Effects of the DAG analogue 1,2-dioctanoyl-sn-glycerol (DiC8) on nicotine- and clothianidin-evoked currents through α-bungarotoxin-insensitive nicotinic acetylcholine receptors expressed on cockroach neurosecretory cells.

We previously demonstrated that the cockroach α-bungarotoxin-sensitive nicotinic acetylcholine receptors, nAChR1 and nAChR2 subtypes, are differently sensitive to intracellular calcium pathways. Here, using whole cell patch-clamp recordings, we studied the effects of the diacylglycerol (DAG) analogue 1,2-dioctanoyl-sn-glycerol (DiC8) on nicotine- and clothianidin-evoked currents under an α-bungarotoxin treatment. Our results demonstrated that DiC8 reduced nicotine and clothianidin evoked currents. 10 µM DiC8 suppressed the increase in nicotine-induced currents which was brought about by application of 5 mM caffeine or 9 mM Ca2+, whereas DiC8 did not affect the decrease in nicotine-induced currents induced by BAPTA. Similarly, bath application of caffeine or 9 mM Ca2+ did not change the clothianidin effects, and the amplitude of clothianidin-induced currents was not affected. However, co-application of both 10 µM DiC8 with 9 mM Ca2+, caffeine or BAPTA reduced clothianidin current amplitudes. We conclude that nicotine and clothianidin differently modulate nAChR1 and nAChR2 subtypes under DiC8 treatment, and that nicotine activates nAChR1, whereas clothianidin activates both nAChR1 and nAChR2 subtypes.

Mitochondrial ROS cause motor deficits induced by synaptic inactivity: Implications for synapse pruning.

Developmental synapse pruning refines burgeoning connectomes. The basic mechanisms of mitochondrial reactive oxygen species (ROS) production suggest they select inactive synapses for pruning: whether they do so is unknown. To begin to unravel whether mitochondrial ROS regulate pruning, we made the local consequences of neuromuscular junction (NMJ) pruning detectable as motor deficits by using disparate exogenous and endogenous models to induce synaptic inactivity en masse in developing Xenopus laevis tadpoles. We resolved whether: (1) synaptic inactivity increases mitochondrial ROS; and (2) chemically heterogeneous antioxidants rescue synaptic inactivity induced motor deficits. Regardless of whether it was achieved with muscle (α-bungarotoxin), nerve (α-latrotoxin) targeted neurotoxins or an endogenous pruning cue (SPARC), synaptic inactivity increased mitochondrial ROS in vivo. The manganese porphyrins MnTE-2-PyP5+ and/or MnTnBuOE-2-PyP5+ blocked mitochondrial ROS to significantly reduce neurotoxin and endogenous pruning cue induced motor deficits. Selectively inducing mitochondrial ROS-using mitochondria-targeted Paraquat (MitoPQ)-recapitulated synaptic inactivity induced motor deficits; which were significantly reduced by blocking mitochondrial ROS with MnTnBuOE-2-PyP5+. We unveil mitochondrial ROS as synaptic activity sentinels that regulate the phenotypical consequences of forced synaptic inactivity at the NMJ. Our novel results are relevant to pruning because synaptic inactivity is one of its defining features.

In vivo injection of α-bungarotoxin to improve the efficiency of motor endplate labeling.

INTRODUCTION: Motor endplates are composed of a motor neuron terminal and muscle fiber and are distributed in skeletal muscle, causing muscle contraction. However, traditional motor endplate staining methods are limited to the observation of partial skeletal muscle. The procedure was time-consuming due to strict incubation conditions, and usually provided unsatisfactory results. We explored a novel method to label motor endplate rapidly by in vivo injection of fluorescent α-bungarotoxin. METHODS: Fifty-two mice were randomly divided into two groups, an experiment group (n = 50), and a contrast group (n = 2). In experiment group, α-bungarotoxin was injected via the caudal vein. The injection dosages were designated as 0.1, 0.2, 0.3, 0.4, and 0.5 µg/g. The experimental mice were divided into five subgroups of ten mice per group. The contrast group was only injected with 200 µL normal saline solution. Bilateral gastrocnemius were acquired for microscope analysis and optical clearing to seek specific fluorescent signal. RESULTS: A dose of 0.3 µg/g of α-bungarotoxin with 1 h conjugation time could display the number and structure of motor endplate in plane view. Compared with the traditional procedure, this method was rapid, convenient, and time-saving. Combined with the optical clearing technique, spatial distribution could also be seen, helping to better understand the stereoscopic view of motor endplate position in skeletal muscle. CONCLUSIONS: In vivo injection of α-bungarotoxin proved effective for studying motor endplate in skeletal muscle.

