Postural modifications and neuronal excitability changes induced by a short-term serotonin depletion during neonatal development in the rat

Abstract

Serotonin (5-HT) plays an important role both in the development and in the recovery of locomotion after spinalization in vertebrates. We investigated the contribution of the serotonergic system to the maturation of the lumbar motoneurons and networks in the neonatal rat. A 5-HT synthesis inhibitor, p-chlorophenylalanine (PCPA), was administered daily from the first postnatal day (P0) onward. This protocol depleted serotonin in the spinal cord within 3-4 d, as demonstrated by immunohistochemistry. PCPA-treated rats exhibited postural changes characterized by lesser flexion at the knee and ankle levels and lesser extension of the hip. Posture was asymmetric, suggesting possible deficits in the interlimb coordination. Intracellular recordings were made at P3-5 from motoneurons innervating different hindlimb muscles, using the in vitro brainstem-spinal cord-nerve-attached preparation. In PCPA-treated rats, the conduction velocity of motoneurons was increased, and their excitability was decreased (because of higher rehobase and input conductance) compared with sham animals. In accordance with postural observations, changes were more pronounced in hip extensor/knee flexor than in ankle extensor motoneurons. The maturation of repetitive firing properties was stopped by PCPA treatment, although PCPA, applied in vitro, had no effect on membrane properties. The spontaneous endogenously generated activity, which is a characteristic of immature networks, was increased in PCPA-treated rats, suggesting that developing lumbar networks are sensitive to 5-HT levels. Serotonin may play a critical role during development in regulating the balance between the excitability of motoneurons and that of interneurons. Interneuronal excitability is crucial for the activity-dependent development of spinal cord networks.

Fig. 1.

In vitro brainstem–spinal cord–sciatic nerve preparation. A, Ventral view of the preparation used in this study. The inset shows a higher magnification of the different nerve branches left attached to the spinal cord that are stimulated to identify motoneurons functionally.Filled circles indicate the approximate location of stimulating electrodes. Branches were identified by their muscular target (B). B, Schematic lateral view of the rat hindlimb showing the location and insertions of different muscles. EDL, Extensor digitorum longus;G–Sol, gastrocnemius–soleus;PBST, posterior biceps, semitendinosus;TA, tibialis anterior. Scale bar, 1 cm.

Fig. 2.

Immunohistochemical confirmation that PCPA injections deplete serotonin in the spinal cord of neonatal rat.A, Schematic transverse section through the right hemicord of a lumbar segment, indicating approximately the location of the microphotographs in B and C (note that these are from different specimens). Dorsal is up, and lateral is to the right. B, Serotonergic fibers found close to lumbar motoneurons in a P0 rat.B1, Transverse section of the lumbar (L5) spinal cord, showing a Lucifer yellow-labeled motoneuron (arrowhead), previously identified as innervating the ankle flexor muscle, and TRITC-labeled (red) serotonergic varicosities and fibers (arrows). B2, Single fluorescent filter exposition of the same neuron showing a serotonergic fiber (arrow) in close proximity to the cell soma. Note that only the fluorescent filter for TRITC was used but that the outlines of the cell soma and nuclei are visible because of their intense labeling.C, Depletion of serotonin induced by PCPA injections. Transverse sections at lumbar (L5) level in P4 sham-injected (C1) and PCPA-injected (C2) animals. Serotonergic fibers (arrows) close to large cell bodies, presumably motoneurons (unlabeled; arrowheads), are numerous in the sham but were very sparse in the PCPA-treated specimen. The double-headed arrow in C1 indicates a blood vessel. Scale bar, 50 μm.

Fig. 3.

Age-related weight increase of sham and PCPA-treated rats. From P3 onward, PCPA-treated animals had a lower daily weight increase than shams (n = 23 for both groups). The weight increase returned to normal soon after the last PCPA injection (P5); the horizontal bar indicates the duration of the treatment.

Fig. 4.

