A cross-sectional study of ageing at the mouse neuromuscular junction and effects of an experimental therapeutic approach for dynapenia

Abstract

Despite prior efforts to understand and target dynapenia (age-induced loss of muscle strength), this condition remains a major challenge that reduces the quality of life in the aged population. We have focused on the neuromuscular junction (NMJ) where changes in structure and function have rarely been systematically studied as a dynamic and progressive process. Our cross-sectional study found neurotransmission at the male mouse NMJ to be biphasic, displaying an early increase followed by a later decrease, and this phenotype was associated with structural changes to the NMJ. A cross-sectional characterization showed that age-induced alterations fell into four age groups: young adult (3–6 months), adult (7–18 months), early aged (19–24 months), and later aged (25–30 months). We then utilized a small molecule therapeutic candidate, GV-58, applied acutely during the later aged stage to combat age-induced reductions in transmitter release by increasing calcium influx during an action potential, which resulted in a significant increase in transmitter release. This comprehensive study of neuromuscular ageing at the NMJ will enable future research to target critical time points for therapeutic intervention.

Graphical Abstract

In this study, age-induced changes at the neuromuscular junction were evaluated in a cross-sectional fashion. Using electrophysiology and immunohistochemistry, the changes from 3 months to 30 months of age in a mouse model were documented. The change in neurotransmission at the neuromuscular junction follows a biphasic trend (bottom row) – first an increase followed by a later decrease. These changes are accompanied by a variety of changes morphologically (top row) to acetylcholine receptor and neurofilament staining. This study identified a time course in which significant changes were observed before degeneration, defining potential critical time windows for prophylactic interventions to treat dynapenia.

Introduction

The current approach to studying ageing is often associated with the study of senescence, but this perspective does not encompass the complex physiological changes through the course of normal ageing. Researchers have started to recognize ageing as a complex and non-pathological process that is more than a gradual single-step degeneration. Additionally, this time-dependent and dynamically changing process has often been misinterpreted as a pathology that manifests itself only at later times in life due to the onset of age-related symptoms. A perspective that targets only the late-onset ageing process can misguide research efforts aiming at developing prophylactic medicine (Board, 2019), as preventing mortality is not the only goal of ageing studies. Indeed, a recent longitudinal study in humans showed that effects of ageing manifest in multiple ways as early as the age of 45, and people who are at the same chronological age often have vastly different age-induced biological metrics (cardiovascular health, motor function, brain volume, etc.) (Elliott et al., 2021). These observations support the idea that there is a difference in the ‘pace of ageing’ among individuals, and these changes can be detected before senescence. Currently, we lack detailed information on the pace of ageing across systems and cells in the body. It is important to understand the cellular and biological changes that occur throughout the ageing process to guide the development of effective interventions. Documenting the pace of ageing is necessary for identifying the proper timing for therapeutic approaches to age-related functional deficits. Here, we have focused on the age-related process of dynapenia (the age-induced loss of muscle strength). Thus, the main objective of this study was to identify age-associated functional and structural changes at the male mouse neuromuscular junction (NMJ). We have also tested the effects of acute exposure to a novel small molecule calcium channel gating modifier on age-induced reductions in neurotransmission.

Age-induced loss of muscle strength (dynapenia) and the neuromuscular junction as a potential therapeutic target

Declining motor function in the elderly population has long been a major focus in geriatric medicine. Age-induced frailty and falls are the leading causes of injury-related death (Burns, 2018; Centers for Disease Control and Prevention, 2019), and risk factors such as muscle weakness, balance deficits and gait instability are responsible for these injuries (Ganz et al., 2007; Granacher et al., 2011; Rubenstein & Josephson, 2002). While muscle wasting might be expected to have a close connection to loss of muscle strength, it is reported to contribute to only 39% of the functional motor deficits, and many attempts at restoring muscle mass in the aged individual have failed to completely restore motor functions (Clark & Manini, 2008, 2012; Clark, Fernhall et al., 2006; Clark, Manini et al., 2006; Delmonico et al., 2009; Kawakami et al., 2001). Motor neuron activity plays a major role in converting intent into action via the neuromuscular system. Therefore, attributing loss of muscle strength purely to muscle wasting is inadequate. Since age-induced loss of muscle mass (sarcopenia) and age-induced loss of muscle strength are now recognized as distinct, a new term, dynapenia, has been introduced to better reference the loss of strength in the aged neuromuscular system (Clark & Manini, 2008, 2012). Dynapenia recognizes both the neurological and muscular deficits with a special focus on the failure of voluntarily activating skeletal muscles (Clark & Manini, 2012). This includes α-motor neuron activity, NMJ structure and function, and skeletal muscle properties. The NMJ is a highly specialized synapse between the motor nerve and the muscle, and it is a key structure where signals from the brain are converted into actions in the muscle. If the transmission from the motor nerve to the muscle membrane cannot sufficiently initiate action potentials in the postsynaptic muscle membrane, muscles fibres will fail to contract. This can be the result of either a decrease in neurotransmitter release or a decrease in postsynaptic receptor number or function. Numerous studies have reported changes in presynaptic neurotransmitter release in the aged NMJ, and these changes often occur in conjunction with changes in postsynaptic acetylcholine receptor (AChR) morphology, axon terminal morphology and muscle fibre integrity (Banker et al., 1983; Deschenes, 2011; Fahim, 1997; Kelly, 1978; Mahoney et al., 2014; Pousinha et al., 2015; West et al., 1991), suggesting an interaction between the pre- and post-synapse is crucial for maintenance and function of the neuromuscular system. Prior studies of rodent NMJ ageing reported conflicting results (Banker et al., 1983; Deschenes, 2011; Fahim, 1997; Gutmann et al., 1971; Kelly, 1978; Mahoney et al., 2014; Pousinha et al., 2015), which may be due to (1) studying either rats or mice of different genetic background, (2) using a non-specific or a broad range of time points for the ‘aged’ group, (3) not separating sex as a variable, and (4) examining different muscles with varying compositions of muscle fibre type and nerve activation pattern (e.g. diaphragm with 50% fast twitch and 50% slow twitch muscle fibre (Lieberman et al., 1973) which is involuntarily activated vs tibialis anterior muscle with 100% fast twitch muscle fibre (Hirofuji et al., 2000) and is voluntarily activated). Taken together, these factors should be considered when comparing the effect of age on the neuromuscular system. In this study, we have decided to report age-induced changes at the NMJ across multiple time points in male mice using a voluntarily activated and primarily fast twitch somatic skeletal muscle (epitrochleoanconeus muscle, ETA; Villarroel-Campos et al., 2022) at multiple time points in the life of a mouse. In addition, we have tested the effect of acute exposure to a novel small molecule treatment strategy aimed at increasing neurotransmission at the ageing NMJ.

