Anthony Albert Grace - US grants

The physiology of dopamine (DA) neuronal systems has been a major topic of investigation due to the hypothesized involvement of these neurons in various clinical and neurological disorders, such as schizophrenia, Parkinson's disease, and the side effects of chronic antipsychotic drug administration. Although in vivo electrophysiological studies have proven valuable in ascertaining the responses of DA neurons to pharmacological intervention in the whole animal, these studies have proven insufficient in uncovering the ionic mechanisms underlying these actions. By using in vitro intracellular recording techniques, is should be possible to characterize the membrane conductance changes which are responsible for regulating spontaneous activity, as well as the ionic mechanism which underlie the responses of DA neurons to drug administration. In order to derive data specific to this neurochemical system, the dopaminergic nature of the neurons recorded must first be established. The direct identification of DA neurons in the slice will be done by combining intracellular injection of fluorescent dyes with catecholamine histofluorescene. Furthermore, morphological characterization of identified DA neurons will be accomplished by intracellular injection of Lucifer yellow and horseradish peroxidase. Injection of dyes into the know postsynaptic target sites of DA neurons prior to recording and intracellular staining will permit comparisons of DA neuron morphology and electrophysiology based upon their sites of termination. The next stage of the study will involve determination of the biophysical properties of DA neurons to establish the electrophysiological bases against which responses to drugs can be measured. Thus, sodium spikes, low threshold and high threshold calcium spikes, and pacemaker depolarizations will be investigated by pharmacological blockade of specific ionic channels. Furthermore, the threshold and voltage dependencies of both active and passive ion fluxes will be determined in order to establish the presence of voltage activated or inactivated conductances. The direct effects of DA on neuronal activity and the involvement of the DA autoreceptor in the regulation of cell firing will be examined by the administration of DA agonists and antagonists. In summary, these studies are designed to identify the nature of DA cell autoregulation of spontaneous activity, with special emphasis on the differential modes of regulation expressed by DA cell subpopulations. It is hoped that these studies will provide insights into methods by which we may pharmacologically manipulate, in a highly specific manner, the activity level of this cell group to aid in the alleviation of a variety of clinical disorders.

Dysfunctions of dopaminergic systems are thought to play a role in the etiology of several neurological and psychiatric disorders, such as Parkinson's disease and schizophrenia. However, models of these disorders to date have often failed to take into account the large amount of homeostatic regulation that comes into play whenever these systems are perturbed, and which serve to reset the system towards normality. Another possible approach to investigating the normal functions of this system and its role in disease would be to examine in detail the modes of autoregulation that modulate the activity of dopamine-containing neurons, with a predisposition to examining how deficits in these processes could precipitate dysfunctional states. We have been investigating the membrane processes involved in the self-regulation of dopamine neuron activity using the in vitro rat brain slice preparation, in which confounding environmental or afferent influences can be better controlled. In this proposal, we plan to extend this investigation into dopamine neuron regulation by studying five processes believed to play a role in their function: 1) identifying the spike generating zones within dopamine neurons and their morphological correlates, and in this way enable the study of dopamine neuron regulation in terms of its functional subcompartments; 2) testing the involvement of glutamatergic afferents in the regulation of dopamine neuron firing pattern; 3) examining the functional mechanism of autoreceptor stimulation and factors involved in regulating its sensitivity, for the purpose of studying the autonomous control exerted by the dopamine neuron on its responsivity to its own neurotransmitter; 4) determining the important factors which play a role in controlling dopamine release from dendritic stores, and thus elucidating whether this release is an autoregulatory process or is mediated, as it is in the striatum, by glutamatergic afferent processes; and 5) analyzing the functional implications of the different peptidergic cotransmitters contained within subpopulations of dopamine neurons, detailing their involvement in cell firing and how their co-release may influence autoreceptor sensitivity and peptide interactions in identified subsets of dopamine neurons, which will be classified by combining retrograde labelling from their projection sites and intracellular staining. The validity of the results to the in situ cell class will be determined by carrying out parallel experiments in vivo, when feasible. This type of combined functional and morphological analysis of individual dopamine neurons may thus facilitate the development of models concerning states of psychiatric dysfunction. Furthermore, by examining in detail how these regulatory processes differ between identified subpopulations of dopamine neurons, it may be possible to derive new therapeutic approaches by pharmacologically targeting these regulatory sites, and thereby avoid the debilitating side effects often associated with the use of direct-acting drugs.

