Paul A. Garris - US grants

DESCRIPTION: The goal of the proposed work is to better understand neuronal compensation in Parkinson's disease. Symptoms of this neurodegenerative disorder are associated with the loss of nigrostriatal dopamine neurons but do not present until deficits are severe. It is postulated that potent adaptive changes in dopamine neurotransmission maintain function during the preclinical or presymptomatic phase. These adaptive mechanisms will be examined in a widely used animal model of Parkinson's disease, the rat with 6-hydroxydopamine lesions. The proposed experiments will study the regulation of extracellular dopamine in the partially denervated striatum, a condition that mimics the preclinical phase. Previous studies using the technique of microdialysis have documented normal concentrations of extracellular dopamine in the lesioned striatum despite losses of up to 80 % of the dopamine terminals. The proposed work will extend these observations by directly investigating the mechanisms responsible for maintaining extracellular dopamine levels. To accomplish this aim, real-time microsensors will be employed to monitor dynamic changes in extracellular dopamine elicited by transient electrical stimulation. In situ rate constants for dopamine release and uptake will be determined from the chemical measurements. Release and uptake are fundamental to dopamine neurotransmission and are the primary determinants of extracellular dopamine concentrations in the brain. A novel hypothesis will be tested. The hypothesis states that normal concentrations of extracellular dopamine are generated in the partially denervated striatum without active compensatory changes in dopamine release and uptake. An understanding of the adaptive changes that maintain dopamine function during the preclinical phase of Parkinson's disease could advance diagnosis and treatment of this disorder as well as provide new insight into other neurodegenerative disease, brain function during the normal aging process and neuronal plasticity.

A grant has been awarded to Dr. Paul A. Garris at Illinois State University
to develop a new instrument for wireless monitoring of neural activity in
the brain of awake, unrestrained animals. The new instrument, called
real-time animal telemetry (RAT), will combine two powerful technologies;
microsensors for spatially and temporally resolved measurements, and digital
telemetry for remote data transmission and system control with high speed
and high fidelity. The primary advantage of RAT will be sub-second
characterization of brain function with minimal perturbation of behavior.
The proposed RAT instrument holds great promise for advancing the study
of brain-behavior relationships.

RAT will be a modular and multifunctional instrument, a design that advances
development and affords flexibility to its application. Several types of
measurement techniques will be incorporated into RAT: Voltammetry which
monitors the chemistry of the brain and electrophysiology which
measures brain bioelectrical activity. Combined, the techniques assess the
release of a neurotransmitter and its postsynaptic effect to obtain a more
integrative view of brain function. Although great strides have recently been made
applying real-time voltammetry and electrophysiology to awake animals,
the connection between sensor and recording equipment is made by a cable tether. Unfortunately, the hard connection affects behavior and hinders or even prevents
investigation of important paradigms such as those involving social interactions
and complex environments. To overcome this problem, the new instrument will
use a wireless link. Moreover, because real-time voltammetric and electrophysiological measurements are very susceptible to transmission artifacts, RAT will use high fidelity digital telemetry.

Ultimately, RAT should be commercially viable instrumentation in the support of
biological research. The incorporation of well-established techniques will
make the proposed RAT instrument attractive to large number of users working
in the neuroscience fields. With further development, there is enormous
potential for RAT to support other existing real-time microsensors. RAT can
also evolve to accommodate sensor technologies that emerge in the future.

