ion channels

Structural and functional characterization of autoimmune antibodies against NMDAR

Project Summary. The goal of this project is to understand the origins and molecular mechanisms underlying the anti-cancer autoimmune response against the N-methyl-D-aspartate receptor (NMDAR) and its correlation with anti-N-methyl-D-aspartate receptor autoimmune encephalitis (NMDARAE). While anti-cancer immune responses can promote tumor elimination, they may also lead to the production of self-reactive antibodies that trigger autoimmune diseases. NMDARAE is the most common form of immune-mediated encephalitis, which results in prominent neuropsychiatric symptoms, including seizures, psychosis, and memory deficits. NMDARs belong to a family of ligand-gated ion channels expressed exclusively in the central nervous system. They are involved in various aspects of brain development and function, including learning and memory. They respond to the neurotransmitter glutamate and a co-agonist, glycine or D-serine, to mediate excitatory neurotransmission, which plays a central role in synaptic plasticity. NMDARAE is associated with ovarian teratomas, where aberrant NMDAR expression is believed to trigger an autoimmune response. In NMDARAE, anti-NMDAR antibodies, as well as B cells and antibody-secreting cells, cross the blood-brain barrier via unknown mechanisms, resulting in the presence of anti-NMDAR antibodies at high titers within the brain and cerebrospinal fluid (CSF). These antibodies target NMDARs, modulating their function and contributing to disease pathology. Emerging evidence, supported by our preliminary data, suggests that NMDARs are also expressed in triple-negative breast cancer (TNBC), extending the relevance of anti-NMDAR autoimmunity beyond ovarian teratomas. In our TNBC mouse model, which ectopically expresses NMDARs (TNBC-NMDAR), we observed the onset of anti-NMDAR autoimmunity, where the produced antibodies cause both anti-tumor activity and symptoms such as lowered seizure threshold, mirroring key features of NMDARAE. Here, we will establish this TNBC mouse model as we develop molecular methods to characterize it. Aim 1 will focus on establishing and characterizing the TNBC- NMDAR mouse model. We will develop a detection method utilizing the intact tetrameric NMDAR channel proteins and a method to isolate B cells expressing B cell receptors against NMDAR from biological samples by using fluorescently labeled intact NMDAR proteins, followed by single-cell RNA sequencing. Aim 2 will utilize single-particle cryo-electron microscopy (cryo-EM) to investigate the interactions between NMDAR and the cloned antibodies, providing insights into epitope recognition, NMDAR subtype specificity, and conformational changes induced by antibody binding. Aim 3 will assess the impact of the cloned antibodies on NMDAR channel activity using electrophysiology. We will also assess anti-tumor activity and NMDARAE onset by each antibody clone. Together, the proposed research will gain insights into the link between anti-cancer anti-NMDAR autoimmunity and NMDARAE. It will also elucidate which functional properties of the cloned antibodies promote anti-tumor activity while contributing to NMDARAE, thereby informing potential therapeutic strategies.

BKCa Channel Contributions to Cerebellar Regulated TSC-Associated Neuropsychiatric Disorders