Dexmedetomidine attenuates inflammatory reaction in the lung tissues of septic mice by activating cholinergic anti-inflammatory pathway.

Dexmedetomidine (Dex) is a highly selective α2-adrenergic receptor agonist that is widely used for sedation in intensive care units and in clinical anesthesia. Dex has also been shown to possess anti-inflammatory benefits. However, the underlying mechanism by which Dex relieves the inflammatory reaction in the lung tissues of septic mice has not been fully elucidated. In this study, we aimed to evaluate the protective effects and possible mechanism of Dex on the sepsis-induced lung inflammatory response in mice. Sepsis was induced in mice models through the intraperitoneal injection of lipopolysaccharide (LPS). The preemptive administration of Dex substantially abated sepsis-induced pulmonary edema, pulmonary histopathological changes, and NF-κB p65 activity. The production of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) at both the mRNA and protein levels was also reduced. Moreover, these effects were significantly blocked by the α7 nicotinic acetylcholine receptor (α7nAChR) antagonist α-bungarotoxin (α-Bgt). α-Bgt aggravated pulmonary edema and pulmonary histopathological changes, as well as increased NF-κB p65 activity and TNF-α and IL-6 expression at both the mRNA and protein levels. The overall results demonstrate that Dex inhibits the LPS-induced inflammatory reaction in the lung tissues of septic mice partly through the α7nAChR-dependent cholinergic anti-inflammatory pathway.

Block of postjunctional muscle-type acetylcholine receptors in vivo causes train-of-four fade in mice.

BACKGROUND: Train-of-four (TOF) fade during nerve-mediated muscle contraction is postulated to be attributable to inhibition of prejunctional nicotinic α3ß2 acetylcholine receptors (nAChRs), while decrease of twitch tension is attributable to block of postjunctional muscle nAChRs. The validity of these presumptions was tested using specific prejunctional and postjunctional nAChR antagonists, testing the hypothesis that fade is not always a prejunctional phenomenon. METHODS: Pentobarbital anaesthetized mice had TOF fade measured after administration of: either 0.9% saline; the prejunctional α3ß2 nAChR antagonist, dihydro-ß-erythroidine (DHßE); the postjunctional nAChR antagonists, α-bungarotoxin (α-BTX) or α-conotoxin GI; and a combination of α-BTX and DHßE; or a combination of α-conotoxin GI and DHßE. RESULTS: Saline caused no neuromuscular changes. Administration of muscle nAChR antagonists, α-BTX or α-conotoxin GI caused significant decrease of twitch tension and TOF fade compared with baseline (P<0.01). DHßE alone caused no change of twitch tension or fade even after 90 min, but its coadministration with α-BTX or α-conotoxin GI significantly accelerated the onset of paralysis and degree of fade compared with α-BTX or α-conotoxin GI alone (P<0.01). CONCLUSIONS: Occupation of postjunctional nAChRs alone by α-BTX or α-conotoxin GI causes fade. As the prejunctional effects of DHßE on fade became manifest only when co-administered with α-BTX or α-conotoxin GI, specific inhibition of prejunctional nAChR alone is not necessary and sufficient to cause fade. Fade observed during repetitive nerve stimulation can be because of block of either postjunctional nAChRs alone, or block of prejunctional and postjunctional nAChRs together.

Electroacupuncture activates enteric glial cells and protects the gut barrier in hemorrhaged rats.

