Ethics statement

This study was carried out in accordance with the European Community guidelines (Directives 86/609/EU and 2010/63/EU, Recommendation 2007/526/CE, European Convention for the Protection of Vertebrate Animals used for Experimental or Other Scientific Purposes ETS 123/Appendix A) and Portuguese Laws on Animal Care (Decreto-Lei 129/92, Portaria 1005/92, Portaria 466/95, Decreto-Lei 197/96, Portaria 1131/97). The protocols used in this study were approved by the Portuguese National Authority (General Direction of Veterinary) and by the Ethics Committee of the Institute of Molecular Medicine.

Animals

Transgenic males B6SJL-TgN(SOD1-G93A) 1Gur/J (Jackson Laboratory, No. 002726), carrying the human SOD1 gene with the G93A point mutation [10] and wild-type B6SJLF1/J females were purchased from Jackson Laboratory (USA) and a colony was established at the rodent facility of the Institute of Molecular Medicine. At time of weaning, littermates were identified through ear punching and separated in different cages according to their gender. The ear tissue extracted during the tagging procedure was then used to run a polymerase chain reaction (PCR) and genotype the progeny [11]. All animals were housed 4-5 animals/cage, under 12h light/12h dark cycle, and received food and water ad libitum.

Experiments were conducted in two age groups: 4-6 and 12-15 weeks-old animals, corresponding to the pre-symptomatic and symptomatic phases of the disease, respectively. Both males and females were used, being the proportion between them similar in all groups (Tables 1-4). Transgenic mice, hereafter referred to as SOD1(G93A) mice, were compared to non-transgenic mice, used as age-matched controls (WT).

Phase of the disease assessment

To establish the age groups correspondent to the pre-symptomatic and symptomatic stage of the disease, mice were tested on a Rotarod. A training period comprising two stages was performed in order to minimize environmental variability. First animals were habituated to handling - initiated four days before the testing day. Then animals were habituated to the Rotarod apparatus and were taught the task itself. For that, mice were placed on the rod at the lowest rotation speed (4 rpm) where they had to maintain for at least 120s, as described in [12]. In the testing day, mice were sequentially assessed at 5 and 10 rpm, for a maximum of 300s each speed (as similarly done by [13]). The latency-to-fall (LF), which corresponds to the time that mice spent on the rotating rod, was recorded. 3 trials were performed per speed with 5 min rest between each trial.

Electrophysiological recordings

Mice were sacrificed by decapitation under halothane anaesthesia. Hemi-diaphragm muscle with the attached phrenic-nerve was isolated and mounted on a 3ml perspex chamber. Tissue was perfused continuously with a gassed physiological saline solution (containing (mM): NaCl 117; KCl 5; NaHCO₃ 25; NaH₂PO₄ 1,2; glucose 11; CaCl₂ 2,5; MgCl₂ 1,2; 95% O₂ and 5% CO₂) kept at room temperature (22-25^°C). Perfusion was maintained at a rate of 3ml/min via a roller pump, and bath volume was kept constant by suction.

Intracellular recordings were performed in the conventional way [14–16]. Glass microelectrodes (10-20 MΩ) filled with 3M KCl were inserted within muscle fibers at the motor endplate and the reference electrode was an Ag–AgCl pellet placed in the bath. Muscle contraction was prevented by treating preparations with 2µM μ-conotoxin GIIIB [17] for 45 to 60min, as done previously by [18]. The toxin selectively blocks voltage dependent sodium channels in the muscle fiber thereby preventing muscle action potential generation. In this experimental condition, neuromuscular transmission occurs at normal quantal content without interference of nicotinic acetylcholine receptors. The phrenic-nerve was stimulated supramaximally at 0.5 Hz with a constant current of 20 µs duration. The evoked activity (EPPs) at each neuromuscular junction was measured during intervals of 10 minutes. MEPPs were acquired in gap free mode during 100s. The resting membrane potential of the muscle fiber was monitored through the entire experiment. Signals were amplified, digitalized and stored on a computer with the Clampex program (pClamp10 Axon Instruments, Foster City, USA), for offline analysis of data.

