Luc Stoppini

A simple method for organotypic cultures of nervous tissue

Hippocampal slices prepared from 2-23-day-old neonates were maintained in culture at the interface between air and a culture medium. They were placed on a sterile, transparent and porous membrane and kept in petri dishes in an incubator. No plasma clot or roller drum were used. This method yields thin slices which remain 1-4 cell layers thick and are characterized by a well preserved organotypic organization. Pyramidal neurons labelled by extra- and intracellular application of horse radish peroxidase resemble by the organization and complexity of their dendritic processes those observed in situ at a comparable developmental stage. Excitatory and inhibitory synaptic potentials can easily be analysed using extra- or intracellular recording techniques. After a few days in culture, long-term potentiation of synaptic responses can reproducibly be induced. Evidence for a sprouting response during the first days in culture or following sections is illustrated. This technique may represent an interesting alternative to roller tube cultures for studies of the developmental changes occurring during the first days or weeks in culture.

Time course of synaptic development in hippocampal organotypic cultures

Using electrophysiological recordings of field potentials, we investigated the time course of synapse formation and maturation in organotypic cultures prepared from neonate animals of different ages. Following explanation, the size of the maximal synaptic responses elicited in area CA1 by stimulation of a small group of CA3 neurons increased progressively during the first three weeks in culture in a way that corresponded to the changes observed in synaptic contact density. Growth of synaptic responses was found to occur much more rapidly in cultures prepared from 8-day-old as compared with 2-day-old rats. Development of synaptic connections between CA3 and CA1 neurones was also faster than between granule cells and CA3 neurones. Acquisition of mature synaptic properties occurred in vitro as indicated by changes in degree of paired-pulse facilitation and the onset of long-term potentiation (LTP) after a few days in culture. The onset of LTP was much faster in cultures prepared from 8-day-old as compared with 2-day-old neonates and corresponded approximately to the 12-14th postnatal day. It is concluded that development proceeds in the cultures with a time course that resembles the in situ situation.

Structural modifications associated with synaptic development in area CA1 of rat hippocampal organotypic cultures

Using morphological techniques, we characterized the developmental reorganization that takes place during the first weeks after explanation in area CA1 of organotypic hippocampal cultures maintained at the interface between medium and a CO2-enriched atmosphere. Pyramidal neurones redistributed from a vertical into an horizontal cell layer in the middle of a three-dimensional culture, with apical dendrites running above the pyramidal layer. Glial cells redistributed into a thin layer at the bottom of the culture, forming an interface between tissue and culture medium. Astrocytes were identified as the most numerous non neuronal cells. No sign of glial proliferation could be observed, except for a transient increase during the first days after explanation. The density of synaptic contacts in the stratum radiatum decreased immediately after explanation and then increased by about 20-fold to reach values in the proximal part of the apical layer after 4 weeks in culture which were only slightly smaller than those measured in 1-month-old rats. The synaptic density in the most distal part of the dendritic layer which receives connections extrinsic to the hippocampus remained significantly lower than in vivo. The ratio of spine to shaft contacts was comparable to that found in vivo. These results indicate that interface type of organotypic cultures can be used as an interesting model for studies of synaptic development in vitro.

Lesion-induced neurite sprouting and synapse formation in hippocampal organotypic cultures

By sectioning, using a razor blade, one- and three-week-old rat hippocampal organotypic cultures, we have tested the possibility that neurite outgrowth and reactive synaptogenesis would take place even after several weeks in culture in this in vitro model. At the light-microscopic level, recovery from the section and formation of a thin scar were observed within six days following the lesion. Immunostainings using neurofilament antibodies showed the presence of numerous degenerative and regenerative images one day after the cut and many fibres crossing the section six days after the lesion. Electrophysiological recordings of synaptic responses elicited across the section indicated the formation of new functional synaptic contacts and complete recovery of transmission within three to six days. Interestingly, functional recovery in three-week-old cultures was found to be significantly slower than in one-week-old tissue. These findings were confirmed at the electron-microscopic level. Evidence was obtained for an effective cleaning of the lesion site by macrophages and astroglial cells, the existence of many degenerative and regenerative images one day after the cut and the presence of new dendrites, axonal fibres and synapses in the area of the section six days after the lesion. All these changes were slower in three- than in one-week-old cultures. These results indicate that organotypic cultures can be used as an interesting model for studies of reactive synaptogenesis.

