New Perspectives in Drug Discovery Using Neuroactive Molecules From the Venom of Arthropods

Arthropods are one of the most ancient groups of animals in earth and their venoms have been responsible for their chemical defense in a very efficient way. Resulting from an in‐ tense and elaborated evolutionary process, venoms produced by arthropods have a very complex repertoire of biologically active molecules. When inoculated in mammals these molecules induce a wide range of systemic effects, including actions in the CNS. In mamma‐ lian CNS, venom compounds may either inhibit or stimulate with affinity and specificity structures such as: ion channels, neurotransmitter receptors and transporters [1-3]. Not sur‐ prisingly, these actions have attracted the attention of many investigators in search of tools to help the understanding of neural mechanisms as well as those in search of novel probes in CNS drug design for the last 20 years [3,4]. In addition to the growing interest in finding new neuroactive compounds, the improvement of proteomic and transcriptome techniques has stimulated great progress in the bioprospecting, enabling and accelerating the testing of new toxins in several animal models. Animal research aiming at the efficacy of peptides and acylpoliamines, isolated from arthropod venoms, have revealed the great potential of these compounds to treat various diseases, such as epilepsy, Parkinson's, Alzheimer's, chronic pain and anxiety disorders


Introduction
Arthropods are one of the most ancient groups of animals in earth and their venoms have been responsible for their chemical defense in a very efficient way. Resulting from an intense and elaborated evolutionary process, venoms produced by arthropods have a very complex repertoire of biologically active molecules. When inoculated in mammals these molecules induce a wide range of systemic effects, including actions in the CNS. In mammalian CNS, venom compounds may either inhibit or stimulate with affinity and specificity structures such as: ion channels, neurotransmitter receptors and transporters [1][2][3]. Not surprisingly, these actions have attracted the attention of many investigators in search of tools to help the understanding of neural mechanisms as well as those in search of novel probes in CNS drug design for the last 20 years [3,4]. In addition to the growing interest in finding new neuroactive compounds, the improvement of proteomic and transcriptome techniques has stimulated great progress in the bioprospecting, enabling and accelerating the testing of new toxins in several animal models. Animal research aiming at the efficacy of peptides and acylpoliamines, isolated from arthropod venoms, have revealed the great potential of these compounds to treat various diseases, such as epilepsy, Parkinson's, Alzheimer's, chronic pain and anxiety disorders According to World Health Organization (WHO), neurological and mental disorders are one of the greatest threats to public health not only for its direct and immediate effects, but also for the progressive nature of these diseases, often leading to disability and death [5]. The symptoms of most of these diseases are often well treated with a several pharmaceuticals, such as antidepressants, anxiolytics, anticonvulsants and analgesics. However, it is well known that neuroactive drugs may induce a complex range of adverse effects that limit the constantly seek new therapeutic strategies, since most of these are inadequate or cause serious side effects [14].
Analgesics and systemic conservative therapies are widely used for pain control. However, in many cases, especially in patients with neuropathic pain, more aggressive treatments are needed, which promote a significant clinical improvement but only in 30-50% of patients [15,16].
Although an injection of arthropod venoms is commonly reported to cause tonic pain and hyperalgesia, there is also evidence suggesting that these venoms might have antinociceptive effects on inflammation. Thus, nowadays, toxins isolated from arthropods are considered powerful tools, since they have congruent targets of the impulse transmission of pain, and may provide an attractive alternative to opioid treatments.

Polypeptide toxins from Scorpion
The most studied Arthropod venom is extracted from the Asian scorpion Mesobuthus martensi Karsch (BmK). It is composed of several toxins, and so far, ten have been described, which produce powerful antinociceptive effects. This is the case of the two β-excitatory antiinsect toxins BmK IT-AP (or Bm33-I) and BmK AngP1, two β-depressant anti-insect toxins BmK dITAP3 and BmK IT2, as well as six toxins yet without consensus classification, BmK AS, BmK AS1, BmK AGAP, BmK Ang M1, BmK AGP-SYPU1 and BmK AGP-SYPU2. These compounds probably belong to a family of peptides NaScTx that are composed of 60-76 amino acid residues with four disulfide bonds, the cysteine positions among these toxins are highly conserved [17,18]. Considering their structures, they might be able to bind to sodium channels impairing depolarization of the action potential in nerve and muscle, resulting in neurotoxicity [18], although it remains to be fully investigated.
The NaScTx family can be classified in at least two major families, α and β, according to the mode of action on Na + channels [19]. The binding of α-toxins delays Na v channel inactivation, while that of β-toxins shifts the membrane potential dependence of channel activation to more negative potentials. α and β-toxins also exhibit pharmacological preferences for mammals or insects sodium channels. Therefore, considering their pharmacological activities, α and β NAScTx can be also divided into three groups: i.