Disturbances within the dopamine system are thought to contribute to the etiology of several disorders in humans, including schizophrenia and Parkinson's disease. Studies investigating the consequences of damage to the dopamine system have revealed the marked degree of compensatory capacity available to these neurons, suggesting that simple changes in dopamine levels are unlikely to underlie major human disorders. Instead, it is likely that dysfunctions within the dopaminergic system occur via disturbances in the regulatory processes controlling their activity. One way to address the role of autoregulation in the normal or abnormal function of an organism is to first damage the system and then examine the compensatory changes that occur. This proposal investigates several aspects of the dopamine system function in the basal ganglia of both intact rats and rats with partial lesions of the dopamine system. This is done using a combination of in vivo and in vitro recording techniques, and centers around four aspects of basal ganglia function: 1) the processes through which dopamine neurons regulate their activity, including. the function of somatodendritic autoreceptors and the factors involved in controlling their firing pattern; 2) how these autoregulatory systems are influenced by striatonigral feedback systems, and how these systems lead to a hyper-responsive state of dopamine neurons in partially lesioned rats; 3) the properties of dopaminoceptive neurons in the striatum and accumbens, their response to dopamine administration, and the influence of dopamine on afferent processes and network properties of these nuclei; and 4) how the basal ganglia influences thalamic function and how this correlates with behavior. By examining the dopamine system in the basal ganglia from dopamine cell regulation through dopamine-mediated actions on feedback and output systems, an integrated view of the basal ganglia function in normal and dysfunctional states may be achieved. In addition to the scientific goals of this research plan, it will provide for a significant level of scientific advancement and professional growth for the applicant to be achieved through collaborations and extended scientific interactions with other investigators. In addition to enabling the development and implementation of advanced neurophysiological techniques, it will provide the time necessary for the PI to expand into new areas of investigation and improve the PI's background knowledge in clinically related research. This proposal is oriented toward providing an integrated view of how the basal ganglia is regulated under normal and compromised conditions. By using basic science techniques to investigate systems thought to be involved in human psychiatric or neurological disorders, this research has the potential of providing information of value to the development of more effective therapeutic approaches to treat such conditions.

DESCRIPTION (Adapted from applicant's abstract): This is a new five-year grant that proposes to use in vivo and in vitro electrophysiological recordings to examine the functional interactions among cortical afferents and the dopamine (DA) system within the nucleus accumbens of rats. Although initial studies into schizophrenia were directed at a role for DA in the etiology of this disorder, interest has recently been focused on a cortical locus of pathology, with emphasis on the prefrontal cortex and the hippocampus. Moreover, owing to the genetic linkage of schizophrenia, one topic of concentration has been the study of neonatal lesions of the ventral hippocampus and its impact system function in the adult organism. One region in which the hippocampus, prefrontal cortex, and the mesolimbic DA system overlap in particular appears to have a functional role if gating prefrontal cortical throughput within this structure. In addition, the DA system appears to play a role as well in that is has been shown by us and others to affect both the afferent processes and the response of nucleus accumbens cells to these inputs. We propose to evaluate this functional interaction and how it may be selectively altered by neonatal disruption according to the following specific aims: (1) to characterize the physiology and pharmacology of the responses evoked in accumbens neurons in vitro by stimulation of prefrontal cortical, hippocampal, and amygdalar afferents. (2) To examine the pharmacology of DA actions on each of these afferent systems in vitro. (3) to characterize the response properties and types of interaction between pairs of afferents to accumbens neurons recorded in vivo. (4) To examine how the DA system affects the afferent-evoked responses and their interactions in vivo, and how these interactions may be altered by psychotropic drugs. The first 4 aims will provide a comprehensive characterization of the nature of the interactions among these cortical afferents and their modulation by DA in the normal adult rat, to provide the necessary baseline data set for the last specific aim: (5) To compare the effects of neonatal versus adult lesions of the ventral hippocampus on the interaction between excitatory afferents to the accumbens neurons and their regulation by DA. In this way, we will gain a better understanding of the nature of these cortical interactions within the nucleus accumbens, how these systems are modulated by DA, and how they may undergo a compensatory reorganization as a consequence of neonatal damage to the ventral hippocampus.

