Exp. Brain Res. 27, 161-179 (1977)

Experimental Brain Research 9

Springer-Verlag 1977

The Rubro-Bulbospinal Path. A Descending System Known to Influence Dynamic Fusimotor Neurones and its Interaction with Distal Cutaneous Afferents in the Control of Flexor Reflex Afferent Pathways T. Jeneskog and H. Johansson Department of Physiology,University of Umefi, Ume~, Sweden

Summary. The inhibitory effect of electrical stimulation in the near-rubral region on polysynaptic segmental as well as ascending pathways activated by the flexor reflex afferents (FRA) in hind limb nerves was studied in chloralose anaesthetized cats. The effective stimulating region totally coincided with the one from which a D zone climbing fibre response may be elicited in the contralateral cerebellar cortex. The descending path was dependent upon an intact dorsolateral spinal funiculus, where also a characteristic volley could be recorded with a surface electrode on short train central stimulation. The suppressive action on the transmission through the F R A pathways was evoked in the absence of a lower lumbar dorsal root potential, and it was concluded that the effect was exerted by postsynaptic inhibition. It was suggested that this descending path, the effects of which resemble those elicited from the dorsal reticulospinal system, is identical to the rubro-bulbospinal path, previously known to influence dynamic fusimotor neurones. The transmission through the F R A pathways was also suppressed by conditioning stimulation of ipsilateral, low threshold distal cutaneous afferents. The time course of this effect was the same as that with central conditioning stimulation. Facilitatory interaction was revealed with double conditioning and it was suggested that the descending path and the distal cutaneous afferents converge upon a common group of interneurones, which postsynaptically inhibit an early (possibly the first one) interneurone in the F R A pathways. As low threshold distal cutaneous afferents supply the primary peripheral input via climbing fibres to the cerebello-cortical D zone, it was concluded that the different stimuli (central or peripheral) which activate a common group of inferior olivary neurones destined for the D zone also activate a common group of segmental inhibitory interneurones.

162

T. Jeneskogand H. Johansson The results are discussed in relation to current concepts of segmental motor control, and it is suggested that the mechanisms studied could be involved in the regulation of stepping. Key words: Rubro-bulbospinal path - Dorsal reticulospinal system - Distal cutaneous afferents - F R A path transmission - Climbing fibre projection

Introduction

A descending system from the mesencephalon which influences selectively dynamic fusimotor neurones has been extensively analyzed by Appelberg and coworkers (Appelberg, 1962, 1967; Appelberg and Molander, 1967; Appelberg and Jeneskog, 1969, 1972; Jeneskog, 1974a-c). Static fusimotor or skeletomotor neurones are not coactivated by this system, which may be activated from a restricted, near-rubral region denoted the MesADC ('mesencephalic area for control of dynamic spindle sensitivity') by Appelberg (1967). Recent results (Jeneskog, 1974a) have indicated that this motor system emerges from cells in the rostral parts of the red nucleus. The path then descends homolaterally in the vicinity of the inferior olive and probably transverses at least one synapse in the low brain stem before it reaches the contralateral dorsolateral funiculus of the spinal cord. At the segmental level, the effects are probably bilaterally exerted (Appelberg and Jeneskog, 1969). The pathway was provisionally denoted the rubro-bulbospinal path (RBSP) by Jeneskog (1974c). The spinal component mediating the dynamic fusimotor effects from the MesADC is, although proceeding in the spinal dorsolateral funiculus, not identical to the rubrospinal or the corticospinal tract (Appelberg and Jeneskog, 1969, 1972). There is, however, another major motor system which is known to descend in the dorsolateral funiculus of the spinal cord, namely the dorsal reticulospinal system (Holmqvist and Lundberg, 1959; Engberg et al., 1968a, b), which influences certain segmental reflex pathways. Baldissera et al. (1972a) studied effects from the mesencephalic tegmentum in descending pathways, and found that after medullary interruption of the rubrospinal tract in bilaterally de-corticated animals, a discharge could still be recorded in the contralateral dorsolateral funiculus on brief train stimulation at the dorsal border of the red nucleus. Single stimuli did not evoke a discharge. From the segmental effects elicited via this stimulation, it was concluded that the discharge recorded represented activity at least partly in the dorsal reticulospinal system. The dorsal reticulospinal system thus has a number of features in common with the rubro-bulbospinal path: 1) it may be activated from the near-rubral region, 2) it descends in the contralateral dorsolateral spinal funieulus, 3) single shocks in the mesencephalon do not evoke a descending discharge, and 4) it reaches the lumbar spinal cord, where its effects are bilaterally exerted. These similarities indicated that the two systems might in fact be identical (Jeneskog, 1974c), and this possibility has now been investigated. T h e evidence to be presented below does indeed indicate a close coincidence between the two paths mentioned.

