Dynamic synchronization of ongoing neuronal activity across spinal segments regulates sensory information flow.

Dynamic synchronization of ongoing neuronal activity across

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spinal segments regulates sensory information flow E. Contreras-Hernández1, D. Chávez1 and P. Rudomin1, 2 1

Department of Physiology, Biophysics and Neurosciences, Center of Research and Advanced Studies of the Instituto Politécnico Nacional, México. 2 El Colegio Nacional, México Running title: Neuronal synchronization and regulation of sensory information Number of figures: 10 Number of tables: 0 Total number words in the text: 9272 Number of words in the abstract: 191 Key words: Neuronal synchronization; dorsal horn neurones; presynaptic inhibition;

Corresponding Author: Prof. P. Rudomin Department of Physiology, Biophysics and Neurosciences Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional Av. Instituto Politécnico Nacional 2508, México DF 07360, México Email: [email protected] Phone: 525 5747 3995

This is an Accepted Article that has been peer-reviewed and approved for publication in the The Journal of Physiology, but has yet to undergo copy-editing and proof correction. Please cite this article as an 'Accepted Article'; doi: 10.1113/jphysiol.2014.288134.

1 This article is protected by copyright. All rights reserved.

Key points 

We have examined in the spinal cord of the anaesthetized cat the relation between ongoing

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correlated fluctuations of dorsal horn neuronal activity and state-dependent activation of inhibitory 



reflex pathways. We found that high levels of synchronization between the spontaneous activity of dorsal horn neurones occur in association with the preferential activation of spinal pathways leading to primary afferent depolarization and presynaptic inhibition relative to activation of pathways mediating Ib postsynaptic inhibition. It is suggested that changes in synchronization of ongoing activity within a distributed network of dorsal horn neurones play a relevant role in the configuration of structured (non-random) patterns of functional connectivity that shape the interaction of sensory inputs with spinal reflex pathways subserving different functional tasks.

Abstract Previous studies on correlation between spontaneous cord dorsum potentials recorded in the

lumbar spinal segments of anaesthetized cats suggested the operation of a population of dorsal horn neurones that modulates, in a differential manner, transmission along pathways mediating Ib non-

reciprocal postsynaptic inhibition and pathways mediating primary afferent depolarization and presynaptic inhibition (Chávez et al., 2012). To have further insight on the possible neuronal mechanisms that underlie this process, we have now measured changes in the correlation between the spontaneous activity of individual dorsal horn neurones and the cord dorsum potentials associated

with intermittent activation of these inhibitory pathways. We found that high levels of neuronal synchronization within the dorsal horn are associated with states of incremented activity along the pathways mediating presynaptic inhibition relative to pathways mediating Ib postsynaptic inhibition. It is suggested that ongoing changes in the patterns of functional connectivity within a distributed

ensemble of dorsal horn neurones play a relevant role in the state-dependent modulation of impulse transmission along inhibitory pathways, among them those involved in the central control of sensory information. This feature would allow the same neuronal network to be involved in different functional tasks.


Abbreviations c, caudal; CC, cross-correlation; CCol, Clarke’s Column; CDPs, cord dorsum potentials; DRPs,

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dorsal root potentials; DLF, dorsolateral fasciculus; GS, gastrocnemius; IFPs, intraspinal field potentials; iVRPs, inhibitory ventral root potentials; JFP, Joint Firing Probability; L, left; nCDPs,

negative cord dorsum potentials; npCDPs, negative-positive cord dorsum potentials; PAD, primary afferent depolarization; PCA, Principal Component Analysis; PBSt, posterior biceps and semitendinosus; r, rostral; R, right; SE, standard error; SP, Superficial Peroneal; SU, Sural; VRPs, ventral root potentials.

Introduction Recent studies have given particular attention to the influence of synchronization between

neuronal spontaneous activity on the functional organization of a variety of neuronal ensembles including the cerebral cortex (Womelsdorf et al., 2007; Tagawa & Hirano, 2012), hippocampal and olfactory glomeruli (Bazelot et al., 2010; Karnup et al., 2006) and retina (Torborg & Feller, 2005). In the spinal cord, these studies deal with changes in the synchronization of spontaneous motoneuronal activity during development (Hanson et al., 2008; O'Donovan et al., 2008) and of changes in

synchronization of dorsal horn neuronal activity induced by different experimental procedures, including nociceptive stimulation (Lidierth and Wall, 1996; Biella et al., 1997; Eblen-Zajjur & Sandkuhler, 1997; Contreras-Hernández et al., 2013). Yet, little attention has been given to the role of neuronal synchronization as a mechanism allowing state-dependent modulation of transmission along

spinal inhibitory pathways, including those involved in the presynaptic control of sensory information, and on how the patterns of neuronal synchronization change during different functional and pathological situations (see Burke, 1999 and Hultborn, 2001). Rudomin et al., (1987) used spike triggered averaging of dorsal root potentials (DRPs) and

ventral root potentials (VRPs) to identify intermediate zone neurones mediating primary afferent depolarization (PAD) and presynaptic inhibition of muscle afferents. They found one set of

interneurones (Class I) whose spontaneous activity was associated with short latency inhibitory ventral root potentials (iVRPs) that were generated without concurrent dorsal root potentials (DRPs)


and another set of neurones (Class II) whose spontaneous activity was instead associated with short latency DRPs and monosynaptic iVRPs. Further studies indicated that Class I neurones included a population of last-order neurones

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mediating non-reciprocal glycinergic postsynaptic inhibition of motoneurones and of interneurones in Clarke’s column (CCol), while Class II neurones comprised GABAergic interneurones acting on the intraspinal terminals of group Ia muscle afferents and spinal motoneurones, where they produced PAD and postsynaptic inhibition, respectively (Rudomin et al., 1990). In addition to the time-locked DRPs and/or iVRPs, Rudomin et al., (1987) found that the action

potentials of Class I interneurones were usually preceded by spontaneous negative cord dorsum potentials (nCDPs), while the action potentials of Class II interneurones were preceded by spontaneous negative-positive potentials (npCDPs). They suggested that separate sets of dorsal horn neurones generated these two types of cord dorsum potentials (CDPs) and that the neurones involved in their generation affected, rather selectively, transmission along the reflex pathways leading to

activation of Class I and Class II interneurones. More recently, Chávez et al., (2012) proposed that the spontaneous nCDPs and npCDPs were

instead generated by a common neuronal ensemble distributed along several lumbar spinal segments and that increased synchronization between the spontaneous neuronal activity within this ensemble lead to a state-dependent modulation of sensory feedback, in this case associated with the preferential

activation of pathways mediating PAD and presynaptic inhibition relative to activation of pathways mediating non-reciprocal postsynaptic inhibition (see also Manjarrez et al., 2000). Since this proposal

was based on the analysis of the spontaneous CDPs generated along the lumbosacral cord by unidentified sets of neurones, it seemed necessary to have more direct information on the changes in the correlation between the ongoing activity of individual dorsal horn neurones during the generation of the spontaneous nCDPs and npCDPs. It was assumed that this approach would provide relevant

information on the functional organization of the dorsal horn neuronal networks involved in the generation of the nCDPs and npCDPs, and allow distinction between the Rudomin et al., (1987) and

the Chávez et al., (2012) proposals. That is, “separate sets of dorsal horn neurones command Class I

and Class II interneurones” versus “changes in synchronization within a distributed dorsal horn neuronal network underlie the preferential activation of Class I and Class II interneurones”.


We found a significant number of neurones within laminae III-VI in the L5-L6 segments that increased their spontaneous activity during both the nCDPs and npCDPs recorded from the same and from the adjacent spinal segments. We also found that the joint firing probabilities and neuronal

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synchronization were in most cases higher during the generation of npCDPs than during the generation of nCDPs. These findings are taken to support the proposal that increased synchronization

in the spontaneous activity within a longitudinally distributed ensemble of dorsal horn neurones underlies preferential activation of the pathways that mediate PAD relative to the activation of the pathways producing Ib postsynaptic inhibition. A preliminary report of these findings has been published in abstract form (Contreras-Hernández et al., 2010).

