Physiology and biophysics

Permanent URI for this collectionhttps://digital.lib.washington.edu/handle/1773/4957

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Miniature treadmills reveal proprioceptive mechanisms in walking Drosophila
(2024-09-09) Pratt, Brandon; Tuthill, John C
As animals navigate in an unpredictable and ever-changing world, their movement is bombarded by unanticipated perturbations. Proprioception, the sense of how the body is articulated and moving in space, enables animals to rapidly overcome these perturbations to avoid predators and to find mates, shelter, and food. However, it has been challenging to study the role of proprioception in adaptive movement because of the lack of tools to precisely perturb animal locomotion and the diminishment of spontaneous locomotion after manipulating proprioceptive neural circuits. To overcome these limitations, I engineered miniature treadmill systems for the genetic model organism, Drosophila melanogaster, that enable robust locomotion in flies lacking proprioceptive feedback, calibrated perturbations to walking, and quantifications of 3D walking kinematics. Using these systems, I found that proprioceptive feedback controls step kinematics across walking speeds, the middle legs of flies correct for asymmetric perturbations, and a class of proprioceptive neurons, called hair plates, facilitate the swing-to-stance transition, as predicted by the connectome. Overall, my dissertation work makes technical advances to reveal fundamental principles of how proprioception supports adaptive locomotion.
An investigation of microtubule-kinetochore attachment mechanisms
(2024-04-26) Murray, Lucas Edward; Asbury, Charles L
The ability to replicate is a defining feature of life. At the center of eukaryotic cell division are a set of protein machines responsible for pulling apart the chromosomes before cells divide. Spindle microtubules grow from the poles of the cell and connect to chromosomes via protein complexes called kinetochores. Kinetochores must maintain tenacious attachments to microtubule tips, even as they assemble and disassemble underneath their grip. Additionally, kinetochores mediate an error correction process to ensure the proper attachments to microtubules are formed before separation of the chromosomes commences. Here, I work to understand how the proteins in the kinetochore work together to maintain attachments to microtubules. I investigate two different mechanisms for microtubule-kinetochore attachment: the conformational wave mechanism and the biased diffusion mechanism. I developed a new optical trapping assay, using it to show that microtubule protofilament morphological and energetic properties can be measured and changed. I investigate the role of protofilament curl enlargement in the attachment and motility of the kinetochore. I develop theoretical models that show that the biased diffusion mechanism can fit experimentally measured detachment rates for assembling and disassembling kinetochores. Finally, I show kinetochores exhibit asymmetry in their sliding friction when they are dragged along microtubule lattices, a new phenomenon for microtubule-kinetochore biophysics. I argue this sliding friction forms the basis for a new mode of error correction during cell division, one that likely holds across most eukaryotic organisms.
The Mechanisms Underlying Sigh Generation in the PreBÃ¶tzinger Complex are Dependent on Neuroglial Interactions
(2022-01-26) Severs, Liza J; Ramirez, Jan-Marino
Rhythm generation is a vital component of life. Rhythmic activity regulates the circadian clock, which controls hormonal cycles, sleep wake states, blood pressure, body temperature, and reaction times. Rhythmogenesis underlies breathing and produces several behaviors -such as eupnea, sighs, and gasps- that are critical to everyday life. Moreover, rhythmic oscillations have emerged throughout the brain that link these coordinated activities together. Gaining a better understanding of rhythm generation and the underlying cell activities that make up these behaviors can help to provide a better understanding of many diseases which originate from the disruption of these cycles. The breathing networks in the medulla are a fantastic model system for studying rhythm generation. These areas maintain their intrinsic rhythm generating properties in vitro for extended periods of time, providing valuable insight into how individual cells and ion channels contribute to producing complex breathing behaviors. The preBÃ¶tzinger Complex (preBÃ¶tC) in the ventral-lateral medulla is one of these rhythmogenic circuits. Within this circuit, three behaviors necessary for normal breathing arise- normal breathing ‘eupnea’, sighing, and gasping. This thesis primarily focuses on the generation of the sigh rhythm.Sighing is a critical element of normal breathing that has vital physiological functions. Sighs are deep augmented breaths that are critical for survival as they prevent collapse of the lungs, clinically referred to as atelectasis, by re-inflating collapsed alveoli. Furthermore, during sleep, sighs trigger arousal to hypoxic conditions and are more common during REM sleep and transitions between different sleep states. Indeed, reduced sighing has been reported in infants who have died from sudden infant death syndrome (SIDS). Sighing is also linked to higher-order brain function and emotions such as love, stress, exasperation, and is clinically tied to several anxiety disorders. These ‘augmented breaths’ occur at a rate of ~12 per hour in adults and much more frequently in infants. Sighs often occur as a superimposed burst overlaying the ongoing eupneic burst, giving the sigh its characteristic biphasic shape. Eupnea consists of three primary phases: inspiration, postinspiration, and active expiration, each hypothesized to be generated by an excitatory rhythmogenic microcircuit in the ventral lateral medulla. Evidence suggests that eupneic inspiration, gasping, and sighing are generated by the same breathing circuit, the preBÃ¶tC. Yet, how the same neural circuit gives rise to two distinct rhythmic activities with separate timing characteristics remains unknown. The primary directive of this thesis is to provide a novel hypothesis for sigh generation.
