Peter Wenner - US grants

[unreadable] DESCRIPTION (provided by applicant): Balancing the strength of excitatory and inhibitory elements within a network is essential for the proper function of the circuit. If excitatory systems dominate, spasticity or epileptiform activity could result. The period of circuit development is of crucial importance because it is then that the balance is first established. Insight to mechanisms that regulate excitatory and inhibitory synaptic strength during development would provide a critical step in our understanding of how networks arrive at this balance. A new form of synaptic plasticity has been identified in cultured neurons, which suggests that neurons homeostatically regulate their level of spiking activity by adjusting the strength of their synaptic inputs (synaptic scaling). The objective of this application is to determine the level of spiking activity in the postsynaptic neuron drives the maturation of synaptic strength during embryonic development of spinal motor networks. To this end we will block spiking activity in the motor network or in individual spinal neurons in ovo in the chick embryo. By comparing the strength of the synaptic inputs in activity-blocked and control neurons we will test the role of activity in the regulation of synaptic strength in an excitatory motoneuron (Specific Aim 1 & 2) and in an identified inhibitory GABAergic interneuron (Specific Aim 3 & 4). This is possible because we have recently characterized a class of interneuron that receives direct input from motoneurons. Using a combination of molecular, electrophysiological, optical, and immunocytochemical techniques we are proposing to begin a comprehensive study to understand the role of activity in the development of excitatory and inhibitory synaptic strength in excitatory and inhibitory spinal neurons. We are proposing that the level of activity regulates the strength of newly formed synaptic connections in the embryo. The results of the study will provide a better understanding of how a network achieves a balance between excitatory and inhibitory elements during development. [unreadable] [unreadable]

Synaptic refinement is the process of pruning back an initially exuberant neuronal projection pattern. This process is thought to be important in allowing developing circuits to adjust to their environment and it eliminates the need to genetically specify every detail of a neuron's connectivity. This process has been studied extensively in excitatory neurons that set up topographic projections to another part of the nervous system (e.g. retinal map formed in the visual cortex). To understand the development of neural networks, however, of we must appreciate how both excitatory and inhibitory elements within a network develop. Recently, inhibitory neurons in the auditory system have been shown to refine their tonotopic projections, demonstrating that inhibitory refinement can occur. The functional meaning and the underlying mechanisms of inhibitory refinement are completely unknown. Further, it had been unknown whether locally projecting inhibitory interneurons undergo refinement. Dr. Wenner's lab has characterized the connections of a locally projecting inhibitory interneuron in the chick embryo spinal cord called the R-interneuron, and have shown that these R-interneurons refine their connections with spinal motoneurons. The PI proposes a set of experiments to assess the function of inhibitory refinement (objective 1) and test whether the refinement process of this inhibitory interneuron is dependent on spiking activity (objective 2) and inhibitory neurotransmitter signaling (objective 3). This will provide important information about the similarities and differences in inhibitory and excitatory synaptic plasticity during development.

The study will give insights into how a developmental balance between excitatory and inhibitory systems is achieved, and a better understanding of why this developmental balance may not be realized in certain disease states. In addition, this project will foster education on several levels within and beyond the PI's lab. meeting. Although recently arrived at Emory University, Dr. Wenner has mentored undergraduate students, including those from underrepresented minorities, in his lab. Dr. Wenner also participates in Atlanta's Brain Awareness Month by visiting K-12 classrooms. The proposed project will allow scientific education for an undergraduate student, graduate student, and a postdoctoral fellow as they participate in research, publishing, and presenting to the local community, as well as an international community at the annual neuroscience

DESCRIPTION (provided by applicant): It is an extraordinary accomplishment that most developing neuronal networks achieve an appropriate level of excitability, during a dynamic period of embryonic development when there are several challenges to a network's excitability (e.g. cells increase in size). Therefore it is not surprising that the incidence of seizure activity is higher in the neonatal period than in any other age group. Neonatal seizures are often the first indication of neurological dysfunction, and are strong predictors of long-term cognitive and developmental impairment. Understanding the rules and mechanisms that underlie the maturation of network excitability are therefore essential. In the last decade an exciting new field has emerged that provides critical insights to understanding the rules that networks follow in order to achieve appropriate levels of activity. Many studies have now shown that networks homeostatically maintain activity levels within an appropriate range by adjusting synaptic strength (homeostatic synaptic plasticity). The vast majority of these studies have blocked network activity in vitro (culture) for days, and although activity doesn't homeostatically recover, changes in synaptic strength are in a compensatory direction. Therefore, it is assumed, although untested, that these changes would normally act to recover activity levels following perturbations in vivo. Compensatory changes in intrinsic cellular excitability (cell's responsiveness to synaptic input) also likely contribute to the homeostatic process, although these changes have received far less attention than synaptic compensations. We have found that changes in cellular excitability are faster and likely more important in the initial recovery of normal activity levels. The objective of this application is to better understand the role of cellular excitability in the process of homeostatically recovering and maintaining network activity in developing networks. We are proposing to perturb network activity in the living embryo, allow for the homeostatic recovery of activity and assess how changes in cellular excitability contribute to this recovery. This will provide a more realistic, comprehensive understanding of the homeostatic regulation of cellular excitability and its role in maintaining appropriate activity levels and maturing network excitability. PUBLIC HEALTH RELEVANCE: We are testing the possibility that a spontaneous network activity that is expressed in embryonic neural circuits regulates the intrinsic cellular excitability in motor and interneurons. In this way, we are studying the maturation of embryonic network excitability.

