Voltage-Gated Sodium Channels and the Disruption of Gastric Vagal Afferent Signaling after Spinal Cord Injury
Open Access
- Author:
- Blanke, Emily
- Graduate Program:
- Neuroscience
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- June 25, 2021
- Committee Members:
- Amy Arnold, Major Field Member
Salvatore Stella, Major Field Member
Andras Hajnal, Major Field Member
Gregory Holmes, Chair & Dissertation Advisor
Victor Ruiz-Velasco, Outside Unit & Field Member
Alistair Barber, Program Head/Chair - Keywords:
- Spinal Cord Injury
Gastric neurons
Whole-cell patch-clamp
Voltage-gated Na+ channels
SCI
NaV
visceral afferent
vagus
vagal afferent
nodose ganglia
NG - Abstract:
- Gastrointestinal (GI) dysfunctions are described as one of the “most debilitating” burdens of spinal cord injury (SCI) because these impairments not only impact the health of SCI individuals, but also greatly limit relationships and social interactions, further diminishing their quality of life. While paralysis is the most striking result of SCI, visceral sensory loss is an equally problematic and debilitating comorbidity. Digestive processes rely on the coordinated actions of the autonomic nervous system (ANS), in particular, the parasympathetic vagus nerve (VN) and the enteric nervous system (ENS). Proper function of the GI tract is critically dependent upon the transmission of sensory stimuli to higher brain areas of the central nervous system (CNS) in order to coordinate ANS function. The VN is the principal link between the periphery and the CNS, and the somas of vagal afferents reside within the nodose ganglia (NG). The vagus remains anatomically intact after SCI; however, the ANS no longer coordinates gastric functions. While the vagal efferents remain intact, they fail to respond spontaneously due to lack of presynaptic stimuli. Our recent data demonstrates post-SCI GI sensory deficits that point to the inability of vagal afferents to transmit chemical information from the GI tract. These results suggest there is pathophysiological remodeling occurring within the vagal afferents and NG. An underlying possible mechanism of vagal afferent dysfunction is gleaned from other insult models and other sensory afferent models post-SCI. Voltage-gated sodium (NaV) channels are shown to change in response to SCI in dorsal root ganglia (DRG), thalamus, dorsal horn, and motoneurons. Furthermore, in response to other insults, vagal afferents show changes in excitability in response to stimuli, and these models also suggest a NaV channel change or other ion channel disruption underly the hypoexcitability observed in the vagal afferents. Within the vagal afferents, the transmission of a neural impulse is heavily dependent on NaV1.8 channels for neurotransmission. This dissertation tests the following overarching hypothesis: following SCI, the functional reduction of vagal afferent neural excitability and gastric reflex sensitivity is mediated through reduced NaV1.8 channel expression and neurotransmission of action potentials in gastric-projecting NG afferents. The first set of experiments confirms vagal afferents from T3-SCI rats cannot respond to mechanical stimulation, as shown previously with chemical stimulation. The VN contains mechano- and chemosensory fibers as well as the motor fibers necessary for the CNS control of GI reflexes. Previous experiments demonstrate the chemosensory component of vagal afferents is disrupted in SCI; however, the mechanosensory component is not investigated. Using immunocytochemistry, we confirm the expression of two common receptors (cholecystokinin a receptor; CCKar and transient receptor potential vanilloid 1; TRPV1) within the gastric-projecting NG neurons. Cell bodies for the vagal afferent fibers are located within the NG and the majority of vagal afferent axons are unmyelinated C-fibers that are stimulated by capsaicin through activation of TRPV1 channels. Vagal afferent fibers also express receptors for GI hormones, including CCK. Immunocytochemical labeling for CCKar and TRPV1 demonstrate expression on dissociated gastric-projecting NG neurons. Using whole nerve recording, we demonstrate vagal afferent response to graded mechanical stimulation of the stomach is significantly attenuated by T3-SCI at 3-day and 3-week recovery. These findings demonstrate that T3-SCI provokes peripheral remodeling and prolonged alterations in the response of vagal afferent fibers to the physiological signals associated with digestion. The second set of experiments is a mechanistic study of the role of NaV1.8 toward the underlying SCI-induced vagal afferent pathophysiology. Whole-cell current- and voltage-clamp recordings are performed on acutely dissociated DiI-labeled gastric NG neurons to measure active and passive properties of C- and A-fibers and NaV1.8 channel biophysical characteristics. Single cell quantitative reverse transcription polymerase chain reaction (qRT-PCR) is utilized to quantify NaV1.7, 1.8, and 1.9 mRNA expression within gastric NG neurons. Acute and chronic SCI do not alter either the active and passive properties, and NaV1.8 channel biophysical properties within dissociated gastric NG neurons. However, the expression of the NaV1.7, 1.8, and 1.9 is downregulated after acute SCI. This led us to hypothesize after SCI, NaV channels trafficking is altered within the peripheral terminals of gastric vagal afferents changing their excitability properties. Taken together these findings implicate a possible mechanism for the SCI-induced gastric dysfunction. The last set of experiments begins to investigate a causal purinergic mechanism of vagal afferent dysfunction. There is strong evidence of sensory afferent neural plasticity that is mediated by purinergic receptor activation in disease states. Other studies demonstrate systemic inflammatory signaling (such as what may occur after SCI) may provoke satellite glial cell (SGC)-mediated overstimulation of purinergic receptors leading to dysregulation of NG neurons. When sensory afferent somas are exposed to inflammatory mediators, such as ATP, SGCs surrounding the somas are activated. They form a syncytium through gap junctions and release ATP onto the somas of the sensory neurons. The ATP and its byproduct ADP activate purinergic P2 receptors, such as P2X3/P2X2/3 and P2Y1, on the neurons which leads to an increase in calcium signaling and neuronal excitability. Using immunohistochemistry, whole-cell patch-clamp recordings, and Ca2+ imaging, we show gastric vagal afferent express P2X3 and P2Y1, ATP stimulation increases internal Ca2+ concentration, and ADP modestly inhibits CaV currents. Our data suggest the gastric vagal afferents have the components and respond to purinergic stimulation for this mechanism of dysregulation to occur. In conclusion, these studies focus on investigating the mechanism underlying SCI-mediated gastric vagal afferent dysfunction. We show the vagal afferent dysfunction is nonspecific to the type of stimuli in vivo. Additionally, we demonstrate that genes coding for NaV1.7, 1.8, and 1.9 are downregulated acutely after SCI within gastric NG neurons; however, NaV1.8 channel is still functional chronically and acutely in dissociated NG cell bodies. Based upon these observations, it is possible that the effects of diminished NaV channel expression may provoke disruption only within the peripheral terminals of gastric vagal afferents thus changing their excitability properties.