Songbird organotypic culture as an in vitro model for interrogating sparse sequencing networks

Sparse sequences of neuronal activity are fundamental features of neural circuit computation; however, the underlying homeostatic mechanisms remain poorly understood. To approach these questions, we have developed a method for cellular-resolution imaging in organotypic cultures of the adult zebra finch brain, including portions of the intact song circuit. These in vitro networks can survive for weeks, and display mature neuron morphologies. Neurons within the organotypic slices exhibit a diversity of spontaneous and pharmacologically induced activity that can be easily monitored using the genetically encoded calcium indicator GCaMP6. In this study, we primarily focus on the classic song sequence generator HVC and the surrounding areas. We describe proof of concept experiments including physiological, optical, and pharmacological manipulation of these exposed networks. This method may allow the cellular rules underlying sparse, stereotyped neural sequencing to be examined with new degrees of experimental control

Axonal regeneration in hippocampal and spinal cord organotypic slice cultures

Under normal conditions, axonal regeneration after lesions is not possible in mature CNS but can occur in embryonic and early postnatal nervous systems. In recent years, a number of possible strategies to enhance axonal regeneration and eventually treat spinal cord and brain injuries have been identified, some of which have been used successfully in animal experiments, but till now there is still no successful treatment available for patients. This problem is partly due to the complexity of the animal experiments which makes it difficult to compare different treatment strategies. In this project, we have used organotypic slice culture models to test the effectiveness of pharmacological compounds that interfere with various signal transduction mechanisms, to promote axonal regeneration. We used the entorhino hippocampal slice cultures to assess regeneration of entorhinal fibers projecting to the dentate gyrus after mechanical lesions and treatment. It was previously shown (Prang et. al., 2001) that there is a marked decrease in regenerating fibers when a lesion is made at 6 7 days in vitro or later in slices derived from postnatal day 5 6 mice. We took this as a control model where there is little spontaneous axonal regeneration, added treatments on the day of lesion and later traced for entorhinal axons with biotinylated dextran amine (BDA). In this study it was shown that compounds acting on the cAMP, PKC and G proteins can promote regeneration. Furthermore, we have identified the inhibition of the PI3 kinase pathway and the IP 3 receptor as potential drug targets that promote axonal regeneration. In order to study axonal growth in a spinal cord environment we have developed a spinal cord longitudinal organotypic slice culture model which allowed us to follow axons along the rostro caudal extension of the spinal cord. Slices of cervical spinal cord were cut in the sagittal plane from early postnatal mice and were maintained in culture for various time periods up to 4 weeks. Histological and immunohistochemical stainings of the cultures have shown that these slice cultures maintain the ventro dorsal polarity of the spinal cord and that an intrinsic fibre projection develops which runs along the rostro caudal extension of the spinal cord slice culture. After mechanical lesion, these fibres have the ability to regenerate spontaneously demonstrating the intrinsic ability of the spinal cord for repair, but this ability is decreased with increasing time in culture. During the culture period the axons became myelinated and expressed synaptic markers. These cultures could thus serve also as a model for myelin formation and synaptogenesis. We have analyzed the potential of axons from longitudinal spinal cord cultures to grow into an adjacent slice of cerebellar tissue. We could show that spinal cord axons do enter the cerebellar slice in particular when early postnatal spinal cord is combined with postnatal cerebellum. Pharmacological treatments were used to enhance axonal growth. Similarly to our findings in the entorhino hippocampal model, cAMP activators and PKC inhibitors promoted axonal growth from the spinal cord to the cerebellum. In cocultures of longitudinal spinal cord slices with cortical slices we have shown that fibers from the cortical slices grew extensively into the spinal cord slice and extended caudally for substantial distances. Our results demonstrate that organotypic slice cultures can be a useful tool to study axonal growth and regeneration. Intrinsic spinal cord axons have a considerable potential for spontaneous regeneration in the early postnatal period and are able to grow both through a mechanical lesion and into another tissue. Moreover, compounds interfering with signal transduction mechanisms, particularly cAMP, PKC, PI3 Kinase, G proteins and IP3 receptors, were able to promote axonal growth and regeneration in diverse slice culture models making them interesting drug candidates for the promotion of axonal regeneration

Automated four-dimensional long term imaging enables single cell tracking within organotypic brain slices to study neurodevelopment and degeneration.

