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SummaryStudies at the cellular and molecular level of magnetoreception – sensing and responding to magnetic fields – is a relatively new research area. As it appears that different mechanisms of magnetoreception in animals evolved from different origins, many questions about the mechanisms remain left open. Here we present new information regarding the Electromagnetic Perceptive Gene (EPG) fromKryptopterus vitreolusthat may serve as part of the foundation to understanding and applying magnetoreception. Using HaloTag coupled with fluorescent ligands and phosphatidylinositol specific phospholipase C (PI-PLC) we show that EPG is associated to the membrane via glycosylphosphatidylinositol (GPI) 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 homologous proteins. It was also revealed that EPG relies on a motif of three phenylalanine residues in order 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 field.Graphical AbstractIn BriefEPG is a magnetoreceptive GPI anchored protein. Critical to its function is a three-phenylalanine motif which allows EPG to sense and respond to EMF. When expressed in mammalian cell, an increase in intracellular calcium is observed using GCaMP6m. This work represents progress towards understanding magnetoreception for use in future technologies.HighlightsEPG is associated to the cell membrane via glycosylphosphatidylinositol anchoringIn mammalian cells, EPG increases intracellular calcium upon EMF stimulationHomologs of EPG from the uPAR/Ly6 family show different responses to EMFA three-phenylalanine motif in EPG is critical to its magnetoreceptive ability

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. While there are many tools available to induce these systems, nature has provided other mechanisms that can be utilized to expand the split protein toolbox. Here we show a novel method for reconstituting split proteins using magnetic stimulation. We have found that the Electromagnetic Perceptive Gene (EPG) changes conformation due to magnetic fields 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 to expand the toolbox of the split protein platform and allow better elucidation of complex biological systems.

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 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 ± 186.35s) compared to controls (644.03 ± 15.06s) 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 suppress seizure activity, and magnetogenetics via EPG may be an effective strategy to alleviate seizure severity in a minimally invasive, closed-loop and cell-specific fashion.

Brain injuries induced by external forces are particularly challenging to model experimentally. In recent decades, the domestic pig has been gaining popularity as a highly relevant animal model to address the pathophysiological mechanisms and the biomechanics associated with head injuries. Understanding cognitive, motor, and sensory aspects of pig behavior throughout development is crucial for evaluating cognitive and motor deficits after injury. We have developed a comprehensive battery of tests to characterize the behavior and physiological function of the Yucatan minipig throughout maturation. Behavioral testing included assessments of learning and memory, executive functions, circadian rhythms, gait analysis, and level of motor activity. We applied traditional behavioral apparatus and analysis methods, as well as state-of-the-art sensor technologies to report on motion and activity, and artificial intelligent approaches to analyze behavior. We studied pigs from 16 weeks old through sexual maturity at 35 weeks old. The results show multidimensional characterization of minipig behavior, and how it develops and changes with age. This animal model may capitulate the biomechanical consideration and phenotype of head injuries in the developing brain and can drive forward the field of understanding pathophysiological mechanisms and developing new therapies to accelerate recovery in children who have suffered head trauma.

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.

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.

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 comprised of 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 which enables rapid switching between active and sham stimulation trials as well as precise control of stimulation delivery.

Brain injuries that are induced by external forces are particularly challenging to model experimentally. In recent decades, the domestic pig has been gaining popularity as a highly relevant animal model to address the pathophysiological mechanisms and the biomechanics associated with head injuries. Understanding cognitive, motor, and sensory aspects of pig behavior throughout development is crucial for evaluating cognitive and motor deficits after injury. We have developed a comprehensive battery of tests to characterize the behavior and physiological function of the Yucatan minipig throughout maturation. Behavioral testing included assessments of learning and memory, executive functions, circadian rhythms, gait analysis, and level of motor activity. We applied traditional behavioral apparatus and analysis methods, as well as state-of-the-art sensor technologies to report on motion and activity, and artificial intelligent approaches to analyze behavior. We studied pigs from 16 weeks old through sexual maturity at 35 weeks old. The results show multidimensional characterization of minipig behavior, and how it develops and changes with age. This animal model may capitulate the biomechanical consideration and phenotype of head injuries in the developing brain and can drive forward the field of understanding pathophysiological mechanisms and developing new therapies to accelerate recovery in children who have suffered head trauma.

