Chemical Activation of Cervical Cell Bodies: Effects on Responses to Colorectal Distension in Lumbosacral Spinal Cord of Rats

Chao Qin,1 Margaret J. Chandler,1 Kenneth E. Miller,2 and Robert D. Foreman1

 1Department of Physiology and  2Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73190


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Qin, Chao, Margaret J. Chandler, Kenneth E. Miller, and Robert D. Foreman. Chemical Activation of Cervical Cell Bodies: Effects on Responses to Colorectal Distension in Lumbosacral Spinal Cord of Rats. J. Neurophysiol. 82: 3423-3433, 1999. We have shown that stimulation of cardiopulmonary sympathetic afferent fibers activates relays in upper cervical segments to suppress activity of lumbosacral spinal cells. The purpose of this study was to determine if chemical excitation (glutamate) of upper cervical cell bodies changes the spontaneous activity and evoked responses of lumbosacral spinal cells to colorectal distension (CRD). Extracellular potentials were recorded in pentobarbital-anesthetized male rats. CRD (80 mmHg) was produced by inflating a balloon inserted in the descending colon and rectum. A total of 135 cells in the lumbosacral segments (L6-S2) were activated by CRD. Seventy-five percent (95/126) of tested cells received convergent somatic input from the scrotum, perianal region, hindlimb, and tail; 99/135 (73%) cells were excited or excited/inhibited by CRD; and 36 (27%) cells were inhibited or inhibited/excited by CRD. A glutamate (1 M) pledget placed on the surface of C1-C2 segments decreased spontaneous activity and excitatory CRD responses of 33/56 cells and increased spontaneous activity of 13/19 cells inhibited by CRD. Glutamate applied to C6-C7 segments decreased activity of 10/18 cells excited by CRD, and 9 of these also were inhibited by glutamate at C1-C2 segments. Glutamate at C6-C7 increased activity of 4/6 cells inhibited by CRD and excited by glutamate at C1-C2 segments. After transection at rostral C1 segment, glutamate at C1-C2 still reduced excitatory responses of 7/10 cells. Further, inhibitory effects of C6-C7 glutamate on excitatory responses to CRD still occurred after rostral C1 transection but were abolished after a rostral C6 transection in 4/4 cells. These data showed that C1-C2 cells activated with glutamate primarily produced inhibition of evoked responses to visceral stimulation of lumbosacral spinal cells. Inhibition resulting from activation of cells in C6-C7 segments required connections in the upper cervical segments. These results provide evidence that upper cervical cells integrate information that modulates activity of distant spinal neurons responding to visceral input.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The neurophysiological basis of visceral sensation is still not understood completely although significant advances regarding segmental processing of visceral afferent inputs to the spinal cord have been made. Previous studies show that cells are most likely excited in segments where the visceral afferent fibers of a visceral organ enter the spinal cord (Hobbs et al. 1992a). Also inputs from organs in close proximity converge on sensory neurons in the spinal cord. For example, nociceptive visceral information from the heart and gall bladder can excite the same spinothalamic tract cells (Ammons et al. 1984). In contrast, multisegmental modulation of visceral afferent processing also occurs when afferent inputs of different visceral organs are separated by several spinal segments. For example, afferent inputs from the urinary bladder, which enter lumbar and sacral spinal segments, suppress activity of the upper thoracic spinothalamic tract (STT) cells that are excited by inputs arising from the heart (Brennan et al. 1989).

We initially reasoned that this inhibition of cell activity in segments distant from the site of visceral input resulted from activation of supraspinal nuclei with descending pathways, such as the nucleus raphe magnus, parabrachial nuclei, and periaqueductal gray. These descending pathways modulate the activity of STT cells receiving visceral and somatic input (Jones 1992). However, our experimental results did not support this reasoning. Visceral and somatic inputs from cardiopulmonary afferent fibers and forelimbs still inhibit activity of lumbosacral STT cells and dorsal horn cells after spinal cord transection at the C1 segment (Hobbs et al. 1992b; Zhang et al. 1996). This observation suggests that supraspinal pathways are not required for this multisegmental interaction. Furthermore we also have shown that the inhibitory effects can be abolished after the spinal cord is transected in lower cervical segments. These observations indicate that neurons of upper cervical segments may be important for viscerosomatic inhibition of neurons in distant segments.

Our transection studies led to the hypothesis that neurons exist in upper cervical segments that either directly or indirectly suppress activity of neurons receiving visceral inputs in the lumbosacral spinal cord. Previous studies report that stimulation of the dorsolateral surface of the cervical spinal cord, either electrically or with glutamate, decreases renal sympathetic nerve activity and spontaneous firing of sympathetic preganglionic neurons (Schramm and Livingstone 1987; Schramm et al. 1988). Although inhibition was observed only after C1 transection, this study supports our hypothesis that activation of neurons in the upper cervical segments produces inhibitory effects on neurons in distant segments.

