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Committee on Neurobiology and Department of Neurobiology, Pharmacology and Physiology, University of Chicago, Chicago, Illinois 60637
Submitted 12 April 2004; accepted in final form 1 June 2004
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ABSTRACT |
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INTRODUCTION |
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). Whereas the location and timing of noxious cutaneous stimulation is precisely detected, visceral pain is typically perceived diffusely in both time and space. Spatial discrimination of visceral pain is particularly imprecise as it is referred to a distant cutaneous or musculoskeletal site. Behavioral and autonomic reactions to visceral stimulation are abolished by spinal transection (Ness and Gebhart 1988b), whereas reactions to cutaneous stimuli typically persist and are often enhanced. In contrast to cutaneous nociceptors, visceral afferents, such as those that innervate the colon, respond in a graded fashion to both innocuous and noxious intensities of stimulation (Sengupta and Gebhart 1994; Su et al. 1997). Thus although a 20-mmHg distension of the colon and rectum is not aversive and a 80-mmHg distension is, many individual primary afferents respond to both stimulation intensities. In contrast, cutaneous afferents that respond to noxious stimuli, nociceptors, do not respond to innocuous stimuli under nonpathological conditions. Correspondingly, it is of interest that humans report uncomfortable feelings after visceral, but not cutaneous, stimulation at intensities below the pain threshold (Strigo et al. 2002).
Colorectal distension (CRD) evokes cardiovascular (pressor response and tachycardia) and somatomotor ("hunching") reactions that increase in magnitude with increasing stimulus pressure (Ness and Gebhart 1988b). Cold block at the first cervical segment eliminates the tachycardia and hunching reactions evoked by CRD and greatly reduces the evoked pressor reaction (Ness and Gebhart 1988b). Thus the lumbosacral cord alone cannot support the behavioral reactions to CRD. Because CRD-evoked reactions are unaffected by decerebration, they must depend on a loop from lumbosacral spinal cord to brain stem and/or high spinal cord and back to lumbosacral spinal cord. Both the ascending and descending limbs of this loop appear to travel in the lateral funiculi (Ness 2000).
Neurons in the medullary raphe magnus (RM) and the adjacent nucleus reticularis magnocellularis (NRMC) project to the dorsal horn, intermediate gray, and central canal region (Basbaum and Fields 1978, 1979; Holstege and Kuypers 1982) and have well-described, bidirectional modulatory effects on cutaneous nociceptive input to dorsal horn cells (Fields et al. 1991; Mason 2001; Sandkuhler 1996). Two types of nonserotonergic neurons are hypothesized to underlie the nociceptive inhibitory and facilitatory effects of RM activation (reviewed in Mason 2001). ON cells are excited by noxious cutaneous stimuli and inhibited by opiates, whereas OFF cells are inhibited by noxious cutaneous stimuli and excited by opiates (Barbaro et al. 1986). ON and OFF cell discharge have been implicated in the enhancement and suppression, respectively, of cutaneous nociceptive sensitivity, particularly that which is opioid receptor-mediated (Barbaro et al. 1986; Cheng et al. 1986; Fields et al. 1983b; Heinricher and Drasner 1991; Heinricher et al. 1992, 1994). A third type of nonserotonergic RM/NRMC neuron, the NEUTRAL cell, is defined by its lack of a response to noxious tail heat and is reportedly unaffected by opioids. However, NEUTRAL cells respond to other noxious cutaneous and visceral stimuli, including CRD, leaving open the possibility that these cells play a role in nociceptive modulation (Brink and Mason 2003; Ellrich et al. 2000; Leung and Mason 1998).
RM and NRMC neurons also modulate sensory input from the viscera. Activation of RM and NRMC can either suppress or facilitate the responses of spinal neurons to noxious visceral input, including CRD (Zhuo et al. 2002). Further, CRD-evoked hunching is modulated bidirectionally by RM and NRMC activation (Zhuo and Gebhart 2002). If RM and NRMC cells contribute to a spinal-brain stem-spinal loop critical to generating the reaction to CRD, then they should respond monotonically to increasing intensities of CRD that elicit behavioral reactions. Therefore in the present study, the responses of RM and NRMC cells to multiple CRD stimulation intensities were compared with the simultaneously evoked behavioral reactions.
