John Disterhoft - US grants

The experiments proposed here are based on and will extend the recent in vitro experiments by Disterhoft and collaborators. They have demonstrated conditioning-specific biophysical alterations in hippocampal CA1 pyramidal neurons of brain slices from nictitating membrane/eye retraction conditioned rabbits. Rabbits will be conditioned in a tone discrimination reversal nictitating membrane paradigm. Hippocampectomized rabbits cannot learn this discrimination reversal successfully. Pseudoconditioned, naive and antidromic activation rabbits will be controls. Extracellular recording from CA1 and CA3 pyramidal cells will be done to determine if hippocampal engagement is unilateral when monaural tone CS's are paired with an ipsilateral puff US. If hippocampal changes are unilateral, one hippocampus can serve as a within-animal control in the brain slice experiments. In vitro hippocampal slices will be prepared and maintained with standard procedures. The experiments will be carried out "blind", without experimenter knowledge of the behavioral training history of the rabbits. Biophysical parameters to be measured include spike amplitude, resting potential, input resistance, sag, AHP magnitude and duration. A current, accommodation, EPSP and IPSP amplitude. The calcium-mediated potassium current, which underlies the slow AHP, and the fast, voltage activated potassium current, IA, will receive particular attention. Pyramidal neurons and the interneurons intermixed with them will be studied to determine if biophysical alterations are found in both cell types after conditioning. CA1 and CA3 regions will be examined to determine if ionic alterations in these functionally connected regions are interdependent. Stages of discrimination reversal acquisition will be varied to determine when biophysical alterations become established during learning. The possibility that cellular changes become consolidated between training sessions will be evaluated by studying slices made immediately after or 24 hours after the training session. Evidence for a correlation between biophysical and behavioral measures across the total learning curve will be sought. The research program will begin probing the cellular substrates of mammalian associative learning in a novel way by using the in vitro brain slice. This approach allows a biophysical analysis of learned alterations in specified cell population not previously possible.

Our laboratory has demonstrated that the dihydropyridine calcium antagonist nimodipine markedly facilitates associative learning in aging rabbits. Further, we have demonstrated in young adult rabbits that among the neural changes induced by classical conditioning is a reduction in the afterhyperpolarization (AHP) that follows a burst of action potentials by hippocampal CA1 neurons. This AHP is generated through the activation of a Ca2+-dependent K+ conductance. Nimodipine antagonism of neuronal calcium conductance may cause a reduction of the AHP in hippocampal neurons of the aging rabbits, thereby including a biophysical change similar to one found in the young adult hippocampus following learning. This pharmacologically- induced reduction of the AHP may in turn facilitate learning. We outline a series of behavioral neurophysiological and biophysical experiments to test this hypothesis. We will also address basic questions regarding how aging might alter the AHP, Ca2+ and K+ currents in CA1 pyramidal neurons. Hippocampus, a brain region affected by aging, will be studied; trace eyeblink conditioning, a hippocampally-dependent associative learning task, will be used. Behavioral pharmacological experiments will define the nimodipine dose/response curve, the retention period of the nimodipine induced learning facilitation, effects on tone conditioned stimulus (CS) sensitivity and control for nonspecific performance enhancement. The possible contribution of enhanced cerebral blood flow, in addition to modulation of neuronal function, will be evaluated by measuring nimodipine enhancement of blood flow and by determining the effects of flunarizine, a non-dihydropyridine Ca2+ channel blocker, on learning in aging rabbits. Single hippocampal neurons will be studied in vivo as an index of nimodipine's action on elements of the conditioned reflex arc during learning. Biophysical experiments will be performed on CA1 pyramidal neurons in brain slices and acutely dissociated neurons to determine the effect of aging on the AHP, Ca2+ and faster K+ currents; on responsitivity to neurotransmitters including acetylcholine, norepinephrine and serotonin; and on size of the AHP reduction after associative learning. Our experimental program is designed to investigate cellular mechanisms by which learning is impaired by aging and by which pharmacological intervention with nimodipine might facilitate learning in aging rabbits. In general, our research is addressed to the alterations in calcium metabolism which are known to occur in the aging brain. The particular compound which we propose to investigate, nimodipine, has been approved by the FDA for clinical use in humans in the US and is already being used in Europe. Therefore, it is likely that these experiments will make a rather direct contribution to the clinical amelioration of learning deficits in the aging human in the near future.