Inflammatory pain regulation through TRPV1 ion channel and phosphoinositide signaling
(2020-02-04) Stratiievska, Anastasiia; Gordon, Sharona E
Inflammatory pain is one of the biggest challenges facing contemporary society. The TRPV1 ion channel is a major player in inflammatory pain. During inflammation TRPV1 is regulated by both modulation of gating and changes in channel number on the plasma membrane. Phosphoinositides are a class of membrane lipids which are directly involved in regulation of channel activity and signaling. PIP2 is a phosphoinositide which affects gating of TRPV1 but whether it activates or inhibits was controversial. We showed that under physiological conditions PIP2 activates TRPV1, while in non-physiological conditions, PIP2 inhibits TRPV1, which resolves the controversy in the literature. The phospholipid PIP3 is a product of a lipid kinase called PI3K, which is also involved in inflammatory pain. During inflammation, increased PI3K activity leads to increased PIP3, which leads to fusion of vesicles containing TRPV1 with the plasma membrane. This mechanism underlies increased sensitivity to pain during inflammation. We identified a novel mechanism for reciprocal regulation between PI3K and TRPV1 during inflammation, which results in potentiation of PI3K activity. A soluble fragment of TRPV1 called the ARD was sufficient to reproduce this effect. To move this work further, we are optimizing a novel opto-PI3K system to achieve better spatial and temporal control of PI3K activity. This will be significant for advancing our understanding of the mechanism of inflammatory pain and opening possibilities for developing new treatments.
The mechanisms and applications of a nucleotide-based heart failure therapy
(2020-02-04) Murray, Jason David; Regnier, Michael
The naturally-occurring nucleotide 2-deoxy-ATP (dATP) is known to increase the amount of force produced by cardiac muscle when used as a substrate in place of ATP. Cytosolic concentration of dATP can be elevated by expressing both subunits of the enzyme ribonucleotide reductase (RNR), and overexpression of RNR in the heart has been previously shown to increase the magnitude of pressure development in the heart as well as increase the rates of contraction and relaxation in transgenic mice as well as virally-transduce rodent and large-animal models. The first aim of this dissertation utilizes Brownian dynamics simulations in concert with small-angle x-ray diffraction analysis of sarcomere structure to demonstrate that dATP induces structural changes in the conformation of myosin that increases its electrostatic affinity for actin and leads to an altered structural confirmation at rest that resembles changes caused by calcium-mediated activation. The second aim applies our therapeutic to a disease model and explores possible changes in skeletal muscle activation through a combinatorial gene therapy for a mouse model of Duchenne muscular dystrophy. Overexpression of RNR in concert with an artificial microdystrophin construct improved fractional shortening in cardiomyocytes but did not alter the overall force or kinetics of hindlimb muscle in either our disease model or in a mouse model of transgenic overexpression of ribonucleotide reductase. The third aim explores methods by which dATP can be elevated. We describe cardiac function after overexpression of Rrm1 and Rrm2B (RNRB), an isoform of the small subunit of RNR that has not been characterized in the heart. RNRB elevates cardiomyocyte dATP in a transgenic mouse model to the same degree as RNR, resulting in increases in cardiac function on multiple biophysical scales. Calcium handling was also altered in cardiomyocytes through an increase in the rate of calcium signal decay during contraction, a novel result which has not been previously described. These changes were reproducible in vitro in adult rat cardiomyocytes overexpressing RNRB through virally-mediated transduction in culture. Identifying a form of RNR that can be stably overexpressed in cardiomyocytes is an important step towards developing a therapeutic based on elevation of dATP.
