Indira M. Raman - US grants

The voltage-gated Na+ currents near threshold voltages will be studied in Purkinje neurons of the cerebellum, with a general interest in understanding how interaction among ionic currents determine action potential threshold and produce different patterns of activity. Purkinje cells form the sole output of the cerebellar cortex and show a range of spiking behaviors. Although their activity plays a central role in motor behavior and motor learning and disruption of their activity results in sever motor deficits, relatively little research has made use of modern electrophysiological techniques to investigate how ionic currents control normal Purkinje cell firing. The proposed experiments will be done on Purkinje cells enzymatically isolated from rat cerebellum. First, using whole-cell voltage clamping and cell attached single-channel recording, the properties of transient, persistent, and the novel resurgent Na+ currents will be described. Next, current-clamp recording will be used to study the parameters that shape threshold in Purkinje cells. Finally, the recorded waveforms of simple spikes, complex spikes, and trains of spikes will be used as voltage-clamp protocols to measure how changes in the relative amplitude and depolarizing strength of each aspect of Na+ current (transient, persistent, and resurgent) can modify threshold and evoke different patterns of spiking.

DESCRIPTION (provided by applicant): Many neurons of the cerebellum are spontaneously active, firing 10 to 100 action potentials per second even in the absence of synaptic input. This high basal activity correlates with information coding mechanisms that differ from those of cells in circuits that are generally quiescent until excited synaptically. For example, in the cerebellar nuclei, long-term changes in the strength of excitatory synaptic inputs are not generated by classical Hebbian rules of coincident synaptic excitation and postsynaptic firing. Instead, synaptic currents are potentiated by patterns of stimulation that combine inhibition and excitation, in a manner that resembles the activity of (inhibitory) Purkinje afferents and (excitatory) mossy fiber afferents predicted to occur during cerebellar associative learning tasks. Such results support the idea that cerebellar circuits have rules for information transfer and storage that distinguish them from other well studied brain regions. The present proposal is motivated by the question of how spontaneous firing sets the stage for plasticity that is independent of spike timing. In the proposed research, experiments will be performed on neurons of the cerebellar nuclei in cerebellar slices of mice. Voltage-clamp and current-clamp recordings of synaptic responses, ionic currents, and action potentials, as well as imaging of Ca signals in nuclear cell dendrites, will be directed toward identifying the mechanisms of potentiation of excitatory synaptic responses to mossy fiber input, as well as toward examining the influence of spontaneous activity in Purkinje afferents and nuclear cells on plasticity. The resulting data will provide general information about the fundamental properties of signal encoding across brain regions, as well as specific information about the ionic mechanisms underlying cerebellar synaptic plasticity under normal and pathophysiological conditions. PUBLIC HEALTH RELEVANCE: In the present work, we are studying cells in the cerebellum, a part of the brain that controls the learning and execution of coordinated muscle movements, and whose electrical and chemical signaling patterns are disrupted in ataxia, dystonia, dyslexia, and autism. Because signaling patterns by brain cells depend on specialized proteins called ion channels, we are studying the properties of these ion channels, with the goal of understanding how their activity leads to long-lasting changes in cerebellar signals that are important for motor learning. These data can be used to make comparisons to disrupted signals in the cerebella of animals that have ataxia as a result of genetic mutations of ion channels, with the goal of understanding what goes wrong under pathophysiological conditions and whether ion channels can serve as a target for therapeutic interventions.

DESCRIPTION (provided by applicant): The proposal seeks partial support for the second biennial Gordon Research Conference (GRC) on the Cerebellum, to be held at Colby-Sawyer College in New London, NH, USA, in August 2013. The cerebellum is a brain region that regulates movements and carries out complex behaviors such as sequencing of actions. Its dysfunction is associated with motor disorders, including ataxias, dystonia, and dyskinesia, as well as cognitive impairments, including dyslexia and autism spectrum disorder. Cerebellar research spans many levels of analysis, encompassing molecular biological, genetic, cellular physiological, systems physiological, anatomical, behavioral, computational, and clinical approaches. Although many researchers engaged in the study of the cerebellum have common research questions and scientific goals, they have few opportunities to meet as a group and discuss their work with investigators who apply diverse methodologies to the common question of how the cerebellar works in normal and pathophysiological conditions. The aims of the Cerebellum GRC are to provide the venue for cerebellar researchers to present and discuss their hypotheses, results, and discoveries; to bring together scientists from multiple career stages and different backgrounds, who otherwise would be unlikely to have the opportunity to interact closely; and to develop scientific relationships that will lead to collaborative work and new approaches to investigating the cerebellum in health and disease. These aims will be achieved by 9 sessions of oral presentations by leaders in the field and up-and-coming junior investigators, ample discussion time with active facilitation of participation by young scientists and trainees, poster presentations for maximal exposure of all attendees' research, and unstructured time between scientific sessions for in-depth, spontaneous discussions. Inclusion of sessions, such as animal models of cerebellar disease, human cerebellar function, and emerging technologies to study the cerebellum promise, not only generate a healthy exchange of ideas but also educate investigators about how their work pertains to and can best be brought to bear on the treatment of neurological and mental disorders.

Project Summary/Abstract Premotor neurons of the cerebellar nuclei (CbN) are spontaneously active neurons that integrate excitatory and inhibitory input, largely from mossy fibers and Purkinje neurons, to generate the output of the cerebellum. The cerebellum facilitates coordinated movement and corrects errors in real time, and also changes to learn new motor patterns over time. These observations raise the question of how synaptic input modulates intrinsic firing in a manner that permits premotor CbN neurons to identify deviations from predicted sensory input and encode appropriate corrective outputs. We are studying the biophysical and synaptic mechanisms that constrain and define patterns of CbN cell firing in response to physiological patterns of synaptic input in vitro and expected or unexpected sensory input during motor behaviors in vivo. Recent work suggests that the degree of inhibitory synchrony from convergent Purkinje cells may dictate cerebellar output in mice in a condition-dependent manner. We will therefore study cerebellar physiology and behavior in mice and in larval zebrafish, to provide a comparative approach, both in vitro and in vivo. Purkinje and CbN cell firing patterns will be monitored both during well-learned, predictable motor behaviors, during unpredicted sensory inputs, and during motor learning such as habituation and associative conditioning. The observations will be related to synaptic studies of the interaction of excitatory with inhibitory inputs, convergence and connectivity, and short- term and long-term plasticity. Such linking of the biophysical and behavioral levels will help explain the mechanisms by which the cerebellar circuit produces adaptive responses to deviations from predictions, thereby facilitating well-executed movement.