The effects of opioids on sacrocaudal afferent (SCA) pathways and the pattern-generating circuitry of the thoracolumbar and sacrocaudal segments of the spinal cord were studied in isolated spinal cord and brain stem-spinal cord preparations of the neonatal rat. The locomotor and tail moving rhythm produced by activation of nociceptive and nonnociceptive sacrocaudal afferents was completely blocked by specific application of the μ-opioid receptor agonist [d-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin acetate salt (DAMGO) to the sacrocaudal but not the thoracolumbar segments of the spinal cord. The rhythmic activity could be restored after addition of the opioid receptor antagonist naloxone to the experimental chamber. The opioid block of the SCA-induced rhythm is not due to impaired rhythmogenic capacity of the spinal cord because a robust rhythmic activity could be initiated in the thoracolumbar and sacrocaudal segments in the presence of DAMGO, either by stimulation of the ventromedial medulla or by bath application of N-methyl-d-aspartate/serotonin. We suggest that the opioid block of the SCA-induced rhythm involves suppression of synaptic transmission through sacrocaudal interneurons interposed between SCA and the pattern-generating circuitry. The expression of μ opioid receptors in several groups of dorsal, intermediate and ventral horn interneurons in the sacrocaudal segments of the cord, documented in this study, provides an anatomical basis for this suggestion.
Mammalian locomotion is characterized by an alternating activation of antagonistic muscles and synchronous activation of synergist muscles within a limb. This pattern of activity and its intersegmental coordination is generated by intrinsic spinal networks known as central pattern generators (CPGs) (see Arshavsky et al. 1997; Gelfand et al. 1988; Hultborn et al. 1998; Rossignol 1996). It is now established that locomotor-like rhythms can be evoked in the isolated spinal cord by various combinations of excitatory amino acids, monoamines, and cholinergic agonists. It is also known that stimulation of low-threshold afferents, particularly those of the 1a and 1b pathways, can have significant effects on the locomotor rhythm. Less well understood, however, are the interactions of high-threshold afferent and nociceptive pathways with locomotor networks. Studies of Lundberg and colleagues on spinal cats treated with L-DOPA and monoamine-oxidase blockers revealed that stimulation of high-threshold flexor reflex afferents (FRA) could produce a locomotor-like rhythm (Jankowska et al. 1967a,b; Lundberg 1979). Support that these afferents had direct access to the locomotor CPG was demonstrated by the ability of FRA stimulation to reset the L-DOPA-induced locomotor-like rhythm (Schomburg et al. 1998; e.g., Hultborn et al. 1998). Furthermore, it was found that application of opioids to spinal cats almost abolished the locomotor-like rhythm induced by L-DOPA/nialamide or by FRA stimulation (Schomburg and Steffens 1995). More recent work has further implicated nociceptive afferents directly in the regulation of locomotor activity by showing that noxious heat stimulation of the foot can trigger locomotor-like rhythmic activity in the spinalized cat given nialamide and L-DOPA (Schomburg et al. 2001).
In the present work, we have investigated the role of opioids in the regulation of hindlimb locomotor-like activity generated by caudal-thoracic and lumbar rhythm generating circuitry (Cazalets at al. 1995; Cowley and Schmidt 1997; Kjaerulff and Kiehn 1996; Kremer and Lev-Tov 1997), and tail-moving rhythmicity generated by sacrocaudal networks (Delvolve et al. 2001; Lev-Tov et al. 2000). In contrast to the earlier work in the adult cat, we performed our studies using an isolated preparation of the spinal cord and brain stem spinal cord of the neonatal rat (Blivis and Lev-Tov 2005; Delvolve et al. 2001; Lev-Tov and Delvolve 2000; Lev-Tov et al. 2000). We have previously shown that tonic stimulation of sacrocaudal afferents (SCAs) is a potent activator of locomotor-like activity in the lumbar and sacral segments. The use of this preparation allowed us to separate the influence of opioids on transmission in the afferent pathway from their effects on the pattern-generating networks themselves. Surprisingly, we found only modest effects of opioids on the locomotor capacity of lumbar spinal networks and little or no effect on the rhythmogenic capacity of sacrocaudal segments. The major effect of exogenously applied opioids was on a powerful afferent pathway from sacrocaudal afferents to lumbar and sacral rhythmogenic networks. In the absence of exogenous drugs, activation of this pathway has an extraordinary capacity to produce rhythmic patterns in the lumbar and sacral cord. Application of μ-opioids to the sacrocaudal segments completely abolished the ability of sacrocaudal pathways to induce locomotor-like activity in the lumbar and tail-moving activity in sacrocaudal segments, suggesting an unexpected and powerful regulation of lumbar and sacral rhythmogenic network activity by opioids. Consistent with this idea, immunocytochemistry revealed widespread expression of the μ-opioid receptor MOR-1 on sacral spinal neurons.
Spinal cord (T6–Co3) or brain stem-spinal cord preparations were isolated from P2–P6 ether-anesthetized rats with or without an intact tail and hindlimbs (Blivis and Lev-Tov 2005; Delvolvé et al. 2001; Lev-Tov and Delvolvé 2000; Lev-Tov et al. 2000). Preparations were transferred to a recording chamber and superfused continuously with an oxygenated Krebs saline (e.g., Delvolvé et al. 2001; Kremer and Lev-Tov 1997; Lev-Tov et al. 2000).
Stimulation and recordings
Suction electrode recordings were obtained from pairs of lumbar and sacral ventral roots using a high-gain DC or 0.1 Hz to 10 kHz AC amplifier. Sharp electrode intracellular recordings were obtained from S2 motoneurons impaled from the ventral or ventrolateral aspect of the cord and identified by the presence of antidromic spikes.
Rhythmic activity was induced by activation of sacrocaudal (SC) pathways using either infrared radiant-heat or punctate stimulation of SC dermatomes, or by electrical stimulation of SCAs in the Co2–S4 dorsal roots. The threshold (T) was defined as the current intensity used to evoke a detectable slow ventral root potential in the recorded SC segment (usually S2) by single pulse constant current stimulation applied to one of the sacrococcygeal dorsal root that produced the rhythm (Co2–S4 dorsal roots) (Delvolvé et al. 2001; Strauss and Lev-Tov 2003). Rhythmic activity was also induced by bath-applied neurochemicals or by bipolar stimulation of the ventromedial medulla (Blivis and Lev-Tov 2005; e.g., Zaporozhets et al. 2004). The neurochemically induced locomotor like rhythm was initiated by bath-applied NMDA/5HT (usually 3–4 and 10–20 μM, respectively) and stabilized in some of the experiments by bath-applied dopamine (20–30 μM). When required, the experimental bath was divided into two compartments by a petroleum jelly (Vaseline) wall to enable selective activation of either the locomotor or SC rhythmogenic networks or to test region-dependent effects of various drugs. The following drugs were used in these experiments: the opioid agonists [d-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin acetate salt (DAMGO) and the opioid antagonist naloxone hydrochloride dehydrate (Sigma).
Data acquisition and statistical analysis
Data were digitized (Digidata 1320A, Axon Instruments) and stored on the computer's hard disk for subsequent analyses (see Gabbay et al. 2002; Gabbay and Lev-Tov 2004; Strauss and Lev-Tov 2003). Population recording data were high-pass filtered at 40–50 Hz (Fig. 1A, top), rectified and low-pass filtered at 5–40 Hz (Fig. 1A, bottom).
Analyzed data samples included five nonoverlapping data segments (60 s each) for each pharmacological treatment in each of the experiments of a given series for the NMDA/5HT-induced rhythms. When the rhythm was induced by stimulation of the ventromedial medulla or sacrocaudal afferents, the recordings obtained during each of the three or four different stimulus trains applied at a given intensity were sampled for each treatment in each experiment in a given series. All compared samples included a similar number of cycles. The adequacy of the sample size was verified by the high significance of the results of the bivariate Fourier analysis of each data segment (99% confidence limit of the respective coherence spectrum, see following text and Fig. 1B).
All data samples were analyzed using Spinalcore, a menu-driven MATLAB-based program for stationary and nonstationary time series analyses developed by Mor and Lev-Tov.
The frequency of the rhythm of each data sample was computed from the peak spectra obtained either by uni- or bivariate Fourier analysis (Fig. 1B, CPSD). The mean phase shifts between any given pair of time series variables of each data sample was computed using a Welch-windows based Fourier bivariate (cross-spectral) analysis (Fig. 1B, CPSD) (e.g., Miller and Sigvardt 1998; Strauss and Lev-Tov 2003). Coherence analysis was performed to measure the strength of association between any analyzed pair of time series variables (Fig. 1B, coherence), and the significance of its peak was tested against 99% confidence limit (Fig. 1B, coherence, solid horizontal line). Frequency data were pooled if one-way ANOVA revealed no significant differences between the data samples (Fig. 1C, cycle time data).
Because all the pairs of time series variables compared in the present study yielded highly significant coherence, the extracted phase data were analyzed using circular statistics to calculate the mean phase-lag and the r vector (rv) describing the concentration of phase-lag values around the mean under each experimental condition (Fig. 1C, φLR, exp. 1 data) (e.g., Delvolve et al. 2001; Gabbay et al. 2002; Gabbay and Lev-Tov 2004; Lev-Tov et al. 2000). Data were pooled if the Watson and Williams test revealed no significant differences between the tested samples (Fig. 1C, φLR, 5 exp. pooled data). Rayleigh's test (Zar 1984) was used to determine whether the phase values were uniformly distributed around the circle (see Delvolve et al. 2001; Gabbay et al. 2002; Gabbay and Lev-Tov 2004). Multi-sample testing was performed to compare the mean phase values of any pair of tested factors (the Watson-Williams test) (Zar 1984).
Immunohistochemistry and confocal microscopy of MOR1 expression
Rats aged P3 (n = 6) and adult (n = 2), were killed with xylazine (10 mg/kg) and ketamine (80 mg/kg ip) and perfused transcardially with 4% paraformaldehyde in 0.1M phosphate buffer solution, pH 7.3 (PBS). The spinal cords were quickly dissected and postfixed overnight in the same fixative. All the experiments reported here were performed in the upper sacral region of the spinal cord (S2–S3). Sections were cut in a cryostat or a Vibratome. Spinal cords were washed in 0.01M PBS (after postfixation), embedded in warm agar-agar (5% in PBS) and cut transversely into 60-μm sections. Sections were collected in wells and immunohistochemistry was performed on free-floating sections to enhance antibody penetration. Sections were initially blocked (for 90 min) with Normal Donkey Serum (1:10 in PBS containing 0.1% of Triton-X-100, PBS-TX) and then incubated with the primary antibody against the μ-opioid receptor MOR-1 (either from Neuromics or Chemicon, raised in rabbit, in a working dilution 1:500). No difference between the two antibodies from these companies was observed. Data presented here were from immunohistochemistry performed using the Neuromics antibody.
The primary antibody was diluted in PBS-TX and incubations were carried out for 20–24 h at room temperature. To assess the specificity of the primary antibody, we also used a μ opioid receptor blocking peptide (Neuromics; No. P10104) at 2 mg/ml concentration. The blocking peptide was mixed with the primary antibody against MOR-1 1 h prior to application at room temperature.
Immunoreactive sites were revealed with a secondary antibody (applied for 3 h) conjugated to FITC (rabbit-FITC, Jackson Labs). The secondary antibody was diluted 1:50. After secondary antibody incubations, the sections were washed in PBS (6 washes of 5 min each) and cover-slipped with a solution of PBS:Glycerol (7:3) to minimize bleaching during image acquisition.
All images were obtained with confocal microscopy using an LSM 510 META (Carl Zeiss, Germany) confocal microscope using an excitation wavelength of 488 nm. To present a low-magnification image of the tissue, two areas were scanned using a ×20 objective and “stitched” together (collage) for the P3 (Fig. 7) and nine areas were scanned for the adult (Fig. 8). The optical thickness at low magnifications was 5 μm and at higher magnifications was 0.9 μm.
Stimulation of nociceptive and nonnociceptive sacrocaudal afferent pathways produces coordinated motor rhythm in the neonatal rat spinal cord
Our previous studies revealed that stimulation of sacrocaudal afferents (SCA) is a potent and robust method for inducing locomotor-like activity in lumbar and tail moving rhythm in sacral segments of the isolated cord of the neonatal rat and mouse in the absence of exogenously applied drugs (Delvolve et al. 2001; Lev-Tov et al. 2000; Whelan et al. 2000). To identify the classes of afferents mediating this effect, we examined the ability of graded stimulation of sacrocaudal afferents or natural nociceptive stimuli to activate the rhythm. Figure 2A shows that regular rhythmic activity could be produced by low-intensity stimulation of sacrocaudal afferents [1.5–2 × threshold (T) in this example and as low as 1.1 and 1.2 T in other experiments performed in this series] as well as by high-intensity stimulation (3–10 T) (see also Delvolve et al. 2001). It seems reasonable to assume that SCA stimulation activated nonnociceptive pathways at the lowest stimulus intensities followed by mixture of nociceptive and nonnociceptive pathways at the highest intensities.
In a series of five experiments, we examined whether natural nociceptive stimuli of the sacrocaudal region is also capable of activating the pattern-generating circuitry of the spinal cord. In this set of experiments, we show that specific activation of pain pathways using infrared radiant heat stimulation of the sacrocaudal skin and transient mechanical (pinch) stimulation of the skin at the base of the tail in isolated spinal cord/tail preparations of neonatal rats produced organized rhythmic motor patterns (Fig. 2B).
μ-opioid receptor agonist blocks the SCA-induced rhythm
Because nociceptive pathways are strongly affected by analgesic opioids and because the proportion of μ-opioid receptors in the spinal cord is much higher than that of the κ- and δ-opioid receptors (Rahman et al. 1998), we examined whether μ-opioids are capable of affecting the rhythm produced by nociceptive and non nociceptive afferent pathways. Figure 3, A and B, shows that 1 μM of the μ-opioid agonist DAMGO completely blocked the rhythm produced by radiant heat (Fig. 3A) or by pinch stimulation (Fig. 3B) of sacrocaudal skin and that the block could be alleviated in both cases by bath application of the opioid antagonist naloxone. Similar results were obtained in each of the five experiments in these series.
