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Exp Brain Res (2003) 148:401–413 DOI 10.1007/s00221-002-1303-6 RESEARCH ARTICLE E. Manjarrez · I. Jimnez · P. Rudomin Intersegmental synchronization of spontaneous activity of dorsal horn neurons in the cat spinal cord Received: 30 May 2002 / Accepted: 2 October 2002 / Published online: 28 November 2002 Springer-Verlag 2002 Abstract Extracellular recordings of neuronal activity made in the lumbosacral spinal segments of the anesthetized cat have disclosed the existence of a set of neurons in Rexed’s laminae III–VI that discharged in a highly synchronized manner during the occurrence of spontaneous negative cord dorsum potentials (nCDPs) and responded to stimulation of low-threshold cutaneous fibers (<1.5T) with mono- and polysynaptic latencies. The cross-correlation between the spontaneous discharges of pairs of synchronic neurons was highest when they were close to each other, and decreased with increasing longitudinal separation. Simultaneous recordings of nCDPs from several segments in preparations with the peripheral nerves intact have disclosed the existence of synchronized spontaneous nCDPs in segments S1–L4. These potentials lasted between 25 and 70 ms and were usually larger in segments L7–L5, where they attained amplitudes between 50 and 150 V. The transection of the intact ipsilateral hindlimb cutaneous and muscle nerves, or the section of the dorsal columns between the L5 and L6, or between the L6 and L7 segments in preparations with already transected nerves, had very small effects on the intersegmental synchronization of the spontaneous nCDPs and on the power spectra of the cord dorsum potentials recorded in the lumbosacral enlargement. In contrast, sectioning the ipsilateral dorsal horn and the dorsolateral funiculus at these segmental levels strongly decoupled the spontaneous nCDPs generated rostrally from those generated caudally to the lesion and reduced the magnitude of the power spectra throughout the whole I. Jimnez · P. Rudomin ()) Department of Physiology, Biophysics and Neurosciences, Centro de Investigacin y Estudios Avanzados del Instituto Politcnico Nacional, Apartado Postal 14-740, Mxico, DF 07000, Mxico e-mail: [email protected] Tel.: +1-525-7477099 Fax: +1-525-7477105 E. Manjarrez Instituto de Fisiologa, Benemrita Universidad Autnoma de Puebla, Mxico frequency range. These results indicate that the lumbosacral intersegmental synchronization between the spontaneous nCDPs does not require sensory inputs and is most likely mediated by intra- and intersegmental connections. It is suggested that the occurrence of spontaneous synchronized nCDPs is due to the activation of tightly coupled arrays of neurons, each comprising one or several spinal segments. This system of neurons could be involved in the modulation of the information transmitted by cutaneous and muscle afferents to functionally related, but rostrocaudally distributed spinal interneurons and motoneurons, as well as in the selection of sensory inputs during the execution of voluntary movements or during locomotion. Keywords Dorsal horn neurons · Neuronal synchronization · Cutaneous afferents · Presynaptic inhibition · Spinal cord Introduction In a previous study (Manjarrez et al. 2000) we analyzed the origin of the spontaneous activity that is recorded from the dorsum of the lumbosacral enlargement of the spinal cord in the anesthetized cat. There were clear indications that the low frequency components of these potentials (3–20 Hz) were produced by a set of spontaneously active neurons located in the dorsal horn, some of which responded with monosynaptic latencies to stimulation of low-threshold cutaneous afferents. During the generation of these spontaneous cord dorsum potentials (nCDPs), the monosynaptic intraspinal field potentials, as well as the dorsal root potentials (DRPs), produced by stimulation of low threshold cutaneous and by Ib muscle afferents, were facilitated, while the intraspinal field potentials and the DRPs produced by stimulation of Ia afferents were depressed. In addition, the spontaneous nCDPs and the fluctuations of monosynaptic reflexes were reduced by conditioning stimulation of cutaneous nerves, suggesting a continuous modulation of the motor 402 output by this set of dorsal horn neurons (see Rudomin and Dutton 1969; Rudomin et al. 1974). In the earlier study (Manjarrez et al. 2000), we suggested that the ensemble of active dorsal horn neurons that leads to the spontaneous nCDPs fires in a highly synchronized manner and is longitudinally distributed throughout several spinal segments. In 1979, Brown and colleagues examined in the spinal cord of the cat the correlation between the spontaneous firing of dorsal horn neurons, and found a significant number of neuron pairs with non-flat cross-correlograms, suggesting the existence of a common input to these neurons. Subsequent studies by Eblen-Zajjur and Sandkuhler (1997) in the rat have confirmed the findings of Brown et al. (1979). In addition, they indicated that the dorsal horn neurons with correlated discharges showed the highest incidence of overlapping receptive fields. However, in these studies, very little information was presented on mechanisms and/or pathways contributing to the intersegmental synchronization of the spontaneous activity of these neurons. One question that emerged from the work of Manjarrez et al. (2000) was the extent to which the arrays of neurons involved in the generation of the spontaneous nCDPs operated mainly within the L5–L7 spinal segments, or whether synchronized spontaneous nCDPs were also generated in other spinal regions. Another question was the extent to which the generation of spontaneous nCDPs was a consequence of the acute transection of muscle and cutaneous nerves that was routinely performed during the dissections. Both issues were taken up recently by Manjarrez et al. (2002c). Preparations with intact peripheral nerves showed spontaneous nCDPs in segments C3– C4 as well as in segments L5–L7. Yet, spectral analysis of the cord dorsum potentials simultaneously