Neuroscience Letters, 113 (1990) 23-28

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Elsevier Scientific Publishers Ireland Ltd. NsL 06851

Interactions between locomotor and respiratory activities during hindlimb stepping on a stationary surface in decerebrate cats Koichi K a w a h a r a and Masayuki Suzuki Department of Information Engineering, Yamagata University, Yonezawa (Japan) (Received 8 January 1990; Revised version received 16 January 1990; Accepted 22 January 1990)

Key words: Still-stepping; Locomotor-respiratory coupling; Correlation analysis; Mesencephalic cat Hindlimb stepping on a stationary surface was evoked by tonic electrical stimulation of the dorso-lateral part of the mesencephalic locomotor region. Such a stepping movement (still-stepping) was characterized by alternating limb loading between the left and right hindlimbs, while the animal maintained a standing posture. Under such conditions, a slight increase in the stimulus intensity produced synchronized stillstepping between the hindlimbs. At that time, respiratory activity, evaluated by recording the diaphragmatic EMG, showed marked changes and was strongly correlated with the stepping frequency. Cross-correlograms between the diaphragmatic and gastrocnemius activities disclosed that locomotor-respiratory coupling increased in strength when the mode of still-stepping changed from the alternating to the synchronized stepping between the hindlimbs.

Previous studies have demonstrated that respiratory rhythm becomes synchronized with the exercise rhythm in humans [2, 5], horses [1], and cats [3, 4]. However, the strength of the coupling between locomotor and respiratory rhythms seems to be weak. Some studies have found no evidence of such locomotor-respiratory synchronization [12, 13]. Recently, we demonstrated that the strength of coupling between respiratory and locomotor rhythms varies depending on the locomotor patterns elicited, especially on whether or not the animals are galloping [6, 8]. This report seems to support the idea that the coupling strength between locomotor and respiratory rhythms varies depending on the condition of the animal. Therefore, our results may partly explain the reason why there are some inconsistencies in the previous findings on locomotor-respiratory synchronization. In this study, we have analyzed the interactions between respiratory and locomotor rhythms during hindlimb stepping on a stationary surface elicited by stimulation of the brain stem in decerebrate cats. This stepping movement on a stationary surface, characterized by alternating limb loading while the animal maintains an upright Correspondence: K. Kawahara, Department of Information Engineering, Yamagata University, Yonezawa 992, Japan. 0304-3940/90/$ 03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd.

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standing posture, is referred to 'still-stepping' of the hindlimb [14, 15]. During the course of our experiment, under such conditions, we found that alternating rhythm between the left and right hindlimbs sometimes became synchronized, similar to a skipping movement. At that time, the respiratory rhythm dramatically changed when the gait of the animal changed from the alternating to the synchronized stepping of the hindlimbs. This study reports on the results of correlation analysis of how the strength of locomotor-respiratory coupling varied depending on the modes of 'stillstepping'. The experimental procedures are described elsewhere in detail [7-1 !]. In short, 4 cats weighing 2.6--3.7 kg were surgically decerebrated at the precollicular-postmammillary level under halothane anesthesia. The head of the animal was fixed in a stereotaxic frame and the limbs were placed on a treadmill belt. After recovery from anesthesia and decerebrate shock, a tungsten microelectrode, insulated with a glass pipet except for the tip, was inserted into the midbrain (PI-P2, L or R4-5, H + 2 - + 0; Horsley-Clarke coordinates), in search of the sites which elicited hindlimb stepping on a stationary surface when electrically stimulated. Hindlimb stepping on a stationary surface was induced by stimulation of the dorsolateral part of the mesencephalic locomotor region [15]. The stimulation consisted of a rectangular pulse of 0.2 ms duration at 50 pulses/s with an intensity of 50-70/~A. Electromyograms (EMGs) were recorded by implantation of bipolar electrodes made of thin (70/~m) copper wires insulated except for the tips into the bilateral gastrocnemius muscles. Diaphragmatic EMGs were also recorded with implanted copper wires. The diaphragmatic and gastrocnemius EMGs were then rectified, R-C integrated (time constant, 0.1 s), and recorded on an FM data recorder. Correlation analysis was performed off-line on the integrated diaphragmatic and bilateral gastrocnemius activities. Autocorrelograms of diaphragmatic and of the left or right gastrocnemius activities showed the mean respiratory and the mean stepping frequency, respectively. Cross-correlograms between the diaphragmatic and gastrocnemius activities were used to estimate the strength of the locomotor-respiratory coupling. At the end of each experiment, the animals were deeply anesthetized with an i.v. injection of pentobarbital sodium and sacrificed. Hindlimb stepping on a stationary surface (still-stepping) was evoked by stimulation of the midbrain in 3 out of the 4 cats tested. In the unsuccessful animal, hindlimb muscle tone was extremely exaggerated due to decerebrate rigidity. Therefore, it was difficult to elicit any locomotor movement by midbrain stimulation. Even in the successfull animals (3 cats), however, the stimulation did not always elicit still-stepping. When the level of hindlimb postural tone was relatively high, the midbrain stimulation did not evoke still-stepping, but produced augmentation of postural tone. When the postural tone of the hindlimb was weakly developed, the stimulation immediately elicited still-stepping without preceding postural changes. One example of still-stepping on a stationary surface is illustrated in Fig. IA. Before the start of stimulation, this cat breathed spontaneously with a mean respiratory interval of about 3.1 s. The alternating rhythmic discharges of the bilateral gastrocnemius muscles began to appear at about 0.9 s after the onset of the stimulation.