Figure 3C shows that the rhythm produced in lumbar and sacrocaudal segments of the cord by electrical stimulation of sacrocaudal afferents (control) was blocked in the presence of DAMGO. The DAMGO-induced block persisted in this experiment following prolongation or intensification of the stimulus train, or after increasing the frequency of the stimulus train (not shown). The block could be alleviated 5 min after addition of low concentrations of the antagonist naloxone to the bath.
We found that DAMGO (1–5 μM) completely blocked the rhythm produced by stimulus trains applied at intensities (StI) <2 T in each of the 12 experiments in this series. Furthermore, the rhythm produced by a stimulus train applied at intensities between 2 and 5 T was completely blocked in 9/12 of the experiments. Comparative analysis of the effects of DAMGO and naloxone on the rhythm revealed that the normalized cycle time (Nc) in the presence of DAMGO and naloxone was slightly but significantly (P < 0.05) longer 1.21 ± 0.44 (at StI < 2 T) and 1.22 ± 0.26 (at 5 T ≥ StI >2 T) than the respective controls 1 ± 0.14 and 1 ± 0.11, Fig. 3D. Analysis of the phase relations (left-right and lumbosacral) revealed that the intensity of stimulation did not affect the phase relations of either the control or the DAMGO/naloxone-treated preparations, thereby enabling pooling of the phase data obtained at the two intensity ranges described in the preceding text. Subsequent analysis of the pooled data revealed that the left-right (φL-R) and lumbosacral (φLS) phase shift in the presence of DAMGO/naloxone were similar to those of the control (Watson and Williams test). Control: φL-R = 181.2 ± 12.8° rv = 0.976 and φLS = 357.1 ± 17° rv = 0.965; DAMGO/naloxone: φL-R = 182.2 ± 15.4° rv = 0.965 and φLS = 357.5 ± 17.5° rv = 0.954.
Another series of control experiments was done to examine whether naloxone itself affects the rhythmogenic capacity of the spinal cord. These experiments revealed that bath application of naloxone (6 experiments) failed to produce spontaneous rhythmic activity in a quiescent cord and that the SCA rhythm did not differ significantly in the presence or absence of naloxone (1-way ANOVA for Nc, Watson and Williams for φ) from the rhythm induced in its absence. Control: NC = 1 ± 0.11, φL-R = 179.1 ± 4.25° rv = 0.997; φLS = 1 ± 15.9° rv = 0.962; naloxone: Nc = 0.99 ± 0.21, φL-R = 179.5 ± 6.19°, rv = 0.994; fLS = 1 ± 11.8° rv = 0.979.
To determine whether the DAMGO block of the SCA-induced rhythm involved a complete suppression of synaptic transmission in the spinal cord, we stimulated the dorsal root of S2 at various intensities and recorded the resultant reflexes from the ipsilateral S2 ventral root, before and after bath-application of DAMGO and in the presence of both DAMGO and naloxone in the experimental chamber. Figure 3E shows that the early mono- and polysynaptic components of the reflex produced either by low- or high-intensity stimulation of the segmental homonymous dorsal root (S2), persisted with no major changes in the presence of DAMGO, whereas the late-polysynaptic component of the reflex (mediated by high-threshold afferents) was shortened in the presence of DAMGO and restored after addition naloxone to the bathing solution. Analysis of six experiments in this series revealed that the normalized amplitude of the early component of the reflex was 1 ± 0.01, 1 ± 0.04, and 1.04 ± 0.07 for the control, DAMGO, and DAMGO/naloxone, respectively. Analysis of the time constant of the decay of the late component (calculated using exponential curve fitting to the late component) showed significant shortening from 3.53 ± 0.53 to 1.34 ± 0.38 s after addition of DAMGO (1-way ANOVA followed by the modified Tukey's method, P < 0.001) and then prolongation to 2.91 ± 1.0 s after addition of naloxone (no different from the control time constant, but significantly longer than the time constant in DAMGO, P < 0.01). These results show that the monosynaptic component of the reflex and its early polysynaptic components persisted in the presence of DAMGO, whereas the late polysynaptic component was reduced (e.g., Sivilotti et al. 1995 for morphine effects on the late NMDA-dependent component of the reflex produced at similar stimulation intensities).
In the next set of seven experiments, we examined the effects of DAMGO on the intracellular potentials produced in motoneurons in response to SCA stimulation. We found that bath application of DAMGO blocked the tonic depolarization, the superimposed oscillation of trans-membrane potential and the associated rhythmic bursts evoked by SCA stimulation (Fig. 4). However, in the presence of DAMGO, each stimulus in the train evoked a polysynaptic depolarizing postsynaptic potential (Fig. 4A, DAMGO; B, DAMGO; IC, top trace and inset), indicating as mentioned in the preceding text, that not all of the polysynaptic afferent-evoked activity was blocked. Continuous injection of depolarizing current to a recorded motoneuron in the presence of DAMGO (Fig. 4B, DAMGO, IC, bottom trace and inset) revealed that the evoked depolarizations were biphasic PSPs composed of an initial small EPSP and a later much larger IPSP (Fig. 4B, DAMGO, IC, bottom record and inset). These potentials are similar to the SCA-evoked biphasic potentials recorded from sacral motoneurons in the presence of the sodium channel blocker QX-314 that we described in an earlier study (Lev-Tov et al. 2000). However, the early excitatory component of the PSPs described in the earlier work was more prominent than the initial component observed in the presence of DAMGO. Bath application of the nonspecific opioid receptor antagonist naloxone restored the tonic depolarization and rhythmic bursts produced by stimulation of SCA (Fig. 4A, bottom records). Similar results were observed in six additional intracellular experiments. We also found a tendency for the motoneuronal input resistance to increase in the presence of DAMGO (1.16 ± 0.21 with respect to control; n = 7), although it did not reach statistical significance (paired t-test).
