γ-Bungarotoxin impairs the vascular endothelial barrier function by inhibiting integrin α5.

γ-bungarotoxin (γ-BGT) is an RGD motif-containing protein, derived from the venom of Bungarus multicinctus, leading to acute death in mice. These RGD motif-containing proteins from snake venom belonging to the disintegrin family can interfere with vascular endothelial homeostasis by directly binding cell surface integrins. Targeting integrins that generate vascular endothelial dysfunction may contribute to γ-BGT poisoning, however, the underlying mechanisms have not been investigated in detail. In this study, the results showed that γ-BGT played a role in -promoting the permeability of the vascular endothelial barrier. Depending on its selective binding to integrin α5 in vascular endothelium (VE), γ-BGT initiated downstream events, including focal adhesion kinase dephosphorylation and cytoskeleton remodeling, resulting in the intercellular junction interruption. Those alternations facilitated paracellular permeability of VE and barrier dysfunction. Proteomics profiling identified that as a downstream effector of the integrin α5 / FAK signaling pathway cyclin D1 partially mediated the cellular structural changes and barrier dysfunction. Furthermore, VE-released plasminogen activator urokinase and platelet-derived growth factor D could serve as potential diagnostic biomarkers for γ-BGT-induced vascular endothelial dysfunction. Our results indicate the mechanisms through which γ-BGT as a novel disintegrin directly interacts with the VE, with consequences for barrier dysfunction.

On characterizing the Red-headed Krait (Bungarus flaviceps) venom: Decomplexation proteomics, immunoreactivity and toxicity cross-neutralization by hetero-specific antivenoms.

The Red-headed Krait (Bungarus flaviceps) is a medically important venomous snake species in Southeast Asia, while there is no specific antivenom available for its envenoming. This study investigated the venom composition through a decomplexation proteomic approach, and examined the immunoreactivity as well as cross-neutralization efficacy of two hetero-specific krait antivenoms, Bungarus candidus Monovalent Antivenom (BcMAV) and Bungarus fasciatus Monovalent Antivenom (BfMAV), against the venom of B. flaviceps from Peninsular Malaysia. A total of 43 non-redundant proteoforms belonging to 10 toxin families were identified in the venom proteome, which is dominated by phospholipases A2 including beta-bungarotoxin lethal subunit (56.20 % of total venom proteins), Kunitz-type serine protease inhibitors (19.40 %), metalloproteinases (12.85 %) and three-finger toxins (7.73 %). The proteome varied in quantitative aspect from the earlier reported Indonesian (Sumatran) sample, suggesting geographical venom variation. BcMAV and BfMAV were immunoreactive toward the B. flaviceps venom, with BcMAV being more efficacious in immunological binding. Both antivenoms cross-neutralized the venom lethality with varying efficacy, where BcMAV was more potent than BfMAV by ~13 times (normalized potency: 38.04 mg/g vs. 2.73 mg/g, defined as the venom amount completely neutralized by one-gram antivenom protein), supporting the potential utility of BcMAV for para-specific neutralization against B. flaviceps venom.

Varespladib (LY315920) rescued mice from fatal neurotoxicity caused by venoms of five major Asiatic kraits (Bungarus spp.) in an experimental envenoming and rescue model.

The venoms of Asiatic kraits (Bungarus spp.) contain various neurotoxic phospholipases A2 (beta-bungarotoxins) which can irreversibly damage motor nerve terminals, resulting in rapidly fatal suffocation by respiratory muscle paralysis or oral airway obstruction. Hence, there is a need of adjunct therapy at the pre-hospital stage to prevent or delay the onset of neurotoxicity, so that antivenom can be given within golden hour before the envenoming becomes antivenom-resistant. This study investigated the efficacy of varespladib, a small molecule PLA2 (phospholipase A2) inhibitor, given as a bolus subcutaneously upon the onset of krait venom-induced paralysis in a mouse experimental envenoming and rescue model, where the severity of neurotoxicity was scored and the survival rate was monitored over 24 h. Varespladib at 10 mg/kg effectively alleviated the neurotoxicity of Bungarus sindanus, Bungarus multicinctus and Bungarus fasciatus venoms, and rescued all mice from venom-induced lethality (100% survival). Varespladib at this dose, however, only partially reduced the neurotoxicity of Bungarus caeruleus and Bungarus candidus venoms, while all challenged mice were dead by 23 h (B. caeruleus) and 12 h (B. candidus). An increased dose of varespladib at 20 mg/kg markedly abated the venom neurotoxicity past 8 h of envenoming, and protected the mice from venom lethality (B. caeruleus: 75% survival; B. candidus: 100% survival). The finding is consistent with previous studies which demonstrated varespladib's inhibitory effect against some snake venoms. The findings suggest varespladib could be repurposed as an emergency drug for prevention or rescue (if given early enough) from the acute, neurotoxic envenoming syndromes caused by various major krait species in Asia.

