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. Author manuscript; available in PMC: 2023 Dec 27.
Published in final edited form as: Exp Brain Res. 2020 Apr 22;238(5):1293–1303. doi: 10.1007/s00221-020-05805-y

Activation of α-adrenoceptors depresses synaptic transmission of myelinated afferents and inhibits pathways mediating primary afferent depolarization (PAD) in the in vitro mouse spinal cord

Elvia Mena-Avila 1, Jonathan J Milla-Cruz 1, Jorge R Calvo 1, Shawn Hochman 2, Carlos M Villalón 3, José-Antonio Arias-Montaño 1, Jorge N Quevedo 1,*
PMCID: PMC10751985  NIHMSID: NIHMS1586904  PMID: 32322928

Abstract

Somatosensory afferent transmission strength is controlled by several presynaptic mechanisms that reduce transmitter release at the spinal cord level. We focused this investigation on the role of α-adrenoceptors in modulating sensory transmission in low-threshold myelinated afferents and in pathways mediating primary afferent depolarization (PAD) of neonatal mouse spinal cord. We hypothesized that the activation of α-adrenoceptors depresses low threshold-evoked synaptic transmission and inhibits pathways mediating PAD. Extracellular field potentials (EFPs) recorded in the deep dorsal horn assessed adrenergic modulation of population monosynaptic transmission, while dorsal root potentials (DRPs) recorded at root entry zone assessed adrenergic modulation of PAD. We found that noradrenaline (NA) and the α1-adrenoceptor agonists phenylephrine and cirazoline depressed synaptic transmission (by 15, 14 and 22%, respectively). DRPs were also depressed by NA, phenylephrine and cirazoline (by 62, 30, and 64%, respectively), and by the α2-adrenoceptor agonist clonidine, although to a lower extent (20%). We conclude that NA depresses monosynaptic transmission of myelinated afferents onto deep dorsal horn neurons via α1-adrenoceptors and inhibits interneuronal pathways mediating PAD through the activation of α1- and α2-adrenoceptors. The functional significance of these modulatory actions in shaping cutaneous and muscle sensory information during motor behaviors requires further study.

Introduction

Presynaptic inhibition (PSI) is a fundamental mechanism for gain control of primary afferent neurotransmission at intraspinal terminals. A common form of PSI is via metabotropic receptor-mediated modulation of transmitter release (Starke et al., 1989; Wu et al., 1997). For primary afferents, this includes actions via presynaptic GABAB (Peng et al., 1989; Salio et al., 2017) and monoamine receptors (Yuan et al., 2009; García-Ramírez et al., 2014; Lu et al., 2018).

One monoamine transmitter involved in the metabotropic control of sensory neurotransmission is NA. The present study focuses on α1- and α2-adrenoceptors extensively expressed in the spinal cord (Day et al., 1997; Stone et al., 1998; Nicholson et al., 2005) and dorsal root ganglia neurons (Shi et al., 2000; Pluteanu et al., 2002; Nicholson et al., 2005).

Selective α1- and α2-adrenoceptor activation is implicated in the depression of synaptic transmission from high-threshold afferents in superficial dorsal horn interneurons (Kawasaki et al., 2003; Sonohata et al., 2004; Yuan et al., 2009). Activation of α1- and α2-adrenoceptors also alters the excitability of lamina II interneurons by preferential α1-adrenoceptor-mediated depolarization of inhibitory, and α2-adrenoceptormediated hyperpolarization of excitatory interneurons, through the activation of Gαq/11 and Gαi/o proteins, respectively (Gassner et al., 2009). In sum, α1- and α2-adrenoceptors are positioned to depress nociceptive transmission by facilitating inhibitory and depressing excitatory circuits. Facilitation of presynaptic GABAA receptors on high-threshold afferent terminals by α1-adrenoceptors (Yuan et al., 2009), is consistent with an ionotropic form of PSI seen at primary afferent axoaxonic synapses that generates PAD (Barker et al., 1972; Rudomin et al., 1999), which can be recorded experimentally as an antidromically-propagating DRP.

