Pflugers Arch - Eur J Physiol DOI 10.1007/s00424-016-1886-6
NEUROSCIENCE
Analysis of spontaneous activity of superficial dorsal horn neurons in vitro: neuropathy-induced changes Carolina Roza 1 & Irene Mazo 1 & Iván Rivera-Arconada 1 & Elsa Cisneros 1 & Ismel Alayón 1 & José A. López-García 1
Received: 13 July 2016 / Revised: 6 September 2016 / Accepted: 26 September 2016 # Springer-Verlag Berlin Heidelberg 2016
Abstract The superficial dorsal horn contains large numbers of interneurons which process afferent and descending information to generate the spinal nociceptive message. Here, we set out to evaluate whether adjustments in patterns and/or temporal correlation of spontaneous discharges of these neurons are involved in the generation of central sensitization caused by peripheral nerve damage. Multielectrode arrays were used to record from discrete groups of such neurons in slices from control or nerve damaged mice. Whole-cell recordings of individual neurons were also obtained. A large proportion of neurons recorded extracellularly showed well-defined patterns of spontaneous firing. Clock-like neurons (CL) showed regular discharges at ∼6 Hz and represented 9 % of the sample in control animals. They showed a tonic-firing pattern to direct current injection and depolarized membrane potentials. Irregular fast-burst neurons (IFB) produced shortlasting high-frequency bursts (2–5 spikes at ∼100 Hz) at irregular intervals and represented 25 % of the sample. They showed bursting behavior upon direct current injection. Of the pairs of neurons recorded, 10 % showed correlated firing. Correlated pairs always included an IFB neuron. After nerve damage, the mean spontaneous firing frequency was unchanged, but the proportion of CL increased significantly (18 %) and many of these neurons Electronic supplementary material The online version of this article (doi:10.1007/s00424-016-1886-6) contains supplementary material, which is available to authorized users. * José A. López-García [email protected] 1
Dpto. Biología de Sistemas, Edificio de Medicina, Universidad de Alcalá, Campus Universitario, 28871 Alcalá de Henares, Madrid, Spain
appeared to acquire a novel low-threshold A-fiber input. Similarly, the percentage of IFB neurons was unaltered, but synchronous firing was increased to 22 % of the pairs studied. These changes may contribute to transform spinal processing of nociceptive inputs following peripheral nerve damage. The specific roles that these neurons may play are discussed. Keywords Multielectrode arrays . Nociception . Pain . Spike trains . SNI
Introduction Neurons located in the superficial dorsal horn (SDH) are directly implicated in the mechanisms of pain transmission through the spinal cord [11]. SDH neurons have been classed using different criteria such as morphology, neurochemical and electrophysiological traits, and local or projecting status [12, 48, 59]. Since more than 90 % of these neurons are located in local circuits [56], several approaches have been used to elucidate the organization of these circuits. It is known that circuits show a modular organization that allows the integration of different types of inputs ending on projection neurons located in lamina I [19, 33, 39]. However, the present knowledge of the types of neurons and their interconnections forming these circuits is limited and we are far from understanding the spinal neural coding for the signaling of painful events. Since 1926, the basic element of a neural code was established as the number of spikes fired in a fraction of time [1, 2]. However, it has long been accepted that a much more complex code arises when neurons are able to emit temporal patterns of spikes whose timing is reliable on a short time scale [21]. The coordination of temporal patterns across a
Pflugers Arch - Eur J Physiol
population of neurons adds another dimension of complexity to the neural code, i.e., synchronization [3, 26]. Technological advances have allowed for simultaneous recording of populations of neurons in different areas of the central nervous system, and complex analyses are being implemented to understand the encoding of different neural events, such as learning and memory [10], object recognition [58], or reward circuits [9]. It is remarkable that spike patterns are conserved in homologous regions across species [36], suggesting a key role of spike trains in neuronal coding. Damage to peripheral nerves alters the processing of nociceptive signals in the spinal cord. This involves changes in the coding of afferent inputs which are assumed to underlie central sensitization and neuropathic pain symptoms. In the past, some studies consistently demonstrated an increased excitability of projection and unidentified deep dorsal horn widedynamic range neurons following neuropathic insult [14, 27, 38, 57]. Changes included increased background activity and prolonged afterdischarges, enhanced responses to lowthreshold mechanical stimuli, and a leftward shift in the curve defining intensity-response relations as well as enlarged receptive fields. In contrast, intracellular recordings from superficial layers, including inhibitory neurons, detect only slight changes in intrinsic properties [5, 15, 53]. In general, the changes that take place in the coding of afferent signals in the SDH following peripheral injury are poorly understood despite their prominent position in the chain of nociceptive transmission. Since firing frequency may be only one variable of neuronal coding, here, we have used multiple recording electrodes to simultaneously assess the firing of small groups of neurons. We have used the spared nerve injury model of neuropathy to assess possible changes in the patterns of action potentials and synchronous discharges which could also amplify nociception by increasing the gain or generating noise in nociceptive lines giving rise to abnormal pain sensations [50]. We also used whole-cell recordings to characterize basic membrane properties and histological techniques to locate recording sites.
Induction of neuropathy and behavioral test Surgery was performed in 22–25-day-old animals under deep anesthesia with 3–4 % isoflurane in 100 % oxygen. For spared nerve injury (SNI), an incision was made in the skin of the lateral surface of the thigh and the sciatic terminal branches were exposed; the common peroneal and the tibial nerves were ligated with 8/0 nylon monofilament and sectioned distal to the ligature, sparing the sural branch. The incision was closed in layers with 5/0 sutures. The animals were housed in groups of 3–4 with access to water and food ad libitum and inspected periodically for infections or abnormal behavior. The up-down paradigm [13] was used to evaluate withdrawal threshold to mechanical stimulation on the sural innervation territory of the hind paw. For the SNI group, behavioral data was collected before the surgery and 2 weeks after induction of neuropathy, immediately prior to the electrophysiological experiment. For control animals, measurements were taken only the day of the electrophysiological experiment. Surgical extraction and maintenance of the longitudinal slice in vitro Mice were anesthetized with urethane (2 g/kg i.p.). The lumbar skin was cut and a dorsal laminectomy from thoracic to sacral vertebrae was performed. The spinal cord with attached roots was dissected free and placed in sucrose-substituted artificial cerebrospinal fluid (ACSF) at 4 °C (see composition below). The meninges were carefully removed, the roots separated from the cord, and the L1-S1 segments of the cord cut out. The cord fragment was glued with cyanoacrylate to a rigid base and positioned in a vibratome with the dorsal side up. A single slice of ∼500 μm containing the dorsal side of the cord together with the attached dorsal roots was obtained. The slice was mounted in the recording chamber with the dorsal side down to provide access to the recording electrodes and maintained at room temperature (22 ± 1 °C) with oxygenated ACSF (95 % O2, 5 % CO2) at a flow of 5 ± 1 ml/min. The composition of the ACSF was (in mM) NaCl 127, KCl 1.9, KH2PO4 1.5, MgSO4 1.3, CaCl2 2, NaHCO3 22, and glucose 10, (pH 7.4). For sucrose-substituted ACSF, NaCl was substituted for equimolar concentration of sucrose.
