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Acetylcholinesterase deficiency contributes to neuromuscular junction dysfunction in
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type I diabetic neuropathy
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Carmen C. Garcia1,3, Joseph G. Potian1, Kormakur Hognason1, Baskaran Thyagarajan1, Lester
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G. Sultatos1, Nizar Souayah2, Vanessa H. Routh1, and Joseph J. McArdle1.
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Department of Pharmacology and Physiology1, and Neuroscience2.
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School-University of Medicine and Dentistry of New Jersey, Newark, New Jersey.
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address, Cátedra de Fisiopatología, Instituto de Medicina Experimental, Universidad Central de
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Venezuela, Caracas, Venezuela.
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Running Head: AChE deficiency and NMJ dysfunction in type I diabetes
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Corresponding author: Joseph J. McArdle, PhD. Department of Pharmacology and Physiology New Jersey Medical School-UMDNJ MSB-I626 185 South Orange Av Newark, NJ 07101-1709 Voice: (973)972-4428 Fax : (973)972-4554 email:
[email protected]
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New Jersey Medical 3
Present
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ABSTRACT
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Diabetic neuropathy (DN) is associated with functional and morphological
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changes of the neuromuscular junction (NMJ) associated with muscle weakness. This
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study examines the effect of type 1 diabetes on NMJ function. Swiss Webster mice were
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made diabetic with three inter-daily ip injections of streptozotocin (STZ). Mice were
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severely hyperglycemic within 7 days after beginning the STZ treatment. While
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performance of mice on a rotating rod remained normal, the twitch tension response of
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the isolated Extensor digitorum longus to nerve stimulation was significantly reduced at 4
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weeks after the onset of STZ-induce hyperglycemia. This mechanical alteration was
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associated with increased amplitude and prolonged duration of miniature endplate
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currents (mEPCs). Prolongation of mEPCs was not due to expression of the embryonic
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acetylcholine receptor but to reduced muscle expression of acetylcholine esterase
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(AChE). Greater sensitivity of mEPC decay time to the selective butyrylcholinesterase
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(BChE) inhibitor PEC suggests that muscle attempts to compensate for reduced AChE
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levels by increasing expression of BChE. These alterations of AChE are attributed to
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STZ-induced hyperglycemia since similar mEPC prolongation and reduced AChE
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expression was found for db/db mice. The reduction of muscle endplate AChE activity
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early during the onset of STZ-induced hyperglycemia may contribute to endplate
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pathology and subsequent muscle weakness during diabetes.
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Key
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streptozotocin, type 1 diabetes
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words :
acetylcholinesterase,
butyrylcholinesterase,
neuromuscular junction,
2
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INTRODUCTION
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Hyperglycemia associated with Diabetes mellitus produces long-term damage
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and failure of various tissues (10). In particular, diabetes-induced neural damage is a
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predominant form of neuropathy in the Western world. Changes of neuromuscular
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transmission would contribute to the progressive weakness of extensor and flexor
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muscles during diabetes (4). Therefore, the goal of this study was to further explore the
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effects of diabetes on the neuromuscular junction (NMJ).
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A limited number of studies have examined the impact of experimental diabetic
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neuropathy on pre- and postsynaptic elements of the NMJ. For example, type 1 diabetic
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rodents have decreased numbers of acetylcholine (ACh) containing vesicles as well as
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degeneration of mitochondria within motor nerve endings. These changes are
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associated with inhibition of transmission across the NMJ (17,18,33).
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postsynaptic level, the pattern of ACh receptor (AChR) distribution is altered and
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discrete AChR islands form on the muscle endplate surface (43). These experimental
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studies are clinically significant since muscle endplate remodeling also occurs during
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human diabetes (49, 56, 57).
At the
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In this study, we examined muscles from mice at various times after the onset of
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streptozotocin (STZ) – induced hyperglycemia. This broad-spectrum antibiotic is toxic to
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pancreatic β cells and produces an experimental model of type 1 diabetes (28).
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Recently, we reported (67) electrophysiological alterations of the NMJ of mice with STZ-
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induced type 1 diabetes. In particular, miniature endplate current (mEPC) amplitude
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increased significantly at early times after the onset of STZ-induced hyperglycemia.
