Browsing by Author "Turner, Ray W."

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    Determinants of Synaptic Integration in Cerebellar Neurons
    (2013-05-10) Engbers, Jordan David Thomas; Turner, Ray W.
    The integration of synaptic inputs by neurons relies on protein channels that conduct specific ions. Cav3 calcium (Ca2+) channels can amplify excitatory postsynaptic potentials (EPSPs) while Ca2+-activated potassium (KCa) channels decrease EPSP amplitude. By comparison, inhibitory postsynaptic potentials (IPSPs) can activate hyperpolarization-activated (HCN) channels that generate a rebound excitatory current at the end of an inhibitory stimulus. This thesis examines how Cav3, KCa, and HCN channels control synaptic integration in cerebellar Purkinje cells and deep cerebellar nuclei (DCN) neurons. These two populations of neurons are central to cerebellar function and represent a dichotomy of synaptic processing, as Purkinje cells receive primarily excitatory inputs, while DCN neurons receive mainly inhibitory inputs. I tested the hypothesis that Cav3-mediated Ca2+ current activates KCa channels to control the summation of parallel fibre EPSPs in Purkinje cells. Patch clamp recordings from in vitro slices of rat cerebellum showed that Cav3 current activates intermediate conductance KCa (KCa3.1) channels, which have previously never been found in central neurons. KCa3.1 channels are activated at hyperpolarized membrane voltages, due to an extended Cav3 channel window current, and suppress summation of low-frequency EPSPs. Dynamic clamp experiments and computer simulations revealed that the Cav3-KCa3.1 complex increases the signal-to-noise ratio for sensory-like parallel fibre inputs undergoing short-term facilitation by selectively suppressing background inputs. In DCN neurons, I tested the hypothesis that Cav3 and HCN channels control the frequency and timing of rebound bursts following inhibition by IPSPs. The results demonstrate that Cav3 and HCN currents are activated during physiological levels of hyperpolarization and modulate rebound bursts. A novel model of a DCN neuron showed that Cav3 current is solely responsible for generation of the rebound burst, while HCN channels increase burst frequency and temporal precision. Together, this research demonstrates how a novel Cav3-KCa3.1 channel complex participates in the processing of excitatory inputs, and identifies a new synergistic interaction between ion channels that enables processing of inhibitory inputs. These findings illustrate the importance of ion channel interactions for signal processing in the cerebellum, with far reaching implications for neural circuits throughout the brain.
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    Dynamic remodeling of the ionic basis of an intrinsic inhibitory response by the phospholipid PIP2
    (2020-09-22) Nicholls, Shane; Turner, Ray W.; Zamponi, Gerald W.; Altier, Christophe
    The excitability of an individual neuron can be directly related to the different types of potassium channels it expresses. Potassium channels that are activated by either calcium or voltage contribute to a wide range of physiological processes and many cognitive impairments if mutated or disrupted. A key mechanism to regulate neuronal activity is to generate afterhyperpolarizations (AHPs). AHPs are brief inhibitory periods that span from a fast (ms) to slow (sec) time frame. A voltage and calcium-gated potassium channel (Kv7) and an intermediate conductance calcium-gated potassium channel (IK) in CA1 hippocampal pyramidal cells hyperpolarize the cell by generating a medium and slow AHP. The availability of at least Kv7 channels can be modulated by a phosphatidylinositol molecule (PIP2). We measured the relative activity of each potassium channel using whole-cell patch recordings in rat hippocampal tissue slices maintained in vitro and in the tsA-201 heterologous expression system. Both channels prove to be activated by calcium increases derived from a combination of Cav1.3 channels and ryanodine receptor 2. However, modulating PIP2 levels produced opposite effects on coexpressed Kv7.2/3 and IK-mediated outward currents. Super resolution microscopy of immunolabeled proteins in cultured hippocampal neurons revealed a novel close association between Kv7.2 and IK potassium channels in somatic and dendritic membranes. The ability for PIP2 to reverse the roles of Kv7.2/3 and IK channels in producing a medium/slow AHP identifies a novel mechanism by which the ionic basis of inhibitory responses can be dynamically modulated to control intrinsic excitability in CA1 pyramidal cells.