BackgroundCombining 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.ObjectiveThis 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).MethodsSeveral 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.ResultsAll 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.ConclusionsOur 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.HighlightsA single rTMS session induced short-term changes but they were not sustainableMulti-session delivery of rTMS paired with sensory stimulation induced long-term plasticityrTMS paired with sensory stimulation induced plasticity via time-dependent mechanismDelivery of sensory stimulation only did not induce long-term plasticity

Magnetoreception, the response to geomagnetic fields is a well described phenomenon in nature. However, it is likely that convergent evolution led to different mechanisms in different organisms. One intriguing example is the unique Electromagnetic Perceptive Gene (EPG) from the glass catfish Kryptopterus vitreolus, that can remotely control cellular function, upon magnetic stimulation in in-vitro and in animal models. Here, we report for the first time the cellular location and orientation of the EPG protein. We utilized a differential labelling technique in determining that the EPG protein is a membrane anchored protein with an N-terminal extracellular domain. The kinetics and diffusion dynamics of the EPG protein in response to magnetic stimulation was also elucidated using single particle imaging and tracking. Pulse chase labelling and Total Internal Reflection Fluorescence (TIRF) imaging revealed an increase in EPG kinetics post magnetic stimulation activation at a single particle level. Trajectory analysis show notably different EPG protein kinetics before and after magnetic stimulation in both 2 (free vs bound particle) and 3 state (free vs intermediate vs bound particle) tracking models. These data serve to provide additional information that support and understand the underlying biophysical mechanisms behind EPG activation by magnetic stimulation. In conclusion, our results provide evidence for the basis of magnetoreception in EPG protein that would aid in future studies that seek to understand this novel mechanism. This study is important for understanding the phenomenon of magnetoreception as well as developing new technologies for magnetogenetics - the utilization of electromagnetic fields to remotely control cellular function. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/329946v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@5f8baorg.highwire.dtl.DTLVardef@cad9corg.highwire.dtl.DTLVardef@5a1950org.highwire.dtl.DTLVardef@133f11d_HPS_FORMAT_FIGEXP M_FIG Graphical TOCElucidation of magnetoreception in a fish derived Electromagnetic Perceptive Gene (EPG), using genetic tagging and single particle tracking with Total Internal Reflection Fluorescence (TIRF) suggests changes in kinetics of membranal motion upon stimulation by magnetic field. C_FIG

Several marine species have developed a magnetic perception that is essential for navigation and detection of prey and predators. One of these species is the transparent glass catfish that contains an ampullary organ dedicated to sense magnetic fields. Here we examine the behavior of the glass catfish in response to static magnetic fields which will provide valuable insight on function of this magnetic response. By utilizing state of the art animal tracking software and artificial intelligence approaches, we quantified the effects of magnetic fields on the swimming direction of glass catfish. The results demonstrate that glass catfish placed in a radial arm maze, consistently swim away from magnetic fields over 20 µT and show adaptability to changing magnetic field direction and location.

Developing synthetic biological devices to allow the noninvasive control of cell fate and function, in vivo can potentially revolutionize the field of regenerative medicine. To address this unmet need, we designed an artificial biological &ldquo, switch&rdquo, that consists of two parts: (1) the electromagnetic perceptive gene (EPG) and (2) magnetic particles. Our group has recently cloned the EPG from the Kryptopterus bicirrhis (glass catfish). The EPG gene encodes a putative membrane-associated protein that responds to electromagnetic fields (EMFs). This gene&rsquo, s primary mechanism of action is to raise the intracellular calcium levels or change in flux through EMF stimulation. Here, we developed a system for the remote regulation of [Ca2+]i (i.e., intracellular calcium ion concentration) using streptavidin-coated ferromagnetic particles (FMPs) under a magnetic field. The results demonstrated that the EPG-FMPs can be used as a molecular calcium switch to express target proteins. This technology has the potential for controlled gene expression, drug delivery, and drug developments.