[unreadable] DESCRIPTION (provided by applicant): Considerable research effort has justifiably been directed towards identifying the mechanisms of amphetamine action. However, whether amphetamine acts on phasic dopaminergic signaling, as has been shown recently for cocaine, another psychostimulant, is not known. Some involvement is anticipated, given the role proposed for this mode of dopaminergic neurotransmission in learning and motivated behavior and amphetamine's use in treating attention deficit hyperactivity disorder and its highly addictive nature. On the other hand, while both drugs inhibit dopamine uptake, amphetamine, but not cocaine, depletes vesicular stores of dopamine. Consequently, amphetamine may not share with cocaine the ability to increase the amplitude of dopamine concentration transients in the nucleus accumbens, presumably elicited by phasic or burst firing and exocytotic release. Examining the link between amphetamine and phasic dopaminergic signaling is significant, because the behaviorally relevant mechanisms by which this psychostimulant acts are not fully elucidated. In particular, documented dissociations between amphetamine's effects on behavior and dialysate dopamine suggest other targets besides tonic dopaminergic signaling. In contrast to phasic dopaminergic signaling, which generates dopamine concentration transients in terminal fields, tonic dopaminergic signaling maintains a low, steady-state or ambient level of brain extracellular dopamine. To address these important issues related to amphetamine action, the present project will investigate the effects of amphetamine on phasic dopaminergic signaling. The first aim will compare the stimulant effects of amphetamine in rats and Syrian hamsters. While separate studies suggest that Syrian hamsters are less sensitive to amphetamine than other rodents including rats, this difference has not been established under the same conditions. By comparing neurochemical measurements in these two species in the subsequent experiments, the behavioral relevance of amphetamine effects on phasic dopaminergic signaling will be uniquely assessed. The second aim will determine the effects of amphetamine on dopamine uptake and exocytotic dopamine release in the nucleus accumbens, which have not been established in vivo. These presynaptic mechanisms regulate dopamine transient amplitude. The third aim will characterize the effects of amphetamine on the frequency and amplitude of dopamine concentration transients in the nuclues accumbens directly using the same approach, fast-scan cyclic voltammetry in freely moving animals, that was previously used to establish cocaine effects on phasic dopaminergic signaling. This research will investigate how amphetamine, which is used both as a therapeutic agent and a drug of abuse, affects the brain. New microsensor technology will be employed in laboratory animals to characterize amphetamine's effect on brain chemistry related to learning and motivaton. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): In this Cutting-Edge Basic Research Award (CEBRA) proposal, an electrical engineer/computer scientist (PI Mohseni) and a neurobiologist/analytical chemist (PI Garris) will collaborate to develop an ultra-small, implantable device for wireless neural monitoring and stimulation in awake animals. This device will advance the investigation of biologic mechanisms of drug addiction by extending recent innovations in fast-scan cyclic voltammetry (FSCV) at a carbon-fiber microelectrode (CFM). By virtue of chemically selective recording with sub-second temporal resolution at a micron-sized probe, FSCV at a CFM is recognized as state-of-the-art for neurotransmitter monitoring. In the last decade, great strides have been made applying FSCV at a CFM to ambulatory rats for studying the role of phasic dopaminergic transmission in goal-directed behaviors, particularly with regard to cocaine reinforcement, and combining chemical and electrical measurements at the same probe for quantifying postsynaptic effects of brain dopamine dynamics. By extending the already attractive analytical attributes of FSCV at a CFM on several fronts, we firmly believe that the device proposed here meets criteria for the CEBRA program. Proposed developments include: (1) replacing the hardwired connection with a digital telemetry link; (2) replacing the external stimulus generator with an on-chip component; (3) significantly reducing overall dimensions, weight, and power consumption; (4) supporting multiple, independently configurable channels for FSCV, electrophysiology, and combined measurements. Collectively, these advances address concerns related to the current use of "large" head-mounted devices as well as tethering animals to equipment, which may alter behavior, act as a noise source, and preclude measurements in smaller, but valuable animal models such as transgenic mice. Moreover, multiple data channels afford the enticing prospect of marrying two powerful technologies for neural monitoring, FSCV at a CFM and multielectrode array single-unit recording, to assess complex circuit-level control of drug-related behavior on an integrative level. The three specific aims are to: (1) using very-large-scale-integration techniques in standard complementary-metal-oxide-semiconductor technology, develop a 16-channel device supporting voltammetry, electrophysiology, and combined measurements, as well as electrical stimulation; (2) test and characterize the device on several levels, including benchtop engineering assessment, in vitro calibration with flow injection analysis, and anesthetized and awake rats; (3) showcase device features in two pilot applications, FSCV dopamine measurements in the awake rat during high-dose administration of the psychostimulant amphetamine and single-unit recording with microwire bundles chronically implanted in a transgenic mouse model. By developing the first wireless integrated circuit supporting chemical and electrical sensing and electrical stimulation, this CEBRA proposal will address key limitations of existing instrumentation for FSCV at a CFM and advance this already powerful neural monitoring technique. PUBLIC HEALTH RELEVANCE: This project will develop a miniaturized wireless device supporting chemical and electrical recording as well as electrical stimulation in the brain of small laboratory animals. This device will advance basic science research investigating neural mechanisms of drug addiction. [unreadable] [unreadable] [unreadable]

