of opioids, acting at pharmacologically defined mu-, delta- and kappa-opioid receptor subtypes, as modulators of synaptic transmission in the CA1 and dentate gyrus regions of the hippocampus, and in another brain region known as the periaqueductal gray. The experiments described in this proposal will utilize whole-cell patch clamp electrophysiological techniques to examine the effects of opioids on individual neurons in rat brain slice preparations. The first set of experiments will focus on the mechanisms through which mu-, delta-, and kappa-opioid receptor agonists act to decrease evoked and spontaneous synaptic transmission in the hippocampal slice. In particular, these experiments will examine the contributions made by distinct voltage-dependent calcium channels, intracellular calcium stores, and nitric oxide in supporting synaptic transmission and the modulation of this process. The second set of experiments will determine the effects of opioid agonists directly on subpopulations of gamma-aminobutyric acid (GABA)-containing neurons (interneurons) in the CA1 region of the hippocampus, and the cellular mechanisms these receptors utilize to affect these cells. These experiments will be aided through the use of differential interference contrast microscopy to identify these neurons in living brain slices. The third set of experiments will determine what mechanisms account for the ability of certain non-opioid peptides to attenuate the behavioral and cellular actions of the opioids. Thus, we will determine the effects of the peptides cholecystokinin (CCK) and neuropeptide FF on neurons found in the hippocampus, and the periaqueductal gray. The investigations described in this grant proposal should improve our understanding of the specific receptor subtypes that the opioids act upon, the cellular mechanisms mediating their responses, the roles opioids may play as neuromodulators in the brain, and the mechanism of their interaction with other non-opioid peptides. in addition, since limbic structures like the hippocampus have been shown to participate in normal and abnormal behavioral states such as learning, epilepsy, and emotional behavior, and the periaqueductal gray has been shown to be important in mediating opioid analgesia, these studies may yield new insights as to the interaction between opioids and these phenomena.

Marijuana (Cannabis sativa) is the most widely used illegal drug of abuse in the U.S. The active ingredients of this drug, which include delta9-tetrahydrocannabinol (THC), are collectively known as cannabinoids (CBs). Several scientific advances in the last decade, including the cloning of specific CB receptors, and the identification of endogenous agonists for these receptors, provide an opportunity to evaluate the function of the CB system in regulating neuronal activity both acutely, and during long-term CB exposure. One of the primary consequences of CB receptor activation is the presynaptic inhibition of the major inhibitory (GABA) and excitatory (glutamate) amino acid neurotransmitters in the mammalian brain. Our own published and preliminary data examining CB actions in brain slices suggest that CB receptor activation causes a pronounced inhibition of GABAergic synaptic transmission in both the hippocampus and the nucleus accumbens. These effects in the hippocampus may be at least partly responsible for the well-known disruption of short-term memory caused by marijuana consumption, whereas the direct effects observed in the nucleus accumbens may be responsible for at least some of the rewarding or pleasurable properties of this drug that are presumed to sustain its use in humans. The goal of the experiments described in thus proposal is to understand the neurobiological substrates upon which CB drugs act to alter CNS function, with particular emphasis on the modulation of synaptic transmission: in these two relevant brain: areas. To this end, these studies will identify the molecular mechanisms through which CBs alter synaptic transmission in these specific neuronal circuits, will determine what role endogenously occurring CBs play in regulating neuronal activity, and will establish whether physiological correlates of CB tolerance and dependence can be identified so that we may further understand the neurobiological consequences of long-term marijuana exposure. These experiments will utilize electrophysiological techniques, combined with differential interference microscopy to identify specific neurons in living brain slices. We will then conduct studies with several recently developed pharmacological agents that disrupt endogenous CB catabolism and re-uptake by neurons. In addition, single-cell anatomical techniques will be incorporated into these studies so that the neurons involved in these interactions can be conclusively identified based upon morphological and histochemical features. These studies will significantly increase our understanding of the neurobiology of CBs, and will provide information that should prove relevant to understanding the cellular consequences of long-term CB exposure in humans.