Leonid G. Ermilov

Immunization with neuronal nicotinic acetylcholine receptor induces neurological autoimmune disease.

Neuronal nicotinic AChRs (nAChRs) are implicated in the pathogenesis of diverse neurological disorders and in the regulation of small-cell lung carcinoma growth. Twelve subunits have been identified in vertebrates, and mutations of one are recognized in a rare form of human epilepsy. Mice with genetically manipulated neuronal nAChR subunits exhibit behavioral or autonomic phenotypes. Here, we report the first model of an acquired neuronal nAChR disorder and evidence for its pertinence to paraneoplastic neurological autoimmunity. Rabbits immunized once with recombinant alpha3 subunit (residues 1-205) develop profound gastrointestinal hypomotility, dilated pupils with impaired light response, and grossly distended bladders. As in patients with idiopathic and paraneoplastic autoimmune autonomic neuropathy, the severity parallels serum levels of ganglionic nAChR autoantibody. Failure of neurotransmission through abdominal sympathetic ganglia, with retention of neuronal viability, confirms that the disorder is a postsynaptic channelopathy. In addition, we found ganglionic nAChR protein in small-cell carcinoma lines, identifying this cancer as a potential initiator of ganglionic nAChR autoimmunity. The data support our hypothesis that immune responses driven by distinct neuronal nAChR subtypes expressed in small-cell carcinomas account for several lung cancer-related paraneoplastic disorders affecting cholinergic systems, including autoimmune autonomic neuropathy, seizures, dementia, and movement disorders.

Passive Transfer of Autoimmune Autonomic Neuropathy to Mice

Autoimmune autonomic neuropathy (AAN) is an acquired, often severe, form of dysautonomia. Many patients with AAN have serum antibodies specific for the neuronal ganglionic nicotinic acetylcholine receptor (AChR). Rabbits immunized with a fusion protein corresponding to the N-terminal extracellular domain of the ganglionic AChR α3 subunit produce ganglionic AChR antibodies and develop signs of experimental AAN (EAAN) that recapitulate the cardinal autonomic features of AAN in man. We now demonstrate that EAAN is an antibody-mediated disorder by documenting sympathetic, parasympathetic, and enteric autonomic dysfunction in mice injected with rabbit IgG containing ganglionic AChR antibodies. Recipient mice develop transient gastrointestinal dysmotility, urinary retention, dilated pupils, reduced heart rate variability, and impaired catecholamine response to stress. The autonomic signs are associated with a reversible failure of nicotinic cholinergic synaptic transmission in superior mesenteric ganglia. Mice injected with IgG from two patients with AAN (of three tested) demonstrated a milder phenotype with evidence of urinary retention and gastrointestinal dysmotility. The demonstration that ganglionic AChR-specific IgG causes impaired autonomic synaptic transmission and autonomic failure in mice implicates an antibody-mediated pathogenesis for AAN. The antibody effect is potentially reversible, justifying early use of immunomodulatory therapy directed at lowering IgG levels and abrogating IgG production in patients with AAN.

Heme oxygenase 2 is present in interstitial cell networks of the mouse small intestine

BACKGROUND & AIMS The interstitial cell (IC) network may be of fundamental importance in regulating gastrointestinal motility. Intestinal smooth muscle cells are depolarized in the absence of ICs, and there are no spontaneous slow waves. The messenger molecules between IC network and smooth muscle are unknown. Exogenous administration of CO relaxes the opossum internal anal sphincter and the guinea pig ileum, and it modulates potassium current and membrane potential of circular smooth muscle cells of the human jejunum. The aim of this study was to determine whether heme oxygenase (HO)-1 and HO-2, enzymes that catalyze the production of CO, are present in the IC network of the mouse small intestine. METHODS Antibodies specific for c-Kit, HO-1, and HO-2 were used for immunohistochemistry. Confocal images were obtained and were volume rendered, and the images were converted into three-dimensional images. RESULTS HO-2-like but not HO-1-like immunoreactivity was found in IC networks associated with the myenteric plexus and the deep muscular plexus. CONCLUSIONS HO-2 but not HO-1 is present in the IC cell network of the mouse small intestine. The enzymatic activity of HO-2 will result in the endogenous production of CO in IC networks of the mouse small intestine.

Prevertebral ganglia and intestinofugal afferent neurones

Intestinofugal afferent neurones (IFANs) are a unique subset of myenteric ganglion neurones that regulate normal gastrointestinal function. The IFANs relaying mechanosensory information to sympathetic neurones of the prevertebral ganglion (PVG) function as volume detectors. It is possible that mechanosensory information arriving in the PVG via axon collaterals of visceral spinal afferent nerves can be modulated entirely within the PVG itself.

Diaphragm muscle sarcopenia in aging mice.