Adrenoceptors are also involved in the modulation of proprioceptive afferents. In cats, electrical recruitment of A6 coerulospinal projections depressed synaptic transmission from group II muscle spindle afferents and produced PAD on the same group II afferents (Riddell et al., 1993; Noga et al., 1992). Discrete actions were subsequently differentiated into α1- and α2-adrenoceptor responses (Hammar et al., 2003). The α1-adrenoceptor agonist phenylephrine facilitated responses in interneurons activated by groups Ia and Ia/Ib muscle afferents, while the α2-adrenoceptor agonist clonidine depressed the activity of the same group of interneurons (Hammar et al., 2003). Whether these actions occur pre- and/or postsynaptically remains to be determined.

Early studies demonstrated that NA slightly reduces the amplitude of afferent-evoked excitatory monosynaptic responses by mechanisms independent of membrane resistance (Garraway et al., 2001a, b). However, the use of high intensity electrical stimuli precluded knowledge on afferent identity. Our more recent studies in the neonatal mouse deep dorsal horn focused on lower threshold afferents from muscle, cutaneous and mixed nerves (García-Ramírez et al., 2014). We observed comparable NA depressant modulatory actions on DRPs evoked by all nerves, but significant depressant actions only on cutaneous- and muscle-evoked EFPs. However, these studies did not determine the identity of adrenoceptor subtypes, nor the pre- or postsynaptic locus of action.

Our interest to explore adrenergic modulation on presynaptic inhibition arose from the lack of evidence on the role of specific subtypes of adrenoceptors in modulating PSI of low-threshold afferent fibers. This study investigates the role of α1- and α2- adrenoceptors in the modulatory depressant actions of NA on synaptic transmission of myelinated afferents and on pathways mediating PAD. Studies on β adrenoceptors were not undertaken, as they are not expressed in low threshold mechanoreceptors (Zheng et al., 2019) nor alter group I synaptic transmission in interneurons (Hammar et al., 2003). We found that NA depresses monosynaptic transmission of myelinated afferents by the activation of α1-adrenoceptors and inhibits interneuronal pathways mediating PAD through the activation of α1- and α2-adrenoceptors.

Methods

Ethics Statement

All the procedures described here comply with the guidelines contained in the National Institutes of Health Guide for the Care and Use of Laboratory Animals (USA) and were approved by the Institutional Animal and Use Committee in the Center for Research and Advanced Studies (Mexico).

Dissection

Experiments were performed in 6–7 day-old BALB/c mice of both genders. Animals were anesthetized with 10% urethane (2 mg/kg, i.p.) before decapitation at the cervical level. The thoracic to lumbar cord was exposed by a dorsal and ventral approach in ice-cold high-sucrose solution (in mM: sucrose 250, NaCl 12, KCl 2.5, D-glucose 25, MgS04 3.0, CaCl2 1.0, NaH2PO4 1.25, NaHCO3 26, gassed with 95% O2 / 5% CO2). A dorsal laminectomy and a ventral vertebrectomy were performed with special care to maintain the paravertebral muscles and both dorsal and ventral spinal roots in continuity with peripheral nerves. The sciatic nerve and branches were dissected free and the rest of the limb was removed. The preparation was pinned ventral side down in a Sylgard-coated Petri dish. A sagittal cord hemisection was performed by means of an insect pin. Peripheral nerves tibial (Tib), deep peroneal (DP), and sural (SU; caudal cutaneous branch) were dissected free and sectioned distally. A hemisected cord with dissected sciatic nerve and branches was transferred to a 5 ml bath chamber with artificial cerebrospinal fluid (ACSF; in mM: NaCl 125, KCl 2.5, D-glucose 25, MgS04 1.0, CaCl2 2.4, NaH2PO4 1.25, and NaHCO3 26). The ACSF was perfused by gravity and recirculated with a peristaltic pump at 10–15 ml/min, maintained at 23°C and gassed with 95% O2 / 5% CO2. The preparation was then allowed to recover for about 1 h before any other manipulation. Careful considerations were undertaken to ensure a lack of time-dependent change in recording quality, including continuously monitoring for threshold afferent activation and stability of DC recordings from dorsal roots.

