Brain Research, 87 (1975) 161-170 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
161
SPINAL O R G A N I Z A T I O N OF S Y M P A T H E T I C I N H I B I T I O N BY SPINAL A F F E R E N T VOLLEYS
FRIEDRICH KIRCHNER*, DOROTHEA KIRCHNER AND CANIO POLOSA Department of Physiology, McGill University, Montreal (Canada)
The reflex effects evoked in sympathetic neurons by volleys in spinal afferent fibers consist of excitatory and inhibitory components. Sato and Schmidt 14 showed that a volley in myelinated afferents entering a given spinal cord segment evokes a spinal reflex discharge from sympathetic neurons of the same segment and smaller discharges from the immediately adjacent segments. The central pathways of the inhibitory reflexes evoked by spinal myelinated afferents have been much less studied than those of the excitatory reflexes evoked by the same afferents. For a while it was believed that these inhibitory reflexes were exclusively mediated by an action on the medulla oblongatalL However, Okada et al. 1°, Beacham and Perl ~, Sato la and Wyszogrodski and Polosa 16 demonstrated that inhibition of pre- or postganglionic sympathetic neurons could be obtained in acute spinal animals by stimulating spinal afferents. This work has clarified some of the properties of sympathetic inhibition by myelinated afferents. Beacham and Perl 1, Sato ~3 and Wyszogrodski and Polosa 16 have described, in the cat with intact neuraxis and in the acute spinal cat, the time course of segmental inhibition, which was shown to be of several hundred msec duration. In addition, it was shown 16 that a postsynaptic inhibitory mechanism is probably involved in the generation of the inhibition, and that the inhibitory effect can spread, even in the acute spinal cat, at a distance from the point of entry of the stimulated root, but with some attenuation. The latter property was not studied quantitatively, however. Sato la has also found, in the acute spinal cat, that a conditioning shock to a dorsal root can depress the early reflex evoked into an adjacent white ramus by stimulating the same dorsal root. It has also been shown that in the acute spinal cat the inhibition evoked by spinal afferent volleys is shorter in duration than in the cat with an intact neuraxis 13,16. It is possible that the organization of afferent inhibition, within the spinal cord, is similar to that of excitation, but not enough experimental data are available so far for a definite answer. In the investigation reported here we have studied, in the acute spinal cat, some of the properties of the segmental and heterosegmental inhibition of sympathetic neurons by myelinated afferent volleys in order to define its distribution along the spinal cord. We have also studied the properties of afferent inhibition in the chronic spinal cat in order to obtain data on the relation* Present address: PhysiologischesInstitut der Universit~tt,Heidelberg, G.F.R.
162 ships between segmental inputs and those from supraspinal levels. It is conceivable, for instance, that the inhibitory action of the segmental afferents could be effected by a modulation of activity in descending fiber tracts. Or, that segmental and descending inputs might converge on to some common neural element and that, as a result of decentralization, connectivity and other properties of the segmental inputs might be modified. The experiments were performed on 15 cats under pentobarbital anesthesia (35 mg/kg intraperitoneally, followed by intravenous booster doses when required). The cats were immobilized with gallamine triethiodide and artificially ventilated with a positive pressure pump. Systemic arterial pressure was continuously monitored. A catheter in the femoral vein was used for drug injection. Rectal temperature was measured and maintained between 36 and 38 °C by means of heating lamps. After laminectomy, a complete transection of the spinal cord was performed at Cs. Five of the cats were kept in good health and studied 4-6 weeks after the transection (chronic spinal). The other 10 were studied within 24 h after the transection (acute spinal). The average systemic arterial pressure was 80 mm Hg in the chronic spinal and 60 mm Hg in the acute spinal cats. The left renal nerve (RN), L2 white ramus (WR) and cervical sympathetic trunk (CST) were prepared and placed on recording electrodes. Recording was biphasic. For stimulation, several intercostal nerves (ICN) and spinal nerves (SN) were prepared. All neural signals were amplified, displayed on an oscilloscope and stored on magnetic tape. In addition, after rectification and conversion into frequency-modulated pulses, they were averaged on-line with a special purpose computer. Recovery curves of the spinal reflex were plotted using the average areas of 10 reflexes calculated by a PDP 11 computer off-line. The area of the conditioned reflex was expressed as percentage of the area of the unconditioned reflex. A control run of 10 unconditioned test stimuli was made before each run of 10 conditioned test stimuli. All stimulation was carried out at a repetition rate of 0.1-0.2 Hz. Stimuli were square pulses of 0.5 msec duration. Test stimuli were between 20 and 40 times threshold (T), T was measured by recording the stimulus-evoked volley in the nerve distal to the stimulating electrode. Stimulus voltage was measured across the stimulating electrodes with an oscilloscope. Onset of inhibition, obtained from recovery curves, is defined as the arithmetic mean of the shortest conditioning-testing interval at which the test reflex was depressed and the immediately preceding C-T interval tested. Duration of inhibition was obtained by a similar procedure. Acute spinal cats. In 10 cats recording was from the renal nerve. A single shock of intensity 4-10 T or greater to T l l intercostal nerve elicited a reflex discharge in the renal nerve. The properties of this spinal or 'early' reflex have been previously described 2. With double shocks, a conditioning stimulus of intensity above the reflex threshold depressed the reflex discharge to a test stimulus (routinely between 20 and 40 T) at various conditioning-testing intervals. Similar depressant effects by homosegmental conditioning were previously described 1,3,~,13. The threshold for the depressant effect was similar to that for the reflex discharge, i.e., if the conditioning stimulus was of intensity lower than the threshold for a reflex discharge, it had no detectable depressant effect on the test reflex. The mean C-T interval at which this
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Fig. I. Acute spinal cat. Recovery curves of early reflex. R N recording. Test shock to Tit ICN. Conditioning shock to T u ICN at two intensities, 6 T (open circles) and 20 T (squares). Ordinate: average area of conditioned reflex as percentage of the average area of the unconditioned reflex. Abscissa: interval between conditioning and testing shock on logarithmic scale. A - C : specimen records at 6 T, A ' - C " at 20 T. Each specimen is the average of 10 reflexes. In each panel, top record is the unconditioned reflex, bottom record is the conditioned reflex. Sweep triggered by conditioning stimulus in A and A', by test stimulus in B, C, B' and C'. C - T intervals are 4 msec in A (no depression), 63 msec in B, 100 msec in C, 16 msec in A ' (no depression), 32 msec in B' and 200 msec in C'.
