Infections with Trypanosoma cruzi are usually lifelong despite generating a strong adaptive immune response. Identifying the sites of parasite persistence is therefore crucial to understanding how T. cruzi avoids immune-mediated destruction. However, this is a major technical challenge, because the parasite burden during chronic infections is extremely low. Here, we describe an integrated approach involving comprehensive tissue processing, ex vivo imaging, and confocal microscopy, which allowed us to visualize infected host cells in murine tissue with exquisite sensitivity. Using bioluminescence-guided tissue sampling, with a detection level of <20 parasites, we showed that in the colon, smooth muscle myocytes in the circular muscle layer are the most common infected host cell type. Typically, during chronic infections, the entire colon of a mouse contains only a few hundred parasites, often concentrated in a small number of cells each containing >200 parasites, which we term mega-nests. In contrast, during the acute stage, when the total parasite burden is considerably higher and many cells are infected, nests containing >50 parasites are rarely found. In C3H/HeN mice, but not BALB/c mice, we identified skeletal muscle as a major site of persistence during the chronic stage, with most parasites being found in large mega-nests within the muscle fibers. Finally, we report that parasites are also frequently found in the skin during chronic murine infections, often in multiple infection foci. In addition to being a site of parasite persistence, this anatomical reservoir could play an important role in insect-mediated transmission and have implications for drug development. IMPORTANCE Trypanosoma cruzi causes Chagas disease, the most important parasitic infection in Latin America. Major pathologies include severe damage to the heart and digestive tract, although symptoms do not usually appear until decades after infection. Research has been hampered by the complex nature of the disease and technical difficulties in locating the extremely low number of parasites. Here, using highly sensitive imaging technology, we reveal the sites of parasite persistence during chronic-stage infections of experimental mice at single-cell resolution. We show that parasites are frequently located in smooth muscle cells in the circular muscle layer of the colon and that skeletal muscle cells and the skin can also be important reservoirs. This information provides a framework for investigating how the parasite is able to survive as a lifelong infection, despite a vigorous immune response. It also informs drug development strategies by identifying tissue sites that must be accessed to achieve a curative outcome.

Background and PurposeA non‐psychoactive phytocannabinoid, cannabidiol (CBD), shows promising results as an effective potential antiepileptic drug in some forms of refractory epilepsy. To elucidate the mechanisms by which CBD exerts its anti‐seizure effects, we investigated its effects at synaptic connections and on the intrinsic membrane properties of hippocampal CA1 pyramidal cells and two major inhibitory interneurons: fast spiking, parvalbumin (PV)‐expressing and adapting, cholecystokinin (CCK)‐expressing interneurons. We also investigated whether in vivo treatment with CBD altered the fate of CCK and PV interneurons using immunohistochemistry.Experimental ApproachElectrophysiological intracellular whole‐cell recordings combined with neuroanatomy were performed in acute brain slices of rat temporal lobe epilepsy in in vivo (induced by kainic acid) and in vitro (induced by Mg2+‐free solution) epileptic seizure models. For immunohistochemistry experiments, CBD was administered in vivo (100 mg·kg−1) at zero time and 90 min post status epilepticus, induced with kainic acid.Key ResultsBath application of CBD (10 μM) dampened excitability at unitary synapses between pyramidal cells but enhanced inhibitory synaptic potentials elicited by fast spiking and adapting interneurons at postsynaptic pyramidal cells. Furthermore, CBD restored impaired membrane excitability of PV, CCK and pyramidal cells in a cell type‐specific manner. These neuroprotective effects of CBD were corroborated by immunohistochemistry experiments that revealed a significant reduction in atrophy and death of PV‐ and CCK‐expressing interneurons after CBD treatment.Conclusions and ImplicationsOur data suggest that CBD restores excitability and morphological impairments in epileptic models to pre‐epilepsy control levels through multiple mechanisms to reinstate normal network function.

