Pfliigers Archiv
Pfltigers Arch. 381, 4 7 - 5 2 (1979)
EuropeanJournal of Physiology 9 by Springer-Verlag 1979
Initiation of Muscle Activity in Spinalized Pigeons During Spinal Cord Cooling and Warming K. G6rke and Fr.-K. Pierau Max-Planck-Institut f/Jr Physiologische und Klinische Forschung, W. G. Kerckhoff-Institut, Parkstrasse 1, D-6350 Bad Nauheim, Federal Republic of Germany
Abstract. 1. The effect of spinal cord temperature
changes on muscle activity was investigated in unanaesthetized intact and chronically spinalized pigeons and in acutely spinalized pigeons which were artificially respirated and lightly anaesthetized with ether. 2. Spinal cord cooling regularly produced an increase in muscle activity and visible muscle tremor in intact and spinalized pigeons. This motor cold defence reaction was less intensive in spinalized animals, but was qualitatively identical in all groups with regard to spindle shaped firing patterns and grouped discharges. 3. Intravenous injection of 6 0 - 1 0 0 mg/kg L-Dopa enhanced the motor response to spinal cord cooling in acutely spinalized pigeons. It is suggested that L-Dopa may act on dopaminergic or noradrenergic neurones in the spinal cord. 4. The results demonstrate that the generation of the motor cold defence response to cooling of the spinal cord in pigeons is basically independent from supraspinal nervous mechanisms. The decreased intensity of cold induced muscle activity in spinalized animals may be attributed to the loss of excitatory or disinhibitory descending inputs to the spinal cord. 5. Spinal cord warming above normal body temperature (41.5~ produced an increase in muscle activity and slow muscle movements. The pattern was qualitatively different from that of the cold induced tremor. Key words: Spinal transection - Temperature stimulation - L-Dopa -- Electromyogram - Bird.
cold defence responses such as vasoconstriction and shivering (for lit. see Simon, 1974) as well as behavioural responses for temperature regulation (Schmidt and Rautenberg, 1975). Furthermore, spinal cord cooling produces shivering in acutely and chronically spinalized dogs (Simon et al., 1966) rabbits (Kosaka and Simon, 1968a, b) and cats (Herdman, 1978) indicating that in mammals shivering is principally generated by a segmental spinal mechanism. Differences in tremor intensity between intact and spinalized animals suggest a supraspinal control of the spinal "tremor generator". In contrast, cold shivering could not be observed during spinal cord cooling in decerebrated or acutely spinalized pigeons (Rautenberg et al., 1972). It was therefore suggested that an intact connection between spinal cord and brain is required for the cold defence response in birds. However, it has also been demonstrated that most of the spinal motoneurones in pigeons increased their discharge rate during spinal cord cooling, (G6rke, 1976) and thus exhibited similar changes of motoneuronal excitability upon local cooling as in cats (Pierau et al., 1976). As a consequence of this similarity of local temperature effects upon motoneurones in pigeons and cats, the motor cold defence response to spinal cord cooling was reinvestigated in acutely and chronically spinalized pigeons. The results demonstrate that spinal cord cooling produces an increase in muscle activity and induces muscle tremor in spinalized pigeons. The cold shivering was intensified by intravenous, injection of L-Dopa which is known to generate locomotion in spinalized cats (Grillner, 1969; Budakowa, 1973).
Methods Introduction
It is well established in mammals and birds that selective cooling of the spinal cord activates autonomic
Adult pigeons (Columba livia) were provided with chronically implanted spinal cord thermodes. Each consisted of an U-shaped polyethylene tube which was inserted into the vertebral canal from T h 2 - C 4 - 5 during ether anaesthesia. After recovery from anaes-
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48
Pflfigers Arch. 381 (1979) intact
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thesia the pigeons were placed on a grooved styrofoam block so as to keep the animals in an upright position. The heads including the eyes were covered with a black pastboard hood which usually prevented the animals from further movements. Spinal cord temperature was controlled by circulating water of different temperatures through the vertebral thermode and was measured with a fine thermoelement inserted between the two shanks of the thermode. Perfusion temperature higher than 44~C need to be avoided because the animals became restless and tried to escape at spinal temperatures above 44~C. The electrical activity of the muscles was detected by means of "Disa"-(l 3 KO 541) or bipolar steel needle electrodes. The resulting activity was recorded on a pen recorder (Brush, U.S.A.) or photographed directly from an oscilloscope. Usually, the m. pectoralis, m. longissimus dorsi and m. triceps brachii were used for EMG recordings. The ECG was monitored throughout the experiments. The body temperature was maintained in the normal range of 41.5 ~C by means of a heating pad and an infrared lamp. For acute spinalization the pigeons, as prepared above, were anaesthetized with ether and artificial respiration was applied by means of an unidirectional air flow of 0.12 l/rain from an intratracheal catheter through an outlet in an abdominal air sac. The anaesthesia was continued by adding ether into the airflow. The spinal cord was transected at C3- 4 without severing the spinal vein. In the final 5 experirnents the blood pressure of the brachial artery was monitored by means of a strain gauge and a bridge circuit. Drugs were injected into the brachial vein. 6 0 - 1 0 0 mg/kg L-Dopa (2 ~ ) in Ringer's solution was applied in 12 pigeons. In order to prolong the LDopa effect, pre-injections of the monoamino-oxidase inhibitor Nialamide (1 ~ ; 50 mg/kg) were made in 5 experiments. For chronical spinalization, spinal transection was performed at Th 4 during ether anaesthesia in 13 pigeons. The spinal thermode was placed in the vertebral canal from Th4 to SC~ (sacrococcygeal). Some animals showed a reduced reflex activity after this manipulation, which may have been caused by some damage of the dorsal roots since in pigeons the glycogen body occupies the extradural space in this part of the vertebral canal As a result, only 8 of the 13 animals prepared reacted with clear reflex responses following mechanical stimulation of the legs. The reported results were obtained from the experiments performed in these 8 animals. The spinalized animals were able to breathe spontaneously, to maintain their body temperature at normal value and to feed and groom themselves; they remained in good condition for up to 5 weeks. No significant differences in EMG-recordings from the m. quadriceps fern. or m. hallnc, brev. were found at different times after spinal transection ( 2 - 35 days).
