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High-threshold primary afferent supply of spinal lamina X neurons

Krotov, Volodymyra,b,*; Tokhtamysh, Anastasiaa; Safronov, Boris V.c,d; Belan, Pavelb,e; Voitenko, Nanaa,e

doi: 10.1097/j.pain.0000000000001586
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The spinal gray matter region around the central canal, lamina X, is critically involved in somatosensory processing and visceral nociception. Although several classes of primary afferent fibers terminate or decussate in this area, little is known about organization and functional significance of the afferent supply of lamina X neurons. Using the hemisected ex vivo spinal cord preparation, we show that virtually all lamina X neurons receive primary afferent inputs, which are predominantly mediated by the high-threshold Aδ- fibers and C-fibers. In two-thirds of the neurons tested, the inputs were monosynaptic, implying a direct targeting of the population of lamina X neurons by the primary nociceptors. Beside the excitatory inputs, 48% of the neurons also received polysynaptic inhibitory inputs. A complex pattern of interactions between the excitatory and inhibitory components determined the output properties of the neurons, one-third of which fired spikes in response to the nociceptive dorsal root stimulation. In this respect, the spinal gray matter region around the central canal is similar to the superficial dorsal horn, the major spinal nociceptive processing area. We conclude that lamina X neurons integrate direct and indirect inputs from several types of thin primary afferent fibers and play an important role in nociception.

Spinal lamina X neurons integrate direct and indirect inputs from several types of thin primary afferent fibers, thus playing an important role in nociception.

aDepartment of Sensory Signaling, Bogomoletz Institute of Physiology, Kiev, Ukraine

bDepartment of Molecular Biophysics, Bogomoletz Institute of Physiology, Kiev, Ukraine

cInstituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal

dNeuronal Networks Group, Instituto de Biologia Molecular e Celular (IBMC), Universidade do Porto, Porto, Portugal

eKiev Academic University, Kiev, Ukraine

Corresponding author. Address: Department of Molecular Biophysics, Bogomoletz Institute of Physiology, 4 Bogomoletz St, Kiev 01024, Ukraine. Tel.: +380442562426. E-mail address: vkrotov@biph.kiev.ua (V. Krotov).

Sponsorships or competing interests that may be relevant to content are disclosed at the end of this article.

Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal's Web site (www.painjournalonline.com).

Received December 03, 2018

Received in revised form March 06, 2019

Accepted March 15, 2019

Online date: April 10, 2019

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1. Introduction

The gray matter surrounding the spinal central canal, which corresponds to Rexed's lamina X, has a complex organization and comprises the dorsal gray commissure, the ventral gray commissure, and the substantia gelatinosa centralis.34 The neurons residing in this layer play important roles in a number of processes including control of motor neuron function1,39 and autonomic regulation.10 However, a special attention is given to lamina X because of its association with the somatosensory integration and visceral nociception.9,11,19,20,30

Despite recent progress in understanding the principles of organization of the spinal sensory network, the mechanisms of information processing in lamina X are still poorly understood. In vivo recordings in the rat and the cat had shown that neurons located within the gray matter region surrounding the central canal respond to noxious stimuli applied to somatic and visceral structures.15,17,29 Somatic receptive fields of the neurons are small but often have both excitatory and inhibitory components.15,29 These and other data imply a complex arrangement of the primary afferent inputs to the spinal neurons located around the central canal. The area occupied by the dendritic arbors of these neurons15,17,29 and the projection fields of some types of thin primary afferent fibers23,28,40 show substantial overlap suggesting a possibility of direct synaptic contacts. However, a physiological evidence for functional significance of direct primary afferent inputs to lamina X neurons, to the best of our knowledge, is currently unavailable. This is caused, at least in part, by functional limitations of the methodological approaches used so far, for example, recording from partially deafferented neurons in the spinal cord slices.6,31

The major aim of the present work was to characterize the monosynaptic and polysynaptic primary afferent inputs to lamina X neurons. We did the tight-seal recordings using the recently developed ex vivo spinal cord preparation with attached dorsal root, which preserved both the neuronal dendritic structure and the primary afferent supply.18 We show that lamina X neurons receive numerous direct and indirect inputs from nociceptive Aδ- and C-fibers, and that excitability of these cells is under polysynaptic inhibitory control.