To address this hypothesis, we examined the effects of chemical activation of upper cervical cell bodies on responses of lumbosacral neurons to visceral input. Extracellular activity was recorded from single spinal neurons in the lumbosacral spinal cord that responded to noxious colorectal distension (CRD). Responses of these neurons were examined before, during, and after the application of glutamate at C1-C2 and C6-C7 segments. For some cells, the effects of glutamate application were examined before and after spinal transection at rostral C1 and C6 segments. Our results showed that activation of cell bodies in upper cervical segments modulated activity of distant neurons that responded to noxious visceral input.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Experiments were done on 59 male and 9 female Sprague-Dawley rats (Charles River, 280-450 g) anesthetized with pentobarbital sodium (60 mg/kg ip). Catheters were inserted into the right carotid artery to monitor blood pressure and into the left jugular vein to inject fluids and drugs. During the experiment, a continuous intravenous infusion of pentobarbital (10-15 mg · kg-1 · h-1 ) maintained anesthesia. Animals were paralyzed with pancuronium bromide (0.4 mg/kg iv) and were given supplemental doses (0.2 mg/kg ip) as needed during the experiment. After the tracheotomy was done, a positive pressure pump was used to provide artificial ventilation (55-60 strokes/min, 4.0- to 5.0-ml stroke volume). A thermostatically controlled heating pad and overhead infrared lamps were used to maintain rectal temperature at 37 ± 1oC.

The L6-S2 spinal segments were exposed for recording spinal neurons responsive to CRD. In some experiments, C1-C2 and C6-C8 spinal segments were exposed to apply glutamate and to make spinal transections. Rats were placed in a stereotaxic headholder and suspended from thoracic vertebrae (T10-T12) and sacral clamps. The dura mater of exposed spinal segments was removed carefully. Skin flaps of the neck were arranged to form a pool for warm normal saline. A small well filled with warm paraffin oil was made with dental impression material to protect the dorsal surface of the lumbosacral spinal cord from dehydration.

Carbon-filament glass microelectrodes were used to record extracellular action potentials of single spinal neurons. We searched for cells from midline to 2 mm lateral and 0-1.6 mm deep in L6-S2 segments. Signals were amplified and fed into a window discriminator. Discriminator output was displayed on an oscilloscope and recorded in a computer with a rate histogram program (1-s bins) to document all responses. Extracellular potentials and simultaneous discharges from the window discriminator were observed closely to make certain that the activity of only one cell was recorded. Cell activity (imp/s) was measured for 10 s during control and during a stimulus. For each neuron, a given stimulus was considered effective if the change in activity was >= 20% of control activity (Hobbs et al. 1992a). Only cells excited or inhibited by colorectal distension were reported in this study.

CRD was produced by inserting a 4- to 5-cm-long flexible latex balloon intraanally into the descending colon and rectum (Ness and Gebhart 1987, 1988). Intracolonic pressure was monitored continuously via an in-line pressure transducer and sphygmomanometer. CRD (80 mmHg, 15 s) was used as a standard noxious stimulus. The CRD pressure was increased from 0 to 80 mmHg in 1-2 s. Latency was measured from the onset of CRD to the onset of the cell response. Great care was taken to differentiate responses to CRD from mechanical movement of cutaneous fields and receptive endings of muscles and tendons of the abdominal wall. Neurons excited or inhibited by CRD at 80 mmHg were tested with this stimulus at least three consecutive times to determine that the cell responses were consistent. Cells were characterized for cutaneous receptive fields with innocuous stimulation, using a camel-hair brush or pressure from a blunt probe, and with noxious pinch of skin and muscles with blunt forceps. Neurons also were characterized for responses to movement of the hindlegs and tail displacement in a clockwise direction.

Glutamate (1 M) was absorbed onto filter paper pledgets (2 × 2 mm) and was placed on the dorsal surface of C1-C2 or C6-C7 segments. Saline control pledgets were applied at the same sites before or after glutamate. Pledgets remained on the cervical cord for 3 min and then the spinal cord was flushed with physiological saline. At least 20 min elapsed between each application. Colorectal distensions were maintained at 80 mmHg for 15 s with ~1-min intervals (30 s in a few cells) during glutamate tests. Additionally, before another pledget was applied, cell activity and blood pressure were at control levels. For some cells, the effects of glutamate on lumbosacral neurons responsive to CRD were determined before and after spinal transection at the rostral C1 and C6 segments. In 10 experiments, a small knife was used to cut the spinal cord at the rostral C1 segment to eliminate effects of supraspinal pathways. In four experiments, the spinal cord was transected at rostral C6 after cutting C1 to determine if the upper cervical spinal cord was required for glutamate effects on lumbosacral cells. Neuronal responses were recorded >= 20 min after each transection. After the experiments were finished, transection sites were examined to make certain the spinal cords were severed completely and to confirm the segment transected.

To mark spinal recording sites, an electrolytic lesion (50 µA DC, anodal for 20 s, cathodal for 20 s) was made at most recording sites after a cell was studied. At the end of the experiment, animals were killed with an intravenous euthanasia-5 solution. The lumbosacral spinal cord was removed and placed in 10% buffered formalin solution. Frozen sections (55-60 µm) of the lumbosacral cord were viewed to find lesion sites in the gray matter of the spinal cord. Locations were drawn on cross sections traced from the cytoarchitectonic scheme of Molander et al. (l984).