This proposal describes a series of experiments designed to investigate the effects of phencyclidine (PCP) on associative learning and its substrates in the central nervous system. Behavioral, neurochemical, and biophysical methods will be used to examine potential cellular mechanisms for PCP-induced learning deficits. PCP, a common street drug of abuse, is a noncompetitive antagonist of the ionophore of the NMDA receptor. The NMDA channel has received considerable attention recently because of its involvement in the induction of neural plasticity. We will assess the effects of PCP on learning, memory, and its underlying neural mechanisms. The eyeblink conditioning task we will use in rabbits has direct behavioral, and presumably neural, parallels in humans. The PCP dose regimens to be used attempt to simulate the consumption patterns of PCP abusers. We hypothesize that activation of the NMDA receptor-complex is critical for associative learning, based on our finding that chronic PCP treatment blocks acquisition. Retention of previously learned tasks will also be tested. PCP binds with high affinity within the NMDA receptor's ionophore, with particularly dense binding concentrated in the hippocampus. We propose to test whether the hippocampus is a substrate for PCP's observed deleterious effects on learning, using two hippocampally-dependent tasks, trace and tone discrimination reversal eyeblink conditioning in rabbits. MK-801 binds to the PCP receptor site within the NMDA ionophore with higher affinity and greater specificity than PCP itself. We have preliminary evidence that eyeblink conditioning causes enhanced [3H]MK-801 binding (an increase in Bmax) in whole hippocampal membrane preparations from trained compared to pseudoconditioned or handled control rabbits. We will repeat and extend these experiments by examining the effects of PCP on [3H]MK-801 binding, and the time course enhanced binding related to specific stages of learning and specific schedules of PCP treatment. Quantitative autoradiographic techniques will be used to determine whether there is cellular specificity of altered binding within hippocampus following conditioning and/or PCP treatment. The slow afterhyperpolarization (AHP), a Ca2+-dependent kappa+ conductance(s), that follows a burst of action potentials in hippocampal CA1 pyramidal cells is reduced after learning. PCP also apparently affects specific kappa+ conductances. Changes in the AHP, in spike accommodation and in specific kappa+ conductances induced by learning and affected by PCP will be evaluated in CA1 pyramidal cells with current-and voltage-clamp recordings in the slice/patch preparation. Effects of PCP and/or learning on NMDA-mediated transmission will also be examined. Our experimental program is designed to characterize the behavioral deficits which PCP abuse causes, as well as to begin to investigate causative factors at the cellular level with biophysical and neurochemical techniques. Since PCP is a major drug of abuse, it is likely that this research program could make a rather direct contribution to understanding and possibly ameliorating the learning deficits which may be a major consequence of PCP abuse.

Eyeblink conditioning in rabbits will be used as a "model behavioral system" in the analysis of the neural substrates of learning. The medial septum, CA1 hippocampus and prefrontal cortex, important substrates for a variety of learning tasks in both humans and other mammals, will be studied. The "trace" eyeblink conditioning task, in which the rabbit must form a short-term memory trace that the tone conditioned stimulus has occurred in order to appropriately control the blink and avoid an airpuff to the eye, will be used. This task depends upon hippocampus for its successful acquisition. Single neuron activity will be recorded in three interconnected stations of the hippocampal system-from medial septum (the principal source of cholinergic drive to the hippocampus proper), from the CA1 subfield of the hippocampus (the major output of the hippocampal trisynaptic circuit), and from medial prefrontal cortex (which receives afferent drive from CA1 both directly, and indirectly via dorsomedial thalamus). The short term memory load will be manipulated by increasing the trace interval to determine if this affects neuronal processing in any of the regions. The relationships between changes in neuronal firing activity and behavioral acquisition will be examined to determine if they occur at specific time point in training or with specific latencies relative to the response within each region. The excitability changes recorded in vivo will be directly correlated with biophysical measurements of cellular substrates for the excitability increases made in vitro in hippocampal and neocortical slices. Rabbits will be trained to different stages on their "neuronal acquisition curves" and the degree of the slow calcium-mediated after hyperpolarization reduction in CA1 pyramidal neurons will be compared in vitro at different stages of training. Comparable measures will be made after training in cortical slices. The septal cholinergic system will be closely examined to determine if this system may play a particularly important role in establishing the postsynaptic excitability changes recorded both in vivo and in vitro from CA1 pyramidal neurons during training. Evidence for spatial concentrations of change will be sought to determine if neurons in each station function homogeneously during and after acquisition of a hippocampally-dependent task and to see if their function changes as the memory load of the task is increased. The proposed studies are relevant to a better understanding of how the hippocampus and a neocortical region to which it projects function during learning in all mammals, including humans. The hippocampus becomes dysfunctional in Alzheimer's disease and often in aging, leading to memory loss. Its dysfunction is also thought to underly some of the cognitive manifestations of schizophrenia. Frontal cortex is especially likely to be traumatized in auto accidents. Prefrontal damages causes disorders of attention, recent memory, initiative and affect. Understanding hippocampal and prefrontal cortical cellular activity during learning in normal brain will facilitate developing pharmacologic and other clinical strategies for treating their functional disruptions.

