Functionally reduced sensorimotor connections form with normal specificity despite abnormal muscle spindle development: the role of spindle-derived neurotrophin 3

Abstract

The mechanisms controlling the formation of synaptic connections between muscle spindle afferents and spinal motor neurons are believed to be regulated by factors originating from muscle spindles. Here, we find that the connections form with appropriate specificity in mice with abnormal spindle development caused by the conditional elimination of the neuregulin 1 receptor ErbB2 from muscle precursors. However, despite a modest ( approximately 30%) decrease in the number of afferent terminals on motor neuron somata, the amplitude of afferent-evoked synaptic potentials recorded in motor neurons was reduced by approximately 80%, suggesting that many of the connections that form are functionally silent. The selective elimination of neurotrophin 3 (NT3) from muscle spindles had no effect on the amplitude of afferent-evoked ventral root potentials until the second postnatal week, revealing a late role for spindle-derived NT3 in the functional maintenance of the connections. These findings indicate that spindle-derived factors regulate the strength of the connections but not their initial formation or their specificity.

Figure 1.

Late muscle spindle marker expression is decreased in ErbB2Δ mutants. In situ hybridization analysis on serial sections of P3 tibialis anterior muscle from an ErbB2Δ mutant (D–F) ErbB2^NULL/FLOX (no CRE) control (A–C) animal with probes for the transcription factors Egr3 (A, D), Er81 (B, E), and neurotrophic factor NT3 (C, F) reveals low levels of Egr3 expression in a single myofiber of the mutant muscle spindle (D); Egr3 is robustly expressed in multiple intrafusal fibers in the control (A). In situ analysis also demonstrates the complete absence of late spindle markers Er81 and NT3 in mutant muscle spindles (E, F) compared with the control (B, C). Immunohistochemical analysis by whole-mount staining of P4 gluteus maximus muscle with antibodies against PV and VGluT1 in ErbB2^FLOX/FLOX (no CRE) control (G) and ErbB2Δ mutant (H) animals demonstrates the relatively simple morphology of the muscle spindle afferent terminal around a single, rudimentary intrafusal fiber in the mutant. I, Quantitative in situ hybridization studies of the TA muscle of the ErbB2Δ mutant demonstrate induction of Egr3 expression (black) in ∼30% (9.6 ± 3.8 SEM; n = 5) of the number of spindles observed in ErbB2 (ErbB2^NULL/FLOX or ErbB2^FLOX/FLOX) controls (31.0 ± 1.9; n = 5). In contrast, no significant Er81 expression (gray) was observed in P3 mutant muscle. Statistical significance: *p < 0.001, ANOVA (Fisher's least significant difference method); there was no statistically significant difference between the number of muscle spindles expressing Egr3 and Er81 in the ErbB2 controls.

Figure 2.

The gross pattern of central projections is normal in ErbB2Δ mutant mice. A–F, The pattern of central projections of proprioceptive afferents in the L5 spinal cord of the ErbB2Δ mutant is similar to that of the ErbB2 ^FLOX/FLOX control at postnatal day 6. The images are z-stack projections of ∼10 scans of 2 μm thickness. A, B, D, E, The L5 dorsal root was filled with Texas Red Dextran (red) to reveal all afferent projections; motor neurons (MN) were retrogradely labeled from the L5 ventral root with Cascade Blue Dextran (blue). Proprioceptive afferents are selectively labeled by PV immunostaining (green). C, F, The spatial distribution of immunostaining for the VGluT1 is normal in the ErbB2Δ mutants. G–L, Proprioceptive afferent terminals, triple-labeled with Texas Red Dextran, PV, and VGlut1on L5 motor neurons in the ErbB2^FLOX/FLOX control (G–I) and ErbB2Δ (J–L) mutant. The insets in the top corners are enlargements of the yellow boxes; synaptic contacts are indicated by the yellow arrows.

Figure 3.

At P21, VGluT1⁺ contacts on motor neurons are reduced in the ErbB2Δ mice. A–D, Single optical plane (step, 0.6 μm) (A, C) and projections of ∼10 optical planes (single optical thickness, 0.6 μm) (B, D) of two L4 motor neurons from a P21 wild-type control (A,B) and P21 mutant (C,D). Motor neurons were identified by ChAT staining (blue); VGluT1 contacts are shown in red. E, Quantitative analysis of VGluT1⁺ contacts per motor neuron soma and corresponding densities in the mutant and wild-type control demonstrate no significant (NS) loss of VGluT1 contacts on motor neuron somata in the mutant and a modest but significant (p < 0.05) decrement of ∼30% in the density of VGluT1⁺ terminals. No significant difference was observed in the size of motor neurons analyzed in the mutant versus control.