Materials and methods Electrical stimulation of the dorsal root Animals Young adult CD-1 female mice (n = 66, 30–57 days old, and body weight 27–49 g) bred at the University Animal House were used. The European Union and State regulations for animal experiments were followed. All experimental protocols were approved by the Universidad de Alcalá Committee on Animal Research and the Regional Government (project license: ES280050001165).
A dorsal root (usually L4) was inserted into a suction electrode for electrical stimulation. Electrical pulses of increasing magnitude were applied in order to activate the different afferent fibers contained in the dorsal root. Neurons were classified according to the lowest stimulus intensity that evoked action potential firing (extracellular recordings, see below) or subthreshold responses (whole-cell recordings, see below). On the basis of previous experiments from our laboratory
Pflugers Arch - Eur J Physiol
performed in isolated sciatic nerve from adult mice [46], neurons responding to 50-μs pulses of intensities 10.000) as obtained with GLO and showed at least one clear peak in the ACH well away from time 0 (see Fig. 2c).
a
DiI / Trypan blue
Ipsilateral
SNI Dapi
b
Contralateral
200 µm
Biocytin / IB4
c
Sensor I II III IV
25 µm
Electrode shank SP / IB4
d
Control
SP / IB4
e
SNI
100 µm
Pflugers Arch - Eur J Physiol Table 1 Proportions of neurons, mean firing frequencies, and firing patterns to depolarizing current vs neuronal type in control and SNI conditions
Control Spike-train patterns Clock-like
% (n)
I. simple
SNI
9 (10 2i)
Mean firing (Hz) 6.4 ± 0.7
53 (59 1i)
2.2 ± 0.2
I. fast burst
25 (28)
I. slow burst
5 (6)
I. mixed burst No spont.
8 (9)
Control + SNI
% (n) 18 (26 1i)*
Mean firing (Hz) 5.3 ± 0.5
Intracellular patterns Tonic
48 (69 2i )
2.4 ± 0.2
Tonic
1.1 ± 0.2
21 (30)
0.8 ± 0.3
Initial bursting
5.7 ± 2
3.5 (5)
2.5 ± 1.1
1.3 (2) 8.3 (12)
3.9 ± 1.1
Single spike
All patterns
I. irregular, i inverted neurons *p < 0.05 Fisher’s exact test
The proportion of CL neurons was significantly greater in the group of SNI-treated animals (26/144) when compared with that in the controls (10/112), this being one of
the major differences found between the groups (see Table 1; p < 0.05; Fisher’s exact test). No differences were found in cell location within the cord (see Fig. 2g)
b
mV
a 0
1ms
1s
20
c 240 nº events
I. Fq. (Hz)
0.5
10 0
e 20 mV 0.2 s
-56 mV
f
g
0
Time (s) 0.5
300
nº of events
d
0
0 0
Time (s) 0.2
Control -56 mV
20 mV
I/II III IV
0.1 s
32 pA
SNI -56 mV 20 mV 0.1 s
200 µm
I/II III IV
-100 pA
Fig. 2 a Original extracellular recording from a typical CL neuron recorded from a control mouse that had a mean discharge frequency of 9.8 Hz. The lower panel shows instantaneous frequency for this neuron. b The spike shape averaged for the period in a. c The corresponding autocorrelogram histogram built for this neuron in a 5-min period. Note the presence of three clearly identified peaks, which indicates that a preferential interspike interval of ∼100 ms is maintained. The GLO-Mat β1 value for this neuron was 0.16. d Original whole-cell recording showing regular spontaneous firing in the absence of current injection recorded from a control preparation (dotted line marks membrane
potential indicated at the left). e The auto-correlogram corresponding to the neuron in d. Note the similarity with autocorrelogram in c corresponding to the extracellular-recorded CL neuron. f Responses of the neuron in d to direct current injection. The upper panel shows an increased tonic firing to depolarizing pulses. The lower panel shows depolarizing sag upon hyperpolarization, followed by an increase in firing frequency at the end of the pulse. Drawings in g show the location of CL neurons from extracellular recordings in slices from control and SNI animals
Pflugers Arch - Eur J Physiol
a
b
*
mV
0 1
2 ms
10 sec
nº of events
240 160 80 0
d
15 10 5 0 0
e
*
20 mV 2s
-64 mV
nº of events
I. Fq. (Hz)
c
20
0.2
10 0 0
g
10 mV
0.1
Time (s)
0.1
0.2
Time (s)
Control 0.2 s I/II III IV
f 20 mV 0.1 s
SNI
200 µm
-68 mV 32 pA -80 pA
I/II III IV
Fig. 3 a An original extracellular recording from a typical IFB neuron whose instantaneous frequency is presented below. Note that each burst consists of a discrete number of AP (2–6) with high intraburst frequency (mean ∼154 Hz) and a very low mean discharge frequency (0.26 Hz). b Two spikes corresponding to a duplet burst indicated with asterisk in a. c The autocorrelogram built for the neuron in a. Note the presence of a narrow peak very close to 0. d Original whole-cell recording of a neuron from a control mouse showing spontaneous firing in bursts of two to three spikes/burst at a maximal frequency of 80 Hz. The two bursts around the
asterisk are enlarged in the lower panel; note that the oscillation in membrane potential occasionally gives rise to burst firing—AP are truncated at −25 mV. e The autocorrelogram for the neuron in d. Note the similarity with autocorrelogram in c corresponding to the extracellular-recorded IFB neuron. f This neuron showed an initial burst firing pattern in response to depolarizing current pulses as well as depolarizing sag and rebound firing to hyperpolarizing pulses. g Drawings depicting the location of IFB neurons from extracellular recordings within control and SNI preparations
or mean firing frequency between the SNI and control groups (Table 1). Three of the neurons recorded with whole-cell patch electrodes in the control animals showed spontaneous activity with a typical CL pattern, as assessed by their ACH and GLO values (an original example is shown in Fig. 2d, e). All of them exhibited tonic firing patterns to depolarizing current pulses (Fig. 2f). These neurons were significantly more depolarized than the remaining tonic neurons (−56.3 ± 1.9 mV at the lowest point of afterspike hyperpolarization vs −64.9 ± 1.8 mV; p < 0.05, Mann-Whitney test). Two of these neurons showed rectification upon hyperpolarization (sag) and high-frequency rebound firing on
depolarization (Fig. 2f). Another neuron did not show rectification and had a slow return to resting potential after hyperpolarizing pulses. According to GLO, all the remaining extracellularly recorded neurons were classed as irregular in a first instance. For a proportion of these neurons, more than 25 % of their action potentials came in bursts as analyzed with BAR. Hence, irregular firing neurons were segregated between irregular simple (IS) or irregular burst (IB). The extent of irregularity among IS and IB neurons was similar in control and SNI mice (see Online Resource 3 for a detailed quantification of the degree of regularity of the different neurons).