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Further analyses demonstrated a significant prolongation of end plate current decay
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time.
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functional relevance of these diabetes-induced changes.
The goal of the present study was to understand the molecular basis and
3
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Our data demonstrate that the increase of mEPC amplitude and decay time for
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the fast-twitch Extensor digitorum longus (EDL) muscle of STZ-treated mice is primarily
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due to a decline of endplate acetylcholinesterase (AChE) expression and activity.
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These data suggest that plasticity of the NMJ allows remodeling of this synapse in an
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attempt to adapt to hyperglycemia/hypoinsulinema-induced suppression of ACh release
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from motor nerve endings. While loss of AChE activity causes muscle weakness (30)
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the initial alteration of this enzyme during diabetes does not impair motor performance in
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the whole animal. However, there is a gradual loss of muscle twitch tension along with a
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reduction in the number of functional motor units (66). Therefore, we conclude that
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diabetes-induced
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pathophysiological changes of the muscle endplate which lead to muscle weakness.
loss
of
endplate
AChE
activity
initiates
a
sequence
of
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MATERIALS and METHODS
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Mice. All experiments were performed in accordance with the National Institutes
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of Health Guide for the Care and Use of Laboratory Animals. Experimental protocols
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were approved by the UMDNJ Institutional Animal Care and Use Committee.
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male Swiss Webster (SW) mice were purchased from Taconic Farms and housed in our
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institutional animal care facility. Mice weighting 25 to 30 gm received 3 inter-daily ip
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injections of streptozotocin (STZ, Sigma-Aldrich, Inc., Saint Louis, MO, USA) according
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to the following schedule:
Adult
day 1, 100 mg/Kg; day 3, 60 mg/Kg; day 5, 60 mg/Kg.
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Control mice received citrate buffer (ip) following the same schedule. Weekly blood
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glucose levels were monitored with a commercial glucometer (One Touch Ultra®).
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Male db/db mice, a model of type 2 diabetes that harbors a spontaneous mutation in the
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leptin receptor, as well as control C57BL/6 mice, were purchased from Jackson
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Laboratory (Bar Harbor, ME). They were sacrificed at 5 to 6 week of age. All rodents
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were housed in a pathogen-free environment with continuous access to food and water
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on a 12 hour light-dark schedule.
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Rotorod analysis. Mice were trained to walk on a horizontal rod rotating at 4 rpm
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(Rotamex; Columbus Instruments, Columbus, OH). This initial phase of training lasted 3
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days and involved three trials of 5 min/trial. On day 4, the rotation rate was progressively
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increased by 4 rpm every 40 Sec until a maximum velocity of 40 rpm was reached or the
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mouse fell from the rod. Mice continued training for an additional three days under these
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conditions. The rotation velocity at the moment mice fell from the rotating rod was
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recorded in control and diabetic mice at 1, 2, and 4 weeks after the onset of
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hyperglycemia. To estimate end rotorod velocity, mice were subjected to three
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trials/week, three times/day and the average terminal velocity calculated.
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In vivo electrodiagnostic studies. Mice were anesthetized with an ip injection of
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anesthetic containing ketamine (80 mg/kg)/xylazine (10 mg/kg). After shaving the
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abdomen and distal hindlimbs, mice were taped prone to a polystyrene foam board. Skin
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temperature was maintained at 32°C with a heating pad. The stimulating electrodes
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were 0.7 mm needles insulated with Teflon (Dantec sensory needle; Dantec, Skovlunde,
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Denmark). The cathode was placed close to the sciatic nerve at the proximal thigh, and
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the anode was placed subcutaneously 1 cm proximal to the anode. Motor responses
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were recorded from a ring electrode (Hush micro digital rings; Alpine Biomed, Fountain
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Valley, CA) that was placed circumferentially around the hindlimb at 1–1.5 cm from the
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stimulating electrode. The reference ring electrode was placed circumferentially around
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the hindlimb, 2 cm distal to the recording electrode; electrical activity was recorded in
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both flexor and extensor compartments of both hindlimbs.
