Responses and Afferent Pathways of Superficial and Deeper C1-C2 Spinal Cells to Intrapericardial Algogenic Chemicals in 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. Responses and Afferent Pathways of Superficial and Deeper C1-C2 Spinal Cells to Intrapericardial Algogenic Chemicals in Rats. J. Neurophysiol. 85: 1522-1532, 2001. Electrical stimulation of vagal afferents or cardiopulmonary sympathetic afferent fibers excites C1-C2 spinal neurons. The purposes of this study were to compare the responses of superficial (depth <0.35 mm) and deeper C1-C2 spinal neurons to noxious chemical stimulation of cardiac afferents and determine the relative contribution of vagal and sympathetic afferent pathways for transmission of noxious cardiac afferent input to C1-C2 neurons. Extracellular potentials of single C1-C2 neurons were recorded in pentobarbital anesthetized and paralyzed male rats. A catheter was placed in the pericardial sac to administer a mixture of algogenic chemicals (0.2 ml) that contained adenosine (10-3 M), bradykinin, histamine, serotonin, and prostaglandin E2 (10-5 M each). Intrapericardial chemicals changed the activity of 20/106 (19%) C1-C2 spinal neurons in the superficial laminae, whereas 76/147 (52%) deeper neurons responded to cardiac noxious input (P < 0.01). Of 96 neurons responsive to cardiac inputs, 48 (50%) were excited (E), 41 (43%) were inhibited (I), and 7 were excited/inhibited (E-I) by intrapericardial chemicals. E or I neurons responsive to intrapericardial chemicals were subdivided into two groups: short-lasting (SL) and long-lasting (LL) response patterns. In superficial gray matter, excitatory responses to cardiac inputs were more likely to be LL-E than SL-E neurons. Mechanical stimulation of the somatic field from the head, neck, and shoulder areas excited 85 of 95 (89%) C1-C2 spinal neurons that responded to intrapericardial chemicals; 31 neurons were classified as wide dynamic range, 49 were high threshold, 5 responded only to joint movement, and no neuron was classified as low threshold. For superficial neurons, 53% had small somatic fields and 21% had bilateral fields. In contrast, 31% of the deeper neurons had small somatic fields and 46% had bilateral fields. Ipsilateral cervical vagotomy interrupted cardiac noxious input to 8/30 (6 E, 2 I) neurons; sequential transection of the contralateral cervical vagus nerve (bilateral vagotomy) eliminated the responses to intrapericardial chemicals in 4/22 (3 E, 1 I) neurons. Spinal transection at C6-C7 segments to interrupt effects of sympathetic afferent input abolished responses to cardiac input in 10/10 (7 E, 3 I) neurons that still responded after bilateral vagotomy. Results of this study support the concept that C1-C2 superficial and deeper spinal neurons play a role in integrating cardiac noxious inputs that travel in both the cervical vagal and/or thoracic sympathetic afferent nerves.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Experimental studies suggest that the clinical expression of neck and jaw pain resulting from ischemic episodes in the heart (Sampson and Cheitlin 1971) appears to involve neuronal processing in the upper cervical segments of the spinal cord. Results from anatomical and electrophysiological studies have shown that primary afferent fibers from the trigeminal nerve converge with upper cervical spinal afferent fibers onto neurons of the C1-C3 segments (Bogduk 1997; Kerr 1961; Pfaller and Arvidsson 1988). Recent studies have shown that stimulation of cardiac afferent fibers excites C1-C3 neurons that receive convergent somatic inputs from the neck and jaw region (Chandler et al. 1996; Zhang et al. 1997). Convergence of visceral and somatic afferents onto these upper cervical neurons provides a potential mechanism to describe the neck and jaw pain that results from an ischemic heart. Previous studies were designed to examine the convergence of somatic receptive fields and visceral input from the heart onto C1-C2 spinothalamic tract cells in primates (Chandler et al. 2000). To expand our understanding about viscerosomatic processing of cardiac inputs in these segments, the responses of superficial and deeper spinal neurons in the C1-C2 segments to algogenic chemical stimulation of cardiac afferent fibers were studied in rats.

Superficial laminae of the spinal dorsal horn have received considerable attention as areas of relay and integration of sensory inputs to the spinal cord since Christensen and Perl (1970) reported that many superficial dorsal horn neurons were excited specifically by somatic nociceptors. Many studies have demonstrated that superficial neurons relay and transmit somatic nociceptive information to the thalamus and other supraspinal regions and are subjected to descending inhibitory influences (Cervero et al. 1977; Light et al. 1986; Wall et al. 1979; Woolf and Fitzgerald 1983). A few investigators showed that superficial neurons receive inputs from visceral organs (e.g., cardiac sympathetic afferents, biliary system, splanchnic nerve, and colorectal afferents), and some characteristics of these neurons differed from those located in deep laminae (Blair et al. 1981; Cervero and Tattersall 1987; Ness and Gebhart 1989). Quantitative differences in the responses of superficial and deeper spinal neurons to cardiac afferent inputs have not been examined previously although early findings in monkeys show that some superficial neurons of the thoracic spinal cord are activated by cardiac sympathetic afferents (Blair et al. 1981).