Rapid modulation of dynamics and computation in neural systems
(2019-05-02) Pang, Rich; Fairhall, Adrienne L
A central goal in theoretical neuroscience is to understand how neural systems perform computations over the continuum of timescales that underlie behavior. In particular, what are the algorithms and mechanisms enabling single-neuron membrane voltage fluctuations, which occur over milliseconds, to produce the dynamics and information processing in behavior that unfold over hours to years? Notably, while the core ionic processes of membrane voltage fluctuations have been largely elucidated and while extensive theories and evidence exist to explain how slow modulation of neural network structures might underlie learning, almost nothing is known about the liminal regime of seconds to minutes that bridges these two timescales. In the work that follows I address three questions in three different systems, each of which centers around neural computations occurring over the timescales of seconds to minutes. I first investigate the navigational decisions made by flying insects during odor tracking, where I show that fruit flies and mosquitoes exhibit a history dependence in their odor-triggered turning responses that is qualitatively similar to an information-maximizing tracking strategy, but not to others. Next, in collaboration with Ari Zolin, Raphael Cohn, and Vanessa Ruta, I analyze the dynamics of dopaminergic neuromodulation of a short-term memory circuit in the fruit fly mushroom body, where we suggest that the fly dopamine system encodes multiplexed representations of a wide diversity of sensory, motor, and valence signals, some of which predict behavior several seconds in the future. Third, I develop a spiking neural network model capable of storing and replaying sequential activity patterns using a heterosynaptic and fast-acting biological plasticity rule, and which reconstructs sequences through the existing recurrent network structure. Collectively, these results elucidate the computational capacities of three distinct systems and shed new light on short-term information processing in neural computations from three novel angles. Finally, in collaboration with Sid Henriksen and Mark Wronkiewicz, I describe a simple network-growth model reproducing several statistical features of mouse brain network connectivity at the mesoscale; while this work does not explicitly address short-term computations, simplified statistical network models will be crucial to eventually understanding how such computations occur within large scale distributed brain networks.
Effects of clinically-relevant electrical stimulation of macaque sensorimotor cortex on neural activity and behavior
(2018-07-31) Bogaard, Andrew Robert; Fetz, Eberhard E
Electrical stimulation is a popular technique for neuromodulation in research, but the scope of approved therapeutic devices is extremely limited. For conditions where direct stimulation of the cortex may alleviate symptoms, such as stroke, clinical trials testing electrical cortical stimulation (CS) have produced mixed effects. These protocols may be less refined than other types of neurostimulation because basic physiological effects are not well understood, and because responses in the cortex are less predictable than other types of neurostimulation (e.g. to nerves). The following dissertation outlines experiments in non-human primates designed to document the physiological and behavioral effects of two types of CS. First, we adopt a tonic form of CS common among clinical trials, transcranial direct current stimulation, and demonstrate how polarity and dose influence neural activity. Second, we test two forms of phasic CS: one that is timed relative to unilateral hand movements in a reaction time task, and another that is triggered by muscle activity. “Activity-dependent” CS such as these can be used to probe the functional roles of dynamic brain signals, or to induce plasticity. Our monkey model provides direct physiological evidence that is often lacking in human research, and reveals that, under certain conditions, CS produces repeatable changes in brain activity by facilitating or disrupting natural physiological processes. These insights may be useful towards the design of future CS-based therapies.
Quantitative Modeling of Cone Signal Combination in Macaque Primary Visual Cortex
(2018-07-31) Weller, J Patrick; Horwitz, Gregory D
Vision at moderate to high light levels begins with the activity in three types of cone photoreceptors located in back of the retina: long-wavelength-sensitive (L-) cones, medium-wavelength-sensitive (M-) cones, and short-wavelength-sensitive (S-) cones. One central goal in the field of color neurophysiology is to understand how modulations in cone activity are transformed as they propagate through the visual system. My graduate work has been dedicated to understanding how cone signals are processed in the primary visual cortex (area V1). In the first chapter, I lay out some of the typical considerations for designing an effective electrophysiology experiment in the context of color vision, such as the spatiotemporal structure of the stimulus, color spaces and stimulus distributions, as well as the quantitative models under consideration. In the second chapter, I discuss some of the pitfalls and advantages of different stimulus distributions and analysis techniques under the linear-nonlinear cascade model, a standard model in the field. In the third chapter, I present a novel single-cell electrophysiology experiment probing the effect of L- and M-cone modulations on the responses of single neurons in V1. In this chapter, I also present data from a new quantitative model for describing neural responses to L- and M-cone modulations. In the final chapter, I consider how we might use the results of these experiments to build strong priors for fitting an appropriate model of L- and M-cone processing in V1.
Cone photoreceptor heterogeneity in the primate retina
(2018-07-31) Baudin, Jacob Alexander; Rieke, Frederick M
Human vision commences when light is transduced to a neural signal. In daylight, this occurs predominantly within cone photoreceptors. Does this transduction occur identically for all light inputs? The work in this thesis addresses this question as well as the role of phototransduction in controlling downstream signals in the visual system. Chapter 2 focuses on heterogeneity in the transduction of light across wavelengths due to differences in short-, medium-, and long- wavelength sensitive cone photoreceptors. Chapter 3 elaborates on heterogeneity in transduction of inputs across visual space arising from differences in cones across retinal eccentricity. Chapter 4 begins to explore an instance where cone signals directly control retinal output. Together, this body of work aims to provide an appreciation for heterogeneity in signal transduction within cones throughout the primate retina and link cone properties to both retinal output and perception.
