Optimization of a Neurotoxin to Investigate the Contribution of Excitatory Interneurons to Speed Modulation In Vivo.

Precise control of speed during locomotion is essential for adaptation of behavior in different environmental contexts [1-4]. A central question in locomotion lies in understanding which neural populations set locomotor frequency during slow and fast regimes. Tackling this question in vivo requires additional non-invasive tools to silence large populations of neurons during active locomotion. Here we generated a stable transgenic line encoding a zebrafish-optimized botulinum neurotoxin light chain fused to GFP (BoTxBLC-GFP) to silence synaptic output over large populations of motor neurons or interneurons while monitoring active locomotion. By combining calcium imaging, electrophysiology, optogenetics, and behavior, we show that expression of BoTxBLC-GFP abolished synaptic release while maintaining characterized activity patterns and without triggering off-target effects. As chx10(+) V2a interneurons (V2as) are well characterized as the main population driving the frequency-dependent recruitment of motor neurons during fictive locomotion [5-14], we validated our silencing method by testing the effect of silencing chx10(+) V2as during active and fictive locomotion. Silencing of V2as selectively abolished fast locomotor frequencies during escape responses. In addition, spontaneous slow locomotion occurred less often and at frequencies lower than in controls. Overall, this silencing approach confirms that V2a excitation is critical for the production of fast stimulus-evoked swimming and also reveals a role for V2a excitation in the production of slower spontaneous locomotor behavior. Altogether, these results establish BoTxBLC-GFP as an ideal tool for in vivo silencing for probing the development and function of neural circuits from the synaptic to the behavioral level.

Tensile Forces Originating from Cancer Spheroids Facilitate Tumor Invasion.

The mechanical properties of tumors and the tumor environment provide important information for the progression and characterization of cancer. Tumors are surrounded by an extracellular matrix (ECM) dominated by collagen I. The geometrical and mechanical properties of the ECM play an important role for the initial step in the formation of metastasis, presented by the migration of malignant cells towards new settlements as well as the vascular and lymphatic system. The extent of this cell invasion into the ECM is a key medical marker for cancer prognosis. In vivo studies reveal an increased stiffness and different architecture of tumor tissue when compared to its healthy counterparts. The observed parallel collagen organization on the tumor border and radial arrangement at the invasion zone has raised the question about the mechanisms organizing these structures. Here we study the effect of contractile forces originated from model tumor spheroids embedded in a biomimetic collagen I matrix. We show that contractile forces act immediately after seeding and deform the ECM, thus leading to tensile radial forces within the matrix. Relaxation of this tension via cutting the collagen does reduce invasion, showing a mechanical relation between the tensile state of the ECM and invasion. In turn, these results suggest that tensile forces in the ECM facilitate invasion. Furthermore, simultaneous contraction of the ECM and tumor growth leads to the condensation and reorientation of the collagen at the spheroid's surface. We propose a tension-based model to explain the collagen organization and the onset of invasion by forces originating from the tumor.