Human lymphocytes from healthy subjects and ALS patients were obtained from the NINDS Biorepository at the Coriell Institute for Medical Research and reprogrammed into iPSCs as previously described using episomal vectors61. Briefly, mammalian expression vectors containing Oct4, Sox2, Klf4, L-Myc, Lin28, and a p53 shRNA were introduced into the lymphocytes using the Adult Dermal Fibroblast Nucleofector™ Kit and Nucleofector™ 2b Device (Lonza) according to the manufacturer’s protocol. The cells were then cultured on mouse feeders until iPSC colonies appeared. The colonies were then expanded and maintained on Matrigel (BD) in mTeSR1 medium (Stem Cell Technologies).
RNA sequencing output was aligned to the GRCh38 Reference Genome and quantified using the STAR aligner.65 Genes were annotated against the GENCODE version 23 Comprehensive Gene Annotation. Quality control was performed using Picard Tools AlignmentSummaryMetrics. Samples passing quality control and having RNA Integrity Number (RIN) > 5 were used in downstream analysis. To identify differentially expressed genes, the R package DESeq2 was used as previously described.66 The function DESeq was used to estimate size factors, estimate dispersion, fit the data to a negative binomial generalized linear model, and generate differential expression statistics using the Wald test. KEGG enrichment analysis was performed for internal analysis using the R package clusterProfiler.67
To determine if Pikfyve inhibition rescues gain-of-function processes in vivo, we measured DPR levels in C9-BAC transgenic mice 58 with or without Apilimod treatment. Although it was not previously reported 58, we observed significantly higher levels of GR+ punctae in hippocampal neurons in C9-BAC mice than controls (Fig. 6j) using a previously-validated poly(GR) antibody 11. These data are consistent with findings in another published C9-BAC mouse model 14, suggesting that poly(GR) may be a common feature of C9-BAC mice. We also detected a low level of poly(GR) in neurons from control mice (Fig. 6j), which may be derived from other repeat regions or proteins with short poly(GR) sequences. Nevertheless, GR+ punctae levels were significantly higher in C9-BAC mouse neurons than in controls (Fig. 6j). Importantly, Apilimod treatment significantly reduced the number of GR+ punctae in hippocampal neurons in C9-BAC mice after 48 hrs (Fig. 6i, j). Therefore, small molecule inhibition of Pikfyve rescues both gain- and loss-of-function disease processes induced by C9ORF72 repeat expansion in vivo.
Shi Y1,2,3, Lin S1,2,3, Staats KA1,2,3, Li Y1,2,3, Chang WH1,2,3, Hung ST1,2,3, Hendricks E1,2,3, Linares GR1,2,3, Wang Y3,4, Son EY5, Wen X6, Kisler K3,4, Wilkinson B3, Menendez L1,2,3, Sugawara T1,2,3, Woolwine P1,2,3, Huang M1,2,3, Cowan MJ1,2,3, Ge B1,2,3, Koutsodendris N1,2,3, Sandor KP1,2,3, Komberg J1,2,3, Vangoor VR7, Senthilkumar K7, Hennes V1,2,3, Seah C1,2,3, Nelson AR3,4, Cheng TY8, Lee SJ8, August PR9, Chen JA10, Wisniewski N10, Hanson-Smith V10, Belgard TG10, Zhang A10, Coba M3,11, Grunseich C12, Ward ME12, van den Berg LH13, Pasterkamp RJ7, Trotti D6, Zlokovic BV3,4, Ichida JK1,2,3.
HEK 293T cells were used to produce retrovirus, lentivirus, and C9ORF72 protein. HEK cells were used for these purposes based on previous published studies using HEK cells in order to produce viral particles and mammalian proteins. HEK cells were obtained from American Type Culture Collection, catalog number CRL-11268. HEK and iPS cells were tested for mycoplasma before, during, and after the study and were negative.