The purposes of this study were to compare the responses of C1-C2 superficial and deeper spinal neurons to chemical noxious stimulation of cardiac/pericardial receptors and determine the afferent pathways that transmit cardiac noxious information to C1-C2 neurons. Results of this study showed that cardiac noxious inputs from both vagal and sympathetic afferent pathways could either excite or inhibit C1-C2 spinal neurons. There are some qualitative and quantitative differences in response characteristics to chemical stimulation of cardiac afferents and in somatic field properties between the superficial and deeper neurons. Excitatory somatic receptive fields for these neurons varied in size and location and usually included the head, neck, and jaw regions. These data lend further support to the concept that C1-C2 neurons play a role in integrating cardiac nociception. A preliminary report of portions of this work has been published in abstract form (Chandler et al. 1998).


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Experiments were performed in 66 male Sprague-Dawley (Charles River, Boston, MA) rats weighing between 280 and 440 g. The Institutional Animal Care and Use Committee of the University of Oklahoma Health Sciences Center approved the experiments performed in this study. Experiments followed the ethical guidelines of the International Association for the Study of Pain and the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources Commission on Life Sciences and National Research Council 1996; Zimmermann 1983). After the animals were 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 saline and drugs. During each experiment, a continuous intravenous infusion of pentobarbital (10-15 mg · kg-1 · h-1 ) was injected to maintain the appropriate level of anesthesia. Animals were paralyzed with pancuronium bromide (0.4 mg/kg ip) and given supplemental doses (0.2 mg/kg ip) as needed to maintain muscle relaxation during the experiment. After tracheotomy, a tube was inserted into the trachea and the animal was ventilated with a positive pressure pump (55-60 strokes/min, 4.0-5.0 ml stroke volume). Arterial pressure and pupil diameter were monitored to determine the anesthesia level. A thermostatically controlled heating pad and overhead infrared lamps were used to maintain rectal temperature at 37 ± 1°C.

Rats were mounted in a stereotaxic headholder and stabilized with a clamp at the T1-T2 vertebrae. After laminectomies were performed to expose the C1-C2 spinal segments for recording cells and C6-C8 segments for transection of the spinal cord, the dura mater was carefully removed. Dental impression material was built up on the tissue and bone surrounding the upper cervical spinal cord to make a small well that was filled with warm paraffin oil to protect the cord from dehydration. In some experiments, the upper cervical spinal cord was covered with warm agar (3-4% in saline) to improve stability. Carbon-filament glass microelectrodes were used to record extracellular action potentials of single spinal neurons in the C1-C2 segments, 1-3 mm lateral from midline and 0.0-1.4 mm deep. The microelectrode was mounted on a micro-manipulator at an angle of 60-70° from the vertical in the caudorostral axis. This angled approach made penetration easier and reduced dimpling of the cord surface during electrode insertion. We searched for spinal cells with spontaneous discharges that were large enough for analysis. Sometime a burst of discharges that later disappeared could be recorded when the microelectrode was close to a neuron. This phenomenon made it possible to find and study responses of neurons that did not have spontaneous activity. The search was restricted to depths of 0-0.35 and 0.36-1.40 mm below the cord surface to isolate superficial and deeper neurons, respectively. The depth selection for the two populations of neurons was based on neuroanatomical work (Molander et al. 1989) and electrophysiological studies (Ness and Gebhart 1989; Woolf and Fitzgerald 1983), which demonstrated that selection of these depths provided a comparison of superficial neurons in laminae I-III with the deeper neurons of laminae IV-X in rats. Also, in the present study, histologic reconstruction of electrolytic lesions in superficial and deeper laminae of the spinal cord demonstrated that the depth of electrode penetration accurately reflected the location within the spinal cord.

A catheter was placed in the pericardial sac using a modification of the procedure described by Euchner-Wamser et al. (1994). The catheter was made of silicone tubing (0.020 ID, 0.037 OD, 14-16 cm length), and ~10 small holes were made in the distal 2 cm. A high midline thoracotomy (left costal cartilages of ribs 1-3) was made to expose the thymus gland and the heart. The thymus gland was opened on the midline, and the catheter was carefully inserted into the pericardial sac over the left ventricle. The catheter was fixed in place by suturing together the two thymus lobes and the layers of the chest wall. In testing the pericardial catheter, solutions were easily injected and removed via a 1-ml syringe connected to the catheter.

Cardiac receptors were stimulated chemically with a modified algogenic chemical mixture to excite both vagal and sympathetic afferent endings (Handwerker and Reeh 1991). The mixture contained bradykinin, serotonin, prostaglandin E2, histamine, and adenosine, all of which may be released during myocardial ischemia (Euchner-Wamser et al. 1994; Foreman 1999; Meller and Gebhart 1992). Drugs were individually dissolved in normal saline to a concentration of 10-3 M and kept frozen. On the day of an experiment, stock solutions were warmed and further diluted in normal saline to concentrations of 10-5 M (except adenosine, 10-3 M). The protocol for administering the mixture of algesic chemicals was to inject warm saline (0.2 ml) into the pericardial sac and withdraw after 60 s to determine volume effects, to inject 0.2 ml of the chemical mixture and withdraw after 60 s, and to use two saline flushes (0.2 ml each) for rinsing the chemicals within the pericardial sac. At least 20 min elapsed between each injection of chemicals.