The goal of this revised competitive renewal application is to understand the function of the elements of a brain circuit which interconnects the hippocampus and cerebellum during learning and consolidation of trace eyeblink conditioning. The working hypothesis is that the hippocampal formation temporarily potentiates the response of the conditioning stimulus (CS) when it is paired with an unconditioned stimulus (US). This potentiated response facilitates the input from the pontine nuclei to the cerebellum and becomes necessary for learning when the temporal demands of the associative task are more complex, as in trace conditioning. Tetrodes for neuron ensemble recording will be placed in: the medial septum (MS) to detect changes related to the cholinergic and inhibitory neuron drive to the hippocampus; area CAl and the subiculum to follow changes in responsiveness of the hippocampal formation; the retrosplenial cortex (RSCtx) to follow the response to the CS through acquisition and consolidation o the CR; pontine nuclei (pN) to measure the timing and amplitude of the tone CS input to the cerebellar circuit and changes in the response to the CS; cerebellar anterior interpositus nucleus (IPA) to record cerebellar circuit output and a neural "assay" o behavioral learning; the ventral anterior thalamus (vaTh) to monitor changes related to feedback from the IPA; prefrontal cortex (PFCtx) and anteroventral thalamus (AvTh) to evaluate the loop through the prefrontal cortex by which the cerebellum is hypothesized to transfer information about the developing conditioned motor response to the hippocampus. Reversible lesions will be made at various points in the circuit to evaluate their contribution to acquisition and consolidation of the eyeblink CR. Retrosplenial and/or prefrontal cortices are hypothesized to serve as long term storage sites for changed activity relevant to the learned association. Modern single neuron ensemble recording and analysis techniques will be used. These will include recording 25-50 single neurons simultaneously in the behaving rabbit with several tetrodes; computerized waveform separation with advanced cluster cutting techniques; neuron-behavior response analysis with poststimulus time histograms, multiple between neuron cross correlations, and multivariate analysis techniques; use of reversible lesions for probing the function of specific sites during learning and consolidation.. It is known that both the cerebellum and the hippocampus are intimately engaged in mediating the eyeblink conditioning task, especially when the stimulus demands are enhanced as in the trace eyeblink conditioning task. The dynamics of this interaction are not understood. Further understanding of how the hippocampal-cerebellar circuit functions in the rabbit brain during learning will have direct parallels to its operation in the human brain. This information has relevance to understanding and treating disorders of learning such as those which occur alter stroke, in aging, in dementing diseases such as Alzheimer's Disease, and in disorders which may involve hippocampal dysfunction such as schizophrenia or epilepsy.

A better understanding of how the neural circuits underlying associative learning and memory change in aging brain and may be related to age- associated learning deficits will be sought. Our strategy will be to study brain system activity in young and aging subjects in parallel with changes in behavior during the acquisition of a well-defined behavioral paradigm and in reference to appropriate control conditions. Functional magnetic resonance imaging (fMRI) will be used in conscious subjects to simultaneously examine conditioning-specific hemodynamic changes during acquisition and consolidation of eyeblink conditioning. The circuitry within the frontal cortex, hippocampus and associated temporal cortex, basal ganglia and cerebellum will be examined. These regions are chosen for emphasis because of their demonstrated involvement in the eyeblink conditioning task in both animals and in humans, and because they are likely to change in important ways during the aging process. The first specific aim is to compare the light conditioned stimulus (CS) and corneal air puff unconditioned stimulus (US) pathways with fMRI at baseline before learning. The second specific aim will be to examine the circuit activated during a 500 msec trace conditioning paradigm which is learned equivalently by young and aging subjects. The third specific aim will be to examine the circuit activated during a 1000 msec trace eyeblink conditioning paradigm in which aging subjects will show a deficit. We hypothesize that this paradigm will more thoroughly activate the frontal cortex and hippocampus/temporal lobe and thus be more likely to detect functional differences due to reduced function in these regions during the aging process. We expect that these experiments will yield significant information regarding the location of changes in hemodynamic activity during associative learning and after aging throughout the frontal cortex-limbic-cerebellar system. The will provide the basis for a noninvasive functional technique to evaluate the effectiveness of potential therapeutic agents for enhancing neural system function during learning and memory in conditions such as normal aging and Alzheimer's Disease. They will also enhance our ability to further understand the neurobiological substrates of associative learning in the human brain.