(a) Super-resolution microscopy images of control iMNs showing colocalization (arrows) of C9ORF72 (green) with EEA1 (red). Scale bar: 5 µm. This experiment was repeated 3 times with similar results. (b) Immunoblot against C9ORF72, EEA1, and LAMP1 on lysates from iPSC-derived motor neurons separated into light (endosomal) and heavy (lysosomal) membrane fractions using percoll gradient centrifugation. This experiment was repeated twice with similar results. (c) Super-resolution microscopy images of LAMP1 immunostaining in iMNs of specified genotypes expressing eGFP or C9ORF72 (isoform A or B)-eGFP. Scale bar: 5 µm. This experiment was repeated 3 times with similar results. (d-f) Number of LAMP1+ vesicles in control (d-f), patient (d), C9ORF72+/− (e), and C9ORF72−/− (f) iMNs overexpressing eGFP or C9ORF72 (isoform A or B)-eGFP. Each grey open circle represents a single iMN, Mean ± s.d. For (d), n=80 (CTRL + GFP), 80 (C9-ALS + GFP), 64 (C9-ALS + isoA), and 61 (C9-ALS + isoB) iMNs quantified from two biologically independent iMN conversions of 3 CTRL or 4 C9-ALS lines. For (e), n=20 (CTRL + GFP), 15 (C9ORF72+/− + GFP), 12 (C9ORF72+/− + isoA), and 13 (C9ORF72+/− + isoB) iMNs f quantified from two biologically independent iMN conversions per condition. For (f), n=20 iMNs quantified from two biologically independent iMN conversions per condition. One-way ANOVA with Tukey correction between CTRL2 and C9ORF72+/− and C9ORF72−/− (e, f), one-way ANOVA with Tukey correction between controls and patient conditions (d). F-value (DFn, DFd): (3, 273)=12.12 (d), (3, 57)=5.64 (e), (3, 77)=6.091 (f). Dotted lines outline iMNs. (g) Representative electron micrographs of control, C9ORF72−/−, and patient iMNs showing lysosomes as electron-dense spherical perinuclear structures (arrows). This experiment was repeated twice with similar results. Scale bar: 1 μm. (h-i) Number of electron-dense spheres per square micron of perinuclear cytosol in control (h-i), C9ORF72−/− (h), and patient iMNs (i) Median ± interquartile range, each data point represents a single cell, Two-sided Mann-Whitney test). For (h), n=20 (CTRL2) and 19 (C9ORF72+/−), and for (i) n=20 (CTRL2) and 26 (C9ORF72 patient) cells quantified from two biologically independent iMN conversions of one line per genotype. (j) Super-resolution microscopy images of Lamp1 immunoreactivity in control and C9-KO mouse spinal neurons. This experiment was repeated twice with similar results. Scale bar: 5 μm. (k) Number of Lamp1+ punctae in Chat+ mouse spinal neurons. Median ± interquartile range, two-tailed t-test. t-value: 3.681. Degrees of freedom: 113. n=59 (CTRL2) and 56 (C9ORF72−/−) cells quantified from sections of two mice per genotype.
Base text for this translation. ___. Wang Meng’ou’s , ed. Tangren xiaoshuo jiaoshi . Taipei: Zhongzheng Shuju, 1983, 2319-38. For other texts and editions see footnote 1. Translations Birch, Cyril. “The Curly-bearded Hero,” in Anthology of Chinese Literature, v. 1, New York, 1965, pp. 314-322. Chai, Ch’u, and Winberg Chai. “The Curly-Bearded Guest,” in A Treasury of Chinese Literature, New York, 1965, pp. 117-124. Hsu Sung-nien. “Biographie d’un preux barbu,” Anthologie de la littérature chinoise.Paris: Delagrave, 1933, pp. 241-6. Levenson, Christopher, tran., The Golden Casket. Harmondsworth, Middlesex: Penguin Books, 1967, pp. 137-47. Lévy, André. “Barbe-bouclée, L’étranger à la barbe et aux favoris bouclés,” in Histoires extraordinaires et récits fantastiques de la Chine ancienne.Paris: Flammarion, 1993, pp. 177-195 (with notes). Lin Yutang. “Curly-Beard,” in Famous Chinese Short Stories. New York: John Day (Cardinal), 1953, pp. 3-22. Schafer, E.H. “Three Divine Women of South China,” CLEAR, 1 (1979), pp. 31-42. Wang, Elizabeth Te-chen, tran. “The Curly-Bearded Guest,” in Wang’s Ladies of the Tang: 22 Classical Chinese Stories. Taipei: Mei Ya Publications, 1973, pp. 133-50.
