Neuromuscular recovery from botulism involves multiple forms of compensatory plasticity.

Introduction: Botulinum neurotoxin (BoNT) causes neuroparalytic disease and death by blocking neuromuscular transmission. There are no specific therapies for clinical botulism and the only treatment option is supportive care until neuromuscular function spontaneously recovers, which can take weeks or months after exposure. The highly specialized neuromuscular junction (NMJ) between phrenic motor neurons and diaphragm muscle fibers is the main clinical target of BoNT. Due to the difficulty in eliciting respiratory paralysis without a high mortality rate, few studies have characterized the neurophysiological mechanisms involved in diaphragm recovery from intoxication. Here, we develop a mouse model of botulism that involves partial paralysis of respiratory muscles with low mortality rates, allowing for longitudinal analysis of recovery. Methods and results: Mice challenged by systemic administration of 0.7 LD50 BoNT/A developed physiological signs of botulism, such as respiratory depression and reduced voluntary running activity, that persisted for an average of 8-12 d. Studies in isolated hemidiaphragm preparations from intoxicated mice revealed profound reductions in nerve-elicited, tetanic and twitch muscle contraction strengths that recovered to baseline 21 d after intoxication. Despite apparent functional recovery, neurophysiological parameters remained depressed for 28 d, including end plate potential (EPP) amplitude, EPP success rate, quantal content (QC), and miniature EPP (mEPP) frequency. However, QC recovered more quickly than mEPP frequency, which could explain the discrepancy between muscle function studies and neurophysiological recordings. Hypothesizing that differential modulation of voltage-gated calcium channels (VGCC) contributed to the uncoupling of QC from mEPP frequency, pharmacological inhibition studies were used to study the contributions of different VGCCs to neurophysiological function. We found that N-type VGCC and P/Q-type VGCC partially restored QC but not mEPP frequency during recovery from paralysis, potentially explaining the accelerated recovery of evoked release versus spontaneous release. We identified additional changes that presumably compensate for reduced acetylcholine release during recovery, including increased depolarization of muscle fiber resting membrane potential and increased quantal size. Discussion: In addition to identifying multiple forms of compensatory plasticity that occur in response to reduced NMJ function, it is expected that insights into the molecular mechanisms involved in recovery from neuromuscular paralysis will support new host-targeted treatments for multiple neuromuscular diseases.

Effects of Juvenile or Adolescent Working Memory Experience and Inter-Alpha Inhibitor Protein Treatment after Neonatal Hypoxia-Ischemia.

Hypoxic-Ischemic (HI) brain injury in the neonate contributes to life-long cognitive impairment. Early diagnosis and therapeutic interventions are critical but limited. We previously reported in a rat model of HI two interventional approaches that improve cognitive and sensory function: administration of Inter-alpha Inhibitor Proteins (IAIPs) and early experience in an eight-arm radial water maze (RWM) task. Here, we expanded these studies to examine the combined effects of IAIPs and multiple weeks of RWM assessment beginning with juvenile or adolescent rats to evaluate optimal age windows for behavioral interventions. Subjects were divided into treatment groups; HI with vehicle, sham surgery with vehicle, and HI with IAIPs, and received either juvenile (P31 initiation) or adolescent (P52 initiation) RWM testing, followed by adult retesting. Error rates on the RWM decreased across weeks for all conditions. Whereas, HI injury impaired global performance as compared to shams. IAIP-treated HI subjects tested as juveniles made fewer errors as compared to their untreated HI counterparts. The juvenile group made significantly fewer errors on moderate demand trials and showed improved retention as compared to the adolescent group during the first week of adult retesting. Together, results support and extend our previous findings that combining behavioral and anti-inflammatory interventions in the presence of HI improves subsequent learning performance. Results further indicate sensitive periods for behavioral interventions to improve cognitive outcomes. Specifically, early life cognitive experience can improve long-term learning performance even in the presence of HI injury. Results from this study provide insight into typical brain development and the impact of developmentally targeted therapeutics and task-specific experience on subsequent cognitive processing.

