Combining Microfluidics,Optogenetics and Calcium Imaging to Study Neuronal Communication In Vitro

In this paper we report the combination of microfluidics, optogenetics and calcium imaging as a cheap and convenient platform to study synaptic communication between neuronal populations in vitro. We first show that Calcium Orange indicator is compatible in vitro with a commonly used Channelrhodopsine-2 (ChR2) variant, as standard calcium imaging conditions did not alter significantly the activity of transduced cultures of rodent primary neurons. A fast, robust and scalable process for micro-chip fabrication was developed in parallel to build micro-compartmented cultures. Coupling optical fibers to each micro-compartment allowed for the independent control of ChR2 activation in the different populations without crosstalk. By analyzing the post-stimuli activity across the different populations, we finally show how this platform can be used to evaluate quantitatively the effective connectivity between connected neuronal populations.     

Increased Neuronal Nuclear Calcium Influx in Neonatal Seizures

We hypothesized that neonatal seizures lead to increased Ca²⁺ influx (nCa²⁺I) in neuronal nuclei of newborn rats and that such increase is nitric-oxide mediated. Neuronal nuclear ⁴⁵Ca²⁺ influx (nCa²⁺I) was measured in neuronal nuclei of 25 10-day-old male rat-pups newborn brains. They were divided into five groups (n = 5/group). (I) control; (II) hypoxia without seizures; (III) hypoxia with seizures; (IV) kainate, 2 mg/kg intraperitoneal (i.p.)-induced seizures and (V) 7-nitroindazole (7-NINA), 1 mg/kg i.p. pretreated, kainate-induced seizures. nCa²⁺I was significantly (P < 0.05) increased following hypoxia or seizures (hypoxic- or kainate-induced). Post-hypoxic seizures further enhanced nCa²⁺I increase induced by hypoxia (P < 0.05). 7-NINA abated the nCa²⁺I increase induced by kainate. We conclude that (1) kainate or hypoxia-induced seizures in newborn rats modify the neuronal nuclear membrane function, resulting in increased nCa²⁺I, (2) seizures exacerbate the hypoxia-induced increased nCa²⁺I incurred after hypoxia and (3) intranuclear calcium surges during kainate-induced neonatal seizures are nitric oxide-mediated.     

Voltage-sensitive Dye Recording from Axons,Dendrites and Dendritic Spines of Individual Neurons in Brain Slices

Understanding the biophysical properties and functional organization of single neurons and how they process information is fundamental for understanding how the brain works. The primary function of any nerve cell is to process electrical signals, usually from multiple sources. Electrical properties of neuronal processes are extraordinarily complex, dynamic, and, in the general case, impossible to predict in the absence of detailed measurements. To obtain such a measurement one would, ideally, like to be able to monitor, at multiple sites, subthreshold events as they travel from the sites of origin on neuronal processes and summate at particular locations to influence action potential initiation. This goal has not been achieved in any neuron due to technical limitations of measurements that employ electrodes. To overcome this drawback, it is highly desirable to complement the patch-electrode approach with imaging techniques that permit extensive parallel recordings from all parts of a neuron. Here, we describe such a technique - optical recording of membrane potential transients with organic voltage-sensitive dyes (V_m-imaging) - characterized by sub-millisecond and sub-micrometer resolution. Our method is based on pioneering work on voltage-sensitive molecular probes ². Many aspects of the initial technology have been continuously improved over several decades ^{3, 5, 11}. Additionally, previous work documented two essential characteristics of V_m-imaging. Firstly, fluorescence signals are linearly proportional to membrane potential over the entire physiological range (-100 mV to +100 mV; ^{10, 14, 16}). Secondly, loading neurons with the voltage-sensitive dye used here (JPW 3028) does not have detectable pharmacological effects. The recorded broadening of the spike during dye loading is completely reversible ^{4, 7}. Additionally, experimental evidence shows that it is possible to obtain a significant number (up to hundreds) of recordings prior to any detectable phototoxic effects ^{4, 6, 12, 13}. At present, we take advantage of the superb brightness and stability of a laser light source at near-optimal wavelength to maximize the sensitivity of the V_m-imaging technique. The current sensitivity permits multiple site optical recordings of V_m transients from all parts of a neuron, including axons and axon collaterals, terminal dendritic branches, and individual dendritic spines. The acquired information on signal interactions can be analyzed quantitatively as well as directly visualized in the form of a movie.     