Pflugers Arch - Eur J Physiol
Among the IB neurons, some produced short-lasting (typically 2–5 spikes) high-frequency bursts (∼100 Hz) and were classed as irregular fast-burst neurons (IFB, an original recording is shown in Fig. 3a, b). Other neurons produced long-lasting (up to 200 spikes) low-frequency bursts (below 30 Hz) and were classed as irregular slowburst neurons (ISB, an original recording example is shown in Fig. 4a, b). IS neurons fired mainly isolated spikes (an original recording is shown in Fig. 4d, e) (see Table 1). IFB neurons had autocorrelograms characterized by a single peak close to time 0 followed by a few isolated events at different time points, which is consistent with the high intraburst frequency and the low mean frequency
a
found in these neurons (see Fig. 3c and Table 1). No differences were found for IFB neurons between the control and SNI groups regarding proportion, mean spontaneous firing frequency, or location (shown in Fig. 3g and Table 1) within the cord. Six spontaneous neurons with IFB patterns, as assessed with BAR, were recorded with whole-cell electrodes (3 controls and 3 SNI, see an original example in Fig. 3d, e). The maximum intraburst instantaneous frequency was very similar in control and SNI (106.3 ± 14.1 Hz, n = 6). When these 6 neurons were depolarized by current injection (at two times rheobase), the maximal firing frequency of the evoked burst (102.3 ± 15.6 Hz) matched the spontaneous intraburst frequency. Neurons of the control group had a
b
mV
0
1 ms
c
10 s
75
nºof events
I. Fq. (Hz)
0.25
50 25
400 200 0
0
d
0
Time (s)
0.5
e
mV
0.5 0
f 10 s
nº of events
I. Fq. (Hz)
10 5 0
g
1 ms 25 20 15 10 5 0 0
i 20 mV
Control
0.5 s -59 mV
Time (s)
1
IS
ISB
I/II III IV
h 20 mV 0.1 s
SNI
200 µm
-59 mV 96 pA
I/II III IV
-150 pA
Fig. 4 a An original extracellular recording from a typical ISB neuron and its corresponding instantaneous frequency (lower panel). Each burst consisted of a large number of AP (>100 spikes) with a mean intraburst frequency of ∼12 Hz and an overall mean discharge frequency of 7 Hz. b The averaged spike shape. c The corresponding auto-correlogram histogram. Note the presence of a flat line with a peak at ∼50 ms which detects certain regularity within the burst. d An original extracellular recording from an irregular simple neuron with its corresponding instantaneous frequency plot (lower panel; mean firing frequency of
1.4 Hz), averaged spike (e), and auto-correlogram (f). Note the presence of a flat line indicative of random interspike interval between consecutive spikes. g An original whole-cell recording from a control neuron with irregular spontaneous firing and spontaneous EPSPs. h This neuron showed tonic firing pattern in response to depolarizing current pulses as well as depolarizing sag and rebound firing to hyperpolarizing pulses. i Drawings depicting the location of ISB (white circles) and IS neurons (black circles) from extracellular recordings in control and SNI preparations
Pflugers Arch - Eur J Physiol
Synchronicity of spontaneous spikes in control and SNI animals Spike synchrony was tested using multiple recordings from MEAs on a total of 129 pairs in control and 136 pairs in SNI by means of cross-correlation histograms. Two major findings came out of this analysis. First, the percentage of correlated pairs was significantly greater in the SNI animals (30/136, 22 %) compared to that in the control (13/129; 10 %; p < 0.01; Fisher’s exact test), this being another major difference between the control and SNI groups. Second, all the correlated pairs found in the control and SNI conditions consisted at least of one IFB neuron. The IFB neurons showing correlation had similar properties to those for which no correlation was found, including the type of input from afferent fibers, number of spikes per burst, mean firing frequency for spontaneous discharges, and mean intraburst frequency. The other neuron of the pair could belong to any class including again IFB neurons (see Fig. 5). Among the 13 pairs recorded in the control, 6 were IFB-IS and 7 were IFB-IFB pairs. For SNI, 10 pairs were IFB-IS, 7 pairs were IFB-CL, 1 was IFS-ISB, and the remaining 11 were IFB-IFB pairs. Representative examples of the types of crosscorrelograms found in our sample are shown in Fig. 5. The most common type was a central symmetrical or asymmetrical
a
*1
*2
*3
u1 u2 u3 u4 10 sec
b
*1
*2
*3
u1 u2 u3 u4 25 ms
25 ms
25 ms
c 50 u1-u2
0.1
0 - 0.1 20
0
0.1
0.1
0 - 0.1
0 - 0.1 20
u2-u4
0
Time 0.1 (s)
u3-u4
Nº of events
Nº of events 0
u1-u4
Nº of events
Nº of events
Nº of events 0 0 - 0.1 8 u2-u3
0 - 0.1
40
40 u1-u3
Nº of events
characteristic firing pattern to depolarizing current pulses consisting on an initial burst of action potentials at the beginning of the pulse and exhibited sag and rebound firing on response to hyperpolarizing pulses (see Fig. 3f). Two SNI neurons of this class showed initial bursting pattern and sag but only subthreshold rebounds. The remaining neuron was the only neuron of our sample with a singlespiking pattern to depolarizing current and did show sag and rebound firing. ISB and IS neurons had ACHs that consisted of a noisy flat line indicating similar probability of firing at any given time from any given spike, although a few ACHs from ISB neurons showed a peak superimposed to the flat line but never reached values for regularity with GLO (Fig. 4c, f). These neurons exhibited mean firing frequencies in the midrange and the IS group was the most abundant class among spontaneously active neurons (Table 1). No differences were found in proportions, firing frequencies, and location within the cord (shown in Fig. 4i and Table 1) between the control and SNI groups. In the SNI group, we found 2 neurons exhibiting bursts starting with high-frequency firing which declined with time. None of the neurons recorded with whole-cell patch electrodes belonged to the ISB type, but 4 of them were of the IS type (2 control and 2 SNI, see an example in Fig. 4g). All of these neurons showed a tonic-firing pattern to intracellular depolarizing pulses. Sag and rebound firing were also commonly observed in IS neurons (see Fig. 4h).
0
0.1
0 - 0.1
0
0.1
Time (s)
Fig. 5 Example of four IFB units (u1–u4) recorded simultaneously in the superficial dorsal horn of a neuropathic mouse. a The spontaneous discharges of each individual unit as lines in a 100-s window. Asterisks represent instances in which the four units fired simultaneously. b The structure of three synchronous events (marked by asterisks in a) in an expanded time base. Spontaneous discharges are represented by their actual spike shapes. c The cross-correlograms obtained for each possible pair in a 5-min period. All histograms have a peak close to 0 indicative of synchrony between the neural pair. The cross-correlogram u1–u3 shows a central trough of 3-ms width
peak of 15–100 ms of width measured at half amplitude. We also observed lateral peaks displaced 5–15 ms from 0 showing widths in the range of 5–50 ms. In some cases, a central trough appeared combined with any of the previous ones. These were usually very narrow (5 ms). All types were found in control and SNI animals. Responses of neurons from control and SNI animals to dorsal root stimulation Extracellular recordings showed strong artifacts due to electrical stimulation of the dorsal root which precluded the possibility of building accurate stimulus-response curves. In fact, 27 % of the total sample of neurons could not be accurately
Pflugers Arch - Eur J Physiol
classified. A majority of the remaining neurons (164/185) responded to dorsal root stimulation and only 21/185 did not show any responses. Responses consisted on the firing of action potentials or the stopping of ongoing spontaneous activity. This later response was classed as Binverted^ and occurred in a small proportion of neurons, all of them having mean firing frequencies >4 Hz and belonging to the CL (n = 3) or the IS class (n = 4). Responsive neurons were classed according to the lowest stimulus intensity that evoked a clearly identifiable response as having LTA-input or HTA-input or C-inputs (Online Resource 2). Table 2 shows the number of neurons responding to the different inputs according to their spontaneous firing class. A comparison of these data clearly shows that neurons receiving LTA-input were significantly more abundant in the SNI group than in the control (34 vs 20 % respectively; p < 0.05 Fisher’s exact test), whereas the proportion of neurons receiving exclusively C-inputs was smaller in the SNI group (3 % in SNI vs 11 % in control; p < 0.05 Fisher’s exact test), this being another major difference between the groups. It is noteworthy that the largest increase in LTA-input after SNI was found among CL neurons (Table 2). As a consequence, the mean firing frequency in the neurons receiving LTA-input of SNI animals was significantly greater than that of the controls (1.4 ± 0.35 in control and 3.2 ± 0.4 Hz in SNI; p < 0.05, Mann-Whitney). No differences were found between the firing frequency of the control and SNI animals in the total sample. Approximately 78 % of neurons recorded with whole-cell patch electrodes (56/72) responded to dorsal root stimulation with EPSPs (38/72) or a mixture of EPSPs and IPSPs (18/72) or with action potentials (21/72). One additional neuron showed only IPSPs. The remaining 22 % was indifferent to afferent stimulation. In the control group, action potentials were elicited by dorsal root stimulation in tonic and initial burst neurons. In the SNI group, action potentials were elicited in delayed firing neurons as well. In the control, 35 % neurons (8/30) responded to electrical stimulation at LTA-input Table 2 Numbers of neurons receiving A-low threshold, Ahigh threshold, and C-input in each neuronal type in control and SNI conditions
Spike-train patterns
Clock-like I. burst (all) I. simple No spont. Total
A-LT
intensity, a percentage that increased to 58 % (19/42) after neuropathy; however, this difference did not reach statistical significance.