In Vitro Neurotoxicity of Chinese Krait (Bungarus multicinctus) Venom and Neutralization by Antivenoms.

Bungarus multicinctus, the Chinese krait, is a highly venomous elapid snake which causes considerable morbidity and mortality in southern China. B. multicinctus venom contains pre-synaptic PLA2 neurotoxins (i.e., ß-bungarotoxins) and post-synaptic neurotoxins (i.e., α-bungarotoxins). We examined the in vitro neurotoxicity of B. multicinctus venom, and the efficacy of specific monovalent Chinese B. multicinctus antivenom, and Australian polyvalent elapid snake antivenom, against venom-induced neurotoxicity. B. multicinctus venom (1-10 µg/mL) abolished indirect twitches in the chick biventer cervicis nerve-muscle preparation as well as attenuating contractile responses to exogenous ACh and CCh, but not KCl. This indicates a post-synaptic neurotoxic action but myotoxicity was not evident. Given that post-synaptic α-neurotoxins have a more rapid onset than pre-synaptic neurotoxins, the activity of the latter in the whole venom will be masked. The prior addition of Chinese B. multicinctus antivenom (12 U/mL) or Australian polyvalent snake antivenom (15 U/mL), markedly attenuated the neurotoxic actions of B. multicinctus venom (3 µg/mL) and prevented the inhibition of contractile responses to ACh and CCh. The addition of B. multicinctus antivenom (60 U/mL), or Australian polyvalent snake antivenom (50 U/mL), at the t90 time point after the addition of B. multicinctus venom (3 µg/mL), did not restore the twitch height over 180 min. The earlier addition of B. multicinctus antivenom (60 U/mL), at the t20 or t50 time points, also failed to prevent the neurotoxic effects of the venom but did delay the time to abolish twitches based on a comparison of t90 values. Repeated washing of the preparation with physiological salt solution, commencing at the t20 time point, failed to reverse the neurotoxic effects of venom or delay the time to abolish twitches. This study showed that B. multicinctus venom displays marked in vitro neurotoxicity in a skeletal muscle preparation which is not reversed by antivenom. This does not appear to be related to antivenom efficacy, but due to the irreversible/pseudo-irreversible nature of the neurotoxins.

Immunoreactivity and neutralization study of Chinese Bungarus multicinctus antivenin and lab-prepared anti-bungarotoxin antisera towards purified bungarotoxins and snake venoms.

Bungarus multicinctus is the most venomous snake distributed in China and neighboring countries of Myanmar, Laos, north Vietnam and Thailand. The high mortality rate of B. multicinctus envenomation is attributed to the lethal components of α-, ß-, γ- and κ- bungarotoxins contained in the venom. Although anti-B. multicinctus sera were produced in Shanghai, Taiwan and Vietnam, the most widely clinic used product was term as B. multicinctus antivenin and manufactured by Shanghai Serum Bio-technology Co. Ltd. In the present investigation, high purity α-, ß- and γ-bungarotoxins were separately isolated from B. multicinctus crude venom. Rabbit anti- α-, ß- and γ-bungarotoxin antisera were prepared by common methods, respectively. LD50 values of α-, ß- and γ-bungarotoxins were systematically determined via three administration pathways (intraperitoneal, intramuscular and intravenous injections) in Kunming mice. LD50 values of ß-bungarotoxin were closely related with injection routines but those of both α- and γ-bungarotoxins were not dependent on the injection routines. Commercial B. multicinctus antivenin showed strong immunoreaction with high molecular weight fractions of the B. multicinctus but weakly recognized low molecular weight fractions like α- and γ-bungarotoxins. Although B. multicinctus antivenin showed immunoreaction with high molecular weight fractions of Bungarus fasciatus, Naja atra, Ophiophagus hannah venoms but the antivenin only demonstrated animal protection efficacy against O. hannah venom. These results indicated that the high molecular weight fractions of the O. hannah played an important role in venom lethality but those of B. fasciatus and N. atra did not have such a role.

