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Neuroscience |
1 Physiology Department, University of Toronto, Toronto, Ontario, Canada
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Abstract |
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(Received 8 September 2005;
accepted after revision 5 December 2005;
first published online 8 December 2005)
Corresponding author M. P. Charlton: Physiology Department, University of Toronto, 1 King's College Circle, Room 3308, Toronto, Ontario, Canada M5S1A8. Email: milton.charlton{at}utoronto.ca
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Introduction |
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Perturbations in the cholesterol level can affect the function of several proteins used in synaptic transmission such as neurotransmitter receptors (Fong & McNamee, 1986; Burger et al. 2000; Sooksawate & Simmonds, 2001; Eroglu et al. 2003), Ca2+ and other channels (Jennings et al. 1999; Romanenko et al. 2002; Kato et al. 2003; Taverna et al. 2004). Some synaptic vesicle proteins, such as synaptotagmin and synaptophysin also interact with cholesterol (Huttner & Schmidt, 2000; Thiele et al. 2000). The interaction of synaptophysin with synaptobrevin/VAMP may depend on interactions of synaptophysin with cholesterol (Mitter et al. 2003). Therefore, the concentration of cholesterol in membranes may be an important determinant of the efficiency of synaptic transmission.
The effects of cholesterol depletion on exocytosis have been studied in PC12 cells, and it was found that depolarization-induced release of dopamine was reduced (Lang et al. 2001; Chamberlain et al. 2001) but conversely, in pancreatic ß cells, exocytosis was increased (Xia et al. 2004). In rat brain synaptosomes, depolarization and Ca2+ ionophore-stimulated loss of glutamate were severely reduced by cholesterol depletion (Gil et al. 2005).
Owing to the involvement of cholesterol with many synaptic proteins including those that control exocytosis, we examined the importance of cholesterol in synaptic transmitter release. We measured quantal transmitter release at crayfish neuromuscular junctions that have presynaptic axons large enough to permit measurement of axon membrane potential and injection of probes. Depletion of cholesterol hyperpolarized axons, and blocked both presynaptic action potentials and excitatory post synaptic potentials (EPSPs). There were no changes in the amplitude or time course of spontaneous miniature EPSPs (mEPSPs) indicating that the block of EPSPs was solely due to block of transmitter release. Transmitter release triggered by focal depolarization (not requiring action potentials) was slightly increased after cholesterol depletion. The frequency of spontaneous quantal transmitter release measured as mEPSPs was increased by cholesterol depletion by a non-Ca2+-dependent mechanism. The frequency of asynchronous Ca2+-stimulated transmitter release was also increased by cholesterol depletion. The effects of cholesterol depletion on action potentials and spontaneous transmitter release were reversed when cholesterol levels were replenished. The data indicate that cholesterol can control, to some extent, the rate of transmitter release independent of Ca2+.
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Methods |
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Crayfish (Procambarus clarkia; length 5–6.5 cm) obtained from Atchafalaya Biological Supply (Raceland, LA, USA) were kept at 15–18°C in de-chlorinated tap water. We used the extensor muscle in the first and second walking legs. To induce rapid natural autotomy of a leg, we grasped the leg and used it to lift the crayfish. Animals were treated in accordance with local animal care guidelines. Dissection and experiments were performed at 20–21°C in normal crayfish (control) saline containing (mM): NaCl 205, KCl 5.4, CaCl2 13.5, MgCl2 2.7 and Hepes 5; pH was adjusted to 7.4 using NaOH. In Ca2+-free saline, all CaCl2 was replaced by equimolar MgCl2. As measured with an osmometer (model 5520, Wescor Inc., Logan, UT, USA) the osmolality of all solutions was 430 ± 10 mmol kg–1. The extensor muscle was exposed by removal of the flexor muscle and overlying sensory nerve (Bradacs et al. 1997). Muscle contractions were reduced by application of gentle stretch to the next distal leg segment.
The extensor muscle is innervated by glutamatergic synapses made by one phasic excitatory axon and one tonic excitatory axon. In order to examine transmitter release from only tonic synapses, we also used the walking leg opener muscle that only has one glutamatergic excitatory tonic and one GABAergic inhibitory axon (Cooper et al. 1995). In experiments on the opener muscle, we blocked inhibitory synaptic transmission with 50 µM picrotoxin added to the bath solution (Golan & Grossman, 1996).