A role for polysialylated neural cell adhesion molecule in lesion-induced sprouting in hippocampal organotypic cultures

By mediating cell-cell interactions, the neural cell adhesion molecule (N-CAM) has been implicated in various events such as axonal pathway formation, neurite outgrowth or synaptic remodelling. One mechanism by which N-CAM could contribute to these events has been proposed to involve modifications of the content of the molecule in polysialic acid. Here we have tested this possibility using an in vitro model of lesion-induced reactive synaptogenesis in hippocampal organotypic cultures. We present evidence that the sprouting reaction triggered by a section of CA3-CA1 connections in these cultures is associated with the expression of the highly sialylated form of N-CAM on regenerating neurites. In addition, we have examined the functional importance of this sialylation mechanism by analysing the effect of treating sectioned cultures with endo-neuraminidase-N which removes the polysialic acid portions of N-CAM. Measurements of the time course of recovery from the lesion, as assessed by the formation of new functional synaptic contacts across the section, showed that removal of the polysialic acid moieties of N-CAM significantly delays the sprouting reaction. The results support the idea that up regulations of highly sialylated forms of N-CAM are of functional importance in neurite sprouting and synapse regeneration in this in vitro model.

International STakeholder NETwork (ISTNET): creating a developmental neurotoxicity (DNT) testing road map for regulatory purposes

Abstract A major problem in developmental neurotoxicity (DNT) risk assessment is the lack of toxicological hazard information for most compounds. Therefore, new approaches are being considered to provide adequate experimental data that allow regulatory decisions. This process requires a matching of regulatory needs on the one hand and the opportunities provided by new test systems and methods on the other hand. Alignment of academically and industrially driven assay development with regulatory needs in the field of DNT is a core mission of the International STakeholder NETwork (ISTNET) in DNT testing. The first meeting of ISTNET was held in Zurich on 23–24 January 2014 in order to explore the concept of adverse outcome pathway (AOP) to practical DNT testing. AOPs were considered promising tools to promote test systems development according to regulatory needs. Moreover, the AOP concept was identified as an important guiding principle to assemble predictive integrated testing strategies (ITSs) for DNT. The recommendations on a road map towards AOP-based DNT testing is considered a stepwise approach, operating initially with incomplete AOPs for compound grouping, and focussing on key events of neurodevelopment. Next steps to be considered in follow-up activities are the use of case studies to further apply the AOP concept in regulatory DNT testing, making use of AOP intersections (common key events) for economic development of screening assays, and addressing the transition from qualitative descriptions to quantitative network modelling.

Live imaging of neural structure and function by fibred fluorescence microscopy

Only a few methods permit researchers to study selected regions of the central and peripheral nervous systems with a spatial and time resolution sufficient to image the function of neural structures. Usually, these methods cannot analyse deep‐brain regions and a high‐resolution method, which could repeatedly probe dynamic processes in any region of the central and peripheral nervous systems, is much needed. Here, we show that fibred fluorescence microscopy—which uses a small‐diameter fibre‐optic probe to provide real‐time images—has the spatial resolution to image various neural structures in the living animal, the consistency needed for a sequential, quantitative evaluation of axonal degeneration/regeneration of a peripheral nerve, and the sensitivity to detect calcium transients on a sub‐second timescale. These unique features should prove useful in many physiological studies requiring the in situ functional imaging of tissues in a living animal.

