Methanolic extract of Rhinella schneideri (Anura: Bufonidae) poison causes ultrastructural changes in nerve terminals of phrenic nerve-diaphragm preparations in mice / Extracto metanólico de Rhinella schneideri (Anura: Bufonidae) causa cambios ultraestructurales en las terminaciones nerviosas de preparaciones de nervio frénico de ratón

Abstract Rhinella schneideri (or Bufo paracnemis), popularly known in Brazil as cururu toad, is also found in other countries in South America. The cardiovascular effects of this poison are largely known and recently was shown that it is capable to affect the neuromuscular junction on avian and mice isolated preparation. In this work, we used transmission electron microscopy to investigate the ultrastructure of the motor nerve terminal and postsynaptic junctional folds of phrenic nerve-hemidiaphragm preparations incubated for either 5 or 60 min with the methanolic extract of R. schneideri (50 µg/mL). In addition, the status of the acetylcholine receptors (AChR) was examined by TRITC-α-bungarotoxin immunofluorescence location at the endplate membrane. The results show that 5 min of incubation with the gland secretion extract significantly decreased (32 %) the number of synaptic vesicles into the motor nerve terminal, but did not decrease the electron density on the top of the junctional folds where nicotinic receptors are concentrated; however, 60 min of incubation led to significant nerve terminal reloading in synaptic vesicles whereas the AChR immunoreactivity was not as marked as in control and after 5 min incubation. Muscle fibers were well-preserved but intramuscular motor axons were not. The findings corroborated pharmacological data since the decrease in the number of synaptic vesicles (5 min) followed by recovery (60 min) is in accordance with the transient increase of MEPPs frequency meaning increased neurotransmitter release. These data support the predominant presynaptic mode of action of the R. schneideri, but do not exclude the possibility of a secondary postsynaptic action depending on the time the preparation is exposed to poison. Rev. Biol. Trop. 66(3): 1290-1297. Epub 2018 September 01.

Resumen Rhinella schneideri (o Bufo paracnemis), conocido popularmente en Brasil como sapo cururu, también se encuentra en otros países de América del Sur. Los efectos cardiovasculares de este veneno son ampliamente conocidos y recientemente se demostró que es capaz de afectar la unión neuromuscular en la preparación aislada de aves y ratones. En este trabajo, utilizamos microscopía electrónica de transmisión para investigar la ultraestructura de la terminación nerviosa motora y pliegues de unión postsináptica de preparaciones de nervio frénico-hemidiafragma incubadas durante 5 o 60 min con el extracto metanólico de R. schneideri (50 μg/mL). Además, se examinó el estado de los receptores de acetilcolina (AChR) mediante la ubicación de inmunofluorescencia de TRITC-α-bungarotoxina en la membrana de la placa terminal. Los resultados muestran que 5 min de incubación con el extracto de secreción de glándula disminuyeron significativamente (32 %) el número de vesículas sinápticas en el terminal del nervio motor, pero no disminuyeron la densidad electrónica en la parte superior de los pliegues de unión donde se concentran los receptores nicotínicos. Sin embargo, 60 min de incubación condujeron a una recarga significativa de los terminales nerviosos en las vesículas sinápticas, mientras que la inmunorreactividad del AChR no fue tan marcada como en el control y después de 5 min de incubación. Las fibras musculares estaban bien conservadas, pero los axones motores intramusculares no. Los hallazgos corroboraron los datos farmacológicos ya que la disminución en el número de vesículas sinápticas (5 min) seguida de recuperación (60 min) está de acuerdo con el aumento transitorio de la frecuencia de MEPPs, lo que significa una mayor liberación de neurotransmisores. Estos datos apoyan el modo de acción presináptico predominante de R. schneideri, pero no excluyen la posibilidad de una acción postsináptica secundaria dependiendo del tiempo en que la preparación esté expuesta al veneno.