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2. Materials and methods

2.1. Ethical approval

All experimental procedures were approved by the local Animal Ethics Committee of the Bogomoletz Institute of Physiology (Kiev, Ukraine) and were performed in accordance with the European Commission Directive (86/609/EEC) and ethical guidelines of the International Association for the Study of Pain.

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2.2. Ex vivo spinal cord preparation

The method for functional studies of lamina X neurons has recently been described.18 In the present work, we used Wistar rats (P9-P11) of both sexes. A rat was quickly decapitated, and the vertebral column was cut out and immersed into oxygenated sucrose solution at 20 to 22°C, which contained (in mM): 200 sucrose, 2 KCl, 1.2 NaH2PO4, 0.5 CaCl2, 7 MgCl2, 26 NaHCO3, and 11 glucose (pH 7.4 when bubbled with 95% O2 and 5% CO2). The spinal cord containing all lumbosacral and several thoracic segments with attached L5 or L5 and L4 dorsal roots was gently removed, and the dura matter was cut. The spinal cord was hemisected (starting from the thoracic segments) along the midline, and the half prepared for the recording was glued (lateral side down) to a metal plate (Fig. 1A).

Figure 1

Figure 1

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2.3. Electrophysiological recordings

For the patch-clamp recording, lamina X neurons in the spinal L4-L5 segments were visualized using the oblique infrared light-emitting diode illumination technique.36,41 The whole-cell and cell-attached recordings were performed at room temperature (20-22°C) in oxygenated solution containing (in mM): NaCl 125, KCl 2.5, CaCl2 2, MgCl2 1, NaH2PO4 1.25, NaHCO3 26, and glucose 10 (pH 7.4, 95% O2 and 5% CO2). The cell-attached recordings were performed to study the spiking activity in intact (nonperfused) neurons. Patch pipettes were pulled from the borosilicate glass using a P-87 horizontal puller (Sutter Instruments, Novato, CA), and after filling with intracellular solution had a resistance of 3 to 5 MΩ. The intracellular solution contained (in mM) 145 K-gluconate, 2.5 MgCl2, 10 HEPES, 2 Na2-ATP, 0.5 Na-GTP, and 0.5 EGTA (pH 7.3). Signals were recorded and low-pass filtered (2.6 kHz) using a MultiClamp 700B amplifier (Molecular Devices, San Jose, CA) and sampled at 10 kHz with Digidata 1320A under control of the pClamp 9.2 software (Molecular Devices). Offset potentials were compensated before seal formation. Liquid junction potentials were not compensated. All chemicals were from Sigma-Aldrich (St. Louis, MO).

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2.4. Dorsal root stimulations

Dorsal roots were stimulated through a suction electrode using an ISO-Flex (AMPI, Jerusalem, Israel) stimulator.18 A 50-µs current pulse of increasing amplitude (10-100 µA) was applied to recruit Aβ and Aδ fibers, whereas a 1-ms pulse (20-150 µA) was used to additionally activate C-afferents. Stimulation frequency was set at 0.1 Hz to avoid the slowing down of conduction in C fibers32 and wind-up14 phenomena. Monosynaptic primary afferent inputs to lamina X neurons were identified on the basis of low failure rates and small latency variations as described previously.24,33 Fibers mediating monosynaptic inputs were classified based on their conduction velocity (CV) and the stimulus strength that elicited postsynaptic responses using criteria developed by Fernandes et al.12 Aδ- fibers were activated by a 50-µs pulse and had their CV between 1.5 and 0.5 m/second. C-fibers were activated by a 1-ms pulse and had CV below 0.5 m/second. Those afferents, which conducted at the Aδ range CVs but were activated only by a 1-ms pulse stimulation, were classified as high-threshold Aδ (HT-Aδ). The slowly conducting (CV < 0.5 m/second) fibers, which were activated by a 50-µs pulse stimulation, were considered as low-threshold C (LT-C) afferents. The afferent CV was calculated by dividing the conduction distance (the length of the root from the opening of the suction electrode to the dorsal root entry zone) by the latency of the monosynaptic response with a 1-ms allowance for synaptic transmission.

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2.5. Data analysis

The data were analyzed using Clampfit 9.2 (Molecular Devices) and MiniAnalysis software (Synaptosoft, Decatur, GA). The data are presented as mean ± SEM with n referring to the number of cells analyzed. Categorical data were analyzed using Fisher exact test. Unless otherwise stated, quantitative data were compared with Kruskal–Wallis nonparametric test followed by Dunn post hoc test. The P < 0.05 was considered as statistically significant for each test.