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Fig. 2. Acute spinal cat. Recovery curves of early reflex. RN recording. Test shock to Tll ICN. Conditioning shock to Tll ICN (filled circles), L~ SN (triangles), Ts ICN (open circles), T4 ICN 50 T (open squares), T4 ICN 80 T (filled squares). Ordinate and abscissa as in Fig. I. Inset: conditioning stimulus intensity required for depression of early reflex (ordinate) at the various levels indicated on abscissa. effect began was 14 msec (range 6.5-24.0, n = 10). The mean duration of the depression was 715 msec (range 150-1600 msec, n = 10). At the peak of the depressant effect the test reflex was 41 ~ of the unconditioned reflex (range 30-61), at a mean C - T interval of 96 msec (range 50-200). These values were obtained with conditioning stimuli of intensity slightly above threshold, i.e., between 8 and 20 T. Fig. 1 shows a typical example of recovery curves of the spinal sympathetic reflex obtained with T l l I C N stimulation and R N recording at two intensities of the conditioning stimulus (6 T, open circles, 20 T, squares). Curves, like the ones shown, were obtained in 8 of the 10 cats. In 2 cats the curves were biphasic. Using a conditioning stimulus of higher intensity (Fig. 1, squares) the depression was greater, began at a similar C - T interval and lasted longer. When the conditioning stimulus was applied to more cranial intercostal nerves or to lumbar spinal nerves, the T l l I C N to R N reflex could still be depressed, but the required intensity of the conditioning stimulus increased with distance of its segmental level from the T l l segment. An example of this finding of an intersegmental spread of the depression is shown in Fig. 2. In the case shown, a conditioning shock to Tll I C N depressed the Tz~ I C N to R N test reflex at 5 T (filled circles). Applying a conditioning stimulus of the same intensity to Lz SN resulted in a depression of smaller intensity (filled triangles). When the conditioning stimulus was applied to Ts ICN, a stimulus intensity of 50 T was required to obtain a relatively weak depression. Finally, when the conditioning stimulus was applied to T4 I C N there was no depressant effect with conditioning stimulus intensities up to 50 T (open squares). Only when the conditioning stimulus intensity was raised to 80 T was a weak depression of very long latency (filled squares) observed. A similar long intersegmental effect at high conditioning stimulus intensity was observed in one other cat only. In 2 cats recording was from L2 WR. Stimulation of L2 SN with 4-10 T intensity
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Fig. 3. Acute spinal cat. Recovery curves of early reflex. L2 WR recording. Test shock to L2 SN. Conditioning shock to L2 SN (filled squares), L1 SN (open circles), TI~ ICN (open squares), Tlo ICN (open squares). Ordinate and abscissa as in Fig. 1. evoked a reflex discharge. The properties of this reflex have been previously described by Sato and Schmidt 14. With double shocks results were obtained which were comparable with those described above for ICN stimulation and R N recording. The results obtained in one cat are shown in Fig. 3. With L2 SN conditioning of intensity 15 T the depression of the L2 SN to L2 WR reflex began at a C - T interval of 10 msec (shorter intervals were not tested in this cat), reached a peak of 34 ~o of control at 160 msec and lasted longer than 320 msec. When a conditioning stimulus of similar intensity (18 T) was applied to L1 SN a depression of similar duration but less intensity was obtained. Applying the conditioning stimulus to T12 ICN and T10 ICN at intensities of 50 T did not evoke a depression of the L2 SN to L~ WR reflex. Similar results were obtained in the other cat. In one cat recording was from the CST. By stimulating T4 ICN with 4-10 T intensity a reflex discharge was evoked, the properties of which have been previously described by Kirchner et al. 7. With double shocks results qualitatively similar to those already described for R N and WR recording were obtained. The results in this cat are shown in Fig. 4. The figure shows the recovery curve of the T4 ICN to CST reflex conditioned by a 15 T stimulus to T4 ICN. The depression begins at 24 msec, lasts 500 msec and the reflex is down to 63 ~o of control at a C - T interval of 200 msec. When the conditioning stimulus was applied to Ts ICN, with intensity 60 T, only a small depressant effect was obtained. No effect was elicited by L2 and L4 SN with intensities up to 200 T or greater. Chronic spinal cats. In this preparation, recording from the R N was made in 4 cats, from the L2 WR in two and from the CST in one. At all these recording sites the early reflex had properties of threshold, latency and duration similar to those observed in the acute preparation. With double shocks, depression of the test reflex was seen as in the acute spinal cats. However, in the chronic spinal cat the threshold for depression was consistently lower than for excitation, so that conditioning stimuli which