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Stimulation within the M e s A D C also evokes climbing fibre responses in the D zone of the contralateral cerebellar cortex, and it has been suggested that the inferior olivary climbing fibre neurones are activated via collaterals from the descending path (Jeneskog, 1974a), thus giving the cerebellar cortex climbing fibre information about the m o t o r c o m m a n d signal (Miller and Oscarsson, 1970). These same climbing fibre neurones are known to be influenced also from the ascending dorsolateral spino-olivocerebellar path, the D L F - S O C P (Miller et al., 1969; Jeneskog, 1974a), which is primarily activated by cutaneous afferents from the distal parts of the limb (Larson et al., 1969). The spinal part of the D L F - S O C P is monosynaptically activated by the primary afferents, but there are several relays in the lower brain stem before the inferior olive (Larson et al., 1969). Supraspinal descending signals might therefore influence this path not only at an olivary level but also at a preolivary level, allowing the olivary neurones to work as comparators of the descending m o t o r c o m m a n d signal and the resulting activity in interneurones of the D L F - S O C P , also activated from the periphery (Miller and Oscarsson, 1970). In this way the olivary neurones would be able to inform the cerebellum about the efficiacy of transmission through a m o t o r center concerned with the particular m o t o r mechanism which is influenced by these descending and peripheral inputs (Oscarsson, 1973). This m o t o r center would thus consist of the above mentioned D L F - S O C P interneurones, and it was therefore of interest to analyze whether stimuli which activate the D L F - S O C P may produce effects similar to those elicited from the descending system. This was tested by comparing the influence of cutaneous nerve stimulation on segmental as well as ascending F R A pathways with the one elicited from the M e s A D C . It was then found that the influence from peripheral and central sites was very similar, and, furthermore, that the pattern of nerves which was most effective in this respect was the same as that also most effective in activating the D L F - S O C P . The effect of peripheral stimulation was not, however, dependent upon suprasegmental levels. Thus the existence of a brain stem m o t o r center as described above, on the basis of the hypothesis of Oscarsson (1973), could neither be proven nor disproven. H o w ever, the descending and ascending systems both have access to 1) the same group of inferior olivary neurones destined for the cerebello-cortical D zone and 2) a c o m m o n group of inhibitory interneurones located in the segment.

Methods The experiments were performed on 20 cats, weighing 2.8-4.5 kg. After induction with halothane (Fluothane, ICI) in an O2-N20-halothane mixture, they were anesthetized with intravenously administered c~-chloralose (60 mg/kg). In some experiments small doses (5 mg/kg) of pentobarbital (Mebumal 6%, ACO) were added intravenously during the course of the experiment. Blood pressure, end tidal CO2, rectal temperature and temperatures in the paraffine pools covering the exposed areas were monitored throughout the experiments. Slow, sometimes intermittent, i.v. infusion of sodium chloride-acetate (ACO) was used to prevent acidosis during the operation, the aim being to keep end tidal CO2 within normal limits (4.0-4.5%). A dextrane solution (Macrodex + Rheomacrodex, Pharmacia) was administered slowly i.v. if the blood pressure tended to fall below 75 mm Hg. In most experiments the animals were paralyzed during the recording period with Flaxedil (Pharma Rhodia) and artificiallyventilated. Body and pool temperatures were kept within

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normal limits with the aid of radiant heat from above and a heat blanket under the belly of the animal. Operation: After tracheal, venous and arterial cannulations two laminectomies were performed. One of them exposed the low thoracic cord (Th12-Th13) where the dura was opened and the dorsal funiculi and the right spinal half were transected and removed for approximately one segment. In some experiments the right ventral quadrant was dissected and prepared for recording of ascending mass discharges. In the second laminectomy (L5-LT) the left S1, L7 and L6 ventral roots were cut distally after opening the dura and, in most experiments, a thin dorsal rootlet usually from left L6 or S1 was dissected for about 15 mm and cut peripherally for recording of dorsal root potentials. Some or all of the following nerves in the left hind limb were dissected: quadriceps (Q), anterior biceps and semimembranosus (ABSm), posterior biceps and semitendinosus (PBSt), gastrocnemius and soleus (GS), caudal femoral cutaneous (CF), saphenous (Saph), sural (Su), cutaneous part of superficial peroneal (SP) and tibial after giving off branches to flexor digitorum and hallucis longus, tibialis posterior and popliteus (Tib). Usually the Tib nerve was left in continuity with the periphery. Two craniotomies were performed, one over the right mesencephalon for electrode penetrations to the rubral region, and the other over the posterior parts of the cerebellum exposing the left paramedian lobule for surface recording. The dura was opened in both craniotomies. Recording: Monosynaptic reflex discharges were recorded in cut ventral roots and evoked by the double volley technique described by Holmqvist and Lundberg (1959). The effect of a conditioning volley in flexor reflex afferents (FRA) - usually high threshold muscle afferents - was measured as an inhibition (to extensor muscles) or a facilitation (to flexor muscles) of the monosynaptic reflex. The numerical value of this influence was called 100% effectiveness of the FRA volley. The result of a conditioning stimulation (central or peripheral) removing part of this influence could thus be plotted quantitatively as a reduced effectiveness of the FRA volley (cf. Figs. 2E, 4A, 4B, 5E and 7B). Evoked activity in the paramedian lobule was recorded via a spring mounted silver ball electrode on the cerebellar surface, with the indifferent electrode placed in the temporal muscles. Climbing fibre responses were thus recorded as characteristic, sharply rising positive potentials. Descending discharges were recorded with a surface electrode on the dorsolateral aspect of the spinal cord in Thl3. Ascending mass discharges were recorded from the right (contralateral to peripheral stimulation) ventral quadrant which was mounted on a bipolar hook electrode. Dorsal root potentials were recorded with a bipolar electrode, one of the poles close to the cord and the other at the cut end of the filament (interelectrode distance about 15 ram). All signals were amplified, suitably filtered and displayed on a storage oscilloscope for visual inspection and simultaneously on another oscilloscope for film recording (Grass Kymograph Camera). In all figures the recordings consist of superimposed traces. In some experiments signals were computer-averaged in order to avoid errors due to spontaneous fluctuations in reflex transmittability and to reveal liminal effects. Stimulation: Peripheral nerves were stimulated via bipolar hook electrodes, except for the Q and Saph nerves, which were stimulated via buried electrodes. Stimulus shocks were 0.1 ms square waves, and the strength of stimulation is given in times threshold (T) for the most excitable fibres in the nerve, as determined by recording the incoming volley at the appropriate dorsal root entry zone. Stimulation in the mesencephalon was performed via stereotaxically guided, glass-insulated platinum-iridium wire electrodes (flee tip 60-90 ~tm, impedance 10-100 kff2 at 1 kHz). The stimulator delivered constant current pulses (cathodal, 0.2 ms square waves) via isolation units. Short trains of pulses at 600 Hz with a maximum current of 100 ~tA were used. Abbreviations: DLF dorsolateral funiculus of spinal cord, DLF-SOCP spino-olivocerebellar path running in dorsolateral spinal funiculus, DR dorsal root, DRP dorsal root potential, F R A flexor reflex afferents, MesADC near-rubral area in mesencephalon from which the rubro-bulbospinal path and the D zone rubro-olivocerebellar path are activated in parallel (cf. Appelberg, 1967; Jeneskog, 1974a), NR red nucleus, PM cerebellar paramedian lobule, RBSP rubro-bulbospinal path, VR ventral root.

