Non-Invasive Electrical Brain Stimulation Montages for Modulation of Human Motor Function

Non-invasive electrical brain stimulation (NEBS) is used to modulate brain function and behavior, both for research and clinical purposes. In particular, NEBS can be applied transcranially either as direct current stimulation (tDCS) or alternating current stimulation (tACS). These stimulation types exert time-, dose- and in the case of tDCS polarity-specific effects on motor function and skill learning in healthy subjects. Lately, tDCS has been used to augment the therapy of motor disabilities in patients with stroke or movement disorders. This article provides a step-by-step protocol for targeting the primary motor cortex with tDCS and transcranial random noise stimulation (tRNS), a specific form of tACS using an electrical current applied randomly within a pre-defined frequency range. The setup of two different stimulation montages is explained. In both montages the emitting electrode (the anode for tDCS) is placed on the primary motor cortex of interest. For unilateral motor cortex stimulation the receiving electrode is placed on the contralateral forehead while for bilateral motor cortex stimulation the receiving electrode is placed on the opposite primary motor cortex. The advantages and disadvantages of each montage for the modulation of cortical excitability and motor function including learning are discussed, as well as safety, tolerability and blinding aspects.     

Adenosine Formation and Release by Embryonic Chick Neurons and Glia in Cell Culture

Adenosine formation and release were studied in 48-h-old cultured ciliary ganglia and confluent peripheral and CNS glial cultures from embryonic chicks. Metabolic poisoning induced by 30 mM 2-deoxyglucose and 2 micrograms/ml oligomycin reduced ATP concentration by 90%. An increase in adenosine accounted for 15-40% of the fall in ATP. Dilazep (3 X 10(-6) M), a nucleoside transport inhibitor, decreased both incorporation of adenosine (an index of nucleoside transport) and release of adenosine by 80-90%. Dilazep trapped the newly formed adenosine intracellularly. A concentration of alpha, beta-methylene ADP that inhibited ecto-5'-nucleotidase by 80-90% did not alter the concentration of adenosine or AMP in neurons, glia, or medium. The results demonstrate that adenosine is formed intracellularly and exported out of the cell via the nucleoside transporter. The participation of ecto-5'-nucleotidase was excluded.     

Contact-Mediated Inhibition Between Oligodendrocyte Progenitor Cells and Motor Exit Point Glia Establishes the Spinal Cord Transition Zone

Rapid conduction of action potentials along motor axons requires that oligodendrocytes and Schwann cells myelinate distinct central and peripheral nervous system (CNS and PNS) domains along the same axon. Despite the importance of this arrangement for nervous system function, the mechanisms that establish and maintain this precise glial segregation at the motor exit point (MEP) transition zone are unknown. Using in vivo time-lapse imaging in zebrafish, we observed that prior to myelination, oligodendrocyte progenitor cells (OPCs) extend processes into the periphery via the MEP and immediately upon contact with spinal motor root glia retract back into the spinal cord. Characterization of the peripheral cell responsible for repelling OPC processes revealed that it was a novel, CNS-derived population of glia we propose calling MEP glia. Ablation of MEP glia resulted in the absence of myelinating glia along spinal motor root axons and an immediate breach of the MEP by OPCs. Taken together, our results identify a novel population of CNS-derived peripheral glia located at the MEP that selectively restrict the migration of OPCs into the periphery via contact-mediated inhibition.     

神经电生理刺激对脑卒中患者脊髓运动神经元的影响

目的:探讨神经电生理刺激对脑卒中患者脊髓运动神经元的影响。方法:2015年9月至2018年2月选择在本院神经内科病房诊治的脑卒中患者160例,根据入院顺序随机分为研究组与对照组,各80例。对照组给予常规治疗,研究组在对照组治疗的基础上给予神经电生理刺激治疗,两组都治疗观察14 d,记录脊髓运动神经功能变化情况。结果:两组治疗后14 d,患者的认知障碍、焦虑抑郁情绪、双下肢无力,步态不稳均得到了改善,研究组的一般情况的改善情况更加明显,主要体现在认知和情绪方面。两组治疗14 d后的脊髓运动神经评分都显著高于治疗前(P0.05),研究组也显著高于对照组(P0.05)。治疗后两组的脑电诱发电位波幅都显著高于治疗前(P0.05),研究组也显著高于对照组(P0.05),两组治疗前后潜伏期对比差异无统计学意义(P0.05)。研究组治疗期间的肺部感染、颅内感染、迟发性颅内血肿等并发症发生率为3.8%,对照组为5.0%,对比差异无统计学意义(P0.05)。随访至今(2019年8月),研究组的预后恢复情况显著好于对照组(P0.05)。结论:电生理刺激在脑卒中患者的应用能促进恢复脊髓运动神经元功能,改善脑电活动功能,提高认知功能,减少并发症和焦虑情绪的发生,改善患者的预后恢复。     

