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J Physiol (2003), 546.3, pp. 789-800
© Copyright 2003 The Physiological Society
DOI: 10.1113/jphysiol.2002.030924
2-laminin deficient terminals |
ABSTRACT |
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(Received 15 August 2002; accepted after revision 1 November 2002; first published online 20 December 2002)
Corresponding author P. Noakes: School of Biomedical Sciences, and Special Research Center for Genomics and Bioinformatics, University of Queensland St Lucia, Queensland 4072, Australia. Email: p.noakes{at}mailbox.uq.edu.au
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INTRODUCTION |
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During development of the skeletal neuromuscular junction (NMJ), a bi-directional exchange of information between nerve and muscle induces both pre- and postjunctional differentiation (Sanes & Lichtman, 1999). Several of the signals responsible for this pre- and postjunctional specialisation have already been identified. For example, nerve-derived agrin plays a key role in regulating postjunctional differentiation (Burgess et al. 1999), while the laminin 
4 chain has been shown to be important for co-localisation of pre- and postjunctional elements (Patton et al. 2001).
The present study focuses on the 2-laminin chain, which has been shown to be a regulator of prejunctional differentiation (Noakes et al. 1995; Libby et al. 1999). 2-Laminin is produced by muscle cells and deposited into the synaptic basal lamina some 12-24 h after contact by the motoneuron growth cone (Sanes et al. 1990; Green et al. 1992; Patton et al. 1997). 2-Laminin is directed to acetylcholine receptor (AChR) rich regions of the basal lamina (Hunter et al. 1989; Martin et al. 1995; Moscoso et al. 1995) where it complexes with 
1-laminin and either 2-, 4- or 5-laminin to form laminins 4 (221), 9 (421) and 11 (521) (Patton et al. 1997). These laminins have been shown to arrest neurite outgrowth and promote differentiation of motor terminals (Porter et al. 1995; Cho et al. 1998; Son et al. 1999).
Despite these extensive expression and localisation studies, the biological function of 2-laminin has been best studied in mice that have had the gene for 2-laminin inactivated. In such mice, examination of the morphology of the neuromuscular junction revealed predominately prejunctional defects, including fewer active zones, diffuse distribution of synaptic vesicles throughout the nerve terminal and an invasion of Schwann cell processes into the junctional cleft (Noakes et al. 1995; Patton et al. 1998). 2-Laminin-deficient mice also show fewer postjunctional folds than the wild-type mice.
In the present study, we investigated the functional consequences of the absence of 2-laminin at NMJs from postnatal day 8 (P8) through to P18. Our results show that evoked and spontaneous neurotransmission is dramatically reduced at mutant terminals from postnatal day 8 onwards. This decrease in transmitter release is shown to correlate with morphological aberrations in mutant terminals.
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METHODS |
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Animals
The present study used wild-type mice (with two normal copies of the 2-laminin gene) or homozygous mutant mice (with no normal copies of the 2-laminin gene). These mice were obtained from the mating of heterozygous males and females, which were maintained on a defined C57BL/6-129SvJ genetic background. The day of birth was termed postnatal day zero (Thieler, 1989). Identification of wild-type and homozygote mice was established by using a DNA tail assay (Hanley & Merlie, 1991; Noakes et al. 1995). All wild-type and mutant mice used in this study were age-matched littermates. Mice used for electrophysiology studies were anaesthetised with a rising concentration of carbon dioxide and then killed by cervical dislocation. Mice used for immunohistochemical and electron microscopy studies were killed by an overdose of nembutal (30 mg kg-1, Boehringer Ingelheim, Germany) followed by cervical dislocation. These procedures were approved by the University of Queensland's Animal Care and Ethics Committee. At least four litter-matched pairs (wild-type and mutant) at each age were used for both the physiological and histological procedures described below.
Electrophysiology
Preparation. Whole mouse diaphragms with intact phrenic nerves were dissected free and pinned to the bottom of a 3 ml bath, on a bed of cured silicone rubber (Sylgard, Dow Corning Corp., USA). Preparations were continuously perfused at the rate of 3 ml min-1 with Tyrode solution of the following composition (mM): NaCl 123.4; KCl 4.7; MgCl2 1.0; NaH2PO4 1.3; NaHCO3 16.3; CaCl2 0.3; and D-glucose 7.8. The temperature of the bath was maintained between 32 and 34 °C. The reservoir supplying the bath was continuously gassed with 95 % O2 and 5 % CO2, and the pH was maintained at 7.3.
Electrical stimulation. The phrenic nerves supplying either the left or right mouse hemi-diaphragm were gently sucked into a glass pipette filled with Tyrode solution. Two Ag-AgCl wires, one within the pipette and the other on the outside, were used as the anode and cathode respectively to stimulate the axons innervating the mouse diaphragm. For both intracellular and extracellular experiments, the phrenic nerve was stimulated with square wave pulses of 0.08 ms duration and 10-15 V strength at a frequency of 0.1 Hz using a Grass Instruments stimulator (SD48) coupled to a Grass stimulus isolator (SIU5).
Intracellular recording. Glass microelectrodes (30 to 50 M
) filled with 2 M KCl were used to record endplate potentials (EPPs), miniature endplate potentials (MEPPs) and the resting membrane potentials (RMPs) in low calcium (0.3 mM) Tyrode solution to reduce the quantal content sufficiently to prevent initiation of action potentials. These electrical signals were amplified using an Axoclamp 2B amplifier with 
1 head stage (Axon Instruments, USA), then digitised (20-40 kHz sampling rate) using a MacLab system with Scope and Chart software (version 3.5.5, A/D Instruments, USA). The digitised signals were then stored on a Macintosh (PowerMac 7500/120) computer for later analysis.