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Fig. 4. Acute spinal cat. Recovery curves of early reflex. CST recording. Test shock to T4 ICN. Conditioning shock to T4 ICN (filled squares), Ts ICN (open circles), L~ SN (open squares) and L4 SN (open squares). Ordinate and abscissa as in Fig. 1. were subthreshold for excitation could be used, obtaining inhibitory effects on the segmental reflex which were uncontaminated by excitation. An example of this purely inhibitory effect is shown in Fig. 5. The figure shows the depression of the T l l I C N to RN reflex by a T l l I C N conditioning stimulus which does not evoke a discharge. A: control. B: at a C - T interval of 100 msec the test reflex is suppressed. Note absence of discharge in response to conditioning stimulus. Conditioning 2 T, testing 20 T. Recovery curves in these chronic spinal cats were qualitatively similar to those obtained in the acute, but a general impression was that for a given stimulus intensity the depression was more intense in the chronic than in the acute spinal cat. With R N recording and T l l I C N conditioning, the depressant effect began at a C - T interval of 12 msec (range 3.5-24, n = 4 ) . Mean duration of the depression was 640 msec (range 260-1300, n = 4 ) . At the peak of the depressant effect, which occurred at a mean C - T interval of 100 msec (range 25-200) the test reflex was down to 23 ~ of control. These results were obtained with conditioning stimulus intensities between 3 and 10 T. Fig. 6 shows recovery curves obtained with R N recording in a chronic spinal cat, 5 weeks after transection. Open circles label the curve obtained with T l l I C N conditioning of intensity 3 T (at 5 T, in this case, there was a complete suppression of the test reflex). With conditioning shock given to Lz SN depression of the test reflex could still be obtained, but a stimulus intensity greater than 3 T was required. When the conditioning stimulus was applied to T4 I C N at intensities up to 20 T no depression was obtained (higher stimulus intensities were not tried in this case). With WR recording and L2 SN conditioning at intensity of 1-2 T (2 cats), the depression of the Lz S N t o L2 W R reflex began at C - T intervals of 18 and 16 msec, the duration of the depression was 300 and 600 msec and at the peak of the depression, at a C - T interval of 100 msec, the reflex was down to 30 and 51 ~ of control. Fig. 7 shows the recovery curves of the L2 SN to L2 W R reflex obtained with conditioning stimuli applied to various segmental levels. A conditioning stimulus of
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Fig. 7. Chronic spinal cat (5 weeksafter Ca transection). Recoverycurves of early reflex.L~WR recording. Test shock to L2 SN. Conditioning shock to L2 SN (open circles), L4 SN (filledcircles),Tz~ ICN (filled squares) and T4 ICN (open squares). Ordinate and abscissa as in Fig. 1. 1 T intensity applied to L2 SN depressed the L2 SN to L2 WR reflex. When the conditioning stimulus was applied to L4 SN and T12 ICN, in order to obtain a depression of similar intensity to that obtained with the L2 SN conditioning, the intensity of the conditioning stimulus had to be raised to 6 T. When the conditioning stimulus was applied to T4 ICN, stimulus intensities up to 60 T had no detectable depressant effect. In the only case with CST recording, double shocks to T4 ICN resulted in the usual depression of the test reflex (Fig. 8). The figure shows the recovery curve of the T4 ICN to CST reflex obtained in this chronic spinal cat with conditioning shocks of 4 T intensity to T4 ICN. Depression began at less than 20 msec and lasted for 400 msec, with its peak at around 100 msec. The only heterosegmental input tested with CST recording was L4 SN. Stimulus intensities up to 200 T evoked no depression of the test reflex. With intensity greater than 200 T a depression was seen, the time course of which is plotted in Fig. 8. These experiments have shown that in the acute and chronic spinal cat the amplitude of a segmental sympathetic reflex evoked by myelinated afferents can be depressed by a homo- or heterosegmental conditioning shock to afferent fibers. It is likely that the phenomenon underlying this depressant effect is an inhibitory process, and not a process of postexcitatory depression and hence it seems appropriate to use the term inhibition to describe this depression. One reason is that the size of the discharge evoked by the conditioning stimulus was usually a fraction (e.g., 20-30 ~ ) of that evoked by the test stimulus, yet often the test discharge was reduced by a fraction greater than that. Moreover, in the chronic spinal animal a depression of the test reflex, of time course similar to that observed in the acute spinal, could be elicited by a conditioning stimulus which evoked no discharge. It is possible that, at least at short C - T intervals, and when the conditioning stimulus evoked a discharge, postexcitatory depression contributed to the generation of the recovery curve 1,x~. In the acute spinal cat the inhibitory effect of the conditioning volley was always preceded by an excitatory effect. This constant association of the two effects might
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Fig. 8. Chronic spinal cat (6 weeksafter C8 transection). Recoverycurves of early reflex. CST recording. Test shock to T4 ICN. Conditioning shock to T4 ICN (filledsquares) and L4 SN (open circles). Ordinate and abscissa as in Fig. 1. suggest, as a simple explanation, the operation of a recurrent inhibitory circuit. However, the observations made with antidromic stimulation and intra- or extracellular recording 4,6,11 rule out this possibility. The data of Lebedev 9, obtained with paired antidromic shocks, are not in contrast with the ones listed above, since they can be accounted for on the basis of differences in time course of recovery of excitability among preganglionic axons of different sizes. Golgi studies by Rethelyi12 show that the axons of presumed sympathetic preganglionic neurons lack recurrent collaterals. Finally, this hypothesis is disproved