174
Brain Research, 575 (1992) 174-179
Elsevier BRES 25110
Phasic modulation of vestibulospinal neuron activity during fictive locomotion in lampreys Nathalie Bussi6res and R6jean Dubuc D~partement de Kinanthropologie, Universit~ du Quebec ft Montreal C.P.8888 succ. A, Montreal, Que. (Canada) and Centre de Recherche en Sciences Neurologiques, Universit~ de Montreal C.P.6128 succ. A, Montreal, Que (Canada)
(Accepted 17 December 1991) Key words: Locomotion; Vestibulospinal neuron; Vestibular nucleus; Vestibular afferent; Lamprey; Brainstem
This study was aimed at characterizing the activity of vestibulospinal neurons recorded intracellularly during fictive locomotion in lampreys. The majority (78%) of identified vestibulospinal neurons showed rhythmic fluctuations of their membrane potential correlated with locomotor discharges recorded in pairs of rostral ventral roots. Of the rhythmically modulated vestibulospinal cells, most (72%) were maximally depolarized during ipsilateral ventral root discharges and showed a minimum during contralateral activity. Other cells (20%) showed an opposite pattern, that is their peak of depolarization occurred during contralateral activity. Finally, a third category of cells (8%) showed a more complex pattern of activity. Two waves of depolarization could occur per locomotor cycle, one during each burst discharge. The pattern of fluctuation recorded in vestibulospinal neurons appears to be related to the side of the spinal cord onto which the cells are projecting. The lamprey central nervous system has become an important model for studying the cellular interactions responsible for the generation and control of certain motor behaviour such as locomotion 6. Different classes of spinal interneurons have been identified and a model for segmental rhythm generation has been proposed 2. Supraspinal control, on the other hand, originates from two main systems: the reticulospinal and the vestibulospinal tracts. Reticulospinal cells make synaptic contacts along the spinal cord with motoneurons and spinal interneutons which are known to play a key role in the generation of locomotion 1'9. The role of vestibulospinal neurons has received less attention (see, however, ref. 13). They are grouped in two nuclei: the octavomotorius intermedius nucleus and the octavomotorius posterior nucleus. Cells of these nuclei receive synaptic contacts from vestibular afferents ~4. Vestibulospinal neurons are known to project only to the most rostral segments of the spinal cord, where they make monosynaptic contacts with motoneurons and spinal interneurons 13. In addition, reticulospinal and vestibulospinal systems are closely linked in the brainstem with the axons of vestibulospinal cells making en passant electrotonic and chemical contacts with reticulospinal cells of the posterior rhombencephalic reticular nucleus (PRRN) 13. Reticulospinal cells receive alternating excitatory and inhibitory inputs during fictive locomotion. Depolariza-
tion occurs during ipsilateral locomotor discharges recorded from rostral ventral roots and hyperpolarization during contralateral activity 5'7. Moreover, it has been shown that these inputs originate at least partly from spinal locomotor networks 5. The aim of the present study was to determine if the vestibulospinal cells are also rhythmically active during fictive locomotion in the lamprey. Fifteen adult lampreys (Ichthyornyzon unicuspis), 25-30 cm in length were anaesthetized with 3-aminobenzoate methanesulfonate (100 mg/1). The dissection was completed in a cold Ringer solution 15 oxygenated (95% 0 2, 5% CO2) and titrated at pH 7.4. Muscles and viscera were removed in order to expose the dorsal side of the brainstem and the rostral spinal cord. The final preparation consisted of the brainstem, the spinal cord, and the supporting cartilage. The preparation was then pinned to a layer of Sylgard 184 D o w Corning covering the bottom of a recording chamber filled with a Ringer solution continuously renewed, oxygenated, and maintained between 5 and 9°C. Locomotor activity was induced by adding N M D A (50 pM) to the perfusing solution and locomotor discharges were recorded with two suction electrodes placed on the dorsal aspect of two rostral and opposed ventral roots (lst to 6th segments of a total of 100). Intracellular recordings of vestibulospinal neurons were carried out with glass microelectrodes
Correspondence: R. Dubuc, D6partement de Kinanthropologie, Universit6 du Qu6bec ~t Montr6al, C.P.8888 succ. A, Montr6al Que., H3C 3P8 Canada. Fax: (1) (514) 343-2111.