Methods

Ethical approval

All animals used in this study were male mice of the C57/BL6 background obtained from Charles River Laboratories, Wilmington, MA, and all of the animals were housed in traditional mouse cages without physical/environmental enhancements and had unlimited access to food and water. Female animals are not included in this study as we have preliminary evidence that they have a different ageing time course. Animal studies were performed according to protocols approved by the University of Pittsburgh Institutional Animal Care and Use Committee (IACUC).

Animals

We have chosen to focus the study on normal ageing, and thus have decided to only include data from mice that are between 3 and 30 months. This range is based on our desire to exclude developmental influences before 3 months of age and to exclude the extreme variability that exists among mice that live beyond 30 months of age (including super agers). This upper limit is based on prior data (The Jackson Laboratory) demonstrating a 50% mortality rate at 28 months of age for the C57/BL6 mouse line. Therefore, we present data at multiple time points from 3 months through 30 months of age.

For this study, we have used surface muscle fibres of the mouse epitrochleoanconeous (ETA) muscle, a thin upper arm muscle that is almost entirely fast-twitch: at the young adult stage, surface fibres in the ETA are either Type IIb (54% of all fibres) or Type IIx (30% of all fast-twitch fibres do not stain for Type IIa or IIb antibodies), and the remaining fibres in the inferior part of ETA are Type IIa (16%) (Rogozhin et al., 2008; Villarroel-Campos et al., 2022). Following euthanasia of the animal (CO₂ inhalation, followed by thoracotomy), the ETA was dissected and prepared as an ex vivo nerve–muscle preparation. Normal mammalian Ringer (NMR) solution (150 mM NaCl, 5 mM KCl, 11 mM glucose, 10 mM HEPES, 1 mM MgCl₂ and 2 mM CaCl₂ at pH 7.4) was used to bathe the preparation together with continuous oxygenation.

Intracellular microelectrode recordings at mouse NMJ

The ETA nerve muscle preparation was stimulated with a suction electrode at 10× threshold, and 1 μM μ-conotoxin GIIIB (Alomone Labs Ltd, Jerusalem, Israel) (Hong & Chang, 1989) was added to block action potential-evoked muscle contraction. Borosilicate electrodes with approximately 40–60 MΩ resistance were filled with 3 M potassium acetate to perform microelectrode recordings. To assess the magnitude of transmitter release at each NMJ, spontaneous miniature endplate potentials (mEPPs) were collected for 1 min followed by 10 evoked endplate potentials (EPPs) at 0.2 Hz. The data were corrected for non-linear summation (McLachlan & Martin, 1981). The quantal content (QC) was calculated by dividing the average amplitude of the EPP by the corresponding average amplitude of mEPPs recorded from the same synapse. QC was used to estimate the number of vesicles (quanta) released with each presynaptic action potential. Short-term plasticity was evaluated following 10 presynaptic action potentials at 50 Hz in each muscle fibre. When evaluating the efficacy of the novel candidate GV-58, a vehicle of 0.05% v/v dimethyl sulfoxide was added into the bath of the nerve–muscle preparation before control recordings, and then 50 μM GV-58 was added into the bath for 30 min before recording from the same muscle fibres to evaluate the effect of GV-58. The EC₅₀ for positive allosteric modulation of Cav2 calcium channels is 7–9 μM (Liang et al., 2012) and based on this, a 50 μM dose has been shown to produce maximal effects in mouse models of neuromuscular disease (Tarr et al., 2013). Therefore, we have used the 50 μM dose in this study. Data were collected using an Axoclamp 900A (Molecular Devices, San Jose, CA, USA) and digitized at 10 kHz for data analysis with pClamp 10 (Molecular Devices). During analysis of the electrophysiology data, a 2000 Hz Gaussian low pass filter was used on both the mEPP and EPP data.