Peripheral nerve injury leads to altered cortical excitation-inhibition balance which is associated with sensory dysfunctions. We tested if non-invasive repetitive transcranial magnetic stimulation (rTMS) which has shown to induce neuronal excitability, and cell-specific magnetic activation via the Electromagnetic-perceptive gene (EPG) which is a novel gene that was identified and cloned from Kryptopterrus bicirrhis and demonstrated to evoke neural responses when magnetically stimulated, can restore cortical excitability. A battery of behavioral tests, fMRI and immunochemistry were performed in the weeks following limb denervation in rats. The results demonstrate that neuromodulation significantly improved long-term mobility, decreased anxiety and enhanced neuroplasticity. The study also identifies the acute post-injury phase as a critical time for intervention. Moreover, the results implicate EPG as an effective cell-specific neuromodulation approach. Together, these results reinforce the growing amount of evidence from human and animal studies that are establishing neuromodulation as an effective strategy to promote plasticity and rehabilitation.

Manganese Enhanced MRI (MEMRI) utilizing different manganese chloride (MnCl2) delivery methods, has yielded valuable architectural, functional and connection information about the brain. MEMRI also has the potential in characterizing neural pathways in the spinal cord. The spinal cord grey matter is anatomically composed of nine distinct cellular laminae, where each of the laminae receives input from a specific type of neuronal population and process or serves as a relay region in a specific sensory or motor pathway. This type of laminar arrangement in the spinal cord is currently only visualized by histological methods. It is of significant interest to determine whether laminar specific enhancement by Mn2+ can be achieved in the spinal cord, as has been reported in the brain and olfactory pathway. Here we focus on using MEMRI to determine the specific laminae of the thoracic region of the spinal cord. We focus on MnCl2 changes in the ascending and descending tracts of the spinal cord. Major factors in applying this technique in the spinal cord are the ability to acquire high-resolution spinal cord images and to determine a noninvasive route of administration which will result in uptake by the central nervous system.We have applied the MEMRI approach by intraperitoneal (i.p). delivery of MnCl2 and imaged lumbar and thoracic spinal cord levels in rats to determine whether T1 weighted MRI can detect spinal cord laminae 48 hours following MnCl2 administration. T1 weighted images of the lower lumbar level were obtained from MnCl2 injected and control rats. Here we demonstrate laminar specific signal enhancement in the spinal cord of rats administered with MnCl2 vs. controls in MRI of the cord with ultra-high, 69 μm in-plane resolution. We also report reduced T1 values over time in MnCl2 groups across laminae I-IX. The regions with the largest T1 enhancements were observed to correspond to laminae that contain either high cell density or large motor neurons, making MEMRI an excellent tool for studying spinal cord architecture, physiology and function in different animal models.

Neurostimulation technologies have been shown to be effective in promoting recovery, alleviating disease symptoms, and enhancing quality of life in different neurological diseases and injuries. There is growing interest in developing neurostimulation as visual prosthetics for diseases that ultimately lead to visual impairments. However, evidence shows that the implantation of electrodes in the visual cortex, retina, and optic nerve which are the primary targets for visual-based neurostimulation, can induce tissue damage unspecific stimulation and low perceptual spatial resolution to reproduce original images. We have recently developed an innovative, non-invasive method to control cellular function using the electromagnetic-perceptive gene (EPG). This magnetogenetic-based technology complements the existing arsenal of neurostimulation technologies and provides a novel approach for cell-specific, temporal-specific, location-specific and non-invasive neurostimulation. We worked towards developing an apparatus that will allow for behavioral testing in awake rats during magnetogenetic stimulation of the visual cortex. We designed and built a new behavioral rodent apparatus that provides direct and controlled contact with the head using non-conductive and non-ferrous materials. This apparatus allows the rat to press one of two levers depending on visual cues and magnetogenetic stimulation. In addition, this new apparatus will allow for testing of behavioral responses to cortical stimulation controlled by other neurostimulation devices, including non-invasive brain stimulation, optogenetics, ultrasound, and electrodes in awake, restrained rats.

