Mechanical stimulation and electrophysiological monitoring at subcellular resolution reveals differential mechanosensation of neurons within networks.

A growing consensus that the brain is a mechanosensitive organ is driving the need for tools that mechanically stimulate and simultaneously record the electrophysiological response of neurons within neuronal networks. Here we introduce a synchronized combination of atomic force microscopy, high-density microelectrode array and fluorescence microscopy to monitor neuronal networks and to mechanically characterize and stimulate individual neurons at piconewton force sensitivity and nanometre precision while monitoring their electrophysiological activity at subcellular spatial and millisecond temporal resolution. No correlation is found between mechanical stiffness and electrophysiological activity of neuronal compartments. Furthermore, spontaneously active neurons show exceptional functional resilience to static mechanical compression of their soma. However, application of fast transient (∼500 ms) mechanical stimuli to the neuronal soma can evoke action potentials, which depend on the anchoring of neuronal membrane and actin cytoskeleton. Neurons show higher responsivity, including bursts of action potentials, to slower transient mechanical stimuli (∼60 s). Moreover, transient and repetitive application of the same compression modulates the neuronal firing rate. Seemingly, neuronal networks can differentiate and respond to specific characteristics of mechanical stimulation. Ultimately, the developed multiparametric tool opens the door to explore manifold nanomechanobiological responses of neuronal systems and new ways of mechanical control.

Neurons differentiate magnitude and location of mechanical stimuli.

Neuronal activity can be modulated by mechanical stimuli. To study this phenomenon quantitatively, we mechanically stimulated rat cortical neurons by shear stress and local indentation. Neurons show 2 distinct responses, classified as transient and sustained. Transient responses display fast kinetics, similar to spontaneous neuronal activity, whereas sustained responses last several minutes before returning to baseline. Local soma stimulations with micrometer-sized beads evoke transient responses at low forces of ∼220 nN and pressures of ∼5.6 kPa and sustained responses at higher forces of ∼360 nN and pressures of ∼9.2 kPa. Among the neuronal compartments, axons are highly susceptible to mechanical stimulation and predominantly show sustained responses, whereas the less susceptible dendrites predominantly respond transiently. Chemical perturbation experiments suggest that mechanically evoked responses require the influx of extracellular calcium through ion channels. We propose that subtraumatic forces/pressures applied to neurons evoke neuronal responses via nonspecific gating of ion channels.

Stimulation and Artifact-Suppression Techniques for In Vitro High-Density Microelectrode Array Systems.

We present novel voltage stimulation buffers with controlled output current, along with recording circuits featuring adjustable high-pass cut-off filtering to perform efficient stimulation while actively suppressing stimulation artifacts in high-density microelectrode arrays. Owing to the dense packing and close proximity of the electrodes in such systems, a stimulation through one electrode can cause large electrical artifacts on neighboring electrodes that easily saturate the corresponding recording amplifiers. To suppress such artifacts, the high-pass corner frequencies of all available 2048 recording channels can be raised from several Hz to several kHz by applying a "soft-reset" or pole-shifting technique. With the implemented artifact suppression technique, the saturation time of the recording circuits, connected to electrodes in immediate vicinity to the stimulation site, could be reduced to less than 150 µs. For the stimulation buffer, we developed a circuit, which can operate in two modes: either control of only the stimulation voltage or control of current and voltage during stimulation. The voltage-only controlled mode employs a local common-mode feedback operational transconductance amplifier with a near rail-to-rail input/output range, suitable for driving high-capacitive loads. The current/voltage controlled mode is based on a positive current conveyor generating adjustable output currents, whereas its upper and lower output voltages are limited by two feedback loops. The current/voltage controlled circuit can generate stimulation pulses up to 30 µA with less than ±0.1% linearity error in the low-current mode and up to 300 µA with less than ±0.2% linearity error in the high-current mode.

Biomimetic hydrogels direct spinal progenitor cell differentiation and promote functional recovery after spinal cord injury.

