Rapid recovery and altered neurochemical dependence of locomotor central pattern generation following lumbar neonatal spinal cord injury.

KEY POINTS: Spinal compression injury targeted to the neonatal upper lumbar spinal cord, the region of highest hindlimb locomotor rhythmogenicity, leads to an initial paralysis of the hindlimbs. Behavioural recovery is evident within a few days and approaches normal function within about 3 weeks. Fictive locomotion in the isolated injured spinal cord cannot be elicited by a neurochemical cocktail containing NMDA, dopamine and serotonin 1 day post-injury, but can 3 days post-injury as readily as in the uninjured spinal cord. Low frequency coordinated rhythmic activity can be elicited in the isolated uninjured spinal cord by NMDA + dopamine (without serotonin), but not in the isolated injured spinal cord. In both the injured and uninjured spinal cord, eliciting bona fide fictive locomotion requires the additional presence of serotonin. ABSTRACT: Following incomplete compression injury in the thoracic spinal cord of neonatal mice 1 day after birth (P1), we previously reported that virtually normal hindlimb locomotor function is recovered within about 3 weeks despite substantial permanent thoracic tissue loss. Here, we asked whether similar recovery occurs following lumbar injury that impacts more directly on the locomotor central pattern generator (CPG). As in thoracic injuries, lumbar injuries caused about 90% neuronal loss at the injury site and increased serotonergic innervation below the injury. Motor recovery was slower after lumbar than thoracic injury, but virtually normal function was attained by P25 in both cases. Locomotor CPG status was tested by eliciting fictive locomotion in isolated spinal cords using a widely used neurochemical cocktail (NMDA, dopamine, serotonin). No fictive locomotion could be elicited 1 day post-injury, but could within 3 days post-injury as readily as in age-matched uninjured control spinal cords. Burst patterning and coordination were largely similar in injured and control spinal cords but there were differences. Notably, in both groups there were two main locomotor frequencies, but injured spinal cords exhibited a shift towards the higher frequency. Injury also altered the neurochemical dependence of locomotor CPG output, such that injured spinal cords, unlike control spinal cords, were incapable of generating low frequency rhythmic coordinated activity in the presence of NMDA and dopamine alone. Thus, the neonatal spinal cord also exhibits remarkable functional recovery after lumbar injuries, but the neurochemical sensitivity of locomotor circuitry is modified in the process.

Inhibition of tumor formation and redirected differentiation of glioblastoma cells in a xenotypic embryonic environment.

BACKGROUND: Tissue microenvironment plays key roles in regulating the progression of aggressive tumors. Tumors are uncommon in the early embryo, suggesting that embryonic tissue microenvironments are nonpermissive for tumors. Yet, the effects of embryonic tissue microenvironments on tumor cells have not been extensively studied. We have, therefore, tested the behavior of human glioblastoma multiforme (GBM) cells transplanted into a central neural tissue microenvironment in the chicken embryo. RESULTS: GBM cells were cultured as spheres to enrich for GBM stem cells (GSCs) and transduced with GFP for identification. Within the proliferative embryonic neural tissue, GSC-enriched GBM cells exhibited reduced proliferation and survival, altered gene expression, and formed no tumors, in marked contrast to their aggressive behavior in vitro and tumor formation in other tissue microenvironments including the chorioallantoic membrane of the chicken embryo and the brain of adult severe combined immunodeficiency (SCID) mice. Surviving cells in the spinal neural tube exhibited tumor-atypical expression profiles of neuron-, glia-, stem cell-, and tumor-related genes. CONCLUSIONS: Embryonic neural tissue provides a poor environment for GBM cell survival and tumor formation, and redirects differentiation toward a more benign phenotype. Understanding the anti-tumorigenic effects of this embryonic tissue microenvironment could provide opportunities to develop novel therapies for GBM treatment.

From wires to waves, a novel sensor system for in vivo pressure monitoring.

Pressure monitoring in various organs of the body is essential for appropriate diagnostic and therapeutic purposes. In almost all situations, monitoring is performed in a hospital setting. Technological advances not only promise to improve clinical pressure monitoring systems, but also engage toward the development of fully implantable systems in ambulatory patients. Such systems would not only provide longitudinal time monitoring to healthcare personnel, but also to the patient who could adjust their way-of-life in response to the measurements. In the past years, we have developed a new type of piezoresistive pressure sensor system. Different bench tests have demonstrated that it delivers precise and reliable pressure measurements in real-time. The potential of this system was confirmed by a continuous recording in a patient that lasted for almost a day. In the present study, we further characterized the functionality of this sensor system by conducting in vivo implantation experiments in nine female farm pigs. To get a step closer to a fully implantable system, we also adapted two different wireless communication solutions to the sensor system. The communication protocols are based on MICS (Medical Implant Communication System) and BLE (Bluetooth Low Energy) communication. As a proof-of-concept, implantation experiments in nine female pigs demonstrated the functionality of both systems, with a notable technical superiority of the BLE.