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METHODS |
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Male Sprague-Dawley rats (n = 56, 300–525 g; Charles River, Portage, MI) were used for all experiments. Rats were divided into two cohorts that differed primarily in the level of anesthesia used. Animals studied at 1% halothane constituted the Hal-hi cohort (n = 15), and those studied at 0.8% were the Hal-lo cohort (n = 41). Additional minor differences in the experimental protocols for the two cohorts are noted in the following text.
Rats were anesthetized with halothane that was maintained at 2% throughout surgery and administered through a Y-tube inserted into the trachea (Hal-hi) or through a nose cone (Hal-lo). The Hal-hi cohort of rats was pretreated with atropine sulfate (40 µg in 0.1 ml sc), whereas animals in the Hal-lo cohort were not. A catheter was inserted into the femoral artery to record blood pressure. Needle electrodes were placed into the thorax bilaterally to record the electrocardiogram, into the biceps femoris to record the electromyographic (EMG) activity of the hindlimb muscles, and into the superficial abdominal musculature to record the hunching evoked by CRD. A craniotomy was made over the cerebellum, and the exposed dura was cut. Core temperature was maintained at 36–38°C via a water-perfused heating pad. After the surgery, the halothane concentration was lowered to 1% (Hal-hi) or to 0.8% (Hal-lo), and the animal was allowed to equilibrate for 60 min.
Electrophysiological methods
Tungsten metal electrodes (A-M Systems, Pullman, WA) were used for all experiments. Microelectrodes were lowered into the region of the RM and NRMC (P 9.9–11.9 mm from bregma, L 0.0–1.0 mm, V 8.5–10.5 mm from cerebellar surface). The unit waveform was acquired at 40 kHz by a CED Micro1401 interface (CED, Cambridge, UK). Spike2 acquisition software (CED) stored the time of the spike and 30 digitized points from the waveform. Individual waveforms were discriminated off-line using template-matching software.
Neurons were isolated by their spontaneous activity. In the Hal-lo cohort, all extracellular units that were successfully isolated, even ones with no obvious response to CRD, were studied. In the Hal-hi cohort, cells that responded robustly to 80 mmHg CRD were arbitrarily chosen for further testing of their responses to lower CRD intensities. Responses of these cells to 80 mmHg CRD distension have been previously reported (Brink and Mason 2003).
Stimulation methods
Cells were tested for their responses to noxious paw heat and multiple intensities of CRD. Heat stimuli were applied using a peltier device (Yale Instrumentation, New Haven, CT) placed on the footpad and toes of the hindpaw. The paw was affixed to the peltier platform (2-cm square) so that it was exposed to the full duration of the stimulus. Each heat stimulus consisted of a 2- to 3-s ramp from 32 to 46–58°C with a 4-s plateau at the peak temperature. The peltier platform then ramped back down to 32°C over the course of 3–7 s. Between thermal stimuli, the peltier platform was maintained at 32°C.
To inflate the colon, the finger portion of a glove was secured onto flexible tubing (Tygon R3603, 2 mm OD). This tubing was connected to a 60-mm syringe with a side port connected to a manometer. The glove finger was coated with petroleum jelly and inserted 7–9 cm into the anus to a point just past the external sphincter muscle. The stimulus consisted of a 20-s inflation to one of four intensities of CRD, three noxious (40, 60, 80 mmHg) and one nonnoxious (20 mmHg). The stimulus had rapid on and off rates (<1 s) and was held at 0 mmHg between stimuli.
Experimental protocol
Because we have previously demonstrated that serotonergic neurons respond weakly, at most, to CRD, we did not study serotonergic cells. Therefore cells that discharged very slowly and regularly—cells that are very likely to be serotonergic—were not recorded. All other cells were tested with repeated (3–5) trials of noxious paw heat that were interleaved with CRD trials. All stimuli were separated by intervals of 5 min. Cells recorded from the Hal-hi cohort (n = 19) were tested with a heat intensity of 56°C, but for cells recorded from Hal-lo rats (n = 75), the peak temperature was chosen to be the minimum that reliably elicited a robust motor withdrawal, typically 49 or 52°C. Initial CRD stimuli were applied at an intensity of 60 mmHg. If cells did not respond to the 60 mmHg CRD, the maximal intensity of 80 mmHg was applied. Cells that responded to 80 or 60 mmHg were then tested for their response to 40 and 20 mmHg distensions. For each stimulus, three to five trials were recorded.