Discrete Distributions of Adenosine Receptors in Mammalian Retina

Binding sites for both the adenosine A1 receptor agonists [3H]phenylisopropyladenosine and [3H]cyclohexyladenosine and the mixed A1-A2 agonist N-[3H]ethylcarboxamidoadenosine [( 3H]NECA) were localized in rabbit and mouse retinas using autoradiographic techniques. These two classes of agonists bound to very different regions of mammalian retinas. A1 agonist binding was localized to the inner retina, particularly over the inner plexiform layer. The binding of [3H]NECA was observed primarily over the retinal pigmented epithelium and the outer and inner segments of photoreceptors. [3H]NECA labeling was not affected either by including a low concentration of unlabeled A1 agonist or by pretreating tissue with N-ethylmaleimide to inhibit ligand binding at A1 sites. While virtually all of the [3H]NECA binding was displaced by an excess of unlabeled NECA, displacement with antagonist or a large excess of cyclohexyladenosine revealed that approximately 30% of the [3H]NECA binding was at non-A1,A2 sites. The majority of the binding in the outer retina thus labeled A2 receptor sites. The unique localizations of the two classes of adenosine receptors suggest different functions in visual processing.     

Modulation of Central Nociceptive Coding by Acupoint Stimulation

It is universally accepted that acupuncture or acupoint stimulation can produce analgesic effect on patients with painful disorders. The past decades has seen remarkable progress in exploring the central mechanisms of acupuncture-induced pain relief, including the neurotransmitter release and expression of particular receptors and genes in the spinal cord and the brain stem regions. Development of new techniques makes it possible to record and image the brain network patterns underlying pain perception and modulation, and to investigate the role of higher-level brain areas in mediating acupuncture analgesia. This review will present the current understanding of the neural network that is implicated in the modulation of pain by acupuncture. Special issue article in honor of Dr. Ji-Sheng Han.     

Stimulation of Mammalian G-protein-responsive Adenylyl Cyclases by Carbon Dioxide