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Fig. 1. Respiratory activity during alternating still-stepping (A) and synchronized still-stepping (B) of the hindlimbs. The stimulus site was located at P2.0, L4.2, H+0.5 (Horsley-Clarke coordinates) both in A and B. The stimulus intensity was 50 #A in A and 60 #A in B. A bold solid line under the integrated right gastrocnemius EMG indicates the period of the midbrain stimulation. Abbreviations: DIA. EMG, diaphragmatic EMG; INT DIA. EMG, integrated diaphragmatic EMG; GAS. EMG(L), left gastrocnemius EMG; INT GAS. EMG(L), integrated left gastrocnemius EMG; GAS. EMG(R), right gastrocnemius EMG; INT GAS. EMG(R), integrated right gastrocnemius EMG.

The duration of the grouped discharges of the gastrocnemius muscle was about 0.25 s, and was shorter than that during locomotion on a moving treadmill. The mean stepping interval was about 0.46 s. Both the mean stepping interval and the duration of gastrocnemius activity during still-stepping were stable and did not change markedly from one animal to the next. During still-stepping, the respiratory interval increased to about 4.1 s; that is, the respiratory frequency decreased. The integrated diaphragmatic activity showed that there were some interactions between the diaphragmatic and gastrocnemius activities. Spikes corresponding to the left and right gastrocnemius grouping discharges were superimposed over the fundamental respiratory bursting discharges of the diaphragm. We then increased the stimulus intensity from 50 #A (Fig. 1A) to 60 #A (Fig. 1B). The stimulus site was the same as that in Fig. 1A. The midbrain stimulation elicited nearly synchronized grouping discharges of the left and right gastrocnemius muscles. This synchronous hindlimb stepping on a stationary surface was similar to a skipping movement. The respiratory activity dramatically changed during such synchronized still-stepping. The diaphragmatic activity showed the grouping discharges synchronized with the gastrocnemius activity; that is, 1:1 entrainment of the respiratory rhythm to the stepping rhythm occurred. In this example, the synchronized stepping terminated in spite of the continuation of the stimulation. Correlation analysis of diaphragmatic and gastrocnemius activities was then done to detect their periodicity and to evaluate quantitatively the strength of the coupling

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between the respiratory and stepping rhythms. An example of the results is illustrated in Fig. 2. Figure 2A shows the results during alternating still-stepping and Fig. 2B those during synchronized stepping. During alternating still-stepping, the autocorrelogram of the diaphragmatic activity showed that the fundamental respiratory frequency was modulated by the left and right gastrocnemius rhythmic discharges. The lag time of the first peak in the cross-correlogram between the left and right gastrocnemius activities in Fig. 2A was about 0.22 s, and was about half of the mean stepping interval. This result reflected the activity alternating between the left and right gastrocnemius muscles during this kind of still-stepping. In contrast, the almost coinciding oscillation between the bilateral gastrocnemius activities and the cross-correlogram between the left and right gastrocnemius activities in Fig. 2B reflected a nonalternating (synchronized) activity during this kind of still-stepping. The almost flat crosscorrelogram between the diaphragmatic and right gastrocnemius activity in Fig. 2A showed that diaphragmatic and gastrocnemius activities were not closely correlated

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Fig. 2. Autocorrelograms (ACG) and cross-correlograms (CCG) obtained during alternating still-stepping (A) and synchronized still-stepping (B). The maximum lag time in the diaphragmatic ACG in A was + 5.0 s. In the remaining correlograms in A, the maximum lag time was +3.5 s. The maximum lag time in all the correlograms in B was + 3.0 s. Abbreviations: DIA ACG, diaphragmatic ACG; G(L) ACG, ACG of the left gastrocnemius activity; G(R) ACG, ACG of the right gastrocnemius activity; G(L}~G(R) CCG, CCG between the left and right gastrocnemius activities; D-G(R) CCG, CCG between the diaphragmatic and right gastrocnemius activities.