After isolation of a unit, noxious paw stimulation was alternated with 40, 60, and 80 mmHg CRD stimulation until three trials of each were obtained. Then three trials of 20 mmHg CRD were collected either in alternation with the preceding stimuli or with trials that paired CRD and heat stimulation. Data from these latter paired trials are not included in the present study. The recording site for at least one cell per animal was labeled by injection of 20 µA hyperpolarizing current for 4 min.
Histology
Animals were overdosed with 5% halothane and perfused with a fixative containing 4% paraformaldehyde and 7% sucrose in 0.1 M phosphate-buffered saline. The brain stem was removed, postfixed for 2–12 h, and then immersed in 30% sucrose in 0.1 M PBS. Coronal sections (50 µm) were cut on a freezing microtome. Sections were mounted on gelatin-coated slides and then stained with cresyl violet. Lesion sites were identified and recovered. Under x50 magnification, the lateral and dorsal distances from midline and the ventral midline edge of the section, respectively, were measured. Sites were assigned an anterior-posterior location by comparison of sections with a standard atlas (Paxinos and Watson 1986). Unlabeled sites were located by their stereotaxic distance from marked recording sites.
The nuclear boundaries used are modified from Newman (1985). We considered RM to include a region 300 µm wide centered on the midline and extending from the base of the brain to a point 1,500 µm dorsal at levels from –11.6 to –10.4 relative to bregma. A box representing this area is illustrated at the top of Fig. 10. A small region between the pyramids (150 µm dorsal, 50 µm lateral on each side) with densely packed, small cells was considered to be raphe pallidus. NRMC was considered to include a region that stretched laterally from RM to the lateral edge of the pyramids and had a dorsal extent of 1,000 µm. Cells dorsal to RM or NRMC were considered to be located in nucleus reticularis gigantocellularis (NRGC).
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Although cells with a slow and regular pattern of discharge that are highly likely to be serotonergic (Li and Bayliss 1998; Mason 1997; Wang et al. 2001) were not studied, an algorithm that physiologically identifies serotonergic and nonserotonergic cells was employed to ensure that all studied cells were indeed nonserotonergic. Thus for each cell, the mean (x) and SD (SDISI) of the interspike interval (in milliseconds) were calculated from a 15-min record of the cell's activity. The function, y (x, SDISI) = 146 –x + 0.98 SDISI, was then used to physiologically classify cells as serotonergic or nonserotonergic on the basis of the rate and variability of the background cellular discharge (Mason 1997). Cells with a function value <0 were classified as serotonergic, and cells with a function value >0 were classified as nonserotonergic. None of the cells studied had a function value of <45. A previously described quantitative method was used to classify all cells as ON, OFF, or NEUTRAL (Leung and Mason 1998).
Analysis of physiological responses
Systolic blood pressure was calculated from the blood pressure recording and instantaneous heart rate was calculated from the inverse of the interval between QRS waves in the EKG. Customized software then converted blood pressure and heart rate from x,y pairs to arrays (10 values/s) using linear interpolation. The blood pressure reaction to either CRD or paw heat was calculated as the peak blood pressure value after stimulus onset (0–20 s) less the mean blood pressure for the 10 s prior to stimulus onset. Similarly, the heart rate reaction to either CRD or paw heat was the peak heart rate value after stimulus onset less the mean heart rate before the stimulus onset.
The hunching evoked by CRD was quantified as the total rectified and integrated abdominal EMG recorded during the stimulus less the total EMG for a corresponding period prior to the stimulus. Motor reactions were normalized to the average reaction evoked by 60 mmHg.