recorded from the lumbar and cervical regions showed very little coherence between them, suggesting they were generated by two independent populations of neurons. The purpose of the present series of observations has been to further characterize the population of dorsal horn neurons located within the lumbosacral regions of the spinal cord that discharges in synchrony with the spontaneous nCDPs, to examine the segmental distribution of the involved networks and, by means of spinal lesions, to disclose the intraspinal pathways mediating their synchronized intersegmental firing. Some of these observations have been published in abstract form (Manjarrez et al. 1998). Materials and methods General procedures Guidelines contained in NIH publication 85–23 (revised in 1985) on the principles of laboratory animal care were followed throughout. The experimental procedures were similar to those followed previously (Manjarrez et al. 2000). Briefly, observations were carried out in adult cats initially anesthetized with pentobarbitone (35 mg/kg weight, i.p.), supplemented during the dissection and recording periods with additional doses of 10 mg/kg i.v. as necessary to maintain an adequate level of anesthesia, which was assessed by verifying that the pupils were constricted, that blood pressure was stable (between 100 and 120 mmHg), and that it was unaffected by noxious stimulation to the skin. In all experiments the lumbosacral and low thoracic spinal segments were exposed and the left L4–S1 ventral roots sectioned. In 18 experiments the left posterior biceps and semitendinosus (PBSt), gastrocnemius (GS), deep peroneus (DP), sural (SU) and superficial peroneus (SP) nerves were dissected free, sectioned and their central ends prepared for stimulation. In four other experiments these nerves were exposed and left intact. A pair of stainless steel needles was inserted into the left foot pad for stimulation. After the surgical procedures, the animals were paralyzed with pancuronium chloride (0.1 mg/kg) and artificially ventilated. Tidal air volume was adjusted to a 4% CO2 concentration in the expired air. Exposed tissues were covered with mineral oil to prevent desiccation and kept at constant temperature (37C) by means of radiant heat. Stimulation and recording Evoked and spontaneous potentials were recorded from the dorsal surface of the lumbosacral spinal cord along several segments. To this end we used pairs of silver ball electrodes, one placed on the surface of the spinal cord and the other on the paravertebral muscles at the same segmental level. The preamplifier filters were set to 0.3 Hz in the low range and 10 kHz in the high range. In all figures, negativity in recordings of cord dorsum potentials is upwards. Action potentials from dorsal horn neurons and field potentials were recorded from the L6–L7 segments using glass micropipettes filled with 1.2 M NaCl solution. The resistance of the microelectrodes varied between 7 and 15 MW. In several experiments simultaneous recordings were made from pairs of dorsal horn neurons in layers III–VI within the L6–L7 segments using two separate micropipettes inserted at angles to each other. The approximate distance between neurons was estimated from the longitudinal separation of the tips of the recording micropipettes measured when both were touching the cord surface. Clearly this is an overestimate because the micropipettes converged at deeper regions. Sampling and averaging of potentials was triggered with pulses generated by a window discriminator activated either by the spontaneous nCDPs recorded from a given (reference) spinal segment, or by stimuli applied to the cutaneous nerves or the ipsilateral foot pad in preparations with intact nerves (single pulses 1.1–1.5 times the strength of the most excitable fibers, determined relative to the threshold of the smallest afferent volley recorded from the cord dorsum, T). The averages were made up of 32–64 successive potentials. All potentials and triggering pulses were stored on a digital tape for further processing. The interaction between the neuronal sets producing the spontaneous nCDPs and neurons responding to stimulation of cutaneous afferents (evoked responses) was examined using the same protocol as Manjarrez et al. (2000). Briefly, every time a spontaneous nCDP crossed a fixed voltage level, set with a window discriminator, a TTL pulse was sent to the input channel of a control device that triggered a sequence of no stimulus, a stimulus at a delay after the nCDP, or a stimulus >500 ms from an nCDP (control). Potentials for each condition were collected separately. On the completion of one cycle of stimulation, the control device prevented initiation of the next cycle until after 2 s. Spectral analysis Spectral analysis of 2-min recordings of cord dorsum potentials obtained in the absence of stimulation of sensory nerves was done in ten experiments, using the Fourier transform of the Chaos Data Analyzer program of the American Institute of Physics Academic Software. Spontaneous cord dorsum potentials were acquired with a sampling rate of 500 Hz. 403 Coincidence histograms The relationship between spontaneous neuronal firing and the spontaneous nCDPs was established for each neuron by aligning the raster displays of the spontaneous action potentials with the peak of the averaged spontaneous nCDPs recorded close to the site of micropipette insertion (see Fig. 1). Coincidence histograms were obtained by adding the raster displays of all neurons (see Figs. 1, 2). Histograms were constructed using 1-ms bins within a range of 200–250 ms. Cross-correlation between action potentials generated by pairs of neurons Cross-correlation between the spontaneous activity of pairs of neurons was calculated using the cross-correlation function (see Fig. 3A, B). To this end we used recordings of the activity of pairs of neurons taken during the occurrence of a spontaneous nCDP. Correlation strength (F) was expressed as the ratio between the peak correlation and the basal correlation (Fig. 3C; see EblenZajjur and Sandkuhler 1997). Spinal