Current approaches for dynamic profiling of single cells rely on dissociated cultures, which lack important biological features existing in tissues. Organotypic slice cultures preserve aspects of structural and synaptic organisation within the brain and are amenable to microscopy, but established techniques are not well adapted for high throughput or longitudinal single cell analysis. Here we developed a custom-built, automated confocal imaging platform, with improved organotypic slice culture and maintenance. The approach enables fully automated image acquisition and four-dimensional tracking of morphological changes within individual cells in organotypic cultures from rodent and human primary tissues for at least 3 weeks. To validate this system, we analysed neurons expressing a disease-associated version of huntingtin (HTT586Q138-EGFP), and observed that they displayed hallmarks of Huntington's disease and died sooner than controls. By facilitating longitudinal single-cell analyses of neuronal physiology, our system bridges scales necessary to attain statistical power to detect developmental and disease phenotypes

Modeling inflammation and oxidative stress in gastrointestinal disease development using novel organotypic culture systems.

Gastroesophageal reflux disease (GERD), Barrett's esophagus (BE), graft-versus-host disease (GVHD), and inflammatory bowel diseases such as ulcerative colitis and Crohn's disease are common human gastrointestinal diseases that share inflammation as a key driver for their development. A general outcome resulting from these chronic inflammatory conditions is increased oxidative stress. Oxidative stress is caused by the generation of reactive oxygen and nitrogen species that are part of the normal inflammatory response, but are also capable of damaging cellular DNA, protein, and organelles. Damage to DNA can include DNA strand breaks, point mutations due to DNA adducts, as well as alterations in methylation patterns leading to activation of oncogenes or inactivation of tumor suppressors. There are a number of significant long-term consequences associated with chronic oxidative stress, most notably cancer. Infiltrating immune cells and stromal components of tissue including fibroblasts contribute to dynamic changes occurring in tissue related to disease development. Immune cells can potentiate oxidative stress, and fibroblasts have the capacity to contribute to advanced growth and proliferation of the epithelium and any resultant cancers. Disease models for GERD, BE, GVHD, and ulcerative colitis based on three-dimensional human cell and tissue culture systems that recapitulate in vivo growth and differentiation in inflammatory-associated microphysiological environments would enhance our understanding of disease progression and improve our ability to test for disease-prevention strategies. The development of physiologically relevant, human cell-based culture systems is therefore a major focus of our research. These novel models will be of enormous value, allowing us to test hypotheses and advance our understanding of these disorders, and will have a translational impact allowing us to more rapidly develop therapeutic and chemopreventive agents. In summary, this work to develop advanced human cell-based models of inflammatory conditions will greatly improve our ability to study, prevent, and treat GERD, BE, GVHD, and inflammatory bowel disease. The work will also foster the development of novel therapeutic and preventive strategies that will improve patient care for these important clinical conditions

Expression of the FGFR2c mesenchymal splicing variant in human keratinocytes inhibits differentiation and promotes invasion