The Kryptopterus bicirrhis (glass catfish) is known to respond to electromagnetic fields (EMF). Here we tested its avoidance behavior in response to static and alternating magnetic fields stimulation. Using expression cloning we identified an electromagnetic perceptive gene (EPG) from the K. bicirrhis encoding a protein that responds to EMF. This EPG gene was cloned and expressed in mammalian cells, neuronal cultures and in rat’s brain. Immunohistochemistry showed that the expression of EPG is confined to the mammalian cell membrane. Calcium imaging in mammalian cells and cultured neurons expressing EPG demonstrated that remote activation by EMF significantly increases intracellular calcium concentrations, indicative of cellular excitability. Moreover, wireless magnetic activation of EPG in rat motor cortex induced motor evoked responses of the contralateral forelimb in vivo. Here we report on the development of a new technology for remote, non-invasive modulation of cell function.

Background: Therapeutic strategies for traumatic brain injury (TBI) in the last three decades have failed to show significant benefit in large scale studies. Given the multitude of pathological mechanisms involved in TBI, strategies focusing on multimodality regimen have gained interest as promising future interventions. Hypothesis: We hypothesized that combining noninvasive transcranial magnetic stimulation (TMS) with rehabilitative training in an environmental enrichment (EE) can facilitate post-TBI recovery in rats via cortical excitability and reorganization. Methods: We subjected rats to controlled cortical impact, and then assigned them to one of four groups: 1. No treatments (TBI), 2. EE after injury (TBI + EE), 3. TMS for one week (TBI + TMS), and 4. TMS for one week combined with EE (TBI + TMS/EE). For TMS, a 10 Hz repetitive TMS protocol was used. Results: At 7 days, TBI + TMS and TBI + TMS/EE groups had significantly increased primary somatosensory cortex local field potential (LFP) compared to TBI and TBI + EE groups (P

Interhemispheric interaction has a major role in various neurobehavioral functions. Its disruption is a major contributor to the pathological changes in the setting of brain injury such as traumatic brain injury, peripheral nerve injury, and stroke, as well as neurodegenerative diseases. Because interhemispheric interaction has a crucial role in functional consequence in these neuropathological states, a review of noninvasive and state-of-the-art molecular based neuromodulation methods that focus on or have the potential to elucidate interhemispheric interaction have been performed. This yielded approximately 170 relevant articles on human subjects or animal models. There has been a recent surge of reports on noninvasive methods such as transcranial magnetic stimulation and transcranial direct current stimulation. Since these are noninvasive techniques with little to no side effects, their widespread use in clinical studies can be easily justified. The overview of novel neuromodulation methods and how they can be applied to study the role of interhemispheric communication in neural injury and neurodegenerative disease is provided. Additionally, the potential of each method in therapeutic use as well as investigating the pathophysiology of interhemispheric interaction in neurodegenerative diseases and brain injury is discussed. New technologies such as transcranial magnetic stimulation or transcranial direct current stimulation could have a great impact in understanding interhemispheric pathophysiology associated with acquired injury and neurodegenerative diseases, as well as designing improved rehabilitation therapies. Also, advances in molecular based neuromodulation techniques such as optogenetics and other chemical, thermal, and magnetic based methods provide new capabilities to stimulate or inhibit a specific brain location and a specific neuronal population.