Neuromuscular junctions considered for analysis had a stable resting membrane potential ranging from -60mV to -80mV. EPP amplitudes were normalized to a membrane potential of -75 mV using the following equation V_NOR = [V_OBS x (-75)]/RMP, where V_NOR indicates the corrected amplitude, V_OBS the recorded amplitude and RMP the recorded resting membrane potential [19–21]. The mean amplitude of EPPs was calculated for each neuromuscular junction by averaging normalized EPP amplitudes of 60 consecutive sweeps. The quantal content of EPPs, was estimated through the ratio between the normalized average of EPPs amplitude and the normalized average of MEPPs amplitude, both acquired during the same period. The threshold for MEPPs detection was set at levels between 0.2 mV and 1mV of amplitude. This maximum amplitude was established in order to exclude giant MEPPs (GMEPPs). The mean amplitude of MEPPs was calculated by averaging the amplitude of 100 consecutive MEPPs. The same procedure was done to assess the mean rise-time of MEPPs and mean decay-time of MEPPs. The mean frequency of MEPPs was measured by counting the number of events acquired during 100s. The distribution of MEPPs amplitudes was approximated to a Gaussian function or a Sum of two Gaussian function and the goodness of fit was evaluated by the square correlation coefficient R². The minimum amplitude for GMEPPs detection was set at 1mV. The mean frequency of GMEPPs was obtained by counting the number of these events acquired during the 100s. To evaluate the probability of GMEPPs occurrence between groups, the mean ratio between frequency of GMEPPs and frequency of MEPPs was calculated for each fiber exhibiting GMEPPs.

Statistical analysis

All data are presented as mean ± standard error mean (SEM) in each group, with n corresponding to the number of animals used or the number of neuromuscular junctions sampled, as indicated in each figure or table.

Statistical significance of differences between means was determined through GraphPad software. The parametric Student’s t test was used whenever both groups presented a normal distribution and equality of variances. Normality was tested through Shapiro-Wilk test (S–W), which is more appropriate for small samples (n<30), and homogeneity of variances tested through F test. Alternatively, the non-parametric Mann–Whitney U-test (M-W) was applied when one of the groups was non-normally distributed and Student’s t test with Welsh correction when the equality of variances condition was not met (F test p<0.05).

Motor phenotype of pre-symptomatic and symptomatic SOD1(G93A)

As shown in Table 1 the mean latency-to-fall of 4-6 weeks old SOD1(G93A) mice did not differ from WT mice neither at 5 rpm (262 ± 14.0s, n=11, and 242±13.4, n=16, respectively, p>0.05, Mann Whitney test), nor at 10 rpm (236±21.8s, n=11, and 257±11.8s, n=16, respectively, p>0.05, Mann Whitney test). Thus, we established the age of 4-6 weeks old as the pre-symptomatic SOD1(G93A) group. In contrast, the performance of 12-15 weeks old SOD1(G93A) mice on the Rotarod task was significantly decreased when compared to WT mice. The mean latency-to-fall, not different between groups at 5 rpm (267±12.3s, n=10, in WT and 246±23.6s n=10, in SOD1(G93A) groups, p>0.05, Mann Whitney test), became significantly reduced by 31% at 10 rpm (263±13.9s, n=10, in WT and 181±31.3s, n=10, in SOD1(G93A) groups, p<0.05, Mann Whitney test). We therefore established this group as the symptomatic SOD1(G93A) mice.

Pre-symptomatic phase of ALS

The evoked activity of pre-symptomatic SOD1(G93A) mice is presented in Figure 1.A and further quantified in table 2. Pre-symptomatic SOD1(G93A) mice presented a mean amplitude of EPPs significantly higher than WT mice (16.6±1.48mV, n=40, in WT group and 24.3±1.61mV, n=40, in SOD1(G93A) group, p<0.05, Mann Whitney test). This difference was maintained after the normalization of muscle fiber resting membrane potential to -75mV (18.3±1.64mV, n=40, in WT and 26.8±1.54mV, n=40, in SOD1(G93A) mice, p<0.05, Mann Whitney test). Similarly, the mean quantal content of SOD1(G93A) mice was significantly enhanced by approximately 31% in relation to WT mice (29.0±1.89, n=40, in WT and 38.1±2.42, n=40, in SOD1(G93A) mice, p<0.05, Mann Whitney test). The mean resting membrane potential did not differ between WT and SOD1(G93A) mice (p>0.05, Mann Whitney test) (Table 2).