175 filled with 4 M KAc (50-100 Mf~) or 20% Lucifer Yellow in 0.1 M LiCI (80-100 Mfl). Signals were sent to an Axoclamp-2A amplifier (0-10 kHz) and stored on magnetic tapes (8 tracks, Vetter DI) with ventral root neurograms. Fig. 1A illustrates the experimental paradigm used for cell identification. Vestibulospinal cells were identified by their monosynaptic response to ipsilateral vestibular nerve stimulation and by their antidromic response to spinal cord stimulation. Mean membrane potential was 65 + 7 mV and mean conduction velocity was 1.0 + 0.6
A
~
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m/s (measured at a stimulation strength of 3 x threshold). Fig. 1B 1 shows the all or none antidromic response of a vestibulospinal cell which appeared as the intensity of spinal cord stimulation was progressively increased. The action potential occurred at fixed latency and followed a frequency of stimulation of 15 Hz. Synaptic responses of the same cell to ipsilateral and contralateral vestibular nerve stimulation are shown in Fig. 1B 2 and Fig. 1B 3. The synaptic response of the vestibulospinal cell to ipsilateral vestibular nerve stimulation (Fig. 1B2) has the characteristics of a monosynaptic response with
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25 ms Fig. 1. A: schematic representation of the stimulation and recording paradigms. For vestibulospinal (VS) cell identification, one glass stimulation electrode was placed on the ipsilateral (i) and the contralateral (co) vestibular nerves (Vest n) to generate monosynaptic inputs. In addition, one glass stimulation electrode was placed on each lateral tract to antidromically activate the cell. Recording suction electrodes were placed on rostral ventral roots (VR) of opposite sides. BI: antidromic responses of a vestibulospinal cell (conduction velocity = 0.7 m/s) to ipsilateral spinal cord stimulation at threshold intensity (top) and at a stimulation frequency of 15 Hz (bottom). B2,B3: responses of the cell to ipsilateral and contralateral vestibular nerve stimulations. Each illustration is made up of 4 superimposed synaptic responses. Single and twin (15 Hz) stimulation pulses were delivered (2/zA). The ipsilateral response has a fast rising component with a small inflection (arrow). C: effects of kynurenic acid (4 mM) and of Ca 2+ free Ringer (Mn 2+ 2mM) on the synaptic responses of a vestibulospinal neuron to ipsilateral vestibular nerve stimulation.
176 a very rapid rise time (0.3 ms) and a latency of less than 3 ms. Moreover, there is a small inflection on the ascending phase of the excitatory postsynaptic potential, suggesting an electrotonic component (see arrow in Fig. 1B2). In addition, the amplitude and the shape of the response to a second stimulation applied 65 ms after a first are constant. The response of the vestibulospinal cell to contralateral vestibular nerve stimulation appears at a longer latency, has a longer rise time, and the amplitude of the second response, to a 15 Hz stimulation, is reduced. In another experiment, the role of excitatory amino acids in this synaptic transmission was studied by adding kynurenic acid (4 mM), a blocker of excitatory amino acid receptors, to the perfusing solution (Fig. 1C). Responses to ipsilateral vestibular nerve stimulation were markedly reduced. Of the two excitatory components composing the control response, only the first persisted in the presence of kynurenic acid, but showed a reduction in amplitude (67%). When Ca 2+ was removed and replaced by Mn 2+ (2 raM), thus abolishing all chemical synaptic transmission, the short latency component which resisted the effect of kynurenic acid was still present and persisted even at stimulation rates of 40 Hz. Taken together, these results suggest that the second excitatory component of the synaptic response and part of the first are resulting from the activation of excitatory amino acid receptors, while most of the first component is resulting from electrotonic transmission. Synaptic contacts between vestibular afferents and cells of the octavomotorius nuclei have been studied morphologically. They are of 3 types: chemical synapses, gap junctions and desmosomes TM. Indeed, our results confirm the presence of a mixed electrical and chemical synapse (see also ref. 13). The activity of 32 vestibulospinal cells has been studied during fictive locomotion of which 25 showed rhythmic fluctuations of their membrane potential correlated with ventral root activity in the rostral spinal cord. The activity of 72% of the rhythmically modulated cells showed a maximum of depolarization during ipsilateral activity and a minimum during contralateral activity. The average peak to peak amplitude of the fluctuations was 3.8 + 2.9 mV (Fig. 2). Some of these cells, like the one illustrated in Fig. 2A1, were sufficiently depolarized during ipsilateral ventral root activity that action potentials were generated. This particular vestibulospinal cell was filled with Lucifer yellow and reconstructed from photomicrographs taken at different focal planes (Fig. 2A3). The cell has a soma of 40/tin in diameter and its dendritic arborization reaches the lateral border of the alar plate and extends partly within the most lateral aspects of the basal plate. The axon is clearly visible projecting down to the spinal cord. This axonal projection was also demonstrated electrophysiologically by ipsilateral spinal
cord stimulation, which triggered an antidromic action potential in the cell (Fig. 2A2). Most cells did not, however, show discharges during fictive locomotor activity. The cell illustrated in Fig. 2B~ had regular oscillations well correlated with a stable locomotor activity. Averages of both membrane potential fluctuations and ventral root discharges triggered on the beginning of activity in the ipsilateral ventral root (Fig. 2B2) show that the depolarization wave in the vestibulospinal cell precedes the beginning of discharges in the ipsilateral ventral root. A similar phase relationship was observed for the other vestibulospinal neurons which were depolarized during ipsilateral locomotor activity. This suggests that vestibulospinal neuron depolarization may parallel closely motoneuronal depolarization in the most rostral spinal segments. All vestibulospinal cells did not have the same type of activity during locomotion. In 20% of the rhythmically active cells, the membrane potential reached a maximum of depolarization during contralateral burst discharge and a minimum during ipsilateral activity. For all cells with this pattern of activity, mean oscillation amplitude was 4.1 + 1.8 mV. Fig. 3A shows such a vestibulospinal neuron depolarized during the contralateral ventral root activity. Averages show that the depolarization wave precedes and persists through the contralateral ventral root discharge (Fig. 3A2). Moreover, the cell was identified by stimulation of the contralateral spinal cord (Fig. 3A3). An antidromic action potential was elicited before any synaptic response, appeared at a fixed latency and was still present at stimulation rates of 35 Hz. On the other hand, the response of the same cell to ipsilateral spinal cord stimulation consisted of excitatory postsynaptic potentials and discharges, which did not follow rates of 15 Hz. Other vestibulospinal neurons (8%) showed a more complex pattern of membrane potential fluctuations during locomotion. Two waves of depolarization could occur, one during the ipsilateral and/or one during the contralateral ventral root activity. An example is provided in Fig. 3B1. Initially, the cell is depolarized during ipsilateral activity, with only a small wave of depolarization during contralateral activity (Fig. 3B2). However, less than a minute later, when the locomotor activity became more vigourous in the contralateral ventral root, this second depolarization wave gained amplitude (Fig. 3B 3 and 3B4). This suggests that the amplitude of the membrane potential fluctuations show a close relationship with the intensity of the locomotor activity in the spinal cord. Interestingly, the cell was antidromically activated from both sides of the spinal cord, indicating a bilateral projection (Fig. 3B5). A similar pattern of activity was observed in 5 other cells recorded in octavomotorius nuclei.