Nerve stimuli were
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monophasic pulses delivered from a Medtronic Keypoint (Medtronic, Minneapolis, MN)
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through a constant current stimulator with fine intensity control. Recordings were
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acquired with Medtronic Keypoint electromyography amplifiers (500 Hz/5 KHz), and
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stored for subsequent analysis. For all studies, the position of the stimulating electrode
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was optimized so that the threshold for evoking a motor response was less than 0.7 mA.
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Stimulus intensity was increased to produce maximal compound muscle action potential
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(CMAP) amplitude. Integration of the maximal CMAP record indicated the maximum
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CMAP area.
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number estimate (MUNE) was then performed at a standard amplifier gain using a
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modification of the technique described previously by McComas et al. (48) and used by
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Shefner (65) in mice. Briefly, at a stimulation rate of 1 Sec-1 the stimulus intensity was
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slowly increased from subthreshold levels until a small all-or-none response was
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evoked. The response was digitally recorded after its stability was established by three
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to four identical repeats. The intensity was slowly augmented until the response
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increased in a quantal fashion. The increased response was again monitored for stability
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before a tracing was acquired for analysis. This process was repeated for a total of 10
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increments. Individual motor unit area was determined by subtracting the CMAP area of
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each response from that of the prior response. The average of the 10 individual values
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yielded an estimate of average single motor unit action potential area. The area of the
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maximum CMAP was divided by the preceding value to yield the MUNE.
Recording of amplitude (peak-peak) and distal incremental motor unit
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In vitro evaluation of muscle performance. The fast twitch Extensor digitorum
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longus (EDL) nerve-muscle preparation was dissected and mounted in a glass chamber
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(Rodnoti Glass technology, Inc., Monrovia, CA) filled with oxygenated (95% O2 – 5%
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CO2) normal Ringer solution (pH 7.4, room temperature) containing (mM) NaCl (135),
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KCl (5), MgCl2 (1), CaCl2 (2), Na2HPO4 (1), NaHCO3 (15), glucose (5.5).
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The EDL nerve was drawn into a suction electrode for indirect activation of
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muscle twitches. One tendon of the muscle was tied to a Grass Force transducer
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connected to a Digidata 1440A (Axon Instruments, Molecular Devices, Sunnyvale, CA).
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This permitted acquisition and analysis with PCLAMP software (Axon Instruments) of
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muscle mechanical responses to nerve stimulation. Isolated preparations were adjusted
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to optimal length for force generation and equilibrated for 15 min prior to supramaximal
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nerve stimulation (1 Hz).
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Physiologic recording at neuromuscular junctions (NMJs). The EDL muscle was
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removed from isoflurane-anesthetized mice and pinned to a Sylgard®-lined chamber
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containing HEPES-Ringer (HR) solution (135 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM
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MgCl2, 5.5 mM dextrose, and 5 mM HEPES; pH 7.3–7.4). Preparations were bathed in
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HR solution containing 0.75 μM μ-conotoxin GIIIB to inhibit muscle action potentials and
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mechanical responses to nerve stimulation. Miniature endplate currents (mEPCs) and
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endplate currents (EPCs, 1 Hz) were recorded at a holding potential of -75 mV with two-
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electrode voltage clamp technique (Axoclamp 2B, Axon Instruments, Foster City, CA).
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PCLAMP software (version 9.2, Axon Instruments) was used for acquisition and
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analyses of endplate currents. Voltage clamp recording minimized concerns introduced
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by diabetes-induced changes of muscle fiber size, membrane capacitance, or input
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resistance. Endplate currents were also recorded in the presence of 1 μM αA
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OIVA[K15N][N16K] from cone snail venom (71) or Waglerin-1 from snake venom (46) in
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order to pharmacologically dissect the contribution of embryonic and adult AChRs to
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endplate currents (72). Some muscles were exposed to either 5 μM phenserine tartrate
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(PT) or 5 μM phenethylcymserine tartrate (PEC) which are selective inhibitors of
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acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), respectively. PT and
7
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PEC were kindly donated by Dr. Nigel H. Greig (National Institute on Aging/ NIH,
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Baltimore, MD, USA).