Spike-timing dependent plasticity and connectivity in primate sensorimotor cortex
(2016-09-22) Seeman, Stephanie; Perlmutter, Steve I
Specific connectivity between populations of neurons gives rise to network function. The plasticity of these connections allows for network learning and adaptation. While much work has explored plasticity and underlying connectivity, there is still much to learn about why particular connections are susceptible to manipulation. Hebbian plasticity has been shown in a variety of neural circuits, yet there are subtle differences in the mechanisms driving these effects at each synapse. Similarly, functional connectivity has been described utilizing varying methods. Ultimately, these bodies of work tile a broad spatiotemporal view of cortical function. Here, we explore plasticity between sensorimotor populations and the underlying connections which serve targeted changes. The variability we observed in our results further highlights that one size does not fit all, and perhaps only by looking at the collective may we begin to understand the complexities of cortical processing. I. Paired-stimulation for spike-timing dependent plasticity in primate sensorimotor cortex Classic studies in vitro have described spike-timing dependent plasticity (STDP) at a synapse: the connection from neuron A to neuron B is strengthened (or weakened) when A fires before (or after) B within an optimal time window. Accordingly, more recent in vivo works have demonstrated behavioral effects consistent with an STDP mechanism; however, many relied on invasive single-unit recordings. The ability to modify cortical connections becomes useful in the context of injury when connectivity, and associated behavior, is compromised. To avoid the need for long-term, stable isolation of single units, one could control timed activation of two cortical sites with paired, electrical stimulation. We tested the hypothesis that STDP could be induced via prolonged paired-stimulation as quantified by cortical evoked potentials (EPs) in sensorimotor cortex of awake, behaving monkeys. Paired-stimulation between two interconnected sites produced robust effects in EPs consistent with STDP; however, only at a subset of tested pairs (2/15). This protocol otherwise produced increases in global network excitability or depression of the conditioned pair. Taken together, these results suggest that paired-stimulation in vivo is a viable method to induce STDP between cortical populations, but that factors beyond activation timing must be kept in mind to produce a site-independent effect. II. Intrinsic functional connectivity of neural populations in forelimb sensorimotor cortex The structure of neocortex is defined by its anatomical and functional connections, from which processing and cognition arise. Functional connectivity exists and may be investigated along a wide spatiotemporal range utilizing a variety of electrophysiological techniques and analyses. Single- and multi-unit recordings can show connectivity of local micro-circuits whereas electrocorticography (ECoG) or functional imaging highlights macro-scale connections across the whole brain. Further, recordings of action potentials compared to local field potentials (LFPs) in ECoG are based in fundamentally different mechanisms generating neural activity and connectivity. We explored meso-scale connectivity in hand area of primate sensorimotor cortex via EPs and band-limited coherence in spontaneous LFP between cortical sites. The strength of both EPs and coherence showed an inverse relationship with inter-site distance as well as regionality between primary motor (M1) and somatosensory (S1) cortex. Despite these similarities, EP and coherence connectivity maps were not well correlated likely due to different underlying mechanisms driving the two signals. Taken together, these results show connectivity structure at a meso-scale, population level similar to that at other scales. Further exploration utilizing different recording techniques or functional connectivity metrics may elucidate additional network structure.
Three lenses into the cardiac dyad: calcium signaling, microtubule trafficking, and junctional sarcoplasmic reticular mobility
(2016-07-14) Drum, Benjamin Mark Loren; Santana, Luis F
The cardiac dyad, the intersection between the sarcoplasmic reticulum and the sarcolemma in cardiac myocytes, is critically important for cellular contraction and function. It is the hub of calcium signaling where ryanodine receptors on the sarcoplasmic reticulum and L-type calcium channels on the sarcolemma interact. If the geometric distance between these two proteins is disrupted, calcium signaling will be impaired. In addition, these proteins are trafficked and maintained by microtubules. In this dissertation, I take a three-pronged approach to understanding the dyad. First, I use a mouse model of Timothy syndrome (TS), a disease created by an overactive L-type calcium channel, to examine the effects of the physiological calcium environment on calcium signaling. We find that the resting level of calcium in the cytosol is increased by 1.6-fold, calcium transients are increased by 1.8-fold, and sarcoplasmic reticulum load is increased by 1.5-fold in TS, leading to arrhythmogenicity. Secondly, I use an adeno-associated virus serotype 9 expressing a plus-end microtubule binding protein (AAV9-EB3-EGFP) to visualize microtubules in real time. I quantify microtubule dynamics in ventricular myocytes and find that oxidative stress in myocardial infarction disrupts microtubules, leading to a decrease in the surface expression of KV4.2 and KV4.3 and decrease in Ito, also leading to arrhythmia by prolonging the action potential. I also show that microtubules and molecular motors, specifically kinesin-1, are responsible for trafficking the β2 subunit of CaV1.2, and that disruption of kinesin-1 results in a loss in current density of CaV1.2 current. I also examine microtubule dynamics in atrial myocytes and use this research to probe the role of BIN1 in linking microtubule dynamics to the cardiac dyad. Lastly, I use an adeno-associated virus expressing triadin, a protein localized to the junctional sarcoplasmic reticulum (jSR) tagged with a photo-activatable GFP (AAV9-TRD-PAGFP) to directly monitor the movement of the jSR under normal physiology and find that 8% of jSR segments exert mobility. This movement is produced by molecular motors and is increased in the setting of myocardial infarction. By exploring the cardiac dyad through the lenses of calcium signaling, microtubule trafficking, and direct jSR movement, I am able to elucidate a multifaceted look at the regulation and function of the dyad as well as its importance in cardiac physiology.
