EXPERIMENTAL

NEUROLOGY

Responses

49,

466-486 (1975)

of Sympathetic Postganglionic Neurons Peripheral Nerve Stimulation in the Pigeon (Columba livia)

to

ROBERT B. LEONARD AND DAVID H. COHEN l Department

of Physiology,

University of Virginia,

Charlottesville, Received

Virginia

.ScIrool of Medicine,

22901

June 27,1975

Reflex changes in heart rate and arterial blood pressure can be elicited in pigeons with high cervical transection by stimulation of brachial or lumbosacral peripheral and spinal nerves. This extends the phenomenon of spinally mediated, somatosympathetic reflexes to another vertebrate class. In a preliminary attempt to explore the spinal circuitry mediating these reflexes, the responses of single sympathetic postganglionic neurons were studied during spinal and peripheral nerve stimulation. With stimulation and recording at the same spinal segment, calculation of the central delay suggests the segmental reflex circuitry may be relatively simple, possibly trisynaptic. As the distance between stimulating and recording sites increases, postganglionic neuronal responsiveness decreases and becomes more variable. However, there is clear evidence that lumbosacral afferents can activate postganglionic neurons at brachial levels, indicating an effective propriospinal circuitry for somatosympathetic reflexes. Experiments on birds with intact spinal cords demonstrate that these spino-spinal pathways are also functional in the intact animal. While the segmental reflex is not different in the intact bird, the propriospinal pathways do behave somewhat differently, possible suggesting tonic central control.

INTRODUCTION We have been developing visually conditioned heart rate change in the pigeon as a vertebrate model system for cellular studies of learning (6)) 1 Supported by NSF grants GB-13816X and GB-35204X and a grant from the Benevolent Foundation of Scottish Rite Freemasonary, Northern Jurisdiction, U.S.A. R. B. Leonard was supported by a fellowship from the Benevolent Foundation of Scottish Rite Freemasonary and P.H.S. Training Grant HL-05815. D. H. Cohen was partially supported by P.H.S. Research Career Development Award HL-16579. This report is based in part on a doctoral dissertation submitted to the University of Virginia by R. B. Leonard. Address reprint requests to: David H. Cohen, Department of Physiology, University of Virginia School of Medicine, Charlottesville, Virginia 22901. 466 Copyright All rights

0 of

1975

by Academic

Press.

reproduction in any form

Inc. reserved.

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and a major phase of this has involved mapping the neural pathways that participate in establishing the conditioned response. To date, this effort has been largely directed toward delineating the visual pathways transmitting the conditioned stimulus information and the descending pathways mediating expression of the cardioacceleratory conditioned response ( 7). However, we have recently initiated analysis of two other segments of the system, the somatosensory pathways transmitting the unconditioned stimulus information (foot-shock), and the pathways mediating the unconditioned heart rate change (cardioacceleration). With respect to the unconditioned stimulus pathway, the relevant dermatomes, peripheral nerves, fiber groups, and dorsal roots have been identified, thus providing a description of the peripheral components of this pathway (23). Further, the spinal distribution of the participating dorsal roots has been anatomically described (25). While little is known directly regarding the unconditioned response pathway, at the periphery it undoubtedly overlaps to a large extent with the final common path for the conditioned response for which a rather detailed description is available (7, 8). It has been shown that this conditioned response is mediated exclusively via the extrinsic cardiac nerves, and, though both the sympathetic and vagal cardiac innervations contribute, the sympathetic component is apparently of greater importance (9). The question addressed in this report is how the coupling between the unconditioned stimulus and unconditioned response pathways occurs. Stated differently, the problem is to determine the circuitry by which lumbosacral somatic afferents gain access to sympathetic preganglionic neurons. This issue is germane to current general interest in somatosympathetic reflexes (21, 30), and the literature in this area indicates that such reflexes are mediated at various neural levels including the spinal cord (3, 31). As a starting point, we chose to focus on the spino-spinal somatosympathetic reflexes, since specification of the involved segmental and propriospinal circuitry would establish a foundation for delineating the more complexly organized pathways involving suprasegmental structures. It will be shown here that spino-spinal somatosympathetic coupling occurs in the bird just as in mammals, including reflex cardioacceleratory and pressor responses. Given this, experiments are then reported which provide an initial exploration of the involved spino-spinal circuitry by describing postganglionic neuronal responses to peripheral nerve stimulation in intact and spinally transected animals. METHODS All experiments involved White Carneaux pigeons which were 2-6 months of age and weighed 400-650 g.

(CoZzamba Zivia)