177
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Fig. 2. AI: rhythmic fluctuations of the membrane potential of a vestibulospinal (VS) cell during fictive locomotion. Locomotor discharges were recorded from the 2nd ipsilateral ventral root (i VR) and the 3rd contralateral ventral root (co VR). A2: identification of the cell by the antidromic response to ipsilateral stimulation of the spinal cord (conduction velocity = 0.7 m/s). Note that the antidromic action potentials persist at a stimulation rate of 30 Hz. A3: reconstruction of the cell showing a clear axonal projection to the ipsilateral spinal cord (VIII n, vestibular nerve; MRRN, middle rhombencephalic reticular nucleus; PRRN, posterior rhombencephalic reticular nucleus). BI: activity of another vestibulospinal cell during fictive locomotion recorded from the 2nd ipsilateral and 5th contralateral ventral roots. Bz: averages of membrane potential fluctuations and of rectified neurograms (n = 14). The averaging was triggered on the onset of ipsilateral ventral root activity. The time scale was normalized so that a locomotor cycle equals 100%. B3: antidromic response of the cell to ipsilateral spinal cord stimulation at threshold intensity (top) and at 15 Hz (bottom).
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Fig. 3. A~: rhythmic fluctuations of the membrane potential of a vestibulospinal (VS) cell depolarized during contralateral locomotor activity. Locomotor discharges were recorded from the 5th ipsilateral ventral root (i VR) and the 6th contralateral ventral root (co VR). A2: averages of membrane potential and of ventral root neurograms (n = 12). m3: responses of the cell to spinal cord stimulations on the ipsilateral (ipsi) and the contralateral (contra) sides. Note that the cell is antidromically activated from the contralateral side. B1,B3: rhythmic modulation of a vestibulospinal cell which shows a more complex pattern of activity during two bouts of fictive locomotion. Ventral root activities were recorded in segment 3 of each side. The amplitude of the depolarization'is proportional to contralateral burst intensity as clearly seen on the averages (B 2, n = 8 and B4, n = 9). B5: antidromic responses elicited by spinal cord stimulation of each side.
T h e p r e s e n t results show that vestibulospinal cells are clearly rhythmically active d u r i n g fictive l o c o m o t i o n in lamprey. T h e m a j o r i t y of the cells r e c o r d e d showed a m a x i m u m of d e p o l a r i z a t i o n d u r i n g ipsilateral spinal activity a n d a m i n i m u m d u r i n g c o n t r a l a t e r a l activity (n = 18). T h e spinal projections of these cells were identified by ipsilateral spinal cord stimulation. T h e i r exact loca-
tion has b e e n d e t e r m i n e d for those filled with Lucifer yellow, in which cases the cell bodies were located at the level of the o c t a v o m o t o r i u s i n t e r m e d i u s nucleus. In other vestibulospinal n e u r o n s , m e m b r a n e p o t e n t i a l fluctuations were in antiphase, that is the cells s h o w e d their maxim u m d e p o l a r i z a t i o n d u r i n g c o n t r a l a t e r a l l o c o m o t o r activity a n d their m i n i m u m d u r i n g ipsilateral activity (n =
179 5). These cells were r e c o r d e d m o r e caudally, at the level of the octavomotorius posterior nucleus, where vestibulospinal neurons have b e e n p r o p o s e d to project their axon to the contralateral spinal cord 11'13. This has b e e n recently confirmed with anatomical tracers 3 (also Bussi6res and D u b u c , unpublished observations). It is noteworthy that Kasicki et al. s described the same type of activity, that is depolarization during contralateral ventral root activity, for reticulospinal neurons such as the M a u t h n e r cell, whose axon projects to the contralateral spinal cord. Finally, some cells had variable phase relation: during a single l o c o m o t o r episode, they could be d e p o l a r i z e d during ipsilateral activity and during contralateral activity at different times or at the same time. Interestingly, these neurons r e c o r d e d at the level of the octavomotorius intermedius nucleus were antidromically activated by spinal cord stimulation on either side. A n a t o m i c a l exp e r i m e n t s with the c o m b i n e d use of two r e t r o g r a d e tracers are in progress to d e t e r m i n e whether such bilaterally projecting vestibulospinal neurons exist within the lamprey brainstem. The v e r t e b r a t e labyrinthine system is i m p o r t a n t for head orientation and postural adjustments. It is presumed that vestibulospinal neurons will play an important role in selecting the a p p r o p r i a t e response to vestibular stimulation (see also ref. 10). They r e p r e s e n t the most direct pathway from the vestibular apparatus to the spinal cord where they m a k e electrotonic and chemical contacts with m o t o n e u r o n s innervating the rostral myo-
