Electrophysiological effects of BDNF and TrkB signaling at type-identified diaphragm neuromuscular junctions.

Previous studies show that synaptic quantal release decreases during repetitive stimulation, i.e., synaptic depression. Neurotrophin brain-derived neurotrophic factor (BDNF) enhances neuromuscular transmission via activation of tropomyosin-related kinase receptor B (TrkB). We hypothesized that BDNF mitigates synaptic depression at the neuromuscular junction and that the effect is more pronounced at type IIx and/or IIb fibers compared to type I or IIa fibers given the more rapid reduction in docked synaptic vesicles with repetitive stimulation. Rat phrenic nerve-diaphragm muscle preparations were used to determine the effect of BDNF on synaptic quantal release during repetitive stimulation at 50 Hz. An ∼40% decline in quantal release was observed during each 330-ms duration train of nerve stimulation (intratrain synaptic depression), and this intratrain decline was observed across repetitive trains (20 trains at 1/s repeated every 5 min for 30 min for 6 sets). BDNF treatment significantly enhanced quantal release at all fiber types (P < 0.001). BDNF treatment did not change release probability within a stimulation set but enhanced synaptic vesicle replenishment between sets. In agreement, synaptic vesicle cycling (measured using FM4-64 fluorescence uptake) was increased following BDNF [or neurotrophin-4 (NT-4)] treatment (∼40%; P < 0.05). Conversely, inhibiting BDNF/TrkB signaling with the tyrosine kinase inhibitor K252a and TrkB-IgG (which quenches endogenous BDNF or NT-4) decreased FM4-64 uptake (∼34% across fiber types; P < 0.05). The effects of BDNF were generally similar across all fiber types. We conclude that BDNF/TrkB signaling acutely enhances presynaptic quantal release and thereby may serve to mitigate synaptic depression and maintain neuromuscular transmission during repetitive activation.NEW & NOTEWORTHY Neurotrophin brain-derived neurotrophic factor (BDNF) enhances neuromuscular transmission via activation of tropomyosin-related kinase receptor B (TrkB). Rat phrenic nerve-diaphragm muscle preparations were used to determine the rapid effect of BDNF on synaptic quantal release during repetitive stimulation. BDNF treatment significantly enhanced quantal release at all fiber types. BDNF increased synaptic vesicle cycling (measured using FM4-64 fluorescence uptake); conversely, inhibiting BDNF/TrkB signaling decreased FM4-64 uptake.

Bicarbonate ion transport by the electrogenic Na+ /HCO3- cotransporter, NBCe1, is required for normal electrical slow-wave activity in mouse small intestine.

BACKGROUND: Normal gastrointestinal motility depends on electrical slow-wave activity generated by interstitial cells of Cajal (ICC) in the tunica muscularis of the gastrointestinal tract. A requirement for HCO3- in extracellular solutions used to record slow waves indicates a role for HCO3- transport in ICC pacemaking. The Slc4a4 gene transcript encoding the electrogenic Na+ /HCO3- cotransporter, NBCe1, is enriched in mouse small intestinal myenteric region ICC (ICC-MY) that generate slow waves. This study aimed to determine how extracellular HCO3- concentrations affect electrical activity in mouse small intestine and to determine the contribution of NBCe1 activity to these effects. METHODS: Immunohistochemistry and sharp electrode electrical recordings were used. KEY RESULTS: The NBCe1 immunoreactivity was localized to ICC-MY of the tunica muscularis. In sharp electrode electrical recordings, removal of HCO3- from extracellular solutions caused significant, reversible, depolarization of the smooth muscle and a reduction in slow-wave amplitude and frequency. In 100 mM HCO3- , the muscle hyperpolarized and slow wave amplitude and frequency increased. The effects of replacing extracellular Na+ with Li+ , an ion that does not support NBCe1 activity, were similar to, but larger than, the effects of removing HCO3- . There were no additional changes to electrical activity when HCO3- was removed from Li+ containing solutions. The Na+ /HCO3- cotransport inhibitor, S-0859 (30µM) significantly reduced the effect of removing HCO3- on electrical activity. CONCLUSIONS & INFERENCES: These studies demonstrate a major role for Na+ /HCO3- cotransport by NBCe1 in electrical activity of mouse small intestine and indicated that regulation of intracellular acid:base homeostasis contributes to generation of normal pacemaker activity in the gastrointestinal tract.