Data for neurons responsive to CRD are presented as means ± SE. Statistical comparisons were made using Student's paired or unpaired t-test. Changes in spontaneous activity and CRD-evoked responses of lumbosacral neurons during glutamate application on the spinal cord generally are presented as percentage decrease or increase of firing rates from control activity. Maximal inhibition or excitation was defined as the percentage of control activity or the mean frequency (imp/s) after the first minute following glutamate application. Comparison of data was considered statistically different if P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Neurons responsive to noxious CRD

A total of 135 L6-S2 neurons were activated by CRD; 87 cells were recorded in left spinal gray matter and 48 cells were on the right side. A comparison of the response characteristics of cells recorded from the right or left sides revealed no significant difference in spontaneous activity or responses to CRD. No difference was observed between the response characteristics of the cells in the male and female rats. Of 126 neurons tested for somatic input, 95 (75%) received convergent inputs from cutaneous receptive fields, hindleg movement, and tail rotation. Cutaneous receptive fields were generally on the ipsilateral scrotum, perianal region, lower back part of the body, and hind limb. Thirty-one neurons received only visceral input from the CRD stimulus.

The discharge rate of 99/135 (73%) lumbosacral cells increased with CRD stimulation. On the basis of the pattern and duration of the evoked responses, these neurons were subdivided further into three groups: excitatory abrupt (EA), excitatory sustained (ES), and excitatory-inhibitory (E-I) (Table 1). Forty-five EA neurons had a short onset latency (<1 s) after the start of CRD. Responses either were maintained at a steady rate or slowly adapted to a lower level of activity throughout the stimulus period. When the CRD was terminated, activity abruptly returned to baseline within 2 s (Fig. 1A). Thirty-five of these 45 neurons were spontaneously active. Somatic input converged on 33 of 42 EA neurons examined (Table 2). Thirteen ES neurons reached maximal discharge rates at or near the termination of the CRD stimulus; responses were sustained for 58.2 ± 10.3 s (range 16-126 s) after termination of the stimulus (Fig. 1B). Eleven of 13 ES neurons had spontaneous activity. Eight of 11 ES neurons examined for somatic fields received convergent somatic inputs (Table 2). Forty-one E-I neurons typically were activated at a short latency (<2 s) and reached an average maximal discharge rate of 40.2 ± 3.8 imp/s. At the termination of CRD, activity abruptly decreased or disappeared for 23.5 ± 5.1 s before recovering to precontrol levels (Fig. 1C). All E-I neurons had spontaneous activity discharging at an average rate of 17.6 ± 2.7 imp/s. Twenty-eight of 38 E-I neurons examined had convergent somatic inputs. (Table 2).


                              
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Table 1. Response characteristics of L6-S2 lumbosacral spinal neurons activated by noxious colorectal distension



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Fig. 1. Response patterns of lumbosacral neurons activated by noxious colorectal distension (CRD, 80 mmHg). A: excitatory abrupt (EA) cell. B: excitatory sustained (ES) cell. C: excitatory-inhibitory (E-I) cell. D: inhibitory abrupt (IA) cell. E: inhibitory sustained (IS) cell. F: inhibitory-excitatory (I-E) cell. Bin width of rate histograms is 1 s in all examples. Time bar is 10 s at the right top of each graph. Duration of colorectal distension is indicated by a horizontal bar at the bottom of each graph in all figures.


                              
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Table 2. Somatic input to L6-S2 lumbosacral spinal neurons activated by colorectal distension

CRD inhibited 36/135 (27%) neurons, which were subdivided further into three groups: inhibitory abrupt (IA), inhibitory sustained (IS), and inhibitory-excitatory (I-E) neurons (Table 1). CRD reduced activity of 20 IA neurons from 12.9 ± 2.1 to 3.4 ± 0.9 imp/s (26% of control) after a short onset latency (<1 s). After termination of CRD, spontaneous activity quickly returned to predistension levels within 2 s (Fig. 1D). Thirteen of 20 IA neurons received convergent somatic inputs (Table 2). Seven IS neurons had an average spontaneous activity of 17.0 ± 5.1 imp/s that was reduced to 4.7 ± 2.7 imp/s (28% of control) by CRD. After CRD was terminated, spontaneous activity slowly returned to predistension levels after 42.4 ± 7.2 s (range 18-64 s; Fig. 1E). Three IS neurons had a long onset latency (13.5 ± 3.5 s), whereas the other four cells had a short onset latency (<l s). Four of six IS neurons tested had convergent somatic inputs (Table 2). Nine I-E neurons decreased spontaneous activity from 16.4 ± 3.5 to 2.9 ± 0.9 imp/s (18% of control) shortly after the onset (<2 s) of CRD. After CRD was terminated, spontaneous activity quickly exceeded prestimulus control within 2 s (Fig. 1F). The poststimulus response was sustained for 34.2 ± 5.1 s (range 13-56 s) with average maximal discharge rate of 26.3 ± 5.1 imp/s. All I-E neurons received convergent somatic input (Table 2).

Effect of glutamate at C1-C2

Effects of glutamate pledgets applied to C1-C2 segments were tested on 75 lumbosacral neurons responsive to CRD. Lesions made at the recording sites were identified for 65 cells (Fig. 2); 41 were found in laminae II-VI, 21 in laminae VII, and 3 in laminae VIII and IX. Lesion sites of the different classes of CRD responsive neurons did not reveal a clear differential distribution of one subgroup compared with other subgroups, although neurons inhibited or inhibited/excited by CRD were more commonly in laminae V and VI.