Each muscle fibre was impaled within 0.5 mm of the endplate region. The position of the microelectrode with respect to the endplate region was confirmed by analysis of the rise time of EPPs and MEPPs. Recording sites with rise times greater than 1.5 ms were rejected. The RMP was continuously monitored throughout the recording period. During our recording sessions initial RMP values were in the range of -85 to -90 mV. Thereafter RMPs decreased to steady values of -55 to -60 mV. The recording was terminated if the RMP, after reaching these steady state values, fluctuated by more than 10 % during the recording period. The recording was also terminated if muscle action potentials were present during the recording. Six sites were selected from each of the four hemi-diaphragms examined in each experimental group. Recordings were made from each NMJ until at least 100 EPPs and 30 MEPPs were recorded.
Depression studies. Trains of 25 stimuli (2 or 20 Hz, 10-15 V strength and 0.08 ms duration) were delivered every 5-10 min to the phrenic nerves of wild-type and 2-laminin-deficient terminals in Tyrode solution containing 2 mM calcium to ensure high levels of transmitter release and 2.5 µM d-tubocurarine to block muscle action potentials. Four to six recording sites were selected from each of four hemi-diaphragms examined in both wild-type and 2-laminin-deficient mice. Preparations were allowed to rest for 5-10 min between trains of stimuli. EPP amplitudes were normalised to the amplitude of the first EPP in the train.
Extracellular recordings. Extracellular recordings of the nerve terminal impulse (NTI), endplate currents (EPCs) and miniature endplate currents (MEPCs) were obtained using micropipettes (20 µm diameter) filled with the Tyrode solution. Focal extracellular recordings were obtained by first placing the electrode loosely over the terminal close to the last node of Ranvier. While stimulating the phrenic nerve, the position of the electrode was adjusted until both EPCs and MEPCs with rise times of less than 1 ms were detected. Care was taken to ensure that the frequency of EPCs and MEPCs did not change as the micropipette was lowered, since electrode pressure can give rise to an increase in spontaneous frequency (Fatt & Katz, 1952; Bennett et al. 1986a,b). Once the neuromuscular junction was located, stimulation was halted for 5 min before recording MEPCs and EPCs to allow the terminals to replenish vesicle pools. Six sites were selected for each muscle with at least 30 MEPCs and 100-200 stimulations recorded at each site. As with intracellular recordings, four hemi-diaphragms from each experimental group were examined.
Data analysis. Intracellular recording sites were included for analysis if at least 100 EPPs and 30 MEPPs were generated during the recording. Extracellular recording sites were included for analysis if the frequency of EPCs and MEPCs did not change due to electrode pressure and if at least one EPC was recorded during the first 10 stimuli. Quantal content (m-), was calculated as:
(m-) = mean EPP amplitude/mean MEPP amplitude,
unless more than 30 % of the stimuli failed to produce a response, in which case m-, was calculated in accordance with Del Castillo & Katz (1954) using the formula:
(m-) = loge(number of stimuli/number of failures).
The binomial parameters of release p- (probability of release) and n (number of release sites) were calculated as described in Robinson (1976). The rise times, decay times, amplitude and frequency of evoked and spontaneous releases were calculated and tabulated for each experimental group. The means were compared using a Student's t test; P < 0.05 was taken to be statistically significant.
Immunohistochemistry
Diaphragm and sternomastoid muscles were removed and pinned out onto the Sylgard-covered base of Petri dishes. The muscles were fixed with 4 % paraformaldehyde in phosphate buffered saline (PBS) then treated with 0.1 M glycine in PBS. The muscles were blocked and permeabilised in 4 % bovine serum albumin (BSA; Sigma Chemicals, Australia) in PBS with 0.05 % Triton X-100. Synaptic vesicles were labeled with mouse anti SV2 (Buckley & Kelly, 1985; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA, USA), which was then detected with a fluorescein-conjugated secondary antibody. Rhodamine-conjugated -bungarotoxin (Molecular Probes, USA) was used to detect postjunctional AChRs. The tissue was then mounted in FluoroGard antifade mounting medium (Bio-Rad, Hercules, USA).
Neuromuscular junctions were viewed using a Bio-Rad MRC 600 scanning confocal microscope. A Z-series of each terminal was collected and projected into a single image using NIH image software (available from http://rsb.info.nih.gov/nih-image/). When collecting images, a 'Setcol' look up table (LUT) was used to ensure that the intensity and background were kept on a linear scale of 1-256 arbitrary intensity units (AIU). Furthermore, wild-type and mutant litter-matched pairs were stained and viewed concurrently to allow for direct comparison. All immunostaining experiments were accompanied by staining controls, which included deletion of both the primary and secondary antibodies. The effect of optical cross bleeding leading to false positive results was tested in control muscle fibres by deleting either the rhodamine -bungarotoxin or the fluorescein-conjugated secondary antibody from the incubation mixture.
Electron microscopy
Diaphragm muscles were removed, fixed in 2.5 % glutaraldehyde, post-fixed in 1 % osmium tetroxide, followed by dehydration through a graded series of acetone, infiltrated and then embedded with Epon resin (ProSciTech, Thuringowa, Queensland, Australia). Transverse sections (60 nm thick) were then cut and stained with uranyl and Reynolds lead citrate (Reynolds, 1963).
