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. 2014 Feb 28;9(2):e89999.
doi: 10.1371/journal.pone.0089999. eCollection 2014.

Serotonin, dopamine and noradrenaline adjust actions of myelinated afferents via modulation of presynaptic inhibition in the mouse spinal cord

David L García-Ramírez  1 , Jorge R Calvo  1 , Shawn Hochman  2 , Jorge N Quevedo  1
Affiliations

Serotonin, dopamine and noradrenaline adjust actions of myelinated afferents via modulation of presynaptic inhibition in the mouse spinal cord

David L García-Ramírez et al. PLoS One. .

Abstract

Gain control of primary afferent neurotransmission at their intraspinal terminals occurs by several mechanisms including primary afferent depolarization (PAD). PAD produces presynaptic inhibition via a reduction in transmitter release. While it is known that descending monoaminergic pathways complexly regulate sensory processing, the extent these actions include modulation of afferent-evoked PAD remains uncertain. We investigated the effects of serotonin (5HT), dopamine (DA) and noradrenaline (NA) on afferent transmission and PAD. Responses were evoked by stimulation of myelinated hindlimb cutaneous and muscle afferents in the isolated neonatal mouse spinal cord. Monosynaptic responses were examined in the deep dorsal horn either as population excitatory synaptic responses (recorded as extracellular field potentials; EFPs) or intracellular excitatory postsynaptic currents (EPSCs). The magnitude of PAD generated intraspinally was estimated from electrotonically back-propagating dorsal root potentials (DRPs) recorded on lumbar dorsal roots. 5HT depressed the DRP by 76%. Monosynaptic actions were similarly depressed by 5HT (EFPs 54%; EPSCs 75%) but with a slower time course. This suggests that depression of monosynaptic EFPs and DRPs occurs by independent mechanisms. DA and NA had similar depressant actions on DRPs but weaker effects on EFPs. IC50 values for DRP depression were 0.6, 0.8 and 1.0 µM for 5HT, DA and NA, respectively. Depression of DRPs by monoamines was nearly-identical in both muscle and cutaneous afferent-evoked responses, supporting a global modulation of the multimodal afferents stimulated. 5HT, DA and NA produced no change in the compound antidromic potentials evoked by intraspinal microstimulation indicating that depression of the DRP is unrelated to direct changes in the excitability of intraspinal afferent fibers, but due to metabotropic receptor activation. In summary, both myelinated afferent-evoked DRPs and monosynaptic transmission in the dorsal horn are broadly reduced by descending monoamine transmitters. These actions likely integrate with modulatory actions elsewhere to reconfigure spinal circuits during motor behaviors.

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Conflict of interest statement

