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Intracellular Double Labeling of Substantia Nigra and Pedunculopontine Neurons in in vitro Slice Preparation
Published in Avital Schurr, Benjamin M. Rigor, BRAIN SLICES in BASIC and CLINICAL RESEARCH, 2020
Patch pipettes are made from thin-walled borosilicated glass capillaries (O.D. 1.5 mm, Sutter) by pulling them in three steps (P-87, Sutter). The pipette has a DC resistance of 5 to 7 MΩ when filled with the standard pipette solution. The standard pipette solution consists of the following ionic compositions (in mM): 145 CsCl; 3 MgCl2; 2 ATP-Na2; 10 HEPES; and 1 to 10 EGTA. The pH 7.2 is adjusted with CsOH; CsCl can be replaced with K-gluconate (145 mM); and pH 7.2 must be adjusted with KOH. To suppress K+ currents, the Ringer’s solution, in which the slice is bathed, can be modified to include 20 mM tetraethylammonium chloride (TEA-Cl) by replacing equimolar NaCl. In order to better isolate calcium currents, ionic compositions (in mM) of the bathing media can be changed to 145 TEA-Cl, 3 CaCl2 or BaCl2, 3 CsCl, 10 HEPES, 6 glucose, 4 4-AP, and 1 μM TTX; pH 7.2 is adjusted with TEA-OH. The osmolarity of the intracellular and extracellular solution is adjusted to 290 to 300 and 270 to 280 mOsm/1 with glucose, respectively.
Muscles of the Upper Airway and Accessory Respiratory Muscles
Published in Alan D. Miller, Armand L. Bianchi, Beverly P. Bishop, Neural Control of the Respiratory Muscles, 2019
Erik van Lunteren, Thomas E. Dick
Repetitive contraction of a muscle may lead not only to loss of force over time, but also to a transient increase in force, so-called force potentiation. Experimentally this can be elicited following a tetanic contraction (posttetanic potentiation), during repetitive twitches (twitch or staircase potentiation), or during the early portion of a fatiguing series of contractions. Rat sternohyoid demonstrates considerably greater force potentiation than the diaphragm.59 In patients with obstructive apnea, there is typically a waxing and waning of pharyngeal muscle activation. Based on the experimental evidence of high force potentiation and low degree of fatigue resistance, fluctuations in pharyngeal muscle force may actually exceed that estimated based on electromyographic recordings. Subjects with sleep apnea develop hypoxemia and hypercapnia, which are known to affect diaphragm contractile function. Cat geniohyoid muscle studied in vivo was found not to alter its isometric twitch kinetics with either chemical perturbation, but had a decline in fatigue resistance with severe hypoxia (but not mild hypoxia or hypercapnia).45 Finally, differences between pharyngeal muscles and the diaphragm have been noted in their susceptibility to the effects of various pharmacological agents. In studies in which both muscles were assessed, aminophylline improved rat diaphragm fatigue resistance but not sternohyoid fatigue resistance,60 vecuronium (a neuromuscular blocking agent) decreased the force of the canine geniohyoid muscle more than that of the diaphragm,26 and tetraethylammonium chloride (a K+ channel blocker) augmented rat sternohyoid more than diaphragm twitch force.61
Affinity Modification: Physical Chemistry
Published in Dmitri G. Knorre, Valentin V. Vlassov, Affinity Modification of Biopolymers, 1989
Dmitri G. Knorre, Valentin V. Vlassov
An expression similar to Equation 59 in the absence of Y was derived by Palumaa et al.207 Their equation may be obtained from Equation 59 putting y0 = 0. The equation was applied to calculate the kinetic parameters and association constants of alkylation of the horse serum acylcholine acylhydrolase (EC 3.1.1.8) with N,N-dimethyl-2-phenylaziridinium ion.207 The set of [PZ]ij values corresponding to different initial concentrations of the reagent (xo)i at times tj was treated by nonlinear least-square regression program using the function: where B0 = (k1/k2)(1 − K/Kx), B1 = 1/KX, B2 = Kr/Kx, and B3 = exp (−k2). Parameters Bi found by a minimization procedure permitted to estimate k1, k2, Kx, and Kr. At 25°C in a phosphate buffer pH 7.5 and ionic strength 0.15 M with (x0)i taken in the range 0.104 to 0.766 M, the following values were obtained: Kx = 1.01 × 104 M−1, Kr = 2.94 × 103 M−1, k1 = 1.02 × 10−4 sec−1, and k2 = 2.01 × 10−4 sec−1. The accuracy of the data (SDs within 1 to 10%) proves the validity of the equation applied. The same authors have applied the full equation (Equation 59) to estimate the affinity of the cationic inhibitors to acylcholine acylhydrolase.358 The kinetic curves of alkylation of the enzyme with N,N-dimethyl-2-phenylaziridinium ion in the presence of different competitors were obtained. Using the function shown in Equation 61 with the same significance of Bo, B2, B3, and B1 = (Kr + y0)/KxKr, the authors have calculated from these data by nonlinear least-square regression program Ky values for several competitors. These values are in good agreement with the Ki values indicated in the brackets for the same compounds found in the study of competitive inhibition of acetylthiocholine hydrolysis by the enzyme. The Ky(Ki) values were found to be for tetramethylammonium chloride: 4.28 × 10−3 (4.36 × 10−3) M−1, for tetraethylammonium chloride: 2.87 × 10−3 (2.84 × 10−3) M−1, and for N-methylacridinium iodide 1.85 × 10−7(1.15 × 10−7)M−1.