Project Summary TSC is associated with neurodevelopmental disability including cognitive disability and autism spectrum disorders (ASD) that make up part of TSC associated neuropsychiatric disorders (TAND). The mechanisms for TAND remain poorly understood but studies have increasingly implicated cerebellar dysfunction in the pathogenesis of cognitive and behavioral deficits in both TSC and other neurodevelopmental disorders. A shared feature is cerebellar Purkinje cell (PC) dysfunction. Changes in intrinsic properties of PCs results in both motor and cognitive/ behavioral changes in disease models and in individuals afflicted by these disorders. Mechanistic underpinnings of these altered properties remain unknown, but a significant emerging body of data implicate ion channel dysfunction as the primary etiology of these deficits. The current proposal seeks to delineate the ion channel contribution to PC dysfunction and to TAND-relevant behaviors. In doing so, these studies will produce significant both short- and long-term impact. Short-term: These proposed studies will provide a mechanistic understanding of the contribution of ion channels to the neuronal dysfunction in the cerebellum that has been demonstrated to be causally linked to abnormal TAND-relevant behaviors. In addition, we will target specific ion channels both genetically and pharmacologically to evaluate the benefits of ion channel restoration on both electrophysiological abnormalities but also the TAND-relevant behaviors observed in the model. Long-term: These studies, thus, provide a framework for subsequent clinically-relevant therapeutic development for TAND. First, these studies will uncover the ability for TAND-relevant behaviors to be improved upon targeting ion channel alterations in TSC. These studies will also define molecular targets on which therapeutic development can be targeted, thereby potentially providing a molecular-informed pipeline for therapeutic development. In addition, these studies will utilize clinically-available, FDA-approved pharmacological agents to target ion channel function and investigate the potential therapeutic benefits for these agents for TAND-relevant behaviors. Thus, these studies will address a core gap in knowledge to achieve a better mechanistic understanding of TAND and to develop therapeutic opportunities to address TAND. These studies will not only reveal previously understudied and novel mechanistic underpinnings for these behaviors but will provide pre-clinical insights into the therapeutic utility of clinically-utilized agents for the treatment of TAND-related behaviors, thus potentially providing both immediate and long-term opportunities for the treatment of TAND. Moreover, although these studies focus on TSC, these mechanisms may prove generalizable beyond TSC and provide a shared basis and therapeutic opportunity for other neuropsychiatric/developmental conditions.

Molecular strategies for resolving differential regulation of dopamine subpopulations

Project Summary/Abstract Dopamine neurons in the ventral tegmental area (VTA) fire action potentials in complex patterns of tonic and phasic activity in response to environmental stimuli and during behavioral tasks. Transcriptomic, anatomical, and functional studies have established that VTA dopamine neurons can be divided into multiple subpopulations with variable gene expression, projection patterns, and response profiles. We recently completed a transcriptomic study that identified genetic markers for three distinct subpopulations of VTA dopamine neurons, and also found evidence for variability in ion channel gene expression between populations that correlated with differences in activity-dependent gene expression. However, much remains unknown regarding how specific genes encoding ion channels, receptors, transcription factors, or other signaling components contribute to the variability in baseline physiological properties observed across the VTA. Here we propose to combine slice electrophysiology recordings of VTA dopamine neurons with post-hoc single-cell sequencing analysis (i.e. patch-seq), which will allow us to directly correlate gene expression and physiological properties in order to identify candidate genes that may be key drivers of the variability between subpopulations. We also propose to validate and utilize a novel dual-recombinase CRISPR/Cas9 system for targeted gene mutagenesis in intersectional neuronal populations, which will provide a mechanism for testing gene function with unprecedented precision. We will use this approach to test the function of two candidate ion channel genes, the potassium channels Kcnh5 and Kcnh7, previously identified in our transcriptomic study as potential contributors to dopamine neuron action potential firing properties. We hypothesize that these genes are important for enabling rapid action potential firing in highly excitable dopamine neurons found in specific subpopulations. As a whole, with this proposal we aim to generate a valuable dataset linking gene expression in VTA dopamine neurons with physiology and subpopulation identification, as well as develop an intersectional gene mutagenesis strategy that can be used throughout the brain to precisely target neuronal subpopulations to test gene function. With this approach, we hope to facilitate future precision targeting of the dopamine system and dopamine-dependent behaviors.

