Optogenetic-guided cortical plasticity after nerve injury.

Peripheral nerve injury causes sensory dysfunctions that are thought to be attributable to changes in neuronal activity occurring in somatosensory cortices both contralateral and ipsilateral to the injury. Recent studies suggest that distorted functional response observed in deprived primary somatosensory cortex (S1) may be the result of an increase in inhibitory interneuron activity and is mediated by the transcallosal pathway. The goal of this study was to develop a strategy to manipulate and control the transcallosal activity to facilitate appropriate plasticity by guiding the cortical reorganization in a rat model of sensory deprivation. Since transcallosal fibers originate mainly from excitatory pyramidal neurons somata situated in laminae III and V, the excitatory neurons in rat S1 were engineered to express halorhodopsin, a light-sensitive chloride pump that triggers neuronal hyperpolarization. Results from electrophysiology, optical imaging, and functional MRI measurements are concordant with that within the deprived S1, activity in response to intact forepaw electrical stimulation was significantly increased by concurrent illumination of halorhodopsin over the healthy S1. Optogenetic manipulations effectively decreased the adverse inhibition of deprived cortex and revealed the major contribution of the transcallosal projections, showing interhemispheric neuroplasticity and thus, setting a foundation to develop improved rehabilitation strategies to restore cortical functions.

Playtime for Cephalopods: Understanding the Significance of Play Behavior in Octopus bimaculoides.

Play is considered to be an essential part of development that supports learning, memory, and the development of flexible behavioral strategies. An increasing amount of non-mammalian species have been discovered to engage in play behavior, but there has been little research into play behavior in cephalopods specifically. Here we studied play behavior of wild-caught, laboratory-housed California Two-Spot Octopuses, Octopus bimaculoides. The behavior observed in these animals is consistent with established criteria of play behavior in animals. The evidence of play in O. bimaculoides provides further insight into the evolutionary development of play behavior as a whole, the characteristics of play in an under-studied species, and also has the potential to be used as a factor in determining the welfare state of octopuses to improve their management in captivity.

3D octopus kinematics of complex postures: Translation to long, thin, soft devices and their potential for clinical use.

INTRO/BACKGROUND: Octopuses are capable of complex arm movements. Unfortunately, experimental barriers and lack of a robust analysis method made it difficult to quantify the three-dimensional (3D) kinematics of soft, flexible bodies, such as the octopus arm. This information is not only crucial for understanding the posture of the animal's arm but also for the development of similarly designed soft, flexible devices. OBJ/GOAL: The primary goal of this work was to create a method to comprehensively quantify complex, 3D postures of octopus (Octopus Bimaculoides) arms in a manner that is conducive and translatable to octopus arm-inspired devices for health monitoring and rehabilitation. METHODS: In this study, 3D underwater motion capture was used to collect kinematic data on both live octopuses and disembodied arms that still had neural activity. A new method was developed to define how arm curvature and regional segments were oriented relative to each other in 3D. This included identification of the bend within a segment along with the computation of the relative orientation between segments, thus permitting the complete quantification of complex arm motions. RESULTS: By comparing vector-based and radius of curvature-based approaches to magnitude of curvature, it was clear that the vector-based approach was less dependent on the length of a segment and that its reported ranges of motion were translatable for outcome measures associated with clinical use. The new approach for the relative orientation of each segment of the octopus arm resulted in the capability of describing the octopus arm in many unique postures, such as straight, simple bending, and complex bending as it utilized the three rotational angles. OUTCOME/IMPACT: This method and its application to octopus arms will yield new information that can be used to better communicate and track not only octopus arm movements but in the development of complex, segmented, soft-bodied devices that can be used in health monitoring and rehabilitation.

A putative design for the electromagnetic activation of split proteins for molecular and cellular manipulation.