This award supports a collaborative effort between PIs at Case Western Research University and Illinois State University to develop new instrumentation in support of neurobiology research. Monitoring neural activity in awake laboratory animals has proven to be a powerful tool for investigating how the brain ultimately controls behavior. Driving this approach are recent advances in microsensors for probing brain function very quickly and on microscopic scales as the behavior occurs. However, neuromonitoring at implanted microsensors remains particularly challenging, as the majority of available measurement systems are hampered by large size, high power requirements, and wired connections between animal and recording equipment. These technological limitations will be overcome by developing the next generation of ultra-small, low-power wireless devices for neurochemical and neuroelectrical monitoring and for neuromodulation using electrical stimulation in freely behaving animals.

State-of-the-art engineering methods called very-large-scale-integration (VLSI) and complementary-metal-oxide-semiconductor (CMOS) technologies will be employed to manufacture the proposed devices. This fabrication process will result in multichannel, multifunctional, wireless devices whose size and weight are suitable for implantation, freeing the animal from exposed, bulky instruments and cables that alter behavior, generate noise artifacts during movement, and limit experimental design. Power consumption will be dramatically reduced as well by the fabrication strategy, enabling the use of miniature batteries as a power source during operation. Once constructed, assembled and packaged into a chronically implantable form, and tested, these devices will be used in animal experiments to investigate the role of dopamine in motivated behavior. This important brain neurotransmitter has been implicated in responding to rewards or the cues that predict rewards, and in altering the long-term functioning of brain circuits involved in motivation.

By overcoming technical limitations of existing instrumentation, these new miniature wireless devices for will advance neurobiological investigations in the primary animal models used for the study of brain-behavior relationships and will extend this line of research to smaller animals whose size has previously limited inquiry. Such devices will ultimately be commercially viable driven by the needs of the larger neuroscience community. Through dissemination of these sensors and collaboration with users the sensor platform can evolve to meet emerging research needs. Additionally, this project will not only train undergraduate and graduate students in both science and engineering in the respective laboratories of the investigators, but it will also foster interdisciplinary training as the two laboratories will extensively and closely work together to develop and apply the new instrumentation. In a broader vein, the award will lead to establishing new working relationship between neurobiologists and engineers in support of both research and education.

DESCRIPTION (provided by applicant): Neurochemical Pattern Generation with Smart Electrical Stimulation Pedram Mohseni1 and Paul A. Garris2 1 Case Western Reserve University 2 Illinois State University In this Small Research Grant (Parent R03) proposal, an electrical engineer/computer scientist (PI Mohseni) and a neurobiologist/analytical chemist (PI Garris) will collaborate to develop the next generation of wireless neurochemical sensing integrated circuits (ICs) by incorporating the additional functionality of chemical pattern generation with electrical stimulation. Real-time chemical microsensors hold great promise for investigating brain function and pathology. Their defining analytical characteristic is the capability to interrogate the activity of a single neuron type, by virtue of identifying the released neurotransmitter. Perhaps the most monumental achievement to date with this measurement modality is dopamine monitoring with subsecond temporal resolution at a brain-implanted, micron-sized probe during goal-directed behavior using fast-scan cyclic voltammetry (FSCV) at a carbon-fiber microelectrode (CFM). Great strides have also been made in wireless ICs supporting this ground-breaking technology. Adding neurochemical pattern generation to extant sensing- only ICs substantively expands the utility of chemical sensing ICs, ultimately laying foundational work for future closed-loop devices. We submit that extending passive chemical measurements to the realm of high-precision, dynamic chemical control is consistent with the R03 scope of development of new research technology. The two specific aims of this proposal are to: (1) develop a neurochemical sensing IC supporting FSCV at a CFM with integrated electrical stimulation capability for neurochemical pattern generation; (2) test and characterize the IC. The engineering innovation is that IC architecture will precisely synchronize the timing of voltammetry and stimulus current generation to avoid temporal overlap and minimize the possibility of stimulus artifacts interfering with FSCV recordings, whenever the stimulator is activated via an external trigger. In addition to benchtop engineering assessment, functionality will be tested in vitro with flow injection analysis and in vivo with anesthetized rats, using diverse neurochemical patterns as templates. The conceptual innovation is transfer function-driven neurochemical pattern generation and subsequent verification of the fidelity of the generated profile by the stimulating-sensing IC. Selection of dopamine as the test analyte in this work is very judicious. Involved in important brain functions and debilitating neuropathologies, dopamine is amenable to FSCV detection, is the most studied neurotransmitter using microsensors, and has well-established transfer functions suitable for time- and amplitude-based pattern generation. Transferable to other neurotransmitters, microsensor strategies, and applications, we emphasize the general versatility of the proposed IC as a more wide-ranging device beyond these development tests with dopamine. Thus, in the long term, the proposed IC will provide an innovative and powerful addition to the neurobiology toolkit for investigating the neural underpinnings of behavior and disease symptoms, and could have additional clinical bearing by providing the framework for ultimately developing new neuromodulation devices for many human neuropathologies. PUBLIC HEALTH RELEVANCE: Neurochemical Pattern Generation with Smart Electrical Stimulation Pedram Mohseni1 and Paul A. Garris2 1 Case Western Reserve University 2 Illinois State University Relevance to Public Health: This project will develop an advanced integrated circuit for measuring and controlling neurotransmitter levels in the brain of laboratory animals. This technology will advance basic biomedical research and the development of neuroprostheses for treating human neuropathologies.