Carbon dioxide is fundamental to the physiology of all organisms. There is considerable interest in the precise molecular mechanisms that organisms use to directly sense CO₂. Here we demonstrate that a mammalian recombinant G-protein-activated adenylyl cyclase and the related Rv1625c adenylyl cyclase of Mycobacterium tuberculosis are specifically stimulated by CO₂. Stimulation occurred at physiological concentrations of CO₂ through increased k_cat. CO₂ increased the affinity of enzyme for metal co-factor, but contact with metal was not necessary as CO₂ interacted directly with apoenzyme. CO₂ stimulated the activity of both G-protein-regulated adenylyl cyclases and Rv1625c in vivo. Activation of G-protein regulated adenylyl cyclases by CO₂ gave a corresponding increase in cAMP-response element-binding protein (CREB) phosphorylation. Comparison of the responses of the G-protein regulated adenylyl cyclases and the molecularly, and biochemically distinct mammalian soluble adenylyl cyclase revealed that whereas G-protein-regulated enzymes are responsive to CO₂, the soluble adenylyl cyclase is responsive to both CO₂ and bicarbonate ion. We have, thus, identified a signaling enzyme by which eukaryotes can directly detect and respond to fluctuating CO₂.Inorganic carbon (C_i)³ is central to prokaryotic and eukaryotic physiology. The predominant biologically active forms of C_i are CO₂ and and their relative contributions to the total C_i pool are pH-dependent. Biological roles for CO₂ and include photosynthetic carbon fixation (1), pH homeostasis (2), carbon metabolism (3), activation of virulence in pathogenic organisms (4), sperm maturation (5), and as an alarmone in Drosophila (6, 7).Given its importance in biology, the identification of CO₂ responsive signaling pathways is key to understanding how organisms cope with fluctuating CO₂. Two seven transmembrane receptors, Gr21a and Gr63a, have been shown to confer CO₂ responsiveness in Drosophila neurons (6, 7). Guanylyl cyclase D expressing olfactory neurons also mediate sensitivity to CO₂ in mice (8). A role for cGMP-activated channels in CO₂ sensing has been observed in CO₂ avoidance behavior in Caenorhabditis (9, 10). Despite these impressive advances, no eukaryotic signaling enzymes unequivocally demonstrated to respond to CO₂ have been identified.The mammalian soluble adenylyl cyclase (sAC) synthesizes the second messenger 3′,5′-cAMP and is directly stimulated by (11–13). Stimulation of sAC by has an unequivocal role in sperm maturation (5, 14–16). sAC is a member of the Class III family of adenylyl cyclases (ACs), a family that also includes the G-protein-regulated ACs and many examples from prokaryotic genomes (17, 18). The Class III ACs can be divided into four subclasses (a–d) based upon polymorphisms within the active site (19). sAC is a member of Class IIIb, a subclass characterized partly by replacement of a substrate binding Asp with Thr. The Class IIIa ACs include the mammalian G-protein-stimulated ACs and numerous prokaryotic examples. These have been previously assumed to be non-responsive to C_i (12).All prokaryotic Class IIIb ACs examined to date respond to C_i including enzymes from organisms as diverse as Anabaena PCC 7120, Mycobacterium tuberculosis, Stigmatella aurantiaca, and Chloroflexus aurantiacus (20, 21). Two Class IIIb ACs, Slr1991 of Synechocystis PCC 6803 and CyaB1 of Anabaena PCC 7120, have been proven to respond to CO₂ and not , giving rise to the idea of AC as a true gas-sensing molecule (22, 23). The finding that Class IIIb ACs respond to CO₂ and not necessitates an examination of the assumption that G-protein-regulated ACs and related prokaryotic enzymes do not respond to C_i.Here we demonstrate, contrary to previous work, that a recombinant G-protein-regulated AC and the Class IIIa Rv1625c AC of M. tuberculosis H37Rv show a pH-dependent response to C_i due to specific stimulation by CO₂ at physiologically relevant concentrations. CO₂ interacted directly with the apoprotein and modulated the activity of both the prokaryotic enzyme and G-protein-regulated AC in vivo. Finally, we contrasted the responses of sAC- and G-protein-regulated ACs to different species of C_i and propose that the mammalian cAMP signaling pathway is able to discriminate between CO₂ and in vivo.     

Phosphorylation of Mitogen-Activated Protein Kinase in Cultured Rat Cortical Glia by Stimulation of Metabotropic Glutamate Receptors

Abstract: Activation of metabotropic glutamate receptors (mGluRs) in glia results in significant physiological effects for both the glia and the neighboring neurons; but in many cases, the mGluR subtypes and signal transduction mechanisms mediating these effects have not been determined. In this study, we report that mGluR activation in primary cultures of rat cortical glia results in tyrosine phosphorylation of several proteins, including p44/p42 mitogen-activated protein kinases, also referred to as extracellular signal-regulated kinases (ERK1/2). Incubation of glial cultures with the general mGluR agonist 1-aminocyclopentane-1 S ,3 R -dicarboxylate and the mGluR group I-selective agonists ( RS )-3,5-dihydroxyphenylglycine (DHPG) and l -quisqualate resulted in increased tyrosine phosphorylation of ERK1/2. The group II-selective agonist (2 S ,2' R ,3' R )-2-(2',3'-dicarboxycyclopropyl)glycine and group III-selective agonist l (+)-2-amino-4-phosphonobutyric acid had no effect on tyrosine phosphorylation. DHPG-induced ERK1/2 phosphorylation could be inhibited by an antagonist that acts at group I or group II mGluRs but not by antagonists for group II and group III mGluRs. Protein kinase C (PKC) activators also induced ERK1/2 phosphorylation, but the PKC inhibitor bisindolylmaleimide I did not inhibit DHPG-induced ERK1/2 phosphorylation at a concentration that inhibited the response to phorbol 12,13-dibutyrate. These data suggest that mGluR activation of ERK1/2 in cultured glia is mediated by group I mGluRs and that this effect is independent of PKC activation. Furthermore, immunoblots with antibodies against various mGluR subtypes show expression of mGluR5, but no other mGluRs in our cultures. Taken together, these results suggest that mGluR5 stimulation results in tyrosine phosphorylation of ERK1/2 and other glial proteins.     