Competing Interests: Shawn Hochman serves as an editor in PLOS ONE. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. Methods.
A, schematic representation of the experimental setup showing the hemisected spinal cord and the sciatic nerve with dissected nerve branches. Extracellular field potentials (EFPs) were recorded in the deep dorsal horn at L4. Microstimulation (μStim) was applied through the same micropipette where the evoked-EFPs were larger. Dorsal root potentials (DRPs) were recorded in dorsal roots L4 and L5. Peripheral nerves were stimulated at strengths based on the recruitment of the most excitable fibers. B, DRP (upper trace) and EFP (lower trace) produced by stimulation of the tibial nerve with strength 4 xT. By convention DRPs are presented with negativity upwards and EFPs with negativity downwards. C. Comparison of evoked responses observed at different stimulation strengths. Ci, afferent volley (upper traces) recorded in the sciatic nerve (Sci-AV) and EFP (lower traces) recorded at L4 by stimulation of the tibial (Tib) nerve. The fastest component of the sciatic afferent volley corresponds to myelinated (A cutaneous/I-III muscle) and the second one to unmyelinated afferents (C cutaneous/IV muscle). Note that at 4xT there is no evidence of a C fiber volley. Cii, DRP (upper traces) and EFP (lower traces) recorded at L4 level. Tib was stimulated with graded strengths from 1.6-10 xT. The short-latency EFP is maximal at 2 xT.
Figure 2
Figure 2. Modulatory actions of monoamines on EFPs and DRPs evoked by stimulation of low-threshold afferents.
Ai, DRPs (upper panel) recorded at L5 dorsal root and EFPs (lower panel) recorded in the dorsal horn at L4 before (black), during (red) and after (blue) bath application of 10 µM 5HT (traces are averages of 16 samples). Expanded segments of DRPs and EFPs are shown in insets. EFPs were recorded in the dorsal horn at 120 µm depth from the cut surface of the hemisected cord. The Tib nerve was stimulated at strength 4 xT. Note the remarkable depression of DRPs and short-latency EFPs in the presence of 5HT and the complete recovery after wash. Aside from the clear reversible depression of the larger short-latency EFP, the second short-latency (arrow) and the long-latency slow decaying EFP are also depressed. Aii–Cii, population averages of time course of monoamine-induced depression of evoked responses. Gray shadows indicate the period of bath application of monoamines. Aii, plot of the time course of effects produced by 5HT on L4-DRP (close circles), L5-DRP (open circles) and short-latency EFPs (open triangles). Arrows indicate the time of bath application of 5HT and the onset of drug wash out. Arrowheads point out the time corresponding to traces illustrated in Ai. Each point represents mean ± SD of 16 consecutive events for EFP and DRP amplitude with respect to control. Bi–Bii and Ci–Cii, description is identical to Figure Ai and Aii except that DA and NA actions are presented, respectively. Note the clear depression of DRPs in the presence of DA and NA and the incomplete recovery after NA wash. Short-latency EFPs are not significantly affected neither by DA nor NA but the smaller second short-latency (arrows) and the long-latency slow decaying EFP are depressed by these monoamines.
Figure 3
Figure 3. Summary graphs of the effects of monoamines on EFPs and DRPs evoked by stimulation of myelinated afferents.
In A–C the bars represent the percentage inhibition (mean ± SD; samples sizes reported inside individual bars) of L4- and L5-DRP and the fast short-latency EFP amplitude with respect to control. A, L4-DRPs and L5-DRPs and EFPs evoked by stimulation of the Tib nerve. B, L4-DRPs and EFPs evoked by the stimulation of the cutaneous nerves SP and Su and C, L4-DRPs and EFPs evoked by the stimulation of the muscle nerves PBSt, St and DP. All the nerves were stimulated with strengths 2-4 xT. For the Tib nerve (A) the effects of 5HT, DA and NA on L4- and L5-DRPs are greatly reduced, as is the effect of 5HT on short-latency EFP (P<0.05). The effects of 5HT, DA and NA on cutaneous and muscle afferent stimulation-evoked DRPs is similarly reduced (B and C; P<0.05) while 5HT and NA also depress evoked EFPs (P<0.05).
Figure 4
Figure 4. 5HT depresses monosynaptic transmission of low threshold afferent fibers.
A, EPSCs (upper panel) recorded on an unidentified L4 dorsal horn neuron and DRPs (lower panel) recorded at L4 dorsal root, before (black) during (red) and after (blue) bath application of 10 µM 5HT. Note the remarkable depression of EPSCs and DRPs, and the recovery after wash. B, EPSCs (upper panel) recorded on an unidentified L4 dorsal horn neuron and DRPs (lower panel) recorded at L5 dorsal root, before (black), during bath application of 1 mM mephenesin (green) and then 10 µM 5HT (red). Note that the monosynaptic component of the EPSC was virtually abolished after bath application of 5HT. C, summary graph of the depression (P<0.05) observed with 5HT and mephenesin + 5-HT on DRPs (filled bars) and EPSCs (open bars). The number of experiments is indicated inside graphed bars.