Protein kinase C bidirectionally modulates Ih and HCN1 surface expression in hippocampal pyramidal neurons
(2015-09-29) Williams, Aaron Douglas; Poolos, Nicholas P
Hyperpolarization-activated, cyclic nucleotide-gated (HCN) ion channels attenuate excitability in hippocampal pyramidal neurons. Loss of HCN channel-mediated current (Ih), particularly that mediated by the HCN1 isoform, occurs with the development of epilepsy. Previously, we showed that following pilocarpine-induced status epilepticus, there are two independent changes in HCN function in dendrites: decreased Ih amplitude associated with a loss of HCN1 surface expression, and a hyperpolarizing shift in voltage-dependence of activation (“gating”). The hyperpolarizing shift in gating was attributed to decreased phosphorylation due to loss of p38 MAP kinase activity and increased calcineurin activity; however, the mechanisms controlling Ih amplitude and HCN1 surface expression under epileptic or normal physiological conditions are poorly understood. I sought to investigate phosphorylation as a mechanism regulating Ih amplitude and HCN1 surface expression (as it does HCN gating) in hippocampal principal neurons under normal physiological conditions. I discovered that inhibition of either tyrosine phosphatases or the serine/threonine phosphatases PP1 and PP2A decreased Ih at maximal activation in hippocampal CA1 pyramidal dendrites and pyramidal-like principal (PLP) neuron somata from naïve rats. Furthermore, I found that inhibition of PP1/PP2A decreased HCN1 surface expression, while tyrosine phosphatase inhibition did not. Protein kinase C (PKC) activation reduced Ih amplitude and HCN1 surface expression, while PKC inhibition produced the opposite effect. PP1/2A inhibition and PKC activation both increased the serine phosphorylation state of the HCN1 protein. The effect of PKC activation on Ih was irreversible. These results indicate that PKC bidirectionally modulates Ih amplitude and HCN1 surface expression in hippocampal principal neurons.
GABA-B receptors regulate extrasynaptic GABA-A receptors
(2015-02-24) Tao, Wucheng; Spain, William J
Classically, GABAB receptors regulate neurotransmission primarily through presynaptic mechanisms that inhibit neurotransmitter release, and thus inhibit GABAA receptor function. Many studies have shown that postsynaptic GABAB receptors have no direct functional effects on GABAA receptors. In this thesis, I describe novel results that indicate postsynaptic GABAB receptors activation enhances GABAA receptor function in dentate gyrus granule cells (DGGCs). During my early experiments on DGGCs, I made the surprising observation that inhibition of GABAB receptors reduced the amplitude of currents mediated by GABAA receptors. Intrigued by this exciting and unexpected result, I shifted my research to address the following three questions: 1) Do GABAB receptors regulate GABAA receptor function? 2) What are the molecular mechanisms responsible for enhancement of GABAA currents following GABAB receptor activation? and 3) What are the signaling pathways involved in this modulation? Based on these questions, I divide my thesis contents into four chapters. Chapter 1 presents an introductory review of GABAA receptors and GABAB receptors and a summary of my thesis. The main parts of this thesis with descriptions of results addressing the three questions above are presented in chapter 2 - 4: In chapter 2, I found that in dentate gyrus granule cells, postsynaptic GABAB receptor activation enhanced extrasynaptic GABAA receptor function with no effect on synaptic GABAA receptors. Specially, this modulation was cell type specific and only occurs in cell type with delta subunit-containing GABAA receptors. Also, this modulation did not occur at resting condition and required increase of ambient GABA concentration; in chapter 3, I found GABAB receptor activation increased surface expression of delta subunit-containing GABAA receptors with no change in its total protein expression or single channel conductance or channel kinetics of GABAA receptors; in chapter 4, I found that GABAB receptor modulation of GABAA receptors required two signaling pathways, one was mediated by PKA and other one was PKC; and these two signaling pathways worked in opposite directions to modulate surface expression of delta subunit-containing GABAA receptors and GABA currents. As DGGCs act as a gate for hippocampus to prevent excessive excitation inputs from entorhinal cortex, the mechanisms (enhancement of tonic inhibition by membrane trafficking of delta subunit-containing GABAA receptors) I found here maybe utilized by DGGCs to enhance their gating role.
