Barbara Sorg - US grants

The repeated, intermittent administration of cocaine and other psychostimulants produces an augmentation in locomotor behavior upon subsequent exposure to these drugs, a phenomenon known as behavioral sensitization. Associated with behavioral sensitization by cocaine is an alteration in dopamine neurotransmission primarily in the mesolimbic dopamine system. This system consists of dopamine cell bodies located in the A10 region of the ventromedial mesencephalon and their projections to the nucleus accumbens. The molecular mechanisms mediating the development of sensitization to cocaine in the mesolimbic dopamine pathways are not characterized, but it is known that changes occurring in the cells within the A10 region are critical for the development of sensitization. As a first step towards examining the phenomenon of sensitization at the molecular level, changes in the levels of mRNAs coding for tyrosine hydroxylase (TH), cholecystokinin (CCK), and neurotensin (NT) will be assessed. All of these gene products are localized in the A10 region and are important modulators of dopamine cell function and therefore may play a crucial role in mediating sensitization. Messenger RNA coding for TH, the rate-limiting enzyme in dopamine synthesis, is decreased in animals which demonstrate behavioral sensitization to cocaine. This effect has been demonstrated early after withdrawal, but whether it is a permanent effect is not known. this proposal will examine the time-course of changes in TH mRNA levels in the A10 region by Northern blot analysis, and whether these changes are accompanied by altered TH enzyme activity in the A10 region and nucleus accumbens, as measured by DOPA accumulation using in vivo microdialysis. Subsequently, it will be determined whether altered TH mRNA levels are a result of reduced TH gene transcription and/or a reduction in TH mRNA half- life. Finally, the same Northern blots used to measure TH mRNA levels will be used to examine the levels of CCK and NT mRNAs in the A10 region at various times following acute and chronic cocaine. These studies will define the changes in gene expression of three molecules that are important for modulating mesolimbic dopamine transmission and thus may provide insight into the altered function of dopamine neurons in response to repeated cocaine exposure. Defining the molecular mechanisms relevant to sensitization by cocaine in an animal model could help explain the mechanisms involved in the development of paranoid psychosis in humans, and may have implications for pharmacological treatment of this disorder.

Repeated exposure to psychostimulants, such as cocaine and amphetamine, produce an augmentation in the motor-stimulant response that increases with time, a phenomenon known as behavioral sensitization. Repeated exposure to stressful stimuli also produces an enhancement in the locomotor response to subsequent psychostimulants. the neurochemical changes underlying behavioral sensitization have focused on the mesocorticolimbic dopamine system, including the dopamine cell bodies in the ventral tegmental area (VTA) and projections to the medial prefrontal cortex (mPFC) and nucleus accumbens (NAcc). Recent cross-sensitization studies using in vivo microdialysis show that, in general, repeated stress or repeated cocaine augments the cocaine- or stress-induced increase in extracellular dopamine levels in the NAcc, while in the mPFC, an apparent tolerance to subsequent stimuli occurs. The augmentation in dopamine levels in the NAcc is known to be associated with behavioral sensitization but the inhibitory role of mPFC dopamine on locomotor activity has not been examined in the context of behavioral sensitization. The present studies propose to test the hypothesis that the apparent tolerance of mPFC neurons to subsequent stimuli contributes to the hyperlocomotion and associated increase in dopamine transmission in the NAcc in rats sensitized to repeated stress or cocaine. the first goal of this proposal is to characterize the changes that occur in extracellular dopamine levels in the mPFC following repeated footshock stress and repeated cocaine. In addition to measurement of basal dopamine levels in rats administered repeated stress and cocaine, in vivo dialysis measurements of mPFC extracellular dopamine levels will be performed following early, middle, and late withdrawal periods to determine if there are temporal changes in the tolerance effect. Secondly, mechanisms that may be mediating the tolerance effect in the mPFC will be examined in both the mPFC terminal field and in the dopamine cell body region in the VTA. The terminal field will be examined by local perfusion of K+, amphetamine, and cocaine through the dialysis probe. Additionally, dopamine reuptake will be tested in vitro in mPFC synaptosomes. Regulation by neurotransmitters in the VTA will be investigated by intra-VTA injection of NMDA, substance P and opioid agonists, and neurotensin, which modulate mPFC dopamine transmission. The final goal of this proposal is to determine the role of mPFC dopamine in the development and expression of behavioral and neurochemical sensitization. The first approach is to destroy mPFC dopamine terminals prior to repeated stress and cocaine treatment, and the second approach is to locally administer D1 and D2 receptor agonists into the mPFC just prior to a cocaine challenge to determine whether these treatments alter the levels of extracellular dopamine in the NAcc and associated increase in locomotor activity. Elucidation of the interactive role of mPFC dopamine in mediating sensitization to stress and cocaine has potential implications for understanding idiopathic as well as stress- and psychostimulant-precipitated psychoses, including paranoid schizophrenia, panic disorder, and posttraumatic stress disorder.