OBJECTIVE: Demyelination that results from disease or traumatic injury, such as spinal cord injury (SCI), can have a devastating effect on neural function and recovery. Many researchers are examining treatments to minimize demyelination by improving oligodendrocyte availability in vivo. Transplantation of stem and oligodendrocyte progenitor cells is a promising option, however, trials are plagued by undirected differentiation. Here we introduce a biomaterial that has been optimized to direct the differentiation of neural progenitor cells (NPCs) toward oligodendrocytes as a cell delivery vehicle after SCI. APPROACH: A collagen-based hydrogel was modified to mimic the mechanical properties of the neonatal spinal cord, and components present in the developing extracellular matrix were included to provide appropriate chemical cues to the NPCs to direct their differentiation toward oligodendrocytes. The hydrogel with cells was then transplanted into a unilateral cervical contusion model of SCI to examine the functional recovery with this treatment. Six behavioral tests and histological assessment were performed to examine the in vivo response to this treatment. MAIN RESULTS: Our results demonstrate that we can achieve a significant increase in oligodendrocyte differentiation of NPCs compared to standard culture conditions using a three-component biomaterial composed of collagen, hyaluronic acid, and laminin that has mechanical properties matched to those of neonatal neural tissue. Additionally, SCI rats with hydrogel transplants, with and without NPCs, showed functional recovery. Animals transplanted with hydrogels with NPCs showed significantly increased functional recovery over six weeks compared to the media control group. SIGNIFICANCE: The three-component hydrogel presented here has the potential to provide cues to direct differentiation in vivo to encourage regeneration of the central nervous system.

A Multi-Functional Microelectrode Array Featuring 59760 Electrodes, 2048 Electrophysiology Channels, Stimulation, Impedance Measurement and Neurotransmitter Detection Channels.

Biological cells are characterized by highly complex phenomena and processes that are, to a great extent, interdependent. To gain detailed insights, devices designed to study cellular phenomena need to enable tracking and manipulation of multiple cell parameters in parallel; they have to provide high signal quality and high spatiotemporal resolution. To this end, we have developed a CMOS-based microelectrode array system that integrates six measurement and stimulation functions, the largest number to date. Moreover, the system features the largest active electrode array area to date (4.48×2.43 mm2) to accommodate 59,760 electrodes, while its power consumption, noise characteristics, and spatial resolution (13.5 µm electrode pitch) are comparable to the best state-of-the-art devices. The system includes: 2,048 action-potential (AP, bandwidth: 300 Hz to 10 kHz) recording units, 32 local-field-potential (LFP, bandwidth: 1 Hz to 300 Hz) recording units, 32 current recording units, 32 impedance measurement units, and 28 neurotransmitter detection units, in addition to the 16 dual-mode voltage-only or current/voltage-controlled stimulation units. The electrode array architecture is based on a switch matrix, which allows for connecting any measurement/stimulation unit to any electrode in the array and for performing different measurement/stimulation functions in parallel.

Electrical Stimulation of Human Mesenchymal Stem Cells on Conductive Nanofibers Enhances their Differentiation toward Osteogenic Outcomes.

Tissue scaffolds allowing the behavior of the cells that reside within them to be controlled are of particular interest for tissue engineering. Herein, the preparation of conductive fiber-based bone tissue scaffolds (nonwoven mats of electrospun polycaprolactone with an interpenetrating network of polypyrrole and polystyrenesulfonate) is described that enable the electrical stimulation of human mesenchymal stem cells to enhance their differentiation toward osteogenic outcomes.

Instructive Conductive 3D Silk Foam-Based Bone Tissue Scaffolds Enable Electrical Stimulation of Stem Cells for Enhanced Osteogenic Differentiation.

Stimuli-responsive materials enabling the behavior of the cells that reside within them to be controlled are vital for the development of instructive tissue scaffolds for tissue engineering. Herein, we describe the preparation of conductive silk foam-based bone tissue scaffolds that enable the electrical stimulation of human mesenchymal stem cells (HMSCs) to enhance their differentiation toward osteogenic outcomes.

Macromol. Rapid Commun. 21/2015.

Back Cover: Tissue scaffolds allowing the behavior of the cells that reside within them to be controlled are of particular interest for tissue engineering. Herein, the preparation of conductive nanofiber-based bone tissue scaffolds are described, made from nonwoven mats of electrospun polycaprolactone with an interpenetrating network of polypyrrole and polystyrenesulfonate. These scaffolds enable the electrical stimulation of human mesenchymal stem cells to enhance their differentiation toward osteogenic outcomes. Further details can be found in the article by J. G. Hardy,* M. K. Villancio-Wolter, R. C. Sukhavasi, D. J. Mouser, D. Aguilar Jr., S. A. Geissler, D. L. Kaplan,* and C. E. Schmidt* on page 1884.