Stimulation and recording

The experimental setup is detailed in García-Ramírez et al. (2014). Briefly, peripheral nerves were stimulated electrically with glass suction electrodes, with 0.2 ms single-shock stimuli every 10 s at strength 2 times the threshold to recruit the most excitable afferent fibers (xT) in the dorsal root (DR). Conditions were set to ensure that stimulation with strength 2 xT of the Tib, DP and SU nerves, recruited the faster conducting myelinated afferents (presumed to be Aβ/I-II), as myelination is incomplete at early postnatal age (see Vejsada et al., 1985). In neonatal mice, the low-threshold afferent volley (AV) evoked by stimulation of peripheral nerves with strength 2 xT is quite spread due to slowed conduction, arising from incomplete myelination, and also substantially further reduced by experimentation conducted at room temperature (Pinto et al., 2008). For this reason, low-threshold, nerve-evoked EFPs may display several short-latency components, all of which were considered as monosynaptic (Figs. 1, 3–5).

Fig. 1.

Fig. 1

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NA depresses low threshold EFPs and DRPs from different sensory origin. a Left panel, EFPs evoked by stimulation of the Tib nerve at 2 xT and recorded at L4, before (black), during (red) and after (blue) bath application of NA (10 μM). The inset shows an expanded segment of EFPs. Right panel, summary plot of NA effects. b Left panel, DRPs recorded from DR L4 (same color code as in a). Right panel, summary plot of effects. c and d Analysis of the effect of NA on EFPs and DRPs evoked by stimulation of Tib (purple), SU (blue) and DP (green) nerves at 2 xT. In all plots, numbers inside boxes indicate the number of experiments. a, p <0.001 vs control; b, p <0.001 vs NA; ns, not statistically different (ANOVA and Tukey’s test)

Fig. 3.

Fig. 3

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The α1-adrenoceptor agonist phenylephrine reduces synaptic transmission of myelinated afferents and inhibits pathways mediating PAD. EFPs and DRPs were evoked by stimulation of the Tib nerve at 2 xT and recorded at L4. a Left panel, EFPs before (black), during (red) and after (blue) bath application of phenylephrine (10 μM). The inset shows a magnified segment of EFPs. Right panel, summary plot of effects. b Same format as in a, for DRPs. Note that phenylephrine depressed EFPs and DRPs. c Left panel, the α1-adrenoceptor antagonist prazosin (20 μM) had no effect on EFPs amplitude per se (purple), but prevented the depressant action of phenylephrine (red). Right panel, summary plot of effects. d Same format as in c, for DRPs. Prazosin prevented the depressant effect of phenylephrine on DRPs. a, p <0.01; b, p <0.001 vs control values (ANOVA and Tukey’s test). phen, phenylephrine; praz, prazosin

Fig. 5.

Fig. 5

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Activation of α2-adrenoceptors inhibits neuronal pathways mediating PAD with no effect on afferent synaptic transmission. EFPs and DRPs were evoked by stimulation of the Tib nerve at 2 xT recorded at L4. a Left panel, EFPs before (black), during (red) and after (blue) bath application of clonidine (10 μM). The inset shows a magnified segment of EFPs. Right panel, summary plot of effects. b Same format as in a, for DRPs. c Left panel, bath application of both the α2-adrenoceptor antagonist atipamezole (1 μM) and clonidine (10 μM) failed to affect EFPs amplitude (purple and red, respectively). Right panel, summary plot of effects. d Same format as in c, for DRPs. a, p <0.01 vs control (ANOVA and Tukey’s test). clon, clonidine, atip, atipamezole

Extracellular field potentials (EFPs) were recorded in the deep dorsal horn with micropipettes (1–2 MΩ) filled with 2 M NaCl. Micropipettes were placed perpendicularly through the cut surface of the L3–5 spinal segment of the cord, between the central canal and the border of the dorsal column (approximately between III–VI Rexed laminae) at depths 60–200 μm, until finding the largest amplitude EFPs. As myelinated afferent fibers are glutamatergic, the monosynaptic component of EFPs reflects the excitatory postsynaptic currents on dorsal horn interneurons activated by a population of afferent fibers (Jahr and Yoshioka,1986; Jessell et al., 1986). The longer, slow-decaying EFPs reflect the field potential of PAD recorded intraspinally.