Methods Ethical approval Cats were bred and housed under veterinarian supervision at the Institutional Animal Care unit

(SAGARPA permission AUT-B-C-0114-007). All experiments were approved by the Institutional Ethics Committee for Animal Research (Protocol no. 126-03) and comply with the ethical policies and regulations of The Journal of Physiology (Drummond, 2009) and of the National Institutes of Health (Bethesda, MD, USA; Animal Welfare Assurance no. A5036-01). The Guide for the Care and Use of Laboratory Animals (National Research Council, 2010) was followed in all cases. General procedures Preparation The experiments were performed in 22 young adult cats of either sex. The animals were initially

anaesthetized with pentobarbitone sodium (40 mg/kg i.p.). The carotid artery, radial vein, trachea and urinary bladder were cannulated. Additional doses of pentobarbitone sodium were given intravenously (5 mgs/kg/hr) to maintain an adequate level of anaesthesia, tested by assessing that withdrawal reflexes were absent, that the pupils were constricted and that arterial blood pressure was between 100 and 120 mm Hg. A solution of 100 mM of sodium bicarbonate with glucose 5% was given i.v. (0.03 ml/min) to prevent acidosis (Rudomin et al., 2007). When necessary, dextran 10% was administered to keep blood pressure above 100 mm Hg. The lumbo-sacral and low thoracic spinal segments were exposed by laminectomy and opening of the dura mater. In all experiments the left Sural (SU) and Superficial Peroneal (SP) nerves and in 3 5 This article is protected by copyright. All rights reserved.

experiments the posterior biceps and semitendinosus (PBSt) and the gastrocnemius (GS) nerves were dissected free and prepared for stimulation. After the main surgical procedures, the animals were transferred to a stereotaxic metal frame allowing immobilization of the head and spinal cord and pools

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were made with the skin flaps to prevent desiccation of the exposed tissues. They were filled with paraffin oil and maintained between 36 and 37°C by means of radiant heat. Subsequently, the animals were paralyzed with pancuronium bromide (0.1 mg/kg) and

artificially ventilated. The tidal volume was adjusted to maintain 4% of CO2 concentration in the expired air. During paralysis, adequacy of anaesthesia was ensured with supplementary doses of anaesthetic (2 mg/kg in an hour) and by assessing that the pupils remained constricted and that heart

rate and blood pressure were not changed following a noxious stimulus (paw pinch). At the end of the experiments, the animals were euthanized with an overdose of pentobarbitone.

The spinal cord was removed leaving the recording micropipette(s) in place. After fixation and dehydration, the lumbosacral segments were placed in a solution of methyl salicylate for clearing and subsequently cut transversally to obtain a section containing the micropipette(s), and photographed. Recording and stimulation Cord dorsum potentials were recorded by means of 4-10 silver ball electrodes placed on the

surface of the L4-L7 segments on both sides of the spinal cord against an indifferent electrode inserted in the paravertebral muscles, using AC amplifiers with filters set from 0.3 Hz to 1 kHz. Glass micropipettes filled with 2M NaCl (2-6 MΩ) were used to record intraspinal field

potentials (IFPs) as well as extracellular neuronal action potentials by means of an Axoclamp 2B amplifier. The recording micropipettes were inserted in the left side of the L5-L6 segments and were gradually displaced downward within the dorsal horn until recording spontaneous action potentials from one or more neurones. Cord dorsum and intraspinal potentials were sampled at 10 KHz, visualized on-line and digitally stored for further analysis with software written in MatLab (MathWorks). In 3 experiments a L6 dorsal rootlet in the left side was dissected, sectioned and its central end

placed on a pair of Ag–AgCl electrodes to record DRPs. In all the other experiments the dorsal roots were left intact to avoid the changes in the synchronization of neuronal activity and in the effectiveness of the PAD pathways induced by the acute dorsal root section (Chávez et al., 2012 and Rudomin et al., 2013). 6 This article is protected by copyright. All rights reserved.

The SU and SP nerves were stimulated with single pulses 0.1 ms duration with strengths ranging from 1.15 to 2.0 times the threshold of the most excitable fibres (T), judged from the CDPs recorded in the L5 and/or L6 segments. The PBSt and GS nerves were stimulated with single pulses 0.1 ms, 2-

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5T strength. In 4 experiments, in addition to electrical stimulation of cutaneous nerves, we used Von Frey filaments of different thickness (North Coast Medical) to determine the threshold and sensory

fields of neuronal responses to mechanical stimuli applied perpendicularly to the skin in the left hindlimb (0.6 to 180 g-F; see Results for more details). A bipolar stimulating electrode was placed over the left dorsolateral fasciculus (DLF) at the

T10-T12 level to test for antidromic activation of the analyzed neurones. This tract includes ascending axons from dorsal horn neurones located in lower lumbar segments that respond to electrical stimulation of low threshold cutaneous afferents and to skin tactile stimulation as well as by high threshold muscle afferents (Lundberg & Oscarsson, 1961). The DLF was stimulated with single 0.1 ms pulses, 1.4-5T (times the strength necessary to generate a descending volley in the lumbosacral cord). No tests were made for neuronal antidromic activation from other ascending tracks or for crossed projections. Selection of spontaneous nCDPs and npCDPs Analysis of the synchronization of neuronal firing with the spontaneous CDPs of specific shapes

required proper selection and classification of the CDPs recorded in a particular spinal segment. To

this end, we first selected the CDPs that exceeded a predetermined amplitude threshold, that was adjusted as low as possible to include a wide range of potentials. Figure 1A provides one example of this procedure. The upper pair of traces show the spontaneous CDPs simultaneously recorded from

the L6 rostral and the L6 caudal left spinal segments (L6rL-CDPs and L6cL-CDPs, respectively),

together with the DRPs (L6-DRPs) and the intraspinal field potentials (L6-IFPs) that were recorded in the L6cL segment within the dorsal horn at 1532 µm depth (Rexed’s lamina V). The L6cL CDPs that exceeded a predetermined threshold level (shown in Figure 1A by the

dotted horizontal line) were selected for further processing. It may be noted that some of the CDPs appeared without associated DRPs (see box 1), while others were generated together with distinct L6DRPs (boxes 2 and 3). Selection of the spontaneous CDPs as either nCDPs or npCDPs was made using a set of 3 dot-templates. The horizontal dots marked with arrows pointing downward in the upper trace in Fig. 1B and E indicate the upper limit of amplitude selection of the CDPs, while the 7 This article is protected by copyright. All rights reserved.

bars with arrows pointing upwards show the lower limit. The dot-templates with opposing arrows forced selection of potentials within a narrow range. Spontaneous nCDPs were selected when dottemplate #3 was positioned at the same level as dot-template #1, that was placed on the baseline just

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before the onset of the negative component (Fig. 1B, upper trace). Spontaneous npCDPs were instead selected when dot-template #3 was placed at the time of the positivity that followed the negative component of the CDP (Fig. 1E). In both cases dot-template #2 set the lower limit of the negative

component of the CDPs. Fig. 1C and D shows superposed traces of two sets of nCDPs and Fig. 1F and G of two sets of

npCDPs of different amplitudes that were selected with the dot-templates. The selected CDPs are displayed together with the simultaneously recorded L6-DRPs and the L6-IFPs. These recordings show that the npCDPs, but not the nCDPs were associated with spontaneous DRPs, as previously

shown by Chávez et al., (2012), and were therefore taken as reliable indicators of PAD. Although the design of the templates allowed a semiautomatic selection of the spontaneous

nCDPs and npCDPs, during data analysis the boundaries of the templates were adjusted for optimal selection. The retrieved nCDPs and npCDPs were in addition visually inspected for a final selection.

CDPs that could not be clearly classified as nCDPs or npCDPs were not considered for further analysis. They comprised 5- 12% of the total sample. Currently, more sophisticated mathematical and computational tools are being implemented to

extract not only time window-locked spontaneous nCDPs and npCDPs, but also CDPs with more complex shapes that may reflect the activation of the same or different neuronal ensembles (Vidaurre et al., 2012; Martin et al., 2013). Selection of neuronal spikes We selected for analysis the spontaneous action potentials of individual neurones mainly located

in the dorsal horn in segments L5-L6 within laminae III-VI. To this end, a 25 Hz high-pass Bessel

filter (8-pole) was used to eliminate slow potentials recorded with the intraspinal micropipette leaving only the neuronal action potentials. Spontaneous spikes exceeding a preset amplitude were selected and sorted by means of the Principal Component Analysis (PCA; see Lewicki, 1998). The aim of PCA is to sort automatically the recorded spikes, grouping those with similar shape. It operates by changing the representation of the spikes from the time-voltage domain to the principal components space. With this procedure the spikes with the same shape are grouped in clusters whose elements 8 This article is protected by copyright. All rights reserved.

show maximum covariance among themselves and minimum correlation with elements from a different cluster. In few cases it was necessary to use a k-means algorithm based in the Euclidean distance to ensure good quality in the classification of the spikes by PCA. It was assumed that action

