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, 524 (9), 1892-919

Role of Primary Afferents in the Developmental Regulation of Motor Axon Synapse Numbers on Renshaw Cells

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Role of Primary Afferents in the Developmental Regulation of Motor Axon Synapse Numbers on Renshaw Cells

Valerie C Siembab et al. J Comp Neurol.

Abstract

Motor function in mammalian species depends on the maturation of spinal circuits formed by a large variety of interneurons that regulate motoneuron firing and motor output. Interneuron activity is in turn modulated by the organization of their synaptic inputs, but the principles governing the development of specific synaptic architectures unique to each premotor interneuron are unknown. For example, Renshaw cells receive, at least in the neonate, convergent inputs from sensory afferents (likely Ia) and motor axons, raising the question of whether they interact during Renshaw cell development. In other well-studied neurons, such as Purkinje cells, heterosynaptic competition between inputs from different sources shapes synaptic organization. To examine the possibility that sensory afferents modulate synaptic maturation on developing Renshaw cells, we used three animal models in which afferent inputs in the ventral horn are dramatically reduced (ER81(-/-) knockout), weakened (Egr3(-/-) knockout), or strengthened (mlcNT3(+/-) transgenic). We demonstrate that increasing the strength of sensory inputs on Renshaw cells prevents their deselection and reduces motor axon synaptic density, and, in contrast, absent or diminished sensory afferent inputs correlate with increased densities of motor axons synapses. No effects were observed on other glutamatergic inputs. We conclude that the early strength of Ia synapses influences their maintenance or weakening during later development and that heterosynaptic influences from sensory synapses during early development regulates the density and organization of motor inputs on mature Renshaw cells.

Keywords: Ia afferent; VAChT; VGLUT1; calbindin; development; motoneuron; parvalbumin; spinal cord.