247 23 Text: 4892 24 25 2 ABSTRACT Infections with Trypanosoma cruzi are usually life-long despite 26 generating a strong adaptive immune response. Identifying the sites of parasite 27persistence is therefore crucial to understand how T. cruzi avoids immune-mediated 28 destruction. However, this is a major technical challenge because the parasite burden 29 during chronic infections is extremely low. Here, we describe an integrated approach 30 involving comprehensive tissue processing, ex vivo imaging, and confocal 31 microscopy, which has allowed us to visualise infected host cells in murine tissue, with 32 exquisite sensitivity. Using bioluminescence-guided tissue sampling, with a detection 33 level of <20 parasites, we show that in the colon, smooth muscle myocytes in the 34 circular muscle layer are the most common infected host cell type. Typically, during 35 chronic infections, the entire colon of a mouse contains only a few hundred parasites, 36 often concentrated in a small number of cells containing >200 parasites, that we term 37 mega-nests. In contrast, during the acute stage, when the total parasite burden is 38 considerably higher and many cells are infected, nests containing >50 parasites are 39 rarely found. In C3H/HeN mice, but not BALB/c, we identified skeletal muscle as a 40 major site of persistence during the chronic stage, with most parasites found in large 41 mega-nests within the muscle fibres. Finally, we report that parasites are also 42 frequently found in the skin during chronic murine infections, often in multiple infection 43 foci. In addition to being a site of parasite persistence, this anatomical reservoir could 44 play an important role in insect-mediated transmission, and have implications for drug 45 development. 46 47 48 49 3 IMPORTANCE Trypanosoma cruzi causes Chagas disease, the most important 50 parasitic infection in Latin America. Major pathologies include severe damage to the 51 heart and digestive tract, although symptoms do not usually appear until decades after 52 infection. Research has been hampered by the complex nature of the disease and 53 technical difficulties in locating the extremely low number of parasites. Here, using 54 highly sensitive imaging technology, we reveal the sites of parasite persistence in 55 experimental mice at single-cell resolution. We show that parasites are frequently 56 located in smooth muscle cells in the circular muscle layer of the colon, and that 57 skeletal muscle cells and the skin can also be important reservoirs. This information 58 provides a framework for investigating how the parasite is able to survive as a life-long 59 infection, despite a vigorous immune response. It also informs drug-development 60 strategies by identifying tissue sites that must be accessed to achieve a curative 61 outcome.62 63 64 65 66 KEY WORDS: Trypanosoma cruzi, Chagas disease, chronic persistence, murine 67 imaging, colon, skeletal muscle, skin 68 69 70 71 72 73 74 4 INTRODUCTION The intracellular protozoan parasite Trypanosoma cruzi is the...

Digestive Chagas disease (DCD) is an enteric neuropathy caused by Trypanosoma cruzi infection. The mechanism of pathogenesis is poorly understood and the lack of a robust, predictive animal model has held back research. We screened a series of mouse models using gastrointestinal tracer assays and in vivo infection imaging systems to discover a subset exhibiting chronic digestive transit dysfunction and significant retention of faeces in both sated and fasted conditions. The colon was a specific site of both tissue parasite persistence, delayed transit and dramatic loss of myenteric neurons as revealed by whole-mount immunofluorescence analysis. DCD mice therefore recapitulated key clinical manifestations of human disease. We also exploited dual reporter transgenic parasites to home in on locations of rare chronic infection foci in the colon by ex vivo bioluminescence imaging and then used fluorescence imaging in tissue microdomains to reveal co-localisation of infection and enteric nervous system lesions. This indicates that long-term T. cruzi-host interactions in the colon drive DCD pathogenesis, suggesting that the efficacy of anti-parasitic chemotherapy against chronic disease progression warrants further pre-clinical investigation.