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Figure 1. The neuromuscular transmission of the SOD1(G93A) mice in the pre-symptomatic phase of the disease.

A) Illustrates raw recordings of 5 EPPs from 4–6 week-old WT and pre-symptomatic SOD1(G93A) mice. B) Shows the frequency histogram of MEPPs amplitudes, which pools the amplitude of all MEPPs recorded at WT (2785 MEPPs) and SOD1(G93A) (2270 MEPPs) fibers. WT – Black bars; SOD1(G93A) – Gray bars. C) Presents the nonlinear regression applied to WT and SOD1(G93A) distributions. As illustrated, both distributions were best fitted with a Gaussian function. In D) are shown examples of a MEPP (<1mV) and a GMEPP (>1mV) and in E) examples of spontaneous events recorded in gap free mode across 10s in WT and SOD1(G93A) neuromuscular junctions.

https://doi.org/10.1371/journal.pone.0073846.g001

As also shown in table 2 and Figure 1, the mean amplitude of MEPPs was significantly higher in SOD1(G93A) mice by approximately 15% when compared to WT mice (0.55±0.02mV, n=40, in WT and 0.63±0.02mV, n=40, in SOD1(G93A) mice, p<0.05, Mann Whitney test). This difference is more outstanding when looking at the distribution of MEPPs amplitudes (Figure 1.B) and to the Gaussian functions that shape the scatter of data (being the values of R² equal to 0.93 and 0.88 for WT and SOD1(G93A) curves respectively) (Figure 1.C). The distribution of data from SOD1(G93A) presents a pronounced skewing to the right, highlighting the shift to larger amplitudes of MEPPs in this group. The frequency of MEPPs in pre-symptomatic SOD1(G93A) mice (0.59±0.06s^-1, n=40) did not differ from WT mice (0.64±0.07s^-1, n=40) (p>0.05, Mann Whitney test). As illustrated in Figure 1.D,E and Table 2, the mean frequency of GMEPPs was significantly increased in SOD1(G93A) mice (0.44±0.08 s^-1, n=33) in relation to the WT mice (0.23±0.06 s^-1, n=30) (p<0.05, Mann Whitney test). Moreover, the mean ratio between the frequency of GMEPPs and the frequency of MEPPs (calculated for each fiber exhibiting GMEPPs) was increased in SOD1(G93A) group (2.27±1.35, n=30) by 82% when compared with WT group (1.86±0.45, n=33) (p>0.05, Mann Whitney test) indicating that the occurrence of a GMEEPs is proportionally higher than a MEPP.

As shown in table 2, the mean rise-time of MEPPs in SOD1(G93A) mice was significantly decreased (1.32±0.07ms, n=40, in WT and 1.11±0.08ms, n=40, in SOD1(G93A) mice, p<0.05, Mann Whitney test) in relation to WT mice. No differences on the mean decay-time of MEPPs between SOD1(G93A) (3.67±0.14ms, n=40) and WT (4.00±0.15ms, n=40) (p>0.05, Mann Whitney test) mice were found.

Symptomatic phase of ALS

The evoked response of symptomatic SOD1(G93A) mice is illustrated in Figure 2.A and quantifications shown in detail in Table 3. The mean amplitude of EPPs (normalized to a resting membrane potential of -75mV and non-normalized) and the mean quantal content of EPPs in SOD1(G93A) did not significantly differ from WT (p>0.05, Mann Whitney test). The mean resting membrane potential was also maintained between groups (p>0.05, Student t test).

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Figure 2. The neuromuscular transmission of the SOD1(G93A) mice in the symptomatic phase of the disease.

A) Illustrates raw recordings of 5 EPPs from adult WT mice and the two groups of symptomatic SOD1(G93A) neuromuscular junctions, SOD1a and SOD1b. B) Shows the frequency histogram of MEPPs amplitudes, that pools the amplitude of all MEPPs recorded at WT (2288 events) and SOD1(G93A) (3674 events) fibers. WT – Black bars; SOD1(G93A) – Gray bars. C) Shows the nonlinear regressions that best shape WT (Gaussian function) and SOD1(G93A) (sum of two Gaussians) data As illustrated, SOD1(G93A) data follows a bimodal distribution with two peak amplitudes, pointing to the existence of 2 groups of neuromuscular junctions. D) After categorizing the two groups, the distributions of MEPPs amplitudes from SOD1a and SOD1b groups were drawn and best-fitted with Gaussian functions. As it is visible, the peak amplitude from both distributions matches the two peak amplitudes from the bimodal curve, validating the grouping. E) Shows examples of spontaneous events recorded in gap free mode across 10s in WT, SOD1a and SOD1b neuromuscular junctions.

https://doi.org/10.1371/journal.pone.0073846.g002

As shown in table 3 and illustrated in Figure 2.B, the mean amplitude of MEPPs did not vary between WT and SOD1(G93A) mice (0.50±0.03mV, n=30, and 0.49±0.03mV, n=39, respectively, p>0.05, Mann Whitney test) indicating no apparent changes in the post-synaptic cell. We noticed, however, that the distribution of SOD1(G93A) MEPPs amplitudes did not follow a Gaussian distribution. Instead, a sum of two Gaussian functions best fitted the distribution of SOD1(G93A) data, as shown in Figure 2.C (first peak at 0.31±0.01 mV and second peak at 0.63±0.01mV, R²=0.996), raising the possibility of two populations of neuromuscular junctions. To investigate this, we established the mean amplitude of SOD1(G93A) MEPPs (0.49mV) as a limit to categorize neuromuscular junctions into two groups – group A with a mean amplitude of MEPPs lower than 0.49mV and group B equal or higher than 0.49 – which will be referred to as SOD1a and SOD1b, respectively, in the following text As illustrated in Figure 2.D, the distributions of MEPPs amplitudes from both groups of neuromuscular junctions were best fitted with Gaussian functions. The peak amplitudes of each group (Group SOD1a peak amplitude at 0.30±0.01 mV, R²=0.90, and Group SOD1b peak amplitude at 0.65±0.01mV, R²=0.97) matched the two peak amplitudes from the bimodal curve, an indicator of proper grouping.

Regarding the frequency of spontaneous events, the mean frequency of MEPPs did not differ between WT and symptomatic SOD1(G93A) mice (p>0.05, Mann Whitney test) (Table 3, Figure 2.E). The mean frequency of GMEPPs was reduced by approximately 27% in SOD1(G93A) mice (0.32±0.10s^-1, n=20) as compared to WT mice (0.44±0.12s^-1, n=16) (p>0.05, Mann Whitney test). Accordingly, the proportion of GMEPPs (ratio between the frequency of GMEPPs and the frequency of MEPPs) was decreased by 23% in SOD1(G93A) group (1.21±0.68, n=20) when compared to WT mice (1.58±0.49, n=16) (p>0.05, Mann Whitney test).

Once the two groups of symptomatic SOD1(G93A) neuromuscular junctions were established, evoked and spontaneous activity were re-analyzed in order to delineate the profile of each group, as shown in table 4. The SOD1a group presented a mean amplitude of EPP significantly reduced (21.0±2.45mV, n=30, in WT and 11.4±2.00mV, n=19, in SOD1a groups, p<0.05, Mann Whitney test). Also, the quantal content of EPPs was slightly decreased in relation to WT (41.3±3.79, n=30, in WT and 32.7±4.40, n=19, in SOD1a, p>0.05, Mann Whitney test), although not statistically significant. The mean frequency of MEPPs was approximately the same in both SOD1a and WT groups (0.61±0.11s^-1, n=19, and 0.76±0.11s^-1, n=30, respectively, p>0.05, Mann Whitney test). However, GMEPPs were more rare in SOD1a (3 out of 19 neuromuscular junctions, 16%) than in WT (16 out of 30 neuromuscular junctions, 53%). In addition, SOD1a exhibited a mean frequency of GMEPPs significantly reduced when compared to WT (0.44±0.12s^-1, n=16 in WT and 0.07±0.03 s^-1, n=3, in SOD1a groups, p<0.05, Student t test with Welsh correction) as well as a lower mean ratio between the frequency of GMEPPs and the frequency of MEPPs (1.58±0.49, n=16, in WT and 0.11±0.07, n=3, in SOD1a groups, p<0.05, Student t test with Welsh correction). SOD1a presented a lower mean amplitude of MEPPs (0.50±0.03mV, n=30 in WT and 0.32±0.02, n=19, in SOD1a groups, p<0.05, Mann Whitney test), a higher mean rise-time of MEPPs (0.98±0.07ms, n=30, in WT and 1.39±0.12ms, n=19, in SOD1a groups, p<0.05, Mann Whitney test), and no differences in the mean decay-time of MEPPs (3.12±0.14ms, n=30, in WT and 3.12±0.17ms, n=19, in SOD1a groups, p>0.05, Mann Whitney test). In contrast, most of the parameters investigated in SOD1b group did not show significant differences (p>0.05, Mann Whitney test or Student t test, depending on the normality of the parameter analyzed; Table 4) when compared to the WT group, the exception being the mean amplitude of MEPPs, which was significantly increased as compared with the WT group (0.50±0.03mV, n=30 in WT and 0.64±0.02, n=20, in SOD1b groups, p<0.05, Mann Whitney test).