Variations in neurotoxicity and proteome profile of Malayan krait (Bungarus candidus) venoms.

Malayan krait (Bungarus candidus) is a medically important snake species found in Southeast Asia. The neurotoxic effects of envenoming present as flaccid paralysis of skeletal muscles. It is unclear whether geographical variation in venom composition plays a significant role in the degree of clinical neurotoxicity. In this study, the effects of geographical variation on neurotoxicity and venom composition of B. candidus venoms from Indonesia, Malaysia and Thailand were examined. In the chick biventer cervicis nerve-muscle preparation, all venoms abolished indirect twitches and attenuated contractile responses to nicotinic receptor agonists, with venom from Indonesia displaying the most rapid neurotoxicity. A proteomic analysis indicated that three finger toxins (3FTx), phospholipase A2 (PLA2) and Kunitz-type serine protease inhibitors were common toxin groups in the venoms. In addition, venom from Thailand contained L-amino acid oxidase (LAAO), cysteine rich secretory protein (CRISP), thrombin-like enzyme (TLE) and snake venom metalloproteinase (SVMP). Short-chain post-synaptic neurotoxins were not detected in any of the venoms. The largest quantity of long-chain post-synaptic neurotoxins and non-conventional toxins was found in the venom from Thailand. Analysis of PLA2 activity did not show any correlation between the amount of PLA2 and the degree of neurotoxicity of the venoms. Our study shows that variation in venom composition is not limited to the degree of neurotoxicity. This investigation provides additional insights into the geographical differences in venom composition and provides information that could be used to improve the management of Malayan krait envenoming in Southeast Asia.

Toxins for decoding interface selectivity in nicotinic acetylcholine receptors.

Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels that play crucial roles in neurotransmission and regulate complex processes in brain functions, including anxiety, learning and memory, food intake, drug addiction, cognition and nociception. To perform these and other functions, a diverse array of nAChR subtypes are generated by homomeric or heteromeric assembly of 17 homologous nAChR subunits. Agonists, acetylcholine and nicotine, bind to the interface formed between two α subunits and between α and non-α subunits to activate the nAChR and allow cation influx. The diversity of subunit interfaces determines the channel properties, the responses to different agonists/antagonists, desensitization and downstream signaling and thus, define specialized properties and functions. Over the last several decades, snake venom neurotoxins have contributed to the purification, localization and characterization of molecular details of various nAChRs. Utkin et al. have described the purification and characterization of αδ-bungarotoxins, a novel class of neurotoxins in a recent paper published in the Biochemical Journal [Biochem. J. (2019) 476, 1285-1302]. These toxins from Bungarus candidus venom preferably bind to α-δ site with two orders of magnitude higher affinity compared with α-γ or α-ε sites. The subtle changes in the structure of αδ-bungarotoxins led to variation in interface selectivity. Such new classes of antagonists will offer us great opportunity to delineate the pharmacophores and design new highly selective antagonists. Thus, their findings provide new impetus to re-evaluate molecular details of pharmacological properties of α-neurotoxins with careful consideration towards subtype-, interface- and species-selectivity.

Venom proteome of Bungarus sindanus (Sind krait) from Pakistan and in vivo cross-neutralization of toxicity using an Indian polyvalent antivenom.