Operant conditioning of cortical cell and muscle response patterns
(2014-10-13) Eaton, Ryan W.; Fetz, Eberhard E.
<bold>Part I:</bold> In primates, corticomotoneuronal (CM) cells have sufficiently strong synaptic linkages to motoneurons to mediate post-spike facilitation in spike-triggered averages of muscle activity. We investigated the degree to which activity of CM cells and their target muscles could be independently controlled by operantly conditioning their relative activation levels. In two Macaca nemestrina monkeys, single cortical neurons were recorded with moveable microwires chronically implanted in the caudal bank of the pre-central gyrus. Rectified EMG of 12 distal forelimb muscles was recorded with sub-cutaneously implanted pairs of wires in each muscle in one monkey from which 35 unique cell-muscle pairs were operantly conditioned. In the other animal, surface electrodes were placed over wrist flexor and extensor muscles (9 unique cell-muscle pairs). Spike-triggered averages of rectified EMG were compiled while the monkeys performed a force target-tracking task about the wrist. Twenty-four CM cells, with post-spike effects at latencies between 6 and 16 ms in one or more forelimb muscles, were selected for activity dissociation conditioning. Monkeys performed an operant conditioning task through which relative activation of the CM cell and a target muscle could be explored (44 unique cell-muscle pairs). Cell and muscle activity controlled the position of a cursor on a screen, with cursor position determined by concurrent cell spike rates (C) and EMG activity of a target muscle (M); one activity assigned the horizontal direction the other, vertical. Monkeys received fruit sauce rewards for holding the cursor in target positions requiring at least four combinations of increased (+) and suppressed (-) activation relative to levels observed during force generation, namely C+M+; C+M-; C-M+ and C-M-. The monkeys learned to reciprocally activate cells and target muscles exhibiting post-spike facilitation (42 out of 44 cell-muscle pairs), in both directions. For muscles with post-spike suppression (4 out of 4 pairs), monkeys learned to co-activate these cell-muscle pairs. These results indicate that cortical cells with direct synaptic linkages to motoneurons can be flexibly activated relative to their target muscles. Further, CM cell-muscle activity dissociations can be rapid, robust, reversible and are subject to volitional control. Calculation of mutual information between CM cell and muscle activation patterns during reciprocal dissociation events, compared to force target-tracking, implicates involvement of other upstream sources. Activity independence between correlationally-linked components within a neural circuit favors strategies for brain computer interface (BCI) control in which individual neurons are each assigned an individual degree-of-freedom of device output. <bold>Part II:</bold> Operant conditioning of neural activity has typically been achieved under controlled behavioral conditions using food reinforcement. To reward cell activity during unconstrained behavior, we sought midbrain sites whose stimulation would support operant responding. Three nemestrina monkeys learned to perform a manual step-tracking task rewarded by fruit sauce. We found sites in nucleus accumbens and surrounding striatum whose stimulation could maintain task performance and verified that response rates increased monotonically with increasing pulse frequency and amplitude. We recorded activity of single neurons with moveable microwires chronically implanted in the precentral gyrus and documented neural modulation with a force-guided target-tracking task. We attempted to condition increased firing rates first with the monkey in the training booth and then during free behavior in the cage using the Neurochip—a head-fixed, autonomous recording and stimulating system. Spikes occurring above baseline rates triggered single or multiple electrical pulses to the reinforcement site (1 mA, 0.2 ms biphasic current pulses). This rate-contingent, unit-triggered stimulation was made available for periods of 1 to 3 minutes separated by 3 to 10 minute time-out periods without stimulation, regardless of cell activity. During in-booth sessions feedback was presented as vertical cursor movement and auditory clicks. During in-cage conditioning, barely audible clicks occurred during each spike-triggered stimulation event. In-booth conditioning produced increases in single neuron firing probability after transition to intracranial reinforcement in 48 of 58 cells. Reinforced cell activity could rise > 5 times that of non-reinforced activity, doubling in most sessions. Activity peaks typically occurred during the first 10 seconds of each time-in period and, for many cells, activity remained elevated above baseline for the full period. In-cage conditioning produced significant increases in post-transition activity in 21 out of 33 sessions. In-cage effects peaked later and lasted longer than in-booth effects but were often comparatively smaller, between 13 and 18 percent above non-reinforced activity. The difference in responding in the two conditioning environments could be due to the dynamic range of candidate cell firing rate, robustness of the reinforcement site and differing levels of attention and competing behaviors in the booth and cage. Controls indicate that stimulation of the reinforcement site did not directly evoke increased cell activity. In several sessions, neighboring, synaptically-linked motor cortex neurons were recorded simultaneously with the trigger cell, revealing network involvement in eliciting conditioned rate changes in the stimulation-triggering neuron.