We thank the NINDS Biorepository at Coriell Institute for providing the following cell lines for this study: ND12133, ND03231, ND01751, ND11976, ND03719, ND00184, ND5280, ND06769, ND10689, ND12099, ND14954, ND08957, ND12100, and ND014587. We thank Helena Chui and Carol Miller at the University of Southern California Alzheimer’s Disease Research Center and Neil Shneider at the Columbia University Medical Center for control and C9ORF72 patient tissue. We thank the Choi Family Therapeutic Screening Facility for chemical screening support and the Translational Imaging Center at USC for imaging support. We thank Max Koppers, Youri Adolfs, Christiaan van der Meer, and Mark Broekhoven for help with mouse breeding and kainate injection experiments. We thank Prof. Satoshi Waguri for providing the M6PR-GFP construct. We thank Christopher Buser for assistance with electron microscopy. We also thank Sam Alworth (DRVision Technologies, LLC), Katja Hebestreit, and Raj Bhatnagar (Verge Genomics), Bob Baloh, Jacqueline O’Rourke, Christopher Donnelly, Chang Tong, Andrew McMahon and Qing Liu-Michael for reagents, technical support, and discussions. E.Y.S. is a Walter V. and Idun Berry Postdoctoral Fellow. K.A.S. was supported in part by a Muscular Dystrophy Association Development Grant. L.M. was supported by NIH grant T32DC009975–04. This work was supported by NIH grants AG039452, AG023084, and NS034467 to B.V.Z. R.J.P. was supported by grants from ALS Foundation Netherlands (TOTALS), Epilepsiefonds (12–08, 15–05), and VICI grant Netherlands Organisation for Scientific Research (NWO). This work was also supported by NIH grants R00NS077435 and R01NS097850, U.S. Department of Defense grant W81XWH-15–1-0187, and grants from the Donald E. and Delia B. Baxter Foundation, the Tau Consortium, the Frick Foundation for ALS Research, the Muscular Dystrophy Association, the New York Stem Cell Foundation, the USC Keck School of Medicine Regenerative Medicine Initiative, the USC Broad Innovation Award, and the Southern California Clinical and Translational Science Institute to J.K.I. J.K.I. is a New York Stem Cell Foundation-Robertson Investigator.
The Li force is observable when it is employed. Unlike the Li force, Neijing is said to be invisible. The "pivot point" essential to Li combat is not necessary in Neijing. At the point of attack, one must ‘song’ (loosen) himself to generate all Neijing energy one possesses and direct this energy stream through one's contact point with an opponent.[5] The contact point only represents the gateway to conduct Neijing energy at the point of attack.[6]
We anticipate three key implications of our findings: 1) ALS/FTD caused by the C9ORF72 repeat expansion requires both gain- and loss-of-function mechanisms, 2) increasing C9ORF72 activity in motor neurons should mitigate disease and provides a new therapeutic target, and 3) PIKFYVE inhibition and other approaches that modulate vesicle trafficking may ameliorate C9ORF72 disease processes in both neurons and myeloid cells. The fact that mutations in FIG4 cause ALS, epilepsy, and Charcot-Marie-Tooth 55 illustrates the broad implications of impaired vesicle trafficking within the CNS. The identification of targets that effectively modulate vesicle trafficking in neurons, glia, and myeloid cells could hold tremendous therapeutic value for C9ORF72 ALS/FTD and other CNS disorders.
Our iMN survival results (Fig. 1c-e) suggest that the repeat expansion alters iMN glutamate sensing. In cortical neurons, homeostatic synaptic plasticity is maintained through endocytosis and subsequent lysosomal degradation of glutamate receptors in response to chronic glutamate signaling 45,46. Defects in this process lead to the accumulation of glutamate receptors on the cell surface 45,46.

Cells were fixed in 6-well culture plates in 2.5 % glutaraldehyde in 0.1M cacodylate buffer, post-fixed in 1% osmium tetroxide for 1 hour and block stained in 1% uranyl acetate in 0.1M acetate buffer pH 4.4 overnight at 4 ˚C. Dehydration was performed in increasing concentrations of ethanol (10%/25%/50%/75%/90%/100%/100%/100%) for 15 minutes each and infiltrated with increasing concentrations of Eponate12 (Ted Pella Inc., Redding, CA, USA), 25% Eponate12 (no catalyst) in ethanol for 3 hours, 50% overnight, 100% for 5 hours, 100% overnight, and polymerized in fresh Eponate12 with DMP-30 for 48 hours at 60 ˚C. Previously marked areas were sawed out, the tissue culture plastic was removed and the selected area sectioned parallel to the substrate at a thickness of 70 nm. Sections at a depth of 3–5 µm were collected on formvar-filmed 50 mesh copper grids and imaged at 80 kV in an FEI 208 Morgagni (FEI is in Hillsboro, OR, USA). Per micrograph, cytosol was used to quantify the number of electron dense spheres that were defined as lysosomes 40.