disease /disorder model; prefrontal lobe /cortex

DESCRIPTION (provided by applicant): Stress is known to be a factor in the etiology or exacerbation of psychiatric disorders, and plays an important role in reinstatement of drug-taking behavior. This proposal will use primarily electrophysiological recordings in anesthetized rats to examine the effects of acute and chronic stress on the regulation of the locus coeruleus (LC) by the amygdalar complex. Norepinephrine and corticotropin releasing hormone have been identified as central factors in the response to stress. In particular, the noradrenergic neurons of the LC are known to have a major role in central mediation of stress responses. These neurons are driven by a number of afferent influences. One set of afferents that appears to have a primary role in stress as it relates to psychiatric disorders and relapse to drug abuse are the projections that involve the amygdala. We propose to examine amygdalar regulation of LC neuron activity, and how this system is modified by acute and chronic stress. Our central hypothesis is that chronic stress causes a sensitization of LC neuron responsivity via its effects on amygdalofugal pathways. This will be done along four specific aims: 1) to examine the physiology of projection systems connecting the basolateral amygdala complex with the LC, 2) to examine the response of this system to acute stressors (footshock), and how these responses are modified following chronic cold exposure, 3) to evaluate the nature of the change in LC neuron responsivity following chronic stress, and 4) to determine whether the alteration in LC responsivity following chronic stress can be altered by selective interruption of these amygdalar efferents. We will use primarily in vivo single unit and intracellular recording methods to examine systems-level interactions, with in vitro recordings used to measure persistent changes in LC neuronal activity and to address specific pathway-related questions as they arise. We will independently verify the behavioral effects of chronic cold exposure by measuring acoustic startle, a response that is sensitive to manipulations of the central amygdala and forebrain NE systems. We hope that a better understanding of amygdalar-coerulear systems will lead to insights into how stress-induced pathophysiology in these pathways can, in part, mediate some of the behavioral symptoms of psychiatric disease and drug-abuse disorders.

DESCRIPTION (provided by applicant): This application seeks funding to address the opportunity at the University of Pittsburgh for a coherent training program on the neurobiology of substance use and abuse. The program will support three post- doctoral trainees and three pre-doctoral trainees at the dissertation stage. It is structured around five core areas of training faculty expertise: 1) Systems neurobiology of motivated behavior and cognition 2) Adolescent developmental biology and substance use vulnerability 3) Molecular modulation of neurotransmitter release and reuptake 4) Nicotine reinforcement and relapse 5) Stress responsive brain circuits. A didactic course will address these core areas of focus. There will also be a formal seminar series drawing prominent scientists whose work is at the cutting edge of research into these areas of focus. Extensive training in grant writing is formally incorporated through a "mentored and reviewed" grant writing course requirement. Presentation and analysis skills will be developed through an in-house seminar series in which works in progress will be presented by trainees or other individuals within the University of Pittsburgh substance use research community. The training program will enhance critical skills necessary for success as independent investigators, and the sum of its components will create multiple nodes of interaction between researchers, thereby increasing the breadth and quality of training of the supported individuals. PUBLIC HEALTH RELEVANCE: This grant will establish a program that will provide enhanced training into the neurobiology of substance use and abuse. The overarching goal is to produce research scientists with a broad base of knowledge in this area, and with the skills needed to succeed as independent investigators.

DESCRIPTION (provided by applicant): This application is for a new R21 under the Cutting-Edge Basic Research Awards (CEBRA) mechanism from NIDA. The majority of studies that examine the consequences of drug abuse and withdrawal focus on the impact of long-term administration of drugs. However, at this stage of use the brain has undergone substantial physiological changes that are typically resistant to effective treatment. On the other hand, it is clear that naive subjects show substantial negative affective responses upon withdrawal from even single doses of a psychostimulant, sufficient to cause the individual to seek more drug to obviate these actions. However, the neuronal circuitry and the physiological changes that underlie such acute actions are unknown. A dominant theory in drug abuse relates to the opponent-process model, in which taking a drug that is associated with a brief positive emotional effect leads to a homeostatic alteration that opposes this action with a long-term negative affective state. Given the involvement of the dopamine system in reward and in anhedonia, we recorded the activity of dopamine neurons in the ventral tegmental area. Our results show that animal models of depression are associated with a decrease in the number of dopamine neurons firing, termed population activity. Moreover, we find a similar effect 18 hours following administration of amphetamine, consistent with this negative affective state. In both animal models of depression and 18 hours after amphetamine, this depression in dopamine neuron activity can be reversed by inactivating the amygdala or by administering the NMDA channel blocker ketamine; a drug that is known to be a rapidly acting antidepressant. Taken together, these data support our central hypothesis: The negative affective state following amphetamine withdrawal, which drives individuals to take additional doses of the drug, is mediated via an amygdala-driven decrease in ventral tegmental area DA neuron firing; moreover, this state is reversed by ketamine administration. We propose to test this model in a rat using acute amphetamine administration and withdrawal according to these Specific Aims: 1) Test the effects of acute amphetamine administration on dopamine neuron activity states measured at different time points after administration. 2) Test whether amygdala inactivation or administration of propranolol or ketamine after amphetamine will prevent the decrease in ventral tegmental area dopamine neuron population activity. 3) Test whether the decrease in dopamine neuron activity state measured at 18 hours post-amphetamine correlates with a change in a progressive ratio scale of amphetamine self-administration, and if this can be altered by propranolol or ketamine pre-treatment. This is a highly novel approach that can provide both insights into the neural circuits underlying the opponent process-driven negative consequences of acute amphetamine intake, as well as a novel treatment that can potentially break the cycle of drug use leading to abuse.