The ability to manipulate cellular function using an external stimulus is a powerful strategy for studying complex biological phenomena. One approach to modulate the function of the cellular environment is split proteins. In this method, a biologically active protein or an enzyme is fragmented so that it reassembles only upon a specific stimulus. Although many tools are available to induce these systems, nature has provided other mechanisms to expand the split protein toolbox. Here, we show a novel method for reconstituting split proteins using magnetic stimulation. We found that the electromagnetic perceptive gene (EPG) changes conformation due to magnetic field stimulation. By fusing split fragments of a certain protein to both termini of the EPG, the fragments can be reassembled into a functional protein under magnetic stimulation due to conformational change. We show this effect with three separate split proteins: NanoLuc, APEX2, and herpes simplex virus type-1 thymidine kinase. Our results show, for the first time, that reconstitution of split proteins can be achieved only with magnetic fields. We anticipate that this study will be a starting point for future magnetically inducible split protein designs for cellular perturbation and manipulation. With this technology, we can help expand the toolbox of the split protein platform and allow better elucidation of complex biological systems.

A conserved phenylalanine motif among Teleost fish provides insight for improving electromagnetic perception.

Magnetoreceptive biology as a field remains relatively obscure; compared to the breadth of species believed to sense magnetic fields, it remains under-studied. Here, we present grounds for the expansion of magnetoreception studies among Teleosts. We begin with the electromagnetic perceptive gene (EPG) from Kryptopterus vitreolus and expand to identify 72 Teleosts with homologous proteins containing a conserved three-phenylalanine (3F) motif. Phylogenetic analysis provides insight as to how EPG may have evolved over time, and indicates that certain clades may have experienced a loss of function driven by different fitness pressures. One potential factor is water type with freshwater fish significantly more likely to possess the functional motif version (FFF), and saltwater fish to have the non-functional variant (FXF). It was also revealed that when the 3F motif from the homolog of Brachyhypopomus gauderio (B.g.) is inserted into EPG - EPG(B.g.) - the response (as indicated by increased intracellular calcium) is faster. This indicates that EPG has the potential to be engineered to improve upon its response and increase its utility to be used as a controller for specific outcomes.

A conserved phenylalanine motif among teleost fish provides insight for improving electromagnetic perception.

Magnetoreceptive biology as a field remains relatively obscure; compared with the breadth of species believed to sense magnetic fields, it remains under-studied. Here, we present grounds for the expansion of magnetoreception studies among teleosts. We begin with the electromagnetic perceptive gene (EPG) from Kryptopterus vitreolus and expand to identify 72 teleosts with homologous proteins containing a conserved three-phenylalanine (3F) motif. Phylogenetic analysis provides insight as to how EPG may have evolved over time and indicates that certain clades may have experienced a loss of function driven by different fitness pressures. One potential factor is water type with freshwater fish significantly more likely to possess the functional motif version (FFF), and saltwater fish to have the non-functional variant (FXF). It was also revealed that when the 3F motif from the homologue of Brachyhypopomus gauderio (B.g.) is inserted into EPG-EPG(B.g.)-the response (as indicated by increased intracellular calcium) is faster. This indicates that EPG has the potential to be engineered to improve upon its response and increase its utility to be used as a controller for specific outcomes.

Single unit electrophysiology recordings and computational modeling can predict octopus arm movement.

The octopus simplified nervous system holds the potential to reveal principles of motor circuits and improve brain-machine interface devices through computational modeling with machine learning and statistical analysis. Here, an array of carbon electrodes providing single-unit electrophysiology recordings were implanted into the octopus anterior nerve cord. The number of spikes and arm movements in response to stimulation at different locations along the arm were recorded. We observed that the number of spikes occurring within the first 100ms after stimulation were predictive of the resultant movement response. Computational models showed that temporal electrophysiological features could be used to predict whether an arm movement occurred with 88.64% confidence, and if it was a lateral arm movement or a grasping motion with 75.45% confidence. Both supervised and unsupervised methods were applied to gain streaming measurements of octopus arm movements and how their motor circuitry produces rich movement types in real time. Deep learning models and unsupervised dimension reduction identified a consistent set of features that could be used to distinguish different types of arm movements. These models generated predictions for how to evoke a particular, complex movement in an orchestrated sequence for an individual motor circuit.

