Biology subjects

Thomas, C. I.

Publications and source records attributed to Thomas, C. I..

10 recordsLinked to original sources

bioRxiv2026

The functional neuronal lipotype is specified by activity and PS synthesis using dietary and lysosomal inputs

Neurons maintain specialized membranes necessary for lifelong circuit function. While neuronal membranes thus contain unique lipidomes, how and why specific lipids become synaptically enriched remains mysterious. Here, we identify a conserved hallmark of mature neurons, phosphatidylserines containing omega-3 fatty acids (n-3 PS), which dynamically accumulate during synaptogenesis from Drosophila to humans. This neuronal lipotype depends on dietary n-3 and lysosomal catabolism mediated by Saposin, the phospholipase PLA2G15, and the lysophospholipid transporter spin/SPNS1. Reducing neuronal PS biosynthesis by targeting the PS synthase (Pss) caused lipidomic, synaptic, and behavioral deficits exacerbated by impaired glycerophospholipid catabolism. While dietary, lysosomal, and lipoprotein manipulations broadly altered n-3 levels, silencing developmental activity-templating neurons selectively reduced n-3 PS. Remarkably, pathogenic Pss variants lacking feedback inhibition similarly reduced n-3 PS and impaired circuit function. As n-3 PS is reduced during neurodegeneration but absent from iPSC-derived neurons, important relationships between lipotype and physiology may not be recapitulated in vitro.

neuroscience↗

bioRxiv2026

Microglia drive demyelination via multiple sclerosis antibodies and BTK signaling

Microglia are the predominant immune cells in multiple sclerosis (MS) demyelinating lesions, where they phagocytose myelin, but whether they destroy myelin or merely scavenge its debris is unknown. Here, we explore whether pathogenic autoantibodies found in MS may induce the phagocytic destruction of myelin by microglia. Applying patient-derived, myelin-targeting antibodies to the mouse cortex, we developed an in vivo model of MS with focal demyelination that depended on epitope specificity and Fc gamma receptor and complement binding. Longitudinal monitoring of microglia-myelin interactions using in vivo two-photon microscopy revealed rapid microglial envelopment of intact myelin driving myelin loss, while single-cell RNA sequencing identified a demyelination-associated microglial signature. Parallel changes were observed in human MS lesions, where microglia enveloped intact myelin and similar genes were upregulated. Inhibition of Brutons tyrosine kinase (BTK) limited microglial transcriptional changes and prevented myelin loss following microglial envelopment. These findings directly implicate microglia in pathological myelin loss and support BTK inhibition as a therapeutic strategy to prevent demyelination by modulating microglia behavior.

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bioRxiv2023

Astrocyte coverage of excitatory synapses correlates to measures of synapse structure and function in primary visual cortex

Most excitatory synapses in the mammalian brain are contacted by astrocytes, forming the tripartite synapse. This interface is thought to be critical for glutamate turnover and structural or functional dynamics of synapses. While the degree of synaptic contact of astrocytes is known to vary across brain regions and animal species, the implications of this variability remain unknown. Furthermore, precisely how astrocyte coverage of synapses relates to in vivo functional properties of individual dendritic spines has yet to be investigated. Here, we characterized perisynaptic astrocyte processes (PAPs) contacting synapses of pyramidal neurons of the ferret visual cortex and, using correlative light and electron microscopy, examined their relationship to synaptic strength and to sensory-evoked Ca2+ activity. Nearly all synapses were contacted by PAPs, and most were contacted along the axon-spine interface (ASI). Structurally, we found that the degree of PAP coverage scaled with synapse size and complexity. Functionally, we found that PAP coverage scaled with the selectivity of Ca2+ responses of individual synapses to visual stimuli and, at least for the largest synapses, scaled with the reliability of visual stimuli to evoke postsynaptic Ca2+ events. Our study shows astrocyte coverage is highly correlated with structural properties of excitatory synapses in the visual cortex and implicates astrocytes as a contributor to reliable sensory activation.

neuroscience↗

bioRxiv2023

A multi-faceted analysis of synapses reveals the role of neuroligin-1 cleavage in presynaptic vesicle accumulation in the lateral amygdala

Neuroligin-1 (NLGN1) is a cell adhesion molecule found at excitatory glutamatergic synapses in the brain which regulates synaptic function and maturation. Extracellular cleavage of NLGN1 by proteases has been shown to control vesicle release in cultured neurons, but nothing is known about the underlying changes to synapse structure that accompany this, or how synapse function is affected in brain tissue. We found that prevention of NLGN1 cleavage through mutation to the extracellular stalk domain increases synaptic vesicle docking and miniature excitatory post-synaptic current frequency at synapses of the lateral amygdala. Using a novel volume electron microscopy (vEM) analysis pipeline based on deep learning extraction of thousands of synapses and vesicles clouds and subsequent spatial analyses, we found that the total pool of synaptic vesicles shifts closer to the synapse in mutants. Furthermore, we observed an increased frequency of incomplete synapses that lack vesicle accumulation, pointing towards disruption of synaptic pruning and accumulation of putatively non-functioning synapses. Our study provides evidence of a structural and functional role of NLGN1 cleavage in native brain tissue, and establishes a foundation for vEM analysis of synapse-vesicle spatial relationships in other animal models of dysfunction and disease.

neuroscience↗

bioRxiv2022

Presynaptic Rac1 controls synaptic strength through the regulation of synaptic vesicle priming.

