1Department of Physiology and 2Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73190
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ABSTRACT |
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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.
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INTRODUCTION |
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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.
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METHODS |
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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 · kg1 · 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.
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RESULTS |
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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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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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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
(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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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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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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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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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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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.
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DISCUSSION |
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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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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
).
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ACKNOWLEDGMENTS |
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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.
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FOOTNOTES |
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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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REFERENCES |
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