Descendingand Peripheral Controlof FRA Pathways

165

Results

Effects on the Monosynaptic Reflex from the Near-Rubral Region: Two sets of stimulating electrodes were utilized in some of the experiments in this series. One set was advanced through the mesencephalon to the ventro-caudal part of the red nucleus (NR), where a selective activation of the rubrospinal tract may be obtained through stimulation of the interposito-rubral fibres (Baldissera et al., 1972b). The other set was placed in the MesADC with the aid of the short latency climbing fibre response recorded in the D zone of the contralateral paramedian lobule (PM) of the cerebellum on short train central stimulation (cf. Jeneskog, 1974a). Although the electrode tips of the different sets were rather close to each other (around 1.0 mm), spread of stimulating current between the two areas was considered negligable, as evidenced by the fact that the effects produced from the two stimulating sites were quite different from each other. This is demonstrated for two simultaneously recorded parameters in Figure 1G-H. Stimulation in the MesADC (70 ~tA) evoked a short latency climbing fibre response in the contralateral PM (Fig. 1G, upper trace), but the NR stimulation (70 ~tA, 1.0 mm more ventrally in the brain stem) did not (Fig.lH, upper trace). Latencies of the climbing fibre responses were always measured from the effective stimulus shock. The lower trace in H shows the typical rubrospinal volley recorded in the dorsolateral lower thoracic cord (DLF), in this case consisting of both directly and indirectly activated rubrospinal fibres (Baldissera et at., 1972b). The MesADC stimulation, on the other hand, evoked a different kind of volley in the thoracic cord. This volley was not evoked by a single stimulus shock, but appeared with the second shock of a train and then growed markedly with a longer train of stimulating pulses, indicating temporal summation in the path. This type of volley is seen in the lower trace of Figure 1G, and it is similar to the volley recorded by Baldissera et al. (1972a) after medullary interruption of the rubrospinal tract, and considered by them to be caused at least partly by fibres belonging to the dorsal reticulospinal system. These two parameters are also illustrated in Figure 1A-F from another experiment, where in addition effects on the monosynaptic reflex was simultaneously recorded. There were typical effects evoked by NR stimuli on the monosynaptic reflexes, i.e. facilitation of the flexor monosynaptic reflex (C compared to A) and inhibition (in this case almost complete) of the extensor monosynaptic reflex (F compared to D). However, the MesADC stimulation did not influence the monosynaptic reflexes of either flexors or extensors, as illustrated in Figure 1B and E respectively, with a conditioning-test interval which was optimal for the NR influence. The effects shown in Figure 1A-F were evoked with a strength of 70 ~tA in MesADC and 50 ~tA in NR. Thresholds for evoking a D zone climbing fibre response from MesADC and a rubrospinal volley from NR were 25 ~tA and 10 ~tA, respectively, in that experiment. It is therefore concluded that a stimulating electrode may be placed in the MesADC and, with suitable current intensities, descending effects may be produced from that area while avoiding a simultaneous activation of the rubrospinal tract (compare also Appelberg et al., 1975).

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Effects f r o m M e s A D C on a Segmental F R A Pathway: Short train electrical

stimulation in the M e s A D C did not influence the excitability of hind limb a m o t o n e u r o n e s (Fig. 1 and A p p e l b e r g et al., 1975), but effectively suppressed reflex effects to a m o t o r nucleus e v o k e d f r o m the flexor reflex afferents ( F R A ) . This is illustrated in Figure 2 with the m o n o s y n a p t i c testing technique. Figure 2 A shows the m o n o s y n a p t i c reflex e v o k e d by d o u b l e shock stimulation of the Q nerve. W h e n this stimulus was p r e c e d e d at a suitable interval by a strong single shock to the GS nerve, there was a m a r k e d depression of the m o n o s y n a p t i c reflex due to activation of F R A (Fig. 2B). T h e effectiveness of this F R A - i n h i b i t i o n was strongly r e d u c e d by a conditioning short train stimulation in the M e s A D C at certain intervals b e f o r e the F R A volley (Fig. 2D), alt h o u g h the central stimulation itself did not influence the a - m o t o n e u r o n e excitability (Fig. 2C). In Figure 2 E the time course of the effectiveness of the F R A volley is plotted against the interval b e t w e e n the first conditioning shock in the M e s A D C and the arrival of the GS volley at the cord. It is seen that the effect of central conditioning starts a r o u n d 12 ms after the first stimulus shock, b e c o m e s maximal at 2 5 - 6 0 ms interval, and then gradually fades off, This effect was p r o d u c e d without a detectable dorsal r o o t potential ( D R P ) in caudal