Integrated software for multi-dimensional analysis of motion using tracking, electrophysiology, and sensor signals.

Tracking followed by analysis of specific point-of-interest from conventional or high-speed video recordings have been widely used for decades in various scientific disciplines such as sport, physiotherapy, and behavioral science. Another method used to characterize movement in 3D involves the use of motion capture systems, which produce files containing a collection of 3D-coordinates and corresponding timestamps. When studying animal or human movement, combining motion tracking with other recording methods-like monitoring muscle activity or sensor signals-can yield valuable insights. However, manual analysis of data from these diverse sources can be time-consuming and prone to errors. To address this issue, this article introduces a new, free, and open-source software developed in MATLAB. This software can be used as-is, or developed further to meet specific requirements. Once the coordinates are imported, multiple tools can be used for data preprocessing, such as to correct mistakes that may have occurred during tracking because of software errors or suboptimal video quality. In addition, the software can import coordinates from multiple cameras and combine them into a unified data series. With these inputs, the software can automatically calculate kinematic parameters and descriptive statistics, generate 2D and 3D animations, and analyze gait cycles, enabling swift and accurate analysis of multidimensional motion data. Moreover, the software can import electrophysiology traces and sensor signals, which can be filtered, rectified, smoothed, and correlated with the kinematic data in various ways. Thanks to its user-friendly graphical user interface, the software is easy to navigate and can be used to analyze complex movements without any need for coding skills. This versatile tool is well-suited for a wide range of experimental contexts, making it a valuable resource for researchers across diverse scientific disciplines.

How to generate graded spinal cord injuries in swine - tools and procedures.

Spinal cord injury (SCI) is a medically, psychologically and socially disabling condition. A large body of our knowledge on the basic mechanisms of SCI has been gathered in rodents. For preclinical validation of promising therapies, the use of animal models that are closer to humans has several advantages. This has promoted the more-intensive development of large-animal models for SCI during the past decade. We recently developed a multimodal SCI apparatus for large animals that generated biomechanically reproducible impacts in vivo. It is composed of a spring-load impactor and support systems for the spinal cord and the vertebral column. We now present the functional outcome of farm pigs and minipigs injured with different lesion strengths. There was a correlation between the biomechanical characteristics of the impact, the functional outcome and the tissue damage observed several weeks after injury. We also provide a detailed description of the procedure to generate such a SCI in both farm pigs and minipigs, in the hope to ease the adoption of the swine model by other research groups.

Vesicular glutamate and GABA transporters sort to distinct sets of vesicles in a population of presynaptic terminals.

Vesicular glutamate transporters (VGLUTs) 1 and 2 are expressed by neurons generally accepted to release glutamate as a neurotransmitter, whereas VGLUT3 appears in populations usually associated with a different classical transmitter. We now demonstrate VGLUT2 as well as the vesicular GABA transporter (VGAT) in a subset of presynaptic terminals in the dentate gyrus of the rat hippocampal formation. The terminals are distributed in a characteristic band overlapping with the outer part of the granule cell layer and the inner zone of the molecular layer. Within the terminals, which make asymmetric as well as symmetric synapses onto the somatodendritic compartment of the dentate granule cells, the 2 transporters localize to distinct populations of synaptic vesicles. Moreover, the axons forming these terminals originate in the supramammillary nucleus (SuM). Our data reconcile previous apparently conflicting reports on the physiology of the dentate afferents from SuM and demonstrate that both glutamate and GABA may be released from a single nerve terminal.

Author Correction: Onecut-dependent Nkx6.2 transcription factor expression is required for proper formation and activity of spinal locomotor circuits.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

In vivo Cell Tracking Using Non-invasive Imaging of Iron Oxide-Based Particles with Particular Relevance for Stem Cell-Based Treatments of Neurological and Cardiac Disease.

Stem cell-based therapeutics is a rapidly developing field associated with a number of clinical challenges. One such challenge lies in the implementation of methods to track stem cells and stem cell-derived cells in experimental animal models and in the living patient. Here, we provide an overview of cell tracking in the context of cardiac and neurological disease, focusing on the use of iron oxide-based particles (IOPs) visualized in vivo using magnetic resonance imaging (MRI). We discuss the types of IOPs available for such tracking, their advantages and limitations, approaches for labeling cells with IOPs, biological interactions and effects of IOPs at the molecular and cellular levels, and MRI-based and associated approaches for in vivo and histological visualization. We conclude with reviews of the literature on IOP-based cell tracking in cardiac and neurological disease, covering both preclinical and clinical studies.

