In 1869, muscarine (Fig. 2) was isolated from European A. muscaria and was for decades believed to be the main active principle (Eugster 1979), from which the compound derives its name. Muscarine is a selective cholinergic agonist suspected to contribute to the overall activity of A. muscaria. Muscarine is both water soluble and thermostable (does not degrade with cooking). It is known to be responsible for reduced heart rate, lowering of blood pressure, vomiting, diarrhoea, bradycardia, bronchorrhea, tearing, bronchospasm (asthmatic-like breathing), salivation, pupil contraction and blurred vision.

Figure 2. 

Structure of muscarine.

Muscazone (Fig. 3) (2-Amino-2-(2-oxo-3H-1,3-oxazol-5-yl)acetic acid) is another compound that has been isolated from European specimens of the fly agaric (Fritz et al. 1965). It is a product of ibotenic acid breakdown by UV radiation. Muscazone has a minor pharmacological activity compared with the other agents (Catalfomo and Eugster 1970).

Figure 3. 

Chemical changes of ibotenic acid to muscazone and muscimol.

Ibotenic acid and muscimol are structurally related. Muscimol, being structurally similar to GABA is a potent GABA_A receptor agonist, while ibotenic acid is an agonist of NMDA glutamate receptors interactions causing the hallucinogenic effects observed during intoxication (Johnston, 2009).

Ibotenic acid, (S)-2-Amino-2-(3-hydroxyisoxazol-5-yl)acetic acid, is a colourless, crystalline substance soluble in water (Fig. 3). It is metabolized by decarboxylation in the stomach, liver, and brain (Nielsen et al. 1985) to equal amount of muscimol which pass the blood-brain barrier (Michelot and Melendez-Howell 2003). Both substances can be detected in urine within 1 h of exposure (Merova et al. 2008). Ibotenic acid, unlike muscimol, is much more dangerous, causing ibotenate-induced seizures and lesions in specific brain regions, similar to Alzheimer’s disease for which it is used in animal test models (Stebelska 2013). Stereotactic intrahippocampal administration of ibotenic acid (5µg/µl) lesioned rats impairs cholinergic transmission, learning and memory performance (related to Alzheimer’s disease) and thus chosen as a suitable model to understand drug efficacy in preventing Alzheimer’s disease pathophysiology (Patocka et al. 2017).

Exposure to chronic stress in young male rats increases hippocampal glutaminergic receptor density or affinity, thus making cornus ammonis neurons more vulnerable to ibotenic acid (Conrad et al. 2007). In Alzheimer’s disease (AD), where HPA (hypothalamic-pituitary-adrenal) activity is elevated, hippocampal NMDA receptor number does not generally decrease. The results on the presence of NMDA sites in the majority of AD cases indicate that receptor density is preserved except in cases where there is extremely severe cell loss. Rats exposed to the same paradigm of chronic restraint do not show increase in hippocampal NMDA or non-NMDA receptor binding (Geddes et al. 1986).

Muscimol (5-(Aminomethyl)-isoxazol-3-ol) was isolated from A. muscaria in the early 1960’s (Takemoto et al. 1964; Eugster, Muller and Good 1965) as a colourless, crystalline substance, readily soluble in cold water (Fig. 3). It appears to be the essential principle of Amanita muscaria since it is present in very high concentration (0.03–0.1%) of fresh mushroom (DeFeudis 1980). Oral, systemic, or intracerebral administration of muscimol undoubtedly affects certain CNS functions and behaviour. Muscimol is a non-selective GABA_A receptor agonist activating both pre- and postsynaptic receptors and partial agonist of GABAc receptors devoid of effects on the GABA-metabolizing enzyme, GABA_A transaminase, and the GABA_A uptake systems which also enters the brain after peripheral injection (Snodgrass 1978). It is reported to be a potent agonist at bicuculline-sensitive, strychnine-insensitive postsynaptic receptors of the mammalian central nervous system. For example, muscimol (3 mg/kg, i.p.) evokes serotonin rise and decreases catecholamine levels in the brain. The compound binds to GABA_A receptors mainly in the areas of the forebrain, including the caudate nucleus and putamen, the thalamus and the hippocampal formation leading to the opening of the receptor associated with the chloride ion channel, which in turn leads to inhibition of neuronal activity, where these receptors are located (Stebelska 2013). Muscimol high-affinity binding in forebrain regions such as caudate – putamen, thalamus, and hippocampus was dependent on distinct population of GABA_A receptors possibly containing subunit 6 and lacking subunit 1 as shown in knockout study in mice (Chandra et al. 2009).