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L6, i n d i c a t i n g t h a t p r e s y n a p t i c i n h i b i t i o n was n o t r e s p o n s i b l e for t h e o b s e r v e d effects. W i t h i n t h e series o f e x p e r i m e n t s 5 - 8 p u l s e s in t h e M e s A D C w e r e sufficient to elicit t h e s u p p r e s s i v e effect o n t h e F R A p a t h w a y . T h e s u p p r e s s i v e a c t i o n b y s t i m u l a t i o n in t h e M e s A D C was e q u a l l y effective w h e n t e s t e d with high t h r e s h o l d c u t a n e o u s (Su) afferents as t h e F R A i n p u t o n to an e x t e n s o r m o n o s y n a p t i c reflex. L i k e w i s e , c o n d i t i o n i n g s t i m u l a t i o n in t h e M e s A D C s u p p r e s s e d t h e f a c i l i t a t i o n o f t h e f l e x o r m o n o s y n a p t i c r e f l e x (e.g. Fig. 3 A ) , using e i t h e r high t h r e s h o l d m u s c l e o r c u t a n e o u s a f f e r e n t s as t h e F R A input. In a few experiments stimulation not only of the rubrospinal tract but also within the MesADC evoked a DRP in lumbar dorsal root filaments with rather low stimulating intensities (around 40 pA, of. Hongo et al., 1972). Those experiments were not included in the present analysis, because in such cases it was not possible to exclude presynaptie influences on the segmental mechanism studied. Engberg et al. (1968a) noted that on medullary activation of the dorsal reticulospinal system, it was sometimes impossible to reveal clear action on the segmental reflex pathways without raising the current intensity above the threshold for evoking a DRP. W h e n e v e r t e s t e d , a r a t h e r s m a l l D L F lesion in t h e l o w e r t h o r a c i c c o r d (left T h 1 3 , cf. M e t h o d s ) i n t e r r u p t e d t h e p a t h f r o m t h e M e s A D C to l u m b a r segments, and a suppressive action on the segmental FRA pathway from the central site c o u l d no l o n g e r b e d e m o n s t r a t e d . Mapping of Effective Region in the Mesencephalon: M a p p i n g t h r o u g h t h e m e s e n c e p h a l o n at t h e r u b r a l level was p e r f o r m e d in a n u m b e r of e x p e r i m e n t s to c o m p a r e t h e e x t e n t of a r e a s effective in eliciting o n t h e o n e h a n d a s h o r t lat e n c y D z o n e c l i m b i n g fibre r e s p o n s e in t h e c o n t r a l a t e r a l P M (i.e. a t y p i c a l effect o f t h e R B S P ) , a n d on t h e o t h e r h a n d a c h a r a c t e r i s t i c d e s c e n d i n g d i s c h a r g e in t h e c o n t r a l a t e r a l D L F a n d t h e s u p p r e s s i o n of F R A effects u p o n t h e m o n o s y n a p t i c r e f l e x (i.e. effects t y p i c a l for t h e d o r s a l r e t i c u l o s p i n a l system). A n e x a m p l e o f such an e x p e r i m e n t is i l l u s t r a t e d in F i g u r e 3 A . T h e g r a p h shows t h r e s h o l d curves for t h e t h r e e d i f f e r e n t p a r a m e t e r s a n d d e m o n s t r a t e s t h e close c o i n c i d e n c e in d e p t h p r o f i l e s b e t w e e n t h e curves. T h e t h r e s h o l d c u r v e for t h e s u p p r e s s i o n of F R A effects o n t h e m o n o s y n a p t i c r e f l e x (PBSt) was o b -

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tained with a conditioning interval of 30 ms between the first stimulus shock in the mesencephalon and the arrival of the F R A (GS nerve, 7T) volley at the cord. A t depth - 4 . 0 there was no longer suppression of the F R A effect, but rather a facilitation of the monosynaptic reflex itself, i.e. possibly a rubrospinal effect with 70 ~tA stimulation. The typical dorsal reticulospinal volley, which was recorded alone on stimulation at depth - 3 . 0 began to be mixed with a rubrospinal discharge on 60 ~tA stimulation at depth -3.5. However, on decreasing the current intensity to 50 ~tA the dorsal reticulospinal volley again appeared alone. The short latency climbing fibre response which was recorded with 60 ~tA stimulation at depth - 3 . 5 but not with 50 ~tA, was replaced by a longer latency response when the electrode was advanced to - 4 . 0 (cf. Jeneskog, 1974a). In this experiment the low threshold area for evoking the dorsal reticulospinal volley seemed to extend somewhat deeper in the brain stem than the low threshold areas for the other two effects studied. However, the areas usually coincided totally, as demonstrated in Figure 3B for the short latency D zone climbing fibre response and the dorsal reticulospinal volley from another experiment, or the area for the descending volley was somewhat larger than the area for any of the other two parameters, but then symmetrically arranged, i.e. the depth with the lowest absolute threshold for the different effects was the same. A similar type of experiment is illustrated in Figure 4A, where the depth profile for the suppressive action on F R A effects is shown together with the areas giving short and long latency climbing fiber responses. In addition the depths from which a D R P was evoked in caudal L6 is illustrated. All p a r a m e ters were recorded with a constant central stimulus intensity (65 ~A) and it is seen that the suppressive action on the F R A effect was evoked from depths coinciding with those also giving the short latency climbing fibre response. The D R P was, however, evoked only from deeper parts of the electrode track. The excitability level of m o t o n e u r o n e s was high in that experiment, demonstrated