DESCRIPTION (provided by applicant): Depression is the most prevalent of the psychiatric disorders, causing higher morbidity and mortality than any other psychiatric disorder. Nonetheless, the neurobiological underpinnings of this disorder are not known. Anhedonia, an inability to derive pleasure from the environment, is a major feature of depression. We focus on the neurobiological basis of anhedonia, which is also present in schizophrenia, bipolar disorder, drug withdrawal, and Parkinson's disease, thereby being consistent with the Research Domain Criteria approach. The dopamine (DA) system has been classically associated with anhedonia, and has been suggested to play a role in depression; however, direct experimental evidence for this association is lacking. Functional imaging studies have shown an association between hyperactivity in area 25 (homologous to rodent infralimbic prefrontal cortex) and hyper-excitability of the amygdala in patients with depression. We have found in our preliminary studies that activation of the infralimbic prefrontal cortex decreases DA neuron activity states, which can be reversed by inactivation of the basolateral amygdala. Moreover, in animal models of depression we found that there is also a decrease in DA neuron activity states that also can be reversed by inactivation of the basolateral amygdala. These data are consistent with a model in which depression-induced hyperactivity of the ilPFC drives an amygdala-dependent down-regulation of DA neuron responsivity, leading to anhedonia. Moreover, ketamine, which has been found clinically to elicit rapid anti-depressant responses, reverses both the decrease in DA neuron activity and the helplessness state specifically in rats showing learned helplessness behavior. This could lead to insights into novel mechanisms of treatment for depression and other disorders in which anhedonia plays a major role. However, to do this we will need a better understanding of the circuitry involved, in particular the circuit by which the basolateral amygdala down-modulates DA neuron activity states. To address our overarching hypothesis, which is in disorders with a negative affective state, hyperactivity within the ilPFC causes an overdrive of the BLA, resulting in an attenuation of VTA DA neuron activity and anhedonia, we will perform experiments to address the following Specific Aims: 1) To examine how infralimbic prefrontal cortical activation affects DA neuron activity states, 2) To examine the pathways through which basolateral amygdala activation attenuates DA neuron activity patterns, 3) To evaluate the involvement of these systems in animal models of depression and anhedonia, and 4) to evaluate how ketamine can reverse the attenuated DA neuron activity and anhedonia in animal models. This will be done using an integrated systems-oriented approach focused on in vivo electrophysiology, optogenetics and behavior. By providing a better understanding of pathways that may underlie anhedonia in depression and other disorders, we will be in a better position to identify drugs with novel and specific mechanisms that may alleviate this crippling disorder.

