To verify the existence of pathways between corticospinal tract neurons and ipsilateral motoneurons indicated in , all descending tract fibers were transected on one side of the spinal cord, and two experimental approaches were used. The first was to seek evidence for facilitation of the activation of reticulospinal tract fibers by conditioning stimulation of PT fibers. If this facilitation was not seen, it would be unlikely that the reticulospinal neurons projecting to the lumbosacral enlargement on the side opposite to the hemisection are excited by corticospinal neurons. However, depression or enhancement of the reticulospinal volleys would indicate interaction. A depression of the volleys might occur if the axons of RS neurons were discharged after activation by stimulation of PT fibers and made refractory. In contrast, if RS neurons were excited but not discharged by the stimulation of PT fibers, then they should be more excitable to activation through collaterals of MLF fibers, which could be reflected in an enhancement by spatial facilitation of the later components of the descending volleys. Observations related to these problems are presented below.
A second experimental approach was to analyze the effects of conditioning stimulation of PT fibers on disynaptic EPSPs and IPSPs evoked in hindlimb motoneurons by stimulation of axons of reticulospinal tract fibers in the MLF (Jankowska et al., 2003
). These effects are described below.
Evidence for PT actions evoked via RS neurons based on records of descending volleys
Descending volleys evoked by stimulation of the medullary pyramid are usually difficult to see at thoracic and lumbar levels because of dispersion of the action potentials in fibers of different conduction velocities but are more readily recorded in the upper cervical segments (Illert et al., 1977
; Lundberg, 1979
). When recording cervical volleys evoked by a train of PT stimuli at 300–400 Hz, in addition to the direct volleys that appeared ~0.8 msec after the stimuli, longer latency temporally facilitated components were seen (, boxes). Because the differences in the onset latencies of the early and late components were ~1 msec, the later components may be attributed to activation of additional neurons located somewhere between the PT and the recording site in the C3 segment. When submaximal PT stimuli were used, these longer latency volleys started to appear after the second or third stimulus and increased in amplitude after each successive stimulus (), as would be expected for temporarily facilitated synaptic actions. Similar effects were seen in all four experiments in which cervical descending volleys were recorded. Our hypothesis is that the additional neurons mediating the longer latency volleys were RS neurons with axons in MLF, and we sought evidence for this by interacting the volleys evoked by PT stimuli with those evoked by MLF stimulation.
Stimulation of the MLF evokes volleys with early and late components (Jankowska et al., 2003
). The early volleys are evoked by direct activation of descending RS axons and have constant amplitudes after each stimulus of the train. The later components of MLF volleys are considered to reflect transsynaptic activation of RS neurons via other RS neurons (Jankowska et al., 2003
), as indicated in ; consistent with this, their amplitudes increase with successive stimuli (). If stimulation of PT fibers evokes EPSPs in the same RS neurons, then the later components should be enhanced by spatial facilitation when both PT and MLF are stimulated together.
shows that this facilitation was readily seen; the late component evoked by the first MLF stimulus was considerably larger when the MLF stimulus was preceded by a train of conditioning PT stimuli. The facilitation was clear in recordings made at both cervical and thoracic levels. Facilitation was also evoked from the right PT in all four experiments in which it was tested. The effects evoked from the right PT were either as strong () or slightly weaker than those from the left PT.
Figure 4 Facilitation of the late components of MLF descending volleys and of disynaptic EPSPs in motoneurons by stimulation of both left and right PT after elimination of corticospinal tract fibers at C2–C3. A, Descending volleys after stimulation of (more ...)
Late components of the descending volleys recorded at a thoracic level following PT and MLF stimuli might be relayed by brainstem reticulospinal neurons () but might also reflect the activation of spinal neurons, for example long propriospinal tract neurons with cell bodies in the C3 and C4 segments that are coexcited by corticospinal and reticulospinal tract fibers (Illert et al., 1981
). To estimate the relative contributions of spinal and brainstem neurons, the facilitation of the later components of the descending volleys was compared before and after lesions of PT fibers in the upper cervical segments. Because PT fibers run in the most dorsal part of the lateral funiculus at the C2 level, they could be transected while leaving intact the reticulospinal tract fibers in the more ventral part of the lateral funiculus and in the ventral funiculus. These lesions were made following the procedure of Lundberg and collaborators (Illert et al., 1975
; Lundberg, 1979
), enlarged until the direct PT volleys recorded a few millimeters caudal to the lesions were abolished. The lesions were made bilaterally in two experiments. The extent of these lesions in two experiments is shown in , and H
, and the resulting disappearance of PT volleys in the lowest panels (E
). Because the PT descending volleys recorded at the C3–C4 segments are evoked at latencies of <1 msec and are superimposed on fairly large stimulus artifacts, they can be more conveniently estimated from the difference records obtained before and after dorsolateral funiculus (DLF) lesions.
