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Reorganization of Ia afferent synapses on motoneurons after peripheral nerve injuries

Haley Titus

June 11th, 2009

Advisor: Dr. Francisco Alvarez

The Spinal Cord and the Stretch Reflex

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Big Picture- Patients lose and do not recover the stretch reflex after peripheral nerve injury.

The Peripheral connections of this circuit are functional after peripheral regeneration:

-the Ia afferent (Ia)

-the axon from the motoneuron (MN)

We decided to study the anatomy of the Central connection

between the Ia afferent and the motoneuron.

Sensory input

Motor output

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Overall Aim and Hypothesis

• This thesis explores the possibility that failure in Ia to motoneuron neurotransmission during stretch evoke activity is in part due to a central structural reorganization of Ia synapses on motoneurons in rat.

• The overall hypothesis is that the density and distribution of Ia synapses on motoneurons is altered after peripheral nerve injuries in rat.

Specific Aim 1

Aim 1 analyzed VGLUT1 content, labeling Ia afferents, on motoneurons in the tibial pools of Lamina IX (LIX) in the rat spinal cord with peripheral tibial nerve injuries in which afferents and motor axons were allowed to reinnervate (resutured) or were prevented from reinnervation (ligated).

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Topography of the Lumbar Spinal Cord in Rats(Molander et al., 1984)

Tibial nerve motoneurons in the Lumbar spinal cord of the Rat

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Vesicular glutamate transporter 1 (VGLUT1) loads excitatory neurotransmitter glutamate into small, clear vesicles.

VGLUT1 synapses in the spinal cord are most frequently originated in sensory mechanoreceptors, including cutaneous and proprioceptors.

What is VGLUT1?

Vesicular glutamate transporter 1 (VGLUT1) in FITC

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VGLUT1 density in Lamina IX of the Lumbar Spinal Cord after peripheral nerve injury in the rat

NeuN – Neuronal Nuclear Protein

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NeuN-IR density in Lamina IX of the Lumbar Spinal Cord after peripheral nerve injury in the rat

VGLUT1 + NeuN

50 µm

Analysis of Synaptic ContactsSynaptic density = # of VGLUT1 contacts per 100μm perimeter of soma

Step =

.5μm

Step =

.5μm

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VGLUT1 contacts on Motoneurons in the Tibial Pool

Specific Aim 1: Results and Discussion

• These data suggest that a significant depletion of VGLUT1 contacts in lamina IX of the rat spinal cord occurs after peripheral nerve injury independent of whether there is reinnervation or not.

• Additionally, the tibial nerve injury transected axons from multiple motoneuron pools targeting three different muscle groups and in aim 1 it was possible that motoneurons analyzed reinnervated the appropriate or inappropriate target.

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Specific Aim 2

Aim 2 analyzed changes in the density of VGLUT1 contacts on the cell bodies of MG motoneurons that appropriately reinnervated into the MG muscle after MG nerve injury or Tibial nerve injury in the rat.

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Density of VGLUT1 contacts on the Soma of Reinnervated MG Motoneurons - Group Analysis for Tibial Nerve Injury (N=4 Rats)

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Interanimal variability for Tibial Nerve Injury – individual animal analysis (N=8-12 motoneurons)

Nerve Injury Models

• In the Tibial nerve cut and resuture injury model, all Ia afferents that travel within the tibial nerve are affected.

• The MG nerve is a branch of the Tibial nerve. In the MG nerve cut and resuture injury model only MG Ia afferents are effected.

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Specific Aim 2: Results

• These data suggest that significant depletion of VGLUT1 contacts around the soma occur after peripheral nerve injury and reinnervation in the rat.

• The depletion appeared to be permanent.

• There was not a statistically significant difference between MG and tibial nerve injury models.

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Specific Aim 3

Aim 3 analyzed the change in density of VGLUT1 on the dendritic arbor of MG motoneurons that appropriately reinnervated into the MG muscle after Tibial nerve injury in the rat.

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Total dendritic arbor analysis

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Sholl dendritic analysis

Specific Aim 3

• These data suggest a reorganization of VGLUT1 contacts on the dendritic arbor in the rat.

• These data suggest there was not a significant depletion in VGLUT1 synaptic contacts in the total dendritic arbor compared to the control after appropriate long term reinnervation of the alpha MG motoneuron.

• These data suggest depletion in VGLUT1 synaptic contacts in the proximal dendritic arbor after appropriate reinnervation of the alpha MG motoneuron.

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Conclusion

•Reorganization of VGLUT1 Ia afferent synapses on motoneuron somatodendritic surfaces occurs after peripheral nerve injuries in the rat.

•There is a loss and lack of recovery of the more proximal VGLUT1 synapses located in the cell somata and in the initial primary dendrite segments. Otherwise known as proximal synaptic stripping.

• Input now targets more distal dendrites.

•These changes appear to be relatively permanent.

•Suggested to play a role in the strength of the central synaptic connection of the monosynaptic stretch circuit.

Acknowledgements

• I would like to express my appreciation for Dr. Francisco Alvarez. He is always optimistic and has been a wonderful teacher and mentor.

• I would like to thank the other members of my committee, Dr. Mark Rich and Dr. Timothy Cope, for their input and support of my project.

• I would like to thank the entire Alvarez Lab for helping me learn new techniques at the bench.

• I would like to thank the entire Cope Lab for their collaborative efforts on this project and the excitement all members brought to every experiment.

• I would like to acknowledge Erin Fulchiero for offering encouragement on a daily basis and being a great friend this past year.

• I would like to thank my entire extended family for encouraging me to work hard and have a passion for what I am trying to accomplish.

• Lastly, I would like to express gratitude for my loving boyfriend Chris Mitchell who has spent a good deal of his free time accompanying me in lab during the evenings and over the weekends for the past year.