Inferring Neuronal Dynamics from Calcium Imaging Data Using Biophysical Models and Bayesian Inference

Calcium imaging has been used as a promising technique to monitor the dynamic activity of neuronal populations. However, the calcium trace is temporally smeared which restricts the extraction of quantities of interest such as spike trains of individual neurons. To address this issue, spike reconstruction algorithms have been introduced. One limitation of such reconstructions is that the underlying models are not informed about the biophysics of spike and burst generations. Such existing prior knowledge might be useful for constraining the possible solutions of spikes. Here we describe, in a novel Bayesian approach, how principled knowledge about neuronal dynamics can be employed to infer biophysical variables and parameters from fluorescence traces. By using both synthetic and in vitro recorded fluorescence traces, we demonstrate that the new approach is able to reconstruct different repetitive spiking and/or bursting patterns with accurate single spike resolution. Furthermore, we show that the high inference precision of the new approach is preserved even if the fluorescence trace is rather noisy or if the fluorescence transients show slow rise kinetics lasting several hundred milliseconds, and inhomogeneous rise and decay times. In addition, we discuss the use of the new approach for inferring parameter changes, e.g. due to a pharmacological intervention, as well as for inferring complex characteristics of immature neuronal circuits.     

Resonantlike Synchronization and Bursting in a Model of Pulse-Coupled Neurons with Active Dendrites

We analyze the dynamical effects of active, linearized dendritic membranes on the synchronization properties of neuronal interactions. We show that a pair of pulse-coupled integrate-and-fire neurons interacting via active dendritic cables can exhibit resonantlike synchronization when the frequency of the oscillators is approximately matched to the resonant frequency of the membrane impedance. For weak coupling the neurons are phase-locked with constant interspike intervals whereas for strong coupling periodic bursting patterns are observed. This bursting behavior is reflected by the occurrence of a Hopf bifurcation in the firingrates of a corresponding rate-coded model.     

Growth of Ectopic Dendrites on Cortical Pyramidal Neurons in Neuronal Storage Diseases Correlates with Abnormal Accumulation of GM2 Ganglioside

Abstract: Ganglioside analysis and quantitative Golgi studies of the cerebral cortex of cats with ganglioside and nonganglioside lysosomal storage diseases reveal a correlation between the amount of accumulated GM2 ganglioside and the extent of ectopic dendrite growth on cortical pyramidal neurons. This correlation was not observed with any of the other gangliosides assayed for, including GM1 ganglioside. These results suggest a specific role for GM2 ganglioside in the initiation of ectopic neurites on pyramidal cells in vivo and are consistent with the developing hypothesis that different gangliosides have specific roles in different cell types dependent upon the receptor or other effector molecules with which they may interact.     

A Biobased Composite Gel Polymer Electrolyte with Functions of Lithium Dendrites Suppressing and Manganese Ions Trapping

Lithium (Li) dendrites in Li anodes, and dissolution and migration of manganese (Mn) ions in LiMn₂O₄ (LMO) cathodes, have hampered these extraordinary electrode materials from being efficiently applied in high performance Li batteries. Here, a novel, bifunctional, biobased composite gel polymer electrolyte (c‐GPE) is created to simultaneously deal with the two critical issues. The skeleton of c‐GPE is constructed from a sandwich structure composed of porous polydopamine spheres and two layers of the environmentally friendly soy protein isolate‐based nanofiber membranes, and the carbonized polydopamine spheres are coated without any binder on the surface of the membranes. After a facile and innocuous preparation process, the skeleton material displays excellent thermal stability and good affinity to liquid electrolyte, which endows c‐GPE with significant functions of effective mitigation of the dissolution of Mn ions, and chelation of the fleeing Mn ions, as well as the dramatic suppression of Li dendrite growth. Consequently, the LMO/Li batteries involving c‐GPE show a great improvement in the cycling stability and rate performance compared with those of the cells based on commercial Celgard 2400. This work will be quite promising to meet the distinct requirements from Li batteries and provide a high‐efficiency and safe biobased GPE for next generation energy storage systems.     