The vagi and spinal cord were prepared for interrupting visceral afferent pathways. The left and right cervical vagus nerves were separated from the carotid artery and silk suture was looped around each nerve trunk. To determine if cervical spinal cells were excited by vagal afferents activated with chemical stimulation of the heart, the algogenic mixture was injected before and after the vagi were transected. The ipsilateral cervical vagus nerve was cut with a scissors after gently pulling the tie around the nerve; if the cell still responded to chemical stimulation of the heart, the contralateral cervical vagus nerve was cut. If the C1-C2 neuron still responded to chemical stimulation of the heart after bilateral vagotomy, the spinal cord was transected at C6-C7 to determine if the cell was activated by cardiac spinal (sympathetic afferent) input.

Cutaneous receptive fields of spinal neurons were tested for responses to innocuous brushing with a camel-hair brush, pressure with a blunt stick, and noxious pinching of skin and muscles with a blunt forceps. Neurons were categorized as follows: wide dynamic range (WDR) cells were excited by brushing the hair and had a greater response to noxious pinching of the somatic field; high-threshold (HT) cells were excited only by noxious pinching of the somatic field; low-threshold (LT) cells were excited primarily by hair movement. If a cutaneous receptive field was not found, moving joints (MJ) of the shoulder and forearm were tested. Outlines and descriptions of receptive fields were recorded manually for all neurons examined.

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 cells were studied. At the end of the experiment, the animals were euthanized with an overdose of intravenous pentobarbital sodium or euthanasia-5 solution. The cervical spinal cord was removed and placed in 10% buffered formalin solution. Frozen sections (55-60 µm) were cut to examine the locations of lesions. Laminae were identified using the cytoarchitectonic scheme of Molander et al. (l989). The segmental locations of C6-C7 transections also were confirmed.

Data are presented as means ± SE. Spontaneous activity of neurons was determined by counting activity for 10 s and then dividing by 10 to obtain impulses per second (imp/s). Changes in neuronal activity (imp/s) were calculated by subtracting the mean of 10 s of control activity from the mean of 10 s of the greatest response to stimulation. For each neuron, a given stimulus was considered effective if the activity changed >= 20% of control activity (Hobbs et al. 1992). Latency to response, time to peak response, time to recovery of control activity, and mean maximal response (imp/s) were measured after pericardial drug administration. Statistical comparisons were made using Student's paired or unpaired t-test and chi 2 analysis. Differences were considered statistically significant at P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Intrapericardial injection of the algogenic mixture changed the activity of 89 of 232 neurons recorded in the left C1-C2 spinal cord and 7 of 21 neurons recorded in the right side. Lesions made at recording sites were identified histologically for 77 neurons that responded to intrapericardial chemicals (Fig. 1). Three patterns of neuronal responses were observed: excitation (E), inhibition (I), and excitation-inhibition (E-I). The percentage of neurons that responded with each pattern was not different for 93 neurons recorded in C1 segment compared with 160 neurons recorded in C2 segment (Table 1). Moreover, no differences between response characteristics to intrapericardial chemicals were found for C1 neurons compared with C2 neurons (Table 2). Of 253 C1-C2 spinal neurons examined, 106 were recorded from superficial dorsal horn (depth, <0.35 mm) and 147 were recorded from the deeper spinal gray matter. Intrapericardial chemicals changed the activity of 20/106 (19%) neurons in the superficial laminae, whereas 76/147 (52%) neurons in the deeper spinal gray matter responded (Fig. 2, A and B). The proportion of responsive neurons in the superficial laminae was significantly less than in the deeper gray matter (P < 0.01).



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Fig. 1. Lesion sites of neurons recorded in C1-C2 spinal cord. Drawings of cervical spinal cord are from Molander et al. (1989). Black circles () represent neurons excited by intrapericardial chemicals. Triangles (black-triangle) represent neurons inhibited by intrapericardial chemicals. Squares () represent neurons excited/inhibited by intrapericardial chemicals. I-X, laminae; CCN, central cervical nucleus; IBN, internal basilar nucleus; LCN, lateral cervical nucleus; LSN, lateral spinal nucleus; Pyr, pyramidal tract.


                              
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Table 1. Responses of C1 and C2 spinal neurons responsive to intrapericardial chemicals


                              
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Table 2. Comparison of response characteristics of C1 and C2 spinal neurons excited or inhibited by intrapericardial chemicals



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Fig. 2. Comparison of superficial and deeper C1-C2 neurons responsive to intrapericardial chemicals. A: superficial neurons. E, excitatory response; I, inhibitory response; E-I, excitatory/inhibitory response; NR, neurons not responding to intrapericardial chemicals. B: deeper neurons. C: number of neurons excited by cardiac afferents. SL-E, short-lasting excitatory response neurons. LL-E, long-lasting excitatory response neurons. D: number of neurons inhibited by cardiac afferents. SL-I, short-lasting inhibitory response neurons. LL-I, long-lasting inhibitory response neurons.