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Fig. 1 Increase of muscle activity in an intact (a) and an acutely spinalized (b) pigeon during spinal cord cooling.EMG-recordings of two different animals from the muscles indicated. Temperature in the vertebral canal: lower lines in a and b. Arrows indicate onset of cooling. Note: the different voltage calibration in a and b. Spikes are partly retouched
Table l. Threshold temperatures of the vertebral canal for the activation of muscle activity during spinal cord cooling and warming in intact, acutely and chronically spinalized pigeons. Only those experiments were regarded in which a distinct threshold could be distinguished
Cooling
Intact
Acutely spinalized
Chronically spinalized
~ = 38,0~ s = 1.4 R= 5 n =12"
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~? = s = R-~ n~-
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Warming 2 = s = R-n~-
44.8~ 1.0 2.3 4
45.1~ 0.6 1.3 6
* One animal exhibited an extraordinary low threshold in the intact (32~ C) as well as in the spinalized condition (28~C) and was not considered in this table. n = number of animals; Yc = mean; s = standard deviation; R = range
Results
a) Intact Pigeons.
In non spinalized pigeons, spinal cord cooling always produced a cold defence response and vigorous shivering similar to the cold response r e p o r t e d b y R a u t e n b e r g (1969). T h e e x a m p l e i n Fig. l a demonstrates the initiation of muscle discharge in the pectoralis muscle some seconds after the onset of spinal cord cooling. Both, the increased muscle activity and the accompanying visible tremor were maintained throughout the period of cooling. The threshold temperature for the increase in muscle activity was between 37~ and 39~ (2= 38,0~ T a b l e 1) w h i c h is i n g o o d a g r e e m e n t w i t h t h e d a t a o f R a u t e n b e r g (1969).
b) Acutely Spinalized Pigeons.
Muscular activation due t o s p i n a l c o r d c o o l i n g c o u l d b e e l i c i t e d i n 13 o u t o f 22
K. G6rke and Fr.-K. Pierau : Muscle Activity of Spinalized Pigeons on Spinal Cooling and Warming
acutely spinalized pigeons. The remaining 9 animals did not react to pinching or to other mechanical stimuli which probably indicates a severe spinal shock. The intravertebral threshold temperature for muscle activation was only little lower (Table I) but the intensity of shivering was usually less than in intact pigeons, as is demonstrated in Fig. 1b. The recordings of Fig. l b also indicate the individual differences of cold induced EMG-activity in different muscles. The pectoralis muscle was usually more activated than the other muscles and showed a more tonic increase of activity. An additional peak of muscle activity often appeared during rewarming, which is also reported for intact and spinalized animals (Simon et al., 1966). The increase of activity of the m. long. dorsi was often of a more phasic type and sometimes ceased during cooling (Fig. lb). As in intact birds, the enhanced muscle activity was accompanied by distinct shivering. During prolonged intense cooling the activation was sometimes reduced. On the other hand, strong twitches occurring in single muscle groups often induced uncoordinated movements of the wings or the legs. This appears to be typical for the effect of spinal cord cooling in spinalized pigeons probably indicating a loss of supraspinal control.
c) Effect of L-Dopa on Acutely Spinalized Pigeons. The previous results suggest that muscle activity and shivering induced by spinal cord cooling is present in spinalized animals, but that the intensity of muscle activity is much smaller than in intact pigeons. The question arises as to whether the quantitative differences of the spinal cooling response was due to the loss of a specific afferent-efferent supraspinal pathway or the consequence of a reduced excitatory input in spinalized animals. In addition, the large decrease of systemic blood pressure after spinal transection may also reduce muscle activity. However, the latter seems not to be the principal cause of the decreased muscle activity since application (0.1 mg/kg i.v.) of the octapeptide Angiotensin regularly increased the blood pressure but had very little effect, if any, on shivering intensity. As it is known that injection of L-Dopa induces the spinal motoric network for locomotion in spinalized cats (Grillner, 1969) this drug was administered in the acutely spinalized pigeons. Intravenous injection of LDopa usually increased muscle activity at normal spinal cord temperature (Fig. 2b) and significantly enhanced the cold induced muscle tremor (Fig. 20. Often the latency of the onset of muscle tremor was much shorter after L-Dopa injection, as is demonstrated in Fig. 2d, even though in this example the velocity of cooling was smaller than in the control period (Fig. 2c). Fig. 2g shows that the pattern of cold tremor in acutely
49 m. Iongissimus
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cooling in a acmely spinalized pigeon, a--f. EMG-recordings of the m. longissimus dorsi at intravertebrat temperatures indicated, e--d = 1 min, e--f = 3 rain after onset of cooling, g: grouped discharges from part f at higher film speed; h: grouped discharges of the same muscle before spinal transection. Spikes are partly retouched
spinalized pigeons after administration of L-Dopa is similar to that of untreated intact animals (Fig. 2h). In the given example the frequency of the grouped discharges of the m. long. dorsi is about 36/s in the spinalized L-Dopa treated pigeon. This falls within the range of the tremor frequency (20-36/s) of the back muscle in intact animals. The effect of L-Dopa depended on the condition of the animals. Immediate increase of the intensity of the cold response was produced by DDopa in 2 intact pigeons. Drug administration during the first 30 min after spinal transection also immediately increased muscle tonus and enabled the animals to respond to spinal cord cooling (3 animals). However, L-Dopa injection 3 - 4 h after spinal transection only once increased muscle activity immediately but in 4 animals an additional injection of DDopa was required to induce a cold defence response. These differences of the L-Dopa action are probably due to the fall in blood pressure after spinatization. Our experiments seem to indicate that blood pressure needs to be elevated from about 50mmHg to normal values of 140mmHg for some time before muscles can be properly activated. Two animals in which repeated injections of L-Dopa
50
Pfliigers Arch. 381 (1979) m. q u a d r i c e p s femoris
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Fig. 3. Increase of muscle activity during spinal cord cooling in a chronically spinalized pigeon (4 days p.o.). EMG-recordings of the m. quadriceps femoris. Temperature in the vertebral canal: fine lines in recordings a--f. g: grouped discharges from part e at higher film speed and 5 times higher amplification. Note: spindle shaped pattern of tonic activity after the beginning of spinal cooling in b and e. Spikes are partly retouched
had no effect on muscle activity were in poor condition after spinal transection; the blood pressure of one of these animals decreased to about 30 mmHg and only increased up to 100 mmHg after administration of LDopa.