Figures

Figure 1
Figure 1. Specificity of calbindin, parvalbumin and VGLUT1 antibodies tested in knockout tissues
All images are in epifluorescence captured at low magnification with a digital camera. with All images are wide-field epifluorescence captured at low magnification with a digital camera. A-B, Parvalbumin and calbindin-immunoreactivity in the cerebellum of wild-type (A1 and B1), parvalbumin knockout (A2) and calbindin knockout (B2) adult mice. Parvalbumin (A1) and calbindin (B1) immunoreactivities are revealed in Purkinje cells of adult wild-type mice and abolished in the cerebellum of their respective knockout animals (A2, B2)(dashed line indicate the Purkinje cell layer). C-D, Parvalbumin and calbindin-immunoreactivities in the spinal cord of adult wild-type (C1 and D1), parvalbumin knockout (C2) and calbindin knockout (D2) mice. Parvalbumin-IR afferent axons are visible in the dorsal columns (DCs) of the spinal cord of wild-type mice (C1, outlined area), but not in parvalbumin knockout mice (C2). Calbindin antibodies reveal in wild-type spinal cords a large number of immunoreactive cells in lamina II (LII, D1) and in the Renshaw cell area (ventral dashed outline, D1). No immunoreactive neurons are observed in spinal cord sections from knockout animals (D2). E, VGLUT1-immunoreactivity in the spinal cord of wild-type (E1), VGLUT1 heterozygote (E2) and VGLUT1 knockout (E3) mice. A normal pattern of VGLUT1-IR boutons is present in spinal cords from wild type (E1) and heterozygotes (E2). There are no VGLUT1-IR boutons detected in VGLUT1 knockout mice (E3). Some blood vessel immunolabeling remains in knockout mice indicating it is due to a cross-reaction of the primary antibody (it is not revealed by secondary antibodies applied to the section in the absence of primary antibodies). This relatively weaker immunoreaction of blood vessels did not interfere with the identification of VGLUT1-IR synaptic puncta on spinal neurons and was variable from animal to animal. It is also a particular feature of the guinea-pig antibody against VGLUT1 we used since other anti-VGLUT1 antibodies raised in different species (rabbit) did not show blood vessel labeling. We used guinea pig antibodies to better combine with other rabbit-raised primary antibodies for dual and triple immunfluorescence. Scale bars: 200 μm in A1, B1, C1, D1 and E1.
Figure 2
Figure 2. Distribution and density of VGLUT1-immunoreactive boutons in LIX and around motoneurons
A-D, Low magnification confocal images of VGLUT1-immunoreactivity in the ventral horn of wild-type (A), Er81(−/−) (B), Egr3(−/−) (C) and mlcNT3(+/−) (D) animals at P20. The dotted yellow line indicates the border between the ventral grey and the white matter. At P20, VGLUT1-IR boutons are largely absent in the ventral horn of Er81(−/−) mice. Compared to wild-type animals, VGLUT1-immunoreactivity in the ventral horn appears decreased in Egr3(−/−) mice and increased in mlcNT3(+/−) animals. E-H, High magnification images of single confocal planes through the somata of NeuN-immunoreactive motoneurons (magenta, Cy3) and associated VGLUT1-IR boutons (green, FITC) in wild-type (E), Er81(−/−) (F), Egr3(−/−) (G) and mlcNT3(+/−) P20 animals (H). Virtually no VGLUT1-IR boutons were observed around motoneurons in Er81(−/−) mice and these were significantly decreased and of smaller size in Egr3(−/−) mice compared to wild-types. In contrast, the number of VGLUT1-IR varicosities in the neuropil and in contact with motoneurons was significantly increased in mlcNT3(+/−) animals. I, Lamina IX (LIX) neuropil density of VGLUT1-IR puncta (per 100 μm2) in mutant animals compared to wild-type littermates at P20. VGLUT1-IR clusters were nearly absent in the LIX neuropil of Er81(−/−) animals. In Egr3(−/−) animals VGLUT1-IR density was partially reduced, while mlcNT3(+/−) animals showed significant increases in density compared to wild-type littermates (asterisks, p<0.001, One-way ANOVA followed by Bonferroni-corrected t-tests). J, Number of VGLUT1-IR contacts per 100 μm of linear perimeter on NeuN-IR motoneuron somata at P20. Compared to wild-type littermates, VGLUT1-IR contacts on motoneurons were almost absent in Er81(−/−) animals (asterisks, p<0.001, Bonferroni-corrected t-tests) and significantly decreased in Egr3(−/−) mice (asterisk, p=0.015, Bonferroni-corrected t-tests). In contrast, VGLUT1-IR contact density increased in both mlcNT3(+/−) mice compared to wild-type littermates at P20 (asterisks, p<0.001, Bonferroni-corrected t-tests). In all the graphs the error bars represent SEM estimates. Scale bars: 200 μm in D (A to D at the same magnification); 10 μm in E (E to G at the same magnification); 20 μm in H.
Figure 3
Figure 3. Calbindin and parvalbumin expression in the ventral horns of wild-type, Er81(−/−), Egr3(−/−) and mlcNT3(+/−) mice