"classic" highly specific for mammals; ii. "α-like toxins" active both on mammals and insects, which are far less specific and less active than the "classical" ones; iii. α-toxins only specific for insects and without any toxicity on mammals, even at high concentrations. Moreover, the insect selective β-toxins have been divided into two groups: the excitatory insect toxins and the depressant insect toxins.
Regarding the β-excitatory anti-insect toxins, BmK IT-AP (Insect Toxin-Analgesic Peptide), which was isolated in 1999, produces a potent antinociceptive effect in mouse-twisting model, after i.v. injection [20]. The same toxin has also been sequenced by another group and named Bm K 33-I [21]. Later, Guan and colleagues [22] identified a novel toxin with analgesic effects, BmK AngP 1 , which shows an evident analgesic effect with simultaneous excitato-ry insect toxicity, but is devoid of any toxicity on mice even at high dosages. The analgesic effect was assessed with a mouse-twisting model. The analgesic effect on mice of the AngP 1 is at least 4-5 times weaker than that of IT-AP, but the toxicity to insects is twice as strong as that of IT-AP [20,22] In relation of depressant toxins isolated from BmK venom, BmK IT2 has been more studied from the venom of BmK (Fig 1).  [33]. More recently, electrophysiological studies have shown that they are able to inhibit Na + currents in NG108-15 cells [34] and to depress TTX-sensitive and TTX-resistant Na + currents in rat small DRG neurons. Interestingly, in rat models, BmK AS1 also displays antinociceptive effects according to [33]. These authors concluded that the effects could be mediated by the modulation of voltage-gated Na + channels and they also suggested that BmK AS and BmK AS1 could form a new family of scorpion insect toxins.
BmK AGAP (antitumor-analgesic peptide), isolated in 2003, had strong inhibitory effect on both viscera and soma pain [35]. To evaluate the extent to which residues of the toxin core contribute to its analgesic activity, nine mutants of BmK AGAP were produced and tested. However, further studies are necessarily to elucidate the mechanism of action as well as to exploit its analgesic activity [36]. In relation to BmK Ang M1 [37], it also was reported to exhibit potential analgesic effect. Moreover, electrophysiological studies showed that BmK AngM1 at the concentration of 1 µM inhibited voltage-dependent Na + current (I Na ) and voltage-dependent delayed rectifier K + current (I K ), but had no effects on transient K + current [37].
It is important to note that the excitatory and depressant anti-insect toxins belong to different groups, which have distinct modes of interaction with receptors. Thus, one can infer that the analgesic effect of these peptides may have a molecular mode and mechanism different from that of insect toxicity. Still, the mechanisms by which these scorpion toxins can modulate pain pathways remain to be clarified. According to [8], four different possibilities might be described: i. peptides act directly Na + channels involved in the pathway of pain,

ii.
peptides modulate indirectly the pain sensation,

iii.
peptides also modulate other targets involved in pain pathway iv. pain alleviation is only apparent and results from misinterpretations that might have occurred from animal models used.

Polypeptide toxins from Spider
Another group of arthropods that have very promising antinociceptive compounds are spiders [41]. In 1996, Roerig & Howse reported the effect of ω-agatoxina IVA (Fig 1) isolated from funnel spider Agelenopsis aperta venom, against thermal stimulation in the tail flick test, when co-administrated with morphine intrathecal. Intrathecal injection of ω-agatoxin IVA (0.2 nmol/kg) also decreased the licking time in both the early and late response phases in a dose-dependent manner in the Formalin test [42]. The use of this peptide as an analgesic could be of particular benefit in patients tolerant or opioid-dependent, since this compound exhibits selectivity for the P/Q Ca 2+ channels [43]. Other spider venom very promissory is the venom of the Brazilian armed spider Phoneutria nigriventer, the purified fraction 3 (PhTx3) contains 6 toxin isoforms (Tx3-1 to -6) [44,45] that target Ca 2+ channels with different affinity patterns. Moreover, one toxin, Tx3-6 (Phα1β), demonstrated that it preferentially blocks the N-type calcium current [46] and produce a potent antinociceptive effect with higher therapeutic index [44]. Dalmolin and colleagues [45] showed that Tx3-3 (purified the same fraction) caused a short-lasting antinociceptive effect in the nociceptive pain test and a long-lasting antinociceptive effect in neuropathic pain models, without producing detectable side effects. However, Tx3-3 did not change the inflammatory pain. Tx3-3 blockade of P/Q-and R-type Ca 2+ channels and inhibit the glutamate release in rat brain cortical synaptosomes [47]. Other neurotoxin isolated from spider Phoneutria nigriventer is Phα1β, which is a potent toxin blocking neuronal voltage-sensitive Ca 2+ channels. This peptide induced longer antiallodynic effect than µ-conotoxin MVIIA and morphine in mice [48].