Ageing and neurotrophic signalling effects on diaphragm neuromuscular function.

The age-related mechanisms underlying sarcopenia are largely unknown. We hypothesize that age-related neuromuscular changes depend on brain-derived neurotrophic factor (BDNF) acting through the tropomyosin-related kinase receptor B (TrkB). Maximal specific force and neuromuscular transmission failure were assessed at 6, 18 and 24 months following control, BDNF or phosphoprotein phosphatase 1 derivative (1NMPP1) treatment in male TrkB(F616A) mice. Phosphoprotein phosphatase-1 derivatives such as 1NMPP1 inhibit TrkB kinase activity as a result of this single amino acid mutation in the ATP binding domain. Maximal twitch and isometric tetanic force were reduced at 24 months compared to 6 and 18 months (P < 0.001). Neuromuscular transmission failure significantly increased at 18 and 24 months compared to 6 months (age × treatment interaction: P < 0.001). Neuromuscular transmission was improved following BDNF at 6 and 18 months and was impaired only at 6 months following 1NMPP1 treatment. Age and inhibition of TrkB kinase activity had similar effects on neuromuscular transmission failure, supporting a critical role for BDNF/TrkB signalling on neuromuscular changes in ageing. These results suggest that an age-related loss of endogenous BDNF precedes reductions in TrkB kinase activity in the diaphragm muscle.

TrkB kinase activity maintains synaptic function and structural integrity at adult neuromuscular junctions.

Activation of the tropomyosin-related kinase receptor B (TrkB) by brain-derived neurotrophic factor acutely regulates synaptic transmission at adult neuromuscular junctions (NMJs). The role of TrkB kinase activity in the maintenance of NMJ function and structure at diaphragm muscle NMJs was explored using a chemical-genetic approach that permits reversible inactivation of TrkB kinase activity in TrkB(F616A) mice by 1NMPP1. Inhibiting TrkB kinase activity for 7 days resulted in significant, yet reversible, impairments in neuromuscular transmission at diaphragm NMJs. Neuromuscular transmission failure following 2 min of repetitive phrenic nerve stimulation increased from 42% in control to 59% in 1NMPP1-treated TrkB(F616A) mice (P = 0.010). Recovery of TrkB kinase activity following withdrawal of 1NMPP1 treatment improved neuromuscular transmission (P = 0.006). Electrophysiological measurements at individual diaphragm NMJs documented lack of differences in quantal content in control and 1NMPP1-treated mice (P = 0.845). Morphological changes at diaphragm NMJs were modest following inhibition and recovery of TrkB kinase activity. Three-dimensional reconstructions of diaphragm NMJs revealed no differences in volume at motor end plates (labeled by α-bungarotoxin; P = 0.982) or presynaptic terminals (labeled by synaptophysin; P = 0.515). Inhibition of TrkB kinase activity by 1NMPP1 resulted in more compact NMJs, with increased apposition of presynaptic terminals and motor end plates (P = 0.017) and reduced fragmentation of motor end plates (P = 0.005). Recovery of TrkB kinase activity following withdrawal of 1NMPP1 treatment resulted in postsynaptic remodeling likely reflecting increased gutter depth (P = 0.007), without significant presynaptic changes. These results support an essential role for TrkB kinase activity in maintaining synaptic function and structural integrity at NMJs in the adult mouse diaphragm muscle.

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.

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 TrkB(F616A) 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.

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.

Impairment of diaphragm muscle force and neuromuscular transmission after normothermic cardiopulmonary bypass: effect of low-dose inhaled CO.