New mechanically-gated ion channels and tethers for touch

Can a single neuron solve MNIST? Neural computation of machine learning tasks emerges from the interaction of dendritic properties

Physiological experiments have highlighted how the dendrites of biological neurons can nonlinearly process distributed synaptic inputs. However, it is unclear how qualitative aspects of a dendritic tree, such as its branched morphology, its repetition of presynaptic inputs, voltage-gated ion channels, electrical properties and complex synapses, determine neural computation beyond this apparent nonlinearity. While it has been speculated that the dendritic tree of a neuron can be seen as a multi-layer neural network and it has been shown that such an architecture could be computationally strong, we do not know if that computational strength is preserved under these qualitative biological constraints. Here we simulate multi-layer neural network models of dendritic computation with and without these constraints. We find that dendritic model performance on interesting machine learning tasks is not hurt by most of these constraints and may synergistically benefit from all of them combined. Our results suggest that single real dendritic trees may be able to learn a surprisingly broad range of tasks through the emergent capabilities afforded by their properties.

Investigating activity-dependent processes in cerebral cortex development and disease

The cerebral cortex contains an extraordinary diversity of excitatory projection neuron (PN) and inhibitory interneurons (IN), wired together to form complex circuits. Spatiotemporally coordinated execution of intrinsic molecular programs by PNs and INs and activity-dependent processes, contribute to cortical development and cortical microcircuits formation. Alterations of these delicate processes have often been associated to neurological/neurodevelopmental disorders. However, despite the groundbreaking discovery that spontaneous activity in the embryonic brain can shape regional identities of distinct cortical territories, it is still unclear whether this early activity contributes to define subtype-specific neuronal fate as well as circuit assembly. In this study, we combined in utero genetic perturbations via CRISPR/Cas9 system and pharmacological inhibition of selected ion channels with RNA-sequencing and live imaging technologies to identify the activity-regulated processes controlling the development of different cortical PN classes, their wiring and the acquisition of subtype specific features. Moreover, we generated human induced pluripotent stem cells (iPSCs) form patients affected by a severe, rare and untreatable form of developmental epileptic encephalopathy. By differentiating cortical organoids form patient-derived iPSCs we create human models of early electrical alterations for studying molecular, structural and functional consequences of the genetic mutations during cortical development. Our ultimate goal is to define the activity-conditioned processes that physiologically occur during the development of cortical circuits, to identify novel therapeutical paths to address the pathological consequences of neonatal epilepsies.

Modulation of oligodendrocyte development and myelination by voltage-gated Ca++ channels

The oligodendrocyte generates CNS myelin, which is essential for normal nervous system function. Thus, investigating the regulatory and signaling mechanisms that control its differentiation and the production of myelin is relevant to our understanding of brain development and of adult pathologies such as multiple sclerosis. We have recently established that the activity of voltage-gated Ca++ channels is crucial for the adequate migration, proliferation and maturation of oligodendrocyte progenitor cells (OPCs). Furthermore, we have found that voltage-gated Ca++ channels that function in synaptic communication between neurons also mediate synaptic signaling between neurons and OPCs. Thus, we hypothesize that voltage-gated Ca++ channels are central components of OPC-neuronal synapses and are the principal ion channels mediating activity-dependent myelination.

The GluN2A Subunit of the NMDA Receptor and Parvalbumin Interneurons: A Possible Role in Interneuron Development

N-methyl-D-aspartate receptors (NMDARs) are excitatory glutamate-gated ion channels that are expressed throughout the central nervous system. NMDARs mediate calcium entry into cells, and are involved in a host of neurological functions. The GluN2A subunit, encoded by the GRIN2A gene, is expressed by both excitatory and inhibitory neurons, with well described roles in pyramidal cells. By using Grin2a knockout mice, we show that the loss of GluN2A signaling impacts parvalbumin-positive (PV) GABAergic interneuron function in hippocampus. Grin2a knockout mice have 33% more PV cells in CA1 compared to wild type but similar cholecystokinin-positive cell density. Immunohistochemistry and electrophysiological recordings show that excess PV cells do eventually incorporate into the hippocampal network and participate in phasic inhibition. Although the morphology of Grin2a knockout PV cells is unaffected, excitability and action-potential firing properties show age-dependent alterations. Preadolescent (P20-25) PV cells have an increased input resistance, longer membrane time constant, longer action-potential half-width, a lower current threshold for depolarization-induced block of action-potential firing, and a decrease in peak action-potential firing rate. Each of these measures are corrected in adulthood, reaching wild type levels, suggesting a potential delay of electrophysiological maturation. The circuit and behavioral implications of this age-dependent PV interneuron malfunction are unknown. However, neonatal Grin2a knockout mice are more susceptible to lipopolysaccharide and febrile-induced seizures, consistent with a critical role for early GluN2A signaling in development and maintenance of excitatory-inhibitory balance. These results could provide insights into how loss-of-function GRIN2A human variants generate an epileptic phenotypes.