Immunohistochemistry

Immunohistochemical staining of proteins at the NMJ was performed after the intracellular recordings from each muscle as described previously (Laghaei et al., 2018). The ETA nerve–muscle preparations were incubated with NMR solution containing 2 μg/ml α-bungarotoxin-Alexa 488 (Thermo Fisher Scientific, Waltham, MA, USA) for 30 min, allowing specific binding to and subsequent visualization of postsynaptic AChRs. The preparation was then washed with NMR solution and fixed with 2% paraformaldehyde for 20 min. Permeabilization and blocking were performed in phosphate buffered saline (PBS) with 2% bovine serum albumin, 2% goat serum, and 0.5% Triton X-100. The primary antibody was prepared in the same blocking and permeabilization buffer. For measurements of neurofilament distribution, we used an antibody against neurofilament-M (1:1000, monoclonal, no. 2H3, DSHB, Iowa City, IA, USA) and the tissue was allowed to incubate overnight at room temperature. The preparation was then washed and incubated for 4–6 h with a secondary antibody(1:1000,goatanti-mouse-Alexa 647, Thermo Fisher Scientific) mixed in the blocking and permeabilization buffer. Afterwards, the preparation was washed in PBS and mounted on slides with the Prolong Gold (Thermo Fisher Scientific) mounting medium. A Leica SP5 spectral confocal microscope (Leica Microsystems, Wetzlar, Germany) was used to image stained NMJs. The resonance scanner mode was used with 0.068 μm pixel size, a 1024 × 1024 pixel array, a 0.6 μm step size, a 4× zoom factor and 64 line averages per frame. An oil immersion objective (HC PL AOD CS2 ×40/1.30 OIL) was used. The laser power was set at 0.3% for 488 nm and 0.05% for 638 nm. All images were collected at 12-bit.

Image analysis

ImageJ (NIH, Bethesda, MD, USA) was used to analyse the confocal images. For the measurement of AChR area, the following steps were used: the image was first converted to 16-bit, followed by auto-thresholding with the method ‘Huang2 White’. Then, the α-bungarotoxin (BTX) signal was selected using the magic wand tool (hand drawn tracing was used when the magic wand failed to distinguish between small islands of stain). The scale was calibrated by including the pixel size of the image in μm. Then the area of the AChR signal was measured using the measure function in ImageJ. The corresponding neurofilament (NF) image was then processed. The contouring of the BTX signal was first transferred onto the NF image using the command ‘Restore Selection’. The image was then converted to 16-bit. The same auto-thresholding criteria were used for the NF images as described above for BTX images. The NF signal outside of the BTX signal was cleared using the ‘Clear Outside’ command. The scale was calibrated by including the pixel size of the image in μm. Then, the selection of the NF signal inside the BTX signal was made by using the combination of command ‘Invert’ and command ‘Create Selection’. Then, the area was measured with the ImageJ command ‘Measure’. The appearance of the unique features such as the presence of fluorescent aggregates, multi-innervation, and axons with swollen segments were documented as ‘yes’ or ‘no’ for these respective properties. Features like end-bulb number and the number of AChR islands were counted and documented accordingly.

Statistical analysis

Statistical Analysis was performed using GraphPad Prism Version 9.4.0 (GraphPad Software, Inc., San Diego, CA, USA). Data are presented as means ± SEM unless otherwise noted. An α of 0.05 was used for all statistical tests.

Results

Individual neuromuscular synapses in a single muscle age differently within one animal

Using intracellular microelectrode recording, endplate potentials (EPPs), miniature endplate potentials (mEPPs) and quantal content (QC) at the NMJ were assessed from individual synapses in animals between 3 and 30 months of age. The results at the 3 month age was reported separately, and subsequently the data were grouped into 3-month intervals starting at month 4 through month 30. We have found that as animals age, the variability in QC within one animal increases. In extreme cases, the QC values in one animal could range from 12 to 120. Therefore, in this study, we have analysed changes between synapses because using animal number as our ‘n’ may obscure the important differences between individual synapses, which appear to age independently of one another. For example, the mean QC for all synapses recorded from one 4-month-old mouse was 66.7 ± 18 (mean ± SD), which is similar to the mean QC from one 20-month-old mouse (62 ± 38), but there was a much greater standard deviation between synapses in the older animal (see Fig. 1B). To avoid giving the impression that the population of synapses in these two animals was similar, we have decided to report values from each synapse, as synapses appear to age independently from others in the same animal, as opposed to reporting mean values from animals. Using this approach, one can appreciate that the QC from individual synapses recorded from young adults can range from 50 to 125, but in aged animals, QC can range between 15 and 150 or more (see Fig. 1B). Since QC is a ratio of EPP/mEPP amplitude, the range of values reported here is independent of differences in input resistance between individual muscle fibres (which can impact individual mEPP and EPP values).

Figure 1. Aged-induced biphasic change in neurotransmission of the NMJ in male mouse ETA muscle.

