Neuronal Activity

Brain-wide cellular resolution measurement of recent neuronal activity.


While cFos is a common neuronal activity marker, its responsiveness to cAMP and other signaling pathways limits its specificity as a marker of neuronal activity per se.

Npas4, however, is exclusively regulated by calcium-dependent signaling downstream of neuronal activity, offering a more precise readout of neuronal activity.

The Neuronal Activity Full-Pipeline service concurrently measures both immediate early gene products, Npas4 and cFos, to provide a robust snapshot of recent neuronal activity. This service is well-suited for uncovering brain-wide signatures linked to recent neuronal activity following genetic, pharmacological, or behavioral manipulations.

Learn more about the advantages of concurrent Npas4 and cFos measurement by visiting our science page.

Neuronal Activity


Whole Brains to Actionable Readouts of Recent Neuronal Activity

Experimental Treatments That Alter Brain Activity Patterns

Perform your favorite behavioral, pharmacological, genetic, or other manipulation, and send us your samples. Or collaborate with us, and we’ll perform the animal treatments for you.

Tissue Clearing and Labeling of Recently Active Neurons

We’ll use our modified iDISCO+ tissue clearing protocol to render intact brain samples optically transparent while labeling recently active neurons with a well-validated cFos antibody in conjunction with our proprietary Npas4 antibody.

Light Sheet Imaging of cFos and Npas4

Cleared and labeled brains are imaged on the Lightsheet Z.1 equipped with the Mesoscale Imaging System™. The Mesoscale Imaging System is optimized for imaging large intact tissues with unprecedented speed and resolution and is compatible with the high refractive index solutions used in tissue clearing protocols.

Translucence’s imaging pipelines capture cellular-resolution, whole-brain images of Npas4 and cFos expression patterns, providing insights into the distribution of recently active neurons.

Stitching of Light Sheet Images

We employ our in-house developed software solution, Stitchy™, for light sheet image stitching. Stitchy is a robust software solution that stitches terabyte-scale datasets 2.5-5.5 times faster than leading commercial software solutions. By eliminating intermediate file formats, Stitchy enables seamless integration with downstream image analysis pipelines.

AI-Powered Whole-Brain Regional Data

The Translucence Teravoxel Toolkit (3TK) software performs AI-powered quantification, applying deterministic filters and segmenting Npas4- and cFos-positive neurons. Our automated quantification pipeline then aligns samples to the Allen reference atlas and generates brain-wide cell counts of neuronal activity across hundreds of identified brain areas.

Actionable data

Brain-Wide Cellular Resolution Snapshots of Recent Neuronal Activity

Browse actionable insights generated by the

Neuronal Activity


Browse data generated by the Neuronal Activity Full-Pipeline service which captures immediate early gene products, Npas4 and cFos, to provide a robust snapshot of recent neuronal activity.

Automated Identification of Npas4 and cFos Cells in iDISCO+ Cleared Brains

Optical slices through an intact mouse brain cleared and stained with antibodies against cFos and Npas4 using our modified iDISCO+ procedure. Our 3TK software uses machine learning and a series of deterministic filters to label immunoreactive neurons in 3D throughout the brain. The color labels are arbitrary to help distinguish individual neurons. Rather than only labeling the bright cells, our AI-powered tools are designed to identify all immunoreactive cells staining intensities and performs measurements of staining intensity for each cell. This provides a much richer dataset than simply counting immunopositive cells above an arbitrary cut-off.

Npas4 Expression is Exquisitely Tuned to Neuronal Activity

Traditional immediate-early genes (IEG’s) have served as great markers of neuronal activity, however they are also regulated via neuromodulators via cAMP and by other paracrine factors.

Npas4 is exquisitely tuned to Ca²+ signaling downstream of neuronal activity providing a purer readout of electrogenic signals.

3TK-mediated Quantification of Npas4 After Light Exposure

Our Translucence Teravoxel ToolKit (3TK) software uses AI-powered workflows to identify individual Npas4(+) neurons throughout the cleared and stained intact mouse brain.

Each brain is computationally warped to the Allen Reference Brain and neurons are registered to an anatomical map to determine Npas4 expression metrics across 100’s of brain regions.

We have collapsed the hierarchical annotations into larger regions to demonstrate brain-wide Npas4 expression levels in 14 brains from mice exposed to light for various durations after housing in the dark for 24h.

Brain-Wide Quantification of Neuronal Activity in 100’s of Brain Regions

These 2D heatmaps are drawn from 3D whole-brain data sets and show the level of Npas4 expression determined from the Npas4 staining intensity of individual neurons.

The Npas4 signal is quantified in 100’s of brain regions over time. We have plotted data from a few exemplar regions in the bottom left.

The volcano plot shows brain regions in blue with statistically significant elevations of Npas4 expression after light exposure.

Npas4 KO Mouse Validation of the Activity Signaling Npas4

Concurrent staining of cFos and Npas4 with the Translucence Neuronal Activity Tissue Clearing Kit in WT and Npas4 KO mouse brains. The mice went through a social exposure task to drive the expression of immediate-early genes. The lack of Npas4 staining in the KO brain demonstrates the specificity of our Activity Signaling recombinant monoclonal anti-Nas4 antibody.

Neuronal Activity Anatomics with cFos and Npas4

Warping individual brains to the Allen Reference Brain allows for registration of individual cFos and Npas4 labels into a common coordinate space. The hierarchical Allen Brain Atlas then allows for automated regional quantification. This heatmap visualizes levels of cFos and Npas4 levels in a subset of brain regions in response to two different drug treatments. Each column is one brain.

The Neuronal Activity Full-Pipeline Service

The Neuronal Activity Full-Pipeline service is an end-to-end service that leverages next-generation 3D histology technologies encompassing tissue clearing, light sheet imaging, and AI-powered quantification to provide comprehensive and high-quality data to support your research endeavors.

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