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potentials with a similar shape and amplitude were generated by the same neurone. Changes in neuronal activity during spontaneous nCDPs and npCDPs Action potentials of individual neurones recorded during 15-30 min periods were used to

disclose changes in spontaneous firing before, during and after the selected spontaneous nCDPs and npCDPs. Rasters of neuronal action potentials were constructed to visualize possible changes in neuronal firing during the CDPs. The neuronal activity displayed in the rasters was added to construct

the corresponding histograms. Changes in neuronal activity during the nCDPs were calculated by means of the normalized

increment (In) in neuronal firing during the spontaneous nCDPs as follows: I𝑛 =

NSpeak − NSbasal NSpeak

Where NSpeak is the total number of spontaneous spikes occurring within a window of 40 ms

centered at the peak of the negative component of all the spontaneous nCDPs recorded from a specific segment. NSbasal is the total number of spikes generated during the same time window placed 40 ms before the onset of the nCDPs (baseline). The value of I for the npCDPs (Inp) was calculated in a similar way. Values of I close to 1 indicate maximal neuronal firing during the CDPs. Joint neuronal firing probabilities The Joint Firing Probability index (JFP) was used to estimate changes in the synchronization of

the spontaneous activity of pairs of individual neurones firing during the nCDPs and npCDPs. This

index is defined as follows: 𝐽𝐹𝑃𝑛𝐶𝐷𝑃 (𝑡) =

𝑁𝑛𝐶𝐷𝑃 (𝑡) 𝑛𝑛𝐶𝐷𝑃

Where 𝐽𝐹𝑃𝑛𝐶𝐷𝑃 is the joint-firing probability index of a specific pair of neurones at the time

interval t, 𝑁𝑛𝐶𝐷𝑃 (𝑡) the number of nCDPs where both neurons fired at the same interval and 𝑛𝑛𝐶𝐷𝑃 is the total number of recorded nCDPs. Similarly, 𝐽𝐹𝑃𝑛𝑝𝐶𝐷𝑃 is defined as the JFP of a pair of neurones firing during the npCDPs. 9 This article is protected by copyright. All rights reserved.

Cross correlation between neuronal action potentials The correlation between the spontaneous firing of pairs of neurones (n1 and n2) during the

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nCDPs was calculated using the shuffle-corrected cross-correlogram function (CC; see Brody, 1999), defined as follows: 𝐶𝐶(𝑛1 , 𝑛2 ) = 〈𝑁1𝑟 ⊙ 𝑁2𝑟 〉 − 𝑃1 ⊙ 𝑃2 Where 〈𝑁1𝑟 ⊙ 𝑁2𝑟 〉 is the mean cross-correlation between the spontaneous spike train functions

𝑁1𝑟 and 𝑁2𝑟 of neurons n1 and n2 during the whole sample of nCDPs, and r the number of nCDPs in the sample. 𝑃1 ⊙ 𝑃2 is the cross-correlation between 𝑃1 and 𝑃2 , i.e., the averages of the activity of neurones n1 and n2 during the nCDPs (𝑃1 = 〈𝑁1𝑟 〉 and 𝑃2 = 〈𝑁2𝑟 〉). Significance limits were established as twice standard deviation (2σ, 95% confidence limit for a Gaussian distribution). CCs for pairs of neurones during the npCDPs were calculated similarly. Statistics Significance of the differences between the joint firing probabilities of pairs of neurones and

between mean firing rates of individual neurones during the nCDPs and npCDPs was calculated using the Wilcoxon test, because the data showed a non-Gaussian distribution (Hollander et al., 2013). For

further assessment of the significance of the observed differences, these values were compared with those obtained with the paired t-test. The dependence between two variables was established using a linear adjustment model. The

Kolmogorov-Smirnov test was used to verify that these variables had a normal probability distribution. The bondage of linear adjustment was calculated using the Pearson correlation coefficient (R). Computations were made using the SigmaPlot software (Systat Software Inc.).

Results Activity of single neurones during spontaneous nCDPs and npCDPs The aim of this set of observations was to examine the changes in spontaneous activity of the

same neurone before, during and after the nCDPs and npCDPs recorded from a given segment. The upper set of traces in Fig. 2A shows superposed dot-template selected spontaneous nCDPs recorded in the L5 caudal left (L5cL) segment, and the traces below a single nCDP together with the 10 This article is protected by copyright. All rights reserved.

simultaneously recorded IFP and a neuronal action potential. As shown by the superposed traces in the left side, the action potentials selected throughout the whole recording period had a similar amplitude and shape, so we assumed they were generated by the same neurone that was located in the

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border between Rexed’s laminae V and VI (red dot in Fig. 8Ba). The individual CDPs were aligned using as reference the peak of the negative component of the

L5cL-nCDPs. As shown by the rasters and the histogram displayed in Fig. 2C and D, this neurone increased its spontaneous firing in synchrony with the L5cL-nCDPs. The time course of the increased activity resembled that of the spontaneous nCDPs. A similar procedure was followed to disclose changes in the spontaneous activity of the same neurone during the generation of the npCDPs. Fig.

2E, G and H shows in addition that this neurone also increased its spontaneous firing during the negative component of the spontaneous L5cL-npCDPs. To find dorsal horn neurones that increased their spontaneous activity both during the nCDPs

and the npCDPs was crucial to test the proposal of Chávez et al., (2012) that temporal changes in the functional connectivity within the same ensemble of dorsal horn neurones underlie the generation of both nCDPs and npCDPs. It thus seemed necessary to show that the procedure used to select the nCDPs and npCDPs was indeed as selective as intended, particularly when dealing with the smallest CDPs where classification as nCDPs or npCDPs could be somewhat ambiguous. To ensure that the separation of the CDPs as nCDPs or npCDPs was reliable, we examined, one

by one, the dot-template selected CDPs. Of 201 L5cL CDPs classified as nCDPs (upper traces in Fig.

2A), only 6 had a small positivity and could be instead considered as npCDPs. There were in addition 12 CDPs that could not be classified as either nCDPs or npCDPs. That is, 91% of the selected potentials were nCDPs. Of 104 CDPs selected as npCDPs (upper set of traces in Fig. 2E), only 4 were nCDPs and 3 remained undefined. This means that 93% of the selected CDPs were npCDPs. Since the selection procedure retrieved CDPs of different amplitudes, we examined the extent

to which the same neurone fired in synchrony with large as well as with small nCDPs and npCDPs. To this end we divided the whole sample of selected nCDPs and npCDPs depicted in Fig. 2A and E in 4 groups of different amplitudes as shown in Fig. 2B and F and we observed the changes in the

neuronal activity within each group (Fig. 2C and G). It may be seen that the neuronal activity was increased during the generation of the largest (in black) as well as during the smallest (in green) nCDPs and npCDPs. 11 This article is protected by copyright. All rights reserved.

In addition to its increased activity during the negative component of the spontaneous nCDPs and npCDPs, this neurone was also activated by electrical stimulation of the SP nerve with single

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pulses 1.4T strength. The latencies of the neuronal responses relative to the arrival of the afferent

volley varied from 1.0 to 4.5 ms (2.75±1.23 ms) suggesting mono- and oligosynaptic activation (Fig.

2 I-K). We also found dorsal horn neurones whose activity was increased only during the spontaneous

nCDPs or during the npCDPs. Figure 3 provides one example of a neurone located in Rexed's lamina

V in the left L6 rostral segment (marked with a red dot in Fig. 8Ca). It may be seen that neuronal activity was increased during the small as well as during the large nCDPs (Fig 3A-D), but not during the npCDPs recorded in the same segment (Fig. 3E-H). This neurone also increased its discharge frequency during the nCDPs recorded in the L5 rostral left (L5rL) and in the caudal left (L5cL) segments but not during the npCDPs recorded in these segments (not illustrated). Since the number of

npCDPs generated during the whole recording period (30 min) was relatively small (n=37), it is possible that longer recording periods would show neuronal activation during the npCDPs. Alternatively, as shown below, the increments of neuronal activity during the nCDPS and/or npCDPs

may also depend on their segmental location. Stimulation of the SP nerve activated this neurone with latencies ranging from 2-5ms (mean