Two types of linear regressions were performed. First, to determine how behavioral and cellular reactions changed with increasing intensities of CRD, each physiological reaction (EMG, tachycardia, and pressor) and the response of each cell type was regressed on intensity. Both the behavioral and cellular reactions were normalized to their response to 60 mmHg CRD (chosen because all cells were tested for their response to 60 mmHg CRD). To make the slope of the resulting regression easier to interpret, CRD stimulation intensity was normalized to 60 mmHg. Thus in the case of a zero intercept, the slope of a perfectly correlated response ("unity slope") would be 1 (rather than 1/60 or 0.0167 if intensity were not normalized). The second regression was done to determine if behavioral reactions could be predicted by the responses of each group of cells described. For these regressions, each behavioral reaction (EMG, tachycardia, and pressor) was regressed on the response of each cell type. Both the behavioral reactions and the cell responses were normalized to their values at 60 mmHg, again allowing for a slope of 1.0 for a perfect correlation. Significant (P < 0.05) slopes and intercepts are reported.
Statistics
Each variable is expressed as a mean ± SE. Statistical tests were performed using Microsoft Excel (Redmond, WA) or SigmaStat (SPSS Science, Chicago, IL).
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RESULTS |
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Baseline values for both heart rate (309.9 ± 4.2 vs. 332.4 ± 1.7 bpm) and blood pressure (116.3 ± 2.3 vs. 132.2 ± 0.5 mmHg) were significantly lower in Hal-hi animals than in Hal-lo animals (t-test, P < 0.001). Whereas baseline blood pressure remained steady throughout the experimental paradigm, baseline heart rate increased after a minimum of 10 CRD stimulations. Because the lowest intensity of CRD stimulation was presented only after presentation of three to five trials of each of the other three intensities, resting heart rate was significantly greater prior to 20 mmHg CRD trials than before the remaining CRD trials (Fig. 1A).
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CRD evoked a somatomotor reaction of the abdominal musculature that has been termed "hunching" (Fig. 1) (Ness and Gebhart 1988b). This somatomotor reaction was only consistently observed in animals maintained at 0.8% halothane. As detailed in METHODS, the abdominal motor reaction to CRD was normalized to each individual rat's average reaction to a 60-mmHg distension. The somatomotor reaction to CRD reached maximal activation at a nearly immediate onset at intensities of 60 and 80 mmHg but was often delayed at lower intensities (Fig. 1A). In addition to having a shorter latency in response to higher stimulation intensities, the motor reaction increased in magnitude with increasing stimulation intensity. The normalized magnitude of the hunching reaction was 1.17 ± 0.14 in response to an 80-mmHg CRD and only 0.18 ± 0.03 to a 20-mmHg CRD. A linear regression of normalized motor reaction across the four CRD intensities tested was significant and revealed the predicted unity slope (P < 0.001).
Characterization of recorded cells: resting discharge and response to noxious heat
A total of 94 cells was recorded from Hal-hi (n = 19) and Hal-lo (n = 75) rats. Cells that were excited, inhibited or unaffected by paw heat were classified as ON (n = 39; 41%), OFF (n = 23; 24%), or NEUTRAL (n = 28; 30%) cells, respectively. Four cells (4%) did not consistently respond to noxious heat or did not discharge at rates of >1 Hz before heat stimulation. These cells were not classified as it was unclear whether they were unaffected or inhibited by heat. The background discharge rate averaged 9.7 ± 1.0 spikes/s, and the mean coefficient of variation of the interspike interval (CV) was 2.93 ± 0.33. These values did not differ across ON, OFF, and NEUTRAL cell classes (ANOVA, P = 0.66). Although the majority of these cells (62/94) discharged in a bursting pattern (CV >1), a minority (n = 19; 20%) had a regular discharge pattern (CV <0.5). Regularly discharging neurons were distributed across each of the cell classes (8 ON cells, 6 OFF cells, 5 NEUTRAL cells) at the same proportion as nonregularly discharging neurons (
2, P = 0.77).