AIM: To investigate whether electroacupuncture ST36 activates enteric glial cells, and alleviates gut inflammation and barrier dysfunction following hemorrhagic shock. METHODS: Sprague-Dawley rats were subjected to approximately 45% total blood loss and randomly divided into seven groups: (1) sham: cannulation, but no hemorrhage; (2) subjected to hemorrhagic shock (HS); (3) electroacupuncture (EA) ST36 after hemorrhage; (4) vagotomy (VGX)/EA: VGX before hemorrhage, then EA ST36; (5) VGX: VGX before hemorrhage; (6) α-bungarotoxin (BGT)/EA: intraperitoneal injection of α-BGT before hemorrhage, then EA ST36; and (7) α-BGT group: α-BGT injection before hemorrhage. Morphological changes in enteric glial cells (EGCs) were observed by immunofluorescence, and glial fibrillary acidic protein (GFAP; a protein marker of enteric glial activation) was evaluated using reverse transcriptase polymerase chain reaction and western blot analysis. Intestinal cytokine levels, gut permeability to 4-kDa fluorescein isothiocyanate (FITC)-dextran, and the expression and distribution of tight junction protein zona occludens (ZO)-1 were also determined. RESULTS: EGCs were distorted following hemorrhage and showed morphological abnormalities. EA ST36 attenuated the morphological changes in EGCs at 6 h, as compared with the VGX, α-BGT and HS groups. EA ST36 increased GFAP expression to a greater degree than in the other groups. EA ST36 decreased intestinal permeability to FITC-dextran (760.5 ± 96.43 ng/mL vs 2466.7 ± 131.60 ng/mL, P < 0.05) and preserved ZO-1 protein expression and localization at 6 h after hemorrhage compared with the HS group. However, abdominal VGX and α-BGT treatment weakened or eliminated the effects of EA ST36. EA ST36 reduced tumor necrosis factor-α levels in intestinal homogenates after blood loss, while vagotomy or intraperitoneal injection of α-BGT before EA ST36 abolished its anti-inflammatory effects. CONCLUSION: EA ST36 attenuates hemorrhage-induced intestinal inflammatory insult, and protects the intestinal barrier integrity, partly via activation of EGCs.

Horseradish peroxidase dye tracing and embryonic statoacoustic ganglion cell transplantation in the rat auditory nerve trunk.

At present severe damage to hair cells and sensory neurons in the inner ear results in non-treatable auditory disorders. Cell implantation is a potential treatment for various neurological disorders and has already been used in clinical practice. In the inner ear, delivery of therapeutic substances including neurotrophic factors and stem cells provide strategies that in the future may ameliorate or restore hearing impairment. In order to describe a surgical auditory nerve trunk approach, in the present paper we injected the neuronal tracer horseradish peroxidase (HRP) into the central part of the nerve by an intra cranial approach. We further evaluated the applicability of the present approach by implanting statoacoustic ganglion (SAG) cells into the same location of the auditory nerve in normal hearing rats or animals deafened by application of ß-bungarotoxin to the round window niche. The HRP results illustrate labeling in the cochlear nucleus in the brain stem as well as peripherally in the spiral ganglion neurons in the cochlea. The transplanted SAGs were observed within the auditory nerve trunk but no more peripheral than the CNS-PNS transitional zone. Interestingly, the auditory nerve injection did not impair auditory function, as evidenced by the auditory brainstem response. The present findings illustrate that an auditory nerve trunk approach may well access the entire auditory nerve and does not compromise auditory function. We suggest that such an approach might compose a suitable route for cell transplantation into this sensory cranial nerve.

B chain is a functional subunit of beta-bungarotoxin for inducing apoptotic death of human neuroblastoma SK-N-SH cells.

beta-Bungarotoxin (beta-Bgt), a presynaptic phospholipase A(2) (PLA(2)) neurotoxin isolated from the venom of Bungarus multicinctus, consists of A chain and B chain. The goal of the present study is to explore the functional contribution of the two subunits to the toxicity of beta-Bgt. beta-Bgt was found to induce apoptotic death of SK-N-SH cells via elevating intracellular Ca(2+) and intracellular ROS production. Moreover, an activation of p38 MAPK was associated with the cytotoxicity of beta-Bgt. SB202190 (p38 MAPK inhibitor), N-acetylcysteine (antioxidant reagent), 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid (BAPTA) (Ca(2+) chelator) and the inhibitors of Ca(2+) release from intracellular depots (ruthenium red and 2-aminoethoxydiphenyl borate) effectively attenuated the cytotoxicity of beta-Bgt. In sharp contrast to the inability of A chain, B chain was able to induce cytotoxic effects on SK-N-SH cells as beta-Bgt did. Abolishment of PLA(2) activity did not significantly alter the cytotoxic activity of beta-Bgt. MK801 (an NMDA receptor antagonist), antibodies against NMDA receptor and 4-aminopyridine (a potassium channel blocker) markedly reduced the cytotoxic effects of beta-Bgt, B chain and catalytically inactivated beta-Bgt. Moreover, antibodies against NMDA receptor blocked the binding of rhodamine-labeled beta-Bgt to SK-N-SH cells. Taken together, our data indicate that B chain is a functional subunit responsible for the cytotoxicity of beta-Bgt, and suggest that the cytotoxicity of beta-Bgt is mediated by NMDA receptor and potassium conductance.