Synapses contain a limited number of synaptic vesicles (SVs) that are released in response to action potentials (APs). Therefore, sustaining synaptic transmission over a wide range of AP firing rates and timescales depends on SV release and replenishment. Although actin dynamics impact synaptic transmission, how presynaptic regulators of actin signaling cascades control SV release and replenishment remains unresolved. Rac1, a Rho GTPase, regulates actin signaling cascades that control synaptogenesis, neuronal development, and postsynaptic function. However, the presynaptic role of Rac1 in regulating synaptic transmission is unclear. To unravel Rac1s roles in controlling transmitter release, we performed selective presynaptic ablation of Rac1 at the mature mouse calyx of Held synapse. Loss of Rac1 increased synaptic strength, accelerated EPSC recovery after conditioning stimulus trains, and augmented spontaneous SV release with no change in presynaptic morphology or AZ ultrastructure. Analyses with constrained short-term plasticity models revealed faster SV priming kinetics and, depending on model assumptions, elevated SV release probability or higher abundance of tightly docked fusion-competent SVs in Rac1-deficient synapses. We conclude that presynaptic Rac1 is a key regulator of synaptic transmission and plasticity mainly by regulating the dynamics of SV priming and potentially SV release probability.

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bioRxiv2021

Selective activation of BK channels in small-headed dendritic spines suppresses excitatory postsynaptic potentials

Dendritic spines are the main receptacles of excitatory information in the brain. Their particular morphology, with a small head connected to the dendrite by a slender neck, has inspired theoretical and experimental work to understand how these structural features affect the processing, storage and integration of synaptic inputs in pyramidal neurons (PNs). The activation of glutamate receptors in spines triggers a large voltage change as well as calcium signals at the spine head. Thus, voltage-gated and calcium-activated potassium channels located in the spine head likely play a key role in synaptic transmission. Here we study the presence and function of large conductance calcium-activated potassium (BK) channels in spines from layer 5 PNs. We find that BK channels are localized to dendrites and spines regardless of their size, but their activity can only be detected in spines with small head volumes ([≤] 0.09 {micro}m3), which reduces the amplitude of two-photon (2P) uncaging (u) excitatory postsynaptic potentials (EPSPs) recorded at the soma. In addition, we find that calcium signals in spines with small head volumes are significantly larger than those observed in spines with larger head volumes. In accordance with our experimental data, numerical simulations predict that synaptic inputs impinging onto spines with small head volumes generate voltage responses and calcium signals within the spine head itself that are significantly larger than those observed in spines with bigger head volumes, which are sufficient to activate spine BK channels. These results show that BK channels are selectively activated in small-headed spines, suggesting a new level of dendritic spine-mediated regulation of synaptic processing, integration, and plasticity in cortical PNs.

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bioRxiv2021

Computational modeling predicts acidic microdomains in the glutamatergic synaptic cleft

At chemical synapses, synaptic vesicles release their acidic contents into the cleft leading to the expectation that the cleft should acidify. However, fluorescent pH probes targeted to the cleft of conventional glutamatergic synapses in both fruit flies and mice reveal cleft alkalinization, rather than acidification. Here, using a reaction-diffusion scheme, we modeled pH dynamics at the Drosophila neuromuscular junction (NMJ) as glutamate, adenosine triphosphate (ATP) and protons (H+) are released into the cleft. The model incorporates bicarbonate and phosphate buffering systems as well as plasma membrane calcium-ATPase (PMCA) activity and predicts substantial cleft acidification but only for fractions of a millisecond following neurotransmitter release. Thereafter, the cleft rapidly alkalinizes and remains alkaline for over 100 milliseconds, as the PMCA removes H+ from the cleft in exchange for calcium ions (Ca2+) from adjacent pre- and post-synaptic compartments; thus recapitulating the empirical data. The extent of synaptic vesicle loading and time course of exocytosis has little influence on the magnitude of acidification. Phosphate, but not bicarbonate buffering is effective at ameliorating the magnitude and time course of the acid spike, while both buffering systems are effective at ameliorating cleft alkalinization. The small volume of the cleft levies a powerful influence on the magnitude of alkalinization and its time course. Structural features that open the cleft to adjacent spaces appear to be essential for alleviating the extent of pH disturbances accompanying neurotransmission. SIGNIFICANCE STATEMENTAcid-base imbalances have surprisingly potent neurological effects highlighting the acute pH sensitivity of many neural mechanisms. Acid-Sensing Ion-Channels (ASICs), which open in response to acid shifts in extracellular pH, are an example of such a mechanism. However, while ASICs open during neurotransmission at conventional glutamatergic synapses, pH-sensitive electrodes and fluorophores show no signs of acidification at these synapses, only alkalinization. To resolve this paradox, we built a computational model which allows a glimpse beyond the experimental limitations of pH-sensitive electrodes and fluorophores. Our model reveals a highly dynamic pH landscape within the synaptic cleft, harboring deep but exceedingly rapid acid transients that give way to a prolonged period of alkalinization, thus reconciling ASIC activation with direct measurements of extracellular pH.

