Age-Dependent Roles of C1q in Ia Afferent Synapse Dynamics Post-Peripheral Nerve Injury: A Comparative Study in Neonates and Adults Pubblico
Bianco, Celia (Spring 2024)
Abstract
Peripheral nerve injuries, especially those involving nerve transections, frequently cause reduced functionality. These injuries lead to the irreversible loss of Ia proprioceptive synapses from their target motoneurons, preventing full functional recovery despite nerve regeneration and muscle reinnervation. Consequently, the monosynaptic muscle stretch reflex disappears, leading to impaired fine motor control in affected individuals. Crush injuries, however, do not cause this permanent loss of Ia afferent synapses, and therefore, the muscle stretch reflex returns. The recovery pattern after crush injuries shifts dramatically during the early postnatal period, a crucial phase for developing weight-bearing movements. Injuries inflicted at this stage result in far greater and more permanent Ia afferent synaptic losses than when this injury occurs during adulthood. Notably, the extent of Ia afferent synaptic loss from neonatal crush injuries closely mirrors that of full nerve transections in adults. These permanent synaptic losses occur despite successful muscle reinnervation and no evidence of peripheral immune cell invasion, which has been implicated in removing these contacts after nerve transections in adults. This early developmental window is marked by an upregulation of complement proteins by microglia, triggering the complement cascade for developmental synaptic pruning to refine motor circuits. Considering the pivotal role of C1q, the complement cascade's initiating protein, in synaptic pruning during both development and pathology, our study investigates C1q's involvement in post-injury synaptic pruning and its differential impact between neonates and adults. We examined two cohorts: neonates subjected to sciatic nerve crush injuries, with and without global C1q deletion, and adults undergoing sciatic nerve transection, including animals with C1q selectively removed from microglia prior to injury, and their genetic control littermates. Our analysis on the quantity of VGLUT1 synaptic boutons on injured motoneurons reveals a stark contrast; unlike in adults, where synaptic contacts decrease after nerve transection regardless of C1q presence, VGLUT1 contacts in neonates are preserved in the absence of C1q. However, these synapses are significantly reduced in size, indicating diminished functionality. This finding emphasizes C1q's distinct role in synaptic pruning following nerve injuries across developmental stages and suggests an age-dependent mechanism of synaptic loss post-injury. It provides new insights into the complex interplay between injury, development, and synaptic plasticity.
Table of Contents
Introduction…………………………………………………………………….……………..…...1
Epidemiology of Nerve Injury…………………………………………………………….1
Degenerative Processes Following Peripheral Nerve Damage……………………………2
Peripheral Nerve Regeneration……………………………………………………………5
Sensory Input Recovery in the Central Nervous System………………………………….8
The Critical Window of Developmental Synapse Pruning………………………………10
Materials and Methods…………………………………………………………………………...12
Transgenic Models and Experimental Groups ………………………………………..…12
Tamoxifen Treatment and PNI Procedures………………………………………………14
Harvesting Tissue for Histological Analysis………………………………………….…16
Histological Processing and Immunohistochemistry……………………………….……18
Analysis of VGLUT1 Densities on 3D Reconstructed MNs…………………….………21
Analysis of VGLUT1 Contact Size…………………………………………………...…22
Quantifying Muscle Reinnervation…………………………………………………...….22
Statistical Comparisons of VGLUT1 Synaptic Density..…………………………..……23
Statistical Comparisons of VGLUT1 Synapses Sizes.………………………………….25
Results…………………………………………………………………………………………....26
C1q is necessary for VGLUT1 synaptic losses after nerve crush injury in neonates........27
The size of preserved synapses decreases in both WTs and C1q global Kos……………32
At the end point used in the study (18 days post-injury) muscle reinnervation is………35
similar and complete, independent of C1q presence.
Microglia express and release C1q after nerve injury in adult spinal cords......................37
C1q is not necessary for VGLUT1 synapse plasticity 14 days after nerve transection….40
in adults
Figures and Tables……………………………………………………………………………….14
Table 1. Mouse Models…………………………………………………………………..14
Table 2. Antibodies Used for Immunohistochemistry…………………………………...20
Table 3. Interanimal Variability in Different Experimental Groups for VGLUT1……...24
Density (one-way ANOVAs)
Table 4. Interanimal Variability in Different Experimental Groups for Synapse Size......25
Figure 1. VGLUT1-IR Synapse Loss, Microglia Reaction and C1q 18 days after….......28
P12 Sciatic Nerve Crush.
Figure 2. Preservation of VGLUT1-IR Synapses Following Nerve Crush Injury in........31
Postnatal Mice Lacking C1q
Figure 3. Neonate Synapse Size After Injury in Wild-Types and C1q Global KOs.........34
Figure 4. Reinnervation of Motor end plates in Wild-Types and C1q Global KOs..........36
Figure 5. Successful Depletion of C1q from Microglia in Adult Conditional C1q KO....39 Mice
Figure 6. Lack of Preservation of VGLUT1-IR Synapses After Nerve Cut Injury...........42
in C1q Conditional KO Adult Mice
Figure 7. Adult Synapse Size is Unaffected by Injury or Presence of C1q.......................43
Discussion......................................................................................................................................43
Future Directions...............................................................................................................46
References......................................................................................................................................48
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