Figure 5
Figure 5. 5HT depresses low threshold-evoked EFPs and DRPs with a different time course.
A, EFPs (upper traces) and DRPs (lower traces) produced by the stimulation of the Tib nerve at 4xT and recorded at L4 dorsal horn and L4 dorsal root, respectively. Overlapped traces were recorded consecutively from 16–40 s (left panel) and from 52–76 s (right panel) after bath application of 5HT (10 µM). Red and blue traces correspond to the first recording for each period of time. Note that between 16–40 s after 5HT application DRPs are progressively depressed while the EFPs remain largely unaltered, and between 52–76 s the depression of DRPs reaches a plateau while EFPs are progressively depressed. Terminal potentials (arrows) are not affected by 5HT. B, comparison of the time course of decay of DRPs (blue circles) and EFPs (red circles). Note that depression of EFPs and DRPs is clearly phase-shifted. Vertical dashed line indicates the onset of EFP depression. Horizontal dashed line indicates DRP depression to 40% of control. Terminal potentials (TP, yellow triangles) remain unaltered in the presence of 5HT. C, summary graph of the effects of 5HT on the amplitude of terminal potentials respect to control. The effects were not statistically different (Wilcoxon test, P<0.05).
Figure 6
Figure 6. Monoamines depress DRPs in a dose-dependent manner while 5HT produces a biphasic effect on EFPs.
Concentration-response curves of 5HT, DA and NA at cumulative concentrations of 0.001, 0.01. 0.1, 1, 10 and 100 µM, on DRPs (Ai, B and C) and EFPs (Aii) evoked by the stimulation of the Tib nerve at 4xT. DRPs are depressed in a dose-dependent manner by the three monoamines. The monosynaptic component of the EFPs is facilitated by 5HT at doses below 1 µM and inhibited with doses of 10 and 100 µM. Each point represents mean ± SE of amplitude respect to control. The curves are constructed from 7, 5, 3 and 6 experiments, as indicated. For the effects of 5HT, DA and NA on DRPs the pIC50s are 6.2±0.1, 6.1±0.7 and 6.0±0.1 (mean ± SE), and the corresponding IC50s are 0.6, 0.8 and 1.0 µM, as indicated. For the effect of 5HT on EFPs the curve was biphasic and IC50 values were not calculated.
Figure 7
Figure 7. Comparing effects of the monoamines on muscle and cutaneous afferent-evoked DRPs and EFPs.
A–C, DRPs (upper traces) and EFPs (lower traces) evoked by the stimulation of the muscle nerve St (left panels) and the cutaneous nerve SP (right panels), both with strengths 4xT. In A–C black traces show control recordings before, and red traces after 5 min of bath application of 5HT, DA and NA 10 µM, respectively. Note that 5HT, DA and NA depressed DRPs, but only 5HT and NA depressed the fastest components of low threshold-evoked EFPs.
Figure 8
Figure 8. The monoamines have no effect on the excitability of myelinated afferent fibers.
A, DRPs recorded at L4 dorsal root and evoked by intraspinal microstimulation (μStim) in control (upper traces) or after application of 10 µM 5HT, DA, or NA each (lower traces). Insets show expanded segments of upper traces. Note that DRPs are preceded by a short-latency compound action potential (CAP). The CAP evoked at the stimulus intensities used was always submaximal. Note that monoamines depressed intraspinal microstimulation evoked-DRPs but not the short-latency CAPs. Traces are averages of 16 consecutive events and the effects of 5HT, DA and NA. These effects were largely reversible during washout of each drug. B, summary graph of the effects seen. 5HT, DA and NA strongly depressed microstimulation-evoked DRPs (P<0.05) but not CAPs. The number of experiments is indicated inside the bars.
Figure 9
Figure 9. Summary interpretation of changes observed.
Drawing highlights putative sites of action of the monoamine transmitters. 1. The short-latency EFP reflects monosynaptic transmission from primary afferents, which is reduced by 5HT and NA, but not DA. As this occurs in the absence of changes in excitability of primary afferents (no change in CAP amplitude), actions are likely exerted via activation of 5HT and NA metabotropic receptors (red and yellow dots, respectively). These receptors will act on signal transduction pathways to reduce synaptic transmission and/or reduce glutamate receptor responsiveness on postsynaptic interneurons. 2. The intraspinal circuit responsible for PAD may involve one or more interposed interneurons, as depicted in the schematic. Monoamines may reduce the excitability of these interneurons and/or reduce transmission between first and higher-order interneurons by activation of 5HT, NA and DA receptors (red, black and yellow dots, respectively) so that fewer interneurons are recruited to produce PAD. 3. 5HT, DA, and NA may act at the last order GABAergic axo-axonic synapse leading to reduced activation of the GABAA-like receptors responsible for producing PAD.
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