NMC4 Short Talk: Resilience through diversity: Loss of neuronal heterogeneity in epileptogenic human tissue impairs network resilience to sudden changes in synchrony

A myriad of pathological changes associated with epilepsy, including the loss of specific cell types, improper expression of individual ion channels, and synaptic sprouting, can be recast as decreases in cell and circuit heterogeneity. In recent experimental work, we demonstrated that biophysical diversity is a key characteristic of human cortical pyramidal cells, and past theoretical work has shown that neuronal heterogeneity improves a neural circuit’s ability to encode information. Viewed alongside the fact that seizure is an information-poor brain state, these findings motivate the hypothesis that epileptogenesis can be recontextualized as a process where reduction in cellular heterogeneity renders neural circuits less resilient to seizure onset. By comparing whole-cell patch clamp recordings from layer 5 (L5) human cortical pyramidal neurons from epileptogenic and non-epileptogenic tissue, we present the first direct experimental evidence that a significant reduction in neural heterogeneity accompanies epilepsy. We directly implement experimentally-obtained heterogeneity levels in cortical excitatory-inhibitory (E-I) stochastic spiking network models. Low heterogeneity networks display unique dynamics typified by a sudden transition into a hyper-active and synchronous state paralleling ictogenesis. Mean-field analysis reveals a distinct mathematical structure in these networks distinguished by multi-stability. Furthermore, the mathematically characterized linearizing effect of heterogeneity on input-output response functions explains the counter-intuitive experimentally observed reduction in single-cell excitability in epileptogenic neurons. This joint experimental, computational, and mathematical study showcases that decreased neuronal heterogeneity exists in epileptogenic human cortical tissue, that this difference yields dynamical changes in neural networks paralleling ictogenesis, and that there is a fundamental explanation for these dynamics based in mathematically characterized effects of heterogeneity. These interdisciplinary results provide convincing evidence that biophysical diversity imbues neural circuits with resilience to seizure and a new lens through which to view epilepsy, the most common serious neurological disorder in the world, that could reveal new targets for clinical treatment.

Activity dependent myelination: a mechanism for learning and regeneration?

The CNS is responsive to an ever-changing environment. Until recently, studies of neural plasticity focused almost exclusively on functional and structural changes of neuronal synapses. In recent years, myelin plasticity has emerged as a potential modulator of neural networks. Myelination of previously unmyelinated axons, and changes in the structure on already-myelinated axons, can have large effects on network function. The heterogeneity of the extent of how axons in the CNS are myelinated offers diverse scope for dynamic myelin changes to fine-tune neural circuits. The traditionally held view of myelin as a passive insulator of axons is now changing to one of lifelong changes in myelin, modulated by neuronal activity and experience. Myelin, produced by oligodendrocytes (OLs), is essential for normal brain function, as it provides fast signal transmission, promotes synchronization of neuronal signals and helps to maintain neuronal function. OLs differentiate from oligodendrocyte precursor cells (OPCs), which are distributed throughout the adult brain, and myelination continues into late adulthood. OPCs can sense neuronal activity as they receive synaptic inputs from neurons and express voltage-gated ion channels and neurotransmitter receptors, and differentiate into myelinating OLs in response to changes in neuronal activity. This lecture will explore to what extent myelin plasticity occurs in adult animals, whether myelin changes occur in non-motor learning tasks, especially in learning and memory, and questions whether myelin plasticity and myelin regeneration are two sides of the same coin.