Figure 4.

The isolated spinal cord preparation and identification of motor neurons. A, Shows a drawing of the lumbar spinal cord dissected together with the sciatic nerve and its major peripheral nerves. Recording and stimulating electrodes were applied to the L4 and L5 lumbar ventral roots (vr-L4 and vr-L5), en passant on the L4 and L5 dorsal root ganglia (DRG-L4 and DRG-L5), and to the following nerves: the deep peroneal nerve supplying the tibialis anterior (TA nerve) and extensor digitorum longus muscles, the superficial peroneal nerve supplying the peroneus longus and brevis (SP nerve), the first muscular branch of the tibial nerve supplying the lateral head of gastrocnemius (LG nerve), soleus and plantaris muscle and the third muscular branch of the tibial nerve supplying the flexor hallucis longus, tibialis posterior, and flexor digitorum longus and the lateral and medial plantar nerves (tibial nerve). In some experiments, an extracellular glass electrode was used to record afferent-evoked field potentials. B, C, Images of TA motor neurons (green) retrogradely labeled in vivo at P0 using cholera toxin B subunit conjugated with Alexa 488 together with a microelectrode filled with Alexa 543 hydrazide (red) cell attached to a TA motor neuron (B) and after rupture of the membrane and intracellular dye-filling (C). D, A two-photon image of lateral gastrocnemius motor neurons retrogradely labeled with cholera toxin B subunit-conjugated Alexa 647 (blue) and showing one cell filled with the intracellular dye after 15 min of whole-cell recording (pink because of red and blue colocalization). E, an antidromically evoked action potential recorded from a wild-type LG motor neuron and evoked by stimulation of the L4 ventral root.

Figure 5.

Temporal relationships between afferent-evoked field potentials, ventral root potentials, and intracellularly recorded EPSPs from muscle-identified motor neurons. A, Simultaneous recording of the extracellular field potentials (Field potential; top) recorded within the motor nucleus at the L4/5 border and the L5 ventral root potential (vr-L5; bottom) in control aCSF (blue traces) and in a low (0.1 mm) calcium aCSF (red traces) to block chemical synaptic transmission. The recordings were made in a wild-type P5 mouse. The traces on the right are shown at an expanded time base indicated by the dotted box on the left panel. The field potential recorded in low-calcium aCSF is the terminal potential (labeled as “Terminal field potential onset” and is marked by red arrowed dotted line) and is generated by action potentials invading primary afferent arbors within the motor nucleus. The synaptic field potential starts where the field potential recorded in normal aCSF deviates from the low calcium trace (labeled as “Synaptic field potential onset” and marked by black arrow). Note that the onset of the synaptic field potential coincides with the onset of the electrotonically recorded ventral root potential (labeled as “Ventral root potential onset” and marked by dotted line with arrowhead). The field potential traces are averages from 250 individual responses (10 Hz at supramaximal stimulus intensity) so that the synaptic field potential amplitude is depressed. The ventral root potential is not averaged. The response to the first stimulus is shown for each condition. The synaptic delay is approximated as the time from the onset of the terminal field (red arrow) to the onset of the synaptic field (black arrow). B, Field potential and ventral root potentials recorded in a P5 ErbB2Δ preparation. The color code is the same as in A. Note that there was no difference in the synaptic delay between wild-type and ErbB2Δ mice. C, Comparison of the timing of the synaptic potential recorded in a tibialis anterior motor neuron (TA mn) evoked by stimulation of the TA muscle nerve (5× T at 0.1 Hz) with the simultaneously recorded L4 ventral root potential (vr-L4) and the afferent volley recorded from the L4 dorsal root ganglion (DRG-L4) in a P4 wild-type mouse. Each recording has been averaged from five traces. Insets above the top (TA mn) and middle (vr-L4) traces show the responses at a slower timescale. Note that, in this cell, the onset of the homonymous synaptic potential precedes the onset of the ventral root potential consistent with its origin monosynaptically. The red arrow under the DRG-L4 trace identifies the timing of the earliest part of the volley that was used to calculate the conduction velocity of the primary afferent axons. D, Graph showing the difference in time between the onset of intracellularly recorded EPSPs from wild type TA and LG motor neurons and the ventral potentials generated by stimulation of the homonymous muscle nerve or the DRG (DRG-L4 for TA and DRG-L5 for LG). The stimulation intensity was 5× T at 0.1 Hz. The onset times were calculated from averages of three to five sweeps. The black symbols show the average and SEM for the data. E, Graph showing the conduction velocities of the primary afferents calculated from the latency from stimulation of the peripheral nerve to the volley recorded in the DRG (Nerve-DRG) or the latencies from stimulation of the peripheral nerve to the onset of the EPSP recorded in the motor neuron (Nerve-MN). There were no statistical differences in the conduction velocities between the wild type (n = 5; blue) and ErbB2Δ (n = 5; red) mice (t test; p = 0.47 for Nerve-DRG; p = 0.38 for Nerve-MN).