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Fig. 2. Lesion sites of cells tested for responses to glutamate drawn on L6-S2 segments of the rat spinal cord. , lesion sites of EA and ES cells responsive to CRD; black-square, E-I cells; open circle , IA and IS cells; , I-E cells. Drawings of lumbosacral spinal cord are from Molander et al. (1984). I-X, laminae; Liss, Lissauer's track; LSN, lateral spinal nucleus; Pyr, pyramidal tract; MG, medial group of large neurons in the intermediate zone; IM, intermediomedial nucleus.

RESPONSES OF NEURONS EXCITED BY CRD. For 56 lumbosacral neurons excited by CRD, glutamate placed on C1-C2 segments decreased activity evoked during CRD of 33 (59%) neurons, increased evoked activity of 11 (20%), and did not affect 12 (21%) (Table 3). No significant difference was found among the different classes of cells excited by CRD (chi 2 analysis). Evoked excitatory responses to CRD decreased from 35.8 ± 2.9 to 26.9 ± 2.9 imp/s (P < 0.05, n = 56) 1 min after glutamate was applied. Duration from the onset of suppressed CRD responses to the return of the evoked cell responses to control levels was 7.7 ± 0.6 min. Glutamate did not change the mean spontaneous activity of these 56 cells (13.8 ± 2.1 vs. 12.3 ± 1.7 imp/s). However, glutamate significantly decreased spontaneous activity from 7.1 ± 1.5 to 2.7 ± 0.8 imp/s in the 33 cells with decreased evoked activity during CRD (P < 0.01). Responses of three neurons (2 EA and 1 E-I) are shown in Fig. 3, A-C, and percent changes of evoked responses are shown in Fig. 3D.


                              
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Table 3. Effects of glutamate pledgets at C1-C2 and C6-C7 segments on the CRD responses of lumbosacral cells excited and excited/inhibited by CRD



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Fig. 3. Effects of glutamate pledgets at C1-C2 segments on activity of lumbosacral cells excited by noxious CRD. A: spontaneous activity and CRD responses of an EA neuron inhibited by glutamate. Glutamate on and off at C1-C2 is indicated by up-arrow  and down-arrow , respectively, in this figure and the subsequent figures. Glu, glutamate. B: CRD-evoked activity of an EA cell without spontaneous activity. C: spontaneous activity and CRD responses of an E-I cell. D: percent changes of excitatory responses of cells (n = 56) to CRD during application of glutamate or saline (n = 8) at C1-C2 as shown by a horizontal bar. **P < 0.001; *P < 0.05.

RESPONSES OF NEURONS INHIBITED BY CRD. For 19 neurons inhibited by CRD, glutamate application at C1 segment increased spontaneous activity of 13 (68%) neurons, decreased activity of 4 (21%), and did not affect 2 (11%) (Table 4). Average spontaneous activity for these cells increased from 15.7 ± 1.9 to 22.7 ± 2.9 imp/s (P < 0.05, n = 19). The duration of excitation averaged 6.3 ± 0.6 min. In cells responding with inhibition to CRD, glutamate at C1-C2 did not change the maximal CRD inhibitory responses between control (11.6 ± 1.1 imp/s) and effects during glutamate application (12.8 ± 2.2 imp/s). Two examples of these neurons (1 IA and 1 I-E) are shown in Fig. 4, A and B. Figure 4C shows the percent change of spontaneous activity during application of glutamate or saline.


                              
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Table 4. Effects of glutamate pledgets at C1-C2 and C6-C7 segment on spontaneous activity of lumbosacral neurons inhibited and inhibited/excited by CRD



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Fig. 4. Effects of glutamate pledgets at C1-C2 segments on activity of lumbosacral cells inhibited by noxious CRD. A: spontaneous activity and CRD responses of an IA cell. B: spontaneous activity and CRD responses of an I-E cell. C: percent changes of spontaneous activity of inhibitory cells (n = 19) responsive to CRD during the application of glutamate or saline (n = 3) at C1-C2. *P < 0.05.

Effects of glutamate at C6-C7

RESPONSES OF NEURONS EXCITED BY CRD. For 18 lumbosacral cells excited by CRD, glutamate placed on C6-C7 segments decreased activity evoked during CRD of 10 neurons, increased the responses of 2 neurons, and did not affect the activity of 6 neurons (Table 3). For cells inhibited by glutamate application, the mean spontaneous activity decreased from 7.0 ± 1.2 to 2.3 ± 1.4 imp/s. The average increase in activity to CRD of 25.4 ± 4.2 imp/s was reduced to 11.5 ± 3.4 imp/s during C6-C7 glutamate application. The duration of reduced excitatory responses to CRD was 7.1 ± 0.8 min. Saline pledgets placed on the C6-C7 segments did not change activity of lumbosacral cells (Fig. 5A).



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Fig. 5. Comparison of effects of glutamate (Glu) at C1-C2 and C6-C7 segments on responses of cells excited by CRD in lumbosacral spinal cord. A: normal saline at C6-C7 on activity of an EA cell. B: glutamate at C1-C2 on same cell. C: glutamate at C6-C7 on same cell. D: reduction of CRD responses by glutamate application at C1-C2 and C6-C7 segment (n = 9). In this graph (D) and in H, each symbol and line represents an individual cell. The black circles on either side of the lines represent mean activity in imp/s during control (left) and after glutamate (right). **P < 0.01. E: normal saline at C1-C2 on an E-I cell. F: glutamate at C1-C2 on this cell. G: glutamate at C6-C7 on same cell. H: reduction of CRD responses by glutamate at C1-C2 but not at C6-C7 (n = 4). *P < 0.05.