A-D, Low magnification confocal images of calbindin-immunoreactive (CB-IR) cells in the ventral horn of wild-type (A), Er81(−/−)(B), Egr3(−/−) (C) and mlcNT3(+/−) (D) animals at P20. The border between white and ventral gray matter is outlined and a white dotted line is drawn horizontally from the dorsal tip of the central canal (cc in A). CB-IR cells in the ventral horn of P20 mice are largely restricted to the Renshaw cell area in the ventral-most regions of LVII and LIX (arrow). A few additional CB-IR neurons (usually with weaker immunofluorescence) are found near the dorsal-ventral border and close to the central canal. Genetic alterations in proprioceptive inputs did not change the number or distribution of CB-IR neurons. E, The number of CB-IR cells per ventral horn in P20 mice remained largely unchanged. A small difference was detected in one Er81(−/−) mutant (asterisks, p<0.01, Bonferroni t-tests compared to average controls). F, Number of CB-IR cells in the ventral most 250 μm of LVII and LIX. Most of these CB-IR cells correspond with RCs and their numbers did not significantly change in any mutant. In E and F the wild-type bar (grey) represents the average estimate from ventral horns pooled from two animals per line (n = 23, 33 and 31 ventral horns in, respectively, the Er81(−/−), Egr3(−/−) and mlcNT3(+/−) samples), while averages in mutant animals (black bars) are represented individually (n = 10-18 ventral horns per animal). Error bars indicate SEM estimates. G-J, Parvalbumin-immunoreactivity (PV-IR) in the ventral horn of P20 wild-type (G), Er81(−/−) (H), Egr3(−/−) (I) and mlcNT3(+/−) (J) mice. PV-IR neurons in P20 mice are distributed throughout the whole ventral horn with a lower density in lateral lamina IX. Similar numbers and distribution of PV-IR cells are found in mutant animals. K, Number of PV-IR cells per ventral horn in P20 mice. Wild-type bar (grey) is the average estimate from control ventral horns from two animals (n = 22, 32 and 33 ventral horns in the Er81(−/−), Egr3(−/−) and mlcNT3(+/−) samples, respectively), while the average mutant animals (black bars) are represented individually (n = 8-22 ventral horns per animal). Error bars in all graphs indicate SEM estimates. Scale bars: 100 μm in A and G (A-D and G-J are at the same magnification).
Figure 4
Figure 4. Normal distribution of VGLUT1 and VAChT contacts on calbindin-IR Renshaw cells
A1, A2, A3, Triple immunofluorescence for calbindin (magenta, Cy3), VGLUT1 (green, FITC) and VAChT (white, Cy5). The images are 2D projections of 4 optical sections (obtained at 20×; 6 μm depth) and show all three immunoreactivities in different combinations. The location of three RCs reconstructed in following panels is indicated in A1 and with asterisks in A2. White dashed lines indicate the edge of the spinal cord gray matter and the boundary between lamina IX and VII. Green dashed line indicates the region with high density of VGLUT1-IR puncta. The dashed grey line indicates the region with the highest concentration of motor axon-derived VAChT-IR synaptic boutons. Each of the three RCs are located in different positions with respect to regions with high density VGLUT1-IR or VAChT-IR boutons. B1, Full reconstruction of RC#2 superimposed on a single optical section (through the RC2 cell body) from a high magnification (60×) tiled image. Contacts from VAChT-IR and VGLUT1-IR boutons are indicated as grey and green circles, respectively. Dashed line is the ventral border of the spinal cord gray matter. B2, Ninety degree rotation of RC#2 showing the section thickness. Dendrites oriented in the longitudinal rostro-caudal axis intersect the section surface faster than dendrites in more or less transverse orientations. Some transversally oriented dendrites reach their natural terminations within the section and usually end with small bi- or trifurcations (see asterisk in B1, B2 and also C). C, Reconstructions of three RCs with VGLUT1-IR (green dots) and VACHT-IR boutons (grey dots) mapped on their dendrites. VAChT-IR contacts are denser on dendrite segments coursing through the VAChT area, while VGLUT1-IR contacts are more frequent in dendrites entering the VGLUT1 area. D, Full reconstruction of RC#2 within Sholl circles. Two dendritic areas are boxed; one is in the first 50 μm Sholl (proximal) from the cell body center and the other at 200-250 Sholl distance (distal). E1, E2, E3 and F1, F2, F3 are Imaris surface renderings of these dendritic segments in three different rotations. Arrowheads indicate different views of the same contacts. The proximal segment (E) has a high density of VAChT-IR contacts, while the distal segment (F) receives predominantly VGLUT1-IR contacts. Scale bars: 100 μm in A1, 50 μm in B1 and 10 μm in E3 (F panels are at the same magnification as E).
Figure 5
Figure 5. Quantification of VGLUT1-IR and VAChT-IR contacts on reconstructed Renshaw cells