d) Chronically SpinaIized Pigeons. The primary complications resulting from acute spinalization such as decreased blood pressure, disability of temperature regulation and acute loss of supraspinal excitatory motor input can be avoided in chronically spinalized animals. However, because of the low transection level (see Methods), the recordings are limited to the thigh muscles which have only limited functional significance for cold shivering. Spinal cord cooling regularly produced an increase in muscle activity and muscle tremor in the chronically spinalized pigeons. The increase in EMG-activity usually started only a few seconds after the beginning of spinal cord cooling (Fig. 3 b) and was often associated with leg movements. The intensity of EMG-activity increased with further cooling (Fig. 3 b - e ) which was accompanied by a visible tremor of the leg. Cha(acteristically, the tonic activity often changed into
spindle shaped bursts (Fig. 3 b - c ) and motor units recruited during cooling tended to fire in grouped discharges (Fig. 3 g). The occurrence of spindle shaped bursts and grouped discharges has been suggested to be a characteristic feature for cold induced tremor (for lit. see Kosaka and Simon, 1968a, b) and is thus a further indication of the identity of shivering in intact and chronically spinalized pigeons, although the threshold temperature for cold activation was lower than in the other two groups (Table 1). The frequency of grouped discharges of the m. quadric, fern. was slightly smaller (16/s in the example of Fig. 3g) than the discharge frequency of the same muscle in intact animals, which varied between 16 and 30/s ( 2 = 2 2 ; s=3.3). The increase of tonic EMG-activity during rewarming (Fig. 3f) resembles the pronounced muscle activity which was often recorded in intact pigeons; similar observations are reported in the literature (Simon et al., 1966). Musde activity was not only increased during cooling but also during warming above normal body temperature in intact as well as in spinalized pigeons. It was impossible to record muscle activity in intact unanaesthetized pigeons at temperatures higher than 44~C, because the animals were agitated. However, in chronically spinalized pigeons, warming above normal body temperature always produced a characteristic regular firing pattern in EMG recordings (Fig. 4). The frequency of this tonic discharge increased with temperature up to 46~ and was maintained throughout the warming period (Fig. 4b). Higher temperatures were not used to avoid damage of spinal cord tissue. However, we never observed irreversible changes of EMG-activity or any other sign of neuronal injury during repeated warming. On the contrary, the spinal cord temperature at which an abrupt onset (increasing temperature) or abolition (decreasing temperature) of tonic muscle activity was found, was constant for any given motor unit in the range of _+0.2 ~C (Table 1). This constancy of threshold temperatures of tonic EMGactivity was a characteristic feature for warm activation. In contrast to the muscle reaction to spinal cord cooling, warming never produced grouped discharges or spindle shaped pattern in the EMG. Accordingly, local warming of the spinal cord above normal body temperature never produced muscle tremor comparable to cold shivering. However, warming always caused slow movements of the legs and the toes in all 8 chronically spinalized pigeons investigated.
Discussion
The present results show that in pigeons the cold defence response to local spinal cooling does not
51
K. GSrke and Fr.-K. Pierau: Muscle Activity of Spinalized Pigeons on Spinal Cooling and Warming
46
~f ~JIJu~``~ui~d~`L~``~`h~`~i`i ~"*il"i~l~i ~jh~j~l~iLiL`L`h~ji~.~u~i~tjj'iui~k~hu'bIIr~h~j` Fig. 4 Tonic activity of the m. flexor hallucis brevis induced by spinal cord warming in an chronically spinalized pigeon. Temperature in the vertebral canal : upper lines in recordings a--c. Note: tonic activity does not adapt during spinal warming. Spikes are partly retouched
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require an intact supraspinal loop (Rautenberg et al., 1972; Necker and Rautenberg, 1975; Hissa and Rautenberg, 1974, 1975). Similar to mammals (Simon et al., 1966; Herdman, 1978) spinalized pigeons respond to local cooling of the spinal cord with an increase of EMG-activity and muscle tremor, which is qualitatively identical with the cold tremor produced by local spinal cooling in intact animals. This is in good agreement with the observations in both spinalized and intact pigeons, that spinal cord cooling enhances monoand polysynaptic spinal reflexes as well as the activity of single motoneurones (G6rke et at., 1975; G6rke, 1976). Thus, the previous finding that the cold sensitivity of spinal motoneurones in pigeons resembles the cold sensitivity of spinal motoneurones in cats (Klussmann and Pierau, 1972; Pierau et al., 1976) conforms to the results of the present investigation. Our results also suggest that the spinal cord of pigeons possesses the capacity of cold defence mechanisms as far as motor activity is concerned. Since the ability for cold induced muscle tremor in mammals is also organized at the segmental level (Simon et al., 1966; Herdman, 1978) one may conclude that the spinal cord of homoiothermic organisms is generally equipped with the neuronal circuitry for the generation of coordinated muscle contractions like cold induced tremor. The increase of cold induced tremor by L-Dopa in acutely spinalized pigeons supports the notion, that the loss of shivering intensity after spinal transection is mainly due to the interruption of an excitatory or disinhibitory descending input. Similar to spinalized cats, L-Dopa may stimulate noradrenergic spinal receptors normally provided by descending noradrenergic neurones (And~n et al., 1966; Grillner, 1969; Budakowa, 1973). A release of the spinal generator for locomotion by this descending input, as discussed by Grillner (1975), seems to be an adaptable model for the spinal '~ generator".
