The neurotoxic activity of the venom produced by Micrurus species is determined by applying a stimulus through the electrodes that are in contact with the biventer cervicis muscle of 4- to 8-day-old male chicks, or with the diaphragm and the phrenic nerve of male mice with body weight between 25g and 35g, which results in muscle contraction under normal conditions.²⁴ To perform this analysis, electrical impulses (0.1Hz for 0.2ms) are applied using a low-frequency stimulator and muscle contractions are recorded with a force displacement transducer that is coupled to recording equipment located in the muscle tissue of an isolated organ perfusion system. After a stabilization period of 20 minutes, a single concentration (0.1, 0.5, 1, 5 or 10 µg/mL) of the venom under study is added to the organ bath. To confirm the complete block of muscle contractions,^8,10 and to establish the concentration of the venom that caused such an effect,²⁷ it is necessary to apply new electrical stimuli and verify the records using a dose-response curve.

Using muscle preparations similar to the biventer cervix muscle of male chicks, it is also possible to evaluate the ability of the venom to induce muscle damage²⁶

To achieve a selective stimulation of the muscle by suppressing neuromuscular activity, the preparations are placed in an organ bath in 10µM d-tubocurarine and the muscle is directly stimulated with electrical impulses of 0.1Hz at a maximum voltage of 0.2ms. The poison is then added to the preparation and left in contact until contraction is blocked or after 3 hours, after which time the tissues are immersed in 10% formaldehyde for histological examination to confirm myotoxicity.^19,23,25,27

Micrurus snake venom, or some of its specific toxins, can alter the transmission of the normal electrical pulse in the neuromuscular junction. This is reflected, on the one hand, in a decrease or blockage of the response to direct electrical stimulation on the muscle and, on the other, in fluctuations in resting membrane potential, such as changes in amplitude, form and frequency, and Wedensky inhibition in some cases; these effects are caused by both prolonged and short exposures to the venom or one of its toxins.^24,28

Similarly, by evaluating muscle contractility after applying electrical stimulation in the presence of acetylcholine (ACh) and the venom under study, it is possible to determine if there are effects on the post-synaptic response to ACh. When there are no alterations in muscle contractility, the effect is considered presynaptic.^24,25

Myotoxicity can be evaluated in an observational way and without the need for a pathological study, assessing the response of the striated muscle when exposed to the venom and the blocking action of the toxins on muscle contracture in the presence of a direct electrical impulse and high concentrations of K⁺ in the organ bath.^26,27

Since there is new information on Micrurus snake venoms and considering the large number of this genus species in Colombia, it is necessary to encourage research that characterizes these substances biochemically and biologically and promotes the development of more specific and useful antivenins to treat snakebite accidents.

In this context, the objective of this review was to present an overview of the neurotoxicity of Micrurus snake venom and its functional characterization using ex vivo analysis methods.

The venom of snakes from the genus Micrurus has neurotoxic components. Lomonte et al.¹⁵ identified the following families of proteins in its proteome: 3FTx (a-neurotoxins), PLA2 (P-neurotoxins), metalloproteases, L-amino-acid oxidases, Kunitz-type serine protease, C-type lectin-like proteins, acetylcholinesterase and hyaluronidases, being the first two the ones with more involvement. The proportion of PLA2 and 3FTx toxins in these venoms is a key element for identifying their main neurotoxic and myotoxic effects.^15,16,18

Table 2 presents some of the neurotoxins identified in snakes of the family Elapidae. It also includes the characteristics of the snake venom of the genus Crotalus durissus due to its neurotoxic and myotoxic behavior.²²

Table 2

Species	Toxin	Type	Reference
Notechis scutaus	Notexin	β-neurotoxins	49
Oxyuranus scutellatus	Taipoxin	β-neurotoxins	30,31,48
Oxyuranus microlepidotus	Paradoxin	β-neurotoxins	34,50
Crotalus durissus terrificus	Crotoxin	β-neurotoxins	28,30,31,51,52
Pseudonaja textiles	Textilotoxin	β-neurotoxins	23,33
Bungarus multicinctus	β-bungarotoxin	β-neurotoxins	28,31
Micrurus frontalis	Frontoxin	α-neurotoxins- 3FTX	35
Micrurus lemniscatus	Lemnitoxin	β-neurotoxins	42
Micrurus dumerilli	MdumPLA2	β-neurotoxins	16
Micrurus mipartitus	MmipPLA2	β-neurotoxins	16

Source: Elaborated based on Hodgson & Wickramaratna.²³

Recent studies in proteomics have demonstrated the predominance of PLA2 and 3FTx toxins in the venom phenotype in species of the genus Micrurus and have shown that their proportions vary widely across the American continent.^{14,16,21,22,41-43} Similarly, Lomonte et al.¹⁵ made a projection of the behavior of these two toxins, finding that PLA2 is more abundant in the southern cone and that 3FTx predominates in Central and North America. However, it is worth mentioning that this projection should be corroborated by a detailed analysis of each of the venoms since the evolutionary and ecological processes of each species are factors that determine the greater proportion of one of these 2 toxins.

