Loss of Npn1 from motor neurons causes postnatal deficits independent from Sema3A signaling

AbstractThe correct wiring of neuronal circuits is of crucial importance for the function of the vertebrate nervous system. Guidance cues like the neuropilin receptors (Npn) and their ligands, the semaphorins (Sema) provide a tight spatiotemporal control of sensory and motor axon growth and guidance. Among this family of guidance partners the Sema3A-Npn1 interaction has been shown to be of great importance, since defective signaling leads to wiring deficits and defasciculation. For the embryonic stage these defects have been well described, however, also after birth the organism can adapt to new challenges by compensational mechanisms. Therefore, we used the mouse lines Olig2-Cre;Npn1cond and Npn1Sema− to investigate how postnatal organisms cope with the loss of Npn1 selectively from motor neurons or a systemic dysfunctional Sema3A-Npn1 signaling in the entire organism, respectively. While in Olig2-Cre+;Npn1cond−/− mice clear anatomical deficits in paw posturing, bone structure, as well as muscle and nerve composition became evident, Npn1Sema− mutants appeared anatomically normal. Furthermore, Olig2-Cre+;Npn1cond mutants revealed a dysfunctional extensor muscle innervation after single-train stimulation of the N.radial. Interestingly, these mice did not show obvious deficits in voluntary locomotion, however, skilled motor function was affected. In contrast, Npn1Sema− mutants were less affected in all behavioral tests and able to improve their performance over time. Our data suggest that loss of Sema3A-Npn1 signaling is not the only cause for the observed deficits in Olig2-Cre+;Npn1cond−/− mice and that additional, yet unknown binding partners for Npn1 may be involved that allow Npn1Sema− mutants to compensate for their developmental deficits

Npn-1 Contributes to Axon-Axon Interactions That Differentially Control Sensory and Motor Innervation of the Limb

The initiation, execution, and completion of complex locomotor behaviors are depending on precisely integrated neural circuitries consisting of motor pathways that activate muscles in the extremities and sensory afferents that deliver feedback to motoneurons. These projections form in tight temporal and spatial vicinities during development, yet the molecular mechanisms and cues coordinating these processes are not well understood. Using cell-type specific ablation of the axon guidance receptor Neuropilin-1 (Npn-1) in spinal motoneurons or in sensory neurons in the dorsal root ganglia (DRG), we have explored the contribution of this signaling pathway to correct innervation of the limb. We show that Npn-1 controls the fasciculation of both projections and mediates inter-axonal communication. Removal of Npn-1 from sensory neurons results in defasciculation of sensory axons and, surprisingly, also of motor axons. In addition, the tight coupling between these two heterotypic axonal populations is lifted with sensory fibers now leading the spinal nerve projection. These findings are corroborated by partial genetic elimination of sensory neurons, which causes defasciculation of motor projections to the limb. Deletion of Npn-1 from motoneurons leads to severe defasciculation of motor axons in the distal limb and dorsal-ventral pathfinding errors, while outgrowth and fasciculation of sensory trajectories into the limb remain unaffected. Genetic elimination of motoneurons, however, revealed that sensory axons need only minimal scaffolding by motor axons to establish their projections in the distal limb. Thus, motor and sensory axons are mutually dependent on each other for the generation of their trajectories and interact in part through Npn-1-mediated fasciculation before and within the plexus region of the limbs

<i>Sema3F</i> mutants show normal behavior in the dark and light open field test.

<p>(A-C) Gross locomotion and exploratory behavior of <i>Sema3F</i> animals is analyzed at the age of 4 weeks in the dark open field. No significant differences are evident in (A) the total distance travelled (14100 ± 1069 cm vs. 13920 ± 1457 cm, p = 0.48), (B) the locomotion velocity (12.90 ± 1.07 cm/s vs. 12.61 ± 1.29 cm/s, p = 0.65) or (C) the number of rearings (95.60 ± 7.99 vs. 78.40 ± 12.40, p = 0.35). Statistical analysis: N = 10 for each group, Mann-Whitney test. * p < 0.05, ** p < 0.005, *** p < 0.001. (D-I) Anxiety related behavior is investigated in the light open field at 4 weeks of age. Overall locomotion or exploratory behavior is not affected in <i>Sema3F</i> mutants in the light open field as determined by (D) the total distance travelled (9505 ± 658.7 cm, N = 21 vs. 9543 ± 809.3 cm, N = 26, p = 0.86), (E) the locomotion velocity (8.586 ± 0.595 cm/s, N = 21 vs. 8.465 ± 0.725, N = 26, p = 0.72) or (F) the number of rearings (60.52 ± 5.57, N = 21, vs. 48.89 ± 6.06, N = 19, p = 0.17). The determination of (G) the distance travelled in the center (1759 ± 172.1 cm, N = 21 vs. 1796 ± 215.8 cm, N = 26, p = 0.66), (H) the time until the first center entry (74.86 ± 19.07 s, N = 21 vs. 106.8 ± 17.59 s, N = 26, p = 0.10) and (I) the number of center visits (69.10 ± 6.09, N = 21 vs. 78.00 ± 9.91, N = 26, p = 0.47) does not reveal any anxiety related behavior in <i>Sema3F</i> mutants. (J-L) Gait of <i>Sema3F</i> animals was analyzed 9 weeks after birth using the CatWalk analysis system. No significant differences were found in (J) the forelimb base of support (14.64 ± 0.73 mm, N = 9 vs. 14.37 ± 0.32 mm, N = 9; p = 1.0), (K) the duty cycle of the forelimbs (59.88 ± 1.43%, N = 9 vs. 57.42 ± 0.86%, N = 9; p = 0.11) or (L) the step pattern of the animals (5.11 ± 0.23, N = 9 vs. 5.07 ± 0.09, N = 9; p = 0.58).Statistical analysis: Mann-Whitney test. * p < 0.05, ** p < 0.005, *** p < 0.001.</p