Factors that affect the onset of action of non-depolarizing neuromuscular blocking agents / 대한마취과학회지

Neuromuscular blockade plays an important role in the safe management of patient airways, surgical field improvement, and respiratory care. Rapid-sequence induction of anesthesia is indispensable to emergency surgery and obstetric anesthesia, and its purpose is to obtain a stable airway, adequate depth of anesthesia, and appropriate respiration within a short period of time without causing irritation or damage to the patient. There has been a continued search for new neuromuscular blocking drugs (NMBDs) with a rapid onset of action. Factors that affect the onset time include the potency of the NMBDs, the rate of NMBDs reaching the effect site, the onset time by dose control, metabolism and elimination of NMBDs, buffered diffusion to the effect site, nicotinic acetylcholine receptor subunit affinity, drugs that affect acetylcholine (ACh) production and release at the neuromuscular junction, drugs that inhibit plasma cholinesterase, presynaptic receptors responsible for ACh release at the neuromuscular junction, anesthetics or drugs that affect muscle contractility, site and methods for monitoring neuromuscular function, individual variability, and coexisting disease. NMBDs with rapid onset without major adverse events are expected in the next few years, and the development of lower potency NMBDs will continue. Anesthesiologists should be aware of the use of NMBDs in the management of anesthesia. The choice of NMBD and determination of the appropriate dosage to modulate neuromuscular blockade characteristics such as onset time and duration of neuromuscular blockade should be considered along with factors that affect the effects of the NMBDs. In this review, we discuss the factors that affect the onset time of NMBDs.

Anatomical Characterization of Vanilloid Receptor 1 (VR1)-positive Primary Afferents in Lower Lumbar Cord in the Rat / 대한해부학회지

Primary afferents sensitive to capsaicin and noxious heat express vanilloid receptor 1(VR1) in both their peripheral and central fibers and terminals. We used multiple immunofluorescence and confocal microscopy to characterize their pattern of termination in rat spinal cord, colocalization of neurochemical markers of primary afferents and other presynaptic receptors. VR1-positive unmyelinated fibers mainly terminate in lamina I, where they co-stain for CGRP, and to a limited extent for SP, and in lamina II, especially its medial half, where they co-stain for IB4. VR1 positive thin myelinated fibers terminate in lamina I and co-stain for the neurochemical tracer CTB, injected in the sciatic nerve. As revealed by simultaneous staining for the synaptic marker synaptophysin, VR1-positive terminals are abundant in lamina I and sparse in lamina II. In L6-S1 spinal cord, VR1-positive fibers and terminals were abundant in Lissauer's tract, lamina I-V, medial collateral path to lamina X, and lateral collateral path to sacral parasympathetic nucleus. Most of VR1 positive fibers in Lissuer's tract and LCP are colocalized with SP. In conclusion, it is suggested that VR1 positive fibers in spinal cord are both peptidergic and non-peptidergic, IB4 positive fibers, mediating both somatic and visceral sensations, and that peptidergic VR1 positive fibers are mainly related with visceral sense.

Presynaptic alpha-7 nicotinic acetylcholine receptors modulate excitatory synaptic transmission in hippocampal neurons / 生理学报

The effects of presynaptic nicotinic acetylcholine receptors (nAChRs) on excitatory synaptic transmission in CA1 pyramidal neurons of the rat hippocampus were examined by blind whole-cell patch clamp recording from hippocampal slice preparations. Local application of the nAChRs agonist dimethylphenyl-piperazinium iodide (DMPP) did not induce a postsynaptic current response in CA1 pyramidal cells. However, DMPP enhanced the frequency and amplitude of spontaneous excitatory postsynaptic current (sEPSC) in these cells in a dose-dependent manner. This enhancement was blocked by the selective nicotinic alpha-7 receptor antagonist alpha-bungarotoxin, but not by the antagonist mecamylamine, hexamethonium or dihydro-beta-erythroidine. The frequency of miniature excitatory postsynaptic current (mEPSC) in CA1 pyramidal neurons was also increased by application of DMPP, indicating a presynaptic site of action of the agonist. Taken together, these results suggest that activation of presynaptic nAChRs in CA1 pyramidal neurons, which contain alpha-7 subunits, potentiates presynaptic glutamate release and consequently modulate excitatory synaptic transmission in the hippocampus.

Autoregulação colinérgica da transmissão neuromuscular / Cholinergic autoregulation of neuromuscular transmission

Autoregulação colinérgica é o nome dado à capacidade da acetilcolina de regular sua própria liberação neuronal. A acetilcolina atua sobre receptores nicotínicos e muscarínicos presentes nos terminais nervosos motores. Tais receptores, em conjunto com outros não colinérgicos, permitem que os terminais colinérgicos sejam regulados por agentes liberados próximos ou distantes dos nervos motores. O acionamento dos receptores nicotínicos e muscarínicos pré- sinápticos determinam, respectivamente, aumento e redução da quantidade de acetilcolina liberada para a fenda sináptica. Estudos farmacológicos sugerem que os receptores nicotínicos pré-sinápticos do terminal nervoso motor sejam do subtipo ganglionar Nn, por se comportarem de forma semelhante aqueles presentes nos gânglios autonômicos. Os muscarínicos, por outro lado, comportam-se farmacologicamente como receptores cardíacos do subtipo M2