Neuromuscular junctions were examined and photographed using a JEOL 1010 transmission electron microscope. Photographic negatives were then scanned at 2400 dots per inch (dpi) using a Hewlett Packard scanner. The digitised images were then analysed using NIH image analysis software. This analysis included the following measurements: the extent of invasion of Schwann cell processes into the junctional cleft; direct nerve terminal muscle apposition (the junctional cleft); the number of synaptic vesicles per terminal profile; the number of mitochondria per terminal profile; and the cross-sectional areas of individual terminal profiles. These data were obtained from four litter-matched pairs of wild-type and 2-laminin-deficient mice at P18 and P21. The means ± S.E.M. are presented, and significance was performed using a paired Student's t test (Table 3).
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RESULTS |
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Both spontaneous and evoked release is reduced in 2-laminin mutants
Since 2-laminin is proposed to play a role in NMJ development, we first examined the physiology of P18 NMJs, after the final stages of NMJ development that occur in the first 2 weeks of postnatal life (Balice-Gordon et al. 1993a,b; Colman et al. 1997; Culican et al. 1998; Sanes & Lichtman, 1999; Marques et al. 2000). At 18 days postnatal, the resting membrane potential was not significantly different in 2-laminin mutants compared with wild-type animals (Table 1), suggesting that there was no change in the passive properties of the muscles' plasma membrane. We next examined the amplitude and frequency of spontaneously occurring MEPPs. As seen in Noakes et al. (1995), the frequency of MEPPs was significantly (P < 0.001; Student's t test) reduced by 79.8 ± 6.9 % (n = 7) in 2-laminin-deficient mice (Table 1). The amplitude of MEPPs however, was not significantly different in mutants (Table 1, Fig. 1B and E). These results suggest a prejunctional defect and confirm our previous suggestion that the postjunctional AChR density is not affected by deletion of 2-laminin (Noakes et al. 1995). There was an increase of 40.8 ± 25.3 % in the MEPP time course (rise and decay times) in 2-laminin-deficient neuromuscular junctions but this was not significant, suggesting either a wider junctional cleft due to Schwann cell invasion and/or lower levels of acetylcholine esterase (AChE) in the junctional cleft (Table 1, Fig. 1A and D).
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Figure 1. Intracellular recordings from 18-day-old wild-type and A and D, examples of spontaneous (4 superimposed traces) and evoked (10 consecutive traces) endplate potentials (EPPs) taken from control (A) and mutant (D) neuromuscular junctions. Such recordings were used to construct frequency amplitude histograms of spontaneous (B and E) and evoked (C and F) peak amplitudes. The histograms shown in B and C were constructed from a single recording site as were the histograms shown in E and F. The distributions of both miniature endplate potential (MEPP) and EPP amplitudes were the same in both wild-type and | ||
We next looked at the amplitude, frequency and time course of evoked release in P18 mice under conditions of low release probability (0.3 mM extracellular calcium). A Poisson distribution of EPP amplitudes was observed in all mutant and some (81.8 %) of the wild-type terminals (Fig. 1C and F). The frequency of recording EPPs at the mutant terminals was significantly (P < 0.001; Student's t test) reduced compared to wild-type terminals such that the failure rate in mutant terminals was 78 ± 16 % compared with 48 ± 20 % in control terminals (62.5 ± 33.3 % increase, Table 1 and Fig. 1C and F).
Measurements of the skeletal muscle fibre diameter in P18 showed a significant (P < 0.0001; Student's t test) decrease in 2-laminin-deficient mice, whereas fibre diameters at P8 were not significantly different (Table 1). At P18, changes in cable properties due to the different size muscle fibres could complicate any direct comparison of spontaneous and evoked transmitter release. We therefore also looked at spontaneous and evoked release in P8 mice just prior to the onset of muscle growth defects in 2-laminin-deficient mice. Comparing the differences in release characteristics between P18 and P8 we observed the same reduction in evoked transmitter release between wild-type and 2-laminin-deficient mice. The time course of release in P8 mice was similar to that observed in 18-day-old mice (Table 1). We also observed no significant change in MEPP amplitude, although the frequency of MEPPs was significantly reduced (P < 0.001; Student's t test; Table 1). The distributions of EPP amplitudes were also comparable between wild-type and mutant mice, even though the failure rate was more than 100 % higher (21 ± 4 % in control and 45 ± 29 % in mutants, P < 0.05; Student's t test) in mutant terminals than in wild-type terminals (Table 1).
Action potential conduction was not affected in 2-laminin mutants
Intracellular recording of EPPs revealed a significant increase in the number of failures in the 2-laminin mutants compared to control littermates (Table 1; Fig. 1). This increase in the number of failures over that seen in control littermates could be due to either failure of the action potential to invade the terminal or failure of depolarisation-secretion coupling in mutant terminals. The peri-neural sheaths of peripheral nerves also express 2-laminin (Patton et al. 1998). This raises the possibility that the observed increase in the failure rate of evoked neurotransmission seen at the 2-laminin-deficient neuromuscular junctions could be due to a failure to conduct an action potential successfully to the motor nerve terminal.
To determine if this high failure rate of evoked release in mutant mice was due to nerve conduction failure, we recorded transmitter release using a focal extracellular recording technique. This technique allowed for the constant monitoring of the NTI during the recording session. We recorded evoked EPCs, MEPCs and the NTI at neuromuscular junctions from wild-type and 2-laminin-deficient mice aged between P8 and P18 days. In both wild-type and mutant mice, the NTI was always present and showed no intermittence or variation during the recording periods (see Fig. 2). This suggests that the increased failure rate observed in the 2-laminin-deficient terminals was not due to failure of the action potential to invade the nerve terminal, but rather to a failure of depolarisation-secretion coupling.