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    Heterogenous Change in Neuronal Bursts Following Recovery from Activity Silencing
    (2018-08-02) Kipp, Alexander Joseph; Colicos, Michael A.; Turner, Ray W.; Davidsen, Jörn
    Silencing of activity in hippocampal neuronal cultures was used to study how dynamic neuronal activity achieves a state of homeostasis, using calcium imaging to detect neuronal firing patterns. Recovering cultures were found to display abnormal activity patterns after 48hrs of exposure to tetrodotoxin, as indicated by paradoxical spike and correlation statistics. It was found that the cultures recovering from activity silencing did not resemble a neuronal system with enhanced excitation, but differed significantly from control experiments. Using a newly developed measure of homogeneity it was found that activity patterns in cultures recovering from silencing were more heterogeneous during bursts, which is in contrast to the current perception that bursting activity is a homogeneous event. It was also observed that there were more active neurons during the recovery period. It is hypothesized that these changes in neuronal system dynamics are brought about due to the insertion and heterogenous manipulation of silent synapses. Results suggest a mechanism through which the interplay between homeostatic scaling, silent synapses and bursting behavior could mediate neuronal network homeostasis.
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    Intermediate conductance calcium-activated potassium channels modulate summation of parallel fiber input in cerebellar Purkinje cells
    (Proceedings of the National Academy of Sciences, 2012-02-14) Engbers, Jordan D .T.; Anderson, Dustin M.; Asmara, Hadhimulya; Rehak, Renata; Mehaffey, W. Hamish; Hameed, Shahid; McKay, Bruce E.; Kruskic, Mirna; Zamponi, Gerald W.; Turner, Ray W.
    Encoding sensory input requires the expression of postsynaptic ion channels to transform key features of afferent input to an appropriate pattern of spike output. Although Ca(2+)-activated K(+) channels are known to control spike frequency in central neurons, Ca(2+)-activated K(+) channels of intermediate conductance (KCa3.1) are believed to be restricted to peripheral neurons. We now report that cerebellar Purkinje cells express KCa3.1 channels, as evidenced through single-cell RT-PCR, immunocytochemistry, pharmacology, and single-channel recordings. Furthermore, KCa3.1 channels coimmunoprecipitate and interact with low voltage-activated Cav3.2 Ca(2+) channels at the nanodomain level to support a previously undescribed transient voltage- and Ca(2+)-dependent current. As a result, subthreshold parallel fiber excitatory postsynaptic potentials (EPSPs) activate Cav3 Ca(2+) influx to trigger a KCa3.1-mediated regulation of the EPSP and subsequent after-hyperpolarization. The Cav3-KCa3.1 complex provides powerful control over temporal summation of EPSPs, effectively suppressing low frequencies of parallel fiber input. KCa3.1 channels thus contribute to a high-pass filter that allows Purkinje cells to respond preferentially to high-frequency parallel fiber bursts characteristic of sensory input.
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    Modeling temperature- and Cav3 subtype-dependent alterations in T-type calcium channel mediated burst firing
    (2021-07-17) Fernandez, Fernando R.; Iftinca, Mircea C.; Zamponi, Gerald W.; Turner, Ray W.
    Abstract T-type calcium channels are important regulators of neuronal excitability. The mammalian brain expresses three T-type channel isoforms (Cav3.1, Cav3.2 and Cav3.3) with distinct biophysical properties that are critically regulated by temperature. Here, we test the effects of how temperature affects spike output in a reduced firing neuron model expressing specific Cav3 channel isoforms. The modeling data revealed only a minimal effect on baseline spontaneous firing near rest, but a dramatic increase in rebound burst discharge frequency for Cav3.1 compared to Cav3.2 or Cav3.3 due to differences in window current or activation/recovery time constants. The reduced response by Cav3.2 could optimize its activity where it is expressed in peripheral tissues more subject to temperature variations than Cav3.1 or Cav3.3 channels expressed prominently in the brain. These tests thus reveal that aspects of neuronal firing behavior are critically dependent on both temperature and T-type calcium channel subtype.
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    Signaling complexes of voltage-gated calcium channels
    (Taylor and Francis, 2011-09) Turner, Ray W.; Anderson, Dustin M.; Zamponi, Gerald W.
    Voltage gated calcium channels are key mediators of depolarization induced calcium entry into electrically excitable cells. There is increasing evidence that voltage gated calcium channels, like many other types of ionic channels, do not operate in isolation, but instead forms signaling complexes with signaling molecules, G protein coupled receptors, and other types of ion channels. Furthermore, there appears to be bidirectional signaling within these protein complexes, thus allowing not only for efficient translation of calcium signals into cellular responses, but also for tight control of calcium entry per se. In this review, we will focus predominantly on signaling complexes between G protein-coupled receptors and high voltage activated calcium channels, and on complexes of voltage-gated calcium channels and members of the potassium channel superfamily.
