Presynaptic perspective: Axonal transport defects in neurodevelopmental disorders.

Disruption of synapse assembly and maturation leads to a broad spectrum of neurodevelopmental disorders. Presynaptic proteins are largely synthesized in the soma, where they are packaged into precursor vesicles and transported into distal axons to ensure precise assembly and maintenance of presynapses. Due to their morphological features, neurons face challenges in the delivery of presynaptic cargos to nascent boutons. Thus, targeted axonal transport is vital to build functional synapses. A growing number of mutations in genes encoding the transport machinery have been linked to neurodevelopmental disorders. Emerging lines of evidence have started to uncover presynaptic mechanisms underlying axonal transport defects, thus broadening the view of neurodevelopmental disorders beyond postsynaptic mechanisms. In this review, we discuss presynaptic perspectives of neurodevelopmental disorders by focusing on impaired axonal transport and disturbed assembly and maintenance of presynapses. We also discuss potential strategies for restoring axonal transport as an early therapeutic intervention.

Alpha herpesvirus exocytosis from neuron cell bodies uses constitutive secretory mechanisms, and egress and spread from axons is independent of neuronal firing activity.

Alpha herpesviruses naturally infect the peripheral nervous system, and can spread to the central nervous system, causing severe debilitating or deadly disease. Because alpha herpesviruses spread along synaptic circuits, and infected neurons exhibit altered electrophysiology and increased spontaneous activity, we hypothesized that alpha herpesviruses use activity-dependent synaptic vesicle-like regulated secretory mechanisms for egress and spread from neurons. Using live-cell fluorescence microscopy, we show that Pseudorabies Virus (PRV) particles use the constitutive Rab6 post-Golgi secretory pathway to exit from the cell body of primary neurons, independent of local calcium signaling. Some PRV particles colocalize with Rab6 in the proximal axon, but we did not detect colocalization/co-transport in the distal axon. Thus, the specific secretory mechanisms used for viral egress from axons remains unclear. To address the role of neuronal activity more generally, we used a compartmentalized neuron culture system to measure the egress and spread of PRV from axons, and pharmacological and optogenetics approaches to modulate neuronal activity. Using tetrodotoxin to silence neuronal activity, we observed no inhibition, and using potassium chloride or optogenetics to elevate neuronal activity, we also show no increase in virus spread from axons. We conclude that PRV egress from neurons uses constitutive secretory mechanisms: generally, activity-independent mechanisms in axons, and specifically, the constitutive Rab6 post-Golgi secretory pathway in cell bodies.

In-Vivo Calcium Imaging of Sensory Neurons in the Rat Trigeminal Ganglion.

Genetically encoded calcium indicators (GECIs) enable imaging techniques to monitor changes in intracellular calcium in targeted cell populations. Their large signal-to-noise ratio makes GECIs a powerful tool for detecting stimulus-evoked activity in sensory neurons. GECIs facilitate population-level analysis of stimulus encoding with the number of neurons that can be studied simultaneously. This population encoding is most appropriately done in vivo. Dorsal root ganglia (DRG), which house the soma of sensory neurons innervating somatic and visceral structures below the neck, are used most extensively for in vivo imaging because these structures are accessed relatively easily. More recently, this technique was used in mice to study sensory neurons in the trigeminal ganglion (TG) that innervate oral and craniofacial structures. There are many reasons to study TG in addition to DRG, including the long list of pain syndromes specific to oral and craniofacial structures that appear to reflect changes in sensory neuron activity, such as trigeminal neuralgia. Mice are used most extensively in the study of DRG and TG neurons because of the availability of genetic tools. However, with differences in size, ease of handling, and potentially important species differences, there are reasons to study rat rather than mouse TG neurons. Thus, we developed an approach for imaging rat TG neurons in vivo. We injected neonatal pups (p2) intraperitoneally with an AAV encoding GCaMP6s, resulting in >90% infection of both TG and DRG neurons. TG was visualized in the adult following craniotomy and decortication, and changes in GCaMP6s fluorescence were monitored in TG neurons following stimulation of mandibular and maxillary regions of the face. We confirmed that increases in fluorescence were stimulus-evoked with peripheral nerve block. While this approach has many potential uses, we are using it to characterize the subpopulation(s) of TG neurons changed following peripheral nerve injury.

RNA Profile of Cell Bodies and Exosomes Released by Tumorigenic and Non-Tumorigenic Thyroid Cells.