The proteome of the Pakistani B. sindanus venom was investigated with reverse-phase HPLC and nano-ESI-LCMS/MS analysis. At least 36 distinct proteins belonging to 8 toxin protein families were identified. Three-finger toxin (3FTx), phospholipase A2 (including ß-bungarotoxin A-chains) and Kunitz-type serine protease inhibitor (KSPI) were the most abundant, constituting ~95% of total venom proteins. The other toxin proteins of low abundance are snake venom metalloproteinase (SVMP), L-amino acid oxidase (LAAO), acetylcholinesterase (AChE), vespryn and cysteine-rich secretory protein (CRiSP). The venom was highly lethal to mice with LD50 values of 0.04 µg/g (intravenous) and 0.15 µg/g (subcutaneous). The 3FTx proteins are diverse, comprising kappa-neurotoxins, neurotoxin-like protein, non-conventional toxins and muscarinic toxin-like proteins. Kappa-neurotoxins and ß-bungarotoxins represent the major toxins that mediate neurotoxicity in B. sindanus envenoming. Alpha-bungarotoxin, commonly present in the Southeast Asian krait venoms, was undetected. The Indian VINS Polyvalent Antivenom (VPAV) was immunoreactive toward the venom, and it moderately cross-neutralized the venom lethality (potency = 0.25 mg/ml). VPAV was able to reverse the neurotoxicity and prevent death in experimentally envenomed mice, but the recovery time was long. The unique toxin composition of B. sindanus venom may be considered in the formulation of a more effective pan-regional, polyspecific antivenom. BIOLOGICAL SIGNIFICANCE: Bungarus sindanus, an endemic krait species distributed mainly in the Sindh Province of Pakistan is a cause of snake envenomation. Its specific antivenom is, however, lacking. The proteomic study of its venom revealed a substantial presence of κ-bungarotoxins and ß-bungarotoxins. The toxin profile corroborates the potent neurotoxicity and lethality of the venom tested in vivo. The heterologous Indian VINS polyvalent antivenom (VPAV) cross-reacted with B. sindanus venom and cross-neutralized the venom neurotoxicity and lethality in mice, albeit the efficacy was moderate. The findings imply that B. sindanus and the phylogenetically related B. caeruleus of India share certain venom epitopes. Research should be advanced to improve the efficacy spectrum of a pan-regional polyspecific antivenom.

Neuromuscular Effects of Common Krait (Bungarus caeruleus) Envenoming in Sri Lanka.

OBJECTIVE: We aimed to investigate neurophysiological and clinical effects of common krait envenoming, including the time course and treatment response. METHODOLOGY: Patients with definite common krait (Bungarus caeruleus) bites were recruited from a Sri Lankan hospital. All patients had serial neurological examinations and stimulated concentric needle single-fibre electromyography (sfEMG) of orbicularis oculi in hospital at 6 wk and 6-9 mth post-bite. PRINCIPAL FINDINGS: There were 33 patients enrolled (median age 35 y; 24 males). Eight did not develop neurotoxicity and had normal sfEMG. Eight had mild neurotoxicity with ptosis, normal sfEMG; six received antivenom and all recovered within 20-32 h. Seventeen patients developed severe neurotoxicity with rapidly descending paralysis, from ptosis to complete ophthalmoplegia, facial, bulbar and neck weakness. All 17 received Indian polyvalent antivenom a median 3.5 h post-bite (2.8-7.2 h), which cleared unbound venom from blood. Despite this, the paralysis worsened requiring intubation and ventilation within 7 h post-bite. sfEMG showed markedly increased jitter and neuromuscular blocks within 12 h. sfEMG abnormalities gradually improved over 24 h, corresponding with clinical recovery. Muscle recovery occurred in ascending order. Myotoxicity was not evident, clinically or biochemically, in any of the patients. Patients were extubated a median 96 h post-bite (54-216 h). On discharge, median 8 days (4-12 days) post-bite, patients were clinically normal but had mild sfEMG abnormalities which persisted at 6 wk post-bite. There were no clinical or neurophysiological abnormalities at 6-9 mth. CONCLUSIONS: Common krait envenoming causes rapid onset severe neuromuscular paralysis which takes days to recover clinically consistent with sfEMG. Subclinical neuromuscular dysfunction lasts weeks but was not permanent. Antivenom effectively cleared venom but did not prevent worsening or reverse neuromuscular paralysis.

