Muscle spindle Ia afferent nerves respond to mechanical stimuli by firing actions potentials with a frequency and pattern that is a complex function of stimulus waveform and intensity. This process occurs as a result of mechanical stimuli being transmitted by intrafusal muscle fibers to Ia afferent terminals, which transduce deformation into a depolarization called the receptor potential. The myelinated Ia axon divides into a number of branches as it approaches the intrafusal muscle fibers. Receptor potentials spread into this branched axonal network and at some point or points axon potentials are initiated. Where this occurs is not known but such sites are presumed to be rich with voltage-gated sodium channels. In order to localize the encoding sites, we are studying the distribution of Tetrodotoxin sensitive voltage-gated sodium channels (V-NaCh) immunocytochemically in cat muscle spindles. We will subsequently record the sites of impulse initiation electrophysiologically.


Small muscle bundles containing both extrafusal muscle fibers, including neuromuscular junctions and muscle spindles were isolated from tail muscle and lightly fixed in 0.5% paraformaldehyde solution for 12 hours. Standard immunocytochemical techniques were used. V-NaCh antibody AP1380-10.1 was provided by Dr. Rock Levinson (University of Colorado Health Sciences Center, Denver). For comparison purposes tissues were simultaneously labeled for panspecific Syntrophin using the antibody 1351 made by Dr. Froehner et al. The Leica confocal microscope was used to view the labeled tissue.


Expected V-NaCh labeling in Extrafusal Muscle & Neuromuscular Junctions:

Extrafusal fibres - thick sectionFig. 1 Thick optical section view of extrafusal muscle fibers with neuromuscular junctions (nmj) showing staining for V-NaCh. The muscle surfaces, sarcolemmas, show staining consistent with the conduction of action potentials in muscle. Neuromuscular junctions also display labeling.



Neuromuscular junction - optical sectionFig. 2 A Higher power view of a nmj labeled for V-NaCh and Syntrophin. A. V-NaCh is concentrated at the synaptic cleft. This region is well known to be rich in V-NaCh. C. A similar region is also rich with Syntrophin. B. Colocalization of V-NaCh (red) and Syntrophin (green) at the synaptic cleft is expected and shows that the V-NaCh antibody is labeling as expected. The yellow regions demonstrate co-localization of high V-NaCh and Syntrophin labeling.

Motor nerve node of RanvierFig. 3 Extended focus view of a node of Ranvier on a motor axon. V-NaCh is concentrated at the node. The relative absence of labeling in the internodal region is further indication that the V-NaCh antibody is labeling structures as expected. Calibration bars are 10 mm.

Muscle Spindle Structure:

Isolated muscle spindleFig. 4a An isolated rat muscle spindle. A low power transmitted light view. The spindle is surrounded by a capsule, which clearly bulges at the sensory region. A bundle of myelinated axons enter the capsule and connect to the sensory terminals. Calibration bar 100 mm.


Sensory Endings - NomarskiFig. 4b A cat muscle spindle sensory region viewed under Nomarski optics. Each intrafusal muscle fiber is wrapped by nerve terminals in an annulo spiral spiral fashion. Calibration bar 10 mm.


V-NaCh Labeling in Muscle Spindle:

Bag fibre 0.6 um optical sectionFig. 5 A cat spindle intrafusal bag fiber, optical section thickness about 0.6 mm, labeled for V-NaCh. V-NaCh labeling is clearly present on the sarcolemma but gaps of staining on the sarcolemma (yellow arrows) occur at locations where sensory terminals are expected to overlie the intrafusal fiber. Sensory terminals are unstained and can not be discerned at all. Sensory axons wwhere observed to be label for V-NaCh at nodes of Ranvier (red arrow) lying near the sensory terminals.

Bag Fibre RenderFig. 6 A 3-dimensional rendering of serial sections of the spindle shown in Fig. 5 clearly showing lack of V-NaCh staining under regions overlaid with sensory terminals.



In muscle spindles we found that the sarcolemmas of intrafusal muscle fibers were well labeled, consistent with conduction of action potentials in these specialized muscle fibers. However, the terminal Ia afferent axons, which are unmyelinated and wrap around the intrafusal muscle fibers, have no discernible labeling. These terminals are known to produce receptor potentials in response to mechanical deformation. Myelinated regions of the same axons displayed strong labeling at nodes of Ranvier. Such nodes close to sensory terminals are sites where action potential firing is likely to be initiated. Future studies are going to map the location of nodes of Ranvier and particularly heminodes in relation to the branching structure of the sensory Ia afferent axons.


The authors wish to thank: