Browsing by Author "Tadross, Michael R"
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Item Open Access A Data-Driven Approach to Uncovering the Neural Dynamics of Anxiety(2022) Hughes, DaltonAnxiety is a behavioral state induced by low-threat, uncertain situations in which perceived danger is diffuse. The anxiety state is then accompanied by increased vigilance and risk assessment to one’s surroundings. Recent studies have shown that the brain regions responsible for encoding anxiety are widely located in the frontal cortex and extended limbic system; however, the network architecture responsible for hypervigilance has yet to be elucidated. Here, I propose to employ a data-driven method of using in vivo recordings of electrical activity across multiple brain regions concurrently as mice freely explore classic ethological anxiety-related behavioral assays and are administered pharmacological agents that modulate the anxiety state. Using novel machine-learning techniques, I have generated neural models that reflect the network-level activity engaged during the performance of these tasks. I have then validated the structure of this anxiety network in its ability to generalize to other anxiety-related tasks and models of disease. I anticipate that this strategy will discover an independent network that is correlated with anxiety-related behaviors. Thus, successful completion of the proposed work will lead to a network-level understanding of anxiety. Furthermore, the framework discovered through this study has the potential to facilitate the development of new revolutionary approaches for anxiety disorders.
Item Open Access A modular switch for spatial Ca2+ selectivity in the calmodulin regulation of CaV channels.(Nature, 2008-02-14) Dick, Ivy E; Tadross, Michael R; Liang, Haoya; Tay, Lai Hock; Yang, Wanjun; Yue, David TCa2+/calmodulin-dependent regulation of voltage-gated CaV1-2 Ca2+ channels shows extraordinary modes of spatial Ca2+ decoding and channel modulation, vital for many biological functions. A single calmodulin (CaM) molecule associates constitutively with the channel's carboxy-terminal tail, and Ca2+ binding to the C-terminal and N-terminal lobes of CaM can each induce distinct channel regulations. As expected from close channel proximity, the C-lobe responds to the roughly 100-microM Ca2+ pulses driven by the associated channel, a behaviour defined as 'local Ca2+ selectivity'. Conversely, all previous observations have indicated that the N-lobe somehow senses the far weaker signals from distant Ca2+ sources. This 'global Ca2+ selectivity' satisfies a general signalling requirement, enabling a resident molecule to remotely sense cellular Ca2+ activity, which would otherwise be overshadowed by Ca2+ entry through the host channel. Here we show that the spatial Ca2+ selectivity of N-lobe CaM regulation is not invariably global but can be switched by a novel Ca2+/CaM-binding site within the amino terminus of channels (NSCaTE, for N-terminal spatial Ca2+ transforming element). Native CaV2.2 channels lack this element and show N-lobe regulation with a global selectivity. On the introduction of NSCaTE into these channels, spatial Ca2+ selectivity transforms from a global to local profile. Given this effect, we examined CaV1.2/CaV1.3 channels, which naturally contain NSCaTE, and found that their N-lobe selectivity is indeed local. Disruption of this element produces a global selectivity, confirming the native function of NSCaTE. Thus, differences in spatial selectivity between advanced CaV1 and CaV2 channel isoforms are explained by the presence or absence of NSCaTE. Beyond functional effects, the position of NSCaTE on the channel's amino terminus indicates that CaM can bridge the amino terminus and carboxy terminus of channels. Finally, the modularity of NSCaTE offers practical means for understanding the basis of global Ca2+ selectivity.Item Open Access A Toolbox for Observing and Modulating the Gut-Brain Axis(2022) Garrett, Aliesha DanielleAn estimated 10% of people worldwide have an enteric nervous system (ENS) related illness including irritable bowel syndrome (IBS), diabetes, colorectal cancer, fecal incontinence, and chronic constipation or diarrhea. Current drug treatments have severe side effects and often do not adequately address symptoms; a new approach is needed. ENS stimulation is a promising therapy for these patients, but a major limitation to this approach is our lack of knowledge. The human ENS is comprised of 5 million neurons and drives the digestive system, but its normal function and connections to the central nervous system (CNS) remain poorly understood. One of the major canonical signaling pathways between the ENS and the CNS is the vagus nerve, but the neural circuits involved are still under investigation. Better understanding of these circuits would provide a potential method of treatment for ENS related illness, with neurostimulation serving as an alternative to pharmaceutical treatments. Herein I describe a project which addresses these needs via development of new imaging tools to better understand the gut-brain axis, as well as demonstrating its utility as a target for