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Neurotrophin 4/5 is required for the normal development of the slow muscle fiber phenotype in the rat soleus

Dario I. Carrasco^* and Arthur W. English

Department of Cell Biology, Emory University School of Medicine, Atlanta, GA 30322, USA

View larger version (156K): [in a new window]   Fig. 1. Sampling scheme used to acquire the fields for data analysis (see Materials and methods). Figure shows a cryosection incubated in antibody BA-D5 from a neurotrophin 4/5 (NT-4/5)-treated soleus muscle of a 4-week-old pup.

View larger version (108K): [in a new window]   Fig. 2. (A) Percentage of BA-D5-immunopositive fibers (slow) and unstained fibers (fast) is shown for 2-, 4- and 6-week-old neurotrophin 4/5 (NT-4/5)-treated soleus and untreated contralateral soleus muscle. Values represent means ± S.E.M. *=P<0.05 vs untreated. (B–E) Representative images taken from histological sections of rat soleus muscle obtained at 4 weeks and 6 weeks of age: untreated contralateral soleus (B,D); NT-4/5-treated soleus (C,E). Each section was stained with monoclonal antibody BA-D5, which is specific to the slow or I/b myosin heavy chain (MyHC) isoform. Immunopositive fibers were visualized with peroxidase-conjugated goat anti-mouse secondary antibody. Scale bar, 50 µm.

View larger version (142K): [in a new window]   Fig. 3. Representative images taken from histological sections of soleus muscle obtained at 4 weeks of age from rats injected with (A,B) saline, (C,D) neurotrophin 4/5 (NT-4/5) and (E,F) TrkB–IgG. Each section was double-stained with monoclonal antibodies A4.840 (I/b) and MY-32 (fast and neonatal). The A4.840-immunopositive fibers were visualized with a fluorescein isothiocyanate (FITC)-conjugated secondary antibody (A,C,E), and the MY-32-immunopositive fibers were visualized with a Texas Red®-conjugated streptavidin secondary antibody (B,D,F). Asterisks mark double-stained fibers. Scale bar, 50 µm.

View larger version (21K): [in a new window]   Fig. 4. Neurotrophin 4/5 (NT-4/5) accelerates the fast-to-slow myosin heavy chain (MyHC) isoform switch in neonatal rat soleus and its signaling is required for the normal development of the soleus muscle fiber phenotype. The percentage of fibers expressing fast MyHC only (fast), both fast and slow MyHC simultaneously (hybrid) and slow MyHC only (slow) are shown for 4-week-old (A,C) and 6-week-old (B,D) rat soleus muscles treated with saline, NT-4/5 or TrkB–IgG. Values represent means ± S.E.M. *=P<0.05 vs saline.

View larger version (15K): [in a new window]   Fig. 5. Brain-derived neurotrophic factor (BDNF) does not affect the fast-to-slow myosin isoform switch in neonatal rat soleus. The percentage of fast, hybrid and slow fibers is shown for 4-week-old rat soleus muscles treated with saline (open bars) and BDNF (filled bars). Values represent means ± S.E.M. Differences are not statistically significant at P<0.05.

View larger version (37K): [in a new window]   Fig. 6. Neurotrophin 4/5 (NT-4/5) mRNA in rat soleus becomes upregulated earlier during postnatal development than do the myosin heavy chain (MyHC) I/b (slow) and MyHC IIA (fast) mRNAs. PCR products of reverse-transcribed RNA extracted from soleus muscles (four at each age) using primer pairs for the 18S rRNA and competimers in conjunction with primer pairs for either (A) MyHC I/b, (B) MyHC IIA or (C) NT-4/5 at postnatal age 4 days (P4), 7 days (P7), 14 days (P14), 21 days (P21) and 28 days (P28). Arrows show the postnatal ages at which increases in MyHC I/b, MyHC IIA and NT-4/5 mRNA synthesis occur. Note that a different set of primer pairs for 18S rRNA and competimers (product size 489 bp) was used to visualize the MyHC IIA product (310 bp). nRT = no reverse transcription. (D) The relative levels of MyHC I/b, MyHC IIA and NT-4/5 mRNAs. Values represent means ± S.E.M. *=P<0.05 vs day 4; =P<0.5 vs day 7.

View larger version (17K): [in a new window]   Fig. 7. Neurotrophin 4/5 (NT-4/5) requires normal neuromuscular synaptic transmission to exert its effect on the neonatal rat soleus. The percentage of fast, hybrid and slow fibers is shown for 4-week-old rat soleus treated with saline, botulinum toxin (BTX) alone and BTX+NT-4/5. Values represent means ± S.E.M. *=P<0.05 vs BTX and BTX+NT-4/5.

View larger version (18K): [in a new window]   Fig. 8. Hypothetical models of retrograde regulation of soleus (SOL) motoneuron properties by neurotrophin 4/5 (NT-4/5). (A) Following injection in the muscle, NT-4/5 forms or activates a retrograde signal that is transported by primary afferent (Ia) neurons from muscle spindles. This signal influences cellular mechanisms involved in the determination of the properties of the synapses of these afferent neurons onto SOL motoneurons and, via this mechanism, accelerates the acquisition of the repetitive pattern of discharge of SOL motoneurons and, consequently, the fast-to-slow myosin heavy chain (MyHC) transformation in the SOL muscle. (B) Alternatively, following injection in the muscle, NT-4/5 forms or activates a retrograde signal that is transported by SOL motoneurons. This signal influences cellular mechanisms involved in the maturation of intrinsic SOL motoneuron properties, i.e. rehobase, afterhyperpolarization (AHP) or input resistance (IR), and, via this mechanism, accelerates the acquisition of the repetitive pattern of discharge of SOL motoneurons and, consequently, the fast-to-slow MyHC transformation in this muscle. = increase; = decrease.