Identification of nicotinic acetylcholine receptor recycling and its role in maintaining receptor density at the neuromuscular junction in vivo

Abstract

In the CNS, receptor recycling is critical for synaptic plasticity; however, the recycling of receptors has never been observed at peripheral synapses. Using a novel imaging technique, we show here that nicotinic acetylcholine receptors (AChRs) recycle into the postsynaptic membrane of the neuromuscular junction. By sequentially labeling AChRs with biotin-bungarotoxin and streptavidin-fluorophore conjugates, we were able to distinguish recycled, preexisting, and new receptor pools at synapses in living mice. Time-lapse imaging revealed that recycled AChRs were incorporated into the synapse within hours of initial labeling, and their numbers increased with time. At fully functional synapses, AChR recycling was robust and comparable in magnitude with the insertion of newly synthesized receptors, whereas chronic synaptic activity blockade nearly abolished receptor recycling. Finally, using the same sequential labeling method, we found that acetylcholinesterase, another synaptic component, does not recycle. These results identify an activity-dependent AChR-recycling mechanism that enables the regulation of receptor density, which could lead to rapid alterations in synaptic efficacy.

Figure 1.

Multiple AChR pools are present at synapses on sternomastoid muscles of living mice. A, In a living adult mouse, a neuromuscular junction of the sternomastoid muscle was labeled with a saturating dose of BTX-biotin followed by a saturating dose of streptavidin (strept)-Alexa 660 (blue, pseudocolor). B, C, The junction was then immediately bathed with a saturating dose of streptavidin-Alexa 488 (green) and TMR-BTX (red). The absence of any labeling (even when camera exposure was increased significantly) indicates that all receptors and biotin sites were saturated by the initial applications. D-G, The same neuromuscular junction imaged 24 h later, after application of new streptavidin-Alexa 488 (green) and TMR-BTX (red) to the sternomastoid muscle. The remaining preexisting receptors were still labeled with BTX-biotin-streptavidin-Alexa 660 (blue, pseudocolor). The streptavidin-Alexa 488 labeling indicates the presence of receptors that retained BTX-biotin sites but lost their streptavidin-Alexa 660. These free sites are unlikely because of the loss of streptavidin on the surface (see Figs. 2, 3, 4, 5) and therefore referred to as recycled receptors. The TMR-BTX labeling indicates receptors that were not bound to either BTX-biotin or streptavidin and are referred to as new receptors. Scale bar, 20 μm.

Figure 2.

Streptavidin does not dissociate from AChR-BTX-biotin clusters on muscle cells in culture. A, In C2C12 myotube cultures, ghost clusters (as described by Kummer et al., 2004) that were no longer associated with myotubes were labeled with BTX-biotin followed by streptavidin-Alexa 594. B, Five days later, the same clusters were treated with streptavidin-Alexa 488 (to identify receptors that retained BTX-biotin and lost streptavidin). C, Overlay of images in A and B. The absence of green labeling in B indicates that there was no dissociation of streptavidin from receptor-bungarotoxin-biotin complexes on ghost clusters after 5 d. D, AChRs on a living C2C12 myotube that were labeled with a saturating dose of BTX-biotin followed immediately by a saturating dose of streptavidin-Alexa 660 (pseudocolor blue) and then imaged 12 h later. E, F, The same cells were then incubated with streptavidin-Alexa 488 (green) to label receptors that retain BTX-biotin and lose streptavidin and BTX-Alexa 594 (red) to label new AChRs. The absence of green labeling in E indicates that no detectable streptavidin-biotin dissociation occurred over this period, whereas the red labeling indicates the addition of new receptors. G, Overlay of D-F. Scale bars, 20 μm.

Figure 3.

Lack of streptavidin (Strept) dissociation from AChE-fasciculin 2-biotin in vivo. A, Example of a living adult mouse neuromuscular junction previously labeled with BTX-biotin (to label AChRs) followed by a saturating dose of streptavidin-Alexa 594 (red) at time 0 and imaged at 4 d. Red signal indicates the preexisting receptors still remaining on day 4. B, Same neuromuscular junction imaged after application of streptavidin-Alexa 488 (green). Green signal indicates the AChRs that were recycled over 4 d. C, Example of an NMJ labeled with fasciculin 2-biotin (to label AChEs) followed by a saturating dose of streptavidin-Alexa 594 (red) at time 0 and imaged at 4 d. Red signal indicates the preexisting acetycholinestrase remaining on day 4. D, The same neuromuscular junction imaged after application of new streptavidin-Alexa 488 (green) and viewed with much higher camera gain than used in B. The weak green signal indicates that there is very little streptavidin dissociation after 4 d. When the same gain was used as in B, the fluorescence was almost undetectable. Scale bars, 20 μm.

Figure 4.