lesions In ten experiments we investigated the effects of acute spinal lesions on the spontaneous nCDPs that were simultaneously recorded from several segments in the lumbosacral enlargement. Fig. 1A–F Synchronized spontaneous and evoked activity of neurons in laminae III–VI. A Raster display of the spontaneous activity of 17 neurons during the occurrence of spontaneous nCDPs. Neuronal activity was recorded between 0.71 and 1.92 mm below the cord surface. All responses were aligned relative to the peak of the spontaneous nCDPs recorded at the site of electrode penetration. The small boxes to the right indicate data obtained from the same interneuron. B Histogram generated by adding the raster displays illustrated in A. C Upper trace Extracellular recording of the activity of a single neuron in lamina IV at 1.26 mm depth during the generation of a spontaneous nCDP; lower The lesions were made under the dissecting microscope by teasing the spinal tissue with two fine forceps between the L6 and L7 or between the L5 and L6 segments. Histology At the end of the experiment the animals were sacrificed with a lethal dose of pentobarbitone, perfused with 10% formalin, and the spinal cord removed. After fixation and dehydration, the lumbosacral segments were placed in a solution of methyl salicylate for clearing and cut transversally to obtain 100-m sections including the lesions (see Wall and Werman 1976). Results Spontaneous activity of neurons In six experiments, spontaneous activity was recorded extracellularly from neurons located within the lumbar segments L6 and L7, in regions corresponding to Rexed’s laminae III–VI (0.7–2.0 mm depth). No tests were performed for their antidromic activation from thoracic segments to determine if these were interneurons or ascending tract neurons (see “Discussion,” however). The trace nCDP simultaneously recorded at the site of the electrode penetration. Note the neuronal burst-like discharge and the extracellular field potential appearing in synchrony with the nCDP. D–F Responses of the same neurons produced by single pulses (1.1–1.5T) applied to the SU or SP nerves. Same format as in A– C. Raster displays in D were ordered according to response latency. In all figures, recordings of cord dorsum potentials are with negativity upward. Recordings of intraspinal potentials are with positivity upward. Voltage calibrations in C and F are for cord dorsum potentials 404 Fig. 2A–F Spontaneous activity and evoked responses of unsynchronized neurons. Same format as that of Fig. 1. Neuronal activity was recorded between 0.75 and 2.0 mm depth. Neuronal recordings in C and F were made in laminae IV at 1.31 mm depth. Voltage calibrations in C and F are for cord dorsum potentials activity of 17 neurons was increased during the occurrence of the spontaneous nCDPs recorded from L6 or L7 (synchronic neurons, see Fig. 1A–C). This is consistent with the observations of Sandkuhler and Eblen-Zajjur (1994) in the anesthetized rat, who also found neurons in laminae III–VI with spontaneous burst-like activity. There were, in addition, 11 neurons located in the same spinal regions whose activity was not synchronized with the spontaneous nCDPs (see Fig. 2A–C). Figure 1A shows the raster displays of the spontaneous discharges of 17 dorsal horn synchronic neurons that were recorded in different experiments during the generation of the spontaneous nCDPs. Recording of neuronal activity was made between 0.7 and 1.92 mm below the cord dorsal surface, 0.7–1.0 mm from the midline. The histogram in Fig. 1B was constructed by adding the activity of all neurons before and during the spontaneous nCDPs recorded close to the site of electrode penetration. The shape of the histogram is similar to that of the spontaneous nCDPs (bottom trace in Fig. 1C). For comparison, Fig. 2A–C shows the raster displays and histogram of the spontaneous activity of neurons whose activity was not clearly synchronized with the spontaneous nCDPs. These neurons were in the same spinal region as the synchronic neurons (0.75–2.0 mm depth). In contrast with the histogram of Fig. 1B, the histogram displayed in Fig. 2B is rather flat, and shows no resemblance to the spontaneous intraspinal field potentials recorded with the same electrode, or with the spontaneous nCDPs (Fig. 2C). Responses to stimulation of low-threshold cutaneous afferents To explain the facilitatory interactions observed between the spontaneous nCDPs and the responses evoked by stimulation of a cutaneous nerve, Manjarrez et al. (2000) proposed that the dorsal horn neurons that were activated during the spontaneous nCDPs also responded to stimulation of low-threshold skin afferents. To test this proposal further, we have examined the responses of the synchronic and non-synchronic neurons to stimulation of low-threshold cutaneous afferents from the SU and SP nerves. Figure 1D shows the raster displays obtained from the responses of 17 dorsal horn synchronic neurons to stimulation of the SP or SU nerves with single pulses 1.1–1.5T. The neuronal activity was aligned relative to the time of arrival of the afferent volley recorded from L6. The raster displays of each neuron were ranked according to their response latencies. It may be seen that the histogram constructed with the whole set of neuronal responses has a shape that resembles that of the evoked intraespinal field and cord dorsum potentials (Fig. 1E, F). As illustrated in Fig. 1D, most neurons responded with onset latencies between 1 and 3 ms after the arrival of the afferent volley. The shortest values (0.7–1.2 ms) suggest monosynaptic activation and the longer values (1.5–3 ms), oligosynaptic connections (see Brown 1981; Koerber and Brown 1982; Willis and Coggeshall 1991). The activation threshold of the synchronic neurons to stimulation of the cutaneous afferents varied between 1.1 and 1.5T, and had no clear relationship to response latency. 