26The parasitic protozoan Toxoplasma gondii becomes encysted in brain and muscle tissue 27 during chronic infection, a stage that was previously thought to be dormant but has been 28 found to be active and associated with physiological effects in the host. Dysregulation of 29 catecholamines in the CNS has previously been observed in chronically-infected animals. In 30 the study described here, the noradrenergic system was suppressed with decreased levels of 31 norepinephrine in brains of infected animals and in infected neuronal cells in vitro. 32 Expression of dopamine β-hydroxylase (DBH), essential for synthesis of norepinephrine 33 from dopamine, was the most differentially-expressed gene in infections in vitro and was 34 down-regulated in infected brain tissue, particularly in the prefrontal cortex and dorsal locus 35 coeruleus/pons region. The down-regulated DBH expression in infected rat 36 catecholaminergic and human neuronal cells corresponded with decreased norepinephrine 37 and increased dopamine. As the DBH suppression was observed in vitro, this effect is not 38 caused by neuroinflammation. Silencing of DBH expression was specific for T. gondii infection 39 and was not observed with CMV infection. The noradrenergic-linked behaviors of sociability 40 and arousal were altered in chronically-infected animals, with a high correlation between 41 DBH expression and infection intensity. These findings together provide a plausible 42 mechanism to explain prior discrepancies in changes to CNS neurotransmitters levels with 43 infection. The suppression of norepinephrine synthesis observed here may, in part, explain 44 behavioural effects of infection, associations with mental illness, and neurological 45 3 consequences of infection such as the loss of coordination and motor impairments associated 46 with human toxoplasmosis. 47 48T. gondii infects warm-blooded animals and is characterised by a transient acute infection 49 wherein vegetative tachyzoite forms rapidly replicate in tissues followed by a persistent 50 chronic infection. Chronic stages of infection can persist for years and potentially the lifetime 51 of the host with the bradyzoite-stage parasites encysted in cells within immunoprivileged 52 tissues, including muscle, eyes, and brain. Several reports have published host behavioral 53 changes with infection. A selective loss of aversion to feline urine and increased motor 54 activity has been observed in rodents, specifically manipulating behavior that will enhance 55 the probability of parasite transmission (1, 2). 56Toxoplasmosis can be a severe disease in immunocompromised individuals and in utero. 57Infection can cause retinochoroiditis and congenital hydrocephalus and cerebral 58 calcifications. T. gondii was recently ranked the second most important food-borne parasite 59 in Europe and is classified as a Neglected Parasitic Infection (CDC, Atlanta) (3). It has also 60 been linked by epidemiological studies to cognitive impairment and major mental illnesses. 61Severe cases are ass...

BackgroundThe protozoan parasite Toxoplasma gondii is one of the most widely distributed and successful parasites. Toxoplasma gondii alters rodent behavior such that infected rodents reverse their fear of cat odor, and indeed are attracted rather than repelled by feline urine. The location of the parasite encysted in the brain may influence this behavior. However, most studies are based on the highly susceptible rodent, the mouse.Methodology/Principal FindingsLatent toxoplasmosis was induced in rats (10 rats per T. gondii strains) of the same age, strain, and sex, after oral inoculation with oocysts (natural route and natural stage of infection) of 11 T. gondii strains of seven genotypes. Rats were euthanized at two months post inoculation (p.i.) to investigate whether the parasite genotype affects the distribution, location, tissue cyst size, or lesions. Tissue cysts were enumerated in different regions of the brains, both in histological sections as well in saline homogenates. Tissue cysts were found in all regions of the brain. The tissue cyst density in different brain regions varied extensively between rats with many regions highly infected in some animals. Overall, the colliculus was most highly infected although there was a large amount of variability. The cerebral cortex, thalamus, and cerebellum had higher tissue cyst densities and two strains exhibited tropism for the colliculus and olfactory bulb. Histologically, lesions were confined to the brain and eyes. Tissue cyst rupture was frequent with no clear evidence for reactivation of tachyzoites. Ocular lesions were found in 23 (25%) of 92 rat eyes at two months p.i. The predominant lesion was focal inflammation in the retina. Tissue cysts were seen in the sclera of one and in the optic nerve of two rats. The choroid was not affected. Only tissue cysts, not active tachyzoite infections, were detected. Tissue cysts were seen in histological sections of tongue of 20 rats but not in myocardium and leg muscle.Conclusion/SignificanceThis study reevaluated in depth the rat model of toxoplasmosis visualizing cyst rupture and clarified many aspects of the biology of the parasite useful for future investigations.

(-)-Englerin A (EA) is a natural product which has potent cytotoxic effects on renal cell carcinoma cells and other types of cancer cell but not non-cancer cells. Although selectively cytotoxic to cancer cells, adverse reaction in mice and rats has been suggested. EA is a remarkably potent activator of ion channels formed by Transient Receptor Potential Canonical 4 and 5 proteins (TRPC4 and TRPC5) and TRPC4 is essential for EA-mediated cancer cell cytotoxicity. Here we specifically investigated the relevance of TRPC4 and TRPC5 to the adverse reaction. Injection of EA (2 mg.kg-1 i.p.) adversely affected mice for about 1 hour, manifesting as a marked reduction in locomotor activity, after which they fully recovered. TRPC4 and TRPC5 single knockout mice were partially protected and double knockout mice fully protected. TRPC4/TRPC5 double knockout mice were also protected against intravenous injection of EA. Importance of TRPC4/TRPC5 channels was further suggested by pre-administration of Compound 31 (Pico145), a potent and selective small-molecule inhibitor of TRPC4/TRPC5 channels which did not cause adverse reaction itself but prevented adverse reaction to EA. EA was detected in the plasma but not the brain and so peripheral mechanisms were implicated but not identified. The data confirm the existence of adverse reaction to EA in mice and suggest that it depends on a combination of TRPC4 and TRPC5 which therefore overlaps partially with TRPC4-dependent cancer cell cytotoxicity. The underlying nature of the observed adverse reaction to EA, as a consequence of TRPC4/TRPC5 channel activation, remains unclear and warrants further investigation.

Toxoplasma gondii is associated with physiological effects in the host. Dysregulation of catecholamines in the central nervous system has previously been observed in chronically infected animals. In the study described here, the noradrenergic system was found to be suppressed with decreased levels of norepinephrine (NE) in brains of infected animals and in infected human and rat neural cells in vitro. The mechanism responsible for the NE suppression was found to be downregulation of dopamine ␤-hydroxylase (DBH) gene expression, encoding the enzyme that synthesizes norepinephrine from dopamine, with downregulation observed in vitro and in infected brain tissue, particularly in the dorsal locus coeruleus/pons region. The downregulation was sex specific, with males expressing reduced DBH mRNA levels whereas females were unchanged. Rather, DBH expression correlated with estrogen receptor in the female rat brains for this estrogen-regulated gene. DBH silencing was not a general response of neurons to infection, as human cytomegalovirus did not downregulate DBH expression. The noradrenergic-linked behaviors of sociability and arousal were altered in chronically infected animals, with a high correlation between DBH expression and infection intensity. A decrease in DBH expression in noradrenergic neurons can elevate dopamine levels, which provides a possible explanation for mixed observations of changes in this neurotransmitter with infection. Decreased NE is consistent with the loss of coordination and motor impairments associated with toxoplasmosis. Further, the altered norepinephrine synthesis observed here may, in part, explain behavioral effects of infection and associations with mental illness.