PAD was inferred from DRPs recorded at DRs L4 or L5 by means of a glass suction electrode (~120 μm tip internal diameter) placed en passant on dorsal roots near the entry zone. DRPs represent the passive antidromic propagation of PAD generated in the central terminals of a population of afferent fibers (Rudomin et al., 1999). The short-latency components recorded from DRs correspond to the orthodromically-arriving AVs, which were not affected by the application of NA (García-Ramírez et al., 2014).

EFPs were recorded with a MultiClamp 700B amplifier (Molecular Devices, USA), filtered at 2 KHz, and digitized at 20 KHz using a Digidata 1322A A/D card (Axon Instruments, USA). DRPs were recorded with custom made AC-coupled amplifiers (band pass filter 0.1 Hz – 3 KHz), or with DC-coupled amplifiers (A-M Systems, USA; band pass filter DC – 3 KHz). Raw data were collected with a pClamp software (v.10.2, Molecular Devices, USA), and stored for off-line analysis.

Drugs

Noradrenaline bitartrate, phenylephrine hydrochloride, cirazoline hydrochloride, prazosin hydrochloride, clonidine hydrochloride, desipramine hydrochloride and atipamezole hydrochloride were purchased from Sigma-Aldrich (St Louis, MO). Stock solutions (10 mM) of all drugs, except NA, were prepared in DMSO and stored at −20°C until needed. To ensure accurate dose-response assessment of NA actions sodium metabisulfite (0.1%) was added to prevent NA oxidation and 10 μM desipramine to block NA uptake (see Krnjevic et al.,1978; Garraway et al., 2001b). All drugs were dissolved in normal ACSF, superfused in the recording chamber from separate gravity-fed reservoirs at known concentrations for 10 min each. Depending on the drug, the concentration range was 1–20 μM. Cumulative concentration-response curves for NA, phenylephrine and clonidine were performed with concentrations 0.001, 0.01. 0.1, 1, 10 and 100 μM.

Data analysis

The magnitude of short-latency EFPs was determined as the amplitude from baseline to the peak. Latencies for EFPs were measured from the shortest afferent volley to the onset inflection of EFPs. As DRPs are preceded by spread AVs and dorsal root reflexes (Fig. 1), the magnitude of DRPs was determined as the area under the curve (AUC) from the depolarization onset up to 200 ms.

All data are expressed as percentage of control values (mean ± SEM), and traces are averages of 12 samples, unless otherwise stated. For statistical comparisons, ANOVA followed by Tukey’s test was employed. All differences were considered significant if p < 0.05.

Results

NA was tested in 41 experiments, recording both DRPs and EFPs. In 23 experiments, DRPs and EFPs were evoked by stimulation of the Tib nerve, and in 18 experiments by stimulation of SU (n = 11) and DP (n = 7) nerves. To test the effect of α-adrenoceptor ligands on EFPs and DRPs (n = 49), stimulation was to the mixed Tib nerve. All nerves were stimulated at strength 2 xT.

Effects of NA on EFPs and DRPs evoked by stimulation of low-threshold afferents from different nerves

To investigate the noradrenergic modulation of synaptic transmission and PAD evoked by stimulation of myelinated afferents from selective nerves, we first examined the effect of NA on the monosynaptic component of EFPs and DRPs recorded at L4 level. NA (10 μM) reduced the monosynaptic component of EFPs evoked by stimulation of the Tib (mixed), SU (cutaneous) and DP (muscle) nerves by 15 ± 2% (n = 23), 17 ± 3% (n = 11) and 17 ± 4% (n = 7; Fig. 1 a and c). The depressant effect was long-lasting and not reversible after 1 h wash, being of similar magnitude for EFPs evoked by stimulation of the three different nerves (Fig. 1c).

