Freeze-Frame Imaging of Dendritic Calcium Signals With TubuTag

The extensive dendritic arbor of neurons is thought to be actively involved in the processing of information. Dendrites contain a rich diversity of ligand- and voltage-activated ion channels as well as metabotropic receptors. In addition, they are capable of releasing calcium from intracellular stores. Under specific conditions, large neurons produce calcium spikes that are locally restricted to a dendritic section. To investigate calcium signaling in dendrites, we introduce TubuTag, a genetically encoded ratiometric calcium sensor anchored to the cytoskeleton. TubuTag integrates cytoplasmic calcium signals by irreversible photoconversion from green to red fluorescence when illuminated with violet light. We used a custom two-photon microscope with a large field of view to image pyramidal neurons in CA1 at subcellular resolution. Photoconversion was strongest in the most distal parts of the apical dendrite, suggesting a gradient in the amplitude of dendritic calcium signals. As the read-out of fluorescence can be performed several hours after photoconversion, TubuTag will help investigating dendritic signal integration and calcium homeostasis in large populations of neurons.

Freeze-frame imaging of dendritic calcium signals with TubuTag

The extensive dendritic arbor of neurons is thought to be actively involved in the processing of information. Dendrites contain a rich diversity of ligand- and voltage-activated ion channels as well as metabotropic receptors. In addition, they are capable of releasing calcium from intracellular stores. Under specific conditions, large neurons produce calcium spikes that are locally restricted to a dendritic section. To investigate calcium signaling in dendrites, we introduce TubuTag, a genetically encoded calcium sensor anchored to the cytoskeleton. TubuTag integrates cytoplasmic calcium signals by irreversible photoconversion from green to red fluorescence when illuminated with violet light. To image the mm-long dendritic tree of pyramidal neurons at subcellular resolution, we used a custom two-photon microscope with a large field of view. As the read-out of fluorescence can be performed several hours after photoconversion, TubuTag will help investigating dendritic signal integration and calcium homeostasis in large populations of neurons.

Morphology of spinal ganglia of different segmentary levels in the domestic dog

The spinal ganglia, which perform the function of the first link on the afferent impulses’ way from the receptors to the central nervous system, recognize internal and external irritations, and are the first to transform them into a nervous impulse. As the representatives of the peripheral nervous system, they are some of the main objects of the studies in contemporary neuromorphology. Based on the results of anatomic, neurohistological, histochemical, morphometric and statistical methods of the studies, we conducted a complex survey, revealing the morphology of spinal ganglia of different segmental levels in the domestic dog. In particular, we determined the differences in the microscopic structure and morphometric parameters of cervical, thoracic, lumbar and sacral spinal ganglia and the ganglia of the cervical and lumbar enlargements in mature domestic dogs. The study showed that the spinal ganglia of domestic dogs can have different skeletotopy, different shape and sizes due to their species peculiarity. Also, the surveyed animals, according to the results of our studies, had the cervical and thoracic spinal ganglia of oval, while the lumbar and sacral – spindle-like shapes. According to the results of morphometry, the area of the spinal ganglia in lengthwise section differed: the smallest area belonged to the thoracic, the largest to the sacral spinal ganglia. The density of neuronal arrangement per 0.1 mm2 of the area of the spinal ganglia correlated with their sizes: the highest parameter was identified for the thoracic spinal ganglia, the lowest – for the sacral. The conducted studies revealed that histo- and cyto-structure of the spinal ganglia is characteristic of notable differentiation of the nervous cells of small sizes. Therefore, we differentiated neurons of the spinal ganglia into large, medium and small. The highest quantity of large neurons was found in the sacral ganglia, and largest amount of medium-sized neurons – in the ganglia of the lumbar enlargement. In other ganglia, small neurons dominated. Correspondingly, different nuclear-cytoplasmic ratio in these neurons was determined, indicating different extent of morphofunctional condition of nervous cells. We determined content of localization and separation of nucleic acids in histostructure of the spinal cord at the tissue and cellular levels.

