Histological Approach to Reconstruction and Identification of Neuronal Processes
¹F. Hao, J. Edgerton, S. Li, and D. Jaeger
¹Department of Biology, Emory University, Atlanta, GA;

Abstract

Currently, there is no effective way to identify axon from dendrite strictly from morphological shape. The ability to use axonal markers will aid in the interpretation of results from neuronal recordings. One way to distinguish processes is via an antibody targeting proteins specific to the axon. Following this idea, this project uses anti-tau-1, and pan-axonal neurofilament marker. Results from the fluorescent labeling will be paired with the morphological reconstruction of the same neuron. This study found anti-tau-1 to be ineffective, and pan-axonal neurofilament marker to be successful. Future studies will involve labeling neurons that have already been recorded in order to study the effect of axonal injuries to electrophysiological properties of Globus Pallidus neurons.

Introduction

This project used monoclonal anti-tau-1 as its primary antibody. Tau is a microtubule-associated protein, and its main function is to stabilize microtubules in the axon where the protein is primarily found. Based on the axon’s unique neurofilament composition, pan-axonal neurofilament marker was also used.

Thus anti-tau-1 and pan-axonal neurofilament marker should provide specific labeling of the axon. Comparing images from immunofluorescence and biocytin staining will identify the axon. This information will help in morphological reconstruction, and modeling.





Figure 1. Demonstration of morphological reconstruction using neurolucida. a & c are photographs taken after biocytin staining. b is the corresponding reconstruction of neuron a, and d corresponds with c. The processes cannot be identified based on these pictures and reconstructions alone.



Figure 2. The anti-tau-1 antibody did not clearly label axons in rat hippocampal slice. The arrows point to the same neuron in the above pictures. The anti-tau- 1 primary antibody (b) only selectively labeled certain somas and axons, as is shown in a double label experiment with anti-calbindin (a). Immunofluorescent images from anti-calbindin show that the staining protocol worked, and that the anti-tau-1 antibody is at fault.



Figure 3. Pan-axonal neurofilament marker successfully labeled axons in slices. In both (a) and (b), clear signals are seen. However, cell bodies are not labeled, and thus this particular immunofluorescent technique must be paired with another labeling procedure to match each axon with its soma.

Methods and Materials

Immunofluorescence: Brain coronal sections were taken from rats and fixed in a 4% paraformaldehyde solution for four hours. Then sections were blocked with 1% normal goat serum and 0.2% Triton x-100 for two hours at room temperature. Following washing, the sections were incubated overnight with the primary antibody anti-tau-1 (Chemicon) at a dilution of 1:500, and 5% normal goat serum. Texas Red dye-conjugated Affinipure goat anti-mouse (Jackson Immunoresearch) was used as the secondary at a concentration of 1:200, and was applied to the slices for two hours. Slices were then mounted onto slides using Vectastain’s mounting solution. Protocol was repeated with Pan-axonal neurofilament marker as the primary antibody.

Biocytin Staining: 0.5% biocytin was added into the intracellular solution during the recording phase. Then 1% hydrogen peroxide, 10% methanol, and 2% albumin were added to the slices for 30 minutes. After rinsing, 0.1% Triton, 2% albumin, and the ABC staining kit was applied to the slices overnight. A Vector Kit was then used to stain the slices for 10 minutes.

Morphological Reconstruction: Neurolucida was used after the Biocytin protocol.

Conclusions and Future Studies

Anti-tau-1 was used to identify appendages in order to better interpret data from electrophysiological recordings.

However, in test brain slices, it failed to label Globus Pallidus neurons. The images were unclear due to high background color.

Pan-axonal neurofilament marker gave a clear label of axons throughout all structures in the brain slices.

In the future, neurons that have been recorded from will be stained with biocytin and pan-axonal neurofilament marker. This will identify each process as either dendrite or axon. This data will be used for building models and statistical analysis of electrophysiological properties.

Resources

Funded by HHMI Grant # 52005873 and RO1 NS039852-05