Diabetic urethropathy compounds the effects of diabetic cystopathy.

PURPOSE: The effects of short-term and long-term diabetes mellitus on urethral function were investigated to determine the contribution of urethral dysfunction to diabetes mellitus voiding dysfunction. MATERIALS AND METHODS: Isovolumetric bladder pressure, urethral perfusion pressure and external urethral sphincter electromyography were measured in urethane anesthetized, female Sprague-Dawley rats (Charles River Laboratories, Wilmington, Massachusetts) 5 or 10 weeks after streptozotocin induced diabetes mellitus. Urethral responses to serial administration of the skeletal muscle blocker alpha-bungarotoxin, the nitric oxide synthase inhibitor N(omega)-nitro-L-arginine and the alpha-adrenergic agonist L-phenylephrine were determined in diabetes mellitus and age matched controls. RESULTS: Peak bladder pressures and contraction amplitudes were significantly decreased in diabetes mellitus rats. Detrusor-sphincter dyssynergia occurred in approximately 30% of diabetes mellitus rats but never in controls. Alpha-Bungarotoxin caused a greater decrease in baseline urethral perfusion pressure in diabetes mellitus rats than in controls (approximately 40% vs approximately 15%). Bladder contraction associated urethral smooth muscle relaxation amplitudes were significantly less in diabetes mellitus rats than in controls. N(omega)-nitro-L-arginine significantly suppressed urethral relaxation in controls but not in diabetes mellitus rats. L-phenylephrine significantly increased baseline urethral perfusion pressure in diabetes mellitus rats but not in controls. The unassociated conditions of insensitivity to N-nitro-L-arginine and hypersensitivity to L-phenylephrine were more common in 10-week diabetes mellitus rats than in control rats. CONCLUSIONS: Diabetes mellitus induced urethropathy is characterized by external urethral sphincter dysfunction, decreased urethral smooth muscle relaxation and nitric oxide responsiveness, and increased urethral smooth muscle responsiveness to alpha(1)-adrenergic agonists. These changes increase outlet resistance and, thereby, decrease voiding efficiency. This exacerbates voiding dysfunction, creating a vicious cycle of progressive lower urinary tract damage and dysfunction. Early intervention targeting outlet resistance may be indicated.

The involvement of central cholinergic system in the pressor effect of intracerebroventricularly injected U-46619, a thromboxane A2 analog, in conscious normotensive rats.

The aim of this study was to determine the involvement of the central cholinergic system in the rise in blood pressure evoked by the thromboxane A2 (TxA2) analog, U-46619, given centrally. Intracerebroventricular (i.c.v.) injections of U-46619 (0.5, 1.0 and 2.0 microg) caused dose- and time-related increases in blood pressure and decreased heart rate in awake rats. U-46619 (1 microg; i.c.v.) also produced an approximately 65% increase in posterior hypothalamic extracellular acetylcholine and choline levels. Pretreatment with SQ-29548 (8 microg; i.c.v.), selective TxA2 receptor antagonist, completely inhibited both the cardiovascular responses and the increase in acetylcholine and choline levels to subsequent injection of U-46619 (1 microg; i.c.v.). Atropine (10 microg; i.c.v.), nonselective muscarinic receptor antagonist, pretreatment did not affect the cardiovascular responses observed after U-46619 (1 microg; i.c.v.). Pretreatment with the nonselective nicotinic receptor antagonist, mecamylamine (50 microg; i.c.v.) attenuated the pressor effect of U-46619 (1 microg; i.c.v.). Higher doses of mecamylamine (75 and 100 microg; i.c.v.) pretreatments did not change the magnitude of the blockade of pressor response to U-46619; however, they abolished the bradycardic effect of U-46619 dose-dependently. Interestingly, pretreatment of rats with methyllycaconitine (10 microg; i.c.v.) or alpha-bungarotoxin (10 microg; i.c.v.), selective antagonists of alpha7 subtype of nicotinic acetylcholine receptors (alpha7nAChRs), partially abolished the pressor response to i.c.v. injection of U-46619 (1 microg). Similar to the mecamylamine data, the use of higher doses of methyllycaconitine (25 and 50 microg; i.c.v.) produced the same magnitude of blockade that was observed after the 10 microg methyllycaconitine pretreatment, but it completely abolished the bradycardic effect of U-46619 (1 microg; i.c.v.) at the dose of 25 microg. The present results show that central administration of U-46619 produces pressor and bradycardic effect and increase in hypothalamic acetylcholine and choline levels by activating central TxA2 receptors. The activation of central nicotinic receptors, predominantly alpha7nAChRs, partially mediates the cardiovascular responses to i.c.v. injection of U-46619.