by the observation that in some experimental conditions the excitatory and inhibitory processes can be uncoupled (ref. 13, Fig. 3C, using peripheral nerve stimulation; in the present experiments by the use of chronic spinal preparations). The longitudinal distribution along the cord of spinal afferent inhibition seems to follow, qualitatively, the same pattern described for the early somatosympathetic reflex by Sato and Schmidt 14. At constant stimulus intensity, the probability of evoking inhibition and the strength and duration of the inhibition decays with the distance of the segmental level of the conditioning stimulus from the segmental level of the test reflex. Conversely, the conditioning stimulus intensity required for inhibiting the test reflex increases with the distance from the segmental level(s) of the test reflex. This spatial pattern was observed with stimulus intensities within the myelinated fiber range. Long distance intersegmental inhibitory effects (e.g., T4 ICN inhibition of the T12 ICN to RN or of the L2 SN to Lz WR and L2 SN inhibition of a T4 ICN to CST reflex) were obtained only with very high stimulus intensities, i.e. in the C fiber range. In the chronic spinal cats the pattern of distribution was similar. The similarities of the spread observed with CST and either RN or L~ WR recording suggest that the distribution in the cephalic and caudal direction is symmetrical. The anatomical basis for the observed pattern of distribution may reside in the connectivity pattern of the primary afferents or of interneurons. Although the pattern of intersegmental spread of inhibition is similar to that of excitation, the time course of inhibition is much longer than that of excitation. The obvious question here
170 is whether the longer persistence of the inhibitory effect is the result o f some property of the inhibitory transmitter, as yet unknown, or of the neuronal circuitry associated with the inhibitory pathway (e.g., reverberatory and delay circuits). In the chronic spinal cats the threshold required for evoking inhibition by spinal afferent stimulation was lowered. In this preparation a conditioning stimulus which did not evoke excitation could inhibit the spinal reflex segmentally, and, at higher intensity, intersegmentally. In this preparation descending fiber tracts from supraspinal levels are degenerated, and the threshold o f inhibition by intraspinal stimulation is also lowered 8. On the other hand, the properties o f excitation by myelinated spinal afferents and intraspinal stimulation do not appear changed. A simple hypothesis to account for some of these findings is the development o f plastic changes at the level o f a c o m m o n inhibitory interneuron situated at the segmental level resulting in stronger connectivity o f intraspinal descending system and segmental afferents or denervation hypersensitivity after degeneration o f supraspinal descending pathway. This investigation was supported by the Medical Research Council o f Canada. F.K. was the holder o f a D A A D Fellowship.
1 BEACHAM,W. S., AND PERL, E. R., Characteristics of a spinal sympathetic reflex, J. Physiol. (Lond.),
173 (1964) 431-448. 2 CoorE, J. H., AND DOWNMAN,C. B. B., Central pathways of some autonomic reflex discharges, J. Physiol. (Lond.), 183 (1966) 714-729. 3 CoorE, J. H., DOWNMAN,C. B. B., AND WEBER, W. V., Reflex discharges into thoracic white rami elicited by somatic and visceral afferent excitation, J'. Physiol. (Lond.), 202 (1969) 147-160. 4 FERNANDEZDE MOLINA,A., KUNO, M., ANDPERL,E. R., Antidromically evoked responses from sympathetic preganglionic neurones, J. Physiol. (Lond.), 180 0965) 321-335. 5 FRANZ, D. N., EVANS, M. H., AND PERL, E. R., Characteristics of viscerosympathetic reflexes in the spinal cat, Amer. J. PhysioL, 221 (1966) 1292-1298. 6 HONGO,T., AND RYALL,R. W., Electrophysiological and microelectrophoretic studies on sympathetic preganglionic neurones in the spinal cord, Acta physiol, scand., 68 (1966) 96-104. 7 KIRCHNER,F., SATO,A., ANDWErOINGER,H., Central pathways of reflex discharges in the cervical sympathetic trunk, Pfliigers Arch. ges. PhysioL, 319 (1970) 1-11. 8 KmCHNER,F., WYSZOGRODSKLI., ANDPOLOSA,C., Some properties of sympathetic neuron inhibition by depressor area and intraspinal stimulation, submitted to Pfliigers Arch. ges. Physiol. 9 LEBEDEV,V. P., Features of axon &lateral horn sympathetic preganglionic neurons of low thoracic spinal cord, SechenovphysioL J. U.S.S.R., 57 (1971) 1647-1655. 10 OKADA,H., NAKAO,O., ANDNISIDA,I., Effect of sciatic stimulation upon the efferent impulses in the long ciliary nerve of the cat, Jap. J. PhysioL, 10 (1960) 327-339. 11 POLOSA,C., The silent period of sympathetic preganglionic neurons, Canad. J. PhysioL Pharmacol., 45 (1967) 1033-1045. 12 RETHELYI,M., Cell and neuropil architecture of the intermediolateral (sympathetic) nucleus of cat spinal cord, Brain Research, 46 (1972) 203-213. 13 SATO,A., Spinal and supraspinal inhibition of somato-sympathetic reflexes by conditioning afferent volleys, Pfliigers Arch. ges. PhysioL, 336 (1972) 121-133. 14 SATO,A., AND SCHMIDT,R. F., Spinal and supraspinal components of the reflex discharges into lumbar and thoracic white rami, J. Physiol. (Lond.), 212 (1971) 839-850. 15 WEIDINGER, H., Das Vasomotorenzentrum in der Medulla Oblongata. Versuche zur Bestirnmung
seiner Lage und Funktion, Habilitationsschrift, Univ. Heidelberg, 1966. 16 WVSZOGRODSKI,I., AND POLOSA,C., The inhibition of sympathetic preganglionic neurons by somatic afferents, Canad. J. Physiol. Pharmacol., 51 (1973) 29-38.