Rhythmogenic capacity of the pattern-generating circuitry is not impaired in the presence of μ-opioid receptor agonists
The action of DAMGO in blocking the SCA-induced rhythm could be explained either by blockade of transmission along the pathway between SCA and the pattern-generating circuitry or by an action on the CPG itself. To clarify this issue, we blocked the SCA-induced rhythm by bath-applied DAMGO and then tried to activate the rhythmogenic networks either by bath application of NMDA/5HT or by stimulation of the ventromedial medulla. Figure 5A shows that when the SCA-induced control rhythm (control) was completely blocked by DAMGO (DAMGO), bath application of NMDA/5HT (3.5 and 10 μM, respectively) produced a robust rhythm in the lumbar and sacral segments (NMDA/5HT). In another series of experiments, we examined whether the parameters of the neurochemically induced rhythms were affected by the presence of DAMGO. The results of one of these experiments are shown in Fig. 5B, and the combined results from four experiments performed in this series are shown in C and D. The cycle time of the rhythm (Fig. 5, B and C, NMDA/5HT Nc = 1 ± 0.03) was significantly prolonged to 1.48 ± 0.18 (ANOVA followed by modified Tukey's method, P < 0.001) in the presence of DAMGO (Fig. 5, B and C, NMDA/5HT, DAMGO) and partially recovered (Nc = 1.18 ± 0.11, significantly shorter than Nc in DAMGO, P < 0.01, but longer than Nc in control, P < 0.05) after addition of naloxone to the preparations (Fig. 5, B and C, NMDA/5HT, DAMGO, naloxone). The left-right and lumbosacral phase relations (φLR and φLS) were not affected (Watson and Williams test) by the presence of DAMGO or DAMGO and naloxone, Fig. 5D and Table 1. Note that the intensity of rhythmic firing is enhanced in the presence of DAMGO, probably reflecting the increase in input resistance observed in some of the recorded motoneurons.
Control experiments (n = 5, not shown) revealed that naloxone alone had no effect on the NMDA/5HT-induced rhythms (Table 1). Thus although the neurochemically induced rhythm slowed during the DAMGO block of SCA-induced bursting, the rhythmogenic capacity of the lumbar and sacral networks remained intact.
We then investigated the segmental site of the DAMGO block of the SCA-induced rhythm and if this region was capable of rhythmogenesis in the presence of DAMGO. For this purpose, we stimulated the ventromedial medulla (VMM), which has been shown to produce locomotor-like (Zaporozhets et al. 2004) and sacrocaudal rhythmicity (Blivis and Lev-Tov 2005) in the absence of bath-applied drugs. In our experiments, the brain stem spinal cord preparation was placed in a dual compartment experimental bath, allowing us to compare the effects of VMM stimulation (black records, Fig. 6) and SCA stimulation (blue records, Fig. 6) on the rhythmic activity produced by different regions of the cord in the presence and absence of DAMGO. As illustrated in Fig. 6, the bath was separated into two compartments by creating a Vaseline barrier at either the lumbosacral junction (L6) or the spino-medullary junction. The results are shown in Fig. 6, A–C.
Application of DAMGO to the sacrocaudal segments (Fig. 6, A and C) blocked the SCA-induced rhythm recorded in the lumbar and sacral segments, whereas stimulation of the ventromedial medulla under these conditions produced robust rhythmic activity in both regions. The DAMGO block of the SCA-induced rhythm was alleviated shortly after addition of naloxone to the sacrocaudal segments. Analysis of the six experiments performed in this series revealed that the cycle time of the VMM-induced rhythm (Nc = 1 ± 0.07) was not affected when DAMGO was applied to the sacrocaudal compartment (Nc = 1.06 ± 0.13) or when naloxone (Nc = 1.08 ± 0.15) was added to the DAMGO-treated segments (Fig. 6C, left, black histograms). The phase between the activities of the left and right hemicords (φL-R) and between the ipsilateral lumbosacral segments (φLS) were also unaltered under these conditions (Watson and Williams test). Control: φL-R = 173.5 ± 12.7°, r = 0.97; φLS = 340 ± 12.9°, r = 0.975; DAMGO: fL-R = 167.7 ± 13.9°, r = 0.971; φLS = 347 ± 11°, r = 0.982; DAMGO, naloxone: fL-R = 178.45 ± 13.59° r = 0.972; φLS = 343.6 ± 9.2°, r = 0.945.