The role of cholinergic networks of the anterior basal and inferior frontal lobes in the predatory behaviour of Sepia officinalis.

The predatory behaviour of the cuttlefish has been the subject of a few detailed studies and can be divided into several stages: prey detection, orientation, positioning, prey-seizing, prey-manipulation and ingestion. Nevertheless, the data about its control by the CNS remain fragmentary. By injecting a cholinergic agonist (nicotine) and antagonists (alpha-bungarotoxin, mecamylamine), the implication of cholinergic networks of the anterior basal and inferior frontal lobes in the control of predatory behaviour are demonstrated. Through these cholinergic networks, the anterior basal lobe takes an important part in the orientation and positioning. The inferior frontal lobe seems to play a role in the control of brachial manipulative and buccal mass activities. The implication of cholinergic networks of the anterior basal and inferior frontal lobes in the predatory behaviour and the pharmacology of nicotinic receptors are discussed.

The antinociceptive effects of alpha7 nicotinic agonists in an acute pain model.

Nicotinic receptors have been found to play a role in modulating pain transmission in the CNS. Activation of cholinergic pathways by nicotine and nicotinic agonists has been shown to elicit antinociceptive effects in a variety of species and pain tests. The involvement of alpha(7) nicotinic receptors in nicotinic analgesia was assessed after spinal (i.t.) and intraventricular (i.c.v.) administration in mice. Dose-dependent antinociceptive effects were seen with the alpha(7) agonist choline after spinal and supraspinal injection using the tail-flick test. Furthermore, alpha(7) antagonists MLA and alpha-BGTX significantly blocked the effects of choline. Dihydro-beta-erythroidine and mecamylamine failed to block choline-induced antinociception. These results strongly support the involvement of alpha(7) subunits in choline's antinociceptive effects. DMXB and 4-OH-DMXB, partial alpha(7) agonists, failed to elicit a significant antinociceptive effect. However, they blocked choline-induced antinociception in a dose-dependent manner following i.t. injection. This antagonism is probably related to their partial agonistic properties of the alpha(7) receptors. These studies suggest that activation of alpha(7) receptors in the CNS elicits antinociceptive effects in an acute thermal pain model.

Spinal mechanisms underlying A-85380-induced effects on acute thermal pain.

Systemic administration of nicotinic receptor (nAChR) agonists is antinociceptive in models of acute pain whereas their intrathecal (i. t.) administration has been reported to be antinociceptive, nociceptive or without effect. It has been hypothesized that the action induced is dependent upon the subtype and location of the nAChR activated. In addition, there is considerable evidence that nAChR ligand-induced antinociception is mediated by other neurotransmitter systems via descending pathways from the brainstem to the spinal cord. The present study investigated the effects of i. t. and systemic administration of A-85380, a novel nAChR agonist, in the paw withdrawal model of acute thermal pain in the rat. Given i.t. , A-85380 (1 and 10 nmol/rat) decreased the latency to paw withdrawal by 2-4 s. This pronociception was accompanied by a spontaneous flinching behavior. Both of these effects were differentially blocked by i.t. pretreatment with the nAChR antagonists mecamylamine (10 nmol)>MLA (100 nmol)>DHbetaE (50% with 1000 nmol) but not by alpha-bungarotoxin (0% at 0.63 nmol). Given systemically, A-85380 (0.56 micromol/kg, i.p.) induced antinociception as indicated by an increased latency to paw withdrawal, an effect differentially altered by i.t. pretreatment with monoaminergic antagonists (100 nmol/rat). While mecamylamine and prazosin had no effect, scopolamine, methysergide and MDL 72222 partially antagonized and idazoxan completely antagonized A-85380-induced antinociception. Finally, as measured by in vivo microdialysis, levels of 5-HT, but not NE, in the i.t. space of the lumber region of the spinal cord were significantly increased following the systemic administration of A-85380. Together these data suggest that the nociceptive properties of spinally administered nAChR agents are not mediated by either an alpha(4)beta(2) or an alpha(7) subtype nAChR, whereas the antinociceptive properties of systemically-administered nAChR agents are mediated by descending noradrenergic, serotonergic and muscarinic inhibitory pathways.

