Greg Gerhardt - US grants

We propose to investigate dynamic presynaptic changes in monoamine-containing afferents that may occur during aging in brain areas known to regulate balance and gait (i.e., striatum and cerebellum). The experiments will utilize a rodent model (Fischer 344 rat) to assess age-related changes in dopaminergic inputs to the striatum and noradrenergic afferents to the cerebellum in intact animals. Following balance and coordination measurements in the young mature and aged animals, in vivo electrochemical methods will be employed to quantitate the magnitude and temporal characteristics of potassium-evoked and electrically-induced release of monoamines. High performance liquid chromatography coupled with electrochemical detection (HPLC-EC) will be used to measure whole tissue levels of nonoamines and their metabolites, and correlate the levels with the monoamine release data. Finally, in situ monoamine-containing brain cell transplants into adult hosts will be used to delineate possible intrinsic versus extrinsic determinants that may be associated with any age-related changes.

The basal ganglia are a group of nuclei in the brain with major responsibility for causing movement of body parts. To date, neuroscientists have been able to collect anecdotal data from these motor nuclei by recording either electrical or chemical events from single cells within the basal ganglia. However, this approach does not tell us what ensembles of cells within the nuclei are doing at one instance to effect a movement. Thus, it is necessary to create and develop recording devices which will allow scientists to measure electrical and chemical events from multiple sites within a nucleus of cells if we are to fully understand how neural circuits operate to cause an arm to move for example. Dr. Greg Gerhardt is taking the initial steps in the development of solid-state microsensors that can be used to monitor the neurochemical substratum of motor regulation. He plans to develop and test biosensors to monitor chemical and electrical events in "real time" in the extracellular environment of striatal neurons. He proposes to develop multichannel silicon-based biosensors for the rapid determination of dopamine, glutamate, and acetylcholine activity in the basal ganglia of laboratory animals. This work is extremely important, being at the cutting edge of research which will allow us to understand the neurochemical mechanisms that underlie execution and control of brain motor circuits.

Dopaminergic neurons in the brain are thought to play an important role in the etiology of schizophrenia. The chronic intake of amphetamine, a dopamine releasing drug, can produce clinical symptoms similar to paranoid schizophrenia, whereas the drugs that are used therapeutically to alleviate the symptoms of schizophrenia are predominantly dopamine receptor blockers. These observations imply that schizophrenia or at least some of the symptoms of this disorder are due to an excess of dopamine in certain areas of the brain. Three major dopaminergic neuronal systems have been described in the brain that may be related to the disorder of schizophrenia; the mesocortical system, the mesolimbic system and the nigrostriatal system. The studies outlined in this proposal will evaluate and compare the regulation of dopamine release from tissue minces and synaptosomes obtained from these three areas of the rat brain. The areas of the brain that will be examined include the dopaminergic nerve terminal regions; the olfactory tubercle (mesolimbic), prefrontal cortex (mesocortical) and striatum (nigrostriatal), and the cell body regions that project to one or more of these areas; the ventral tegmental area (mesocortical and mesolimbic areas) and the substantia nigra (nigrostriatal). Initial studies will be performed to characterize the basal and potassium stimulated release of endogenous dopamine form these brain areas. Then experiments will be performed to evaluate the presynaptic autoreceptor regulation of dopamine release in these five brain regions by performing dose response curves with the D2 selective dopamine agonists, pergolide and quinpirole; the D1 selective dopamine agonist SKF 38393; the non- selective dopamine antagonist, haloperidol; the D2 selective agonists, sulpiride, the D1 selective antagonist, Schering 23390 and the dopamine releasing drug, d-amphetamine. A second series of studies will evaluate the effects of chronic drug treatments on dopamine release from the five brain areas. The drugs that will be administered chronically include, the direct acting dopamine agonists, pergolide (D2), SKF 38393 (D1) and apomorphine (D1,D2); the nonselective dopamine antagonist haloperidol, the D2 selective antagonist sulpride, the D1 selective antagonist Schering 23390 and the dopamine releasing drug, amphetamine. Following the chronic administration of these compounds the autoreceptor regulation of the release of endogenous dopamine in the five dopaminergic brain regions will be evaluated. Evaluating the autoreceptor control of dopamine release in selective areas of the brain should expand our knowledge on the possible biochemical dysfunctions and treatments of the disorder schizophrenia.

This revised competing renewal involves work previously funded by a R29 (First Award). The objective of this proposal is to characterize the mechanisms that underlie dynamic presynaptic changes in dopamine (DA)- and norepinephrine (NE)-containing afferents that occur during aging in brain areas known to regulate balance and gait. This work uses Fischer 344 rats as an animal model to assess age-related changes in dopaminergic inputs to the striatum and nucleus accumbens, as well as noradrenergic afferents to the cerebellum in intact animals. In addition, young and aged in oculo transplants of both substantia nigra and locus coeruleus are studied. These brain areas are known to be critically involved in movement. First, in vivo electrochemical recordings in anesthetized rats, using monoamine selective Nafion-coated electrodes and high-speed chronoamperometric recordings (5-25Hz), will be employed to quantitate the magnitude and high-resolution temporal characteristics of monoamine release in young and aged animals. In particular, amphetamine-and n-methyl-d-aspartate (NMDA)-induced release as compared to potassium-evoked release will be explored to investigate different types of presynaptic release processes in aging. The diffusion/clearance properties of locally-applied DA will also be investigated to more directly explore potential changes in high-affinity neuronal uptake and diffusion of DA which may occur with senescence. In addition, some measurements will be carried out in unrestrained freely- moving animals in order to investigate potential anesthetic effects associated with age-induced changes. Intracranial microdialysis experiments will be employed utilizing elevated dialysate levels of potassium, NMDA, and amphetamine to further substantiate the validity of the in vivo electrochemical protocols, and to explore potential changes in monoamine metabolites in the aged brain. Balance, coordination and motor learning tests will be performed in 14-16 month old animals and potassium-evoked overflow of DA will be tested in these animals to investigate if behavioral tests correlate with alterations in DA overflow. Secondly, potassium-and amphetamine-evoked overflow of NE will be carried out in the cerebellum of the different age groups analogous to the studies of DA to explore if NE afferents are changed in aging. Ne diffusion/clearance will be explored in the different age groups to more directly investigate high-affinity neuronal uptake processes in aging. Balance, coordination and motor learning tests will be performed in 14-16 month old animals to investigate if behavioral changes are correlated with alterations in potassium-evoked NE overflow. Finally, in oculo monoamine-containing brain cell transplants will be used as a model system to explore release processes from both young and aged locus coeruleus and substantia nigra brain grafts, and to investigate potential presynaptic changes that are extrinsically-versus intrinsically determined in aging.