Responses to intrapericardial chemicals

Intrapericardial injections of algogenic chemicals increased the average activity of 48/96 (50%) C1-C2 spinal neurons from 8.1 ± 1.3 to 21.6 ± 2.6 imp/s. These neurons were subdivided into two groups based on the time of recovery to control activity after chemicals were removed: neurons responsive to intrapericardial chemicals <50 s were classified as short-lasting excitatory (SL-E, n = 22) and neurons with responses >50 s were classified as long-lasting excitatory (LL-E, n = 26, Fig. 3, A and B). Table 3 compares the characteristics of SL-E and LL-E neurons responsive to intrapericardial chemicals. Time to reach the peaks of the responses in LL-E neurons was significantly longer than those in SL-E cells (P < 0.05), whereas the latency to onset of the evoked responses was not different. The duration of evoked responses after intrapericardial chemicals were removed was significantly shorter in SL-E neurons (17.3 ± 3.5 imp/s, range 0-49 s) compared with the responses of LL-E neurons (134.3 ± 13.8 imp/s, range 56-325 s, P < 0.01). Maximal excitatory change in activity, however, was not different for the two groups of neurons. No spontaneous activity was seen in 7/22 (32%) SL-E neurons and in 1/26 (5%) LL-E neurons.



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Fig. 3. Response patterns of C1-C2 spinal neurons to intrapericardial chemicals (IC) and saline injections. A: SL-E response. B: LL-E response. C: SL-I response. D: LL-I response. E and F: excitatory/inhibitory response.


                              
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Table 3. Classes of C1-C2 spinal neurons responsive to intrapericardial chemicals

Chemical injections in the pericardial sac reduced spontaneous activity of 41/96 (43%) neurons from 14.8 ± 2.3 to 6.5 ± 1.4 imp/s (44% of control). Spontaneous activity of these neurons (14.8 ± 2.3 imp/s, n = 41) was significantly higher than spontaneous activity of neurons excited by intrapericardial injection (8.1 ± 1.3 imp/s, n = 48; P < 0.02). Similar to the E neurons, I neurons responsive to intrapericardial chemicals were subdivided into two groups: SL-I (n = 18) and LL-I (n = 23) neurons (Fig. 3, C and D, and Table 3). Time to reach the peaks of inhibitory responses of SL-I neurons was significantly shorter than those in LL-I neurons (P < 0.01). Spontaneous activity of LL-I neurons slowly returned to control levels with an average time to recovery of 119.6 ± 14.1 s (range 51-302 s), which was significantly longer than SL-I neurons (20.7 ± 3.8 s, range 1-50 s; P < 0.01). Control spontaneous activities and maximal inhibitory responses were not different for SL-I and LL-I neurons.

Of 96 neurons that responded to intrapericardial chemicals, 7 deeper neurons had an E-I response. Six E-I neurons exhibited excitation followed by inhibition after chemicals were withdrawn (Fig. 3E), whereas another E-I neuron exhibited early inhibition before intrapericardial chemicals were removed (Fig. 3F). Characteristics of E-I neurons are given in Table 3.

A comparison of the response characteristics of superficial and deeper spinal neurons responsive to intrapericardial injections of the chemical mixture is presented in Table 4. In superficial gray matter, excitatory responses to cardiac afferent input were more likely to be LL-E than SL-E (Fig. 2C, P < 0.05), whereas no significant difference between LL-E and SL-E neurons was found in deeper laminae. Additionally, latencies to excitatory responses of superficial LL-E neurons were significantly shorter compared with LL-E neurons in deeper spinal cord (Table 4, P < 0.05). In contrast, no significant differences between the occurrence of SL-I and LL-I neurons were found in either superficial or deeper laminae (Fig. 2D), and no differences were found for other characteristics of neurons inhibited by intrapericardial chemicals (Table 4).


                              
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Table 4. Comparison of characteristics of superficial and deeper C1-C2 spinal neurons responsive to intrapericardial chemicals

Convergence of somatic receptive field and cardiac input

Somatic receptive fields were found for 226/253 (89%) C1-C2 neurons tested for their responses to intrapericardial chemicals; 98 neurons were superficial and 128 neurons were in the deeper dorsal horn. Comparisons of receptive field properties of neurons in superficial and deeper dorsal horn are presented in Fig. 4. The majority of superficial (73/98; 74%) and deeper (111/128; 87%) neurons received nociceptive inputs (HT and WDR) from somatic fields (Fig. 4, A and B). Superficial neurons were significantly less likely to have HT receptive fields compared with neurons in the deeper dorsal horn neurons (P < 0.01), whereas superficial neurons were significantly more likely to have WDR receptive fields compared with deeper neurons (P < 0.05). Furthermore superficial neurons were significantly more likely to have LT receptive fields (24/98; 24%) than the deeper dorsal horn neurons (7/128; 6%; P < 0.01). Additionally, 10 deeper neurons responded only to joint movement (MJ), whereas one superficial cell was an MJ neuron.



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Fig. 4. Comparison of characteristics of somatic field properties of somatic and cardiosomatic neurons in superficial and deeper spinal cord. A: all superficial neurons examined for cardiac input. B: all deeper neurons examined for cardiac input. C: properties of somatic and cardiosomatic neurons in superficial spinal cord. D: properties of somatic and cardio-somatic neurons in deeper spinal cord. LT, low threshold; WDR, wide dynamic region; HT, high threshold; MJ, moving joint.