tomes and some spinal interneurons 13. The results presented here show that vestibulospinal neurons receive rhythmic inputs that k e e p them in tune with the locom o t o r activity occurring in the rostral spinal cord. The source of this m o d u l a t i o n is not known. In fact, little is known about the inputs to the vestibulospinal cells, except for those from vestibular afferents 13'14. In the case of reticulospinal neurons, this rhythmic m o d u l a t i o n which occurs during fictive locomotion has been shown to come at least partly from spinal cord inputs 5. It is possible that a similar situation occurs in vestibulospinal neurons. M o d u l a t i o n of reflex transmission during locomotion allows the animal to a d a p t its m o v e m e n t s to the external environment (for a review see ref. 12). M e m b r a n e potential fluctuations in vestibulospinal neurons during fictive locomotion m a y be i m p o r t a n t in the modulation of vestibular reflexes by favouring certain periods of the cycle over others for input transmission. We have, in fact, recently shown that such m o d u l a t i o n occurs for transmission of vestibular inputs to reticulospinal neurons during fictive locomotion in lampreys 4.
1 Buchanan, J.T., Identification of interneurons with collateral, caudal axons in the lamprey spinal cord: synaptic interactions and morphology, J. Neurophysiol., 47 (1982) 961-975. 2 Buchanan, J.T. and Grillner, S., A newly identified class of excitatory premotor interneurons in the lamprey spinal cord, Acta Physiol. Scand., 128 (1986) 13A. 3 Bussi6res, N. and Dubuc, R., Rhythmic modulation of vestibulospinal neurones during fictive locomotion in lampreys, Third IBRO World Cong. Neurosci., 3 (1991) 486. 4 Bussi~res, N. and Dubuc, R., Phasic modulation of transmission from vestibular inputs to reticulospinal neurones during fictive locomotion in lampreys, Soc. Neurosci. Abstr., 21 (1991) 1025. 5 Dubuc, R. and Grillner, S., The role of spinal cord inputs in modulating the activity of reticulospinal neurons during fictive locomotion in the lamprey, Brain Res., 483 (1989) 196-200. 6 Grillner, S., Wall6n, P., Brodin, L. and Lansner, A., Neuronal network generating locomotor behaviour in lamprey, Annu. Rev. Neurosci., 14 (1991) 169-199. 7 Kasicki, S. and Grillner, S., Miitler cells and other reticulospinal neurones are phasically active during fictive locomotion in the isolated nervous system of the lamprey, Neurosci. Lett., 69 (1986) 239-243. 8 Kasicki, S., Grillner, S., Ohta, Y., Dubuc, R. and Brodin, L., Phasic modulation of reticulospinal neurons during fictive locomotion and other types of spinal motor activity in the lamprey,
Brain Res., 484 (1989) 203-216. 9 0 h t a , Y. and Grillner, S., Monosynaptic excitatory amino acid transmission from the posterior rhombencephalic reticular nucleus to spinal neurons involved in the control of locomotion in lamprey, J. Neurophysiol., 62 (1989) 1079-1089. 10 Orlovsky, G.N., Deliagina, T.G., Grillner, S. and Wall6n, P., Natural vestibular stimulation evokes static and dynamic responses in lamprey reticulospinal neurons, Acta Physiol. Scand., 138 (1990) 13A. 11 Ronan, M., Origins of the descending spinal projections in petromyzontid and myxinoid agnathans, J. Comp. Neurol., 281 (1989) 54-68. 12 Rossignol, S., Lund, J.P. and Drew, T., The role of sensory inputs in regulating patterns of rhythmical movements in higher vertebrates. In A. Cohen, S. Rossignol and S. GriUner (Eds.), Neural Control of Rhythmic Movements in Vertebrates, Wiley, New York, 1988, pp. 201-283. 13 Rovainen, C.M., Electrophysiology of vestibulospinal and vestibuloreticulospinal systems in lampreys, J. Neurophysiol., 42 (1979) 745-765. 14 Stefanelli, A. and Caravita, S., Ultrastructural features of the synaptic complex of the vestibular nuclei of Lampetra planeri (Bloch), Z. Zellforsch., 108 (1970) 282-296. 15 Wickelgren, W.O., Physiological and anatomical characteristics of reticulospinal neurones in lamprey, J. Physiol., 270 (1977) 89-114.
