Fig 2: The UCN2-CRHR2 axis promotes axonal growth of cultured SCMNs and NMJ regeneration after acute damage. (a) Representative images of neurofilament staining used as reporter for axon length in SCMNs cultured for 24 h either in normal culture medium (NC) or NC supplemented with UCN2, A2B or their combination. (b) Quantification of the average axonal length after the indicated treatments. Three independent experiments were performed, and at least 100 neurons per condition measured. Bars represent mean ± SD. Statical analysis was performed with ANOVA followed by post hoc Tukey test; *** p < 0.001, ** p < 0.01, ns p > 0.05. (c) Scheme illustrating the treatment protocol, consisting of daily i.m. injections of either vehicle or recombinant UCN2 for 72 h after α-LTx treatment, followed by functional and structural assessment of NMJ status. (d) Electrophysiological assessment of NMJ function by evoked EPPs recordings following either vehicle or UCN2 administration. Each bar represents mean ± SD. n = 4 animals, 11 fibres. * p < 0.05, ns p > 0.05. ANOVA followed by post hoc Tukey test. (e) Representative immunostaining and (f) quantification performed on soleus muscles after intoxication and treatment with either vehicle or UCN2. Regenerated NMJs are positive for both pre- and post-synaptic markers (VAMP-1 in green and nAChR in red, respectively). White asterisks indicate still degenerated NMJs (absent or faint VAMP1 signal). Right panels are magnifications of the indicated NMJs. Scale bars: 50 µm, 20 µm for magnification. Each bar in (f) represents the mean ± SD percentage of recovered NMJs. ** p < 0.01 by Student’s t-test.

Fig 3: The CRHR2 receptor participates in the structural and functional recovery of the NMJ. (a) Scheme illustrating the treatment protocol, consisting of daily i.m. injections of either vehicle or the CRHR2 antagonist A2B up to 96 h after α-LTx injection, and the analysis performed to assess the functional and structural status of the NMJ. (b) Electrophysiological recordings of evoked EPPs from intoxicated soleus muscles after either vehicle of A2B administration. Each bar represents mean ± SD. n = 4 animals, 11 fibres. ** p < 0.01, ns p > 0.05. ANOVA followed by post hoc Tukey test. (c) Representative immunostaining and (d) quantification performed on soleus muscles after intoxication with α-LTx and treatment with either vehicle or A2B. Regenerated NMJs display both pre- and post-synaptic markers (VAMP-1 in green and nAChR in red, respectively). White asterisks indicate still degenerated NMJs (lack or little VAMP-1 signal). The right panel shows a magnification. Scale bars: 50 µm. Bars in (d) represent the mean± SD percentage of regenerated NMJs. ** p < 0.01 by Student’s t-test.