Dennis A. Turner, MD - US grants

DESCRIPTION: (Investigator's Abstract) The objective of this proposal is to analyze the anatomical and physiological integration of neurons from dissociated fetal hippocampus which have been grafted into normal and lesioned adult hippocampus. The synaptic integration of the transplants into the hippocampal circuitry of the recipient will be assessed in the following specific aims. The first aim is to directly test the hypothesis that transplants of dissociated fetal hippocampus become integrated into the normal host hippocampus and develop synaptic connections with host neurons. This will be accomplished by pre-labeling fetal neurons with Rhadomaine microspheres to identify the grafted neurons in an in vitro slice preparation so that extracellular and intracellular physiological recording techniques can be utilized to assess synaptic integration of identified grafted cells. The density and quality of synaptic interactions will be used to evaluate the physiological integration of the grafted neurons which will be filled with the intracellular stain, Lucifer yellow, to precisely assess the degree of their anatomical integration into the host parenchyma. Ingrowth of graft processes into appropriate and/or inappropriate cytoarchitectonic areas will be used to measure the anatomical integration of the grafted neurons. In a second series of experiments the principal investigator will establish the specificity and strength of physiological connectivity between graft and host by selectively removing only the grafted neurons from hippocampal slices during physiological recording sessions. To accomplish this the grafted neurons will be pre-labeled with a laser sensitive photo-activated dye, chlorin e6 attached to latex microspheres. After physiological recording are made in hippocampal slices the tissue will be exposed to laser illumination from an infrared laser which will produce selective loss of only the labeled grafted neurons. It is stated that this approach will directly remove only grafted neurons and their synaptic connections and lead to a specific test of the physiological mechanism of graft interaction with the host hippocampus. In the final specific aim the investigators will test the hypothesis that synaptic integration and survival of fetal hippocampal grafts varies as a function of both the severity and time course of lesions to the host brain. This will be accomplished by using unilateral kainic acid lesions involving the CA3 region of the hippocampus as a more severe target lesion to remove the Schaffer collateral input onto the dendrites of CA1 pyramidal neurons and to compare the effects of this lesion on graft survival and integration with a less extensive and more selective novel laser induced lesion of CA3 neurons possessing commissural projections. This second lesion will be produced by infrared laser illumination of one hippocampus in which the commissural projecting CA3 neurons have been retrogradely labeled with photo-activated dye containing microspheres injected into the contralateral hippocampus. Replacement of synaptic circuitry in the CA1 area will be analyzed after grafting of pre-labeled fetal hippocampal cells into the denervated CA1 region at various time points after either lesion. Analysis of the time course of enhancement of graft integration will critically test the hypotheses that both neurotrophic factors induce by the lesions and vacated synaptic sites resulting from the denervation influence graft survival and integration.

This project will develop methods of Bayesian statistical analysis for mixture distributions in studies of mechanisms of neuronal synaptic activity. Physiological analysis of synaptic mechanisms (such as short- or long-term potentiation) is based on presumptions regarding activity at individual synaptic sites, yet such individual sites can rarely be analyzed directly. Thus, to infer baseline activity and dynamic changes at individual sites from physiological recordings, statistical/neurophysiological models have been developed which include the output from multiple sites and cater for background noise. These models assume that the recorded cell output represents a random mixture distribution composed of synaptic signal 'components,' where each 'component' represents one or more synaptic sites. Bayesian mixture models and methods of analysis using (uncertain) mixtures of (uncertain numbers of) components will be explored in this context. This pilot project will aim to benchmark and validate the novel Bayesian approaches in the context of experimental data from synapses with small numbers of neural transmitter sites. Technically, research will formulate prior distributions for model parameters based on available synaptic noise data, physiological evidence, and experimental conditions; study simulations to examine operating characteristics and explore the sensitivity and robustness to prior and model specifications; derive inferences about physiological parameters; determine methods of assessment of goodness of fit; make comparisons with existing and traditional approaches; and develop computer software development for implementation of the modelling techniques. The project represents a collaborative, cross-disciplinary initiative to develop novel methods for analysis and statistical inference concerning the mechanisms governing electrochemical signal transmission at neural junctions in animal nervous systems. Interaction between the theoretical development and physiological data will play a critical role in identifying and resolving uncertainties about physiological processes and mechanisms in nervous systems, assessing site to site variability in neural signals, synaptic output, identifying critical synaptic characteristics, and assessing changes under differing experimental and physiological conditions. The project will involve substantial analysis of existing and forthcoming physiological data sets in order to provide assessments of the validity of the statistical models and the derived inferences with respect to underlying physiology. The immediate objective is to validate the new statistical approaches proposed by demonstrating their application and improvements over existing statistical analyses in synaptic response analysis. Ultimately these methods are expected to contribute to fundamental understanding of signalling mechanisms in human and other nervous systems.