405 Fig. 3A–C Correlation between spontaneous firing of pairs of synchronic neurons. A Cross-correlation histogram of spontaneous activity of two synchronic neurons located within lamina IV at 1.26 and 1.25 mm depth, separated by 2 mm along the rostrocaudal axis. B Cross-correlation histogram of two synchronic neurons located between laminae IV and V at 1.45 and 1.36 mm depth and longitudinally separated by 7.4 mm. C Strength of correlation (F) of four pairs of neurons versus longitudinal separation between the recording micropipettes. For further explanations see text Figure 2D, E shows the raster display and histogram obtained from the responses of 11 non-synchronized neurons to stimulation of the SP or SU nerves (1.1– 1.5T). These neurons responded with latencies ranging from 1.9 to 8.3 ms (Fig. 2D). There was no significant difference in the activation thresholds of synchronic (1.2€0.2T) and non-synchronic neurons (1.2€0.5T) (P>0.05). firing relative to the other neuron during the generation of the spontaneous nCDPs was lower. Altogether it was possible to have stationary recordings of the activity of four pairs of synchronic neurons. As shown in Fig. 3C, the correlation strength (F) between the spontaneous activity of these four pairs of synchronic neurons during the spontaneous nCDPs decreased with increasing separation between the tips of the two recording micropipettes, but was still detectable by 7.4 mm. Although correlation strength is not a direct measure of the coefficient of correlation, these data indicate that the synchronization between neuronal activity at the time of the spontaneous nCDPs decreases with increasing separation between neurons. Cross-correlation between the activity of pairs of neurons The results described in the preceding sections have indicated that a substantial number of dorsal horn neurons discharge in synchrony during the generation of the spontaneous nCDPs in the same spinal segment. This raised the question as to the extent to which longitudinal distance between neurons affected the magnitude of their cross-correlation. To this end we made simultaneous recordings of the spontaneous activity of pairs of synchronic neurons located in laminae III–VI within the L7 and L6 segments by means of two micropipettes whose tips were separated one from the other between 2 and 7.4 mm along the rostrocaudal axis in different experiments. Figure 3A shows the cross-correlation histogram of the activity of two dorsal horn synchronic neurons located in the more ventral part of lamina IV that were separated by 2 mm. This histogram had a distinct peak relative to the basal values, indicating highly correlated activation during the generation of the spontaneous nCDPs (see Inbar et al. 1979). The calculated correlation strength (F) for this pair of neurons was 5.2. For comparison, the histogram in Fig. 3B shows the cross-correlation histogram of a pair of synchronic neurons located between laminae IV and V, which were separated by 7.4 mm. This pair of neurons had a lower correlation strength (2.4). This means that the probability of having one neuron Intersegmental synchronization of the spontaneous nCDPs Segmental patterns of spontaneous nCDPs We have suggested previously (Manjarrez et al. 2000) that the set of dorsal horn neurons that fire in synchrony with the spontaneous nCDPs is distributed throughout several spinal segments in a longitudinal array. The data depicted in Fig. 3C suggest that this array may not extend more than 10 mm rostrally and 10 mm caudally in the L6– L7 segments. However, the small number of neurons examined precludes a proper estimate of the longitudinal extension of this array. To gain more information on the longitudinal extension of the synchronized activity of the dorsal horn neurons during the occurrence of the spontaneous nCDPs, simultaneous recordings of cord dorsum potentials were made from several spinal segments (usually from S1–L4). Figure 4 shows samples of spontaneous nCDPs that were recorded in the same preparation simultaneously from four spinal segments (L4–L7). In some cases they were larger in L4 and L5 than in L6 and L7 (open square in 406 Fig. 4A–F Synchronized cord dorsum potentials in several spinal segments. The figure shows samples of nCDPs simultaneously recorded from four different spinal segments in a single experiment within a 10-min time period. Vertical dotted lines show onset of the earliest potential in each array of synchronous discharges. Symbols indicate different patterns of intersegmental nCDPs. For further explanations see text Fig. 4A), while in other cases they were larger in L6 and L5 than in L4 or L7 (open square in Fig. 4B), or in L7 and L6 than in L5 and L4 (filled diamond in Fig. 4F). Synchronized spontaneous nCDPs could also appear in three (filled circles in Fig. 4C, E) or in two segments (filled squares in Fig. 4C, F), and also remain confined within one segment only (either in L6, open diamonds in Fig. 4A, B, or in L7, open circles in Fig. 4D, E). Although we have not performed a statistical analysis of the likelihood of occurrence of specific patterns of the synchronized intersegmental nCDPs, some of them appeared to be generated more often of what one may expect from random variations (see Roerig and Feller 2000). To illustrate this point, in Fig. 5A we have superimposed recordings obtained from the same experiment as that of Fig. 4, in which the spontaneous nCDPs appeared simultaneously in four spinal segments (L4–L7). Figure 5B shows instead recordings in which the spontaneous nCDPs appeared only in three segments (L4, L5 and L6), together with a small slow positive wave in L7. The nCDPs displayed in Fig. 5C appeared mainly in segments L4 and L5, while the potentials illustrated in Fig. 5D appeared only in segment L7 and were timelocked to a small, slow positive wave in segments L4–L6. Averaging procedures were used to disclose those nCDPs that appear in synchrony in different spinal segments. To this end, the nCDPs recorded at a given location (reference nCDPs) were fed to a window discriminator. A single pulse was generated whenever the reference nCDPs exceeded a predetermined value, usually set to include responses above 30% of maximal amplitude. These pulses were used to trigger the averaging of