DESCRIPTION: (Adapted from the Investigator's Abstract) Chemical Intolerance (CI) in humans is defined as the inability to tolerate environmental chemicals due to experience of symptoms associated with those chemicals. Although CI is prevalent in individuals claiming that prior chemical exposures produced their intolerance, the underlying etiology remains unknown. A high percentage of individuals reporting CI present with psychiatric symptomatology, and some investigators have suggested that CI is an atypical form of posttraumatic stress disorder (PTSD). The proposed work emphasizes the parallels between PTSD and CI in the development of a potential animal model for CI. Animal models of four phenomena appear most suitable for examining PTSD and, therefore, CI: 1) sensitization of the central nervous system (CNS), 2) conditioned fear, 3) extinction of a conditioned fear response and 4) avoidance of a conditioned stimulus. In support of CI as a PTSD-like phenomenon, recent studies in this laboratory have found that rats given repeated daily inhalation of low levels of formaldehyde (Form) demonstrated long-term cross-sensitization to cocaine-induced locomotion. In addition, daily Form treated rats exhibited increased avoidance to subsequent Form, and a reduced ability to extinguish a conditioned fear response to an odor paired with foot shock. Since Form does not penetrate beyond the upper airway, it is likely that Form serves as a stressor to produce sensitization. For the proposed studies, four exposure doses of Form will be employed. The first specific aim will test the hypothesis that repeated Form exposure produces increases in stress/anxiety responses after subsequent Form presentation in the same or different environment from which original exposures occurred. Serum corticosterone levels and behaviors reflective of anxiety/stress will be monitored during and after re-exposure to Form. The second specific aim will examine the hypothesis that repeated Form induces sensitization to later Form presentation and cross-sensitization to cocaine via the hypothalamic-pituitary-adrenal (HPA) axis. Cross-sensitivity to cocaine will be monitored behaviorally, and nucleus accumbens dopamine levels will be measured by in vivo microdialysis. Form sensitization to itself will be measured by assessment of nucleus accumbens dopamine levels and also behaviorally by monitoring avoidance responses to later Form presentation. This aim will also perform adrenalectomy in one-half of rats to determine whether an intact HPA axis is required for Form-induced effects. The third specific aim will test the hypothesis that repeated Form exposure produces a decreased ability to extinguish a conditioned fear response. The specificity of the effect (context vs odor or tone) will also be explored. The study of behavioral and neurochemical sensitization, conditioned fear and extinction in Form-exposed rodents will provide a mechanistically-based animal model system for studying the development and maintenance of CI in humans.