Tumor cells release exosomes, extracellular vesicle containing various bioactive molecules such as protein, DNA and RNA. The analysis of RNA molecules packaged in exosomes may provide new potential diagnostic or prognostic tumor biomarkers. The treatment of radioiodine-refractory aggressive thyroid cancer is still an unresolved clinical challenge, and the search for biomarkers that are detectable in early phase of the disease has become a fundamental goal for thyroid cancer research. By using transcriptome analysis, this study aimed to analyze the gene expression profiles of exosomes secreted by a non-tumorigenic thyroid cell line (Nthy-ori 3.1-exo) and a papillary thyroid cancer (TPC-1-exo) cell line, comparing them with those of cell bodies (Nthy-ori 3.1-cells and TPC-1-cells). A total of 9107 transcripts were identified as differentially expressed when comparing TPC-1-exo with TPC-1-cells and 5861 when comparing Nthy-ori 3.1-exo with Nthy-ori 3.1-cells. Among them, Sialic acid-binding immunoglobulin-like lectins 10 and 11 (SIGLEC10, SIGLEC11) and Keratin-associated protein 5 (KRTAP5-3) transcripts, genes known to be involved in cancer progression, turned out to be up-regulated only in TPC-1-exo. Gene ontology analysis revealed significantly enriched pathways, and only in TPC-1-exo were the differential expressed genes associated with an up-regulation in epigenetic processes. These findings provide a proof of concept that some mRNA species are specifically packaged in tumor-cell-derived exosomes and may constitute a starting point for the identification of new biomarkers for thyroid tumors.

Fission-independent compartmentalization of mitochondria during budding yeast cell division.

Lateral diffusion barriers compartmentalize membranes to generate polarity or asymmetrically partition membrane-associated macromolecules. Budding yeasts assemble such barriers in the endoplasmic reticulum (ER) and the outer nuclear envelope at the bud neck to retain aging factors in the mother cell and generate naïve and rejuvenated daughter cells. However, little is known about whether other organelles are similarly compartmentalized. Here, we show that the membranes of mitochondria are laterally compartmentalized at the bud neck and near the cell poles. The barriers in the inner mitochondrial membrane are constitutive, whereas those in the outer membrane form in response to stresses. The strength of mitochondrial diffusion barriers is regulated positively by spatial cues from the septin axis and negatively by retrograde (RTG) signaling. These data indicate that mitochondria are compartmentalized in a fission-independent manner. We propose that these diffusion barriers promote mitochondrial polarity and contribute to mitochondrial quality control.

3-Dimensional Ultrastructural Analysis of the Rat Pinealocyte: Presence of Secretory Bulbous Projections Delineated from the Cell Body by Junctional Complexes.

INTRODUCTION: The superficial pineal gland of the Sprague Dawley rat is a neuroendocrine structure secreting the hormone melatonin. By use of block face scanning electron microscopy, our aim here was to identify the 3-dimensional ultrastructure of the gland. METHODS: A series of 2,731 block face images of the rat pineal tissue, 30 nm in thickness, was obtained in a Teneo volume scanning electron microscope and used for 3-dimensional reconstruction by use of the TrakEM2-plugin in the ImageJ software. Thin sections of the tissue were cut for transmission electron microscopy. RESULTS: Our analyses revealed cellular bulbous processes, containing 50-100 nm clear vesicles, that emerged from a neck-like area at the cell body of the pinealocyte. These bulbous processes extend into small canaliculi located in the center of parenchymal folliculi of the gland as well as into the perivascular spaces. Junctional complexes, comprising both gap and tight junctions, connected the lateral cellular membranes of the pinealocytes, where the bulbous processes emerged from the cell bodies. The canaliculi were, via the extracellular space, connected to the perivascular spaces. DISCUSSION: The junctional complexes reported here would prevent a substance, released from the vesicles in the bulbous processes, from targeting the cell body from which they emerge. In line with previous combined morphological and biochemical demonstrations of glutamate located in clear vesicles of bulbous processes in the rat pineal gland, our data ultrastructurally support the concept that bulbous processes could participate in a paracrine glutamatergic inhibition of the melatonin secretion in the pineal gland. CONCLUSION: Bulbous secretory projections separated from the cell body by a junctional complex represents a new feature of neuroendocrine cells.