the cord dorsum potentials in all segments. Fig. 5A–D Intersegmental patterns of spontaneous nCDPs. Data derived from the same set of records as that of Fig. 4. A–D Superposed records of the spontaneous nCDPs that were simultaneously recorded from four different spinal segments. These potentials were selected on the basis of their intersegmental patterns. Traces were aligned according to the onset time of the nCDPs recorded in one segment (L6 in A and B, L5 in C and L7 in D). The time relation between the reference nCDPs and the potentials recorded in the other segments was preserved in all cases. Vertical dotted lines indicate estimated onset times of the reference potentials. For further explanations see text 407 Fig. 6A–L The intersegmental distribution of synchronized spontaneous nCDPs in the lumbosacral enlargement is not affected by peripheral nerve transection. A–C Superposed traces of averaged spontaneous nCDPs simultaneously recorded in four different segments before and after transection of cutaneous and muscle nerves in the ipsilateral hindlimb. The triggering pulses for the averaging were generated by the spontaneous nCDPs recorded in segments L4, L5, L6 or L7–S1, as indicated.The vertical arrows show the estimated onset of the L5 and L6 nCDPs. In A, the other arrow points to the nCDP recorded after the nerve transection. D–F Superposed averages of spontaneous nCDPs triggered by cord dorsum potentials recorded in different segments and of spontaneous nCDPs followed by stimulation of the ipsilateral foot pad with pulses 1.2T taken before peripheral nerve transection. G–I Cord dorsum potentials produced by stimulation of the foot pad with pulses of different strengths, as indicated. J–L Superposed power spectra of cord dorsum potentials recorded from different segments before and after peripheral nerve section. For further explanations see text Figure 6A–C shows the superposed averages of the spontaneous nCDPs recorded from four different segments in a preparation with intact hindlimb nerves and after some of these nerves were sectioned (see below). The intersegmental distribution of the spontaneous nCDPs varied according to which nCDPs were used as reference for the averaging procedure. In the experiment of Fig. 6, when the triggering pulses were generated by the L5 or L6 nCDPs, large synchronized nCDPs were recorded in segments L5 and L6 and small potentials were recorded more caudally or rostrally (Fig. 6A, B). In contrast, when the averages were obtained using as reference the nCDPs recorded in the border between L7 and S1, the nCDPs remained confined within that region (Fig. 6C). It thus seems that in this particular experiment the neuronal arrays involved in the generation of the L4– L6 spontaneous nCDPs were different from those involved in the generation of the L7–S1 nCDPs. In other experiments the averages obtained using as reference the spontaneous L6 or L7 nCDPs disclosed time-locked nCDPs in segments L5 to S1 (Fig. 7A, B). Time shifts between spontaneous nCDPs Differences in onset latencies between averaged nCDPs recorded from different segments were difficult to estimate because of their slow rising phase. Yet, as shown by the arrows in Fig. 6A, when triggering the averages with the L5 nCDPs, the potentials generated in L5 seemed to appear 4.6 ms before those recorded in L6. In contrast, when the averages were obtained using as reference the L6 nCDPs, the time shift between the L5 and L6 nCDPs was rather small (Fig. 6B). Latency shifts between the nCDPs recorded in different segments were also observed without averaging. As shown in Fig. 5A, the L5 and L6 nCDPs had a similar latency and preceded the L4 and L7 nCDPs by 5.8 and 9.0 ms, respectively. However, when the nCDPs appeared only in the L4–L6 segments, they started at about the same time in the three segments (Fig. 5B). It is likely that nCDPs with similar onset times were generated by a common input. On the other hand, the time shifts between spontaneous nCDPs recorded from different segments could indicate that these potentials were generated in one segment and propagated to other segments. Yet, they could be also generated by a common input and the 408 Fig. 7A–J Sectioning the ipsilateral dorsolateral funiculus and the ipsilateral dorsal horn reduces segmental synchronization of spontaneous nCDPs. Data obtained from preparations with acute transection of ipsilateral muscle and cutaneous hindlimb nerves. A– E Effects on the averaged spontaneous nCDPs by a lesion comprising the ipsilateral dorsolateral funiculus between segments L6 and L7. Averages were obtained by triggering with pulses generated by the spontaneous L6 or L7 nCDPs, as indicated. The extent of the lesion is shown in C. F–J Effects produced by sectioning the ipsilateral dorsal horn and columns. Histology shown in H. For further explanations see text latency shifts would result from differences in conduction time to the different segments. As reported by Manjarrez et al. (2000), the first (monosynaptic) component of the cord dorsum responses evoked by stimulation of cutaneous afferents was facilitated when preceded by spontaneous nCDPs. We have found that the magnitude of the facilitation of the evoked responses recorded in different segments varied according to which nCDPs were used as reference for the averaging. In the experiment of Fig. 6, facilitation of the monosynaptic component of the evoked responses recorded in L5 and L6 was larger when using as reference the L7–S1 potentials (Fig. 6F) than when using the L5 or the L6 potentials (Fig. 6D, E). In other experiments (not illustrated) the evoked nCDPs recorded in these segments were all facilitated when preceded by spontaneous nCDPs, regardless of the segmental origin of the nCDPs used as reference for the averaging. Evoked CDPs In addition to the spontaneous nCDPs, we also recorded the cord dorsum potentials produced by stimulation of the foot pad in preparations with intact hindlimb nerves, or by stimulation of either the ipsilateral SU or SP nerve in preparations with acutely transected nerves. In these