The authors are grateful to D. Cyr, S. Doucet, G. Filosi, C. Gauthier, D. Lauzier and J. Provencher for the excellent technical assistance provided. We are also grateful to Dr. S. Alford for his helpful comments on this manuscript. This work was supported by Canadian MRC (Grant 10703), FRSQ, FCAR (Qu6bec) and The Bunting Research Foundation. N.B. receives a studentship from FCAR and the NCE for Neural Regeneration and Functional Recovery.
Brain Research, 575 (1992) 174-179
Elsevier BRES 25110
Phasic modulation of vestibulospinal neuron activity during fictive locomotion in lampreys Nathalie Bussi6res and R6jean Dubuc D~partement de Kinanthropologie, Universit~ du Quebec ft Montreal C.P.8888 succ. A, Montreal, Que. (Canada) and Centre de Recherche en Sciences Neurologiques, Universit~ de Montreal C.P.6128 succ. A, Montreal, Que (Canada)
(Accepted 17 December 1991) Key words: Locomotion; Vestibulospinal neuron; Vestibular nucleus; Vestibular afferent; Lamprey; Brainstem
This study was aimed at characterizing the activity of vestibulospinal neurons recorded intracellularly during fictive locomotion in lampreys. The majority (78%) of identified vestibulospinal neurons showed rhythmic fluctuations of their membrane potential correlated with locomotor discharges recorded in pairs of rostral ventral roots. Of the rhythmically modulated vestibulospinal cells, most (72%) were maximally depolarized during ipsilateral ventral root discharges and showed a minimum during contralateral activity. Other cells (20%) showed an opposite pattern, that is their peak of depolarization occurred during contralateral activity. Finally, a third category of cells (8%) showed a more complex pattern of activity. Two waves of depolarization could occur per locomotor cycle, one during each burst discharge. The pattern of fluctuation recorded in vestibulospinal neurons appears to be related to the side of the spinal cord onto which the cells are projecting. The lamprey central nervous system has become an important model for studying the cellular interactions responsible for the generation and control of certain motor behaviour such as locomotion 6. Different classes of spinal interneurons have been identified and a model for segmental rhythm generation has been proposed 2. Supraspinal control, on the other hand, originates from two main systems: the reticulospinal and the vestibulospinal tracts. Reticulospinal cells make synaptic contacts along the spinal cord with motoneurons and spinal interneutons which are known to play a key role in the generation of locomotion 1'9. The role of vestibulospinal neurons has received less attention (see, however, ref. 13). They are grouped in two nuclei: the octavomotorius intermedius nucleus and the octavomotorius posterior nucleus. Cells of these nuclei receive synaptic contacts from vestibular afferents ~4. Vestibulospinal neurons are known to project only to the most rostral segments of the spinal cord, where they make monosynaptic contacts with motoneurons and spinal interneurons 13. In addition, reticulospinal and vestibulospinal systems are closely linked in the brainstem with the axons of vestibulospinal cells making en passant electrotonic and chemical contacts with reticulospinal cells of the posterior rhombencephalic reticular nucleus (PRRN) 13. Reticulospinal cells receive alternating excitatory and inhibitory inputs during fictive locomotion. Depolariza-
tion occurs during ipsilateral locomotor discharges recorded from rostral ventral roots and hyperpolarization during contralateral activity 5'7. Moreover, it has been shown that these inputs originate at least partly from spinal locomotor networks 5. The aim of the present study was to determine if the vestibulospinal cells are also rhythmically active during fictive locomotion in the lamprey. Fifteen adult lampreys (Ichthyornyzon unicuspis), 25-30 cm in length were anaesthetized with 3-aminobenzoate methanesulfonate (100 mg/1). The dissection was completed in a cold Ringer solution 15 oxygenated (95% 0 2, 5% CO2) and titrated at pH 7.4. Muscles and viscera were removed in order to expose the dorsal side of the brainstem and the rostral spinal cord. The final preparation consisted of the brainstem, the spinal cord, and the supporting cartilage. The preparation was then pinned to a layer of Sylgard 184 D o w Corning covering the bottom of a recording chamber filled with a Ringer solution continuously renewed, oxygenated, and maintained between 5 and 9°C. Locomotor activity was induced by adding N M D A (50 pM) to the perfusing solution and locomotor discharges were recorded with two suction electrodes placed on the dorsal aspect of two rostral and opposed ventral roots (lst to 6th segments of a total of 100). Intracellular recordings of vestibulospinal neurons were carried out with glass microelectrodes
Correspondence: R. Dubuc, D6partement de Kinanthropologie, Universit6 du Qu6bec ~t Montr6al, C.P.8888 succ. A, Montr6al Que., H3C 3P8 Canada. Fax: (1) (514) 343-2111.