A, Four reconstructed RCs, three with cell bodies located lateral (RC 1), medial (RC 7) or dorsal (RC 8) to the region containing a high density of VAChT-IR boutons and one RC with the cell body in the middle of this region (RC 2). The positions of VAChT-IR contacts are marked in black and VGLUT1 contacts in grey. The highest numbers of contacts occur in dendrite segments entering the VAChT and VGLUT1 areas, independent of distance to cell body, order of dendrite or thickness. B, Distribution of contacts along the dendrites of the four cells shown in A. Y-axis is the percentage of contacts of each class (i.e., percentage of VAChT-IR contacts in each bin with respect to all VAChT contacts), found at different distance bins. X-axis represents path distance; “50” means contacts from 0 (dendrite origins) to 50 μm path distance (in any dendrite), “100” is for contacts located >50 to 100, and so on. Cells located medial, lateral and dorsal to the VAChT regions have variable distributions of VAChT contacts because differences in dendrite locations. Dendrites entering the VAChT region receive a large number of contacts, while dendrites away from this region receive very few. In contrast, RCs with cell bodies in the middle of the VAChT region (i.e., RC 2) display balanced distributions of high numbers of VAChT-IR contacts in all proximal dendrites that then decay with distance. In general, few VAChT-IR contacts are found further than 150 μm from the cell body. VGLUT1-IR contacts are also found in higher frequency in dendrite regions entering the area with high density of VGLUT1-IR boutons. By difference to VAChT-IR contacts they are also frequent in distal dendrites. C, Density of VAChT-IR and VGLUT1-IR contacts (boutons per 100 μm2 of dendrite surface) at different distance bins in the same four cells. D, Average frequency and density of contacts for the eight RCs reconstructed. In the left panel the location of all RCs are indicated on an “average” section profile with the approximate VGLUT1 and VAChT areas indicated. Middle panel represents the average distribution of VAChT- and VGLUT1-IR contacts at different distance bins in the eight RCs (error bars indicate SEMs; not all RCs had dendrite segments at all distances and the number of RCs included in each data point is indicated in the right panel). VAChT-IR contacts decrease in frequency more abruptly than VGLUT1-IR contacts. The distribution of available surface at different distances in indicated in the inset histogram. As a result, on average, the density of VAChT-IR contacts falls abruptly after 150 μm distance from the cell body, while the density of VGLUT1-IR contacts is maintained in the distal dendrite. The largest overlap between VAChT-IR and VGLUT1-IR contacts occurs in the first 100 μm of dendrites.
Figure 6
Figure 6. VGLUT1-immunoreactive contacts on Renshaw cells in the ventral horn of wild-type, Egr3(−/−) and mlcNT3(+/−) P20 mice
A1, B1, C1, Low magnification confocal images of VGLUT1-IR boutons (green, FITC) and CB-IR RCs (magenta, Cy3) in the ventral horn at P20 in wild-type (A1), Egr3(−/−) (B1) and mlcNT3(+/−) (C1) animals. The yellow dotted line indicates the border between the ventral horn and the white matter. A2, B2, C2, Superimposed VGLUT1-IR (green) and CB-IR neurons (magenta, Cy3). Boxes indicate areas shown at high magnification in A3, B3, and C3. A3-5, B3-5, C3-5, VGLUT1-IR boutons on P20 CB-IR RCs surface rendered in Imaris software and shown in three different rotations. Arrowheads indicate the same VGLUT1-IR contacts on RC dendrites at three different rotations. Compared to wild-types there are fewer VGLUT1-IR contacts in Egr3(−/−) RCs and more in mlcNT3(+/−). Scale bars: 100 μm in A1 (A1-A2, B1-B2 and C1-C2 are all at the same magnification); 20 μm in A3 (A3, B3 and C3 are at the same magnification).
Figure 7
Figure 7. Density of VGLUT1-immunoreactive contacts on mature Renshaw cells in wild-type mice compared to mutant mice
A, Three-dimensional reconstructions of RCs from P20 wild-type (A1), Egr3(−/−) (A2), and mlcNT3(+/−) (A3) mice. VGLUT1-IR contacts (dots) are plotted on the dendrites of the traced cells. B, Estimates of VGLUT1-IR contacts per 10 μm of linear dendrite on CB-IR RCs of wild-type, Egr3(−/−) and mlcNT3(+/−) mice of P15 (light grey bars), P20 (black bars) and adult (dark grey bars) postnatal ages. Renshaw cells from wild-type animals show densities and changes with maturation similar to those reported in Mentis et al. (2006) and Siembab et al. (2010). In wild-type and Egr3(−/−) animals, RCs display statistically significant decreases in VGLUT1-IR contact density from P15 to adult (p<0.001, one-way ANOVA). In contrast, VGLUT1-IR contact density increased significantly from P15 to adult in mlcNT3(+/−) mice (p=0.041, one-way ANOVA). Compared to their age-matched wild-types, RCs in Egr3(−/−) animals always had statistically significant lower density of VGLUT1-IR contacts, while in mlcNT3(+/−) mice show significant increases: Lines