Enriched environment housing starting at birth induces neuroanatomical rearrangements of spinal motor pools.

<p>At 12 weeks of age, motor neurons were retrogradely labeled by injection of Alexa Fluor-conjugated CTBs into the dorsal (red) or ventral (green) muscles of the distal forelimb. (A) The number of retrogradely traced motor neurons in the respective motor pool was comparable in wildtypes and mutants of all housing conditions (dorsal pool: p = 0.83; ventral pool: p = 0.68; N ≥ 3 for each group, one-way ANOVA). (B) Motor pools were reconstructed from labeled motor neurons of the brachial spinal cord. The schematics show the projection of all motor neurons along the anterior-posterior axis. The first and last outline of the ventral horn grey matter are indicated (C) In normal housing conditions the medial motor pool of adult animals is significantly larger in <i>Sema3F</i> mutants compared to their wildtype littermates. In contrast, the lateral motor pool remains unchanged (ventral: 0.00069 ± 0.00012, N = 7 vs. 0.00124 ± 0.00015, N = 5; p < 0.05; dorsal: 0.00089 ± 0.00015, N = 7 vs. 0.00102 ± 0.00027, N = 5; p = 0.66; Student’s t-test). (D) A specific scattering of the pool is evident in the dorsal-ventral direction, while the medial-lateral dimensions of the pool are not affected (dorsal-ventral: 0.13 ± 0.0076, N = 7 vs. 0.18 ± 0.0114, N = 5, p < 0.01; medial-lateral: 0.11 ± 0.0061, N = 7 vs. 0.12 ± 0.0080, N = 5, p = 0.73, Student’s t-test). (E + F) After housing in an enriched environment starting at birth, plastic rearrangements become evident and no motor pool shows a significantly altered area between wildtype and mutant animals (area dorsal: 0.00102 ± 0.00021, N = 5 vs. 0.00113 ± 0.00050, N = 3, p = 0.81; area ventral: 0.00084 ± 0.00024, N = 5 vs. 0.00065 ± 0.00007, N = 3, p = 0.57; scattering medial-lateral: 0.1149 ± 0.0068, N = 5 vs. 0.0979 ± 0.0074, N = 3, p = 0.16; scattering dorsal-ventral: 0.1372 ± 0.0108, N = 5 vs. 0.1506 ± 0.0104, N = 3, p = 0.44, Student’s t-test). (G) Enriched environment starting at 4 weeks does not induce these changes. Here, only the lateral motor pool appears normal while the medial motor pool is still significantly larger in mutants compared to wildtype littermates (dorsal: 0.00065 ± 0.00006, N = 8 vs. 0.00066 ± 0.00015, N = 3, p = 0.94; ventral: 0.00045 ± 0.00004, N = 8 vs. 0.00113 ± 0.00041, N = 3, p < 0.05, Student’s t-test). (H) The analysis of the specific scattering reveals an extension of the pool in dorsal-ventral direction (medial-lateral: 0.1068 ± 0.0021, N = 8 vs. 0.1121 ± 0.004982, N = 3, p = 0.26; dorsal-ventral: 0.1157 ± 0.0031, N = 8 vs. 0.1642 ± 0.0375, N = 3, p < 0.05, Student’s t-test). (I and J) Already at 4 weeks after birth the plastic rearrangements of the medial motor pool due to enriched environment housing are evident. While the medial pool shows a significant dorsal-ventral scattering in normally housed animals (I) (area dorsal: 0.00141 ± 0.00030, N = 5 vs. 0.00132 ± 0.00021, N = 5, p = 0.81; area ventral: 0.00124 ± 0.00020, N = 5 vs. 0.00371 ± 0.00071, N = 5, p < 0.05; scattering dorsal-ventral: 0.1587 ± 0.0139, N = 5 vs. 0.2486 ± 0.0204, N = 5, p < 0.01, Student’s t-test), in animals that were housed in an enriched environment starting at birth the pool has normal dimension (J) (area dorsal: 0.00106 ± 0.00023, N = 5 vs. 0.00151 ± 0.00032, N = 5, p = 0.29; area ventral: 0.00130 ± 0.00023, N = 5 vs. 0.00212 ± 0.00049, N = 5, p = 0.17; scattering dorsal-ventral: 0.1564 ± 0.0109, N = 5 vs. 0.1923 ± 0.0154, N = 5, p = 0.09, Student’s t-test). * p < 0.05, ** p < 0.005, *** p < 0.001.</p