3,4-diaminopyridine reverses paralysis in botulinum neurotoxin-intoxicated diaphragms through two functionally distinct mechanisms.

Botulinum neurotoxins (BoNTs) are exceedingly potent neurological poisons that prevent neurotransmitter release from peripheral nerve terminals by cleaving presynaptic proteins required for synaptic vesicle fusion. The ensuing neuromuscular paralysis causes death by asphyxiation. Although no antidotal treatments exist to block toxin activity within the nerve terminal, aminopyridine antagonists of voltage-gated potassium channels have been proposed as symptomatic treatments for botulism toxemia. However, clinical evaluation of aminopyridines as symptomatic treatments for botulism has been inconclusive, in part because mechanisms responsible for reversal of paralysis in BoNT-poisoned nerve terminals are not understood. Here we measured the effects of 3,4-diaminopyridine (DAP) on phrenic nerve-elicited diaphragm contraction and end-plate potentials at various times after intoxication with BoNT serotypes A, B, or E. We found that DAP-mediated increases in quantal content promote neurotransmission from intoxicated nerve terminals through two functionally distinguishable mechanisms. First, DAP increases the probability of neurotransmission at non-intoxicated release sites. This mechanism is serotype-independent, becomes less effective as nerve terminals become progressively impaired, and remains susceptible to ongoing intoxication. Second, DAP elicits persistent production of toxin-resistant endplate potentials from nerve terminals fully intoxicated by BoNT/A, but not serotypes B or E. Since this effect appears specific to BoNT/A intoxication, we propose that DAP treatment enables BoNT/A-cleaved SNAP-25 to productively engage in fusogenic release by increasing the opportunity for low-efficiency fusion events. These findings have important implications for DAP as a botulism therapeutic by defining conditions under which DAP may be clinically effective in reversing botulism symptoms.

In vitro and ex vivo screening of candidate therapeutics to restore neurotransmission in nerve terminals intoxicated by botulinum neurotoxin serotype A1.

Botulinum neurotoxins (BoNTs) are exceedingly potent neurological poisons that block cholinergic release in the peripheral nervous system and cause death by asphyxiation. While post-exposure prophylaxis can effectively eliminate toxin in the bloodstream, there are no clinically effective treatments to prevent or reverse disease once BoNT has entered the neuron. To address the need for post-symptomatic countermeasures, we designed and developed an in vitro assay based on whole-cell, patch-clamp electrophysiological monitoring of miniature excitatory post-synaptic currents in synaptically active murine embryonic stem cell-derived neurons. This synaptic function-based assay was used to assess the efficacy of rationally selected drugs to restore neurotransmission in neurons comprehensively intoxicated by BoNT/A. Based on clinical reports suggesting that elevated Ca2+ signaling promotes symptomatic relief from botulism, we identified seven candidate drugs that modulate presynaptic Ca2+ signaling and assessed their ability to reverse BoNT/A-induced synaptic blockade. The most effective drugs from the screen were found to phasically agonize voltage-gated calcium channel (VGCC) activity. Lead candidates were then applied to ex vivo studies in BoNT/A-paralyzing mouse phrenic nerve-hemidiaphragm (PND) preparations. Treatment of PNDs with VGCC agonists after paralytic onset transiently potentiated nerve-elicited muscle contraction and delayed progression to neuromuscular failure. Collectively, this study suggests that Ca2+-modulating drugs represent a novel symptomatic treatment for neuromuscular paralysis following BoNT/A poisoning.

The Clinical Epidemiology and 30-Day Outcomes of Emergency Department Patients With Acute Kidney Injury.