Although the capacity for coordinated muscle tremor appears to be maintained in acutely and chronically spinalized animals, the pattern of shivering often changed to coarse movements and contractions of single muscle groups in our experiments as well as those in dogs (Simon et al., 1966) and rabbits (Kosaka and Simon, 1968a). This also supports the view that the descending control is responsible for the subtile coordination of muscle tremor. The negative results of Rautenberg et al. (1972) who could not induce cold shivering in spinalized or decerebrated pigeons might be attributed to deep anaesthesia or less controlled conditions of the animals, but the published data are incomplete. The inhibition of cold shivering by hypothalamic cooling (Necker and Rautenberg, 1975) might result from a block of descending excitatory pathways or activation of supraspinal inhibition. The inhibition of cold shivering by intrahypothalamic injections of catecholamines (Hissa and Rautenberg, 1975) seems to support the latter view. In intact animals the increase in EMG-activity during spinal warming above normal body temperature was accompanied by agitated movement. Since the threshold temperature was at about 44 ~C, we interpreted this effect as the result from noxious heat stimulation. However, our results on spinalized pigeons indicate that the effect of local warming on motor activity is at least in part a direct effect of temperature on the motoneurones themselves. This is in agreement with the observation that some motoneurones increase their firing rate with spinal cord warming (G6rke, 1975). In decerebrated cats a number of tonic motoneurones were also activated by local spinal warming (Rau, 1970) suggesting that the temperature for maximal firing rate of some tonic motone~rones is higher than normal body temperature. In contrast, most of the phasic motoneurones increase their activity with spinal cord cooling (Klussmann, 1969). One of the most obvious
52
electrophysiological differences between both populations is the more pronounced after hyperpolarization in tonic motoneurones, which seems to determine the slower but regular discharge of tonic motoneurones (Eccles et al., 1958). However, th~ duration and amplitude of after hyperpolarization of motoneurones is decreased by warming (Pierau and Klussmann, 1971), which in turn might cause the neurones to increase their firing frequency and may partly explain why numerous tonic motoneurones are activated at higher temperatures. At present the question remains open as to whether the different pattern of muscle contraction during warm and cold activation is connected with the different temperature sensitivity of tonic and phasic motoneurones as revealed by local cooling and warming (Klussmann et al., 1972). Acknowledgements. We thank Mrs. M. Hofmann for technical assistance and Drs. E. Simon and J. B. Mercer for helpful discussions.
References And~n, N. E., Jukes, M. G. M., Lundberg, A., Vyklicky, L.: The effect of Dopa on the spinal cord. I. Influence on transmission from primary afferents. Acta Physiol. Scand. 67, 373-386 (1966) Budakowa, N. N. : Stepping movements in the spinal cat due to Dopa administration. Fiziol. Zh. SSSR. 59, 1190--1198 (1973) Eccles, J. C., Eccles, R. M., Lundberg, A. : The action potentials of the alpha motoneurones supplying fast and slow muscles. J. Physiol. (Lond.) 142, 275-291 (1958) G6rke, K. : Effect of spi~al cord temperature on spontaneous activity of spinal motoneurones in birds and reptiles. Pfliigers Arch. 362, R94 (1976) G6rke, K., Necker, R., Rautenberg, W.: Neurophysiological investigation of spinal reflexes at different temperatures of the spinal cord in birds and reptiles. Pfliigers Arch. 359, 269-271 (1975) Grillner, S. : Supraspinal and segmental control of static and dynamic c~-motoneurones in the cat. Acta Physiol. Scand., Suppl. 327, 1 34 (1969) Grillner, S.: Locomotion in vertebrates: Central mechanisms and reflex interaction. Physiol. Rev. 55, 247-304 (1975) Herdman, S. J. : Recovery of shivering in spinal cats. Exp. Neurol. 59, 177-189 (1978)
Pfltigers Arch. 381 (1979) Hissa, R., Rautenberg, W. : The infuence of centrally applied Noradrenaline on shivering and body temperature in the pigeon. J. Physiol. (Lond.) 238, 421-435 (1974) Hissa, R., Rautenberg, W. : Thermoregulatory effects of intrahypothalamic injections of neurotransmitters and their inhibitors in the pigeon. Comp. Biocbem. Physiol. 51A, 319-326 (1975) Klussmann, F. W. : Der Einfluf3 der Temperatur auf die afferente und efferente motorische Innervation des Rtickenmarks. Pflfigers Arch. 305, 295-315 (1969) Klussmann, F. W., Pierau, Fr.-K. : Extrahypothatamic deep body thermosensitivity. In: Essays on temperature re~tlatiou (J. Bligh and R. E. Moore, eds.). Amsterdam-London: North Holland Publ. Comp. 1972 Klussmann, F. W., Pierau, Fr.-K., Spaan, G.: Frequency and synchronization of spinal alpha motoneurones during shivering. Posebna Izdanja - XII. Odjelhene Medicinskih Nauka, Knija 4 (1972) Kosaka, M., Simon, E. : K~iltetremor wacher, chronisch spinalisierter Kaninchen im Vergleich zum K~iltezittern intakter Tiere. Pfltigers Arch. 302, 333-356 (1968a) Kosaka, M., Simon, E. : Der zentralnerv6se spinale Mechanismus des K/iltezitterns. Pflfigers Arch. 302, 357-373 (1968b) Necker, R., Rautenberg, W.: Effect of spinal deafferentation on temperature regulation and spinal thermosensitivity in pigeons. Pfl/igers Arch. 360, 287-299 (1975) Pierau, Fr.-K., Klussmann, F. W. : Spinal excitation and inhibition during local spinal cooling and warming. J. Physiol. (Paris) 63, 380- 382 (1971) Pierau, Fr.-K., Klee, M. R., Klussmann, F. W. : Effect of temperature on postsynaptic potentials of cat spinal motoneurones. Brain Res. 114, 2 1 - 3 4 (1976) Rau, B. : Der Einflul3 der R/ickenmarkstemperatur auf die Entladungsfrequenz und die rekurrente Hemmung tonischer Motoneurone. Dissertation, Justus Liebig-Universit/it, Gie13en 1970 Rautenberg, W. : Die Bedeutung der zentralnerv6sen Thermosensitivifiit ffir die Temperaturregulation der Taube. Z. vergl. Physiol. 62, 235-266 (1969) Rautenberg, W., Necker, R., May, B. : Thermoregulatory responses of the pigeon to changes of the brain and the spinal cord temperatures. Pfliigers Arch. 338, 31-42 (1972) Simon, E.: Temperature regulation: The spinal cord as a site of extrahypothalamic thermoregulatory functions. Rev. Physiol. Biochem. Pharmacol. 71, 1 - 76 (1974) Simon, E., Klussmann, F. W., Rautenberg, W., Kosaka, M.: K~iltezittern bei narkotisierten spinalen Hunden. Pfl/igers Arch. ges. Physiol. 291, 187-204 (1966) Schmidt, I., Rautenberg, W.: Instrumental thermoregulatory behavior in pigeons. J. Comp. Physiol. 101, 225-235 (1975) Received December 19, 1978
Pfltigers Arch. 381, 4 7 - 5 2 (1979)
EuropeanJournal of Physiology 9 by Springer-Verlag 1979
Initiation of Muscle Activity in Spinalized Pigeons During Spinal Cord Cooling and Warming K. G6rke and Fr.-K. Pierau Max-Planck-Institut f/Jr Physiologische und Klinische Forschung, W. G. Kerckhoff-Institut, Parkstrasse 1, D-6350 Bad Nauheim, Federal Republic of Germany
Abstract. 1. The effect of spinal cord temperature
changes on muscle activity was investigated in unanaesthetized intact and chronically spinalized pigeons and in acutely spinalized pigeons which were artificially respirated and lightly anaesthetized with ether. 2. Spinal cord cooling regularly produced an increase in muscle activity and visible muscle tremor in intact and spinalized pigeons. This motor cold defence reaction was less intensive in spinalized animals, but was qualitatively identical in all groups with regard to spindle shaped firing patterns and grouped discharges. 3. Intravenous injection of 6 0 - 1 0 0 mg/kg L-Dopa enhanced the motor response to spinal cord cooling in acutely spinalized pigeons. It is suggested that L-Dopa may act on dopaminergic or noradrenergic neurones in the spinal cord. 4. The results demonstrate that the generation of the motor cold defence response to cooling of the spinal cord in pigeons is basically independent from supraspinal nervous mechanisms. The decreased intensity of cold induced muscle activity in spinalized animals may be attributed to the loss of excitatory or disinhibitory descending inputs to the spinal cord. 5. Spinal cord warming above normal body temperature (41.5~ produced an increase in muscle activity and slow muscle movements. The pattern was qualitatively different from that of the cold induced tremor. Key words: Spinal transection - Temperature stimulation - L-Dopa -- Electromyogram - Bird.