NA (10 μM) also decreased the AUC of simultaneously recorded DRPs evoked by the stimulation of Tib, SU and DP nerves by 62 ± 3% (n = 23), 59 ± 5% (n = 11) and 44 ± 2% (n = 7) of control values, respectively (Fig. 1b and d). The magnitude of depression of DRPs evoked by stimulation of different nerves was similar, and the effect was only partially reversed after 1 h wash.

As the effect produced by NA on EFPs and DRPs from different sensory origin were equally depressed, for the rest of the experiments we stimulated the Tib nerve.

NA-induced depression of myelinated-evoked EFPs and DRPs is mediated by α-adrenoceptors

To test whether the NA depressant effect was mediated by α-adrenoceptors, we constructed concentration-response curves. The inhibition by NA of EFPs was concentration-dependent, with maximum inhibition 26 ± 10% and IC50 188 nM (pIC50 6.72 ± 0.28). The α1-adrenoceptor agonist phenylephrine mimicked the effect of NA on EFPs with maximum inhibition 25 ± 6% and IC50 779 nM (pIC50 6.11 ± 0.42). In contrast, the α2-adrenoceptor agonist clonidine had no effect. These results suggest that the inhibition of synaptic transmission produced by NA is mediated by α1-adrenoceptors (Fig. 2a).

Fig. 2.

Fig. 2

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The NA depressant effect on synaptic transmission and PAD is mediated by α-adrenoceptors. a and b, Effect of NA, the α1-adrenoceptor agonist phenylephrine, and the α2-adrenoceptor agonist clonidine on EFPs and DRPs. Ligands were applied in a cumulative manner. The agonist phenylephrine mimicked the effect of NA on EFPs, whereas clonidine had no effect (a). Depression of DRPs by NA was reproduced by phenylephrine and clonidine (b). Note that clonidine was less efficacious for depressing DRPs. The curves drawn are the best-fit to a logistic equation (n = 4). Values for IC50 and maximum inhibition are given in the text

The depression of the DRPs by NA (Fig. 2b) was also concentration-dependent with maximum inhibition 70 ± 8% and IC50 191 nM (pIC50 6.71 ± 0.27). DRPs were depressed by the α1-adrenoceptor agonist phenylephrine with maximum inhibition 67 ± 7% and IC50 188 nM (pIC50 6.72 ± 0.28), and by the α2-adrenoceptor agonist clonidine with maximum inhibition 38 ± 5% and IC50 2.6 μM (pIC50 5.58 ± 0.82). These results suggest that NA inhibits interneuronal pathways mediating PAD by activating α2- and α1-adrenoceptors.

Activation of α1-adrenoceptors reduces synaptic transmission of myelinated afferents and inhibits neuronal pathways mediating PAD

Bath application of the α1-adrenoceptor agonist phenylephrine (10 μM), reduced the amplitude of Tib-evoked EFPs by 14 ± 2% (Fig. 3a) and DRPs AUC by 30 ± 2% (n = 19, Fig. 3b). These effects were long-lasting and remained after 1 h wash. Depression of EFPs and DRPs by phenylephrine (10 μM), was prevented by prior application of the α1-adrenoceptor antagonist prazosin (20 μM) with values −5 ± 4% and −13 ± 5% of controls, respectively (Figs. 3c and d).

Depression of EFPs and DRPs by the α1-adrenoceptor agonist cirazoline (10 μM) was 22 ± 4% and 64 ± 3%, respectively (n = 11), mimicking the effect of NA and phenylephrine (Fig. 4; see Discussion). The cirazoline effect was also long-lasting and not fully reversible.

Fig. 4.