Nine of 10 lumbosacral neurons excited by CRD and inhibited by glutamate at the C6-C7 segment also were inhibited by glutamate at the C1-C2 segment. An example is shown in Fig. 5, B and C. One E-I neuron was inhibited by glutamate only at C6-C7. A comparison of the effects of glutamate at C1-C2 with that of glutamate at C6-C7 on CRD-evoked responses of nine lumbosacral cells is shown in Fig. 5D. When glutamate pledgets were applied at either C1-C2 or C6-C7, average maximal inhibition of spontaneous activity was not significantly different between these segments (5.7 ± 2.4 to 0.4 ± 0.2 imp/s vs. 3.7 ± 1.4 to 1.1 ± 0.7 imp/s). The mean reduction of excitatory responses to CRD also was not different (23.9 ± 4.6 reduced to 9.8 ± 2.5 imp/s during glutamate application at C1-C2 vs. 21.9 ± 4.3 reduced to 9.8 ± 3.2 during glutamate application at C6-C7).

Four of six lumbosacral neurons that were not affected by glutamate application at C6-C7 were inhibited by glutamate application at C1-C2; activity of two cells was not affected by application of glutamate at either location. An example of CRD responses of a cell that responded to C1-C2 glutamate but not to C1-C2 saline or C6-C7 glutamate is shown in Fig. 5, E and G. A comparison of effects of glutamate on CRD-evoked responses of four lumbosacral cells that were inhibited by C1-C2 glutamate but not by C6-C7 glutamate is shown in Fig. 5H.

RESPONSES OF NEURONS INHIBITED BY CRD. Of six lumbosacral cells inhibited by CRD and excited by glutamate at C1-C2, glutamate at C6-C7 excited four cells, inhibited one cell, and did not affect the activity of one cell (Table 4). For the four cells excited by glutamate at C6-C7, spontaneous activity increased from 8.1 ± 2.1 to 13.9 ± 2.6 imp/s. Before C6-C7 glutamate application, CRD decreased activity from 8.1 ± 2.0 to 1.8 ± 1.0 imp/s. During glutamate application at C6-C7, CRD decreased activity of six cells from 13.9 ± 2.6 to 2.3 ± 1.7 imp/s, P < 0.05 compared with inhibitory CRD responses before C6-C7 glutamate. The spontaneous activity of one cell inhibited by C6-C7 glutamate decreased from 6.9 to 0 imp/s.

Influences of transection at rostral C1 on effects of glutamate at C1-C2

Ten cells in 10 experiments were recorded before and after the spinal cords were transected at the rostral C1 segments to eliminate effects of supraspinal pathways. An example of a neuron inhibited by glutamate at C1-C2 before and after C1 transection is shown in Fig. 6, A and C. After C1 transection, average spontaneous activity did not change (9.5 ± 1.9 vs. 9.0 ± 2.0 imp/s), but the average excitatory responses to CRD increased from 29.1 ± 4.5 to 42.3 ± 5.5 imp/s (P < 0.05) after transection. Thus lumbosacral neurons were under significant descending inhibition from supraspinal sites.



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Fig. 6. Effects of spinal transections at rostral C1 and rostral C6 on spontaneous activity and evoked responses of an E-A cell responsive to noxious CRD. A: glutamate at C1-C2 before C1 transection. B: glutamate at C6-C7 before C1 transection. C: glutamate at C1-C2 after cut at rostral C1; D: glutamate at C6-C7 after cut at rostral C1; E: glutamate at C6-C7 after cut at rostral C6.

Before C1 transection, glutamate application at C1-C2 decreased excitatory CRD-responses in 8 of 10 cells and increased excitatory CRD-responses in 2 cells. After C1 transection, glutamate at C1-C2 segments still reduced excitatory responses to CRD for seven of eight neurons; C1 transection abolished glutamate inhibition of one cell. One of two cells excited by glutamate at C1-C2 still was excited by C1-C2 glutamate after C1 transection; glutamate excitation of another cell was abolished by C1 transection.

For seven lumbosacral cells that still were inhibited by C1-C2 glutamate after C1 transection, C1-C2 glutamate reduced spontaneous activity from 8.7 ± 2.6 to 3.8 ± 2.6 imp/s before transection and from 10.0 ± 2.8 to 4.1 ± 2.9 imp/s after transection. Before C1 transection, C1-C2 glutamate reduced activity increases to CRD from 28.2 ± 6.0 to 12.3 ± 4.6 imp/s. After C1 transection, C1-C2 glutamate reduced activity increases to CRD from 41.8 ± 5.7 to 27.6 ± 4.1 imp/s. The mean duration of the glutamate reduction of CRD effects was 6.7 ± 1.3 min before C1 transection and 5.7 ± 1.2 min after transection. These data showed that spontaneous activity and the responses of neurons to CRD still were inhibited by glutamate at C1-C2 after transection at rostral C1 segment. Comparison of inhibitory effects on CRD-evoked excitatory responses by glutamate before and after transection at C1 is shown as the change in percentage of the CRD response in Fig. 7A and as the change in imp/s in Fig. 7B.