175 filled with 4 M KAc (50-100 Mf~) or 20% Lucifer Yellow in 0.1 M LiCI (80-100 Mfl). Signals were sent to an Axoclamp-2A amplifier (0-10 kHz) and stored on magnetic tapes (8 tracks, Vetter DI) with ventral root neurograms. Fig. 1A illustrates the experimental paradigm used for cell identification. Vestibulospinal cells were identified by their monosynaptic response to ipsilateral vestibular nerve stimulation and by their antidromic response to spinal cord stimulation. Mean membrane potential was 65 + 7 mV and mean conduction velocity was 1.0 + 0.6
A
~
coVestn /,,1
m/s (measured at a stimulation strength of 3 x threshold). Fig. 1B 1 shows the all or none antidromic response of a vestibulospinal cell which appeared as the intensity of spinal cord stimulation was progressively increased. The action potential occurred at fixed latency and followed a frequency of stimulation of 15 Hz. Synaptic responses of the same cell to ipsilateral and contralateral vestibular nerve stimulation are shown in Fig. 1B 2 and Fig. 1B 3. The synaptic response of the vestibulospinal cell to ipsilateral vestibular nerve stimulation (Fig. 1B2) has the characteristics of a monosynaptic response with
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25 ms Fig. 1. A: schematic representation of the stimulation and recording paradigms. For vestibulospinal (VS) cell identification, one glass stimulation electrode was placed on the ipsilateral (i) and the contralateral (co) vestibular nerves (Vest n) to generate monosynaptic inputs. In addition, one glass stimulation electrode was placed on each lateral tract to antidromically activate the cell. Recording suction electrodes were placed on rostral ventral roots (VR) of opposite sides. BI: antidromic responses of a vestibulospinal cell (conduction velocity = 0.7 m/s) to ipsilateral spinal cord stimulation at threshold intensity (top) and at a stimulation frequency of 15 Hz (bottom). B2,B3: responses of the cell to ipsilateral and contralateral vestibular nerve stimulations. Each illustration is made up of 4 superimposed synaptic responses. Single and twin (15 Hz) stimulation pulses were delivered (2/zA). The ipsilateral response has a fast rising component with a small inflection (arrow). C: effects of kynurenic acid (4 mM) and of Ca 2+ free Ringer (Mn 2+ 2mM) on the synaptic responses of a vestibulospinal neuron to ipsilateral vestibular nerve stimulation.
176 a very rapid rise time (0.3 ms) and a latency of less than 3 ms. Moreover, there is a small inflection on the ascending phase of the excitatory postsynaptic potential, suggesting an electrotonic component (see arrow in Fig. 1B2). In addition, the amplitude and the shape of the response to a second stimulation applied 65 ms after a first are constant. The response of the vestibulospinal cell to contralateral vestibular nerve stimulation appears at a longer latency, has a longer rise time, and the amplitude of the second response, to a 15 Hz stimulation, is reduced. In another experiment, the role of excitatory amino acids in this synaptic transmission was studied by adding kynurenic acid (4 mM), a blocker of excitatory amino acid receptors, to the perfusing solution (Fig. 1C). Responses to ipsilateral vestibular nerve stimulation were markedly reduced. Of the two excitatory components composing the control response, only the first persisted in the presence of kynurenic acid, but showed a reduction in amplitude (67%). When Ca 2+ was removed and replaced by Mn 2+ (2 raM), thus abolishing all chemical synaptic transmission, the short latency component which resisted the effect of kynurenic acid was still present and persisted even at stimulation rates of 40 Hz. Taken together, these results suggest that the second excitatory component of the synaptic response and part of the first are resulting from the activation of excitatory amino acid receptors, while most of the first component is resulting from electrotonic transmission. Synaptic contacts between vestibular afferents and cells of the octavomotorius nuclei have been studied morphologically. They are of 3 types: chemical synapses, gap junctions and desmosomes TM. Indeed, our results confirm the presence of a mixed electrical and chemical synapse (see also ref. 13). The activity of 32 vestibulospinal cells has been studied during fictive locomotion of which 25 showed rhythmic fluctuations of their membrane potential correlated with ventral root activity in the rostral spinal cord. The activity of 72% of the rhythmically modulated cells showed a maximum of depolarization during ipsilateral activity and a minimum during contralateral activity. The average peak to peak amplitude of the fluctuations was 3.8 + 2.9 mV (Fig. 2). Some of these cells, like the one illustrated in Fig. 2A1, were sufficiently depolarized during ipsilateral ventral root activity that action potentials were generated. This particular vestibulospinal cell was filled with Lucifer yellow and reconstructed from photomicrographs taken at different focal planes (Fig. 2A3). The cell has a soma of 40/tin in diameter and its dendritic arborization reaches the lateral border of the alar plate and extends partly within the most lateral aspects of the basal plate. The axon is clearly visible projecting down to the spinal cord. This axonal projection was also demonstrated electrophysiologically by ipsilateral spinal
cord stimulation, which triggered an antidromic action potential in the cell (Fig. 2A2). Most cells did not, however, show discharges during fictive locomotor activity. The cell illustrated in Fig. 2B~ had regular oscillations well correlated with a stable locomotor activity. Averages of both membrane potential fluctuations and ventral root discharges triggered on the beginning of activity in the ipsilateral ventral root (Fig. 2B2) show that the depolarization wave in the vestibulospinal cell precedes the beginning of discharges in the ipsilateral ventral root. A similar phase relationship was observed for the other vestibulospinal neurons which were depolarized during ipsilateral locomotor activity. This suggests that vestibulospinal neuron depolarization may parallel closely motoneuronal depolarization in the most rostral spinal segments. All vestibulospinal cells did not have the same type of activity during locomotion. In 20% of the rhythmically active cells, the membrane potential reached a maximum of depolarization during contralateral burst discharge and a minimum during ipsilateral activity. For all cells with this pattern of activity, mean oscillation amplitude was 4.1 + 1.8 mV. Fig. 3A shows such a vestibulospinal neuron depolarized during the contralateral ventral root activity. Averages show that the depolarization wave precedes and persists