468

LEONARD

Dcntortstration

of Spine-Spinal

AND

COHEN

Somatosynpathetic

Reflexes

Surgical Preparation. Three birds were initially used to explore the possibility of spino-spinally mediated cardioacceleration ; additional observations were then made on the numerous animals used for the electrophysiological experiments described below. Only birds which survived spinal transection with active spinal reflexes and mean arterial blood pressures exceeding 60 mm Hg were studied. For spinal transection, anesthesia was induced with sodium pentobarbital (35 mg/kg, ip) . Blood pressure was measured from a brachial artery with a Statham P23Dc pressure transducer. To aid in maintaining arterial blood pressure, 6% dextran in pigeon physiological salt solution (17) was infused into a brachial vein at 1.0-1.5 ml/hr. In some cases norepinephrine was also delivered in the infusion fluid at 1-2 pg/kg/hr. To prevent movement during transection and subsequent stimulation, the animals were immobilized with Flaxedil (2-3 mg/hr) or with a combination of Flaxedil ( 1 mg/hr) and Metubine Iodide (0.1 mg/hr) delivered in the infusion fluid. They were ventilated with a constant-volume respirator; body temperature was maintained with either a thermostatically-controlled heating pad or an infrared lamp ; and heart rate and blood pressure were monitored throughout. A laminectomy was performed to expose the cervical 3-4 intersegment, and the spinal cord was slowly transected over a longitudinal extent of 1 mm. [ Huber’s (IS) nomenclature for the pigeon spinal segments is used throughout.] A small amount of tissue was cauterized by passing current through an Insl-X coated, stainless, steel insect pin (#OO), and the cauterized tissue was then gently aspirated. This sequence of cauterization and aspiration was repeated until the cord was completely transected. (Preliminary experiments indicated that most birds survive this procedure with stable arterial blood pressures.) Following transection, the wound edges and head-holder pressure points were liberally and repeatedly infiltrated with 1% lidocaine hydrochloride, and general anesthesia was discontinued. In most cases the segment of the spinal cord including the transected region was removed at the termination of the experiment, fixed in 10% formalin, embedded in paraffin, and serially sectioned longitudinally at 20 pm. Every section was then stained with a modified Kliiver-Barrera procedure (24), and the completeness of the transection was verified. Stimdation. The sciatic nerve was exposed by opening the synsacrum at the level of the hip. It was freed of connective tissue and mounted on 70% platinum-30% iridium hook electrodes at a distance of 1.7-2.0 cm from the spinal cord. The nerve was then covered with a mixture of

SOMATOSYMPATHETIC

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469

petroleum jelly and mineral oil, and was stimulated at 60 Hz with monophasic, 0.5-msec pulses from either a Nuclear-Chicago Constant-Current Stimulator or a Grass S8 Stimulator with SIU 5 Stimulus Isolation Units. Arterial blood pressure, instantaneous heart rate, and timing marks were displayed on a Grass Model 7 Polygraph. Observations of reflex autonomic responses were made 2-36 hr following transection. Respartses

of PastgangIianic

Newom

Surgical Preparation. Fifteen animals surviving spinal transection with active spinal reflexes and mean arterial blood pressures exceeding 60 mm Hg were studied. The general preparation and transection procedures were as described above. In addition, however, a dorsal midline incision was made along the back. The pectoral girdle muscles were cut at their origins, and the muscles along the dorsal and transverse vertebral processes were removed. Steel plates approximately 1.3 cm in length were fastened to the fused thoracic vertebral processes with alpha cyanoacrylate adhesive (Aron Alpha). A flat head screw was then fastened across the plates, and attaching this screw to the stereotaxic frame fixed the vertebral column rigidly. The brachial plexus was exposed by reflecting the scapula, separating the girdle muscle, and removing the cervical floating rib and associated musculature. Spinal nerves 13 and 14 were largely cleared of connective tissue from the dorsal root ganglion to the anastomosis of the brachial plexus and were mounted on platinum-iridium hook electrodes approximately 1 cm from the spinal cord. The sciatic nerve was prepared as described above, and all nerves were covered with a mixture of petroleum jelly and mineral Oil. The sympathetic ganglion associated with the fourteenth spinal segment was exposed beneath the ventral root. [At cervical and thoracic levels in the pigeon the ganglia are applied directly to the ventral surface of the roots and nerve at or slightly distal to the position of the dorsal root ganglion (22).] This ganglion is the primary source of cardioaccelerator fibers in the pigeon (26). Its caudal portion was freed of most connective tissue, but the innermost layer surrounding the ganglion was cleared only sufficiently to allow microelectrode penetration. In most experiments, penetrations were made at the dorsolateral aspect of the ganglion at its caudal pole, and the microelectrode travelled ventromedially and rostrally through the ganglion. At the termination of several experiments the completeness of transection was verified histologically as described above. Fifteen other birds with intact spinal cords were also studied. These were treated in much the same manner as the spinally transected animals,

470

LEONARD

AND

COHEN

except anesthesia was induced and maintained with urethane (1.5 g/kg, ip), and blood pressure was not monitored. Stimulation and Recording. For intact animals, recording was initiated upon completion of the surgical preparation, while for spinally transected animals, at least 5 hr was allowed to elapse between completion of the surgery and initiation of recording. Peripheral and spinal nerves were stimulated with monophasic, 0.5-msec pulses. The nerves were stimulated continuously at < 0.2 Hz during electrode penetrations using intensities sufficient to activate all components of the compound action potential (23). Two nerves could be alternately stimulated continuously, and the specific combination stimulated was varied throughout the experiment. The activity of single postganglionic neurons was recorded with 4 M NaCl micropipettes having DC tip resistances of 5-20 Ma. Differential amplification was used to reduce EKG signals, and the reference electrode was an Ag/ AgCl ball placed close to the sympathetic ganglion. Conventional techniques were used for further amplification and display. Postganglionic

Field Potentials

Evoked

by Pregaflglionic

Sti+nulation

Three birds, anesthetized with urethane as above, were prepared for stimulation of the column of Terni [preganglionic cell column (26) ] and recording in sympathetic ganglion 15. Spinal segment 15 was exposed by a laminectomy, and sympathetic ganglion 15 was prepared as described above for ganglion 14. Bipolar stainless steel electrodes were used for spinal stimulation. These consisted of electrolytically sharpened, #00 insect pins coated with Insl-X and having tip separations of 1.0-1.5 mm. They were oriented longitudinally by the midline and lowered to a position ventral to the column of Terni. Monophasic pulses were delivered as the electrode was moved dorsally through the preganglionic cell column. Field potentials in sympathetic ganglion 15 were recorded with 4M NaCl micropipettes having DC tip resistances of l-3 Ma. They were differentially amplified and averaged using a DEC PDP-12 computer. Ten or more evoked field responses were averaged at each dorsoventral position of the spinal stimulating electrode. RESULTS Evideme

for Spine-Spitzally

Mediated

Somatosympathetic

Reflexes

Figure 1A illustrates heart rate and blood pressure responses to sciatic nerve stimulation several hours following spinal transection. Despite high cervical transection, it is clear that reflex cardioaccelerator and pressor responses occur, though of lower magnitude than those observed in the intact animal (23). Furthermore, these response magnitudes are graded

