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 [1-3]. 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 . 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 usage in some patients or may even function as a factor of impairment in people’s quality of life. According to , none of antiepileptic drugs discovered in the last 20 years, was efficient to cure or even suppress seizures in epileptic patients. Therefore, there is a continued need for the discovery of novel drugs to treat most neurological and mental disorders .
This chapter will target the discussion of recent contributions of research on the compounds of arthropod venom, for the discovery of novel tools to study the functioning of the structures of mammalian CNS, as well as the supply of novel alternatives to the treatment of neurological disorders. Among the major compounds, it will be highlighted those with the analgesic, anxiolytic, antiepileptic and neuroprotective effects, with emphasis on the most promising on preclinical or clinic evaluation.
2. Main targets of the neuroactive compounds isolated from arthropod venoms
Venom isolated from bee, scorpion and spider have been used to the treatment of various diseases in Chinese and Korean traditional medicine, such as epilepsy, stroke, facial paralysis, arthritis, rheumatism, back pain, cancerous, tumors, and skin diseases [8-10]. Moreover, venoms of arthropod animals have been used to study various physiopathological processes, and also offer opportunity to design and develop new therapeutic drugs [3,11,12] .
Arthropod venoms are rich in biologically active substances with different physiological actions, specially the neurotoxins. So far, identified neurotoxins generally comprise the classes of peptides or acylpolyamines, acting with affinity and specificity over excitatory or inhibitory neurotransmissions (for revision see . The actions of these compounds include the interaction with Na+, K+ and Ca2+ ion channels, agonism or antagonism of metabotropic and ionotropic receptors for neurotransmitters as the excitatory neurotransmitter glutamate. At the presynaptic level, several studies have shown the interaction of arthropod neurotoxins with protein transporters of neurotransmitters, resulting in the facilitation or inhibition of their uptake.
3. Antinociceptive effects
Of extreme importance for the organism, pain is an indicator of corporal integrity and has been considered since January 2000, by the Joint Commission on Accreditation on Healthcare Organizations (JCAHO) as the fifth vital sign that should be assessed and recorded together with other signals immediately after birth. According to the International Association for the Study of Pain (IASP), pain is defined as an unpleasant sensation and emotional experience associated with actual or potential tissue damage. However, approximately one third of world population suffers from pathological persistent or recurrent pain, which is a common complaint in patients with different diseases, and exerts great impact on their social life . In these cases, treatment is a challenge for researchers and health professionals who constantly seek new therapeutic strategies, since most of these are inadequate or cause serious side effects .
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.
3.1. Polypeptide toxins from Scorpion
The most studied Arthropod venom is extracted from the Asian scorpion
The NaScTx family can be classified in at least two major families, α and β, according to the mode of action on Na+ channels . The binding of α-toxins delays Nav 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:
“classic” highly specific for mammals;
“α-like toxins” active both on mammals and insects, which are far less specific and less active than the “classical” ones;
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 . The same toxin has also been sequenced by another group and named Bm K 33-I . Later, Guan and colleagues  identified a novel toxin with analgesic effects, BmK AngP1, which shows an evident analgesic effect with simultaneous excitatory 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 AngP1 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). Intraplantar injection of BmK IT2 inhibited thermal hyperalgesia in carrageenan-treated rats and significantly prolonged paw withdrawal latency in normal rats . This toxin also displays an inhibitory effect on the C component of the rat nociceptive flexion reflex by subcutaneous injection in vivo . Peripheral or spinal delivery of BmK IT2 suppressed formalin-induced nociceptive behaviors and c-Fos expression in spinal cord [25,26]. Both BmK IT2 and Bm K dIT-AP3 (depressant Insect Toxin-Analgesic Peptide 3) are toxic for insects, but not for mammals , and shows 86.7% of sequence similarity . BmK dIT-AP3 also induces analgesia in the mouse-twisting model . Using whole-cell patch clamp, it has been shown that BmK dIT-AP3 inhibits Nav currents of rat dorsal root ganglion (DRG) neurons, blocking more selectively the tetrodotoxin-resistant (TTX-R) component of the Na+ currents. These results suggest that the inhibition of the rat nociceptive flexion reflex by BmK dITAP3 may be attributed to modulation of the DRG’s voltage-gated Na+ channels .
Wang and colleagues  isolated a new antinociceptive peptide, named BmK AGP-SYPU1. Recombinant BmK AGP-SYPU1 showed similar analgesic effects on mice compared to natural when assayed using a mouse-twisting model . More recently, BmK AGP-SYPU2 was purified and tested, also in mouse-twisting model. Sequence determination showed that the mature BmK AGP-SYPU2 peptide is composed of 66 amino acid residues, and BmK AGP-SYPU2 is identical to BmK alpha2 and BmK alphaTX11.