through the contralateral ventral root discharge (Fig. 3A2). Moreover, the cell was identified by stimulation of the contralateral spinal cord (Fig. 3A3). An antidromic action potential was elicited before any synaptic response, appeared at a fixed latency and was still present at stimulation rates of 35 Hz. On the other hand, the response of the same cell to ipsilateral spinal cord stimulation consisted of excitatory postsynaptic potentials and discharges, which did not follow rates of 15 Hz. Other vestibulospinal neurons (8%) showed a more complex pattern of membrane potential fluctuations during locomotion. Two waves of depolarization could occur, one during the ipsilateral and/or one during the contralateral ventral root activity. An example is provided in Fig. 3B1. Initially, the cell is depolarized during ipsilateral activity, with only a small wave of depolarization during contralateral activity (Fig. 3B2). However, less than a minute later, when the locomotor activity became more vigourous in the contralateral ventral root, this second depolarization wave gained amplitude (Fig. 3B 3 and 3B4). This suggests that the amplitude of the membrane potential fluctuations show a close relationship with the intensity of the locomotor activity in the spinal cord. Interestingly, the cell was antidromically activated from both sides of the spinal cord, indicating a bilateral projection (Fig. 3B5). A similar pattern of activity was observed in 5 other cells recorded in octavomotorius nuclei.
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Fig. 2. AI: rhythmic fluctuations of the membrane potential of a vestibulospinal (VS) cell during fictive locomotion. Locomotor discharges were recorded from the 2nd ipsilateral ventral root (i VR) and the 3rd contralateral ventral root (co VR). A2: identification of the cell by the antidromic response to ipsilateral stimulation of the spinal cord (conduction velocity = 0.7 m/s). Note that the antidromic action potentials persist at a stimulation rate of 30 Hz. A3: reconstruction of the cell showing a clear axonal projection to the ipsilateral spinal cord (VIII n, vestibular nerve; MRRN, middle rhombencephalic reticular nucleus; PRRN, posterior rhombencephalic reticular nucleus). BI: activity of another vestibulospinal cell during fictive locomotion recorded from the 2nd ipsilateral and 5th contralateral ventral roots. Bz: averages of membrane potential fluctuations and of rectified neurograms (n = 14). The averaging was triggered on the onset of ipsilateral ventral root activity. The time scale was normalized so that a locomotor cycle equals 100%. B3: antidromic response of the cell to ipsilateral spinal cord stimulation at threshold intensity (top) and at 15 Hz (bottom).
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T h e p r e s e n t results show that vestibulospinal cells are clearly rhythmically active d u r i n g fictive l o c o m o t i o n in lamprey. T h e m a j o r i t y of the cells r e c o r d e d showed a m a x i m u m of d e p o l a r i z a t i o n d u r i n g ipsilateral spinal activity a n d a m i n i m u m d u r i n g c o n t r a l a t e r a l activity (n = 18). T h e spinal projections of these cells were identified by ipsilateral spinal cord stimulation. T h e i r exact loca-
tion has b e e n d e t e r m i n e d for those filled with Lucifer yellow, in which cases the cell bodies were located at the level of the o c t a v o m o t o r i u s i n t e r m e d i u s nucleus. In other vestibulospinal n e u r o n s , m e m b r a n e p o t e n t i a l fluctuations were in antiphase, that is the cells s h o w e d their maxim u m d e p o l a r i z a t i o n d u r i n g c o n t r a l a t e r a l l o c o m o t o r activity a n d their m i n i m u m d u r i n g ipsilateral activity (n =
179 5). These cells were r e c o r d e d m o r e caudally, at the level of the octavomotorius posterior nucleus, where vestibulospinal neurons have b e e n p r o p o s e d to project their axon to the contralateral spinal cord 11'13. This has b e e n recently confirmed with anatomical tracers 3 (also Bussi6res and D u b u c , unpublished observations). It is noteworthy that Kasicki et al. s described the same type of activity, that is depolarization during contralateral ventral root activity, for reticulospinal neurons such as the M a u t h n e r cell, whose axon projects to the contralateral spinal cord. Finally, some cells had variable phase relation: during a single l o c o m o t o r episode, they could be d e p o l a r i z e d during ipsilateral activity and during contralateral activity at different times or at the same time. Interestingly, these neurons r e c o r d e d at the level of the octavomotorius intermedius nucleus were antidromically activated by spinal cord stimulation on either side. A n a t o m i c a l exp e r i m e n t s with the c o m b i n e d use of two r e t r o g r a d e tracers are in progress to d e t e r m i n e whether such bilaterally projecting vestibulospinal neurons exist within the lamprey brainstem. The v e r t e b r a t e labyrinthine system is i m p o r t a n t for head orientation and postural adjustments. It is presumed that vestibulospinal neurons will play an important role in selecting the a p p r o p r i a t e response to vestibular stimulation (see also ref. 10). They r e p r e s e n t the most direct pathway from the vestibular apparatus to the spinal cord where they m a k e electrotonic and chemical contacts with m o t o n e u r o n s innervating the rostral myo-
tomes and some spinal interneurons 13. The results presented here show that vestibulospinal neurons receive rhythmic inputs that k e e p them in tune with the locom o t o r activity occurring in the rostral spinal cord. The source of this m o d u l a t i o n is not known. In fact, little is known about the inputs to the vestibulospinal cells, except for those from vestibular afferents 13'14. In the case of reticulospinal neurons, this rhythmic m o d u l a t i o n which occurs during fictive locomotion has been shown to come at least partly from spinal cord inputs 5. It is possible that a similar situation occurs in vestibulospinal neurons. M o d u l a t i o n of reflex transmission during locomotion allows the animal to a d a p t its m o v e m e n t s to the external environment (for a review see ref. 12). M e m b r a n e potential fluctuations in vestibulospinal neurons during fictive locomotion m a y be i m p o r t a n t in the modulation of vestibular reflexes by favouring certain periods of the cycle over others for input transmission. We have, in fact, recently shown that such m o d u l a t i o n occurs for transmission of vestibular inputs to reticulospinal neurons during fictive locomotion in lampreys 4.