Superficial and deeper neurons were separated into somatic or cardiosomatic groups based on the absence or presence of cardiac responses (Fig. 4, C and D). Superficial neurons excited by noxious somatic stimulation (HT and WDR) were more likely to receive only somatic input (54/73; 74%) than neurons of the deeper dorsal horn (50/111; 45%; P < 0.01). Furthermore HT and WDR superficial neurons with only somatic input were more likely to be classified as WDR (41/54; 76%) than neurons receiving convergent input from the heart (9/19; 47%; P < 0.05). It should be noted that both superficial and deeper cells with LT receptive fields responded only to somatic stimulation and not to cardiac afferent stimulation (Fig. 4, C and D). In addition, no differences in somatic field types were found for cardiosomatic neurons that were classified according to their responses to intrapericardial chemicals (Table 5).


                              
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Table 5. Somatic input onto C1-C2 spinal neurons responsive to intrapericardial chemicals

Stimulation of the somatic fields from the head, neck, and shoulder areas excited 85 of the 96 neurons that responded to intrapericardial chemical injections. Ten of the neurons did not have a detectable somatic field, and one I neuron was not tested for somatic input. A comparison of the sizes of the somatic fields revealed additional differences between neurons recorded in the superficial and deeper gray matter. The areas of somatic fields were measured and were assigned to the following categories: small (ipsilateral, long axis <= 3 cm), medium (ipsilateral, long axis >3 cm), and bilateral fields. Representative samples are illustrated in Fig. 5, A-C. For the superficial cardiosomatic neurons, 10/19 (53%) had small somatic fields and 4/19 (21%) had bilateral fields (Fig. 5D). In contrast, 19/61 (31%) of the deeper neurons had small fields and 28/61 (46%) had bilateral fields. These differences between somatic field sizes of superficial and deeper neurons were significant (P < 0.05). No differences were found for the medium-sized fields of the superficial (26%) and deeper (23%) neurons.



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Fig. 5. Comparison of somatic field size of superficial and deeper neurons responsive to intrapericardial chemicals. A: examples of small somatic fields. B: examples of medium fields. C: examples of bilateral fields. D: comparison of somatic field size of superficial and deeper neurons responsive to intrapericardial chemicals.

Effects of cervical vagotomy

To determine if upper cervical spinal cells were excited by chemical activation of cardiac vagal afferents, the chemical mixture was injected before and after the ipsilateral and then the contralateral cervical vagus nerves were cut. Transection of the ipsilateral vagus nerve interrupted cardiac noxious input to 8/30 (27%, 6 E, 2 I) neurons (Fig. 6). Examples of an E and an I neuron are shown in Fig. 7, A and B, respectively. No significant differences were found in response characteristics of E and I neurons (Tables 6 and 7, respectively) that still responded to intrapericardial chemicals after transection of the ipsilateral vagus nerve.



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Fig. 6. Diagram showing effects of cervical vagotomy and transection at C6-C7 segments on responses of C1-C2 neurons to intrapericardial injection of chemicals. ICV, ipsilateral cervical vagotomy. CCV, contralateral cervical vagotomy. A, abolished; R, responded; L, cell lost after intervention.



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Fig. 7. Examples of effects of cervical vagotomy and spinal transection on spontaneous activity and evoked responses of C1-C2 neurons to IC. A and B: a C1 neuron excited and a C2 neuron inhibited by intrapericardial chemicals. Their responses were abolished by ICV. C and D: a C2 neuron excited and a C2 neuron inhibited by intrapericardial chemicals. Their responses were abolished by sequential CCV (bilateral vagotomy). E: a C2 neuron excited by intrapericardial chemicals. Its response was abolished by transection at lower cervical spinal cord.


                              
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Table 6. Excitatory responses of C1-C2 neurons to intrapericardial chemicals after cervical vagotomy and C6-C7 transection


                              
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Table 7. Inhibitory responses of C1-C2 neurons to intrapericardial chemicals after cervical vagotomy and C6-C7 transection

For 22 (15 E, 7 I) neurons that still responded to cardiac noxious input after ipsilateral vagotomy, sequential transection of the contralateral vagus nerve (bilateral vagotomy) eliminated responses to intrapericardial chemicals in 3 E and 1 I neurons. Figure 7, C and D, shows responses of an E and an I neuron that were abolished by bilateral cervical vagotomy. Since four E and two I neurons were lost during contralateral vagotomy, eight E and four I neurons still responded to intrapericardial chemicals (Fig. 6). Tables 6 and 7 show a comparison of spontaneous activity and evoked responses of C1-C2 spinal neurons to intrapericardial chemicals after bilateral vagotomy. Although evoked responses of E neurons that still responded to cardiac afferent input after bilateral vagotomy (n = 8) were significantly lower than evoked responses before ipsilateral cervical vagotomy (n = 21, Table 6), no significant differences between pre- and postvagotomy responses were found in those 8 E neurons (8.9 ± 1.6 vs. 7.6 ± 2.2 imp/s, n = 8).