A, average of quantal content from 3 to 30 months of age. Dotted lines in each subsection represents the averaged value of quantal content. Data are plotted using the mean values recorded from 12–15 synapses in each animal at the indicated age ranges (n = 3 animals for 3 months; n = 5 for 3–6 months; n = 3 for 7–9 months; n = 4 for 10–12 months; n = 3 for 13–15 months; n = 6 for 16–18 months; n = 9 for 19–21 months; n = 5 for 22–24 months; n = 13 for 25–27 months; n = 8 for 28–30 months). B, comparison of quantal content across the 4 ageing epochs (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001). The numbers (n) for each plot were as follows: Young Adult 3–6 months, n = 78 synapses from 8 animals (SD = 17.94); Adult 7–18 months, n = 179 synapses from 16 animals (SD = 23.53); Early Aged 19–24 months, n = 131 synapses from 14 animals (SD = 30.75); Later Aged 25–30 months, n = 217 synapses from 21 animals (SD = 26.45). C, sample traces of mEPPs and EPPs recorded from each epoch (for presentation purposes, the stimulus artifacts have been truncated). D, comparison of EPP amplitudes recorded from the 4 epochs (**P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001) (SD are 0.11, 0.11, 0.09, 0.12 for the 4 epochs, respectively). E, comparison of mEPP amplitude recorded across 4 epochs (*P ≤ 0.05) (SD are 7.32, 7.82, 10.5, 9.27 for the 4 epochs, respectively). A one-way ANOVA with Tukey’s post hoc test was used for all the statistical analyses.

The age-induced change in neurotransmission at the NMJ is biphasic in male mice

Across the ageing time course, we detected a significant decrease in the amplitude of mEPPs recorded from synapses in animals that were older (19–30 months) as compared to synapses in young adult animals (3–6 months of age). In addition, our recordings showed a significant increase in EPP amplitude when comparing synapses from animals that were 3–18 months old as compared to synapses from animals that were 19–24 months old, and a significant decrease in EPP amplitude after 25 months of age. We further quantified these results by plotting changes in neurotransmitter release using quantal content (QC) as a quantification for synaptic strength. We found the change in QC from 3–30 months to be biphasic: a gradual increase from 7–24 months, followed by a decrease from 25–30 months. The increase from months 7–24 followed a two-step pattern: an initial increase in the 7–18 month age group (as compared to the 3–6 month age group), as well as a second larger increase in the 19–24 month age group. (Fig. 1A). These changes led us to separate the data into four epochs: a 3–6 months epoch termed ‘Young Adult’; a 7–18 months epoch termed ‘Adult’; a 19–24 months epoch termed ‘Early Aged’; and a 25–30 months epoch termed ‘Later Aged’ (Fig. 1B). It is worth noting that these epochs pertain only to changes in neurotranmission in male C57/BL6 mouse NMJs – our preliminary data on neurotransmission changes in female C57/BL6 mouse NMJs appear to follow a separate time course from the males (data not included), and will be the subject of a future study. We have applied the same epochs to our plots of mEPP (Fig. 1D) and EPP (Fig. 1E) data. We found a significant decrease in mEPP amplitude when comparing the Young Adult epoch with Early Aged epoch or the Later Aged epoch. As for EPPs, the Later Aged epoch exhibited a significantly decreased EPP amplitude compared to the other three epoches, while the Early Aged epoch showed a significant increase in EPP amplitude as compared to the Adult epoch.

Morphological changes in postsynaptic receptor and motor axon neurofilament staining during ageing at the NMJ of male mice

Using confocal imaging of fluorescent stains for presynaptic nerves (labelled with an antibody against neurofilament-M) and postsynaptic AChRs (labelled with fluorescently labelled α-bungarotoxin; BTX) at the NMJ, we were able to visualize the structural changes at the NMJ during ageing. We used the same four ageing epochs to group our data as described above. We first measured the change in the area of both AChRs and innervating motor axons (Fig. 2A–D). We found that the area of endplates (stained using BTX to label AChRs) showed a significant decrease between the Young Adult and Adult epochs, followed by a further significant decrease in the Early Aged epoch. This decreasing trend did not continue in the Later Aged epoch, where we detected an increase compared to the Early Aged epoch (Fig. 2E). When we measured the area of the innervating motor axon (as labelled using the neurofilament antibody), we found a significant decrease from Young Adult to Adult and Early Aged epochs, but the area did not change from the Early Aged to the Later Aged epoch (Fig. 2F). We next investigated the change in motor axon occupancy (as indicated by neurofilament staining) inside AChR-labelled endplates, and we found that the occupancy significantly decreased when comparing the Young Adult with Adult, Early Aged and Later Aged epochs but was unchanged after the Adult epoch (Fig. 2G).

Figure 2. Age-induced morphological changes of the NMJ in male mouse ETA muscle.

A–D, representative images from animals of 3 months old (Young Adult) (A), 16 months old (Adult) (B), 20 months old (Early Aged) (C), and 25 months old (Later Aged) (D) demonstrating the morphology of AChR (left; stained with BTX), motor axon (middle; stained with antibody against neurofilament-medium, NF-M), and the overlap of the AChR and motor axon signals (right; AChR in red and motor axon in cyan). E, comparison of AChR area (μm²) across the 4 ageing epochs (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001) (SD are 99.96, 106.2, 78.58, 111.3 for the 4 epochs, respectively). F, comparison of motor axon (NF-M staining) area within the area defined by the AChR staining in the 4 epochs (**P ≤ 0.01; ****P ≤ 0.0001) (SD are 71.02, 46.86, 26.43, 31.17 for the 4 epochs, respectively). G, comparison of the percentage occupancy of AChR with motor axons (NF-M staining) (***P ≤ 0.001; ****P ≤ 0.0001) (SD are 13.54, 9.532, 7.295, 6.01 for the 4 epochs, respectively). The numbers (n) for each plot were as follows: Young Adult, n = 84 synapses from 6 animals; Adult, n = 173 synapses from 10 animals; Early Aged, n = 68 synapses from 4 animals; Later Aged, n = 165 synapses from 9 animals. A one-way ANOVA with Tukey’s post hoc test was used for all the statistical tests.