experiments, stimulus strength was adjusted between 1.1 and 1.3T to produce responses of about the same amplitude as the spontaneous nCDPs. Figure 6G, H and I shows the cord dorsum potentials produced by stimulation of the ipsilateral foot pad with pulses 1.15, 1.2 and 1.25T strength. These responses appeared in the four segments, but were clearly larger in the L5 and L6 segments. A similar rostrocaudal distribution was obtained following stimulation of the SP or SU nerves, in confirmation of previous studies (Bernhard 1953; Bernhard and Widen 1953; Koerber and Brown 1982). It should be noted in Fig. 6 that the rostrocaudal distribution of the evoked responses (Fig. 6G–I) was similar to that of the spontaneous nCDPs triggered by the L5 and L6 nCDPs illustrated in Fig. 6A, B. Effects of spinal cord lesions on the intersegmental synchronization of the spontaneous nCDPs Most of the observations performed in this study were made in preparations in which several muscle and cutaneous nerves were acutely transected during the dissection (see “Materials and methods”). This raised the 409 Fig. 8A–C Effects of spinal lesions on the frequency power spectra recorded from different spinal segments. Data obtained from preparations with ipsilateral acute transection of muscle and cutaneous hindlimb nerves. Upper graphs in each panel show the power versus frequency spectra recorded at various segmental levels, as indicated. Power spectra are expressed as a percentage of maximum before the spinal lesion. The lower sets of graphs show the areas of the power spectra. Thick lines before, fine lines after, a spinal lesion between segments L6 and L7. A Effects produced by a lesion to the ipsilateral dorsal columns. For histology see open circle in Fig. 9A. B Effects produced by a lesion comprising the ipsilateral dorsal horn (see filled triangle in Fig. 9C). C Effects produced by a dorsal hemisection (see square in Fig. 9E). For further explanations see text question as to the extent to which acute nerve transection was responsible for the limited longitudinal distribution of the spontaneous nCDPs within the lumbar enlargement. In four experiments we recorded the spontaneous nCDPs from preparations in which the left PBSt, GS, SU, SP and DP nerves were dissected free, but left intact (see “Materials and methods”). We found that transection of these nerves had a relatively small effect on the amplitude and segmental distribution of the synchronized nCDPs (Fig. 6A–C). In three preparations in which these nerves were cut during the dissection, we found that sectioning the ipsilateral dorsal columns between the L5 and L6 segments also had very small effects on the intersegmental synchronization of the spontaneous nCDPs. This lesion was aimed to interrupt not only residual sensory inputs from the periphery, but also the possible synchronization due to transsegmental conduction of action potentials generated by primary afferent depolarization in the intraspinal terminals of the sensory fibers (see Rossignol et al. 1998). In contrast to the ineffectiveness of these lesions, we found that transection of the ipsilateral dorsal horn (three experiments) and/or the dorsolateral funiculus (three experiments) markedly reduced the intersegmental synchronization of the spontaneous nCDPs. As shown in Fig. 7A–E, after a lesion to the ipsilateral dorsolateral funiculus, the spontaneous nCDPs recorded rostrally to the lesion were largely (but not completely) uncoupled from the potentials recorded caudally to the lesion. The uncoupling became more pronounced when the lesion also included the dorsal horn (Fig. 7F–J), and was almost complete following a dorsal bilateral hemisection (not illustrated; see Fig. 9E for histology). 410 Fig. 9A–F Summary of the changes in the segmental distribution of frequency power spectra produced by spinal lesions. Data obtained from preparations with ipsilateral transected hindlimb nerves. A, C, E Segmental distribution of the normalized areas of the frequency power spectra. Integration procedures as in Fig. 8. B, D and F The same but after sectioning the ipsilateral dorsal columns (B), the ipsilateral dorsal horn (D) and the dorsal spinal half (F), between L6 and L7. Vertical dotted lines indicate the segmental level of the spinal lesion. Data and histological reconstruction of spinal lesions from individual experiments are indicated by the same symbol Power spectra experiment of Fig. 6, the power spectrum of the potentials recorded in the L5 and L6 segments was highest in the low frequency range (between 2 and 4 Hz; see Fig. 6J–L). In other experiments (Fig. 8A) the power spectrum of the potentials recorded at L6 showed two peaks, one at 5 and the other at 10 Hz, while at L5 and L7 the power spectra showed one peak only (at about 7 Hz). In the experiment of Fig. 8B the 5-Hz peak was of about the same magnitude in segments L6–S1, while the 10-Hz component was largest in L7 and S1. Finally, in the experiment of Fig. 8C, the 5- and 10-Hz components were largest in L6. The factors determining the features of the power spectra of the cord dorsum potentials recorded in different preparations have not been elucidated. In two preparations with intact hindlimb nerves we observed that transection of the left PBSt, SU and SP produced a small increase of the power spectra of the spontaneous cord dorsum potentials in segment L6 and practically no changes in the adjacent segments (Fig. 6J– L) To facilitate the analysis of the effects produced by the spinal lesions, the power spectra were integrated. Clearly this procedure precludes detection of changes in a specific frequency range. Yet, it provides qualitative information In a previous study made in anesthetized preparations with acutely transected muscle and cutaneous nerves (see Manjarrez et al. 2000), we reported that the cord dorsum potentials recorded from L6 or L7 had dominant frequencies between 3 and 20 Hz. These frequency spectra appear to be basically the same as those recorded in anesthetized preparations with intact hindlimb nerves (see Manjarrez et al. 2002c). Simultaneous recordings