DESCRIPTION (provided by applicant): The central hypothesis of this project focuses on protective strategies for enhancing neuronal metabolism during and after short-term hypoglycemia. Several related mechanisms will be studied using physiological and mitochondrial imaging techniques in acute hippocampal slices, following hypoglycemia. In particular, we will directly assess neuronal-glial metabolic inter-relationships by analyzing whether the "lactate shuttle" is altered as a function of age. We will also directly measure the presence or absence of mitochondrial permeability transition as a function of age. Because slice metabolism varies as a function of slice oxygenation, age of the tissue and slice conditions (i.e., interface versus submerged slice conditions), direct oxygen tension measurements will be performed in the tissue using a Clark-style oxygen microelectrode at the same depth as the electrical recordings. The oxygen tension monitoring will ensure that metabolic substrate provision is controlled appropriately within the slice. Neuronal-glial metabolic interactions will be analyzed using glial poisoning with fluoroacetate to estimate direct neuronal contribution to glycolysis and aerobic metabolism, by substituting lactate and pyruvate. Pyruvate will be assessed as a potential treatment for preventing hypoglycemic damage. Occurrence and prevention of mitochondrial permeability transition with these stresses will also be assessed, using cyclosporin A and newer analogs such as N-Me-VaI-CSA. Analysis of these mechanisms which lead to enhanced susceptibility to neuronal damage following hypoglycemia are likely to lead to enhanced treatment. Mechanisms of neuronal injury are expected to vary depending on the duration and severity of the hypoglycemia, and the age of the animal from which hippocampal slices were harvested. These in vitro slice models of hypoglycemia will contribute greatly to the understanding of neuronal metabolism, and particularly the metabolic interactions between neurons and glia as a function of lifespan.

[unreadable] DESCRIPTION (provided by applicant): [unreadable] The central hypothesis of this project focuses on metabolic neuroprotection mechanisms, which can maintain neuronal metabolism during and following acute hypoglycemia. Several different forms of metabolic enhancement will be studied using physiological and mitochondrial imaging techniques in acute hippocampal slices, following various levels of hypoglycemia or energy deprivation. Both glucose and oxygen levels will be tightly monitored in the tissue slices for actual tissue levels. Several mechanisms likely contribute to enhance acute cell death and susceptibility to hypoglycemia, the susceptibility to which varies considerably across the lifespan, from the neonatal period to aging. Neuroprotective mechanisms to be studied include provision of intermediate metabolites, such as pyruvate, lactate or ketone bodies, enhancement of glycogen stores in glial cells, and exploration as to decreased need for glucose in neonatal individuals. Because slice metabolism varies as a function of slice oxygenation, age of the tissue and slice conditions (i.e., interface versus submerged slice conditions), direct oxygen tension measurements will be performed in the tissue using a Clark-style oxygen microelectrode at the same depth as the electrical recordings. The oxygen tension monitoring will ensure that metabolic substrate provision is controlled appropriately within the slice. Similarly, glucose levels in the tissue will be monitored using micro-iontophoresis electrode techniques. These studies will reveal both the effects of graded hypoglycemia in the tissue as well as a number of metabolic neuroprotective strategies, which may be extended to dietary treatments to buffer intermittent hypoglycemia. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Aging can be associated with a decreased ability to respond to metabolic challenges resulting, for example, in fatigue on cognitive tasks or increased susceptibility to substrate deprivation, such as relative hypoglycemia or hypoxia. We hypothesize that cellular and molecular changes in the regulation of intrinsic CNS bioenergetics (i.e., neuronal-glial interactions, aerobic and anaerobic metabolic pathways, substrate availability, etc.) during aging can impair adaptive responses to sustained energy demand. We propose to study prolonged metabolic demand and fatigue in isolated brain tissue from aged animals, in the absence of systemic factors such as poor vasculature or systemic substrate supply, to identify intrinsic changes in neuronal metabolism and neuronal-glial interactions. In vitro brain slices obtained from aged animals retain the in vivo metabolic characteristics of that age, as well as the intrinsic circuits and other factors leading to regulation of metabolism on a local tissue scale. Preliminary experiments indicate that neuronal function and mitochondrial redox state in aging hippocampus are more vulnerable to metabolic stress, such as lowered glucose levels and prolonged synaptic stimulation, compared to tissue from younger animals, suggesting that aged individuals may have reduced ability to support an increased rate of oxidative metabolism for an extended period of time. We will evaluate neuronal fatigue and neuronal-glial interactions during prolonged metabolic stress by studying the energetic relationships between oxygen utilization, mitochondrial redox state, and neuronal activity, using direct tissue lactate, glucose and Po2 measurements, NAD(P)H fluorescence, and neuronal responses in hippocampus. These techniques will be used during prolonged synaptic stimulation (increased metabolic demand) and conditions of limited substrate delivery. These results will facilitate understanding how local tissue responses and bioenergetics affect metabolism in aging. The understanding of the mechanisms underlying neuronal fatigue may indicate novel targets for treatment which may enhance performance on sustained cognitive tasks. PUBLIC HEALTH RELEVANCE: This proposal seeks to understand how metabolism in the brain changes with aging, assessing both mechanisms underlying fatigue to persistent responses and possible treatment directions. The goal is to assess components of oxidative and glycolytic metabolism, particularly during sustained metabolic demand, over minutes, which are intrinsic to neurons and glia. The in vitro slice preparation proposed here allows assessment of local metabolic interactions directly in brain tissue, without direct involvement of the vascular system and systemic provision of substrates.