DESCRIPTION (provided by applicant): A Closed-Loop Microsystem for Neuromodulation of Reward Circuitry Pedram Mohseni1 and Paul A. Garris2 1 Case Western Reserve University 2 Illinois State University While great strides have been made in our understanding of the basic neurobiology of addiction, many outstanding questions still remain. There is also a grave necessity to develop new treatments, as current options exhibit high recidivism. For good reason, particular attention has focused on the role of dopamine neurons in compulsory drug taking and addictive behavior. One emergent hypothesis is that abused substances usurp reward-processing circuits by hyperactivating phasic dopamine signaling, which leads to altered synaptic plasticity and the overvaluation of cues predicting drug availability. Driving the pursuit of this potentially unifying hypothesis are recent technical advances in microsensors, transgenic animals, and optogenetics. Collectively, these powerful approaches permit high-fidelity dopamine monitoring and ultra-fine molecular control over dopamine neurons and their targets. However, there is a dearth in the current state of technology for dynamic, state-dependent control. Such technology would actively link neuromonitoring and neurostimulation in closed-loop manner to permit extant neural activity and a priori criteria determine the desired outcome. The long-term objective of this research is thus to realize closed-loop devices supporting research in drug abuse and clinical therapies for treating addiction. To this end, an electrical engineer/computer scientist (PI Mohseni) and a neurobiologist/analytical chemist (PI Garris) will collaborate on the present Cutting-Edge Basic Research Awards (CEBRA) proposal to develop a computation and control integrated circuit (IC) for such linking of neuromonitoring and neurostimulation. This IC will incorporate fast- scan cyclic voltammetry (FSCV) at a carbon-fiber microelectrode (CFM), a state-of-the-art neuromonitoring technique with exquisite temporal, spatial, and chemical resolution, and principal component regression (PCR), a chemometrics approach for resolving single analytes from complex neurochemical profiles. The three specific aims are to: (1) develop a sensing, computation, and control IC supporting FSCV at a CFM; (2) test and characterize the IC; (3) pilot dopamine-sensing-based feedback control with the IC for neutralizing activated phasic dopamine signaling. We submit that developing this IC is a transformative step for addiction research, by laying the foundation for novel implantable microsystems supporting applications in biomedical research and ultimately for smart therapeutic neuroprostheses in the clinical realm. Feedback control based on the ability of FSCV and PCR to interrogate the neurochemical activity of a single neuron-type also represents a significant technical advance toward the development of closed-loop devices. This research is additionally innovative, because an IC for neurochemical feedback control has not been realized, and the proposed IC will be capable of modulating both static and dynamic neurochemical activity and accommodating a broad repertoire of analytes important in addiction. This project will also train one doctoral student each in the fields of electrical engineering-computer science and neurobiology-analytical chemistry during its two-year duration.