The motoneurone axon was stimulated via a platinum–iridium wire embedded in a silicon rubber tube into which the proximal end of the leg was inserted and another wire external to the tube that contacted the bath solution. These wires were connected to a stimulator (A-M Systems Isolated Pulse Stimulator, Model 2100, Everett, WA, USA).
Sharp microelectrodes (5–10 M
) filled with 3 M KCl and standard electrophysiological techniques were used to impale the extensor muscle fibres and phasic axon to record the synaptic responses (EPSPs) and presynaptic action potentials (APs), respectively. Extracellular loose macropatch recordings at individual boutons or groups of boutons were made using a bevelled heat-polished electrode (2–5 M) filled with control saline. Synaptic boutons were visualized with the aid of the vital dye 4-(4-diethylaminostyryl)-N-methylpyridiniumiodide (4-Di-2-Asp; Molecular Probes, Eugene, OR, USA) applied for 4 min at 2 µM.
Data collection and analysis
Signals were amplified 10- to 1000-fold by an electrometer (Model IE-201, Warner Instrument Corp, Hamden, CT, USA) and low-pass filtered (2 kHz, Bessel 4-pole, Model LPF202, Warner Instrument Corp). Signals were digitized (10 kHz) (Digidata 1200, Axon Instruments, Union City, CA, USA) and stored on a personal computer using WinWCP (Strathclyde Electrophysiology Software, University of Strathclyde, Glasgow, UK). mEPSPs representing spontaneous transmitter release were digitized at 20 kHz by a PowerLab/4sp (ADInstruments, Round Rock, TX, USA) data acquisition system. Files were analysed using Minianalysis (Synaptosoft, Decatur, GA, USA); mEPSPs were counted manually and the amplitudes were determined automatically by the program. Data were transferred to SigmaPlot 8.0 for producing graphs and SigmaStat 3.0 (SPSS, Chicago, IL, USA) for statistical analysis. Data were tested for significance using non-paired t test, unless otherwise noted as paired t test or non-parametric Mann-Whitney U test. The amplitude distribution of mEPSPs is skewed (Van der Kloot, 1991); therefore the non-parametric Kolmogorov-Smirnov test was used. All experiments were repeated five times unless noted otherwise.
Cholesterol assay
To assay for experimentally induced changes in membrane cholesterol content, we used the fluorescent probe filipin which binds to membrane cholesterol (Kruth & Vaughan, 1980). Cyclodextrin-treated or control preparations were fixed with a 3% formaldehyde solution for 1 h followed by 1.5 mg ml–1 glycine for 10 min to quench the fixative and then incubated for 2 h in 0.05 mg ml–1 filipin. One leg of a pair from the same animal was used as a control and the other was treated with 10 mM MßCD or 10 mM Ch-MßCD for 30 min. The controls were not treated with cyclodextrins but were incubated in filipin together with the contralateral leg. To examine cholesterol recovery, preparations were treated with 10 mM MßCD for 15 min, then washed with control saline for 5 min and then the saline was replaced with 2.5 mM Ch-MßCD for 10 min; for control preparations, 2.5 mM MßCD was used instead of 2.5 Ch-MßCD for the last 10 min. The preparations were examined with an upright fluorescence microscope (Nikon Optiphot) using a UV filter set (385 nm excitation, 40 nm dichroic, 430 nm long-pass filter) and a 40 x UV water dipping objective (Olympus). Fluorescence images were captured with an intensified CCD video camera (PTI model IC-110; Montmouth Junction, NJ, USA) and an Axon Lightning frame grabber using Axon Imaging Workbench software (Axon Instruments). The intensifier voltage, gain and black level of the camera were constant for all fluorescence measurements. Each picture was an average of 32 frames. Intensity values from five pictures were averaged for each preparation. Fluorescence intensity (PI) was determined by measuring the average pixel intensity (approximately 1000 pixel area) of the brightest region of the axon (approximately at the centre of the field). To eliminate variability caused by background fluorescence, the pixel intensity of a region adjacent to the axon was subtracted from the area of interest.