i The hippocampus is critically involved in the early stages of declarative learning, and its function and capacity are degraded during normal aging that causes age-associated learning impairments. It has been repeatedly demonstrated that a cellular biomarker of this age-associated learning deficit is the enlarged Ca2+-dependent postburst afterhyperpolarization (AHP) that reduces the intrinsic excitability of CA1 pyramidal neurons in aged subjects. Thus, we have hypothesized that restoring intrinsic excitability of aged CAI neurons to a young-like state by reducing the AHP using genetic manipulations would rescue the age- related learning deficits. Hence we have designed a research program to identify the candidate proteins for genetic manipulation with the use of recombinant adeno-associated viral (AAV) vectors. In the initial 3.5 years of this MERIT award, we have determined that 1) Ca2+ accumulation in the cytosol evoked with trains of action potentials is greatly elevated in aged CA1 neurons and may underlie the enlarged AHP in these neurons; 2) Ca2+ buffer capacity is increased in aged CAI neurons, potentially as a cellular mechanism to counteract the increased Ca2+ accumulation; 3) CREB activation (an important cellular mechanism for protein synthesis necessary for learning and for AHP reduction) is impaired in hippocampus of aged rats; and 4) L-type Ca2+ channel (LTCC) expression on the surface of CAI neurons is elevated in aged rats, which provides a molecular mechanism for the reported increased Ca2+ influx through LTCC in aged CAI neurons. Based on these findings, we have identified Ca2+ binding proteins, CREB, and LTCC as candidates to rescue the age-related deficits by manipulating their function with AAV vectors. We have created AAV vectors targeting CREB and LTCC, and will continue the systematic characterization of their potential as therapeutics for restoring the age-related deficits. The candidate Ca2+ binding protein genes to manipulate will be determined from protein microarray experiments (a new powerful method to screen expression level changes in hundreds of proteins), and confirmed through literature review and further molecular (e.g., western blot) assays. In addition, we will identify the source(s) ofthe elevated Ca2+ accumulation in aged CAI neurons using Ca2+ imaging with two-photon laser scanning microscopy; and thus, reveal additional potential therapeutic targets for intervention. Our goals remain unchanged: to confirm that the AHP is the key regulator of intrinsic excitability and that targeted molecular methods to reduce the AHP in CAI neurons in aged subjects will lead to successful learning. Continued success will indicate that the protein being manipulated is a viable candidate to target as a therapeutic intervention point for age- associated learning impairments. This research program has clear relevance to understanding and treating neurodegenerative diseases such as Alzheimer's Disease, in which aging is the principal risk factor. RELEVANCE (See instructions): Behavioral, calcium imaging, molecular and biophysical experimental approaches will be used to investigate the role of neuronal calcium processing in control of learning in young and, aging rats. The goal is to determine if molecular genetic interventions developed from these approaches reverse age-associated learning impairments in rats. Successful experiments will have direct translatability to humans, as molecular genetic approaches are being developed to treat neurodegeneration in aging humans and the hippocampus- dependent eyeblink conditioning task has direct parallels between experimental animals and humans. ; r i IFCT/PFPFOPMAMnP .';iTF/<;\ r\f aHHitinnal cnat-o ic noorlorl iico

DESCRIPTION (provided by applicant): The cerebellum and its associated brainstem circuitry are required for learning and retaining simple conditioned reflexes of striated muscles, and the hippocampus is required to acquire and temporarily store stimulus associations for more complicated conditioning paradigms, such as trace conditioning. The overall goals of this proposal are to continue to characterize the neural substrates of these intermediate events in the hippocampus and other forebrain regions and, additionally, to begin to localize the brain regions underlying permanent storage of memory using hippocampally-dependent trace eyeblink conditioning in the rabbit. Our analysis of this circuit should lead to a more thorough understanding of the interrelationships among the cerebellum, hippocampus, and hippocampally-related forebrain structures, including the caudomedial prefrontal cortex (cmPFC), cortical association areas and the basal ganglia. Our aims will test a common hypothesis that the "memory trace" is transferred from the hippocampus to the cortex during acquisition and consolidation of learned responses. Our recent single neuron recordings indicate that the hippocampus and caudomedial prefrontal cortex mediate only a temporary role in learning the hippocampus-dependent trace eyeblink conditioned response. We propose that an increased excitability of association cortex neurons provides a necessary facilitation of pontine / mossy fiber inputs to the cerebellum, and that the basal ganglia provide necessary feedback from the cerebellum to the forebrain required to establish the neocortical changes. Single neuron activity will be recorded from large numbers of cells in rabbits during learning with multiple, independently moveable tetrodes to characterize activity at several key sites on the developing conditioned reflex arc. Inactivation will be done with reversible lesions, combined with single neuron recording and behavioral measurements, to evaluate the role of some regions. Our experimental program will begin testing the hypothesis that memories are stored in neocortical regions in a process dependent upon the engagement of the hippocampus and basal ganglia earlier in the learning process. Our data will have considerable relevance to better understanding the processes by which learning occurs in mammalian brain, including in human brain as we and others have shown important parallels in the processes by which humans and experimental animals acquire eyeblink conditioning. The results of these experiments will also be useful in the design of more appropriate treatments for learning deficits in young and aging individuals.

DESCRIPTION (provided by applicant): This new proposal is a request for funding of a broadly-based postdoctoral and pre-doctoral Mechanisms of Aging and Dementia Training Program from the Northwestern University Institute for Neuroscience (NUIN). Funding to support four advanced pre-doctoral candidates, after they have begun full-time thesis research, and four postdoctoral trainees, in early or later stages of training, is requested. This training program has developed from a multi-disciplinary group of investigators whose work focuses on the mechanisms of aging and dementia, in particular Alzheimer s Disease, with approaches spanning molecular, cellular, systems, behavioral, neuropsychological and clinical neuroscience, including four with current NIH MERIT awards, one with a Javits award and one who is a Howard Hughes Investigator. Seven of the 23 faculty preceptors are women, including the Associate Director. These faculty are affiliated with NUIN, the Cognitive Neurology and Alzheimer s Disease Center that has an NIA-supported Alzheimer?s Disease Center grant, and the Buehler Center on Aging. The program will be directed by John Disterhoft, Ph.D., with the assistance of Sandra Weintraub, Ph.D. (Associate Director), an Internal Steering Committee and an External Advisory Committee. The four postdoctoral and four pre-doctoral trainees will conduct their research under the guidance of 23 preceptors from 10 departments of 2 schools on the Chicago and Evanston campuses of Northwestern University. Postdoctoral trainees will be selected on the basis of previous training and a research plan. Pre-doctoral trainees will be selected from NUIN and the Medical Scientist Ph.D. Training Program on the basis of course performance, rotation, and the relevance of the proposed dissertation research. Special consideration will be given to trainees whose research plans are interdisciplinary and carried out in more than one preceptor laboratory. A concerted effort will be made to recruit women and minorities. The program will offer a broad range of interdisciplinary research and training opportunities in both fundamental and clinical approaches to aging and dementia research. The preceptor faculty will assist and monitor trainee progress through formal advising and evaluations, through the classroom, and through informal discussions. In addition to providing research training, the program will help trainees develop skills in written and oral communication, grant writing, networking, and career development. Instilling a clear awareness of ethical issues facing neuroscientists and responsible conduct in science will be another training goal.