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3. Results

3.1. Intrinsic properties

A population of lamina X neurons was heterogeneous in respect to the intrinsic firing properties. Cells in our sample showed 5 distinct discharge patterns (Fig. 1B). The most frequent pattern was tonic (49%) followed by adapting (27%), single-spike (6%), delayed (9%), and bursting (9%) discharges. These groups of neurons also differed in their passive membrane properties (Table 1). Statistical analysis revealed that the membrane input resistance and capacitance in adapting neurons were significantly different from those in tonic (P < 0.05), delayed (P < 0.01), and bursting (P < 0.01) neurons. The resting membrane potential in single-spike neurons was lower than in tonic and bursting groups (P < 0.01). The bursting neurons had the largest membrane capacitance, which differed significantly from that in tonic, adapting, and single-spike cells (P < 0.05). Thus, bursting group is likely to represent a population of the largest lamina X neurons.

Table 1

Table 1

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3.2. Primary afferent inputs

The inputs were studied in experiments in which the dorsal roots were first stimulated with a 50-µs pulse (10-100 µA) to recruit Aβ-, Aδ-, and LT-C-afferents, and then with a 1-ms pulse (10-150 µA) to additionally activate HT-Aδ- and C-afferents (Fig. 2A).

Figure 2

Figure 2

A striking feature of the neurons located in lamina X was a lack of the low-threshold Aβ/δ- primary afferent input. None of the neurons tested (n = 100) responded to the root stimulation with a 50-µs pulse of less than 50 µA. At the saturating Aδ-fiber stimulations (50 µs, 100 µA), the excitatory responses were seen in about one-third of the neurons (Fig. 2Ba, left). The monosynaptic excitatory postsynaptic currents (EPSCs) were identified in only 10% of the neurons; they were mediated through Aδ- (n = 8) and LT-C (n = 7) fibers and were always accompanied by the polysynaptic EPSCs. The polysynaptic EPSCs only were seen in 19% of the neurons. The largest number of cells responding to the short-pulse stimulation was in the tonic group (38%), whereas single-spike and delayed firing neurons did not show any input (Fig. 2Ba, right).

Virtually all neurons tested (90%, 92 of 103) responded to the saturating C-fiber stimulation (1 ms, 150 µA) of the root (Fig. 2Bb, left). Direct primary afferent inputs were identified in 66% of the neurons (Fig. 2Bb, right). The highest percentage of neurons showing monosynaptic EPSCs was in the bursting and tonic groups, whereas the single-spike neurons were deprived of direct inputs from the nociceptive afferents (Fig. 2Bb, right).

Based on the experiments with the C-fiber range stimulation, we examined the pattern of the monosynaptic input distribution in the groups of neurons with distinct intrinsic firing properties (Fig. 2Ca and Cb). In general, most direct inputs to lamina X neurons were mediated by the C- and HT-Aδ- primary afferent fibers (Fig. 2Ca). The largest number of the C-fiber inputs per neuron was seen in the tonic group, whereas most HT-Aδ inputs were found in the bursting neurons (P < 0.05, Fig. 2Ca). The bursting group also had the highest percentage of neurons with the HT-Aδ–fiber inputs (Fig. 2Cb, P < 0.05).

Apart from the EPSCs, the dorsal root stimulations elicited a polysynaptic inhibitory component of the response in 48% of lamina X neurons (Fig. 3). In most cases (41%), the inhibitory postsynaptic currents (IPSCs) followed EPSCs, but in a few neurons (7%), the pure inhibitory responses were recorded. Inhibitory inputs were predominantly evoked by the C-fiber stimulations; the Aδ range stimuli elicited IPSCs in only 8 cells. With the exception of the single-spike neurons, which being deprived of monosynaptic excitatory inputs (Fig. 2Bb, right) generated pure inhibitory responses (Fig. 3), all cell types did not show significantly different patterns of inhibitory inputs (Fig. 3). This robust inhibition mediated through nociceptive primary afferents can play an essential role in control of lamina X neurons under physiological conditions.

Figure 3

Figure 3

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3.3. Evoked and spontaneous action potentials

The integrating properties of lamina X neurons were studied by recording evoked and spontaneous action potentials in the cell-attached mode (Fig. 4Aa and Ba). These recordings were performed before the formation of the whole-cell mode giving us a possibility to first analyze firing in an intact (nonperfused) neuron.