Figure 6B shows the effects of adding DAMGO to the rostral compartment (including the brain stem and the cervico-thoraco-lumbar segments of the spinal cord). Under these conditions (Fig. 6C), the VMM-induced rhythm was slowed significantly (1-way ANOVA followed by Tukey's method P < 0.05; the normalized cycle time (Nc) was 1.35 ± 0.33 compared with 1 ± 0.07 of control) and recovered (Nc = 1.14 ± 0.26) after addition of naloxone (DAMGO naloxone) to the rostral compartment (Fig. 6, B and C, middle). The mean phase shifts were not affected under these conditions. Control: φL-R = 187.1 ± 12°, r = 0.978; φLS = 348.4 ± 7.1°, r = 0.992; DAMGO: φL-R = 181.1 ± 9.2°, r = 0.987; φLS = 350.8 ± 9.7°, r = 0.986; DAMGO/naloxone: φL-R = 187.2 ± 10.6°, r = 0.93; φLS = 347.7 ± 13.5°, r = 0.973.
The effects of DAMGO on the SCA-induced rhythm under these conditions were more variable with a tendency to slow the rhythm that did not reach statistical significance (1-way ANOVA, Fig. 6C). The normalized cycle times (Nc) were 1 ± 0.13, 1.16 ± 0.37, and 1.23 ± 0.36 for control, DAMGO, and DAMGO/naloxone, respectively. The mean phase shifts under these conditions were also not affected by the opioid agonist and antagonists. Control: φL-R = 182.8 ± 7.5°, r = 0.991; φLS = 0 ± 13.5°, r = 0.973; DAMGO: φL-R = 181.7 ± 9.4°, r = 0.987; φLS = 11.3 ± 22.4°, r = 0.926; DAMGO/naloxone: φL-R = 181.5 ± 12.4°, r = 0.977; φLS = 5.1 ± 27.2°, r = 0.894.
Finally, we established that DAMGO, or a combination of DAMGO and naloxone, applied specifically to the brain stem (Vaseline barrier at the spino-medullary junction), had no effect on the rhythm induced by either SCA or VMM stimulation (histograms of the cycle times are shown in Fig. 6C, right). VMM stimulation: control, Nc = 1 ± 0.6; φL-R = 178.5 ± 5.8°, r = 0.995; φLS = 345.7 ± 19.4°, r = 0.944; DAMGO, Nc = 0.99 ± 0.12; φL-R = 180.1 ± 7.1°, r = 0.992; φLS = 350.4 ± 11.6°, r = 0.98; DAMGO/naloxone, Nc = 1.03 ± 0.18; φL-R = 180.7 ± 7.7°, r = 0.991; φLS = 351.7 ± 13.2°, r = 0.974. SCA stimulation: Control, Nc = 1 ± 0.05; φL-R = 178.8 ± 9.9°, r = 0.985; φLS = 345 ± 19.3°, r = 0.944; DAMGO, Nc = 1.06 ± 0.12; φL-R = 183.8 ± 6.4°, r = 0.994; φLS = 350.4 ± 11.6°, r = 0.98; DAMGO/naloxone, Nc = 1.1 ± 0.24; φL-R = 177.7 ± 8.7°, r = 0.988; φLS = 351.7 ± 13.2°, r = 0.974.
In summary, our results show that the DAMGO block of the SCA-induced rhythm occurred within the sacrocaudal segments of the spinal cord but that it did not compromise the rhythmogenic capacity of the thoracolumbar or sacrocaudal regions. Confirmation that the DAMGO-sensitive sacrocaudal interneurons are not required for lumbar rhythmogenesis was demonstrated by persistence of virtually unaltered VMM-induced rhythmic activity after their surgical removal (Fig. 6D). In three different experiments in this series, the normalized cycle time was 1 ± 0.08 and 0.96 ± 0.11 before and after the transection, respectively. The alternating left-right pattern was also not affected by the transection φL-R = 182.3 ± 4.32° r = 0.997 and 181.8 ± 3.4° r = 0.998, in the presence and absence of the SC segments, and the alternating flexor-extensor pattern persisted, with a minor but significant phase shift between the ipsilateral L2 and L5 from 203.8 ± 5.9° r = 0.995, to 186.2 ± 10.38° r = 0.984, probably due to the proximity of the recorded L5 to mid-L6 transaction plane.
Distribution of μ-opioid receptors in the gray matter of the sacrocaudal segments of the spinal cord
In the final set of experiments, we used immunocytochemistry for the μ-opioid receptor MOR-1 to establish the existence of DAMGO-sensitive interneurons in the sacrocaudal segments. These experiments were performed on both neonatal (P3) and adult rat spinal cords. The specificity of the MOR-1 immunoreactivity was demonstrated by the abolition of immunofluorescence by preincubation of the MOR-1 antibody with its antigenic peptide (see Figs. 7and 8) at both ages tested.
