Leiwe MN, Fujimoto S, Baba T, Moriyasu D, Saha B, Sakaguchi R, Inagaki S, Imai T. Automated neuronal reconstruction with super-multicolour Tetbow labelling and threshold-based clustering of colour hues. Nat Commun 2024;15(1):5279. doi: 10.1038/s41467-024-49455-y.

 

Background: Mapping densely labelled neuronal circuits is limited by the difficulty of distinguishing overlapping neurites using light microscopy and by the laborious nature of manual tracing. Conventional multicolour fluorescence methods such as Brainbow and Tetbow use only three fluorescent proteins, producing too few colour variations to separate neighbouring neurons effectively.

 

Hypothesis: This study hypothesized that combining super-multicolour Tetbow labelling using more than three fluorescent proteins with an automated hue-based analysis pipeline could accurately reconstruct densely labelled neuronal circuits without relying on physical continuity.

 

Methods: The authors developed the QDyeFinder pipeline, which uses spectral unmixing and quantitative analysis of seven fluorescent proteins to extract neurite colour vectors. Neurolucida 360 was employed for soma detection and automated neurite tracing, generating fragments that were clustered by colour similarity using the custom dCrawler algorithm. Validation involved manual reconstructions for ground truth comparisons in cortical and olfactory bulb samples.

 

Results: Seven-colour Tetbow labelling provided superior neuronal discriminability (>99.9%) compared to conventional three-colour methods. Automated detection identified over 15,000 neurite fragments that were accurately clustered into individual neurons at an optimal threshold (Th(d) = 0.2). QDyeFinder successfully reconstructed dendritic and axonal morphologies, including neurites spanning multiple brain sections.

 

Conclusions: Super-multicolour labelling combined with the QDyeFinder pipeline enables fully automated, hue-based neuronal reconstruction across large volumes, overcoming limitations of continuity-dependent tracing and significantly advancing scalable connectomics.

Zhang Y, Bizanti A, Harden SW, Chen J, Bendowski K, Hoover DB, Gozal D, Shivkumar K, Heal M, Tappan S, Cheng ZJ. Topographical mapping of catecholaminergic axon innervation in the flat-mounts of the mouse atria: a quantitative analysis. Sci Rep. 2023 Apr 7;13(1):4850. doi: 10.1038/s41598-023-27727-9.

 

Background: The sympathetic nervous system (SNS) regulates cardiac functions such as heart rate, contractility and conduction velocity. However, a comprehensive three-dimensional (3D) map of cardiac sympathetic innervation remains lacking. Earlier studies using sectioned or partial heart preparations disrupted axonal continuity and prevented large-scale morphological analysis. Therefore, a detailed topographical description of catecholaminergic innervation in the mouse atria is essential for understanding sympathetic regulation of cardiac physiology and remodeling.

 

Hypothesis: This study hypothesized that sympathetic postganglionic catecholaminergic axons display distinct and quantifiable topographical distributions across different atrial regions, with region-specific variations in axon density and organization.

 

Methods: The authors used whole-mount atrial preparations from C57BL/6J mice. Tissues were processed for tyrosine hydroxylase (TH) immunolabeling, imaged with confocal microscopy and analyzed using Neurolucida 360 for 3D tracing and digitization of catecholaminergic axons. Neurolucida Explorer was employed for morphometric and density analyses across defined atrial regions.

 

Results: Four to five major TH-immunoreactive bundles entered the atria at consistent sites and branched into overlapping projection fields. Axon density was greatest near the sinoatrial node in the right atrium and at the left atrium–pulmonary vein junction. Approximately 18–30% of intrinsic cardiac ganglion neurons were TH-positive, but few showed direct axonal innervation. Dense sympathetic fibers also surrounded blood vessels and adipocytes.

 

Conclusions: This study provided the first comprehensive quantitative map of catecholaminergic innervation in mouse atria, revealing pronounced regional asymmetry and preferential right- and left-sided projections. These findings establish an anatomical foundation for future functional and pathological analyses of cardiac sympathetic control.

