The role of C1q in Ia afferent synapse removal and motoneuron regeneration after peripheral nerve injuries during early postnatal development. Open Access

Abstract

After mild nerve crush injuries in young developing animals, the synapses on

motoneurons from muscle length sensing Ia afferents are permanently removed, in contrast to

what occurs after the same injuries in adults, which recover these synapses. This impairs the

ability of motoneurons to receive direct proprioceptive information during movement and

respond to muscle stretch. During development, microglia facilitate motor circuitry refinement

by increasing the release of protein C1q to remove unnecessary Ia synapses. This process is

important for establishing Ia afferent connections specifically with homonymous and synergistic

motoneurons and prevent synapse formation on antagonist motoneurons that will lead to cocontraction

of muscles with opposite actions around joints. Thus, we hypothesized that the

upregulation of the protein C1q during development leads to a larger loss of Ia synapses after a

sciatic nerve crush in young developing animals. To investigate the role of C1q after a sciatic

nerve crush, we compared Ia synapse losses in a C1q knockout model (C1q KO) to wild types,

both receiving a sciatic nerve crush at postnatal day (P12). We assessed Ia synapse densities by

immunostaining for the vesicular glutamate transporter isoform 1 (VGLUT1) which is a specific

marker of Ia synapses on motoneurons. Eighteen days post sciatic crush injury (P30 of age) both

wild type and C1q KO had a significant depletion in VGLUT1 density on injured motoneuron

cell bodies compared to uninjured with equal effect sizes. At the same timepoint, in both C1q

KO and wild type mice there was also a significant reduction in VGLUT1 synaptic area of equal

size. Preliminarily data obtained 48 days post-injury (P60 age) show recovery of VGLUT1

synapses in C1q KO and wild type mice injured motoneurons; however, additional replicates are

necessary to derive conclusions. To conclude, the absence of C1q does not spare VGLUT1

synapses 18 days after a sciatic nerve crush injury in mice at P12, while C1q’s role 48 days postinjury

remains unclear until further quantifications. Future experiments will focus on

investigating the timepoints of microglial interaction in the absence of C1q and in examining the

functionality of these synapses both at P30 and P60.

Table of Contents

Introduction.……………………………………………………………………………………...1

The Complement Pathway...................................................................................................2

Peripheral Nerve Injuries & Regeneration.........................................................................4

Synaptic plasticity in the CNS.............................................................................................6

Hypothesis............................................................................................................................7

Materials & Methods.…………………………………………………………………………...9

Transgenic Models and Experimental Groups..................................................................9

Peripheral Nerve Injury Surgeries.....................................................................................9

Harvesting Tissue for Histological Analysis......................................................................10

Histological Processing and Immunohistochemistry.........................................................10

Analysis of VGLUT1 Densities on 3D Reconstructed Motoneurons................................13

Analysis of VGLUT1 synaptic bouton sizes.......................................................................16

Analysis of Muscle Reinnervation.....................................................................................18

Statistical Comparisons of VGLUT1 Synaptic Density and Synapse size........................18

Statistical Comparisons of Muscle Reinnervation............................................................20

Results…………………………………………………………………………………………...21

Wild type and C1q KO mice injured at P12 ......................................................................21

complete reinnervation of NMJs by motor axons 18 days post-injury (P30 age)

Ia synapses over injured motoneurons are similarly depleted in ....................................24

C1q KO mice and wild type mice 18 days after injury (P30 age)

Ia synaptic size is similar reduced in wild type and C1q KO mice...................................30

VGLUT1 synapse densities do not differ between injured and uninjured.......................32

motoneurons in wild type or C1qKO animals 48 days after injury at P60.

VGLUT1 boutons may retain smaller sizes 48 days after injury,.....................................34

but the data is unconclusive

Figures and Tables …………………………………………………………………………......9

Table 1. Mouse Models and Numbers................................................................................9

Table 2. Antibodies used for Immunohistochemistry and other reagents...................12

Table 3. Statistics data table for Figures 5D1 and 5D2...................................................24

Table 4. Statistics data table for Figure 6D......................................................................29

Table 5. Statistics data table for Figure 6F......................................................................31

Figure 1. Classical pathway of the complement cascade initiated by…………………...3

C1q release by microglia

Figure 2. Myotatic Stretch Reflex………………………………………………………..6

Figure 3. Methods for VGLUT1 density quantification..................................................14

Figure 4. Method for VGLUT1 synaptic size quantification..........................................17

Figure 5. NMJ reinnervation is complete by P30, 18 days..............................................23

post sciatic nerve crush (Data added to Bianco 2024)

Figure 6. Changes in VGLUT1 density and synaptic sizes at P30...................................26

18 days after a sciatic nerve crush injury at P12

Figure 7. Regeneration of VGLUT1 synapses and synaptic size at P60..........................35

48 days after a sciatic nerve crush injury at P12

Discussion……………………………………………………………………………................37

Future Directions………………………………………………………………………...42

References………………………………………………………………………………….......44

About this Honors Thesis

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School	Emory College
Department	Neuroscience and Behavioral Biology
Degree	B.S.
Submission	Honors Thesis
Language	English
Research Field	Biology, Cell Biology, Molecular Biology, Neuroscience
Keyword	C1q Peripheral Nerve Injury Complement Cascade Synaptic plasticity
Committee Chair / Thesis Advisor	Alvarez, Francisco , Emory University
Committee Members	Cafferty, Patrick , Emory University Roesch, Leah , Emory University

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