Snake and Spider Toxins Induce a Rapid Recovery of Function of Botulinum Neurotoxin Paralysed Neuromuscular Junction.

Botulinum neurotoxins (BoNTs) and some animal neurotoxins (ß-Bungarotoxin, ß-Btx, from elapid snakes and α-Latrotoxin, α-Ltx, from black widow spiders) are pre-synaptic neurotoxins that paralyse motor axon terminals with similar clinical outcomes in patients. However, their mechanism of action is different, leading to a largely-different duration of neuromuscular junction (NMJ) blockade. BoNTs induce a long-lasting paralysis without nerve terminal degeneration acting via proteolytic cleavage of SNARE proteins, whereas animal neurotoxins cause an acute and complete degeneration of motor axon terminals, followed by a rapid recovery. In this study, the injection of animal neurotoxins in mice muscles previously paralyzed by BoNT/A or /B accelerates the recovery of neurotransmission, as assessed by electrophysiology and morphological analysis. This result provides a proof of principle that, by causing the complete degeneration, reabsorption, and regeneration of a paralysed nerve terminal, one could favour the recovery of function of a biochemically- or genetically-altered motor axon terminal. These observations might be relevant to dying-back neuropathies, where pathological changes first occur at the neuromuscular junction and then progress proximally toward the cell body.

Isolation and characterization of α-elapitoxin-Bf1b, a postsynaptic neurotoxin from Malaysian Bungarus fasciatus venom.

Bungarus fasciatus is one of three species of krait found in Malaysia. Envenoming by B. fasciatus results in neurotoxicity due to the presence of presynaptic and postsynaptic neurotoxins. Antivenom, either monovalent or polyvalent, is the treatment of choice in systemically envenomed patients. In this study, we have isolated a postsynaptic neurotoxin which we named α-elapitoxin-Bf1b. This toxin has an approximate molecular weight of 6.9 kDa, with LCMS/MS data showing that it is highly homologous with Neurotoxin 3FTx-RI, a toxin identified in the Bungarus fasciatus venom gland transcriptome. α-Elapitoxin-Bf1b also shared similarity with short-chain neurotoxins from Laticauda colubrina and Pseudechis australis. α-Elapitoxin-Bf1b produced concentration- and time-dependent neurotoxicity in the indirectly-stimulated chick biventer cervicis muscle preparation, an effect partially reversible by repetitive washing of the preparation. The pA2 value for α-elapitoxin-Bf1b of 9.17 ± 0.64, determined by examining the effects of the toxin on cumulative carbacol concentration-response curves, indicated that the toxin is more potent than tubocurarine and α-bungarotoxin. Pre-incubation of Bungarus fasciatus monovalent and neuro polyvalent antivenom failed to prevent the neurotoxic effects of α-elapitoxin-Bf1b in the chick biventer cervicis muscle preparation. In conclusion, the isolation of a postsynaptic neurotoxin that cannot be neutralized by either monovalent and polyvalent antivenoms may indicate the presence of isoforms of postsynaptic neurotoxins in Malaysian B. fasciatus venom.

The effect of temperature on the effects of the phospholipase A2 neurotoxins ß-bungarotoxin and taipoxin at the neuromuscular junction.

Snake venom neurotoxins with phospholipase A2 affect the neuromuscular junction with three distinct phases. There is a transient decrease in twitch height, followed by a facilitatory phase and finally a progressive blockade. It has been suggested that the initial phase is a direct consequence of the binding of the toxins to nerve terminals. This study was designed to determine whether the initial phase is present under conditions that would reduce the enzyme activity of the toxins. At 27 °C, ß-bungarotoxin and taipoxin exhibited all three phases, i.e. 5-6 min after exposure to the preparation, twitch height was significantly reduced (P < 0.5) to 50 ± 4% and 64 ± 9% of control respectively. This was followed by facilitation and subsequent blockade. However, at 20 °C, neither toxin exhibited the first phase while the second phase, although reduced, clearly occurred and the blocking activity of these toxins always appeared. The data clearly demonstrate that the initial fall is temperature dependent as reducing the temperature from 27 °C to 20 °C blocks the first phase. As the second phase still occurs the toxins must have bound to their target. Therefore, the first phase cannot simply be a toxin binding step.