DESCRIPTION (provided by applicant): It has proven difficult to recreate perceptually accurate and realistic touch and pressure sensation following thalamic stimulation in humans. Previous human experiments have used primarily single-site microstimulation with regular patterns of stimulation, which consistently results in a perception of tingling or paresthesias, and only occasionally a more natural sensation. We propose to perform multi-site microstimulation in human sensory thalamus during intra-operative experiments, while performing thalamic DBS surgery. During these experiments we will first test pairs of electrodes in a 16 or 32-channel Adtech microwire array and a Tucker- Davis acute, switching headstage. We have developed a switching unit to rapidly connect various pairs of electrodes for microstimulation. Using this switching unit connected to the headstage we can rapidly evaluate multiple pairs of electrodes for the evoked sensory response. These experiments are a natural extension of our large experience in recording with these microwire arrays from motor thalamus and subthalamic nucleus during a complex behavioral task (n = 52 patients). Then, we will next apply patterned microstimulation (10 - 50 5A) at dual or multiple microwire combinations in the sensory thalamus, to recreate the evoked actual mechanical skin input. Patterns will initially consist of a decelerating stimulus with an increasing interpulse interval (like an adapting primary sensory response), an accelerating stimulus with progressively shorter interpulse intervals, and a constant pulse sequence, with the same number of pulses applied over a 1 sec period. The patient's perception of the thalamic stimulation will then be critically assessed for these three patterns, while progressively altering the amplitude, the duration (from 100 to 1000 msec) and the location of the stimulation (to different pairs). These experiments will both help reveal the critical patterning of the natural evoked responses in sensory thalamus, as well as provide a potential substrate for insertion of realistic sensory inputs for development of a sensory prosthetic device. The R21 mechanism will be used to develop the programmed multisite microstimulation protocols and to assess the patient-derived concept of realistic perception, in direct comparison to a graded touch signal. Further tests will use a DBS macro-electrode, but with patterned microstimulation, to assess whether it is possible to extend the microwire data to a larger field. PUBLIC HEALTH RELEVANCE: This project will study how the brain processes sensation and the perception of sensation, in the human thalamus. This study will be important to understanding diseases where sensation is abnormally altered, such with nerve damage. This research may help to understand how an artificial sensory signal could be interpreted as a normal sensation, if the proper spatial and temporal pattern can be recreated. These results may lead to a sensory prosthesis to replace sensation where the nervous system has been damaged, for restoration of function.