Fig. 4

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The α1-adrenoceptor agonist cirazoline reduces synaptic transmission of myelinated afferents and inhibits pathways mediating PAD. EFPs and DRPs were evoked by stimulation of the Tib nerve at 2 xT and recorded at L4. a Left panel, EFPs before (black), during (red) and after (blue) bath application of cirazoline (10 μM). The inset shows a magnified segment of EFPs. Right panel, summary plot of effects. b Same format as in a, for DRPs. Note that cirazoline reduces both EFPs and DRPs. a, p <0.01; b, p <0.001 vs control (ANOVA and Tukey’s test). cira, cirazoline

Activation of α2-adrenoceptors inhibits neuronal pathways mediating PAD with no effect on low-threshold afferent synaptic transmission

As shown in Fig. 2, the α2-adrenoceptor agonist clonidine depresses Tib-evoked DRPs with no effect on simultaneously recorded EFPs. Fig. 5 shows that bath application of clonidine (10 μM) had no effect on EFPs (−4 ± 3%), but decreased DRPs by 20 ± 4% (n = 7). The depressant effects of clonidine on DRPs were long-lasting and not reversible up to 60 min recording. To block the effect of the α2-adrenoceptor agonist, we used the α2-adrenoceptor antagonist atipamezole (1 μM). Figure 5c shows that neither the antagonist (−1 ± 2%, n = 6) nor the agonist (−2 ± 6%, n = 6) produced a significant effect on EFPs. Fig. 5d illustrates that atipamezole per se produced no effect on DRPs (2 ± 4%, n = 6), but prevented DRP depression induced by clonidine (−1 ± 6%, n = 6). Therefore, depression of DRPs by clonidine occurs through the activation of α2-adrenoceptors.

Discussion

We show that NA reduces synaptic transmission by activating α1-adrenoceptors in myelinated afferent terminals, and inhibits the interneuronal pathway mediating PAD by the activation of both α1- and α2-adrenoceptors, possibly located on interneurons mediating PAD. The reduction in PAD by α1-adrenoceptors could be partly due to a decrease in afferent transmission. Specifically, the α1-adrenoceptor agonists phenylephrine and cirazoline mimicked the depressant effect of NA on EFPs and DRPs, and the effect of phenylephrine on EFPs and DRPs was prevented by prazosin, a selective antagonist of α1-adrenoceptors. Depression of DRPs by the α2-adrenoceptor agonist clonidine, and blockade by the antagonist atipamezole indicate the presence of α2-adrenoceptors on pathways mediating PAD. The lack of effect of the α2-adrenoceptor agonist clonidine on EFPs suggests that α2-adrenoceptors have no role in modulating the synaptic efficacy of myelinated afferents (see Fig. 6).

Fig. 6.

Fig. 6

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Schematic representation of the possible location of α1- and α2-adrenoceptors modulating synaptic transmission of low-threshold afferents and neuronal pathways mediating PAD. a The PAD circuit comprises a tri-synaptic pathway: glutamatergic low-threshold afferents (green), glutamatergic first-order interneurons (green) and a last-order GABAergic interneurons (pink), contacting afferent fibers axoaxonically. Descending noradrenergic fibers are represented in red. b Magnification of the circuit shown in a. Synaptic transmission of low-threshold afferents is reduced by the activation of α1-adrenoceptors at presynaptic terminals (purple). PAD is inhibited by the activation of α1- (purple) and α2-adrenoceptors (blue), possibly expressed by the first (+) and/or last (−) order interneurons (see Discussion)

Previously, García-Ramírez et al. (2014) observed that NA (10 μM) had no significant effect on low-threshold Tib-evoked EFPs, but led to a small yet significant reduction in the low-threshold evoked in other cutaneous- and muscle-evoked EFPs. Here, we also found that NA reduced synaptic transmission by slightly greater magnitude presumably by the use of the NA transporter (NET) inhibitor desipramine (O’Neill et al., 2010). In addition, to reduce degradation we used NA freshly prepared, an antioxidant agent, and performed experiments under low light conditions (Barrière et al., 2008; Tartas et al., 2010).