Nerve injury induces long-term changes in neuronal activity in the primary somatosensory cortex (S1), which has often been implicated as the origin of sensory dysfunction. However, the cellular mechanisms underlying this phenomenon remain unclear. C-fos is an immediate early gene, which has been shown to play an instrumental role in plasticity. By developing a new platform to image real-time changes in gene expression in vivo, we investigated whether injury modulates the levels of c-fos in layer V of S1, since previous studies have suggested that these neurons are particularly susceptible to injury. The yellow fluorescent protein, ZsYellow1, under the regulation of the c-fos promoter, was expressed throughout the rat brain. A fiber-based confocal microscope that enabled deep brain imaging was utilized, and local field potentials were collected simultaneously. In the weeks following limb denervation in adult rats (n = 10), sensory stimulation of the intact limb induced significant increases in c-fos gene expression in cells located in S1, both contralateral (affected, 27.6 ± 3 cells) and ipsilateral (8.6 ± 3 cells) to the injury, compared to controls (n = 10, 13.4 ± 3 and 1.0 ± 1, respectively, p value

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.

Background. Peripheral nerve injury leads to changes in neuronal activity in the contralateral and ipsilateral primary somatosensory cortices (S1), which may lead to enduring sensory dysfunction and pain. Plasticity in the barrel and visual cortices has been shown to occur in a layer-specific manner. However, little is known about the layer specific changes associated with limb injury. Objective. To determine the layer-specific changes in neuronal activity associated with short-term plasticity induced by peripheral nerve injury in the rat. Methods. In vivo electrophysiology recordings (multiunit activity and local field potential) and high-resolution functional magnetic resonance imaging techniques were applied to characterize neuronal and hemodynamic responses across the depth of S1 contralateral and ipsilateral to the injury. Results. Within 60 minutes following injury, atypical increases in neuronal and hemodynamic responses in the deprived S1, ipsilateral to the noninjured limb, were observed in response to stimulation of the noninjured limb. The most prominent increases in neuronal activity in the deprived S1 occurred in layer V. Conclusion. Layer V neurons provide the major output of S1 and they send and receive transcallosal input. Thus, the immediate changes in neuronal firing patterns in layer V induced by the injury, can adversely affect the activity of subcortical regions and also interfere with normal cortical processing and interhemispheric communication. Therefore, a rehabilitation strategy that targets layer V neurons activity and starts immediately after the injury may benefit the functional outcome.

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.

Repetitive Transcranial Magnetic Stimulation (rTMS) has been successfully used as a non-invasive therapeutic intervention for several neurological disorders in the clinic as well as an investigative tool for basic neuroscience. rTMS has been shown to induce long-term changes in neuronal circuits in vivo. Such long-term effects of rTMS have been investigated using behavioral, imaging, electrophysiological, and molecular approaches, but there is limited understanding of the immediate effects of TMS on neurons. We investigated the immediate effects of high frequency (20 Hz) rTMS on the activity of cortical neurons in an effort to understand the underlying cellular mechanisms activated by rTMS. We used whole-cell patch-clamp recordings in acute rat brain slices and calcium imaging of cultured primary neurons to examine changes in neuronal activity and intracellular calcium respectively. Our results indicate that each TMS pulse caused an immediate and transient activation of voltage gated sodium channels (9.6 ± 1.8 nA at -45 mV, p value < 0.01) in neurons. Short 500 ms 20 Hz rTMS stimulation induced action potentials in a subpopulation of neurons, and significantly increased the steady state current of the neurons at near threshold voltages (at -45 mV: before TMS: I = 130 ± 17 pA, during TMS: I = 215 ± 23 pA, p value = 0.001). rTMS stimulation also led to a delayed increase in intracellular calcium (153.88 ± 61.94% increase from baseline). These results show that rTMS has an immediate and cumulative effect on neuronal activity and intracellular calcium levels, and suggest that rTMS may enhance neuronal responses when combined with an additional motor, sensory or cognitive stimulus. Thus, these results could be translated to optimize rTMS protocols for clinical as well as basic science applications.

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.

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.