Effects of transection at C6-C7 segments

For 12 neurons (8 E and 4 I) that still responded to intrapericardial chemicals after bilateral vagotomy, transections at C6-C7 segments were performed to determine if these neurons were activated by cardiac spinal (sympathetic) input. A comparison of response characteristics of these neurons after spinal transection is shown in Tables 6 and 7; two neurons (1 E and 1 I) were lost during transection. Spontaneous activity of I neurons after transection at C6-C7 (n = 3) was significantly lower than control activity before or after ipsilateral vagotomy (n = 9 and 7, respectively, P < 0.05), whereas no differences in spontaneous activity of E neurons were found. Figure 7E shows an example of an excitatory response that was abolished by transection at C6-C7 segment of spinal cord. Spinal transection at C6-C7 abolished the evoked responses to intrapericardial chemicals in all E and I neurons tested. Thus responses of 10/22 (45%) neurons to intrapericardial chemicals depended on spinal (sympathetic afferent) pathways (Fig. 6).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Results of this study showed that intrapericardial injections of algogenic chemicals activated 38% of C1-C2 spinal neurons. Of this population, a significantly larger portion of the neurons in the deeper laminae responded to this chemical stimulus than neurons in the superficial laminae (52 vs. 19%). These neurons exhibited excitation (50%), inhibition (43%), and excitation/inhibition, which could be either short- or long-lasting. Both vagal and sympathetic afferent pathways were responsible for transmitting nociceptive information from the heart to upper cervical spinal neurons (Fig. 8). In addition, 84% of neurons responsive to intrapericardial chemicals also received convergent noxious somatic input from head, ears, neck, and shoulder areas. Superficial neurons tended to have smaller somatic receptive fields than neurons in the deeper laminae.



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Fig. 8. Neural pathways by which cardiac noxious stimulation activates C1-C2 spinal neurons.

Activation of cardiac afferents

To examine effects of cardiac noxious input on C1-C2 spinal neurons, intrapericardial administration of algogenic chemicals was used as a tool to intensify cardiac nociceptor responses to noxious stimuli. Previous studies have shown that a mixture of algogenic chemicals excites nociceptors in a skin-nerve preparation more potently and with less tachyphylaxis than a single substance (Handwerker and Reeh 1991) and excites cervical spinothalamic tract (STT) neurons (Chandler et al. 2000) and thoracic spinal neurons (Euchner-Wamser et al. 1994). Furthermore these algogenic chemicals excite both sympathetic and vagal afferents (Meller and Gebhart 1992). It is reasonable that stimulating cardiac/pericardial receptors with a mixture of chemicals would be more effective than single algogenic substances, although we did not compare the effects of individual compounds in this study.

Somatic receptive fields

In the present study, the majority of C1-C2 superficial and deeper neurons (81%) received nociceptive (HT and WDR) input from somatic fields. These results agree with studies in cervical, thoracic, or lumbosacral spinal neurons of rat and cat (Cervero and Tattersall 1987; Dado et al. 1994; Ness and Gebhart 1987). The present study showed that superficial neurons were significantly less likely to have HT and more likely to have LT receptive fields than the deeper dorsal horn neurons. However, the somatic field properties of superficial and deeper neurons that received convergent input from cardiac afferents were similar. Thus the differences observed in the total population are primarily attributed to superficial neurons that received only somatic input. These observations agree generally with the study by Cervero and Tattersall (1987), who reported that somatic neurons receive specific cutaneous inputs from LT mechanoreceptors or from nociceptors.

Superficial neurons responsive to cardiac input tended to have smaller somatic fields than deeper neurons. This organization is similar to that described for dorsal horn neurons responding to stimulation of splanchnic nerves in lower thoracic spinal cord in cats (Cervero and Tattersall 1987). However these results differ from those of Ness and Gebhart (1989), who showed that superficial dorsal horn neurons excited by colorectal distension have larger cutaneous convergent receptive fields than neurons in deeper spinal laminae of thoracolumbar spinal cord in rats. This difference may be due to differences in the definition of receptive field size and the preparation (e.g., spinal segments, recording electrodes, search stimulus for selection of neurons, anesthesia, etc.).

Cardiac pain during myocardial ischemia is referred to somatic areas on the chest and extends to the shoulder, arms, upper back, and neck and even to the head and lower jaw (Sampson and Cheitlin 1971). Ruch (1961) suggested that visceral pain is referred to somatic structures because visceral and somatic sensory inputs entering the same segments converge on the same neurons. Angina referred to the chest, shoulder, and arms generally is attributed to activation of sympathetic afferent fibers that enter upper thoracic spinal segments (Kuo et al. 1984; Sampson and Cheitlin 1971; White 1957); primate STT neurons in these segments are strongly excited by stimulation of cardiac sympathetic afferents (Blair et al. 1981; Hobbs et al. 1992) and primarily inhibited by vagal afferent input (Ammons et al. 1983). In contrast, afferent input from both vagal and cardiopulmonary sympathetic afferent fibers primarily excited C1-C3 STT cells in primates (Chandler et al. 1996, 2000) and spinal neurons in rats (Fu et al. 1992; Zhang et al. 1997). These neurons have excitatory somatic receptive fields often located on neck and jaw regions. The results of the present study are consistent with previous findings. In summary, the somatic response patterns of the upper cervical neurons provide further evidence for a neural mechanism of referred pain that originates in the heart but is perceived in the neck and head.