treatment of gastrointestinal (GI) illness, specifically cancer-associated cachexia. Leaders in enteric neuroscience note that the continued inconsistencies in GI electrotherapies are driven by a fundamental lack of understanding of gut innervation and circuitry. New tools to directly observe colonic innervation and neuronal response, as well as a map of the whole peripheral nervous system, will reveal crucial targets for stimulation and enable more efficient targeting selection for neurostimulation or other local interventions, which will reduce off target effects and improve efficacy. To address these issues, I have developed an intravital window for direct imaging of the colon, enabling observation of colonic ENS response to stimulation in vivo for the first time. Additionally, I have developed an embryonic window, allowing visualization of embryonic GI development from E9.5 through birth. Finally, I have generated a mouse peripheral nerve map based on Diffusion Tensor Magnetic Resonance Imaging (DT MRI). Using novel scan parameters and post-processing algorithms, I identified nerve fibers throughout the body and generated quantitative tractography which specifically highlights GI innervation via the vagus nerve. Cachexia is a multi-systemic syndrome which produces weight loss, muscle atrophy, adipose wasting, fatigue, and anorexia. Affecting an estimated 1% of the global population and up to 80% of all cancer patients, cachexia is fatal in roughly 30% of cases and is incurable. Cancer-associated cachexia (CAC) is particularly devastating as in addition to resulting in decreased quality of life, CAC reduces tolerance and efficacy of cancer treatments and higher overall mortality. As many as half of all cancer deaths are attributed to CAC. There are currently no clinically meaningful treatments for CAC, despite attempts to employ dietary support, physical therapy, anti-inflammatory medication, appetite stimulants, and other supportive therapies. Herein I describe potential therapeutic approach for treatment of CAC via vagal perturbation – either by vagotomy or ultra-low frequency vagal block with an implanted stimulator. This intervention significantly attenuates weight loss, skeletal muscle atrophy, anorexia, urea cycle dysregulation, and circulating inflammatory cytokine elevation. Most importantly, it increases survival time in mice injected with tumor cells, suggesting this could be a clinically meaningful approach for treatment of CAC.
Item Open Access Behavioral state and stimulus strength regulate the role of somatostatin interneurons in stabilizing network activity.(bioRxiv, 2024-09-10) Cammarata, Celine M; Pei, Yingming; Shields, Brenda C; Lim, Shaun SX; Hawley, Tammy; Li, Jennifer Y; St Amand, David; Brunel, Nicolas; Tadross, Michael R; Glickfeld, Lindsey LInhibition stabilization enables cortical circuits to encode sensory signals across diverse contexts. Somatostatin-expressing (SST) interneurons are well-suited for this role through their strong recurrent connectivity with excitatory pyramidal cells. We developed a cortical circuit model predicting that SST cells become increasingly important for stabilization as sensory input strengthens. We tested this prediction in mouse primary visual cortex by manipulating excitatory input to SST cells, a key parameter for inhibition stabilization, with a novel cell-type specific pharmacological method to selectively block glutamatergic receptors on SST cells. Consistent with our model predictions, we find antagonizing glutamatergic receptors drives a paradoxical facilitation of SST cells with increasing stimulus contrast. In addition, we find even stronger engagement of SST-dependent stabilization when the mice are aroused. Thus, we reveal that the role of SST cells in cortical processing gradually switches as a function of both input strength and behavioral state.Item Open Access Ca2+ channel nanodomains boost local Ca2+ amplitude.(Proc Natl Acad Sci U S A, 2013-09-24) Tadross, Michael R; Tsien, Richard W; Yue, David TLocal Ca(2+) signals through voltage-gated Ca(2+) channels (CaVs) drive synaptic transmission, neural plasticity, and cardiac contraction. Despite the importance of these events, the fundamental relationship between flux through a single CaV channel and the Ca(2+) signaling concentration within nanometers of its pore has resisted empirical determination, owing to limitations in the spatial resolution and specificity of fluorescence-based Ca(2+) measurements. Here, we exploited Ca(2+)-dependent inactivation of CaV channels as a nanometer-range Ca(2+) indicator specific to active channels. We observed an unexpected and dramatic boost in nanodomain Ca(2+) amplitude, ten-fold higher than predicted on theoretical grounds. Our results uncover a striking feature of CaV nanodomains, as diffusion-restricted environments that amplify small Ca(2+) fluxes into enormous local Ca(2+) concentrations. This Ca(2+) tuning by the physical composition of the nanodomain may represent an energy-efficient means of local amplification that maximizes information signaling capacity, while minimizing global Ca(2+) load.Item Open Access Electrogenetics: Genetically Encoded