SOMATOSYMPATHETIC

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FIG. 1. Reflex heart rate and blood pressure responses in a spinally transected pigeon before and after right cardiac nerve transection. (A) The response to sciatic nerve stimulation (SOO-msec train of 0.5 msec pulses at 60 Hz) before cardiac nerve transection. (B) The response to such stimulation after right cardiac nerve transection. Top trace, arterial blood pressure in mm Hg; middle trace, I-set time marks; bottom trace, instantaneous heart rate in beats/min. The heavy bar on the time base denotes stimulus duration.

with stimulus intensity, and it appears that effective stimulus intensities are below those required for C-fiber activation (23). The time required for somatic reflexes to recover from spinal shock varied from less than 1 hr to 3 hr, and somatically elicited autonomic reflexes could not be detected during this period. Somatosympathetic reflexes reappeared after vigorous flexion-withdrawal and continued to become more active over a period of l-2 hr. In order to demonstrate that the spino-spinal reflex tachycardia was mediated by the cardioaccelerator nerve rather than release of adrenal catecholamines, the main branch of the right cardiac nerve was sectioned in several birds. Figure 1B shows the effect of such cardiac nerve transection on the reflex tachycardia at the same sciatic nerve stimulation intensity used for Fig. 1A. The response magnitude is clearly reduced markedly, and the pretransection latency of 1.5 set is increased to 3.5-4.0 sec. Postganglionic Unit Responsesin Spinal Animuls General Responsiveness.Most action potentials were biphasic negativepositive in conformation with a dominant negative phase; however, both triphasic and biphasic waveforms with dominant positivity were recorded. The spike durations were long, the dominant portion of the waveform

472

LEONARD AND COHEN

sometimes exceeding 2 msec. A large majority of the units were spontaneously active; however, their frequencies were low ( < l/set) and often irregular, with periods of silence followed by periods of low frequency activity. While the possibility of a correlation between this pattern and the cardiac or respiratory cycle was not systematically examined, no obvious relationships were observed. Although the number of spontaneously active units unresponsive to stimulation of any nerve was not quantitatively determined in all experiments, when examined it was found to be 45%. In responsive units single nerve shocks usually elicited only a single spike, although an occasional unit responded with two spikes. In addition, many units did not respond to every stimulus, and the number of response failures increased markedly with stimulation frequencies above 0.2 Hz. With stimulus trains of higher frequency most units would respond to only the first few stimuli. Consequently, the frequency used in searching for units or determining their responsecharacteristics was < 0.2 Hz. A total of 61 units responding to stimulation of one or more nerves was recorded in spinal animals. Of this total population, only 29 were tested with stimulation of all three nerves. Failure to hold units through the entire protocol due to instability of the ganglion was the main reason; additionally, in some experiments not all of the nerves were prepared for stimulation. To assesspossible sampling bias this might have introduced, we compared the proportion of units responding to stimulation of each nerve in (a) the population tested with all three nerves, and (b) the population tested with stimulation of only one or two nerves. This comparison indicated that a significantly lower percentage of units was responsive to stimulation of nerve 13 (x’( 1) = 9.3, P < O.OI), and the sciatic nerve (x’( 1) = 4.9, P < 0.05) in the population tested with all three nerves. Thus, there appears to have been a higher probability of locating units unresponsive to stimulation of a particular nerve in experiments where all three nerves were stimulated. (In these experiments, the increased time spent at each recording site in all likelihood increased the probability of locating a unit responsive to stimulation of at least one nerve, as well as the probability of locating units without spontaneous activity.) However, since these two populations did not differ in other responseecharacteristics, such as latency, it would seem that the bias was only toward sampling unresponsive units, and that the populations of responsive units were equivalent. Of the population tested with stimulation of all three nerves, 76% responded to nerve 14, 66% to nerve 13, and 41% to sciatic nerve stimulation. There was no difference between the proportion of units responsive to stimulation of nerves 13 and 14. However, significantly more units responded to nerve 14 than to sciatic stimulation (x2( 1) = 5.7, P < 0.02).

SOMATOSYMPATHETIC

REFLEXES

473

More units also responded to nerve 13 than to sciatic stimulation, but this difference was not statistically significant. Response Latency. There were considerable differences among units in response latency to stimulation of any given nerve. Figure 2 shows histograms of the number of units responding at various latencies to stimulation of each nerve. The mean latency for each was: nerve 14, 29.3 msec, range 3.6-72.3 msec; nerve 13, 41.9 msec, range 13.3-73.5 msec; sciatic nerve, 50.1 msec, range 18.kWO.l msec. A trend toward increased mean latency with increased separation of stimulation and recording sites is evident and is bettpr shown in Fig. 3. The latency difference between responses to stimulation of nerves 13 and 11 is significant [t (65) = 2.63, P < 0.051, as is the difference between nerve 11 and the sciatic nerve [t(52) = 3.61, P < 0.05]. However, the difference for nerve 13 and the sciatic nerve is not statistically significant. In the spinal animals, ten units were recorded that responded to stimulation of all three nerves, and Fig. 3 illustrates the responses of one such unit. The mean latencies of these units are shown in Fig. 3, and they do not differ from those of the population responding to stimulation of any nerve. However, the population responding to stimulation of all three nerves does

FIG. 2. Latency nerve stimulation Latencies to nerve

distributions of the responses of ganglion 14 neurons to somatic in spinal animals. (A) Latencies to nerve 14 stimulation, (B) 13 stimulation. (C) Latencies to sciatic nerve stimulation.