of cord dorsum potentials from different spinal segments in anesthetized preparations with intact or acutely transected muscle and cutaneous nerves have now indicated that within the lumbosacral segments the low frequency components have a restricted rostrocaudal distribution (see Figs. 6J–L, 8 and Manjarrez et al. 2002c). In two preparations the region of maximal power was confined within the L6 and S1 segments (Fig. 8A, B), and in a third preparation it was located between the L5 and L7 segments (Figs. 6J–L, 8C, 9A, C, E). Another feature that emerged from the analysis of these power spectra was that the dominating frequencies were not the same in all segments. For example, in the 411 of changes in the whole frequency range. Figure 8A shows that a small lesion comprising the ipsilateral dorsal column between segments L6 and L7 made in a preparation with acutely transected ipsilateral hindlimb nerves had very little effect on the frequency spectrum of the spontaneous cord dorsum potentials, both rostrally and caudally to the lesion (see also Fig. 9A, B). In contrast, as illustrated in Figs. 8B and 9D, sectioning the dorsolateral funiculus in the ipsilateral side had more pronounced effects on the power spectrum, particularly on the low frequency components of the potentials recorded from L7 and S1, that is, caudally to the lesion. A dorsal bilateral transection had even more dramatic effects, both rostrally and caudally to the lesion (Figs. 8C, 9F). Discussion Features of the synchronized spontaneous activity of dorsal horn neurons The present study has indicated that in Rexed’s laminae III–VI of the lumbosacral spinal cord of the anesthetized cat, there is a substantial number of neurons that fire in synchrony during the generation of the spontaneous nCDPs. These findings are in agreement with our previous proposal based on the analysis of field potentials (Manjarrez et al. 2000) that the neurons that discharge during the generation of the spontaneous nCDPs are located within the same region where low-threshold cutaneous afferents terminate. We also found neurons in the same spinal regions that did not fire in synchrony with the spontaneous nCDPs. Since these neurons also responded to stimulation of cutaneous nerves, the absence of correlated activity could be due to the small excitatory drive, below firing threshold, received at the time of generation of the spontaneous nCDPs (see O’Donovan 1999). However, since the available samples were rather non-homogeneous, they might also represent another functional type of neuron. Our data also indicate that the population of neurons with synchronized spontaneous activity is longitudinally distributed along several lumbosacral spinal segments, in a similar manner to the neurons responding to stimulation of cutaneous nerves (see Fig. 6, and Bernhard 1953; Bernhard and Widen 1953; Koerber and Brown 1982). However, in agreement with the findings of Lidierth and Wall (1998) in the rat, the spontaneous synchronous discharge of these neurons appears not to depend on the input supplied by primary afferents, because sectioning several ipsilateral hindlimb muscle and cutaneous nerves (see Fig. 6), as well as the dorsal columns between L6 and L7, had very little effect on the segmental distribution of the synchronized nCDPs. It thus seems that the synchronization of the spontaneous activity of the laminae III–VI neurons emerges from local interactions, as suggested by the finding that a section of the ipsilateral dorsal horn and of the ipsilateral dorsolateral funiculus largely uncouples the spontaneous nCDPs generated rostrally from those generated caudally to the lesion (Fig. 7; see also Kerkut and Bagust 1995; Lidierth and Wall 1998). Yet, these may not be the only pathways mediating the intersegmental synchronization of the neuronal activity (see Kremer and Lev-Tov 1997). In this regard, it should be noted that the transections comprising the dorsal horn and the dorsolateral funiculus also reduced the magnitude of the integrated frequency power spectra of the spontaneous cord dorsum potentials recorded both rostrally and caudally to the lesion (Figs. 8, 9). This could be due to the reduction of mutually excitatory, self-potentiating interactions between adjacent sets of neurons that increase the excitability of the whole ensemble and synchronize their spontaneous activity (see Lidierth and Wall 1996; O’Donovan 1999; Roerig and Feller 2000). The generation of a particular segmental combination of synchronized nCDPs could be due to the activation of specific sets of tightly coupled arrays of neurons. The excitation of any neuron within a given array, either by sensory or by spinal inputs, would activate, in a rather stereotyped manner, the neurons belonging to that particular ensemble (see Silberberg et al. 2002). This explains the remarkable similarity between the segmental distribution of the responses produced by stimulation of the ipsilateral foot pad and the spontaneous nCDPs recorded before as well as after the transection of muscle and cutaneous nerves in the ipsilateral hindlimb (see Fig. 6A, C, G–I). In the absence of synchronized descending influences, such as those generated during the execution of voluntary movements (Baker et al. 1999), or during real or fictive locomotion (Arshavsky et al. 1997; Grillner 1981), the activation of the different arrays would be random, as suggested by the data depicted in Fig. 4, which show that the different intersegmental patterns of nCDPs may occur more or less independently during the same recording period. In the anesthetized cat, this activity appears to be confined within the lumbosacral segments and has little relation to the neuronal arrays involved in the generation of the spontaneous nCDPs in the cervical segments (Manjarrez et al. 2002c). Yet, these two arrays could become synchronized by common inputs in other conditions, such as during locomotion (Kremer and Lev-Tov 1997; Pearson 2000). Morphological substrates for intersegmental synchronization of nCDPs Although there are many studies on the different types of neurons in the dorsal horn, information on their intra- and intersegmental connectivity is rather limited (see Willis and Coggeshall 1991 for review), and does not allow disclosure of the neurons that are involved in the intersegmental synchronization of the spontaneous nCDPs. 