Receptor subtypes mediating depressor responses to microinjections of nicotine into medial NTS of the rat.

Microinjections (50 nl) of nicotine (0.01-10 microM) into the nucleus of the solitary tract (NTS) of adult, urethan-anesthetized, artificially ventilated, male Wistar rats, elicited decreases in blood pressure and heart rate. Prior microinjections of alpha-bungarotoxin (alpha-BT) and alpha-conotoxin ImI (specific toxins for nicotinic receptors containing alpha7 subunits) elicited a 20-38% reduction in nicotine responses. Similarly, prior microinjections of hexamethonium, mecamylamine, and alpha-conotoxin AuIB (specific blockers or toxin for nicotinic receptors containing alpha3beta4 subunits) elicited a 47-79% reduction in nicotine responses. Nicotine responses were completely blocked by prior sequential microinjections of alpha-BT and mecamylamine into the NTS. Complete blockade of excitatory amino acid receptors (EAARs) in the NTS did not attenuate the responses to nicotine. It was concluded that 1) the predominant type of nicotinic receptor in the NTS contains alpha3beta4 subunits, 2) a smaller proportion contains alpha7 subunits, 3) the presynaptic nicotinic receptors in the NTS do not contribute to nicotine-induced responses, and 4) EAARs in the NTS are not involved in mediating responses to nicotine.

Protection against alpha-bungarotoxin poisoning by immunization with synthetic toxin peptides.

The purpose of the present work was to determine the ability of BgTX peptides, corresponding to the various loops and exposed regions of alpha-bungarotoxin (BgTX) and representing regions that are recognized by B and/or T cells, to stimulate protective immunity in mice against in vivo challenge with BgTX. The BgTX LD50 values in non-immune mice or mice that had been immunized with proteins and peptides unrelated to BgTX were: Balb/c, 0.128 microgram/g; SJL, 0.156 microgram/g. Immunization of Balb/c and SJL mice with each of the synthetic peptides in its free form afforded considerable protection against BgTX poisoning. Peptides L1 (residues 3-16), L2 (residues 26-41) and C-tail (residues 66-74) of BgTX were the most protective and mice immunized with these peptides survived LD50 values that were three times higher than control mice. Immunization with an equimolar mixture of the three peptides was even more protective and these mice survived even higher challenge doses of BgTX (4.6-fold higher than LD50 of controls; i.e. protection index, PI = 4.6). An OVA conjugate carrying all three peptides, when used as an immunogen, conferred extremely high protection (PI > or = 18.1) which was almost double the protection obtained by BgTX immunization (PI = 9.7). Thus, the conjugate of the three peptides should serve as an effective vaccine against BgTX poisoning. Furthermore, these results with BgTX peptides should serve as a prototype for the design and synthesis of peptide vaccines against other members of this large family of toxins which include both long and short neurotoxins as well as cytotoxins.

alpha-Bungarotoxin sensitization in experimental autoimmune myasthenia gravis.

A mouse model of MG, termed experimental autoimmune myasthenia gravis (EAMG), can be obtained after immunization with Torpedo acetylcholine receptor (AChR). Although many studies have detailed the consequence of AChR antibodies binding at the neuromuscular junction and the difficulty in obtaining obvious clinical signs, less attention has been focused on the possibility of amplifying the muscular block in order to discriminate between immunized and healthy animals. In the present studies we observe that a single inoculation of alpha-bungarotoxin (alpha-bgt) can amplify the neuromuscular block revealed by repetitive nerve stimulation, and induce in EAMG mice a stable muscular weakness state lasting for at least 169 hours instead of 95 hours in normal mice. This model could provide an excellent tool for evaluating drugs active on neuromuscular transmission.