474

LEONARD

AND

14

COHEN

13

SC

NERVE

FIG. 3. Mean response latencies to somatic nerve stimulation in spinal animals. Solid bars indicate mean latencies of units responding to stimulation of each nerve. Lined bars indicate mean latencies of units that responded to stimulation of all three nerves. The standard error is shown by the vertical line above each bar. SC = sciatic nerve.

differ from the general population, in that there are no significant differences among responselatencies to stimulation of the various nerves. With repetitive stimulation at 0.2 Hz, considerable latency variability was observed in some units, as well as response failures. Furthermore, units with longer responselatencies to stimulation tended to show greater variability. Although it was originally thought that units responding to sciatic stimulation showed greater variability, analysis of the coefficients of variation of units responding to all three nerves failed to confirm this. The proportion of response failures should give another indication of response variability and driving security. While the greatest difference in failure rate was seen comparing nerve 14 with sciatic nerve stimulation, this difference did not reach statistical significance. Postganglionic Unit Response in Animals with Intact Spinal Cords General Responsiveness. The spike conformations and spontaneous activity were similar to those observed in spinal animals. Again, most units reponded with only a single spike, though there was a slight increase in the number responding with two spikes. Many units responded with a second spike at long latencies when nerve 13 or 14 was stimulated, and other units responded to sciatic stimulation at long latencies only (> 2.50 msec). Figure 5 illustrates this for sciatic nerve stimulation with a multiple-unit recording from ganglion 14. The response to a single nerve shock is characterized by two bursts of unit activity. First, low amplitude spike activity and a broadening of the baseline are evident at 70-125 msec, with a large amplitude unit responding at 120 msec. Second, a larger responseecan be

SOMATOSYMPATHETIC

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475

C

FIG. 4. Example of a long latency unit in ganglion 14 that responded to stimulation of all three somatic nerves. (A) Response to nerve 14 stimulation. (B) Response to nerve 13 stimulation. (C) Response to sciatic nerve stimulation. The horizontal calibration indicates 10 msec and the vertical calibration 100 pV.

seen at 210-360 msec, with two large unit responsesat 285 and 335 msec. This second response is not observed in spinal animals, suggesting supraspinal mediation. A total of 61 units responding to stmulation of one or more nerves was recorded in intact animals. Of this total population, 49 were held long enough to test their response to all three nerves. This increase over 29 such units in the spinal animals resulted from a modification in the experimental protocol where this information was obtained at the expense oE the number of response observations. In addition, all three nerves were prepared for stimulation in all experiments in the intact animals. The proportion of units responding to stimulation of each nerve was equivalent in the overall population of 61 units and the population of 49 units evaluated to all three nerves. The percentage of Lmits responding to stimulation of each was: nerve 14, 59% ; nerve 13, 71%; sciatic nerve, 24%. The proportion for the sciatic nerve differs significantly from those for both nerve 13 [x?(l) = 23.2, P < O.OOl] and nerve 14 [x’( 1) = 12.3, P < 0.0011, while the difference between 13 and 14 is not significant. Response Latency. Figure 6 shows the latency distributions for units responding to stimulation of each nerve. The mean latencies were : nerve 14, 33.9 msec, range 3.7-80.7 msec; nerve 13, 36.3 msec, range 5.3-87.2 msec; sciatic nerve, 78.2 msec, range 53.6-115.2 msec. As in spinal animals there

476

LEONARD

FIG. 5. Multiple unit response sciatic nerve shock. The vertical 50 msec.

AND

COHEN

from sympathetic ganglion 14 in response calibration is 50 pV, and the horizontal

to a single calibration

is a trend toward increaseed latency with increased distance between stimulation and recording sites (Fig. 7). The mean response latency with sciatic nerve stimulation is significantly longer than that to stimulation of either nerve 13 [t(54)= 5.07, P < 0.051 or nerve 14 [t(42)= 5.13, P < 0.051. The difference between nerve 13 and 14 is not statistically significant. Six units that responded to stimulation of all three nerves were recorded in the intact animals. While a trend toward increasing latency is suggested in Fig. 7, the differences among nerves are not significant. In contrast to the spinal animals, a comparison between this group and the general population indicates a significantly long latency to stimulation of nerve 13 [t(47) = 2.84, P < 0.051 or nerve 14 [t(35) = 2.57, P < 0.051 in the units responding to stimulation of all three nerves. A comparison of the number of response failures to repeated stimulation was undertaken for the total population as one indication of response

FIG. 6. Latency nerve stimulation Latencies to nerve

distributions of the response of ganglion 14 neurons to in intact animals. (A) Latencies to nerve 14 stimulation. 13 stimulation. (C) Latencies to sciatic nerve stimulation.

somatic (B)

NERVE

FIG. 7. Mean response latencies to somatic nerve stimulation in intact animals. Solid bars indicate mean latencies of units that responded to stimulation of all three nerves. The standard error is shown by the vertical line above each bar. SC = sciatic nerve.