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Fig. 7. Comparison of effects of glutamate on excitatory responses of lumbosacral neurons to CRD before and after transection at different segments of spinal cord. A: percent change of CRD responses during glutamate at C1-C2 before and after cut at rostral C1 (n = 7). B: change in discharge rate (imp/s) of CRD responses during glutamate at C1-C2 before and after cut at rostral C1 (n = 7). C: percent change of CRD responses during glutamate at C6-C7 before and after cut at rostral C1 and C6 (n = 4).

Influences of transection of spinal cord on effects of glutamate at C6-C7

To determine if glutamate activation of neurons in the C6-C7 segments required rostral cervical segments to produce effects on lumbosacral cells, a sequential transection at rostral C6 was made in four animals. Spontaneous activity and CRD-evoked responses of four lumbosacral neurons still were inhibited by glutamate at C6-C7 after the transection was made at the rostral C1 segment (Fig. 6, B and D). However, transection at rostral C6 abolished inhibitory effects on these lumbosacral cells when glutamate was applied to C6-C7 segments (Fig. 6E). Before transection at C1, glutamate applied at C6-C7 reduced spontaneous activity of lumbosacral cells from 6.5 ± 2.3 to 2.4 ± 1.4 imp/s and reduced evoked responses to CRD from 29.4 ± 10.3 to 12.4 ± 6.2 imp/s (n = 4). The duration from onset of suppressed CRD responses to the return of the CRD response to control level was 5.8 ± 0.4 min. After rostral C1 transection, spontaneous activity was reduced from 6.5 ± 1.7 to 1.6 ± 0.9 imp/s, and excitatory responses to CRD were reduced from 36.6 ± 8.3 to 11.5 ± 5.7 imp/s when glutamate was applied at C6-C7. The duration of inhibition produced by glutamate was 5.3 ± 0.6 min. After rostral C6 transection, C6-C7 glutamate application did not change spontaneous activity (6.9 ± 4.5 imp/s vs. 5.8 ± 3.5 imp/s) and evoked responses to CRD (40.5 ± 12.2 imp/s vs. 40.6 ± 12.2 imp/s) of lumbosacral cells. Comparison of the changes induced by glutamate at C6-C7 before and after transection at rostral C1 and C6 is shown in Fig. 7C.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Lumbosacral spinal neurons responsive to CRD

We confirmed and extended the work of Ness and Gebhart (1987), who examined the effects of CRD on cells in the medial L6-S1 spinal segments. They subdivided neurons into four classes based on their response characteristics to CRD (75-80 mmHg, 20 s): short-latency abrupt (SL-A), short-latency sustained (SL-S), long latency (LL), and inhibited (INHIB) neurons. The major difference between the present study and their work was that our search area was expanded to include the entire mediolateral area of the L6-S2 spinal cord, which is consistent with the distribution of Fos expression in the lumbosacral spinal cord following noxious colorectal distension (Traub et al. 1992). We found many cells that responded with both excitation and inhibition to CRD. On the basis of these findings, we used a different classification system to include response patterns of excitation and inhibition in the same cells, i.e., excitatory-inhibitory (E-I) and inhibitory-excitatory (I-E). A comparison of the two classifications shows that excitatory responses of our EA and ES subgroups are similar to the SL-A, SL-S, and LL subgroups, and IS is similar to INHIB neurons. In the present study, inhibitory effects might be more visible because average spontaneous activity for neurons was higher than that of the previous study (Ness and Gebhart 1987). The reasons for the difference in spontaneous activity between the two studies might be due to the expanded search area in spinal cord and difference in anesthetic technique.

Results of this study showed that CRD excited or excited/inhibited 73% of the sampled lumbosacral cells and inhibited or inhibited/excited 27% of the sampled neurons. These results are consistent with the study of Ness and Gebhart (1987), who showed that 82% of cells were excited and 18% of cells were inhibited by CRD in the lumbosacral spinal cord. In contrast, Cadden and Morrison (1991) report that CRD suppresses activity in 83% of the neurons that were selected if they responded to mechanical stimulation of the ipsilateral hindpaw. The major difference in results might be the selection process used to identify the cells that were included in each protocol. In the present study, lumbosacral spinal cells were studied if they responded to CRD. In the study by Cadden and Morrison (1991) neurons were selected if they responded to stimulation of distal somatic fields, i.e., the hindpaw. We have shown in monkey studies that afferent inputs from visceral organs most commonly excite STT cells receiving proximal somatic input (Foreman 1993; Hobbs et al. 1992a). The cells recorded in the present study most commonly had somatic receptive fields that included the tail and proximal somatic structures. Therefore it was much more likely that these neurons would receive excitatory input during CRD.

Glutamate activation of C1-C2 neurons

Activation of cells in C1-C2 segments with glutamate changed spontaneous activity and CRD-evoked responses of lumbosacral neurons without requiring supraspinal pathways. Usually glutamate application to upper cervical segments inhibited lumbosacral neurons that were excited by CRD and excited neurons that were inhibited by CRD. All classes of lumbosacral neurons that were subdivided according to CRD response characteristics were affected by glutamate activation of the upper cervical cells.