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Figure 2. Extracellular recordings from 18-day-old wild-type and A and D, examples of spontaneous (4 superimposed traces) and evoked (10 consecutive traces) endplate currents (EPCs) taken from control (A) and mutant (D) neuromuscular junctions. Note the invariable presence of the nerve terminal impulse (NTI) in both wild-type and mutant recordings. Frequency amplitude histograms of spontaneous (B and E) and evoked (C and F) peak amplitudes are shown. The histograms shown in B and C were constructed from a single recording site as were the histograms shown in E and F. The distributions of MEPC amplitudes were similar in both wild-type and | ||
Neither the amplitude nor the shape of the MEPCs were significantly different in 2-laminin-deficient terminals at any age examined (Table 2; Fig. 2). The frequency of MEPCs was also reduced in 2-laminin terminals. Furthermore, the decay time of MEPCs was slightly but not significantly longer in 2-laminin-deficient terminals at P18. Evoked release was significantly (P < 0.05; Student's t test) reduced in 2-laminin-deficient terminals at both ages studied (Table 2; Fig. 2). When amplitude-frequency histograms were constructed for each recording site, a reduction in the amplitude of EPCs for 2-laminin-deficient terminals was revealed (Fig. 2). This shift is demonstrated in Fig. 2C and F where the mean EPC amplitude was 0.22 ± 0.05 mV for wild-type and 0.13 ± 0.07 mV for mutants.
2-Laminin-deficient terminals show greater synaptic depression
Noakes et al. (1995) showed reduced levels of synapsins I and II and reduced vesicle clustering at the active zones of the 2-laminin-deficient terminals. This reduction in synapsin levels and thus vesicle clustering might reduce the ability of mutant terminals to sustain constant levels of release during high-frequency stimulation. We examined the capacity of wild-type and mutant terminals to maintain a constant state of transmitter release rate during high frequency stimulation. Trains of stimuli were delivered at a frequency of 2 or 20 Hz in Tyrode solution containing 2 mM calcium and 2.5 µM d-tubocurarine to maintain the EPP amplitude below threshold for initiating action potentials and thus inhibit muscle contraction. Under these conditions, transmitter release showed a rapid depression over the first five stimuli, and then reached a plateau (Fig. 3A and B). This rapid fading to a plateau is typical in the presence of AChR antagonists such as d-tubocurarine due to the blockade of a positive feedback loop (Bowman et al. 1988). In the present study, when the stimuli were delivered at a frequency of 2 Hz, 2-laminin-deficient terminals showed similar levels of depression to the wild-type terminals (Fig. 3A). However, when the stimuli were delivered at a frequency of 20 Hz, transmitter release in the 2-laminin-deficient terminals was depressed to a much greater degree than in wild-type terminals (Fig. 3B). The paired-pulse ratio of the first two stimuli in the train was significantly lower in 2-laminin-deficient terminals compared to wild-type terminals (0.82 ± 0.07 in control and 0.47 ± 0.05 in mutant, P < 0.001; n = 23 release sites from four animals in control and 21 release sites from four animals in mutants), further strengthening the suggestions that 2-laminin deficiency induces prejunctional abnormalities. The steady state release level reached after the initial rapid depression was also significantly lower in 2-laminin-deficient terminals (0.55 ± 0.04 in control and 0.33 ± 0.05 in mutants, P < 0.005, Student's t test) suggesting a reduced vesicle-recycling rate.
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Figure 3. A, trains of stimuli were delivered to wild-type ( | ||
The quantal content is reduced in 2-laminin-deficient terminals
The results of the present study suggest a prejunctional rather than postjunctional defect leading to the reduction in evoked and spontaneous transmitter release. As such, we performed a quantal analysis of the binomial parameters of release in both mutant and wild-type terminals under conditions of low release probability (0.3 mM calcium) to determine if the reduction in evoked transmitter release was due to a general reduction in the probability of transmitter release at each release site or a reduction in the number of release sites. We first used the intracellular data to calculate the mean quantal content (m-) in mutant and wild-type terminals. Due to the high failure rate observed in both wild-type and mutant mice, m- was calculated as the natural log of the fraction of stimuli that failed to produce a response (see Methods, Del Castillo & Katz, 1954). Transmitter release in both wild-type and mutant terminals was highly intermittent and variable between release sites. Such non-uniformity of transmitter release efficacy has been well documented in several different preparations in wild-type animals (Bennett et al. 1986a,b; Walmsley et al. 1988; Lavidis & Bennett, 1992, 1993). Quantal content values at each age were normalised against the average quantal content in wild-type mice at that age. At both 8 and 18 days postnatal, we observed a significant (P < 0.0001; Student's t test) reduction in the quantal content of 2-laminin-deficient terminals (Fig. 4A).
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Figure 4. The quantal content was reduced in A, graph of the normalised quantal content (m-) in | ||
Due to the high failure rate of transmitter release at most terminals in 0.3 mM calcium, a binomial analysis of transmitter release was only possible in the most active nerve terminals. In agreement with our intracellular data, m- in 2-laminin-deficient terminals was approximately 35 % of the m- in wild-type terminals (P < 0.0001; Student's t test, Fig. 4B). The reduction in m- in mutant terminals was due to a 56 ± 6 % reduction in n (P < 0.05, Student's t test) without any significant change in p- (Fig. 4C). The differences in the m-, n and p- between mutant and control terminals were comparable at both ages studied.
Morphological examination confirms physiological observations
We next examined the arrangement of pre- and postjunctional elements in wild-type and 2-laminin-deficient terminals using immunohistochemistry and electron microscopy. Vesicles were labelled with antibodies to the neural antigen SV2 (Buckley & Kelly, 1985) and AChRs were labelled with rhodamine--bungarotoxin (-BTX). The co-localisation of pre- and postjunctional elements was examined at P8 and P18 in litter-matched wild-type and mutant pairs.