1 Buchanan, J.T., Identification of interneurons with collateral, caudal axons in the lamprey spinal cord: synaptic interactions and morphology, J. Neurophysiol., 47 (1982) 961-975. 2 Buchanan, J.T. and Grillner, S., A newly identified class of excitatory premotor interneurons in the lamprey spinal cord, Acta Physiol. Scand., 128 (1986) 13A. 3 Bussi6res, N. and Dubuc, R., Rhythmic modulation of vestibulospinal neurones during fictive locomotion in lampreys, Third IBRO World Cong. Neurosci., 3 (1991) 486. 4 Bussi~res, N. and Dubuc, R., Phasic modulation of transmission from vestibular inputs to reticulospinal neurones during fictive locomotion in lampreys, Soc. Neurosci. Abstr., 21 (1991) 1025. 5 Dubuc, R. and Grillner, S., The role of spinal cord inputs in modulating the activity of reticulospinal neurons during fictive locomotion in the lamprey, Brain Res., 483 (1989) 196-200. 6 Grillner, S., Wall6n, P., Brodin, L. and Lansner, A., Neuronal network generating locomotor behaviour in lamprey, Annu. Rev. Neurosci., 14 (1991) 169-199. 7 Kasicki, S. and Grillner, S., Miitler cells and other reticulospinal neurones are phasically active during fictive locomotion in the isolated nervous system of the lamprey, Neurosci. Lett., 69 (1986) 239-243. 8 Kasicki, S., Grillner, S., Ohta, Y., Dubuc, R. and Brodin, L., Phasic modulation of reticulospinal neurons during fictive locomotion and other types of spinal motor activity in the lamprey,
Brain Res., 484 (1989) 203-216. 9 0 h t a , Y. and Grillner, S., Monosynaptic excitatory amino acid transmission from the posterior rhombencephalic reticular nucleus to spinal neurons involved in the control of locomotion in lamprey, J. Neurophysiol., 62 (1989) 1079-1089. 10 Orlovsky, G.N., Deliagina, T.G., Grillner, S. and Wall6n, P., Natural vestibular stimulation evokes static and dynamic responses in lamprey reticulospinal neurons, Acta Physiol. Scand., 138 (1990) 13A. 11 Ronan, M., Origins of the descending spinal projections in petromyzontid and myxinoid agnathans, J. Comp. Neurol., 281 (1989) 54-68. 12 Rossignol, S., Lund, J.P. and Drew, T., The role of sensory inputs in regulating patterns of rhythmical movements in higher vertebrates. In A. Cohen, S. Rossignol and S. GriUner (Eds.), Neural Control of Rhythmic Movements in Vertebrates, Wiley, New York, 1988, pp. 201-283. 13 Rovainen, C.M., Electrophysiology of vestibulospinal and vestibuloreticulospinal systems in lampreys, J. Neurophysiol., 42 (1979) 745-765. 14 Stefanelli, A. and Caravita, S., Ultrastructural features of the synaptic complex of the vestibular nuclei of Lampetra planeri (Bloch), Z. Zellforsch., 108 (1970) 282-296. 15 Wickelgren, W.O., Physiological and anatomical characteristics of reticulospinal neurones in lamprey, J. Physiol., 270 (1977) 89-114.
The authors are grateful to D. Cyr, S. Doucet, G. Filosi, C. Gauthier, D. Lauzier and J. Provencher for the excellent technical assistance provided. We are also grateful to Dr. S. Alford for his helpful comments on this manuscript. This work was supported by Canadian MRC (Grant 10703), FRSQ, FCAR (Qu6bec) and The Bunting Research Foundation. N.B. receives a studentship from FCAR and the NCE for Neural Regeneration and Functional Recovery.
