1Department of Integrative Medical Biology, Umeå University, S-901 87 Umeå, Sweden; 2Department of Integrative Physiology, National Institute for Physiological Sciences, Myodaiji, Okazaki 444-8585; and 3Department of Physiology, Kyorin University School of Medicine, Shinkawa, Mitaka, Tokyo 181-8611, Japan
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ABSTRACT |
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Alstermark, B., T. Isa, Y. Ohki, and Y. Saito. Disynaptic Pyramidal Excitation in Forelimb Motoneurons Mediated Via C3-C4 Propriospinal Neurons in the Macaca fuscata. J. Neurophysiol. 82: 3580-3585, 1999. In contrast to findings in the cat, it recently has been shown that disynaptic pyramidal EPSPs only rarely are observed in forelimb motoneurons of the macaque monkey in the intact spinal cord or after a corticospinal transection in C5. This finding has been taken to indicate that the disynaptic pyramidal excitatory pathway via C3-C4 propriospinal neurons (PNs) is weakened through phylogeny when the monosynaptic cortico-motoneuronal connection has been strengthened. We reinvestigate this issue with special focus on the possibility that the inhibitory control of the C3-C4 PNs may be stronger in the macaque monkey than in the cat. The effect in forelimb motoneurons of electrical stimulation in the contralateral pyramid was investigated in anesthetized macaque monkeys (Macaca fuscata). We confirmed the low frequency of disynaptic pyramidal EPSPs in forelimb motoneurons. However, after intravenous injection of strychnine, disynaptic EPSPs could be evoked in 39 of 41 forelimb motoneurons recorded after lesion of the corticospinal fibers in C5. After a corresponding lesion in C2, disynaptic pyramidal EPSPs were observed in 2 of 25 motoneurons. In contrast to previous reports, we conclude that C3-C4 PNs can mediate disynaptic pyramidal excitation in high frequency of occurrence to forelimb motoneurons in the C6-C8 segments and that this transmission is under a stronger inhibitory control than in the cat. Thus, the hypothesis that the disynaptic excitatory cortico-motoneuronal pathway via the C3-C4 PNs is weakened in parallel with the strengthened monosynaptic connection through phylogeny is not supported by the present findings.
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INTRODUCTION |
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In the cat, Illert et al. (1977)
first demonstrated that disynaptic pyramidal excitation in forelimb
motoneurons can be mediated via spinal interneurons with cell bodies
located in the C3-C4 segments (cf. also Alstermark and Sasaki 1985
;
Illert and Wiedemann 1984
). This premotoneuronal system
was denoted the C3-C4
propriospinal system and it has since been analyzed in detail (cf.
Alstermark and Lundberg 1992
). In behavioral studies
using either selective spinal cord lesions or transneuronal labeling,
it was shown that C3-C4
propriospinal neurons (PNs) can mediate the descending command for
forelimb target reaching (Alstermark and Kümmel
1990
; Alstermark et al. 1981
). So far the
C3-C4 propriospinal system
is unique because it is the only example of a command mediating
interneuronal system in the spinal cord for voluntary movements.
In primates, interest has focused on the monosynaptic corticospinal
pathway to motoneurons, which is lacking in the cat (cf. Phillips and Porter 1977). Therefore it was of
considerable interest when non-monosynaptic excitation was first
demonstrated in man (Baldissera and Pierrot-Deseilligny
1989
; Malmgren and Pierrot-Deseilligny 1988
).
However, the interpretation by Gracies et al. (1994)
that disynaptic pyramidal excitation can be mediated by PNs located rostral to the forelimb segments has been criticized strongly by
Maier et al. (1998)
. In the macaque monkey, Maier
et al. (1998)
found that electrical stimulation in the
contralateral pyramid evoked disynaptic excitatory postsynaptic
potentials (pyramidal EPSPs) only rarely compared with the cat. They
concluded that their "results provide little evidence for
significant corticospinal excitation of motoneurons via a system of
C3-C4 propriospinal neurons in the monkey."
It is interesting that Bortoff and Strick (1993), who
studied the corticospinal termination in the Cebus monkey (which is phylogenetically more advanced than the macaque monkey) found a heavy
termination laterally in Rexeds laminae VI-VII in the C3-C4 segments exactly
where the C3-C4 PNs are
located in the cat (Illert et al. 1978
). Bortoff
and Strick (1993)
carefully stated that "Although
as much as 30% of the corticospinal input terminates in the ventral
horn of the Cebus monkey, it is clear that the majority of
corticospinal terminations are located within the intermediate zone of
the spinal cord... . Thus, the importance of corticospinal
projections to interneurons should not be underemphasized."