Cardiopulmonary bypass (CPB) is associated with significant postoperative morbidity, but its effects on the neuromuscular system are unclear. Recent studies indicate that even relatively short periods of mechanical ventilation result in significant neuromuscular effects. Carbon monoxide (CO) has gained recent attention as therapy to reduce the deleterious effects of CPB. We hypothesized that 1) CPB results in impaired neuromuscular transmission and reduced diaphragm force generation; and 2) CO treatment during CPB will mitigate these effects. In adult male Sprague-Dawley rats, diaphragm muscle-specific force and neuromuscular transmission properties were measured 90 min after weaning from normothermic CPB (1 h). During CPB, either low-dose inhaled CO (250 ppm) or air was administered. The short period of mechanical ventilation used in the present study ( approximately 3 h) did not adversely affect diaphragm muscle contractile properties or neuromuscular transmission. CPB elicited a significant decrease in isometric diaphragm muscle-specific force compared with time-matched, mechanically ventilated rats ( approximately 25% decline in both twitch and tetanic force). Diaphragm muscle fatigability to 40-Hz repetitive stimulation did not change significantly. Neuromuscular transmission failure during repetitive activation was 60 +/- 2% in CPB animals compared with 76 +/- 4% in mechanically ventilated rats (P < 0.05). CO treatment during CPB abrogated the neuromuscular effects of CPB, such that diaphragm isometric twitch force and neuromuscular transmission were no longer significantly different from mechanically ventilated rats. Thus, CPB has important detrimental effects on diaphragm muscle contractility and neuromuscular transmission that are largely mitigated by CO treatment. Further studies are needed to ascertain the underlying mechanisms of CPB-induced neuromuscular dysfunction and to establish the potential role of CO therapy.

Modeling dendritic arborization based on 3D-reconstructions of adult rat phrenic motoneurons

Stereological techniques that rely on morphological assumptions and direct three-dimensional (3D) on focal measurements have been previously used to estimate the dendritic surface areas of phrenic motoneurons (PhrMNs). Given that97% of a motoneuron’s receptive area is provided by dendrites, dendritic branching and overall extension are physiologically important in determining the output of their synaptic receptive fields. However, limitations intrinsic to shape-based estimations and incomplete labeling of dendritic trees by retrograde techniques have hindered systematic approaches to examine dendritic morphology of PhrMNs. In this study, a novel method that improves dendritic filling of PhrMNs in lightly-fixed samples was used. Confocal microscopy allowed accurate 3D reconstruction of dendritic arbors from adult rat PhrMNs. Following pre-processing,segmentation was semi-automatically performed in 3D, and direct measurements of dendritic surface area were obtained. A quadratic model for estimating dendritic tree surface area based on measurements of primary dendrite diameter was derived (r2 =0.932; p<0.0001). This method may enhance interpretation of motoneuron plasticity in response to injury or disease by permitting estimations of dendritic arborization of PhrMNs since measurements of primary dendrite diameter can be reliably obtained from a number of retrograde labeling techniques

El área superficial de las dendritas en motoneuronas frénicas (PhrMNs) ha sido estimada anteriormente mediante técnicas estereológicas basadas en suposiciones geométricas, y medida en tres dimensiones (3D) utilizando microscopía confocal. Dado que el 97% del área receptora de una motoneurona corresponde a sus dendritas, la ramificación y extensión dendrítica son fisiológicamente importantes para determinar la salida de sus campos receptivos. Sin embargo, limitaciones inherentes a las estimaciones basadas en morfología neuronal y la tinción incompleta de los árboles dendríticos mediante técnicas retrógradas han dificultado los estudios sistemáticos de la morfología dendrítica en PhrMNs. En este estudio, se utilizó una nueva técnica que mejora la tinción dendrítica de las PhrMNs en preparaciones fijadas ligeramente. La reconstrucción dendrítica en 3D se logró con gran precisión utilizando microscopía confocal en PhrMNs de ratas adultas. Luego de una etapa de pre-procesamiento, la segmentación de los árboles dendríticos se realizó semi-automáticamente en 3D y usando mediciones directas del área superficial, se derivó un modelo cuadrático para estimar dicha área partiendo del diámetro de la dendrita primaria (r2 = 0.932; p<0.0001). Este método podría mejorar la evaluación de la plasticidad neuronal en respuesta a trauma u otras enfermedades permitiendo la estimación de la arborización dendrítica en PhrMNs, ya que el diámetro de la dendrita primaria puede obtenerse confiablemente de numerosas técnicas de tinción retrógrada