Accelerated EM Connectome Reconstruction using 3D Visualization and Segmentation Graphs

Recent advances in automatic image segmentation and synapse prediction in electron microscopy (EM) datasets of the brain enable more efficient reconstruction of neural connectivity. In these datasets, a single neuron can span thousands of images containing complex tree-like arbors with thousands of synapses. While image segmentation algorithms excel within narrow fields of views, the algorithms sometimes struggle to correctly segment large neurons, which require large context given their size and complexity. Conversely, humans are comparatively good at reasoning with large objects. In this paper, we introduce several semi-automated strategies that combine 3D visualization and machine guidance to accelerate connectome reconstruction. In particular, we introduce a strategy to quickly correct a segmentation through merging and cleaving, or splitting a segment along supervoxel boundaries, with both operations driven by affinity scores in the underlying segmentation. We deploy these algorithms as streamlined workflows in a tool called Neu3 and demonstrate superior performance compared to prior work, thus enabling efficient reconstruction of much larger datasets. The insights into proofreading from our work clarify the trade-offs to consider when tuning the parameters of image segmentation algorithms.

An Integration of Deep Learning and Neuroscience for Machine Consciousness

Conscious processing is a useful aspect of brain function that can be used as a model to design artificial-intelligence devices. There are still certain computational features that our conscious brains possess, and which machines currently fail to perform those. This paper discusses the necessary elements needed to make the device conscious and suggests if those implemented, the resulting machine would likely to be considered conscious. Consciousness mainly presented as a computational tool that evolved to connect the modular organization of the brain. Specialized modules of the brain process information unconsciously and what we subjectively experience as consciousness is the global availability of data, which is made possible by a nonmodular global workspace. During conscious perception, the global neuronal work space at parieto-frontal part of the brain selectively amplifies relevant pieces of information. Supported by large neurons with long axons, which makes the long-distance connectivity possible, the selected portions of information stabilized and transmitted to all other brain modules. The brain areas that have structuring ability seem to match to a specific computational problem. The global workspace maintains this information in an active state for as long as it is needed. In this paper, a broad range of theories and specific problems have been discussed, which need to be solved to make the machine conscious. Later particular implications of these hypotheses for research approach in neuroscience and machine learning are debated.

Biography of Professor Eber Landau and his contribution to anthropology, anatomy and neurology at the universities in Estonia, Lithuania and Switzerland

This article provides an overview of the scientific activities of Professor Eber Landau (1878–1959) in Estonia, Lithuania, France, and Switzerland. In addition to providing chronological data about the academic life of Prof. Landau, in particular at the Universities of Tartu, Bern, Kaunas, and Lausanne, the article emphasizes his contribution to the development of anatomy, anthropology, histological techniques, neurohistology, and neurology. In the latter two fields, the eponyms Landau reflex, or radiopronator superius reflex, and synarmotic cells of Landau, a subtype of non-traditional large neurons in the granular layer of the cerebellar cortex, have engraved his name in medical history.

Intracerebral Inoculation of Mouse-Passaged Saffold Virus Type 3 Affects Cerebellar Development in Neonatal Mice

ABSTRACTSaffold virus (SAFV), a human cardiovirus, is occasionally detected in infants with neurological disorders, including meningitis and cerebellitis. We recently reported that SAFV type 3 isolates infect cerebellar glial cells, but not large neurons, in mice. However, the impact of this infection remained unclear. Here, we determined the neuropathogenesis of SAFV type 3 in the cerebella of neonatal ddY mice by using SAFV passaged in the cerebella of neonatal BALB/c mice. The virus titer in the cerebellum increased following the inoculation of each of five passaged strains. The fifth passaged strain harbored amino acid substitutions in the VP2 (H160R and Q239R) and VP3 (K62M) capsid proteins. Molecular modeling of the capsid proteins suggested that the VP2-H160R and VP3-K62M mutations alter the structural dynamics of the receptor binding surface via the formation of a novel hydrophobic interaction between the VP2 puff B and VP3 knob regions. Compared with the original strain, the passaged strain showed altered growth characteristics in human-derived astroglial cell lines and greater replication in the brains of neonatal mice. In addition, the passaged strain was more neurovirulent than the original strain, while both strains infected astroglial and neural progenitor cells in the mouse brain. Intracerebral inoculation of either the original or the passaged strain affected brain Purkinje cell dendrites, and a high titer of the passaged strain induced cerebellar hypoplasia in neonatal mice. Thus, infection by mouse-passaged SAFV affected cerebellar development in neonatal mice. This animal model contributes to the understanding of the neuropathogenicity of SAFV infections in infants.IMPORTANCESaffold virus (SAFV) is a candidate neuropathogenic agent in infants and children, but the neuropathogenicity of the virus has not been fully elucidated. Recently, we evaluated the pathogenicity of two clinical SAFV isolates in mice. Similar to other neurotropic picornaviruses, these isolates showed mild infectivity of glial and neural progenitor cells, but not of large neurons, in the cerebellum. However, the outcome of this viral infection in the cerebellum has not been clarified. Here, we examined the tropism of SAFV in the cerebellum. We obtained anin vivo-passaged strain from the cerebella of neonatal mice and examined its genome and its neurovirulence in the neonatal mouse brain. The passaged virus showed high infectivity and neurovirulence in the brain, especially the cerebellum, and affected cerebellar development. This unique neonatal mouse model will be helpful for elucidating the neuropathogenesis of SAFV infections occurring early in life.