Lipid Composition of Neuronal Cell Bodies and Neurites from Cultured Dorsal Root Ganglia

Abstract: The lipid composition of neuronal somata and neuritic processes of cultured root ganglia has been determined. Neuronal soma contained 37% of dry weight as lipid (15.4% cholesterol, 4.8% galactolipid, and 57.1% phospholipid). The major phospholipids were phosphatidylcholine and phosphatidyl ethanolamine. Galactolipids consisted of cerebroside and sulfatide in molar ratio 2:1. The neuronal soma contained tetrasialo-, disialo-, and monosialoganglioside. In contrast, neurites contained 15% of the dry weight as lipid (22.1% cholesterol, 7.7% galactolipid with cerebroside and sulfatide in molar ratio 2:1, and 56.4% total phospholipid). The neuritic galactolipid content was higher, as was the percentage of sphingomyelin, and phosphatidyl serine. The higher cholesterol content in neuritic lipid reflected the higher percentage of plasma membrane in this compartment. The ganglioside pattern of neurites was distinct from that of the neuronal soma and consisted entirely of gangliosides GQ1b, GT1b, GD1b, GD1a, and GD3, with no monosialogangliosides. The results indicate a preferential phospholipid and glycolipid sorting to the neuritic plasma membrane that may be related to the distinctive functions of this neuronal compartment.     

Depletion of Neuronal Endoplasmic Reticulum Calcium Stores by Thapsigargin: Effect on Protein Synthesis

Abstract: We have used thapsigargin (TG), a specific, irreversible inhibitor of endoplasmic reticulum (ER) Ca²⁺-ATPases, and caffeine, an agonist of the ryanodine receptor, to study the effect of emptying of ER calcium stores on protein synthesis in neuronal cells. TG at 1 µ M caused a permanent inhibition of protein synthesis in hippocampal slices from 3-week-old rats but no inhibition in slices prepared from 2-month-old animals. Caffeine at 10 m M caused a reduction of protein synthesis in both 3-week- and 2-month-old rats immediately after exposure, but complete recovery of protein synthesis occurred within 30 min after treatment. In neuronal cells, TG produced an almost complete inhibition of protein synthesis that was only partially reversed over a 24-h recovery period. TG did not significantly affect neuronal ATP levels or energy charge. Fifty percent inhibition of protein synthesis was achieved with ∼5 n M TG. Recovery of protein synthesis after TG treatment was significantly hindered when serum was omitted from the medium after TG exposure, suggesting that serum promotes recovery of ER calcium homeostasis. It is concluded that TG is a suitable tool for the study of the mechanisms of protein synthesis inhibition after transient cerebral ischemia. The possibility that disturbances in ER calcium homeostasis may contribute to the pathological process of ischemic cell death is discussed.     

Combining Brain Imaging with Microarray: Isolating Molecules Underlying the Physiologic Disorders of the Brain

Many diseases of the nervous system cause dysfunction by impairing neuronal physiology more than by altering brain anatomy--including age-related cognitive decline, most psychiatric disorders, and even the earliest stages of Alzheimer's disease. The absence of clear anatomical markers makes it difficult to identify targeted cells, which in turn impedes attempts to isolate the pathogenic molecules that cause physiologic disruption. Here we show how brain imaging and microarray can be used as complimentary techniques that together can characterize the cellular and molecular aspects of this class of diseases.     

Calcium Dependency of Muscarinic and Nicotinic Agonist-Induced ATP and Catecholamine Secretion from Porcine Adrenal Chromaffin Cells

The secretion of catecholamines and ATP induced by cholinergic agonists and its dependence on extracellular Ca2+ were studied in cultured porcine adrenal chromaffin cells. Both nicotine and methacholine (a selective muscarinic agonist) induced secretion and increases in cytosolic free Ca2+ concentration ([Ca2+]in), although the activation of nicotinic receptors produced responses that were larger than those produced by activation of muscarinic receptors. The secretion and the increase in [Ca2+]in evoked by nicotine were completely dependent on extracellular Ca2+ and were blocked by prior depolarization of the cells with high extracellular K+ levels. In addition, nicotine induced significant 45Ca2+ influx. In contrast, the secretion and the increase in [Ca2+]in evoked by methacholine were partially dependent on extracellular Ca2+; methacholine also induced 45Ca2+ influx. Prior depolarization of the cells with high extracellular K+ levels did not block methacholine-induced secretion. In general, nicotinic responses were mediated by Ca2+ influx through voltage-dependent pathways. In contrast, muscarinic responses were dependent on both Ca2+ influx through an unknown mechanism that could not be inactivated by high K+ concentration-induced depolarization and presumably also intracellular Ca2+ mobilization.     