? DESCRIPTION (provided by applicant): Current understanding of Alzheimer's disease focuses on accumulation of amyloid and tau proteins, enhanced disease progression with vascular factors (ie, APoE), a large reduction in metabolism and substrate/energy supply to the brain, significant changes in neurovascular coupling, neuronal damage leading to memory and cognitive abnormalities, cholinergic cell loss, and diffuse brain atrophy. Though a large number of treatments are in trials, the underlying basis of Alzheimer's disease remains unclear. Thus, similar to dopamine replacement therapy for Parkinson's disease, the clinical focus for Alzheimer's disease has been to treat symptoms (ie, memory) rather than the underlying (unknown) cause. Thus, available human treatments focus on acetylcholinesterase inhibition to improve cholinergic function and memory. Other clinical approaches to enhance cholinergic cell function and memory loss in Alzheimer's patients have involved nerve growth factor [NGF] gene therapy into nucleus basalis Meynert and deep brain stimulation [DBS] applied to the fornix, both currently in trials. However, human fornix stimulation has also been noted to show widespread metabolic changes in the brain. Though the focus of this DBS approach has been on memory enhancement, fornix and septal stimulation also induces cholinergic stimulation, which can affect blood vessel reactivity and neurovascular coupling and improve metabolism throughout the brain. We hypothesize that fornix DBS stimulation is also causing secondary septal stimulation of cholinergic nuclei, affecting neurovascular coupling and blood flow, in addition to direct hippocampal physiological effects. Septal stimulation would lead to diffuse cholinergic enhancement of hippocampal function, causing changes in excitatory transmission, neurovascular coupling and enhanced substrate/metabolic supply to the brain, likely improving the widespread vascular changes noted in Alzheimer's disease. We propose to study both physiological and vascular effects of fornix/septal stimulation in a mouse model of Alzheimer's disease that shows a clear, progressive deterioration with representative histological changes (ie, plaques and tangles) over months [CVN-AD].

Abstract DBS therapy for Parkinson's disease is now the primary surgical approach for Parkinson's disease, recently FDA approved at 4 years after onset of disease. However, this therapy is still limited to treatment of a subset of motor symptoms (ie, tremor, rigidity, bradykinesia and dyskinesias) and requires considerable postoperative clinical adjustment to program and maintain function. A number of improvements to DBS for PD are being tested, including changes in patterns of stimulation and specific targets. However, a major new approach involves internal parameter adjustment using a surrogate physiological marker of clinical symptoms, useful for confirming initial electrode placement, programming, and also long-term optimization of parameters. Several research systems have been suggested and are in testing for development of closed loop systems, including systems based on recording beta-band oscillations. Closed loop control involving recording a surrogate signal relevant to PD could improve DBS therapy on several time scales, including short-term dynamics (ie, over minutes), initial programming (over weeks to months), and long-term, depending on the time course of response to STN DBS. In addition to spontaneous beta band recording we have also implemented direct evoked potential recording using the stimulating DBS electrode, requiring suppression of the DBS-induced artifact. These intraoperative DBS recordings during STN DBS implants have revealed a complex evoked potential likely reflecting GPe/GPi activation, which may provide an excellent surrogate marker. This complex evoked potential changes over a short-term time period as the treatment effect of STN DBS comes on, indicating that the evoked potential likely reflects DBS effects on a larger motor circuit as the circuit dynamically is altered to an improved state. We hypothesize that this surrogate marker (in addition to beta band oscillations) may provide a key feedback signal for scalar, graded (proportional) closed loop DBS control, highly relevant to DBS effects on PD circuitry. To test this hypothesis we will perform long-term recording of this signal from humans (in either STN or GPe/GPi) together with DBS stimulation (in STN and/or GPi), using a novel DBS recording/stimulation device (Medtronics RC+S). These clinical experiments will focus on a small, pilot clinical study (n = 6 patients) to implant bilateral STN + GPe/GPi DBS electrodes in Parkinson's patients eligible for DBS using conventional stereotactic localization, connecting to Medtronics RC+S IPGs. Patients will benefit from either ordinary STN or GPi DBS stimulation and then we will also test the possibility of synergism between the two electrodes for clinical efficacy. Additionally, we will analyze the motor efficacy of both an external (using recording and modifying the parameters manually) and internal (using an algorithm for providing parameters automatically) scalar, closed loop response to these recorded surrogate markers. We will take advantage of the graded nature of the spontaneous and evoked responses to construct a proportional control feedback system, and to specifically delineate the time constants of the closed loop system to be able to define optimally damped control of PD symptoms. These experiments will provide a number of novel outcomes, including a direct, within-person comparison of STN and GPe/GPi DBS efficacy, development of an optimal surrogate parameter for detecting DBS efficacy using spontaneous and evoked physiological responses in direct comparison to clinical symptoms, and defining an optimal, scalar feedback, proportional control system for treatment on various time scales.