Evaluation of Presynaptic Action of Depolarizing Neuromuscular Blocking Agents with Single Twitch Response in Vitro / 대한마취과학회지

BACKGROUND: This study was performed to evaluate the presynaptic effects of depolarizing neuromuscular blocking drugs by using slow and fast frequencies of indirect stimulation on partial twitch depression in vitro. METHODS: A rat phrenic nerve hemidiaphragm was dissected and was mounted in an organ bath containing an oxygenated Krebs solution. The phrenic nerve was stimulated supramaximally and the twitch response (0.1 Hz) was stabilized for at least 30 minutes. T200/T1 ratio (twitch height of the 200th stimuli divided by that of the first stimuli) at frequencies of 0.2, 0.5, 1.0, and 2.0 Hz using a drug concentration which provided approximately 20% twitch depression at 0.1 Hz was calculated. To compare T200/T1 ratios with TOF ratios, a 2.0 Hz TOF response was measured immediately after the 200th stimuli at either frequency of stimulation. RESULTS: T200/T1 ratios produced by succinylcholine (SCC) and decamethonium (C10) were located between alpha-bungarotoxin (ABX) and hexamethonium (C6), however, significant differences among the four drugs were found at 2.0 Hz. The propensity for a decrease in T200/T1 ratios at 2.0 Hz might differ from this study: C6 > C10 > SCC > ABX. T200/T1 ratios at 2.0 Hz were not different from TOF ratios. CONCLUSIONS: It is concluded that small doses of C10 have a greater presynaptic activity than that of SCC, when the observed effects in this study were compared with the result of ABX acting predominantly at postsynaptic receptors and C6 acting predominantly at presynaptic receptors.

The Effect of Frequency of Stimulation on Partial Twitch Depression in a Rat Phrenic Nerve Hemidiaphragm Preparation / 대한마취과학회지

Background: This study was designed to determine whether presynaptic receptor blockade could be differentiated from postsynaptic receptor blockade by examining the effect of increasing frequencies of indirect stimulation on partial twitch depression in vitro rat phrenic nerve hemidiaphragm preparations. Methods: After isolating rat phrenic nerve hemidiaphragm preparation, T200/T1 ratio (twitch height of the 200th stimuli divided by that of the 1st stimuli) at frequencies of 0.2, 0.5, 1.0, and 2.0 Hz using a drug concentration which provided approximately 20% twitch depression at 0.1 Hz was calculated. To compare T200/T1 ratios with TOF ratios, 2.0 Hz TOF response was measured immediately after 200th stimuli at either frequency of stimulation. Results: Hexamethonium caused a marked decrease in T200/T1 ratio at 0.5~2.0 Hz of stimulation, whereas alpha-bungarotoxin caused no change in T200/T1 ratios at up to 2.0 Hz of stimulation. The T200/T1 ratios produced by d-tubocurarine, vecuronium, mivacurium, and rocuronium located intermediate between alpha-bungarotoxin and hexamethonium, however significant differences among four drugs were found at 2.0 Hz. The propensity for decrease in T200/T1 ratios at 2.0 Hz might differ from this study: hexamethonium >d-tubocurarine >rocuronium >mivacurium = vecuronium >alpha-bungarotoxin. T200/T1 ratios at 2.0 Hz were not different from TOF ratios. Conclusions: When the observed effects in this study were provided with result of alpha-bungarotoxin acting predominantly at postsynaptic receptors and hexamethonium acting predominantly at presynaptic receptors, the effects of nondepolarizing muscle relaxants at each binding site could be differentiated by examining the T200/T1 ratios at 2.0 Hz.

Train-of-Four Fade Related to Potency during Recovery from Neuromuscular Block in Isolated Forearm / 대한마취과학회지

