Despite recent progress in fluorescence microscopy techniques, electron microscopy (EM) is still superior in the simultaneous analysis of all tissue components at high resolution. However, it is unclear to what extent conventional fixation for EM using aldehydes results in tissue alteration. Here we made an attempt to minimize tissue alteration by using rapid high-pressure freezing (HPF) of hippocampal slice cultures. We used this approach to monitor fine-structural changes at hippocampal mossy fiber synapses associated with chemically induced long-term potentiation (LTP). Synaptic plasticity in LTP has been known to involve structural changes at synapses including reorganization of the actin cytoskeleton and de novo formation of spines. While LTP-induced formation and growth of postsynaptic spines have been reported, little is known about associated structural changes in presynaptic boutons. Mossy fiber synapses are assumed to exhibit presynaptic LTP expression and are easily identified by EM. In slice cultures from wildtype mice, we found that chemical LTP increased the length of the presynaptic membrane of mossy fiber boutons, associated with a de novo formation of small spines and an increase in the number of active zones. Of note, these changes were not observed in slice cultures from Munc13-1 knockout mutants exhibiting defective vesicle priming. These findings show that activation of hippocampal mossy fibers induces pre- and postsynaptic structural changes at mossy fiber synapses that can be monitored by EM.

BackgroundMammary gland morphogenesis involves ductal elongation, branching, and budding. All of these processes are mediated by stroma - epithelium interactions. Biomechanical factors, such as matrix stiffness, have been established as important factors in these interactions. For example, epithelial cells fail to form normal acinar structures in vitro in 3D gels that exceed the stiffness of a normal mammary gland. Additionally, heterogeneity in the spatial distribution of acini and ducts within individual collagen gels suggests that local organization of the matrix may guide morphogenesis. Here, we quantified the effects of both bulk material stiffness and local collagen fiber arrangement on epithelial morphogenesis.ResultsThe formation of ducts and acini from single cells and the reorganization of the collagen fiber network were quantified using time-lapse confocal microscopy. MCF10A cells organized the surrounding collagen fibers during the first twelve hours after seeding. Collagen fiber density and alignment relative to the epithelial surface significantly increased within the first twelve hours and were a major influence in the shaping of the mammary epithelium. The addition of Matrigel to the collagen fiber network impaired cell-mediated reorganization of the matrix and increased the probability of spheroidal acini rather than branching ducts. The mechanical anisotropy created by regions of highly aligned collagen fibers facilitated elongation and branching, which was significantly correlated with fiber organization. In contrast, changes in bulk stiffness were not a strong predictor of this epithelial morphology.ConclusionsLocalized regions of collagen fiber alignment are required for ductal elongation and branching suggesting the importance of local mechanical anisotropy in mammary epithelial morphogenesis. Similar principles may govern the morphology of branching and budding in other tissues and organs.

Erwin Schrödinger pointed out in his 1944 book “What is Life” that one defining attribute of biological systems seems to be their tendency to generate order from disorder defying the second law of thermodynamics. Almost parallel to his findings, the science of complex systems was founded based on observations on physical and chemical systems showing that inanimate matter can exhibit complex structures although their interacting parts follow simple rules. This is explained by a process known as self-organization and it is now widely accepted that multi-cellular biological organisms are themselves self-organizing complex systems in which the relations among their parts are dynamic, contextual and interdependent. In order to fully understand such systems, we are required to computationally and mathematically model their interactions as promulgated in systems biology. The preponderance of network models in the practice of systems biology inspired by a reductionist, bottom-up view, seems to neglect, however, the importance of bidirectional interactions across spatial scales and domains. This approach introduces a shortcoming that may hinder research on emergent phenomena such as those of tissue morphogenesis and related diseases, such as cancer. Another hindrance of current modeling attempts is that those systems operate in a parameter space that seems far removed from biological reality. This misperception calls for more tightly coupled mathematical and computational models to biological experiments by creating and designing biological model systems that are accessible to a wide range of experimental manipulations. In this way, a comprehensive understanding of fundamental processes in normal development or of aberrations, like cancer, will be generated.

Angiogenic sprouts at the leading edge of an expanding vascular plexus are recognised as major regulators of the structure of the developing network. Early in sprout development, a vascular lumen is often evident which communicates with the parent vessel while the distal tip is blind-ended. Here we describe the temporal evolution of blind-ended vessels (BEVs) in a small wound made in the panniculus carnosus muscle of a mouse viewed in a dorsal skin-fold window-chamber model with intra-vital microscopy during the most active period of angiogenesis (days 5-8 after injury). Although these structures have been mentioned anecdotally in previous studies, we observed BEVs to be frequent, albeit transient, features of plexus formation. Plasma leakage into the surrounding extracellular matrix occurring from these immature conduits could play an important role in preparing hypoxic tissue for vascular invasion. Although sprout growth is likely to be regulated by its flow environment, the parameters regulating flow into and through BEVs have not been characterised in situ. Longitudinal data from individual animals show that the number of BEVs filled with plasma alone peaks at day 7, when they can exceed 150 μm in length. Additionally, BEVs greater than 40 μm in length are more likely to be filled with stationary erythrocytes than with plasma alone. Using a mathematical model, we show how the flux of 150kD fluorinated (FITC-) dextran through an individual plasma-filled BEV is related to its geometry being determined primarily by its surface area; by fitting theoretical intensity values to experimental data we assess the permeability of the vessel to FITC-dextran. Plasma skimming provides a mechanistic explanation for the observation that BEVs with larger surface area are more likely to recruit erythrocytes.

The granule cells of the dentate gyrus give rise to thin unmyelinated axons, the mossy fibers. They form giant presynaptic boutons impinging on large complex spines on the proximal dendritic portions of hilar mossy cells and CA3 pyramidal neurons. While these anatomical characteristics have been known for some time, it remained unclear whether functional changes at mossy fiber synapses such as long-term potentiation (LTP) are associated with structural changes. Since subtle structural changes may escape a fine-structural analysis when the tissue is fixed by using aldehydes and is dehydrated in ethanol, rapid high-pressure freezing (HPF) of the tissue was applied. Slice cultures of hippocampus were prepared and incubated in vitro for 2 weeks. Then, chemical LTP (cLTP) was induced by the application of 25 mM tetraethylammonium (TEA) for 10 min. Whole-cell patch-clamp recordings from CA3 pyramidal neurons revealed a highly significant potentiation of mossy fiber synapses when compared to control conditions before the application of TEA. Next, the slice cultures were subjected to HPF, cryosubstitution, and embedding in Epon for a fine-structural analysis. When compared to control tissue, we noticed a significant decrease of synaptic vesicles in mossy fiber boutons and a concomitant increase in the length of the presynaptic membrane. On the postsynaptic side, we observed the formation of small, finger-like protrusions, emanating from the large complex spines. These short protrusions gave rise to active zones that were shorter than those normally found on the thorny excrescences. However, the total number of active zones was significantly increased. Of note, none of these cLTP-induced structural changes was observed in slice cultures from Munc13-1 deficient mouse mutants showing severely impaired vesicle priming and docking. In conclusion, application of HPF allowed us to monitor cLTP-induced structural reorganization of mossy fiber synapses.