cold defence responses such as vasoconstriction and shivering (for lit. see Simon, 1974) as well as behavioural responses for temperature regulation (Schmidt and Rautenberg, 1975). Furthermore, spinal cord cooling produces shivering in acutely and chronically spinalized dogs (Simon et al., 1966) rabbits (Kosaka and Simon, 1968a, b) and cats (Herdman, 1978) indicating that in mammals shivering is principally generated by a segmental spinal mechanism. Differences in tremor intensity between intact and spinalized animals suggest a supraspinal control of the spinal "tremor generator". In contrast, cold shivering could not be observed during spinal cord cooling in decerebrated or acutely spinalized pigeons (Rautenberg et al., 1972). It was therefore suggested that an intact connection between spinal cord and brain is required for the cold defence response in birds. However, it has also been demonstrated that most of the spinal motoneurones in pigeons increased their discharge rate during spinal cord cooling, (G6rke, 1976) and thus exhibited similar changes of motoneuronal excitability upon local cooling as in cats (Pierau et al., 1976). As a consequence of this similarity of local temperature effects upon motoneurones in pigeons and cats, the motor cold defence response to spinal cord cooling was reinvestigated in acutely and chronically spinalized pigeons. The results demonstrate that spinal cord cooling produces an increase in muscle activity and induces muscle tremor in spinalized pigeons. The cold shivering was intensified by intravenous, injection of L-Dopa which is known to generate locomotion in spinalized cats (Grillner, 1969; Budakowa, 1973).
Methods Introduction
It is well established in mammals and birds that selective cooling of the spinal cord activates autonomic
Adult pigeons (Columba livia) were provided with chronically implanted spinal cord thermodes. Each consisted of an U-shaped polyethylene tube which was inserted into the vertebral canal from T h 2 - C 4 - 5 during ether anaesthesia. After recovery from anaes-
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48
Pflfigers Arch. 381 (1979) intact
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thesia the pigeons were placed on a grooved styrofoam block so as to keep the animals in an upright position. The heads including the eyes were covered with a black pastboard hood which usually prevented the animals from further movements. Spinal cord temperature was controlled by circulating water of different temperatures through the vertebral thermode and was measured with a fine thermoelement inserted between the two shanks of the thermode. Perfusion temperature higher than 44~C need to be avoided because the animals became restless and tried to escape at spinal temperatures above 44~C. The electrical activity of the muscles was detected by means of "Disa"-(l 3 KO 541) or bipolar steel needle electrodes. The resulting activity was recorded on a pen recorder (Brush, U.S.A.) or photographed directly from an oscilloscope. Usually, the m. pectoralis, m. longissimus dorsi and m. triceps brachii were used for EMG recordings. The ECG was monitored throughout the experiments. The body temperature was maintained in the normal range of 41.5 ~C by means of a heating pad and an infrared lamp. For acute spinalization the pigeons, as prepared above, were anaesthetized with ether and artificial respiration was applied by means of an unidirectional air flow of 0.12 l/rain from an intratracheal catheter through an outlet in an abdominal air sac. The anaesthesia was continued by adding ether into the airflow. The spinal cord was transected at C3- 4 without severing the spinal vein. In the final 5 experirnents the blood pressure of the brachial artery was monitored by means of a strain gauge and a bridge circuit. Drugs were injected into the brachial vein. 6 0 - 1 0 0 mg/kg L-Dopa (2 ~ ) in Ringer's solution was applied in 12 pigeons. In order to prolong the LDopa effect, pre-injections of the monoamino-oxidase inhibitor Nialamide (1 ~ ; 50 mg/kg) were made in 5 experiments. For chronical spinalization, spinal transection was performed at Th 4 during ether anaesthesia in 13 pigeons. The spinal thermode was placed in the vertebral canal from Th4 to SC~ (sacrococcygeal). Some animals showed a reduced reflex activity after this manipulation, which may have been caused by some damage of the dorsal roots since in pigeons the glycogen body occupies the extradural space in this part of the vertebral canal As a result, only 8 of the 13 animals prepared reacted with clear reflex responses following mechanical stimulation of the legs. The reported results were obtained from the experiments performed in these 8 animals. The spinalized animals were able to breathe spontaneously, to maintain their body temperature at normal value and to feed and groom themselves; they remained in good condition for up to 5 weeks. No significant differences in EMG-recordings from the m. quadriceps fern. or m. hallnc, brev. were found at different times after spinal transection ( 2 - 35 days).