Electrophysiology(2022) Weaver, IsaacThere are about 86 billion neurons in the human brain, connected by trillions of synapses. Deciphering the electrical signaling between neurons is key to understanding the brain in both health and disease. As understanding begins with observation, the past decade has brought significant investment in scalable, stable neural recording technologies. An ideal recording platform would have the ability to record from thousands of neurons simultaneously with millisecond temporal precision and knowledge of the genetic identity of each cell—all while being low-cost, scalable, and amenable to simple data storage. Moreover, deciphering how disease progression remodels neural ensembles requires recordings with months-long stability. To date, no recording technology offers these features in combination. Here, we present an approach that aims to address these limitations. We conceived genetically encoded electrophysiology, in which we establish a covalent link between genetically tagged neurons and surface modified electrodes via novel engineered conductive polymers. The approach retains millisecond temporal resolution native to electrophysiology while combining genetic specificity of tagged neurons. This method utilizes a covalent reaction between the HaloTag protein and its chemical ligand, which was successfully used by the Tadross lab for cell-type specific delivery of drugs. We detail the development of each component of genetically encoded electrophysiology, beginning with engineered conductive polymers and incorporation of HaloTag binding ligand. Through different assays, we verify both successful polymerization onto electrodes and HaloTag ligand availability for HaloTag protein binding. We further demonstrate the concept in cultured neurons using custom microelectrode arrays and development platform. We provide proof-of-concept data to support our approach and demonstrate its feasibility. We discuss the implications of future work which could build on the proof-of-concept technology to refine the approach, optimize it for use in culture and adapt it to in vivo—animal—recordings.
Item Embargo GRIP Display: A One-Pot Library Display Platform for the Directed Evolution of Proteins(2023) Goldenshtein, VictoriaLibrary display technologies have enabled the development of peptides with affinity for a given substrate. Such affinity-capture reagents have driven progress in many fields, from basic biochemistry to neuropharmacology. A major limitation in the development of neuro-pharmaceuticals has been an inability to examine how the behavioral effects of drugs are mediated by each of the distinct yet intermingled cell types in any given brain region. DART (Drugs Acutely Restricted by Tethering) is the first method to overcome this technical barrier, enabling the delivery of therapeutics to a precise genetically defined neuronal cell type. At the core of DART’s specificity is a capture of a chemical Rx-HTL (HaloTag Ligand conjugated to a drug) by a genetically encoded HTP (HaloTag protein), creating an artificial dosing window. Although the technology has already revealed novel neurobiological insights, a narrow dosing window currently limits DART to neurobiological questions where dose can be tightly controlled, such as via intracranial infusion over a small brain volume. Our goal is to adapt the principles of directed evolution and library display to improve the dosing window of DART and enable its brain-wide delivery. Moreover, using the same principles, we aim to develop an orthogonal DART pair for multiplexed delivery of any combination of drugs to two distinct cell types. The underlying principle of a library display tool is a physical linkage between phenotype (a protein) and genotype (its corresponding nucleotide sequence). This conjugated mRNA, encoding the displayed protein, serves as a unique identifier for each variant. Over the past three decades, several display systems have been developed, each with a unique set of limitations. Typically, there is a tradeoff between the stability of this linkage and the number of unique variants (library size). Thus, no existing platform offers the desired trifecta of linkage stability, library size, and product yield. This work introduces a novel in vitro protein display technology called GRIP Display (Gluing RNA to Its Protein) that permits the generation and simultaneous screening of vast protein libraries (~10^14 variants) against a target of interest, with minimal genetic cross-talk, significant selection enrichment, and one-step simple experimental protocol. Here, we demonstrate 1) the development of GRIP Display and its utility in the optimization of a large binding tunnel of HTP to enhance the covalent capture of its chemical ligand; 2) the development of high-affinity orthogonal HTP/HTL pairs with minimal cross-reactivity; 3) a rational design of a novel peptide/RNA interaction to promote the avidity of binding and create a “single read” display technology GRIP.2. GRIP Display represents a valuable resource for the protein engineering community, and can substantially advance the range of neurobiological questions amenable to DART.