161
SPINAL O R G A N I Z A T I O N OF S Y M P A T H E T I C I N H I B I T I O N BY SPINAL A F F E R E N T VOLLEYS
FRIEDRICH KIRCHNER*, DOROTHEA KIRCHNER AND CANIO POLOSA Department of Physiology, McGill University, Montreal (Canada)
The reflex effects evoked in sympathetic neurons by volleys in spinal afferent fibers consist of excitatory and inhibitory components. Sato and Schmidt 14 showed that a volley in myelinated afferents entering a given spinal cord segment evokes a spinal reflex discharge from sympathetic neurons of the same segment and smaller discharges from the immediately adjacent segments. The central pathways of the inhibitory reflexes evoked by spinal myelinated afferents have been much less studied than those of the excitatory reflexes evoked by the same afferents. For a while it was believed that these inhibitory reflexes were exclusively mediated by an action on the medulla oblongatalL However, Okada et al. 1°, Beacham and Perl ~, Sato la and Wyszogrodski and Polosa 16 demonstrated that inhibition of pre- or postganglionic sympathetic neurons could be obtained in acute spinal animals by stimulating spinal afferents. This work has clarified some of the properties of sympathetic inhibition by myelinated afferents. Beacham and Perl 1, Sato ~3 and Wyszogrodski and Polosa 16 have described, in the cat with intact neuraxis and in the acute spinal cat, the time course of segmental inhibition, which was shown to be of several hundred msec duration. In addition, it was shown 16 that a postsynaptic inhibitory mechanism is probably involved in the generation of the inhibition, and that the inhibitory effect can spread, even in the acute spinal cat, at a distance from the point of entry of the stimulated root, but with some attenuation. The latter property was not studied quantitatively, however. Sato la has also found, in the acute spinal cat, that a conditioning shock to a dorsal root can depress the early reflex evoked into an adjacent white ramus by stimulating the same dorsal root. It has also been shown that in the acute spinal cat the inhibition evoked by spinal afferent volleys is shorter in duration than in the cat with an intact neuraxis 13,16. It is possible that the organization of afferent inhibition, within the spinal cord, is similar to that of excitation, but not enough experimental data are available so far for a definite answer. In the investigation reported here we have studied, in the acute spinal cat, some of the properties of the segmental and heterosegmental inhibition of sympathetic neurons by myelinated afferent volleys in order to define its distribution along the spinal cord. We have also studied the properties of afferent inhibition in the chronic spinal cat in order to obtain data on the relation* Present address: PhysiologischesInstitut der Universit~tt,Heidelberg, G.F.R.