Glutamate has been used to activate cell bodies in the cervical spinal cord in previous studies (Sandkuhler et al. 1993; Schramm and Livingstone 1987) because it does not affect axons of passage (Goodchild et al. 1982). Some concern exists that glutamate may cause depolarization block of cells if it is applied for long periods (Lipski et al. 1988), but this was not a major factor in this study. Glutamate was applied repeatedly to cervical spinal segments without attenuating effects on spontaneous and evoked activity of lumbosacral neurons. Furthermore a c-fos immunocytochemical study (Jones 1998) has shown that microinjections of glutamate into the upper cervical spinal cord increase the number of neurons in gray matter that express Fos-like immunoreactivity. Because Fos-like immunoreactivity is a marker for cell activity, the increased number of neurons expressing Fos suggests that glutamate increases cell discharge rate and does not cause depolarization block.

Glutamate was applied to C1-C2 segments because our previous studies suggest that upper cervical neurons may relay information that inhibits the activity of lumbosacral neurons (Hobbs et al. 1992b; Zhang et al. 1996). Inhibition of the activity of lumbosacral spinothalamic tract neurons and dorsal horn neurons by electrical stimulation of splanchnic or cardiopulmonary afferent fibers is eliminated after spinal transection between C4 and C6 segments but not after transection at rostral C1. It is reasonable that, in the present study, upper cervical propriospinal neurons activated by glutamate could be candidates for relaying inhibitory effects of thoracic afferent input to lumbosacral neurons.

Anatomic studies show that some neurons in the upper cervical spinal cord have descending projections to the lumbosacral spinal cord in monkeys (Burton and Loewy 1976), in cats (Matsushita et al. 1979; Yezierski et al. 1980), and in rats (Menetrey et al. 1985; Miller et al. 1998). In the rat study by Miller et al. (1998), retrogradely labeled cells were located in the C1-C2 segments after horseradish peroxidase or flurorogold was injected unilaterally or bilaterally into the L5-S1 segments. The labeled neurons were located in the nucleus proprius, ventral horn, central gray region (area X) and lateral cervical and spinal nuclei. In addition, microinjections of glutamate into the upper cervical spinal cord of rats significantly reduce the total number of lumbar neurons demonstrating Fos-like immunoreactivity when noxious heat is applied to the hindpaw (Jones 1998). These findings support electrophysiological evidence that descending systems from upper cervical spinal cord could modulate nociceptive input entering the lumbosacral spinal cord (Hobbs et al. 1992b; Zhang et al. 1996).

Supraspinal and cervical descending modulation

Excitatory CRD-evoked responses of lumbosacral neurons significantly increased after the spinal cord was transected at the rostral C1 segment, which suggested that these neurons were under tonic descending inhibition. This observation agrees with previous studies showing that the evoked responses of thoracic and lumbosacral viscerosomatic neurons are under considerable tonic descending inhibition from supraspinal nuclei (Akeyson et al. 1990; Cervero 1983; Ness and Gebhart 1987; Tattersall et al. 1986).

In contrast to the inhibition of lumbosacral cells that we observed in the intact preparation, glutamate activation of cervical cells inhibited renal sympathetic activity only after the spinal cord was transected near the C1 segment (Schramm and Livingstone 1987). One explanation for this difference is that the rostral ventrolateral medulla plays a key role in modulating upper cervical spinal cord effects on sympathetic nerve activity (Poree and Schramm 1992a). Therefore this activity may suppress upper cervical cells until that inhibition is removed by the transection. Poree and Schramm (1992b) also show that thoracic spinal neurons responsive to splanchnic stimulation are inhibited by glutamate activation of cervical cells in spinalized rats; however, these effects were not tested in the intact preparation. On the basis of our findings, it is possible that thoracic spinal neurons might be inhibited by activation of cervical neurons when the spinal cord is intact.

Neural mechanisms of descending effects of C1-C2 glutamate

In addition to inhibition of CRD-evoked activity of lumbosacral cells, activation of descending propriospinal pathways from C1-C2 segments excited cells that were inhibited by CRD. It is proposed that these cells might be inhibitory interneurons in the lumbosacral segments and be part of the population of neurons in the superficial dorsal horn and lamina V that have immunoreactivity for inhibitory neurotransmitters GABA and enkephalin (Glazer and Basbaum 1981; Magoul et al. 1987; Ruda et al. 1986). Several possibilities might explain our results (Fig. 8). First, inhibitory interneurons in lumbosacral segments could inhibit the activity of neurons excited by CRD, which would include neurons that have been shown to be the origin of the spinothalamic tract (Ness and Gebhart. 1987) and the postsynaptic dorsal column tract (Al-Chaer et al. 1996a,b). Second, inhibitory interneurons might be inhibited indirectly by other interneurons that receive directly CRD input, which then would result in disinhibition of the neurons excited by CRD. Disinhibition of neurons excited by CRD would intensify the transmission of noxious information in the spinal cord. Furthermore C1-C2 glutamate could activate cervical inhibitory circuits that, in turn, suppressed the activity of neurons excited by CRD in lumbosacral segments. Because the effects of C1-C2 glutamate stimulation on lumbosacral cells produced similar decreases in the number of impulses/second before and after C1 spinal transection, most of the inhibition originated from the upper cervical spinal cord and did not require supraspinal pathways. The smaller percent change in inhibition of the CRD response after C1 transection might suggest that supraspinal pathways contributed to the inhibition; however, most likely the difference was due to the increased CRD responses that resulted from release of tonic descending inhibition after the transection.