Fabrication and Validation of Sub-Cellular Carbon Fiber Electrodes.

Multielectrode arrays for interfacing with neurons are of great interest for a wide range of medical applications. However, current electrodes cause damage over time. Ultra small carbon fibers help to address issues but controlling the electrode site geometry is difficult. Here we propose a methodology to create small, pointed fiber electrodes (SPFe). We compare the SPFe to previously made blowtorched fibers in characterization. The SPFe result in small site sizes [Formula: see text] with consistently sharp points (20.8 ± 7.64°). Additionally, these electrodes were able to record and/or stimulate neurons multiple animal models including rat cortex, mouse retina, Aplysia ganglia and octopus axial cord. In rat cortex, these electrodes recorded significantly higher peak amplitudes than the traditional blowtorched fibers. These SPFe may be applicable to a wide range of applications requiring a highly specific interface with individual neurons.

NAP (davunetide) modifies disease progression in a mouse model of severe neurodegeneration: protection against impairments in axonal transport.

NAP (davunetide) is a novel neuroprotective compound with mechanism of action that appears to involve microtubule (MT) stabilization and repair. To evaluate, for the first time, the impact of NAP on axonal transport in vivo and to translate it to neuroprotection in a severe neurodegeneration, the SOD1-G93A mouse model for amyotrophic lateral sclerosis (ALS) was used. Manganese-enhanced magnetic resonance imaging (MRI), estimating axonal transport rates, revealed a significant reduction of the anterograde axonal transport in the ALS mice compared to healthy control mice. Acute NAP treatment normalized axonal transport rates in these ALS mice. Tau hyperphosphorylation, associated with MT dysfunction and defective axonal transport, was discovered in the brains of the ALS mice and was significantly reduced by chronic NAP treatment. Furthermore, in healthy wild type (WT) mice, NAP reversed axonal transport disruption by colchicine, suggesting drug-dependent protection against axonal transport impairment through stabilization of the neuronal MT network. Histochemical analysis showed that chronic NAP treatment significantly protected spinal cord motor neurons against ALS-like pathology. Sequential MRI measurements, correlating brain structure with ALS disease progression, revealed a significant damage to the ventral tegmental area (VTA), indicative of impairments to the dopaminergic pathways relative to healthy controls. Chronic daily NAP treatment of the SOD1-G93A mice, initiated close to disease onset, delayed degeneration of the trigeminal, facial and hypoglossal motor nuclei as was significantly apparent at days 90-100 and further protected the VTA throughout life. Importantly, protection of the VTA was significantly correlated with longevity and overall, NAP treatment significantly prolonged life span in the ALS mice.

Genetic tools to manipulate MRI contrast.

Advances in molecular biology in the early 1970s revolutionized research strategies for the study of complex biological processes, which, in turn, created a high demand for new means to visualize these dynamic biological changes noninvasively and in real time. In this respect, MRI was a perfect fit, because of the versatile possibility to alter the different contrast mechanisms. Genetic manipulations are now being translated to MRI through the development of reporters and sensors, as well as the imaging of transgenic and knockout mice. In the past few years, a new molecular biology toolset, namely optogenetics, has emerged, which allows for the manipulation of cellular behavior using light. This technology provides a few particularly attractive features for combination with newly developed MRI techniques for the probing of in vivo cellular and, in particular, neural processes, specifically the ability to control focal, genetically defined cellular populations with high temporal resolution using equipment that is magnetically inert and does not interact with radiofrequency pulses. Recent studies have demonstrated that the combination of optogenetics and functional MRI (fMRI) can provide an appropriate platform to investigate in vivo, at the cellular and molecular levels, the neuronal basis of fMRI signals. In addition, this novel combination of optogenetics with fMRI has the potential to resolve pre-synaptic versus post-synaptic changes in neuronal activity and changes in the activity of large neuronal networks in the context of plasticity associated with development, learning and pathophysiology.