In addition to toxins calcium modulators, compounds isolated from spider that interact with other ionic channels have shown great potential. A new class of peptide toxins named is the Huwentoxin I (HWTX-I, Fig 1) that is the most abundant toxic component in the crude venom of the Chinese bird spider Ornithoctonus huwena. Whole-cell patch clamp records revealed that HWTX-I selectively inhibits N-type Ca 2+ channels in NG108-15 cells, and it also can block transmitter release from nerve endings by preventing depolarization induced by calcium influx [38] Antinociception effect of the HWTX-I in formalin test was greater and lasted two-fold longer time compared to morphine [39]. Furthermore, Tao and collaborators [40] demonstrated that intrathecal administration of HWTX-I is effective in antinociception in the rat model of rheumatoid arthritis more effective than ibuprofen.
Several studies have reported that intrathecal administration of non-selective blockers of Ca 2+ channels shows antinociceptive effects in animals tested with thermal stimuli: hot plate and tail flick. According to [49], N and P/Q Ca 2+ channels are probably involved in nociceptive behavior induced by formalin injection in rats, while the L-type channels has no effect. N-and P/Q-type Ca 2+ channels are expressed specifically in the nervous system, and they have a major importance in controlling the excitation of spinal neurons from sensory afferents of inflamed tissues, relieving inflammatory pain.
A new class of peptide toxins named π-theraphotoxin-Pc1a (π-TRTX-Pc1a; also known as psalmotoxin-1 (PcTx1) was isolated from the venom of the spider neotropical Psalmopoeus cambridgei ( Fig.1). π-TRTX-Pc1a is the most potent and selective blocker of ion channels sensitive to acid -ASICa [50]. These channels play important roles in pathological conditions such as cerebral ischemia or epilepsy, as well as being responsible for the sensation of pain that accompanies tissue acidosis and inflammation [51]. Since external acidification is a major factor in pain associated with inflammation (hematosis muscle and cardiac ischemia, or cancer), these neurotoxins can be used to control the pain sensation triggered by these channels [52]. π-TRTX-Pc1a was shown to be an effective analgesic, comparable to morphine, in rat models of acute and neuropathic pain when injected directly in Central Nervous System [53] and intranasal administration of this peptide resulted in neuroprotection of neurons in a mouse model of ischemic stroke even when administered hours after injury [54].
Other important target in the search for new analgesics isolated from spider venoms are Na V channels, since modulatory compounds of these channels are the dominant pharmacological species in spider venoms, although still poorly characterized. In this context, Intrathecal administration of β-TRTX-Gr1b (formerly GsAFI), a peptide obtained from venom of Grammostola spatulata, the Chilean pink tarantula spider, induced analgesia in a variety of rat pain models such as the tail flick latency test, hot plate threshold test, von Frey threshold test, and formalin pain test, without any confounding side-effects. Moreover, the β-TRTX-Gr1b peptide did not exhibit cross tolerance with morphine [55].
Further on spider venoms, Purotoxin-1 (PT1) was recently isolated the, from the venom of the Central Asian spider Geolycosa sp [56]. PT1 is a 35-residue peptide with four disulfide bonds, and it exerts a potent analgesic effect in rat models of acute and chronic inflammatory pain by injection of either carrageenan or Freund's complete adjuvant, respectively. PT1 was also effective in reducing the number of nocifensive events triggered by the injection of capsaicin or formalin (only second phase) [56]. This molecule also inhibits P2X3 receptors in a powerful and selective manner. These ATP-activated receptors are largely expressed in mammalian sensory neurons play a key role in the pain perception. Thus, PT1 appears to be a promising lead compound for the development of analgesics that target these receptors [56].

Polypeptide toxins from Bees and Wasps
Bee venom has been traditionally used to relieve pain and treat chronic pain diseases (for revision see [57]). Moreover, acupoint stimulation into the subcutaneous region (acupuncture) rather than other injection sites may be important for the antinociceptive effects of this venom. There is increasing evidence suggesting that bee venom has antinociceptive effects on visceral nociceptive effects, mechanical and thermal hyperplasia, formalin-induced pain behavior and collagen-induced arthritic pain, as well as knee osteoarthritis (OA)-related pain [58][59][60][61][62][63]. BV contains at least 18 active components, including enzymes, peptides, and biogenic amines, which have a wide variety of pharmaceutical properties, and so multiple mechanisms associated to antinociceptive effects have been suggested, such as activation of the central and spinal opioid receptor and α 2 -adrenergic receptor, as well as activation of the descending serotonergic pathways (for revision see [64]).