The altered isoform switching of the fibroblast growth factor receptor 2 (FGFR2) and aberrant expression of the mesenchymal FGFR2c isoform in epithelial cells is involved in cancer progression. We have recently described that the ectopic expression of FGFR2c in normal human keratinocytes induces epithelial-mesenchymal transition and leads to invasiveness and anchorage-independent growth. Here, we extended our analysis to the effects of this FGFR2c forced expression on human keratinocyte differentiation and stratification. Our findings demonstrated that, differently from cells overexpressing the epithelial splicing variant FGFR2b, keratinocytes ectopically expressing FGFR2c are not able to form a monolayer and display decreased expression of early differentiation markers. This impaired ability to enter the differentiation program is related to the up-modulation of the transcription factor ΔNp63. In addition, FGFR2c-expressing keratinocytes undergo defective stratification and invasion of the collagen matrix in 3D organotypic cultures, further suggesting their tumorigenic potential. Taken together, our results support the hypothesis that the receptor switching and the consequent appearance of the mesenchymal FGFR2c variant in the epithelial context would drive early steps of carcinogenesis, unbalancing the p63/FGFR interplay, and altering the paracrine response to the microenvironment

A biomimetic pancreatic cancer on-chip reveals endothelial ablation via ALK7 signaling

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive, lethal malignancy that invades adjacent vasculatures and spreads to distant sites before clinical detection. Although invasion into the peripancreatic vasculature is one of the hallmarks of PDAC, paradoxically, PDAC tumors also exhibit hypovascularity. How PDAC tumors become hypovascular is poorly understood. We describe an organotypic PDAC-on-a-chip culture model that emulates vascular invasion and tumor-blood vessel interactions to better understand PDAC-vascular interactions. The model features a 3D matrix containing juxtaposed PDAC and perfusable endothelial lumens. PDAC cells invaded through intervening matrix, into vessel lumen, and ablated the endothelial cells, leaving behind tumor-filled luminal structures. Endothelial ablation was also observed in in vivo PDAC models. We also identified the activin-ALK7 pathway as a mediator of endothelial ablation by PDAC. This tumor-on-a-chip model provides an important in vitro platform for investigating the process of PDAC-driven endothelial ablation and may provide a mechanism for tumor hypovascularity.R01 EB000262 - NIBIB NIH HHS; TL1 TR001410 - NCATS NIH HHS; UC4 DK104196 - NIDDK NIH HHS; UH3 EB017103 - NIBIB NIH HHSPublished versio

Cold plasma-treated ringer’s saline: a weapon to target osteosarcoma

Osteosarcoma (OS) is the main primary bone cancer, presenting poor prognosis and difficult treatment. An innovative therapy may be found in cold plasmas, which show anti-cancer effects related to the generation of reactive oxygen and nitrogen species in liquids. In vitro models are based on the effects of plasma-treated culture media on cell cultures. However, effects of plasma-activated saline solutions with clinical application have not yet been explored in OS. The aim of this study is to obtain mechanistic insights on the action of plasma-activated Ringer’s saline (PAR) for OS therapy in cell and organotypic cultures. To that aim, cold atmospheric plasma jets were used to obtain PAR, which produced cytotoxic e ects in human OS cells (SaOS-2, MG-63, and U2-OS), related to the increasing concentration of reactive oxygen and nitrogen species generated. Proof of selectivity was found in the sustained viability of hBM-MSCs with the same treatments. Organotypic cultures of murine OS confirmed the time-dependent cytotoxicity observed in 2D. Histological analysis showed a decrease in proliferating cells (lower Ki-67 expression). It is shown that the selectivity of PAR is highly dependent on the concentrations of reactive species, being the differential intracellular reactive oxygen species increase and DNA damage between OS cells and hBM-MSCs key mediators for cell apoptosis.Peer ReviewedPostprint (published version