Merino-Galán L, Zamarbide M, Belloso-Iguerategui A, Alonso-Moreno MC, Gago B, Reinares-Sebastián A, Blesa J, Dumitriu D, Quiroga-Varela A, Rodríguez-Oroz MC. Resilience of striatal synaptic plasticity over early structural adaptations in premotor parkinsonism. NPJ Parkinsons Dis 2025;11(1):146. doi: 10.1038/s41531-025-00994-1.

 

Background: Parkinson’s disease involves progressive dopaminergic neuron loss in the substantia nigra and striatal dopamine depletion. Before motor symptoms appear, compensatory synaptic and structural adaptations may help maintain neural function, but their timing and mechanisms are unclear. Understanding these early changes in striatal spiny projection neurons (SPNs) is crucial for identifying premotor plasticity processes.

 

Hypothesis: This study hypothesized that early dopaminergic dysfunction induced by α-synuclein (A53T) overexpression impairs striatal synaptic plasticity and elicits compensatory structural remodeling of SPN dendrites before motor deficits emerge.

 

Methods: The authors used adult rats inoculated bilaterally in the substantia nigra with AAV vectors overexpressing A53T human α-synuclein and analyzed dopaminergic, synaptic and structural changes at 72 hours, 1, 2 and 4 weeks post-inoculation. Synaptic plasticity was assessed by FASS-LTP and neurotransmitter levels by HPLC. Dendritic spine morphology and dendritic arbor complexity were examined using high-resolution confocal microscopy and three-dimensional reconstruction with Neurolucida 360, while ultrastructural analyses were performed with electron microscopy.

 

Results: Dopamine content declined as early as 72 hours, accompanied by inhibition of chemical LTP, which partially recovered at four weeks. At this stage, dopaminergic neuron loss and fiber swelling became significant without motor deficits. Dendritic spine density decreased, particularly in thin spines, while mushroom spine head volume increased. Fewer spines contained smooth endoplasmic reticulum, though its relative area enlarged. SPNs displayed greater dendritic branching and complexity.

 

Conclusions: The results indicate that early dopaminergic dysfunction impairs striatal synaptic plasticity but triggers structural compensations in SPNs that may preserve network function. These adaptive spine and dendritic changes represent key homeostatic mechanisms sustaining striatal resilience during premotor parkinsonism.

Walker CK, Greathouse KM, Tuscher JJ, Dammer EB, Weber AJ, Liu E, Curtis KA, Boros BD, Freeman CD, Seo JV, Ramdas R, Hurst C, Duong DM, Gearing M, Murchison CF, Day JJ, Seyfried NT, Herskowitz JH. Cross-platform synaptic network analysis of human entorhinal cortex identifies twf2 as a modulator of dendritic spine length. J Neurosci 2023;43(20):3764-3785. doi: 10.1523/JNEUROSCI.2102-22.2023.

 

Background: Synaptic dysfunction and dendritic spine alterations are early and critical events in Alzheimer’s disease (AD), particularly within the entorhinal cortex, which plays a key role in memory processing. Proteomic studies have identified numerous molecular changes in AD, but linking these to specific cellular phenotypes remains a challenge.

 

Hypothesis: This study hypothesized that integrating synaptic proteomic data with dendritic spine morphology metrics enables identification of proteins that regulate dendritic spine structure in the human entorhinal cortex.

 

Methods: The authors analyzed post mortem entorhinal cortex tissue from control, cognitively resilient and AD individuals. Dendritic spines on pyramidal neurons were imaged after Golgi–Cox staining and reconstructed using Neurolucida 360, with quantitative analyses performed in Neurolucida Explorer. Parallel proteomic profiling of synaptosomal fractions was performed using mass spectrometry and weighted co-expression network analysis, followed by CRISPR activation experiments in cultured neurons.

 

Results: AD samples exhibited reduced overall dendritic spine density compared with controls, while cognitively resilient cases maintained densities similar to controls. Co-expression analysis identified a protein module correlated with thin spine length, with Twinfilin-2 (TWF2) as its central hub. Increasing TWF2 expression in neurons selectively lengthened thin spines without altering density.

 

Conclusions: These findings identify TWF2 as a modulator of dendritic spine length and validate an integrated proteomic–morphometric approach to discover synaptic regulators in AD.