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Fig. 1 Increase of muscle activity in an intact (a) and an acutely spinalized (b) pigeon during spinal cord cooling.EMG-recordings of two different animals from the muscles indicated. Temperature in the vertebral canal: lower lines in a and b. Arrows indicate onset of cooling. Note: the different voltage calibration in a and b. Spikes are partly retouched
Table l. Threshold temperatures of the vertebral canal for the activation of muscle activity during spinal cord cooling and warming in intact, acutely and chronically spinalized pigeons. Only those experiments were regarded in which a distinct threshold could be distinguished
Cooling
Intact
Acutely spinalized
Chronically spinalized
~ = 38,0~ s = 1.4 R= 5 n =12"
2 = 37.6~ s = 2.4 R = 7.6 n---- 8*
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~? = s = R-~ n~-
2 = s = R= n=
44.8~ 0.6 2.0 8
Warming 2 = s = R-n~-
44.8~ 1.0 2.3 4
45.1~ 0.6 1.3 6
* One animal exhibited an extraordinary low threshold in the intact (32~ C) as well as in the spinalized condition (28~C) and was not considered in this table. n = number of animals; Yc = mean; s = standard deviation; R = range
Results
a) Intact Pigeons.
In non spinalized pigeons, spinal cord cooling always produced a cold defence response and vigorous shivering similar to the cold response r e p o r t e d b y R a u t e n b e r g (1969). T h e e x a m p l e i n Fig. l a demonstrates the initiation of muscle discharge in the pectoralis muscle some seconds after the onset of spinal cord cooling. Both, the increased muscle activity and the accompanying visible tremor were maintained throughout the period of cooling. The threshold temperature for the increase in muscle activity was between 37~ and 39~ (2= 38,0~ T a b l e 1) w h i c h is i n g o o d a g r e e m e n t w i t h t h e d a t a o f R a u t e n b e r g (1969).
b) Acutely Spinalized Pigeons.
Muscular activation due t o s p i n a l c o r d c o o l i n g c o u l d b e e l i c i t e d i n 13 o u t o f 22
K. G6rke and Fr.-K. Pierau : Muscle Activity of Spinalized Pigeons on Spinal Cooling and Warming
acutely spinalized pigeons. The remaining 9 animals did not react to pinching or to other mechanical stimuli which probably indicates a severe spinal shock. The intravertebral threshold temperature for muscle activation was only little lower (Table I) but the intensity of shivering was usually less than in intact pigeons, as is demonstrated in Fig. 1b. The recordings of Fig. l b also indicate the individual differences of cold induced EMG-activity in different muscles. The pectoralis muscle was usually more activated than the other muscles and showed a more tonic increase of activity. An additional peak of muscle activity often appeared during rewarming, which is also reported for intact and spinalized animals (Simon et al., 1966). The increase of activity of the m. long. dorsi was often of a more phasic type and sometimes ceased during cooling (Fig. lb). As in intact birds, the enhanced muscle activity was accompanied by distinct shivering. During prolonged intense cooling the activation was sometimes reduced. On the other hand, strong twitches occurring in single muscle groups often induced uncoordinated movements of the wings or the legs. This appears to be typical for the effect of spinal cord cooling in spinalized pigeons probably indicating a loss of supraspinal control.
c) Effect of L-Dopa on Acutely Spinalized Pigeons. The previous results suggest that muscle activity and shivering induced by spinal cord cooling is present in spinalized animals, but that the intensity of muscle activity is much smaller than in intact pigeons. The question arises as to whether the quantitative differences of the spinal cooling response was due to the loss of a specific afferent-efferent supraspinal pathway or the consequence of a reduced excitatory input in spinalized animals. In addition, the large decrease of systemic blood pressure after spinal transection may also reduce muscle activity. However, the latter seems not to be the principal cause of the decreased muscle activity since application (0.1 mg/kg i.v.) of the octapeptide Angiotensin regularly increased the blood pressure but had very little effect, if any, on shivering intensity. As it is known that injection of L-Dopa induces the spinal motoric network for locomotion in spinalized cats (Grillner, 1969) this drug was administered in the acutely spinalized pigeons. Intravenous injection of LDopa usually increased muscle activity at normal spinal cord temperature (Fig. 2b) and significantly enhanced the cold induced muscle tremor (Fig. 20. Often the latency of the onset of muscle tremor was much shorter after L-Dopa injection, as is demonstrated in Fig. 2d, even though in this example the velocity of cooling was smaller than in the control period (Fig. 2c). Fig. 2g shows that the pattern of cold tremor in acutely
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cooling in a acmely spinalized pigeon, a--f. EMG-recordings of the m. longissimus dorsi at intravertebrat temperatures indicated, e--d = 1 min, e--f = 3 rain after onset of cooling, g: grouped discharges from part f at higher film speed; h: grouped discharges of the same muscle before spinal transection. Spikes are partly retouched
spinalized pigeons after administration of L-Dopa is similar to that of untreated intact animals (Fig. 2h). In the given example the frequency of the grouped discharges of the m. long. dorsi is about 36/s in the spinalized L-Dopa treated pigeon. This falls within the range of the tremor frequency (20-36/s) of the back muscle in intact animals. The effect of L-Dopa depended on the condition of the animals. Immediate increase of the intensity of the cold response was produced by DDopa in 2 intact pigeons. Drug administration during the first 30 min after spinal transection also immediately increased muscle tonus and enabled the animals to respond to spinal cord cooling (3 animals). However, L-Dopa injection 3 - 4 h after spinal transection only once increased muscle activity immediately but in 4 animals an additional injection of DDopa was required to induce a cold defence response. These differences of the L-Dopa action are probably due to the fall in blood pressure after spinatization. Our experiments seem to indicate that blood pressure needs to be elevated from about 50mmHg to normal values of 140mmHg for some time before muscles can be properly activated. Two animals in which repeated injections of L-Dopa
50
Pfliigers Arch. 381 (1979) m. q u a d r i c e p s femoris
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Fig. 3. Increase of muscle activity during spinal cord cooling in a chronically spinalized pigeon (4 days p.o.). EMG-recordings of the m. quadriceps femoris. Temperature in the vertebral canal: fine lines in recordings a--f. g: grouped discharges from part e at higher film speed and 5 times higher amplification. Note: spindle shaped pattern of tonic activity after the beginning of spinal cooling in b and e. Spikes are partly retouched
had no effect on muscle activity were in poor condition after spinal transection; the blood pressure of one of these animals decreased to about 30 mmHg and only increased up to 100 mmHg after administration of LDopa.