PLA2 is a superfamily of enzymes composed of 16 groups that are classified according to their type into: secreted PLA2 (sPLA2), cytosolic PLA2 (cPLA2), calcium-independent PLA2 (iPLA2), platelet activating factor (PAF), lipoprotein-associated PLA2 (LpPLA2s), adipose PLA2 (AdPLA2s) and lysosomal PLA2 (LPLA2s). The first two are especially important because they are involved in the inflammatory and degenerative response of the central nervous system.⁵³ sPLA2 are present in different classes of venoms and are classified in four main subtypes, and types 1 and 2 are found in snake venoms, mainly in elapids, vipers and crotalids; these enzymes are single-chain polypeptides and have a molecular mass of 13-15 kDa, with approximately 7 disulfide bonds. Finally, cPLA2 has a molecular mass of 40-100 kDa and depends on calcium for functioning.⁵³

Even though the exact neurotoxic route of PLA2 is not known, at least three mechanisms of action by which these enzymes exert their presynaptic toxic activity have been described: 1) the neurotoxin induces phospholipid hydrolysis of the presynaptic membrane and prevents it from interacting with the acetylcholine vesicles, 2) the damage described in the cell membranes favors the excessive influence of Ca++ inside the cells, which causes an exaggerated release of the acetylcholine neurotransmitter, as well as the alteration of its recycling process and its subsequent depletion, and 3) its enzymatic activity is left aside, so the protein complexes that favor the coupling of the vesicle and the presynaptic membrane are blocked.^{16,23,47,54-56}

3FTx are non-enzymatic polypeptide structures made up of between 60 and 74 amino acid residues that are among the main components of elapid venoms.⁵⁷ Its structure has 3 loops of p-sheets that extend from a small globular hydrophobic core that is linked by 4 preserved disulfide bonds; this structure resembles that of a hand with 3 fingers, which is why they are known as three-finger toxins.^57,58

Neurotoxicity (main effect of coral snake venom), cytotoxicity and cardiotoxicity are some of the pharmacological effects identified in 3FTx. However, it should be noted that its biological activity varies depending on the affinity it has with the receptors^59-61 (Table 3).

Table 3

3FTx type		Mechanism of action	Example
Neurotoxins	α	α 1 and/or α 7 nAChR antagonists.	α-bungarotoxins
	K	They recognize different subtypes of neuronal α 3β4 nAChR	K-bungarotoxins
	MT	They selectively bind to mAChR.	Dendroaspis angusticeps MT1
Cardiotoxins		They form ionic pores in lipid membranes.	Cardiotoxin V4II from Naja mossambica
α -cardiotoxins and others alike		They bind to β1 and β2 adrenergic receptors.	CTX9, CTX14, CTX15, CTX21 and CTX23 from Ophiophagus Hannah
Non-conventional		Weak neurotoxins with nanomolar affinity to AChR α1 (reversible binding) and α7 (poorly reversible binding)	Candoxin
Acetylcholinesterase inhibitors		They bind to acetylcholinesterase	Fasciculins
L-type Ca2+ channel blockers		They block L-type Ca2+ channel in skeletal and heart muscles	Calciseptine
Platelet aggregation inhibitors		They interfere with the interaction between fibrinogen and the glycoprotein IIB-IIIa receptor (αIIBβ3).	Dendroaspins

nAChR: nicotinic acetylcholine receptor; mAChR: muscarinic acetylcholine receptors; MT: muscarinic toxin; CTX: cardiotoxin

Depending on the amino acid sequence, 3FTx neurotoxins can be classified into two types: short-chain neurotoxins (type I) and long-chain neurotoxins (type II), both with a molecular mass varying between 6 and 9 kDa.^58,61 Short-chain a-neurotoxins are the main responsible for the neurotoxic effect of elapid venoms due to their high- affinity binding for nicotinic acetylcholine receptor (nAChR) and the inhibitory control they exert on this receptor without affecting the release of neurotransmitters from the presynaptic terminal.^59,62,63

Recent studies on the classical biochemistry of elapid venom composition in Colombia have made major progress in the description of the ontogenetic characteristics and phylogenetic trees of these species.^16,19,21 In this regard, multiple research works assess the peripheral neurotoxic and myotoxic activity of the venom of M. dissoleucus, M. mipartitus,¹⁹M. lemniscatus, Micrurus frontalis and M. surinamensis,¹³ or the PLA2 of the species Micrurus nigrocinctus.⁶⁴

Specifically, for Australian elapids (Pseudechis spp.), Hart et al.⁶⁵ indicated that binding of neurotoxins to nAChR is more effective in preparation from chick muscle than in human and rat skeletal muscles. This difference is explained because the synaptic junctions are almost absent in birds, even though neuromuscular junctions in avian tissues are almost the same size as in rats; this makes nAChR more susceptible to post-synaptic neurotoxins and more sensitive to the neurotoxic action of elapid venoms.⁶⁵

Although the neurotoxic activity of Micrurus snake venom has not been sufficiently studied, several methodologies have been used as complementary techniques besides organ baths, e.g. cell culture models, that can contribute to the understanding of the actions of the full venom and its main components (PLA2 and 3FTx). For example, hippocampal tissue has been used to prove viability and conservation of cellular function (integrity of mitochondria and intracellular calcium variability) after the administration of different doses of venom from these species,⁶⁶ to establish toxin binding to transmembrane receptors,⁶⁷ and determine electroencephalographic and neuropathological changes in behavioral studies of animal models.⁶⁸