Figure 6.

Monosynaptic connections between primary afferents and motor neurons are preserved but reduced in strength in ErbB2Δ mice. A, B, Images of a wild-type (A) and an ErbB2Δ (B) LG motor neuron recorded from and subsequently filled with Alexa 543 hydrazide (shown in red). The images are z-stack projections of either 40 (A) or 44 (B) scans of 1.9 μm optical thickness. The identification of the cell as an LG motor neuron was established by double labeling with the retrograde tracer CTb–647 (blue) as illustrated in the two insets on the bottom left of each image. Note that the double-labeled cells identified by the arrows are pink (blue and red), indicating colocalization, whereas the dye-filled cells shown in the main part of the image are red. Immunohistochemistry against VGluT1 revealed several VGluT1⁺ terminals apposed to the motor neuron dendrites. These can be seen in the large insets in both figures (white arrows), which show an expanded region off the dendrites indicated by the dotted yellow box. Identification of VGluT1⁺ terminal appositions was made on individual optical sections. Each image is a z-stack projection of 18 sections each 0.92 μm thick. D, Dorsal; V, ventral; L, lateral; M, medial. C, Intracellularly recorded EPSPs together with simultaneously recorded ventral root potential in response to stimulation of the LG nerve (5× T, average of 5 stimuli at 0.1 Hz) in wild-type and ErbB2Δ mice (P5). The amplitude of the monosynaptic response was measured for all records at 3 ms (indicated) after the onset of the EPSP or the ventral root potential (see Results). The black arrowheads indicate the stimulus artifact. Note that both the EPSP and the monosynaptic component of the ventral root potential are smaller in the ErbB2Δ motor neurons compared with WT (indicated by the red arrows). D, Bar graphs showing the average amplitude of the ventral root potentials (left bars) and the intracellularly recorded EPSPS (right bars) in wild-type (blue) and ErbB2Δ (red) animals in response to stimulation of the sites indicated below the plot. DR, Dorsal root; TA, tibialis anterior nerve; LG, lateral gastrocnemius nerve. The average intracellular response of the monosynaptic EPSP is shown for five wild-type (blue) and six ErbB2Δ (red) LG and TA motor neurons (per, peripheral nerve). Statistical significance: *p < 0.05, **p < 0.025, t test.

Figure 7.

Selective elimination of NT3 expression in NT3Δ mutants does not result in reduced afferent-evoked monosynaptic ventral root potentials at P5. In situ hybridization analysis of serial sections of P1 (A–F) hindlimb from NT3^FLOX/FLOX/Egr3^CRE/CRE mutant (D–F), NT3^FLOX/FLOX (no CRE) control (A–C) animals with probes for the transcription factors Eg3 (A, D), Er81 (B, E), and NT3 (C, F) reveals selective elimination of NT3 from mutant muscle spindles. G, Averaged extracellular recordings from the L5 ventral root after stimulation of the L5 dorsal root at 5× T from three groups of animals (1 control and 2 mutants) at P5. The frequency of stimulation was 0.1 Hz. Note that the amplitude of the monosynaptic component the ventral root potential (arrows) did not differ significantly between the controls (NT3^FLOX/FLOX; no Cre) and either of the two mutants (NT3^FLOX/FLOX/Egr3^CRE/CRE or NT3^FLOX/FLOX/myf5^CRE). H, Graph showing the average response from three preparations from each group of animals. There was no significant difference between the groups (p = 0.10, ANOVA). I, Recordings from the same classes of animal as in G but at P14. Note that the monosynaptic reflex was greatly reduced in the mutants compared with the controls. J, Graph showing the averaged amplitude of the monosynaptic response from three animals per group. There was a significant reduction in the monosynaptic reflex for both mutants compared with that of the control group (*p < 0.01, ANOVA Fisher's test).