162 ships between segmental inputs and those from supraspinal levels. It is conceivable, for instance, that the inhibitory action of the segmental afferents could be effected by a modulation of activity in descending fiber tracts. Or, that segmental and descending inputs might converge on to some common neural element and that, as a result of decentralization, connectivity and other properties of the segmental inputs might be modified. The experiments were performed on 15 cats under pentobarbital anesthesia (35 mg/kg intraperitoneally, followed by intravenous booster doses when required). The cats were immobilized with gallamine triethiodide and artificially ventilated with a positive pressure pump. Systemic arterial pressure was continuously monitored. A catheter in the femoral vein was used for drug injection. Rectal temperature was measured and maintained between 36 and 38 °C by means of heating lamps. After laminectomy, a complete transection of the spinal cord was performed at Cs. Five of the cats were kept in good health and studied 4-6 weeks after the transection (chronic spinal). The other 10 were studied within 24 h after the transection (acute spinal). The average systemic arterial pressure was 80 mm Hg in the chronic spinal and 60 mm Hg in the acute spinal cats. The left renal nerve (RN), L2 white ramus (WR) and cervical sympathetic trunk (CST) were prepared and placed on recording electrodes. Recording was biphasic. For stimulation, several intercostal nerves (ICN) and spinal nerves (SN) were prepared. All neural signals were amplified, displayed on an oscilloscope and stored on magnetic tape. In addition, after rectification and conversion into frequency-modulated pulses, they were averaged on-line with a special purpose computer. Recovery curves of the spinal reflex were plotted using the average areas of 10 reflexes calculated by a PDP 11 computer off-line. The area of the conditioned reflex was expressed as percentage of the area of the unconditioned reflex. A control run of 10 unconditioned test stimuli was made before each run of 10 conditioned test stimuli. All stimulation was carried out at a repetition rate of 0.1-0.2 Hz. Stimuli were square pulses of 0.5 msec duration. Test stimuli were between 20 and 40 times threshold (T), T was measured by recording the stimulus-evoked volley in the nerve distal to the stimulating electrode. Stimulus voltage was measured across the stimulating electrodes with an oscilloscope. Onset of inhibition, obtained from recovery curves, is defined as the arithmetic mean of the shortest conditioning-testing interval at which the test reflex was depressed and the immediately preceding C-T interval tested. Duration of inhibition was obtained by a similar procedure. Acute spinal cats. In 10 cats recording was from the renal nerve. A single shock of intensity 4-10 T or greater to T l l intercostal nerve elicited a reflex discharge in the renal nerve. The properties of this spinal or 'early' reflex have been previously described 2. With double shocks, a conditioning stimulus of intensity above the reflex threshold depressed the reflex discharge to a test stimulus (routinely between 20 and 40 T) at various conditioning-testing intervals. Similar depressant effects by homosegmental conditioning were previously described 1,3,~,13. The threshold for the depressant effect was similar to that for the reflex discharge, i.e., if the conditioning stimulus was of intensity lower than the threshold for a reflex discharge, it had no detectable depressant effect on the test reflex. The mean C-T interval at which this
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Fig. I. Acute spinal cat. Recovery curves of early reflex. R N recording. Test shock to Tit ICN. Conditioning shock to T u ICN at two intensities, 6 T (open circles) and 20 T (squares). Ordinate: average area of conditioned reflex as percentage of the average area of the unconditioned reflex. Abscissa: interval between conditioning and testing shock on logarithmic scale. A - C : specimen records at 6 T, A ' - C " at 20 T. Each specimen is the average of 10 reflexes. In each panel, top record is the unconditioned reflex, bottom record is the conditioned reflex. Sweep triggered by conditioning stimulus in A and A', by test stimulus in B, C, B' and C'. C - T intervals are 4 msec in A (no depression), 63 msec in B, 100 msec in C, 16 msec in A ' (no depression), 32 msec in B' and 200 msec in C'.
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Fig. 2. Acute spinal cat. Recovery curves of early reflex. RN recording. Test shock to Tll ICN. Conditioning shock to Tll ICN (filled circles), L~ SN (triangles), Ts ICN (open circles), T4 ICN 50 T (open squares), T4 ICN 80 T (filled squares). Ordinate and abscissa as in Fig. I. Inset: conditioning stimulus intensity required for depression of early reflex (ordinate) at the various levels indicated on abscissa. effect began was 14 msec (range 6.5-24.0, n = 10). The mean duration of the depression was 715 msec (range 150-1600 msec, n = 10). At the peak of the depressant effect the test reflex was 41 ~ of the unconditioned reflex (range 30-61), at a mean C - T interval of 96 msec (range 50-200). These values were obtained with conditioning stimuli of intensity slightly above threshold, i.e., between 8 and 20 T. Fig. 1 shows a typical example of recovery curves of the spinal sympathetic reflex obtained with T l l I C N stimulation and R N recording at two intensities of the conditioning stimulus (6 T, open circles, 20 T, squares). Curves, like the ones shown, were obtained in 8 of the 10 cats. In 2 cats the curves were biphasic. Using a conditioning stimulus of higher intensity (Fig. 1, squares) the depression was greater, began at a similar C - T interval and lasted longer. When the conditioning stimulus was applied to more cranial intercostal nerves or to lumbar spinal nerves, the T l l I C N to R N reflex could still be depressed, but the required intensity of the conditioning stimulus increased with distance of its segmental level from the T l l segment. An example of this finding of an intersegmental spread of the depression is shown in Fig. 2. In the case shown, a conditioning shock to Tll I C N depressed the Tz~ I C N to R N test reflex at 5 T (filled circles). Applying a conditioning stimulus of the same intensity to Lz SN resulted in a depression of smaller intensity (filled triangles). When the conditioning stimulus was applied to Ts ICN, a stimulus intensity of 50 T was required to obtain a relatively weak depression. Finally, when the conditioning stimulus was applied to T4 I C N there was no depressant effect with conditioning stimulus intensities up to 50 T (open squares). Only when the conditioning stimulus intensity was raised to 80 T was a weak depression of very long latency (filled squares) observed. A similar long intersegmental effect at high conditioning stimulus intensity was observed in one other cat only. In 2 cats recording was from L2 WR. Stimulation of L2 SN with 4-10 T intensity
