Title : Activity - dependent regulation of the binomial parameters p and n at the mouse 1 neuromuscular junction in vivo 2 3 4

27 Block of neurotransmission at the mammalian neuromuscular junction (NMJ) triggers an 28 increase in the number of vesicles released (quantal content). The increase occurs whether nerve 29 and muscle activity are both blocked by placement of a tetrodotoxin (TTX) containing cuff on 30 the nerve or whether muscle activity is selectively blocked by injection of alpha bungarotoxin 31 (BTX). We used analysis of variance to examine whether the mechanism underlying the 32 increase in quantal content differed between the two types of activity blockade. We found that 33 TTX-induced blockade increased probability of release (p), while BTX-induced blockade 34 increased the number of releasable vesicles (n). The lack of increase in p when postsynaptic 35 activity was blocked with BTX suggests that block of presynaptic activity triggers the increase. 36 To determine whether n is regulated by mismatch of pre-and postsynaptic activity introduced by 37 BTX injection we combined BTX and TTX and still found an increase in n. We conclude block 38 of acetylcholine binding to acetylcholine receptors during spontaneous release triggers the 39 increase in n. 40 41 42 Activity regulates p an n 3 INTRODUCTION 43 Chronic manipulations of activity trigger a series of changes in synaptic function that 44 maintain firing rates of networks within certain boundaries and have thus been termed 45 homeostatic regulation of synaptic function. In one commonly studied form, homeostatic 46 synaptic plasticity is triggered by a decrease in activity and results in an increase in excitatory 47 synaptic strength (Moulder et al. 2006; Rich and Wenner 2007; Turrigiano 2008). 48 The mammalian neuromuscular junction (NMJ) is a classic excitatory synapse ideally 49 suited to studies of homeostatic regulation of synaptic strength in vivo since there is only one 50 presynaptic input and one neurotransmitter. The presence of only one input allows for studies of 51 evoked release and quantal content that are not possible at central synapses. The first report of 52 what would now be termed homeostatic regulation of synaptic strength was in 1971 at the 53 neuromuscular junction. In the study, limb immobilization triggered an increase in postsynaptic 54 acetylcholine receptors (AChRs) that was paralleled by an increase in quantal amplitude 55 (Robbins and Fischbach 1971). Subsequent studies using tetrodotoxin (TTX) to block nerve 56 activity in vivo found an increase in quantal content at the mouse NMJ (Snider and Harris 1979; 57 Tsujimoto and Kuno 1988; Tsujimoto et al. 1990; Wang et al. 2004). It has also been found that 58 block of AChRs with α bungarotoxin (BTX) at the NMJ in vivo triggers an increase in quantal 59 content (Molenaar et al. 1991; Plomp et al. 1992; 1994). 60 Application of TTX to the nerve and block of AChRs with BTX are fundamentally 61 different ways of blocking synaptic transmission. TTX application blocks spiking of both nerve 62 and muscle while block of AChRs only blocks spiking of the muscle. Application of BTX 63 blocks binding of acetylcholine to AChRs during spontaneous release of transmitter while TTX 64 does not affect this process. In the chick spinal cord block of transmitter receptors, but not block 65 of spiking, triggers an increase in quantal amplitude (Wilhelm and Wenner 2008). Available 66 evidence at the mammalian NMJ suggests that TTX and BTX increase quantal content via 67 distinct mechanisms, but the two methods of blocking synaptic transmission have never been 68 directly compared. The increase in quantal content triggered by TTX is evident only in solution 69 containing low extracellular calcium (Wang et al. 2004) whereas the increase in quantal content 70 following BTX is present at normal extracellular Ca (Molenaar et al. 1991; Plomp et al. 1992; 71 1994). In the present study we demonstrate that TTX and BTX increase quantal content by 72 Activity regulates p an n 4 distinct mechanisms. Block of distinct aspects of synaptic activity trigger the increases in 73 quantal content triggered by TTX and BTX. 74 75 Activity regulates p an n 5 METHODS 76 Ethical Approval 77 All procedures involving animals were approved by the Wright State LACUC committee. 