[unreadable] DESCRIPTION (provided by applicant): This application is a request for continued funding of a broadly based postdoctoral and predoctoral Mechanisms of Aging and Dementia Training Program from the Northwestern University Institute for Neuroscience (NUIN). Funding to support four advanced predoctoral candidates, after they have begun full time thesis research, and four postdoctoral trainees, in early or later stages of training, is requested. This training program has developed from a multidisciplinary group of investigators whose work focuses on the mechanisms of aging and dementia, including Alzheimer's Disease, Parkinson's Disease and Amyotrophic Lateral Sclerosis, with approaches spanning molecular, cellular, systems, behavioral, neuropsychological and clinical neuroscience, including four with current NIH MERIT awards, and one Howard Hughes Investigator. Seven of 24 faculty preceptors are women, including the Associate Director. These faculty are affiliated with NUIN, the Cognitive Neurology and Alzheimer's Disease Center, that has an NIA supported Alzheimer's Disease Center grant, the Buehler Center on Aging and the Udall Parkinson's Disease Center. The program will be directed by John Disterhoft, PhD with the assistance of Sandra Weintraub, PhD (Associate Director), an internal Steering Committee and an External Advisory Committee. The four postdoctoral and four predoctoral trainees will conduct their research under the guidance of 24 preceptors from 10 departments of 2 schools on the Chicago and Evanston campuses of Northwestern University. Postdoctoral trainees will be selected on the basis of previous training and a research plan. Predoctoral trainees will be selected from NUIN and the Medical Scientist PhD Training Program on the basis of course performance, rotation and the relevance of proposed dissertation research. Special consideration will be given to trainees whose research plans are interdisciplinary and carried out in more than one preceptor laboratory. A concerted effort will be made to recruit women and minorities. The program will offer a board range of interdisciplinary research and training opportunities in both the fundamental and clinical approaches to aging and dementia research. The preceptor faculty will assist and monitor trainee progress through formal advising and evaluations, through the classroom and through informal discussions. In addition to providing research training, the program will help trainees develop skills in written and oral communication, grant writing, networking, and career development. Instilling a clear awareness of ethical issues facing neuroscientists and responsible conduct in science will be another training goal. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The aims of this research program are to use the whisker barrel cortex and pons as "anchor points" to examine information processing loops within the forebrain during whisker signaled trace eyeblink conditioning (EBC). These aims will be done to test our hypothesis that permanent representations of the conditioning network are located in SI cortex, entorhinal (EC) and/or perirhinal (PR) cortex, and caudate nucleus. Five specific aims will determine if there are increases in activity suitable to potentiate the input to the pontine nuclei that mediate forebrain facilitation of cerebellar dependent learning. 1. Recordings from the pontine nuclei will determine if activity patterns from neurons after conditioning change relative to baseline and to pseudoconditioning. The forebrain afferents to pontine regions that change will also be defined with retrograde tract tracing. 2. Barrel columns in somatosensory cortex will be studied to determine how neurons in different layers of this sensory neocortex change during learning and after memory consolidation. Descriptions of the structure and function of the whisker barrel microcircuit are used to predict the sequence of change that will be seen. Single neuron activity in thalamic barreloids will define the role of thalamic input to the cortex during learning. 3. Reversible lesions in SI barrel cortex will be done after behavioral consolidation to confirm and extend our pilot data indicating that such lesions block the performance of consolidated conditioned responses. 4. EC interfaces hippocampus and SI cortex via PR and may transfer functional changes between them during learning and consolidation. We will test the hypothesis that EC is required for consolidation of trace CRs with reversible lesions. An EC layer analysis will be done with single neuron recording. Since PR is the cortical node between EC and somatosensory cortex, it will be explored as a site for consolidated responses if the EC experiments are negative. 5. Single neuron recordings of different neuron classes in caudate nucleus during learning will be completed to determine the role of this region in mediating the procedural aspects of trace EBC. Reversible lesions will be done to determine if the caudate nucleus is required for retention of trace CRs after behavioral consolidation and the activity patterns of caudate neurons will be studied after consolidation of trace EBC. The well defined somatotopic arrangement of the whisker representation in the rabbit sensory cortex makes it a useful region on which to focus while examining neocortical mechanisms mediating consolidation and memory storage following hippocampal and temporal neocortical processing of a learned response signaled by a whisker stimulation CS. A common hypothesis is that permanent storage of learning resides in the neocortex following hippocampal processing. Data supporting this hypothesis are rare. This proposed research program will address this issue by defining nodes mediating a well studied forebrain mediated learned response. PUBLIC HEALTH RELEVANCE: The well defined map of the whisker representation in the rabbit sensory cortex makes it useful for examining neocortical mechanisms mediating consolidation and memory storage of a learned response signaled by whisker stimulation. Our eyeblink conditioning paradigm will allow us to examine the cortical and striatal mechanisms involved in conditioned responding. This learning should have common mechanisms with the neural basis of maladaptive behaviors such as drug addiction, and the data gathered here will also be useful for understanding and treating deficits in learning and memory in young and aging persons.