Endogenous Adenosine Modulation of 22Na Uptake by Rat Brain Synaptosomes

To evaluate if endogenous extracellular adenosine influences sodium channel activity in nerve terminals, we investigated how manipulations of extracellular adenosine levels influence ²²Na uptake by rat brain synaptosomes stimulated with veratridine (VT). To decrease extracellular adenosine levels, adenosine deaminase (ADA) that converts adenosine into an inactive metabolite was used. To increase extracellular adenosine levels, we used the adenosine deaminase inhibitor erythro-9(2-hydroxy-3-nonyl) adenine (EHNA), as well as the inhibitor of adenosine transport, nitrobenzylthioinosine (NBTI). ADA (0.1–5U/ml) caused an excitatory effect on ²²Na uptake stimulated by veratridine, which was abolished in the presence of the adenosine deaminase inhibitor erythro-9(2-hydroxy-3-nonyl) adenine (EHNA, 25M). Both the adenosine uptake inhibitor nitrobenzylthioinosine (NBTI, 1–10M) and the adenosine deaminase inhibitor EHNA (10–25M) inhibited ²²Na uptake by rat brain synaptosomes. It is suggested that adenosine is tonically inhibiting sodium uptake by rat brain synaptosomes.     

Identification of Mammalian Brain and Spinal Cord Opioid Receptor by Photoaffinity Labeling

Abstract

A radioiodinated photoreactive enkephal in derivative, ¹²⁵I(D-Ala² p-N₃-Phe⁴-Met⁵) enkephalin, was used to photoaffinity label the opioid receptor from the membranes of four mammalian brains (without cerebellum) and spinal cords. These included the cat, rabbit, guinea pig and mouse. The photolabeled membranes were analyzed by sodium dodecyl sulfate gel electrophoresis. A 43,000-daltons protein was specifically photolabeled in all the membranes tested, as the specific labeling of this protein was inhibited in the presence of 14.5 uM of (D-Ala² Met⁵) enkephalin. These data suggest that the 43,000-daltons protein is a binding protein of the opioid receptor in the different mammalian neural tissues.     

Closing of Venus Flytrap by Electrical Stimulation of Motor Cells

Electrical signaling and rapid closure of the carnivorous plant Dionaea muscipula Ellis (Venus flytrap) have been attracting the attention of researchers since XIX century, but the exact mechanism of Venus flytrap closure is still unknown. We found that the electrical stimulus between a midrib and a lobe closes the Venus flytrap leaf by activating motor cells without mechanical stimulation of trigger hairs. The closing time of Venus flytrap by electrical stimulation of motor cells is 0.3 s, the same as mechanically induced closing. The mean electrical charge required for the closure of the Venus flytrap leaf is 13.6 µC. Ion channel blockers such as Ba²⁺, TEACl as well as uncouplers such as FCCP, 2,4-dinitrophenol and pentachlorophenol dramatically decrease the speed of the trap closing. Using an ultra-fast data acquisition system with measurements in real time, we found that the action potential in the Venus flytrap has a duration time of about 1.5 ms. Our results demonstrate that electrical stimulation can be used to study mechanisms of fast activity in motor cells of the plant kingdom.Key Words: action potential, electrophysiology, electrical signaling, Venus flytrap, motor cells     

Modulation of Tryptophan Hydroxylase Activity by Phospholipids: Stimulation Followed by Inactivation

The activity of tryptophan hydroxylase from the rat brainstem was stimulated rapidly three- to fourfold by the addition of phosphatidylinositol or phosphatidylserine. However, the activity of the enzyme once stimulated was decreased gradually by subsequent incubation with the phospholipid at 37 degrees C, reaching a level below the original activity after 1 h of incubation. The presence of ferrous ion almost perfectly protected the enzyme against this phospholipid inactivation. The activity of the enzyme inactivated by incubation with the phospholipid was not only restored, but also increased further by incubation at 37 degrees C with ferrous ion and dithiothreitol. Gel filtration analysis revealed that the enzyme stimulated by phosphatidylinositol was eluted in a void volume together with the phospholipid vesicles, but the enzyme inactivated by incubation with phosphatidylinositol was eluted at a later region apart from the vesicles. These results, taken together, suggest the possible involvement of cellular membranes in the regulation of tryptophan hydroxylase in the central nervous system.     