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bioRxiv2021

A binocular synaptic network supports interocular response alignment in visual cortical neurons

In the visual system, signals from the two eyes are combined to form a coherent representation through the convergence of synaptic input populations onto individual cortical neurons. As individual synapses originate from either monocular (representing one eye) or binocular (representing both eyes) cortical networks, it has been unclear how these inputs are integrated coherently. Here, we imaged dendritic spines on layer 2/3 binocular cells in ferret visual cortex with in vivo two-photon microscopy to examine how monocular and binocular synaptic networks contribute to the interocular alignment of orientation tuning. We found that binocular synapses varied in degree of congruency, namely response correlation between left and right eye visual stimulation. Binocular congruent inputs were functionally distinct from binocular noncongruent and monocular inputs, exhibiting greater tuning selectivity and connection specificity. Using correlative light and electron microscopy, we found no difference in ultrastructural anatomy and instead, observed strength in numbers using a simple model simulating aggregate synaptic input. This model demonstrated a predominate contribution of binocular congruent inputs in sculpting somatic orientation preference and interocular response alignment. Our study suggests that, in layer 2/3 cortical neurons, a binocular network is responsible for forming a coherent representation in individual neurons through recurrent intracortical interactions.

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bioRxiv2019

Presynaptic development is controlled by the core active zone proteins CAST/ELKS

Many presynaptic active zone proteins have multiple regulatory roles that vary during distinct stages of neuronal circuit development. However, our understanding how presynaptic active zone proteins regulate synapse development during neuronal circuit maturation is in its early stages. Although CAST/ELKS are presynaptic active zone core proteins, their roles in synapse development in the mammalian central nervous system remain enigmatic. To unravel CAST/ELKS roles in glutamatergic synapse development, we analyzed how their loss during the early stages of circuit maturation impacted the calyx of Held presynaptic terminal development and function. We found a reduction in presynaptic surface area and volume, but an increase in active zone size. Additionally, we found a reduction in all presynaptic Cav2 channel subtype currents. Surprisingly, these changes did not impair synaptic transmission. We propose that CAST/ELKS are involved in pathways regulating presynaptic morphological properties and Cav2 channel subtype levels during early stages of neuronal circuit maturation.

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bioRxiv2019

Presynaptic mitochondria volumes and complexity of subsynaptic distribution increase during development at a high-fidelity synapse

The calyx of Held, a large glutamatergic presynaptic terminal in the auditory brainstem undergoes developmental changes to support the high action-potential firing rates required for auditory information encoding. In addition, calyx terminals are morphologically diverse which impacts vesicle release properties and synaptic plasticity. Mitochondria influence synaptic plasticity through calcium buffering and are crucial for providing the energy required for synaptic transmission. Therefore, it has been postulated that mitochondrial levels increase during development and contribute to the morphological-functional diversity in the mature calyx. However, the developmental profile of mitochondrial volumes and subsynaptic distribution at the calyx of Held remains unclear. To provide insight on this, we developed a helper-dependent adenoviral vector (HdAd) that expresses the genetically encoded peroxidase marker for mitochondria, mito-APEX2, at the mouse calyx of Held. We developed protocols to detect labeled mitochondria for use with serial block face scanning electron microscopy to carry out semi-automated segmentation of mitochondria, high-throughput whole terminal reconstruction and presynaptic ultrastructure in mice of either sex. Subsequently, we measured mitochondrial volumes and subsynaptic distributions at the immature postnatal day 7 (P7) and the mature (P21) calyx. We found an increase of mitochondria volumes in terminals and axons from P7 to P21 but did not observe differences between stalk and swelling subcompartments in the mature calyx. Based on these findings, we propose that mitochondrial volumes and synaptic localization developmentally increase to support high firing rates required in the initial stages of auditory information processing.\n\nSignificance StatementElucidating the developmental processes of auditory brainstem presynaptic terminals is critical to understanding auditory information encoding. Additionally, morphological-functional diversity at these terminals is proposed to enhance coding capacity. Mitochondria provide energy for synaptic transmission and can buffer calcium, impacting synaptic plasticity; however, their developmental profile to ultimately support the energetic demands of synapses following the onset of hearing remains unknown. Therefore, we created a helper-dependent adenoviral vector with the mitochondria-targeting peroxidase mito-APEX2 and expressed it at the mouse calyx of Held. Volumetric reconstructions of serial block face electron microscopy data of immature and mature labeled calyces reveal that mitochondrial volumes are increased to support high firing rates upon maturity.

neuroscience↗