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with each other. In contrast, the clear oscillation with the same intervals between peaks as those of gastrocnemius activities in the cross-correlogram between the diaphragmatic and right gastrocnemius activities in Fig. 2B showed that 1:1 entrainment of the respiratory rhythm to the stepping rhythm. These results raised the possibility that coupling between the respiratory and the hindlimb stepping rhythm generators was weak during alternating still-stepping but became stronger during synchronized still-stepping. This study has demonstrated that the strength of coupling between respiratory and stepping rhythms dramatically changed when the mode of still-stepping changed from alternating to synchronized stepping. We previously demonstrated that the strength of locomotor-respiratory coupling varies depending on locomotor patterns and that the coupling strength becomes stronger during galloping than during walking and trotting [6, 8]. A gallop is characterized by almost synchronized hindlimb movements; that is, the left and right hindlimbs strike the surface of a treadmill belt almost concurrently. In the synchronized still-stepping described in this study, both hindlimbs skipped on the stationary surface of a treadmill belt almost concurrently. Therefore, the present results may provide some evidence supporting our previous finding that locomotor-respiratory coupling is strong during a gallop, in which both hindlimbs strike the surface of the treadmill almost concurrently. We would like to express our sincere thanks to Prof. Y. Miyamoto, Department of Information Engineering, Yamagata University, for his continuous encouragements of this study. We also thank Mr. Y. Nakazono, Department of Physiology, Sapporo Medical College, and Ms. Y. Yamauchi, Department of Information Engineering, Yamagata University, for their valuable assistance in the animal experiments. This study was partly supported by a grant from the Ministry of Health and Welfare of Japan and from the Ministry of Education and Culture of Japan (01570055). 1 Bramble, D.M. and Carrier, D.R., Running and breathing in mammals, Science, 219 (1983) 251-256. 2 Bechbache, R.R. and Duffin, J., The entrainment of breathing frequency by exercise rhythm, J. Physiol. Lond., 272 (1971) 553-561. 3 Iscoe, S., Respiratory and stepping frequencies in conscious exercising cats, J. Appl. Physiol., 51 (1981) 835-839. 4 lscoe, S. and Polosa, C., Synchronization of respiratory frequency by somatic afferent stimulation, J. Appl. Physiol., 40 (1976) 138-148. 5 Jasinkas, C.L., Wilson, B.L. and Hoare, J., Entrainment of breathing rate to movement frequency during work at two intensities, Respir. Physiol., 42 (1980) 199-209. 6 Kawahara, K., Coupling between central pattern generators: locomotor-respiratory coupling in decerebrate cats, Trends Biol. Cybern., (1990) in press. 7 Kawahara, K., Kumagai, S., Nakazono, Y. and Miyamoto, Y., Analysis of entrainment of respiratory rhythm by somatic afferent stimulation in cats using phase response curves, Biol. Cybern,, 58 (1988) 235-242. 8 Kawahara, K., Kumagai, S., Nakazono, Y. and Miyamoto, Y., Coupling between respiratory and stepping rhythms during locomotion in decerebrate cats, J. Appl. Physiol., 67 (1989) 110-115. 9 Kawahara, K., Nakazono, Y., Kumagai, S., Yamauchi, Y. and Miyamoto, Y., Parallel suppression of extensor muscle tone and respiration by stimulation of pontine dorsal tegmentum in decerebrate cat, Brain Res., 473 (1988) 81-90.

28 10 Kawahara, K., Nakazono, Y., Yamauchi, Y. and Miyamoto, Y., Coupling between respiratory and locomotor rhythms during fictive locomotion in decerebrate cats, Neurosci. Lett., 103 (1989) 326-332. 11 Kawahara, K., Yamauchi, Y., Nakazono, Y. and Miyamoto, Y., Spectral analysis on low frequency fluctuation in respiratory rhythm in the decerebrate cat, Biol. Cybern., 61 (I 989) 265-270. 12 Kay, J.D.S., Peterson, E.S. and Vejby-Christensen, H., Breathing in man during steady state exercise on a bicycle at two pedalling frequencies and during treadmill walking, J. Physiol. Lond., 251 (1975) 645-656. 13 Kelman, G.R. and Watson, A.W.S., Effect of added dead-space on pulmonary ventilation during submaximal, steady-state exercise, Q. J. Exp. Physiol,, 58 (1973) 305-313. 14 Mori, S., Aoki, M., Kawahara, K. and Sakamoto, T,, Level setting of postural tonus and initiation of locomotion by MLR stimulation. In J. Szent~tgothai, M. Palkovits and J. H~imori (Eds.), Regulatory Functions of the CNS, Pergamon Press, Oxford, 1981, pp. 179-182. 15 Mori, S., Kawahara, K. and Sakamoto, T., Supraspinal aspect of locomotion in the mesencephalic cat. In A. Roberts and B. Roberts (Eds.), Neural Origin of Rhythmic Movements, Soc. Exp. Biol., 1983, pp. 445~,68.

Interactions between locomotor and respiratory activities during hindlimb stepping on a stationary surface in decerebrate cats.

Hindlimb stepping on a stationary surface was evoked by tonic electrical stimulation of the dorso-lateral part of the mesencephalic locomotor region. ...
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