BACKGROUND: Acute kidney injury (AKI) is associated with increased mortality and dialysis in hospitalized patients but has been little explored in the emergency department (ED) setting. OBJECTIVE: The objective of this study was to describe the risk factors, prevalence, management, and outcomes in the ED population, and to identify the proportion of AKI patients who were discharged home with no renal-specific follow-up. DESIGN: This is a retrospective cohort study using administrative and laboratory databases. SETTING: Two urban EDs in Vancouver, British Columbia, Canada. PATIENTS: We included all unique ED patients over a 1-week period. METHODS: All patients had their described demographics, comorbidities, medications, laboratory values, and ED treatments collected. AKI was defined pragmatically, based upon accepted guidelines. The cohort was then probabilistically linked to the provincial renal database to ascertain renal replacement (transplant or dialysis) and the provincial vital statistics database to obtain mortality. The primary outcome was the prevalence of AKI; secondary outcomes included (1) the proportion of AKI patients who were discharged home with no renal-specific follow-up and (2) the combined 30-day rate of death or renal replacement among AKI patients. RESULTS: There were 1651 ED unique patients, and 840 had at least one serum creatinine (SCr) obtained. Overall, 90 patients had AKI (10.7% of ED patients with at least one SCr, 95% confidence interval [CI], 8.7%-13.1%; 5.5% of all ED patients, 95% CI, 4.4%-6.7%) with a median age of 74 and 70% male. Of the 31 (34.4%) AKI patients discharged home, 4 (12.9%) had renal-specific follow-up arranged in the ED. Among the 90 AKI patients, 11 died and none required renal replacement at 30 days, for a combined outcome of 12.2% (95% CI, 6.5%-21.2%). LIMITATIONS: Sample sizes may be small. Nearly half of ED patients did not obtain an SCr. Many patients did not have sequential SCr testing, and a modified definition of AKI was used.

MISE EN CONTEXTE: L'insuffisance rénale aiguë (IRA) est associée à une mortalité accrue et à un recours plus fréquent à l'hémodialyse chez les patients hospitalisés. Toutefois, l'IRA a très peu été étudiée dans le cadre du service des urgences. OBJECTIF DE L'ÉTUDE: Dresser le portrait des facteurs de risque, de la prévalence, de la prise en charge et des conséquences de l'IRA au sein d'une population de patients admis aux urgences. Établir la proportion de patients atteints d'IRA qui ont par la suite été renvoyés à la maison sans aucun suivi en néphrologie. MODÈLE D'ÉTUDE: Il s'agit d'une étude de cohorte rétrospective menée à partir des bases de données administratives et de laboratoire des hôpitaux concernés. CADRE DE L'ÉTUDE: L'étude s'est tenue dans deux services d'urgence de Vancouver (CB) au Canada. PARTICIPANTS: Nous avons inclus tous les patients ayant été admis aux urgences au cours d'une période d'une semaine. MÉTHODOLOGIE: Les données démographiques, les comorbidités, la liste des médicaments prescrits, les résultats de laboratoire et les traitements administrés lors du séjour aux urgences ont été colligés pour chacun des participants. L'IRA a été définie avec pragmatisme, conformément aux lignes directrices acceptées. La cohorte a ensuite été couplée de façon probabiliste à la base de données provinciale sur l'insuffisance rénale afin d'évaluer l'incidence de thérapies de remplacement rénal (dialyse ou greffe) et la base de données statistique provinciale pour obtenir le taux de mortalité. Le critère de jugement principal était la prévalence de l'IRA ; les critères de jugement secondaires incluaient la proportion de patients atteints d'IRA retournés à la maison sans prescription de suivi en santé rénale ainsi que le taux combiné de mortalité ou d'établissement d'une thérapie de remplacement rénal à l'intérieur de 30 jours chez ces mêmes patients. RÉSULTATS: Au total, 1 651 patients se sont présentés aux urgences au cours de la période étudiée. Au moins une mesure de la créatinine sérique (SCr) avait été effectuée pour 840 d'entre eux. Dans l'ensemble, 90 patients souffraient effectivement d'IRA, (10,7% des patients avec au moins une mesure de SCr [IC à 95%: 8,7 à 13,1%] ; 5,5% de tous les patients [IC à 95%: 4,4 à 6,7%]). Il s'agissait de patients majoritairement de sexe masculin (70%) et leur âge médian était de 74 ans. Des 31 patients souffrant d'IRA (34,4%) retournés à la maison, on a prévu un suivi en santé rénale pour seulement quatre (12,9%) d'entre eux pendant leur séjour aux urgences. Parmi les 90 patients souffrant d'IRA, 11 sont décédés et aucun n'a eu besoin d'une thérapie de remplacement de la fonction rénale dans les 30 jours suivant la visite aux urgences, ce qui représente un résultat combiné de 12,2% (IC à 95%: 6,5 à 21,2%). LIMITES DE L'ÉTUDE: Le faible échantillonnage et le fait qu'aucune mesure de la SCr n'ait été effectuée pour près de la moitié des patients ayant séjourné aux urgences. De plus, plusieurs patients pour qui on avait procédé à une mesure de la SCr n'ont pas eu de mesure séquentielle, et une définition modifiée de l'insuffisance rénale a été utilisée pour réaliser l'étude.