Item Open Access Mechanism of local and global Ca2+ sensing by calmodulin in complex with a Ca2+ channel.(Cell, 2008-06-27) Tadross, Michael R; Dick, Ivy E; Yue, David TCalmodulin (CaM) in complex with Ca(2+) channels constitutes a prototype for Ca(2+) sensors that are intimately colocalized with Ca(2+) sources. The C-lobe of CaM senses local, large Ca(2+) oscillations due to Ca(2+) influx from the host channel, and the N-lobe senses global, albeit diminutive Ca(2+) changes arising from distant sources. Though biologically essential, the mechanism underlying global Ca(2+) sensing has remained unknown. Here, we advance a theory of how global selectivity arises, and we experimentally validate this proposal with methodologies enabling millisecond control of Ca(2+) oscillations seen by the CaM/channel complex. We find that global selectivity arises from rapid Ca(2+) release from CaM combined with greater affinity of the channel for Ca(2+)-free versus Ca(2+)-bound CaM. The emergence of complex decoding properties from the juxtaposition of common elements, and the techniques developed herein, promise generalization to numerous molecules residing near Ca(2+) sources.Item Open Access Molecular endpoints of Ca2+/calmodulin- and voltage-dependent inactivation of Ca(v)1.3 channels.(J Gen Physiol, 2010-03) Tadross, Michael R; Ben Johny, Manu; Yue, David TCa(2+)/calmodulin- and voltage-dependent inactivation (CDI and VDI) comprise vital prototypes of Ca(2+) channel modulation, rich with biological consequences. Although the events initiating CDI and VDI are known, their downstream mechanisms have eluded consensus. Competing proposals include hinged-lid occlusion of channels, selectivity filter collapse, and allosteric inhibition of the activation gate. Here, novel theory predicts that perturbations of channel activation should alter inactivation in distinctive ways, depending on which hypothesis holds true. Thus, we systematically mutate the activation gate, formed by all S6 segments within Ca(V)1.3. These channels feature robust baseline CDI, and the resulting mutant library exhibits significant diversity of activation, CDI, and VDI. For CDI, a clear and previously unreported pattern emerges: activation-enhancing mutations proportionately weaken inactivation. This outcome substantiates an allosteric CDI mechanism. For VDI, the data implicate a "hinged lid-shield" mechanism, similar to a hinged-lid process, with a previously unrecognized feature. Namely, we detect a "shield" in Ca(V)1.3 channels that is specialized to repel lid closure. These findings reveal long-sought downstream mechanisms of inactivation and may furnish a framework for the understanding of Ca(2+) channelopathies involving S6 mutations.Item Open Access Sequential ionic and conformational signaling by calcium channels drives neuronal gene expression.(Science, 2016-02-19) Li, Boxing; Tadross, Michael R; Tsien, Richard WVoltage-gated CaV1.2 channels (L-type calcium channel α1C subunits) are critical mediators of transcription-dependent neural plasticity. Whether these channels signal via the influx of calcium ion (Ca(2+)), voltage-dependent conformational change (VΔC), or a combination of the two has thus far been equivocal. We fused CaV1.2 to a ligand-gated Ca(2+)-permeable channel, enabling independent control of localized Ca(2+) and VΔC signals. This revealed an unexpected dual requirement: Ca(2+) must first mobilize actin-bound Ca(2+)/calmodulin-dependent protein kinase II, freeing it for subsequent VΔC-mediated accumulation. Neither signal alone sufficed to activate transcription. Signal order was crucial: Efficiency peaked when Ca(2+) preceded VΔC by 10 to 20 seconds. CaV1.2 VΔC synergistically augmented signaling by N-methyl-d-aspartate receptors. Furthermore, VΔC mistuning correlated with autistic symptoms in Timothy syndrome. Thus, nonionic VΔC signaling is vital to the function of CaV1.2 in synaptic and neuropsychiatric processes.Item Embargo Synaptic Control of Dopamine as a Driver of Reward Learning(2023) Burwell, Sasha Carmelle VeraVentral tegmental area dopamine (VTADA) neurons fire in a manner consistent with Reward Prediction Error, with better-than-expected and worse-than-expected outcomes correlating with bursts and pauses, respectively. Burst and pause firing dynamics are believed to be responsible for driving associative learning, yet interrogating this causality, and understanding how these firing patterns are synaptically created within endogenous neural circuits, has been technically difficult. Utilizing a novel tool, DART (drug acutely restricted by tethering), paired with a multiplexed cue-reward associative learning task and in vivo neural recordings, I explore which classes of endogenous synaptic inputs to VTADA neurons create their canonical firing dynamics, and their role in the associated reward learning behaviors. My key finding is that antagonizing GABAA receptors on VTADA neurons decreases the pauses in firing these cells exhibit, but also accelerates extinction learning in response to unexpected reward omission. In the same mice, the manipulation had no impact on conditioning to a novel cue-reward pairing, indicating that positive-valence learning was unperturbed. This dissertation work provides critical insight into the neural circuitry underlying adaptive behaviors by creating a new framework for understanding conditioning and extinction as anti-correlated behaviors, and by establishing a novel role for direct inhibitory GABAA signaling to VTADA cells in conditioned conviction.