3.5±1.1ms), compatible with di- and oligosynaptic links (Fig. 3I-K). All together we examined the changes in the spontaneous activity of 126 neurones during the

generation of spontaneous nCDPs and npCDPs. About half of these neurones (61) increased their activity during the negative components of both the nCDPs and npCDPs, 21 neurones were activated only during the nCDPs, 6 only during the npCDPs and 38 neurones showed no changes in activity during the generation of either the nCDPs or the npCDPs. The intraspinal location of the whole sample of neurones presently examined is shown in Fig. 8. Changes of neuronal activity during nCDPs and npCDPs generated in different segments The observations presented in the preceding section described the changes in neuronal firing associated with the nCDPs and npCDPs recorded in one segment only. However, as shown in Fig. 1 spontaneous CDPs could appear simultaneously in several segments. It thus seemed of interest to examine the changes in neuronal activity during the nCDPs and npCDPs generated in different lumbar 12 This article is protected by copyright. All rights reserved.

segments. We assumed that this approach would provide information on the functional connectivity of the examined neurones with neurones located in different segments. The data depicted in Fig. 4 illustrate the changes in the spontaneous activity of two neurones

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whose action potentials were simultaneously recorded with a single micropipette inserted in the left side of rostral L6 (L6rL) within lamina V (see histology). Fig. 4A and B displays two separate sets of npCDPs and nCDPs recorded from several segments together with the associated IFPs and neuronal

action potentials (red arrow for Neurone 1 and black arrow for Neurone 2). The graphs in figures 4C

and D show the segmental distribution of the changes in spontaneous activity of Neurone 1, measured at the time of the negative phase of the nCDPs and npCDPs generated in different segments, as indicated in Fig. 4A, during two successive recording periods of 15 min each. The blue and red

horizontal bars show the normalized increments (In and Inp ) of neuronal firing during the nCDPs and npCDPs, respectively (see Methods). Values of I close to 1 indicate maximal neuronal firing during

the CDPs. Changes in neuronal activity related to the CDPs recorded in the left (L) and right side (R) of the spinal cord are plotted separately. As illustrated in Fig. 4C, during the first 15 min recording period, the activity of neurone 1 was

mostly increased during the npCDPs recorded in the L5cL and L6rL segments (red bars), while the

activity recorded during the nCDPs was increased in those and in other segments as well, both on the left and right sides (blue bars). These patterns changed during the second 15 min recording period

(Fig. 4D). Although the activity of neurone 1 was still strongly increased during the L5cL and L6rL npCDPs and also during the L5cL nCDPs, it was also increased during the nCDPs and npCDPs

recorded from more rostral segments, particularly in the right side (L4cR and L5rR). The activation patterns of Neurone 2 were quite different from those of Neurone 1. During the

first 15 min, the activity of Neurone 2 was increased at the time of the nCDPs and npCDPs generated in the L5 and L4 segments in the left side (Fig. 4E). In contrast, during the second 15 min period, the neuronal activity was mostly increased during the npCDPs generated in more caudal segments (Fig.

4F). It thus seems that although the increased activity of individual neurones during the nCDPs and

npCDPs recorded from different segments showed significant variations during successive recording periods, some of these associations were more stable and persisted for longer recording periods than others (e.g. increased activation of Neurone 1 during the L5cL nCDPs and npCDPs and increased 13 This article is protected by copyright. All rights reserved.

activity during the L6rL npCDPs in Fig. 4C and D, as well as increased activation of Neurone 2 during the L5rL nCDPs in Fig. 4E and F). Similar temporal variations in the association of neuronal firing with the CDPs recorded from

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different segments were found in all cases. This raised the question on the extent to which changes seen in a single neurone were shared with other neurones, because in a given recording period uncorrelated neuronal activity would be expected to contribute less to the population responses than correlated activity (see Rudomin and Madrid, 1972). Joint neuronal firing during spontaneous nCDPs and npCDPs Chávez et al., (2012) found that the spontaneous npCDPs recorded simultaneously from

contiguous segments (e.g., L5 and L6) were usually more correlated than the corresponding nCDPs. Hence, we reasoned that pairs of neurones whose activity was increased during both types of CDPs would be more synchronized during the npCDPs than during the nCDPs. Changes in the activity of pairs of neurones during the nCDPs and npCDPs were estimated with the Joint Firing Probability index (JFP; see Methods). To this end we examined the changes in joint activity of 21 pairs of neurones both activated

during the nCDPs and npCDPs. In 4 cases the paired neuronal activity was recorded with one micropipette and in 17 cases the neuronal activity was recorded with two micropipettes placed as

close as possible (less than 1 mm) within the same segment. Figure 5A summarizes the changes in the JFPs of all pairs of neurones calculated at different

time intervals (t1-t6) during the nCDPs and the npCDPs recorded in the same segment of intraspinal recording of neuronal activity (L5 or L6) or in the adjacent segment, as indicated. The JFPs obtained

during the npCDPs were plotted in increasing order (red dots) together with the JFPs of the corresponding nCDPs (black dots). We found that in most cases the JFPs obtained just before (t3) and

during the negative phase of the npCDPs (t4) were higher than the JFPs obtained during the nCDPs. Fig. 5B displays the mean and standard error (SE) of the whole sample of JFPs illustrated in Fig.

5A. Data obtained from the same and from adjacent segments were combined. It may be seen that the

mean JFPs observed at times t3 and t4 were also higher during the npCDPs (red dots) than during the nCDPs (black dots), in contrast with the JFPs calculated before (t1 and t2) and after (t5 and t6) the CDPs that were rather similar.


The lower set of graphs in Fig. 5B displays the p-values calculated with the paired t-test (squares) and the Wilcoxon test (circles) at different times during the nCDPs and npCDPs. With both tests the p-values were lowest at t3 and t4 (p= 2.08x10-3 for t3 and p= 1.91x10-5 for t4 paired t-test;

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p=0.092 for t3 and p=0.073 for t4 Wilcoxon test). We consider that these p-values represent reliable differences in the joint neuronal firing just before and during the npCDPs and nCDPs (t3 and t4), because there is a clear decrease up to one order of magnitude respect to p-values calculated before (t1 and t2) and after (t5 and t6) the CDPs (see Vaux, 2012; Nuzzo, 2014). We also asked the question on whether the differences in the JFPs observed during the npCDPs

and nCDPs could be related to the level of neuronal excitability, inferred from the baseline discharge rates of the examined neurones. Fig. 5C shows the changes in the mean frequency of the spontaneous activity of the neurones that fired in synchrony during the nCDPs and the npCDPs. The neuronal baseline mean discharge rates (measured at t1, that is 120 ms before the peak of the nCDPs and of the

npCDPs) were similar and relatively low (0.16 ± 0.02 and 0.15 ± 0.02 Hz, respectively). Thereafter

the neuronal discharge frequency increased and attained maximum values at t4, the time of the negative peak (0.327 ± 0.033 and 0.468 ± 0.05 Hz during the nCDPs and npCDPs, respectively). The observed differences in the spontaneous discharge rate at the onset and during the negative

peak of the nCDPs and npCDPs were also statistically significant as shown by the lower graphs in Fig. 5C. With both tests the p-values were lowest at t3 and t4 (p= 4.48x10-5 for t3 and p= 7.61x10-5

for t4, paired t-test; p= 0.094 at t3, and p=0.067 at t4, Wilcoxon test). These observations indicate that although the discharge rates of individual neurones and the

probabilities of joint neuronal firing were increased at the time of the onset and peak of the nCDPs and npCDPs, they were rather similar before the onset of the CDPs and were significantly higher during the npCDPs than during the nCDPs. We found in addition that the baseline discharge frequency of neurones showing increased

activity during the nCDPs only (0.15±0.07 Hz at t1) and during the npCDPs only (0.11±0.05 Hz at t1) was quite similar to the baseline discharge rates of neurones showing increased activity during both nCDPs and npCDPs. Therefore, the inability of these neurones to discharge during both the nCDPs

and npCDPs may not be ascribed to a reduced background activity.


Cross-correlograms of neuronal activity during nCDPs and npCDPs We have assumed that the increased joint firing of dorsal horn neurones observed during the npCDPs leads to more effective activation of the PAD-mediating pathways. Since the joint firing

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index only provides an estimate of the overall changes in neuronal activity, to have an additional assessment of the changes in neuronal temporal synchronization during the generation of the nCDPs and npCDPs, we calculated the cross-correlation between the spontaneous activity of pairs of neurones (CC, see Methods). Fig. 6 provides some representative examples of the CCs obtained from the activity generated

by pairs of neurones during nCDPs and npCDPs. The CCs illustrated in Fig. 6A-D were obtained from simultaneous recordings of 3 neurones. The activity of neurone n1 was recorded with a single

micropipette inserted in the L5cL segment at 1636 µm depth and the activity of neurones n2 and n3 was recorded with another micropipette inserted at 1762 µm depth from the cord dorsum, about 1 mm rostral to the other micropipette. The histograms show the CCs between neurons n1 and n2 and between neurones n1 and n3 during the L5cL nCDPs and npCDPs, as indicated.