The limits imposed in primate vision by transduction in cone photoreceptors
(2014-10-13) Angueyra, Juan; Rieke, Frederick M
Experientially, our ability to visually perceive the world seems extraordinary, yet we keep asking ourselves: "could perception be better?" This dissertation centers around this question, exploring how the biophysics of photoreceptors impose constraints on the visual system and dictate certain aspects of perception. The first chapter explores the sources of noise in cone phototransduction and identifies open-close transitions of the cGMP-gated channels as a dominant source of noise. This noise source also escapes the light-adaptation mechanisms that control gain, establishing a particular scenario that determines the shape of threshold-vs.-intensity curves in single cones and ultimately in humans. The second chapter investigates how cones adapt during eye movements, uncovering that adaptation has at least two different time scales that influence the encoding of mean luminance and modulation around the mean luminance separately. This will lead to the construction of a biophysical model that only when endowed with two separate light-adaptation mechanisms is able to reproduce responses to a wide array of stimuli. It is my hope that this model can be used as a tool to explore the constraints imposed by cone signals on the rest of the retinal circuitry and that it can help clarify how computations are implemented in downstream neural circuits.
Cortical surface recurrent brain-computer interfaces
(2013-11-14) Zanos, Stavros; Fetz, Eberhard E
The output of a "traditional" brain computer-interface (BCI) is the operation of an effector mechanism, like a cursor or a prosthetic arm. In contrast, the output of a recurrent brain-computer interface (rBCI) is electrical stimulation delivered directly into the central nervous system (CNS). Recurrent BCIs have been used to artificially bridge two separate sites in the CNS whose communication may have been interrupted. They have also been used to associate activity of a site in the CNS with stimulation of another site, to produce synaptic plasticity between the two sites. To date, rBCIs have utilized intracortical implants to record neural activity and deliver electrical stimuli, which have problems that limit their clinical applicability. These limitations can be addressed by cortical surface electrodes, subdural or epidural, that can capture electrocorticography (ECoG) signals and deliver electrical cortical surface stimulation. We first examine the recording capabilities of cortical surface arrays. We study the relationship of ECoG signals with motor behavior and EMG activity from upper extremity muscles. We demonstrate that EMG activity can be decoded from multichannel ECoG, and document the gradual decrement in decoding performance over several months of recording. Second, we examine the stimulation capabilities of cortical surface arrays. We characterize the effects of repetitive stimulation on the electrode-tissue interface, by measuring electrode impedance. We determine the impact of stimulation on cortical excitability, by measuring stimulus-evoked motor responses. Finally, we examine the effect of stimulation on spontaneous cortical activity, as evidenced by ECoG power at different frequencies. Third, we investigate a cortical surface rBCI system to study the role of sensorimotor beta oscillations in synaptic plasticity. Stimulation at a cortical site was triggered from specific phases of beta (15-25 Hz) oscillatory episodes of ECoG recorded from a different site. The effects of conditioning stimulation on cortical connectivity were determined through cortically-evoked potentials and ECoG phase coherence. We document a short-term change in cortical connectivity following beta oscillations that is mediated by synaptic modification and follows a Hebbian-like rule. Our findings on properties of cortical surface recording and stimulation will promote translation of these techniques to clinical applications. Our demonstration of changes in cortical connectivity induced by a cortical surface rBCI furthers our understanding of cortical oscillations and provides a paradigm for activity-dependent cortical plasticity using these less invasive implants.
The role of coupled gating of L-type calcium channels in arrhythmogenesis in Timothy syndrome (LQT8)
(2012-09-10) Cheng, Edward Peiyang; Santana, Luis F
L-type Ca<super>2+</super> (CaV1.2) channels shape the cardiac action potential waveform and are essential for excitation-contraction (EC) coupling in the heart. We find that a transient subpopulation of 2 to 6 CaV1.2 channels gate concertedly via a novel coupled gating modality. In the presence of the scaffolding protein AKAP150: PKC&alpha, calmodulin inhibition, and the G406R mutation that causes Timothy syndrome (TS), also known as long QT syndrome type 8 (LQT8), increase the probability of coupled gating. As for the mechanism of coupled gating, we propose that CaV1.2 channels interact with each other via their carboxy tails when calmodulin is dislodged from the IQ domain, and coupled gating requires AKAP150 to bind to the CaV1.2 carboxy tails via its leucine zipper domain and to act as a scaffold. To study further the role of coupled gating and AKAP150 in arrhythmogenesis in LQT8, we created a LQT8 transgenic mouse expressing cardiac-specific CaV1.2-LQT8-tRFP channels. Importantly, we then crossed these LQT8 transgenics with AKAP150<super>-/-</super> mice, and the LQT8/ AKAP150<super>-/-</super> mice show a rescue of the wild-type (WT) phenotype. Compared to WT and LQT8/ AKAP150<super>-/-</super> ventricular myocytes, LQT8 ventricular myocytes have delayed inactivation in whole-cell ICa, and there is increased frequency in coupled gating and open time in cell-attached iCa. These myocytes also have prolonged action potential duration and arrhythmogenic voltage fluctuations, such as early and delayed afterdepolarizations. With respect to EC coupling, LQT8 ventricular myocytes have increased [Ca<super>2+</super>] transient amplitudes and more frequent arrhythmogenic spontaneous Ca<super>2+</super> releases. On the whole animal level, LQT8 mice have prolonged QTC interval and more frequent incidence of Torsades de Pointes ventricular tachycardia than WT or LQT8/ AKAP150<super>-/-</super>. In conclusion, AKAP150 mediated coupled gating of CaV1.2 channels plays a central role in the pathophysiology of LQT8, causing local perturbation on EC coupling that lead to arrhythmogenesis.