Sarcopenia, defined as muscle weakness and fiber atrophy, of respiratory muscles such as the diaphragm (DIAm) has not been well characterized. The DIAm is the main inspiratory muscle and knowledge of DIAm sarcopenia is important for establishing the effects of aging on respiratory function. We hypothesized that aging is associated with a loss of DIAm force and reduced fiber cross-sectional area (CSA), and that these changes vary across fiber types. DIAm sarcopenia was assessed in young (5 month; n = 11) and old (23 month; n = 12) wild-type mice reflecting ~100 and 75% survival, respectively. In addition, DIAm sarcopenia was evaluated in BubR1(H/H) mice (n = 4) that display accelerated aging (~60% survival at 5 months) as a result of expression of a hypomorphic allele (H) of the mitotic checkpoint protein BubR1. Maximum specific force (normalized for CSA) of the DIAm was 34% less in old mice and 57% lower in BubR1(H/H) mice compared to young mice. Mean CSA of type IIx and/or IIb DIAm fibers was 27% smaller in old wild-type mice and 47% smaller in BubR1(H/H) mice compared to young mice. Mean CSA of type I or IIa fibers was not different between groups. Collectively these results demonstrate sarcopenia of the DIAm in aging wild-type mice and in BubR1(H/H) mice displaying accelerated aging. Sarcopenia may limit the ability of the DIAm to accomplish expulsive, non-ventilatory behaviors essential for airway clearance. As a result, these changes in the DIAm may contribute to respiratory complications with aging.

Structure-activity relationships in rodent diaphragm muscle fibers vs. neuromuscular junctions

The diaphragm muscle (DIAm) is a highly active muscle of mixed fiber type composition. We hypothesized that consistent with greater activation history and proportion of fatigue-resistant fibers, neuromuscular transmission failure is lower in the mouse compared to the rat DIAm, and that neuromuscular junction (NMJ) morphology will match their different functional demands. Minute ventilation and duty cycle were higher in the mouse than in the rat. The proportion of fatigue-resistant fibers was similar in the rat and mouse; however the contribution of fatigue-resistant fibers to total DIAm mass was higher in the mouse. Neuromuscular transmission failure was less in mice than in rats. Motor end-plate area differed across fibers in rat but not in mouse DIAm, where NMJs displayed greater complexity overall. Thus, differences across species in activation history and susceptibility to neuromuscular transmission failure are reflected in the relative contribution of fatigue resistant muscle fibers to total DIAm mass, but not in type-dependent morphological differences at the NMJ.

The three-dimensional structure of myenteric neurons in the guinea-pig ileum.

Myenteric neurons of the guinea-pig ileum were intracellularly filled with the fluorescent dye Lucifer Yellow, optically sectioned with a confocal microscope and volume reconstructed to recreate 3-D images of the cells. The resulting images provide information not evident from regular microscopy. The somata varied in cross-section from flat-oval to nearly circular, and their surface membranes were marked by invaginations and protrusions significantly increasing the surface area of the somatic membrane. The neurons could be divided into four morphological classes: Dogiel type I, Dogiel type II, filamentous, and intermediate. There was no clear correlation between cell class and the shape of the soma in cross-section. The dendritic processes of all the neurons studied extended in an orad-caudad or circumferential direction of the bowel wall. When the filled neurons were viewed edge-on, the spatial arrangement of the processes was confined to a plane that had a thickness less than the thickness of the parent soma. The broad, short dendrites of Dogiel type I neurons were oval or nearly circular in cross-section. Directly measured quantitative data were obtained for the volume and surface area of the somata and visible processes. The structural details reported herein are likely to have important implications regarding the functional properties of individual enteric ganglion neurons and circuits of enteric ganglion neurons.

The novel TrkB receptor agonist 7,8‐dihydroxyflavone enhances neuromuscular transmission

Neurotrophin signaling at the neuromuscular junction modulates cholinergic transmission and enhances neuromuscular transmission via the tropomyosin‐related kinase receptor subtype B (TrkB).A novel flavonoid, 7,8‐dihydroxyflavone (7,8‐DHF), selectively activates TrkB receptors. Using TrkBF616A mice that are susceptible to specific inhibition of TrkB activity by 1NMPP1, we show that neuromuscular transmission is enhanced by 7,8‐DHF (∽32%) via activation of TrkB in diaphragm muscle. The small molecule 7,8‐DHF may constitute a novel therapy to improve neuromuscular function. Muscle Nerve, 2012

Safety factor for neuromuscular transmission at type-identified diaphragm fibers

The safety factor (SF) for neuromuscular transmission varies across limb muscles of different fiber‐type composition. Using intracellular recordings in rat diaphragm fibers, we found that SF varies across muscle fiber types (even within a single muscle), being larger for type IIx or IIb fibers than for type I or IIa fibers. Fiber‐type differences in activation history or mechanical load may contribute to differences in SF and are important determinants of neuromuscular plasticity. Muscle Nerve, 2007

Ageing and neurotrophic signalling effects on diaphragm neuromuscular function

Sarcopenia of the diaphragm muscle, i.e. loss of muscle force and size with increasing age, may contribute to respiratory impairment in old age but the exact mechanisms underlying this are currently unknown. Across the lifespan in mice there is worsening neuromuscular function of the diaphragm muscle, specifically reduced force and impaired neuromuscular transmission. We tested the hypothesis that age‐related changes to the diaphragm muscle depend on brain‐derived neurotrophic factor (BDNF), acting through its high affinity receptor. BDNF improves neuromuscular transmission in the diaphragm muscle into early old age, but not older ages. Inhibition of BDNF signalling impairs neuromuscular transmission only in young adult mice. Our results suggest that the loss of endogenous BDNF precedes reduced activity of the high affinity receptor tropomyosin‐related kinase receptor B in the ageing mouse diaphragm muscle.