Additional morphological features identified during ageing of the male mouse NMJ

During our morphological analysis, we also documented several additional features at the ageing NMJ. The occurrence of some of these features was associated with specific epochs. One such feature is multi-innervation (more than one axon innervating a single NMJ; Fig. 3A). The percentage of synapses that were multi-innervated increased significantly when comparing the Young Adult to Adult, Early Aged, and Later Aged epochs (Table 1). Another interesting phenomenon we observed was the frequent occurrence of swollen motor nerve axon segments, which previously has been reported to appear less frequently in the tibialis anterior muscle (Valdez et al., 2010). These swollen segments of axons were detected as enlargements in the neurofilament staining within parts of an axon entering a motor nerve terminal (Fig. 3B). We quantified the percentage of axons with swollen axon segments and found a significant increase in the Later Aged epoch as compared with all younger age epochs (Table 1). Next, we documented an occasional morphological alteration at the ends of the branched neurofilament staining within the motor nerve terminal. These alterations were characterized as circular endings (or end bulbs), as compared to the more common sharp or stubby endings (Fig. 3C). We quantified the numbers of these end bulbs and found a significant increase in the Later Aged epoch as compared with all younger age epochs (Table 1). During our measurements of the AChR area, the presence of small fluorescent aggregates outside the endplate region that resembled debris was occasionally spotted (Fig. 3D). This was later confirmed to be non-specific autofluorescence, emitting photons over a wide range of the visible spectrum. The presence of these autofluorescent aggregates was documented and quantified as a percentage of synapse images in which these were observed. We found an increase in synapse images that contained these autofluorescent aggregates near endplates in Adult, Early Aged and Later Aged synapses as compared with Young Adult synapses, and a further significant increase in Later Aged synapses as compared to Adult synapses (Table 1). Next, we quantified the number of AChR islands (isolated continuous AChR staining regions within one NMJ) in each epoch and found a significant increase in BTX island number within each endplate in the Later Aged epoch as compared with the previous three younger epochs (Table 1). On rare occasions, we also observed the phenomenon of neurofilament staining within motor axons extending away from endplates in the Later Aged epoch (Fig. 3E).

Figure 3. Confocal microscopy images demonstrating additional morphological features at the ageing NMJ in male mouse ETA muscle.

A, sample image of a synapse receiving multiple innervating axons (green arrows; stained with antibody against neurofilament). B, sample image of a swollen segment (cyan arrow) in the motor axon (stained with antibody against neurofilament). C, sample image of end-bulbs (yellow arrows) in a synapse (AChR stained with bungarotoxin (BTX) in red and motor axon stained with antibody against neurofilament in cyan). D, sample image with aggregates (red arrows; autofluorescence) and multiple islands of AChR (white asterisk; stained with bungarotoxin). E, sample image illustrating neurofilament stain within motor axons extending beyond the BTX staining (magenta arrow) and multiple islands of AChR (white asterisk; red BTX stain).

Table 1.

Quantification on additional morphological features at the ageing NMJs in mouse ETA muscle

	Percentage of multi-innervated synapses	Percentage of axons with swollen segments	Neurofilament end-bulb number	Percentage of synapses surrounded by aggregates	Number of acetylcholine receptor islands
Young adult	3.57 (18.67) *†‡	65.57 (47.91) ‡ (P < 0.001)	1.01 (1.29) ‡ (P < 0.001)	2.38 (15.34) *†‡	5.66 (3.04) ‡ (P = 0.0008)
Adult	16.34 (37.09) § (P = 0.04)	66.01 ± (47.52) ‡ (P < 0.001)	1.57 (1.69) ‡ (P = 0.002)	23.53 (42.56) § (P = 0.002) ‡ (p < 0.001)	5.15 (3.35) ‡ (P < 0.001)
Early Aged	21.74 (41.55) § (P = 0.01)	79.71 ± (40.51) ‡ (P = 0.04)	1.59 (1.58) ‡ (P = 0.01)	34.78 (47.98) § (P < 0.001)	5.52 (2.57) ‡ (P = 0.0008)
Later Aged	17.47 (38.09) § (P = 0.02)	94.58 (22.71) §*†	2.40 (2.36) §*†	50.00 (50.15) § (P < 0.001) *	7.43 (4.04) §*†

Changes in morphological features in each epoch. Values are means (SD).

Each symbol represents statistical significance against a specific epoch: § against Young Adult; n = 84 in synapse number from 6 animals in this epoch

^*against Adult; n = 154 in synapse number from 9 animals in this epoch

^†against Early Aged; n = 69 in synapse number from 4 animals in this epoch

^‡against Later Aged; n = 166 in synapse number from 9 animals in this epoch. One-way ANOVA with Tukey’s post hoc test was used for all the statistical tests.