and driving security. There was a significant increase in the proportion of failures in responseto sciatic nerve as compared to nerve 14 stimulation [x?(l) = 5.3, P < 0.051. While there were also more response failures with the sciatic nerve than nerve 13 stimulation, this difference was not statistically significant, nor was the comparison of nerves 13 and 14. A Further Obsermtion on Latemy Variability. Although the objective in these experiments was to obtain extracellular data on postganglionic neurons, in several instances, cells were penetrated and held sufficiently long to observe several responsesto stimulation of one or more nerves. Since the electrodes had rather large tip diameters and were filled with NaCl, the results of these intracellular penetrations must be viewed with extreme caution, and, in fact, most cells deteriorated rather rapidly. However, given these qualifications, such data were suggestive of one possibly important source of response latency variability. Figure 8 illustrates one such penetration in an animal with an intact spinal cord. Panel A shows the response to sciatic nerve stimulation and indicates considerable PSP summation before firing threshold is reached, including an instance of response failure. Panel B shows responsesof the same unit to stimulation of nerve 13. In the lower superimposed traces, a difference in the latency to the first spike is evident. In comparing the upper and lower traces of B, the second spike appears to have a longer

variability

latency

in the lower

trace,

although

the synaptic

potential

latencies

are

approximately the same. In the other trace of the superimposed pair the second spike fails, but the synaptic potentials occur at approximately the same latency. Similar observations of a spike occurring at variable latencies

478

LEONARD

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COHEN

FIG. 8. Intracellular record of the response of a postganglionic neuron (ganglion 14) to stimulation of two somatic nerves. (A) Response to sciatic nerve stimulation. Lower sweep triggered by a spontaneous action potential. (B) Several responses to nerve 13 stimulation. The horizontal calibration indicates 20 msec for A and B. The vertical calibration indicates 10 mV for A, and 5 mV for B.

on a relatively constant latency EPSP were made in several units responding only once to a single stimulus. These observations are suggestive that the variability imposed at presynaptic to postganglionic synapses contributes significantly to the extracellularly determined response variability, though the limitations of these intracellular recordings must but be recognized. Conzparison of Spinal afld Intact Results General Responsivenessof Postganglionic Units. The proportion of units responding to stimulation of each nerve was compared in spinal and intact animals for the subpopulation of units tested to all three nerves. There was no difference in the proportion of units responding to either nerve 13 or 14. On the other hand, there was a significant decrease in the proportion responding to stimulation of the sciatic nerve in intact animals [x2( 1) = 3.8, P < 0.051. Response Latency. A similar comparison was undertaken on the mean response latencies to stimulation of each nerve. The mean latencies with stimulation of nerves 13 and 14 were similar in intact and spinal animals (cf. Figs. 3 and 7). However, the increased latency to sciatic nerve stimulation in intact animals was significant [t(36) = 3.73, P < 0.051. The mean latencies of the subpopulation of units that responded to stimulation of all three nerves in each preparation were also compared. The latencies

SOMATOSYMPATIIE’~IC

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REFLEXES

FIG. 9. Ganglion 1.5 response (lo-sweep average) to spinal stimulation in the region of the column of Terni of segment 15. Top trace: Response evoked with stimulating electrodes in the dorsal column 300 pm dorsal to the column of Terni. Middle trace: Response evoked with stimulating electrodes in the column of Terni. Bottom trace: Response evoked with stimulating electrodes 300 pm ventral to the column of Terni. Horizontal calibration indicates 2 msec, and the vertical calibration 100 pV. The arrow denotes stimulus onset.

of this subpopulation were longer in intact animals for each nerve [nerve 13--t( 14) = 2.43, P < 0.05 ; nerve 14-t( 14) =2.34, P < 0.05 ; sciatic nerve --t( 14) = 2.39, P < O.O.S]. However, a comparison of the proportion of failures within both spinal and intact preparations yielded no significant differences. Pregarzglionic to Postganglionic Activation

Time

Since experimental anatomical material indicates that preganglionic neurons from several spinal segments project upon ganglion 14 (26), several additional experiments were undertaken to determine the time from activation of preganglionic neurons to the response of postganglionic neurons of the same segment. An estimate of this time is important in interpreting the segmental somatosympathetic reflex times. These experiments were conducted at segment 15, since the number of preganglionic neurons is greater at this segment. Figure 9 shows a series of averaged evoked potentials in ganglion 15 in response to spinal stimulation. For the upper trace the stimulating

electrode

was dorsal

to the column

of Terni,

and only

a late potential was observed. When the stimulating electrode was in the column (middle

of Terni, a distinct short latency potential appeared at 2.9 msec trace). With the stimulating electrode ventral to the column of

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SOMATOSYMPATAETIC

REFLEXES

Terni, this potential largely disappears. Thus, these experiments indicate that the preganglionic to postganglionic delay is approximately 2.9 msec. DISCUSSION Spinally Mediated Cardiovasrzllar Responses to Afferent Nerve Sthm& hon. Few studies have dealt with spinally mediated changes in cardiac