Responses to intrapericardial chemicals

Every spinal neuron recorded in the upper cervical segment was tested for its responsiveness to intrapericardial injections of algogenic chemicals. Results showed that these injections changed activity of 38% of the C1-C2 spinal neurons: 19% of the total number of neurons examined were excited, 16% were inhibited, and 3% were excited/inhibited. In contrast, a study in primates showed that algogenic chemical agents into the pericardial sac altered the activity of 68% of C1-C2 spinothalamic tract neurons; 55% of the total number of neurons examined were excited and 13% were inhibited (Chandler et al. 2000). One explanation for the differences between the two studies is that in monkeys recordings were exclusively from C1-C2 spinothalamic tract neurons (Chandler et al. 2000). Upper thoracic STT cells in monkeys (Ammons et al. 1985) and spinoreticular tract cells in cats (Bolser et al. 1989) also are primarily excited by chemical stimulation of cardiac receptors. It is important to note that unidentified thoracic spinal neurons in cats sometimes were inhibited by chemical stimulation of the heart, whereas identified spinoreticular neurons were either excited or unaffected by a chemical stimulus. The mixed responses of unidentified neurons in cats (Bolser et al. 1989) agree with the variability of responses seen in the present study in rats. It is not surprising that a functionally heterogeneous population of neurons produced variable responses to a uniform stimulus, since they might serve as excitatory or inhibitory interneurons as well as neurons projecting to various brain nuclei or to more caudal spinal segments (Clement et al. 2000; Malick et al. 2000; Qin et al. 1999; Zhang et al. 2000). It also should be pointed out that species differences might account for some of the disparities between the results from rats, cats, and primates.

Functional characteristics of response patterns

The long- and short-lasting response patterns of C1-C2 spinal neurons to intrapericardial administration of algogenic substances were similar to the responses reported for the small population of thoracic spinal neurons in a previous study in rats (Euchner-Wamser et al. 1994). We propose that the long-lasting responses most likely play a role in visceral nociception. The long-lasting responses were most closely correlated with the long-lasting responses observed for chemosensitive vagal and sympathetic afferent fibers and dorsal root ganglion cells that are predominately unmyelinated (Armour et al. 1994; Baker et al. 1980; Kaufman et al. 1980; Nerdrum et al. 1986). Because chemosensitive, and not mechanosensitive, afferent neurons are sensitized with prostaglandins (Nerdrum et al. 1986), intrapericardial injections of the mixture of algogenic chemicals containing PGE2 might have enhanced the responses in LL-E or LL-I cells. In contrast to the nociceptive role of the LL types of neurons, the SL-E or SL-I neurons might have received inputs from afferent fibers that are more responsive to mechanical stimulation and are more likely fibers in the A-delta range (Baker et al. 1980). Mechanosensitive sympathetic afferents respond to chemical injections of bradykinin, but the responses are shorter lasting and are not sensitized with PGE2 (Nerdrum et al. 1986). Mechanosensitive vagal afferents respond to bradykinin secondary to a change in hemodynamics, but they do not respond to direct stimulation with bradykinin (Kaufman et al. 1980).

Response patterns of superficial and deep neurons

Characteristics of responses to intrapericardial injections of algogenic chemicals were different for C1-C2 spinal neurons located in superficial or deeper laminae. Latencies to the excitatory responses of superficial neurons were significantly shorter than latencies to the responses of cells in deeper laminae. Additionally, in the superficial laminae, excitatory responses of neurons to cardiac afferent input were more likely to be LL-E than SL-E, whereas no significant differences were found in responses of neurons in the deeper laminae. Quantitative differences between superficial and deeper viscerosomatic neurons also have been reported in the thoracolumbar spinal segments of rats when colorectal distension was used as the noxious visceral stimulus (Ness and Gebhart 1989). We suggested above that the SL-E responses of spinal neurons might be due to the activation of mechanosensitive afferent fibers. Since most neurons in the superficial laminae were unresponsive to intrapericardiac chemicals, it is possible that very few mechanosensitive afferent fibers terminate in the superficial dorsal horn. This possibility is supported also by evidence that the excitatory responses of SL-E neurons tended to be less robust than the responses of SL-E neurons in the deeper dorsal horn. This observation agrees with that of Ness and Gebhart (1989), who found that maximum responses for short-latency abrupt neurons in deep lamine were significantly greater than for superficial neurons.

Comparison to electrical stimulation of cardiopulmonary afferent fibers

Electrical stimulation of cardiopulmonary sympathetic and cervical vagal afferent fibers in rats produces excitatory responses in most ipsilateral C1-C3 cells (Fu et al. 1992; Zhang et al. 1997). In contrast, stimulation of cardiac receptors with intrapericardiac chemicals produced approximately equal numbers of excitatory and inhibitory responses in C1-C2 spinal neurons. One possible explanation for these differences is the types of afferent fibers activated by these different forms of stimulation. Injections of the algogenic chemicals most likely excited only chemosensitive and polymodal afferent fibers from the heart that travel in both the vagal and sympathetic afferent pathways (Meller and Gebhart 1992; Nerdrum et al. 1986). Electrical stimulation of an individual nerve trunk simultaneously excites various types of nerve fibers, including mechanical, thermal, and chemosensitive afferents, and also generates a relatively synchronous volley to the spinal neurons. The single route of transmission, the synchronous volley in the afferent fibers, and the mixture of fiber types might account for the predominately excitatory effects of electrical stimulation. Another explanation for the differences might be the bilateral activation of vagal and sympathetic afferents with chemical stimulation. Electrical stimulation of the vagus nerve ipsilateral to C1-C2 spinal neurons primarily produced excitation, whereas inhibition occurred in ~50% of C1-C2 neurons that responded to contralateral vagal input (Fu et al. 1992). Thus in the present study, it is possible that bilateral activation of afferent fibers with chemical stimulation could result in either excitation or inhibition depending on the dominance of inputs from ipsilateral or contralateral pathways.