The Pig as a Translational Animal Model for Biobehavioral and Neurotrauma Research.

In recent decades, the pig has attracted considerable attention as an important intermediary model animal in translational biobehavioral research due to major similarities between pig and human neuroanatomy, physiology, and behavior. As a result, there is growing interest in using pigs to model many human neurological conditions and injuries. Pigs are highly intelligent and are capable of performing a wide range of behaviors, which can provide valuable insight into the effects of various neurological disease states. One area in which the pig has emerged as a particularly relevant model species is in the realm of neurotrauma research. Indeed, the number of investigators developing injury models and assessing treatment options in pigs is ever-expanding. In this review, we examine the use of pigs for cognitive and behavioral research as well as some commonly used physiological assessment methods. We also discuss the current usage of pigs as a model for the study of traumatic brain injury. We conclude that the pig is a valuable animal species for studying cognition and the physiological effect of disease, and it has the potential to contribute to the development of new treatments and therapies for human neurological and psychiatric disorders.

Magnetogenetic stimulation inside MRI induces spontaneous and evoked changes in neural circuits activity in rats.

The ability to modulate specific neural circuits and simultaneously visualize and measure brain activity with MRI would greatly impact understanding brain function in health and disease. The combination of neurostimulation methods and MRI in animal models have already shown promise in elucidating fundamental mechanisms associated with brain activity. We developed an innovative magnetogenetics neurostimulation technology that can trigger neural activity through magnetic fields. Similar to other genetic-based neuromodulation methods, magnetogenetics offers cell-, area- and temporal-specific control of neural activity. However, the magnetogenetics protein (Electromagnetic Preceptive Gene (EPG)) are activated by non-invasive magnetic fields, providing a unique way to target neural circuits by the MRI gradients while simultaneously measure their effect on brain activity. EPG was expressed in rat's visual cortex and the amplitude of low-frequency fluctuation (fALFF), resting-state functional connectivity (FC), and sensory activation was measured using a 7T MRI. The results demonstrate that EPG-expressing rats had significantly higher signal fluctuations in the visual areas and stronger FC in sensory areas consistent with known anatomical visuosensory and visuomotor connections. This new technology complements the existing neurostimulation toolbox and provides a mean to study brain function in a minimally-invasive way which was not possible previously.

Closed-loop neurostimulation via expression of magnetogenetics-sensitive protein in inhibitory neurons leads to reduction of seizure activity in a rat model of epilepsy.

On-demand neurostimulation has shown success in epilepsy patients with pharmacoresistant seizures. Seizures produce magnetic fields that can be recorded using magnetoencephalography. We developed a new closed-loop approach to control seizure activity based on magnetogenetics using the electromagnetic perceptive gene (EPG) that encodes a protein that responds to magnetic fields. The EPG transgene was expressed in inhibitory interneurons under the hDlx promoter and kainic acid was used to induce acute seizures. In vivo electrophysiological signals were recorded. We found that hDlx EPG rats exhibited a significant delay in the onset of first seizure (1142.72 ± 186.35 s) compared to controls (644.03 ± 15.06 s) and significantly less seizures (4.11 ± 1.03) compared to controls (8.33 ± 1.58). These preliminary findings suggest that on-demand activation of EPG expressed in inhibitory interneurons suppresses seizure activity, and magnetogenetics via EPG may be an effective strategy to alleviate seizure severity in a closed-loop, and cell-specific fashion.

Proposed three-phenylalanine motif involved in magnetoreception signalling of an Actinopterygii protein expressed in mammalian cells.