dynamics following somatic nerve stimulation, although some attention has been directed toward changes in arterial blood pressure. For example, Sherrington (31) reported that hindlimb nerve stimulation in the chronically spinal dog produces large pressor responses, and Brooks (3) described small increases in both heart rate and blood pressure with sciatic nerve stimulation in spinal cats. While Fernandez de Molina and Per1 (12) were able to confirm the pressor responseto hindlimb nerve stimulation in the spinal cat, heart rate changes were generally not observed. The present data establish conclusively that reflex tachycardia can be elicited by peripheral nerve stimulation in the spinally transected pigeon, and a major component of this response is mediated by the right cardiac nerve. The contribution of the adrenal or general sympathetic catecholamine release appears to be a lower magnitude, longer latency cardioacceleration, confirming the results of Brooks (3). As found by Coote and Downman (lo), somatic reflexes recoved first, but once established the reflex tachycardia showed little or no further increases over the remaining observation period (cf. 3). Numerous studies on cats have establishedthat somatosympathetic reflex coupling can be mediated at multiple levels, including spinal, medullary, and suprapontine (30). Our observations that peripheral nerve stimulation elicits sequential bursts of activity in sympathetic ganglion 14 suggest a similar organization in the pigeon. While this ganglion contains the majority of the cells of origin of the cardioaccelerator fibers in the pigeon (26), the units studied in the present experiments were not specifically identified as such. Thus, the results do not allow the strong conclusion that reflex tachycardia is mediated at multiple levels. However, they do support multiple levels of sympathetic reflex coupling, and it seems likely that reflex tachycardia would be included among these sympathetic responses. Responsiveness of Single Postgunglionic Nezrvotu to Afferent Nerve Stiw&ztion. Further confirmation of spinally mediated, somatosympathetic

reflexes in the pigeon is provided by our finding that postganglionic neurons are activated by somatic nerve stimulation in animals with high cervical transection. Moreover, the responsiveness of postganglionic neurons to stimulation of various somatic nerves indicates that this spino-spinal coupling extends beyond the segmentallevel, and supports the finding of spinally

nlediated reflex tachycardia to stimulation of lumbosacral nerves. However, this responsiveness to stimulation of different somatic nerves does not necessarily imply a generalized sympathetic activation. Indeed, differences among the proportions of units responding to stimulation of different nerves suggest some local sign. For example, the considerably smaller proportion of postganglionic neurons responding to stimulation of all three nerves indicates some specificity, as does the increase in responsiveness and decrease in mean refles latency as the number of spinal segments between recording and stimulating sites decreases. These findings are generally consistent with the mammalian literature, where it is also reported that with increasing segmental distance between stimulating and recording sites there is a decrease in postganglionic responsiveness and an increase in the response latency ( 11, 29 j . Another consistent feature of our findings was the considerable variability in reflex latency both within and among units. Beacham and Per1 (2) and Janig and Schmidt (20) also described such latency variation, and the Beacham and Per1 (2) results are of particular interest in this regard, since their recordings were from single units in the white rami. Thus, their data do not include variability introduced at the preganglionic to postganglionic synapse and reflect the variability in a purely segmental pathway. Scgmcntal Rcflcxes. Although it is established that ganglion 14 in the pigeon receives preganglionic input from several spinal segments (26)) it might be assumed that the shortest latency postganglionic responses to stimulation of nerve 14 represent conduction through an exclusively segmental pathway. WhiIe it is difficult to subdivide that latency distribution from our experiments (Fig. 2A), there is a suggestion of a distinct response cluster between 3.6 and 7.9 msec which may represent segmental transmission. Given this, one can then calculate an estimate of the segmental central delay. Since the p05 -t ganglionics are activated by stimulating fibers that conduct at 10 m/set or slower, the afferent conduction time to the spinal cord would be at least 1 msec. The time required for preganglionic activation of postganglionic neurons can be taken as 2.9 msec on the basis of our findings from stimulation of the preganglionic column. From these estimates and the mean responseslatency of the most rapidly activated subpopulation of postganglionic units, a central delay of l&1.9 msec is obtained. This value is quite close to that calculated from the Beacham and Per1 data (2) using the preganglionic conduction velocities reported by Fernandez de Molina, Kuno, and Per1 (13). While sucll a central delay is longer than that of the fastest somatic spinal reflexes, it still suggeststhat the segmental spinal circuitry mediating

482

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the shortest latency somatosympathetic reflexes may be reasonably simple. Since our anatomical evidence excludes the possibility of a monosynaptic dorsal root projection upon preganglionic neurons (ZS), this circuit must be at least disynaptic, and an estimated central delay of 1.8 msec makes it unlikely that more than three central synapses are involved. (It might be noted that a possible contribution of ventral root afferents (5) camlot presently be assessed, since the spinal terminal fields of these fibers have not been described.) In contrast, the central delay calculated from the mean response latency of the total postganglionic population responsive to stimulation of nerve 14 is 29 msec (Fig. 2A), considerably longer than previously reported central delays (2, 11, 29). The most likely explanation for this result is the participation of various extrasegmental pathways. In fact, several arguments can be advanced to suggest that activation of many ganglion 14 neurons by nerve 14 stimulation involves transmission through such pathways. First, anatomical evidence indicates that preganglionic neurons in several spinal segments project upon ganglion 14 (26), and dorsal root fibers entering segment 14 project to several of these segments (25). Second, many investigators report that stimulation of hindlimb nerves does not consistently elicit responses in high thoracic preganglionic neurons (2, 30), suggesting that the postganglionic neurons responsive to stimulation of all three nerves in our study were probably activated by preganglionic neurons caudal to segment 14. Third, the mean response latency to nerve 14 stimulation in the population of units responsive to stimulation of all three nerves approximates that of the total population. This implies that the general population includes many units with characteristics similar to those of the more restricted population and, therefore, that they too are activated by preganglionic neurons of more caudal spinal segments. Propriospinal Pathways. The apparent complexity of the propriospinal conduction pathways, as suggested by the latency means and variances, precludes any precise numerical analysis of conduction times. In addition, differences between spinal and intact animals with respect to responsiveness and Iatencies further confounds such an analysis, save for the segmenta response characteristics which did not differ. In spinally transected birds, the shortest latency responses to nerve 13 stimulation were 9.7 msec longer than those to stimulation of nerve 14, the mean latency difference being 12.6 msec. Regardless of whether total latency, central delay, or some variant of these values is used to express the increased conduction time, it remains considerably longer than generally reported in the literature (e.g., 2, 11). In contrast, in the intact animals the latency difference for the fastest responses to nerve 13 and 14 stimulation was onfy 1.6 msec with a mean latency difference of 2.4 msec, both