Figure 4

Figure 4

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3.3.1. Evoked action potentials

The Aδ-fiber range stimulation (50 µs, 100 µA) of the dorsal root induced a spike in only 8% of neurons (Fig. 4Ac). This was in agreement with the results of our whole-cell experiments showing a few inputs from Aδ-afferent and LT-C–afferent fibers to lamina X neurons. However, the root stimulation at intensities activating high-threshold afferents (1 ms, 150 μA) evoked spikes in 34% of the neurons (Fig. 4Aa, Ab, and Ad). In most cases (17 of 28 cells tested in both cell-attached and whole-cell configurations), the response consisted of one spike, which was triggered by the long-latency EPSP components mediated by C-fibers (Fig. 4Ab). The highest percentage of responding neurons was in the groups with the neurons exhibiting delayed and tonic patterns of intrinsic firing. These experiments have revealed a critical role of the high-threshold long-latency excitatory inputs in activating lamina X neurons.

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3.3.2. Spontaneous action potentials

Although a moderate number of neurons discharged in response to the root stimulation, a surprisingly high percentage of cells showed spontaneous spiking activity. The ongoing discharges at frequencies higher than one spike per 30 seconds were observed in two-thirds of lamina X neurons (Fig. 4Bb, left). Most tonic and bursting neurons showed spontaneous firing activity, whereas most of the single-spike and delayed neurons were silent (P < 0.01, Fig. 4Bb, right).

Thus, the differences in the primary afferent inputs, evoked, and spontaneous discharges seen in the neurons with distinct intrinsic firing properties are likely to reflect an involvement of specific populations of lamina X neurons in diverse signal processing circuitries.

Figure 5

Figure 5

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4. Discussion

In this study, we used a hemisected ex vivo spinal cord preparation with preserved dorsal root to study the intrinsic firing properties, primary afferent inputs, and spontaneous discharges in the neurons located in the gray matter region around the central canal. Our data show that (1) the population of these neurons is heterogeneous in respect to their intrinsic firing properties; (2) most lamina X neurons receive functional monosynaptic inputs from the high-threshold Aδ- and C-afferents; (3) about a half of the neurons also show a primary afferent-driven polysynaptic inhibition; (4) the neurons can be excited by the nociceptive primary afferent input; and (5) most of the neurons generate ongoing discharges caused by the activity in the local neuronal network. Thus, neurons surrounding the central canal are active elements of the intrinsic spinal network, which are directly targeted by nociceptive afferent fibers and process diverse modalities of peripheral input.

We took advantage of the technique which has recently been developed for the functional examination of lamina X neurons.18 By hemisecting the spinal cord, we created an access to the neurons residing in the gray matter around the central canal for the visually guided patch-clamp recording. The hemisected lumbosacral cord with attached dorsal root preserved the entire rostrocaudal extension of the dendritic tree (except for its commissural branches) of lamina X neurons, their ipsilateral primary afferent input and the local neuronal network. This technique of the tight-seal recording from deep spinal neurons has been developed as an alternative to the standard slice preparation, in which the neurons are subjected to a considerable deafferentation. We did successful recordings using animals at postnatal days P9-P11; thus, the hemisected spinal cord preparation is appropriate for studying physiology of deep spinal neurons in young rodents.

However, because our preparation did not preserve the peripheral receptive fields, testing of natural stimuli was not possible. Therefore, electrical stimulation of dorsal root was used to characterize the activation threshold of primary afferents. Under these conditions, the activation threshold of a given afferent fiber depended on its axon diameter and degree of myelination. Furthermore, it should be noted that the descending pathways are not completely developed by postnatal days P9-P1113 and may not remain intact in our preparation.

Lamina X neurons are involved in a number of physiological processes. Our recordings were restricted to the cells located in the L4-L5 lumbar segments, in which lamina X is virtually deprived of the sympathetic or parasympathetic preganglionic neurons involved in the autonomic control.10,18 According to the previous studies, the neurons modulating output of spinal motoneurons are not abundant in this part of lamina X.1,39 For these reasons, the neurons from our study are most likely to belong to the circuits processing somatosensory and visceroceptive information.9,11,15,17,19,20,26,27,29