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Fig. 4A and B. Comparison of mesencephalic areas effective in eficiting cerebellar climbing fibre responses and various segmental effects. Graph in A shows the effectiveness of the FRA volley (ABSm nerve, 10T) on the monosynaptic reflex (GS nerve) plotted against the depth in the mesencephalon of the conditioning stimulation (7 shocks, 600 Hz, 65 ~tA, 50 ms interval). The two bars in the PM column show the depths from which short latency (upper) and long latency (lower) climbing fibre responses were evoked in the D zone of the contralateral PM with the same stimulation as above. Bars in the DRP column and in the VR column delimit the areas from which a dorsal root potential (caudal rootlet of L6) and a ventral root discharge, respectively, were evoked in the same experiment with stimulation as above. B Shows the results from another experiment presented in the same way. Monosynaptic reflex from Q nerve, FRA volley from GS nerve (6T), mesencephalic stimulation 9 shocks, 600 Hz, 70 ~tA, 35 ms interval. There was no DRP recording in that experiment

b y t h e fact t h a t a v e n t r a l r o o t d i s c h a r g e c o u l d b e e v o k e d f r o m c e r t a i n d e p t h s o f t h e track. T h e D R P a n d t h e v e n t r a l r o o t d i s c h a r g e w e r e e v o k e d f r o m t h e s a m e d e p t h s of t h e track, a n d this a r e a s e e m s to b e t h e o n e f r o m which t h e r u b r o spinal t r a c t was a c t i v a t e d in t h a t e x p e r i m e n t with t h e p a r t i c u l a r s t i m u l a t i n g intensity. F i g u r e 4B shows a n o t h e r e x p e r i m e n t o f t h e s a m e kind. M a p p i n g was p e r f o r m e d with 70 ~tA s t i m u l a t i o n in t h e m e s e n c e p h a l o n a n d in this case o n l y t h e s h o r t l a t e n c y D z o n e c l i m b i n g f i b r e r e s p o n s e was e v o k e d b y t h e c e n t r a l s t i m u l a t i o n . T h e s u p p r e s s i o n of t h e effect o f the F R A v o l l e y on t h e m o n o s y n a p t i c r e f l e x was n o t v e r y s t r o n g in this e x p e r i m e n t , b u t t h e effective m e s e n c e p h a l i c a r e a is s e e n to c o i n c i d e with t h e o n e also giving t h e s h o r t lat e n c y D z o n e c l i m b i n g fibre r e s p o n s e . A s in t h e e x p e r i m e n t i l l u s t r a t e d in F i g u r e 4 A , t h e r u b r o s p i n a l t r a c t was p r e s u m a b l y a c t i v a t e d f r o m t h e d e e p e r p a r t s o f t h e e l e c t r o d e track.

Suppression of FRA Effects on the Monosynaptic Reflex by Stimulation of Cutaneous Nerves: T h e R B S P is closely c o n n e c t e d to t h e D L F - S O C P ( J e n e s kog, 19743), a n d so it was o f i n t e r e s t also to a n a l y z e w h e t h e r a c t i v a t i o n o f this l a t t e r s y s t e m c o u l d p r o d u c e effects similar to t h o s e elicited b y M e s A D C - a c t i v a t i o n o f t h e d o r s a l r e t i c u l o s p i n a l s y s t e m (cf. I n t r o d u c t i o n ) . This s e e m e d to b e t h e case, a n d t h e e v i d e n c e is p r e s e n t e d in F i g u r e s 5 a n d 6 f r o m t h e s a m e exp e r i m e n t as F i g u r e 2. F i g u r e 5 A shows t h e m o n o s y n a p t i c reflex e v o k e d b y s t i m u l a t i o n o f t h e Q n e r v e , a n d B t h e i n h i b i t o r y effect on this r e f l e x b y a p r e c e d i n g s t r o n g single

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shock to the GS nerve. The effectiveness of this F R A inhibition was strongly reduced by a single low threshold (2T) shock to the Tib nerve at certain intervals before the F R A volley, exemplified in Figure 5D with 50 ms interval. Figure 5C shows that the conditioning shock to the Tib nerve with the same interval did not influence the size of the monosynaptic reflex itself. The graph in Figure 5E illustrates the effectiveness of the F R A volley when conditioned by a 2T single shock to the Tib nerve at different intervals before the arrival of the GS volley at the cord. The depressant effect starts at around 10 ms interval, is