Figure 8.

Muscle-identified motor neurons retain specific inputs from primary afferents in ErbB2Δ mutant mice. A, Comparison of the potentials evoked by homonymous (blue trace, LG) and antagonist (red trace, TA) muscle nerve stimulation at 5 T intensity averaged from five stimuli applied once every 10 s. The blue and red arrows indicate the onset of the EPSPs. B, Recordings of homonymous and antagonist evoked synaptic potentials in an ErbB2Δ LG motor neuron. The conditions and stimulation parameters were the same as for the wild-type records in A. C, D, Bar graph showing the average latency of the synaptic potential in evoked in LG (C) and TA (D) motor neurons by stimulation of different nerves for wild-type (blue bars) and ErbB2Δ (red bars) mice. The black bars show the latency of the EPSP after simulation of the DRG. Error bars are SEM. E, The left panels show five superimposed intracellular responses from a wild-type LG motor neuron after stimulation of the homonymous LG nerve. Two different frequencies of stimulation are illustrated (0.1 Hz, top; and 10 Hz, bottom). Five sequential color-coded trials are illustrated. Note that the onset of the EPSP after stimulation of the homonymous nerve results in very little variation (“jitter”) in the latency at the two different frequencies (as indicated by the dotted line). In contrast, stimulation of the antagonist nerve (right panels) results in more variation in the latency particularly at 10 Hz. The color-coded arrows denote the onset of the EPSP in each trial. F, Intracellular responses from an LG ErbB2Δ motor neuron under identical testing. Note that, at 10 Hz, both WT and ErbB2Δ motor neurons exhibited reduced amplitude in EPSPs as expected, presumably attributable to neurotransmitter depletion. G, H, Bar graphs for wild-type (G) and ErbB2Δ (H) motor neurons comparing the average latency jitter of the homonymous and antagonist nerve-evoked EPSPs. There was no significant difference in the latency-jitter EPSP after the homonymous nerve stimulation for either wild-type or ErbB2Δ motor neurons at all stimulation frequencies. In contrast, stimulation of the antagonist nerve resulted in significantly more jitter at stimulation frequencies of 1 and 10 Hz (*p < 0.05, **p < 0.01, ANOVA Fisher's test).

Figure 9.

Motor neurons receive primary afferent boutons from homonymous but not from antagonistic muscle afferents in control and ErbB2Δ mice. A–D, Confocal images from the same transverse section of a hemisected wild-type spinal cord in the L4/L5 region showing retrograde labeling of TA motor neurons with CTb488 (A, in green), LG motor neurons labeled with CTb–647 (B, in white), motor neurons (mns) retrogradely labeled with Texas Red Dextran (C, in red), and VGluT1 immunoreactivity (D, in blue). The images in the E series are from a wild-type LG motor neuron. E1, A z-stack projection (total scan depth, 9.1 μm) of the motor neuron retrogradely labeled with Texas Red Dextran. The insets show the soma of the motor neuron double labeled with Texas Red Dextran and CTb–647 (red and white) and labeled with CTb–647 alone (white), confirming the identity of the motor neuron as LG. The dotted yellow rectangle is shown at higher magnification with various combinations of fluorochromes in the E2–E4 series. Each image is a single optical scan (0.69 μm thickness). E2 shows VGluT1 immunoreactivity (blue) and the Texas Red Dextran-labeled dendrite of the LG motor neuron. The circle identifies a putative primary afferent bouton labeled with VGluT1, which is also labeled with CTb–647 (E3) as seen in the merged image (E5), indicating that it is a bouton from an afferent originating in the LG muscle. E4, No VGut1⁺ boutons were labeled with the tracer (CTb488, green) from the antagonist muscle (TA). The series of images F1–F5 are from an LG ErbB2 motor neuron and were acquired in the same way as those of the images shown in E1–E5.