NaV1.9 channels in muscle afferent neurons and axons.

The exercise pressor reflex (EPR) is activated by muscle contractions to increase heart rate and blood pressure during exercise. While this reflex is beneficial in healthy individuals, the reflex activity is exaggerated in patients with cardiovascular disease, which is associated with increased mortality. Group III and IV afferents mediate the EPR and have been shown to express both tetrodotoxin-sensitive (TTX-S, NaV1.6, and NaV1.7) and -resistant (TTX-R, NaV1.8, and NaV1.9) voltage-gated sodium (NaV) channels, but NaV1.9 current has not yet been demonstrated. Using a F--containing internal solution, we found a NaV current in muscle afferent neurons that activates at around -70 mV with slow activation and inactivation kinetics, as expected from NaV1.9 current. However, this current ran down with time, which resulted, at least in part, from increased steady-state inactivation since it was slowed by both holding potential hyperpolarization and a depolarized shift of the gating properties. We further show that, following NaV1.9 current rundown (internal F-), application of the NaV1.8 channel blocker A803467 inhibited significantly more TTX-R current than we had previously observed (internal Cl-), which suggests that NaV1.9 current did not rundown with that internal solution. Using immunohistochemistry, we found that the majority of group IV somata and axons were NaV1.9 positive. The majority of small diameter myelinated afferent somata (putative group III) were also NaV1.9 positive, but myelinated muscle afferent axons were rarely labeled. The presence of NaV1.9 channels in muscle afferents supports a role for these channels in activation and maintenance of the EPR. NEW & NOTEWORTHY Small diameter muscle afferents signal pain and muscle activity levels. The muscle activity signals drive the cardiovascular system to increase muscle blood flow, but these signals can become exaggerated in cardiovascular disease to exacerbate cardiac damage. The voltage-dependent sodium channel NaV1.9 plays a unique role in controlling afferent excitability. We show that NaV1.9 channels are expressed in muscle afferents, which supports these channels as a target for drug development to control hyperactivity of these neurons.

Inhibition of α9α10 nicotinic acetylcholine receptors prevents chemotherapy-induced neuropathic pain.

Opioids are first-line drugs for moderate to severe acute pain and cancer pain. However, these medications are associated with severe side effects, and whether they are efficacious in treatment of chronic nonmalignant pain remains controversial. Medications that act through alternative molecular mechanisms are critically needed. Antagonists of α9α10 nicotinic acetylcholine receptors (nAChRs) have been proposed as an important nonopioid mechanism based on studies demonstrating prevention of neuropathology after trauma-induced nerve injury. However, the key α9α10 ligands characterized to date are at least two orders of magnitude less potent on human vs. rodent nAChRs, limiting their translational application. Furthermore, an alternative proposal that these ligands achieve their beneficial effects by acting as agonists of GABAB receptors has caused confusion over whether blockade of α9α10 nAChRs is the fundamental underlying mechanism. To address these issues definitively, we developed RgIA4, a peptide that exhibits high potency for both human and rodent α9α10 nAChRs, and was at least 1,000-fold more selective for α9α10 nAChRs vs. all other molecular targets tested, including opioid and GABAB receptors. A daily s.c. dose of RgIA4 prevented chemotherapy-induced neuropathic pain in rats. In wild-type mice, oxaliplatin treatment produced cold allodynia that could be prevented by RgIA4. Additionally, in α9 KO mice, chemotherapy-induced development of cold allodynia was attenuated and the milder, temporary cold allodynia was not relieved by RgIA4. These findings establish blockade of α9-containing nAChRs as the basis for the efficacy of RgIA4, and that α9-containing nAChRs are a critical target for prevention of chronic cancer chemotherapy-induced neuropathic pain.

EXPRESS: Voltage-dependent sodium (NaV) channels in group IV sensory afferents.