Response patterns to graded intensities of CRD
Of 94 RM and NRMC cells studied, most (n = 84) responded to graded intensities of CRD with one of six basic response patterns. A majority of RM and NRMC cells (n = 66) either increased (n = 40) or decreased (n = 26) their discharge to multiple intensities of CRD tested. Such cells could be further divided into a graded response pattern (n = 35), where the response magnitude increased with increasing stimulation intensity, or a flat response pattern (n = 31), where the response magnitude was not different across supra-threshold stimulation intensities. Cells with a graded response pattern were termed graded positive (n = 20; Gr-pos; Fig. 2) if their discharge increased with increasing stimulation intensity and graded negative (n = 15; Gr-neg; Fig. 3) if their discharge decreased with increasing stimulation intensity. Similarly, cells with a flat response pattern were classified as either flat positive (n = 20; Fl-pos; Fig. 4) or flat negative (n = 11; Fl-neg; Fig. 5) depending on whether CRD evoked an increase or decrease in discharge. Finally, 18 cells had opposing responses to different intensities of CRD (Fig. 6), either excited by low stimulation intensities and inhibited by high stimulation intensities (switch negative; n = 9; Sw-neg) or vice versa (switch positive; n = 9; Sw-pos).
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Most Fl-pos (n = 20) and Fl-neg (n = 11) cells responded similarly to CRD intensities >20 mmHg CRD but did not respond to 20 mmHg CRD (Figs. 4 and 5). Figures 4A and 5A show the mean normalized responses to each intensity of CRD stimulation for Fl-pos and -neg cell populations, respectively. Fl-pos responses to 20 mmHg ranged from no response to 210.3 spikes and those to 80 mmHg ranged from no response to 239.0 spikes. Fl-neg responses to 20 mmHg ranged from –8.7 to –146.3 spikes and those to 80 mmHg ranged from no response to –454.3 spikes. Thresholds for these neurons could not be determined due to their nonlinear coding properties. Linear regression analysis revealed no significant slope for either Fl-pos or -neg cell responses (Figs. 4C and 5C).
For nine Sw-pos and nine Sw-neg cells, lower and higher intensities of CRD evoked opposing responses. Figure 6 shows mean responses for an individual Sw-pos cell (Fig. 6A) and one Sw-neg cell (Fig. 6B) to each intensity of CRD. A crossing point was calculated from a linear interpolation between the smallest inhibitory and excitatory responses. The switch between inhibitory and excitatory responses occurred between 20 and 40 mmHg (n = 6), 40 and 60 mmHg (n = 7), or 60 and 80 mmHg (n = 4) stimuli. One cell was inhibited by 40 and 80 mmHg CRD and excited by 60 mmHg. The crossing points for Sw-pos and -neg cells were not different (t-test, P = 0.45) and averaged 48.6 ± 4.4 mmHg. A linear regression analysis revealed that the normalized responses of both Sw-pos (P < 0.001, not shown) and Sw-neg cells (P = 0.03, not shown) increased significantly as CRD intensity increased. The intercept of the Sw-pos cell regression was significant (P = 0.009) and corresponded to a crossing point of 35.9 mmHg, similar to that calculated above. The intercept of the Sw-neg cell regression was a trend (P = 0.09) and corresponded to a crossing point of 44.1 mmHg.
Cells with their maximal response to lowest intensity of CRD tested
A small group of six cells showed its largest change in discharge at the lowest intensity of CRD applied to that cell (3 at 40 mmHg and 3 at 20 mmHg). Of these six cells, four were classified as ON to heat and as Fl-pos with regard to CRD (Fig. 7). The final cells in this group were also ON cells but one was classified as Sw-neg and one had no response to CRD.
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Some neurons (n = 25) responded after the offset of the CRD stimulus differently from how they responded during the stimulus (Figs. 7 and 8). Most of these cells had either an excitatory (n = 9; Fig. 7 and 8B) or an inhibitory response (n = 6; Fig. 8A) that followed no change in discharge recorded during the stimulus. These responses occurred after most stimulation intensities tested (23/34: 68%) but were more prevalent and greater in magnitude at higher intensities of CRD. Ten of the 25 neurons had opposing responses during and after CRD stimulus application with 7 inhibited during the stimulus and excited afterward (Fig. 8B) and 3 excited during the stimulus and inhibited afterward (not shown). In these cells, the poststimulus response occurred after 75% of the stimulation intensities tested and was nearly as likely to occur after an opposing change in discharge (43%) as after no change in discharge (57%) recorded during the stimulus.