Postural deficits in PCPA-treated rats.A, Dorsal view of a sham-injected (left) and a PCPA-injected (right) animal (P6), illustrating the difference in posture between the two groups. The postural asymmetry of the PCPA-treated specimen is demonstrated by a hyperextension of the right hindlimb. B, Graphs illustrating the positions of hindfeet relative to the navel (N, horizontal dotted line) and the body axis (vertical line). Each graph represents six observations (labeled from 1 to6). Angles between the body axis and the left (αl) or the right (αr) foot axis were measured. C, Postural asymmetry (C1), postural stability (C2), and rostrocaudal position of the toes relative to the navel (C3) (positive values mean rostral) for the sham-injected (left columns; n = 9) and PCPA-injected (right columns; n= 9) specimens. *p < 0.05; **p< 0.001; Student's t tests. Scale bars, 2 cm.

Fig. 5.

Changes in motoneuron excitability in PCPA-treated rats. Motoneuron conductance (A) and rheobase (B) are higher in PCPA-treated rats than in shams. C, Motoneuron rheobase and input conductance are positively correlated only in PCPA-treated specimens. D, Average conductance in functionally identified motoneurons of sham- or PCPA-treated specimens. Graphs show a trend toward higher rheobase and input conductance after PCPA treatment. The motoneurons innervating the ankle extensor muscles are the least affected. The number of neurons is indicated inbrackets. *p < 0.05; **p < 0.01; Student's ttests.

Fig. 6.

Effects of serotonin depletion on the conduction velocity. A, Conduction velocities from functionally identified motoneurons from sham- and PCPA-treated animals at P3–5 (averages for the whole population are indicated bydashed and solid lines, respectively; no significant difference between the two populations;p > 0.05). The number of neurons recorded in each group is indicated. *p < 0.05; Student's t test. B, Conduction velocity and input conductance are positively correlated in the two groups (PCPA-treated, solid line: r = 0.72,p < 0.001; sham, dashed line:r = 0.51, p < 0.05) but do not differ significantly from each other.

Fig. 7.

Serotonin depletion affects the maturation of repetitive firing properties. A, Discharge patterns observed in motoneurons after current injection at twice the rheobase: a single action potential or a doublet (top trace), a transient spike train (middle trace), or a sustained discharge throughout the pulse duration (bottom trace). Figure adapted from Vinay et al. (2000a). B, Proportion of G–Sol motoneurons generating the different patterns shown in A. Thenumber of recorded neurons in each population is indicated at the top of each bar. The data for P0–2 were obtained from noninjected specimens (Vinay et al., 2000a). C, Mean number of action potentials (top graph) during 500-msec-long repetitive discharge in G–Sol motoneurons of sham- and PCPA-injected specimens, plotted against the magnitude of depolarizing current (normalized to rheobase). There is no statistical difference between the regression lines.

Fig. 8.

Bath application of PCPA did not change electrical properties of motoneurons. A, Comparison of some membrane properties (input resistance and rheobase,left column) and steady-state discharge properties (number of action potentials and discharge frequency, right column) before and during bath application of PCPA (400 μm, 100 mg/kg). Recordings were obtained from lumbar motoneurons (n = 5) at P3–5. B, Examples of discharge patterns before (top trace) and during (middle trace) PCPA application. Sustained firing was induced by depolarizing square pulses at twice the rheobase (bottom trace).

Fig. 9.

Increase of spontaneous activity in serotonin-depleted animals. A, Spontaneous activity of a PBST motoneuron in a PCPA-treated specimen (P4). Intracellular recordings were made at resting potential (A1) and after a steady depolarization by current injection (1.4 nA) to reveal the inhibitory and excitatory nature of the postsynaptic potentials (A2). B, Quantification of the spontaneous activity in motoneurons from sham-injected (triangle) or PCPA-injected (square) specimens. The area under postsynaptic potentials, above the resting potential, was measured over 30 consecutive 1 sec bins and plotted against the time after dissection. A significant difference between mean spontaneous activity in PCPA-treated and sham motoneurons was found (p < 0.01; Student'st test). There was no correlation between spontaneous activity and time after dissection in either group.