A Reconstituted System for Studying Kinetochore-Microtubule Attachments
(2012-09-10) Franck, Andrew; Asbury, Charles
Before physically dividing into two nascent daughter cells, a cell must first duplicate, organize and segregate its entire genome. During mitosis, chromosomes are aligned along an axis of symmetry and then pulled apart by force-generating elements in the cell. Advances in genetic, biochemical and biophysical tools have enabled us to identify and study key components of this force-generating apparatus. Here, I will focus on interactions between chromosome-bound organelles called kinetochores and microtubules, dynamic protein filaments. My goal was to better understand how kinetochores harness the work generated by dynamic microtubules to drive chromosome movement. To do so, I used purified kinetochore components and employed biophysical techniques adapted from the study of single molecules. With this system I was able to reconstitute fundamental biological phenomena: persistent kinetochore-like attachments to growing and shortening microtubules, cooperative interactions between components that enhance attachment strength and force-dependent regulation of microtubule dynamics. Using a combination of single molecule techniques and modeling, I have also initiated study into the biophysical mechanisms underlying kinetochore-microtubule attachment. This work has given us valuable insights as to how these essential mitotic machines function within living, dividing cells.
A new view of the radial geometry in muscle: myofilament lattice spacing controls force production and energy storage.
(2012-09-10) Williams, Charles David; Daniel, Thomas L
Muscle is highly organized in both the axial direction and the radial direction, or the direction of contraction and the direction orthogonal to it. As muscle generates force, it does so in both the axial and radial directions. Lattice spacing, which is the radial spacing between its contractile filaments, increases as muscle shortens. Historically, the effects of these processes have not been accounted for in our conceptual or mathematical models of muscle contraction. We develop a computational model of the half-sarcomere that is fully three dimensional and thus replicates the processes which occur in muscle's radial direction. This model employs a novel cross-bridge model which uses multiple springs, both extensional and angular. Where prior cross-bridge models use a change in rest length to generate force, our multi-spring model uses a lever arm mechanism similar to myosin's. Using this model and experiments with isolated skinned muscle, we show that changes in lattice spacing increase the slope of the length tension curve by more than 20%. The length-tension curve describes the relationship between a muscle's sarcomere lengths and the maximum force which it can generate. The length-tension curve has been attributed to changing degrees of the overlap of thick and thin filaments as sarcomere length varies. A steep slope on the length-tension curve is necessary for passive stability of muscular systems, such as cardiac muscle, which operate at short sarcomere lengths. These systems rely on the small length changes which accompany a new load to tailor the force produced for the new load. Without the steeper slope produced by varying lattice spacings, an external mechanism of force regulation would be necessary to provide system stability. Additional model results show that substantial energy is stored in deformation of the cross-bridges during maximum activation, more than twice that which is stored in deformation of the thick and thin filaments. This energy is highly correlated with force produced in the radial direction, itself of the same order of magnitude as the axial force produced. These relative levels of axial and radial forces are themselves a confirmation of previous experimental measurements of radial force. The stored energy may play a role in powering rapid, one-off explosive movements such as prey striking by Mantis shrimp and tongue extension in toads.
The Interaction of Lingo-1 and Amyloid Precursor Protein
(2012-09-10) de Laat, Rian C.; Bothwell, Mark A
Proteolytic cleavage of amyloid precursor protein (APP) generates the amyloid β peptide (Aβ), the main component of cortical and subcortical plaques in Alzheimer's disease (AD). APP can be processed at the cell surface or within endosomes after endocytosis, and via an amyloidogenic or non-amyloidogenic pathway. Along the amyloidogenic pathway, APP is first cleaved by β-secretase followed by γ-secretase to produce Aβ. The non-amyloidogenic pathway involves cleavage by α-secretase and then γ-secretase. Aβ generation is thought to occur in a variety of organelles where APP, β- and γ-secretase reside. Proteins that regulate endocytosis and trafficking can thus control the qualitative proteolysis of APP, and consequently may be associated with pathophysiology of AD. One such protein is Lingo-1, which promotes APP trafficking to the lysosome and concomitant degradation, independent of the secretory pathway. In this manner, Lingo-1 may function as a control mechanism for APP levels.

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