SynBot: An open-source image analysis software for automated quantification of synapses

Savage, Justin T; Ramirez, J.J.; Risher, W. Christopher; Irala, Dolores; Eroğlu, Çağla

doi:10.1101/2023.06.26.546578

Cited by 8 publications

(6 citation statements)

References 68 publications

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“…A 4x4 tile scan with 4, 0.33 µM step Z-stacks were obtained. Synapse images were analyzed using SynBot 54 . Synapses were identified by the colocalization of pre- (Vglut2) and post- (PSD95) synaptic puncta.…”

Section: Star Methodsmentioning

confidence: 99%

Neuron Derived Cytokine Interleukin-34 Controls Developmental Microglia Function

Devlin,

Nguyen,

Grullon

et al. 2024

Preprint

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Neuron-microglia interactions dictate the development of neuronal circuits in the brain. However, the factors that support and broadly regulate these processes across developmental stages are largely unknown. Here, we find that IL34, a neuron-derived cytokine, is upregulated in development and plays a critical role in supporting and maintaining neuroprotective, mature microglia in the anterior cingulate cortex (ACC) of mice. We show that IL34 mRNA and protein is upregulated in neurons in the second week of postnatal life and that this increase coincides with increases in microglia number and expression of mature, homeostatic markers, e.g., TMEM119. We also found that IL34 mRNA is higher in more active neurons, and higher in excitatory (compared to inhibitory) neurons. Genetic KO of IL34 prevents the functional maturation of microglia and results in an anxiolytic phenotype in these mice by adulthood. Acute, low dose blocking of IL34 at postnatal day (P)15 in mice decreased microglial TMEM119 expression and increased aberrant microglial phagocytosis of thalamocortical synapses within the ACC. In contrast, viral overexpression of IL34 early in life (P1-P8) caused early maturation of microglia and prevented microglial phagocytosis of thalamocortical synapses during the appropriate neurodevelopmental refinement window. Taken together, these findings establish IL34 as a key regulator of neuron-microglia crosstalk in postnatal brain development, controlling both microglial maturation and synapse engulfment.

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Section: Star Methodsmentioning

confidence: 99%

Neuron Derived Cytokine Interleukin-34 Controls Developmental Microglia Function

Devlin,

Nguyen,

Grullon

et al. 2024

Preprint

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“…Confocal z-stacks of the ACC and DMS were imaged at 60× magnification and 1.64x zoom on an Olympus Fluoview 3000 confocal laser-scanning microscope. Genotype-blind analyses were conducted using an ImageJ macro called SynBot (https://github.com/Eroglu-Lab/Syn_Bot) to count colocalized synaptic puncta [65]. This quantification method accurately estimates the number of synapses by measuring co-localization rather than stained pre- or postsynaptic proteins.…”

Section: Methodsmentioning

confidence: 99%

“…However, whether this deficit persists in adulthood, particularly in the ACC (Fig 1A ), is unknown. To determine whether the lack of TSP1/2 reduces excitatory synapse density in layers 1, 2/3, and 5 of the adult ACC (Fig 1B ), we utilized a quantitative assay to label and count intracortical excitatory synaptic structures [64,65]. To do so, we quantified the close apposition of pre-synaptic marker, VGlut1, and post-synaptic marker, PSD95, in the adult WT and TSP1/2-KO mice (Fig 1C).…”

Section: Loss Of Tsp1/2 Reduces Excitatory Synapse Numbers In the Adu...mentioning

confidence: 99%

Astrocytic thrombospondins 1 and 2 are required for cortical synapse development controlling instrumental performance

Lawal,

Ulloa Severino,

Wang

et al. 2024

Preprint

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During development, controlled synaptogenesis is required to form functioning neural circuits that underlie cognition and behavior. Astrocytes, a major glial-cell type in the central nervous system (CNS), promote synapse formation by secreting synaptogenic proteins. Thrombospondins 1 and 2 (TSP1/2), which act through their neuronal receptor α2δ-1, are required for proper intracortical excitatory synaptogenesis. In the adult brain, the loss of α2δ-1 impairs training-induced excitatory synaptogenesis in the anterior cingulate cortex (ACC), and this impairment leads to increased effort-exertion during high-effort tasks. Here, we tested whether TSP1 and TSP2 are required for controlling effort during operant conditioning by using a lever press for food reward training in mice. Surprisingly, we found that constitutive loss of TSP1/2 significantly reduced lever pressing performance when the effort required for a food reward was increased, a phenotype opposite of α2δ-1 loss. Loss of TSP1/2 reduced excitatory synapse number significantly in adult brains. However, in the ACC of TSP1/2 knockout mice, there was still training-induced excitatory synaptogenesis, likely through the upregulation of TSP4, a TSP isoform that is also synaptogenic. Unexpectedly, we also found a significant increase in inhibitory synapse number and function in the ACC of TSP1/2 knockout mice, which was eliminated after training. Finally, we found that astrocyte-specific ablation of TSP1/2 in developing but not adult astrocytes is sufficient to reduce performance during high-effort tasks. Taken together, our study highlights the importance of developmental astrocyte-derived synaptogenic cues TSP1 and 2 in establishing excitatory and inhibitory circuits that control effort during operant conditioning in adults.

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“…Regions of astrocytes in the primary visual cortex were imaged at high magnification (60× objective, 1.64× zoom) and resolution (0.34 μM step size, 3.06 μM z-stack) on an Olympus FV 3000 microscope. SynBot, an ImageJ-based software developed by the Eroglu lab ( Savage et al, 2023 Preprint ) was utilized to quantify synapses using the following parameters: Each image was subdivided into three 1.02-μM substacks, z-projected, and the synaptic puncta identified by manual thresholding and pixel-based colocalization. The synaptic puncta were normalized to the area of astrocyte to give synaptic density.…”

Section: Methodsmentioning

confidence: 99%

δ-Catenin controls astrocyte morphogenesis via layer-specific astrocyte–neuron cadherin interactions

Tan,

Bindu,

Hardin

et al. 2023

Journal of Cell Biology

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Astrocytes control the formation of specific synaptic circuits via cell adhesion and secreted molecules. Astrocyte synaptogenic functions are dependent on the establishment of their complex morphology. However, it is unknown if distinct neuronal cues differentially regulate astrocyte morphogenesis. δ-Catenin was previously thought to be a neuron-specific protein that regulates dendrite morphology. We found δ-catenin is also highly expressed by astrocytes and required both in astrocytes and neurons for astrocyte morphogenesis. δ-Catenin is hypothesized to mediate transcellular interactions through the cadherin family of cell adhesion proteins. We used structural modeling and biochemical analyses to reveal that δ-catenin interacts with the N-cadherin juxtamembrane domain to promote N-cadherin surface expression. An autism-linked δ-catenin point mutation impaired N-cadherin cell surface expression and reduced astrocyte complexity. In the developing mouse cortex, only lower-layer cortical neurons express N-cadherin. Remarkably, when we silenced astrocytic N-cadherin throughout the cortex, only lower-layer astrocyte morphology was disrupted. These findings show that δ-catenin controls astrocyte–neuron cadherin interactions that regulate layer-specific astrocyte morphogenesis.

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SynBot: An open-source image analysis software for automated quantification of synapses

Cited by 8 publications

References 68 publications

Neuron Derived Cytokine Interleukin-34 Controls Developmental Microglia Function

Neuron Derived Cytokine Interleukin-34 Controls Developmental Microglia Function

Astrocytic thrombospondins 1 and 2 are required for cortical synapse development controlling instrumental performance

δ-Catenin controls astrocyte morphogenesis via layer-specific astrocyte–neuron cadherin interactions

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