Onecut-dependent Nkx6.2 transcription factor expression is required for proper formation and activity of spinal locomotor circuits.

In the developing spinal cord, Onecut transcription factors control the diversification of motor neurons into distinct neuronal subsets by ensuring the maintenance of Isl1 expression during differentiation. However, other genes downstream of the Onecut proteins and involved in motor neuron diversification have remained unidentified. In the present study, we generated conditional mutant embryos carrying specific inactivation of Onecut genes in the developing motor neurons, performed RNA-sequencing to identify factors downstream of Onecut proteins in this neuron population, and employed additional transgenic mouse models to assess the role of one specific Onecut-downstream target, the transcription factor Nkx6.2. Nkx6.2 expression was up-regulated in Onecut-deficient motor neurons, but strongly downregulated in Onecut-deficient V2a interneurons, indicating an opposite regulation of Nkx6.2 by Onecut factors in distinct spinal neuron populations. Nkx6.2-null embryos, neonates and adult mice exhibited alterations of locomotor pattern and spinal locomotor network activity, likely resulting from defective survival of a subset of limb-innervating motor neurons and abnormal migration of V2a interneurons. Taken together, our results indicate that Nkx6.2 regulates the development of spinal neuronal populations and the formation of the spinal locomotor circuits downstream of the Onecut transcription factors.

Unique luminal localization of VGAT-C terminus allows for selective labeling of active cortical GABAergic synapses.

Neurotransmitter uptake into synaptic vesicles is mediated by vesicular neurotransmitter transporters. Although these transporters belong to different families, they all are thought to share a common overall topology with an even number of transmembrane domains. Using epitope-specific antibodies and mass spectrometry we show that the vesicular GABA transporter (VGAT) possesses an uneven number of transmembrane domains, with the N terminus facing the cytoplasm and the C terminus residing in the synaptic vesicle lumen. Antibodies recognizing the C terminus of VGAT (anti-VGAT-C) selectively label GABAergic nerve terminals of live cultured hippocampal and striatal neurons as confirmed by immunocytochemistry and patch-clamp electrophysiology. Injection of fluorochromated anti-VGAT-C into the hippocampus of mice results in specific labeling of GABAergic synapses in vivo. Overall, our data open the possibility of studying novel GABA release sites, characterizing inhibitory vesicle trafficking, and establishing their contribution to inhibitory neurotransmission at identified GABAergic synapses.

Locomotor central pattern generator excitability states and serotonin sensitivity after spontaneous recovery from a neonatal lumbar spinal cord injury.

The spinal locomotor central pattern generator (CPG) in neonatal mice exhibits diverse output patterns, ranging from sub-rhythmic to multi-rhythmic to fictive locomotion, depending on its general level of excitation and neuromodulatory status. We have recently reported that the locomotor CPG in neonatal mice rapidly recovers the ability to produce neurochemically induced fictive locomotion following an upper lumbar spinal cord compression injury. Here we address the question of recovery of multi-rhythmic activity and the serotonin-sensitivity of the CPG. In isolated spinal cords from control and 3 days post-injury mice, application of dopamine and NMDA elicited multi-rhythmic activity with slow and fast components. The slow component comprised 10-20 s episodes of activity that were synchronous in ipsilateral or all lumbar ventral roots, and the fast components involved bursts within these episodes that displayed coordinated patterns of alternation between ipsilateral roots. Rhythm strength was the same in control and injured spinal cords. However, power spectral analysis of signal within episodes showed a reduced peak frequency after recovery. In control spinal cords, serotonin triggered fictive locomotion only when applied at high concentration (30 µM, constant NMDA). By contrast, in about 50% of injured preparations fictive locomotion was evoked by 2-3 times lower serotonin concentrations (10-15 µM). This increased serotonin sensitivity was correlated with post-injury changes in the expression of specific serotonin receptor transcripts, but not of dopamine receptor transcripts.

Development of a Multimodal Apparatus to Generate Biomechanically Reproducible Spinal Cord Injuries in Large Animals.