Effect of ageing on short-term plasticity of transmitter release at male mouse NMJ synapses

Next, we evaluated the effect of ageing on short-term synaptic plasticity at ageing NMJs. We performed a high frequency motor axon train stimulation using 10 action potentials at 50 Hz to induce short-term synaptic plasticity (tetanic depression and/or potentiation). The evoked responses (EPPs) following each stimulus in the train were recorded and normalized to the amplitude of the EPP after the first stimulus. The average time course of EPP changes during this 50 Hz train stimulation for each ageing epoch is shown in Fig. 4A. We only detected a significant difference between the Young Adult epoch and the three older epochs, with no significant difference between Adult, Early Aged, or Later Aged epochs (Fig. 4A). By the 10th stimulus in the train, there were no significant differences at any ageing epoch. However, because we observed a large variability in QC across some epochs (Fig. 1B), and the probability of transmitter release at transmitter release sites is known to strongly influence short-term synaptic plasticity, we also explored the possibility that the magnitude of short-term plasticity might be correlated with quantal content. We performed a linear regression analysis for plots of quantal content and short-term plasticity magnitude after the 3rd and 10th action potential in the train to encompass both the early tetanic potentiation (3rd stimulus in the train) and the late tetanic depression (10th stimulus in the train) observed over the course of the tetanic stimulation (Fig. 4B). In both cases, there is a very weak correlation between QC and short-term plasticity. The Adult and Later Aged epochs did show a significant correlation between quantal content and response after 10th action potential in the train, however, the R² was relatively low in both cases, and the relationship was shallow between QC and short-term synaptic plasticity. These data indicate that there is not a steep relationship between QC and short-term plasticity across the ageing time course.

Figure 4. Age-induced changes on short-term plasticity in the NMJ of male mouse ETA muscle.

A, responses after 50 Hz train stimulation (200 ms) normalized to the response after the first stimulus. Short-term synaptic plasticity (age-induced changes at each stimulus number) in Young Adult synapses was significantly different (one-way ANOVA with Tukey’s post hoc test) from Adult and Later Aged on the 2nd stimulus (P = 0.05, P = 0.02, respectively), 3rd stimulus (P = 0.005, P = 0.005, respectively), 4th stimulus (P = 0.008, P = 0.004, respectively) and 5th stimulus (P = 0.03, P = 0.01, respectively); Young Adult epoch was significantly different (one-way ANOVA with Tukey’s post hoc test) from the Adult epoch on the 6th stimulus (P = 0.02). B, normalized responses after the 10th stimulus were plotted as a function of quantal content. There is a significant relationship between quantal content and normalized 10th response in Adult (P = 0.0001; R² = 0.16; linear regression) and Later Aged (P = 0.0002; R² = 0.11; linear regression) epochs.

Effect of acute exposure to GV-58 on weak synapses at male mouse NMJ

Lastly, we evaluated the efficacy of a novel candidate small molecule therapeutic on neurotransmitter release in aged NMJs (25- to 30-month-old animals, which contained synapses with significantly reduced neurotransmission). We detected a significant increase in quantal content in these aged synapses after 30 min of GV-58 exposure (Fig. 5A). On average, 50 μM GV-58 was able to elicit a 1.8-fold increase in release of neurotransmitter (on average, QC was 53.7 before, and 76.1 after GV-58 exposure) at NMJ synapses in the Later Aging epoch (Fig. 5B).

Figure 5. Effect of acute GV-58 on weakened NMJs of ETA muscle in mice aged 25–30 months.

A, sample traces of EPPs before (red; vehicle exposed) and after 30 min of 50 μM GV-58 exposure (blue; GV-58 exposed). B, change in quantal content before and after a 30 min GV-58 exposure (****P ≤ 0.0001; Student’s paired t test). On average, there was a 1.8-fold increase in quantal content. C, paired comparison of mEPP sizes before and after a 30 min GV-58 exposure (ns, P = 0.6841; paired t test; SD = 0.09, vehicle; 0.12, GV-58). D, paired comparison of EPP sizes before and after a 30 min GV-58 exposure (****P ≤ 0.0001; paired t test; SD = 8.45, vehicle; 13.47, GV-58).

Discussion

We have conducted a multi-time-point cross-sectional study, investigating the changes in morphology and neurotransmission at the mouse NMJ. We found the change in neurotransmitter release to be biphasic: first increasing during an Early Aged epoch, followed by a later decline during a Later Aged epoch. Within this general trend, we found synapses age independently within the same animal, with some synapses appearing very healthy in the Later Aged epoch, while others were significantly impaired. In contrast, short-term synaptic plasticity did not seem to be affected by age. Additionally, we characterized morphological changes to postsynaptic receptors and presynaptic neurofilament in motor axons at the NMJ over the ageing time course. Lastly, our novel small molecule therapeutic, GV-58, increased neurotransmitter release by 1.8-fold at weakened NMJ synapses present in later ageing. These findings provide insight into the physiological process that underlies ageing at the NMJ, and these age-induced changes can be observed at individual synapses. This information can provide guidance on selecting appropriate experimental time points that will be important for future studies.

Synapses age independently within the same animal

In this study, we demonstrated that with age, the variability between synapses in terms of transmitter release and morphology becomes very large. This highlights the conclusion that taking average values from many synapses in each animal can obscure independent and dynamic changes that occur in individual synapses over the ageing time course. For example, in the Early Aged epoch, which is usually considered the pre-symptomatic phase (Chugh et al., 2020), there is an overall increase in QC. Previous studies on effects of exercise in young and aged muscles concluded there was independent sensitivity to changes among different synapses and muscles (Deschenes et al., 2018; Deschenes et al., 2020). However, at this age there are synapses that show weak neurotransmission and have impaired morphology, while others have enhanced function and appear to have normal morphology. This pre-symptomatic stage might be an optimal time point to consider therapeutic intervention.