DESCRIPTION: (Adapted From The Applicant's Abstract) Repeated exposure to psychostimulants or stress produces a progressive and enduring increase in psychostimulant-induced locomotor activity in rodents, a phenomenon referred to as behavioral sensitization. Extracellular dopamine levels in the nucleus accumbens are enhanced in rats sensitized to stress or cocaine, while extracellular dopamine levels in the medial prefrontal cortex (mPFC) are diminished. Thus, dopamine in the mPFC demonstrates tolerance rather than sensitization in response to a subsequent cocaine or stress challenge. Since dopamine in the mPFC inhibits mPFC excitatory amino acid (EAA) efferents to subcortical sites and produces inhibition of stimulated locomotion, recent studies in this laboratory determined whether the tolerance of mPFC dopamine responsiveness to cocaine or stress may contribute to sensitization of locomotor activity. The effects of intra-mPFC microinjection of d-amphetamine on cocaine-stimulated locomotor activity was measured in control and cocaine sensitized rats, and the results suggest an important contribution by the mPFC for the expression of cocaine sensitization. The experiments are designed to test the hypotheses that 1) tolerance of the mPFC dopamine response in sensitized rats contributes to the expression of behavioral sensitization, and this response may be importantly modulated by glutamate in the mPFC, and 2) repeated stress and/or cocaine pretreatment alter the ability of dopamine agonists administered into the mPFC to regulate subsequent cocaine-induced activity. The proposed work will examine regulation of the expression of stress- and cocaine-induced behavioral sensitization by the mPFC with emphasis on regulation of mPFC dopamine tolerance and its contribution to behavioral output. For all studies, the treatment groups will be naive, daily sham shock, foot shock, saline or cocaine. The first specific aim focuses on the modulation of stress- and cocaine- induced tolerance of dopamine in the mPFC by glutamate. The second specific aim will examine modulation of sensitization by local administration of dopamine receptor agonists into the mPFC. The third specific aim will determine which specific pathway(s) may be altered in the regulation of behavioral sensitization to stress and cocaine with regard to EAA projections from the mPFC to the nucleus accumbens vs. those efferents from the mPFC to the ventral tegmental area (VTA). Incorporation of the mPFC circuitry into sensitization studies should lead to an increased understanding of the mechanisms by which stress predisposes individuals to initiation and reinstatement of drug taking behavior and psychoses.

DESCRIPTION: (provided by the applicant) Repeated cocaine exposure induces neural plasticity as implied by the development of dependence and sensitization. An under explored but critical aspect of cocaine-dependent plasticity is the impact of cocaine on proteins involved in synaptic remodeling during drug-seeking behaviors. This proposal focuses on the proteins that regulate the extracellular matrix (ECM). These proteins are critical for dynamic processes involved in synaptic reorganization during learning. We envision that the ECM acts as a scaffold to optimally align pre- and postsynaptic elements, which must be transiently degraded during synaptic remodeling. Since drug abuse is believed to involve a learning process, molecules involved in remodeling should be altered in brain areas implicated in drug abuse. This notion is supported by recent studies reporting morphological changes in brain regions critical for drug-taking behavior. We hypothesize that these morphological changes require shifts in the expression of ECM proteins, which are dependent on the regulators, matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs). Recent work in our laboratories has demonstrated that activity of the enzyme MMP-9 in the hippocampus is correlated with active learning of a water maze spatial learning task. In addition, acute cocaine treatment increases MMP-9 activity in the nucleus accumbens and medial prefrontal cortex, with a concomitant decrease in the ventral tegmental area. In contrast, only small changes were found in the substantia nigra and striatum, suggesting a specificity of cocaine's effects on plasticity of mesocorticolimbic pathways. The proposed studies will assess the level of expression of MMPs and TIMPs critical for remodeling processes believed to occur during learning and extinction of a cocaine conditioned place preference (CPP) task. These studies will determine which brain regions exhibit plastic changes associated with the pairing of contextual information with cocaine, whether or not the same brain sites are involved in the extinction of cocaine CPP behavior, and if these molecules can be further altered once initial learning of the CPP task has taken place. We postulate that repeated cocaine initially produces synaptic rearrangement in specific brain regions linked to drug craving and addiction, and that changes in MMPs/TIMPs are indicators of this rearrangement. Moreover, subsequent to repeated cocaine treatment, there may be an attenuation or loss of neural plasticity in these brain sites that contributes to the long-lasting nature of addiction.