Like LSD, muscimol and ibotenic acid induce a generalized increase of serotonin but only muscimol keeps the serotonin concentration increased in midbrain and hypothalamus after pre-treatment with p-ehlorophenylalanine (a serotonin synthesis inhibitor). Muscimol and LSD cause a decrease of the catecholamines, as on the contrary ibotenic acid increases the catecholamine concentration (Konig-Bersin et al. 1970).

A low dose of muscimol injected at doses of 0.5–1 mg/kg i.p. affects the EEG of cats and rabbits (De Carolis et al. 1969). These observations further support a localization of action of muscimol in the brain rather than in the peripheral nervous system.

Recently it has been suggested that GABA is involved in morphine analgesia. The injection of 0.15 to 0.2 mg/kg of muscimol i.v., lowered ED₅₀ dose of morphine in mice and rats. Muscimol given alone, at doses up to 2.0 mg/kg (i.v.) failed to cause analgesia in mice or rats. However, when injected intravenously 10 min before morphine at a dose of 0.15 mg for morphine analgesia in mice from 4.1 mg/kg (s.c.) to 1.6 mg/ ED₅₀ is highly significant (Biggio et al. 1977).

Reversible inactivation of brain areas is an useful method for inferring brain-behaviour relationships. Infusion of GABA or of the GABA receptor agonist muscimol is considered one interesting reversible inactivation method because it may not affect fibres of passage and may therefore be compared to axon-sparing types of lesions (Majchrzak et al. 2000). Concluding that reversible inactivation techniques significantly contribute to the knowledge of “where and when” neuronal events for learning and memory take place in the brain.

In concern to affecting memory, intra-hippocampal infusion of muscimol increased the percent of neurons active in cornus amonius (CA3) significantly, improving rats’ learning and memory abilities in both normal and AD-type rats suggesting that intensification of GABAergic processes may be an useful pharmacotherapeutic strategy in early memory decline in AD (Pilipenko et al. 2015). Infusions of muscimol into the dorsal hippocampus in male rats produce impairments in fear learning (at a dose of 0.5 mg of muscimol per hemisphere) (Corcoran and Maren 2001) and working memory (at a dose of 0.03–0.06 µg of muscimol) (Mao and Robinson 1998). These findings suggest that because muscimol is a potent GABA_A agonist, it is likely that hippocampal infusions of muscimol modulate learning through increased neural inhibition of the hippocampus.

Тhe activation of the GABA_A receptor by muscimol modulates the hypothalamic–pituitary–gonadal (HPG) axis increasing kisspeptin expression through stimulating KiSS-1 mRNA expression, in the hypothalamic neurons. Kisspeptin is a neuropeptide closely linked to the reproductive function of multiple species. Surprisingly, the natural GABA compound had no effect on KiSS-1 gene expression, in contrast to muscimol (Kanasaki et al. 2017).