The records in –D show that temporal facilitation of late components of MLF volleys occurred and that spatial facilitation was evoked when a single MLF stimulus was delivered after a train of conditioning stimuli applied within either the left or the right pyramidal tracts. The arrows indicate the late components evoked by single MLF stimuli recorded from the thoracic segments. As in , facilitation from the ipsilateral (left) PT was more potent than facilitation by two or even three MLF stimuli. Facilitation from the contralateral (right) PT was also substantial. The records in were obtained after lesions of the dorsal parts of the lateral funiculi.
In one of the experiments, the effects of the conditioning PT stimuli were tested not only on the descending volleys but also on EPSPs evoked in motoneurons. In all 10 motoneurons recorded intracellularly after DLF lesions, PT stimuli (both ipsilateral and contralateral) continued to facilitate disynaptic EPSPs evoked from MLF (see below), as illustrated in . In this case, the effects have to be relayed via commissural neurons, and to bring these to threshold, a train of two or three MLF stimuli were required. Accordingly, the train of stimuli to the PT was delivered before the final stimulus of a train of stimuli to the MLF, rather than to a single MLF stimulus. Thus, both of these control experiments indicate that facilitatory effects of PT stimuli are associated with actions on RS rather than on propriospinal neurons in the C3–C4 segments or, more generally, on any spinal neurons located caudal to these segments.
In contrast to the late components of the MLF descending volleys, the early components of the volleys were either depressed or unaffected by PT stimuli. The depression can be explained by refractoriness: if collaterals of PT neurons excited reticulospinal neurons with axons in MLF sufficiently strongly to discharge them, they should be refractory to direct activation by MLF stimuli at short conditioning testing intervals. The results of three experiments in which amplitudes of the early and later components of the same MLF volleys were plotted as a function of the intervals between the last PT stimulus of a train and an MLF stimulus are shown in –C and exemplified in . The black symbols in A–C show that the early (i.e., direct) components of the descending MLF volleys recorded at the thoracic and lumbar levels were decreased by approximately one-half at optimal conditioning–testing intervals but did not disappear. The maximal decrease of the thoracic volleys occurred at conditioning–testing intervals of 2–2.5 msec. We estimate that ~1.5–1.8 msec might be needed for RS neurons to be activated by nerve impulses in collaterals of corticospinal tract fibers after PT stimuli (allowing ~0.5 msec for conduction time along these collaterals, 0.3 msec for synaptic delay, and up to 1 msec for the rise time of the asynchronously evoked EPSPs before action potentials are generated) and that the conduction time between the RS neuron somata and the site of application of the stimuli in MLF might be another 0.2 msec. The period during which the early MLF volleys were decreased is thus appropriate to coincide with the refractory periods of the axons of RS neurons. Several factors may explain why the volleys were only partly decreased but not abolished, for instance if the activation of reticulospinal neurons by PT stimulation was asynchronous, or only a fraction of RS neurons was activated (and therefore refractory), or if corticospinal neurons might provide input to only a proportion of the RS neurons that have axons in the MLF. The proportionally smaller decreases of the earliest components of MLF volleys recorded at a cervical level (compare simultaneously recorded thoracic and cervical volleys in ) suggest that a smaller proportion of the RS neurons that terminate within the cervical or upper thoracic segments is discharged by corticospinal neurons.
Figure 5 Effects of a stimulation of the left PT on descending volleys evoked by subsequent stimulation of the MLF. A and B plot the time course of changes in peak-to-peak amplitudes of volleys evoked by a single stimulus (or the first of a train) applied in the (more ...)
If the facilitation of the late components of MLF volleys is attributable to PT actions on RS neurons, then they ought to be reduced during the expected refractory period after activation of RS neurons like the early components. This was indeed the case, as indicated by the gray data points in , and D. The onset of the depression of the late components was sharper, if not earlier, than the onset of the depression of the early components. The degree of depression was likewise similar, to approximately one-half of control amplitude.
Although these results are generally in keeping with activation of RS neurons by PT fibers, they are less clear cut than the results of similar tests on the activation of vestibulospinal neurons by stimuli applied in the cerebellum (Matsuyama and Jankowska, 2004
) where the experimental situation was much simpler. The main complicating factor in the present study was that activation of RS neurons by PT fibers was relatively weak. Accordingly, a train of PT stimuli was needed, and the effects of the final PT stimulus of a train were often superimposed on weak depressive (on direct volleys) and stronger facilitatory (on later volleys) effects of the penultimate stimulus. For the early components of the volleys, this resulted in two periods of depression related to the refractory periods after the third and fourth stimuli of the PT train, as indicated in –C
by open and filled symbols, respectively.