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There was an association between the response pattern to CRD and the cell type—ON, OFF, or NEUTRAL (2, P = 0.03). Because cell type is based on a cell's response to noxious heat, this association was essentially between the response to heat and the pattern of neuronal responses to multiple CRD intensities. Two associations were apparent. First, the proportion of NEUTRAL cells with Gr-pos responses (11/18, 61%) was twice that of the entire sample (25/80, 31% of all cells that could be classified as ON, OFF, or NEUTRAL and responded to CRD). Second, flat responding cells were more likely to respond to heat and CRD in the same direction (18/30, 60%) than in opposite directions. Only 38% (30/80) of the entire population of CRD-responsive and heat-classified cells responded to heat and noxious CRD in the same direction.
In a number of instances, cell responses to two (n = 34), three (n = 3), or four (n = 2) intensities of noxious heat were tested. Because most cells were only tested with two intensities of heat, classification of these cells' response pattern is somewhat speculative. Noxious heat evoked a graded type of response pattern in 18 cells and a flat type of response pattern in 14 cells. Although a cell excited by a lower heat intensity and inhibited by a higher intensity of heat (the Sw-neg pattern) was never observed, one cell was excited by 55°C heat and inhibited by 52°C heat. This distribution of response patterns evoked by noxious heat was not significantly different from that of responses to CRD (2, P = 0.24). However, of the 29 cells that had graded, flat, or switch-type responses to both heat and CRD, most (16/29) had the same response pattern, regardless of direction, to both CRD and noxious heat (2 = 0.03).
Regressions of behavioral reactions on cellular responses
As an intuitive, albeit indirect, test of the underlying hypothesis, we used a linear regression analysis to test whether the magnitude of CRD-evoked reactions could be predicted from the responses of each cell type. If a group of neurons contributes to a behavioral reaction, a significant correlation between the cell class response and the behavioral reaction would be expected. Therefore cell responses and behavioral reactions were normalized to the mean response to a 60-mmHg distension and linear regressions were calculated. Graded-type cell responses were highly predictive of each behavioral reaction with r2 values ranging from 0.30 to 0.61. The responses of flat type cells were least predictive with the greatest r2 value being 0.09 (see right of Fig. 9A). Switch-type cells were intermediate in predictive value with r2 values ranging from 0.14 to 0.42.
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Location of recorded cells
The recording sites were located within RM (n = 75), NRMC (n = 12), raphe pallidus (n = 1), and the dorsally located NRGC (n = 5; Fig. 10). One recording site could not be found. Neurons were recorded throughout the rostrocaudal extent of RM and NRMC, with the largest number being concentrated at the level of the facial nucleus (Fig. 10).
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DISCUSSION |
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All six of the cell classes described here could contribute to the behavioral reactions evoked by CRD either directly, as in the case of positively responding cells, or indirectly via a sign-inverting interneuron in the case of negatively responding cells. Graded-responding cells are most clearly implicated in this role and linear regression analyses revealed that their activity had the greatest predictive value for CRD-evoked reactions. Flat-responding cells may contribute a nonlinear gain component to the reactions, strongly augmenting reactions to stimuli above a threshold. Because most cells with flat type responses responded to distensions of 
40 mmHg but not to those of 20 mmHg, these cells may specifically modulate pain reactions.
Cells that respond in opposing directions to stimuli of low and high intensity have not been previously described in the medullary raphe but have been observed in neurons of the dorsal motor nucleus of the vagus (Zhang et al. 2002). When two stimulation intensities—one "physiological" and one noxious—were tested, parasympathetic motor neurons responded in opposing directions to low- and high-intensity gastric distensions. The authors speculate that such a response pattern allows single neurons to play opposing roles in gastric relaxation and vomiting—reflexes evoked by physiological and noxious distensions, respectively. A similar interpretation is unlikely to apply to the switch type cells recorded in the present study because noxious intensities evoked opposing responses in 12 of 18 of these cells. Instead it is possible that RM and NRMC cells with switch type response patterns modulate innocuous and mild or moderate noxious input differently from how they modulate moderate or extreme noxious input.