Melittin is a small protein containing 26 amino acid residues and is the major bioactive component in BV (Fig.1). This polypeptide readily integrates into and disrupts both natural and synthetic phospholipid bilayers [65,66]. Melittin also enhances the activity of PLA 2 [67] and has a variety of effects on living cells possibly through the disruption of the membrane [68]. The decrease in cyclooxygenase (COX)-2 and phospholipase PLA 2 expression and the decrease in the levels of tumor necrosis factor alpha (TNF-α), interleukin (IL)-1, IL-6, nitric oxide (NO) and oxygen reactive species (ROS) are suggested to be associated with the antiarthritis effect of melittin [69]. This peptide has also been thought to play a role in production of anti-nociceptive and anti-inflammatory effects [64]. In addition, Merlo and colleagues [70] demonstrated the antinociceptive activity of the melittin in experimental models of nociceptive and inflammatory pain. Interestingly, melittin failed to increase the latency for the nociceptive response in the hot-plate model and in the first phase of the formalin test, revealing that melittin presents an activity that resembles more that of anti-inflammatory drugs and less that of centrally acting drugs [70]. Nevertheless, the molecular and cellular mechanisms underlying the anti-nociceptive effects of melittin are not entirely clear and remain to be further clarified by further experimental studies [57].
Addition of melittin, adolapin has been isolated from BV and it demonstrated a potent analgesic effect in mouse-twisting model and the Randall-Sellito's test [71]. The anti-inflammatory activity of adolapin was evaluated and it had a pronounced activity in the following tests: carrageenan, PG, adjuvant rat hind paw edema and adjuvant polyarthritis. The effects of adolapin are presumably due to its ability to inhibit the prostaglandin synthesis via inhibition of cyclooxygenase activity [71,72].
Venoms of wasps also have analgesic peptides. Mortari and colleagues [73] isolated a compound with antinociceptive activity from the venom of the Brazilian social wasp Polybia occidentalis. The isolated peptide is a neurokinin named Thr 6 -Bradykinin. This neurokinin is a small peptide consisting of nine amino acid residues, Arg-Pro-Pro-Gly-Phe-Thr-Pro-Phe-Arg-OH, which exhibits a high degree of homology with bradykinin (BK), except for the substitution of Thr for Ser in position 6 at BK. As a result, small changes in their secondary structures are observed [74]. This modification has been regarded as responsible for increasing B 2 receptor affinity and potency of Thr 6 -BK in relation to BK in vitro and in vivo [74,75]. Thr 6 -BK antinociceptive effect was dose-and time-dependent, when injected directly into the CNS of rats in hot-plate and tail-flick tests, and it was three times more potent than morphine and 4 times more potent than BK in tail-flick test. Thr 6 -BK induced antinociception by activating presynaptic B 2 receptors, which activate descending adrenergic pathways. Studies investigating the role of kinins in the CNS provide new information on the supraspinal system of the pain control, whose modulation may represent a new strategy to control painrelated pathologies [76]. Besides peptides, some studies have evaluated the analgesic activity of acylpolyamines that can be used as new alternative drugs for the treatment of chronic pain, as well as tools for the study of the functional role of the AMPA/kainate receptors in the processing of nociceptive pain [77]. In this regard, intrathecal administration of different doses of these toxins blocked thermally induced allodynia [78] and hyperalgesia [79]. The effect of these neuro-toxins may suggest a possible involvement of AMPA receptors in the spinal cord during the nociceptive excitatory stimulation [80,81].

Antiepileptic and neuroprotective effects
Neurodegenerative disorders comprise a wide range of conditions mostly characterized by a progressive loss of neuronal function and neuronal cell death. The incidence of these diseases in population differs greatly. In conditions such as Parkinson disease and Alzheimer, the number of cases significantly increases in elderly, whereas epileptic patients are mostly children and adolescents. Many processes may trigger neuronal cell death, such as trauma, stroke, tumors, infections, genetic factors and biochemical alterations. Among the latest, the alterations in Ca 2+ -mediated signaling is thought to play a key role in many neurodegenerative disorders and the increase in intracellular Ca 2+ concentration might alter neuronal membrane potential [82]. Moreover, the hyperactivation of excitatory transmission mediated mostly by L-glutamate and its ionotropic receptors; kainate, AMPA and NMDA, is responsible for the excessive cationic influx that depolarizes neuronal cells and lead to sustained hyperexcitation observed in brain pathologies such as epilepsy [83]. This increase in glutamatergic activity often referred to as glutamate excitotoxicity [84], might also involve non-receptor neurochemical events such as failure in glutamate uptake system, which ends with an increase in the availability of this neurotransmitter in the synaptic cleft [85,86]. The importance of L-glutamate in neurological disorders relies on the fact that this neurotransmitter is release in the great majority of fast synapses in CNS [84,83]. In this context, many molecules mostly peptides and acylpolyamines, acting on ion channels, receptors and transporters were isolated from arthropod venoms, remarkably spiders, scorpions and wasps [3]. According to [82], polyamines are non-specific antagonist of ligand-gated ion channels, acting at glutamatergic and Ach receptors in an uncompetitive way, that is, the receptor must be activated in order to occur the blockade. This mode of action might diminish the side effects of newly designed medicines, since it blocks only the activated receptors, but does not prevent their opening.