It may be seen that the synchronization between the action potentials of neurones n1 and n2 (Fig. 6A

and B) and between neurones n1 and n3 (Fig. 6C and D) peaked at zero lag, decayed gradually with increasing lags and was almost negligible at ± 20 ms. Yet, it was higher during the npCDPs than during the nCDPs. The CCs between neurones n2 and n3 were also higher during the npCDPs than

during the nCDPs (not illustrated). The histograms depicted in Fig. 6E-H were obtained from two neurones (nA and nB) recorded

in another experiment, in this case with two micropipettes inserted in the L5cL segment, one at 1180

and the other at 1760 µm depth. The CCs were symmetrical around zero lag and higher during the npCDPs than during the nCDPs recorded in the same segment where the electrodes were inserted (L5cL; Fig. 6E, F). This was in contrast with the CCs obtained during the CDPs recorded rostrally

(L5rL) that were higher during the nCDPs than during the npCDPs (Fig. 6G, H). We have examined the CCs of 12 pairs of neurones located in the L5 segment and of 7 pairs of

neurones located in the rostral part of the L6 segment. 8/12 of the L5 pairs (67%) showed higher CC during the L5-npCDPs than during the L5-nCDPs. The activity of 7/12 of the L5 neurone pairs was also increased at the time of the CDPs recorded from the adjacent L6 segment. Of these, 5/7 pairs (70%) showed higher CCs during the L6-npCDPs than during the L6-nCDPs. The activity of 3/5 pairs 16 This article is protected by copyright. All rights reserved.

of L6 neurones (60%) was also more synchronized during the L6-npCDPs than during the L6-nCDPs, while 4 pairs (57%) showed higher CC during the L5-npCDPs than during the L5-nCDPs. We may therefore conclude that the higher JFPs and CCs between neurone pairs observed

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during the npCDPs than during the nCDPs recorded in the same or in the adjacent segment indicate a higher temporal synchronization of neuronal activity. Neuronal responses produced by electrical stimulation of cutaneous nerves Manjarrez et al., (2003) showed that dorsal horn neurones firing in synchrony with spontaneous

CDPs responded to electrical stimulation of low threshold cutaneous afferents. Since at that time no

distinction was made between neurones discharging during nCDPs and/or npCDPs, it now seemed important to examine possible differences between the responses of neurones activated in synchrony during nCDPs and/or npCDPs and neurones that showed no changes in firing during these CDPs. In 111/126 neurones we examined the responses produced by electrical stimulation of low

threshold SP and/or SU fibres. 37/52 neurones whose activity was increased during both the nCDPs

and npCDPs, 9/19 neurones whose activity was increased during the nCDPs but not during the npCDPs and 4/6 neurones activated during npCDPs but not during the nCDPs responded to stimulation of low threshold SU or SP afferents (1.15-1.8T). This was in contrast with the responses

of neurones whose activity did not change during the nCDPs or npCDPs, where only 5/34 responded to electrical stimulation of these cutaneous nerves. Fig. 7A shows the distribution of the onset latencies of the neuronal responses produced by

electrical stimulation of the SP nerve with single pulses 1.1 to 1.8 T, measured relative to the arrival of the afferent volley to the spinal cord (see double headed arrow). While some neurones (5/33) responded to SP stimulation with monosynaptic latencies (range 0.9-1.0 ms), most of them (28/33) responded only with polysynaptic latencies (range 1.4-23 ms). There were no substantial differences in the onset latencies of the responses to SP nerve stimulation of neurones firing during both the nCDPs and npCDPs and of neurones firing only during the nCDPs or npCDPs. The onset latencies of the neuronal responses to stimulation of the SU nerve stimulation were in the polysynaptic range (218 ms; see Fig. 7B). In 8 experiments, in addition to the responses produced by stimulation of cutaneous afferents, we examined the responses of 18 neurones to stimulation of the GS and PBSt nerves with stimuli ranging between 2-5T. The onset latencies of the responses of 7 neurones to cutaneous (1.7-3.7 ms) 17 This article is protected by copyright. All rights reserved.

and muscle (9-13 ms) nerve stimulation suggested polysynaptic activation. 5 neurones in this group showed increased activation during both the nCDPs and npCDPs, and one neurone increased its activity only during the npCDPs. These neurones were located in more central and deeper regions of

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the gray matter (see Fig. 8Ba and D). These observations suggest that the neurones whose activity was increased both during the

spontaneous nCDPs and npCDPs responded more frequently to electrical stimulation of low-threshold cutaneous afferents than neurones whose activity was not changed during the CDPs. To test this possibility further, and considering that the increased neuronal firing during the CDPs varied with the segmental origin of these potentials (Fig. 4), we asked whether there was any relation between the

mean number of segments where a given neurone increased its activity during the nCDPs and/or npCDPs recorded in different segments with the proportion of neurones responding to electrical stimulation of low-threshold cutaneous nerves. As shown in Fig. 7C, these two parameters were positively correlated (0.87 of linear adjustment

bondage, see statistics), meaning that neurones associated with few spontaneous CDPs responded less frequently to stimulation of low threshold cutaneous afferents than those neurones associated with CDPs in more segments. Yet, we found no correlation between neuronal activation during the spontaneous nCDPs and npCDPs and the mono- or oligosynaptic latencies of the evoked SP and/or

SU responses. Neuronal responses produced by mechanical stimulation of the skin In 4 experiments we examined the responses of 23 neurones to stimulation of the skin with Von-

Frey filaments. Of 12 neurones whose activity was increased both during the nCDPs and npCDPs, 11 responded to mechanical stimulation of the skin in the left hindlimb. Their threshold varied between medium and high force levels (0.6-180 gm-F). Of four neurones whose activity increased during the nCDPs but not during the npCDPs, three also responded to mechanical stimulation with activation thresholds varying between 2 and 6 gm-F. Two of the seven neurones that showed no change in spontaneous firing during the nCDPs and npCDPs responded to mechanical stimulation with strengths of 1.4 gm-F. All the neurones activated by tactile stimuli showed phasic responses when the Von-Frey filament touched the skin, but not when it was removed, suggesting that these were activated by rapidly-adapting cutaneous receptors (e.g. Meissner or Pacinian corpuscles; see Gilman, 2012).


The diagrams in Fig. 7D depict the sensory fields of the neurones responding to mechanical stimulation of the skin. These fields were determined by probing the skin with Von-Frey hairs and noting whether or not there was a neuronal response. Some neurones had relatively small sensory

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fields, while others had larger fields, but no differences could we find between the neuronal thresholds to mechanical stimulation and laminar location. Although the location of these sensory fields agrees in general with those previously described by other investigators (Brown & Fuchs, 1975; Brown et al., 1975; Light & Durkovic, 1984), there were some discrepancies in the sense that the spinal location of the examined neurones tended to be more lateral than that reported in the above studies (see Fig. 7E).

It is possible that these discrepancies were due to different experimental conditions (i.e., spinal versus anaesthetized preparations), or else that we selected for analysis neurones with relatively low rate of spontaneous firing (see Brown et al., 1975). Altogether these observations indicate that there were no significant differences in the sensory

fields of neurones whose activity was increased both during the nCDPs and npCDPs and neurones that showed increased firing during either the nCDPs or the npCDPs. Spinal location of the examined neurones As detailed in Methods, at the end of the experiment the glass micropipette was cut leaving the

intraspinal portion in place. In all experiments except two, the section containing the recording micropipette was fully recovered and photographed (see Fig. 8A). Of 126 neurones we could determine the approximate intraspinal location of 117 neurones using the depth readings made during the experiment that were positioned along the track left by the recording micropipette. The location of 57 neurones whose activity was increased during both the nCDPs and npCDPs

(n+, np+) is shown in Fig. 8B. Most of them (37) responded to stimulation of cutaneous and/or muscle nerves (1.15-1.8T) with mono- or polysynaptic latencies (Fig 8Ba). Neurones that failed to respond to stimulation of cutaneous nerves had a similar intraspinal distribution (Fig. 8Bb, yellow dots). The 19 neurones whose activity increased only during the nCDPs (n+, np0) and the 6 neurones

whose activity increased only during the npCDPs (n0, np+) showed a similar distribution (Figs. 8Ca and Fig. 8Cb). About half of these neurones also responded to stimulation of cutaneous nerves. Fig.