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values being shorter than those reported by Coote, Downman and Weber ( 11). The shortest latency sciatic response in spinally transected animals was 15.2 msec longer than that to nerve 14 stimulation, and the mean difference was 20.8 msec. These contrast with the respective values of 44.3 and 50.0 msec in the intact bird. While it is not possible to interpret these values unambiguously at this point, the general ordinal relationship frequently described in the literature is maintained. That is, with recording from ganglion 14, the shortest latency responses are obtained with nerve 14 stimulation, these latencies successively increasing as the stimulating . . site IS moved to nerve 13 and then to the sciatic nerve. Conzparison of Spittally Transected and Nontransected Animals. The rationale for undertaking experiments on nontransected birds was twofold. First, our ultimate objective is to specify the spino-spinal pathways mediating the unconditioned stimulus-unconditioned response coupling in our system, and there is the inevitable concern that spinal transection may alter the behavior of this circuitry. Second, pigeons with high cervical transection are difficult to prepare and maintain, and we anticipated that a considerably larger sample of unit data could be obtained in intact animals if the analysis were restricted to responselatencies observed in transected preparations. Transection had no apparent effect on segmental responses, but it did appear to alter the response characteristics with sciatic nerve stimulation. However, one reservation is that general anesthesia was discontinued in transected animals while intact preparations were maintained on urethane throughout the experiments. In the spinal animals there was a higher proportion of responsive postganglionic neurons, and these responded at shorter latencies than in intact preparations. Some arguments were developed in an earlier section suggesting that the neurons responsive to stimulation of all three nerves were in fact activated via preganglionics located caudal to segment 14. The mean latencies of these units to stimulation of each nerve were significantly longer in the nontransected animals; in contrast, the short latency responsesto nerve 14 stimulation were unchanged. This suggests the possible existence of a descending supraspinal influence preferentially affecting the propriospinal pathways. The specific nature of such a descending pathway is presently unknown, although descending pathways inhibiting spinal sympathetic circuitry have been identified (10, 19). Con&id&g Cornnrents. The present findings suggest the following conclusions. (a) Spinospinally mediated somatosympathetic coupling clearly exists in the pigeon, including reflex increases in heart rate. (b) The segmental circuitry for such coupling, though not monosynaptic, is relatively simple and possibly trisynaptic. (c) Coupling over many segments

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is possible, implying an effective propriospinal circuitry. (d) This multisegmental coupling manifests some local sign. (e) The long propriospinal pathways may also be under tonic supraspinal control, While the data demonstrated an orderly progression in reflex latency with increasing segmental distance between stimulating and recording levels, it was not possible to make detailed inferences on the properties of the involved propriospinal pathways. The primary reason was difficulty in adequately describing or controlling the intraspinal conduction pathways potentially available for such reflex coupling. This can, however, be achieved by direct recording from preganglionic neurons, since that would eliminate problems of multisegmental convergence that occur with postganglionic recording. Given this, one could then attempt a more rigorous anatomical and physiological identification of both the propriospinal and the segmental interneuronal circuitries. The latter is of particular importance, since it is highly likely that propriospinal, as well as descending supraspinal, pathways gain access to the preganglionic neurons through such segmental interneurons (28). There are several additional observations in the literature that may well have significance for investigating this interneuronal and propriospinal circuitry. First, activation of somatic motoneurons by stimulation of visceral afferents has been reported (l), as has activation of autonomic efferents (4, 27). Second, visceral afferents from the rami communicantes and the splanchnic nerve converge with somatic afferents upon spinal interneurons (14, 16). Third, such convergence has been observed in fibers of the ventral funiculus (14, 1.5), suggesting that propriospinal or longer ascending neurons receive both visceral and somatic inputs as well. This interaction and convergence upon spinal interneurons raises the possibility that subsequent investigations of the interneuronal and propriospinal systems involved in somatosympathetic interactions may differ little from the more classical investigations of somatic spinal physiology. Finally, with respect to our specific learning model, the specification of this interneuronal and propriospinal circuitry is essential, not only for an understanding of the coupling between lumbosacral afferents and preganglionic neurons influencing heart rate, but also for the eventual understanding of how the descending pathways mediating expression of the conditioned cardioacceleratory response gain access to these preganglionic neurons. REFERENCES 1. ALDERSON, A. M., and C. B. B. DOWNMAN. 1966. Supraspinal thoracic reflexes of somatic and visceral origin. Arch. Ital. Biol. 2. BEACHAM, W. S., and E. R. PERL. 1964. Background and reflex sympathetic preganglionic neurons in the spinal cat. J. Physiol.

inhibition of 104: 309-327. discharge of 172: 400-416.

