Jason R. Pugh - US grants

[unreadable] DESCRIPTION (provided by applicant): While it has been clearly demonstrated that the cerebellum is involved in motor and associative learning, it is remains unclear where and how this learning takes place within the cerebellum. Many studies have focused on the synaptic physiology and electrophysiological properties of Purkinje cells, but very few studies have investigated the role of deep cerebellar nuclear (DCN) neurons in the cerebellar circuit. Behavioral studies of eyelid conditioning have suggested that at least a portion of cerebellar learning takes place or is stored outside the cerebellar cortex, likely in the DCN. However, little is known about the properties of excitatory synapses in the DCN and there has been no direct evidence of synaptic plasticity at this site. This work will provide insight on the function of excitatory synapses in the DCN by making a more detailed study of the basic properties of these synapses than has been done previously. We will begin by investigating the kinetics, pharmacological sensitivity, and voltage dependence of EPSCs, as well as the short-term plasticity of EPSCs during trains of high frequency stimulation. This information will help in understanding how information is transmitted and processed at excitatory synapses in the DCN. In addition, we will investigate whether long-term plasticity is possible at excitatory synapses in the DCN, and if so, the conditions and mechanisms that produce potentiation or depression of EPSCs. These data will provide insight on how information is processed and stored by the cerebellum and in the DCN in particular. [unreadable] [unreadable]

? DESCRIPTION (provided by applicant): The long term goal of this project is to understand how presynaptic receptors, specifically GABAA and GABAB receptors, influence synaptic transmission, cellular computation, and learning in neuronal circuits. GABAA and GABAB receptors are co-expressed in the presynaptic membrane of many excitatory synapses in the central nervous system where they have been shown to modulate synaptic transmission. However, previous studies have generally studied the effects of each receptor type in isolation and with static conditions. It is not currently known how co-activation of these receptors, as is likely to happen in vivo, will modulate synaptic transmission. We propose that expression of presynaptic GABA receptors is a plastic property of the synapse and presynaptic GABA receptors function together to temporally modulate synaptic transmission. Previous work has shown that axons of cerebellar granule cells express both presynaptic GABAA and GABAB receptors, which increase and decrease, respectively, glutamate release at parallel fiber synapses (Dittman and Regehr, 1996; Stell et al., 2007; Pugh and Jahr, 2011; Dellal et al., 2012). Using a combination of whole-cell patch clamp electrophysiology and two-photon laser scanning microscopy (including calcium imaging), this project will demonstrate that, rather than opposing one another, presynaptic GABAA and GABAB receptors function together to temporally modulate transmission and enhance short-term plasticity, a result not possible from activation of either receptor alone. Furthermore, this work will show that expression of presynaptic GABA receptors is regulated by long-term changes in synaptic strength. This work will reveal novel forms of synaptic plasticity and presynaptic receptor function. Regulation of excitatory transmission by presynaptic GABA receptors may be a general mechanism of balancing excitation and inhibition in the central nervous system and disruption of this system may contribute to the etiology and treatment of neurological conditions associated with synaptic imbalances, such as autism, schizophrenia and epilepsy. Aim 1: Determine the effects of GABAA and GABAB receptor co-activation on vesicle release. Hypothesis: Rather than opposing one another, differences in kinetics and affinity of GABAA and GABAB receptors allow them to work together to produce a biphasic effect on release probability. Aim 2: Determine the conditions for presynaptic GABAA and GABAB receptor plasticity. Hypothesis: Presynaptic GABAA and GABAB receptor expression is modulated in opposition to long-term changes in synapse strength. Aim 3: Determine the effects of presynaptic GABAA and GABAB receptors on postsynaptic firing and associative (postsynaptic) plasticity. Hypothesis: Activation of presynaptic GABA receptors enhances short-term facilitation and depression during trains of high frequency-activity, narrowing the duration of postsynaptic firing and the window for long-term plasticity at parallel fiber synapses. Relevance: A precise balance of excitation and inhibition is required for proper functioning of neuronal circuits while imbalances have been associated with several neurological conditions, including autism, schizophrenia, and epilepsy. This work will determine how neurotransmission at excitatory synapses is regulated by the inhibitory neurotransmitter, GABA. This work will lay the groundwork of basic synaptic physiology for future studies investigating dysregulation of excitation and inhibition in these conditions.

PROJECT SUMMARY/ABSTRACT Dystrophin deficiency, which occurs in Duchenne muscular dystrophy (DMD), results in muscle wasting. As dystrophin is expressed in the brain, its deficiency also contributes to neurological symptoms in DMD patients. Cerebellar Purkinje cells in mouse models of DMD have weaker inhibitory synaptic connections and compromised climbing-fiber-evoked plasticity. This implicates cerebellar dysfunction as a contributing factor to the neurological pathophysiology of DMD. Yet, how cerebellar dysfunction ultimately explains DMD neurological symptoms remains incompletely understood. Increasing evidence points to the importance of plasticity gating to maintain a reserve capacity for learning. In Purkinje cells, a candidate gating mechanism of plasticity induction is inhibition from molecular layer interneurons, which suppresses the evoked calcium response to climbing fiber excitation that triggers induction of long-term depression (LTD). Therefore, the objective of this study is to test for a potential synergistic role of inhibitory synapse weakening and compromised LTD in dystrophinopathy and determine if increasing GABAA receptor responsiveness specifically in Purkinje cells restores a high threshold for plasticity induction and thus provide a potential therapeutic strategy to ameliorate cerebellar dysfunction. We will employ a multidisciplinary approach encompassing the use of ex vivo and in vivo functional recordings in Purkinje-cell-autonomous dystrophin-deficient mice, cell-type specific neuropharmacological perturbations, and behavioral analyses. Through two aims, we will test correlative and causative links between aberrant neural circuit responsiveness, spurious plasticity, and cerebellar learning abnormalities. Completion of these aims will contribute novel insights into plasticity regulation, the etiology of neurological impairment in DMD, and potential avenues for treating cerebellum-related symptoms of this disorder.