Patients with intermittent claudication suffer from both muscle pain and an exacerbated exercise pressor reflex. Excitability of the group III and group IV afferent fibers mediating these functions is controlled in part by voltage-dependent sodium (NaV) channels. We previously found tetrodotoxin-resistant NaV1.8 channels to be the primary type in muscle afferent somata. However, action potentials in group III and IV afferent axons are blocked by TTX, supporting a minimal role of NaV1.8 channels. To address these apparent differences in NaV channel expression between axon and soma, we used immunohistochemistry to identify the NaV channels expressed in group IV axons within the gastrocnemius muscle and the dorsal root ganglia sections. Positive labeling by an antibody against the neurofilament protein peripherin was used to identify group IV neurons and axons. We show that >67% of group IV fibers express NaV1.8, NaV1.6, or NaV1.7. Interestingly, expression of NaV1.8 channels in group IV somata was significantly higher than in the fibers, whereas there were no significant differences for either NaV1.6 or NaV1.7. When combined with previous work, our results suggest that NaV1.8 channels are expressed in most group IV axons, but that, under normal conditions, NaV1.6 and/or NaV1.7 play a more important role in action potential generation to signal muscle pain and the exercise pressor reflex.

Functional expression of α7-nicotinic acetylcholine receptors by muscle afferent neurons.

The exercise pressor reflex (EPR) is generated by group III and IV muscle afferents during exercise to increase cardiovascular function. Muscle contraction is triggered by ACh, which is metabolized into choline that could serve as a signal of exercise-induced activity. We demonstrate that ACh can induce current in muscle afferents neurons isolated from male Sprague-Dawley rats. The nicotinic ACh receptors (nAChRs) appear to be expressed by some group III-IV neurons since capsaicin (TRPV1) and/or ATP (P2X) induced current in 56% of ACh-responsive neurons. α7- And α4ß2-nAChRs have been shown to be expressed in sensory neurons. An α7-nAChR antibody stained 83% of muscle afferent neurons. Functional expression was demonstrated by using the specific α7-nAChR blockers α-conotoxin ImI (IMI) and methyllycaconitine (MLA). MLA inhibited ACh responses in 100% of muscle afferent neurons, whereas IMI inhibited ACh responses in 54% of neurons. Dihydro-ß-erythroidine, an α4ß2-nAChR blocker, inhibited ACh responses in 50% of muscle afferent neurons, but recovery from block was not observed. Choline, an α7-nAChR agonist, elicited a response in 60% of ACh-responsive neurons. Finally, we demonstrated the expression of α7-nAChR by peripherin labeled (group IV) afferent fibers within gastrocnemius muscles. Some of these α7-nAChR-positive fibers were also positive for P2X3 receptors. Thus choline could serve as an activator of the EPR by opening α7-nAChR expressed by group IV (and possible group III) afferents. nAChRs could become pharmacological targets for suppressing the excessive EPR activation in patients with peripheral vascular disease.

Physiological slowing and upregulation of inhibition in cortex are correlated with behavioral deficits in protein malnourished rats.

Protein malnutrition during early development has been correlated with cognitive and learning disabilities in children, but the neuronal deficits caused by long-term protein deficiency are not well understood. We exposed rats from gestation up to adulthood to a protein-deficient (PD) diet, to emulate chronic protein malnutrition in humans. The offspring exhibited significantly impaired performance on the 'Gap-crossing' (GC) task after reaching maturity, a behavior that has been shown to depend on normal functioning of the somatosensory cortex. The physiological state of the somatosensory cortex was examined to determine neuronal correlates of the deficits in behavior. Extracellular multi-unit recording from layer 4 (L4) neurons that receive direct thalamocortical inputs and layers 2/3 (L2/3) neurons that are dominated by intracortical connections in the whisker-barrel cortex of PD rats exhibited significantly low spontaneous activity and depressed responses to whisker stimulation. L4 neurons were more severely affected than L2/3 neurons. The response onset was significantly delayed in L4 cells. The peak response latency of L4 and L2/3 neurons was delayed significantly. In L2/3 and L4 of the barrel cortex there was a substantial increase in GAD65 (112% over controls) and much smaller increase in NMDAR1 (12-20%), suggesting enhanced inhibition in the PD cortex. These results show that chronic protein deficiency negatively affects both thalamo-cortical and cortico-cortical transmission during somatosensory information processing. The findings support the interpretation that sustained protein deficiency interferes with features of cortical sensory processing that are likely to underlie the cognitive impairments reported in humans who have suffered from prolonged protein deficiency.