Evidence for ipsilateral PT actions evoked via RS neurons and commissural lamina VIII interneurons based on intracellular records from motoneurons
Reticulospinal tract neurons evoke disynaptic EPSPs and IPSPs via commissural neurons in a particularly high proportion of contralateral GS and Sart motoneurons, respectively (Jankowska et al., 2003
). Intracellular recordings were therefore sought principally from GS and Sart motoneurons but also included other motoneuron types to test whether any PSPs evoked in them from MLF could be facilitated by PT stimuli. The experimental arrangement was as outlined in , and intracellular recordings were made from motoneurons ipsilateral to the spinal hemisection. Disynaptic PSPs were evoked by two or three MLF stimuli and, after adjusting the intensity of these stimuli such that at least the first one was sub- or near-threshold, were interacted with PT stimuli. Trains of PT stimuli were used for conditioning because, as shown above, two to four stimuli were needed to facilitate synaptic activation of reticulospinal neurons. The EPSPs and IPSPs in motoneurons were the result of spatial and temporal facilitation at the level of both reticulospinal and spinal commissural interneurons.
Potent facilitation of EPSPs of MLF origin could be evoked from both the left and right pyramids, as illustrated in . The effects of PT stimuli were in this case tested on EPSPs evoked at such a low stimulus intensity (25 μA) () that they showed marginal temporal facilitation. Distinct temporal facilitation was evoked when stronger MLF stimuli (50 μA) () were used. Conditioning stimulation of the left or the right PT coupled with MLF stimulation evoked EPSPs that were greatly facilitated (), exceeding the amplitude of those evoked by a train to the MLF at 50 μA. Because the PT stimuli at this intensity did not evoke any PSPs when delivered alone (), facilitation must have occurred at a premotoneuronal level.
Figure 6 Facilitation of disynaptic EPSPs evoked from the MLF by conditioning stimulation of the pyramids. Top records in each panel are intracellular potentials from a GS motoneuron on the left side of the spinal cord; the bottom records are from cord dorsum. (more ...)
In order for facilitation to occur, several conditions had to be fulfilled. Facilitation was found when the test EPSPs were near threshold and, as illustrated in , was much more potent when trains of four (rather than three) PT stimuli were used. The frequency of the train of PT stimuli was critical, because facilitation was weak with trains at 200 Hz but was more effective with trains at 300 or 400 Hz. The difference between effects of 200 and 300 Hz stimuli is illustrated in . Finally, the time intervals between the final stimuli of the trains to the PT and MLF had to be appropriate.
Figure 7 Parameters of PT stimulation needed for effective facilitation of synaptic actions evoked from MLF. The top records in each panel are from a GS motoneuron on the left side of the spinal cord. The bottom records are from the cord dorsum. A–C, Small (more ...)
Two example plots of the time course of facilitation of the EPSPs are shown in . The most consistent feature of these plots was that the peak of the facilitation occurred at an interval of ~5 msec between the final stimulus of the train to the PT and the MLF stimulus (as in ). The peak effect thus occurred ~1 msec later than the peak of facilitation of the MLF volleys that are illustrated in . The precise onset of the facilitation was difficult to define, even in the plots with the sharpest rising slopes (as in ). It began at conditioning–testing intervals of ~2–3 msec (i.e., closely following the time of onset of the facilitation of MLF volleys). Failure of facilitation, which would correspond to the refractory period (at intervals 1.5–2.5 msec) in the plots of the MLF volleys in , was not seen. However, some facilitation could have been induced by the third as well as the fourth PT stimulus, which might mask any effects of the refractory period after the fourth stimulus. The time courses of facilitation of EPSPs recorded in nine other motoneurons and of IPSPs recorded in another motoneuron were similar.
Figure 8 Time course of facilitation of disynaptic PSPs evoked from MLF by stimulation of pyramidal tract fibers. A, B, Time course of facilitation of EPSPs evoked in GS motoneurons on the left side of the spinal cord after stimulation of the left and right pyramids (more ...)