Item Open Access Systematic mapping of the state dependence of voltage- and Ca2+-dependent inactivation using simple open-channel measurements.(J Gen Physiol, 2010-03) Tadross, Michael R; Yue, David TThe state from which channel inactivation occurs is both biologically and mechanistically critical. For example, preferential closed-state inactivation is potentiated in certain Ca(2+) channel splice variants, yielding an enhancement of inactivation during action potential trains, which has important consequences for short-term synaptic plasticity. Mechanistically, the structural substrates of inactivation are now being resolved, yielding a growing library of molecular snapshots, ripe for functional interpretation. For these reasons, there is an increasing need for experimentally direct and systematic means of determining the states from which inactivation proceeds. Although many approaches have been devised, most rely upon numerical models that require detailed knowledge of channel-state topology and gating parameters. Moreover, prior strategies have only addressed voltage-dependent forms of inactivation (VDI), and have not been readily applicable to Ca(2+)-dependent inactivation (CDI), a vital form of regulation in numerous contexts. Here, we devise a simple yet systematic approach, applicable to both VDI and CDI, for semiquantitative mapping of the states from which inactivation occurs, based only on open-channel measurements. The method is relatively insensitive to the specifics of channel gating and does not require detailed knowledge of state topology or gating parameters. Rather than numerical models, we derive analytic equations that permit determination of the states from which inactivation occurs, based on direct manipulation of data. We apply this methodology to both VDI and CDI of Ca(V)1.3 Ca(2+) channels. VDI is found to proceed almost exclusively from the open state. CDI proceeds equally from the open and nearby closed states, but is disfavored from deep closed states distant from the open conformation. In all, these outcomes substantiate and enrich conclusions of our companion paper in this issue (Tadross et al. 2010. J. Gen. Physiol. doi:10.1085/jgp.200910308) that deduces endpoint mechanisms of VDI and CDI in Ca(V)1.3. More broadly, the methods introduced herein can be readily generalized for the analysis of other channel types.Item Embargo The Cholinergic Interneurons in the Nucleus Accumbens Mediate the Rewarding Properties of Opioid Drugs(2024) Yousefzadeh, S. AryanaThe majority of drugs of abuse exert their reinforcing properties through dopamine. However, opioid drugs continue to be rewarding in dopamine-depleted mice, suggesting that there is a dopamine-independent pathway underlying the reinforcing properties of opioids. The circuit mechanism of this dopamine-independent pathway remains unknown. Here, we isolated the behavioral contributions made by opioid receptors on cholinergic interneurons in the nucleus accumbens. To avoid the compensatory confounds of genetic manipulations, we used Drug Acutely Restricted by Tethering (DART) to selectively deliver the opioid receptor antagonist, naloxone, to the cholinergic interneurons in the nucleus accumbens. Drug Acutely Restricted by Tethering (DART) operates by genetically programming a designated group of cells to capture a specific drug, thereby concentrating the drug’s action on the chosen subset of cells while having minimal effects on neighboring non-target cells. Here, we developed an opioid-DART toolset and used it to examine the role of cholinergic interneurons in various behavioral assays. We find that antagonism of opioid receptors on cholinergic interneurons impedes opioid-induced positive reinforcement while having no role in opioid-induced analgesia, identifying cholinergic interneurons in the nucleus accumbens as key nodes driving the rewarding properties of opioid drugs. Our findings challenge the dopamine-centric model and suggest new strategies for addressing opioid use disorder by targeting the cholinergic pathway.