Exogenously administered alpha-bungarotoxin binds to embryonic chick spinal cord: implications for the toxin-induced arrest of naturally occurring motoneuron death.

Administration of alpha-bungarotoxin and other curare-like drugs during embryogenesis arrests motoneuron death which normally occurs in the spinal cord from day 6 to day 10 of embryogenesis. The accepted explanation is that such motoneuron rescue is mediated by inhibition of neuromuscular transmission following the blockade of nicotinic cholinoceptors at the neuromuscular junction. In this study we investigated a further possibility, namely that motoneuron rescue might also involve the blockade of alpha-bungarotoxin-sensitive sites within the spinal cord. The kinetic profile of [125I]alpha-bungarotoxin binding was examined in the brachial and lumbar regions of chick spinal cord at embryonic day 15. Binding was specific and apparently saturable within the range 1-34 nM reaching a maximum after 45 min. Specific binding involved a single class of non-interacting sites with a KD of 8.0 nM and a Bmax of 106 +/- 12 fmol/mg of protein. Nicotine displaced specific [125I]alpha-bungarotoxin binding in a concentration-dependent manner. Furthermore, specific binding dissociated slowly in the absence of nicotine. Autoradiographs localizing [125I]alpha-bungarotoxin binding in embryonic spinal cord revealed that, at embryonic day 15, specific toxin binding sites could be detected throughout the gray matter. In contrast, at embryonic day 6, the ventral horn contained the majority of specific binding sites. Exogenously administered [125I]alpha-bungarotoxin reached and bound to nicotine-sensitive sites in the spinal cord at embryonic day 7. To conclude, these data demonstrate that central nicotine-sensitive sites which bind [125I]alpha-bungarotoxin in a saturable and specific manner were present at the beginning of the critical motoneuron death phase of neurogenesis and that they were accessible to exogenously administered toxin. It is proposed that the [125I]alpha-bungarotoxin binding characterized here is to a class of putative alpha-bungarotoxin-sensitive nicotinic cholinoceptors. These studies raise the possibility that alpha-bungarotoxin blockade of such putative nicotinic cholinoceptors within the spinal cord may contribute to toxin-induced arrest of naturally occurring motoneuron death.

Neuronal degeneration induced by stereotaxic injection of beta-bungarotoxin into rat brain.

The presynaptic protein neurotoxin beta-bungarotoxin (beta-bgt) caused degeneration of neurons, following stereotaxic administration into rat brain. Focal lesions were induced with as little as 0.1 ng (5 fmol) of beta-bgt; indicating that the toxin is over 10(6) times more potent than kainic acid. More extensive studies in the septo-hippocampal system showed beta-bgt lesions affected both cell bodies and nerve terminals and were not neurotransmitter-specific. The endogenous phospholipase A2 activity of beta-bgt did not simply account for the creation of lesions since an acidic phospholipase A isoenzyme from Vipera russellii was virtually inactive in causing neuronal damage.

On the appearance of acetylcholine receptors in denervated rat diaphragm, and its dependence on nerve stump length.

Acetylcholine (ACh) sensitivity and extrajunctional receptor distribution of the rat diaphragm were closely monitored during the early period following denervation. Both contracture in response to 10 microgram/ml of ACh and extrajunctional binding of [125I]alpha-bungarotoxin ([125I]alpha-BTX)) were first detectable 30 h after cutting the phrenic nerve in the thorax. If the nerve were cut more proximally, leaving a 3.5 cm distal nerve stump, the same level of ACh contracture and [125I]alpha-BTX binding did not appear until 40 h after operation. This 10-h delay was far longer than the 3-h delay in transmission failure reportedly dependent on stump length. The earliest detectable extrajunctional [125I]alpha-BTX binding appeared throughout the entire muscle fiber, and was not localized to the endplate region as would be expected if degeneration in the nerve terminal induced new receptors. However, later significant increases in [125I]alpha-BTX binding at the endplate region could have resulted from such degeneration. All these results are consistent with neurotrophic regulation of muscle ACh receptors, working via a mechanism involving axonal transport.