Fig 1.
MS and GTO morphology in both neonatal and mature mouse soleus muscles are accurately reported using PV-Cre; R26-tdT and Adv-Cre; R26-tdT mouse models.
Examples of native tdTomato fluorescence in representative muscle spindles (A-C, G-I) and GTOs (D-F, J-L) from each of the mouse models utilized in this study. VGLUT1 expression was largely restricted to the proprioceptive receptor terminals, while tdTomato expression was observed in both proprioceptive receptor terminals and their respective axons. Scale bars represent 50 μm.
Fig 2.
Quantitative analysis of proprioceptive receptors within the mouse soleus muscle.
(A) Schematic representation of the extramuscular bifurcation of the soleus nerve into a proximal thin branch and a distal thick branch and the typical distribution of MS and GTOs within the muscle. (B) A survey of 27 muscles from PV-Cre; R26-tdT mice and Adv-Cre; R26-tdT mice (P3 to P7 and P19 to P20, respectively) found the soleus muscle to contain 11.3 ± 0.4 MS and 5.2 ± 0.2 GTOs (mean ± SEM). (C) Alone, the thin proprioceptive branch of the soleus nerve was found to contain 1.6 ± 0.2 MS and 2.6 ± 0.2 GTOs. (D) Proprioceptive receptor class populations within the mouse soleus varied independently (Spearman correlation rs(25) = -0.1).
Fig 3.
Whole-mount muscle preparation enabled detailed examination of intact proprioceptive innervation of the mouse soleus.
(A) Composite 20X confocal image from a P5 female PV-Cre; R26-tdT mouse soleus. Representative composite 60X confocal images of a GTO and two MS are shown in (B-D), identified by red boxes. (B) Invariably, GTOs were supplied by a single Ib afferent. (C) Example of a MS supplied by two afferents. (D) Example of a MS supplied by three afferents. Scale bars represent 100 μm.
Fig 4.
Quantitative analysis of proprioceptive afferents supplying the mouse soleus muscle.
(A) The number of proprioceptive afferents supplying a given MS ranged from 1 to 5 (n = 135 neonatal and mature MS supplied by 295 proprioceptive afferents in total). Most often, MS were supplied by 2 proprioceptive afferents. (B) Total proprioceptive afferent population per muscle was weakly correlated with total number of receptors (Spearman correlation rs(7) = 0.498). (C) Total number of MS per muscle displayed a modest negative correlation with the number of average afferents per MS (Spearman correlation rs(7) = -0.536).
Fig 5.
Morphological identification of spindle afferents in the mature mouse soleus.
Representative composite 60X confocal images obtained from P19-20 Adv-Cre; R26-tdT soleus muscles using the whole-mount muscle preparation. Proprioceptive ending types are labeled in red. P indicates primary afferent ending, and S indicates secondary afferent ending. (A) Example of a MS supplied by two afferents. The primary afferent forms large annulospirals around intrafusal bag fibers, while the secondary afferent terminates primarily in the region of smaller chain fibers. (B) Example of a MS supplied by two primary afferents terminating within close proximity of each other. (C) Example of a MS supplied by four proprioceptive afferents. Scale bars represent 100 μm.
Table 1.
Shift in proprioceptive afferent configuration observed with increasing number of associated afferents per muscle spindle.
Fig 6.
Axon diameter for both morphologically classified spindle afferent subtypes.
(A) Histogram of axon diameters for afferents that were classified as primary or secondary endings based on morphological criteria. (B) Logistic regression model used to predict proprioceptive afferent subtypes based on diameter. Black dots represent the diameters of afferents that were classified as primary afferents based on morphologic criteria and therefore they have a 100% probability of being a primary afferent. The open circles illustrate the morphologically classified secondary endings, which have a 0% probability of being a primary ending. The gray line indicates the probability of an afferent being a primary based on diameter. The dotted lines illustrate the cut-off diameters (2.46 and 2.91 μm) for classifying afferents. Axons with diameters left of the first dotted line (diameter < 2.46 μm) were classified as group II afferents, axons with diameters between the dotted lines could not be classified based on diameter alone, and axons with diameters to the right of the second dotted line (diameter > 2.91 μm) were classified as Ia afferents.