Parkinson's disease (PD) is a devastating neurodegenerative disease which affects approximately 1 million people in this country. The chief symptoms include advancing slowness of movement (bradykinesia), tremor, rigidity, and postural imbalance. PD involves degeneration of the neurons that make dopamine, a chemical necessary for normal brain function. The diagnosis is entirely clinical: there are no routine laboratory tests available that can establish the diagnosis with certainty. PD has been considered to be an acquired disorder of unknown etiology. Treatment until 1989 has been entirely symptomatic - treating the symptoms but not the root cause of the disease. Very recent research has identified an enzyme defect in platelet mitochondria of patients with Parkinson's disease. The discovery of this defect in complex I activity suggests new investigative and therapeutic strategies, but first it is important to confirm the finding in a larger number and broader range of patients. This project proposes to examine platelet mitochondrial enzyme activity in: (1) a larger group of patients with typical Parkinson's disease (Is this enzyme assay sensitive?); (2) patients with "parkinson-plus" disorders, who have similar symptoms but also additional signs of central nervous system degeneration (Is this enzyme assay specific?), (3) first degree (blood) relatives of patients with PD (Does the enzyme defect appear to be inherited via the mitochondria (maternal) or is it autosomal dominant with reduced penetrance (the trait may be inherited from either parent, but not all who inherit it get sick?)); and (4) healthy, age-matched controls. Two other enzyme activities will also be studied as a control for assessing the overall quality of the isolated platelet mitochondria. Because a large quantity of platelets is required for these special enzyme activities, these will be obtained by plateletpheresis, in which blood is removed from the body, the platelets are separated out, and the remainder of the blood is returned to the volunteer. Although platelets have not been recognized to have functional defects in patients with PD, this project will address this question by screening many patients with PD with functional tests (e.g., rate of aggregation, response to activators) performed on a small sample of peripheral blood obtained in the routine manner. This research should establish the range of complex I activity in each disorder and the sensitivity and specificity of this finding. Family studies will establish the occurrence and pattern of inheritance. Platelet functions studies will establish whether these widely-available tests could be used for screening persons at-risk or those with a questionable diagnosis. Although development of a diagnostic test would be a major clinical advance, the more important potential result of this work would be to point the way to new therapeutic strategies. Experimental therapeutics are under way in other genetic diseases to replace or stimulate defective enzymes; such a strategy could also be pursued for PD.

This application will examine the in vivo actions of a newly described putative dopaminotrophic (DA) factor, glial cell-derived neurotrophic factor (GDNF) in rats. The proposal will test the hypotheses that GDNF is a specific trophic factor for developing, mature and aged midbrain DA neurons, that GDNF will support human as well as rat DA circuits, and that GDNF will counteract the deleterious effects of dopaminergic neurotoxins. It is proposed to examine actions of GDNF in grafts of fetal neuroblasts placed in oculo. Effects of neuron age will be determined by comparing GDNF activity in developing, mature and aged DA grafts. Specificity will be determined by comparing GDNF activity in DA-, NE- and 5HT-containing grafts. Target influences will be addressed by examining GDNF activity in DA double grafts with striatal, hippocampal or iris targets. Finally, GDNF activity will be evaluated in xenografts of fetal human DA neuroblasts in oculo. It is also proposed to examine actions of GDNF on dopaminergic pathways in intact rats and mice. GDNF will be administered intracranially in 3-, 18-, and 24-month old rats to study effects of CNS age. Interactions of GDNF with dopaminergic neurotoxins, such as 6OHDA (rats) and MPTP (mice) will be evaluated both with "protection" and with facilitation of regeneration protocols. Target appropriateness will be evaluated in situ. Finally, GDNF will be studied in mature and immature syngeneic and allogeneic intracranial grafts and in immature human xenografts to gain further insight into the utility of this molecule to augment growth, differentiation, maintenance, and survival of DA neurons. Actions of GDNF will be evaluated using behavioral, histochemical and in vivo electrochemical techniques. Neurochemical and electrophysiological protocols will also be utilized.

This program project grant renewal application has two major research foci: (1) studies on central nervous and cardiovascular changes in noradrenergic transmission that accompany senescence and 2) cholinergic mechanisms that may underlie age-related declines in cognitive function. One basic objective, historically the major focus during the previous six years of the program, continues to be the study of the mechanism of age-related changes in the pharmacological properties of the central nervous and cardiovascular systems. We will study age-related changes in drug responsiveness in both experimental animal models and in man. The molecular mechanisms for such changes, in term of receptors, of second messenger molecules and of the regulation of presynaptic function will be pursued biochemically using both animal and human tissues. Physiological correlates will be examined using techniques of electrical recording, in vivo chronoamperometry, and electrical stimulation. Morphological correlates in both neuronal and non-neuronal elements will be evaluated. Tissue transplantation will be utilized to determined the intrinsic versus extrinsic nature of any age-related central nervous changes. The major hypothesis to be pursued is that per-and/or postsynaptic changes in CNS synaptic circuitry or in peripheral autonomic neuroeffector mechanism will underlie many of the alterations in adrenergic drug responsivity seen with aging. Our second research focus, studies on central nervous synaptic changes which may underlie cognitive dysfunction, represents the major new research initiative if the program. Major efforts in this program will be devoted to examination of aged-related changes in cholinergic circuitry, effects of neuronotrophic factors and transplants, cellular models of memory such as long-term potentiation (LTP) and putative molecular mechanisms of LTP such as phosphorylation of synaptic proteins. Again in admixture of behavioral, electrophysiological, electrochemical and biochemical approaches will be utilized. Our efforts will cover a broad spectrum ranging from molecular genetic approaches to studies of neuronotrophic agents, to participation on a clinical trial using NGF infusion intracerebrally into patients with Alzheimers Disease.

The Center's research has been directed towards strategies for replacement of dopaminergic neurons in the substantia nigra in animal models of Parkinson's disease. Among the strategies developed and evaluated during the previous funding period have been; (1) transplantation of embryonic neurons that have the potential to produce a dopaminergic innervation, (2) infusions of factors that are trophic for dopaminergic neurons, and (3) genetic alteration of cells to make dopamine or growth factors for subsequent transplantation. The central focus of this competing renewal application will be a multidisciplinary study of the recently identified putative dopaminotrophic factor glial cell line-derived neurotrophic factor (GDNF) A second focus of this center will be to further examine properties of transplants of fetal neurons and other types of peripheral cells. This Center will conduct studies of effects of exogenous GDNF on normal mature, aged, lesioned and brain cell-transplanted rodents. In addition, experiments on the roles of endogenous GDNF will be carried out. Experiments to define the biochemical mechanisms of GDNF's trophic activities, in terms of oxidative stress and mitochondrial respiratory enzymes, will also be carried out. Immunological properties of brain cell grafts, and interaction of GDNF with CNS immune mechanisms will be delineated. We will also initiate studies on a second TGF-beta family member, OP-1, which may have a positive effect on dopamine neurons. These studies require an infrastructure of collaborative investigators. First, research strategies must be developed, using the techniques of molecular and developmental biology. Second, the strategies must be evaluated in animal models, using the Center's combined expertise in measuring enzymatic mechanisms, dopamine metabolism and release, neuroanatomical changes, production and transport of mRNA and proteins, electrophysiological change, and normalization of behavioral function after lesion. These studies, which combine brain cell transplantation and trophic factor administration, may ultimately lead to optimal approaches for replacement or preservation of ventral mesencephalic dopamine neurons.

This revised level II Research Scientist Development Award (KO2) application involves in vivo electrochemical and microdialysis studies of the extracellular regulation and neurochemistry of dopamine (DA) in the basal ganglia of both normal and lesioned rats and monkeys. These studies address the role of DA in motor behavior and the inherent plasticity and compensatory neurochemical properties of damaged dopaminergic neurons. First, extracellular regulation of potassium-evoked overflow of DA and clearance/diffusion of DA, L-Dopa and D-Dopa will be investigated in the striatum, nucleus accumbens, substantia nigra and ventral tegmental area of the unilateral 6-OHDA-lesioned Fischer 344 rat, using high-speed (5-25 Hz) chronoamperometry coupled with pressure ejection of drugs from micropipettes. Microdialysis studies of the extracellular regulation of potassium-evoked overflow of DA in both normal and 6-OHDA-lesioned rat striatum and substantia nigra be used to further confirm the validity of the electrochemical studies and to investigate changes in the metabolic regulation of DA in the damaged striatum. As part of his scientific development, the applicant will work on the development of semiconductor- based microphobes that can be used for combined in vivo electrochemical and electrophysiological recordings. Secondly, as part of the applicant's further scientific development, extracellular regulation of DA and potassium-evoked overflow of DA will be investigated in the caudate nucleus,putamen and nucleus accumbens of unilateral MPTP-lesioned Rhesus monkeys. In addition, the effects of a glial-derived neurotrophic factor (GDNF), a new dopaminotrophic factor that promotes the survival of DA neurons, will be studied following unilateral infusion of this growth factor into the substantia nigra of unilateral MPTP-treated Rhesus monkeys. All of the proposed studies will contribute to the scientific development of the applicant and a further neurochemical understanding of the plasticity and compensatory properties of DA neurons, and how this relates to DA's role in movement and movement disorders.

In recent years, microsensors (5-30 microns in diameter) have been developed to investigate rapid (11000 Hz) chemical signaling in biological systems. Recent electrochemical recording methods such as highspeed chronoamperometry (1-25Hz), fast-scan cyclic voltammetry (10 Hz and 100-1000 v/sec scan rates) and amperometry (1000 Hz resolution) have been developed for such recordings. The study of rapid chemical signaling requires the use of microelectrodes that are coupled to sensitive instrumentation that is capable of recording and converting the small changes in current that are due to chemical reactions at the surfaces of the microsensors. A few laboratory designed instruments and some commercial instruments are available. However, no low noise "turnkey" system that is capable of recording, displaying, analyzing data and outputting results is available to the scientific community. In addition, some commercial instrumentation often requires a combination of other laboratory instruments as seen in Figure la (adapted from Stamford et al. 1992). Presently, there is no self contained microcomputer-based instrument that is available to scientists that encompasses the capabilities to record rapid electrochemical signals using fast-scan cyclic voltammetry, highspeed chronoamperometry, 1000-6000Hz amperometry, more standard electrochemical methods, and new modifications of the rapid electrochemical recording methods. A major goal of this proposal is to carry out the Phase I (design) and Phase II (modification and refinement) development of an automated multi-channel recording system as depicted in Figure lb. Such an instrument, which we call FAST for Fast Analytical Sensing Technology, will be useful to students, postdoctoral fellows and principal investigators in the fields of cell biology, chemistry, biology, neuroscience, pharmacolog y, physiology, chemoreception, marine biollogy, psychology and other biological sciences. The second goal of this proposal is to further develop microsensors for the selective, rapid, and sensitive measures of dopamine, norepinephrine, serotonin, nitric oxide and glutamate in biological systems. Both carbon fiber based probes and semiconductor based probe designs will be developed. Further sensor development is needed to enhance the recording capabilities of currently available instrumentation and the proposed FASTin vivo electrochemical recording system. ~

This project involves neurochemical studies of the effects of glial-derived neurotrophic factor (GDNF), a novel dopaminotrophic molecule that promotes the survival of DA neurons, on DA neurons in the rat basal ganglia. The effects of GDNF will be studied by unilaterally infusing this growth factor into the substantia nigra of 6-OHDA lesioned rats, and into 6-OHDA-lesioned rats that have received fetal ventral mesencephalic brain grafts. In these animals, in vivo electrochemical and microdialysis measurements will be used to study the extracellular release and uptake of dopamine (DA) in tahe striatum and substantia nigra. These studies will examine the role of DA in athe neuronal circuits involved with motor behavior, and the compensatory neurochemical changes seen following the treatment of damaged DA neurons with GDNF. First, we will examine the extracellular regulation of potassium-evoked overflow of DA, the effects of nomifensine and GBR- 12909 on K+ - evoked overflow of DA, and the clearance/diffusion of DA in the striatum and substantia nigra of unilateral 6-OHDA-lesioned Fischer 344 rats that have been treated with vehicle or GDNF. High-speed (5-25 Hz) chronoamperometry and fast scan cyclic voltammetry recordings coupled with pressure ejection of drugs from micropipettes will be used to investigate the dynamics of DA release and uptake. Microdialysis studies of the extracellular regulation DA and potassium-evoked DA release will be studied. In addition, the effects of nomifensine or GBR-12909 on potassium-evoked overflow of DA in the striatum and substantia nigra will be used to further confirm the validity of the electrochemical studies, and to investigate changes in the metabolic regulation of DA ina the GDNF- treated animals. Secondly, high-speed electrochemical recordings will be performed in the substantia nigra and striatum of unanesthetized freely- moving rats. Normal rats and animals that have received unilateral 6- OHDA-lesions to the basal ganglia that have been treated with vehicle or GDNF will be studied. These studies will investigate the potential adverse effect of anesthesia ont the dynamic properties of normal DA neurons and damaged DA neurons following GDNF treatment. Third, in vivo electrochemical and microdialysis studies of DA release will be studied in 6-OHDA-lesioned rats that have received transplants of fetal ventral mesencephalon with and without treatment with GDNF. Finally, Project 1 will provide support for the in vivo electrochemical and HPLC-EC measures of DA and DA metabolites that are proposed in Projects 2,3,4, and 5.