A small number of RM and NRMC cells responded most strongly to the lowest CRD intensity tested. While it is possible that such cells positively modulate defecatory function, this is unlikely as half of these cells had their greatest response to 40 mmHg CRD, a stimulus that is well above the physiological range for eliciting defecation (Gebhart and Sengupta 1996; Ness and Gebhart 1988b). These cells are also unlikely to negatively modulate defecation because increasing CRD intensities that are progressively more effective in suppressing defecation evoke smaller responses. A remaining possibility is that cells with their strongest response to lower CRD distensions combine with the other types of cells described in the preceding text to "code" for intensity within the noxious range.
Response to 20 mmHg CRD
In the anesthetized rat, RM neurons respond almost exclusively to noxious stimulation and rarely respond to innocuous cutaneous stimulation (Fields et al. 1983a; Leung and Mason 1998). Therefore the response of RM neurons to 20 mmHg CRD, a nonnoxious and nonaversive stimulus in the awake rat, observed in the present study is surprising. It is possible that while RM neurons respond only to noxious cutaneous stimulation, they respond indiscriminately to noxious and innocuous visceral stimulation. Unlike cutaneous afferents, most visceral afferents, including those innervating the colon, respond to both innocuous and noxious stimulation intensities (Sengupta and Gebhart 1994; Su and Gebhart 1998). Within the spinal cord, 20 mmHg CRD elicits c-fos activation in a set of dorsal horn neurons that are in the same location as c-fos positive cells observed after 80 mmHg CRD (Traub et al. 1992).
Spinal input to CRD-responsive cells in RVM
Although RM and NRMC receive little direct input from the spinal cord, CRD related input, like all somatosensory input, to RM and NRMC must ultimately arise from dorsal horn cells. Lumbosacral (L6–S1) and thoracolumbar (T13–L2) dorsal horn cells respond to graded CRD stimulation with a graded increase or decrease in discharge (Ness and Gebhart 1987 1988a, 1989; Qin et al. 1999). Thus these cells resemble the Gr-pos and -neg cells observed in RM and NRMC in the present study. The mean threshold stimulus intensities needed to activate Gr-pos (7 mmHg) and Gr-neg cells (13 mmHg) are intermediate between the mean thresholds of spinal "short-latency abrupt" (3 mmHg) and "short-latency sustained" (17 mmHg) (Ness and Gebhart 1987). Because the form of RM and NRMC cell responses to CRD could also derive from the combined input from these two spinal cell classes (see following text) (see also Brink and Mason 2003), it is likely that short-latency abrupt and sustained spinal cells are the major source of input to RM/NRMC Gr-pos and -neg cells.
Dorsal horn cells with characteristics resembling those of the switch and flat type responding cells have not been reported. It is interesting that spinal cells that are inhibited by CRD have a threshold between 35 and 40 mmHg (Ness and Gebhart 1987) and could therefore contribute directly to the Fl- and Sw-neg cell classes. With an intervening sign-inverting interneuron, these CRD-inhibited spinal cells could also contribute to the response profile of Fl- and Sw-pos cells.
We have observed RM cells with biphasic firing responses to CRD (excited during the stimulus and inhibited afterwards or vice versa, see Fig. 8B). Similar biphasic responses have also been reported for spinal neurons that respond to visceral stimulation (Chandler et al. 2002; Qin et al. 1999, 2004). In contrast, noxious cutaneous stimuli rarely elicit biphasic responses in either RM (Brink and Mason 2003) or the spinal dorsal horn. Responses that continue after stimulus offset are most parsimoniously the result of input from spinal neurons that respond to CRD with sustained responses. For instance, a RM cell that is inhibited during the stimulus and excited afterward could receive input from spinal cells with inhibitory and short-latency sustained excitatory responses. Alternatively, it is possible that the biphasic responses to visceral stimulation reflect a difference in the afferent input before and after stimulus application. In other words, after the stimulus offset, the viscera may be in a dynamic state of relaxation that differs qualitatively from the resting state. It is also of interest to note that biphasic spinal responses to visceral stimulation are only observed in spinal neurons recorded at the segmental level of the visceral input. For instance, a minority of cervical neurons respond biphasically to cervical, but not thoracic, esophageal distension (Qin et al. 2004). Similarly, CRD only evokes biphasic responses in lumbosacral spinal neurons. Thus RM neurons with biphasic responses to CRD must ultimately receive information from CRD-responsive neurons in the lumbosacral cord.
