Axon Initial Segments- Key Loci of Diseases of the Nervous System 

Ransdell Lab

Lead PI: Dr. Joey Ransdell

Aliyah Grijalva 

Nasim Hosseinifarjam 

Sam Brown

Sachindri Gunasekara

The axon initial segment (AIS) is a distinct region in neurons vital for initiating action potentials. This is due to the high concentration of voltage-gated potassium and sodium channels along with scaffolding proteins like Ankyrin G, which maintain structural integrity and are critical for the appropriate localization and distribution of ion channels at the AIS. Ion channel expression at the AIS affects the functionality and efficiency of neuronal action potential generation. Changes to the AIS can drastically change the excitability of neurons. Investigating the composition of the neuronal AIS across cell types can be used to better understand the mechanisms underlying disorders of the nervous system.

The AIS is frequently implicated in neurological diseases. AIS length and composition have all been linked to numerous neurological diseases such as Autism Spectrum disorder (ASD), epilepsy, cerebellar ataxias, and other conditions. Reduced expression levels of voltage gated sodium channels (Nav) at the AIS can reduce action potenial generation and increase action potenial threshold voltages; resulting in attenuated  neuronal electrical signals.

In this project, we are working to investigate the AIS of cerebellar Purkinje neurons using both Cre-negative (-) and Cre-positive (+) transgenic mouse models. To identify and characterize AIS properties, we are using immunofluorescent labeing. Specifically, we are measuring the immunofluorescent signatures generated by voltage-gated sodium channel and ankyrin G expression at the Purkinje neuron AIS.  

Assess the molecular organization of the Purkinje neuron AIS in various transgenic mouse models. 

Action Potenials

Neurons communicate through action potentials, which are initiated at the axon initial segment.. The propagation of action potentials are reliant on voltage gated ion channels which enable rapid communication throughout the brain. Because of their vital role neuronal signaling, disruptions in this process are strongly linked to neurological disorders.

Axon Inital Segments

The AIS  is built and structured by ankyrin-G and is found to contain high concentrations of proteins like voltage gated ion channels. The AIS is located just beyond the axon hillock. Without the AIS, action potentials could not be generated and neuronal communication would stop.

Voltage Gated Sodium Channels (Nav)

Nav channels are densely located on the membrane of the axon initials segment. The opening of these channels leads to an influx of sodium ions entering the cell, leading to depolarization, the rapid rise of potential in action potentials. The density of the channels allows action potentials to be effectively initiated by the AIS, loss of the channels would result in far less reliable action potentials.

Ankyrin-G

Ankyrin-G is an important protein in the AIS that structures it by anchoring and organizing voltage-gated ion channels and other vital components in the membrane of the AIS. Disruption in ankyrin-G would cause mislocalization of voltage-gated ion channels. Ankyrin-G is also essential for the initial formation of the AIS. Deletion of ANK3, which encodes ankyrin-G, would result in a complete loss of the AIS.

Transgenic Model

A TSC1 FL/WT Cre− female mouse and a ROSA HOM; Cre+ female mouse were used. Cre recombinase excises a loxP-STOP-loxP (LSL) cassette, enabling tdTomato expression in the Cre+ mouse to label Purkinje neurons. In the Cre− mouse, tdTomato is not expressed, so Purkinje neurons are instead identified by calbindin immunofluorescence.

Brain Dissection and Slicing

Transcardial perfusion is performed. Then the brain is dissected and removed from the body, and the cerebellum is cut out. Through cryosectioning, 25 µm sagittal cerebellar slices are cut and put on positively charged slides.

Immunofluorescence Labeling

TSC1 FL/WT Cre-

Brain tissue from a 66-day-old female mouse was washed in PBS, blocked with (0.5% goat serum/0.25% TX), and incubated with primary antibodies (pan-Nav 1:1000, ankyrin-G 1:500, calbindin 1:20,000) for 24 h. After secondary antibody incubation (Alexa 488 1:250, CF647 1:500, CF568 1:250), tissues were washed and imaged using a Zeiss 710 confocal microscope.

ROSA HOM; Cre+

Brain tissue from 74-day-old mice was washed, blocked with (7.5% goat serum/0.25% TX), and incubated overnight with primary antibodies (pan-Nav 1:1000, ankyrin-G 1:500). After secondary incubation (Alexa 488 1:250, CF647 1:500), tissues were washed and imaged using a Zeiss 710 confocal microscope.

Analyzing Data

Distance and gray value (fluorescence intensity) was taken from the AIS of both Ank G and Pan Nav through Fiji Image J . Then background noise was subtracted from the grey value, and a 5-point sliding mean was calculated for fluorescence. Using GraphPad Prism 10, the 5-point sliding mean values were then plotted along distance for Ank G and Pan Nav for all 4 cells. 

This project establishes a standardized framework for visualizing and quantifying the Axon Initial Segment in Purkinje neurons. By using Ankyrin G to show the AIS and Pan Nav, for its importance in generating action potentials, we can successfully map the distribution of these proteins and quantify them on the AIS. The results show that the immunofluorescence imaging and analysis can successfully capture the structural framework of the AIS across different purkinje cell markers (Cre+ and Cre-). This methodology provides a foundation for future studies to research the AIS and its involvement in disease.

I am grateful to Dr. Joey Ransdell for his mentorship and guidance. I also want to thank Nasim Hosseinifarjam for her teachings  and Achintya Kumar Jena for providing me with the Cre+ images. 

de Ruiter MM;De Zeeuw CI;Hansel C;, M. (n.d.). Voltage-gated sodium channels in cerebellar Purkinje cells of Mormyrid Fish. Journal of neurophysiology. https://pubmed.ncbi.nlm.nih.gov/16598064/

Foust, A., Popovic, M., Zecevic, D., & McCormick, D. A. (2010, May 19). Action potentials initiate in the axon initial segment and propagate through axon collaterals reliably in cerebellar Purkinje neurons. The Journal of neuroscience : the official journal of the Society for Neuroscience. https://pmc.ncbi.nlm.nih.gov/articles/PMC2990270/

Grider, M. H. (2023, May 8). Physiology, action potential. StatPearls [Internet]. https://www.ncbi.nlm.nih.gov/books/NBK538143/

Guy-Evans, O. (2025, June 17). What is action potential?. Simply Psychology. https://www.simplypsychology.org/what-is-action-potential.html

Huang, C. Y.-M., & Rasband, M. N. (2018, May). Axon initial segments: Structure, function, and disease. Annals of the New York Academy of Sciences. https://pmc.ncbi.nlm.nih.gov/articles/PMC5992072/

Jenkins, P., & Bender, K. (2025). Axon initial segment structure and function in health and disease | physiological reviews | american physiological society. https://journals.physiology.org/doi/full/10.1152/physrev.00030.2024 

Through this research, I have gained skills in teamwork, technology, and communication. For teamwork, I had to work closely with a graduate student, Nasim Hosseinifarjam, She taught me many of the methods I needed for the project to succeed. For technology, I learned how to use a Confocal microscope and relevant computer programs. And finally, for communication, I regularly consulted with both my faculty mentor and the graduate student for guidance and support.