Selective negative allosteric modulators (NAMs), targeting α5 subunit-containing GABA A receptors (GABA A Rs) as potential therapeutic targets for disorders associated with cognitive deficits, including Alzheimer’s disease (AD), continually fail clinical trials. We investigated whether this was due to the change in the expression of α5 GABA A Rs, consequently altering synaptic function during AD pathogenesis. Using medicinal chemistry and computational modeling, we developed aqueous soluble hybrids of 6,6-dimethyl-3-(2-hydroxyethyl) thio-1-(thiazol-2-yl)-6,7-dihydro-2-benzothiophene-4(5H)-one, that demonstrated selective binding and high negative allosteric modulation, specifically for the α5 GABA A R subtypes in constructed HEK293 stable cell-lines. Using a knock-in mouse model of AD ( APP NL−F/NL−F ), which expresses a mutant form of human amyloid-β (Aβ), we performed immunofluorescence studies combined with electrophysiological whole-cell recordings to investigate the effects of our key molecule, α5-SOP002 in the hippocampal CA1 region. In aged APP NL−F/NL−F mice, selective preservation of α5 GABA A Rs was observed in, calretinin- (CR), cholecystokinin- (CCK), somatostatin- (SST) expressing interneurons, and pyramidal cells. Previously, we reported that CR dis-inhibitory interneurons, specialized in regulating other interneurons displayed abnormally high levels of synaptic inhibition in the APP NL−F/NL−F mouse model, here we show that this excessive inhibition was “normalized” to control values with bath-applied α5-SOP002 (1 μM). However, α5-SOP002, further impaired inhibition onto CCK and pyramidal cells that were already largely compromised by exhibiting a deficit of inhibition in the AD model. In summary, using a multi-disciplinary approach, we show that exposure to α5 GABA A R NAMs may further compromise aberrant synapses in AD. We, therefore, suggest that the α5 GABA A R is not a suitable therapeutic target for the treatment of AD or other cognitive deficits due to the widespread neuronal-networks that use α5 GABA A Rs.

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Digestive Chagas disease (DCD) is an enteric neuropathy caused by Trypanosoma cruzi infection. There is a lack of evidence on the mechanism of pathogenesis and rationales for treatment. We used a mouse model that recapitulates key clinical manifestations to study how infection dynamics shape DCD pathology, and the impact of treatment with the front-line drug benznidazole. Curative treatment at 6 weeks post-infection resulted in sustained recovery of GI transit function, whereas sub-curative treatment led to infection relapse and gradual return of DCD symptoms. Neuro-immune gene expression profiles shifted from chronic inflammation to a tissue repair signature after cure, accompanied by increased glial cell activity and regenerative neurogenesis in the myenteric neuronal plexus. Delaying treatment until 24 weeks post-infection led to a partial reversal of the DCD phenotype, suggesting the accumulation of permanent tissue damage over the course of chronic infection. Our study shows that murine DCD pathogenesis is sustained by chronic T. cruzi infection and is not an inevitable consequence of acute stage denervation. The risk that irreversible enteric neuromuscular tissue damage and dysfunction will develop highlights the importance of prompt diagnosis and treatment. Finally, these findings support the concept of treating asymptomatic T. cruzi infected individuals with benznidazole to prevent DCD development.

Gastrointestinal (GI) disease affects a substantial subset of chronic Chagas disease (CD) patients, but the mechanism of pathogenesis is poorly understood. The lack of a robust, predictive animal model of chronic T. cruzi infection that exhibits functional digestive disease has held back research. To address this, we combined GI tracer assays and bioluminescence in vivo infection imaging systems for diverse parasite strains to discover models exhibiting chronic digestive transit dysfunction. We identified the colon as a specific site of both tissue parasite persistence and delayed transit. Digestive CD mice exhibited significant retention of faeces in both sated and fasted conditions. Histological and immunofluorescence analysis of the enteric nervous system (ENS) revealed a dramatic reduction in the number of neurons and a loss of immunoreactivity of the enteric neural network in the colon. This model therefore recapitulates key clinical manifestations of human digestive CD. We also exploited dual bioluminescent-fluorescent parasites to analyse rare chronic infection foci in the colon at the single cell level, revealing co-localisation with ENS lesions. This indicates that long-term T. cruzi-host interactions in the colon drive pathogenesis and thus chronic disease may be preventable using anti-parasitic chemotherapy.