Studies at the cellular and molecular level of magnetoreception-sensing and responding to magnetic fields-are a relatively new research area. It appears that different mechanisms of magnetoreception in animals evolved from different origins, and, therefore, many questions about its mechanisms remain left open. Here we present new information regarding the Electromagnetic Perceptive Gene (EPG) from Kryptopterus vitreolus that may serve as part of the foundation to understanding and applying magnetoreception. Using HaloTag coupled with fluorescent ligands and phosphatidylinositol specific phospholipase C we show that EPG is associated with the membrane via glycosylphosphatidylinositol anchor. EPG's function of increasing intracellular calcium was also used to generate an assay using GCaMP6m to observe the function of EPG and to compare its function with that of homologous proteins. It was also revealed that EPG relies on a motif of three phenylalanine residues to function-stably swapping these residues using site directed mutagenesis resulted in a loss of function in EPG. This information not only expands upon our current understanding of magnetoreception but may provide a foundation and template to continue characterizing and discovering more within the emerging field.

MRI biosensor for protein kinase A encoded by a single synthetic gene.

PURPOSE: Protein kinases including protein kinase A (PKA) underlie myriad important signaling pathways. The ability to monitor kinase activity in vivo and in real-time with high spatial resolution in genetically specified cellular populations is a yet unmet need, crucial for understanding complex biological systems as well as for preclinical development and screening of novel therapeutics. METHODS: Using the hypothesis that the natural recognition sequences of protein kinases may be detected using chemical exchange saturation transfer magnetic resonance imaging, we designed a genetically encoded biosensor composed of eight tandem repeats of the peptide LRRASLG, a natural target of PKA. RESULTS: This sensor displays a measurable change in chemical exchange saturation transfer signal following phosphorylation by PKA. The natural PKA substrate LRRASLG exhibits a chemical exchange saturation transfer-magnetic resonance imaging contrast at +1.8 and +3.6 ppm, with a >50% change after phosphorylation with minutes-scale temporal resolution. Expression of a synthetic gene encoding eight monomers of LRRASLG yielded two peaks at these chemical exchange saturation transfer frequencies. CONCLUSION: Taken together, these results suggest that this gene may be used to assay PKA levels in a biologically relevant system. Importantly, the design strategy used for this specific sensor may be adapted for a host of clinically interesting protein kinases.

Ipsilateral cortical fMRI responses after peripheral nerve damage in rats reflect increased interneuron activity.

In the weeks following unilateral peripheral nerve injury, the deprived primary somatosensory cortex (SI) responds to stimulation of the ipsilateral intact limb as demonstrated by functional magnetic resonance imaging (fMRI) responses. The neuronal basis of these responses was studied by using high-resolution fMRI, in vivo electrophysiological recordings, and juxtacellular neuronal labeling in rats that underwent an excision of the forepaw radial, median, and ulnar nerves. These nerves were exposed but not severed in control rats. Significant bilateral increases of fMRI responses in SI were observed in denervated rats. In the healthy SI of the denervated rats, increases in fMRI responses were concordant with increases in local field potential (LFP) amplitude and an increased incidence of single units responding compared with control rats. In contrast, in the deprived SI, increases in fMRI responses were associated with a minimal change in LFP amplitude but with increased incidence of single units responding. Based on action potential duration, juxtacellular labeling, and immunostaining results, neurons responding to intact forepaw stimulation in the deprived cortex were identified as interneurons. These results suggest that the increases in fMRI responses in the deprived cortex reflect increased interneuron activity.

Peripheral Nerve Injury Induces Changes in the Activity of Inhibitory Interneurons as Visualized in Transgenic GAD1-GCaMP6s Rats.