posttraumatic stress disorder; olfactory stimulus; behavioral habituation /sensitization; conditioning; chemical stimulation; model design /development; disease /disorder model; odors; hypothalamic pituitary adrenal axis; formaldehyde; chemical hypersensitivity; cocaine; fear; physiologic stressor; psychological stressor; chemosensitizing agent; behavioral /social science research tag; laboratory rat; adrenalectomy;

[unreadable] DESCRIPTION (provided by applicant): We speculate that the long-lasting nature of cocaine and other drug addiction may be due to drug-induced changes that ultimately produce a loss of plasticity in specific brain regions involved with craving and relapse. A novel conceptualization for the treatment of cocaine addiction is that restoration of neural plasticity during the period when the individual is learning to stay away from drugs (the extinction phase) may prevent relapse to drug-seeking or drug-taking behavior. The proposed studies focus on providing a new framework for suppressing relapse by manipulating the extinction period of drug-seeking behavior. Thus, we may take advantage of a critical window during which learning to avoid drug seeking/taking may be enhanced by simultaneous induction of neural plasticity. Electroconvulsive therapy (ECT) or seizure (ECS) is now viewed as successful in treating severe depression because of its ability to increase neural plasticity in the brain. Key molecules thought to be involved in ECS-induced plasticity are growth factors. Because extinction is new learning, alteration of growth factors during the extinction phase of cocaine-induced conditioned place preference (CPP) may reduce the reinstatement of drug-seeking behavior in rats. Our studies show that ECS given during the extinction phase suppresses reinstatement of cocaine-primed CPP compared with controls. The same ECS treatments delivered prior to the extinction phase did not alter reinstatement. The primary goal of these studies is to optimize the parameters of ECS treatment to produce maximal suppression of cocaine-primed reinstatement. The proposed studies center on the main hypothesis that ECS treatment is most effective in suppressing cocaine-primed reinstatement when given during the extinction phase. Specific Aim 1 will determine the effects of seizure duration and number of days of ECS treatment on reinstatement of cocaine-primed CPP. Seizure duration will be altered by changing the pulse dose and duration of ECS and depth of anesthesia during ECS treatments. Specific Aim 2 will optimize the timing of ECS treatment for its ability to suppress reinstatement of cocaine-primed CPP. The timing of ECS will be shifted to before, during or after the extinction phase. This Aim will also test ECS timing (in hours) relative to extinction to determine if the timing of ECS is important for the suppression of reinstatement. Our studies indicate that ECT in humans may be an option for some cases of cocaine addiction, as it is for severe depression in humans. However, our long term interest is to understand the critical neurobiological consequences of ECS that could lead to treatment for cocaine addiction. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Persistent drug-taking behavior involves consolidation of memory for the drug and drug-associated cues and contexts. When a memory is reactivated (retrieved), that memory becomes labile and susceptible to disruption by amnestic agents present at the time of reactivation. Drug abuse studies in rodents indicate that reconsolidation can be disrupted, and this is manifest as suppressed drug-seeking behavior when animals are subsequently primed with the same stimulus used to reactivate the memory. However, most studies have focused on drug-induced conditioned place preference (CPP), in which only a few drug injections are given;thus, the memories may be easier to disrupt. Few labs have focused on the rat self-administration model, which has higher face-validity for human addiction. Self-administration studies showed that reconsolidation of a memory for the drug cue or context can be disrupted by certain agents. However, no studies have disrupted reconsolidation of the memory associated with the drug itself, when the drug is present during reactivation and subsequent reinstatement. This is significant because the drug induces powerful reinstatement in rats and augments relapse in humans. We focus on matrix metalloproteinases (MMPs), which are emerging as key molecules in the neuroplasticity of learning and memory. MMPs are a family of metallopeptidases that direct changes in synaptic morphology via degradation of the extracellular matrix (ECM). We previously showed that an inhibitor of MMPs blocked reconsolidation of a cocaine-associated memory in CPP studies, and that MMP-9 activity was elevated in the medial prefrontal cortex (mPFC) upon reactivation of this memory. Exciting preliminary self-administration studies show that injection of an MMP inhibitor into the mPFC during reactivation of a cocaine-primed memory suppresses later cocaine-primed reinstatement. However, we do not know if the suppressed responding is due to disruption of reconsolidation, nor do we know which MMPs are involved. Also, no studies have defined the impact of MMP inhibition on membrane excitability in the mPFC in cocaine self-administering rats to assess how MMP inhibitors may alter mPFC output. We propose that MMP inhibitors can modify previous cocaine-induced changes in plasticity and impose new plasticity on synapses during the reconsolidation process. We will test the central hypothesis that a cocaine-associated memory is diminished with MMP inhibitors given during cocaine-primed reinstatement, and that this diminished expression of memory occurs by a decreased ratio of excitatory to inhibitory currents in the mPFC. Specific Aim 1 will determine the extent to which inhibition of MMPs disrupts reconsolidation of cocaine-associated memory in cocaine self-administering rats. Specific Aim 2 will define the impact of MMP inhibition on excitatory/inhibitory synaptic input and membrane excitability in the mPFC in cocaine self-administering rats. These studies will have a positive impact on the drug abuse field because they will determine the potential for using MMP inhibitors to disrupt reconsolidation of cocaine memories that may underlie chronic relapse. PUBLIC HEALTH RELEVANCE: The proposed studies will determine the extent to which cocaine-associated memories are able to be disrupted in a rat self-administration model. Disruption of these memories is expected to suppress the motivation to seek or take cocaine. These studies have high translational potential in humans because successful disruption of learned drug-associated memories would help break the cycle of relapse in human cocaine addicts.