The Plasma Membrane Polarity Is Higher in the Neuronal Growth Cone than in the Cell Body of Hippocampal and Cerebellar Granule Neurons.

The polarity of the biological membrane, or lipid order, regulates many cellular events. It is generally believed that the plasma membrane polarity is regulated according to cell type and function, sometimes even within a cell. Neurons have a variety of functionally specialized subregions, each of which bears distinct proteins and lipids, and the membrane polarity of the subregions may differ accordingly. However, no direct experimental evidence of it has been presented to date. In the present study, we used a cell-impermeable solvatochromic membrane probe NR12A to investigate the local polarity of the plasma membrane of neurons. Both in hippocampal and cerebellar granule neurons, growth cones have higher membrane polarity than the cell body. In addition, the overall variation in the polarity value of each pixel was greater in the growth cone than in cell bodies, suggesting that the lateral diffusion and/or dynamics of the growth cone membrane are greater than other parts of the neuron. These tendencies were much less notably observed in the lamellipodia of a non-neuronal cell. Our results suggest that the membrane polarity of neuronal growth cones is unique and this characteristic may be important for its structure and function.

The Reduction of the Cell Nuclear Size in the Cell Body Space During the Differentiation Might Be Cell Lineage Specific (A Retrospective Morphological Note).

The cell body space occupied by the nucleus decreased during the cell differentiation of the granulocytic cell lineage in CML (Chronic Myeloid Leukemia) patients. In contrary, in patients suffering from CLL (Chronic Lymphocytic Leukemia), the cell body space occupied by the nucleus during the cell differentiation of the lymphocytic lineage did not decrease despite the reduction of the cell size. Thus, the cell body space occupied by the cell nucleus during the differentiation was characteristic for each of these cell lineages.

A Deep Learning Approach for Neuronal Cell Body Segmentation in Neurons Expressing GCaMP Using a Swin Transformer.

Neuronal cell body analysis is crucial for quantifying changes in neuronal sizes under different physiological and pathologic conditions. Neuronal cell body detection and segmentation mainly rely on manual or pseudo-manual annotations. Manual annotation of neuronal boundaries is time-consuming, requires human expertise, and has intra/interobserver variances. Also, determining where the neuron's cell body ends and where the axons and dendrites begin is taxing. We developed a deep-learning-based approach that uses a state-of-the-art shifted windows (Swin) transformer for automated, reproducible, fast, and unbiased 2D detection and segmentation of neuronal somas imaged in mouse acute brain slices by multiphoton microscopy. We tested our Swin algorithm during different experimental conditions of low and high signal fluorescence. Our algorithm achieved a mean Dice score of 0.91, a precision of 0.83, and a recall of 0.86. Compared with two different convolutional neural networks, the Swin transformer outperformed them in detecting the cell boundaries of GCamP6s expressing neurons. Thus, our Swin transform algorithm can assist in the fast and accurate segmentation of fluorescently labeled neuronal cell bodies in thick acute brain slices. Using our flexible algorithm, researchers can better study the fluctuations in neuronal soma size during physiological and pathologic conditions.

Bacterial cell-size changes resulting from altering the relative expression of Min proteins.

The timing of cell division, and thus cell size in bacteria, is determined in part by the accumulation dynamics of the protein FtsZ, which forms the septal ring. FtsZ localization depends on membrane-associated Min proteins, which inhibit FtsZ binding to the cell pole membrane. Changes in the relative concentrations of Min proteins can disrupt FtsZ binding to the membrane, which in turn can delay cell division until a certain cell size is reached, in which the dynamics of Min proteins frees the cell membrane long enough to allow FtsZ ring formation. Here, we study the effect of Min proteins relative expression on the dynamics of FtsZ ring formation and cell size in individual Escherichia coli bacteria. Upon inducing overexpression of minE, cell size increases gradually to a new steady-state value. Concurrently, the time required to initiate FtsZ ring formation grows as the size approaches the new steady-state, at which point the ring formation initiates as early as before induction. These results highlight the contribution of Min proteins to cell size control, which may be partially responsible for the size fluctuations observed in bacterial populations, and may clarify how the size difference acquired during asymmetric cell division is offset.