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Fig. 8. Schematic diagram of neural mechanisms that could explain effects on the activity of lumbosacral cells when glutamate is applied at C1-C2 or C6-C7 segments. black-square and , propriospinal cells in cervical spinal cord that inhibit and excite the activity of lumbosacral cells, respectively. , inhibitory interneurons in lumbosacral spinal cord. open circle , in lumbosacral segment, projection neuron. +, excitatory connections; -, inhibitory connections. (see text for detail)

Effects of glutamate at C6-C7 segments

In general, glutamate application at C6-C7 segments changed spontaneous activity and CRD-evoked responses of lumbosacral cells similar to the effects of C1-C2 glutamate. We predicted that the glutamate effects should be less at the lower cervical segments because inhibitory effects on lumbosacral cells by thoracic spinal input required only the upper cervical segments (Hobbs et al. 1992b; Zhang et al. 1996). However, inhibitory effects of C6-C7 glutamate were not statistically different from the effects of glutamate activation of C1-C2 neurons. These effects remained after rostral C1 spinal transection. In contrast, subsequent transection of the spinal cord just rostral to the glutamate stimulation site eliminated glutamate-evoked inhibition from C6-C7 segments. This transection most likely interrupted the axons of lower cervical cells that have ascending projections to upper cervical segments (Molenaar and Kuypers 1975, 1978). Information to upper cervical segments from glutamate-activated neurons of lower segments then could activate inhibitory and/or excitatory propriospinal cells to produce descending inhibitory effects directly on lumbosacral cells or indirectly via inhibitory interneurons (Fig. 8).

Physiological application

Propriospinal neurons originating from the upper cervical spinal cord could independently modulate background activity and noxious evoked responses to visceral inputs in lumbosacral dorsal horn neurons. One functional importance is that this intraspinal modulatory mechanism helps in the ability of an animal or human to localize and discriminate more precisely a noxious stimulus. For example, the inhibitory effect of cell activity in distant segments improves the signal-to-noise ratio of the STT system, thereby enhancing excitatory information that reaches specific somatotopic regions in the ventral posterior lateral nucleus of the thalamus (Foreman 1995).

Results of the present study showed that activation of cells in the upper cervical spinal cord could adjust visceral sensory processing in distant segments without requiring supraspinal pathways. These adjustments in sensory processing had some characteristics that were similar to diffuse noxious inhibitory controls (DNIC), i.e., inhibition of dorsal horn convergent cells when noxious stimulation is applied to widespread somatic areas of the body and to visceral structures (Cadden and Morrison 1991; LeBars et al. 1979a,b, 1986). Because DNIC requires supraspinal connections, the pathway producing inhibitory effects reported in this study is different because inhibition remained after rostral C1 transection. It should be pointed out that the CRD-evoked inhibition in convergent neurons was not examined before and after spinal transection (Cadden and Morrison 1991). On the basis of the results of the present study, it is possible the inhibitory effects of the visceral stimulus of convergent neurons also can be manifested through spinal mechanisms. It also is reported that, in spinalized animals, some propriospinal modulatory processes act on convergent neurons and are triggered by noxious stimulation, but the responses were less potent and had shorter duration than DNIC (Cadden et al. 1983). In that study, transections and xylocaine infusions were made in the region of the upper cervical spinal cord where we activated neurons with glutamate to inhibit the CRD-evoked responses of lumbosacral cells. As a result, part of the population of cells that might have produced inhibition were most likely damaged and/or separated from their descending axons (Cadden et al. 1983).

We propose that the inhibitory processes observed in this study may be a part of the more general phenomenon called "nocigenic inhibition," in which a nociceptive stimulus utilizes segmental and propriospinal as well as supraspinal levels of interaction to produce inhibition of responses resulting from a nociceptive test stimulus (Ness and Gebhart 1991a,b). Our finding in lumbosacral neurons is generally consistent with the results obtained in thoracolumbar neurons by Ness and Gebhart (1991a). One difference is that similar distributions in the variable CRD responses to C1-C2 glutamate were found in all classes of lumbosacral neurons in the present study. However, Ness and Gebhart (1991a) report that all thoracolumbar SL-A neurons were inhibited by noxious pinch in distant cutaneous sites, whereas the response patterns of SL-S cells were variable. One possible explanation for this difference is that glutamate and noxious pinch of the forelimb skin may activate different groups of neurons in the upper cervical segments. In summary, the results of this study lead to the suggestion that neurons in the upper cervical spinal cord can serve as a potential filter, processor, and integrator of sensory information between visceral organs separated by distance and function. It also is conceivable that the incidence and efficacy of intraspinal propriospinal antinociceptive effect were as strong as that from a classical supraspinal region of endogenous antinociception, such as the midbrain periaqueductal gray (Jones 1992).


    ACKNOWLEDGMENTS

The authors thank Drs. J. P. Farber, S. L. Jones, and B. Greenwood for helpful comments as well as D. Holston and C. J. Jou for technical assistance and preparation of the figures.

This study was supported by National Institute of Neurological Disorders and Stroke Grant NS-35471.


    FOOTNOTES

Address for reprint requests: R. D. Foreman, Dept. of Physiology, University of Oklahoma Health Sciences Center, P.O. Box 26901, Oklahoma City, OK 73190.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 1 April 1999; accepted in final form 17 August 1999.


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ABSTRACT
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0022-3077/99 $5.00 Copyright © 1999 The American Physiological Society