In P18 wild-type mice, the pre- and postjunctional elements were very precisely co-localised. Wild-type terminal branches formed varicose, pretzel-like shapes with AChR hotspots directly beneath the brightly stained vesicle clusters (Fig. 5A). P18 mutant terminals, however, showed a lower level of organisation than the wild-type terminals. Mutant terminals were often plaque-like in shape with few distinct SV2-positive varicosities opposite postjunctional AChR hotspots.
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Figure 5. Localisation of synaptic vesicles and postjunctional acetylcholine receptors (AChRs) at control and mutant neuromuscular junctions Shown are relative fluorescent staining intensity profiles taken from control and mutant terminals that were doubly labelled for synaptic vesicles with anti-SV2 (left panels) and acetylcholine receptors with | ||
Similar results were seen in NMJs from P8 mice (Fig. 5B). Wild-type terminals were branched and varicose with precise co-localisation of pre- and postjunctional elements. Mutant terminals, however, were more plaque-like in shape with fewer SV2-positive varicosities and AChR hotspots than wild-type mice. These immunohistochemical results indicate that the area available for release (as defined by SV2-positive varicosities closely associated with AChR hotspots) is reduced in 2-laminin-deficient terminals. This result is consistent with our functional analysis indicating fewer active release sites in 2-laminin-deficient terminals.
We also examined pre- and postjunctional ultrastructural differences between wild-type and 2-laminin-deficient mice at P18 to P20, as was done previously for P8 through to P15 (Noakes et al. 1995). Our electron micrographs of wild-type and 2-laminin-deficient terminals showed the following differences. Firstly, the nerve terminal profile area in 2-laminin-deficient mice was significantly smaller when compared to control mice (P < 0.05, Table 3). As a likely consequence of this reduced size of 2-laminin terminal profiles, the length of nerve-muscle (N-M) apposition, the total number of synaptic vesicles and the number of mitochondria were also reduced in 2-laminin mutants (see Table 3).
Second, the percentage Schwann cell invasion (defined as the percentage of the N-M apposition length that is penetrated by Schwann cell processes) was greater in 2-laminin mutants. In approximately 50 % of the mutant nerve terminal profiles examined (n = 44), Schwann cell process had completely separated the nerve and muscle (i.e. 100 % invasion). The remaining approximately 50 % of terminals showed a wide range of Schwann cell invasion, in some cases (2/44) showing 0 % invasion (Table 3). Third, we observed fewer junctional folds in 2-laminin mutant terminals, with many of these folds lying under Schwann cells (N-S-M apposition) rather than directly under the open nerve terminal profile (N-M). We also observed a decrease in percentage of vesicles clustered in the half of the terminal closest to the muscle (juxta-membrane half).
The above results, together with our previous morphological analyses at P8 (Noakes et al. 1995), support our physiological observations, which suggest fewer effective prejunctional release sites at NMJs from 2-laminin mutants compared to control mice. Our findings also show that the percentage of Schwann cell invasion into the junctional cleft (~75 %) at NMJs from P18 2-laminin-deficient mice was comparable to that seen at P8 (Table 3; Noakes et al. 1995). This could, in part, account for why we do not see a significant reduction in the mean quantal content between the ages of P8 and P18.
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DISCUSSION |
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2-Laminin has been shown to regulate prejunctional differentiation of the skeletal NMJ (Noakes et al. 1995; Libby et al. 1999). Previous analysis of P8 to P15 2-laminin-deficient mice showed predominantly prejunctional aberrations, including fewer active zones, a diffuse distribution of synaptic vesicles, a reduced frequency of MEPPs and invasion of Schwann cell processes into the junctional cleft (Noakes et al. 1995). 2-Laminin-deficient mice also have fewer postjunctional folds than their wild-type littermates (Noakes et al. 1995). In the present study, we have expanded this analysis out to include P18 wild-type and 2-laminin mutant mice. We have also provided the first fully functional analysis of the consequences of these pre- and postjunctional aberrations in 2-laminin-deficient mice between the ages of P8, when mutant mice are the same size and weight as their wild-type littermates, and P18, when the mutant mice are significantly smaller than wild-type littermates.
Changes in spontaneous release suggest a prejunctional defect
As noted previously (Noakes et al. 1995), the frequency of spontaneous transmitter release was significantly lower in 18-day-old 2-laminin-deficient mice compared to their wild-type littermates. Similar reductions in the frequency of spontaneous release were also observed in P8 mutant mice. Despite the significant reductions in spontaneous frequency in both P8 and P18 2-laminin-deficient mice, the amplitudes of the MEPPs were not significantly different. The reduced frequency of MEPPs observed in the present study and the previous study by Noakes et al. (1995) suggests a prejunctional difference between wild-type and 2-laminin-deficient mice (Robitaille et al. 1989).
Evoked release is reduced in 2-laminin mutants
In addition to the reduced frequency of MEPPs, the present study also observed a reduction in evoked release in 2-laminin-deficient terminals at both P8 and P18. During single impulse stimulation, a decrease in the average quantal content in 2-laminin-deficient mice was observed, largely due to an increase in the number of stimuli that failed to produce a response. Extracellular recordings allowed us to simultaneously measure both the NTI and the EPC. These recordings show that reduction in quantal content (m-) was not due to failure of action potential propagation in 2-laminin mutant nerve terminals. This suggests that the increased failure rate in mutants is due to a breakdown in the depolarisation- secretion coupling mechanism.