Identification of CaV channel types expressed in muscle afferent neurons.

Cardiovascular adjustments to exercise are partially mediated by group III/IV (small to medium) muscle afferents comprising the exercise pressor reflex (EPR). However, this reflex can be inappropriately activated in disease states (e.g., peripheral vascular disease), leading to increased risk of myocardial infarction. Here we investigate the voltage-dependent calcium (CaV) channels expressed in small to medium muscle afferent neurons as a first step toward determining their potential role in controlling the EPR. Using specific blockers and 5 mM Ba(2+) as the charge carrier, we found the major calcium channel types to be CaV2.2 (N-type) > CaV2.1 (P/Q-type) > CaV1.2 (L-type). Surprisingly, the CaV2.3 channel (R-type) blocker SNX482 was without effect. However, R-type currents are more prominent when recorded in Ca(2+) (Liang and Elmslie 2001). We reexamined the channel types using 10 mM Ca(2+) as the charge carrier, but results were similar to those in Ba(2+). SNX482 was without effect even though ∼27% of the current was blocker insensitive. Using multiple methods, we demonstrate that CaV2.3 channels are functionally expressed in muscle afferent neurons. Finally, ATP is an important modulator of the EPR, and we examined the effect on CaV currents. ATP reduced CaV current primarily via G protein ßγ-mediated inhibition of CaV2.2 channels. We conclude that small to medium muscle afferent neurons primarily express CaV2.2 > CaV2.1 ≥ CaV2.3 > CaV1.2 channels. As with chronic pain, CaV2.2 channel blockers may be useful in controlling inappropriate activation of the EPR.

NaV1.8 channels are expressed in large, as well as small, diameter sensory afferent neurons.

Sensory neurons in the dorsal root ganglia (DRG) express a subset of voltage dependent sodium channels (NaV) including NaV1.1, 1.6, 1.7, 1.8 and 1.9. Previous work supported preferential localization of NaV1.8 channels to small-medium diameter, nociceptive afferent neurons. However, we recently published evidence that NaV1.8 was the dominant NaV channel expressed in the somas of small, medium and large diameter muscle afferent neurons, which is consistent with other reports. Here, we extend those results to show that NaV1.8 expression is not correlated with afferent neuron diameter. Using immunocytochemistry, we found NaV1.8 expression in ~50% of sensory afferent neurons with diameters ranging from 20 to 70 µm. In addition, electrophysiological analysis shows that the kinetic and inactivation properties of NaV1.8 current are invariant with neuron size. These data add further support to the idea that NaV1.8 contributes to the electrical excitability of both nociceptive and non-nociceptive sensory neurons.

Identification of specific sensory neuron populations for study of expressed ion channels.

Sensory neurons transmit signals from various parts of the body to the central nervous system. The soma for these neurons are located in the dorsal root ganglia that line the spinal column. Understanding the receptors and channels expressed by these sensory afferent neurons could lead to novel therapies for disease. The initial step is to identify the specific subset of sensory neurons of interest. Here we describe a method to identify afferent neurons innervating the muscles by retrograde labeling using a fluorescent dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate). Understanding the contribution of ion channels to excitation of muscle afferents could help to better control excessive excitability induced by certain disease states such as peripheral vascular disease or heart failure. We used two approaches to identify the voltage dependent ion channels expressed by these neurons, patch clamp electrophysiology and immunocytochemistry. While electrophysiology plus pharmacological blockers can identify functional ion channel types, we used immunocytochemistry to identify channels for which specific blockers were unavailable and to better understand the ion channel distribution pattern in the cell population. These techniques can be applied to other areas of the nervous system to study specific neuronal groups.

