Fig 1.
Example of typical location of α-motoneurons innervating LG muscle in L5 spinal segment.
The spinal grey matter borderline is marked. Retrogradely labeled motoneurons (framed) are shown enlarged below. Motoneurons were labeled with True Blue fluorescence tracer injected into the LG muscle belly.
Fig 2.
The direct M1 responses and H3-reflexes recorded in the rat soleus muscle during stimulation sessions of low-threshold proprioceptive fibers in the tibial nerve.
A. Examples of the raw data averaged after 7200 burst repetitions. Arrows indicate stimulus artifacts. B. The mean areas of the H3-reflexes (blue line) and M1-responses (green line) during consecutive days of stimulation in six rats. Four 3 min samples daily were taken for the analysis: at the beginning and at the end of the first and fourth stimulation sessions. The data are expressed as a percentage of Mmax values for individual animals.
Fig 3.
Identification of glutamatergic (VGLUT1; indigo) and cholinergic (VAChT; green) terminals abutting on LG α-motoneuron (α-MN) labeled by means of immunofluorescence (IF).
LG MN perikaryon and proximal parts of dendrites were identified with True Blue (turquoise; A, C, E and G) and all synaptic terminals apposing LG α-MN were identified by synaptophysin IF (red). A-C present single optical section (0.21 μm thick); framed areas on C (merge of A-B) are shown with higher magnification in D to demonstrate contiguity of VAChT or VGLUT1 varicosities with the edge of the α-MN perikaryon. E-H are stacks of 20 optical sections of the same α-MN to show 3D reconstruction of the glutamatergic (indigo) and cholinergic (green) terminals among all synaptic terminals (synaptophysin) abutting on LG α- MN. G–merge of E and F. H—glutamatergic (magenta) and cholinergic (yellow) terminals shown to contact α-MN surface were accepted for quantification while the other (white) terminals, which did not fulfill these criteria, were not analyzed.
Table 1.
The number of TB-identified LG α-MNs subjected to the analysis of VGLUT1- and VAChT IF synaptic terminals apposing their cell bodies.
(N)—the number of animals per group. The numbers of analyzed synaptic terminals are shown in square brackets.
Fig 4.
Changes in the number (A) and in the aggregate volume (B) of VGLUT1 IF synaptic terminals contacting LG α-MNs after seven days of stimulation of Ia fibers in the tibial nerve. Data are reported as mean +/- SEM. Both the number and aggregate volume of VGLUT1 terminals were increased on the stimulated comparing to sham-stimulated side (*p = 0.03 and *p< 0.05, respectively, Wilcoxon test).
Fig 5.
A. The distribution of VGLUT1 IF boutons of different volume apposing to LG α-motoneurons after seven days of low-threshold stimulation of proprioceptive fibers in the tibial nerve. B. Intensity of VGLUT1 IF signal in boutons apposing to LG α-MNs plotted in function of their volume on the sham-stimulated and electrically-stimulated side. Data are reported as mean +/- SEM. Stimulation of Ia fibers resulted in an increased number of VGLUT1 IF terminals of the smallest size at the expense of the largest (volume > 10 μm3) terminals (*p<0.03, **p<0.005, Mann-Whitney U test). An intensity of the signal tended to increase in the largest boutons.
Fig 6.
The effect of unilateral stimulation of low-threshold proprioceptive afferents in the tibial nerve on the number (A) and aggregate volume (B) of VAChT IF terminals contacting LG α-MNs. Data are reported as mean+/- SEM. The number of VAChT IF terminals increased by 26% after stimulation compared to sham-stimulated side (*p< 0.03, Wilcoxon test).
Fig 7.
A. The distribution of VAChT IF boutons of different volume apposing LG α-motoneurons after seven days of stimulation of low-threshold proprioceptive fibers in the tibial nerve. B. Intensity of VAChT IF signal in boutons apposing LG α-MNs plotted in function of their volume on the sham- and stimulated- side. Data are reported as mean +/- SEM. Stimulation of Ia fibers resulted in an increased intensity of signal in VAChT IF boutons (* p<0.05, Wilcoxon test).