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. 2014 Aug 5;9(8):e104081.
doi: 10.1371/journal.pone.0104081. eCollection 2014.

Adenosine A2A receptors activation facilitates neuromuscular transmission in the pre-symptomatic phase of the SOD1(G93A) ALS mice, but not in the symptomatic phase

Filipe Nascimento  1 , Paula A Pousinha  1 , Alexandra M Correia  2 , Rui Gomes  3 , Ana M Sebastião  1 , Joaquim A Ribeiro  1
Affiliations

Adenosine A2A receptors activation facilitates neuromuscular transmission in the pre-symptomatic phase of the SOD1(G93A) ALS mice, but not in the symptomatic phase

Filipe Nascimento et al. PLoS One. .

Abstract

Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease leading to motor neuron dysfunction resulting in impairment of neuromuscular transmission. A2A adenosine receptors have already been considered as a potential therapeutical target for ALS but their neuromodulatory role at the neuromuscular junction in ALS remains to be clarified. In the present work, we evaluated the effects of A2A receptors on neuromuscular transmission of an animal model of ALS: SOD1(G93A) mice either in the pre-symptomatic (4-6 weeks old) or in the symptomatic (12-14 weeks old) stage. Electrophysiological experiments were performed obtaining intracellular recordings in Mg2+ paralyzed phrenic nerve-hemidiaphragm preparations. Endplate potentials (EPPs), quantal content (q. c.) of EPPs, miniature endplate potentials (MEPPs) and giant miniature endplate potential (GMEPPs) were recorded. In the pre-symptomatic phase of the disease (4-6 weeks old mice), the selective A2A receptor agonist, CGS 21680, significantly enhanced (p<0.05 Unpaired t-test) the mean amplitude and q.c. of EPPs, and the frequency of MEPPs and GMEPPs at SOD1(G93A) neuromuscular junctions, the effect being of higher magnitude (p<0.05, Unpaired t-test) than age-matched control littermates. On the contrary, in symptomatic mice (12-14 weeks old), CGS 21680 was devoid of effect on both the amplitude and q.c. of EPPs and the frequency of MEPPs and GMEPPs (p<0.05 Paired t-test). The results herein reported clearly document that at the neuromuscular junction of SOD1(G93A) mice there is an exacerbation of A2A receptor-mediated excitatory effects at the pre-symptomatic phase, whereas in the symptomatic phase A2A receptor activation is absent. The results thus suggest that A2A receptors function changes with ALS progression.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. CGS 21680 facilitation of evoked activity is exacerbated in pre-symptomatic mice; (A) representative time-course change of mean EPP amplitude throughout CGS 21680 (5 nM) perfusion and (B) representation of EPP amplitude increase in 4–6 weeks old WT (n = 5) and pre-symptomatic mice (n = 10) upon A2A receptor activation (CGS 21680 at 5 nM); (C) concentration-response changes in mean EPP amplitude in the presence of CGS 21680 (3 nM: n = 7, WT, n = 7, SOD1G93A; 5 nM: n = 14, WT, n = 13, SOD1G93A; 10 nM: n = 7, WT, n = 5, SOD1G93A) whose effect was blocked by SCH 58261 at 50 nM (n = 5, WT, n = 4, SOD1G93A); (D) raw recording of spontaneous release fluctuations from a 4–6 weeks old WT and pre-symptomatic SOD1G93A neuromuscular junction promoted by CGS 21680 (5 nM); effect of CGS 21680 (5 nM) perfusion regarding (E) MEPP frequency (n = 10, WT, n = 9, SOD1(G93A), (F) quantal Content of EPPs (n = 13, WT, n = 12, SOD1(G93A)) and (G) GMEPP frequency (n = 10, WT, n = 11, SOD1(G93A)) in pre-symptomatic SOD1(G93A) mice and respective healthy controls; *p<0.05 Unpaired t-test; ∂p<0.05 one-way ANOVA with Tukey’s pos-hoc; #p<0.05 Paired t-test (as compared with control value before drug perfusion); control corresponds to 100% in all cases.
Figure 2
Figure 2. A2A receptor modulation is lost in symptomatic SOD1(G93A) mice endplates; (A) representative average time-course of mean EPP amplitude change during CGS 21680 (5 nM) bathing and (B) illustrative mean EPP profile facilitation in 12–14 weeks old control (n = 6) and symptomatic mice (n = 6); (C) dose-response alterations in mean EPP amplitude by CGS 21680 (3 nM: n = 8, WT, n = 10, SOD1G93A; 5 nM: n = 10, WT, n = 7, SOD1G93A; 10 nM: n = 11, WT, n = 7, SOD1G93A) were blocked by SCH 58261 at 50 nM in WT mice (n = 4, WT, n = 4, SOD1G93A); (D) SCH 58261 (50 nM) did not affect evoked activity throughout data acquisition (n = 4, WT, n = 7, SOD1G93A); (E) raw recording of spontaneous release variations from a 12–14 weeks old WT and symptomatic SOD1G93A endplate upon CGS 21680 (5 nM) perfusion; effect of A2A receptor activation by CGS 21680 (5 nM) on (F) MEPP frequency (n = 6, WT, n = 5, SOD1(G93A)) (G) GMEPP frequency (n = 4, WT, n = 4, SOD1(G93A)) and (H) quantal content of EPPs (n = 9, WT, n = 7, SOD1(G93A)); *p<0.05 Unpaired t-test; ∂p<0.05 one-way ANOVA with Tukey’s pos-hoc; #p<0.05 Paired t-test (as compared with control value before drug perfusion); control corresponds to 100% in all cases.
Figure 3
Figure 3. Comparison of A2A receptor function upon disease progression in SOD1(G93A) mice and healthy controls; average time course of mean EPP amplitude facilitation by CGS 21680 (5 nM) in (A) pre-(n = 10) and symptomatic (n = 6) SOD1(G93A) rodents and (B) 4–6 weeks (n = 5) and 12–14 weeks (n = 6) old WT mice; effect of CGS 21680 perfusion at 5 nM on: (C) quantal content of EPPs (4–6 weeks old: n = 13, WT, n = 12, SOD1(G93A); 12–14 weeks old: n = 9, WT, n = 7, SOD1(G93A)); (D) MEPP frequency (4–6 weeks old: n = 10, WT, n = 9, SOD1(G93A); 12–14 weeks old: n = 6, WT, n = 5, SOD1(G93A)); and (E) GMEPP frequency (4–6 weeks old: n = 10, WT, n = 11, SOD1(G93A); 12–14 weeks old: n = 4, WT, n = 4, SOD1(G93A)) in both phases of the study from SOD1(G93A) mice and respective healthy controls; *p<0.05 Unpaired t-test; ∂p<0.05 one-way ANOVA with Tukey’s pos-hoc; #p<0.05 Paired t-test (as compared with control value before drug perfusion); control corresponds to 100% in all cases.
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This work was supported by Fundação do Ministério da Ciência e Tecnologia de Portugal [Grant PTDC/SAU-FAR/118787/2010] (http://www.fct.pt/index.phtml.pt). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.