Project 2 of this revised program project grant application involves functional neurochemical assessments using in vivo electrochemical recording techniques, microdialysis methods and high performance liquid chromatography coupled with electrochemical detection (HPLC-EC) to study dopamine (DA) neuronal systems in the striatum of young adult (5-9 year old), adult (13-16 year old) and late middle-age/aged (20-24 year old) Rhesus monkeys. This project will perform functional studies of neurochemical changes to the monkey striatum during aging in conjunction with the behavioral, histological and immunohistochemical studies of Project 1 and functional MRI-imaging studies in Project 3. First, in vivo electrochemical and microdialysis methods will be used to investigate potassium-evoked overflow of DA and p-tyramine-induced displacement of DA. These studies will be carried out in the caudate nucleus, putamen, and nucleus accumbens of the three age groups of monkeys. In addition to the evoked released of DA, basal levels of dopamine and dopamine metabolites will be studied using microdialysis. Secondly, both in vivo electrochemical and microdialysis studies will be used to investigate the functional properties of the dopamine transporter (DAT) located on dopaminergic nerve terminals. In vivo electrochemical methods will be used to look at DA uptake and microdialysis methods will be used to investigate the effects of GBR12909 and nomifensine on potassium-evoked release of dopamine in the striatum and nucleus accumbens of the young and aging monkeys. Project 1 will also provide histological measures of TH immunoreactive neurons. mRNA levels of the DAT, and cell counts of DA-containing neurons in the substantia nigra of the young adult and aging monkeys. In addition, Projects 1 and 3 will collaborate with this project to investigate motoric performance in the young adult and aging animals. In order to determine whether any age-related changes in motoric behavior and functional MRI imaging correlate with changes in the neurochemistry of DA, Finally, microdialysis measures of basal levels of DA and potassium-evoked overflow of DA will be performed in middle-aged monkeys that have received intraventricular (ICV) injections of vehicle or glial-derived neurotrophic factor (GDNF). In addition, studies of DA uptake will be performed using in vivo electrochemistry in the vehicle and GDNF-treated animals. These studies address the role of DA in motor behavior and the inherent plasticity and compensatory neurochemical properties of aging dopaminergic neurons in nonhuman primates.

DESCRIPTION: Aging of neuronal systems is a complex process that likely involves functional changes in neuronal elements that determine cell death. Data from numerous studies support the hypothesis that the dopamine (DA) neuronal system appears be a key player that is altered in movement disorders, such as Parkinson's disease, and changes in DA function may, impart, relate to movement abnormalities seen in aged animals and humans. This competing renewal plans to examine the dynamics of dopamine transporter (DAT) function during aging, which may be involved with some of the functional changes that occur with DA neurons in senescence. In addition, in an attempt to recover or rescue DA neurons and restore movement functions through the DA system, the principal investigator plans to assess the potential regenerative/protective effects of two putative dopaminotrophic factors, glial cell-derived neurotrophic factor (GDNF) and Neurturin, on dopamine neurons in young and aged Fischer 344 rats. Previous studies from the principal investigator's lab have demonstrated that a consistent alteration in aging processes appears to involve a change in the functional properties of the dopamine transporter (DAT) located on cell soma, axons and dendrites of dopaminergic neurons. Since it is well established that dopamine in the substantia nigra plays a major role in movement behavior, and alterations in DA function in the substantia nigra may play a major role in age-related alterations in motor performance in man and animals, they hypothesize that GDNF, and possibly a close peptide relative, Neurturin, will restore function to aged neurons that reside in the substantia nigra of aged rats. In addition, GDNF and Neurturin may be capable of minimizing age-induced degeneration of dopamine neurons. The principal investigator hypothesizes that these trophic factors will increase dopamine content, dopamine release and uptake. He will employ in vivo electrochemistry recordings to evaluate the DAT, and microdialysis to assess changes in extracellular dopamine and dopamine metabolites following treatments with GDNF and Neurturin. These studies should lead to a better understanding of the functional properties of aged DA neurons and the effects on DA neuronal function seen following treatments with the trophic factors, GDNF and Neurturin. In addition to the symptomatic effects of trophic factor treatment, the principal investigator plans to establish whether age related changes in dopamine can be prevented by treatment with GDNF and Neurturin. All changes in DAT and dopaminergic parameters as a function of aging and trophic factor treatment will be correlated with the behavioral status of the animals.

The major goal of these studies is to design and develop ceramic- based multisite microsensors. At the present time, microsensors are being used primarily in the area of neuroscience to study neurochemicals and to measure the electrical properties of brain cells. The advantages of microsensors are the abilities to rapidly (

Dr. Gerhardt will direct the operation of this Core. This Core will involve the training and teaching of one Post Doctoral Scholar and one Graduate Assistant. These individuals will be recruited through a national search. Once recruited, both will work with the Project of the Center.

The neuroestorative and neuroprotective tropic actions of GDNF on midbrain dopamine (DA) neurons provide a promising therapeutic approach for the treatment of Parkinson's disease. As with many tropic factors, optimum success of this tropic factor depends on focal and controlled delivery of the protein to affect the function of DA neurons, without producing unwanted side effects. It is our central hypothesis that chronic intracerebroventricular or intraputamenal GDNF willproduce effects on damaged DA neurons with greater efficacy, potency and reduced side effects as compared to other methods of delivery. 1. We propose to use a novel indwelling pump that can deliver GDNF chronically in the freely-moving MPTP-lesioned monkey to evaluate two sites of delivery: the lateral ventricle and the putamen. 2. We will also investigate washout of GDNF and re-instatement of the trophic factor in both infusion paradigms. The different highly integrated Projects and Cores will provide key data regarding the functional effects of chronic GDNF treatments to DA neurons, and form the foundation for a Parkinson's Disease Center of Excellence. Project 1 will use microdialysis, in vivo electrochemistry and postmortem HPLC-EC methods to study the nigrostriatal pathway of unilateral MPTP-lesioned monkeys that have received chronic infusions of GDNF. Project 2 will investigate the behavioral consequences of the GDNF infusions and study potential functional changes to DA neurons using fMRI methods. Project 3 will carryout tract tracing and immunohistochemical measures of DA neurons in the same groups of chronically treated monkeys. The Cores A-D will all provide needed support for the experiments outline in Projects 1-3. These studies will answer key questions regarding the chronic delivery of a trophic factor in freely-moving and behaving rhesus monkeys. Such data could lay the foundation for the use of trophic molecules for the treatment of Parkinson's disease in humans.