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Fig. 3. Acute spinal cat. Recovery curves of early reflex. L2 WR recording. Test shock to L2 SN. Conditioning shock to L2 SN (filled squares), L1 SN (open circles), TI~ ICN (open squares), Tlo ICN (open squares). Ordinate and abscissa as in Fig. 1. evoked a reflex discharge. The properties of this reflex have been previously described by Sato and Schmidt 14. With double shocks results were obtained which were comparable with those described above for ICN stimulation and R N recording. The results obtained in one cat are shown in Fig. 3. With L2 SN conditioning of intensity 15 T the depression of the L2 SN to L2 WR reflex began at a C - T interval of 10 msec (shorter intervals were not tested in this cat), reached a peak of 34 ~o of control at 160 msec and lasted longer than 320 msec. When a conditioning stimulus of similar intensity (18 T) was applied to L1 SN a depression of similar duration but less intensity was obtained. Applying the conditioning stimulus to T12 ICN and T10 ICN at intensities of 50 T did not evoke a depression of the L2 SN to L~ WR reflex. Similar results were obtained in the other cat. In one cat recording was from the CST. By stimulating T4 ICN with 4-10 T intensity a reflex discharge was evoked, the properties of which have been previously described by Kirchner et al. 7. With double shocks results qualitatively similar to those already described for R N and WR recording were obtained. The results in this cat are shown in Fig. 4. The figure shows the recovery curve of the T4 ICN to CST reflex conditioned by a 15 T stimulus to T4 ICN. The depression begins at 24 msec, lasts 500 msec and the reflex is down to 63 ~o of control at a C - T interval of 200 msec. When the conditioning stimulus was applied to Ts ICN, with intensity 60 T, only a small depressant effect was obtained. No effect was elicited by L2 and L4 SN with intensities up to 200 T or greater. Chronic spinal cats. In this preparation, recording from the R N was made in 4 cats, from the L2 WR in two and from the CST in one. At all these recording sites the early reflex had properties of threshold, latency and duration similar to those observed in the acute preparation. With double shocks, depression of the test reflex was seen as in the acute spinal cats. However, in the chronic spinal cat the threshold for depression was consistently lower than for excitation, so that conditioning stimuli which
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Fig. 7. Chronic spinal cat (5 weeksafter Ca transection). Recoverycurves of early reflex.L~WR recording. Test shock to L2 SN. Conditioning shock to L2 SN (open circles), L4 SN (filledcircles),Tz~ ICN (filled squares) and T4 ICN (open squares). Ordinate and abscissa as in Fig. 1. 1 T intensity applied to L2 SN depressed the L2 SN to L2 WR reflex. When the conditioning stimulus was applied to L4 SN and T12 ICN, in order to obtain a depression of similar intensity to that obtained with the L2 SN conditioning, the intensity of the conditioning stimulus had to be raised to 6 T. When the conditioning stimulus was applied to T4 ICN, stimulus intensities up to 60 T had no detectable depressant effect. In the only case with CST recording, double shocks to T4 ICN resulted in the usual depression of the test reflex (Fig. 8). The figure shows the recovery curve of the T4 ICN to CST reflex obtained in this chronic spinal cat with conditioning shocks of 4 T intensity to T4 ICN. Depression began at less than 20 msec and lasted for 400 msec, with its peak at around 100 msec. The only heterosegmental input tested with CST recording was L4 SN. Stimulus intensities up to 200 T evoked no depression of the test reflex. With intensity greater than 200 T a depression was seen, the time course of which is plotted in Fig. 8. These experiments have shown that in the acute and chronic spinal cat the amplitude of a segmental sympathetic reflex evoked by myelinated afferents can be depressed by a homo- or heterosegmental conditioning shock to afferent fibers. It is likely that the phenomenon underlying this depressant effect is an inhibitory process, and not a process of postexcitatory depression and hence it seems appropriate to use the term inhibition to describe this depression. One reason is that the size of the discharge evoked by the conditioning stimulus was usually a fraction (e.g., 20-30 ~ ) of that evoked by the test stimulus, yet often the test discharge was reduced by a fraction greater than that. Moreover, in the chronic spinal animal a depression of the test reflex, of time course similar to that observed in the acute spinal, could be elicited by a conditioning stimulus which evoked no discharge. It is possible that, at least at short C - T intervals, and when the conditioning stimulus evoked a discharge, postexcitatory depression contributed to the generation of the recovery curve 1,x~. In the acute spinal cat the inhibitory effect of the conditioning volley was always preceded by an excitatory effect. This constant association of the two effects might