Discussion We set out to get insight into the working of superficial dorsal horn neurons and how they may contribute to shape the nociceptive output message. Using SNI animals allowed us to look for differences with controls that may unveil novel signs of central sensitization following a peripheral neuropathic lesion. Our experiments combine simultaneous extracellular recordings from different neurons with whole-cell recordings performed in the superficial layers of the dorsal horn, in the medial and central regions of the L4 segment where common peroneal and tibial nerves project. Our SNI animals showed a systematic loss of IB4 and substance P labeling in this area and developed mechanical hyperalgesia as previously described [18, 55]. Nerve section may produce changes in glomeruli and synaptic function [4]. However, neuronal loss does not seem to occur in this or previous studies [40]. Spike trains in dorsal horn neurons Here, we report the presence of a variety of neurons in the superficial dorsal horn which can be distinguished by the traits of their spontaneous activity. These are CL, IFB, ISB, IS, and subthreshold neurons. Previous characterizations with intracellular recordings presented a similar typology in neonatal spinal cords [28, 29, 34]. The proportion of spontaneously firing neurons appears to decay with age such that at 3 weeks, ∼60 % of the neurons are reported to become silent [28], in line with the present data in which 80 % of the neurons recorded with whole-cell patch electrodes show no spontaneous spiking. Taking into account the small proportion of neurons with suprathreshold spontaneous firing in adult spinal cords, the use of MEAs clearly increases detection probability of
A-HT
C
NC
NE
Control
SNI
Control
SNI
Control
SNI
Control
SNI
Control
SNI
0 8 12 2 22
12 1i* 14 19 4 49*
2 1i 10 15 1i 3 30
4 19 21 1i 3 47
2 1i 5 5 0 12
0 0 2 1i 2 4
4 8 19 4 35
8 3 22 3 36
2 3 8 0 13
2 1 5 0 8
LT low threshold HT high threshold, NC nonclassifiable neurons, NE nonelectrically evoked neurons, I. irregular, i inverted neurons *p < 0.05 Fisher’s exact test
Pflugers Arch - Eur J Physiol
spiking neurons. Here, we are particularly interested in characterizing these spontaneous neurons.
Clock-like and irregular simple neurons CL neurons fire action potentials at a rather high frequency (∼6 Hz) and constant intervals. Neurons of this kind have been reported before in the spinal cord of rodents using in vivo [31, 51] and in vitro approaches [28, 32, 34]. According to firing frequency and location, our CL neurons resemble the socalled tonic or rhythmic neurons described in lamina I from neonatal mice [22, 28, 34]. These neurons belong to local circuits and a proportion of them receive direct subthreshold excitatory inputs from Aβ afferents [22]. Whereas some of these neurons fire with extreme regularity, others just fulfill the minimum criteria for inclusion in the category according to our analytical algorithms. Therefore, a continuum can be envisaged between the regularity of CL neurons and the irregularity of IS neurons, at least those with similar rates of discharge. Although the sample of neurons recorded with whole-cell electrodes is limited, we did not find any major or systematic differences between CL and IS neurons concerning their electrophysiological properties other than a certain depolarization of CLs. However, previous studies from our laboratory suggest that similar neurons in deeper layers of the cord may possess a high density of HCN channels helping their rhythmicity [44]. Experiments by Luz et al. suggest that this kind of regular rhythm may be endogenously generated but it is also possible that neuromodulators may influence rhythmicity [34]. Experimental maneuvers such as addition of substance P can turn irregular neurons into rhythmically firing [31], and therefore, it is foreseeable that changes in the milieu following nerve injury may favor the expression of this class of activity as found in the present work for SNI animals. Some of the high-frequency CL and IS neurons show inverted responses to primary afferent stimulation. Neurons with similar behavior have been proposed to be inhibitory interneurons playing a role as gatekeepers such that synaptic input silences the neuron and releases the inhibition that it exerts on another pathway [12, 32]. Other CL neurons respond to A and/or C primary afferent stimulation with a brief increase in the frequency of their background activity and are therefore indistinguishable from the typical responses of spinal neurons. A certain parallelism can be traced between these CL and the so-called pulsing neurons described in certain cortical areas [35] which have been proposed to encode signals in a more reliable way than do irregular neurons, particularly when the message is encoded by spike counts. Postsynaptic neurons are proposed to achieve a higher signal-to-noise ratio in counting the number of spikes that they receive.