Confocal imaging of AChR-bungarotoxin-biotin-streptavidin (Strept) complexes in intracellular compartments. Sternomastoid muscles were labeled in living mice with BTX-biotin and saturated with streptavidin-Alexa 488. After 4 d, the muscles were fixed, sectioned longitudinally, and then processed for immunocytochemical detection of AChRs with an Alexa 594-conjugated secondary antibody. A, Example of a stack of 20 confocal images (0.5 μm intervals) of an NMJ that was initially labeled with BTX-biotin-streptavidin-Alexa 488 and collapsed onto a single plane. The small green fluorescence puncta could represent either the accumulation of fluorescent streptavidin still attached to AChR-BTX-biotin complexes or the accumulation of fluorescent streptavidin in intracellular compartments.B, Immunostaining with anti-AChR and secondary antibody conjugated to Alexa 594. These puncta could represent the presence of receptors that are newly synthesized, internalized and recycling, or internalized and in the process of degradation. C, Overlay of the Alexa 488 fluorescence in green and the anti-AChR fluorescence in red. D-F, Detail from the boxed regions in A-C. Arrows indicate colocalization of original streptavidin-Alexa 488 (green) and AChR (red) in intracellular puncta, showing that the AChR-BTX-biotin-streptavidin complexes are internalized. Red arrowheads indicate intracellular puncta that contained only receptors (likely unlabeled receptors that are in the process of insertion or BTX-biotin-labeled receptors in the process of recycling). Green arrowheads indicate intracellular puncta that contained only streptavidin-Alexa 488. G-O, Example of a neuromuscular junction on a muscle section (labeled as described above) immunostained for AChRs and EEA1. G-I, Eight confocal slices at 2 μm intervals were collapsed onto a single image plane. In the overlay image, green indicates anti-AChR immunostaining, and red indicates anti-EEA1 immunostaining. J-L, The boxed regions of G-I shown at higher power. M-O, z-stack of fluorescence intensity taken at various depths along the white lines in J-L. The top of the section is to the right, and the width of these panels corresponds to a total depth of 20 μm.

Figure 5.

Confocal images of AChR-BTX-biotin complexes in intracellular compartments. A, Example of a neuromuscular junction labeled with BTX-biotin followed by anti-biotin-Alexa 488 or 594. This staining was a positive control to demonstrate that this antibody is able to effectively label biotin in complex with AChR. B, Example of a neuromuscular junction that was labeled with biotin-bungarotoxin followed by a saturating dose of streptavidin (strept)-Alexa 488 and then immediately fixed. C, When the same synapse was immunostained with anti-biotin-Alexa 594, no red fluorescence was observed. The absence of staining with anti-biotin indicates that this antibody can only interact with BTX-biotin when it has been stripped of streptavidin. D, Overlay of images in B and C. E, Example of a synapse on a muscle initially incubated with BTX-biotin-streptavidin and then fixed and sectioned 4 d later and immunostained with anti-AChR and an Alexa 594-conjugated secondary antibody. F, Same synapse as in E labeled with anti-biotin-Alexa 488. G, Overlay of images in E and F. H-J, Boxed areas from E-G at higher magnification and viewed at a camera gain sufficient to saturate the signal from synaptic AChRs to accentuate the signal coming from the internal spots. Arrowheads indicate spots of fluorescence that stained positive for AChR, but not for biotin, and arrows indicate spots where both labels were colocalized. Scale bar, 20 μm.

Figure 6.

Time-lapse imaging of the appearance of recycled receptors. A, In a living adult mouse, a neuromuscular junction of the sternomastoid muscle was labeled with a low dose of BTX-biotin followed by a saturating dose of streptavidin (strept)-Alexa 488 (green). B, The junction was then immediately bathed with a saturating dose of streptavidin-Alexa 594 (red) and viewed at substantially higher gain. The absence of any red labeling indicates that all biotins were initially saturated with streptavidin-Alexa 488. C-F, The same neuromuscular junction imaged every 2 h for 8 h after application of new streptavidin-Alexa 594 (red) to the sternomastoid muscle before each time point. Scale bar, 20 μm. Similar results were obtained at 10 synapses from three mice.

Figure 7.

Quantification of original, recycled, and new receptors at the neuromuscular junction after transient activity blockade. These pseudocolor images provide a linear representation of the density of AChRs (white-yellow, high density; red-black, low density). A, Example of a mouse neuromuscular junction labeled with a saturating dose of BTX (btx)-biotin followed immediately by a saturating dose of streptavidin (strep)-Alexa 594, imaged, and then reimaged 4 d later. The total fluorescence intensity (a measure of the total number of AChRs) was expressed as 100% at the initial labeling (left panel, day 0) and normalized to this on each successive view. Second panel from left, Preexisting receptors remaining after 4 d when >50% of the initial fluorescence is lost. To quantitate the recycled receptors, streptavidin-Alexa 594 (red) fluorophore was then added to the muscle. Third panel from left, Sum of preexisting and recycled receptors after 4 d. New receptors were identified by next adding a saturating concentration of BTX-biotin followed by a saturating dose of streptavidin-Alexa 594 (red), as shown in the fourth panel. synth., Synthesized receptors. The fluorescence from these new receptors is added to the preexisting and recycled receptors to determine the total receptor density after 4 d. B, Summary of the amount of original receptors remaining after various times and the proportion of new and recycled receptors that were inserted into the synapse, obtained from many junctions with the approach shown in A. Each data point represents the mean percentage of original fluorescence intensity ± SEM (error bars).

Figure 8.

Quantification of original, recycled, and new receptors at the neuromuscular junction after a transient and maintained activity blockade. A, Assessment of the extent of recycling 8 h after an initial transient blockade of activity with BTX-biotin. B, Assessment of the extent of recycling 8 h after a maintained blockade of activity with curare. C, Summary of results obtained from many synapses studied as in A and B. The data show the mean ± SEM (error bars). For all four conditions, the difference between the two experimental groups was significant (p < 0.001). These experiments were not performed beyond 8 h because of mouse mortality from the long-term curare treatment. It thus was not possible to measure recycling and new receptors at the same synapses. The insertion of new receptors was therefore measured in a parallel experiment using BTX-Alexa 594. strept, Streptavidin.