The venom of the orb-web spider Nephilia clavata was one of the first venoms studied during the 80s, which resulted in the identification of small compounds named acylpolyamines, among whose we may find jorotoxin (JSTX), one of the first glutamate receptor uncompetitive antagonists [83,84]. Together with JSTX, another polyamines such as argiopin from the venom of the spider Argiope lobata [85] and philantotoxin (PhTx) from the venom of the solitary wasp Philanthus triangulum [86]. Following the structural characterization and studies in insect or crustaceans, the reports on the action of these polyamines in mammalian CNS started to take place, mostly during the 90s [87]. JSTX-1 and JSTX-3 are synthetic analogues of JSTX. The first inhibits kainate-induced seizures, whereas the latter block glutamate release and hippocampal epileptic discharges [88,89]. Later, JSTX-3 was shown to inhibit the formation of superoxide dismutase-1 (SOD-1) aggregates that lead mutant motor neurons (mSOD-1) to death during the familiar form of the neurodegenerative disease, amyotrophic lateral sclerosis [90]. The authors concluded that increased Ca 2+ influx mainly through AM-PA/kainate glutamate receptors make mutant neurons more vulnerable to damage and therefore, JSTX-3 is an interesting neuroprotective agent in this model.
The fraction of the venom of the spider Agelenopsis aperta containing argiotoxin, was first demonstrated to have anticonvulsant in NMDA-induced and audiogenic seizures [91]. The synthetic analogue of argiotoxin, Arg-636, is a selective antagonist of NMDA receptors binding to the Mg 2+ binding site at the receptor with anticonvulsant and neuroprotective actions. In addition, from the venom of A. aperta, another NMDA receptor blocker, Agatoxin 489 was reported as anticonvulsant against kainate-induced seizures and its synthetic analogue Agel-505, was able to block cationic currents in oocytes transfected with NMDA receptor cDNA [92].
Aside from antagonizing glutamate receptors, arthropod neurotoxins may exert anticonvulsant and neuroprotective effects targeting other neurotransmitter systems. The venom of the Brazilian spider Phoneutria nigriventer has been extensively studied over the past 20 years. Neurotoxins isolated from the venom of P. nigriventer, such as PhTx-3 (Tx-3) were reported to inhibit Ca 2+ dependent-glutamate release [47]. Tx3-3 and Tx3-4 also inhibit voltage-activated Ca 2+ channels of P/Q type [93] and recently their neuroprotective activity was tested. According to [94], Tx3-3 and Tx3-4 protected hippocampal slices against damage and cell death induced by ischemic insult resulted from low oxygen and low glucose. Moreover, PhTx3, Tx3-3, and Tx3-4, inhibited cell loss in retinal slices submitted to the same ischemic protocol [95]. Another Brazilian species lives in Cerrado, the colonial spider Parawixia bistriata and has many neuroactive molecules with different modes of action [96]. Parawixin-1 was the first isolated neurotoxin from P. bistriata venom. In experiments using rat retinas, submitted to ischemic insult, the intravitreal injection of Parawixin inhibited cell loss [97], probably through a potent and specific enhancing action on glutamate transporters type EAAT2 [98]. Another neurotoxin isolated from the venom of P. bistriata, Parawixin II, formerly, FrPbAII, inhibited GABA and glycine uptakes in synaptosomes from rat cerebral cortices. In addition, the administration of Parawixin II into the vitreous humor of Wistar rats protected retinal neurons against ischemic insult resulted from an increase in the intra ocular pressure [96]. Data also show that Parawixin II blocked seizures induced by the injection of GABAergic antagonists, bicuculline [99], pentylenetetrazole (PTZ) and picrotoxin, as well as pilocarpine and kainic acid [100]. It is worth noting that the acute injection of Parawixin II does not alter rat behavior in the open field and repeated central injection does not impair acquisition and learning in the Morris water maze. Finally, Parawixin II induces ataxia in the rotarod in doses far higher than effective doses, indicating good therapeutic indexes [100].
Also from South America, the Chilean giant pink tarantula Grammostola spatulata paralyzes its preys by injecting a mixture of toxins that blocks ion channels [101]. w-Grammotoxin SIA was isolated from the venom of G. spatulata and the potent blocking effect over N-, P-, and Q-type but not L-type of voltage gated calcium channels was reported [102]. The antagonistic activity of w-grammotoxin over voltage dependent calcium channels is considered a therapeutic option to be used in neurodegenerative disorders such as ischemia.
The African tarantula Hysterocrates gigas known as the giant baboon spider, inhabits the rain forests of West Africa. The isolation of the venom of H. gigas, resulted in the identification of the peptide SNX-482 that blocks R-type voltage dependent calcium channels [103].