At P3, MOR-1 immunoreactivity was widely distributed throughout the cord with the strongest labeling in laminae I–II of the dorsal horn. MOR-1+ cellular profiles were observed throughout the transverse extent of the cord with a particularly dense concentration in the ventromedial area (laminae VII and VIII). Large-diameter cells in lamina IX, presumably motoneurons, were also labeled. The immunolabeling was observed in the somatic and dendritic cytoplasm but was excluded from the nucleus. In the adult, fewer cellular profiles were immunoreactive than in the neonate. As in the neonate, the brightest fluorescence was detected in laminae I–II. MOR1+ neurons were prominent in the ventral part of the cord and large immunoreactive cells were observed in the region of the motor nuclei. The interneurons in Rexed laminae VII and VIII are of particular interest since these are thought to be the origin of spinothalamic, spinocerebellar and/or spinoreticular tracts (Arsenio-Nunes and Sotelo 1985; Fields et al. 1975; Trevino et al. 1972; Yamada et al. 1991). Representative examples are shown in Figs. 7 and 8. Expression of the μ opioid receptors and/or some of its splice variants have been previously shown to be localized in the cytoplasm, dendrites, and axons of spinal cord neurons (Abbadie et al. 2000; Ding et al. 1996; Zhang et al. 2006). We also confirm that several types of neurons within lamina V, VI, VII, and IX express MOR-1 immunoreactivity.
The present work examined the interaction between sacrocaudal afferent pathways and the pattern-generating circuitry in the thoracolumbar and the sacrocaudal cord. We showed that locomotor and sacrocaudal rhythmicity could be produced by stimulation of SCA at different intensities and by using radiant heat and punctate stimulation of SC skin to specifically activate nociceptive SCA pathways. These SCA-induced rhythmic patterns were completely blocked by bath application of the μ-opioid receptor agonist DAMGO. Previous studies of spinal cats have revealed that the motor rhythm produced in the presence of L-DOPA and nialamide by stimulation of various FRA pathways and the spontaneous rhythmic patterns produced under these conditions were severely depressed or completely blocked after administration of the enkephalin DAMGO to the preparations (Schomburg and Steffens 1995). In contrast, we found that the rhythmogenic capacity of the thoracolumbar and sacrocaudal networks was not impaired in the presence of DAMGO because these networks could be readily activated by VMM stimulation or bath-applied neurochemicals despite the DAMGO block of the SCA-induced rhythm.
Can the DAMGO blocked interneurons be considered as constituents of the locomotor CPGs?
We have shown previously that the SCA induced locomotor-like rhythm is mediated by interneurons located in the sacrocaudal segments. This was shown by the complete block of the SCA induced locomotor like rhythm by application of a low-calcium solution to the sacral segments and its recovery in the presence of low calcium after focal ejection of calcium onto SC segments and by the block of the rhythm by application of the non-NMDA receptor antagonist CNQX to the sacrocaudal segments (Strauss and Lev-Tov 2003). The findings of the present work support this observation and suggest that the DAMGO block of the SCA rhythm is mediated by the effects of the drug on these interneurons. We consider unlikely that these interneurons are part of the locomotor CPG because locomotor-like activity generated by stimulation of the brain stem in lumbar segments is unaffected by removal of the sacral segments.
The most likely interpretation of our findings is that the DAMGO-blocked interneurons in the sacral cord are part of the pathway from sacrocaudal afferents to the locomotor network in the lumbar segments. Consistent with this idea, we found that the SCA-induced rhythm was blocked only when DAMGO was applied to the SC segments of the spinal cord. Moreover, the rhythm produced by SCA stimulation was not affected after addition of DAMGO to the rostral compartment of the experimental bath, which included the lumbar segments.