Irregular bursting neurons A proportion of ∼30 % of the neurons in our sample fired spontaneously in bursts of variable duration and intraburst frequency. These two characteristics were linked together, as firing frequency within short bursts was significantly higher than that of long bursts. Irregular fast-burst neurons Two previous studies reported neurons firing in a fashion similar to that of our IFB neurons [47, 54]. These studies, performed in vivo, described superficial dorsal horn neurons that fired spontaneously and evoked bursts of two/three spikes at very high frequencies (∼500 Hz) over the background of a very low mean firing frequency considering prolonged periods. Interestingly, these neurons were associated to the transmission of nociceptive signals because they responded to nociceptive stimulation of their receptive fields and to activation of C-fibers. The neurons that we have classed under IFB include bursts up to seven spikes. In our hands, some of these neurons responded to dorsal root stimulation in the A-LT intensity range. According to our whole-cell recordings, these neurons also fired in bursts to depolarizing current pulses, suggesting that their spontaneous short bursts may be mediated to a large extent by intrinsic properties of the neurons. The most striking finding that we report in connection with this neuronal type is its involvement in synchronous events. Both burst firing and synchronicity are important elements of neuronal coding in the temporal dimension favoring space and time summation and providing for a reliable way to transmit synaptic information [30]. An intriguing possibility is that this conjunction of factors may provide for a strong source of synchronous excitation such as that required for the generation of primary afferent depolarization [17]. Irregular slow-burst neurons The ISB neurons constitute a small part of our population (
NEUROSCIENCE
Analysis of spontaneous activity of superficial dorsal horn neurons in vitro: neuropathy-induced changes Carolina Roza 1 & Irene Mazo 1 & Iván Rivera-Arconada 1 & Elsa Cisneros 1 & Ismel Alayón 1 & José A. López-García 1
Received: 13 July 2016 / Revised: 6 September 2016 / Accepted: 26 September 2016 # Springer-Verlag Berlin Heidelberg 2016
Abstract The superficial dorsal horn contains large numbers of interneurons which process afferent and descending information to generate the spinal nociceptive message. Here, we set out to evaluate whether adjustments in patterns and/or temporal correlation of spontaneous discharges of these neurons are involved in the generation of central sensitization caused by peripheral nerve damage. Multielectrode arrays were used to record from discrete groups of such neurons in slices from control or nerve damaged mice. Whole-cell recordings of individual neurons were also obtained. A large proportion of neurons recorded extracellularly showed well-defined patterns of spontaneous firing. Clock-like neurons (CL) showed regular discharges at ∼6 Hz and represented 9 % of the sample in control animals. They showed a tonic-firing pattern to direct current injection and depolarized membrane potentials. Irregular fast-burst neurons (IFB) produced shortlasting high-frequency bursts (2–5 spikes at ∼100 Hz) at irregular intervals and represented 25 % of the sample. They showed bursting behavior upon direct current injection. Of the pairs of neurons recorded, 10 % showed correlated firing. Correlated pairs always included an IFB neuron. After nerve damage, the mean spontaneous firing frequency was unchanged, but the proportion of CL increased significantly (18 %) and many of these neurons Electronic supplementary material The online version of this article (doi:10.1007/s00424-016-1886-6) contains supplementary material, which is available to authorized users. * José A. López-García [email protected] 1
Dpto. Biología de Sistemas, Edificio de Medicina, Universidad de Alcalá, Campus Universitario, 28871 Alcalá de Henares, Madrid, Spain
appeared to acquire a novel low-threshold A-fiber input. Similarly, the percentage of IFB neurons was unaltered, but synchronous firing was increased to 22 % of the pairs studied. These changes may contribute to transform spinal processing of nociceptive inputs following peripheral nerve damage. The specific roles that these neurons may play are discussed. Keywords Multielectrode arrays . Nociception . Pain . Spike trains . SNI
Introduction Neurons located in the superficial dorsal horn (SDH) are directly implicated in the mechanisms of pain transmission through the spinal cord [11]. SDH neurons have been classed using different criteria such as morphology, neurochemical and electrophysiological traits, and local or projecting status [12, 48, 59]. Since more than 90 % of these neurons are located in local circuits [56], several approaches have been used to elucidate the organization of these circuits. It is known that circuits show a modular organization that allows the integration of different types of inputs ending on projection neurons located in lamina I [19, 33, 39]. However, the present knowledge of the types of neurons and their interconnections forming these circuits is limited and we are far from understanding the spinal neural coding for the signaling of painful events. Since 1926, the basic element of a neural code was established as the number of spikes fired in a fraction of time [1, 2]. However, it has long been accepted that a much more complex code arises when neurons are able to emit temporal patterns of spikes whose timing is reliable on a short time scale [21]. The coordination of temporal patterns across a
Pflugers Arch - Eur J Physiol
population of neurons adds another dimension of complexity to the neural code, i.e., synchronization [3, 26]. Technological advances have allowed for simultaneous recording of populations of neurons in different areas of the central nervous system, and complex analyses are being implemented to understand the encoding of different neural events, such as learning and memory [10], object recognition [58], or reward circuits [9]. It is remarkable that spike patterns are conserved in homologous regions across species [36], suggesting a key role of spike trains in neuronal coding. Damage to peripheral nerves alters the processing of nociceptive signals in the spinal cord. This involves changes in the coding of afferent inputs which are assumed to underlie central sensitization and neuropathic pain symptoms. In the past, some studies consistently demonstrated an increased excitability of projection and unidentified deep dorsal horn widedynamic range neurons following neuropathic insult [14, 27, 38, 57]. Changes included increased background activity and prolonged afterdischarges, enhanced responses to lowthreshold mechanical stimuli, and a leftward shift in the curve defining intensity-response relations as well as enlarged receptive fields. In contrast, intracellular recordings from superficial layers, including inhibitory neurons, detect only slight changes in intrinsic properties [5, 15, 53]. In general, the changes that take place in the coding of afferent signals in the SDH following peripheral injury are poorly understood despite their prominent position in the chain of nociceptive transmission. Since firing frequency may be only one variable of neuronal coding, here, we have used multiple recording electrodes to simultaneously assess the firing of small groups of neurons. We have used the spared nerve injury model of neuropathy to assess possible changes in the patterns of action potentials and synchronous discharges which could also amplify nociception by increasing the gain or generating noise in nociceptive lines giving rise to abnormal pain sensations [50]. We also used whole-cell recordings to characterize basic membrane properties and histological techniques to locate recording sites.
Induction of neuropathy and behavioral test Surgery was performed in 22–25-day-old animals under deep anesthesia with 3–4 % isoflurane in 100 % oxygen. For spared nerve injury (SNI), an incision was made in the skin of the lateral surface of the thigh and the sciatic terminal branches were exposed; the common peroneal and the tibial nerves were ligated with 8/0 nylon monofilament and sectioned distal to the ligature, sparing the sural branch. The incision was closed in layers with 5/0 sutures. The animals were housed in groups of 3–4 with access to water and food ad libitum and inspected periodically for infections or abnormal behavior. The up-down paradigm [13] was used to evaluate withdrawal threshold to mechanical stimulation on the sural innervation territory of the hind paw. For the SNI group, behavioral data was collected before the surgery and 2 weeks after induction of neuropathy, immediately prior to the electrophysiological experiment. For control animals, measurements were taken only the day of the electrophysiological experiment. Surgical extraction and maintenance of the longitudinal slice in vitro Mice were anesthetized with urethane (2 g/kg i.p.). The lumbar skin was cut and a dorsal laminectomy from thoracic to sacral vertebrae was performed. The spinal cord with attached roots was dissected free and placed in sucrose-substituted artificial cerebrospinal fluid (ACSF) at 4 °C (see composition below). The meninges were carefully removed, the roots separated from the cord, and the L1-S1 segments of the cord cut out. The cord fragment was glued with cyanoacrylate to a rigid base and positioned in a vibratome with the dorsal side up. A single slice of ∼500 μm containing the dorsal side of the cord together with the attached dorsal roots was obtained. The slice was mounted in the recording chamber with the dorsal side down to provide access to the recording electrodes and maintained at room temperature (22 ± 1 °C) with oxygenated ACSF (95 % O2, 5 % CO2) at a flow of 5 ± 1 ml/min. The composition of the ACSF was (in mM) NaCl 127, KCl 1.9, KH2PO4 1.5, MgSO4 1.3, CaCl2 2, NaHCO3 22, and glucose 10, (pH 7.4). For sucrose-substituted ACSF, NaCl was substituted for equimolar concentration of sucrose.
Materials and methods Electrical stimulation of the dorsal root Animals Young adult CD-1 female mice (n = 66, 30–57 days old, and body weight 27–49 g) bred at the University Animal House were used. The European Union and State regulations for animal experiments were followed. All experimental protocols were approved by the Universidad de Alcalá Committee on Animal Research and the Regional Government (project license: ES280050001165).