The arboreal tarantula Psalmopoeus cambridgei is an aggressive spider that lives in the tropical forests of Trindad. As mentioned before, PcTx-1 (π-theraphotoxin-Pc1a) present in P. cambridgei venom is the only gating modifier of ASICs [50]. In addition to pain inhibitor, it exerts an interesting neuroprotective and a possible antidepressant activity due to the involvement of ASICs in cell excitability. A drop in pH from neutral 7.4 to more acid extracellular environments, lead to opening of ASICs Na + or Ca 2+ permeable pore, membrane depolarization and increase in Ca 2+ intracellular concentration [104].
In the light of these facts, Yang and coworkers [105] investigated the neuroprotective activity of PcTx in neurons from newborn piglets submitted to a model of asphyxia-induced cardiac arrest. Data show that the administration of PcTx before the hypoxia-ischemia insult partially prevents the death of neurons in putamen, the most vulnerable encephalic area in this model. The addition of MK-801, a NMDA antagonist, in combination with PcTx exerted better results in cell survival, but in low doses of MK-801. In addition to protection of neuronal cells, treatment with PcTx accelerated neurologic recovery. These results point PcTx as a very unique neurotoxin that should be used as tool in the investigation of processes underlying neuroprotection as well as the design of novel neuroprotective agents.
Bees and wasps are part of the group of the insects, whose stings release a cocktail of toxins, including enzymes, peptides and biogenic amines [106]. Toxins in bee venom have received attention for their properties as anti-inflammatory agents, and in many countries, physicians even prescribe bee stings as treatment of rheumatologic diseases. Recently, Doo and colleagues [107] showed that the bee venom when injected in rats with induced Parkinson disease prevent dopamine neurons cell death, possibly by the inhibition of Jun activation.
Regarding solitary wasps, the most studied wasp species is the European beewolf, Philanthus triangulum, the natural predators of honeybees. The adult individuals of this species are herbivores, whereas the larvae eat the paralyzed bees brought to the colony by foraging wasps. The isolation of venom contents begun in the early 80s and revealed that among other classes of molecules, P. triangulum venom contains potent acylpolyamines [86]. Philanthotoxins, like other acylpolyamines are mostly potent and selective antagonists of vertebrate and invertebrate glutamate receptors, particularly AMPA receptors [108]. The first isolated and most studied philathotoxin is PhTX-433 and its synthetic analogue, PhTx-343, which antagonize Ach and glutamate ionotropic receptors. The neuroprotective activity of PhTx-343 was tested in cerebellar granule cells culture challenged with NMDA and kainate toxicity and compared to that of Arg-636 [109]. Data showed that both polyamines protected cultures against damage, but Arg-636 was found to be less potent than PhTx-343 against kainate-induced damage. The structural change in PhTx-343 increased its potency, but in higher doses, toxic side effects, were observed.
Due to their lack of selectivity, the use of philanthotoxins as pharmaceuticals may have been limited, and so many modified synthetic analogues were designed for medical treatment purposes, so far [82]. However, the use of philanthotoxins and other polyamines as tools in research investigation has aided the understanding of several synaptic mechanisms. As it has been recently shown using Ca 2+ -permeable AMPA receptors expressed in HEK cells. According to [110] the block of these AMPA receptors by PhTx-74, a synthetic analogue of PhTX-433 will reflect structural and biophysical parameters of the channel, such as its subunit composition and mean conductance, respectively. In addition, the investigation of the antagonistic activity of PhTx-343 over ACh receptors showed that the interaction of the toxin with nicotinic receptors is largely voltage dependent, slow and uncompetitive, a similar mode by which they block glutamate ionotropic channels [111].
Going further on wasp venoms, the anticonvulsant and/or neuroprotective effects of molecules in the venom of two Brazilian social species of the genus Polybia, were investigated. According to Cunha and co-workers [112] and Mortari and co-workers [113], the non-enzymatic fraction of the venoms of Polybia ignobilis and Polybia occidentalis inhibit seizures evoked by the injection of several chemoconvulsants in Wistar rats. The neuroactive molecules present in the venom of P. ignobilis and P. occidentalis are now in phase of structurefunction investigation.
Finally, neurotoxins from scorpion venoms have been subject of a wide range of works, mostly approaching the identification of voltage-dependent ion channel activators/blockers. The neuroprotective and/or anticonvulsant activity of these peptides, in turn have received a few lines of investigation [3] despite the ancient use of these animals whole or parts, in the popular medicine in oriental countries, like China [20]. One of the most studied species is the Asian scorpion Buthus martensi Karsch whose venom has several neuroactive peptides, among whose, we may find BmK AEP, which was the first anticonvulsant peptide isolated from scorpion venoms. According to [28], the injection of BmK AEP blocked seizures induced by the injection of coriaria lactone in doses causing no visible side effects [114]. Further isolation of venom of B. martensi led to the identification of other peptides, such as BmK AS and BmK Ts and other mostly with analgesic activity. According to Zhao and co-workers [115] BmK AS, a sodium channel modulator at site-4 receptor, inhibited PTZ induced behavioral and electroencephalographic seizures and decreased mean score of pilocarpine-induced seizures. Moreover, these authors showed that BmK AS does not impair locomotion or motor behavior.