DESCRIPTION (provided by applicant): Learning and memory impairments often accompany aging. Frequently, such impairments are related to age- related dementias. A substantial proportion of these, however, are attributable to so-called normal aging. Normal age-related cognitive dysfunction is most apparent in hippocampus-dependent behaviors. Interestingly, however, some aged individuals exhibit intact mnemonic function, whereas others are severely impaired, despite being the same chronologic age. Though the behavioral and physiological deficits associated with age- related cognitive decline are well characterized, the substrates for such individual variability remain unknown. The aim of the proposed research is to evaluate the possibility that hippocampal synapses in aged impaired animals are gradually weakened such that distal synapses no longer have the same influence on neuronal output as more proximal ones. The proposed degradation of distance-dependent synaptic scaling in aged impaired animals would significantly disrupt synaptic integration and consequently impair hippocampal function. The basic strategy of the proposed research is to diagnose aged rats as either impaired or unimpaired using two hippocampus-dependent tasks, and then examine the morphology, receptor expression, function, and plasticity of their synapses in hippocampal CA1 pyramidal neurons. Rats will be behaviorally characterized using the Morris water maze and trace eyeblink conditioning, which assay spatial and temporal learning, respectively. The analyses of synapses will be accomplished by combining conventional and immunogold serial section electron microscopy with whole-cell patch-clamp recordings from CA1 pyramidal neurons. The major prediction is that the proportion or number of perforated synapses in aged impaired rats relative to aged unimpaired rats will be reduced, and this will be accompanied by a reduction in the expression of synaptic AMPA-type and NMDA-type receptors, particularly among distal synapses. The whole-cell patch- clamp recordings will determine whether distal synapses and proximal synapses have the same influence on neuronal output in aged unimpaired rats, and whether such location-independence is disrupted in their aged impaired counterparts. Finally, an examination of the induction and reversal of long-term potentiation and depression in behaviorally characterized aged rats will provide insight into whether dysfunctional synaptic regulation contributes to the proposed reduction in distance-dependent synaptic scaling, and whether it correlates with age-related cognitive decline. All together, the experiments in this proposal will provide fundamental insights into synapses in the aging brain, and whether their form and function relate to cognitive capacity. Additionally, determining the nature and locus of deficient synaptic function in aged animals will facilitate the design of preventative measures intended to make aging more "succesful". PUBLIC HEALTH RELEVANCE The substrates underlying individual variability in the aged population remain unknown. The experiments in this proposal will provide fundamental insights into synapses in the aging brain, and whether their form and function relate to cognitive capacity. Additionally, determining the nature and locus of deficient synaptic function in aged animals will facilitate the design of preventative measures intended to make aging more "succesful".

DESCRIPTION (provided by applicant): This proposal is a request for continued funding of a broadly based postdoctoral and predoctoral Mechanisms of Aging and Dementia Training Program from the Northwestern University Interdepartmental Neuroscience Program (NUIN). Funding to support four advanced predoctoral candidates, after they have begun full time thesis research, and four postdoctoral trainees, in early or later stages of training, is requested. This training program has developed from a multidisciplinary group of investigators whose work focuses on the mechanisms of aging and dementia, including Alzheimer's Disease, Parkinson's Disease and Amyotrophic Lateral Sclerosis, with approaches spanning molecular, cellular, systems, behavioral, neuropsychological and clinical neuroscience. These faculty are affiliated primarily with NUIN and the Cognitive Neurology and Alzheimer's Disease Center, that has an NIA supported Alzheimer's Disease Center grant, and the Udall Parkinson's Disease Center. The program will be directed by John Disterhoft, PhD, with the assistance of Sandra Weintraub, PhD (Associate Director), an internal Steering Committee and an External Advisory Committee. The four postdoctoral and four predoctoral trainees will conduct their research under the guidance of 32 preceptors from 10 departments of 3 schools on the Chicago and Evanston campuses of Northwestern University. Postdoctoral trainees will be selected on the basis of previous training and a research plan. Predoctoral trainees will be selected from NUIN and the other participating PhD programs on the basis of course performance, rotation evaluations, and the relevance of proposed dissertation research. Special consideration will be given to trainees whose research plans are interdisciplinary and carried out in more than one preceptor laboratory. A concerted effort will be made to recruit minorities. The program will offer a broad range of interdisciplinary research and training opportunities in both the fundamental and clinical approaches to aging and dementia research. The preceptor faculty will assist and monitor trainee progress through formal advising and evaluations, through the classroom and through informal discussions. In addition to providing research training, the program will help trainees develop skills in written and oral communication, grant writing, networking, and career development. Instilling a clear awareness of ethical issues facing neuroscientists and responsible conduct in science will be another training goal.