Comparison between phases

To investigate how the neuromuscular transmission evolved from pre-symptomatic to symptomatic SOD1(G93A) mice we further compared both phases of the disease (Table 5) taking into account SOD1a and SOD1b neuromuscular junctions. As shown, SOD1a neuromuscular junctions revealed a marked reduction in the mean amplitude of EPPs (24.3±1.61mV, n=40, in pre-symptomatic SOD1(G93A) and 11.4±2.00mV, n=19, in SOD1a, p<0.05, Mann Whitney test) and no significant variation of the mean quantal content of EPPs (p>0.05, Mann Whitney test), when compared to pre-symptomatic SOD1(G93A) mice.

Regarding spontaneous activity, the SOD1a group presented a decrease in the mean amplitude of MEPPs (0.63±0.02mV, n=40, in pre-symptomatic SOD1(G93A) and 0.32±0.02, n=19, in SOD1a, p<0.05, Mann Whitney test) as well as increased rise-time of MEPPs (1.11±0.08ms, n=40, in WT and 1.39±0.12, n=19, in SOD1a, p<0.05, Mann Whitney test) and lower decay-time of MEPPs (3.67±0.14ms, n=40, in WT and 3.12±0.17ms, n=19, in SOD1a, p<0.05, Student t test). The mean frequency of MEPPs remained unchanged but both mean frequency of GMEPPs and mean ratio (between the frequency of GMEPPs and frequency of MEPPs) were significantly reduced (p<0.05, Student t test with Welsh correction). Interestingly, in any of the quantified parameters did the SOD1b neuromuscular junctions differ in a statistically significant way (p<0.05, Mann Whitney test or Student t test depending on the normality of the parameter analyzed, Table 5) from the pre-symptomatic SOD1(G93A).

Functional differences between symptomatic SOD1(G93A) males and females have been reported [8]. However, the two identified populations of neuromuscular junctions (SOD1a and SOD1b) cannot be attributed to gender differences since SOD1a and SOD1b neuromuscular junctions were observed in either males (11 SOD1a and 10 SOD1b) or females (8 SOD1a and 10 SOD1b) and the proportion of females and males in SOD1a and SOD1b groups was similar (Tables 4-5). Furthermore, both SOD1a and SOD1b endplates were identified in the same hemi-diaphragm. Indeed, 7 of the 11 symptomatic SOD1(G93A) mice presented both groups SOD1a and SOD1b neuromuscular junctions, being these 3 females and 4 males. The age ranges in both SOD1a and SOD1b groups were also fully overlapping (Tables 4 and 5).

The negative trend seen in SOD1(G93A) between different time points contrasts with the positive trend from WT across age. As shown in Table 6, adult WT showed an increased mean quantal content (29.0±1.89, n=40, in young WT and 41.3±3.79, n=30, in adult WT, p<0.05, Mann-Whitney test), in accordance with what has been previously described [22] Regarding spontaneous activity, the mean amplitude of MEPPs was maintained between ages while the mean rise-time (1.32±0.07, n=40, in young WT and 0.98±0.07, n=30, in adult WT, p<0.05, Mann-Whitney test) and mean decay-time (4.00±0.15, n=40, in young WT and 3.12±0.14, n=30, in adult WT, p<0.05, Mann-Whitney test) of MEPPs appeared significantly reduced in adult WT (Table 6), compatible with age-related post-synaptic membrane maturation [23]. These changes highlight the need for age-matched controls use whenever comparing alterations in symptomatic and pre-symptomatic ALS mice models.