The present study also observed an increase in synaptic depression during high frequency (20 Hz) trains of stimuli despite there being no significant difference in synaptic depression during low frequency (2 Hz) trains. The rapid depression of transmitter release to a steady plateau during trains of stimulation is due partly to a blockade of a positive feedback loop (Bowman et al. 1988), and partly to the ability of the nerve terminal to replenish the supply of synaptic vesicles (Wilson, 1979; Bennett, 2001). Given the results of our ultrastructural studies showing a reduction in the total number of vesicles and in the percentage of vesicles in the juxta-membrane half of the terminals, it is possible that the increased depression seen during 20 Hz trains of stimuli may be due to reduced delivery of vesicles to the release area, which is most likely to be the result of a reduction in synapsins I and II (Rosahl et al. 1995; Noakes et al. 1995; Hilfiker et al. 1999). The observation that synaptic depression was only affected at 20 Hz, and not at 2 Hz suggests that at the lower stimulation frequencies, the observed synaptic depression at 2 Hz is the result of the d-tubocurarine-dependent inhibition of a positive feedback loop rather than reduced synaptic vesicle availability. The reduced density of synaptic vesicles seen in these 2-laminin-deficient terminals does not appear to have a functional role during low stimulation frequencies. This suggests that the reduction in quantal content seen during single impulse stimulation was more likely to be due to a reduction in the number of available release sites than a reduction in the probability of release from any individual release site due to poor vesicle delivery.
2-Laminin deficient terminals have fewer active release sites
Analysis of the binomial parameters of release confirmed that the reduction in m- was due primarily to a reduction in n in 2-laminin-deficient terminals, with no significant change in p-. The difference between wild-type and mutant terminals was evident when transmitter release was examined by intracellular methods (examining release from the whole terminal) or extracellular methods (release from some of the terminal, usually from the most active region close to the last node of Ranvier). Active zones found closer to the last node of Ranvier have a higher probability of quantal release (Bennett & Lavidis, 1979; Grinnell & Herrera, 1980; Bennett et al. 1986a). Our present data indicate the maintenance of the most active release sites, and loss of release sites with low probabilities of transmitter release. While the shape of the EPP amplitude histograms was the same in both wild-type and 2-laminin terminals, the EPC amplitude histograms were often more skewed towards higher values in the wild-type terminals. Since the large EPCs are the product of simultaneous release of one quantum of transmitter from several separate active zones, the higher frequency of large EPC amplitudes observed in the wild-type animals suggests a greater number of release sites contributing to the event.
Immunohistochemical staining of wild-type and 2-laminin-deficient terminals showed fewer SV2-positive varicosities and AChR hotspots in 2-laminin-deficient terminals compared to their wild-type littermates at both P8 and P18. While these SV2-positive varicosities are too large to represent single active zones, it is probable that most if not all transmitter release occurs at these SV2-positive varicosities. As such, the reduced number of SV2 positive varicosities observed in the 2-laminin-deficient terminals supports the functional observations that in mutant animals there are fewer active release sites.
Previous examination of the ultrastructure of 2-laminin-deficient terminals in P8-P15 mice also indicated smaller terminals with more Schwann cell invasion in the 2-laminin-deficient mice (Noakes et al. 1995). In the present study, we also examined the ultrastructure of NMJs in P18 wild-type and 2-laminin-deficient mice. In these older animals, the terminal profiles of 2-laminin mutants were smaller than the wild-type terminal profiles. In accordance with the smaller size of the terminal profiles, 2-laminin terminals had a reduced length of nerve-muscle apposition, and fewer vesicles. The reduced length of nerve-muscle apposition in 2-laminin mutants was exacerbated by the increased Schwann cell process invasion seen in 2-laminin mutant terminals.
In P8-P15 2-laminin-deficient terminals, Schwann cells covered approximately 70 % of the nerve-muscle apposition length (Noakes et al. 1995). Similar results were observed for P18 2-laminin-deficient terminals in the present study. This increased encroachment of Schwann cells into the junctional clefts of 2-laminin-deficient terminals would result in a reduced area being available for transmitter release. These reductions in the area available for transmitter release (65-75 %) are comparable to the reductions in the binomial parameters m- and n (65 and 55 %, respectively). This correlation between the morphological and physiological results suggest that the decrease in evoked transmitter release seen in 2-laminin-deficient terminals may be due to increased Schwann cell invasion limiting the area available for successful neurotransmission (i.e. prejunctional neurotransmitter crossing the cleft and binding to a postjunctional receptor). This suggestion is supported by observations in the amphibian NMJ that have noted a correlation between the number, density and size of prejunctional active zones and transmitter release efficacy (Davey & Bennett, 1982; Bennett et al. 1987; Tremblay et al. 1989). In particular, Bennett et al. (1987) suggested that the decreased area of synaptic contacts in distal regions of the terminals branches (where release probability is low, Bennett et al. 1986a) may be due to the encroachment of Schwann cells between pre- and postjunctional elements at more distal ends of the terminal.
Does activity inhibit Schwann cell invasion?
Together, the physiology, immunohistochemistry and electron microscopy results indicate a reduction in number of release sites in 2-laminin-deficient terminals. Furthermore, this reduced number of release sites observed in the 2-laminin-deficient mice appear partly to involve increased invasion of the junctional cleft by Schwann cell processes. Patton et al. (1998) showed that laminin 11 (521) actively inhibits Schwann cell invasion into the junctional cleft. 2-Laminin-deficient mice do not show any expression of laminin 11 in the junctional cleft, which may account for the encroachment of Schwann cells into the junctional cleft seen in these animals (Noakes et al. 1995; Patton et al. 1997). However, the Schwann cells only invade approximately 65-75 % of the length of 2-laminin-deficient terminals. Furthermore, the degree of invasion does not increase between the ages of 8 and 18 days postnatal (Table 3; Noakes et al. 1995). This suggests that some additional signal is present in the remaining approximately 30 % of the 2-laminin-deficient terminals that prevents the Schwann cells from invading those areas.