This proposal aims to enhance diversity within the biomedical workforce by supporting the development of six recent baccalaureate graduates from underrepresented backgrounds to be competitive for neuroscience PhD and/or MD/PhD programs in the best research-oriented universities in the country. The ?Northwestern University Interdepartmental Neuroscience Postbaccalaureate Research Education Program (NU IN-PREP)? represents a cross-departmental initiative that will be administered by leading faculty within the Northwestern University Interdepartmental Neuroscience (NUIN) PhD Program. Because neuroscience is highly interdisciplinary and applicants are from varied academic backgrounds, IN-PREP will create individualized plans for skill building experiences tailored to each scholar's training objectives, thus providing the best preparation for success in graduate school. Experienced IN-PREP mentors, in collaboration with the IN-PREP Director, will work with trainees to customize a one year research program focused on developing critical thinking skills, research techniques, data analysis, and scientific communication. Preparation for graduate school admissions will include a GRE workshop, advice on writing personal statements and interviewing skills. Coursework will include one advanced undergraduate or graduate course to complement the trainee's previous coursework, mini-courses in cellular physiology and cell and molecular biology, and scientific communication workshops. Intensive advising using Individual Development Plans will be done to ensure that skill building, research training and graduate school applications are progressing. IN-PREP will take advantage of the centralized infrastructure of NUIN, which will work closely with IN-PREP to provide administrative support and a broad range of training opportunities, including: guidance for graduate school admissions, engagement with NUIN graduate students at seminars, the annual retreat, and training events in one of six institutional NIH- funded neuroscience training programs. IN-PREP scholars will integrate into the broader neuroscience community by attending the annual national and Chicago Chapter meetings of the Society for Neuroscience. Cohort building activities include participation in select events sponsored by the IMSD-funded Collaborative Learning and Integrated Mentoring in the Biosciences Program; two joint socials with University of Chicago PREP trainees; and a joint scientific conference with PREP trainees from the University of Chicago, Mayo Clinic and University of Michigan. IN-PREP will be administered by the Director, two co-Directors, a Program Assistant, and a Steering Committee. Program evaluation and fine tuning will be done annually and an External Advisory Committee will evaluate IN-PREP during the second and fourth year of the funding cycle. The milestones of NU IN-PREP are for at least 80% of trainees to be admitted to a top ranked neuroscience graduate program and for at least 80% of trainees to attain their PhD degrees within 6 years of graduate school matriculation. The one year NU IN-PREP program has been designed to achieve these milestones.

This project concerns the etiological origins of sporadic Alzheimer's disease (AD). While plaques and tangles still define AD, it is thought that oligomeric forms of A? and tau play major roles in the pathogenic mechanism. What triggers the buildup of these toxins, however, and whether the etiological triggers affect toxin impact, remain unknown. The long-term goal of this project therefore is to understand the etiological origins of sporadic AD, with a strategic focus on the pathobiology of AD risk factors. Its central hypothesis is that risk factors have a common ability to stimulate pathogenic A?O buildup, but that each has its own signature regarding the nature and clinical outcome of this buildup. The important relationship between risk factors and A?O pathobiology has not been studied before in unbiased, non-transgenic models. The immediate focus is on two metabolic AD risk factors: hypercholesterolemia (HypC) and type 2 diabetes (T2D). Investigations will address three aims and will provide results that test the central hypothesis and a prediction of potential diagnostic value. AIM 1 ? Determine the pathobiology that makes HypC and T2D act as risk factors for sporadic AD. The working hypothesis is that the dysfunctional metabolic states of HypC and T2D are AD risk factors because they promote the buildup of pathogenic A?Os. AIM 2 ? Establish a mechanistic principle to explain why AD manifests as heterogeneous phenotypes. The working hypothesis is that although the risk factors HypC and T2D each promote buildup of pathogenic A?Os, there is an etiology-sensitive signature to that buildup that influences the cognitive outcome. AIM 3 ? Establish in vivo biomarkers that can diagnose the etiology-sensitive status of A?O neuropathology. The working hypothesis is that tandem imaging of brain A?Os and brain function will optimally diagnose AD in a manner that is etiology-sensitive. Rabbit will be used as a non-transgenic model. Pilot data show that substantial memory dysfunction is caused by diets associated with HypC and T2D. This dysfunction is linked to A?O buildup, and it manifests with an etiology-specific signature. New analytics will establish the impact of HypC and T2D on brain region-selective memory performance, functional and molecular MRI, AD-linked neuropathology, and buildup of distinct A?O species. The approach is the first rigorous investigation into the onset of A?O pathobiology in an animal model unbiased by transgene expression. The expected outcome is establishment of a molecular mechanism to connect risk factors HypC and T2D to sporadic AD. Results are expected, moreover, to establish a platform for future mechanistic studies of the now-burgeoning list of AD risk factors, illustrate the significance of etiology to AD diagnosis and precision medicine, and accelerate development of effective AD treatments and prevention strategies.

