Neonatal infraorbital nerve crush-induced CNS synaptic plasticity and functional recovery

Abstract

Infraorbital nerve (ION) transection in neonatal rats leads to disruption of whisker-specific neural patterns (barrelettes), conversion of functional synapses into silent synapses, and reactive gliosis in the brain stem trigeminal principal nucleus (PrV). Here we tested the hypothesis that neonatal peripheral nerve crush injuries permit better functional recovery of associated central nervous system (CNS) synaptic circuitry compared with nerve transection. We developed an in vitro whisker pad-trigeminal ganglion (TG)-brain stem preparation in neonatal rats and tested functional recovery in the PrV following ION crush. Intracellular recordings revealed that 68% of TG cells innervate the whisker pad. We used the proportion of whisker pad-innervating TG cells as an index of ION function. The ION function was blocked by ∼64%, immediately after mechanical crush, then it recovered beginning after 3 days postinjury and was complete by 7 days. We used this reversible nerve-injury model to study peripheral nerve injury-induced CNS synaptic plasticity. In the PrV, the incidence of silent synapses increased to ∼3.5 times of control value by 2-3 days postinjury and decreased to control levels by 5-7 days postinjury. Peripheral nerve injury-induced reaction of astrocytes and microglia in the PrV was also reversible. Neonatal ION crush disrupted barrelette formation, and functional recovery was not accompanied by de novo barrelette formation, most likely due to occurrence of recovery postcritical period (P3) for pattern formation. Our results suggest that nerve crush is more permissive for successful regeneration and reconnection (collectively referred to as "recovery" here) of the sensory inputs between the periphery and the brain stem.

Keywords: brain stem; nerve damage; rat; silent synapses; trigeminal.

Fig. 1.

Photograph of an in vitro trigeminal pathway preparation on the third day after right infraorbital nerve (ION) crush. Note that the right TG and ION are thinner than the left control. WP, whisker pad; TG, trigeminal ganglion; SC, superior colliculus.

Fig. 2.

Classification of TG cells in rat pups. A and B: biphasic electrical pulse (top traces) induces an action potential (AP) with different shape. Note that F-type (A) cell shows a narrow AP and afterhyperpolarization (AHP) and S-type cell, wide AP and AHP. C: comparison of half-duration of AP and AHP between F- and S-type TG cells showing significant difference between the two classes.

Fig. 3.

Identification of whisker pad-innervating TG cells. A and B: stimulation of ION induces an AP with fixed latency from both F- and S-type TG cells. Note that each record is formed from 5 superimposed traces. C: plot latencies from both types of TG cells against postnatal days showing that there are no developmental changes in latency. D: averaged latency of F- and S-type TG cells showing no significant difference between them.

Fig. 4.

Effects of ION crush. A: the percentage of recorded S-type TG cells from normal TG (control) and those at different ages after ION crush. There is no significant difference between them, indicating that mechanical crush affects both types of TG cells. B: the proportion of recorded whisker pad-innervating cells shows the time course of ION functional recovery. Immediately after the crush, about ⅔ ION fibers are blocked, and functional recovery starts after postinjury 3 days and finishes at postinjury day 7.

Fig. 5.

Deafferentation-induced silent synapses are reversible after ION functional recovery. A: example record of functional synapses from a trigeminal principal nucleus (PrV) neuron. B: example record of silent synapses from a PrV neuron. C: plot of failure rate at holding potential of +60 mV and −70 mV for normal PrV showing silent synapses from two cells (denoted by asterisks) with high failure rate at −70 mV (lacking AMPA receptor-mediated EPSCs). D: at postinjury days 2–3, more PrV cells reveal silent synapses. E: at postinjury days 5–7, just a few PrV cells show silent synapses. F: the incidence of recorded silent synapses demonstrates that deafferentation-induced silent synapses are reversible after ION functional recovery.

Fig. 6.

ION crush-induced disruption of barrelettes in the PrV is not reversible. The PrV is a peanut-shaped nucleus, and the barrelettes are confined to the ventral half of the nucleus. In the brain stem, most sensory afferent terminals are positive for vesicular glutamate transporter 1 (VGluT1). VGluT1-labeled TG fiber terminals form whisker-related patches (barrelettes) in the sham side PrV at both postnatal day 3 (A) and 10 (C). Three (B) and ten (D) days after ION crush, the patchy terminal distribution is disrupted. Cytochrome oxidase staining reveals barrelette patterns in P3 (E) and P10 (G) sham-operated side better. The barrelettes are absent in nerve crushed side at P3 (F) and P10 (H). DCN, dorsal cochlear nucleus; TrV, trigeminal tract; MV, motor trigeminal nucleus. Asterisks outline the ventral, barrelette region of the PrV. In G, whisker barrelette rows a-e are marked. Scale bar = 250 μm.

Fig. 7.

Astrocytosis in response to ION crush. A–D show low- and high-power micrographs of GFAP labeling in the sham side PrV (A, C) and ION-crushed side PrV (B, D) on P3. E–H: similar series of micrographs showing GFAP labeling at P10. Note that increased glial response is diminished on the ION crush side (F, H) compared with that seen at P3 (B, D). I–L show representative camera lucida drawings of individual astrocytes from control (I, K) and nerve-crushed (J, L) PrV at P3 (I, J) and P10 (K, L). Asterisks outline the barrelette region of the PrV. In F, scale bar = 250 μm for all low-power micrographs, and in G, scale bar = 20 μm for all high-power micrographs; in L, scale bar = 10 μm for all camera lucida drawings. Sholl analysis of number of intersections is shown in M, and number of astrocyte process branch points is shown in N. Bar graphs in O and P show comparisons of astrocyte numbers and GFAP staining intensity, respectively.

Fig. 8.

Microglial response to ION crush. A–D show low- and high-power micrographs of Iba-1 immunolabeling in the sham-operated side PrV (A, C) and ION-crushed side PrV (B, D) on P3. E–H: similar series of micrographs showing Iba-1 immunolabeling at P10. Note that increased glial response is diminished on the ION crush side (F, H) compared with that seen at P3 (B, D). I–L show representative camera lucida drawings of individual astrocytes from control (I, K) and nerve-crushed (J, L) PrV at P3 (I, J) and P10 (K, L). Asterisks outline the barrelette region of the PrV. In B, scale bar = 250 μm for all low-power micrographs, and in G, scale bar = 20 μm for all high-power micrographs; in L, scale bar = 10 μm for all camera lucida drawings. Bar graphs in M and N show comparisons of microglia numbers and sampled microglia size (including the processes), respectively.