Mouse Panx1 Is Dispensable for Hearing Acquisition and Auditory Function

Panx1 forms plasma membrane channels in brain and several other organs, including the inner ear. Biophysical properties, activation mechanisms and modulators of Panx1 channels have been characterized in detail, however the impact of Panx1 on auditory function is unclear due to conflicts in published results. To address this issue, hearing performance and cochlear function of the Panx1−/− mouse strain, the first with a reported global ablation of Panx1, were scrutinized. Male and female homozygous (Panx1−/−), hemizygous (Panx1+/−) and their wild type (WT) siblings (Panx1+/+) were used for this study. Successful ablation of Panx1 was confirmed by RT-PCR and Western immunoblotting in the cochlea and brain of Panx1−/− mice. Furthermore, a previously validated Panx1-selective antibody revealed strong immunoreactivity in WT but not in Panx1−/− cochleae. Hearing sensitivity, outer hair cell-based “cochlear amplifier” and cochlear nerve function, analyzed by auditory brainstem response (ABR) and distortion product otoacoustic emission (DPOAE) recordings, were normal in Panx1+/− and Panx1−/− mice. In addition, we determined that global deletion of Panx1 impacts neither on connexin expression, nor on gap-junction coupling in the developing organ of Corti. Finally, spontaneous intercellular Ca2+ signal (ICS) activity in organotypic cochlear cultures, which is key to postnatal development of the organ of Corti and essential for hearing acquisition, was not affected by Panx1 ablation. Therefore, our results provide strong evidence that, in mice, Panx1 is dispensable for hearing acquisition and auditory function

Akt-dependent Pp2a activity is required for epidermal barrier formation during late embryonic development

Acquisition of epidermal barrier function occurs late in mouse gestation. Several days before birth a wave of barrier acquisition sweeps across murine fetal skin, converging on dorsal and ventral midlines. We investigated the molecular pathways active during epidermal barrier formation. Akt signaling increased as the barrier wave crossed epidermis and Jun was transiently dephosphorylated. Inhibitor experiments on embryonic explants showed that the dephosphorylation of Jun was dependent on both Akt and protein phosphatase 2A (Pp2a). Inhibition of Pp2a and Akt signaling also caused defects in epidermal barrier formation. These data are compatible with a model for developmental barrier acquisition mediated by Pp2a regulation of Jun dephosphorylation, downstream of Akt signaling. Support for this model was provided by siRNA-mediated knockdown of Ppp2r2a (Pr55α or B55α), a regulatory subunit of Pp2a expressed in an Akt-dependent manner in epidermis during barrier formation. Ppp2r2a reduction caused significant increase in Jun phosphorylation and interfered with the acquisition of barrier function, with barrier acquisition being restored by inhibition of Jun phosphorylation. Our data provide strong evidence that Ppp2r2a is a regulatory subunit of Pp2a that targets this phosphatase to Jun, and that Pp2a action is necessary for barrier formation. We therefore describe a novel Akt-dependent Pp2a activity that acts at least partly through Jun to affect initial barrier formation during late embryonic epidermal development

Mesenchymal Stem Cells Promote Oligodendroglial Differentiation in Hippocampal Slice Cultures

We have previously shown that soluble factors derived from mesenchymal stem cells (MSCs) induce oligodendrogenic fate and differentiation in adult rat neural progenitors (NPCs) in vitro. Here, we investigated if this pro-oligodendrogenic effect is maintained after cells have been transplanted onto rat hippocampal slice cultures, a CNS-organotypic environment. We first tested whether NPCs, that were pre-differentiated in vitro by MSC-derived conditioned medium, would generate oligodendrocytes after transplantation. This approach resulted in the loss of grafted NPCs, suggesting that oligodendroglial pre-differentiated cells could not integrate in the tissue and therefore did not survive grafting. However, when NPCs together with MSCs were transplanted in situ into hippocampal slice cultures, the grafted NPCs survived and the majority of them differentiated into oligodendrocytes. In contrast to the prevalent oligodendroglial differentiation in case of the NPC/MSC co-transplantation, naive NPCs transplanted in the absence of MSCs differentiated predominantly into astrocytes. In summary, the pro-oligodendrogenic activity of MSCs was maintained only after co-transplantation into hippocampal slice cultures. Therefore, in the otherwise astrogenic milieu, MSCs established an oligodendrogenic niche for transplanted NPCs, and thus, co-transplantation of MSCs with NPCs might provide an attractive approach to re-myelinate the various regions of the diseased CNS. Copyright (C) 2009 S. Karger AG, Base