Botulinum and Tetanus Neurotoxin-Induced Blockade of Synaptic Transmission in Networked Cultures of Human and Rodent Neurons.

Clinical manifestations of tetanus and botulism result from an intricate series of interactions between clostridial neurotoxins (CNTs) and nerve terminal proteins that ultimately cause proteolytic cleavage of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins and functional blockade of neurotransmitter release. Although detection of cleaved SNARE proteins is routinely used as a molecular readout of CNT intoxication in cultured cells, impaired synaptic function is the pathophysiological basis of clinical disease. Work in our laboratory has suggested that the blockade of synaptic neurotransmission in networked neuron cultures offers a phenotypic readout of CNT intoxication that more closely replicates the functional endpoint of clinical disease. Here, we explore the value of measuring spontaneous neurotransmission frequencies as novel and functionally relevant readouts of CNT intoxication. The generalizability of this approach was confirmed in primary neuron cultures as well as human and mouse stem cell-derived neurons exposed to botulinum neurotoxin serotypes A-G and tetanus neurotoxin. The sensitivity and specificity of synaptic activity as a reporter of intoxication was evaluated in assays representing the principal clinical and research purposes of in vivo studies. Our findings confirm that synaptic activity offers a novel and functionally relevant readout for the in vitro characterizations of CNTs. They further suggest that the analysis of synaptic activity in neuronal cell cultures can serve as a surrogate for neuromuscular paralysis in the mouse lethal assay, and therefore is expected to significantly reduce the need for terminal animal use in toxin studies and facilitate identification of candidate therapeutics in cell-based screening assays.

Methylmercury-Dependent Increases in Fluo4 Fluorescence in Neonatal Rat Cerebellar Slices Depend on Granule Cell Migrational Stage and GABAA Receptor Modulation.

Methylmercury (MeHg) disrupts cerebellar function, especially during development. Cerebellar granule cells (CGC), which are particularly susceptible to MeHg by unknown mechanisms, migrate during this process. Transient changes in intracellular Ca(2+) (Ca(2+) i) are crucial to proper migration, and MeHg is well known to disrupt CGC Ca(2+) i regulation. Acutely prepared slices of neonatal rat cerebellum in conjunction with confocal microscopy and fluo4 epifluorescence were used to track changes induced by MeHg in CGC Ca(2+) i regulation in the external (EGL) and internal granule cell layers (IGL) as well as the molecular layer (ML). MeHg caused no cytotoxicity but did cause a time-dependent increase in fluo4 fluorescence that depended on the stage of CGC development. CGCs in the EGL were most susceptible to MeHg-induced increases in fluo4 fluorescence. MeHg increased fluorescence in CGC processes but only diffusely; Purkinje cells rarely fluoresced in these slices. Neither muscimol nor bicuculline alone altered baseline fluo4 fluorescence in any CGC layer, but each delayed the onset and reduced the magnitude of effect of MeHg on fluo4 fluorescence in the EGL and ML. In the IGL, both muscimol and bicuculline delayed the onset of MeHg-induced increases in fluo4 fluorescence but did not affect fluorescence magnitude. Thus, acute exposure to MeHg causes developmental stage-dependent increases in Ca(2+) i in CGCs. Effects are most prominent in CGCs during development or early stages of migration. GABAA receptors participate in an as yet unclear manner to MeHg-induced Ca(2+) i dysregulation of CGCs.