Project Summary/Abstract The distributed brain network of the hippocampus supports memory and related cognitive abilities. Disruptions of this network occur in many neurological disorders such as epilepsy, brain injury, and neurodegenerative disease. Brain stimulation targeting the human hippocampal network can produce long-lasting improvements of memory ability, with corresponding increases in brain-activity markers of network function. However, mechanisms for this beneficial network-level neuroplasticity caused by brain stimulation remain unknown. Mechanistic knowledge is essential to optimize how and where to stimulate the hippocampal network in order to maximize the resulting memory benefits. This project will investigate the cellular mechanisms for the effects of brain stimulation on the hippocampal network. We will capitalize on the property that activity of regions of the hippocampal network synchronize in the theta frequency band (5-8Hz) to test for mechanistic homology in the effects of stimulation on human versus rodent hippocampal networks. In humans undergoing neurosurgery for intractable epilepsy and in awake, behaving rodents, we predict that electrical stimulation will have greater effects on hippocampal network function when it is delivered with increasing levels of synchronization to the ongoing hippocampal theta activity rhythm. Thus, we will test whether the effects of manipulating the synchrony between brain stimulation and hippocampal theta activity are comparable in humans and rodents. The effects of stimulation will be assessed using measures of hippocampal network functional connectivity and paired-associate memory performance that can be performed similarly in both species. We will then conduct in vitro electrophysiology experiments in rodent brain slices obtained after stimulation in order to identify cellular mechanisms for the effects of stimulation. We predict that stimulation parameters that increase hippocampal network function will increase cellular excitability, as measured via the postburst afterhyperpolarization, of dorsal hippocampal CA1 pyramidal neurons. Viral manipulation of CREB expression, which is necessary for changes in excitability, will be used to causally test the role of dorsal hippocampal CA1 excitability in the effects of stimulation on hippocampal network function. These research objectives are in close alignment with the focus of RFA-NS-18-018 on establishing cellular mechanisms for the effects of brain stimulation on neuronal circuits. Findings will uniquely uncover cellular mechanisms by which brain stimulation beneficially impacts distributed brain networks and corresponding cognitive abilities. These mechanistic insights could propel brain-stimulation treatments for memory impairments caused by disruption of the hippocampal network.

Supplement Project Summary/Abstract The purpose of this administrative supplement for our ongoing grant ?Cellular mechanisms of hippocampal network neuroplasticity generated by brain stimulation? (R01-NS11380) is to use the innovative translational brain stimulation methods developed under the ongoing parent grant to test whether and how they rescue memory impairments in the next-generation rodent model of Alzheimer?s disease (AD), TgF344-AD. AD produces memory impairment by affecting the function of the distributed network of the hippocampus. Our ongoing project investigates the mechanisms whereby electrical brain stimulation targeting the hippocampal network can improve its function. By performing companion in vivo electrophysiological experiments in healthy young adult rodents and in human neurosurgical cases with depth electrodes in regions homologous to those implanted in the rodents, the ongoing project takes a highly translational approach to identify similarities across species in how the hippocampal network responds to brain stimulation. This approach thereby enhances the relevance to human function of the mechanistic insights offered by rodent in vivo and in vitro electrophysiology experiments performed for the ongoing project. This administrative supplement will expand our translational model of hippocampal network brain stimulation to address memory impairment due to AD. We first will behaviorally characterize young (5-6 mo.) and aged (20-23 mo.) F344 wild-type and aged TgF344-AD rats (a rodent model of AD) using the spatial Morris water maze task. Aged F344 rats will be categorized based on performance of this task into age-unimpaired (AU) and age-impaired (AI) subgroups, such that comparisons among the four groups (young, AU, and AI F344 rats and aged TgF344-AD rats) will be able to differentiate variation in stimulation efficacy based on aging, on typical aging-related memory impairment, and on AD pathology. In each of these groups, we will then compare the effects of locking stimulation to the ongoing phase of the hippocampal theta rhythm (versus non-phase-locked control conditions) on hippocampal network in vivo electrophysiology and on performance on the paired associate learning (PAL) touchscreen task, which is hippocampal dependent in both rodents and humans. Across-group comparisons will be used to determine whether phase-locked stimulation is maximally beneficial for hippocampal network electrophysiology and PAL performance in TgF344-AD rats relative to AI rats, with comparisons between AI and AU groups and between AU and young groups used to differentiate the effects of AD from those of aging with versus without memory impairment. Notably, although brain stimulation has shown moderate efficacy for memory impairment in aging and AD, very little data are available regarding mechanisms. These experiments will thus yield important and highly novel data on how brain stimulation targeting the hippocampal network influences its function in animals with AD. Results will be useful in tailoring brain stimulation for memory rescue in human AD patients owing to the highly translational nature of the experimental animal brain stimulation model that we have developed.