Nerve Stretch Injury Induced Pain Pattern and Changes in Sensory Ganglia in a Clinically Relevant Model of Limb-Lengthening in Rabbits

We used a model of tibial lengthening in rabbits to study the postoperative pain pattern during limb-lengthening and morphological changes in the dorsal root ganglia (DRG), including alteration of substance P (SP) expression. Four groups of animals (naïve; OG: osteotomized only group; SDG/FDG: slow/fast distraction groups, with 1 mm/3 mm lengthening a day, respectively) were used. Signs of increasing postoperative pain were detected until the 10th postoperative day in OG/SDG/FDG, then they decreased in OG but remained higher in SDG/FDG until the distraction finished, suggesting that the pain response is based mainly on surgical trauma until the 10th day, while the lengthening extended its duration and increased its intensity. The only morphological change observed in the DRGs was the presence of large vacuoles in some large neurons of OG/SDG/FDG. Cell size analysis of the S1 DRGs showed no cell loss in any of the three groups; a significant increase in the number of SP-positive large DRG cells in the OG; and a significant decrease in the number of SP-immunoreactive small DRG neurons in the SDG/FDG. Faster and larger distraction resulted in more severe signs of pain sensation, and further reduced the number of SP-positive small cells, compared to slow distraction.

Age Does Matter: A Pilot Comparison of Placenta-Derived Stromal Cells for in utero Repair of Myelomeningocele Using a Lamb Model

Introduction: Fetal amniotic membranes (FM) have been shown to preserve spinal cord histology in the fetal sheep model of myelomeningocele (MMC). This study compares the effectiveness of placenta-derived mesenchymal stromal cells (PMSCs) from early-gestation versus term-gestation placenta to augment FM repair to improve distal motor function in a sheep model. Methods: Fetal lambs (n = 4) underwent surgical MMC creation followed by repair with FM patch with term-gestation PMSCs (n = 1), FM with early-gestation PMSCs (n = 1), FM only (n = 1), and skin closure only (n = 1). Histopathology and motor assessment was performed. Results: Histopathologic analysis demonstrated increased preservation of spinal cord architecture and large neurons in the lamb repaired with early-gestation cells compared to all others. Lambs repaired with skin closure only, FM alone, and term-gestation PMSCs exhibited extremely limited distal motor function; the lamb repaired with early-gestation PMSCs was capable of normal ambulation. Discussion: This pilot study is the first in vivo comparison of different gestational-age placenta-derived stromal cells for repair in the fetal sheep MMC model. The preservation of large neurons and markedly improved motor function in the lamb repaired with early-gestation cells suggest that early-gestation placental stromal cells may exhibit unique properties that augment in utero MMC repair to improve paralysis.

Regulation of AMPA and NMDA receptor-mediated EPSPs in dendritic trees of thalamocortical cells

Two main excitatory synapses are formed at the dendritic arbor of first-order nuclei thalamocortical (TC) neurons. Ascending sensory axons primarily establish contacts at large proximal dendrites, whereas descending corticothalamic fibers form synapses on thin distal dendrites. With the use of a multicomparment computational model based on fully reconstructed TC neurons from the ventroposterolateral nucleus of the cat, we compared local responses at the site of stimulation as well as somatic responses induced by both α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR)- and N-methyl-d-aspartate receptor (NMDAR)-mediated currents. We found that AMPAR-mediated responses, when synapses were located at proximal dendrites, induced a larger depolarization at the level of soma, whereas NMDAR-mediated responses were more efficient for synapses located at distal dendrites. The voltage transfer and transfer impedance were higher for NMDAR than for AMPAR activation at any location. For both types of synaptic current and for both input locations at the dendritic arbor, somatic responses were characterized by a low variability despite the large variability found in local responses in dendrites. The large neurons had overall smaller somatic responses than small neurons, but this relation was not found in local dendritic responses. We conclude that in TC cells, the dendritic location of small synaptic inputs does not play a major role in the amplitude of a somatic response, but the size of the neuron does. The variability of response amplitude between cells was much larger than the variability within cells. This suggests possible functional segregation of TC neurons of different size.