A dorsal root (usually L4) was inserted into a suction electrode for electrical stimulation. Electrical pulses of increasing magnitude were applied in order to activate the different afferent fibers contained in the dorsal root. Neurons were classified according to the lowest stimulus intensity that evoked action potential firing (extracellular recordings, see below) or subthreshold responses (whole-cell recordings, see below). On the basis of previous experiments from our laboratory
Pflugers Arch - Eur J Physiol
performed in isolated sciatic nerve from adult mice [46], neurons responding to 50-μs pulses of intensities 10.000) as obtained with GLO and showed at least one clear peak in the ACH well away from time 0 (see Fig. 2c).
a
DiI / Trypan blue
Ipsilateral
SNI Dapi
b
Contralateral
200 µm
Biocytin / IB4
c
Sensor I II III IV
25 µm
Electrode shank SP / IB4
d
Control
SP / IB4
e
SNI
100 µm
Pflugers Arch - Eur J Physiol Table 1 Proportions of neurons, mean firing frequencies, and firing patterns to depolarizing current vs neuronal type in control and SNI conditions
Control Spike-train patterns Clock-like
% (n)
I. simple
SNI
9 (10 2i)
Mean firing (Hz) 6.4 ± 0.7
53 (59 1i)
2.2 ± 0.2
I. fast burst
25 (28)
I. slow burst
5 (6)
I. mixed burst No spont.
8 (9)
Control + SNI
% (n) 18 (26 1i)*
Mean firing (Hz) 5.3 ± 0.5
Intracellular patterns Tonic
48 (69 2i )
2.4 ± 0.2
Tonic
1.1 ± 0.2
21 (30)
0.8 ± 0.3
Initial bursting
5.7 ± 2
3.5 (5)
2.5 ± 1.1
1.3 (2) 8.3 (12)
3.9 ± 1.1
Single spike
All patterns
I. irregular, i inverted neurons *p < 0.05 Fisher’s exact test
The proportion of CL neurons was significantly greater in the group of SNI-treated animals (26/144) when compared with that in the controls (10/112), this being one of
the major differences found between the groups (see Table 1; p < 0.05; Fisher’s exact test). No differences were found in cell location within the cord (see Fig. 2g)
b
mV
a 0
1ms
1s
20
c 240 nº events
I. Fq. (Hz)
0.5
10 0
e 20 mV 0.2 s
-56 mV
f
g
0
Time (s) 0.5
300
nº of events
d
0
0 0
Time (s) 0.2
Control -56 mV
20 mV
I/II III IV
0.1 s
32 pA
SNI -56 mV 20 mV 0.1 s
200 µm
I/II III IV
-100 pA
Fig. 2 a Original extracellular recording from a typical CL neuron recorded from a control mouse that had a mean discharge frequency of 9.8 Hz. The lower panel shows instantaneous frequency for this neuron. b The spike shape averaged for the period in a. c The corresponding autocorrelogram histogram built for this neuron in a 5-min period. Note the presence of three clearly identified peaks, which indicates that a preferential interspike interval of ∼100 ms is maintained. The GLO-Mat β1 value for this neuron was 0.16. d Original whole-cell recording showing regular spontaneous firing in the absence of current injection recorded from a control preparation (dotted line marks membrane
potential indicated at the left). e The auto-correlogram corresponding to the neuron in d. Note the similarity with autocorrelogram in c corresponding to the extracellular-recorded CL neuron. f Responses of the neuron in d to direct current injection. The upper panel shows an increased tonic firing to depolarizing pulses. The lower panel shows depolarizing sag upon hyperpolarization, followed by an increase in firing frequency at the end of the pulse. Drawings in g show the location of CL neurons from extracellular recordings in slices from control and SNI animals
Pflugers Arch - Eur J Physiol
a
b
*
mV
0 1
2 ms
10 sec
nº of events
240 160 80 0
d
15 10 5 0 0
e
*
20 mV 2s
-64 mV
nº of events
I. Fq. (Hz)
c
20
0.2
10 0 0
g
10 mV
0.1
Time (s)
0.1
0.2
Time (s)
Control 0.2 s I/II III IV
f 20 mV 0.1 s
SNI
200 µm
-68 mV 32 pA -80 pA
I/II III IV
Fig. 3 a An original extracellular recording from a typical IFB neuron whose instantaneous frequency is presented below. Note that each burst consists of a discrete number of AP (2–6) with high intraburst frequency (mean ∼154 Hz) and a very low mean discharge frequency (0.26 Hz). b Two spikes corresponding to a duplet burst indicated with asterisk in a. c The autocorrelogram built for the neuron in a. Note the presence of a narrow peak very close to 0. d Original whole-cell recording of a neuron from a control mouse showing spontaneous firing in bursts of two to three spikes/burst at a maximal frequency of 80 Hz. The two bursts around the
asterisk are enlarged in the lower panel; note that the oscillation in membrane potential occasionally gives rise to burst firing—AP are truncated at −25 mV. e The autocorrelogram for the neuron in d. Note the similarity with autocorrelogram in c corresponding to the extracellular-recorded IFB neuron. f This neuron showed an initial burst firing pattern in response to depolarizing current pulses as well as depolarizing sag and rebound firing to hyperpolarizing pulses. g Drawings depicting the location of IFB neurons from extracellular recordings within control and SNI preparations
or mean firing frequency between the SNI and control groups (Table 1). Three of the neurons recorded with whole-cell patch electrodes in the control animals showed spontaneous activity with a typical CL pattern, as assessed by their ACH and GLO values (an original example is shown in Fig. 2d, e). All of them exhibited tonic firing patterns to depolarizing current pulses (Fig. 2f). These neurons were significantly more depolarized than the remaining tonic neurons (−56.3 ± 1.9 mV at the lowest point of afterspike hyperpolarization vs −64.9 ± 1.8 mV; p < 0.05, Mann-Whitney test). Two of these neurons showed rectification upon hyperpolarization (sag) and high-frequency rebound firing on
depolarization (Fig. 2f). Another neuron did not show rectification and had a slow return to resting potential after hyperpolarizing pulses. According to GLO, all the remaining extracellularly recorded neurons were classed as irregular in a first instance. For a proportion of these neurons, more than 25 % of their action potentials came in bursts as analyzed with BAR. Hence, irregular firing neurons were segregated between irregular simple (IS) or irregular burst (IB). The extent of irregularity among IS and IB neurons was similar in control and SNI mice (see Online Resource 3 for a detailed quantification of the degree of regularity of the different neurons).