Peptide-directed assembly of functional supramolecular polymers for biomedical applications: electroactive molecular tongue-twisters (oligoalanine-oligoaniline-oligoalanine) for electrochemically enhanced drug delivery.

We report the preparation and characterization of films of electroactive supramolecular polymers based on non-electroactive oligoalanines and electroactive oligoanilines. Fibroblasts adhered to and proliferated on the films, and the delivery of the clinically relevant anti-inflammatory drug dexamethasone phosphate could be enhanced upon the application of an electrical stimulus.

Electrical stimulation of human mesenchymal stem cells on biomineralized conducting polymers enhances their differentiation towards osteogenic outcomes.

Tissue scaffolds allowing the behaviour of the cells that reside on them to be controlled are of particular interest for tissue engineering. Herein we describe biomineralized conducting polymer-based bone tissue scaffolds that facilitate the electrical stimulation of human mesenchymal stem cells, resulting in enhancement of their differentiation towards osteogenic outcomes.

Conducting polymer-based multilayer films for instructive biomaterial coatings.

AIM: To demonstrate the design, fabrication and testing of conformable conducting biomaterials that encourage cell alignment. MATERIALS & METHODS: Thin conducting composite biomaterials based on multilayer films of poly(3.4-ethylenedioxythiophene) derivatives, chitosan and gelatin were prepared in a layer-by-layer fashion. Fibroblasts were observed with fluorescence microscopy and their alignment (relative to the dipping direction and direction of electrical current passed through the films) was determined using ImageJ. RESULTS: Fibroblasts adhered to and proliferated on the films. Fibroblasts aligned with the dipping direction used during film preparation and this was enhanced by a DC current. CONCLUSION: We report the preparation of conducting polymer-based films that enhance the alignment of fibroblasts on their surface which is an important feature of a variety of tissues.

Biodegradable electroactive polymers for electrochemically-triggered drug delivery.

We report biodegradable electroactive polymer (EAP)-based materials and their application as drug delivery devices. Copolymers composed of oligoaniline-based electroactive blocks linked to either polyethylene glycol or polycaprolactone blocks via ester bonds were synthesized in three steps from commercially available starting materials and isolated without the need for column chromatography. The physicochemical and electrochemical properties of the polymers were characterized with a variety of techniques. The ability of the polymers to deliver the anti-inflammatory drug dexamethasone phosphate on the application of electrochemical stimuli was studied spectroscopically. Films of the polymers were shown to be degradable and cell adhesive in vitro. Such EAP-based materials have prospects for integration in implantable fully biodegradable/bioerodible EAP-based drug delivery devices that are capable of controlling the chronopharmacology of drugs for future clinical application.

Assessing forelimb function after unilateral cervical SCI using novel tasks: limb step-alternation, postural instability and pasta handling.

Cervical spinal cord injury (cSCI) can cause devastating neurological deficits, including impairment or loss of upper limb and hand function. A majority of the spinal cord injuries in humans occur at the cervical levels. Therefore, developing cervical injury models and developing relevant and sensitive behavioral tests is of great importance. Here we describe the use of a newly developed forelimb step-alternation test after cervical spinal cord injury in rats. In addition, we describe two behavioral tests that have not been used after spinal cord injury: a postural instability test (PIT), and a pasta-handling test. All three behavioral tests are highly sensitive to injury and are easy to use. Therefore, we feel that these behavioral tests can be instrumental in investigating therapeutic strategies after cSCI.

The role of central amygdala dopamine in disengagement behavior.