It is possible that the signal that prevents Schwann cell invasion in these areas is transmitter release activity. Synaptic activity is known to modulate Schwann cell activity (Jahromi et al. 1992; Robitaille, 1995; Rochon et al. 2001). Furthermore, work by Bennett and coworkers (Bennett & Lavidis, 1979; Bennett et al. 1986a,b) showed that transmitter release activity varies between consecutive release sites. We propose that the level of transmitter release activity in the 30 % of the 2-laminin-deficient terminals that remain clear of Schwann cell processes is sufficiently high to prevent Schwann cells from invading the cleft in those areas. The level of activity in the 70 % of 2-laminin mutant terminals engulfed by Schwann cell processes may be insufficient to prevent Schwann cell invasion. In wild-type terminals, however, the presence of laminin 11 actively inhibits the invasion of the Schwann cell processes, thus allowing less active release sites to form and remain functionally intact (Patton et al. 1998).
In summary, the present study shows that evoked and spontaneous transmitter release is significantly reduced in 2-laminin-deficient terminals. We also showed that 2-laminin-deficient terminals exhibit greater synaptic depression at high frequencies of stimulation. These functional aberrations in transmitter release are shown to originate from prejunctional, morphological irregularities in the 2-laminin mutant terminals.
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REFERENCES |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
| Balice-Gordon RJ, Chua CK, Nelson CC & Lichtman JW (1993a). Gradual loss of synaptic cartels precedes axon withdrawal at developing neuromuscular junctions. Neuron 11, 801-815 | [Medline] |
| Balice-Gordon RJ , & Lichtman JW (1993b). In vivo observations of pre and postsynaptic changes during the transition from multiple to single innervation at developing neuromuscular junctions. J Neurosci 13, 834-855 | [Abstract] |
| Bennett MR, (2001). Mechanisms of depression of transmitter release at synapses. Neuroreport 12, A15-20 | [Medline] |
| Bennett MR , & Lavidis NA (1979). The effect of calcium ions on the secretion of quanta evoked by an impulse at nerve terminal release sites. J Gen Physiol 74, 429-456 | [Abstract] |
| Bennett MR, Jones P & Lavidis NA (1986a). The probability of quantal secretion along visualized terminal branches at amphibian (Bufo marinus) neuromuscular synapses. J Physiol 379, 257-274 | [Abstract] |
| Bennett MR, Jones P & Lavidis NA (1986b). Transmitter secretion varies between visualized release sites at amphibian neuromuscular junctions. Neurosci Letters 65, 311-315 | |
| Bennett MR, Lavidis NA & Armson FM (1987). Changes in the dimensions of release sites along terminal branches at amphibian neuromuscular synapses. J Neurocytol 16, 221-237 | [Medline] |
| Bowman WC, Marshall IG, Gibb AJ & Harbone AJ (1988). Feedback control of transmitter release at the neuromuscular junction. Trends Pharmacol Sci 9, 16-20 | [Medline] |
| Buckley K , & Kelly RB (1985). Identification of a transmembrane glycoprotein specific for secretory vesicle of neural and endocrine cells. J Cell Biol 100, 1284-1294 | [Abstract] |
| Burgess RW, Nguyen QT, Son YJ, Lichtman JW & Sanes JR (1999). Alternatively spliced isoforms of nerve- and muscle-derived agrin: their roles at the neuromuscular junction. Neuron 23, 33-44 | [Medline] |
| Cho SI, Ko J, Patton BL, Sanes JR & Chiu AY (1998). Motor neurons and schwann cells distinguish between synaptic and extrasynaptic isoforms of laminin. J Neurobiol 37, 339-358 | [Medline] |
| Colman H, Nabekura J & Lichtman JW (1997). Alterations in synaptic strength preceding axon withdrawal. Science 275, 356-361 | |
| Culican SM, Nelson CC & Lichtman JW (1998). Axon withdrawal during synapse elimination at the neuromuscular junciton is accompanied by disassembly of the postsynaptic specialisation and withdrawal of schwann cell processes. J Neurosci 18, 4953-4965 | [Abstract/Full Text] |
| Davey DF , & Bennett MR (1982). Variation in the size of synaptic contacts along developing and mature motor terminal branches. Brain Res 281, 11-22 | [Medline] |
| Del Castillo J , & Katz B (1954). Quantal components of the end-plate potential. J Physiol 124, 560-573 | |
| Fatt P , & Katz B (1952). Spontaneous subthreshold activity at motor nerve endings. J Physiol 117, 109-128 | |
| Green TL, Hunter DD, Chan W, Merlie JP & Sanes JR (1992). Synthesis and assembly of the synaptic cleft protein s-laminin by cultured cells. J Biol Chem 267, 2014-2022 | [Abstract] |
| Grinnell AD , & Herrera AA (1980). Physiological regulation of synaptic effectiveness at frog neuromuscular junctions. J Physiol 307, 301-317 | [Abstract] |
| Hanley T , & Merlie JP (1991). Transgene detection in unpurified mouse tail DNA by polymerase chain reaction. Biotechniques 10, 56 | [Medline] |
| Hilfiker S, Pieribone VA, Czernik AJ, Kao HT, Augustine GJ & Greengard P (1999). Synapsins as regulators of neurotransmitter release. Philos Trans R Soc Lond B 354, 269-279 | |
