Prenatal exposure to elevated NT3 disrupts synaptic selectivity in the spinal cord

Abstract

Monosynaptic connections between muscle spindle (Ia) afferents and motoneurons (MNs), the central portion of the stretch reflex circuit, are highly specific, but the mechanisms underlying this specificity are primarily unknown. In this study, we report that embryonic overexpression of neurotrophin-3 (NT3) in muscles disrupts the development of these specific Ia-MN connections, using transgenic (mlc/NT3) mice that express elevated levels of NT3 in muscles during development. In mlc/NT3 mice, there is a substantial increase in the amplitudes of monosynaptic EPSPs evoked by Ia afferents in MNs as measured with extracellular recordings from ventral roots. Despite this increased functional projection of Ia afferents, there is no obvious change in the anatomical density of Ia projections into the ventral horn of the spinal cord. Intracellular recordings from MNs revealed a major disruption in the pattern of Ia-MN connections. In addition to the normal connections between Ia afferents and MNs supplying the same muscle, there were also strong monosynaptic inputs from Ia afferents supplying unrelated muscles, which explains the increase seen in extracellular recordings. There was also a large variability in the strength of Ia input to individual MNs, both from correct and incorrect Ia afferents. Postnatal muscular administration of NT3 did not cause these changes in connectivity. These results indicate that prenatal exposure to elevated levels of NT3 disrupts the normal mechanisms responsible for synaptic selectivity in the stretch reflex circuit.

Figure 1.

Representative excitatory EPSPs (black traces) recorded extracellularly from lumbar spinal cord ventral roots L3–L5 in response to stimulation of the Q or Add muscle nerve in wild-type and mlc/NT3 mice at P7. Ventral root recordings monitor the average response in all MNs projecting in the root. At L3 and L4, orthodromic action potentials were evoked in some MNs, obscuring the EPSPs. EPSP amplitudes were therefore measured by fitting the rising phase of each response with a model trace (red), as described in Materials and Methods.

Figure 2.

Central projections of sensory afferents in wild-type (left) and mlc/NT3 (right) mice at P7. Two examples for each genotype are provided to show the variability in density of Ia projections. The ROI used to measure the density of Ia projections is shown enclosed by a white dashed line. The ROI used to measure the background intensity is shown as a white dotted circle. Scale bar, 150 μm.

Figure 3.

The specificity of monosynaptic connections between Q and Add Ia afferents and MNs is disrupted in mlc/NT3 mice. Representative EPSPs (black traces) in Q and Add MNs were elicited by stimulation of the Q and Add muscle nerves in P7 mice. Model traces (red) were used to measure the monosynaptic component of each response. Note the presence of large monosynaptic components in unrelated (Q → Add and Add → Q) EPSPs in mlc/NT3 mice. These EPSPs are absent in wild-type mice.

Figure 4.

Average amplitudes (±1 SE) of monosynaptic EPSPs recorded in MNs after stimulation of their homonymous and unrelated muscle inputs in P7 mice. In both the Q–Add and FDL–APF groups, homonymous EPSPs are large in both wild-type and mlc/NT3 mice and are not significantly different from each other. Only mlc/NT3 mice have large unrelated EPSPs, however, and the amplitudes of these EPSPs is significantly larger than those in wild-type mice (p < 0.001).

Figure 5.

Fluctuations in synaptic latencies of homonymous and unrelated EPSPs in wild-type and mlc/NT3 mice. Each panel shows >30 superimposed single traces (black) of the same EPSP. The latency of each single trace was measured separately (see Materials and Methods), and a histogram of the individual latencies is shown in red below the traces. Unrelated EPSPs in wild-type mice have a longer and more variable latency than homonymous EPSPs. The latencies and latency fluctuations of unrelated EPSPs in mlc/NT3 mice, however, are the same as for homonymous EPSPs, providing strong evidence that they are mediated monosynaptically.

Figure 6.

Specificity of Ia–MN EPSPs in individual mice, as measured by the SI (see Results for details) in individual mice. Synaptic specificity is high in both wild-type and adm/NT3 mice, but SIs are significantly lower in mlc/NT3 mice.

Figure 7.

Peripheral axons of sensory and motor neurons do not project to multiple muscles in mlc/NT3 mice (P7). Stimulation of dorsal root L3 evoked compound action potentials in both the Q and Add nerves (top 2 traces), indicating that both nerves contained sensory axons. Stimulation of one muscle nerve, however, evoked no action potentials in the other nerve, indicating that no sensory (or motor) nerve projected in both nerves.

Figure 8.

Amplitude histograms of monosynaptic EPSPs in wild-type and mlc/NT3 mice. Both conventional bar histograms (A) and cumulative frequency histograms (B) are presented for each data set. Amplitudes of homonymous EPSPs are more tightly clustered around the mean in wild-type (black bars and lines) compared with mlcNT3 (red bars and lines) mice. The SDs of EPSP amplitudes for each homonymous data set are shown as horizontal lines in B with the same color code used in the histograms. The SDs for homonymous EPSPs in wild-type and mlc/NT3 mice are significantly different from one another (p < 0.01; see Materials and Methods).

Figure 9.

Homonymous and unrelated EPSP amplitudes are positively correlated in individual MNs in mlc/NT3 but not wild-type mice. Each point represents EPSP amplitudes (A–C) or resting potential (C) in a single MN. The absence of a correlation between EPSP amplitude and resting potential (C) suggests that the positive correlation between homonymous and unrelated EPSPs (B) is not an artifact of the quality of the intracellular recording.