Materials
Methyl-ß-cyclodextrin (MßCD), hydroxypropyl-
-cyclodrextrin (HPCD), the cholesterol–MßCD (Ch- MßCD) complex and filipin were purchased from Sigma-Aldrich (Oakville, ON, Canada). Cyclodextrin compounds were stored in powder form; MßCD and HPCD at room temperature and Ch-MßCD and filipin at –20°C. The acetoxymethyl ester form of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM) was purchased from Calbiochem-Novabiochem (San Diego, CA, USA). BAPTA tetrapotassium salt (BAPTA-K4) was purchased from Molecular Probes.
Application of cyclodextrin and cholesterol–cyclodextrin
Immediately before experiments, cyclodextrins were added to a saline that contained a lower concentration of NaCl (175 mM instead of 205 mM) to obtain the correct osmolarity. Cyclodextrin was applied to muscles and synapses for 30 min while recordings were made. Several concentrations of MßCD were used, but most experiments were performed at 10 mM. In order to bring the bath to a final concentration of 10 mM MßCD, a 1-ml solution of 20 mM MßCD was made and was exchanged with 1 ml solution from the 2-ml bath. The solution was stirred five times at regular intervals during experiments.
Intracellular calcium chelation
The permeant Ca2+ chelator BAPTA-AM was applied at 50 µM in saline containing 0.2% DMSO. In some experiments, BAPTA-K4 (400 mM) was injected ionophoretically into axons via a sharp microelectrode with –2 nA, for 0.5 s at 1 Hz and injection was maintained during the entire recording.
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Results |
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MßCD can change the appearance of mouse skeletal muscle cells (Pouvreau et al. 2004) and rat nasal mucosa (Asai et al. 2002). Therefore, we wondered whether MßCD could be used on Crustacean tissue without causing excessive damage. Moreover, ß-cyclodextrin can be cytotoxic and can cause haemolysis of human erythrocytes (Ohtani et al. 1989). Damage by MßCD may be time-dependent because a 2-h incubation of mouse L-cell fibroblasts with 10 mM MßCD was not toxic but longer incubations were toxic (Kilsdonk et al. 1995). To check the possibility that MßCD causes a significant level of damage to the preparation, we examined the gross structure of muscle fibres and axons used in the electrophysiological studies. After 30 min in the presence of 10 mM MßCD, no apparent change occurred in appearance of muscle fibres or phasic and tonic axons (Fig. 1) visualized with a 40 x water dipping objective. To test for possible swelling of boutons, we ionophoretically injected Lucifer yellow into the phasic axon to allow visualization of boutons under a confocal microscope. The mean bouton diameter did not change significantly over a 30-min treatment period (control, 3.2 ± 0.5 µm; MßCD, 3.2 ± 0.5 µm; one bouton in each of three preparations). While there were no obvious gross changes in muscle fibres, nerves or synaptic boutons after application of MßCD, we could not rule out the possibility of changes in ultrastructure.
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Although MßCD extracts cholesterol from mammalian membranes (Yancey et al. 1996) and some invertebrate cells (Jouni et al. 2002; Zhuang et al. 2002), we wished to verify that it can deplete cholesterol in our preparation. To determine whether MßCD extracts cholesterol from crayfish axons we assayed changes in cholesterol content with the fluorescent polyene antibiotic filipin, which has been used to probe sterols in both vertebrate (Mukherjee et al. 1998) and invertebrate cells (Rolls et al. 1997; Harris et al. 2001; Merris et al. 2003). Filipin associates with membrane cholesterol and therefore can be used as an indicator of the concentration of cholesterol; membranes with high cholesterol content will bind more filipin and will be more fluorescent than those with low cholesterol content (Kruth & Vaughan, 1980).
Muscles from a pair of legs from the same animal were fixed and stained with filipin (see Methods) following 30-min incubation with control saline or saline containing 10 mM MßCD. Axonal staining was visualized by fluorescence microscopy. Smaller axon branches were stained faintly and boutons were not visible, presumably because of the low sensitivity of the fluorescent probe. Staining of the muscle fibre membrane was also faint relative to the primary branch of the axonal membrane. Therefore, we used the primary branch of the axon to measure the effects of MßCD.