Importance of being Nernst: Synaptic activity and functional relevance in stem cell-derived neurons.

Functional synaptogenesis and network emergence are signature endpoints of neurogenesis. These behaviors provide higher-order confirmation that biochemical and cellular processes necessary for neurotransmitter release, post-synaptic detection and network propagation of neuronal activity have been properly expressed and coordinated among cells. The development of synaptic neurotransmission can therefore be considered a defining property of neurons. Although dissociated primary neuron cultures readily form functioning synapses and network behaviors in vitro, continuously cultured neurogenic cell lines have historically failed to meet these criteria. Therefore, in vitro-derived neuron models that develop synaptic transmission are critically needed for a wide array of studies, including molecular neuroscience, developmental neurogenesis, disease research and neurotoxicology. Over the last decade, neurons derived from various stem cell lines have shown varying ability to develop into functionally mature neurons. In this review, we will discuss the neurogenic potential of various stem cells populations, addressing strengths and weaknesses of each, with particular attention to the emergence of functional behaviors. We will propose methods to functionally characterize new stem cell-derived neuron (SCN) platforms to improve their reliability as physiological relevant models. Finally, we will review how synaptically active SCNs can be applied to accelerate research in a variety of areas. Ultimately, emphasizing the critical importance of synaptic activity and network responses as a marker of neuronal maturation is anticipated to result in in vitro findings that better translate to efficacious clinical treatments.

Isolation and characterization of atrioventricular nodal cells from neonate rabbit heart.

BACKGROUND: The properties of the atrioventricular (AV) node in the neonate heart and its role in unique pediatric cardiac arrhythmias such as junctional ectopic tachycardia (JET) are poorly understood. This is due in large part to the dearth of information on the structure and physiology of the AV node in the immature myocardium. METHODS AND RESULTS: Sinoatrial nodal cells (SANCs), AV nodal tissues, and myocytes (AVNCs) were obtained from neonatal (10-day-old) rabbits, and the histological, immunohistological, and electrophysiological properties were characterized in detail. Masson's trichrome histological staining clearly delineated AV nodal structures including the inferior nodal extension, compact node, and the bundle of His region. AV tissue sections and AVNCs were immunolabeled against neurofilament 160 (NF160), connexin 43 (Cx43), hyperpolarization-activated, cyclic nucleotide modulated channel (HCN4), sodium/calcium exchanger, ryanodine receptor, sarcoplasmic/endoplasmic reticulum Ca(2+) pump (SERCA), and phospholamban (PLB). In AVNCs with triple-positive NF160, SERCA, and PLB labeling, SERCA and PLB were found with high degrees of colocalization. The majority (59%) of NF160-positive AVNCs were found to coexpress HCN4. NF160 and HCN4 expression was found to be even higher in SANCs, where 88% of SANCs exhibited coexpression. Spontaneous action potentials recorded from isolated neonatal AVNCs were uniformly of the atrionodal type, showing none of the action potential heterogeneities found in the mature heart. Current recordings found the hyperpolarization-activated funny current (I(f)) in 55% (11 of 21 cells) of AVNCs, consistent with the immunocytochemistry results. CONCLUSIONS: This represents the first detailed electrophysiological and immunohistological report of the neonatal AV node and lays the groundwork for a better understanding of heart rate regulation and unique arrhythmias in the neonate heart.