BACKGREOUND: The speed of action of nondepolarizing muscle relaxants might be correlated with the drug potency and the receptor-plasma concentration gradient. However, because of dissociation constants (KDs) being masked by plasma concentration following systemic administration, we investigated the degrees C of train-of-four (TOF) fade at various stages of the measured first twitch height of TOF (T1) during recovery related to potency of various muscle relaxants using isolated forearm technique. METHODS: Thirty two volunteers of the conscious healthy adults who were not receiving any medication which might have influence neuromuscular transmission were involved in this study. The electrodes were applied over the ulnar nerve at the wrist and supramaximal transcutaneous single twitch stimulation (0.1 Hz) delivered by a peripheral nerve stimulator (Innervator, Fisher & Paykel, New Zealand) using a 0.2 ms square wave pulses was applied. The twitch response of the thumb adductor was measured mechanomyographically using a 2 kg Load Cell strain gauge (Model No. 505H, RS Components Ltd., UK) with a thumb piece modification. Following a 5 min. period of stabilization, forearm pneumatic tourniquet was inflated to 300 mmHg in order to occlude the systemic circulation. The equipotent dose (ED95 0.1) of various muscle relaxants diluted in 20 ml of saline was injected into a vein on the dorsal hand respectively: rocuronium 30 microgram/kg, atracurium 25 microgram/kg, mivacurium 8 microgram/kg and vecuronium 4 microgram/kg. Eight volunteers received each dose. The forearm tourniquet was released at 3 min after drugs given. The neuromuscular block was allowed to recovery spontaneously, and at 25, 50, 75 and 100% recovery of control twitch height, TOF stimulation (2 Hz for 2 s) was administered in order to compare TOF ratios between musle relaxants. RESULTS: In the aminosteroid compounds, the TOF ratios of rocuronium were greater than those of vecuronium at all of the assessment points during recovery. In the benzylisoquinolium compounds, the TOF ratios of atracurium were greater than those of mivacurium at 25 and 50% recovery of control twitch height. CONCLUSION: The degree of TOF fade during recovery is related to drug potency at presynaptic receptors separated from the effects of plasma drug concentration.

The Train-of-Four Ratio Profile During Onset and Offset Following Administration of Neuromuscular Blocking Agents / 대한마취과학회지

BACKGROUND: The train of four (TOF) stimulation is valuable to study pharmacodynamics associated with the interaction between muscle relaxants and receptors in the neuromuscular junction. TOF fade expresses presynaptic effect diminished output of transmitters. The aim of this study was to examine differences in presynaptic effects of different relaxants by measuring the TOF ratio during the onset and offset of block. METHODS: Eighty four healthy adult patients of ASA grades I or II were included in the study. The muscle relaxants studied were vecuronium (0.08 mg/kg), atracurium (0.5 mg/kg), mivacurium (0.15 mg/kg), rocuronium (0.6 mg/kg) and succinylcholine (1.0 mg/kg, 0.1 mg/kg, 0.2 mg/kg, 0.4 mg/kg). The TOF ratios were measured at approximate height of first response in the TOF (T1) of 75, 50 and 25% during onset and offset. Especially its ratios were measured at first depress of T1 during onset and its corresponding T1 during offset following administration of subclinical doses of succinylcholine. RESULTS: In the non-depolarizing muscle relaxants, TOF fade is more evident during offset than onset (p<0.05). The extent of fade varies between muscle relaxants. The greatest TOF fade has been shown in rocuronium during onset. In the succinylcholine, the TOF fade is apparent during onset and related to doses given (p<0.05). However the significant TOF fade is not seen during offset. CONCLUSIONS: All muscle relaxants, including both depolarizing and nondepolarizing agent, have predominantly postsynaptic and presynaptic effects. Furthermore, the fact that moderate TOF fade after subclinical doses of succinylcholine occurred obviously during onset of block is possibly indicating a greater presynaptic receptor blocking action.

Evidence for an 2-presynaptic blocking activity of levamisole in the isolated rat vas deferens

The effects of levamisole on the sympathetic transmission and the mechanism underlying these effects were investigated in the field- stimulated rat vas deferens. Levamisole [5-80 muM] potentiated the contraction of the vas deferens induced by field stimulation at low and high frequencies [2, 20 Hz] in a concentration-dependent manner. The extent of potentiation at low frequency was greater than that at high frequency. However, noradrenaline-induced contractions were only potentiated at high concentrations of levamisole [40, 80 muM]. The potentiating effect of levamisole on electrically evoked muscle twitches was completely abolished in the presence of yohimbine and only a slight potentiation was obtained with high concentrations of levamisole. Moreover, in the presence of levamisole [20 muM], the concentration-response curve of clonidine was shifted to the right and the ED50 of clonidine was increased by three-fold. Levamisole was capable of recovering the electrically-evoked muscle contraction after being abolished by cocaine. These data provided a strong evidence for an alpha2-presynaptic blocking activity of levamisole in the isolated rat vas deferens and confirmed its reported uptake blocking activity, only at high concentration