Changes in VGLUT1 Synaptic Connectivity and Presynaptic Inhibition Following Nerve Crush Open Access

Schultz, Adam (2016)

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Group IA afferents innervate muscle spindles in the periphery and form monosynaptic connections with motoneurons (MNs) in the spinal cord. This connection generates the stretch reflex, which is susceptible to changes following nerve injury. After nerve transection 60% of regenerated MNs lose their stretch-evoked EPSPs, resulting in a near complete loss of the stretch reflex. In contrast, after nerve crush, stretch-evoked EPSPs are maintained in regenerated MNs and recorded muscle forces during the stretch exceed 140% baseline values. This difference between crush and transection might be partially explained by better preservation of IA afferent connections on MNs and also by a decrease in the amount of presynaptic inhibitory control that modulate these connections. To test these hypotheses we used immunoreactivity of Vesicular Glutamate Transporter 1 (VGLUT1) and the 65 kDa isoform of glutamic acid-decarboxylase (GAD65) as markers of IA afferent contacts and presynaptic inhibition, respectively, and analyzed their numbers and characteristics on MNs that are regenerating (21 days post crush) or have reinnervated muscle (3 months) after nerve crush. The left tibial nerves of adult Wistar rats were subjected to a crush nerve injury while the MNs innervating the medial gastrocnemius muscle were retrogradely labeled with cholera toxin b (CtB, Alexa Fluor 555). Spinal cords were extracted, sectioned, processed for triple-color fluorescence (CtB-red, VGluT1-green and GAD65-infrared) and imaged with confocal microscopy. The dendritic arbors of individual MNs were then digitally traced, and VGLUT1 and GAD65 terminals plotted on them and counted. Our results show a slight loss of dendritic VGLUT1 terminals at both 21 day and 3 month post-crush (respectively: -33%, p<0.01 and 26%, p<0.05). This contrasts with the over 60% losses estimated in the same region of dendrite after transection. In addition, we found a significant decrease in the number of GAD65 terminals per VGLUT1 terminal in both 21 day and 3 month post-crush animals (respectively: -32%, p<0.01 and -20%, p<0.05). These results support the hypothesis that better preservation of IA afferent connections and a loss of presynaptic control could be partially responsible for the maintenance of the stretch reflex and the increase in reflex forces after nerve crush.

Table of Contents

Introduction. 1

Methods. 4

Nerve Injury and Injections. 4

Histology and Immunocytochemistry. 4

Confocal Imaging and Neuron Reconstruction. 5

Quantification. 5

Surface-to-Surface Analysis. 6

Statistical Analyses. 7

Results. 8

VGLUT1 Terminal Densities. 8

GAD65 Terminal Densities. 15

Surface Area Reconstructions. 17

Figures and Tables. 9

Table 1. 9

Figure 1. 10

Figure 2. 11

Figure 3. 12

Figure 4. 14

Figure 5. 16

Figure 6. 20

Discussion. 21

References. 25

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