Figure 1.
A novel setup allows monitoring of AChR turnover in live mice.
A: Photograph of the setup showing the anaesthetized 125I-BGT-injected mouse mounted onto the lead support shielding left from right body half. A lead shield with a 16.5 mm wide perforation at the height of the TA muscle limits the detected 125I-emission to that of the lower hindlimb. B: Schematic drawing illustrating the emission path from injected lower hindlimb to the Germanium semiconductor sensor. C: Typical emission spectrum as detected upon injection of 125I-BGT. The integrals below peak 1 were used for quantification of AChR turnover. D: Decay of a non-injected 125I-BGT-standard. Dots, measured values; dotted line, exponential fit based on measured values; continuous line, theoretical decay curve of 125I.
Figure 2.
Most intramuscularly injected 125I-activity rapidly becomes available systemically.
125I-BGT was injected into TA muscles of mice. 125I-emission was repetitively measured immediately after injection (time point 0) and subsequently at time points as indicated. A: Residual 125I-BGT-emission in the injected hindlimb as a function of time after pulse labeling. Dots, measured values; continuous line, two-term exponential fit; dotted lines, extrapolations for the two different exponential terms. This data series had four measurements shortly after 125I-BGT injection. A t1/2 of 49 min was calculated for the expulsion of 125I-activity from the injected TA muscle during the first day after pulse labeling. B: 125I-BGT-emission in the injected hindlimb (black dots) and the ispilateral foreleg (white dots) normalized to the value measured immeditaley after pulse labeling in the injected hindlimb as a function of time after pulse labeling. Continuous lines show a two-term exponential fit and a ‘Bateman’ function for the hindlimb and the foreleg, respectively. The latter describes the result of the two exponential processes, (i) infiltration of 125I-BGT upon release from the hindlimb and (ii) subsequent exponential decay with the same half-life as in the hindlimb. The dotted line indicates the extrapolation for the fit of the expulsion component. Note, that systemically available 125I-BGT accumulates in foreleg muscles and shows a similar decay as in the injected hindlimb muscle.
Figure 3.
The novel approach reveals three levels of metabolic stabilization of AChRs.
A–D: Muscles were either left innervated (innervated) or the were denervated immediatedly prior to (acutely denervated) or five days before pulse labeling with 125I-BGT (long-term denervated). Time point of pulse labeling is t0. Graphs in A–D show the residual 125I-emission in the injected hindlimbs as a function of time after pulse labeling. Dots, measured values (mean ± SD; n-values: control n = 10, acutely denervated n = 5, long-term denervated n = 4); lines, two-term exponential fits. Data are normalized to the mean values measured on day 1 (A), day 2 (B), day 3 (C) or day 7 (D) to highlight the progressive loss of the short-lived AChR population with increasing time after pulse labeling. During the first week of chase muscles were measured at intervals of 24 hours. Already at the day 1 measurement less than 10−5% of unbound 125I-BGT was left. E–F: Muscles were innervated or denervated as in A–D, but instead of 125I-BGT BGT-AF647 was injected at t0. Ten days later BGT-AF555 was injected to label newly formed AChRs. Subsequently, in vivo imaging was performed and synapses were automatically segmented and analyzed. E: Representative maximum z-projections of microscopic fields showing signals of BGT-AF647 (‘old receptors’), BGT-AF555 (‘new receptors’) and overlay of both (‘old receptors in green’, ‘new receptors’ in red) on day ten after labeling with BGT-AF647. Scale bar, 50 µm. F: Quantification of the fraction of pixels with dominant ‘new receptor’ label of the entire NMJ pixels as a function of innervation/denervation status of the analyzed muscle. Mean ± SEM (n-values: control n = 3, acutely denervated n = 4, long-term denervated n = 3). * P<0.05 according to Welch test.
Table 1.
Most 125I activity is excreted by the animal during the first day after pulse labeling.
Table 2.
AChRs exhibit three different half-lives, depending on the state of innervation.
Figure 4.
Two nerve-activity-dependent signals mediate full metabolic stabilization of AChRs.
A: Scheme summarizing the findings of the 125I-BGT approach. Upon delivery of AChRs to the synapse, the presence of the nerve-activity-dependent signal, S1, triggers the prolongation of the receptor half-life from ∼1 to ∼8 days. When not stabilized receptors are degraded. During or at the end of the first week another nerve-activity-dependent signal, S2, is needed to extend the receptor half-life to now ∼13 days. B: Enlargements from Fig. 3E indicate the preferential incorporation of newly formed AChRs at the periphery of NMJs. Pictures show NMJs of innervated (left), acutely denervated (middle) or long-term denervated (right) muscles. ‘Old receptors’ are shown in green, ‘new receptors’ in red. Note, that with increasing time of denervation-induced absence of AChR stabilization the domain where ‘new receptors’ prevail progressively expands from the periphery of NMJs towards the center. Scale bar, 10 µm. C: Transmission electron micrograph through a NMJ. The picture shows the cross-section of one arbor (exemplified by the dotted rectangle in B, left panel). The upper left half shows the presynaptic terminal, the lower right half shows the muscular, post-synaptic apparatus. 1, primary synaptic cleft; 2, secondary synaptic cleft; G, Golgi apparatus; M, mitochondria; N, subsynaptic nucleus; SV, synaptic vesicles. Scale bar, 1 µm. The region of the Golgi apparatus (for better visibility contrast-corrected) is enlarged in the insert. D: Scheme illustrating the hypothesized lateral movement of AChRs within the NMJ and the presumptive spatial localization of S1 and S2. Red rectangles, AChRs; spheres, exocytic (left) and recycling vesicles (middle and right).