Functional implications
As mentioned in the INTRODUCTION, hunching and cardiovascular reactions to CRD are greatly attenuated or blocked by spinalization but unaffected by decerebration (Gebhart and Sengupta 1996; Ness and Gebhart 1988b). These results suggest that the brain stem and/or upper cervical cord are a critical component of a lumbosacral-bulbo/cervical-lumbosacral pathway necessary for behavioral reactions to CRD. A brain stem or cervical site that could participate in such a pathway must meet two requirements: neurons must respond to CRD and activation of the site must be able to facilitate CRD-evoked behavioral reactions.
The lateral reticular nucleus (LRN) meets the first but not the second of these requirements. LRN neurons respond to noxious CRD (Ness et al. 1998). However, all LRN responses to CRD are excitatory, suggesting that LRN's modulatory influence is unidirectional. Indeed, LRN activation inhibits but does not facilitate nociceptive transmission (Janss and Gebhart 1988). Thus while a minority of LRN neurons project to the spinal cord (Liu et al. 1989) and have been implicated in nociceptive modulation (Janss and Gebhart 1988), it is unlikely that CRD responsive neurons in LRN are critical to forming CRD-evoked behavioral reactions.
RM and NRMC neurons are excellent candidates for the required intermediaries in a lumbosacral-bulbo/cervical-lumbosacral pathway that generates the behavioral reactions to CRD. Cells in RM and NRMC respond to CRD at intensities that evoke cardiovascular and visceromotor reactions. As shown in the preceding text, the responses of many RM and NRMC cells are highly correlated with the behavioral reactions evoked by CRD (see Fig. 9). Furthermore, the behavioral and cellular responses evoked by 80 mmHg CRD stimulation are either attenuated or facilitated by RM stimulation depending on the site and intensity of stimulation, suggesting that RM is the source of both pro- and anti-nociceptive modulation (Zhuo and Gebhart 2002; Zhuo et al. 2002). There is corresponding evidence that spinal neurons can be either facilitated or inhibited by the brain stem. Specifically, visceroresponsive dorsal horn neurons that are disinhibited by a high cervical cold block are inhibited by RM stimulation, whereas spinal cells that are disfacilitated by cold block are excited by RM stimulation (Holt et al. 1991; Knuepfer and Holt 1991). Thus RM is likely one of the brain stem sites, perhaps the primary site, which provides descending facilitatory (as well as inhibitory) modulation to CRD-responsive circuits in the spinal cord. A similar pro-nociceptive role RM and NRMC has been demonstrated in the generation of enhanced reactions to noxious cutaneous input observed during conditions of persistent (inflammatory) pain (Porreca et al. 2001, 2002).
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GRANTS |
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FOOTNOTES |
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Address for reprint requests and other correspondence: P. Mason, Dept. of Neurobiology, Pharmacology, and Physiology, University of Chicago, MC 0926, 947 E. 58th St., Chicago, IL 60637 (E-mail: p-mason{at}uchicago.edu).
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INTRODUCTION
) and cells with negative responses are represented by downward triangles (
), while cells that were unaffected (right) are represented with stars (
). The diagram (top) represents a line drawing of a section at –11.0 from bregma; · · · , the area that is enlarged in each of the photomicrographs. The labeled boxes show the regions considered to contain RM and NRMC cells. In the interest of clarity, all cell locations are shown on the right side of the brain, in some cases at a site exactly contralateral to their true location. In a few cases, the location of cells has been adjusted by <100 µm to see individual symbols. The numbers indicate the distance from bregma for each section within a row. VII, facial nucleus; NTB, nucleus of the trapezoid body; p, pyramid; RP, raphe pallidus.
) and Hal-hi (
) animals. The hunching reaction for each animal was normalized (to its reaction to 60 mmHg) and then averaged for Hal-lo animals (right).