Biphasic changes in quantal content during ageing

The inconsistent results from different research groups studying the effect of ageing on NMJ neurotransmission (Banker et al., 1983; Deschenes, 2011; Fahim, 1997; Gutmann et al., 1971; Kelly, 1978; Mahoney et al., 2014; Pousinha et al., 2015) could be due to using different animal species (rats vs. mice); different sexes; choosing different muscle groups (involuntarily activated diaphragm vs. voluntarily activated somatic muscles, or slow-twitching vs. fast-twitching), or focusing on different ages of the animal. Our electrophysiological recordings from a voluntarily activated fast-twitch somatic muscle over the entire time course of ageing identified a biphasic change in quantal content: an increase from Young Adult to Early Aged, followed by a decrease from Early Aged to Later Aged. Separating these epochs can help better interpret ageing as a complex and dynamic process, rather than a purely degenerative process. This kind of detailed categorization has been reviewed recently (Deschenes et al., 2022). The early increase in neurotransmission at male NMJs in our study is consistent with some previous studies (Banker et al., 1983; Deschenes, 2011; Fahim, 1997; Kelly, 1978; Mahoney et al., 2014), while the later decrease is also consistent with other previous reports (Chugh et al., 2020; Pousinha et al., 2015). The significant decrease in mEPP amplitude from the Young Adult epoch to the Early and Later aged epochs suggests that the density of AChRs may be reduced in ageing. Furthermore, our morphological studies also reveal a significant decrease in postsynaptic AChR area with ageing. Taken together, we hypothesize that the increase in quantal content during early ageing could be the result of a presynaptic homeostatic compensation following a reduction in AChR number (Wang & Rich, 2018; Wang et al., 2004; Wang et al., 2010; Wang et al., 2018). We further hypothesize that this compensation may be lost, or unable to counter age-induced deficits in the Later Aged epoch, causing the subsequent decrease in transmitter release. Further study of homeostatic plasticity at the aged NMJ could explore these mechanisms. Lastly, our finding that there is a biphasic change in the magnitude of neurotransmission during ageing highlighted the fact that age-induced physiological changes can be detected relatively early in the ageing process. The time points of the occurrence of homeostatic changes might serve as critical time windows for prophylactic treatments. Therefore, this cross-sectional analysis may help guide future design of anti-ageing approaches in the neuromuscular system.

Morphological changes during ageing of the NMJ

In our morphological analysis, we first studied the change in postsynaptic endplate area, which we found decreased when comparing Young Adult, Adult and Early Aged epochs, and then returned to the Adult epoch level during Later Aged. The endplate area occupied by receptor could influence the postsynaptic response during the evoked and spontaneous transmitter release and trigger homeostatic plasticity (as described above). A significant decrease in end plate area over the ageing time course between Adult and Early Aged epochs has previously been shown in the tibialis anterior muscle in mice (Cheng et al., 2013). An increase in end plate area in Later Aged has also been reported in aged rat NMJ (Prakash & Sieck, 1998). However, the mechanisms underlying the early decrease followed by an increase in end plate area in ageing warrant additional investigation.

In addition, we documented a reduction in neurofilament staining within axons as ageing progressed (most prominent between Young Adult and Adult phases, but also decreasing in Later Aged). Curiously, many other unique morphological features, such as the presence of multiple innervation (consistent with previous reports; Valdez et al., 2010), swollen axons (consistent with previous reports; Valdez et al., 2010), neurofilament bulbs, and postsynaptic receptor islands (fragmentation; consistent with previous reports; Cheng et al., 2013; Slater, 2020; Willadt et al., 2016), were increased over the ageing time course. It is possible that these indicators represent either compensatory mechanisms associated with homeostatic plasticity at the NMJ, or degradation processes in the ageing NMJ. With regard to the staining for neurofilament over the ageing time course, segments of the motor axons often appear to be ‘swollen’ (Fig. 3B) and the percentage of axons with swollen segments increased significantly in the Later Aged epoch. Furthermore, neurofilament staining also revealed ‘end bulbs’ within the NMJ. These motor axon end-bulbs often had a hollow centre, suggesting a curled structure. We have quantified the number of end-bulbs in each synapse and found that there is a significant increase in end-bulb number in the Later Aged epoch; these end-bulbs have previously only been reported once in an early report regarding NMJ ageing (Rosenheimer & Smith, 1985). All of these features could be attributed to an increase in neurofilament accumulation with age. In fact, there have been previous studies linking increased serum level of neurofilaments to ageing and neurodegenerative disease (Bacioglu et al., 2016; Barro et al., 2018; Disanto et al., 2017; Gaiottino et al., 2013; Lu et al., 2015; Mattsson et al., 2017; Meeter et al., 2016; Novakova et al., 2017; Piehl et al., 2018; Reiber, 1994; Siller et al., 2019; Weydt et al., 2016; Wilke et al., 2016), and serum neurofilament levels have been shown to increase gradually in ageing (Kaeser et al., 2021; Khalil et al., 2020; Koini et al., 2021). This apparent over-production of neurofilament may either be a consequence of a compensatory mechanism or an age-induced accumulation that is associated with degradation.

One additional interesting observation was the increased presence of autofluorescent aggregates as ageing progressed. These aggregates resemble lipofuscin in appearance and properties (wide fluorescence emission range; Fahim & Robbins, 1982; Kun et al., 2018). It has previously been reported that the appearance of lipofuscin is associated with ageing (Terman & Brunk, 1998, 2004, 2005; Terman et al., 2010).