412 Lidierth and Wall (1996) have concluded that the isolated spinal cord of the anesthetized rat contains an oscillatory mechanism that is synchronized by way of intrinsic axons in the Lissauer tract and produces lowvoltage, low-frequency, DRPs on all lumbar dorsal roots (see also Lidierth and Wall 1998). It is therefore possible that the synchronization of the spontaneous activity of the neurons in laminae III–VI observed in our experiments during the spontaneous nCDPs is due, at least in part, to the inputs they receive from neurons in lamina II. However, using the Golgi method, Matsushita (1969) found in the cat that cells of lamina II not only connect with other cells in laminae II–VI, but also that cells in laminae IV and V are connected with neighboring cells in laminae III–VI. In addition, Schneider (1992) and Schneider et al. (1995) have described in the spinal cord of the hamster cells in laminae III–V with local axons and mutual excitatory and inhibitory interconnections (Schneider 2001), as well as neurons with deep axons that usually bifurcate into dorsal and caudal daughter branches up to 2.5 mm long, giving off collaterals ventral to the cell body and dendrites. It is therefore possible that the local axon interneurons are involved in the generation of spontaneous nCDPs that remain confined within the same segment, while the intersegmental synchronization is mediated, at least in part, by the deep axon neurons. Recent evidence supports the existence of mixed synapses (chemical and electrical) on neurons throughout the spinal cord of adult rats in Rexed’s laminae III–IX in cervical, thoracic and lumbar segments (Rash et al. 1996). If present in the spinal cord of the adult cat, it is not unlikely that electrical synapses also contribute to the synchronization of the spontaneous interneuronal discharges. In this case, gap junctions could provide additional pathways for bidirectional excitatory communication between neurons (see Tresch and Kiehn 2000; Roerig and Feller 2000). One pending issue relates to the pathways and mechanisms involved in the termination of the discharges of neurons in layers III–VI during the spontaneous nCDPs. Available evidence suggests that these neurons have excitatory as well as inhibitory connections with other neurons in the region (Schneider 2001). It is not unlikely that these inhibitory inputs play an important role in the termination of the episodes of synchronized spontaneous activity of the neurons, as suggested by the finding that stimulation of cutaneous nerves transiently depresses the generation of spontaneous nCDPs (see raster displays in Fig. 1D and Manjarrez et al. 2000), and also by the finding that in some cases the spontaneous nCDPs generated in one segment appear together with positive waves in other segments (Fig. 5B, D), probably because of activation of inhibitory neurons. Yet, depression of neuronal excitability by previous activity could also play a relevant role in this process (see O’Donovan 1999). Functional role of the intersegmental coupling of spontaneous activity of dorsal horn neurons Further understanding of the functional role of the synchronization of dorsal horn neurons during the nCDPs will require information on their axonal projections, that is, if they have only local connections or if their axons also contact neurons with ascending projections (see below and Schneider 1992). In this context, it should be mentioned that in a preliminary study Manjarrez et al. (2002a) recorded a series of potentials in the contralateral posterior sigmoid gyrus that appeared about 14 ms after the L6 spontaneous nCDPs, and were abolished by sectioning the dorsal columns and the ipsilateral dorsolateral funiculus at the thoracic level, suggesting involvement of the spinocervical tract (see Bryan et al. 1973; Willis and Coggeshall 1991). During the occurrence of spontaneous nCDPs in L6, the cortical potentials evoked by electrical stimulation of cutaneous nerves or by mechanical stimulation of the skin (Manjarrez et al. 2002b), as well as the segmental monosynaptic reflexes (Manjarrez et al. 2000), were facilitated. Whether the cortical and segmental actions associated with the generation of the spontaneous nCDPs were mediated by the same neurons (e.g., ascending tract neurons with local collaterals) or by separate sets of neurons, both receiving a common input, remains an open question. It is also important to determine the location and extension of the receptive fields in response to natural stimulation of the skin of the neurons that are synchronized and non-synchronized with the spontaneous nCDPs. According to Brown et al. (1979), all pairs of dorsal horn neurons in the lumbosacral cord of the cat with non-flat cross-correlograms had overlapping receptive fields. These results agree with those reported by Eblen-Zajjur and Sandkuhler (1997) for the rat. It thus seems reasonable to assume that the neurons that fire in synchrony during the spontaneous nCDPs presently analyzed could have overlapping receptive fields. This would increase their influence on spinal reflex pathways when naturally activated. The coupling between the spontaneous activity of dorsal horn neurons could allow the constitution of a distributed assembly, which encodes a stimulus by means of correlated increases of neuronal discharge rates (Aertsen et al. 1989). There is evidence suggesting that synchronization plays a functional role in the coding of a cutaneous stimulus. Long-lasting radiant skin heating induced in the vast majority of neuron pairs an increase in the strength of synchronization (Eblen-Zajjur and Sandkuhler 1997). Acknowledgements We thank Profs. E. Jankowska and A. LevTov for their useful comments on the manuscript, D. Chvez, C.A. Garca and S. 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