Patients and models of temporal lobe epilepsy have fewer inhibitory interneurons in the dentate gyrus than controls, but it is unclear whether granule cell inhibition is reduced. We report the loss of GABAergic inhibition of granule cells in the temporal dentate gyrus of pilocarpine-induced epileptic rats. In situ hybridization for GAD65 mRNA and immunocytochemistry for parvalbumin and somatostatin confirmed the loss of inhibitory interneurons. In epileptic rats, granule cells had prolonged EPSPs, and they discharged more action potentials than controls. Although the conductances of evoked IPSPs recorded in normal ACSF were not significantly reduced and paired-pulse responses showed enhanced inhibition of granule cells from epileptic rats, more direct measures of granule cell inhibition revealed significant deficiencies. In granule cells from epileptic rats, evoked monosynaptic IPSP conductances were Ͻ40% of controls, and the frequency of GABA A receptor-mediated spontaneous and miniature IPSCs (mIPSCs) was Ͻ50% of controls. Within 3-7 d after pilocarpine-induced status epilepticus, miniature IPSC frequency had decreased, and it remained low, without functional evidence of compensatory synaptogenesis by GABAergic axons in chronically epileptic rats. Both parvalbumin-and somatostatin-immunoreactive interneuron numbers and the frequency of both fast-and slow-rising GABA A receptor-mediated mIPSCs were reduced, suggesting that loss of inhibitory synaptic input to granule cells occurred at both proximal/somatic and distal/dendritic sites. Reduced granule cell inhibition in the temporal dentate gyrus preceded the onset of spontaneous recurrent seizures by days to weeks, so it may contribute, but is insufficient, to cause epilepsy.

f Clostridium difficile is the leading cause of infectious nosocomial diarrhea. The pathogenesis of C. difficile infection (CDI) results from the interactions between the pathogen, intestinal epithelium, host immune system, and gastrointestinal microbiota. Previous studies of the host-pathogen interaction in CDI have utilized either simple cell monolayers or in vivo models. While much has been learned by utilizing these approaches, little is known about the direct interaction of the bacterium with a complex host epithelium. Here, we asked if human intestinal organoids (HIOs), which are derived from pluripotent stem cells and demonstrate small intestinal morphology and physiology, could be used to study the pathogenesis of the obligate anaerobe C. difficile. Vegetative C. difficile, microinjected into the lumen of HIOs, persisted in a viable state for up to 12 h. Upon colonization with C. difficile VPI 10463, the HIO epithelium is markedly disrupted, resulting in the loss of paracellular barrier function. Since similar effects were not observed when HIOs were colonized with the nontoxigenic C. difficile strain F200, we directly tested the role of toxin using TcdA and TcdB purified from VPI 10463. We show that the injection of TcdA replicates the disruption of the epithelial barrier function and structure observed in HIOs colonized with viable C. difficile. C lostridium difficile is an anaerobic, spore-forming bacterium that is the leading cause of infectious nosocomial diarrhea and is responsible for over 14,000 deaths annually (1). Human exposure to C. difficile results in a range of manifestations, from asymptomatic colonization, to diarrhea, to lethal toxic megacolon. Various models have been used to study C. difficile infection (CDI), including in vitro models using transformed cell lines and a variety of in vivo models (2-5). In vitro cell culture models are limited in their ability to recapitulate complexities of the human gastrointestinal tract, and detailed, real-time study of the mucosal epithelium during infection in an animal model is technically challenging.Human intestinal organoids (HIOs) are three-dimensional spheroids of human epithelium generated through directed differentiation of human pluripotent stem cells (hPSCs), which include human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs). HIOs contain both mesenchymal and epithelial tissues that are structurally arranged around a central luminal cavity. The epithelial compartment of the HIO possesses an array of small intestinal cell types, including absorptive enterocytes and secretory Paneth, goblet, and enteroendocrine cells, in addition to Lgr5 ϩ intestinal stem cells (6). HIOs have been used to model features of embryonic development, viral infection, and inflammatory bowel disease (7-9). Due to their similarity to the human gastrointestinal tract, HIOs serve as a tractable and physiologically relevant model of the human intestine.We sought to use HIOs to study the interaction between C. difficile and complex human epit...

Temporal lobe epilepsy is the most common type of epilepsy in adults, and its underlying mechanisms are unclear. To investigate how the medial entorhinal cortex might contribute to temporal lobe epilepsy, we evaluated the histology and electrophysiology of slices from rats 3-7 d after an epileptogenic injury (pilocarpine-induced status epilepticus). Nissl staining, NeuN immunocytochemistry, and in situ hybridization for GAD65 mRNA were used to verify the preferential loss of glutamatergic neurons and the relative sparing of GABAergic interneurons in layer III. From slices adjacent to those that were used for anatomy, we obtained whole-cell patch recordings from layer II medial entorhinal cortical neurons. Recordings under current-clamp conditions revealed similar intrinsic electrophysiological properties (resting membrane potential, input resistance, single spike, and repetitive firing properties) to those of controls. Spontaneous IPSCs were less frequent (68% of controls), smaller in amplitude (57%), and transferred less charge (51%) than in controls. However, the frequency, amplitude, and rise time of miniature IPSCs were normal. These findings suggest that after epileptogenic injuries the layer II entorhinal cortical neurons receive less GABA A receptor-mediated synaptic input because presynaptic inhibitory interneurons become less active. To investigate the possible consequences of reduced spontaneous inhibitory input to layer II neurons, we recorded field potentials in the dentate gyrus, their major synaptic target. At 5 d after pilocarpine-induced status epilepticus the spontaneous field potentials recorded in vivo were over three times more frequent than in controls. These findings suggest that an epileptogenic injury reduces inhibition of layer II neurons and results in excessive synaptic input to the dentate gyrus.

In the study presented here, we aimed to describe the epidemiological, clinical and pathological findings of 51 canine cases with histologically-verified diagnoses of primary cardiac hemangiosarcoma (HSA). The medical data for each dog, including signalment, presenting complaints, physical examination findings, results of various diagnostic testing performed and method of treatment, were checked. In addition, all 51 cases were re-examined pathologically. The tumor occurred most frequently in older Golden Retrievers, followed by Maltese dogs and Miniature Dachshunds. Mass lesions of HSA were found more commonly in the right auricle (RAu) (25/51) and right atrium (RA) (21/51), and the RA masses were significantly (P<0.001) larger than the RAu masses. The echocardiographic detection rate of masses in the RAu group (60%; 15/25) was significantly lower than that in the RA group (95%; 20/21). Survival time was significantly (P<0.05) longer for 5 dogs that received adjuvant chemotherapy after tumor resection than for 12 dogs that did not. In this series, the Maltese (9/51) and Miniature Dachshund (7/51), as well as the Golden Retriever, were represented more frequently than other breeds. The lower echocardiographic detection rate of RAu masses compared with RA masses may be related to tumor size and/or location. The significantly longer survival time for dogs receiving adjuvant chemotherapy indicates that postoperative chemotherapy could be useful for dogs with cardiac HSA.