8D shows the intraspinal location of 35 neurones whose spontaneous activity was not changed during the nCDPs or npCDPs. Of these, only 6 responded to the cutaneous and/or muscle nerve stimulation


in contrast with the responses of neurones whose activity increased during the nCDPs and/or the npCDPs (see Table in Fig. 8 for details). Altogether, these observations indicate that there was no preferential distribution of the

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intraspinal location of the neurones according to their correlation with the nCDPs and/or the npCDPs. They also show that neurones responding with mono or polysynaptic latencies to stimulation of cutaneous nerves had a similar intraspinal location. None of the studied neurones could be antidromically activated by stimulation of the ipsilateral

DLF with supramaximal strengths (that is, with latencies below 1.5 ms relative to the arrival of the descending volley to the segment of neuronal location). Neurones were however activated with longer latencies (1.5 to 20 ms). Although the tested neurones had no axons projecting to distant segments through the left DLF, the activation of tract neurons with axons ascending in the dorsal columns or with crossed projections could not be excluded. Inhibitory interactions between spontaneous and evoked CDPs During the course of the present series of experiments we noted that the episodes of increased

neuronal activity observed during the spontaneous nCDPs or npCDPs were preceded and followed by silent periods lasting 30-70 ms (see rasters and histograms displayed in Figs. 2C, D and G, H and Fig. 3C, D). Since these silent periods also appeared in the superposed traces of nCDPs and npCDPs recorded in the same experiment (see upper traces in Fig. 2A and E), as well as in other experiments, we assumed that these changes could be due to inhibitory actions that were shared by many neurones

in the ensemble. The nCDPs and npCDPs depicted in Figs. 1-3 were retrieved using dot-templates that forced

selection of CDPs within a narrow baseline. Therefore, it seemed possible that the silent periods that preceded the selected nCDPs and npCDPs were imposed by the baseline selection procedure. Fig. 9A-C shows a series of superposed CDPs selected without the baseline constrains enforced

by the dot templates. These potentials were recorded in the left side of the L6 segment and show a clear reduction of spontaneous CDPs before as well as after the selected nCDPs (Fig. 9A) and npCDPs (Fig. 9B and C). These findings suggest already that the silent periods were not random but were probably due to activation of inhibitory pathways by the same neuronal ensemble that contributes to the negative component of the CDPs.


In a previous study Manjarrez et al., (2000) showed that the spontaneous CDPs were inhibited for about 100 ms after stimulation of low-threshold cutaneous afferents. We have now examined the changes in the cutaneous-evoked CDPs and DRPs during the silent period that follows the

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spontaneous nCDPs and npCDPs (30-70 ms). Fig. 9D-F illustrates the depression of the CDPs and DRPs produced by electrical stimulation of

the left hindlimb plantar skin to 50% and 74% of their control values when applied with a delay of 50 ms after the peak of the nCDPs, at a time when the negative component had already subsided and

there were no DRPs. As expected, the CDPs and DRPs produced by stimulation of cutaneous afferents were also inhibited (to 31% and 70% of their control values) when tested 50 ms after the negative peak of the npCDPs, already during their positive phase (Fig. 9G-I). We confirmed this finding in other 3 experiments by showing that 30-70 ms after the nCDPs and

npCDPs the dorsal horn field and cord dorsum potentials produced by electrical stimulation of lowthreshold cutaneous fibres were inhibited (see also García et al., 2004). Discussion A distributed organization of the dorsal horn neuronal ensemble As mentioned in the introduction, Rudomin et al., (1987) proposed that separate sets of dorsal

horn neurones modulated the activity of Class I and Class II inhibitory neurones. The neuronal circuitry explaining this proposal is illustrated in Fig. 10A. In this case, activation of Class I glycinergic interneurones by one set of dorsal horn neurones (marked in blue) would lead to Ib nonreciprocal postsynaptic inhibition in motoneurones (MN) and Clarke’s column neurones (CCol). Another set of dorsal horn neurones (marked in red) would activate Class II GABAergic interneurones that produce PAD in muscle spindle afferents and postsynaptic inhibition in motoneurones. The observations presented in this study suggest instead that depending on the level of

synchronization between the dorsal horn neurones, the same network can lead to a selective modulation of the activity of Class I and Class II interneurones. This proposal is based on the findings that the same dorsal horn neurone may increase its spontaneous firing during the negative component of both the nCDPs and the npCDPs (Fig. 2), and that most pairs of individual neurones within this population exhibit higher probabilities of joint firing as well as increased synchronization during the generation of spontaneous npCDPs than during the nCDPs (Figs. 5 and 6). 21 This article is protected by copyright. All rights reserved.

We have assumed, as a working hypothesis, that the patterns of synchronization in the ongoing activity of the dorsal horn neurones reflect, to some extent, specific arrangements of functional connectivity within this neuronal ensemble (see Fries, 2005 and Womelsdorf et al., 2007), and that the

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observed changes in the level of neuronal synchronization, albeit small, can be indicators of changes in the functional organization of the neuronal ensemble (Softky, 1995). During states of low synchronization of the dorsal horn neurones, there would be a predominant activation of the pathways

mediating Ib postsynaptic inhibition, as illustrated in the diagram of Fig. 10B. Since the pathways mediating PAD require a higher temporal and spatial summation to be activated (see Eccles et al., 1963), increased spatial and temporal synchronization within the dorsal horn network would then

provide an additional and more effective excitatory input on Class II interneurones, some mediating PAD of muscle spindle afferents (see Rudomin et al., 1987), as well as on last order dorsal horn interneurones mediating PAD of cutaneous afferents (see Jankowska et al., 1981), as illustrated in Fig. 10C. If, as suggested by the present observations, the activity Class I and Class II interneurones is

influenced by the same set of dorsal horn neurones, how to explain the spontaneous activation of Class I neurones without a concurrent PAD documented by Rudomin et al., (1987)?. We believe that

this situation can be accounted for by assuming mutual inhibitory interactions between Class I and Class II interneurones, as illustrated in the diagrams of Fig 10B and C. The inhibition of the responses of dorsal horn neurones and of the DRPs produced by cutaneous inputs that is observed after the nCDPs had subsided (see Fig. 9D-F), could be due, at least in part, to a glycinergic inhibition

(Rudomin et al., 1990) exerted by Class I interneurones on the dorsal horn neurones mediating PAD of cutaneous afferents. The inhibition exerted by Class I interneurones on Class II interneurones would in turn reduce the PAD of muscle spindle afferents. This would explain the generation of inhibitory ventral root potentials without DRPs associated to the activation of Class I interneurones reported by Rudomin et al., (1987). By the same token, we have assumed that Class II interneurones, in addition to their GABAergic

action on afferent fibres, inhibit Class I interneurones, as they do with motoneurones (Rudomin et al, 1987). Hence, during the preferential recruitment of the pathways mediating PAD there would be a transient inhibition of Class I interneurones.


In addition to this simplified scheme, one should consider possible inhibitory interactions between different sets of dorsal horn neurones, including the commissural neurones (see Biella et al., 1997; Petkó et al., 2004; Jankowska et al., 2007; Bannatyne et al., 2009; Petitjean et al., 2012). These

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inhibitory interactions are envisaged as a mechanism that could contribute to the generation of structured (non-random) patterns of functional neuronal connectivity (Rodríguez et al., 2011). Once the spontaneous activity of the different sets of neurones becomes synchronized, the dorsal

horn neuronal network would behave as a unified ensemble of mutually interacting elements that may acquire particular configurations of functional connectivity and develop specific interactions with other reflex pathways. This feature would allow the same neuronal network to be involved in different functional tasks, as it has been suggested for spinal generators of locomotion patterns (see Chiel et al., 2009). Yet, it should be pointed out that this mode of operation of the dorsal horn neuronal network

does not exclude, but rather complements, the possibility that separate sets of neurones also have direct connections with Class I interneurones and Class II interneurones as illustrated in Fig. 10A, based on the original proposal of Rudomin et al., (1987). Identity of the neurones activated in synchrony with nCDPs and npCDPs Most neurones whose spontaneous activity was increased during the nCDPs and/or npCDPs

responded to electrical stimulation of low-threshold cutaneous afferents with mono- and/or oligosynaptic latencies. They were mainly located in laminae IV-VI, in agreement with previous findings of Manjarrez et al., (2000). These neurones were also more susceptible to be activated by skin stimulation with Von-Frey filaments (with medium to high force levels) or by electrical

stimulation of low threshold cutaneous afferents (1.15-1.8 T), in contrast with neurones whose spontaneous activity was not associated with the spontaneous nCDPs or npCDPs. Since the axonal projections of dorsal horn neurones usually cover only a few millimeters