d) Chronically SpinaIized Pigeons. The primary complications resulting from acute spinalization such as decreased blood pressure, disability of temperature regulation and acute loss of supraspinal excitatory motor input can be avoided in chronically spinalized animals. However, because of the low transection level (see Methods), the recordings are limited to the thigh muscles which have only limited functional significance for cold shivering. Spinal cord cooling regularly produced an increase in muscle activity and muscle tremor in the chronically spinalized pigeons. The increase in EMG-activity usually started only a few seconds after the beginning of spinal cord cooling (Fig. 3 b) and was often associated with leg movements. The intensity of EMG-activity increased with further cooling (Fig. 3 b - e ) which was accompanied by a visible tremor of the leg. Cha(acteristically, the tonic activity often changed into
spindle shaped bursts (Fig. 3 b - c ) and motor units recruited during cooling tended to fire in grouped discharges (Fig. 3 g). The occurrence of spindle shaped bursts and grouped discharges has been suggested to be a characteristic feature for cold induced tremor (for lit. see Kosaka and Simon, 1968a, b) and is thus a further indication of the identity of shivering in intact and chronically spinalized pigeons, although the threshold temperature for cold activation was lower than in the other two groups (Table 1). The frequency of grouped discharges of the m. quadric, fern. was slightly smaller (16/s in the example of Fig. 3g) than the discharge frequency of the same muscle in intact animals, which varied between 16 and 30/s ( 2 = 2 2 ; s=3.3). The increase of tonic EMG-activity during rewarming (Fig. 3f) resembles the pronounced muscle activity which was often recorded in intact pigeons; similar observations are reported in the literature (Simon et al., 1966). Musde activity was not only increased during cooling but also during warming above normal body temperature in intact as well as in spinalized pigeons. It was impossible to record muscle activity in intact unanaesthetized pigeons at temperatures higher than 44~C, because the animals were agitated. However, in chronically spinalized pigeons, warming above normal body temperature always produced a characteristic regular firing pattern in EMG recordings (Fig. 4). The frequency of this tonic discharge increased with temperature up to 46~ and was maintained throughout the warming period (Fig. 4b). Higher temperatures were not used to avoid damage of spinal cord tissue. However, we never observed irreversible changes of EMG-activity or any other sign of neuronal injury during repeated warming. On the contrary, the spinal cord temperature at which an abrupt onset (increasing temperature) or abolition (decreasing temperature) of tonic muscle activity was found, was constant for any given motor unit in the range of _+0.2 ~C (Table 1). This constancy of threshold temperatures of tonic EMGactivity was a characteristic feature for warm activation. In contrast to the muscle reaction to spinal cord cooling, warming never produced grouped discharges or spindle shaped pattern in the EMG. Accordingly, local warming of the spinal cord above normal body temperature never produced muscle tremor comparable to cold shivering. However, warming always caused slow movements of the legs and the toes in all 8 chronically spinalized pigeons investigated.
Discussion
The present results show that in pigeons the cold defence response to local spinal cooling does not
51
K. GSrke and Fr.-K. Pierau: Muscle Activity of Spinalized Pigeons on Spinal Cooling and Warming
46
~f ~JIJu~``~ui~d~`L~``~`h~`~i`i ~"*il"i~l~i ~jh~j~l~iLiL`L`h~ji~.~u~i~tjj'iui~k~hu'bIIr~h~j` Fig. 4 Tonic activity of the m. flexor hallucis brevis induced by spinal cord warming in an chronically spinalized pigeon. Temperature in the vertebral canal : upper lines in recordings a--c. Note: tonic activity does not adapt during spinal warming. Spikes are partly retouched
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require an intact supraspinal loop (Rautenberg et al., 1972; Necker and Rautenberg, 1975; Hissa and Rautenberg, 1974, 1975). Similar to mammals (Simon et al., 1966; Herdman, 1978) spinalized pigeons respond to local cooling of the spinal cord with an increase of EMG-activity and muscle tremor, which is qualitatively identical with the cold tremor produced by local spinal cooling in intact animals. This is in good agreement with the observations in both spinalized and intact pigeons, that spinal cord cooling enhances monoand polysynaptic spinal reflexes as well as the activity of single motoneurones (G6rke et at., 1975; G6rke, 1976). Thus, the previous finding that the cold sensitivity of spinal motoneurones in pigeons resembles the cold sensitivity of spinal motoneurones in cats (Klussmann and Pierau, 1972; Pierau et al., 1976) conforms to the results of the present investigation. Our results also suggest that the spinal cord of pigeons possesses the capacity of cold defence mechanisms as far as motor activity is concerned. Since the ability for cold induced muscle tremor in mammals is also organized at the segmental level (Simon et al., 1966; Herdman, 1978) one may conclude that the spinal cord of homoiothermic organisms is generally equipped with the neuronal circuitry for the generation of coordinated muscle contractions like cold induced tremor. The increase of cold induced tremor by L-Dopa in acutely spinalized pigeons supports the notion, that the loss of shivering intensity after spinal transection is mainly due to the interruption of an excitatory or disinhibitory descending input. Similar to spinalized cats, L-Dopa may stimulate noradrenergic spinal receptors normally provided by descending noradrenergic neurones (And~n et al., 1966; Grillner, 1969; Budakowa, 1973). A release of the spinal generator for locomotion by this descending input, as discussed by Grillner (1975), seems to be an adaptable model for the spinal '~ generator".