The neurorestorative and neuroprotective tropic actions of GDNF on midbrain dopamine (DA) neurons provide a promising therapeutic approach for the treatment of Parkinson's disease. It is our central hypothesis that chronic intracerebroventricular or intraputamenal GDNF will produce effects on damaged DA neurons with greater efficacy, potency and reduced side effects as compared to other methods of delivery. We propose to use a novel indwelling pump that can deliver GDNF chronically in the freely-moving unilateral 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesions monkey to evaluate two sites of delivery: the lateral ventricle and the putamen. Prior studies have shown that single infusions of GDNF produce neuroprotection and neurorestorative effects to DA neurons, which are localized to the substantia nigra and not the striatum of rats and monkeys. We hypothesize, based on preliminary studies, tha chronic delivery of GDNF will restore DA function in regions of the substantia nigra and striatum, producing a greater reversal of parkinsonian behavior than previously seen with single injections. Project 1 of this program project grant application will perform quantitative neurochemical assessments using in vivo micordialysis, in vivo electrochemical, and high performance liquid chromatography coupled with electrochemical detection (HPLC-EC) to study DA neuronal systems in the striatum (putamen and caudate nucleus) and substantia nigra of young adult Rhesus monkeys that have received chronic GDNF infusions. Washout studies will be performed to determine the longevity of the chronic GDNF effects. Re-instatement studies will also be performed to determine if GDNF can sustain or increase DA funciton following washout and be safely re-administered. These neurochemical changes to the monkey striatum during chronic delivery of GDNF will be performed in conjunction with behavioral, immunohistochemical, tract tracing and functional MRI (fMRI) studies of the same monkeys in conjunction with Project 2 and 3. These studies should lead to a greater understanding of the effects of focal and sustained delivery of a potent DA tropic factor to damaged DA neurons. They will likely lay the foundation for focal and chronic delivery of trophic factors to patients with Parkinson's disease.

DESCRIPTION (from applicant's abstract): In schizophrenia, it has been hypothesized that sensory gating abnormalities may be an underlying inherited deficit in the individuals brain that alters its sensitivity to repeated stimuli, i.e., sensory gating. Patients with schizophrenia are often heavy smokers and it is currently believed that a short-lived beneficial therapeutic affect it achieved through smoking on brain sensory processing. Although this affect is involved with the nicotinic cholinergic effects of nicotine, the finding of rapid desensitization which occurs to nicotinergic receptors suggests that another more long-lived modulatory may be involved with the therapeutic effects of nicotine in these patients. The purpose of the application will be to investigate the hypotheses that nicotinergic activation of hippocampal circuitry will lead to changes in sensory gating that are mediated, in part, through increases in extracellular levels of nitric oxide in discrete anatomical regions of the hippocampus. These studies will be conducted using a recently developed microvoltammetric sensor that is very selective and sensitive for direct spatially and temporally resolved measures of NO changes in the extracellular space of the brain. These studies will investigate the effects of cholinergic agnonists and antagonists of NO release in the extracellular space of the rat hippocampus slices, anethesized rats and free-moving animals. In addition, the potential interrelationships between sensory gating and NO signaling will be investigated measuring evolved potentials and NO release in the same animal. The studies should contribute to a better understanding of AChergic mechanisms that may be involved in NO signaling in the rat hippocampus, and potential role(s) of NO in schizophrenia.

ABSTRACT

9987807
Greg A. Gerhardt
University of Kentucky
Ceramic-Based Multisite Microelectrodes for Electrochemical and Electrophysiological Recordings

There is a great need in many areas of science for small devices that can be used to understand the chemical signaling that occurs between organisms and cells. Such microelectrodes can be used for experiments involving chemical communication in biology, which is a fundamental process that has been poorly understood. However, the development of microelectrodes has been hampered by the difficulty in manufacturing extremely small devices that are strong and affordable. The goal of this award is to develop a multisite ceramic-based microelectrode that is sufficiently inexpensive to be disposable and can be applied to chemical recordings in biological systems such as plants, animals and even the ocean.
The present award has two major specific aims. First, a series of biological recordings in the rat brain will be done using an existing microelectrode prototype. Following these initial measurements, several site mask designs will be implemented to determine the design that optimizes the recording site geometry for chemical measurements. In addition, procedures will be investigated that allow for the formation of microelectrodes using thinner materials. Second, a new generation of ceramic-based sensors will be developed to investigate the recording properties of platinum iridium (Pt-Ir) and carbon recording sites. All of these will contribute to the development of a new generation of affordable microelectrodes for chemical measures in biological systems.
The ceramic-based microelectrodes that will be developed with this award have a number of potential uses in biology. The new microelectrodes will be shared with other scientists to explore these potential uses. In addition to collaborations with scientists, the microelectrodes will be used in numerous education programs that center on the dynamics of chemical signaling in biological systems. These include programs to educate local businesses and high schools about the uses of rapid chemical measures in biological systems, a program for high school students to participate in summer research, and a short course, held at Woods Hole, MA, that involves the training of graduate students, postdoctoral students and faculty in the use of the microelectrodes.

The neuroestorative and neuroprotective tropic actions of GDNF on midbrain dopamine (DA) neurons provide a promising therapeutic approach for the treatment of Parkinson's disease. As with many tropic factors, optimum success of this tropic factor depends on focal and controlled delivery of the protein to affect the function of DA neurons, without producing unwanted side effects. It is our central hypothesis that chronic intracerebroventricular or intraputamenal GDNF willproduce effects on damaged DA neurons with greater efficacy, potency and reduced side effects as compared to other methods of delivery. 1. We propose to use a novel indwelling pump that can deliver GDNF chronically in the freely-moving MPTP-lesioned monkey to evaluate two sites of delivery: the lateral ventricle and the putamen. 2. We will also investigate washout of GDNF and re-instatement of the trophic factor in both infusion paradigms. The different highly integrated Projects and Cores will provide key data regarding the functional effects of chronic GDNF treatments to DA neurons, and form the foundation for a Parkinson's Disease Center of Excellence. Project 1 will use microdialysis, in vivo electrochemistry and postmortem HPLC-EC methods to study the nigrostriatal pathway of unilateral MPTP-lesioned monkeys that have received chronic infusions of GDNF. Project 2 will investigate the behavioral consequences of the GDNF infusions and study potential functional changes to DA neurons using fMRI methods. Project 3 will carryout tract tracing and immunohistochemical measures of DA neurons in the same groups of chronically treated monkeys. The Cores A-D will all provide needed support for the experiments outline in Projects 1-3. These studies will answer key questions regarding the chronic delivery of a trophic factor in freely-moving and behaving rhesus monkeys. Such data could lay the foundation for the use of trophic molecules for the treatment of Parkinson's disease in humans.

nitric oxide; nicotinic receptors; schizophrenia; acetylcholine; sensory mechanism; neuropharmacology; hippocampus; carbachol; nicotine; autoradiography; laboratory rat; immunocytochemistry; magnetic resonance imaging;

DESCRIPTION: (provided by the applicant) The capability to record neurotransmitter release in totally unrestrained animals to study the effects of drugs of abuse in prenatal animals, rat adolescent models of drug abuse and transgenic mice, has been limited, or even unattainable. We propose to develop a technology we call Neurochem Chip. This technology involves the development of microsensors that are capable of nearly specifically measuring neurochemicals such as glutamate and GABA, using photolithographically designed microelectrodes and a self-referencing recording approach. In this Stage 1 proposal, Neurochem Chips will be integrated to polyimide flex cables in order to carry out recordings of neurotransmitter release in the frontal cortex, striatum and hippocampus in tethered, awake behaving rats. Pilot studies and prior work support that Neurochem Chip can reliably measure L-glutamate using a self-referencing recording paradigm. However, refinements in technology are needed in order to carry out such recordings in the awake behaving animal to lay the foundation for the development of a totally untethered recording system involving telemetry technology. In addition, a new prototype sensor that will allow for the simultaneous measures of both glutamate and GABA will be investigated. In Stage 2, the recording circuitry will be miniaturized and a total telemetric system will be developed so that large numbers of rats and mice can be recorded. In general, these technologies will allow for the rapid screening of the effects of drugs of abuse on brain neurotransmitter systems, acutely and chronically, in both adult and developing rats and mice which cannot be carried out using current technology. In addition, Neurochem Chip can possibly be developed to measure a large number of other neurotransmitters.