Rodents are widespread animal models in spinal cord injury (SCI) research. They have contributed to obtaining important information. However, some treatments only tested in rodents did not prove efficient in clinical trials. This is probably a result of significant differences in the physiology, anatomy, and complexity between humans and rodents. To bridge this gap in a better way, a few research groups use pig models for SCI. Here we report the development of an apparatus to perform biomechanically reproducible SCI in large animals, including pigs. We present the iterative process of engineering, starting with a weight-drop system to ultimately produce a spring-load impactor. This device allows a graded combination of a contusion and a compression injury. We further engineered a device to entrap the spinal cord and prevent it from escaping at the moment of the impact. In addition, it provides identical resistance around the cord, thereby, optimizing the inter-animal reproducibility. We also present other tools to straighten the vertebral column and to ease the surgery. Sensors mounted on the impactor provide information to assess the inter-animal reproducibility of the impacts. Further evaluation of the injury strength using neurophysiological recordings, MRI scans, and histology shows consistency between impacts. We conclude that this apparatus provides biomechanically reproducible spinal cord injuries in pigs.

Pharmacology of neurotransmitter transport into secretory vesicles.

Many neuropsychiatric disorders appear to involve a disturbance of chemical neurotransmission, and the mechanism of available therapeutic agents supports this impression. Postsynaptic receptors have received considerable attention as drug targets, but some of the most successful agents influence presynaptic processes, in particular neurotransmitter reuptake. The pharmacological potential of many other presynaptic elements, and in particular the machinery responsible for loading transmitter into vesicles, has received only limited attention. The similarity of vesicular transporters to bacterial drug resistance proteins and the increasing evidence for regulation of vesicle filling and recycling suggest that the pharmacological potential of vesicular transporters has been underestimated. In this review, we discuss the pharmacological effects of psychostimulants and therapeutic agents on transmitter release.

Changes in vesicular transporters for gamma-aminobutyric acid and glutamate reveal vulnerability and reorganization of hippocampal neurons following pilocarpine-induced seizures.

The reorganizations of the overall intrinsic glutamatergic and gamma-aminobutyric acid (GABA)-ergic hippocampal networks as well as the time course of these reorganizations during development of pilocarpine-induced temporal lobe epilepsy were studied with in situ hybridization and immunohistochemistry experiments for the vesicular glutamate transporter 1 (VGLUT1) and the vesicular GABA transporter (VGAT). These transporters are particularly interesting as specific markers for glutamatergic and GABAergic neurons, respectively, whose expression levels could reflect the demand for synaptic transmission and their average activity. We report that 1) concomitantly with the loss of some subpopulations of VGAT-containing neurons, there was an up-regulation of VGAT synthesis in all remaining GABA neurons as early as 1 week after pilocarpine injection. This enhanced synthesis is characterized by marked increases in the relative amount of VGAT mRNAs in interneurons associated with increased intensity of axon terminal labeling for VGAT in all hippocampal layers. 2) There was a striking loss of mossy cells during the latent period, demonstrated by a long-term decrease of VGLUT1 mRNA-containing hilar neurons and associated loss of VGLUT1-containing terminals in the dentate gyrus inner molecular layer. 3) There were aberrant VGLUT1-containing terminals at the chronic stage resulting from axonal sprouting of granule and pyramidal cells. This is illustrated by a recovery of VGLUT1 immunoreactivity in the inner molecular layer and an increased VGLUT1 immunolabeling in the CA1-CA3 dendritic layers. These data indicate that an increased activity of remaining GABAergic interneurons occurs during the latent period, in parallel with the loss of vulnerable glutamatergic and GABAergic neurons preceding the reorganization of glutamatergic networks.

Cellular reactions and compensatory tissue re-organization during spontaneous recovery after spinal cord injury in neonatal mice.

Following incomplete spinal cord injuries, neonatal mammals display a remarkable degree of behavioral recovery. Previously, we have demonstrated in neonatal mice a wholesale re-establishment and reorganization of synaptic connections from some descending axon tracts (Boulland et al.: PLoS One 8 (2013)). To assess the potential cellular mechanisms contributing to this recovery, we have here characterized a variety of cellular sequelae following thoracic compression injuries, focusing particularly on cell loss and proliferation, inflammation and reactive gliosis, and the dynamics of specific types of synaptic terminals. Early during the period of recovery, regressive events dominated. Tissue loss near the injury was severe, with about 80% loss of neurons and a similar loss of axons that later make up the white matter. There was no sign of neurogenesis, no substantial astroglial or microglial proliferation, no change in the ratio of M1 and M2 microglia and no appreciable generation of the terminal complement peptide C5a. One day after injury the number of synaptic terminals on lumbar motoneurons had dropped by a factor of 2, but normalized by 6 days. The ratio of VGLUT1/2+ to VGAT+ terminals remained similar in injured and uninjured spinal cords during this period. By 24 days after injury, when functional recovery is nearly complete, the density of 5-HT+ fibers below the injury site had increased by a factor of 2.5. Altogether this study shows that cellular reactions are diverse and dynamic. Pronounced recovery of both excitatory and inhibitory terminals and an increase in serotonergic innervation below the injury, coupled with a general lack of inflammation and reactive gliosis, are likely to contribute to the recovery. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 928-946, 2017.