indicate statistical comparisons at P15 (continuous line), P20 (dotted line), adult (dashed line)(asterisks indicate significance; p<0.05 in post-hoc Dunn’s comparison). In mature mlcNT3(+/−) mice (P20 and adult), VGLUT1-IR contact density on RCs more than doubled compared to wild-types. During postnatal maturation VGLUT1-IR densities significantly decreased in wild-types and Egr3(−/−) mutants and increased in mlcNT3(+/−) mice (p<0.001 One-Way ANOVA on Ranks). C, Densities recorded in three different Sholl bins (aerial distance from the center of the cell body) of 50 μm distances. The densities (within each genotype) were not significantly different among the three different Sholl bins, indicating that the changes in VGLUT1-IR contact density were equally distributed throughout the proximal dendrite.
Figure 8
Figure 8. VAChT-immunoreactive contacts on Renshaw cells in the ventral horn of wild-type, Er81(−/−) Egr3(−/−), and mlcNT3(+/−) P20 mice
A1, B1, C1, D1, Low magnification confocal images of VAChT-IR boutons (green, FITC) and CB-IR RCs (magenta, Cy3) in the ventral horn of a P20 wild-type (A1), Er81(−/−) (B1), Egr3(−/−) (C1), and mlcNT3(+/−) (D1) animal. VAChT-IR staining is increased in LIX and in the RC area of Egr3(−/−) and Er81(−/−) mice compared to wild-types. The yellow dotted line indicates the border between the ventral horn and white matter. A2, B2, C2, D2, Superimposed VAChT-IR (green) and CB-IR (magenta, Cy3). Boxes indicate areas of high magnification shown in A3, B3, C3, and D3. A3-5, B3-5, C3-5, D3-5, VAChT-IR contacts on P20 CB-IR RCs surface rendered in Imaris software and shown in three different rotations. Arrowheads indicate VAChT-IR contacts on RC dendrites, shown in different rotations. There appears to be more VAChT-IR contacts on RCs in Er81(−/−) (B3) and Egr3(−/−) (C3) mice compared to wild-type control (A1) animals. In contrast, RCs in mlcNT3(+/−) (D3) mice have fewer VAChT-IR contacts than wild-types. Scale bars: Scale bars: 100 μm in A1 (A1-A2, B1-B2 C1-C2 and D1-D2 are all at the same magnification); 20 μm in A3 (A3, B3 C3 and D3 are at the same magnification).
Figure 9
Figure 9. Density of VAChT-immunoreactive contacts on Renshaw cells in wild-type and mutant mice
A, Three dimensional reconstructions of RCs from P20 wild-type (A1), Er81(−/−) (A2), Egr3(−/−) (A3), and mlcNT3(+/−) (A4) mice. VAChT-IR contacts (dots) are plotted on the traced dendritic arbors. B, Density of VAChT-IR contacts per 10 μm of linear RC dendrite in Er81(−/−), Egr3(−/−), and mlcNT3(+/−) compared to age-matched wild-type controls. During postnatal development RCs from Er81(−/−) animals showed a significant decrease in VAChT-IR contact density from P15 to P20 (p<0.05 in post-hoc Dunn’s comparisons), while it increased in Egr3(−/−) mice from P15 to P20 and adult (p=0.001, one-way ANOVA on Ranks). The density of VAChT-IR contacts was significantly increased at all ages in RCs from Er81(−/−) and Egr3(−/−) animals compared to wild-types (asterisks, p<0.05 in post-hoc Dunn’s comparison). In contrast, VAChT-IR contact densities in mlcNT3(+/−) were significantly lower compared to wild-type animals (asterisks, P15 and P20, p<0.05 in post-hoc Dunn’s comparisons). C, The density of contacts from VGLUT1-IR and VAChT-IR boutons were negatively correlated in a sample population of RCs from preparations in which both synaptic inputs where simultaneously analyzed in single RCs using triple immunofluorescence.
Figure 10
Figure 10. VGLUT2-immunoreactive contacts on Renshaw cells do not change in any mutation
A, Low magnification confocal images showing VGLUT2-immunoreactivity (green, FITC) in the ventral horn of wild-type (A1), Er81(−/−) (A2), Egr3(−/−) (A3), and mlcNT3(+/−) (A4) mice at P20. The white dotted line represents the border between the ventral horn and the white matter. VGLUT2-IR bouton density is unchanged. B, Confocal image of the ventral horn of a P20 Er81(−/−) spinal cord immunolabeled for CB (magenta, Cy3) and VGLUT2 (green). C, High magnification two-dimensional projection of the CB-IR RC indicated in a box in B. Boxes indicate the dendritic regions shown at higher magnification in D1-D2. D1-D2, High magnification confocal images of VGLUT2-IR contacts (green, arrowheads) on the dendrites of this CB-IR RC (red). E, Reconstruction of this same CB-IR RC showing VGLUT2-IR contacts on the dendrites. F, VGLUT2-IR contact density (per 10 μm of linear dendrite) in P20 wild-type, Er81(−/−), Egr3(−/−), and mlcNT3(+/−)animals. No significant differences were detected in the density of VGLUT2-IR contacts (p=0.997, one-way ANOVA). The blue line represents the density of VAChT-IR contacts and the green line the density of VGLUT1-IR contacts, both on P20 wild-type RCs. Scale bars: 100 μm in A4 and B (A2-A4 are at the same magnification); 20 μm in C; 10 μm in D2 (D1 at the same magnification.

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