The preparations treated with MßCD had fainter axonal staining compared to the untreated preparations (Fig. 2A, control, 42.0 ± 3.7 pixel intensity (PI); 10 mM MßCD, 6.6 ± 1.2 PI, P < 0.001, n= 5), indicating that cholesterol was depleted with a 30-min treatment of 10 mM MßCD. We determined the change in fluorescence when 10 mM Ch-MßCD was used to add extra cholesterol to membranes. A 30-min treatment with 10 mM Ch-MßCD increased axon fluorescence as compared to untreated preparations (Fig. 2B, control, 39.3 ± 3.6 PI; 10 mM Ch-MßCD, 58.4 ± 5.4 PI, P= 0.02, n= 5). This is consistent with the observation that Ch-MßCD containing 7 : 1 MßCD/cholesterol, as used here, adds cholesterol to membranes (Christian et al. 1997). Finally, following the application of MßCD, the Ch-MßCD complex was used to replenish cholesterol in cells. When 10 mM MßCD was applied for 15 min followed by 2.5 mM Ch-MßCD for 10 min (recovery treatment), fluorescence was higher compared to preparations treated for 15 min with 10 mM MßCD and 10 min with 2.5 mM MßCD (control treatment) (Fig. 2C, control, 14.0 ± 3.4 PI; recovery, 35.6 ± 3.5 PI, P= 0.002, n= 5). The recovery preparations had similar pixel intensity to untreated preparations (P > 0.05). Therefore, MßCD reduced the level of cholesterol in crayfish cell membranes while Ch-MßCD increased membrane cholesterol levels and also replenished neuronal cholesterol.
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Next we tested the effects of MßCD on evoked transmitter release from phasic synapses. Presynaptic axons were stimulated by extracellular current pulses at 0.016 Hz delivered to the proximal end of the phasic axon. A muscle fibre was impaled with a microelectrode near a string of phasic boutons and evoked transmitter release was assayed as the amplitude of EPSPs. In control experiments with stimulation at 0.016 Hz, EPSPs were depressed to 75% of the initial amplitude in 30 min and after 60 min to about 66% of the original amplitude (Fig. 3A). This low frequency depression is characteristic of phasic synapses on the crayfish extensor muscle (Silverman-Gavrila et al. 2005). When MßCD was applied after 30 min, there was a rapid decrease in EPSP amplitude that far exceeded the normal rate of depression. After application of MßCD, EPSPs were reduced to 10% of maximum in 30 min. The decline of EPSP amplitude was much faster with 20 mM MßCD (data not shown). There were no obvious changes in the shape of EPSPs but the peak of the EPSP was delayed by 1 ms after 20–30 min in the presence of MßCD (Mann-Whitney U test, P < 0.05, n= 5). This increase in delay may be due to slower propagation of the presynaptic AP. (inset to Fig. 3A).
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To determine whether the transmission block is due to irreversible damage, we tested whether the evoked response could recover. After 15 min of treatment with MßCD, control saline was reintroduced but there was little, if any, recovery of EPSP amplitude (Fig. 3B). Reapplication of cholesterol after MßCD treatment can lead to the recovery of some physiological processes, including exocytosis in rat basophil leukaemia cells (RBL-2H3 cells) (Kato et al. 2003) and the distribution of Cav2.1 calcium channels and SNAREs in rat brain synaptosomes (Taverna et al. 2004). When MßCD was replaced by 2.5 mM Ch-MßCD complex there was a partial EPSP recovery within 5 min (Fig. 3C) but application of 10 mM Ch-MßCD led to a complete EPSP recovery (Fig. 3D). Thus MßCD-induced EPSP block is reversible. Moreover, replacement of cholesterol was sufficient to cause reversal of the effects of MßCD. While we cannot rule out the possibility that MßCD extracted lipids or other moieties besides cholesterol, the reversal of the effects by cholesterol means that alteration of membrane cholesterol content was probably the main effect of MßCD.
To check for non-specific pharmacological effects of cyclodextrins, we applied HPCD, which has a low affinity for cholesterol (Ohtani et al. 1989), and found that evoked transmitter release was not affected (Fig. 3E, P > 0.05, n= 5). Therefore, the removal of cholesterol was the central mechanism of MßCD-induced reduction in evoked transmission. The locus of this effect could be presynaptic, postsynaptic or in both cells.
mEPSP amplitude not affected by MßCD
The reduction in EPSP amplitude could be caused by a reduction in sensitivity of muscle glutamate receptors (Eroglu et al. 2003) or alteration in their channel kinetics. For instance, cholesterol levels could affect the performance of muscle Ca2+ channels (Launikonis & Stephenson, 2001; Pouvreau et al. 2004). To determine whether the main effects of MßCD are presynaptic or postsynaptic we recorded mEPSPs which are the responses to individual spontaneously released quanta of transmitter. We measured the mean amplitude, decay constant and rise time of 50 mEPSPs obtained before and during the last 5 min in the presence of 10 mM MßCD for each of five experiments. Data from a representative experiment are shown in Fig. 4A. The decay time constant before (
control) and after (MßCD) a 30-min treatment with 10 mM MßCD did not change (control, 3.9 ± 0.3 ms; MßCD, 4.6 ± 0.3 ms, P > 0.05). Similarly, the rise time from 10% to 90% of maximum amplitude did not change with the addition of 10 mM MßCD (control, 0.07 ± 0.01 ms; 10 mM MßCD, 0.07 ± 0.01 ms, P > 0.05). The mean mEPSP amplitude before treatment was 241.0 ± 0.01 µV and after 30 min in the presence of 10 mM MßCD, the mean mEPSP amplitude was 243.0 ± 0.009 µV. As shown in Fig. 4B, mEPSP amplitude distribution was not affected by MßCD (P > 0.05, Kolmogorov-Smirnov test). Similar analysis showed that there was no change in time course and amplitude of mEPSPs in four additional experiments. These results indicate that the sensitivity of the muscle to quantal glutamate release was not affected by cholesterol removal. It is therefore likely that MßCD affects evoked EPSPs by reducing evoked transmitter release.
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MßCD blocks presynaptic action potential
Cholesterol can affect components of the resting potential of cells (Cornelius, 2001; Romanenko et al. 2002) and we found that application of 10 mM MßCD caused the resting potential of the primary branch of the phasic axon to hyperpolarize from –71.5 ± 0.5 mV to –80.3 ± 3.6 mV (P < 0.05, n= 5). To evaluate the importance of this hyperpolarization, we applied (in separate experiments) low K+-containing saline (2.5–3 mM instead of 5.4 mM) to obtain an average hyperpolarization of 9.3 mV (n= 5). With this hyperpolarization there was a much smaller reduction in EPSP to 40% after 30 min compared to that found with MßCD, and the presynaptic axon AP was not blocked. Therefore, hyperpolarization of the axon is probably only a partial explanation for the MßCD-induced EPSP block of transmitter release. Application of 10 mM Ch-MßCD after 10 mM MßCD caused the axon resting potential to depolarise above the normal resting potential by 4.2 ± 1 mV (n= 3; data not shown).
Recordings of presynaptic axon APs showed that they were sometimes blocked when MßCD had blocked evoked transmitter release. The mechanism of AP blockade probably involves increased resting conductance because rough measurements of axon conductance made with a single sharp electrode and balanced bridge showed that with 4- to 10-mV hyperpolarizing test pulses, resting conductance increased 1.6 ± 0.2-fold (P < 0.05, n= 5) after application of 10 mM MßCD. It is possible that MßCD had more profound effects on AP propagation in fine branches that would not be immediately detected in larger branches where recordings were made. To study this in more detail we made simultaneous intracellular axonal (sharp electrode) and extracellular focal recordings with electrodes positioned over presynaptic boutons. The focal recordings resolved the AP in the primary and terminal branches at or near the bouton as well as a larger current caused by the action of transmitter on the muscle receptors (EPSC). When 10 mM MßCD was applied for 10 min, the EPSC was vastly diminished and the local AP had disappeared but there was still a robust signal from the primary branch and a robust intracellular AP in the axon. By 30 min both the focal AP and the intracellular AP were greatly diminished. Subsequent application of 10 mM Ch-MßCD restored APs and transmitter release (Fig. 5). Similar results were obtained in three preparations. Therefore, blockade of axonally evoked transmitter release by cholesterol extraction is due to blockade of AP propagation and invasion of presynaptic terminals. Progressive reduction of AP propagation velocity probably explains the increased delay between stimulation and appearance of an EPSP (inset to Fig. 3A).