钙敏感受体(CaSR)基因986、990多态性与尿石症的相关性研究

目的:探讨钙敏感受体(Ca SR)基因单核昔酸多态性与泌尿系结石的关系。方法:选取90例黑龙江地区的泌尿系结石患者及90例健康对照者外周血标本中的基因组DNA,采用PCR(聚合酶链反应)结合DNA测序,检测并分析Ca SR基因的单核苷酸多态性位点的分布。结果:泌尿系结石组和对照组Ca SR基因第986位、990位频率分布符合Hardy-Weinberg定律,其基因型分布频率在泌尿系结石患者和健康对照者中差异无统计学意义(P0.05),但在泌尿系结石患者组内Ca SR第990位GG纯合子和RG杂合子出现频率明显偏高,差异有统计学意义(P0.05)。结论:Ca SR基因第7外显子第986、990多态性位点与泌尿系结石的形成无直接相关性,但第7外显子第990位A/G单核苷酸多态性可能与泌尿系结石的形成密切相关。     

Reinforcement Learning Recruits Somata and Apical Dendrites across Layers of Primary Sensory Cortex

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Role of Cyclic GMP in the Regulation of Neuronal Calcium and Survival by Secreted Forms of β-Amyloid Precursor

Abstract: The Alzheimer's disease (AD) β-amyloid precursor proteins (βAPPs) are large membrane-spanning proteins that give rise to the βA4 peptide deposited in AD amyloid plaques. βAPPs can also yield soluble forms (APP^ss) that are potently neuroprotective against glucose deprivation and glutamate toxicity, perhaps through their ability to lower the intraneuronal calcium concentration ([Ca²⁺]_i). We have investigated the mechanism through which APP^ss exert these effects on cultured hippocampal neurons. The ability of APP^ss to lower rapidly [Ca²⁺]_i was mimicked by membrane-permeable analogues of cyclic AMP (cAMP) and cyclic GMP (cGMP), as well as agents that elevate endogenous levels of these cyclic nucleotides. However, only cGMP content was increased by APP^s treatment, and specific inhibition of cGMP-dependent protein kinase (but not cAMP-dependent kinase) blocked the activity of APP^ss. A membrane-permeable analogue of cGMP (8-bromo-cGMP) also mimicked the ability of APP^ss to attenuate the elevation of [Ca²⁺]_i by glutamate, apparently through inhibition of NMDA receptor activity. In addition, 8-bromo-cGMP afforded protection against glucose deprivation and glutamate toxicity, and the protection by APP^ss against glucose deprivation was blocked by an inhibitor of cGMP-dependent kinase. Together, these data suggest that APP^ss mediate their [Ca²⁺]_i-lowering and excitoprotective effects on target neurons through increases in cGMP levels.     

Disposition of Gangliosides and Sialosylglycoproteins in Neuronal Membranes

Abstract Labeled gangliosides and glycoproteins were obtained by incubation of homogenized neuronal perikarya from rat brain with CMP-[³H] N -acetyl neuraminic acid. The highest degree of labelling was observed in a subcellular fraction that also showed the highest specific activities for several ganglioside glycosyltransferases. The [³H] sialosylglycoconjugates of this fraction remained associated with the membranes after treatment with 1 m -KCl, 125 m m -EDTA, repeated freezing and thawing, or controlled sonication, but were solubilized by sodium deoxycholate (DOC) at a concentration high enough to solubilize the choline phospholipids. About 75% of the neuraminidase-labile sialosyl residues of these labeled endogenous gangliosides and glycoproteins were protected from the action of added neuraminidase or pronase or both enzymes added together. The protection was not abolished by pretreatment of the membranes with high ionic strength or with EDTA but was abolished by sonication or low concentration of DOC. Between 50 and 80% of the neuraminidase-labile sialosyl residues of the gangliosides of the neuronal perikaryon membrane fraction labeled in vivo by an intracerebral injection of N -[³H]acetylmannos-amine were, at 3 h after the injection, also protected from the action of added neuraminidase. The protection was abolished by the addition of DOC. In contrast with the behavior of the labeled glycoconjugates of this neuronal perikaryon fraction, the gangliosides and sialosylglycoproteins from intact synaptosomes were accessible to neuraminidase. It is suggested that most gangliosides and sialosylglycoproteins are sialosylated as intrinsic components of the neuronal perikaryon membrane fraction and that at some stage of the process of transport through the axon and incorporation into the synaptic plasma membrane they change their accessibility to added enzymes.     

Imaging practices and radiation doses from imaging in radiotherapy

Modern radiotherapy treatments require frequent imaging for accurate patient positioning relative to the therapeutic radiation beam. Imaging practices in five Finnish radiotherapy clinics were assessed and discussed from the patient dose optimization point of view. The results show that imaging strategies are not jointly established and variations exist. The organ absorbed doses depend on imaging technique and imaging frequency. In particular, organ doses from the cone beam computed tomography can have very large variations (a factor of 10–50 in breast imaging and factor of 5 in prostate imaging). The cumulative imaging organ dose from the treatment can vary by a factor of ten or more for the same treatment, depending on the chosen technique and imaging frequency. Awareness and optimization of the imaging dose in image-guided radiotherapy should be strengthened.     