Synaptic vesicle distribution and release at rat diaphragm neuromuscular junctions.

Synaptic vesicle release at the neuromuscular junction (NMJ) is highly reliable and is vital to the success of synaptic transmission. We examined synaptic vesicle number, distribution, and release at individual type-identified rat diaphragm NMJ. Three-dimensional reconstructions of electron microscopy images were used to obtain novel measurements of active zone distribution and the number of docked synaptic vesicles. Diaphragm muscle-phrenic nerve preparations were used to perform electrophysiological measurements of the decline in quantal content (QC) during repetitive phrenic nerve stimulation. The number of synaptic vesicles available for release vastly exceeds those released with a single stimulus, thus reflecting a relatively low probability of release for individual docked vesicles and at each active zone. There are two components that describe the decline in QC resulting from repetitive stimulation: a rapid phase (<0.5 s) and a delayed phase (<2.5 s). Differences in the initial rapid decline in QC were evident across type-identified presynaptic terminals (fiber type classification based on myosin heavy chain composition). At terminals innervating type IIx and/or IIb fibers, the initial decline in QC during repetitive stimulation matched the predicted depletion of docked synaptic vesicles. In contrast, at terminals innervating type I or IIa fibers, a faster than predicted decline in QC with repetitive stimulation suggests that a decrease in the probability of release at these terminals plays a role in addition to depletion of docked vesicles. Differences in QC decline likely reflect fiber-type specific differences in activation history and correspond with well-described differences in neuromuscular transmission across muscle fiber types.

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.

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 alpha3 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.

PACAP modulation of the colon-inferior mesenteric ganglion reflex in the guinea pig.

We investigated the effect of pituitary adenylate cyclase activating peptide (PACAP) on the colon-inferior mesenteric ganglion (IMG) reflex loop in vitro. PACAP27 and PACAP38 applied to the IMG caused a prolonged depolarization and intense generation of fast EPSPs and action potentials in IMG neurones. Activation of PACAP-preferring receptors (PAC1-Rs) with the selective agonist maxadilan or vasoactive intestinal peptide (VIP)/PACAP (VPAC) receptors with VIP produced similar effects whereas prior incubation of the IMG with selective PAC1-R antagonists PACAP6-38 and M65 inhibited the effects of PACAP. Colonic distension evoked a slow EPSP in IMG neurones that was reduced in amplitude by prolonged superfusion of the IMG with either PACAP27, maxidilan, PACAP6-38, M65 or VIP. Activation of IMG neurones by PACAP27 or maxadilan resulted in an inhibition of ongoing spontaneous colonic contractions. PACAP-LI was detected in nerve trunks attached to the IMG and in varicosities surrounding IMG neurones. Cell bodies with PACAP-LI were present in lumbar 2-3 dorsal root ganglia and in colonic myenteric ganglia. Colonic distension evoked release of PACAP peptides in the IMG as measured by radioimmunoassay. Volume reconstructed images showed that a majority of PACAP-LI, VIP-LI and VAChT-LI nerve endings making putative synaptic contact onto IMG neurones and a majority of putative receptor sites containing PAC1-R-LI and nAChR-LI on the neurones were distributed along secondary and tertiary dendrites. These results suggest involvement of a PACAP-ergic pathway, operated through PAC1-Rs, in controlling the colon-IMG reflex.

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.