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Next we determined whether cholesterol extraction blocks Ca2+ channels and exocytosis independent of presynaptic APs. To circumvent the requirement for presynaptic APs we depolarized synaptic boutons by passing current through a focal electrode connected to a bridge headstage. The same focal electrode depolarized the synaptic bouton and detected transmitter release as the EPSC (Parnas et al. 1994). In the same experiment, transmitter release was also triggered conventionally by axonal stimulation. Figure 6 shows a result typical of five experiments. Under control conditions both axonal and focal stimulation caused an EPSC. However, when MßCD was applied for 30 min, axonal stimulation failed to evoke an EPSC but the EPSC induced by focal stimulation was increased in amplitude. It is therefore clear that neither Ca2+ channels nor exocytosis were blocked by cholesterol extraction as performed here. Given that the resting conductance of the presynaptic cell increased in the presence of MßCD, it is likely that there was less depolarization due to focal current after administration of MßCD and therefore some step in evoked transmitter release was actually enhanced.
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We next sought to determine whether spontaneous transmitter release, which involves some but not all of the steps in Ca2+-triggerred exocytosis (Hua et al. 1998), would be sensitive to cholesterol depletion. We recorded mEPSPs caused by spontaneous release of single quanta in control or MßCD-containing saline and counted events that were well above the noise level. As noted previously, the amplitude and time course of spontaneous mEPSPs were not affected by MßCD. The control mEPSP frequency was stable for 60 min and ranged from 0.033 to 0.33 Hz. When MßCD was applied there was a 5-fold increase in the rate of spontaneous release within the first 10 min rising to a 6-fold increase in the following 20 min (Fig. 7A and B, P < 0.001, n= 5). When control saline was reapplied after 15 min, there was a slow reduction in mEPSP frequency to about 50% of the maximum in 15 min (Fig. 7C, P < 0.008, n= 5). However, when 10 mM MßCD was replaced by 2.5 mM Ch-MßCD, the frequency of mEPSPs returned to baseline levels within 5 min (Fig. 7D, P > 0.05, n= 5) Application of 2.5 mM Ch-MßCD did not change the rate of spontaneous release on its own (data not shown). Application of HPCD, which has a low affinity for cholesterol (Ohtani et al. 1989), had no effect on the frequency of spontaneous transmitter release (Fig. 7E, P > 0.05, n= 5). To test the hypothesis that hyperpolarization of the presynaptic membrane potential by MßCD increases the frequency of spontaneous release, a low K+-containing saline was used to hyperpolarize the axon by a comparable level but there was no change in spontaneous transmitter release (P > 0.05, n= 5). Therefore, MßCD-induced hyperpolarization cannot account for the increase in mEPSP frequency.
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To test the hypothesis that the MßCD-induced increase in spontaneous release is due to influx of extracellular Ca2+, we performed a set of experiments in low Ca2+-containing saline. We attempted to use EGTA to reduce the ionized calcium concentration but this sometimes damaged the cells. Therefore we used saline with no added Ca2+ and no chelator to achieve a low Ca2+ environment. However, the Ca2+ concentration was low enough to completely block evoked transmission from phasic synapses. When 10 mM MßCD was applied in low Ca2+-containing saline there was a 7.6-fold increase in spontaneous mEPSP frequency (Fig. 9) similar to that obtained in normal saline (13.5 mM).
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To obtain greater Ca2+ buffering we injected BAPTA-K4 ionophoretically into the phasic axons in another set of experiments. About 1 h after beginning the injection, evoked transmitter release was blocked although presynaptic APs were robust. Injection continued throughout the experiment. There was no effect of BAPTA-K4 injection on mEPSP frequency. When evoked transmitter release had been blocked, 10 mM MßCD was applied and the increase in spontaneous release frequency was similar to controls without BAPTA injection (Fig. 9). Similar results were obtained at tonic synapses on the opener muscle. Here, after BAPTA injection (low Ca2+ saline), application of 10 mM MßCD caused an increase in spontaneous transmitter release similar to that obtained without the chelator (Fig. 9). These data indicate that an increased intracellular calcium concentration is not required for the effects of 10 mM MßCD on mEPSP frequency. Therefore the increase in the frequency of spontaneous transmitter release caused by MßCD did not require intracellular or extracellular calcium.