Although we stimulated cutaneous (SU) and muscle (DP) nerves to produce EFPs and DRPs, in most experiments we used the mixed nerve Tib, more suitable for dissection and thick enough to be placed on suction electrodes for stimulation. To estimate the effects of NA and α-adrenoceptor agonists on transmitter release, we recorded the monosynaptic component of EFPs, rather than monosynaptic EPSPs on individual interneurons. This enables investigating the global effect of NA and agonists on synaptic transmission from a population of low-threshold afferent fibers.

We observed the depressant effects of NA and α-adrenoceptor agonists to be maintained at least 1 h after drug removal (Harvey et al., 2006). This could be due to long-lasting effects implicating signal pathways difficult to reverse. In fact, long-lasting activity-independent facilitation or depression of spinal reflexes have been observed previously with serotonin (Machacek et al. 2001; Shay et al. 2005).

Possible mechanisms of the reduction in transmitter release from low-threshold afferents by NA

As NA and the α1-adrenoceptor agonists phenylephrine and cirazoline depressed EFPs, modulatory adrenergic actions must be mediated by presynaptic α1-adrenoceptors on primary afferents contacting first order interneurons (Figs. 1a and c, 2a, 3a and 4a). Therefore, any mechanism leading to calcium decrease in afferent terminals may reduce synaptic transmission. The expression of α1-adrenoceptors have been reported in IB4+, calcitonin gene related peptide (CGRP)+ and NF200+ afferents (Drummond et al., 2014), and more recently Zheng et al. (2019) reported the presence of mRNA for the α1A- and α1D subtypes in low threshold mechanoreceptors.

The α1-adrenoceptor agonist phenylephrine reduced high-threshold primary afferents transmission on lamina II interneurons by activating GABAergic interneurons. This inhibition might involve PAD of high-threshold afferents (Yuan et al., 2009). However, since α1-adrenoceptor agonists depress DRPs, a decrease of myelinated afferent transmission is generated by the activation of presynaptic α1-adrenoceptors (Figs. 3 and 4) and not by an increase in GABAA receptor-mediated PAD.

Possible mechanisms involved in the inhibition by α-adrenoceptors of pathways mediating PAD

We found that NA and both α1- and α2-adrenoceptor agonists (phenylephrine and clonidine) could independently depress DRPs (Figs. 2b, 3b and 5b), with the agonist effects being prevented by their respective antagonists prazosin (Fig. 3d) and atipamezole (Fig. 5d). Depression of DRPs could be accounted for by the reduction in the afferent input or inhibition of pathways mediating PAD.

Nonetheless, that clonidine reduces DRPs with no effect on EFPs (Fig. 5) suggests an inhibitory action on pathways mediating PAD. Unfortunately, with our experimental approach is unlikely to determine whether the α-adrenoceptors are located on first- or last-order interneurons in pathways mediating PAD (Zimmerman et al., 2019), and whether they are located on the somata or on afferent terminals (see Fig. 6). In summary, our results indicate that α1- and a2-adrenoceptors are located postsynaptically on the interneurons mediating PAD (Fig 6) and their activation produces a relative facilitation of synaptic transmission associated to a decrease of PSI related to PAD.

Afferent fibers giving and receiving PAD when stimulating low-threshold afferent fibers in the neonatal mouse

Information about the organization of pathways mediating PAD in rodents is scarce. In analogy with the cat, we considered that, in general, stimulation of low-threshold muscle (group I-II) and cutaneous (Aβ) afferents produces PAD with a similar pattern (Brink et al., 1984; Riddell et al., 1993; Rudomin et al., 1999). Stimulation of myelinated afferents in the mixed Tib nerve produces thus PAD in groups I, II and low-threshold cutaneous afferents. It is not clear to what extent stimulation of low-threshold afferents produces PAD on high-threshold (Aδ and C) afferents (Rudomin et al., 1999), and Zimmerman et al. (2019) recently reported that in the mouse stimulation of Aδ and C afferents produces PAD in Aβ afferents through glutamatergic VGlut3+ neurons. In accord with a previous study (García-Ramírez et al., 2014), we found that NA depressed EFPs and DRPs to the same extent for both muscle and cutaneous nerves, indicating that NA affects similarly low-threshold evoked synaptic transmission and PAD, regardless of low-threshold afferent identity (Fig. 1).