Descending and Peripheral Control of FRA Pathways

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maximal at 30 to 6 0 - 7 0 ms and then gradually fades off. This time course of reduced effectiveness of the F R A volley is closely similar to the one obtained with conditioning in the M e s A D C (cf. Fig. 2E). At very short intervals (less than 10 ms) there was a direct facilitation of the extensor monosynaptic reflex by the Tib shock, but this was not further studied. The time course and the relative size of the D R P recorded in a caudal dorsal rootlet of L6 and evoked by the 2T Tib nerve volley is also schematically indicated in the graph of Figure 5E. It is seen that the time course of the D R P and that of the depression of the effect of the F R A volley are rather different, the D R P being maximal at around 15 ms, while the depressant effect is maximal around 3 0 - 6 0 ms, when the D R P has already faded off. In a number of experiments it has been observed that this suppressive effect was evoked by afferents in the Tib nerve activated at only 1.1 T. In these cases there was no appreciable D R P recorded in mid-S1 filaments until the Tib stimulus was raised to 1.3-1.4 T. A similar depression of the effectiveness of the F R A volley was demonstrated also for the F R A facilitation of flexor monosynaptic reflexes. Furthermore, low threshold Tib nerve stimulation depressed the F R A action on extensor and flexor monosynaptic reflexes also when using high threshold cutaneous (Su) afferents as the F R A input. With regard to these results, it was of interest to consider if the central and peripheral systems exerted their effects via different mechanisms, or if they possibly shared a common segmental inhibitory pathway. This last alternative is supported by the results presented in Figure 6. Figure 6A and B show the monosynaptic reflex from the Q nerve and its inhibition by a F R A volley in the GS nerve, respectively. This reflex was conditioned in Figure 6C by a short train stimulation in the M e s A D C with an interval of 50 ms and a strength (in this case 50 ~A) adjusted to give a just limihal effect. A weak single shock to the Tib nerve (1.3T, 50 ms interval) in fact further depressed the reflex slightly (Fig. 6D). However, when these central and peripheral liminal stimuli were combined, as shown in Figure 6F, there was a clearcut reduced effectiveness of the F R A volley, although the combined conditioning stimuli did not alter the monosynaptic reflex directly, Figure 6E compared to Figure 6A. In order to demonstrate that the effects elicited by the Tib nerve stimulation were probably not due to muscle afferents but to cutaneous afferents from the foot, bipolar electrical stimulation was performed via needle electrodes inserted into the central pad of the left hindlimb in one experiment, where the Tib nerve was left in continuity with the periphery. Two different effects could then be demonstrated on single shock stimulation of the pad with different stimulus strengths, either a suppression of the F R A influence on the monosynaptic reflex or an inhibition of the monosynaptic reflex (extensor muscle) itself. Both effects were elicited at intervals around 50 ms between pad stimulation and the test reflex stimulation. The effect of Tib nerve stimulation on the F R A reflex path to a motor nucleus remained unchanged after total spinalization at the level of L5, showing that no long spinal or supraspinal reflex arcs are involved.

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Fig. 7A and B. Comparison of the capacity of low threshold cutaneous afferents in different skin nerves to depress the transmission through the segmental FRA pathway. A Difference (mean and S.D.) between conditioned and unconditioned reflexes in arbitrary units. Unconditioned reflex is the difference between the height of the monosynaptic reflex itself and the height of the FRA influenced monosynaptic reflex. Conditioned reflex is the difference between the height of the monosynaptic reflex itself and the height of the cutaneous nerve conditioned, F R A influenced monosynaptic reflex. Number of tests with each nerve indicated below in the diagram. B Ordinate is the relative effectiveness of the FRA volley when conditioned with different cutaneous afferents (for procedure, see Methods). Values for individual tests with each nerve indicated as dots. Short horizontal lines are mean values for each nerve. Conditioning-test interval 40-50 ms. Strength of conditioning stimulation 1.1-1.35T

I n t h r e e e x p e r i m e n t s five d i f f e r e n t c u t a n e o u s n e r v e s w e r e t e s t e d a n d c o m p a r e d for t h e i r i n f l u e n c e on t h e s e g m e n t a l F R A p a t h w a y . T h e y w e r e for p r o x i m a l p a r t s o f t h e leg t h e C F a n d S a p h nerves, a n d for t h e distal p a r t s of t h e leg t h e Su, SP a n d T i b nerves. T h e results o f t h e s e e x p e r i m e n t s a r e p r e s e n t e d in t h e d i a g r a m s of F i g u r e 7. I n 7 A is s h o w n t h e a b s o l u t e effect (arb. units) of a single c o n d i t i o n i n g v o l l e y in t h e d i f f e r e n t c u t a n e o u s n e r v e s ( m e a n a n d S.D. of tests) on t h e t r a n s m i s s i o n t h r o u g h t h e F R A p a t h w a y . T h e g r a d u a l i n c r e a s e o f effect f r o m p r o x i m a l to distal n e r v e s i n d i c a t e s t h a t t h e r e c e p t i v e field of t h e c u t a n e ous afferents is of c o n s i d e r a b l e i m p o r t a n c e . This is also s h o w n in 7B, w h e r e t h e r e l a t i v e effect (for p r o c e d u r e , s e e M e t h o d s ) o f t h e d i f f e r e n t n e r v e s is p l o t ted. D o t s a r e t h e i n d i v i d u a l tests a n d s h o r t h o r i z o n t a l lines t h e m e a n for e a c h n e r v e . O n e p o i n t o f i n t e r e s t s h o w n in F i g u r e 7B is t h a t l o w t h r e s h o l d a f f e r e n t s (up to 1.35T in t h e tests) in t h e two n e r v e s with r e c e p t i v e fields i n c l u d i n g t h e f o o t (Tib a n d SP) c o u l d b e so effective as to d e p r e s s t h e t r a n s m i s s i o n t h r o u g h t h e F R A p a t h w a y totally. I n t h e s e e x p e r i m e n t s it c o u l d also b e d e m o n s t r a t e d t h a t t h e s u p p r e s s i v e effect f r o m m e s e n c e p h a l i c s t i m u l a t i o n c o u l d b e f a c i l i t a t e d n o t o n l y f r o m t h e Tib n e r v e b u t also f r o m t h e SP a n d t h e Su nerves. H o w e v e r ,

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from the proximal nerves (Saph and CF) it was not possible to elicit a facilitation of the mesencephalic suppressive effect. The maximum stimulation of cutaneous nerves was in these experiments 1.5T. The experiments presented in Figure 7 include both FRA facilitation of the flexor monosynaptic reflex and FRA inhibition of the extensor monosynaptic reflex.