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3. BROOKS, C. M. 1933. Reflex activation of the sympathetic system in the spinal cat. Amer. J. Pkgsiol. 106: 251-266. 4. BROWN, A. M., and A. MALLIANI. 1971. Spinal sympathetic reflexes initiated by coronary receptors. J. Physiol. 212: 685-705. 5. COGGESHALL, R. E., J. D. COULTER, and W. D. WILLIS, JR. 1974. Unmyelinated axons in the ventral roots of the cat lumbosacral enlargement. J. Camp. h’c~rrol. 153 : 39-58. 6. COHEN, D. H. 1969. Development of a vertebrate experimental model for cellular neurophysiologic studies of learning. Co&. Ref. 4: 61-80. 7. COHEN, D. H. 1974. The neural pathways and informational flow mediating a In “Limbic and Autonomic conditioned autonomic response, pp. 223-275. Plenum Press, New York. Nervous Systems Research.” L. V. DiCa ra [Ed.]. 8 COHEN, D. H. 1974. Analysis of the final common path for heart rate conditioning, pp. 117-135. In “Cardiovascular Psychophysiology.” P. A. Obrist, A. H. Black, J. Brener, and L. V. DiCara [Eds.]. Aldine, Chicago. 9. COHEN, D. H., and L. H. PITTS. 1968. Vagal and sympathetic components of conditioned cardioacceleration in the pigeon. Brain Rrs. 9: 15-31. 10. COOTE, J. H., and C. B. B. DOWNMAN. 1966. Central pathways of some autonomic reflex discharges. J. Physiol. 183: 714-729. 11. COOTE, J. H., C. B. B. DOIVNMAN, and W. V. WEBER. 1969. Reflex discharges into thoracic white rami elicited by somatic and visceral afferent excitation. J. Physiol. 202: 147-159. 12. FERNANDEZ DE MOLINA, A., and E. R. PERL. 1965. Sympathetic activity and the systemic circulation in the spinal cat. J. Physiol. 181: 82-102. 13. FERNANDEZ DE MOLINA, A., M. KUNO, and E. R. PERL. 1965. Antidromically evoked responses from sympathetic preganglionic neurons. J. PIzysiol. 180 : 321-335. 14. FIELDS, H. L., G. A. MEYER, and L. D. PAKTRIDCE, JR. 1970. Convergence of visceral and somatic input onto spinal neurons. E.Q. Nczlvol. 26: 36-52. 15. FIELDS, H. L., L. D. PARTRIDGE, JR., and D. L. WINTER. 1970. Somatic and visceral receptive field properties of fibers in ventral quadrant white matter of the cat spinal cord. J. Ncnrophysiol. 33: 817-837. 16. HAXOCK, M. B., D. D. RIGMONTI, and R. N. BRYAN. 1973. Convergence in the lumbar spinal cord of pathways activated by splanchnic nerve hindlimb cutaneous nerve stimulation. Exp. Neural. 38: 337-348. 17. HESS, A., G. PILAR, and J. N. WEAKLY. 1969. Correlation between transmission and structure in avian ciliary ganglion synapses. J. Physiol. 202: 339-354. 18. HUBER, J. F. 1936. Nerve roots and nuclear groups in the spinal cord of the pigeon. J. Cow@. Newel. 65: 43-91. 19. ILLERT, M., and M. GABRIEL. 1972. Descending pathways in the cervical cord of cats affecting blood pressure and sympathetic activity. Pfliig. ~rclr. 335: 109-124. 20. J~NIG, W., and R. F. SCHMIDT. 1970. Single unit responses in the cervical sympathetic trunk upon somatic nerve stimulation. P&g. Arch. 314: 199-216. 21. KOIZUMI, K., and C. M. BROOKS. 1972. The integration of autonomic system reactions: a discussion of autonomic reflexes, their control and their association with somatic reactions. Erg&. Physiol. 67 : l-68. 2.2. LANGLEY, J. N. 1904. On the sympathetic system of birds, and on the muscles which move the feathers. J. Physiol. 30: 221-252.

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23. LEONARD, R. B., and D. H. COHEN. The peripheral unconditioned stimulus pathway in a model learning system involving conditioned heart rate change in the pigeon. J. Comp. Physiol. Psychol. (In press). 24. LEONARD, R. B., and D. H. COHEN. 197.5. A cytoarchitectonic analysis of the spinal cord of the pigeon (Columbia Ziviu). J. Cow@. Neural. 163 : 159-179. 25. LEONARD, R. B., and D. H. COHEN. 1975. Spinal terminal fields of dorsal root fibers in the pigeon (Columbia liviu). I. Camp. Neural. 163 : 181-192. 26. MACLKINALD, R. L., and D. H. COHEN. 1970. Cells of origin of sympathetic preand postganglionic cardioacceleratory fibers in the pigeon. 1. Camp. Neural. 140 : 343-358. 27. MALLIANI, A., M. PAGANI, G. RECORDATI, and P. J. SCHWARTZ. 1970. Evidence for a spinal sympathetic regulation of cardiovascular function. Experientia 26 : 965-966. 28. PETRAS, J. M., and J. F. CUMMINGS. 1972. Autonomic neurons in the spinal cord of Rhesus monkey: A correlation of the findings of cytoarchitectonics and sympathectomy with fiber degeneration following dorsal rhizotomy. J. Camp. New-ok 146 : 198-218. 29. SATO, A., and R. F. SCHMIDT. 1971. Spinal and supraspinal components of the reflex discharges into lumbar and thoracic white rami. I. Physiol. 212: 839-850. 30. SATO, A., and R. F. SCHMIDT. 1973. Somatosympathetic reflexes: afferent fibers, central pathways, discharge characteristics. Physiol. Rev. 53 : 916-947. 31. SHERRINCTON, C. S. 1906. “The Integrative Action of the Nervous System.” Charles Scribner’s Sons, New York.