Synchrony and Synaptic Signaling in Cerebellar Circuits

The cerebellum permits a wide range of behaviors that involve sensorimotor integration. We have been investigating how specific cellular and synaptic specializations of cerebellar neurons measured in vitro, give rise to circuit activity in vivo. We have investigated these issues by studying Purkinje neurons as well as the large neurons of the mouse cerebellar nuclei, which form the major excitatory premotor projection from the cerebellum. Large CbN cells have ion channels that favor spontaneous action potential firing and GABAA receptors that generate ultra-fast inhibitory synaptic currents, raising the possibility that these biophysical attributes may permit CbN cells to respond differently to the degree of temporal coherence of their Purkinje cell inputs. In vivo, self-initiated motor programs associated with whisking correlates with asynchronous changes in Purkinje cell simple spiking that are asynchronous across the population. The resulting inhibition converges with mossy fiber excitation to yield little change in CbN cell firing, such that cerebellar output is low or cancelled. In contrast, externally applied sensory stimuli elicits a transient, synchronous inhibition of Purkinje cell simple spiking. During the resulting strong disinhibition of CbN cells, sensory-induced excitation from mossy fibers effectively drives cerebellar outputs that increase the magnitude of reflexive whisking. Purkinje cell synchrony, therefore, may be a key variable contributing to the “positive effort” hypothesized by David Marr in 1969 to be necessary for cerebellar control of movement.

From the first spark to catching your breath: A love story in neuroscience

What are the things that draw us to a particular field of science and what is it that keeps us there? For Dr. Bahia, there was a particular attraction to sensory nerves; the monitors of the worlds inside and outside of our bodies. In this talk, Dr. Bahia will outline her career path as a neuroscientist resulting in the title of Research Associate. She will also talk about the longest project she has participated in, 'exploring the role of ion channels in sensory nerves' (rupress.org/jgp/article/147/6/451/43495/The-exceptionally-high-reactivity-of-Cys-621-is)

Under Pressure: the role of PIEZO ion channels in interoception

PIEZO ion channels detect force in cellular membranes. They are expressed in a wide variety of mammalian tissues, including the vasculature, lymphatic system, and the nervous system. We have found that PIEZO2 in sensory neurons is required for the mechanical senses of touch and proprioception, but our understanding of internal organ sensing, interoception, is far behind. I will describe our findings on the role of PIEZO ion channels in the lesser-known interoceptive senses in multiple organ systems.

The contribution of different information channels to different facets of empathy

Receptor Costs Determine Retinal Design

Our group is interested in discovering design principles that govern the structure and function of neurons and neural circuits. We record from well-defined neurons, mainly in flies’ visual systems, to measure the molecular and cellular factors that determine relevant measures of performance, such as representational capacity, dynamic range and accuracy. We combine this empirical approach with modelling to see how the basic elements of neural systems (ion channels, second messengers systems, membranes, synapses, neurons, circuits and codes) combine to determine performance. We are investigating four general problems. How are circuits designed to integrate information efficiently? How do sensory adaptation and synaptic plasticity contribute to efficiency? How do the sizes of neurons and networks relate to energy consumption and representational capacity? To what extent have energy costs shaped neurons, sense organs and brain regions during evolution?

Differential Resilience of Neurons and Networks with Similar Behavior to Perturbation

Both computational and experimental results in single neurons and small networks demonstrate that very similar network function can result from quite disparate sets of neuronal and network parameters. Using the crustacean stomatogastric nervous system, we study the influence of these differences in underlying structure on differential resilience of individuals to a variety of environmental perturbations, including changes in temperature, pH, potassium concentration and neuromodulation. We show that neurons with many different kinds of ion channels can smoothly move through different mechanisms in generating their activity patterns, thus extending their dynamic range.