Ca2+-stimulated asynchronous transmitter release
To determine whether cholesterol extraction affects Ca2+-stimulated asynchronous transmitter release, we applied a Ca2+ ionophore, ionomycin, after treatment of opener muscles with MßCD. In control experiments, ionomycin by itself caused the frequency of spontaneous transmitter release to increase by over 6-fold within 3 min of application (Fig. 10A; 0.10 ± 0.4 to 0.63 ± 0.2 Hz, P < 0.05, n= 4). In another set of experiments, 10 mM MßCD was applied and the frequency of spontaneous release increased 4.6-fold. When ionomycin was then applied, the frequency increased further by over 6-fold. A sample experiment is shown in Fig. 10B and C. Thus MßCD treatment multiplied the effectiveness of ionomycin by over 4-fold.
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Discussion |
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The lack of any apparent postsynaptic effect of MßCD simplified the interpretation of our data and allowed us to concentrate on presynaptic effects of cholesterol depletion.
Cholesterol depletion blocks AP-evoked transmitter release
The effect of cholesterol removal on action potential propagation has not been characterized previously. Cholesterol depletion alters the function of several channels and pumps. For example, there is an increase in the activity of the Na+–K+ pump when cholesterol is added to membranes (Cornelius, 2001). If an electrogenic Na+–K+ pump had been slowed then depolarization would ensue, and this was not observed. Reduction of Na+–K+ pump activity would cause an increase in [Na+]i and loss of excitability. K+ and Ca2+ channels are also affected by cholesterol modulation (Romanenko et al. 2002; Xia et al. 2004). The block of APs may have been caused by the opening of ion channels and not just a change in pump activity as we found that cholesterol depletion resulted in increased axonal conductance.
Cholesterol depletion does not block Ca2+-dependent transmitter release
The blockade of presynaptic APs complicates the study of evoked transmitter release because presynaptic Ca+ channels are not activated. We avoided this issue by directly depolarizing the boutons with a focal electrode. Transmitter release evoked this way was increased after application of MßCD indicating that cholesterol depletion does not block calcium channel activity or downstream processes. These findings are consistent with earlier work which showed that 10 mM MßCD does not alter Ca2+ influx in rat brain synaptosomes (Taverna et al. 2004); however 30 mM MßCD did reduce Ca2+ influx. Furthermore, the ionomycin-induced increase in asynchronous quantal release was approximately what would be expected if calcium-induced release had a multiplicative relationship with MßCD-induced quantal release.
Cholesterol depletion increases spontaneous transmitter release
Cholesterol depletion enhanced the rate of spontaneous release at both phasic (extensor muscle) and tonic (opener muscle) synapses (Figs 7–9), which is consistent with our findings that show that evoked release is enhanced. It is therefore possible that cholesterol controls a step in exocytosis common to both evoked and spontaneous release.
Various cellular perturbations can result in an increase in spontaneous release including increased calcium influx, stretch/hyperosmolarity and second messenger pathways. However we have eliminated some of these possibilities. Depolarization of the crayfish axon increases the rate of spontaneous release, presumably by increasing calcium influx (Wojtowicz & Atwood, 1984). Hyperpolarization might also enhance transmitter release by Ca2+ influx through hyperpolarization and cyclic nucleotide channels (HCNC) (Zhong & Zucker, 2004); however, when we hyperpolarized the axon by an equivalent amount (8–12 mV) using a low K+-containing saline, there was no significant change in the rate of spontaneous release. Our observation that the effects of cholesterol depletion on spontaneous release were not Ca2+ dependent (Fig. 9) also argues against hyperpolarization-mediated Ca2+ entry as a cause of increased transmitter release. Accumulation of Na+ owing to reduced Na+–K+ pump activity could affect transmitter release by displacing Ca2+ from intracellular sites or by allowing build-up of [Ca2+]i by reversing the Na+–Ca2+ exchanger (Mulkey & Zucker, 1992; Zhong et al. 2001); however, this is unlikely to explain the increase in spontaneous transmitter release with MßCD because BAPTA did not block the increase.