Brain-targeting delivery of MMB4 DMS using carrier-free nanomedicine CRT-MMB4@MDZ
Published in Drug Delivery, 2021
Yimeng Du, Jing Gao, Hui Zhang, Xiaohui Meng, Dong Qiu, Xiang Gao, Aiping Zheng
All the reagents were used as received. Midazolam was purchased from Astellas Pharma Inc. (Tokyo, Japan). MMB4 DMS was supplied by Beijing Institute of Pharmacology and Toxicology (Beijing, China). The cyclic peptide CRT (sequence: CRTIGPSVC) was chemically synthesized by the Chinese Peptide Co., Ltd. (Hangzhou, China). The ChromPure mouse Tf was purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). Methanol, isoamyl acetate (IA), trifluoroacetic acid (TFA), tetraethylammonium chloride (TEAC), and sodium dodecyl sulfate (SDS) were purchased from Sigma-Aldrich (St. Louis, MO). Acetic acid was purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). MTT kits were purchased from Immutopics Ltd. (San Clemente, CA). Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), and penicillin/streptomycin were purchased from Gibco (Dublin, Ireland). Other reagents, if not specified, were obtained from Chinese Medicine Group.
Effects of modulation on sodium and potassium channel currents by extremely low frequency electromagnetic fields stimulation on hippocampal CA1 pyramidal cells
Published in Electromagnetic Biology and Medicine, 2021
Yu Zheng, Pei Xia, Lei Dong, Lei Tian, Chan Xiong
Cutting solution (in mM): 90 sucrose, 87.2 NaCl, 2.5 KCl, 7 MgCl2, 0.5 CaCl2, 1.25 NaH2PO4, 25 NaHCO3, and 16.7 glucose. Artificial cerebrospinal fluid (ACSF) (in mM): 120 NaCl, 2.5 KCl, 2 MgSO4 · 7H2O, 2 CaCl2, 1.25 NaH2PO4 · 2H2O, 26 NaHCO3 and 10 glucose. The extracellular solution for whole-cell recordings was the same as ACSF, in the control group and experimental group for INa recordings, the extracellular solution was supplemented with 30 tetraethylammonium chloride (TEA-Cl) and 2 mM 4-aminopyridine (4-AP) to block the IA and IK, and 0.1 mM CdCl2 to block all Ca2+ channels current. For IA recording, the extracellular solution was supplemented with 1 μM TTX to block INa, 30 mM TEA-Cl to block IK, and 0.1 mM CdCl2 to block all Ca2+ channels current. For IK recording, the extracellular solution was supplemented with 1 μM TTX to block INa, 2 mM 4-AP to block IA, and 0.1 mM CdCl2 to block all Ca2+ channels. In experimental group 2, the extracellular solution was supplemented with 30 mM TEA-Cl, 2 mM 4-AP, 1 μM TTX and 0.1 mM CdCl2 to block the IK, IA, INa and all Ca2+ channels current, respectively. In the control and experimental group, the intracellular pipette solution used for INa containing (in mM) CsCl 70, CsF 70, HEPES 10, EGTA 10, Na2ATP 3, MgCl2 2, and the pH was adjusted to 7.2 by CsOH. The intracellular pipette solution used for IA and IK containing (in mM) KCl 65, KF 65, HEPES 10, EGTA 10, Na2ATP 3, MgCl2 2, CaCl2 2, and the PH was adjusted to 7.2 by KOH.
Evaluation of the Presence and Functional Importance of Nucleoside Transporters in Lacrimal Gland for Tear Disposition of Intravenously Injected Substrate in Rabbits
Published in Current Eye Research, 2021
Hanuman Prasad Sharma, Nabanita Halder, Sundararajan Baskar Singh, T. Velpandian
Ribavirin and dipyridamole standards were purchased from TCI, India. Tetraethylammonium chloride (TEA) and sulfadimethoxine (SDM) were used as internal standard (IS) for ribavirin and dipyridamole, respectively, and procured from Sigma-Aldrich, USA. Other chemicals were of the highest purity grade and were from their respective sources.