DESCRIPTION (provided by applicant): The ability to attenuate drug-associated memories in drug addicts is important because this attenuation is expected to suppress the cycle of relapse to drugs. Persistent drug-taking behavior involves consolidation of memory for the drug and drug-associated cues and contexts. When a memory is reactivated (retrieved), that memory becomes labile and susceptible to disruption by amnestic agents (e.g., protein synthesis inhibitors) present at the time of reactivation. Drug abuse studies in rodents indicate that reconsolidation can be disrupted, and this is manifest as suppressed drug-seeking behavior when animals are subsequently primed with the same stimulus used to reactivate the memory. Most studies have focused on drug-induced conditioned place preference (CPP); since only a few drug injections are given with CPP, the memories may be relatively easy to disrupt. However, few labs have focused on the rat self-administration model, which has higher face validity for human addiction. To date, no self-administration studies have attempted to disrupt reconsolidation of the memory associated with the drug itself, when the drug is present during reactivation and subsequent reinstatement. This is significant because the drug induces powerful reinstatement in rats and augments relapse in humans. We present for the first time data showing that administration of amnestic agents into the medial prefrontal cortex (mPFC) during reactivation of a cocaine- associated memory suppresses subsequent cocaine-primed reinstatement when amnestic agents are no longer present. The focus of this proposal is on one of these agents, an inhibitor of matrix metalloproteinases (MMPs). MMPs belong to a family of metalloendopeptidases that can direct changes in synaptic morphology via their effects on the extracellular matrix (ECM). Some components of the ECM are densely organized into perineuronal nets (PNNs) that ensheath primarily inhibitory interneurons in the cortex. Based on our work and that of others, we believe that MMPs are involved in maintaining as well as diminishing cocaine-related memories. We hypothesize that components of the ECM within PNNs must be transiently degraded by MMPs during synaptic remodeling to permit the reconsolidation of memory. We will test our hypothesis in three Specific Aims: Specific Aim 1 will determine the extent to which inhibition of MMPs in the mPFC suppresses MMP activity and increases PNN density and PNN glycoprotein levels. Specific Aim 2 will determine the extent to which MMP inhibition in the mPFC disrupts reconsolidation of cocaine-associated memories in self-administering rats. Specific Aim 3 will determine the impact of MMP inhibition on c-Fos activation in PNN-containing interneurons in the mPFC and whether dynamic changes in PNNs are a key mechanistic step for MMP effects on cocaine-associated memories. These studies will have a positive impact on the drug abuse field because they will determine the potential for using MMP inhibitors to disrupt reconsolidation of cocaine memories that may underlie chronic relapse. PUBLIC HEALTH RELEVANCE: The proposed studies will determine the extent to which cocaine-associated memories are able to be disrupted in a rat self-administration model. Disruption of these memories is expected to suppress the motivation to seek or take cocaine. These studies have high translational potential in humans because successful disruption of learned drug-associated memories would help break the cycle of relapse in human cocaine addicts.