Functional segregation of adrenoceptors in synaptic transmission of low- and high-threshold afferents

The activation of α1- and α2-adrenoceptors reduces high-threshold-evoked responses (Sullivan et al., 1987; Kawasaki et al., 2003; Yuan et al., 2009), and α1-adrenoceptor activation reduces synaptic transmission of low-threshold afferents (Figs. 2, 3 and 4). However, we found that activation of α2-adrenoceptors failed to affect synaptic transmission of low-threshold afferents (Figs. 2 and 5). Hence, there seems to be a functional segregation of α1- and α2-adrenoceptors in the modulation of synaptic transmission between low- and high-threshold afferents. The differential expression of α1- and α2-adrenoceptors on low- and high-threshold afferents could explain the different effects produced by NA, although this appears not to be the case (Shi et al., 2000; Dawson et al., 2011; Drummon et al., 2014).

One limitation of our experimental approach could be related to the differential expression of α-adrenoceptors on different types of low-threshold afferents or on different subpopulations of interneurons, including those mediating PAD. For example, in heterologous expression systems, α1-adrenoceptor subtypes show different affinity for NA, with a rank order α1D > α1B > α1A (Shibata et al., 1995). The α1-adrenoceptor agonists phenylephrine and cirazoline have different affinities for each adrenoceptor subtype (Minneman et al., 1994), with a rank order α1D > α1A> α1B for phenylephrine (Buckner et al., 2002), and α1A > α1D> α1B for cirazoline (Horie et al., 1995). We found a different extent of inhibition by phenylephrine or cirazoline of synaptic transmission and PAD, with cirazoline being more efficacious than phenylephrine. This may suggest a differential expression of α1-adrenoceptor subtypes on afferent fibers and interneurons mediating PAD (Figs. 3 and 4). However, at the concentrations used, we cannot assure the selective activation of α1-adrenoceptor subtypes by cirazoline.

Similarly, in heterologous expression systems the affinity of α2-adrenoceptors for NA depends on the α2-adrenoceptor subtype expressed, with a rank order α2A > α2C > α2B. This pharmacological profile differs for clonidine, with affinity α2A > α2B > α2C (Pihlavisto et al., 1998), and the use of clonidine as the only α2-adrenoceptor agonist (Fig. 5) precluded therefore the identification of specific α2-adrenoceptor subtypes.

The chemical structures of dopamine and NA are similar, and previous studies suggested a functional action of NA at members of the D2-like receptor family (Newman-Tancredi et al., 1997; Czermak et al., 2006; Root et al., 2015). Thus, possible actions of NA on dopamine receptors cannot be excluded, but the effect of selective agonists supports that the observed actions are mediated by α-adrenoceptors.

Functional implications

The reduction in PAD by NA would lead, in principle, to an increase in the synaptic efficacy of low-threshold afferents. This may provide a compensatory mechanism when occurring in conjunction with a reduction of synaptic transmission. A reduction in PAD-related PSI by NA would reinforce proprioceptive feedback during movement, consistent with the view that NA facilitates circuits engaged in motor behaviors. It is thus possible that noradrenergic modulation of both synaptic transmission and pathways mediating PAD contributes to the fine tuning of the synaptic transmission from low-threshold sensory information.

The profuse noradrenergic innervation to the spinal cord and the functional changes displayed after spinal cord injury (Harvey et al., 2006; Rank et al., 2011) emphasize the importance of investigating the physiological role of the noradrenergic system in synaptic transmission and pathways mediating PAD. The functional significance of noradrenergic modulatory actions in shaping cutaneous and muscle sensory information during motor behaviors requires further study.

Knowledge about the α-adrenoceptor subtypes involved could be physiologically relevant and provide basis for therapeutic strategies to improve locomotion and reduce spasticity after spinal cord injury (Stewart et al., 1991; Rémy-Néris et al., 1999).

Footnotes

Conflict of interest The authors declare that they have no conflict of interest.

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