BmK AS had a strong analgesic effect on both visceral and somatic pain [29,30]. It relieves formalin-induced two-phase spontaneous flinching response and carrageenan-induced mechanical hyperalgesia, probably by modulating the voltage-gated Na+ channels of sensory neurons [31,32]. Moreover, BmK AS showed activity nearly equivalent to that of morphine. Later, a new peptide that possesses 86.3% of similarity with BmK AS was identified. Both polypeptides have 66 amino acids cross-linked by four disulfide bridges . In addition, these two peptides show a poor similarity with other known types of scorpion toxins. BmK AS and AS1 are not toxic against mammals and only have a weak toxicity to insects. BmK AS, then BmK AS1, have been found to significantly stimulate the binding of [3H]-ryanodine to partially purified ryanodine receptors . More recently, electrophysiological studies have shown that they are able to inhibit Na+ currents in NG108-15 cells  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 . 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 . 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 . In relation to BmK Ang M1 , 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 (INa) and voltage-dependent delayed rectifier K+ current (IK), but had no effects on transient K+ current .
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 , four different possibilities might be described:
peptides act directly Na+ channels involved in the pathway of pain,
peptides modulate indirectly the pain sensation,
peptides also modulate other targets involved in pain pathway
pain alleviation is only apparent and results from misinterpretations that might have occurred from animal models used.
3.2. Polypeptide toxins from Spider
Another group of arthropods that have very promising antinociceptive compounds are spiders . In 1996, Roerig & Howse reported the effect of ω-agatoxina IVA (Fig 1) isolated from funnel spider
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
Several studies have reported that intrathecal administration of non-selective blockers of Ca2+ channels shows antinociceptive effects in animals tested with thermal stimuli: hot plate and tail flick. According to , N and P/Q Ca2+ 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 Ca2+ 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
Other important target in the search for new analgesics isolated from spider venoms are NaV 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
Further on spider venoms, Purotoxin-1 (PT1) was recently isolated the, from the venom of the Central Asian spider
3.3. Polypeptide toxins from Bees and Wasps
Bee venom has been traditionally used to relieve pain and treat chronic pain diseases (for revision see ). 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 [58-63]. 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 α2-adrenergic receptor, as well as activation of the descending serotonergic pathways (for revision see ).
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 PLA2  and has a variety of effects on living cells possibly through the disruption of the membrane . The decrease in cyclooxygenase (COX)-2 and phospholipase PLA2 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 anti-arthritis effect of melittin . This peptide has also been thought to play a role in production of anti-nociceptive and anti-inflammatory effects . In addition, Merlo and colleagues  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 . 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 .
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 . 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  isolated a compound with antinociceptive activity from the venom of the Brazilian social wasp
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 . In this regard, intrathecal administration of different doses of these toxins blocked thermally induced allodynia  and hyperalgesia . The effect of these neurotoxins may suggest a possible involvement of AMPA receptors in the spinal cord during the nociceptive excitatory stimulation [80,81].
4. 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 Ca2+-mediated signaling is thought to play a key role in many neurodegenerative disorders and the increase in intracellular Ca2+ concentration might alter neuronal membrane potential . 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 . This increase in glutamatergic activity often referred to as glutamate excitotoxicity , 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 . According to , 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
The fraction of the venom of the spider
Aside from antagonizing glutamate receptors, arthropod neurotoxins may exert anticonvulsant and neuroprotective effects targeting other neurotransmitter systems. The venom of the Brazilian spider
Also from South America, the Chilean giant pink tarantula
The African tarantula
The arboreal tarantula
In the light of these facts, Yang and coworkers  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 . 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  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,
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 . 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 Ca2+-permeable AMPA receptors expressed in HEK cells. According to  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 .
Going further on wasp venoms, the anticonvulsant and/or neuroprotective effects of molecules in the venom of two Brazilian social species of the genus
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  despite the ancient use of these animals whole or parts, in the popular medicine in oriental countries, like China . One of the most studied species is the Asian scorpio
The venom of the Mexican scorpion
5. 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 year-old, 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 . A recent survey shows that generalized anxiety disorder, posttraumatic stress disorder, social anxiety disorder and panic disorder are highly predictive of suicidal idealization . 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 pre-frontal 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 .
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 . 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 . 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
Considering alternative targets for mood stabilizers, interesting results were obtained with PcTx, isolated from the venom of the spider
The aggressive Brazilian social wasp
6. 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 ). 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,
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
7. 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.
The authors thank Msc Juliana Castro e Silva for help in preparing the figure.