Rewiring Neuronal Circuits: A New Method for Fast Neurite Extension and Functional Neuronal Connection.

Brain and spinal cord injury may lead to permanent disability and death because it is still not possible to regenerate neurons over long distances and accurately reconnect them with an appropriate target. Here a procedure is described to rapidly initiate, elongate, and precisely connect new functional neuronal circuits over long distances. The extension rates achieved reach over 1.2 mm/h, 30-60 times faster than the in vivo rates of the fastest growing axons from the peripheral nervous system (0.02 to 0.04 mm/h)28 and 10 times faster than previously reported for the same neuronal type at an earlier stage of development4. First, isolated populations of rat hippocampal neurons are grown for 2-3 weeks in microfluidic devices to precisely position the cells, enabling easy micromanipulation and experimental reproducibility. Next, beads coated with poly-D-lysine (PDL) are placed on neurites to form adhesive contacts and pipette micromanipulation is used to move the resulting bead-neurite complex. As the bead is moved, it pulls out a new neurite that can be extended over hundreds of micrometers and functionally connected to a target cell in less than 1 h. This process enables experimental reproducibility and ease of manipulation while bypassing slower chemical strategies to induce neurite growth. Preliminary measurements presented here demonstrate a neuronal growth rate far exceeding physiological ones. Combining these innovations allows for the precise establishment of neuronal networks in culture with an unprecedented degree of control. It is a novel method that opens the door to a plethora of information and insights into signal transmission and communication within the neuronal network as well as being a playground in which to explore the limits of neuronal growth. The potential applications and experiments are widespread with direct implications for therapies that aim to reconnect neuronal circuits after trauma or in neurodegenerative diseases.

Reversing adhesion with light: a general method for functionalized bead release from cells.

Coated beads retain great importance in the study of cell adhesion and intracellular communication; we present a generally applicable method permitting spatiotemporal control of bead adhesion from cells. Herein we demonstrate in vitro release of a poly-d-lysine (PDL) layer from anionic polystyrene beads, allowing complete bead release from rat cortical neurons post-adhesion.

Rapid Mechanically Controlled Rewiring of Neuronal Circuits.

CNS injury may lead to permanent functional deficits because it is still not possible to regenerate axons over long distances and accurately reconnect them with an appropriate target. Using rat neurons, microtools, and nanotools, we show that new, functional neurites can be created and precisely positioned to directly (re)wire neuronal networks. We show that an adhesive contact made onto an axon or dendrite can be pulled to initiate a new neurite that can be mechanically guided to form new synapses at up to 0.8 mm distance in <1 h. Our findings challenge current understanding of the limits of neuronal growth and have direct implications for the development of new therapies and surgical techniques to achieve functional regeneration. Significance statement: Brain and spinal cord injury may lead to permanent disability and death because it is still not possible to regenerate neurons over long distances and accurately reconnect them with an appropriate target. Using microtools and nanotools we have developed a new method to rapidly initiate, elongate, and precisely connect new functional neuronal circuits over long distances. The extension rates achieved are ≥60 times faster than previously reported. Our findings have direct implications for the development of new therapies and surgical techniques to achieve functional regeneration after trauma and in neurodegenerative diseases. It also opens the door for the direct wiring of robust brain-machine interfaces as well as for investigations of fundamental aspects of neuronal signal processing and neuronal function.

Local membrane deformation and micro-injury lead to qualitatively different responses in osteoblasts.

Micro-damage of bone tissue is known to regulate bone turnover. However, it is unknown if individual bone cells can differentiate between membrane deformation and micro-injury. We generated osteoblasts from mouse bone marrow or bone morphogenetic protein 2-transfected C2C12 cells. Single cells were mechanically stimulated by indentation with the atomic force microscopy probe with variable force load either resulting in membrane deformation only, or leading to membrane penetration and micro-injury. Changes in the cytosolic free calcium concentration ([Ca (2+)] i) in fluo4-AM loaded cells were analyzed. When deformation only was induced, it resulted in an immediate elevation of [Ca (2+)] i which was localized to the probe periphery. Multiple consecutive local Ca (2+) responses were induced by sequential application of low level forces, with characteristic recovery time of ~2 s. The duration of [Ca (2+)] i elevations was directly proportional to the tip-cell contact time. In contrast, cell micro-injury resulted in transient global elevations of [Ca (2+)] i, the magnitude of which was independent of the tip-cell contact time. Sequential micro-injury of the same cell did not induce Ca (2+) response within 30 s of the first stimulation. Both local and global Ca (2+)elevations were blocked in Ca (2+)-free media or in the presence of stretch-activated channel blocker Gd (3+). In addition, amount of Ca (2+) released during global responses was significantly reduced in the presence of PLC inhibitor Et-18-OCH 3. Thus, we found qualitative differences in calcium responses to mechanical forces inducing only membrane deformation or deformation leading to micro-injury.

Large dynamic range digital nanodot gradients of biomolecules made by low-cost nanocontact printing for cell haptotaxis.

A novel method is introduced for ultrahigh throughput and ultralow cost patterning of biomolecules with nanometer resolution and novel 2D digital nanodot gradients (DNGs) with mathematically defined slopes are created. The technique is based on lift-off nanocontact printing while using high-resolution photopolymer stamps that are rapidly produced at a low cost through double replication from Si originals. Printed patterns with 100 nm features are shown. DNGs with varying spacing between the dots and a record dynamic range of 4400 are produced; 64 unique DNGs, each with hundreds of thousands of dots, are inked and printed in 5.5 min. The adhesive response and haptotaxis of C2C12 myoblast cells on DNGs demonstrated their biofunctionality. The great flexibility in pattern design, the massive parallel ability, the ultra low cost, and the extreme ease of polymer lift-off nanocontact printing will facilitate its use for various biological and medical applications.

Deconvolution of calcium fluorescent indicator signal from AFM cantilever reflection.

Atomic force microscopy (AFM) can be combined with fluorescence microscopy to measure the changes in intracellular calcium levels (indicated by fluorescence of Ca²âº sensitive dye fluo-4) in response to mechanical stimulation performed by AFM. Mechanical stimulation using AFM is associated with cantilever movement, which may interfere with the fluorescence signal. The motion of the AFM cantilever with respect to the sample resulted in changes of the reflection of light back to the sample and a subsequent variation in the fluorescence intensity, which was not related to changes in intracellular Ca²âº levels. When global Ca²âº responses to a single stimulation were assessed, the interference of reflected light with the fluorescent signal was minimal. However, in experiments where local repetitive stimulations were performed, reflection artifacts, correlated with cantilever motion, represented a significant component of the fluorescent signal. We developed a protocol to correct the fluorescence traces for reflection artifacts, as well as photobleaching. An added benefit of our method is that the cantilever reflection in the fluorescence recordings can be used for precise temporal correlation of the AFM and fluorescence measurements.