DESCRIPTION (provided by applicant): This Stage II CEBRA application involves the development of Neurochem Chip microelectrodes for neurochemical recordings of neurotransmitter release in rats and mice. The long-term goals of this project are to develop Neurochem Chips for rapid in vivo recordings of glutamate, GABA, acetylcholine and choline in the CNS of freely moving rats and mice. In developing the technologies, we will first implement and improve the technologies of Neurochem Chips in rats and mice. First, we plan to optimize the recording approaches in the anesthetized rat and mouse. This will involve microelectrode development in corporation with Thin Film Technologies, Hybrid Circuits, Inc., and Cleveland Medical Devices, Inc. Instrumentation development will occur in cooperation with PCB Assembly, Inc. We will carry out extensive in vitro and in vivo recordings of glutamate, GABA, choline and acetylcholine release and uptake in the prefrontal cortex, striatum and hippocampus of anesthetized rats. There are numerous microelectrode, instrumentation, and recording details that must first be optimized to develop reliable Neurochem Chips. These microelectrodes recordings will be compared with microdialysis measures of glutamate and choline, to try and further validate the techniques. As this grant progresses, the refined and adapted technologies will be applied to studies in awake behaving rats and mice. Freely moving tethered systems will be developed for 4-channel measures in awake rats and 2-channel recordings in mice. In addition, a biotelemetric system will be developed to record from rats with a totally untethered system. This should contribute to 1) increased signal-to-noise of the recordings, 2) increased freedom of movement of the rats, and 3) the potential for monitoring large numbers of rats. The major goal of these studies is to develop Neurochem Chips that are reliable, sensitive and selective for the rapid (second-by-second) determination of neurochemicals in the extracellular space of the brain. This technology could have a major impact on basic science studies of neurotransmitter systems involving drug abuse, disease models and behavior. This CEBRA Stage II mechanism is needed, due to the number of technical issues that must be addressed before these techniques can be disseminated to other researchers.

This award supports the development of microelectrode array technologies that can be used to directly monitor, on a second-by-second basis, neurotransmitter signaling in the brains of laboratory animals. The devices are formed on ceramic wafers analogous to microelectrode chips. The resulting microelectrode arrays can be mass fabricated for general scientific use and can be configured to directly measure second-by-second changes in the neurotransmitters glutamate, acetylcholine and GABA and other molecules. The present technology can be carried out in anesthetized animal preparations, brain slices and cellular systems. In addition, the technology can be adapted for studies in awake behaving animals so that neurotransmitter changes in the brain can be directly related to behavior and function. When perfected, this technology will be widely used by basic scientists in the fields of neuroscience and neurobiology, and may contribute to other applications with scientists developing biosensor devices. The development of the ceramic-based microelectrode array technology could allow for linking behavior with brain function and a further understanding of learning and memory, epilepsy, movement processes, drug abuse, brain systems and a variety of neuronal systems that all use glutamate, acetylcholine and GABA as their primary neurotransmitters. A variety of improvements to microelectrode design include 1) enhanced selectivity for the neurotransmitters glutamate, acetylcholine and GABA; 2) development of methodology to implement the recording technology inexpensively so that other laboratories can afford this state-of-the-art technology; 3) development of the technology for awake animals so that investigators can study the direct links between brain systems and behavior; and 4) development of nanotechnology approaches to enhance the recording properties of the microelectrodes.

Quantitative Neuropharmacoloqical.Studies of the Effects of Chronic Delivery of GDNF in Rhesus Monkeys The neurorestorative and neuroprotective trophic actions of GDNF on midbrain DA neurons provide a promising but controversial therapeutic approach for the treatment of Parkinson's disease (PD). Based on past work, we hypothesize that GDNF is affecting DA neurons through at least 3 major mechanisms: 1) Upregulation of existing DA neurons - direct effects on tyrosine hydroxylase and related proteins; 2) Repair of DA neurons - improved dendritic and neuronal fiber connections; and 3) Neurogenesis and/or gliogenesis. Project 1 will investigate DA function and repair of DA neurons, in conjunction with Projects 2 and 3. It is our central hypothesis that dual-site chronic intraputamenal/intranigral delivery of GDNF will improve function and restoration of damaged DA neurons with greater efficacy, potency and reduced side effects. In addition, we predict, based on preliminary data, that GDNF treatment in milder parkinsonian animals, a model of early stage PD patients, will lead to optimal restoration of the nigrostriatal DA neuronal system. We propose to use an indwelling pump that can deliver GDNF chronically in the freely-moving unilateral 1-methyl-4-phenyl- 1,2,3,6-tetrahydropyridine (MPTP)-lesioned middle-aged female monkeys. Project 1 of this revised program project grant will perform quantitative neurochemical assessments using in vivo microdialysis, Western immunoblot assays, immunohistochemical studies and high performance liquid chromatography coupled with electrochemical detection (HPLC-EC) to study DA neuronal systems in the striatum (putamen and caudate nucleus) and substantia nigra of Rhesus monkeys that have received chronic GDNF infusions. These neurochemical changes to the monkey striatum during chronic delivery of GDNF will be performed in conjunction with behavioral, immunohistochemical, histological and functional MRI (fMRI) studies of the same monkeys in conjunction with Projects 2 and 3. In addition, Project 1 will begin studies of glutamate regulation in the striatum, substantia nigra and globus pallidus of 6-OHDA- lesioned rats and the effects of GDNF infusion on glutamate regulation in the basal ganglia. This is as a new area of scientific growth of the Center. The investigation of the role of glutamate in relation of.DA function will improve our understanding of the essential role of this neurotransmitter to basal ganglia function in PD.