(Brown, 1982; Bannatyne et al., 2009), we have assumed that the dorsal horn neurones involved in the generation of the nCDPs and npCDPs are connected with other neurones in the ipsilateral and contralateral dorsal horn in the same and neighboring segments and probably also with Class I and/or Class II interneurones in deeper layers. Although we still have very limited information on the axonal projections of the dorsal horn neurones that we found to be associated with the generation of the

nCDPs and npCDPs, we know that these neurones were not antidromically activated by stimulation of the ipsilateral dorsolateral fasciculi, a finding suggesting they had no ascending axonal branches 23 This article is protected by copyright. All rights reserved.

reaching the low thoracic segments via this pathway, but crossed or dorsal column projections were not tested which limits the conclusion. A substantial number of neurones that increased their activity during both the nCDPs and

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npCDPs were within lamina IV-VI (Fig. 9B), some of them in the same location as the dorsal horn commissural interneurons with group II and cutaneous inputs (Bannatyne et al., 2006). Hence, it is possible that some of the dorsal horn neurones that increased their spontaneous firing during both the npCDPs and the nCDPs, and responded to low-strength electrical stimulation of cutaneous nerves with polysynaptic latencies, were commissural neurons arising and projecting mostly in Rexed’s laminae III-IV that contributed to the bilateral neuronal synchronization presently observed (see Pétko et al., 2004). Clearly, a more complete functional identification of the dorsal horn interneurones whose

activity is increased during the spontaneous nCDPs and npCDPs is required for a proper appraisal of their involvement as modulators of transmission along a variety of inhibitory pathways. That is, in addition to use their spontaneous activity as a criterion for their selection, it seems necessary to have a

more precise characterization of their location within the spinal laminae as well as of their responses to stimulation of low and high threshold of cutaneous and muscle afferents and of their axonal projections, as was done by Bannatyne et al., (2006) for the characterization of the dorsal horn commissural interneurones mediating group II actions. It would be desirable to complement this characterization with immuno-histochemical studies to categorize the neurones by their expression of different neuropeptides, as it has been done by Polgár et al., (2013), Hughes et al., (2012) and more recently by Ashrafi et al., (2014) and by Fink et al., (2014). Concluding remarks Using activity of units and local field potentials recorded in the cortex of cats and monkeys,

Womelsdorf et al., (2007) concluded that the synaptic effectiveness of functional connections

increases during periods of high rhythmic oscillations in the gamma range (30-100Hz), and could be a means to regulate information flow in complex neuronal circuits (see also Fries, 2005 and Nácher et al., 2013). As pointed out by Womelsdorf et al., (2007) synchronization between two groups of

neurones is likely to facilitate interactions between them, particularly when their temporal interaction windows open at the same time, that is, when the rhythmic synchronization within the groups is also synchronized between the groups. 24 This article is protected by copyright. All rights reserved.

The present set of observations supports the existence in the spinal cord of similar mechanisms of control of information flow by neuronal synchronization. This control appears to be exerted by a longitudinally, bilaterally distributed ensemble of functionally interconnected sets of neurones.

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Increased synchronization between the spontaneous activity of neurones in this ensemble may lead to the preferential activation of the presynaptic inhibitory pathways that modulate the information transmitted by cutaneous afferents to other segmental and supraspinal networks. We suggest that the patterns of functional connectivity between neurones in this ensemble are

continuously modified by sensory and descending influences. They may acquire different, nonrandom, structured configurations in response to nociceptive stimulation or after acute nerve lesions

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extraction method for signal classification applied to cord dorsum potential detection. J Neural Eng 9, 1-12. Womelsdorf T, Schoffelen JM, Oostenveld R, Singer W, Desimone R, Engel AK, & Fries P

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1612. Author contributions Conception and design of experiments: P.R, E.CH and D.Ch. Collection, analysis and

interpretation of data E.CH., D. Ch. and P.R. Drafting of the article and reviewing it critically for important intellectual content: P.R., E.CH and D.Ch. All the authors have contributed to this study. Experiments were performed at the Department of Physiology, Biophysics and Neurosciences, Center of Research and Advanced Studies of the Instituto Politécnico Nacional, México. All authors

approved the final version of the manuscript for publication. Funding This work was partly supported by CONACyT grant 50900. E.CH. was holder of a CONACyT

fellowship for doctoral studies. Acknowledgements We thank Drs. S. Glusman and I. Jiménez and for their valuable comments to the manuscript

and to E. Hernández and C. León for their valuable technical support and to E. Rosales for secretarial assistance. We also thank Dr. P. Sánchez for providing us with the recordings illustrated in Fig. 9A-C.


Figure Legends Figure 1. Dot-template selection of spontaneous nCDPs and npCDPs. A, spontaneous CDPs recorded from the rostral and caudal regions of the left L6 segment (L6rL-CDPs and L6cL-CDPs),

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together with the L6-DRPs and the L6-IFPs. L6cL-CDPs exceeding a preset amplitude (shown by dotted horizontal line) were selected for further processing, together with the associated CDPs, DRPs

and IFPs (see boxes). B, upper trace shows a spontaneous L6cL-nCDP together with the selecting dot templates. Arrows indicate the dot-template bounds. C and D, superposed traces of selected L6cL-

nCDPs and associated DRPs and IFPs. E-G, same as B-D but dot-templates adjusted to select L6cLnpCDPs. Note that negative DRPs appeared together with the L6cL-npCDPs but not with L6cLnCDPs. IFPs were recorded from the left L6 segment at 1532 µm depth (around Rexed’s laminae V). Negativity upward for CDPs and DRPs and downward for IFPs. Further details in text. Figure 2. Neurone showing increased firing during spontaneous nCDPs and npCDPs. A,

superposed L5cL spontaneous nCDPs selected with the dot-templates (upper traces) together with a

single nCDP and associated IFP with spike. Superposed traces on the left show spikes selected with

PCA. B and C, L5cL-nCDPs separated in 4 groups of different amplitudes and rasters of the associated neuronal action potentials (see colors). D, histograms obtained from the raster display

shown in C. E-H, same as A-D, for L5cL-npCDPs. I-K, neuronal responses produced by electrical stimulation of the SP nerve with single pulses 1.4T applied once per second. Mean onset latency of neuronal responses 2.7±1.2 ms. Neurone located between Rexed’s laminae V and VI (red dot in Fig.

8Ba). Means of superposed traces in A, E and I are shown in white. Further explanations in text. Figure 3. Neurone showing increased firing during spontaneous nCDPs but not during

npCDPs. Same format as in Fig. 2. A, superposed spontaneous L6rL-nCDPs together with a single nCDP and associated IFP. B, superposed traces of L6rL-nCDPs of different amplitudes (see colors).

C, rasters of action potentials associated with L6rL-nCDPs of different amplitudes, as indicated. D, histograms obtained from the raster display shown in C. E-H, same as A-D, for L6rL-npCDPs. Note increased neuronal activity during the spontaneous nCDPs but not during the npCDPs. I-K, responses produced by SP nerve stimulation with single pulses 1.5T. Evoked neuronal responses had a mean onset latency of 2.5± 1.3 ms. Neurone located in Lamina V (red dot in Fig. 8Ca). Further explanations in text.


Figure 4. Changes in spontaneous activity of dorsal horn neurones associated with the nCDPs and npCDPS generated in different spinal segments. Data obtained from two neurones whose activity was simultaneously recorded with the same micropipette. A, B samples of spontaneous