| Hunter DD, Shah V, Merlie JP & Sanes JR (1989). A laminin-like adhesive protein concentrated in the synaptic ceft of the neuromuscular junction. Nature 338, 229-234 | [Medline] |
| Jahromi BS, Robitaille R & Charlton MP (1992). Transmitter release increases intracellular calcium in perisynaptic Schwann cells in situ. Neuron 8, 1069-1077 | [Medline] |
| Lavidis NA , & Bennett MR (1992). Probabilistic secretion of quanta from visualised sympathetic nerve varicosities in mouse vas deferens. J Physiol 454, 9-26 | [Abstract] |
| Lavidis NA , & Bennett MR (1993). Probabilistic secretion of quanta from successive sets of visualized varicosities along single sympathetic nerve terminals. J Auto Nervous Sys 43, 41-50 | |
| Libby RT, Lavallee CR, Balkema GW, Brunken WJ & Hunter DD (1999). Disruption of laminin beta2 chain production causes alterations in morphology and function in the CNS. J Neurosci 19, 9399-9411 | [Abstract/Full Text] |
| Marques MJ, Conchello JA & Lichtman JW (2000). From plaque to pretzel: Fold formation and acetylcholine receptor loss at the developing neuromuscular junction. J Neurosci 20, 3663-3675 | [Abstract/Full Text] |
| Martin PT, Ettinger AJ & Sanes JR (1995). A synaptic localization domain in the synaptic cleft protein laminin beta 2 (s-laminin). Science 269, 413-416 | |
| Moscoso LM, Merlie JP & Sanes JR (1995). N-cam, 43k-rapsyn, and s-laminin mRNSs are concentrated at synaptic sites in muscle fibers. Mol Cell Neurosci 6, 80-89 | [Medline] |
| Noakes PG, Gautam M, Mudd J, Sanes JR & Merlie JP (1995). Aberrant differentiation of neuromuscular junctions in mice lacking s-laminin/laminin 2. Nature 374, 258-262 |
[Medline] |
| Patton BL, Chiu AY & Sanes JR (1998). Synaptic laminin prevents glial entry into the synaptic cleft. Nature 393, 698-701 | [Medline] |
| Patton BL, Cunningham JM, Thyboll J, Kortesmaa J, Westerblad H, Edstrom L, Tryggvason K & Sanes JR (2001). Properly formed but improperly localized synaptic specializations in the absence of laminin alpha4. Nat Neurosci 4, 597-604 | [Medline] |
| Patton BL, Miner JH, Chiu AY & Sanes JR (1997). Distribution and function of laminins in the neuromuscular system of developing, adult and mutant mice. J Cell Biol 139, 1507-1521 | [Abstract/Full Text] |
| Porter BE, Weis J & Sanes JR (1995). A motoneuron-selective stop signal in the synaptic protein s-laminin. Neuron 14, 549-559 | [Medline] |
| Reynolds ES, (1963). The use of lead citrate at high pH as an electron opaque stain in electron microscopy. J Cell Biol 17, 208-212 | |
| Robinson J, (1976). Estimation of parameters for a model of transmitter release at synapses. Biometrics 32, 61-8 | [Medline] |
| Robitaille R, (1995). Purinergic receptors and their activation by endogenous purines at perisynaptic glial cells of the frog neuromuscular junction. J Neurosci 15, 7121-7131 | [Abstract] |
| Robitaille R , & Tremblay JP (1989). Frequency and amplitude gradients of spontaneous release along the length of the frog neuromuscular junction. Synapse 3, 291-307 | [Medline] |
| Rochon D, Rousse I & Robitaille R (2001). Synapse-glia interactions at the mammalian neuromuscular junction. J Neurosci 21, 3819-3829 | [Abstract/Full Text] |
| Rosahl TW, Spillane D, Missler M, Herz J, Selig DK, Wolff JR, Hammer RE, Malenka RC & Sudhof TC (1995). Essential functions of synapsins i and ii in synaptic vesicle regulation. Nature 375, 488-493 | [Medline] |
| Sanes JR, Hunter DD, Green TL & Merlie JP (1990). S-laminin. Cold Spring Harb Symp Quant Biol 55, 419-430 | [Medline] |
| Sanes JR , & Lichtman JW (1999). Development of the vertebrate neuromuscular junction. Annu Rev Neurosci 22, 389-442 | [Abstract/Full Text] |
| Son YJ, Patton BL & Sanes JR (1999). Induction of presynaptic differentiation in cultured neurons by extracellular matrix components. Eur J Neurosci 11, 3457-3467 | [Medline] |
| Thieler K, (1989). The House Mouse. Atlas of Embryonic Development. Springer-Verlag, New York | |
| Tremblay JP, Robitaille R, Martineau O, Labrecque C & Fahim MA (1989). Proximodistal gradients of the postjunctional folds at the frog neuromuscular junction: a scanning electron microscopic study. Neuroscience, 30, 535-50 | [Medline] |
| Walmsley B, Edwards FR & Tracey DJ (1988). Nonuniform release probabilities underlie quantal synaptic transmission at a mammalian excitatory central synapse. J Neurophysiol 60, 889-908 | [Abstract] |
| Wilson DF, (1979). Depression, facilitation, and mobilization of transmitter at the rat diaphragm neuromuscular junction. Am J Physiol 237, C31-37 | [Medline] |
Acknowledgements
This work was supported by the National Health and Medical Research Council of Australia, the Motor Neuron Disease Institute Australia and the Ramacotti Foundation. We thank Ms Mary White and Elke Seppanen for genotyping the mice, and Dr Mark Bellingham for providing valuable critical feedback on the manuscript.
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) and 
) preparations in 4 mM calcium at a frequency of 2 Hz. At this frequency, no difference was observed between mutant and wild-type terminals in the level of synaptic depression. B, trains of stimuli were delivered to wild-type (