Unilateral nigrostriatal dopamine depletion in animals induces contralateral sensorimotor deficits that are like symptoms associated with Parkinson's disease (PD). Unilateral nigrostriatal dopamine depletion also causes a contralateral deficit in disengagement behavior (e.g., ability to stop an ongoing activity to orient/attend to a new stimulus). This disengagement deficit has been shown to be resistant to treatments that rescued other motor and somatosensory deficits. Thus, disengagement behavior may involve unique sensorimotor information integration potentially important for attentional allocation and may rely strongly on a mechanism that includes extranigrostriatal circuitry. The central nucleus of the amygdala (CeA) and its connections with the nigral dopamine system have been reported to modulate cognitive processes dependent substantially on attentional allocation. CeA dopamine function might be also important for disengagement behavior. In Experiment 1, rats received microinfusions of 6-hydroxydopamine unilaterally to induce dopamine terminal loss in the CeA and were tested for disengagement behavior in addition to several sensorimotor functions. These rats showed deficits in contralateral disengagement behavior and an asymmetry in adhesive dot removal from the paws, but not in forelimb use in a cylinder or amphetamine rotation. In Experiment 2, rats received D1 or D2 antagonists into the CeA unilaterally prior to behavioral tests. The D1 antagonist disrupted disengagement behavior without affecting the other sensorimotor tests examined. The D2 antagonist had no effects on any of the behaviors tested. Our results suggest that CeA dopamine function is involved in modulation of disengagement behavior.

Rodent Models and Behavioral Outcomes of Cervical Spinal Cord Injury.

Rodent spinal cord injury (SCI) models have been developed to examine functional and physiological deficits after spinal cord injury with the hope that these models will elucidate information about human SCI. Models are needed to examine possible treatments and to understand histopathology after SCI; however, they should be considered carefully and chosen based on the goals of the study being performed. Contusion, compression, transection, and other models exist and have the potential to reveal important information about SCI that may be related to human SCI and the outcomes of treatment and timing of intervention.

Assessing forelimb function after unilateral cervical spinal cord injury: novel forelimb tasks predict lesion severity and recovery.

Cervical spinal cord injury (cSCI) can cause devastating neurological deficits, including impairment or loss of upper limb and hand function. Recently there has been increasing interest in cervical spinal cord injury models because the majority of spinal cord injuries are at cervical levels. Here we examined spontaneous functional recovery of adult rats with either laminectomy or lateral hemisection of the cervical spinal cord at C3-C4. Behavioral tests were carried out, including the forelimb locomotor scale (FLS), a postural instability test (PIT), a pasta-handling test that has been used to assess forepaw digit function and latency to eat, forelimb use during vertical-lateral wall exploration in a cylindrical enclosure, and vibrissae-elicited forelimb placing tests. In addition, a forelimb step-alternation test was developed to assess functional recovery at 12 weeks post-injury. All tests detected cSCI-induced deficits relative to laminectomy. Interestingly, the severity of deficits in the forelimb step-alternation test was associated with more extensive spinal damage, greater impairment, and less recovery in the FLS and other tests. For the pasta-handling test we found that rats with a milder cervical injury (alternators) were more likely to use both forepaws together compared to rats with a more severe injury (non-alternators). In addition, using the PIT, we detected enhanced function of the good limb, suggesting that neural plasticity on the unaffected side of the spinal cord may have occurred to compensate for deficits in the impaired forelimb. These outcome measures should be useful for investigating neural events associated with cSCI, and for developing novel treatment strategies.

Effects of accommodation training on accommodation and depth of focus in an eye implanted with a crystalens intraocular lens.

PURPOSE: To investigate objective measures of the effects of accommodative training of a pseudophakic eye implanted with a Crystalens AT-52SE (eyeonics Inc) intraocular lens (IOL) on reading performance, accommodation, and depth of focus. METHODS: Objective dynamic measures of accommodation, pupil size, and depth of focus were quantified from wavefront measures before and after 1 week of accommodative training that began 29 months after implantation of an accommodating IOL in one patient. Depth of focus was estimated from 50% cut-off of peak performance levels for defocus curves that were computed from the image quality metric VSOTF based on ocular wavefront aberrations. RESULTS: The patient reported improved near vision reading performance after completing the training procedure. After training, there was a shift in conjugate focus in the hyperopic direction, yet the depth of focus increased significantly for near objects. Simulated retinal images and the calculated modulation transfer function of the eye both demonstrated improved quality for near vision after training. CONCLUSIONS: The subjective report of improved near vision after training was correlated with improvement of objective measures. Depth of focus increased for near objects with attempts to accommodate after training. This change was linked to increases in aberrations and pupil size and occurred despite the conjugate focus shifting in the hyperopic direction. These results demonstrate that accommodative training may be useful in improving near vision in patients with accommodating IOLs.