C. elegans germ granules sculpt both germline and somatic RNAome.

Germ granules are membrane-less organelles essential for small RNA biogenesis and germline development. Among the conserved properties of germ granules is their association with the nuclear membrane. Recent studies demonstrated that LOTUS domain proteins, EGGD-1 and EGGD-2 (also known as MIP-1 and MIP-2 respectively), promote the formation of perinuclear germ granules in C. elegans. This finding presents a unique opportunity to evaluate the significance of perinuclear localization of germ granules. Here we show that loss of eggd-1 causes the coalescence of germ granules and formation of abnormal cytoplasmic aggregates. Impairment of perinuclear granules affects certain germline classes of small RNAs including Piwi-interacting RNAs. Transcriptome profiling reveals overexpression of spermatogenic and cuticle-related genes in eggd-1 hermaphrodites. We further demonstrate that disruption of germ granules activates HLH-30-mediated transcriptional program in somatic tissues. Collectively, our findings underscore the essential role of EGGD-1 in germ granule organization and reveal an unexpected germ granule-to-soma communication.

Visualization of trans homophilic interaction of clustered protocadherin in neurons.

Clustered protocadherin (Pcdh) functions as a cell recognition molecule through the homophilic interaction in the central nervous system. However, its interactions have not yet been visualized in neurons. We previously reported PcdhγB2-Förster resonance energy transfer (FRET) probes to be applicable only to cell lines. Herein, we designed γB2-FRET probes by fusing FRET donor and acceptor fluorescent proteins to a single γB2 molecule and succeeded in visualizing γB2 homophilic interaction in cultured hippocampal neurons. The γB2-FRET probe localized in the soma and neurites, and FRET signals, which were observed at contact sites between neurites, eliminated by ethylene glycol tetraacetic acid (EGTA) addition. Live imaging revealed that the FRET-negative γB2 signals rapidly moved along neurites and soma, whereas the FRET-positive signals remained in place. We observed that the γB2 proteins at synapses rarely interact homophilically. The γB2-FRET probe might allow us to elucidate the function of the homophilic interaction and the cell recognition mechanism.

Transient impacts of UV-B irradiation on whole body regeneration in a colonial urochordate.

Within the chordates, only some colonial ascidians experience whole body regeneration (WBR), where amputated small colonial fragments containing blood-vessels have the capability to regenerate the entire functional adult zooid within 1-3 weeks. Studying WBR in small colonial fragments taken at different blastogenic stages (the weekly developmental process characteristic to botryllid ascidians) from the ascidian Botrylloides leachii, about half of the fragments were able to complete regeneration (cWBR) three weeks following separation, about half were still in uncomplete, running regeneration (rWBR), and only a small percentage died. cWBR significantly increased in fragments that originated from a late blastogenic stage compared to an early stage. Most B. leachii populations reside in shallow waters, under variable daily natural UV irradiation, and it is of interest to elucidate irradiation effects on development and regeneration. Here, we show that UV-B irradiation resulted in enhanced mortality, with abnormal morphological changes in surviving fragments, yet with non-significant cWBR vs. rWBRs. Further, UV-B irradiation influenced the proportion of blood cells (morula cells, hemoblasts) and of multinucleated cells, a new WBR-associated cell type. At 24-h post-amputation we observed enhanced expression of ß-catenin (a signaling pathway that plays indispensable roles in cell renewal and regeneration), H3 and PCNA in all cell types of non-irradiated as compared to irradiated fragments. These elevated levels were considerably reduced 9-days later. Since WBR is a highly complex phenomenon, the employment of specific experimental conditions, as UV-B irradiation, alongside blastogenesis (the weekly developmental process), elucidates undisclosed facets of this unique biological occurrence such as transient expression of signature genes.

End-binding protein 1 promotes specific motor-cargo association in the cell body prior to axonal delivery of dense core vesicles.

Axonal transport is key to neuronal function. Efficient transport requires specific motor-cargo association in the soma, yet the mechanisms regulating this early step remain poorly understood. We found that EBP-1, the C. elegans ortholog of the canonical-microtubule-end-binding protein EB1, promotes the specific association between kinesin-3/KIF1A/UNC-104 and dense core vesicles (DCVs) prior to their axonal delivery. Using single-neuron, in vivo labeling of endogenous cargo and EBs, we observed reduced axonal abundance and reduced secretion of DCV cargo, but not other KIF1A/UNC-104 cargoes, in ebp-1 mutants. This reduction could be traced back to fewer exit events from the cell body, where EBP-1 colocalized with the DCV sorting machinery at the trans Golgi, suggesting that this is the site of EBP-1 function. EBP-1 calponin homology (CH) domain was required for directing microtubule growth on the Golgi, and mammalian EB1 interacted with KIF1A in an EBH-domain-dependent manner. Loss- and gain-of-function experiments suggest a model in which both kinesin-3 binding and guidance of microtubule growth at the trans Golgi by EBP-1 promote motor-cargo association at sites of DCV biogenesis. In support of this model, tethering either EBP-1 or a kinesin-3/KIF1A/UNC-104-interacting domain from an unrelated protein to the Golgi restored the axonal abundance of DCV proteins in ebp-1 mutants. These results uncover an unexpected role for a microtubule-associated protein and provide insights into how specific kinesin-3 cargo is delivered to the axon.