Theoretical Study of Antagonists and Inhibitors of Mammalian Adenosine Deaminase: II. Isomeric Aza-deazaanalogues of Adenosine

Isomeric aza-deazaanalogues of adenosine and their N1-protonated forms (except for that of 8-aza-1-deazaadenosine) were studied by computer modeling to find a relationship between their molecular structures and the properties as substrates for the mammalian adenosine deaminase. The atomic charge distribution and maps of electrostatic potential around their van der Waals molecular surface were calculated using the ab initioSTO-3G method. The conformational studies were carried out by the MM⁺ method of molecular mechanics. The previously proposed mechanism of the substrate acceptance in the active site of mammalian adenosine deaminase was refined, and the potential substrate properties were predicted for two previously unstudied adenosine analogues, 5-aza-9-deazaadenosine and 8-aza-3-deazaadenosine.     

Efficient Down-Regulation of Glia Maturation Factor Expression in Mouse Brain and Spinal Cord

Long-lasting siRNA-based down-regulation of gene of interest can be achieved by lentiviral-based expression vectors driving the production of short hairpin RNA (shRNA). We investigated an attractive therapeutic approach to target the expression of proinflammatory GMF by using lentiviral vector encoding GMF-specific shRNA to reduce GMF levels in the spinal cord and brain of mice. To determine the effect of GMF-shRNA on GMF protein levels, we performed quantitative ELISA analysis in brain and in thoracic, cervical and lumbar regions of spinal cord from mice followed by GMF-shRNA (G-shRNA) or control shRNA (C-shRNA) treatments. Our results show a marked reduction of GMF protein levels in brain and spinal cord of mice treated with GMF-shRNA compared to control shRNA treatment. Consistent with the GMF protein analysis, the immunohistochemical examination of the spinal cord sections of EAE mice treated with GMF-shRNA showed significantly reduced GMF-immunoreactivity. Thus, the down-regulation of GMF by GMF-shRNA was efficient and wide spread in CNS as evident by the significantly reduced levels of GMF protein in the brain and spinal cord of mice.     

Autoradiographic Localization and Characterization of Adenosine Receptor Subtypes in Mammalian Brain

Abstract

Quantitative receptor autoradiography studies have shown that adenosine A₁ receptors are heterogeneously distributed in the rat brain with high concentrations found in the forebrain and cerebellum. In contrast, high affinity A₂ receptors appear to be exclusively localized in the striatum. These observations are discussed in relation to the putative neuromodulatory role of the purine in central neurotransmission.     

Modulation of Cortical Oscillations by Low-Frequency Direct Cortical Stimulation Is State-Dependent

Cortical oscillations play a fundamental role in organizing large-scale functional brain networks. Noninvasive brain stimulation with temporally patterned waveforms such as repetitive transcranial magnetic stimulation (rTMS) and transcranial alternating current stimulation (tACS) have been proposed to modulate these oscillations. Thus, these stimulation modalities represent promising new approaches for the treatment of psychiatric illnesses in which these oscillations are impaired. However, the mechanism by which periodic brain stimulation alters endogenous oscillation dynamics is debated and appears to depend on brain state. Here, we demonstrate with a static model and a neural oscillator model that recurrent excitation in the thalamo-cortical circuit, together with recruitment of cortico-cortical connections, can explain the enhancement of oscillations by brain stimulation as a function of brain state. We then performed concurrent invasive recording and stimulation of the human cortical surface to elucidate the response of cortical oscillations to periodic stimulation and support the findings from the computational models. We found that (1) stimulation enhanced the targeted oscillation power, (2) this enhancement outlasted stimulation, and (3) the effect of stimulation depended on behavioral state. Together, our results show successful target engagement of oscillations by periodic brain stimulation and highlight the role of nonlinear interaction between endogenous network oscillations and stimulation. These mechanistic insights will contribute to the design of adaptive, more targeted stimulation paradigms.     

Stimulation by Monovalent Cations of Adenosine Triphosphatase Activity in Extracts of Petiole Tissues3

Adenosine triphosphatase activity has been studied in fractionatedextracts of isolated phloem and xylem tissues of Heracleum mantegazzianumSomm. et Lev. and of petioles of Helianthus annuus L. Enzymeactivity in the microsomal fraction is maximal with ATP as substrate.Monovalent cations stimulate activity, but only below pH 7·0.Divalent cations are inhibitory. Stimulation of ATPase activityby monovalent cations is increased in preparations which haveeither been derived from acetone powders or pre-treated withdithiothreitol or cysteine.