Neuronal Survival Factor from Bovine Brain Is Identical to Neuron-Specific Enolase

Neuronal survival factors in the central nervous system were investigated by using a primary culture of embryonic rat neocortical neurons. Bovine hippocampus was homogenized, and the supernatant from high-speed centrifugation was used as the starting material. At the step of DE-52 ion-exchange chromatography, neuronal survival activity was recovered in two fractions, fraction 14 (F14) and fraction 23 (F23). Antisera to the crude F14 and F23 fractions were raised in rabbits. These two antisera completely inhibited the neurotrophic activity of both fractions. Western blotting analysis revealed that anti-F14 antiserum recognized mainly a 30-kDa protein in F14 and anti-F23 antiserum recognized mainly a 44-kDa protein in F23. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis of F23, the 44-kDa protein was cut out from the gel and partial amino acid sequences of the protein fragments were determined. A GenBank data bank indicated that the amino acid sequence of the fragment was identical to that of neuron-specific enolase (NSE). In our assay system, commercially available NSE itself possessed neuronal survival activity for the cultured neocortical neurons. The effects of NSE and F23 were inhibited completely by anti-NSE polyclonal antibody. Furthermore, highly purified NSE supported the survival of cultured neurons in a dose-dependent manner, and the neurotrophic effect was inhibited by monoclonal antibody to the NSE. These results strongly suggest that NSE is one of the neuronal survival factors in the central nervous system.     

Cellular Acetylcholine Content and Neuronal Differentiation

Abstract: N18TG2 neuroblastoma clone is defective for biosynthetic neurotransmitter enzymes; its inability to establish functional synapses is overcome in the neuroblastoma × glioma 108CC15, where acetylcholine synthesis is also activated. These observations suggest a possible relation between the ability to produce acetylcholine and the capability to advance in the differentiation program and achieve a fully differentiated state. Here, we report the characterization of several clones after transfection of N18TG2 cells with a construct containing a cDNA for rat choline acetyltransferase (ChAT). The ability of these clones to synthesize acetylcholine is demonstrated by HPLC determination on cellular extracts. In the transfected clones, northern blot analysis shows increased expression of mRNAs for a specific neuronal protein associated with synaptic vesicles, synapsin I. Fiber outgrowth of transfected clones is also evaluated to establish whether there is any relation between ChAT levels and morphological differentiation. This analysis shows that the transfected clone 1/2, not expressing ChAT activity, displays a very immature morphology, and its ability to extend fibers also remains rather poor in the presence of "differentiation" agents such as retinoic acid. In contrast, clones 2/4, 3/1, and 3/2, exhibiting high ChAT levels, display higher fiber outgrowth compared with clone 1/2 in both the absence and the presence of differentiating agents.     

Phylogenetic Analysis and Molecular Evolution of Guanine Deaminases: From Guanine to Dendrites

Guanine deaminase (GDA; guanase) is a ubiquitous enzyme that catalyzes the first step of purine metabolism by hydrolytic deamination of guanine, resulting in the production of xanthine. This hydrolase subfamily member plays an essential role in maintaining homeostasis of cellular triphosphate nucleotides for energy, signal transduction pathways, and nitrogen sources. In mammals, GDA protein levels can play a role in neuronal development by regulating dendritic arborization. We previously demonstrated that the most abundant alternative splice form of GDA in mammals, termed cypin (cytosolic PSD-95 interactor), interacts with postsynaptic density proteins, regulates microtubule polymerization, and increases dendrite number. Since purine metabolism and dendrite development were previously thought to be independent cellular processes, this multifunctional protein serves as a new target for the treatment of cognitive disorders characterized by aberrant neuronal morphology and purine metabolism. Although the enzymatic activity of GDA has been conserved during evolution from prokaryotes to higher eukaryotes, a detailed evolutionary assessment of the principal domains in GDA proteins has not yet been put forward. In this study, we perform a complete evolutionary analysis of the full-length sequences and the principal domains in guanine deaminases. Furthermore, we reconstruct the molecular phylogeny of guanine deaminases with neighbor-joining, maximum-likelihood, and UPGMA methods of phylogenetic inference. This study can act as a model whereby a universal housekeeping enzyme may be adapted to act also as a key regulator of a developmental process.