Peripheral nerve injury induces cortical remapping that can lead to sensory complications. There is evidence that inhibitory interneurons play a role in this process, but the exact mechanism remains unclear. Glutamate decarboxylase-1 (GAD1) is a protein expressed exclusively in inhibitory interneurons. Transgenic rats encoding GAD1-GCaMP were generated to visualize the activity in GAD1 neurons through genetically encoded calcium indicators (GCaMP6s) in the somatosensory cortex. Forepaw denervation was performed in adult rats, and fluorescent Ca2+ imaging on cortical slices was obtained. Local, intrahemispheric stimulation (cortical layers 2/3 and 5) induced a significantly higher fluorescence change of GAD1-expressing neurons, and a significantly higher number of neurons were responsive to stimulation in the denervated rats compared to control rats. However, remote, interhemispheric stimulation of the corpus callosum induced a significantly lower fluorescence change of GAD1-expressing neurons, and significantly fewer neurons were deemed responsive to stimulation within layer 5 in denervated rats compared to control rats. These results suggest that injury impacts interhemispheric communication, leading to an overall decrease in the activity of inhibitory interneurons in layer 5. Overall, our results provide direct evidence that inhibitory interneuron activity in the deprived S1 is altered after injury, a phenomenon likely to affect sensory processing.

Establishing an Octopus Ecosystem for Biomedical and Bioengineering Research.

Many developments in biomedical research have been inspired by discovering anatomical and cellular mechanisms that support specific functions in different species. The octopus is one of these exceptional animals that has given scientists new insights into the fields of neuroscience, robotics, regenerative medicine, and prosthetics. Research with this species of cephalopods requires the set-up of complex facilities and intensive care for both the octopus and its ecosystem that is critical for the project's success. This system requires multiple mechanical and biological filtering systems to provide a safe and clean environment for the animal. Along with the control system, specialized routine maintenance and cleaning are required to effectively keep the facility operating long term. It is advised to provide an enriched environment to these intelligent animals by changing the tank's landscape, incorporating a variety of prey, and introducing challenging tasks for them to work through. Our results include MRI and a whole-body autofluorescence imaging as well as behavioral studies to better understand their nervous system. Octopuses possess unique physiology that can impact many areas of biomedical research. Providing them with a sustainable ecosystem is the first crucial step in uncovering their distinct capabilities.

Bioelectromagnetic Platform for Cell, Tissue, and In Vivo Stimulation.

Magnetogenetics is a new field that utilizes electromagnetic fields to remotely control cellular activity. In addition to the development of the biological genetic tools, this approach requires designing hardware with a specific set of demands for the electromagnets used to provide the desired stimulation for electrophysiology and imaging experiments. Here, we present a universal stimulus delivery system comprising four magnet designs compatible with electrophysiology, fluorescence and luminescence imaging, microscopy, and freely behaving animal experiments. The overall system includes a low-cost stimulation controller that enables rapid switching between active and sham stimulation trials as well as precise control of stimulation delivery thereby enabling repeatable and reproducible measurements.

Multi-session delivery of synchronous rTMS and sensory stimulation induces long-term plasticity.

BACKGROUND: Combining training or sensory stimulation with non-invasive brain stimulation has shown to improve performance in healthy subjects and improve brain function in patients after brain injury. However, the plasticity mechanisms and the optimal parameters to induce long-term and sustainable enhanced performance remain unknown. OBJECTIVE: This work was designed to identify the protocols of which combining sensory stimulation with repetitive transcranial magnetic stimulation (rTMS) will facilitate the greatest changes in fMRI activation maps in the rat's primary somatosensory cortex (S1). METHODS: Several protocols of combining forepaw electrical stimulation with rTMS were tested, including a single stimulation session compared to multiple, daily stimulation sessions, as well as synchronous and asynchronous delivery of both modalities. High-resolution fMRI was used to determine how pairing sensory stimulation with rTMS induced short and long-term plasticity in the rat S1. RESULTS: All groups that received a single session of rTMS showed short-term increases in S1 activity, but these increases did not last three days after the session. The group that received a stimulation protocol of 10 Hz forepaw stimulation that was delivered simultaneously with 10 Hz rTMS for five consecutive days demonstrated the greatest increases in the extent of the evoked fMRI responses compared to groups that received other stimulation protocols. CONCLUSIONS: Our results provide direct indication that pairing peripheral stimulation with rTMS induces long-term plasticity, and this phenomenon appears to follow a time-dependent plasticity mechanism. These results will be important to lead the design of new training and rehabilitation paradigms and training towards achieving maximal performance in healthy subjects.