Excitatory-inhibitory balance of synaptic input is shifted by enriched environment housing.

<p>(A) Example of excitatory (vGlut1) and inhibitory (vGAT) synapses on retrogradely labeled motor neurons of 12 week old animals. (B) The number of inhibitory synapses on traced motor neurons remains unchanged between wildtypes and mutants of all housing conditions (normal housing (wt: 133.5 ± 10.52, mut: 127.3 ± 28.76), enriched environment starting at birth (wt: 122.5 ± 3.85, mut: 125.0 ± 6.63), and enriched environment starting at 4 weeks (wt: 119.4 ± 12.0, mut: 113.2 ± 2.15); N = 3 for each group, p = 0.91, one-way ANOVA). (C) Between <i>Sema3F</i> wildtypes and mutants, the number of excitatory synapses is not significantly altered (NH: wt: 8.32 ± 0.90, mut: 8.13 ± 0.67, p = 0.87; EEbirth: wt: 15.99 ± 2.51, mut: 16.81 ± 3.88, p = 0.75; EE4: wt: 8.70 ± 0.93, mut: 8.43 ± 0.24; N = 3 for each group, Students t-test), however, after enriched environment housing starting at birth the number of excitatory synapses were significantly increased when compared to normal housing conditions or enriched environment starting at 4 weeks (NH vs. EEbirth: wt: p = 0.040, mut: p = 0.022; EEbirth vs. EE4: wt: p = 0.047, mut: p = 0.023, N = 3 for each group, Students t-test). Statistical analysis: N = 3 for each group, Students t-test. * p < 0.05, ** p < 0.005, *** p < 0.001. Scale bar: 20 μm.</p

Motor coordination of <i>Sema3F</i> mice after housing in different environmental conditions.