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The sensitivity of the motor end-plate to iontophoretically applied ACh was
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evaluated in the Triangularis sterni (TS) muscle in which endplates are readily observed
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(31, 45). Square pulses through a pipette containing 3 M ACh and having a resistance
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of 200 MOhms delivered ACh onto muscle fibers; endplates regions were identified by
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the presence of miniature endplate potentials. To prevent ACh leakage and undesirable
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desensitization of the AChR, a constant breaking current of 10 to 30 nA was applied to
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the pipette tip.
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response to nCoul of charge passed through the ACh pipette (46).
ACh sensitivity was calculated as the ratio of membrane potential
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Quantitative reverse transcription PCR. RNA was isolated from the EDL muscle
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using Qiagen RNeasy Fibrous Tissue Mini Kit reagents (Qiagen, Valencia, CA). One μg
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of RNA was converted to cDNA with the SuperScript III First-Strand Synthesis System
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for RT-PCR (Invitrogen, Grand Island, NY) according to the manufacturer's instructions.
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Primers to amplify AChE (Mm 00477275_m1), AChR γ subunit (Mm00437417_m1), and
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the
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AppliedBiosystems (ABI). AChE probes were considered to detect all mRNA isoforms as
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they bind to sequences encoding the esterase domain of the enzyme. Real-time PCR
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was performed with the Taq PCR Master Mix Kit (Qiagen, Valencia, CA). Each sample
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was run in duplicate using the Light Cycler II (Roche) programmed with a denaturation
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step at 95°C followed by 35 cycles of 15 Sec at 95°C and a final 60 Sec extension at
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60°C. Relative expression was calculated by the “Delta-delta ct method” (42).
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housekeeping
gene
GAPDH
(Mm99999915_g1)
were
purchased
from
8
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Muscle endplate imaging: Motor endplates of EDL muscles were equilibrated for
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30 min with 5 μM Alexa-647 αA OIVA[K15N][N16K] which binds selectively to embryonic
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AChRs (71). Due to technical difficulty of dissecting EDL muscles from mouse pups,
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diaphragm muscle from four day old mice was stained as positive control for Alexa-647
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αA OIVA[K15N][N16K].
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preparations were visualized using an upright fluorescence microscope (Olympus
211
BX61WI). The presence of endplates was confirmed by a subsequent labeling of all
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AChRs with 1 μM FITC α-Bungarotoxin for 30 min. Microscopic images were acquired
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and analyzed with Metamorph Software (Molecular Devices, Downingtown, PA).
After a 20 min wash in toxin-free physiological saline,
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EDL muscles were stained for AChE with the method of Koelle and Horn (35) as
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modified by Gautron et al. (22). Acetylthiocholine acid was the substrate for the staining
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reaction. AChE labeling was performed at room temperature. After a 30 min reaction
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period, preparations were washed with physiological saline.
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selected endplates were immediately collected using an upright microscope (Olympus
219
BX61WI).
Images of randomly
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AChE activity: EDL muscles were dissected, weighed, and homogenized in 9
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volumes of 100 mM sodium phosphate buffer (pH 7.4). AChE activity was measured by
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the Ellman reaction at 24o in a Shimadzu UV2550 Spectrophotometer (Kyoto, Japan), as
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described by Sultatos et al. (70). The reaction volume in the cuvette was 1 ml, and
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contained 0.44 mM acetylthiocholine and 0.1 mM 5,5’-dithio-bis(2-nitrobenzoic acid).
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The reaction was initiated by addition of 50 μl of homogenate, and the increase in optical
227
density at 412 nm (which was an indicator of thiocholine production) was followed for 10
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min. The slopes of these rate curves were determined by linear regression analyses
229
using Sigmaplot 8 (Systat Software Inc., Chicago, IL).
9
230 231
Statistics. Biochemical and electrophysiological data were analyzed using
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GraphPad Prism Software. Data are presented as means + standard error. One way
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ANOVA with Tukey post hoc test compared experimental and control mean values; P