Further FRA Pathways Influenced from the MesADC and from Distal Cutaneous Nerves: The pathway from the FRA to primary afferents was studied with the DRP as test. Conditioning stimulation in the MesADC (yielding no DRP in itself) or of a distal cutaneous nerve (Tib, SP or Su) was then found to depress the FRA-evoked DRP, whether it was evoked by high threshold afterents in a muscle or a cutaneous nerve. Very low threshold afferents in the distal cutaneous nerves were responsible for the action on the FRA-DRP, since it could be demonstrated with a stimulation strength of 1.2T. Furthermore, it was revealed that a DLF lesion abolished the influence on the FRA-DRP from the MesADC, but left the influence from the cutaneous nerves unaffected. Thus, the inhibitory action from low threshold cutaneous afferents on to primary afferent terminals seems to rely only on segmental mechanisms, but the descending action is dependent upon an intact DLF, just as shown for the action on the path from the FRA to motor nuclei. Transmission from the FRA to ascending pathways in the spinal cord was studied by recording mass discharge elicited by the FRA in the contralateral (to descending path and peripheral nerve) ventral quadrant. These ascending discharges were depressed by MesADC stimulation as well as by stimulation (down to 1.1T) of the distal cutaneous nerves (Su, SP and Tib). Proximal nerves (Saph and CF) were tested in only one experiment and were then found to be quite ineffective although the distal nerves were effective also in that experiment. The suppressive action from the MesADC and from distal cutaneous nerves on to FRA pathways described in this section was studied with conditioning-test intervals from 30 to 55 ms from first conditioning shock to arrival of the FRA volley at the cord.

Discussion

The primary aim of the present investigation was to define the mesencephalic region from which dorsal reticulospinal effects may be elicited (Baldissera et al., 1972a), and to compare this region with the MesADC. The dorsal reticulospinal system is known to depress excitatory and inhibitory actions evoked by the flexor reflex afferents (FRA) in polysynaptic segmental reflex and ascending paths (Engberg et al., 1968a), presumably by postsynaptic inhibition in the first order FRA interneurones (Engberg et al., 1968b). Due to the fact that near-rubral stimulation may activate both the rubrobulbospinal path (RBSP) and the rubrospinal tract strict functional criteria were used in the present investigation to locate the central stimulating electrodes, and to reveal whether a certain motor system was activated by a central stimulus or not. Concerning the rubrospinal tract, all or some of the following

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T. Jeneskogand H. Johansson

three criteria were used: 1) a descending volley in the contralateral low thoracic cord elicited by single stimulating shocks, and capable of following high frequency stimulation with constant latency and effectiveness (Baldissera et al., 1972b), 2) the typical facilitation of flexor monosynaptic reflexes (Hongo et al., 1969) and 3) a DRP in lumbar segments with low stimulating intensities (Hongo et al., 1972). The absence of these criteria were taken to indicate that the rubrospinal tract was not co-activated by a particular mesencephalic stimulation. To define the region from which the RBSP was activated by electrical stimulation (i.e. the MesADC), we relied on the short latency climbing fibre response that is evoked in the D zone of the contralateral cerebellar cortex parallel to activation of descending path (Jeneskog, 1974a). Single shock stimuli via this electrode should not evoke a descending discharge (Appelberg and Jeneskog, 1969; Baldissera et al., 1972a; Appelberg et al., 1975), because this would indicate co-activation of the rubrospinal tract. The segmental mechanism most extensively studied to demonstrate dorsal reticulospinal effects was the suppression of transmission through the FRA pathway on to motor nuclei. This suppressive action should be dependent upon an intact DLF. It should have a characteristic time course and it should be evoked in the absence of a lower lumbar DRP (Engberg et al., 1968a; Baldissera et al., 1972a). These criteria were considered sufficent for the conclusion that a certain central stimulation elicited dorsal reticulospinal effects. Additional mechanisms studied were the transmission from the FRA to primary afferent terminals and to ascending spinal pathways. These mechanisms are known also to be influenced by the dorsal reticulospinal system (Engberg et al., 1968a). Threshold mappings were performed in electrode tracks through the mesencephalon at the rubral level. These studies confirmed those of Baldissera et al. (1972a) with regard to the finding that dorsal reticulospinal effects may be produced from a region slightly dorsal to the one from which the hind limb component of the rubrospinal tract is activated with moderate current intensities. It was furthermore revealed that the effective region for eliciting dorsal reticulospinal effects totally coincided with the MesADC, i.e. the region from which the D zone rubro-olivocerebellar path and the RBSP (Jeneskog, 1974a) are also activated. This was shown with two different techniques. Firstly, when tracking through the mesencephalon with a constant current strength, the extent of the effective regions for the two effects (D zone climbing fibre response and suppression of segmental FRA influence) was totally coinciding (Fig. 4) and secondly, the points of lowest threshold were at the same depth (Fig. 3). Furthermore, the lowest absolute threshold was the same for the two effecs or differed less than 1:5 ~tA, in these cases with the higher threshold for dorsal reticulospinal effects. This may be explained by the fact that only clear suppressive actions were considered, and thus true threshold effects could probably sometimes be evoked with slightly lower current intensities. The typical descending volley which has been supposed to represent activity in the dorsal reticulospinal system (Baldissera et al., 1972a) was studied less carefully but all available evidence indicates that it was evoked from the same