Amongst four reproducible hit compounds, we identified a PIKFYVE kinase inhibitor (YM201636) that significantly increased C9ORF72 patient iMN survival (n=2 patients) (Fig. 6b, c and Supplementary Fig. 15a). PIKFYVE is a lipid kinase that converts phosphatidylinositol 3-phosphate (PI3P) into phosphtidylinositol (3,5)-bisphosphate (PI(3,5)P2)51(Fig. 6f). PI3P is primarily generated by PI3-kinases recruited to early endosomes by RAB5, and PI3P anchors EEA1 to early endosomes to drive endosomal maturation 52(Fig 6f). Following endosomal maturation into lysosomes, PI3P drives fusion of lysosomes with autophagosomes 53. PIKFYVE regulates PI3P levels by converting PI3P into PI(3,5)P2 52, which disfavors lysosomal fusion with endosomes and autophagosomes 53,54. Therefore, inhibition of PIKFYVEincreases autophagosome-lysosome fusion 53 and may compensate for reduced C9ORF72 activity and other disease processes by increasing PI3P levels to facilitate removal of glutamate receptors or DPRs (Fig. 6f). Interestingly, FIG4 is a phosphatase that opposes PIKFYVE kinase by converting PI(3,5)P2 to PI3P and loss-of-function mutations in FIG4 cause ALS 55. Thus, genetic evidence suggests that PIKFYVE inhibition may be capable of modulating ALS disease processes in humans.
J.K.I. and P.A. are co-founders of Acurastem, Inc. P.A. is an employee of Icagen Corporation. J.K.I. and P.A. declare that they are bound by confidentiality agreements that prevent them from disclosing details of their financial interests in this work. S-J.L. is a founder of DRVision Technologies and T-Y.C. is an employee of DRVision Technologies. A.Z. and J.A.C. are co-founders of Verge Genomics and V.H-S., N.W., and T.G.B. are employees of Verge Genomics.

We also found that Reduced C9ORF72 activity also induces iMN hypersensitivity to DPRs by impairing their clearance. This uncovers a more direct form of cooperative pathogenesis between gain- and loss-of-function mechanisms in C9ORF72 ALS/FTD. Through a potentially similar mechanism, reduced C9orf72 levels can also facilitate cytoplasmic TDP-43 accumulation in mouse neurons 20.

We thank the NINDS Biorepository at Coriell Institute for providing the following cell lines for this study: ND12133, ND03231, ND01751, ND11976, ND03719, ND00184, ND5280, ND06769, ND10689, ND12099, ND14954, ND08957, ND12100, and ND014587. We thank Helena Chui and Carol Miller at the University of Southern California Alzheimer’s Disease Research Center and Neil Shneider at the Columbia University Medical Center for control and C9ORF72 patient tissue. We thank the Choi Family Therapeutic Screening Facility for chemical screening support and the Translational Imaging Center at USC for imaging support. We thank Max Koppers, Youri Adolfs, Christiaan van der Meer, and Mark Broekhoven for help with mouse breeding and kainate injection experiments. We thank Prof. Satoshi Waguri for providing the M6PR-GFP construct. We thank Christopher Buser for assistance with electron microscopy. We also thank Sam Alworth (DRVision Technologies, LLC), Katja Hebestreit, and Raj Bhatnagar (Verge Genomics), Bob Baloh, Jacqueline O’Rourke, Christopher Donnelly, Chang Tong, Andrew McMahon and Qing Liu-Michael for reagents, technical support, and discussions. E.Y.S. is a Walter V. and Idun Berry Postdoctoral Fellow. K.A.S. was supported in part by a Muscular Dystrophy Association Development Grant. L.M. was supported by NIH grant T32DC009975–04. This work was supported by NIH grants AG039452, AG023084, and NS034467 to B.V.Z. R.J.P. was supported by grants from ALS Foundation Netherlands (TOTALS), Epilepsiefonds (12–08, 15–05), and VICI grant Netherlands Organisation for Scientific Research (NWO). This work was also supported by NIH grants R00NS077435 and R01NS097850, U.S. Department of Defense grant W81XWH-15–1-0187, and grants from the Donald E. and Delia B. Baxter Foundation, the Tau Consortium, the Frick Foundation for ALS Research, the Muscular Dystrophy Association, the New York Stem Cell Foundation, the USC Keck School of Medicine Regenerative Medicine Initiative, the USC Broad Innovation Award, and the Southern California Clinical and Translational Science Institute to J.K.I. J.K.I. is a New York Stem Cell Foundation-Robertson Investigator.