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Fig. 8. Chronic spinal cat (6 weeksafter C8 transection). Recoverycurves of early reflex. CST recording. Test shock to T4 ICN. Conditioning shock to T4 ICN (filledsquares) and L4 SN (open circles). Ordinate and abscissa as in Fig. 1. suggest, as a simple explanation, the operation of a recurrent inhibitory circuit. However, the observations made with antidromic stimulation and intra- or extracellular recording 4,6,11 rule out this possibility. The data of Lebedev 9, obtained with paired antidromic shocks, are not in contrast with the ones listed above, since they can be accounted for on the basis of differences in time course of recovery of excitability among preganglionic axons of different sizes. Golgi studies by Rethelyi12 show that the axons of presumed sympathetic preganglionic neurons lack recurrent collaterals. Finally, this hypothesis is disproved by the observation that in some experimental conditions the excitatory and inhibitory processes can be uncoupled (ref. 13, Fig. 3C, using peripheral nerve stimulation; in the present experiments by the use of chronic spinal preparations). The longitudinal distribution along the cord of spinal afferent inhibition seems to follow, qualitatively, the same pattern described for the early somatosympathetic reflex by Sato and Schmidt 14. At constant stimulus intensity, the probability of evoking inhibition and the strength and duration of the inhibition decays with the distance of the segmental level of the conditioning stimulus from the segmental level of the test reflex. Conversely, the conditioning stimulus intensity required for inhibiting the test reflex increases with the distance from the segmental level(s) of the test reflex. This spatial pattern was observed with stimulus intensities within the myelinated fiber range. Long distance intersegmental inhibitory effects (e.g., T4 ICN inhibition of the T12 ICN to RN or of the L2 SN to Lz WR and L2 SN inhibition of a T4 ICN to CST reflex) were obtained only with very high stimulus intensities, i.e. in the C fiber range. In the chronic spinal cats the pattern of distribution was similar. The similarities of the spread observed with CST and either RN or L~ WR recording suggest that the distribution in the cephalic and caudal direction is symmetrical. The anatomical basis for the observed pattern of distribution may reside in the connectivity pattern of the primary afferents or of interneurons. Although the pattern of intersegmental spread of inhibition is similar to that of excitation, the time course of inhibition is much longer than that of excitation. The obvious question here
170 is whether the longer persistence of the inhibitory effect is the result o f some property of the inhibitory transmitter, as yet unknown, or of the neuronal circuitry associated with the inhibitory pathway (e.g., reverberatory and delay circuits). In the chronic spinal cats the threshold required for evoking inhibition by spinal afferent stimulation was lowered. In this preparation a conditioning stimulus which did not evoke excitation could inhibit the spinal reflex segmentally, and, at higher intensity, intersegmentally. In this preparation descending fiber tracts from supraspinal levels are degenerated, and the threshold o f inhibition by intraspinal stimulation is also lowered 8. On the other hand, the properties o f excitation by myelinated spinal afferents and intraspinal stimulation do not appear changed. A simple hypothesis to account for some of these findings is the development o f plastic changes at the level o f a c o m m o n inhibitory interneuron situated at the segmental level resulting in stronger connectivity o f intraspinal descending system and segmental afferents or denervation hypersensitivity after degeneration o f supraspinal descending pathway. This investigation was supported by the Medical Research Council o f Canada. F.K. was the holder o f a D A A D Fellowship.
1 BEACHAM,W. S., AND PERL, E. R., Characteristics of a spinal sympathetic reflex, J. Physiol. (Lond.),
173 (1964) 431-448. 2 CoorE, J. H., AND DOWNMAN,C. B. B., Central pathways of some autonomic reflex discharges, J. Physiol. (Lond.), 183 (1966) 714-729. 3 CoorE, J. H., DOWNMAN,C. B. B., AND WEBER, W. V., Reflex discharges into thoracic white rami elicited by somatic and visceral afferent excitation, J'. Physiol. (Lond.), 202 (1969) 147-160. 4 FERNANDEZDE MOLINA,A., KUNO, M., ANDPERL,E. R., Antidromically evoked responses from sympathetic preganglionic neurones, J. Physiol. (Lond.), 180 0965) 321-335. 5 FRANZ, D. N., EVANS, M. H., AND PERL, E. R., Characteristics of viscerosympathetic reflexes in the spinal cat, Amer. J. PhysioL, 221 (1966) 1292-1298. 6 HONGO,T., AND RYALL,R. W., Electrophysiological and microelectrophoretic studies on sympathetic preganglionic neurones in the spinal cord, Acta physiol, scand., 68 (1966) 96-104. 7 KIRCHNER,F., SATO,A., ANDWErOINGER,H., Central pathways of reflex discharges in the cervical sympathetic trunk, Pfliigers Arch. ges. PhysioL, 319 (1970) 1-11. 8 KmCHNER,F., WYSZOGRODSKLI., ANDPOLOSA,C., Some properties of sympathetic neuron inhibition by depressor area and intraspinal stimulation, submitted to Pfliigers Arch. ges. Physiol. 9 LEBEDEV,V. P., Features of axon &lateral horn sympathetic preganglionic neurons of low thoracic spinal cord, SechenovphysioL J. U.S.S.R., 57 (1971) 1647-1655. 10 OKADA,H., NAKAO,O., ANDNISIDA,I., Effect of sciatic stimulation upon the efferent impulses in the long ciliary nerve of the cat, Jap. J. PhysioL, 10 (1960) 327-339. 11 POLOSA,C., The silent period of sympathetic preganglionic neurons, Canad. J. PhysioL Pharmacol., 45 (1967) 1033-1045. 12 RETHELYI,M., Cell and neuropil architecture of the intermediolateral (sympathetic) nucleus of cat spinal cord, Brain Research, 46 (1972) 203-213. 13 SATO,A., Spinal and supraspinal inhibition of somato-sympathetic reflexes by conditioning afferent volleys, Pfliigers Arch. ges. PhysioL, 336 (1972) 121-133. 14 SATO,A., AND SCHMIDT,R. F., Spinal and supraspinal components of the reflex discharges into lumbar and thoracic white rami, J. Physiol. (Lond.), 212 (1971) 839-850. 15 WEIDINGER, H., Das Vasomotorenzentrum in der Medulla Oblongata. Versuche zur Bestirnmung
seiner Lage und Funktion, Habilitationsschrift, Univ. Heidelberg, 1966. 16 WVSZOGRODSKI,I., AND POLOSA,C., The inhibition of sympathetic preganglionic neurons by somatic afferents, Canad. J. Physiol. Pharmacol., 51 (1973) 29-38.