Robust acoustic trapping and perturbation of single-cell microswimmers illuminate three-dimensional swimming and ciliary coordination.

We report a label-free acoustic microfluidic method to confine single, cilia-driven swimming cells in space without limiting their rotational degrees of freedom. Our platform integrates a surface acoustic wave (SAW) actuator and bulk acoustic wave (BAW) trapping array to enable multiplexed analysis with high spatial resolution and trapping forces that are strong enough to hold individual microswimmers. The hybrid BAW/SAW acoustic tweezers employ high-efficiency mode conversion to achieve submicron image resolution while compensating for parasitic system losses to immersion oil in contact with the microfluidic chip. We use the platform to quantify cilia and cell body motion for wildtype biciliate cells, investigating effects of environmental variables like temperature and viscosity on ciliary beating, synchronization, and three-dimensional helical swimming. We confirm and expand upon the existing understanding of these phenomena, for example determining that increasing viscosity promotes asynchronous beating. Motile cilia are subcellular organelles that propel microorganisms or direct fluid and particulate flow. Thus, cilia are critical to cell survival and human health. The unicellular alga Chlamydomonas reinhardtii is widely used to investigate the mechanisms underlying ciliary beating and coordination. However, freely swimming cells are difficult to image with sufficient resolution to capture cilia motion, necessitating that the cell body be held during experiments. Acoustic confinement is a compelling alternative to use of a micropipette, or to magnetic, electrical, and optical trapping that may modify the cells and affect their behavior. Beyond establishing our approach to studying microswimmers, we demonstrate a unique ability to mechanically perturb cells via rapid acoustic positioning.

Vector-mediated PTEN deletion in the adult dentate gyrus initiates new growth of granule cell bodies and dendrites and expansion of mossy fiber terminal fields that continues for months.

Embryonic and early postnatal deletion of the gene phosphatase and tensin homolog (PTEN) results in neuronal hypertrophy, formation of aberrant neural networks and spontaneous seizures. Our previous studies document that deletion of PTEN in mature neurons also causes growth of cortical neuron cell bodies and dendrites, but it is unknown how this growth alters connectivity in mature circuits. Here, we explore consequences of deleting PTEN in a focal area of the dentate gyrus in adult male and female mice. PTEN deletion was accomplished by injecting AAV-Cre unilaterally into the dentate gyrus of double transgenic mice with lox-P sites flanking exon 5 of the PTEN gene and stop/flox tdTomato in the Rosa locus (PTENf/f/RosatdTomato). Focal deletion led to progressive increases in the size of the dentate gyrus at the injection site, enlargement of granule cell bodies, and increases in dendritic length and caliber. Quantitative analysis of dendrites by Golgi staining revealed dramatic increases in spine numbers throughout the proximo-distal extent of the dendritic tree, suggesting that dendritic growth is sufficient to induce new synapse formation by input neurons with intact PTEN expression. Tract tracing of input pathways to the dentate gyrus from the ipsilateral entorhinal cortex and commissural/associational system revealed that laminar specificity of termination of inputs is maintained. Mossy fiber axons from PTEN-deleted granule cells expanded their terminal field in CA3 where PTEN expression was intact and supra-granular mossy fibers developed in some mice. These findings document that persistent activation of mTOR via PTEN deletion in fully mature neurons re-initiates a state of robust cell-intrinsic growth, upending connectional homeostasis in fully mature hippocampal circuits.

Overexpression of EphB6 and EphrinB2 controls soma spacing of cortical neurons in a mutual inhibitory way.