Consistent with PIKFYVE being the relevant target in the iMN survival assay, Apilimod increased C9ORF72 patient, but not control, iMN survival in either neurotrophic withdrawal conditions (Fig. 6d) or excess glutamate (n=4 patients, Supplementary Fig. 15f (n=3 controls, Supplementary Fig. 15g). Automated neuron tracking software independently verified Apilimod efficacy on C9ORF72 patient iMNs (Supplementary Fig. 15h). As further confirmation that PIKFYVE was the active target, ASO-mediated suppression of PIKFYVE also rescued C9ORF72 patient iMN survival (Fig. 6d and Supplementary Fig. 15i). In addition, we synthesized a structural analog of Apilimod with a reduced ability to inhibit PIKFYVE kinase activity in a biochemical assay using purified PIKFYVE protein (Fig. 6b and Supplementary Fig. 15j, 16). The reduced activity analog was significantly less effective at rescuing C9ORF72 patient iMN survival (Fig. 6e). Thus, small molecule inhibition of PIKFYVE can rescue patient motor neuron survival.

The repeat expansion suppresses the production of C9ORF72 protein by inhibiting transcription 3,4,6,7,9,17, raising the possibility that haploinsufficiency for C9ORF72 activity triggers disease pathogenesis. Consistent with this hypothesis, elimination of C9orf72 activity alters myeloid cell behavior in mice 14,18,19 and in vitro studies suggest that C9ORF72 activity may enhance autophagy 20,21.
Live imaging of iMNs expressing a M6PR-GFP fusion protein that localizes to M6PR+ vesicles 44 confirmed that C9ORF72 patient and C9ORF72-deficient iMNs possess increased numbers of M6PR+ vesicle clusters, and that overexpression of C9ORF72 isoform A or B rescues this phenotype (Supplementary Fig. 9c-g and Supplementary Videos 5-9). Clusters did not disperse over the time course of the assay, suggesting that they are relatively stable and not in rapid flux (Supplementary Videos 5-9). In addition, M6PR+ puncta moved with a slower average speed in C9ORF72 patient and C9ORF72+/− iMNs than controls (Supplementary Fig. 9h, i). Thus, reduced C9ORF72 levels lead to fewer lysosomes in motor neurons in vitro and in vivo, and this may be due in part to altered trafficking of M6PR+ vesicles.

An intronic GGGGCC repeat expansion in C9ORF72 is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), but its pathogenic mechanism remains unclear. Here we use human induced motor neurons (iMNs) to show that repeat-expanded C9ORF72 is haploinsufficient in ALS. We show that C9ORF72 interacts with endosomes and is required for normal vesicle trafficking and lysosomal biogenesis in motor neurons. Repeat expansion reduces C9ORF72 expression, triggering neurodegeneration through two mechanisms: accumulation of glutamate receptors leading to excitotoxicity, and impaired clearance of neurotoxic dipeptide repeat proteins derived from the repeat expansion. Thus, cooperativity between gain- and loss-of-function mechanisms leads to neurodegeneration. Restoring C9ORF72 levels or augmenting its function with constitutively active RAB5 or chemical modulators of RAB5 effectors rescues patient neuron survival and ameliorates neurodegenerative processes in both gain- and loss-of function C9ORF72 mouse models. Thus, modulating vesicle trafficking can rescue neurodegeneration caused by the C9ORF72 repeat expansion. Coupled with rare mutations in ALS2, FIG4, CHMP2B, OPTN, and SQSTM1, our results reveal mechanistic convergence on vesicle trafficking in ALS/FTD.
IPSC-MNs at differentiation D35 were harvested in cold Hypotonic buffer (20 mM HEPES pH 7.4, 10 mM KCl, 2 mM MgCl2, 1 mM EDTA, 1mM EGTA, 1 mM DTT and protease inhibitor cocktail (Roche)) and lysed by passing through G25 needles 25 times and then spun down at 700 x g for 10min at 4℃. The Supernatant was loaded onto pre-made 30% Percoll solution and re-centrifuged at 33,000 RPM using Beckman rotor SWI55 for 50min at 4℃. 300 ul aliquots were taken from top to bottom as fractions and all the collected samples were boiled with SDS-PAGE sample buffer and analyzed by western blot.