Opioid receptors, SCA-induced rhythmicity, and pattern generation
We now consider the site and mechanisms of the opioid block of the SCA-induced rhythm. The superficial layers of the dorsal horn and especially lamina II—the substantia gelatinosa—are thought to be major sites for relaying and modulating the nociceptive input (Kumazawa and Perl 1978). These regions are abundant with various types of opioid peptides (Miller and Seybold 1987; Pierce et al. 1998; Sar et al. 1978), binding sites and immunoreactivity for cloned μ, δ, and κ opioid receptors (Arvidsson et al. 1995; Atwe and Kuhar 1977; Besse et al. 1990; Dado et al. 1993; Mansour et al. 1995). Opioid analgesia is thought to be mediated by a combination of pre- and postsynaptic mechanisms (Yaksh 1997). Presynaptic effects include decreased release of transmitter from afferent terminals (Chang et al. 1989; Hori et al. 1992; Jessell and Iversen 1977; Kohno et al. 1999; Kondo et al. 2005; Suarez-Roca and Maixner 1992) due to (inferred from studies of dorsal root ganglia neurons) modulation of membrane conductance associated with G-protein-mediated activation of intracellular pathways either directly or via second messengers (Chen et al. 1988; Moises et al. 1994; Reisine et al. 1996; Wilding et al. 1995). Postsynaptic effects included substantial hyperpolarization of lamina II interneurons and an increase in membrane conductance mediated by inward rectifying potassium channels coupled to G proteins (Eckert and Light 2002; Grudt and Williams 1995; Schneider et al. 1998). Similar pre- and postsynaptic mechanism may account for the opioid block of the SCA-induced rhythm that is produced by nociceptive as well as by nonnociceptive input (e.g., Schmidt et al. 1991; Schomberg and Steffens 1995). The sites of the opioid block of the SCA-induced rhythm depend on the distribution of opioid receptors on constituents of the SCA pathways.
In the present work, we studied the expression of MOR1 in the gray matter of the sacrocaudal segments and confirmed that the superficial dorsal horn layers of the neonate and especially those of the adult showed the highest MOR1 expression. In the neonate, MOR1 immunoreactivity was widely expressed on neurons throughout the spinal cord including intermediate gray interneurons, neurons in the vicinity of the central canal, and interneurons and motoneurons in the ventral horn (see Kumazawa et al. 2003 for direct action of opioids on facial motoneurons). Are these interneurons associated with SCA pathways?
Relatively little is known about the pathways between SCA and the locomotor CPGs (Blivis and Lev-Tov 2005; Blivis et al. 2006; Strauss and Lev-Tov 2003). As mentioned in the preceding text, the projection/relay interneurons of these pathways must be located in the SC segments of the spinal cord (Strauss and Lev-Tov 2003). The axons of these interneurons are known to cross the SC cord and ascend rostrally to the thoracolumbar segments mainly through the ventral white matter funiculi (VF). This is because the locomotor rhythm was virtually abolished after a midsagittal section of the SC segments or after a VF lesion (Strauss and Lev-Tov 2003). Recently we have attempted to identify the location of these projection/relay neurons by in vitro retrograde fluorescent labeling studies of the cut VF at the caudal end of the lumbar cord. The labeled interneurons were located contralateral to the fill, mostly in the intermediate gray and around the central canal. Some labeled cells were found in the dorsal and ventral horns (Blivis et al. 2006). The laminar locations and the trajectories of these neurons are similar to those of projection neurons of the spinothalamic, spinoreticular, and spinocerebellar tracts (Arsenio-Nunes and Sotelo 1985; Fields et al. 1975; Trevino et al. 1972; Xu and Grant 1990, 2005; Yamada et al. 1991) or of ascending propriospinal pathways. As mentioned in the preceding text, our immunohistochemical studies revealed that interneurons with similar laminar locations to these neurons expressed MOR1, so that the DAMGO-induced block of the SCA rhythm may have occurred either at the level of these relay/projection neurons and/or at the level of the dorsal horn interneurons interposed between the afferents and their projection neurons. Further studies of the expression of MOR1 on groups of identified SC interneurons the axons of which project to the lumbar cord via the contralateral VF, and characterization of their physiological properties using optical recordings, are required to clarify these issues.
Our study has shown that opioid receptors are potent modulator of rhythmic patterns produced in the isolated brain stem spinal cord preparations of the neonatal rats by stimulation of sacrocaudal afferents and that the opioid effects on the SCA-induced rhythms are mediated interneurons interposed between the SCA and the pattern-generating circuitry. We also showed that μ-opioid receptor agonists moderately slow the rhythm produced by bath-applied neurochemicals or by VMM stimulation. This modulation may reflect some direct effects of opioids on the locomotor CPGs. Our study and previous studies of the spinal cat (Schomberg and Steffens 1995) have shown that the opioid effects occurred on both nociceptive and nonnociceptive pathways. Opioids have also been shown to exhibit strong modulatory effects on the respiratory pattern-generating circuitry (Gray et al. 1999; Mellen et al. 2003) and on various nonpain related systems and functions (reviewed by Mason 2005). A complex set of pathways originating at the periequaductal gray, converging onto the ventromedial medulla and descending to various regions of the dorsal horn have been reported to initiate endogenous opioid release or activate nonopioid pathways associated with modulation of pain (e.g., Mason 2005). Collectively, our findings raise the possibility that these descending pathways are used not only for pain modulation but also affect afferent-dependent motor activities and regulate the function of central pattern generators in the spinal cord.
This work was supported by Israel Science Foundation Grant 129/04 to A. Lev-Tov, by US-Israel Binational Science Foundation Grants 2001010 and 2005020 to A. Lev-Tov and M. J. O'Donovan, and National Institute of Neurological Disorders and Stroke intramural program funding to G. Z. Mentis and M. J. O'Donovan.
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