Although the capacity for coordinated muscle tremor appears to be maintained in acutely and chronically spinalized animals, the pattern of shivering often changed to coarse movements and contractions of single muscle groups in our experiments as well as those in dogs (Simon et al., 1966) and rabbits (Kosaka and Simon, 1968a). This also supports the view that the descending control is responsible for the subtile coordination of muscle tremor. The negative results of Rautenberg et al. (1972) who could not induce cold shivering in spinalized or decerebrated pigeons might be attributed to deep anaesthesia or less controlled conditions of the animals, but the published data are incomplete. The inhibition of cold shivering by hypothalamic cooling (Necker and Rautenberg, 1975) might result from a block of descending excitatory pathways or activation of supraspinal inhibition. The inhibition of cold shivering by intrahypothalamic injections of catecholamines (Hissa and Rautenberg, 1975) seems to support the latter view. In intact animals the increase in EMG-activity during spinal warming above normal body temperature was accompanied by agitated movement. Since the threshold temperature was at about 44 ~C, we interpreted this effect as the result from noxious heat stimulation. However, our results on spinalized pigeons indicate that the effect of local warming on motor activity is at least in part a direct effect of temperature on the motoneurones themselves. This is in agreement with the observation that some motoneurones increase their firing rate with spinal cord warming (G6rke, 1975). In decerebrated cats a number of tonic motoneurones were also activated by local spinal warming (Rau, 1970) suggesting that the temperature for maximal firing rate of some tonic motone~rones is higher than normal body temperature. In contrast, most of the phasic motoneurones increase their activity with spinal cord cooling (Klussmann, 1969). One of the most obvious
52
electrophysiological differences between both populations is the more pronounced after hyperpolarization in tonic motoneurones, which seems to determine the slower but regular discharge of tonic motoneurones (Eccles et al., 1958). However, th~ duration and amplitude of after hyperpolarization of motoneurones is decreased by warming (Pierau and Klussmann, 1971), which in turn might cause the neurones to increase their firing frequency and may partly explain why numerous tonic motoneurones are activated at higher temperatures. At present the question remains open as to whether the different pattern of muscle contraction during warm and cold activation is connected with the different temperature sensitivity of tonic and phasic motoneurones as revealed by local cooling and warming (Klussmann et al., 1972). Acknowledgements. We thank Mrs. M. Hofmann for technical assistance and Drs. E. Simon and J. B. Mercer for helpful discussions.
References And~n, N. E., Jukes, M. G. M., Lundberg, A., Vyklicky, L.: The effect of Dopa on the spinal cord. I. Influence on transmission from primary afferents. Acta Physiol. Scand. 67, 373-386 (1966) Budakowa, N. N. : Stepping movements in the spinal cat due to Dopa administration. Fiziol. Zh. SSSR. 59, 1190--1198 (1973) Eccles, J. C., Eccles, R. M., Lundberg, A. : The action potentials of the alpha motoneurones supplying fast and slow muscles. J. Physiol. (Lond.) 142, 275-291 (1958) G6rke, K. : Effect of spi~al cord temperature on spontaneous activity of spinal motoneurones in birds and reptiles. Pfliigers Arch. 362, R94 (1976) G6rke, K., Necker, R., Rautenberg, W.: Neurophysiological investigation of spinal reflexes at different temperatures of the spinal cord in birds and reptiles. Pfliigers Arch. 359, 269-271 (1975) Grillner, S. : Supraspinal and segmental control of static and dynamic c~-motoneurones in the cat. Acta Physiol. Scand., Suppl. 327, 1 34 (1969) Grillner, S.: Locomotion in vertebrates: Central mechanisms and reflex interaction. Physiol. Rev. 55, 247-304 (1975) Herdman, S. J. : Recovery of shivering in spinal cats. Exp. Neurol. 59, 177-189 (1978)
Pfltigers Arch. 381 (1979) Hissa, R., Rautenberg, W. : The infuence of centrally applied Noradrenaline on shivering and body temperature in the pigeon. J. Physiol. (Lond.) 238, 421-435 (1974) Hissa, R., Rautenberg, W. : Thermoregulatory effects of intrahypothalamic injections of neurotransmitters and their inhibitors in the pigeon. Comp. Biocbem. Physiol. 51A, 319-326 (1975) Klussmann, F. W. : Der Einfluf3 der Temperatur auf die afferente und efferente motorische Innervation des Rtickenmarks. Pflfigers Arch. 305, 295-315 (1969) Klussmann, F. W., Pierau, Fr.-K. : Extrahypothatamic deep body thermosensitivity. In: Essays on temperature re~tlatiou (J. Bligh and R. E. Moore, eds.). Amsterdam-London: North Holland Publ. Comp. 1972 Klussmann, F. W., Pierau, Fr.-K., Spaan, G.: Frequency and synchronization of spinal alpha motoneurones during shivering. Posebna Izdanja - XII. Odjelhene Medicinskih Nauka, Knija 4 (1972) Kosaka, M., Simon, E. : K~iltetremor wacher, chronisch spinalisierter Kaninchen im Vergleich zum K~iltezittern intakter Tiere. Pfltigers Arch. 302, 333-356 (1968a) Kosaka, M., Simon, E. : Der zentralnerv6se spinale Mechanismus des K/iltezitterns. Pflfigers Arch. 302, 357-373 (1968b) Necker, R., Rautenberg, W.: Effect of spinal deafferentation on temperature regulation and spinal thermosensitivity in pigeons. Pfl/igers Arch. 360, 287-299 (1975) Pierau, Fr.-K., Klussmann, F. W. : Spinal excitation and inhibition during local spinal cooling and warming. J. Physiol. (Paris) 63, 380- 382 (1971) Pierau, Fr.-K., Klee, M. R., Klussmann, F. W. : Effect of temperature on postsynaptic potentials of cat spinal motoneurones. Brain Res. 114, 2 1 - 3 4 (1976) Rau, B. : Der Einflul3 der R/ickenmarkstemperatur auf die Entladungsfrequenz und die rekurrente Hemmung tonischer Motoneurone. Dissertation, Justus Liebig-Universit/it, Gie13en 1970 Rautenberg, W. : Die Bedeutung der zentralnerv6sen Thermosensitivifiit ffir die Temperaturregulation der Taube. Z. vergl. Physiol. 62, 235-266 (1969) Rautenberg, W., Necker, R., May, B. : Thermoregulatory responses of the pigeon to changes of the brain and the spinal cord temperatures. Pfliigers Arch. 338, 31-42 (1972) Simon, E.: Temperature regulation: The spinal cord as a site of extrahypothalamic thermoregulatory functions. Rev. Physiol. Biochem. Pharmacol. 71, 1 - 76 (1974) Simon, E., Klussmann, F. W., Rautenberg, W., Kosaka, M.: K~iltezittern bei narkotisierten spinalen Hunden. Pfl/igers Arch. ges. Physiol. 291, 187-204 (1966) Schmidt, I., Rautenberg, W.: Instrumental thermoregulatory behavior in pigeons. J. Comp. Physiol. 101, 225-235 (1975) Received December 19, 1978