Muscimol was also used as a prototype substance for the design of THIP (Gaboxazole, 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol hydrochloride,) an izoxazole investigated as insomnia and seizure medication but withdrawn from phase 3 clinical trials, due to efficacy and side effects problems (Johnston et al. 2009). The GABA receptors agonist used in a double-blind study administered orally (5–10 mg per day) to ten patients with Huntington’s disease did not result in improvement of motor or cognitive functions but significantly ameliorated chorea in the most severely hyperkinetic patient, and it was associated with the appearance of dystonic features, electroencephalographic changes, and behavioural alterations in five patients. Moreover, adverse effects as increased irritability, agitation and lethargy, lack of attention, loss of appetite, and insomnia occurred in five patients and appeared to be dose related in each instance (Shoulson et al. 1978). However, the constant failure to prove muscimol’s potential effect as an anticonvulsant only indicates that the GABA disturbances connected with motor deficits does not alone account for the clinical features of the ongoing disorders.

Potentiation of inhibitory mechanisms may be important to neuronal protection from the effects of ischaemia. The GABA_A agonist effects of muscimol showed protective role in a dose-dependent manner in both rat and rabbit microsphere embolism model of ischaemia (Lyden and Hedges 1992). In a model of forebrain ischaemia, muscimol given 7 days before the onset, protected the cortex, hippocampus, substantia nigra, striatum and thalamus (Sternau et al. 1989), suggesting that the damaging effects from forebrain ischemia may be a result of excessive excitability or loss of inhibitory influence.

Sonnenberg (1988) found that gastric cancer occurred more frequently in patients who had ischaemic heart disease or cerebrovascular disease, and concluded that gastric cancer and diseases related to hypertension share a common etiologic factor. Prolonged administration of the GABA_A receptor-agonist muscimol (i.p. injections of 0.5 mg/kg body weight) attenuated the enhancement of N-methyl-N-nitro-N-nitrosoguanidine (MNNG)-induced gastric carcinogenesis in spontaneously hypertensive rats (SHR) on the 52^th week, resulting in a significant reduction in the incidence of gastric cancer (Tatsuta et al. 1992) was again connected with GABA agonists activity over the control of anterior pituitary hormones; the sympathetic and parasympathetic nervous system. Using muscimol as a GABA_A receptors agonist proved its protective role in treatment of oral squamous cell carcinoma (Jing Ma et al. 2016).

Last but not least A. muscaria, like the other mushrooms from the genus, contain a vast amount of biologically active compounds with proven antioxidant activity: proteins and peptides (glutathione and ergothioneine), phenolic compounds (flavonoids, lignans, oxidized polyphenols, phenolic acids, stilbenes and tannins), vitamins and derivatives (ascorbic acid, ergosterol, tocopherols, carotenoids), and minerals (zinc and selenium). Their antioxidant properties and ability to scavenge free radicals have been further demonstrated in studies using rodent models with hepatic injury, induced by either streptozotocin (STZ), carbon tetrachloride (CCl₄), or D-galactosamine (D-GalN). For example, the activation of GABA_A receptor inhibits stem cell proliferation but protects differentiated cells from injures (Wang et al. 2017). Muscimol treatment decreases the formation of pseudo bile ductiles and the enlargement of hepatocytic canaliculi in GalN-treated rats revealing that a complex GABA signalling system exists in the rat liver. Its activation protects the liver against toxic injury (Wang et al. 2017).

The colouring of A. muscaria is due to a complicated mixture of pigments. Muscarufin and muscaflavin are terphenylquinone derivatives which give the yellow colouring. The betalain group composed of numerous betalamic acid condensates (muscapurpurin and muscaaurins) with different amino acids, ibotenic or stizolobic acid are responsible for the typical red-orange colour of the caps of A. muscaria (Michelot & Melendez-Howell, 2003). By repeated chromatography the pigment mixture has been fractionated into at least ten components, i. e. the orange muscaaurins, the yellow muscaflavin, the red-violet muscapurpurin and the red muscarubrin (Stintzing et al. 2007).

Among polysaccharides, glucans are the most abundant and widely distributed carbohydrates in the fungal cell wall. A fucomannogalactan (Ruthes et al. 2013) and a (1→3),(1→6)-linked β-D-glucan (Kiho et al. 1994) were isolated from A. muscaria fruiting bodies and their biological activities were further studied against pain and inflammation as well as antitumor activity. All of these activities are a subject of further studies.