Short-term plasticity during ageing of the NMJ

Because the magnitude of transmitter released dynamically changed over ageing, we used a measurement of short-term synaptic plasticity to gain insight into underlying changes within transmitter release sites. Young adult mouse NMJs are known to contain about 700 transmitter release sites (or active zones) (Chen et al., 2012; Laghaei et al., 2018; Nishimune, 2012; Nishimune et al., 2004), and with action potential stimulation, the NMJ releases about 100–150 synaptic vesicles (Laghaei et al., 2018). Prior work in various disease states at the rodent NMJ has shown that tetanic potentiation at the NMJ can increase dramatically when diseased synapses are weakened by mechanisms that lead to a reduction in the probability of release at each active zone, especially when there is a reduction in presynaptic calcium entry (Atchison, 1988; Flink & Atchison, 2003; Tarr et al., 2014). We performed a tetanic stimulation using 10 stimuli at 50 Hz and found no significant differences when comparing adult synapses to early ageing and later ageing phases. The unaltered short-term plasticity across ageing suggests that the early increase and the later decrease in quantal content during ageing is not explained by changes in the probability of transmitter release within each active zone, as this would be predicted to alter short-term synaptic plasticity. Alternatively, it is possible that the age-induced changes in quantal content are underpinned by changes in the number of functional active zones that contribute to transmitter release, but that the properties of transmitter release at each active zone is unchanged during the process of ageing. A study of single active zone function in ageing is warranted and could explore these hypotheses.

Effect of acute exposure to GV-58 on aged NMJs

With 30 min of exposure to GV-58, we were able to elicit a 1.8-fold increase in quantal content in aged synapses. GV-58 is a calcium channel gating modifier that prolongs the open time of the CaV2 family of calcium channels that regulate transmitter release at neuromuscular active zones (Katz et al., 1996; Uchitel et al., 1992). Our data support the therapeutic potential of GV-58 as a neurotransmitter release enhancer which could strengthening weakened transmission, potentially restoring reduced neuromuscular activity in ageing. Previously, the effect of acute GV-58 exposure on two mouse neuromuscular disease models (Lambert–Eaton myasthenic syndrome and spinal muscular atrophy) was examined (Ojala et al., 2023; Tarr et al., 2013, 2014). In both cases, the effects of GV-58 were similar to what we reported here for aged NMJs (s1.8-fold increase in the magnitude of transmitter release). It is also true that GV-58 will increase the magnitude of transmitter release in healthy synapses to the same degree, but this in not relevant to nerve-evoked muscle contraction because all neuromuscular synapses are already above threshold after motor action potential activity; as such, increased transmitter release has no impact on nerve-evoked muscle contraction (Ojala et al., 2023).

Given the similarity in the mechanisms of calcium-triggered transmitter release between human and mouse NMJs (Boehm et al., 2020), we hypothesize that GV-58 exposure would enhance synaptic transmission at human NMJs, as well. Moreover, we believe that the benefit of GV-58 does not only reside in short-term application, but also in long-term benefits. Previous studies have shown that 2 h of resistance training exercise each day was able to delay or prevent age-induced changes to the mouse NMJ (Nishimune et al., 2014). This may be caused by a synaptic activity-induced trophic effect that could maintain the NMJ (Funakoshi et al., 1995; Gomez-Pinilla et al., 2002; Gyorkos & Spitsbergen, 2014; McCullough et al., 2011; McCullough et al., 2013; Wehrwein et al., 2002). We hypothesize that chronic administration of GV-58 could function as a pharmacological stimulant of synaptic activity that could enhance the function of the NMJ, potentially sustaining neuromuscular structure and activity during ageing. Chronic GV-58 treatment could be explored as a non-invasive and less physically demanding solution to dynapenia for the elderly. Additionally, given the beneficial effects of exercise in maintaining neuromuscular stability in the elderly population (for review see Badawi & Nishimune, 2020), GV-58 may act synergistically with exercise to strengthen aged NMJs and attenuate age-related NMJ dysfunction. Our data support future studies of the effects of chronic stimulation with a small molecule calcium channel agonist therapeutic such as GV-58 during the ageing process.

Supplementary Material

Supporting information

Additional supporting information can be found online in the Supporting Information section at the end of the HTML view of the article. Supporting information files available:

Key points.

• Age-induced frailty and falls are the leading causes of injury-related death and are caused by an age-induced loss of muscle strength due to a combination of neurological and muscular changes.

• A cross-sectional approach was used to study age-induced changes to the neuromuscular junction in a mouse model, and physiological changes that were biphasic over the ageing time course were found.

• Changes in physiology at the neuromuscular junction were correlated with alterations in neuromuscular junction morphology.

• An acutely applied positive allosteric gating modifier of presynaptic voltage-gated calcium channels was tested as a candidate therapeutic strategy that could increase transmitter release at aged neuromuscular junctions.

• These results provide a detailed time course of age-induced changes at the neuromuscular junction in a mouse model and test a candidate therapeutic strategy for weakness.

Biography

Yizhi (Nick) Li is currently a PhD candidate in the Centre for Neuroscience graduate program at the University of Pittsburgh. Prior to joining this PhD program, he received his Bachelor of Science degree from the Department of Neuroscience at the University of Pittsburgh and worked as a research associate studying synaptic plasticity. He adopted ageing at the neuromuscular junction as his research model to further his research on synaptic plasticity. His goal is to elucidate the role of neuromuscular junction in age-related muscle weakness.

Data availability statement

Data are available to qualified investigators by contacting the corresponding author.