The degree of Li force one can employ in kung fu depends on several variables such as resilience of muscles, strength of bones, speed and timing of attack and so on. An effective way to enhance the Li force is to exercise one's muscles and bones by applying increasing pressure on them (weight training, gym exercises, etc.).[2] The stronger one's muscles and bones become, the more powerful and skillful the level of kung fu is.[3]
Local field potentials (LFPs) were recorded from iPSC-derived motor neurons on days 17–21 in culture in 6-well multielectrode chips (9 electrodes and 1 ground per well) using a MultiChannel Systems MEA-2100 multielectrode array (MEA) amplifier (ALA Scientific) with built-in heating elements set to 37°C. Cells were allowed to acclimate for 5 minutes after chips were placed into the MEA amplifier, and after glutamate addition (10 μM final concentration). For 1 μM Apilimod treatments, chips were incubated for 35 min in a humidified incubator in the presence of the particular drug, then returned to the MEA amplifier and acclimated for 5 min before beginning recordings. For each condition, recordings (5 min baseline, 10 min glutamate and/or drug, 40 kHz sampling rate) were filtered between 1–500 Hz, and average LFP frequency per well was determined using the accompanying MC Rack software.
Since glutamate receptor activation and neuronal firing both induce calcium influx, we determined their relative contributions to the increased Gcamp6 activation by. using the ion channel inhibitors TTX and TEA to block neuronal firing. C9ORF72+/− iMNs still displayed more frequent Gcamp6 activation than C9ORF72+/+ iMNs (Supplementary Fig. 13a), indicating that part of the hyperexcitability is due to increased glutamate receptor activation. To determine which receptors were responsible for the increased glutamate response, we tested small molecule agonists of specific glutamate receptor subtypes. Activation of NMDA, AMPA, and kainate receptors was higher in C9ORF72+/− iMNs than controls (Supplementary Fig. 13a).
Although studies in mice have indicated that C9orf72 activity is important for myeloid cell function 14,18, a report in mouse neurons and zebrafish suggests that C9orf72 isoform A may modulate poly-Q Ataxin-2 toxicity 20, and studies have implicated C9ORF72 in regulating autophagy 20,31,32,38, our study provides the first direct evidence showing that gain- and loss-of-function C9ORF72 mechanisms cooperate to cause the degeneration of human motor neurons. Recent studies have shown that C9ORF72 isoform A, but not isoform B, can form a functional complex with SMCR8 and WDR41 20. However, since both C9ORF72 isoforms rescue C9-ALS iMN degeneration in our assay, other mechanisms or protein interactions may underlie the rescue of patient iMNs.
Importantly, our work establishes a new approach for suppressing DPR protein toxicity and blocking C9ORF72 pathogenesis: restoring or replacing C9ORF72 activity. Although high levels of C9ORF72 isoform A may have slightly detrimental effects on control motor neuron survival, we have only observed this in neurons without C9ORF72 repeat expansion. Thus, we would not anticipate a harmful effect of forced C9ORF72 expression in C9ORF72 patients. In addition, a better understanding of the effects of forced C9ORF72 expression could inform safe development of this therapeutic strategy. For example, determining if C9ORF72 accelerates turnover of DPR aggregates by stimulating autophagy could lead help to identify new therapeutic targets.
Local field potentials (LFPs) were recorded from iPSC-derived motor neurons on days 17–21 in culture in 6-well multielectrode chips (9 electrodes and 1 ground per well) using a MultiChannel Systems MEA-2100 multielectrode array (MEA) amplifier (ALA Scientific) with built-in heating elements set to 37°C. Cells were allowed to acclimate for 5 minutes after chips were placed into the MEA amplifier, and after glutamate addition (10 μM final concentration). For 1 μM Apilimod treatments, chips were incubated for 35 min in a humidified incubator in the presence of the particular drug, then returned to the MEA amplifier and acclimated for 5 min before beginning recordings. For each condition, recordings (5 min baseline, 10 min glutamate and/or drug, 40 kHz sampling rate) were filtered between 1–500 Hz, and average LFP frequency per well was determined using the accompanying MC Rack software.
The Li force is observable when it is employed. Unlike the Li force, Neijing is said to be invisible. The "pivot point" essential to Li combat is not necessary in Neijing. At the point of attack, one must ‘song’ (loosen) himself to generate all Neijing energy one possesses and direct this energy stream through one's contact point with an opponent.[5] The contact point only represents the gateway to conduct Neijing energy at the point of attack.[6]
×