Venom proteomic characterization and relative antivenom neutralization of two medically important Pakistani elapid snakes (Bungarus sindanus and Naja naja).

Intra- and interspecific variation in venom composition has been shown to have a major effect upon the efficacy of antivenoms. Due to the absence of domestically produced antivenoms, Pakistan is wholly reliant upon antivenoms produced in other countries, such as India. However, the efficacy of these antivenoms in neutralising the venoms of Pakistani snakes has not been ascertained. This is symptomatic of the general state of toxicological research in this country, which has a myriad of highly toxic and medically important venomous animals. Thus, there is a dire need for knowledge regarding the fundamental proteomics of these venoms and applied knowledge of the relative efficacy of foreign antivenoms. Here we present the results of our proteomic research on two medically important snakes of Pakistan: Bungarus sindanus and Naja naja. Indian Polyvalent Antivenom (Bharat Serums and Vaccines Ltd), which is currently marketed for use in Pakistan, was completely ineffective against either Pakistani species. In addition to the expected pre- and post-synaptic neurotoxic activity, the venom of the Pakistan population of N. naja was shown to be quite divergent from other populations of this species in being potently myotoxic. These results highlight the importance of studying divergent species and isolated populations, where the same data not only elucidates clinical problems in need of immediate attention, but also uncovers sources for novel toxins with potentially useful activities. BIOLOGICAL SIGNIFICANCE: Pakistan Bungarus sindanus and Naja naja venoms are differentially complex. Naja naja is potently myotoxic. Neither venom is neutralized by Indian antivenom. These results have direct implications for the treatment of envenomed patients in Pakistan. The unusually myotoxic effects of Naja naja demonstrates the value of studying remote populations for biodiscovery.

Contribution of α7 nicotinic receptor to airway epithelium dysfunction under nicotine exposure.

Loss or dysfunction of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) leads to impairment of airway mucus transport and to chronic lung diseases resulting in progressive respiratory failure. Nicotinic acetylcholine receptors (nAChRs) bind nicotine and nicotine-derived nitrosamines and thus mediate many of the tobacco-related deleterious effects in the lung. Here we identify α7 nAChR as a key regulator of CFTR in the airways. The airway epithelium in α7 knockout mice is characterized by a higher transepithelial potential difference, an increase of amiloride-sensitive apical Na(+) absorption, a defective cAMP-dependent Cl(-) conductance, higher concentrations of Na(+), Cl(-), K(+), and Ca(2+) in secretions, and a decreased mucus transport, all relevant to a deficient CFTR activity. Moreover, prolonged nicotine exposure mimics the absence of α7 nAChR in mice or its inactivation in vitro in human airway epithelial cell cultures. The functional coupling of α7 nAChR to CFTR occurs through Ca(2+) entry and activation of adenylyl cyclases, protein kinase A, and PKC. α7 nAChR, CFTR, and adenylyl cyclase-1 are physically and functionally associated in a macromolecular complex within lipid rafts at the apical membrane of surface and glandular airway epithelium. This study establishes the potential role of α7 nAChR in the regulation of CFTR function and in the pathogenesis of smoking-related chronic lung diseases.

Three-finger toxins, a deadly weapon of elapid venom--milestones of discovery.

Three-finger toxins (TFTs) are the main venom components of snakes from Elapidae family. Amino acid sequences of more than five hundreds TFTs are determined; these toxins form one of the largest protein families present in snake venoms. The first TFT α-bungarotoxin was isolated almost half a century ago and so far it remains a valuable tool in the study of nicotinic acetylcholine receptors. TFTs possess diverse biological activities; for example, α-neurotoxins bind specifically with high affinity to nicotinic acetylcholine receptors, while cytotoxins induce non-specific lysis in great variety of cells. These toxins are widely used as instruments in different branches of life sciences. In this review the main landmarks in TFT study are considered. These are the discovery and isolation of TFTs, determination of their structure and mode of action as well as evolution and relationship within the family.

An imaging assay to analyze primary neurons for cellular neurotoxicity.