[unreadable] DESCRIPTION (provided by applicant): [unreadable] [unreadable] The neurorestorative and neuroprotective trophic actions of GDNF on midbrain dopamine (DA) neurons provide a promising but controversial therapeutic approach for the treatment of Parkinson's disease. We specifically plan to address the following major issues in the next 5 years of support for the University of Kentucky Udall Center: 1) We hypothesize that GDNF is affecting DA neurons through at least 3 major mechanisms: A) Upregulation of existing DA neurons - direct effects on tyrosine hydroxylase and related proteins (Project 1); B) Repair of DA neurons and connections - improved dendritic and neuronal fiber connections (Projects 2 and 3); and C) Neurogenesis and/or gliogenesis (Project 3). We will investigate these three mechanisms in the proposed experiments in nonhuman primate models of PD. 2) We propose to use a novel indwelling pump that can deliver GDNF chronically in the freely-moving MPTP-lesioned monkey to evaluate multisite delivery of GDNF (putamen vs. putamen + substantia nigra) to improve the functional restoration of DA systems. 3) Recent studies in PD patients involving a Phase 2 clinical trial have yielded controversial data regarding potential GDNF antibody formation and preliminary toxicology studies in rhesus monkeys support a potential toxicity of GDNF to cerebellar Purkinje cells and granule cells, when chronically administered in high doses (100 Lig/day) and subsequently withdrawn. In conjunction with Projects 2 and 3, we will investigate the potential toxic effects of GDNF on dopamine and non-dopamine containing cells in the CNS. In addition, we will investigate potential antibody formation from chronic GDNF administration. 4) Finally, we will use an improved delivery approach, to investigate the effects of GDNF infusion on milder parkinsonism in unilateral MPTP-treated monkeys, to investigate if earlier intervention of GDNF treatment may contribute to better functional recovery of the nigrostriatal system. We will utilize a recently developed stable, unilateral milder parkinsonian monkey model for these studies, which may be an "earlystage" model of PD. The different highly integrated Projects and Cores will provide key data regarding the functional effects of chronic GDNF treatments to DA neurons, mechanisms of action of GDNF both positive and negative, and will provide data that may be useful to gain a better understanding of the effects of trophic factors on dead or dying neurons. [unreadable] [unreadable] [unreadable] Project 1 [unreadable] [unreadable] Title: Neuropharmacological Studies [unreadable] [unreadable] PI: Greg Gerhardt, PhD [unreadable] [unreadable] DESCRIPTION (provided by applicant): [unreadable] [unreadable] Quantitative Neuropharmacological .Studies of the Effects of Chronic Delivery of GDNF in Rhesus Monkeys. The neurorestorative and neuroprotective trophic actions of GDNF on midbrain DA neurons provide a promising but controversial therapeutic approach for the treatment of Parkinson's disease (PD). Based on past work, we hypothesize that GDNF is affecting DA neurons through at least 3 major mechanisms: 1) Upregulation of existing DA neurons - direct effects on tyrosine hydroxylase and related proteins; 2) Repair of DA neurons - improved dendritic and neuronal fiber connections; and 3) Neurogenesis and/or gliogenesis. Project 1 will investigate DA function and repair of DA neurons, in conjunction with Projects 2 and 3. It is our central hypothesis that dual-site chronic intraputamenal/intranigral delivery of GDNF will improve function and restoration of damaged DA neurons with greater efficacy, potency and reduced side effects. In addition, we predict, based on preliminary data, that GDNF treatment in milder parkinsonian animals, a model of early stage PD patients, will lead to optimal restoration of the nigrostriatal DA neuronal system. We propose to use an indwelling pump that can deliver GDNF chronically in the freely-moving unilateral 1-methyl-4-phenyl- 1,2,3,6-tetrahydropyridine (MPTP)-lesioned middle-aged female monkeys. Project 1 of this revised program project grant will perform quantitative neurochemical assessments using in vivo microdialysis, Western immunoblot assays, immunohistochemical studies and high performance liquid chromatography coupled with electrochemical detection (HPLC-EC) to study DA neuronal systems in the striatum (putamen and caudate nucleus) and substantia nigra of Rhesus monkeys that have received chronic GDNF infusions. These neurochemical changes to the monkey striatum during chronic delivery of GDNF will be performed in conjunction with behavioral, immunohistochemical, histological and functional MRI (fMRI) studies of the same monkeys in conjunction with Projects 2 and 3. In addition, Project 1 will begin studies of glutamate regulation in the striatum, substantia nigra and globus pallidus of 6-OHDA lesioned rats and the effects of GDNF infusion on glutamate regulation in the basal ganglia. This is as a new area of scientific growth of the Center. The investigation of the role of glutamate in relation of DA function will improve our understanding of the essential role of this neurotransmitter to basal ganglia function in PD. [unreadable] [unreadable]

It is our central hypothesis that changes in the functional properties of dopamine (DA) neurons in the basal ganglia of the CNS contribute to age-related declines in motoric function in aged animals and humans. Prior studies from our laboratory and others support the hypothesis that age-related declines in motoric function are not associated with a robust decline in DA neurons. Rather, functional properties of DA regulation and release are affected in DA-containing areas. A striking finding from our recent studies is that somatodendritic release of DA, evoked by d-amphetamine and measured with in vivo microdialysis, is greatly decreased in the substantia nigra of aged monkeys. Using MRI guided stereotaxic surgery, this Project will use recently developed methods that allow for repeated in vivo microdialysis measures in anesthetized monkeys. This method has been combined with postmortem Western blot immunoassays and HPLC-EC methods for studies of the putamen, caudate nucleus, GPe and SN of young adult (5-9 year old), adult (13-16 year old) and late middleage/aged (20-25 year old) rhesus monkeys. This project will perform functional studies of neurochemical changes to the monkey basal ganglia during aging in conjunction with the behavioral, histological and immunohistochemical studies of Project by Gash and anatomical and functional MRIimaging studies in Project by Zhang. In addition, recent results from our laboratory support that glial cell linederived neurotrophic factor (GDNF) may be capable of restoring function to aged DA neurons. Our second series of neurochemical studies will be performed in late middle-aged/aged monkeys that have received chronic intranigral infusions of vehicle or GDNF, in an attempt to restore or enhance the function of aged DA neurons. These studies address the role of DA in decreased motor behavior in aging and the neurochemical properties of aged dopaminergic neurons in nonhuman primates.