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depths and with similar thresholds as the D zone climbing fibre response and the suppressive action on the transmission through the F R A pathway. It was furthermore demonstrated that the transmission from the F R A to primary afferent terminals as well as to ascending spinal pathways was also depressed by stimulation in the MesADC. Thus, all F R A mechanisms studied in the present investigation seem to be under inhibitory control from a descending system activated from t h e MesADC. The inhibitory control is presumed to be exerted at an interneuronal level rather than presynaptically, because all the effects may be produced without a detectable D R P in lower lumbar segments. From these results it is tempting to make the interpretation that stimulation of the same elements in the near-rubral region activates not only the RBSP but also the dorsal reticulospinal system. Such an interpretation is also supported by the fact that both paths proceed in the DLF. This interpretation, furthermore, leads to the suggestion that the rubro-bulbospinal path and the dorsal reticulospinalsystem may be identical In that case we are facing a descending motor system capable of, on the one hand, activating dynamic fusimotor neurones and, on the other, inhibiting mechanisms influenced segmentally from the FRA. The previous demonstration that the RBSP activates the same inferior olivary climbing fibre neurones as does the D L F - S O C P (Miller et al., 1969; Jeneskog, 1974a) led us to study whether activation of this latter system could also elicit segmental effects similar to those from the M e s A D C (cf. Introduction). This could be a way to test the hypothesis that a certain climbing fibre path (i.e. a certain group of climbing fibre neurones and the cerebello-cortical zone to which they project) is concerned with a certain motor function (Oscarsson, 1973). The hypothesis would be supported if it could be shown that different stimuli, in this case centrally or peripherally applied, which activate the same climbing fibre neurones also influence the same motor mechanisms in an identical way. The D L F - S O C P is primarily activated by low threshold cutaneous afferents from the distal parts of the limb (Larson et al., 1969) and in the present series low intensity stimulation of the tibial nerve was used most extensively to activate this system, the index of its action being the D zone climbing fibre response. It was then revealed that very low intensity stimulation (down to 1.1 times threshold) of the tibial nerve suppressed the segmental influence of the F R A on the monosynaptic reflex, without affecting the monosynaptic reflex itself. The stimulus intensity required was thus as low as the one needed for activation of the D L F - S O C P (plantaris nerve 1.2T with single shocks, 1.1T with a short train according to Larson et al., 1969). The time course of the tibial nerve influence was the same as that of the descending influence from the MesADC. Is then the tibial nerve mediated suppression of segmental F R A effects caused by postsynaptic inhibition in interneurones, or is it a presynaptic mechanism? This is an important question, because cutaneous afferents are known to give presynaptic inhibition not only to other cutaneous afferents (component I of cutaneously evoked DRPs) but also to the F R A (component II) as demonstrated by Carpenter et al. (1963). However, in the experiments

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where very low intensity stimulation of the tibial nerve was effective, there was only a small component I-DRP evoked and the component II could not be demonstrated in mid-S1 filaments until the strength of stimulation was raised to around 2.0 times threshold. Thus the intensity used for nerve stimulation in the present investigation is considered sufficiently weak not to evoke a segmental influence on the FRA mechanisms by presynaptic inhibition, because such effects should have been revealed with the conditioning-test intervals used. Another explanation for the cutaneous nerve effect could be a postexcitatory depression of the FRA pathway, due to the fact that low threshold cutaneous afferents might "use" the FRA path interneurones and thus leave them in a depressed state. However, such an explanation seems less likely because of the different capacity of the cutaneous nerves tested, the distal ones being highly effective and the proximal ones practically ineffective. Hence, it is assumed that the mechanism behind the peripheral nerve influence is a postsynaptic inhibition via an inhibitory interneurone on to the FRA reflex pathway studied. Furthermore, the other two FRA paths studied in the present investigation, i.e. the transmission from the FRA to primary afferent terminals and to ascending spinal pathways, were likewise depressed by very low threshold tibial nerve stimulation. Thus all FRA influenced mechanisms which could be inhibited by activation of the RBSP could be inhibited also by stimulation of low threshold cutaneous afferents. It was shown that with combined conditioning stimulation in the MesADC and to the tibial nerve the effects upon the FRA reflex path did not summate algebraically but the two conditioning stimuli facilitated each other. This facilitation could be demonstrated both when each conditioning stimulus gave a just supraliminal effect and when they were adjusted to be subliminal when used separately. The cutaneous nerve influence remained after spinalization at L5, thus excluding long spinal or supraspinal reflex loops. It is therefore assumed that the RBSP and low threshold cutaneous afferents from the distal parts of the limb have access to and converge on to common interneurones, which postsynaptically inhibit polysynaptic FRA pathways (Fig. 8). The influence on the inhibitory interneurone from skin afferents is not necessarily monosynaptic although indicated in that way in the Figure (vide infra). Thus, contrary to the view discussed by ten Bruggencate and Lundberg (1974) and by Lundberg (1975) describing the dorsal reticulospinal inhibition as a primary event, it is now proposed, that there may be a convergence from primary afferents and descending motor systems not only directly to motoneurones, but a convergence from certain central and peripheral sources also in the control of reflex paths ultimately reaching the motoneurones. To strengthen the notion that the tibial nerve influence described reflects the ability of tibial nerve stimulation to activate the DLF-SOCP, in three experiments several cutaneous nerves were compared for their possible action on the different FRA paths. The pattern which emerged was that cutaneous afferents from the distal parts of the limb, i.e. the tibial, the superficial peroneal and occasionally the sural, were most effective in producing this inhibition of the FRA paths, while the nerves related to more proximal parts of the limb, i.e. the saphenous and the caudal femoral, were practically ineffective. This pattern is closely similar to the pattern of nerves which may activate the

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