Expanding the Toolkit for In Vivo Imaging of Axonal Transport

Using transgenic fluorescent mice, detailed protocols are described to assess in vivo axonal transport of signaling endosomes and mitochondria within motor and sensory axons of the intact sciatic nerve in live animals.

Using our protocol, we can visualize and quantitatively assess the dynamics of signaling endosomes and mitochondria within axons from motor and sensory neurons of live anesthetized mice. This method that can be used to understand the basal physiology of axons in vivo and reveal important patho-mechanism driving peripheral neurodegeneration in mouse model of disease. Our technique can be used to assess the effect of potential treatments such as pharmacological agents and gene therapies on axonal transport in live motor and sensory neurons.

This technique can be used to simultaneously image different organelles in a specific peripheral nerve axon and relay the study of motor disease models as well as aging. Start the preparations by pulling a graded glass micropipette for optimal intramuscular injections into smaller muscles. For the surgery, secure a sterile surgical drape on a heat mat set to 37 degrees Celsius and position and focus the operating microscope.

Then, unpack all the required surgical tools and instruments as described in the manuscript. When the preparations are done, transfer the anesthetized mouse to the mouthpiece located in a separate area of the surgical space and ensure both the corneal and pedal withdrawal reflexes are absent before shaving the fur off the surgical area. Then, remove the shaved fur from the mouse using the sticky side of surgical tape.

Next, place the mouse on weighing scales to record the pre-surgical weight. Then, use a cotton swab to apply eye lubricant and transfer the mouse and mouthpiece to the surgical area. To inject the tibialis anterior, or TA muscle, place the mouse on the back and stretch out the hind limb at 10 degrees from the midline.

When the hind limb is in the correct position, place a surgical tape across the foot. Sterilize the shaved area using a 70%ethanol or equivalent solution. Draw the working atoxic fragment of tetanus, neurotoxin, or HcT solution into a pulled glass micropipette.

Next, make a small incision over the muscle of interest in the area that corresponds with the motor and plate regions.Then. pierce the external fascia on the muscle to inject the HcT. Leave the micropipette in position for five to 10 seconds before slowly withdrawing.

Post the injection, close the incisions with one to two stitches. Then, transfer the mouse to an isolated recovery cage under observation for 30 minutes. Prepare to expose the sciatic nerve by arranging the surgical tools and instruments around the surgical area.

Create a wedge by cutting parafilm into a narrow rectangle of width one centimeter with an angled tip to aid the imaging process. Once the preparations are done, transfer the mouse to the mouthpiece in the surgical area and use surgical tape to secure the head to the mouthpiece. Extend and secure the targeted hind limb at 45 degrees from the midline with surgical tape.

If corneal and pedal withdrawal reflexes are absent, cut the skin overlying the sciatic nerve using scissors. Then, remove the overlying biceps femoris muscle and musculature or connective tissue without damaging the sciatic nerve and blood vessels. When the intact sciatic nerve is sufficiently exposed, apply pre-warmed sterile saline to the area around the sciatic nerve to prevent desiccation.

Then, use curved forceps to disrupt the deep-lying connective tissue and place the pre-prepared parafilm wedge underneath the nerve. Place saline-soaked cotton wool on the exposed area before moving the mouse on top of the heat mat into the induction chamber filled with isoflurane and oxygen. For the imaging, place a 22-by-64 millimeter cover glass on the customized microscope stage and secure the position of the cover glass with a tape.

After applying immersion oil to the objective, connect the microscope stage to the inverted microscope and slowly raise the oil-immersed objective to contact the cover glass. When the setup is ready, secure the anesthetic mouthpiece onto the microscope stage using tape. Then, remove the cotton wool from the sciatic nerve and transfer the mouse from the induction chamber to the mouthpiece with the exposed nerve facing the cover glass.

Secure the mouse's head to the mouthpiece, and while maintaining the lowest adequate level of anesthesia, gently lift the mouse by its tail to add sterile saline to the cover slip near the exposed sciatic nerve. With the help of the oculars, locate the sciatic nerve to determine the optimal focal point and select an area of interest containing motile axonal organelles. Next, switch to the computer software by clicking the Acquisition button and selecting an area of interest.

Use digital zoom to obtain an 80-fold magnification and rotate the selected area to visualize the axons horizontally Click the Regions box and select a rectangular region of interest. In the Acquisition Mode, set the frame size to a minimum of 1024-by-1024 pixels and commence time lapse acquisition of 100 to 1, 000 frames. Capture a minimum of 10 motile cargoes from the three axons per mouse.

In the study, in vivo motor axon-specifc labeling was achieved using transgenic mice. The enhanced green florescence protein, or eGFP expression, in cholinergic motor axons was observed from a live, anesthetized ChAT. eGFP mouse.

With the alternative method, tdTomato expression in a freshly excised nerve from a ChAT. tdTomato mouse was attained without additional tissue processing. Additionally, axons were identified by injecting tracers or markers such as HcT or adenoviruses encoding eGFP into skeletal muscles.

The representative analysis demonstrates a time lapse image series representing in vivo axonal transport of signaling endosomes in live motor neurons of an HB9. GFP mouse. The GFP had a more granular pattern in HB9.

GFP axons. The breeding of Mito. CFP mice with ChAT.

tdTomato mice enabled the visualization of mitochondria in the motor axons. Moreover, the Node of Ranvier could also be identified. The HcT injection into the muscles of the Mito.

CFP mice allowed concurrent visualization of signaling endosomes and mitochondria within the same axons in vivo. Anterogradely and retrogradely moving organelles, as well as stalled organelles, were observed. Reduced anesthesia during the imaging process is advantageous because it can limit the impact of large breathing artifacts, and thus simplify the tracking and analysis of axonal transport.

Sciatic nerves can be instructive for RNA and protein analysis to correlate the organelle dynamics with key components of the axonal transport machinery, such as motor proteins and cargo-specific adapters.