DESCRIPTION (provided by applicant): The goal of this Training Program is to prepare promising graduate students and postdoctoral fellows for successful careers in the neurobiology of aging. We will provide broad-based training in modern concepts in the neurobiology of aging with an emphasis on cellular and molecular mechanisms underlying aging processes and possible therapeutic interventions. The unifying focus of the training faculty is our interest in understanding both normal and pathological mechanisms by which the nervous system responds to changes that occur with age and emerging concepts in therapeutic intervention. This Training Program provides the formal framework for faculty in different departments, who share a common interest in the molecular- cellular basis of brain aging and translational research, to provide in-depth training in aging. The emphasis of the Training Faculty complement each other: some focus on processes that occur in the brain during normal aging, others emphasize the neuropathological processes of diseases and injuries that predominate in the aging brain, and there is a combined interest in applying this knowledge to treating neural disorders of aging. Thus, trainees will learn from faculty who utilize a broad spectrum of state-of-the-art methodological approaches to probe critical questions in aging research. Our successes attest to our abilityto identify, attract, train and place promising young investigators in the area of the neurobiology of aging. Now more than ever before, we are particularly qualified to train graduate students and postdoctoral fellows. The national need for training new investigators in the area of aging is clear due to our changing demography and the increasing average lifespan. Normal aging or pathology of the nervous system underlies much age-associated morbidity. Only if we better understand the basic mechanisms that regulate the aging of the brain and apply this to the treatment ofthe elderly, can we hope to improve the qualityof life during the latter half ofthe lifespan. Our ability to attract and train graduate students and postdoctoral fellows to meet the challenges in the area of the neurobiology of aging will be greatly enhanced by this Training Program. RELEVANCE: This Program will train the next generation of scientists to carryout research on the neurobilogy of aging. Dramatic improvements in the treatment of age-related illnesses such as Alzheimer's disease, Parkinson's disease, stroke, epilepsy and others are need for the aging world population. Only through training of such research areas can we hope to make strides in new treatments for illnesses that cost us billions.

Microdialysis, which is used for brain recording technique, requires the analysis of neurochemical molecules. The micro-fluidics neurochemical analyzer that will be developed will enhance brain and spinal cord recordings with microdialysis by rapidly quantifying multiple neurochemicals with greatly reduced cost and complexity. The instrument, after optimization, is expected to measure neurochemicals such as glutamate, choline, acetylcholine, GABA, glucose and lactate in samples collected from the brain and spinal cord. The resulting inline detectors, coupled to a modern computer-instrument, are expected to have a time resolution of one measurement per second, detecting biologically-relevant levels of neurochemicals. The new analyzer will significantly impact neurosciences, where large numbers of scientists are limited in their use of microdialysis to study the temporal properties of neurotransmission in the brain by the current analytical methodology: A simple literature search yields more than 12,000 articles identified by the term brain microdialysis. The new instrument will have increased time resolution, enhanced sensitivity, rapid sample throughput and lower cost of operation and utilization than current equipment. All of these factors will contribute to an instrument that will be widely adopted to accelerate the efforts of biological researchers and clinicians. Demonstrations related to chemical recordings will be used in several outreach programs for K-12 educational ventures in Kentucky. For more information please contact the PI.

DESCRIPTION (provided by applicant): This CEBRA R21 application involves the further development, characterization and use of an advanced technology to study second-by-second changes in L-glutamate in specific brain regions in rats using enzyme- based microelectrode arrays (MEAs) during self-administration of cocaine. This grant combines the strengths of a recently developed recording technology for glutamate measures (Gerhardt lab) with known expertise in drug self-administration (Bardo lab). This grant will adapt and further develop an innovative technique for addiction research, as per the primary goals of the CEBRA program. The new technology can be potentially used by numerous investigators to better understand the role(s) of neuronal and possibly non- neuronal glutamate in addiction. The current proposal will develop and characterize a recently developed recording technology in awake and behaving rats to determine if cocaine self-administration is related to tonic and/or phasic release of glutamate in the nucleus accumbens core and infralimbic prefrontal cortex. The mechanistic framework for the proposed experiments rests on information about the homeostatic regulation of glutamate levels in critical reward-relevant brain regions. While a variety of neurotransmitter systems are involved in drug abuse, particularly dopamine, recent evidence indicates that glutamatergic tone in nucleus accumbens and related cortico-limbic structures is involved in the long-lasting consequences of stimulant exposure (Kalivas, 2009). Our overarching hypothesis is that psychomotor stimulants lead to enhanced neuronal glutamate release in response to subsequent administration of drugs. The proposal is innovative from both health and technological perspectives. While the changes in glutamate homeostasis have been proposed to underlie cocaine self-administration and reinstatement (Kalivas, 2009), extracellular glutamate measurements have been studied primarily by microdialysis, which does not have the temporal and spatial resolution required to evaluate both tonic and brief phasic pulses of glutamate that characterize neuronal release. Our preliminary results show that the MEA methods in awake animals can measure both tonic and rapid phasic levels of glutamate and that the events may provide different and very important information regarding regulation of glutamate circuitry in the normal brain and in addiction.

PROJECT SUMMARY / ABSTRACT Alzheimer?s disease (AD) progression and normal aging are complex and often heterogeneous processes involving functional changes to neuronal and glial elements that are not just related to neuronal cell loss [1-4, 143]. Prior in vitro and in vivo data support that aberrant regulation of glutamate neuronal systems can be a contributor to neuronal degeneration in aging and likely a contributor to the age-dependent development of AD [1, 4, 6-8]. We think that because there is a dearth of treatment and monitoring options our study will help in the future development of therapeutics and non-invasive spectroscopic monitoring techniques for AD. Recently, we have successfully adapted our enzyme-based microelectrode arrays (MEAs), which are designed to precisely measure tonic and phasic neurotransmitter release in discrete brain structures in awake animals, for use in aged rodents and AD mouse models [54-56, 79, 112]. A major knowledge gap in AD and aging involves changes in the excitatory/inhibitory balance between glutamate (Glu) and GABA release and regulation [9-15]. We believe that the use of our new recording technology will improve our understanding of the glutamate/GABA interplay in an AD model and in normal aging. Our recent studies using this superior technology now allows the measurements of tonic and phasic glutamate levels found that rats >18 months of age have normal or elevated basal Glu levels compared to younger rats. Thus, some animals have elevated glutamate while others do not. As outlined in NIH Notice NOT-AG-18-051 & related announcement PAR-19-070, issues surrounding age and its role in dementia are critical in furthering our understanding of the metabolic and pathological changes that affect signaling in neuronal circuits and networks. We will use young and aged normal and the APP?NLh/?NLh x PS1P264L/P264L knock-in mouse (APP/PS1 KI) that does not overexpress APP or PS1, or use artificial promoters, making it an ideal system for the study of how aging affects the development of AD-related neuropathology [86- 90]. We will use our novel methods to simultaneously record GABA and glutamate signaling in either the CA1 region of the hippocampus or Frontal Cortex. We will first behaviorally characterize all animals so that we can determine potential correlations between the behavioral performance of mice and glutamate and GABA release and/or regulation in the hippocampus (Morris water maze test) and frontal cortex (spatial memory variant of Morris water maze test) [115] and performed as per [116]. Finally, mice that are studied will undergo a pathological examination to evaluate neurodegeneration by evaluating reactive gliosis may be present by looking at Iba1 and GFAP. Collectively, these studies will allow us to compare the effects of normal aging in male and female to the effects seen from a mouse model of AD on the balance of glutamate and GABA signaling and its relationship(s) to cognitive function in both male and female animals towards the development of novel therapeutics to possibly treat the development and progression of AD.