<p>(A-F) Motor coordination deficits were analyzed using the ladder rung test. (A) Under normal housing conditions, <i>Sema3F</i> mutants need significantly more time to cross the ladder with irregularly spaced bars than littermate controls at each time point tested (4 weeks: 11.74 ± 0.86 s, N = 14 vs. 25.58 ± 2.56 s, N = 12, p < 0.001; 8 weeks: 9.48 ± 0.75 s, N = 14 vs. 18.92 ± 1.64 s, N = 12, p < 0.001; 12 weeks: 9.41 ± 0.67 s, N = 13 vs. 18.31 ± 1.89 s, N = 12, p < 0.001, Improvement mut 4–12 weeks: p = 0.03). (B) <i>Sema3F</i> mutants also show a significantly increased number of slips (4 weeks: 1.00 ± 0.22, N = 14 vs. 3.11 ± 0.66, N = 12, p < 0.05; 8 weeks: 1.10 ± 0.23, N = 14 vs. 2.61 ± 0.56, N = 12, p < 0.05; 12 weeks: 0.82 ± 0.14, N = 13 vs. 2.03 ± 0.37, N = 12, p < 0.05). (C) After enriched environment housing starting at birth the motor performance of <i>Sema3F</i> mutants reaches wildtype levels at 8 weeks after birth (4 weeks: 10.37 ± 0.76 s, N = 10 vs. 16.74 ± 1.71, N = 9, p < 0.005; 8 weeks: 12.63 ± 0.90 s, N = 10 vs. 15.85 ± 1.54 s, N = 9, p = 0.14; 12 weeks: 11.00 ± 0.98 s, N = 10 vs. 13.48 ± 1.19 s, N = 9, p = 0.07). (D) These animals never show a significant difference in the number of slips compared to wildtype littermates (4 weeks: 1.00 ± 0.21, N = 10 vs. 1.67 ± 0.49, N = 9, p = 0.36; 8 weeks: 0.83 ± 0.16, N = 10 vs. 1.07 ± 0.17, N = 9, p = 0.31; 12 weeks: 0.47 ± 0.09, N = 10 vs. 0.59 ± 0.15, N = 9, p = 0.50). (E) Enriched environment housing starting at 4 weeks after birth does not improve the motor performance of <i>Sema3F</i> mutants. (4 weeks: 15.19 ± 1.17 s, N = 19 vs. 34.06 ± 2.09 s, N = 11,p < 0.001; 8 weeks: 12.61 ± 1.01 s, N = 19 vs. 23.91 ± 2.17 s, N = 11, p < 0.001; 12 weeks: 11.75 ± 0.62 s, N = 19 vs. 21.55 ± 2.22 s, N = 11, p < 0.001). (F) Also the number of slips from the ladder is significantly increased at each time point after enriched environment housing starting at 4 weeks (4 weeks: 1.81 ± 0.24, N = 19 vs. 5.30 ± 0.59, N = 11, p < 0.001; 8 weeks: 1.00 ± 0.15, N = 19 vs. 2.24 ± 0.32, N = 11, p < 0.005; 12 weeks: 1.00 ± 0.24, N = 19 vs. 2.394 ± 0.3140, N = 11, p < 0.005). Statistical analysis: Mann-Whitney test. * p < 0.05, ** p < 0.005, *** p < 0.001. (G-I) Improved performance in the grid walk test is not caused by alterations in general locomotion or exploratory behavior. At the age of 12 weeks, animals that were housed in normal or enriched housing conditions starting at birth do not reveal any significant differences in the open field test as determined by (G) the total distance that was traveled (NH: 14190 ± 867.2 cm, N = 16 vs. 13350 ± 951.9 cm, N = 20; EE: 13110 ± 871.3 cm, N = 23 vs. 12160 ± 983.2 cm, N = 20; ANOVA: p = 0.53), (H) the locomotion speed (NH: 12.62 ± 0.96 cm/s, N = 16 vs. 12.22 ± 0.95 cm/s, N = 20; EE: 11.73 ± 0.81 cm/s, N = 23 vs. 10.79 ± 0.89 cm/s, N = 20; ANOVA: p = 0.54) and the total number of rearings (NH: 79.00 ± 9.41, N = 16 vs. 95.85 ± 8.82, N = 20; EE: 78.17 ± 8.16, N = 23 vs. 73.25 ± 8.28, N = 20; ANOVA: p = 0.27). Statistical analysis: One-way ANOVA. * p < 0.05, ** p < 0.005, *** p < 0.001.</p

Electromyography reveals innervation defects in <i>Sema3F</i> mutants under all housing conditions.

<p>(A) After stimulation of the <i>musculocutaneous</i> nerve, wildtype animals show a signal in the <i>biceps brachii</i> (green box) while the <i>triceps brachii</i> is not activated (red box) and only the stimulation artefact is visible. In <i>Sema3F</i> mutants the stimulation of the <i>musculocutaneous</i> nerve leads to the activation of <i>biceps brachii</i> and <i>triceps brachii</i> muscles at the same time. This was observed in all housing conditions (normal housing, enriched environment starting at birth and enriched environment starting at 4 weeks). (B) Quantification of activation signals. The table displays the total number of tested animals and the number of animals showing a signal in the respective muscle after activation of the <i>musculocutaneous</i> nerve.</p

Most spinal motor neurons are not protected by PNNs.

<p>(A) In the adult spinal cord only few motor neurons retrogradely labeled from the distal ventral forelimb show PNNs (arrow). (B—D) Examples of motor neurons with no, weak, or strong PNNs, respectively. (E) At 4 weeks of age, when the critical period for adaptive plasticity is closed, more than 65% of traced motor neurons are not covered by PNNs, regardless of the housing conditions (NH: wt: 63.6 ± 3.4%; mut: 70.4 ± 3.0%; EE: wt: 66.6 ± 6.8%, mut: 71.6 ± 9.4%; p = 0.43). Weak PNNs are found on less than 30% (NH: wt: 30.7 ± 2.5%; mut: 27.2 ± 2.5%; EE: wt: 29.0 ± 5.3%, mut: 25.9 ± 8.6%; p = 0.72) and less than 5% of motor neurons show strong PNNs (NH: wt: 5.7 ± 1.7%; mut: 2.4 ± 0.6%; EE: wt: 4.4 ± 2.0%, mut: 2.6 ± 0.8%; p = 0.06). Statistical analysis: N = 3 for each group, one-way ANOVA. * p < 0.05, ** p < 0.005, *** p < 0.001. Scale bar: 20 μm.</p