Differential Resilience of Neurons and Networks with Similar Behavior to Perturbation. (Simultaneous translation to Spanish)

Both computational and experimental results in single neurons and small networks demonstrate that very similar network function can result from quite disparate sets of neuronal and network parameters. Using the crustacean stomatogastric nervous system, we study the influence of these differences in underlying structure on differential resilience of individuals to a variety of environmental perturbations, including changes in temperature, pH, potassium concentration and neuromodulation. We show that neurons with many different kinds of ion channels can smoothly move through different mechanisms in generating their activity patterns, thus extending their dynamic range. The talk will be simultaneously translated to spanish by the interpreter Liliana Viera, MSc. Los resultados tanto computacionales como experimentales en neuronas individuales y redes pequeñas demuestran que funcionamientos de redes muy similares pueden pueden resultar de conjuntos bastante dispares de parámetros neuronales y de las redes. Utilizando el sistema nervioso estomatogástrico de los crustáceos, estudiamos la influencia de estas diferencias en la estructura subyacente en la resistencia diferencial de los individuos a una variedad de perturbaciones ambientales, incluidos los cambios de temperatura, pH, concentración de potasio y neuromodulación. Mostramos que neuronas con muchos tipos diferentes de canales iónicos pueden moverse suavemente a través de diferentes mecanismos para generar sus patrones de actividad, extendiendo así su rango dinámico. La conferencia será traducida simultáneamente al español por la intérprete Liliana Viera MSc.

Physiological importance of phase separation: a case study in synapse formation

Synapse formation during neuronal development is critical to establish neural circuits and a nervous system1. Every presynapse builds a core active zone structure where ion channels are clustered and synaptic vesicles are released2. While the composition of active zones is well characterized2,3, how active zone proteins assemble together and recruit synaptic release machinery during development is not clear. Here, we find core active zone scaffold proteins SYD-2/Liprin-α and ELKS-1 phase separate during an early stage of synapse development, and later mature into a solid structure. We directly test the in vivo function of phase separation with mutants specifically lacking this activity. These mutant SYD-2 and ELKS-1 proteins remain enriched at synapses, but are defective in active zone assembly and synapse function. The defects are rescued with the introduction of a phase separation motif from an unrelated protein. In vitro, we reconstitute the SYD-2 and ELKS-1 liquid phase scaffold and find it is competent to bind and incorporate downstream active zone components. The fluidity of SYD-2 and ELKS-1 condensates is critical for efficient mixing and incorporation of active zone components. These data reveal that a developmental liquid phase of scaffold molecules is essential for synaptic active zone assembly before maturation into a stable final structure.

Dynamic causal communication channels between neocortical areas

COSYNE 2022

Calcium/cation-mechanosensing ion channels activity are crucial mediators for mechanical induction of radial glia

The impact of light source properties, neural morphology and the distribution of light-gated ion channels on the effective spatial resolution of optogenetic stimulation

Optical investigation of excitability and ion channels in layer-5 neocortical pyramidal neurons

Voltage-gated Caᵥ2.2 calcium ion channels in Trpv1 nerve endings in skin contribute to the release of proinflammatory mediators

Biophysically detailed cortical neuron models with genetically-defined ion channels

FENS Forum 2024

Blood pressure pulsations modulate olfactory bulb activity via mechanosensitive ion channels

FENS Forum 2024

Channelome: A comprehensive resource for voltage-gated ion channels

FENS Forum 2024

Culturing postnatal mouse neurons of the deep cerebellar nuclei to investigate the functional expression of TRP ion channels in the cerebellum

FENS Forum 2024

Differential distribution of key regulatory ion channels in excitatory synapses of the epileptic human brain revealed by freeze-fracture replica analysis

FENS Forum 2024

Role of Alzheimer's disease in the neurosensory hypoacusia associated with alteration in voltage-gated potassium ion channels

FENS Forum 2024

Statin effects on recombinant and natively expressed thermo-sensitive transient receptor potential ion channels

FENS Forum 2024