Tetrodotoxin-resistant voltage-dependent sodium channels in identified muscle afferent neurons.

Muscle afferents are critical regulators of motor function (Group I and II) and cardiovascular responses to exercise (Group III and IV). However, little is known regarding the expressed voltage-dependent ion channels. We identified muscle afferent neurons in dorsal root ganglia (DRGs), using retrograde labeling to examine voltage-dependent sodium (Na(V)) channels. In patch-clamp recordings, we found that the dominant Na(V) current in the majority of identified neurons was insensitive to tetrodotoxin (TTX-R), with Na(V) current in only a few (14%) neurons showing substantial (>50%) TTX sensitivity (TTX-S). The TTX-R current was sensitive to a Na(V)1.8 channel blocker, A803467. Immunocytochemistry demonstrated labeling of muscle afferent neurons by a Na(V)1.8 antibody, which further supported expression of these channels. A portion of the TTX-R Na(V) current appeared to be noninactivating during our 25-ms voltage steps, which suggested activity of Na(V)1.9 channels. The majority of the noninactivating current was insensitive to A803467 but sensitive to extracellular sodium. Immunocytochemistry showed labeling of muscle afferent neurons by a Na(V)1.9 channel antibody, which supports expression of these channels. Further examination of the muscle afferent neurons showed that functional TTX-S channels were expressed, but were largely inactivated at physiological membrane potentials. Immunocytochemistry showed expression of the TTX-S channels Na(V)1.6 and Na(V)1.7 but not Na(V)1.1. Na(V)1.8 and Na(V)1.9 appear to be the dominant functional sodium channels in small- to medium-diameter muscle afferent neurons. The expression of these channels is consistent with the identification of these neurons as Group III and IV, which mediate the exercise pressor reflex.

The effect of striatal dopaminergic grafts on the neuronal activity in the substantia nigra pars reticulata and subthalamic nucleus in hemiparkinsonian rats.

The electrophysiological correlates of parkinsonism in the basal ganglia have been well studied in patients with Parkinson's disease and animal models. Separately, striatal dopaminergic cell transplantation has shown promise in ameliorating parkinsonian motor symptoms. However, the effect of dopaminergic grafts on basal ganglia electrophysiology has not thoroughly been investigated. In this study, we transplanted murine foetal ventral mesencephalic cells into rats rendered hemiparkinsonian by 6-hydroxydopamine injection. Three months after transplantation, extracellular and local field potential recordings were taken under urethane anaesthesia from the substantia nigra pars reticulata and subthalamic nucleus along with cortical electroencephalograms and were compared to recordings from normal and hemiparkinsonian controls. Recordings from cortical slow-wave activity and global activation states were analysed separately. Rats with histologically confirmed xenografts showed behavioural improvement measured by counting apomorphine-induced rotations and with the extended body axis test. Firing rates in both nuclei were not significantly different between control and grafted groups. However, burst firing patterns in both nuclei in the slow-wave activity state were significantly reduced (P < 0.05) in rats with large surviving grafts, compared to hemiparkinsonian controls. The neuronal firing entropies and oscillations in both nuclei were restored to normal levels in the large-graft group. Electroencephalogram spike-triggered averages also showed normalization in the slow-wave activity state (P < 0.05). These results suggest that local continuous dopaminergic stimulation exerts a normalizing effect on the downstream parkinsonian basal ganglia firing patterns. This novel finding is relevant to future preclinical and clinical investigations of cell transplantation and the development of next-generation therapies for Parkinson's disease that ameliorate pathophysiological neural activity and provide optimal recovery of function.