Cholesterol depletion may enhance vesicle fusion by altering the biophysical properties of the membranes. Under physiological conditions, cholesterol has a rigidifying effect on the lipid bilayer (Ohvo-Rekila et al. 2002). Therefore the removal of cholesterol in the present study is likely to increase membrane fluidity and this may promote vesicle fusion. Therefore increasing membrane fluidity by cholesterol depletion potentially enhances the rate of transmitter release. Whether this effect is directly on the two membranes involved or on their protein constituents is unknown. The membrane-embedded protein syntaxin may help to form an early fusion pore (Han et al. 2004) and may be affected by cholesterol and membrane physical properties.
Acclimation and membrane properties
One way that poikilothermic animals adapt to temperature changes is by modifying membrane fluidity. Membranes of cold-acclimated crustaceans are more fluid than those of warm-acclimated animals (Lehti-Koivunen & Kivivuori, 1998; Cuculescu et al. 1999). Furthermore, the performance of crustacean neuromuscular systems can be altered by acclimation to different temperatures; neuromuscular systems from cold-acclimated animals are less affected by cold temperatures than those from warm-acclimated animals (Stephens & Atwood, 1982; White, 1983). While the role of cholesterol in regulation of membrane fluidity during acclimation is unclear, there is a reduction in lipid saturation with cold acclimation (Cossins, 1976; Pruitt, 1988; Cuculescu et al. 1999).
Other studies
Our results differ from those found in some systems in which the relation between cholesterol and exocytosis was studied (reviewed by Pfrieger, 2003). For example, the effects of cholesterol depletion on dopamine release at PC12 cells have been studied by Lang et al. (2001) and by Chamberlain et al. (2001). Both studies reported that cholesterol depletion drastically reduced release of dopamine triggered by depolarization, Ca2+ loading or ATP. Similarly, in rat brain synaptosomes, K+-induced depolarization and Ca2+ ionophore-stimulated loss of glutamate were severely reduced by cholesterol depletion (Gil et al. 2005). In contrast, in pancreatic ß cells, exocytosis was increased after cholesterol extraction (Xia et al. 2004). Additionally, in rat hippocampal slices, Koudinov & Koudinova (2001) showed that 2.5 mM MßCD applied for 20 min did not affect evoked field EPSPs but did block maintenance of long-term potentiation.
The importance of lipid-rich membrane areas in localization of critical exocytotic proteins has been emphasized (Lang et al. 2001; Chamberlain et al. 2001; Taverna et al. 2004; reviewed by Salaün et al. 2004). We have yet to investigate whether protein redistribution occurs in presynaptic terminals after cholesterol depletion. The importance of cholesterol in synapse formation and stability has been shown by Goritz et al. (2005).
Implications
Altered cholesterol levels and lipid rafts have been implicated in numerous neurodegenerative disorders (Koudinov & Koudinova, 2005) including Niemann-Pick disease, which is associated with a reduced level of plasma membrane cholesterol (Tashiro et al. 2004). Changes in cholesterol levels have also been linked to Alzheimer's disease (Chochina et al. 2001). Understanding the physiological implications of alterations in cholesterol content may also prove useful in understanding the basic role of biophysical changes in membrane properties in exocytosis.
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References |
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) and treated experiments (•). Control traces show a gradual depression that is characteristic of phasic synaptic transmission at the crayfish neuromuscular junction. A, bath application of 10 mM MßCD for 30 min reduced EPSP amplitude to 10% of the initial value while control EPSPs were 66% of initial amplitude. Inset, sample EPSPs obtained in a treatment experiment at times indicated. B, washout of 10 mM MßCD with control saline did not lead to recovery of EPSP amplitude. C, 15 min after the addition of 10 mM MßCD, exchange with 2.5 mM MßCD led to a partial recovery of EPSP amplitude (to 39% of initial EPSP value). D, application of 10 mM Ch-MßCD 30 min after addition of 10 mM MßCD led to complete EPSP recovery. E, application of 10 mM H
), MßCD applied in saline containing low calcium or BAPTA-AM or after injection of BAPTA-K4 (phasic axon injection), caused an increase mEPSP frequency comparable to that found when MßCD was applied in normal Ca2+-containing saline (5- to 8-fold frequency increase, n= 5 for each condition, P < 0.05). In the opener muscle excitor preparation (
), MßCD was applied in low Ca2+-containing saline in combination with BAPTA-K4 axon injection and mEPSP frequency was increased by an amount similar to that found in experiments performed in control saline with no chelator (5- to 6-fold frequency increase, n= 5 for each condition, P < 0.05).