The development of high-content screening technologies including automated immunostaining, automated image acquisition and automated image analysis have enabled higher throughput of cellular imaging-based assays. Here we used high-content imaging to thoroughly characterize the cultures of primary rat cerebellar granule neurons (CGNs). We describe procedures to isolate and cultivate the CGNs in 96-well and 384-well format, as well as a procedure to freeze and thaw the CGNs. These methods allow the use of CGNs in 96-well format analyzing 2500 samples per experiment using freshly isolated cells. Down-scaling to 384-well format and freezing and thawing of the CGNs allow even higher throughput. A cellular assay with rat CGN cultures was established to study the neurotoxicity of compounds in order to filter out toxic compounds at an early phase of drug development. The imaging-based toxicity assay was able to reveal adverse effects of compounds on primary neurons which were not detected in neuroblastoma or other cell lines tested.

Escape behaviour: reciprocal inhibition ensures effective escape trajectory.

When a zebrafish makes a fast escape response, Mauthner cells directly activate contralateral spinal interneurons which feed reciprocal inhibition to motorneurons on the stimulated side. Ablation of these interneurons in transgenic animals impairs escape responses, indicating their crucial role in survival.

Presence of alpha7 nicotinic acetylcholine receptors on dorsal root ganglion neurons proved using knockout mice and selective alpha-neurotoxins in histochemistry.

In complex tissues where multiple subtypes of nicotinic acetylcholine receptors (nAChRs) are expressed, immunohistochemistry has been the most popular tool for investigation of nAChR subunit distribution. However, recent studies with nAChR subunit knockout mice demonstrated that a large panel of antibodies is unsuitable. Thus, we aimed to develop a histochemical method for selective labeling of alpha7 nAChR with neurotoxins, utilizing alpha7 nAChR-transfected cells, dorsal root ganglia (DRG) and spinal cord from wild-type and knockout mouse. The specificity of Alexa Fluor 488-conjugated alpha-bungarotoxin (Alexa-alphaBgt) was demonstrated in binding to alpha7-transfected cells inhibited by long-chain alpha-cobratoxin (CTX), but not short-chain alpha-neurotoxin II (NTII). In contrast, binding to Torpedo muscle-type nAChRs and to motor end plates in mouse tongue sections was prevented by both CTX and NTII. In tissue sections of DRG, expressing all neuronal nAChR subunits, only CTX precluded Alexa-alphaBgt labeling of neurons, with no staining for alpha7 nAChR knockout tissue. It proved that alpha7 nAChRs are the major alphaBgt-binding sites in mouse DRG. Corresponding results were obtained for terminals in the spinal cord. Thus, we present a protocol utilizing Alexa-alphaBgt and non-labeled CTX/NTII that allows specific histochemical detection of alpha7 nAChR with a spatial resolution at the level of single axon terminals.

Development of microglia in the chick embryo spinal cord: implications in the regulation of motoneuronal survival and death.

The role of microglia during normal development of the nervous system is still not well understood. In the present study, a chick embryo model was used to examine the development of microglia in the spinal cord and characterize their changes in response to naturally occurring and pathological death of motoneurons (MNs). The microglial response to MN axotomy and the effects of microglial activation on MN survival were also studied. We found that: 1) macrophages/microglial cells were present in the spinal cord at early developmental stages (E3) and that they were recruited after normal and induced MN apoptosis; 2) although many microglial cells were seen phagocytosing apoptotic bodies, a proportion of dying cells were devoid of engulfing microglia; 3) axotomy of mature MNs was accompanied by microglial activation in the absence of MN death; 4) excitotoxic (necrotic) MN death provoked a rapid and massive microglial recruitment with phagocytic activity; 5) lipopolysaccharide-induced microglial activation in vivo resulted in the death of immature, but not mature, microglia; and 6) overactivation of microglia modulated the survival of mature MNs, either by killing them or by enhancing their vulnerability to die in response to a mild injury. Taken together, these observations indicate that normal microglia do not play an active role in triggering apoptosis of developing MNs. Rather, they act as phagocytes for the removal of dying cells during the process of programmed cell death.