The venom of the Mexican scorpion Centruroides limpidus limpidus, was fractionated and many activators of voltage-gated ion channel ligands were identified [116]. An exception is Cll9, which stands for Centruroides limpidus limpidus toxin nr 9. Cll9 is a 63-residue peptide that has a divergent mode of action; it inhibits sodium channels in superior cervical ganglion neurons and [117]. When injected in Wistar rats via i.c.v., Cll9 inhibited behavioral and electroencephalographic seizures evoked by the microinjection of penicillin into the basolateral amygdala. It is worth noticing that Cll9 has no effect on arthropods such as crickets or crayfish like many sodium channels modulators found in scorpion venoms.

Actions on mood disorders
According to the World Health Organization, depression, one of the most important mood disorders, affects up to 5-10% of people worldwide at any time of their lives. Patients with a diagnosed mood disorder are more likely to be women, in productive years, 20 to 40 yearold, and will need in most cases, psychotherapy and/or pharmacological intervention. The costs of these psychiatric and/or psychological disorders are immense, since they affect people regardless of education or socioeconomic status, accounting in the worse cases, for a huge number of suicides. In the United States up to 95% of all suicides, involve mentally ill people, accounting for 1.3% of all deaths [118]. A recent survey shows that generalized anxiety disorder, posttraumatic stress disorder, social anxiety disorder and panic disorder are highly predictive of suicidal idealization [118]. Many aspects of the pathophysiology of mood disorders, as well as the regulation of normal mood states remain unknown. However, with the improvement of techniques for research and diagnosis, such as positron emission tomography, magnetic resonance and multiple channels recording electroencephalogram, soon researchers will be capable to identify structures and neurochemical mechanism involved in the regulation of mental states, including mood. So far, we know that limbic structures, such as the amygdala, hippocampus, hypothalamus and prefrontal cortex control the emotional aspects of brain function. There are plenty of connections among these structures, which might be involved in the onset of mood disorders [119].
Pharmacological treatment of mood disorders consists in daily intake of anti-depressants, anxiolytics or anti-psychotics, most of which cause a wide set of undesired side effects that impose restrictions to patients quality of life. In this regard novel drugs prescribed for mood disorder, such as serotonin uptake inhibitors might be better tolerated and safer than classical drugs, such as monoamine oxidase inhibitors. Among the observed undesired effects we can cite; dizziness, sedation, sexual dysfunction and suicidal though, a paradoxical effect of serotonin uptake inhibitors [119]. Aside from tolerability, medicines used as treatment of depression for example, take too long to produce effect in only a minority of patients; 35-45% of treated patients [120]. Therefore, there is a still great need for novel alternatives to be used both, in basic and clinical science.
The neurochemistry of mood disorders is complex and there is a list of candidates for targets of mood stabilizers, such as adrenaline, GABA, serotonin and glutamate receptors and transporters. There is not many works relating neurotoxins from arthropods and mood disorders, but the few works available showed that in some cases, these molecules might contribute for the development of novel drugs.
The venom of the Brazilian colonial spider Parawixia bistriata was fractionated and tested in many animal models of epilepsy, neurodegeneration and anxiety. According to [121], the microinjection of Parawixin 2 (formerly FrPbAII) in the dorsal hippocampus of male Wistar rats increased the time spent in the open arms of the elevated plus maze. Moreover, rats exposed to the light-dark choice apparatus spent more time in the light side of the box, similarly to what observed for diazepam or nipecotic acid, a GABA transporter-1 (GAT-1) inhibitor [121]. In another investigation of P. bistriata venom contents, Saidemberg and co-workers [122] isolated PwTx-I and tested the inhibitory activity of this neurotoxin and its enantiomers on mammalian monoamine oxidases (MAO)-A and -B. According to these authors, PwTx-I, acted as non-competitive inhibitors of MAO-A and MAO-B. MAO metabolizes monoamines dopamine, serotonin and adrenaline, terminating monoaminergic transmission. Inhibitors or MAO (MAOi) have been extensively used as mood stabilizers and currently they have received attention due to their protective activity against age-induced neurodegenerative disorders [123].
Considering alternative targets for mood stabilizers, interesting results were obtained with PcTx, isolated from the venom of the spider P. cambridgei, a selective blocker of ASICs. Data showed that both PcTx and amiloride attenuated the stress-induced hyperthermia, whereas only the administration of PcTx increased number of punished crosses measured in the four-plate test. These results indicate that both blockers could attenuate autonomic anxiety parameters, but only PcTx exerted effects on the behavioral anxiety parameters [124].