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    State-dependent neuromodulation of mammalian spinal networks
    (2018-08-28) Sharples, Simon Arthur; Whelan, Patrick J.; Teskey, Gordon C.; Borgland, Stephanie Laureen; Turner, Ray W.
    It has been known for centuries that the brain is not necessary for the generation of movements that allow for animals to walk. For many farmers, the sight of a chicken running around the farm yard following decapitation was fairly common. At the turn of the 20th century Charles Sherrington and Thomas Graham Brown proposed that circuits located within the spinal cord are responsible for the generation of rhythmic movements of the limbs for walking. With over a century of scientific investigation of rhythmically active circuits of vertebrates and invertebrate species, we have learned that rhythmic movements for locomotion, breathing and chewing are controlled by neuronal circuits called central pattern generators. Large emphasis has been directed toward dissecting the central pattern generator circuits for walking. Locomotor movements are remarkably adaptable and respond to not only external demands imposed by the environment but also internal needs of the animal. Thus, the underlying circuits that generate these diverse movements also need to be flexible to readily adjust in response to imposed demands. Nevertheless, the mutability of these rhythm generating circuits is not well understood. Neuromodulation endows spinal circuits with flexible properties that allow motor outputs to be adaptable to modify ongoing movement. I initially started to study how one neuromodulator, dopamine, controls rhythmic network activities of the spinal cord for walking in in the neonatal mouse in vitro (Sharples et al., 2015). My preliminary studies demonstrated that modulation of rhythmic circuits might have state-dependent effects on locomotor rhythms which is consistent with work conducted in invertebrates (Marder et a., 2014). In this thesis, I explored the diverse actions of modulators on spinal networks across varying states of excitability. This is important because changes in behavioural state or pathology result in alterations in network excitability. In general, I demonstrated that neural networks of the spinal cord are degenerate in their ability to generate locomotor rhythms and that neuromodulators can tune this ability through both degenerate and redundant mechanisms.
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    Subcellular Distribution and Function of Cardiac Ryanodine Receptor in Ventricular Myocytes and Hippocampal Neurons
    (2018-01-23) Hiess, Florian; Chen, S. R. Wayne; Turner, Ray W.; ter Keurs, Henk E D J; Rho, Jong M; Tibbits, Glen F; Nguyen, Minh Dang; Tyberg, John V.
    Cardiac ryanodine receptors (RyR2s) are intracellular calcium (Ca2+) release channels most abundantly expressed in the heart and brain. They are clustered in the endo/sarcoplasmic reticulum (ER/SR) membrane to form elementary units for Ca2+ release. The distribution of these units determines the spatiotemporal profile and stability of ER/SR Ca2+ release. Thus, RyR2 distribution is believed to be essential in cellular processes, such as excitation-contraction coupling and learning and memory. The distribution of RyR2s has been extensively studied in cells/tissues using anti-RyR2 antibody immunostaining. However, sample preparation required for immunostaining may affect cellular structures, besides rendering the cells/tissues non-functional. Hence, the functional relevance of the distribution of RyR2 clusters in live cells/tissue is unclear. We have generated a knock-in mouse model that expresses green fluorescence protein (GFP)-tagged RyR2s. These mice allow us to monitor cellular/subcellular distribution of RyR2 in live cells/tissues by virtue of GFP fluorescence. To improve the detection of GFP-RyR2, we developed a novel GFP-specific probe based on anti-GFP single domain antibodies (nanobodies). Fluorescence imaging was employed to study Ca2+ release and the distribution of GFP-RyR2 in the interior and periphery of live ventricular myocytes and in intact hearts isolated from GFP-RyR2 expressing mice. We found highly-ordered arrays of stationary GFP-RyR2 clusters in the interior of cardiomyocytes in the z-line zone. In contrast, irregular and dynamic distribution of GFP-RyR2 clusters was observed in the periphery of cardiomyocytes. Imaging of intact GFP-RyR2 brain sections revealed a widespread distribution of RyR2 in various brain regions, most prominently in regions involved in spatial learning and memory, such as the hippocampus. To investigate the functional role of RyR2 in this region, we performed electrophysiological studies using hippocampal slices prepared from knock-in mice harboring a cardiac arrhythmia-associated human RyR2 mutation (R4496C) with enhanced channel activity. We found that enhanced RyR2 function reduces long-term potentiation (LTP) in Schaffer collateral inputs to CA1 pyramidal cells. Thus, RyR2 plays a critical role in LTP at these synapses. Behavioral studies on RyR2 mutant mice further supported the role of RyR2 in learning and memory. Overall, these results reveal, the distribution of RyR2 clusters and its functional significance in living ventricular myocytes and hippocampal neurons.