Pflugers Arch - Eur J Physiol
Among the IB neurons, some produced short-lasting (typically 2–5 spikes) high-frequency bursts (∼100 Hz) and were classed as irregular fast-burst neurons (IFB, an original recording is shown in Fig. 3a, b). Other neurons produced long-lasting (up to 200 spikes) low-frequency bursts (below 30 Hz) and were classed as irregular slowburst neurons (ISB, an original recording example is shown in Fig. 4a, b). IS neurons fired mainly isolated spikes (an original recording is shown in Fig. 4d, e) (see Table 1). IFB neurons had autocorrelograms characterized by a single peak close to time 0 followed by a few isolated events at different time points, which is consistent with the high intraburst frequency and the low mean frequency
a
found in these neurons (see Fig. 3c and Table 1). No differences were found for IFB neurons between the control and SNI groups regarding proportion, mean spontaneous firing frequency, or location (shown in Fig. 3g and Table 1) within the cord. Six spontaneous neurons with IFB patterns, as assessed with BAR, were recorded with whole-cell electrodes (3 controls and 3 SNI, see an original example in Fig. 3d, e). The maximum intraburst instantaneous frequency was very similar in control and SNI (106.3 ± 14.1 Hz, n = 6). When these 6 neurons were depolarized by current injection (at two times rheobase), the maximal firing frequency of the evoked burst (102.3 ± 15.6 Hz) matched the spontaneous intraburst frequency. Neurons of the control group had a
b
mV
0
1 ms
c
10 s
75
nºof events
I. Fq. (Hz)
0.25
50 25
400 200 0
0
d
0
Time (s)
0.5
e
mV
0.5 0
f 10 s
nº of events
I. Fq. (Hz)
10 5 0
g
1 ms 25 20 15 10 5 0 0
i 20 mV
Control
0.5 s -59 mV
Time (s)
1
IS
ISB
I/II III IV
h 20 mV 0.1 s
SNI
200 µm
-59 mV 96 pA
I/II III IV
-150 pA
Fig. 4 a An original extracellular recording from a typical ISB neuron and its corresponding instantaneous frequency (lower panel). Each burst consisted of a large number of AP (>100 spikes) with a mean intraburst frequency of ∼12 Hz and an overall mean discharge frequency of 7 Hz. b The averaged spike shape. c The corresponding auto-correlogram histogram. Note the presence of a flat line with a peak at ∼50 ms which detects certain regularity within the burst. d An original extracellular recording from an irregular simple neuron with its corresponding instantaneous frequency plot (lower panel; mean firing frequency of
1.4 Hz), averaged spike (e), and auto-correlogram (f). Note the presence of a flat line indicative of random interspike interval between consecutive spikes. g An original whole-cell recording from a control neuron with irregular spontaneous firing and spontaneous EPSPs. h This neuron showed tonic firing pattern in response to depolarizing current pulses as well as depolarizing sag and rebound firing to hyperpolarizing pulses. i Drawings depicting the location of ISB (white circles) and IS neurons (black circles) from extracellular recordings in control and SNI preparations
Pflugers Arch - Eur J Physiol
Synchronicity of spontaneous spikes in control and SNI animals Spike synchrony was tested using multiple recordings from MEAs on a total of 129 pairs in control and 136 pairs in SNI by means of cross-correlation histograms. Two major findings came out of this analysis. First, the percentage of correlated pairs was significantly greater in the SNI animals (30/136, 22 %) compared to that in the control (13/129; 10 %; p < 0.01; Fisher’s exact test), this being another major difference between the control and SNI groups. Second, all the correlated pairs found in the control and SNI conditions consisted at least of one IFB neuron. The IFB neurons showing correlation had similar properties to those for which no correlation was found, including the type of input from afferent fibers, number of spikes per burst, mean firing frequency for spontaneous discharges, and mean intraburst frequency. The other neuron of the pair could belong to any class including again IFB neurons (see Fig. 5). Among the 13 pairs recorded in the control, 6 were IFB-IS and 7 were IFB-IFB pairs. For SNI, 10 pairs were IFB-IS, 7 pairs were IFB-CL, 1 was IFS-ISB, and the remaining 11 were IFB-IFB pairs. Representative examples of the types of crosscorrelograms found in our sample are shown in Fig. 5. The most common type was a central symmetrical or asymmetrical
a
*1
*2
*3
u1 u2 u3 u4 10 sec
b
*1
*2
*3
u1 u2 u3 u4 25 ms
25 ms
25 ms
c 50 u1-u2
0.1
0 - 0.1 20
0
0.1
0.1
0 - 0.1
0 - 0.1 20
u2-u4
0
Time 0.1 (s)
u3-u4
Nº of events
Nº of events 0
u1-u4
Nº of events
Nº of events
Nº of events 0 0 - 0.1 8 u2-u3
0 - 0.1
40
40 u1-u3
Nº of events
characteristic firing pattern to depolarizing current pulses consisting on an initial burst of action potentials at the beginning of the pulse and exhibited sag and rebound firing on response to hyperpolarizing pulses (see Fig. 3f). Two SNI neurons of this class showed initial bursting pattern and sag but only subthreshold rebounds. The remaining neuron was the only neuron of our sample with a singlespiking pattern to depolarizing current and did show sag and rebound firing. ISB and IS neurons had ACHs that consisted of a noisy flat line indicating similar probability of firing at any given time from any given spike, although a few ACHs from ISB neurons showed a peak superimposed to the flat line but never reached values for regularity with GLO (Fig. 4c, f). These neurons exhibited mean firing frequencies in the midrange and the IS group was the most abundant class among spontaneously active neurons (Table 1). No differences were found in proportions, firing frequencies, and location within the cord (shown in Fig. 4i and Table 1) between the control and SNI groups. In the SNI group, we found 2 neurons exhibiting bursts starting with high-frequency firing which declined with time. None of the neurons recorded with whole-cell patch electrodes belonged to the ISB type, but 4 of them were of the IS type (2 control and 2 SNI, see an example in Fig. 4g). All of these neurons showed a tonic-firing pattern to intracellular depolarizing pulses. Sag and rebound firing were also commonly observed in IS neurons (see Fig. 4h).
0
0.1
0 - 0.1
0
0.1
Time (s)
Fig. 5 Example of four IFB units (u1–u4) recorded simultaneously in the superficial dorsal horn of a neuropathic mouse. a The spontaneous discharges of each individual unit as lines in a 100-s window. Asterisks represent instances in which the four units fired simultaneously. b The structure of three synchronous events (marked by asterisks in a) in an expanded time base. Spontaneous discharges are represented by their actual spike shapes. c The cross-correlograms obtained for each possible pair in a 5-min period. All histograms have a peak close to 0 indicative of synchrony between the neural pair. The cross-correlogram u1–u3 shows a central trough of 3-ms width
peak of 15–100 ms of width measured at half amplitude. We also observed lateral peaks displaced 5–15 ms from 0 showing widths in the range of 5–50 ms. In some cases, a central trough appeared combined with any of the previous ones. These were usually very narrow (5 ms). All types were found in control and SNI animals. Responses of neurons from control and SNI animals to dorsal root stimulation Extracellular recordings showed strong artifacts due to electrical stimulation of the dorsal root which precluded the possibility of building accurate stimulus-response curves. In fact, 27 % of the total sample of neurons could not be accurately
Pflugers Arch - Eur J Physiol
classified. A majority of the remaining neurons (164/185) responded to dorsal root stimulation and only 21/185 did not show any responses. Responses consisted on the firing of action potentials or the stopping of ongoing spontaneous activity. This later response was classed as Binverted^ and occurred in a small proportion of neurons, all of them having mean firing frequencies >4 Hz and belonging to the CL (n = 3) or the IS class (n = 4). Responsive neurons were classed according to the lowest stimulus intensity that evoked a clearly identifiable response as having LTA-input or HTA-input or C-inputs (Online Resource 2). Table 2 shows the number of neurons responding to the different inputs according to their spontaneous firing class. A comparison of these data clearly shows that neurons receiving LTA-input were significantly more abundant in the SNI group than in the control (34 vs 20 % respectively; p < 0.05 Fisher’s exact test), whereas the proportion of neurons receiving exclusively C-inputs was smaller in the SNI group (3 % in SNI vs 11 % in control; p < 0.05 Fisher’s exact test), this being another major difference between the groups. It is noteworthy that the largest increase in LTA-input after SNI was found among CL neurons (Table 2). As a consequence, the mean firing frequency in the neurons receiving LTA-input of SNI animals was significantly greater than that of the controls (1.4 ± 0.35 in control and 3.2 ± 0.4 Hz in SNI; p < 0.05, Mann-Whitney). No differences were found between the firing frequency of the control and SNI animals in the total sample. Approximately 78 % of neurons recorded with whole-cell patch electrodes (56/72) responded to dorsal root stimulation with EPSPs (38/72) or a mixture of EPSPs and IPSPs (18/72) or with action potentials (21/72). One additional neuron showed only IPSPs. The remaining 22 % was indifferent to afferent stimulation. In the control group, action potentials were elicited by dorsal root stimulation in tonic and initial burst neurons. In the SNI group, action potentials were elicited in delayed firing neurons as well. In the control, 35 % neurons (8/30) responded to electrical stimulation at LTA-input Table 2 Numbers of neurons receiving A-low threshold, Ahigh threshold, and C-input in each neuronal type in control and SNI conditions
Spike-train patterns
Clock-like I. burst (all) I. simple No spont. Total
A-LT
intensity, a percentage that increased to 58 % (19/42) after neuropathy; however, this difference did not reach statistical significance.
