Abstract
Mice with transgenic insertion of code for enhanced green fluorescent protein (EGFP) at the locus for glutamic acid decarboxylase 67 (GAD67), one of two key enzymes for the synthesis of γ-aminobutyric acid (GABA) were used to test whether the morphological properties of these neurons show plasticity with nerve injury. Physiological properties and the delivery of intracellular label to EGFP-expressing lamina II neurons was done using whole-cell patch-clamp in spinal cord slices from sham and chronic constriction injury (CCI) mice. As well, whole cell recordings were made of non-EGFP labeled cells to ascertain changes in overall inhibitory signaling following CCI. The EGFP labeled neurons in both sham and CCI mice exhibited islet, central and vertical cell morphological profiles but no radial cell profiles were observed. The length of cell dendrites was found to be significantly shorter in CCI mice for all cell profile types. The longest neurites averaged 155.96 ± 18.29 μm in CCI mice compared to 334.93 ± 29.48 μm in sham control mice. No change was observed in either passive or evoked membrane properties of EGFP-expressing neurons in CCI versus sham mice. Meanwhile, the frequency of miniature inhibitory post-synaptic currents of non-EGFP expressing spinal lamina II neurons was significantly reduced. These results suggest that reduced inhibitory output from GABA neurons occurs with nerve injury in part due to altered cell morphology.
Highlights
Spinal Lamina II has long been known to play a role in modulating incoming sensory afferent information (Light and Perl, 1979; Sugiura et al, 1986); and this process is becoming better defined as specific subpopulations of excitatory and inhibitory neurons and their connectivity is becoming revealed (Peirs et al, 2015; Todd, 2017)
The withdrawal threshold to mechanical stimulation showed a significant decrease in the paw ipsilateral to the constriction injury (CCI) compared with the contralateral paw (Figure 1) and to withdrawal threshold in sham mice by 1 day after surgery; and this difference lasted at least 7 days similar to that previously observed in the laboratory (Zhang et al, 2013)
Spinal cord slices from L4 to L6 were acutely prepared and used to investigate membrane and discharge properties of GABA neurons identified in spinal lamina II by visualization of green fluorescence
Summary
Spinal Lamina II has long been known to play a role in modulating incoming sensory afferent information (Light and Perl, 1979; Sugiura et al, 1986); and this process is becoming better defined as specific subpopulations of excitatory and inhibitory neurons and their connectivity is becoming revealed (Peirs et al, 2015; Todd, 2017). Anatomical studies have shown that roughly one third of lamina II neurons contain the inhibitory neurotransmitter γ-aminobutyric acid (GABA) (Todd and Sullivan, 1990; Polgar et al, 2003). GABA is synthesized from glutamate by the enzyme glutamic acid decarboxylase (GAD) and so can be used as a marker for these inhibitory interneurons. Aδ-fiber and C-fiber terminals, mediating presynaptic inhibition (Todd, 2010); and GABA is mediates post-synaptic inhibition to spinal dorsal horn neurons (Magoul et al, 1987; Bernardi et al, 1995). Spinal application of the GABAA receptor antagonist bicuculline produces behavioral signs touch evoked pain (Yaksh, 1989; Malan et al, 2002) and enhanced reflex activity (Sivilotti and Woolf, 1994); while GABA receptor agonists decrease pain-like behavior after nerve injury (Malan et al, 2002)
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