Using the optimal stimulus parameters and intervals (4–7 msec) between conditioning stimuli applied within the left (ipsilateral) pyramid and test stimuli to the right (contralateral) MLF, facilitation of disynaptic EPSPs could be demonstrated in all motoneurons tested. These included 15 GS, 5 Q, 1 PBST, 1 Sart, and 3 unidentified motoneurons. The facilitated EPSPs evoked by the third MLF stimulus of a train, measured from the area within a time window of 1.5–2.0 msec from the onset, had amplitudes up to 8–10 times larger than the areas of test EPSPs. The mean increase was 3.04 ± 0.38 times (mean ± SEM; n = 25) (). Conditioning stimulation of the right (contralateral) pyramid produced a similar degree of facilitation (increasing EPSPs evoked in the same 25 motoneurons 2.75 ± 0.38 times). Disynaptic EPSPs were also facilitated in 10 additional motoneurons recorded after lesions of the descending corticospinal tract fibers between the C2 and C3 segments, indicating that the facilitation occurred supraspinally. Records from one of these neurons are illustrated in –H.
Figure 9 Comparison of the effectiveness of facilitation of PSPs evoked from the right MLF in motoneurons on the left (A) and right (B) side of the spinal cord by conditioning stimulation of the left and right PT. The bars represent the mean amount of facilitation (more ...)
The records in show that IPSPs evoked in motoneurons by the MLF stimuli were also facilitated by ipsilateral PT stimuli. In this case, a train of three near-maximal stimuli to the MLF evoked small disynaptic IPSPs (), but distinct larger IPSPs appeared on joint stimulation of the MLF and a train of stimuli to the left or right PT at 150 μA (), whereas PT stimuli by themselves were ineffective (). Facilitation was found in all of the 16 motoneurons tested, including 2 GS, 3 Q, and 11 Sart motoneurons. On average, the facilitated IPSPs were 4.83 ± 0.87 times larger than the test IPSPs (). The effects of stimulation of the left and right pyramids were compared in six of these motoneurons. There were no significant differences between the effects evoked from left and right side, although there was a tendency for the IPSPs that followed stimulation of the ipsilateral (in this case, left) PT to be somewhat larger ().
Facilitation of disynaptic IPSPs evoked from MLF by conditioning stimulation of pyramids. Top records in each panel are from a GS motoneuron on the left side. Bottom records are from cord dorsum. Otherwise, the format is the same as in .
Evidence for ipsilateral PT actions involving RS neurons but not commissural lamina interneurons
Reticulospinal tract neurons may act on various populations of spinal interneurons in addition to lamina VIII commissural interneurons, some of which are premotor interneurons that act on motoneurons located on the same side of the spinal cord (Takakusaki et al., 1989
; Davies and Edgley, 1994
). Another possible route for ipsilateral actions evoked by corticospinal neurons thus might be via ipsilaterally projecting RS neurons and ipsilaterally projecting interneurons. To explore this possibility, the effects of stimulation of the right pyramid were tested on 26 motoneurons located on the right side of the spinal cord in preparations in which the spinal cord was hemisected on the left side (the same preparations in which the effects from the left pyramids described above were investigated, as indicated in the diagram of ). Under these conditions, facilitation of disynaptic EPSPs and IPSPs evoked by MLF stimulation was more difficult to estimate because the MLF stimuli sometimes evoked monosynaptic EPSPs in motoneurons (as in the records in –C
). However, in motoneurons in which they were distinct, disynaptic EPSPs (n
= 6) and IPSPs (n
= 9) were increased 1.13 ± 0.13 and 2.10 ± 0.45 times, respectively, by conditioning stimulation of the right (ipsilateral) PT. The degree of facilitation of EPSPs and IPSPs mediated by ipsilateral premotor interneurons thus appeared to be somewhat weaker than the facilitation of PSPs mediated by commissural interneurons (, compare A
Figure 11 Facilitation of disynaptic EPSPs and IPSPs in motoneurons on the right side of the spinal cord. Top records in each panel are intracellular records from a motoneuron on the right side of the cord. A–C, From a GS motoneuron; D–F, from a (more ...)
Although facilitation of disynaptic EPSPs by conditioning stimulation of the left and right PT was similar in motoneurons on the right (), facilitation of disynaptic IPSPs was much more potent from the left PT than from the right PT (). Thus, stimulation of corticospinal tract fibers from the left PT (which descended on the intact right side of the spinal cord) increased these IPSPs more than 10-fold (, ), and a strong effect was seen in almost all motoneurons. In contrast, these IPSPs were facilitated to about the same extent as the disynaptic EPSPs by stimulation of the right PT. The asymmetry in these observations also provides evidence that the effects of stimuli that we used may be attributed to the fibers in the stimulated pyramid, even if stronger stimuli might have activated some fibers on the opposite side.