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npCDPs and nCDPs recorded from different segments, displayed together with the raw and filtered L6rL- IFPs keeping neuronal action potentials (histology shows recording site). Arrows point at the

action potentials of neurone 1 (red) and neurone 2 (black). C, normalized increments (In and Inp) of activity of neurone 1 during spontaneous nCDPs (blue bars) and npCDPs (red bars) generated in different segments in the left (L) and right side (R) of the spinal cord during a first 15 min recording period. D, same during a second recording period of 15 minutes. E and F, as C and D for neurone 2. Further explanations in text. Figure 5. Changes in joint firing probabilities of dorsal horn neurones during spontaneous

nCDPs and npCDPs. A, joint firing index (JFP) of pairs of neurones calculated at different time

intervals (t1–t6) during the spontaneous nCDPs and npCDPs recorded in the same segment as that of neurone location (left L5 or L6) and in the adjacent segment. The npCDPs-JFPs are plotted in

increasing order (red dots) together with the corresponding nCDPs-JFPs (black dots). The inserts show mean L6rL nCDPs (black) and npCDPs (red) recorded in one experiment together with superposed gray bars indicating the time windows for JFPs calculations. Note that at t3 and t4 most JFPs were higher during the npCDPs than during the nCDPs, regardless of their segmental location

relative to neuronal recording segment (same or adjacent). B, mean JFPs and standard error (SE) obtained at different times during the nCDPs (black) and the npCDPs (red). Data obtained from the same and from adjacent segments are combined. C, mean discharge rates and SE of neuronal firing

during the nCDPs and npCDPs. Lower sets of graphs in panels B and C show the p-values calculated with the paired t-test (black squares) and with the Wilcoxon test (gray circles). Further explanations in text. Figure 6. Changes in cross-correlation between the action potentials of pairs of neurones

during nCDPs and npCDPs. A-C, A pair of recording micropipettes were inserted within the dorsal

horn in the L5cL segment. One recorded the action potentials of neurone n1 and the other the action potentials of neurones n2 and n3. A and B, CCs of the activity of neurones n1 and n2 during the L5cL nCDPs and npCDPs. C-D, same for neurones n1 and n3. Note higher neuronal synchronization during the npCDPs than during the nCDPs. E and F, CCs of neurones nA and nB recorded in a different 32 This article is protected by copyright. All rights reserved.

experiment with two separate micropipettes, both inserted in segment L5cL. As in A-D, synchronization of neuronal firing was higher during the npCDPs than during the nCDPs generated in the same segment of neuronal recordings (L5cL). G-H, CCs of same neurones nA and nB during the

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nCDPs and npCDPs generated in the rostral part of segment L5 (L5rL). In this case the neuronal CC was higher during the nCDPs than during the npCDPs. Limits of significance are indicated with dashed lines. See text for further details. Figure 7. Neuronal responses produced by electrical stimulation of cutaneous nerves and

by mechanical stimulation of the skin. A and B, onset latencies of neuronal activation following SP and SU nerve stimulation with single pulses 1.15-1.8T applied at 1 Hz. C, number of segments in

which the examined neurones increased their spontaneous firing during the nCDPs and/or npCDPs, divided by the total number of recorded segments (abscissa) versus percentage of neurones activated by low strength (1.15-1.8T) electrical stimulation of the SU or SP nerves (ordinates). Linear adjustment and 95% confidence band are shown (see Methods). D, neuronal sensory fields disclosed with Von Frey filament stimulation of the hindlimb skin. E, spinal location of neurones responding to

tactile skin stimulation. Color codes in A, B, D and E indicate increased neuronal activation during

nCDPs and npCDPs (n+, np+), only during nCDPs (n+, np0), only during npCDPs (np+, 0) or no changes (n0, np0). Further explanations in text. Figure 8. Intraspinal distribution of neuronal recording sites. A, photography of spinal

section with recording micropipette. B, intraspinal location of 57 neurones whose spontaneous firing was increased during both nCDPs and npCDPs (n+, np+). Ba shows neurones responding to stimulation of muscle and cutaneous nerves and Bb neurones whose responses were not tested or

failed to respond. Note that most neurones were around Rexed’s laminae IV-VI. Ca, location of 19 neurones that increased activity only during nCDPs (n+, np0). Cb, location of 6 neurones firing only

during npCDPs (n0, np+). D, location of 35 neurones whose activity was unchanged during nCDPs or npCDPs (n0, np0). Color codes in the Table indicate latency (monosynaptic, MS or polysynaptic, PS) of responses produced by stimulation of cutaneous and muscle nerves as well as number of examined neurones. In Ba, the red ellipse encloses neurones responding to stimulation of muscle nerves. Red

dots in Ba and Ca show location of the neurones illustrated in Figs. 2 and 3, respectively. Note that most neurones associated with nCDPs and/or npCDPs responded to stimulation of cutaneous nerves, in contrast with neurones whose activity was not changed during nCDPs and npCDPs. Further explanations in text. 33 This article is protected by copyright. All rights reserved.

Fig. 9. Inhibitory interactions between spontaneous and evoked activity of dorsal horn neurones. A-C, three sets of superposed spontaneous L6-CDPs selected without baseline constrains. A, spontaneous nCDPs and B, C spontaneous npCDPs. D, L6-CDPs and L6-DRPs produced by single

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stimuli 3.2T strength applied once per second through a pair of needles inserted in the left foot pad. E, template selected L6-nCDPs. F, test CDPs and DRPs produced by stimuli applied 50 ms after the

peak of the “conditioning” nCDPs. Note reduction of footpad responses even though the nCDPs occurred without associated negative DRPs (see high gain DRP trace below). G-I, same as D-F. Test stimulus was applied during the positive phase of the L6-npCDPs that were generated together with negative DRPs (see high gain trace below). Note depression of the CDPs and DRPs produced by footpad stimulation. Percentage changes of test responses relative to control are indicated. Traces D-I are means of 32 responses. Further explanations in text. Figure 10: Diagrams of separate versus common networks of dorsal horn neurones

controlling Class I and Class II interneurones. A, separate sets of dorsal horn local neurones

modulate the activity of Class I and Class II interneurones as proposed by Rudomin et al., (1987). Activation of Class I glycinergic interneurones by one set of dorsal horn neurones (marked in blue) facilitates transmission along the pathways mediating Ib non-reciprocal postsynaptic inhibition in motoneurones (MN) and Clarke’s column neurones (CCol). Another set of dorsal horn neurones (marked in red) activates Class II GABAergic interneurones that synapse with Ia afferents and motoneurones. B and C, diagrams based on the present observations. They assume that the same

distributed network of dorsal horn neurones differentially modulates activity of Class I and Class II interneurones. B shows that during low levels of dorsal horn neuronal synchronization, Class I interneurones (in blue) are preferentially activated relative to Class II interneurones (in pink) and

generate nCDPs practically without DRPs (lower set of records). C shows that increased synchronization between dorsal horn neurones increases activation of dorsal horn neurones (inactive neurones in white) and recruits Class II interneurones (marked in red), leading to preferential activation of pathways mediating PAD and presynaptic inhibition and generate the npCDPs and DRPs as shown by the lower set of records. Activation of Class I interneurones without concurrent DRPs

(lower traces in B) is assumed to result from reciprocal inhibitory interactions between Class I and

Class II interneurones and between Class I neurones and dorsal horn neurons, as indicated by interrupted lines and question marks. Note that in B and C different sets of last order GABAergic


interneurones are assumed to produce PAD in group Ia muscle and cutaneous afferents (see

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Jankowska et al., 1981). See text for further details.


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B

L5rL L5rR L5cL

-

L6rL L6rR L6rL IFP L6rL IFP filtered

+

2 mm + -

100 ms

Neurone 1

C

80 m V

L5cR

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A

0-15 min L

R

Neurone 2

D

E

16-30 min

L4c

0-15 min

F

16-30 min

0

1 1

0

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same

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0.4

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0.0

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B t2

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t6

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0

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t2

t3

t4

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20

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t6

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10

10

1.0

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0

t3

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0.6

0

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0.6

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Joint firing probability

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1.0

80 m V

A

0.5

paired t-test wilcoxon

0.5

0.0

0.0

-120

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-40

0

40

80 (ms)


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0

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n1 vs n2

n1 vs n3

L5cL nCDPs

0.35 0.3

L5cL npCDPs 0.08

A

B

0.07

L5cL npCDPs

C

D

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-20 -10 0 10 20 ms

0.06

0.25

Counts

L5cL nCDPs

0.05

0.2

0.04

0.15

0.03

0.1

0.02

0.05

0.01

0

0

2

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-20 -10 0 10 20 ms

nA vs nB L5cL nCDPs

E

nA vs nB

L5cL npCDPs

F

L5rL nCDPs 6 5

G

L5rL npCDPs

H

Counts

1.5 4 3

1

2

0.5 1 0

0 -20 -10 0 10 20 ms

-20 -10 0 10 20 ms


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-20 -10 0 10 20 ms

100 m V

number of neurones

6 5

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4 3

0

C

6 5 4 3 2 1 0

2 1

3 2 1

L6rL SU

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0

10

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3

0

10

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30

2 1 0

0

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n=11

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L6rL SP

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b

+pn +n

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b

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I IV V VI

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Cutaneous monosynaptic (MS) Cutaneous polysynaptic (PS) Cutaneous PS & muscle PS muscle PS no response not tested Total

n0, np0 n+,np+ 4 28 5 0 14 6 57


n+,np0 2 7 0 0 10 0 19

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npCDP

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31% 70% + -

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100 ms

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V 40m V 40m

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