Spinal organization

Intrapericardial chemicals activated 52% of the deeper C1-C2 spinal neurons in the present study, which was significantly more than the 19% of superficial neurons activated by the chemical stimulus. These findings differed from those of Cervero and Tattersall (1987), who found that ~65% of both the superficial and deeper neurons in the lower thoracic spinal gray matter of cats responded to electrical stimulation of splanchnic nerves. The differences may be due in part to the use of electrical stimulation of splanchnic afferent fibers as well as to the segmental location of the cells and the different species. We have shown previously that electrical stimulation of cardiopulmonary sympathetic afferent fibers excites all thoracic spinothalamic tract cells, but <70% of the cells are excited with chemical stimulation of the heart (Ammons et al. 1985). The lower percentage of neurons activated in C1-C2 segments compared with neurons in lower thoracic segments might be due partially to the chemical stimulus used in the present study.

The different response characteristics also might be explained by variations in organization of visceral afferent fibers in upper cervical segments compared with thoracic segments. Sympathetic cardiac afferent fibers enter primarily in the T2-T6 spinal segments and not the upper cervical segments (Hopkins and Armour 1989; Kuo et al. 1984; White 1957). The specific ascending pathway by which cardiac sympathetic afferent input reaches upper cervical segments has not been resolved. It is possible that cardiac afferent information reaches C1-C2 neurons via ascending pathways from thoracic to upper cervical spinal cord (Molenaar and Kuypers 1975, 1978). We have shown in a previous study that cardiopulmonary sympathetic input to C1-C3 segments travels bilaterally in the ventrolateral quadrants of the rat spinal cord but does not involve dorsal columns (Zhang et al. 1997). Furthermore stimulation of vagal afferent inputs produces different effects in high cervical segments compared with thoracic segments. Stimulation of vagal afferent fibers primarily excites C1-C2 STT cells in primates and spinal neurons in rats but suppresses the activity of primate C4-S1 STT neurons and rat lumbosacral cells (Ammons et al. 1983; Chandler et al. 1991, 1996; Fu et al. 1992; Hobbs et al. 1989; Ren et al. 1989). Different patterns of termination in upper cervical segments for cardiac information might partially account for different effects observed in C1-C2 neurons compared with responses to visceral inputs in thoracic neurons. No anatomical studies have been done to elucidate the terminations of cardiac afferent information in the upper cervical segments.

Relative contribution of vagal and sympathetic afferents

Intrapericardial administration of algogenic chemicals most likely excites both vagal and sympathetic afferent fibers (Meller and Gebhart 1992); therefore both of these pathways were candidates for transmission of nociceptive cardiac information to C1-C2 neurons. In this study, bilateral vagotomy abolished the responses of 12/22 (55%) neurons that were not lost during the procedure, and transection of C6-C7 segments abolished the responses of the remainder of the neurons (45%). These data are consistent with the concept that both vagal and sympathetic inputs influence processing of nociceptive sensory information in C1-C2 neurons of rats. These results differ, however, from those reported by Chandler et al. (2000), which showed that vagal fibers were the primary pathway for transmission of nociceptive information from the heart to C1-C2 STT cells of the primate. The differences may be due to specific selection of STT cells as opposed to the heterogeneous population examined in the present study. In addition, there may be a species difference. The evidence that vagal afferent fibers transmit nociceptive information from the heart to the upper cervical segments agrees with the speculation from clinical observations that the vagus nerve may be involved with the perception of cardiac pain referred to the neck and jaw (Foreman 1999; Lindgren and Olivecrona 1947; Meller and Gebhart 1992; White and Bland 1948).

In summary, this study revealed quantitative differences in the responses of C1-C2 superficial and deeper spinal neurons to noxious chemical stimulation of cardiac afferents. Furthermore both cardiac vagal and spinal (sympathetic) afferents transmitted nociceptive information to upper cervical spinal neurons. Spinal neurons in C1-C2 segments, particularly deeper neurons, participate in integrating cardiac nociception.


    ACKNOWLEDGMENTS

The authors thank Drs. J. P. Farber and R. W. Blair for helpful comments as well as D. Holston and C. J. Jou for excellent technical assistance and preparation of the figures.

This work 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, PO Box 26901, Oklahoma City, OK 73190 (E-mail: robert-foreman{at}ouhsc.edu).

Received 20 September 2000; accepted in final form 18 December 2000.


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ABSTRACT
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0022-3077/01 $5.00 Copyright © 2001 The American Physiological Society