The aggressive Brazilian social wasp Agelaia vicina, builds huge nests where with over a million of individuals. The neurobiological activity of the venom of A. vicina, was investigated. Oliveira and colleagues [125] showed that the central injection of the non-enzymatic fraction of the venom induced catalepsy in Wistar compared to the neuroleptic drug haloperidol, a nonselective D2 dopamine antagonist. This effect was reversed by the injection of theophylline or ketamine. The fractionation of the venom led to the identification of two peptides, AvTx-7, mastoparan, and AvTx-8. The investigation of AvTx-8 mode of action in vivo, was performed in a model of panic induction through the activation of GABAergic pathways connecting mesencephalic substantia nigra pars reticulate to superior colliculus [126]. These experiments showed that intranigral microinjection of AvTx-8 inhibited the panic like response induced by the GABAergic blockade of superior colliculus. These effects were similar to those of baclofen, a GABA B agonist, but differed from the effects of muscimol, a GABA A agonist. Since post-synaptic GABA B is a metabotropic receptor complex with a potassium channel, AvTx-8 could act in many different sites that would end in channels opening and hyperpolarization of neuronal membrane.

Tools for the study of the functioning of the CNS: learning and memory
Neurotoxins isolated from arthropod are important tools to study of the normal function of the CNS, especially in the structure-function research of the ion channels and the interaction the blockers and modulators in the regulation of the learning and memory (for revision see [127]). In this context, the principal compound used in study of the mechanism of the learning and memory in models of experimental animals is the apamin. Apamin is a short peptide (18 aa) isolated from the venom of honeybee, Apis mellifera. It is generally accepted that apamin selectively blocks small conductance calcium-activated potassium channels (SK or K ca ), although evidences point to an allosteric modulation of opening rather than the block of the pore [128]. Upon an increase in intracellular calcium, SK channels will open and allow an outward current of potassium ions that is responsible for the hyperpolarization phase of the action potentials. Most studies on structure-function of SK channels were conducted using apamin blockade. The homomeric or heteromeric expression of these channels occurs in higher brain areas such as the neocortex, hippocampus and sub-cortical areas such as thalamus and basal ganglia as well as in cerebellum and brainstem. Substantial data SK channels show the involvement of SK channels in processes of learning and memory, and apamin blockade of SK lead to an increase in cellular excitability, facilitates synaptic plasticity and memory processes run by the hippocampus. In addition, apamin induces alterations in dendritic morphology that might counteract aging and neurodegenerative processes that lead to cognitive and memory impairment [129]. In fact, SK channels co-localize with Ca 2+ -permeable NMDA receptors in the CA1 region of the hippocampus and the entry of calcium in the cell through these receptors might activate SK that will hyperpolarize membrane. The blockade of SK channels will modulate hippocampal excitability that is essential in memory processes such as long-term potentiation a commonly observed event of synaptic plasticity. Due to its actions, the use of apamin as a tool in research has been consolidated. In addition, the therapeutic use of apamin, in order to maintain hippocampal function and avoid the deleterious effects of aging in memory and cognitive processes have also been proposed [129].
Besides apamin, modulators peptides of the potassium channel isolated from scorpion also have been tested in models of the learning and memory. The good examples are: Charybdotoxin isolated from scorpion Leiurus quinquestriatus, Kaliotoxin isolated from Androctonus mauretanicus and Iberiotoxin from Buthus tasmulus. Charybdotoxin is a potent selective inhibitor of high (large or big) conductance Ca 2+-activated potassium channels (KCa1.1, BK, or maxi-K), as well as a Kv1.3 channel [130]. Kaliotoxin is a specific inhibitor of Kv1.1 and Kv1.3 [131] and Iberiotoxin is a selective inhibitor of KCa1.1 channels (formerly BK) [132]. These peptides induced an improvement effect in passive avoidance test and olfactory discrimination task [133,134].

Final remarks
The stories of voltage-gated, ligand-gated ion channels and venom toxins are very closely tied. Indeed, the isolation and structural characterization of venom molecules provided a plethora of tools that have been used in the investigation of ion channels structure-function relationships. With the aid of arthropod toxins, remarkably, scorpionic toxins, the characterization of sodium channels was possible. Spider and wasps polyamines, in turn are considered unique ligands of glutamatergic and cholinergic ionotropic receptors. Regarding to peptides and small proteins, arthropod venoms possess an arsenal of these molecules that remain largely unknown and consequently, their pharmacological potential is left unexplored.
Due to the mode of action of neurotoxins, their affinity and selectivity for neuronal structures, many researchers consider them as probes to novel drugs design and development. However, despite of the thousands of patents made with neurotoxins in the past 30 years, very few molecules came to commercialization. [32] Chen, J., Feng, X. H., Shi, J., Tan, Z. Y., Bai, Z. T., Liu, T., et al. (2006). The anti-nociceptive effect of BmK AS, a scorpion active polypeptide, and the possible mechanism on specifically modulating voltage-gated Na+ currents in primary afferent neurons. Peptides, 27(9), 182-92.