To establish functional circuitry, neurons settle down in a particular spatial domain by spacing their cell bodies, which requires proper positioning of the soma and establishing of a zone with unique connections. Deficits in this process are implicated in neurodevelopmental diseases. In this study, we examined the function of EphB6 in the development of cerebral cortex. Overexpression of EphB6 via in utero electroporation results in clumping of cortical neurons, while reducing its expression has no effect. In addition, overexpression of EphrinB2, a ligand of EphB6, also induces soma clumping in the cortex. Unexpectedly, the soma clumping phenotypes disappear when both of them are overexpressed in cortical neurons. The mutual inhibitory effect of EphB6/ EphrinB2 on preventing soma clumping is likely to be achieved via interaction of their specific domains. Thus, our results reveal a combinational role of EphrinB2/EphB6 overexpression in controlling soma spacing in cortical development.

Brain-wide dendrites in a near-optimal performance of dynamic range and information transmission.

Dendrites receive and process signals from other neurons. The range of signal intensities that can be robustly distinguished by dendrites is quantified by the dynamic range. We investigate the dynamic range and information transmission efficiency of dendrites in relation to dendritic morphology. We model dendrites in a neuron as multiple excitable binary trees connected to the soma where each node in a tree can be excited by external stimulus or by receiving signals transmitted from adjacent excited nodes. It has been known that larger dendritic trees have a higher dynamic range. We show that for dendritic tress of the same number of nodes, the dynamic range increases with the number of somatic branches and decreases with the asymmetry of dendrites, and the information transmission is more efficient for dendrites with more somatic branches. Moreover, our simulated data suggest that there is an exponential association (decay resp.) of overall relative energy consumption (dynamic range resp.) in relation to the number of somatic branches. This indicates that further increasing the number of somatic branches (e.g. beyond 10 somatic branches) has limited ability to improve the transmission efficiency. With brain-wide neuron digital reconstructions of the pyramidal cells, 90% of neurons have no more than 10 dendrites. These suggest that actual brain-wide dendritic morphology is near optimal in terms of both dynamic range and information transmission.

Responses of pyramidal cell somata and apical dendrites in mouse visual cortex over multiple days.

The apical dendrites of pyramidal neurons in sensory cortex receive primarily top-down signals from associative and motor regions, while cell bodies and nearby dendrites are heavily targeted by locally recurrent or bottom-up inputs from the sensory periphery. Based on these differences, a number of theories in computational neuroscience postulate a unique role for apical dendrites in learning. However, due to technical challenges in data collection, little data is available for comparing the responses of apical dendrites to cell bodies over multiple days. Here we present a dataset collected through the Allen Institute Mindscope's OpenScope program that addresses this need. This dataset comprises high-quality two-photon calcium imaging from the apical dendrites and the cell bodies of visual cortical pyramidal neurons, acquired over multiple days in awake, behaving mice that were presented with visual stimuli. Many of the cell bodies and dendrite segments were tracked over days, enabling analyses of how their responses change over time. This dataset allows neuroscientists to explore the differences between apical and somatic processing and plasticity.

Repeated Measurement of Microglia-Dendritic Spine Interactions Using Multi-Photon Imaging.

In recent decades, mounting evidence has shown that microglia play a vital role in maintaining synapses throughout life. This maintenance is done via numerous microglial processes, which are long, thin, and highly motile protrusions from the cell body that monitor their environment. However, due to the brevity of the contacts and the potentially transient nature of synaptic structures, establishing the underlying dynamics of this relationship has proven difficult. This article describes a method of using rapidly acquired multiphoton microscopy images to track microglial dynamics and microglia:synapse interactions and the fate of the synaptic structures following those interactions. First, we detail a method for capturing multiphoton images at 1-min intervals for approximately 1 hr and how that process can be done at multiple time points. We then discuss how best to prevent and account for any drifting of the region of interest that can occur during the imaging session and how to remove excessive background noise from those images. Finally, we detail the annotation process for dendritic spines and microglial processes using plugins in MATLAB and Fiji, respectively. These semi-automated plugins allow tracking of individual cell structures, even if both microglia and neurons are imaged in the same fluorescent channel. This protocol presents a method of tracking both microglial dynamics and synaptic structures, in the same animal, at multiple time points, giving the user information on process speed, branching, tip size, location, and dwell time, as well as any dendritic spine gains, losses, and size changes. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Rapid multiphoton image capture Basic Protocol 2: Image preparation using MATLAB and Fiji Basic Protocol 3: Dendritic spine and microglial processes annotation using ScanImage and TrackMate.