? DESCRIPTION (provided by applicant): This application is a resubmission of a new R21 from NIMH. The introduction of antipsychotic drugs for the treatment of schizophrenia has revolutionized the management of this disorder. Nonetheless, these drugs suffer from poor overall efficacy across symptom classes and devastating side effects. The primary mechanism for all drugs used in treating schizophrenia is based on Dr receptor blockade. However, there is little evidence for pathology within the dopamine system itself; instead, the belief is that the dopamine system is being dysregulated by upstream structures. One synapse that has garnered attention is the parvalbumin GABAergic synapse. Indeed, parvalbumin loss is among the most robust findings in the schizophrenia brain. Using an animal model based on developmental disruption, we found that a loss of parvalbumin in the ventral hippocampus corresponds to hyperactivity in this structure, which indirectly causes the dopaminergic neurons to be hyper-responsive to stimuli. This model is consistent with numerous imaging studies in the schizophrenia patient brain. As a consequence, a number of drugs including the mGluR2/3 agonist by Lilly, the PDE10 inhibitor by Pfizer have been developed that target this circuit have been tested for efficacy. However, despite promising preclinical evidence, all have failed to translate to the clinic, leading a number of pharmaceutical companies to cease drug discovery in this arena. However, unlike the preclinical experiments, these drugs were tested in human schizophrenia patients that had been exposed to decades of antipsychotic drug treatment, and then withdrawn for only one week before testing the novel target agent. It has been long known that repeated D2 receptor blockade leads to dopamine supersensitivity, and in fact antipsychotic drug withdrawal supersensitivity psychosis has been reported in the clinic. We posit that prior D2 antagonist antipsychotic drug treatment will interfere with the ability of novel compounds to show clinical efficacy via induction of D2 supersensitivity. Our results show that a drug that specifically targets GABAergic synapses in the hippocampus, a GABA A alpha 5 positive allosteric modulator, has potent and specific actions in the MAM developmental disruption model of schizophrenia. However, following a brief course of antipsychotic drug treatment and withdrawal, this drug is no longer effective in normalizing the dopamine system. We propose to test whether this also occurs with other novel target agents using this MAM model [and the mechanism underlying this change.] Furthermore, we will test whether the dopamine partial agonist drug aripiprazole, an effective antipsychotic drug that may not induce supersensitivity, can be used as a transitional agent in testing novel target drugs in the absence of supersensitivity. We believe that such experiments are critical in understanding why drugs have failed to translate from preclinical studies into clinical investigations, and to chart a course whereby future studies can be conducted without such endogenous confounds in their design.

ABSTRACT Schizophrenia is among the most devastating of the psychiatric disorders, robbing more than 1% of the population worldwide of quality of life. While schizophrenia tends to run in families, the disorder is not completely genetically determined, and depends on an interaction with the environment. Among the most robust findings in schizophrenia is the nearly 30% decrease in parvalbumin (PV)-labeled GABAergic interneurons, particularly in the limbic hippocampus. This loss is paralleled by hyperactivity in the limbic hippocampus, which we have found in animal models drives increased dopamine system responsivity. It has been known that early life stress and anxiety often precedes the psychotic break, and moreover adolescent stress in rats is known to lead to PV neuron loss. Interestingly, stress can damage PV interneurons only when it occurs during adolescence; afterwards, the PV neurons develop perineuronal nets that protect them from damage. We have previously employed a developmental disruption model of schizophrenia based on gestational administration of a mitotoxin. Interestingly, these rats show increased anxiety and heightened stress response during adolescence, before the emergence of a dysregulated dopamine system. If we mimic this adolescent condition by combined stressors, we can induce a MAM-like phenotype in normal rats. Therefore, we posit that susceptibility to stressors that occur prior to the formation of perineuronal nets lead to damage of vHipp PV interneurons, vHipp hyperactivity, and the emergence of the hyperdopaminergic state in the adult. We further suggest that the increased stress responsivity in susceptible adolescents may be due to a failure of the prelimbic prefrontal cortex to limit the impact of stress. Finally, given that females have a later onset of schizophrenia and less pathology, we posit that adolescent stress will not impact female rats to the degree seen in males. We will assess this model via the following Specific Aims: 1) Determine the development of stress-induced pathology in normal adult male and female rats, and how this correlates with PNN and PV interneuron staining; 2) Determine the role of prepubertal plPFC lesions and BLA activity on stress reactivity, anxiety, and footshock-induced PV loss/DA hyperresponsivity in peripubertal and adult rats, and the role of plPFC inhibition of the BLA in the pathology; and 3) Examine whether re-opening the critical period or PNN disruption in the adult can cause the rat to regain stressor-induced PV neuron loss and DA system hyperresponsivity, and if this is driven by the BLA. In this way, we hope to be able to provide a link between genetic/gestational susceptibility and environmental factors that occur early in life, initiating a cascade of events that lead to the emergence of schizophrenia later in life. This will provide insights not only into the pathophysiology of schizophrenia, but also into establishing predictive criteria for psychosis susceptibility and ultimately into prevention.