Distribution of vesicular glutamate transporters 1 and 2 in the rat spinal cord, with a note on the spinocervical tract.

To evaluate whether the organization of glutamatergic fibers systems in the lumbar cord is also evident at other spinal levels, we examined the immunocytochemical distribution of vesicle glutamate transporters 1 and 2 (VGLUT1, VGLUT2) at several different levels of the rat spinal cord. We also examined the expression of VGLUTs in an ascending sensory pathway, the spinocervical tract, and colocalization of VGLUT1 and VGLUT2. Mainly small VGLUT2-immunoreactive varicosities occurred at relatively high densities in most areas, with the highest density in laminae I-II. VGLUT1 immunolabeling, including small and medium-sized to large varicosities, was more differentiated, with the highest density in the deep dorsal horn and in certain nuclei such as the internal basilar nucleus, the central cervical nucleus, and the column of Clarke. Lamina I and IIo displayed a moderate density of small VGLUT1 varicosities at all spinal levels, although in the spinal enlargements a uniform density of such varicosities was evident throughout laminae I-II in the medial half of the dorsal horn. Corticospinal tract axons displayed VGLUT1, indicating that the corticospinal tract is an important source of small VGLUT1 varicosities. VGLUT1 and VGLUT2 were cocontained in small numbers of varicosities in laminae III-IV and IX. Anterogradely labeled spinocervical tract terminals in the lateral cervical nucleus were VGLUT2 immunoreactive. In conclusion, the principal distribution patterns of VGLUT1 and VGLUT2 are essentially similar throughout the rostrocaudal extension of the spinal cord. The mediolateral differences in VGLUT1 distribution in laminae I-II suggest dual origins of VGLUT1-immunoreactive varicosities in this region.

A Neonatal Mouse Spinal Cord Compression Injury Model.

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal cord. A major current area of research is focused on the mechanisms of adaptive plasticity that underlie spontaneous or induced functional recovery following SCI. Spontaneous functional recovery is reported to be greater early in life, raising interesting questions about how adaptive plasticity changes as the spinal cord develops. To facilitate investigation of this dynamic, we have developed a SCI model in the neonatal mouse. The model has relevance for pediatric SCI, which is too little studied. Because neural plasticity in the adult involves some of the same mechanisms as neural plasticity in early life(1), this model may potentially have some relevance also for adult SCI. Here we describe the entire procedure for generating a reproducible spinal cord compression (SCC) injury in the neonatal mouse as early as postnatal (P) day 1. SCC is achieved by performing a laminectomy at a given spinal level (here described at thoracic levels 9-11) and then using a modified Yasargil aneurysm mini-clip to rapidly compress and decompress the spinal cord. As previously described, the injured neonatal mice can be tested for behavioral deficits or sacrificed for ex vivo physiological analysis of synaptic connectivity using electrophysiological and high-throughput optical recording techniques(1). Earlier and ongoing studies using behavioral and physiological assessment have demonstrated a dramatic, acute impairment of hindlimb motility followed by a complete functional recovery within 2 weeks, and the first evidence of changes in functional circuitry at the level of identified descending synaptic connections(1).

[Glutamate, glutamine and ischaemia in the central nervous system]. / Glutamat, glutamin og iskemi i sentralnervesystemet.

BACKGROUND: This review presents basic knowledge on glutamate and glutamine homeostasis in the central nervous system and relates this knowledge to some aspects of cerebral ischaemia. RESULTS AND INTERPRETATION: The amino acid glutamate is the main excitatory neurotransmitter in the central nervous system. Following stimulation of the postsynaptic glutamate receptors, glutamate must rapidly be removed from the synaptic cleft. Whereas several other neurotransmitters are taken up directly into the presynaptic nerve terminal, glutamate is mainly transported into the surrounding glial cells. Glial glutamate can be amidated to glutamine, and since glutamine is a precursor of glutamate without being a neurotransmitter itself, it can be returned to the presynaptic neuron without eliciting new synaptic signals. In the nerve terminal, glutamine can be converted back to glutamate, thereby completing the postulated glutamate-glutamine cycle. During ischaemia, this cycle is impaired as it depends on energy-consuming membrane transport proteins and enzymes.