In a recent study, scientists from the Indian Institute of Science Education and Research (IISER) Pune, Raman Research Institute (RRI), Bangalore, and University of Hyderabad developed a simple assay to show that neurons are shaped by attachment to the substrate underneath them.
In certain respects, neurons behave similar to a bloodhound searching for a scent. Long projections of nerve cells, called axons, traverse through tissues to find partners with whom they can form synapses. As these axons travel, neurons often experience pulling forces due to the normal growth of tissues. Such pulling forces can force axons to elongate and shorten, shaping the network. “The sciatic nerve can experience local stretch of up to 30% during regular limb movement,” says Aurnab Ghose, Associate Professor at IISER Pune and the corresponding author of the study.
Neurons have a property called “prestress”. Try cutting a taut rubber band – it breaks and instantly rebounds. This taut rubber band is said to have “prestress” or pre-existing stress, even before an external load is applied. “Maintenance of prestress is critical for signaling in neurons,” says Ghose, “Imagine a rope held between two points. If the rope is taut, pulling one end results in an instantaneous response at the other end. If the rope is slack, then the propagation of the stress is damped.”
How do cells get prestressed? Two major cytoskeletal structures inside neurons are actin and microtubules. Actin is 7 – 10 nm thick, usually present in bundles of 10 – 300 actin filaments, and can contract with the help of other proteins inside cell. The pre-stressed state of a cell arises from the ‘contractility’ of the actin fibers. Microtubules are thick (~50 nm) hollow tubular structures which act like struts inside a cell to resist compression. These two structures together maintain and support the cell structure.
Previous studies to test prestress in neurons have employed harsh methods, using microneedles or lasers to cut the neurons which make it difficult to distinguish if the effects observed are due to the prestress or physical damage to neurons.
In the present study, the researchers use two novel non-invasive methods to test contractility of neurons. In one method, they used an enzyme (Trypsin) which dissolves proteins that connect a neuron to its substrate. In the second method, they used a patterned substrate where instead of a continuous layer, the substrate consists of isolated islands of extracellular matrix proteins.
The researchers isolated and grew neurons from chick embryos on plates coated with poly-D-lysine substrate. When they treated the neurons with Trypsin , the neurons reduced in length and transformed from curved to straight within just 10 – 20 seconds. This suggests that neurons naturally contract and try to achieve minimum length, unless they are opposed by attachments to the underlying substrate. They also found that this was dependent on actin, not microtubules.
Ghose’s team found that different neurons respond differently to detachment from the substrate – with some neurons shortening at a faster rate compared to others. There were also differences in contractility within a single neuron. The authors propose that this could arise from internal differences in contractility. To understand this, try cutting a few rubber bands with different elasticities – each will rebound differently.
Apart from signaling, contractility in neurons can also contribute to patterning of the brain. “Contractility has been linked to buckling of membranes, resulting in the creation of folds, which are characteristic of the cortex, and can lead to patterning in the brain,” says Namrita Gundiah, Associate Professor at Indian Institute of Science (IISc, Bangalore) who was not associated with the study. She adds, “This study addresses a fundamental question of how contractility in neurons influences neuron networks and patterning in the brain using a simple and elegant method”.