Discussion We set out to get insight into the working of superficial dorsal horn neurons and how they may contribute to shape the nociceptive output message. Using SNI animals allowed us to look for differences with controls that may unveil novel signs of central sensitization following a peripheral neuropathic lesion. Our experiments combine simultaneous extracellular recordings from different neurons with whole-cell recordings performed in the superficial layers of the dorsal horn, in the medial and central regions of the L4 segment where common peroneal and tibial nerves project. Our SNI animals showed a systematic loss of IB4 and substance P labeling in this area and developed mechanical hyperalgesia as previously described [18, 55]. Nerve section may produce changes in glomeruli and synaptic function [4]. However, neuronal loss does not seem to occur in this or previous studies [40]. Spike trains in dorsal horn neurons Here, we report the presence of a variety of neurons in the superficial dorsal horn which can be distinguished by the traits of their spontaneous activity. These are CL, IFB, ISB, IS, and subthreshold neurons. Previous characterizations with intracellular recordings presented a similar typology in neonatal spinal cords [28, 29, 34]. The proportion of spontaneously firing neurons appears to decay with age such that at 3 weeks, ∼60 % of the neurons are reported to become silent [28], in line with the present data in which 80 % of the neurons recorded with whole-cell patch electrodes show no spontaneous spiking. Taking into account the small proportion of neurons with suprathreshold spontaneous firing in adult spinal cords, the use of MEAs clearly increases detection probability of
A-HT
C
NC
NE
Control
SNI
Control
SNI
Control
SNI
Control
SNI
Control
SNI
0 8 12 2 22
12 1i* 14 19 4 49*
2 1i 10 15 1i 3 30
4 19 21 1i 3 47
2 1i 5 5 0 12
0 0 2 1i 2 4
4 8 19 4 35
8 3 22 3 36
2 3 8 0 13
2 1 5 0 8
LT low threshold HT high threshold, NC nonclassifiable neurons, NE nonelectrically evoked neurons, I. irregular, i inverted neurons *p < 0.05 Fisher’s exact test
Pflugers Arch - Eur J Physiol
spiking neurons. Here, we are particularly interested in characterizing these spontaneous neurons.
Clock-like and irregular simple neurons CL neurons fire action potentials at a rather high frequency (∼6 Hz) and constant intervals. Neurons of this kind have been reported before in the spinal cord of rodents using in vivo [31, 51] and in vitro approaches [28, 32, 34]. According to firing frequency and location, our CL neurons resemble the socalled tonic or rhythmic neurons described in lamina I from neonatal mice [22, 28, 34]. These neurons belong to local circuits and a proportion of them receive direct subthreshold excitatory inputs from Aβ afferents [22]. Whereas some of these neurons fire with extreme regularity, others just fulfill the minimum criteria for inclusion in the category according to our analytical algorithms. Therefore, a continuum can be envisaged between the regularity of CL neurons and the irregularity of IS neurons, at least those with similar rates of discharge. Although the sample of neurons recorded with whole-cell electrodes is limited, we did not find any major or systematic differences between CL and IS neurons concerning their electrophysiological properties other than a certain depolarization of CLs. However, previous studies from our laboratory suggest that similar neurons in deeper layers of the cord may possess a high density of HCN channels helping their rhythmicity [44]. Experiments by Luz et al. suggest that this kind of regular rhythm may be endogenously generated but it is also possible that neuromodulators may influence rhythmicity [34]. Experimental maneuvers such as addition of substance P can turn irregular neurons into rhythmically firing [31], and therefore, it is foreseeable that changes in the milieu following nerve injury may favor the expression of this class of activity as found in the present work for SNI animals. Some of the high-frequency CL and IS neurons show inverted responses to primary afferent stimulation. Neurons with similar behavior have been proposed to be inhibitory interneurons playing a role as gatekeepers such that synaptic input silences the neuron and releases the inhibition that it exerts on another pathway [12, 32]. Other CL neurons respond to A and/or C primary afferent stimulation with a brief increase in the frequency of their background activity and are therefore indistinguishable from the typical responses of spinal neurons. A certain parallelism can be traced between these CL and the so-called pulsing neurons described in certain cortical areas [35] which have been proposed to encode signals in a more reliable way than do irregular neurons, particularly when the message is encoded by spike counts. Postsynaptic neurons are proposed to achieve a higher signal-to-noise ratio in counting the number of spikes that they receive.
Irregular bursting neurons A proportion of ∼30 % of the neurons in our sample fired spontaneously in bursts of variable duration and intraburst frequency. These two characteristics were linked together, as firing frequency within short bursts was significantly higher than that of long bursts. Irregular fast-burst neurons Two previous studies reported neurons firing in a fashion similar to that of our IFB neurons [47, 54]. These studies, performed in vivo, described superficial dorsal horn neurons that fired spontaneously and evoked bursts of two/three spikes at very high frequencies (∼500 Hz) over the background of a very low mean firing frequency considering prolonged periods. Interestingly, these neurons were associated to the transmission of nociceptive signals because they responded to nociceptive stimulation of their receptive fields and to activation of C-fibers. The neurons that we have classed under IFB include bursts up to seven spikes. In our hands, some of these neurons responded to dorsal root stimulation in the A-LT intensity range. According to our whole-cell recordings, these neurons also fired in bursts to depolarizing current pulses, suggesting that their spontaneous short bursts may be mediated to a large extent by intrinsic properties of the neurons. The most striking finding that we report in connection with this neuronal type is its involvement in synchronous events. Both burst firing and synchronicity are important elements of neuronal coding in the temporal dimension favoring space and time summation and providing for a reliable way to transmit synaptic information [30]. An intriguing possibility is that this conjunction of factors may provide for a strong source of synchronous excitation such as that required for the generation of primary afferent depolarization [17]. Irregular slow-burst neurons The ISB neurons constitute a small part of our population (