Interleukin-6 promotes sprouting and functional recovery in lesioned organotypic hippocampal slice cultures

Interleukin (IL)‐6 is a pro‐inflammatory cytokine now widely recognized to contribute to the molecular events that follow CNS injury. Little is known, however, about its action on axonal sprouting and regeneration in the brain. We addressed this issue using the model of transection of Schaffer collaterals in mice organotypic hippocampal slice cultures. Transection of slice cultures was associated with a marked release of IL‐6 that could be neutralized by an IL‐6 blocking antibody. We monitored functional recovery across the lesion by recording synaptic responses using a multi‐electrode array. We found that application of IL‐6 antibodies to the cultures after lesioning significantly reduced functional recovery across the lesion. Furthermore, the level of expression of the 43‐kDa growth‐associated protein (GAP‐43) was lower in slices treated with the IL‐6 neutralizing antibody than in those treated with a control IgG. Conversely, addition of exogenous IL‐6 to the culture medium resulted in a dose‐dependent enhancement of functional recovery across the lesion and a higher level of expression of GAP‐43. Co‐culture of CA3 hemi‐slices from thy1‐YFP mice with CA1 hemi‐slices from wild‐type animals confirmed that IL‐6‐treated co‐cultures exhibited an increased number of growing fluorescent fibres across the lesion site. Taken together these data indicate that IL‐6 plays an important role in CNS repair mechanisms by promoting regrowth and axon regeneration.

Development of Human Nervous Tissue upon Differentiation of Embryonic Stem Cells in Three‐Dimensional Culture

Researches on neural differentiation using embryonic stem cells (ESC) require analysis of neurogenesis in conditions mimicking physiological cellular interactions as closely as possible. In this study, we report an air‐liquid interface‐based culture of human ESC. This culture system allows three‐dimensional cell expansion and neural differentiation in the absence of added growth factors. Over a 3‐month period, a macroscopically visible, compact tissue developed. Histological coloration revealed a dense neural‐like neural tissue including immature tubular structures. Electron microscopy, immunochemistry, and electrophysiological recordings demonstrated a dense network of neurons, astrocytes, and oligodendrocytes able to propagate signals. Within this tissue, tubular structures were niches of cells resembling germinal layers of human fetal brain. Indeed, the tissue contained abundant proliferating cells expressing markers of neural progenitors. Finally, the capacity to generate neural tissues on air‐liquid interface differed for different ESC lines, confirming variations of their neurogenic potential. In conclusion, this study demonstrates in vitro engineering of a human neural‐like tissue with an organization that bears resemblance to early developing brain. As opposed to previously described methods, this differentiation (a) allows three‐dimensional organization, (b) yields dense interconnected neural tissue with structurally and functionally distinct areas, and (c) is spontaneously guided by endogenous developmental cues. STEM CELLS 2009;27:509–520

Neutralization of the membrane protein Nogo-A enhances growth and reactive sprouting in established organotypic hippocampal slice cultures.

The reduced ability of central axons to regenerate after injury is significantly influenced by the presence of several molecules that inhibit axonal growth. Nogo‐A is one of the most studied and most potent of the myelin‐associated growth inhibitory molecules. Its neutralization, as well as interference with its signalling, allows for enhanced axonal sprouting and growth following injury. Using differentiated rat organotypic hippocampal slice cultures treated for 5 days with either of two different function‐blocking anti‐Nogo‐A antibodies, we show an increase in CA3 fibre regeneration after lesion. In intact slices, 5 days of anti‐Nogo‐A antibody treatment led to increased sprouting of intact CA3 fibres that are positive for neurofilament 68. A transcriptomic approach confirmed the occurrence of a growth response on the molecular level upon Nogo‐A neutralization in intact cultures. Our results demonstrate that Nogo‐A neutralization for 5 days is sufficient for the induction of growth in mature CNS tissue without the prerequisite of an injury. Nogo‐A may therefore act as a tonic growth suppressor/stabilizer in the adult intact hippocampus.