Because most in vitro studies of intrinsic firing properties and nociceptive afferent inputs focused on neurons in the superficial and deep dorsal horns,43 our knowledge about the properties of spinal neurons surrounding the central canal is very limited. Here, we demonstrate that their population is heterogeneous and exhibits intrinsic firing properties resembling those of lamina I neurons.12,21,25,35 Approximately one-tenth of the neurons near the central canal showed the bursting discharge pattern, which in the superficial lamina I is related to a subclass of the anterolateral tract projection neurons.25,35 Therefore, one cannot exclude the possibility that these neurons with a unique response profile belong to the group of projection neurons residing in lamina X.7,8,18,42,43 In contrast to the superficial dorsal horn,12,21,25 lamina X did not contain rhythmic firing neurons; spontaneous discharges observed in our study were driven by the ongoing activity in the local neuronal network. It should be noted that the incidence of spontaneous discharges correlated with the type of intrinsic firing pattern being highest in the tonic and bursting neurons and lowest in the single-spike and delayed neurons. Thus, specific groups of lamina X neurons are likely to be involved in distinct local neuronal circuitries.

We have found that most of the neurons near the central canal receive inputs from the Aδ- and C-primary afferents, many of which are nociceptive. A possibility of the direct primary afferent inputs to lamina X neurons could be predicted from early morphological studies, showing that the central projections of thin fibers23,28,40 may reach the area occupied by the dendritic arbors of the neurons surrounding the central canal.15,17,29 In the present work, we provide physiological evidence that monosynaptic primary afferent inputs to lamina X neurons are abundant, diverse, and have functional significance. These neurons are directly supplied by 4 classes of thin afferent fibers. Based on their CVs and electrical activation thresholds (amplitudes and durations of the stimulating pulse), these afferents belong to the Aδ, HT-Aδ, LT-C, and C types (Fig. 5). The dorsal root stimulation also evoked the polysynaptic excitatory component, which increased the overall efficacy of the input. A similar pattern of the primary afferent supply was observed in the superficial dorsal horn neurons from lamina I.12,22 Furthermore, we did not reveal any monosynaptic or polysynaptic inputs mediated by the low-threshold Aβ/δ- afferents, and therefore, it is likely that lamina X neurons are preferentially involved in processing of sensory information from the high-threshold nociceptive afferents. This predominantly nociceptive supply of the neurons correlates with an important role of lamina X in processing of painful signals9,11,15,17,19,20,26,27,29 and with abundance of the peptidergic afferent terminals in the zone near the central canal.16

The primary afferent-driven inhibition was observed in about a half of lamina X neurons. In most cases, it was mediated through high-threshold Aδ- and C-type afferents (Fig. 5). A combination of inhibition and excitation was most frequently seen in the bursting, tonic, and adapting neurons. Such inhibitory inputs function as an afferent/network-driven negative feedback terminating induced depolarization and limiting the number of action potentials evoked by the peripheral stimuli. Thus, robust inhibitory control is likely to play an important role in regulating the output characteristics of these neurons under physiological conditions. Excitability of lamina X neurons is also regulated by a number of transmitter/receptor systems.2–6,37,38 Pathological alterations in these systems, for example, leading to disinhibition, may substantially increase the responsiveness of lamina X neurons to the nociceptive peripheral stimuli.

In conclusion, spinal neurons surrounding the central canal show heterogeneous intrinsic firing properties and receive abundant direct inputs from the nociceptive afferent fibers. Many of these neurons fire in response to the high-threshold primary afferent stimulation. Thus, despite their diverse functions, lamina X neurons show many features common to the pain-processing neurons from the superficial dorsal horn.

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Conflict of interest statement

The authors declare no competing financial interests.

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Acknowledgements

The authors thank Mr. Andrew Dromaretsky for the technical assistance.

B.V. Safronov was supported by the FEDER funds through the COMPETE 2020 (POCI), Portugal 2020, and by the FCT project PTDC/NEU-NMC/1259/2014 (POCI-01-0145-FEDER-016588). P. Belan was supported by the National Academy of Sciences of Ukraine (NASU); grant NASU # ІІ—1-12, grant NASU #67/15-Н. N. Voitenko was supported by the NASU Biotechnology grant and NASU grant # ІІ—1-12.

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Supplemental video content

Video content associated with this article can be found online at http://links.lww.com/PAIN/A784.

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References

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Keywords:

Spinal cord; Lamina X neurons; Primary afferent inputs; High-threshold Aδ- and C-fibers; Monosynaptic contacts; Polysynaptic inhibition

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