78 79 Mice 80 For a previous study (Wang et al. 2005) we used ClCn1 mice obtained from 81 Jackson labs (Bar Harbor, ME). Although no mutant mice were used in this study, in order to 82 compare to our previous results we used unaffected litter mates of the strain. Unaffected 83 littermates consisted of mice that were heterozygous for the ClC mutation and mice that carried 84 no copy of the mutation. Since no unaffected siblings have myotonia by EMG it appears that 85 heterozygous mice are unaffected (Wang et al. 2005). 86 87 TTX cuff application and BTX injection 88 Two to 4 month-old mice were anesthetized with intraperitoneal injection of chloral 89 hydrate. Use of chloral hydrate for rodent anesthesia has been called into question (Silverman 90 and Muir 1993; Baxter et al. 2009). Concerns that have been raised include respiratory 91 depression (Flecknell 1996) and adynamic ileus (Fleischman et al. 1977). We have used chloral 92 hydrate to anesthetize mice for the past 12 years. If chloral hydrate caused significant respiratory 93 depression in mice we would expect to have a high rate of overdose. We have found exactly the 94 opposite to be true. In our hands chloral hydrate causes significantly less death due to overdose 95 than injection of ketamine/xylazine. While adynamic ileus was reported in rats this has not been 96 our experience in mice; we have not seen poor appetite, abdominal distention or lack of weight 97 gain following administration. Finally, it has been suggested that chloral hydrate is not optimal 98 as it is a hypnotic, rather than anesthetic agent (Hall et al. 2001) (See however, (Flecknell 1996)). 99 We have not noted pain behavior during surgery such as vocalization or movement in response 100 to incision to suggest that mice are experiencing inadequate anesthesia. We conclude that while 101 choral hydrate may not be optimal for use in rats, it is safe and efficacious in our strain of mice. 102 One reason for the difference between our experience and those of others may be the 103 considerable strain variation in the response to chloral hydrate in rodents (Flecknell 1996). As 104 our surgeries involve toxins we work in a biosafety hood and thus find it easier to use injected 105 anesthetic rather than inhaled anesthetic. 106 Activity regulates p an n 6 Following anesthesia the hip area was sterilized with betadine and a 1 cm incision was 107 made over the sciatic nerve. TTX cuffs were fashioned from silastic tubing and were placed 108 around the left sciatic nerve and attached to an Alzet osmotic pump placed subcutaneously in the 109 back (model 1002, infusing at 0.25 ul/hr, Durect, Cupertino, CA) containing 850 μM 110 tetrodotoxin dissolved in 0.9% NaCl. The tubing to the pump was filled with saline so that after 111 surgery the foot muscles were not paralyzed. This allowed us to confirm that the nerve had not 112 been injured during surgery. Following surgery mice were given one dose of subcutaneous 113 buprenorphine for analgesia. By the next day mice were moving freely, eating and drinking and 114 exhibited no pain behavior. The next morning the mouse was checked for loss of ability to 115 spread its toes to confirm that nerve block had developed. Prior to sacrifice at 8-9 days the nerve 116 block was checked again. Mice were sacrificed using CO2 inhalation and the tibialis anterior 117 muscle was removed. Bungarotoxin injection was performed on anesthetized mice using a 31 118 gauge Hamilton syringe. After anesthesia the area over the tibialis anterior was shaved and 119 washed with betadine prior to injection. 3 ul of 1 ug/ul αbungarotoxin was injected. Four to 5 120 days after injection, mice were sacrificed and quantal content was measured. 121 122 Electrophysiologic recording 123 Mice were sacrificed using CO2 inhalation and the tibialis anterior muscle was removed. 124 For most experiments, the recording chamber was continuously perfused with Ringer solution 125 containing (in millimoles per liter) NaCl, 118: KCl, 3.5; CaCl2, 2; MgSO4, 0.7; NaHCO3, 26.2; 126 NaH2PO4, 1.7; glucose, 5.5 (pH 7.3-7.4, 20-22C) equilibrated with 95% O2 and 5% CO2. For 127 lowering the probability of release for analysis of variance, recordings were done in 1 mM Ca 128 solution in which the Mg concentration was kept at 0.7 mM. Endplate recordings were 129 performed as previously described (Wang et al. 2004; Wang et al. 2006; Wang et al. 2010). 