DESCRIPTION (provided by applicant): Mechanisms of homeostatic plasticity ensure that cells maintain spiking activity levels within a physiologically relevant window. This form of plasticity prevents cells from falling silent or from becoming hyperexcitable. The homeostatic control of spiking activity is critically important to nervous system function, as demonstrated by the severity of conditions like seizure, where such homeostasis is not achieved. Homeostatic plasticity is thought to underlie the robustness of network behavior, and allow for the recovery of behaviors following perturbations. The last 15 years have seen a dramatic rise in studies of homeostatic plasticity, which have now demonstrated multiple mechanisms, which contribute to the resilience of spiking activity following perturbations. By far the most studied mechanism of homeostatic plasticity has been referred to as synaptic scaling. When spiking activity in cultured neurons is blocked for days, all of a cell's excitatory synapses strengthen, or scale up. Current thinking in the field suggests that synaptic scaling is triggered by reduced spiking activity, whic then acts to recover normal spiking levels. In this proposal we challenge this very basic, but largely untested assumption. We hypothesize that synaptic scaling is triggered by reductions in synaptic transmission at individual synapses to homeostatically maintain synaptic transmission, rather than as a means to homeostatically regulate a cells spiking activity. We will test this hypothesis using a combination of optogenetics and multi-electrode array recordings of cultured neurons. We will ask in Aim 1 excitatory upscaling is triggered by reduced glutamatergic transmission? Then we will ask in Aim 2 if excitatory downscaling is triggered by increased glutamatergic transmission? In Aim 3 we will determine if alterations in spiking or transmission trigger GABAergic scaling. If the results favor neurotransmission as the trigger for synaptic scaling, this could provide a transformative shift in the focus of synaptic scaling away from the activity-centric perception that is currently pervasive. This proposal will use a combination of new techniques that allow the assessment and control of spiking activity levels to ask fundamentally important questions about homeostatic plasticity. The results will have significant implications for understanding how circuits change following injury and disease.

Abstract It is an extraordinary accomplishment that most developing neuronal networks achieve an appropriate level of excitability, during a dynamic period of embryonic development when there are several challenges to a network's excitability. Errors in such a complicated process can lead to alterations in the excitability of neonatal spinal circuit, which can be observed behaviorally as myoclonus, hypertonia, recurrent tremor, and spasticity. Understanding the rules and mechanisms that underlie the maturation of network excitability are therefore essential. An exciting new field has emerged, which provides critical insights to understanding the rules that networks follow in order to achieve appropriate levels of activity. Many studies have now shown that perturbations to network activity trigger changes in synaptic strength which are thought to homeostatically recover and maintain activity levels within an appropriate range. Compensatory changes in intrinsic cellular excitability (cell's responsiveness to synaptic input) also likely contribute to the homeostatic process, although these changes have received far less attention than synaptic compensations. By taking advantage of the accessibility of the chick embryo we have been able to follow an actual homeostatic recovery of activity (embryonic movements). Because of this, we have been able to identify a critical and previously unrecognized homeostatic mechanism where changes in resting membrane potential mediate the initial homeostatic recovery of perturbed activity levels in the living embryonic spinal cord. We will identify the mechanism underlying this compensation in the first aim of the grant. Based on a recent study and our proteomic analysis from our previous grant period, we are proposing to examine an unexpected critical relationship between mitochondrial function and homeostatic plasticity in aim 2. Finally in aim 3 we will carry out this work in the genetically advantageous mouse model system. Our study will identify the mechanisms of homeostatic plasticity in the living system and will begin to elucidate the calcium triggers for these forms of plasticity. The work can instruct pharmacological interventions that ameliorate hyperexcitability associated with neurodevelopmental disorders, and help us better understand the function of homeostatic plasticity.

Homeostatic plasticity represents a set of mechanisms that is thought to ensure that neural spiking levels and/or synaptic strength are maintained within a physiologically operational window. When neural activity or neurotransmission is chronically altered for days in cultured neurons or some developing networks, changes in synaptic strength and cellular excitability are expressed that appear to compensate for the perturbation. The autonomic nervous system homeostatically controls many different bodily functions, such as blood pressure and temperature. However, following neural injury or in disease states (e.g. hypertension) these functions are maintained at levels outside the normal range, and this is often associated with chronically altered sympathetic drive. These observations have led us to ask if chronic changes in sympathetic drive could trigger homeostatic mechanisms that are either unable to recover the original activity levels or actually contribute to the inappropriate levels of sympathetic activity? In this application we are taking advantage of the expertise of the Wenner and Hochman labs to address this question (Wenner - homeostatic plasticity in developing systems, Hochman - sympathetic nervous system and an ex vivo sympathetic preparation). We are proposing to chronically inhibit sympathetic drive in the living adult mouse and then examine an ex vivo sympathetic preparation following treatment to identify homeostatic mechanisms that are expressed. We will carry this ex vivo analysis out in a preparation that captures the peripheral arm of the sympathetic circuitry, which also includes the target organ regulating vasomotor function (blood vessels). We will do this in both the adult and at earlier developmental stages when the sympathetic circuitry is maturing and when homeostatic plasticity mechanisms are typically observed. The results will provide a better understanding of conditions that produce chronic sympathetic dysfunction and will be important in designing strategies to correct these autonomic dysfunctions.