130 Briefly, the tibialis anterior muscle was pinned in a dish and stained with 10 μM 4-(4131 diethylaminostyryl)-N-methylpyridinium iodide (4-Di-2ASP; Invitrogen, Carlsbad, CA). In 132 most experiments muscle fibers were crushed away from the endplate band to eliminate 133 contractions following nerve stimulation and two electrode voltage clamp was used to set the 134 holding potential to –45 mV (Wang et al. 2010). To detect miniature endplate currents (MEPCs) 135 in muscle injected with BTX fibers were held at -100 mV. In these experiments contraction was 136 prevented by 1 uM μ conotoxin GIIIB (Peptide Institute, Osaka Japan). For all experiments, 137 Activity regulates p an n 7 quantal content was calculated by dividing peak EPC current amplitude by peak MEPC current 138 amplitude. For lowering the probability of release during 50 Hz repetitive stimulation, 139 recordings were done in low (1 mM) Ca solution in which the Mg concentration was kept at 140 0.7 mM. 141 Determination of the role of P/Q Ca channels in evoked release was performed by 142 measuring mean EPC amplitude before and after application of ω-agatoxin-IVA (Peptide 143 Institute, Osaka Japan) in both control and BTX blocked muscles. After recording EPC 144 amplitude from at least 15 endplates in a tibialis anterior muscle in the absence of ω-agatoxin145 IVA, each muscle was placed in 2 mls of Ringer solution containing 1 μM ω-agatoxin-IVA for 1 146 hour. The solution was continuously bubbled with 95% O2 and 5% CO2. After 1 hour, the 147 muscle was put into a recording chamber that was slowly perfused with Ringer solution 148 containing no ω-agatoxin-IVA. EPCs were recorded from at least 15 endplates in each muscle. 149 All endplate recordings following the application of ω-agatoxin-IVA were made within 40 150 minutes of placing the muscle in the recording chamber. During this 40-minute period mean 151 endplate current (EPC) amplitude did not increase because of wash out of ω-agatoxin-IVA. 152 153 Analysis of Variance 154 Analysis of variance was performed as previously described (Wang et al. 2010). For 155 measurement of EPC variance, at least 20 stimulations of the nerve were performed at 0.5 Hz. 156 However, even at this low rate of stimulation there was slight depression of the EPC during the 157 first 3 pulses, especially after administration of BTX. This depression led to overestimation of 158 EPC variance so the first 3 EPCs were discarded from measurement of EPC variance leaving at 159 least 17 values for analysis. The average number of EPCs measured to determine EPC variance 160 was 22. Using a higher number of EPCs for analysis variance at individual endplates had little 161 effect on estimates of mean variance for a given muscle. The mean variance and quantal content 162 from each muscle was then averaged between muscles to get final values, which were plotted. 163 Endplate recordings in which there was movement artifact or a change in EPC amplitude with 164 time were discarded since such artifacts led to large increases in variance. For each muscle the 165 average m and Var(m) were calculated. The average of m and Var(m) for each muscle were then 166 averaged to generate plots. 167 Activity regulates p an n 8 Contribution of MEPC variance to EPC variance was ignored since MEPC variance is 168 small relative to EPC variance (Wang et al. 2010). Interpretation of analysis of variance is 169 confounded by several assumptions made during the analysis. We have previously examined 170 assumptions made in analysis of variance and found that they were either valid or did not affect 171 interpretation of data from the mouse NMJ (Wang et al. 2010). 172 173 Morphological analysis of sprouting 174 After sacrifice by perfusion with 4% paraformaldehyde, the tibialis anterior muscle was 175 removed and postfixed in 4% paraformaldehyde for 1 hour, cryoprotected in 15 % sucrose 176 solution overnight, and frozen in liquid nitrogen. Muscles were sectioned (20 μm thickness) and 177 acetylcholine receptors in motor endplates were labeled with rhodamine-conjugated α178 bungarotoxin (Molecular Probes). Axons and motor terminals were labeled with a mouse 179 monoclonal antibody against the phosphorylated heavy fragment of neurofilament protein 180 (SMI31, 1:400, Sternberger Monoclonal). Synaptic vesicles were labeled using a mouse 181 polyclonal antibody directed against the SV2 protein (1:40, Developmental Studies Hybridoma 182 Bank, University of Iowa). Labeling of both primary monoclonal antibodies was visualized 183 using a fluorescein-conjugated donkey anti mouse secondary antibody (1:100, Jackson 184 Immunoresearch Laboratories). Z-axis stacks of images at sequential focal planes (0.5 μm 185 separation) of NMJs were obtained using a Fluoview FV 1000 confocal microscope and a 60x 186 objective (Olympus optical). Illustrated images are flat-plane in focus projections obtained from 187 z-series images using Fluoview software. 188 189