We have reinvestigated whether disynaptic pyramidal excitation in forelimb motoneurons can be mediated via C3-C4 PNs in the macaque monkey. It will be shown that this interneuronal system also exists in primates and that the inhibitory control of the C3-C4 PNs is stronger than in the cat.
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METHODS |
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Preparation
Experiments were performed on four macaque monkeys (Macaca
fuscata) with body weights 3-5 kg. The animals were sedated with 0.1 ml/kg Xylazine followed by initial anesthesia with 0.2 ml/kg Ketamine, 0.5-1.0% halothane during surgery, and later up to 100 mg/kg -chloralose. The anesthesia was regularly supplemented with
small doses of ~0.5 mg/kg pentobarbital sodium (Nembutal) to maintain
a stable anesthetic depth (regular respiration and blood pressure ~80
mmHg). During recording, pancuronium bromide (Myoblock) was introduced
and artificial respiration started. Respiratory rate was adjusted to
keep pCO2 ~3.8%. In addition, i.v. injections with
Ringer-glucose including lactate, atropine, and prednisolon were given
to maintain a good general condition of the animals. Rectal temperature
was maintained at ~37°C. The deep radial (DR), median (Med), and
ulnar (Uln) nerves were dissected and mounted on silver electrodes or
burried electrodes for stimulation. Laminectomy was performed to expose
the spinal cord segments C2-T1. Craniotomy was
performed to place the pyramidal electrode. The experiments were
approved by the ethical committee of the National Institute for the
Physiological Sciences, Japan and were performed in accordance with the
NIH guideline for the Care and Use of Laboratory Animals.
Recording and stimulation
In this study we focused on the pyramidal excitation in forelimb
motoneurons in the C6-C8
segments. Of the 85 motoneurons recorded, 35 were DR, 8 Med, 1 Uln, and
41 unidentified. The antidromically identified motoneurons had spike
amplitudes 60-100 mV. Unidentified motoneurons with low input
resistance <2 M and spike heights of
60 mV during depolarization
were found in the vicinity of the DR, Med, and Uln motor nuclei defined
by the antidromic field potentials. Furthermore, some unidentified
motoneurons received heteronymous Ia EPSPs from the DR, Med, or Uln nerves.
The effect of electrical stimulation of the corticospinal fibers in the
contralateral pyramid was investigated by intracellular recordings from
19 forelimb motoneurons (5 DR and 14 unidentified) in the intact spinal
cord, 41 (21 DR, 2 Med, and 18 unidentified) after a
C5 lesion (cf. below), and 25 motoneurons (9 DR,
6 Med, 1 Uln, and 9 unidentified) after a C2 lesion (cf.
below). Intracellular recordings were made with sharp glass
microelectrodes with tip diameters of 1-2 µm filled with 2 M
K-citrate with impedances 3-5 M. Cord dorsum recordings were made
with a silver ball electrode at the dorsal root entry zone in the same
segment as the intracellular recordings. The corticospinal axons were
stimulated electrically in the contralateral pyramid, at the medullary
level, using cathodal pulses (0.2 ms duration; 100-200 µA strength).
Strychnine injection
Intravenous injections of strychnine (1 mg/ml) were regularly administered during intracellular recordings; the standard dose was 0.1 mg/kg. Approximately twenty injections of strychnine were made during one experiment that lasted ~18-20 h. The general condition of the animal remained good although the blood pressure increased transiently for ~2 min after a single injection.
Spinal cord lesions
The corticospinal lesion was evaluated by recording the
descending pyramidal volley with a cord dorsum electrode (cf.
Illert et al. 1977). The direct pyramidal volley can be
seen in Fig. 2B1. After the C5 lesion
of the dorsal part of the lateral funiculus, the major part of the
negativity of the pyramidal volley was abolished (Fig. 2B2).
The histological control is shown in Fig. 2A. We
intentionally spared the ventral part of the lateral funiculus because
the axons of the C3-C4 PNs
are known to be located in this region in the cat (Illert et al.
1978
). Some corticospinal fibers with direct projection to
motoneurons are located in this region (Bortoff and Strick
1993
). Note that part of the synaptic volley (Fig. 2B1, arrow) remained after the C5
lesion (B2). It is likely that part of this synaptic volley
is due to activation of the
C3-C4 PNs (cf.
DISCUSSION). The C2 lesion was made
in the same manner as the C5 lesion and is
illustrated in Fig. 3A. The direct pyramidal volley (Fig.
3B1) was completely abolished (Fig. 3B2). This
lesion was made in the monkeys that had the C5
lesion and after repeated strychnine injections. Measurements of
segmental latencies were made from the direct pyramidal volley recorded
before lesions.
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RESULTS |
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Intact spinal cord before and after strychnine injections
Figure 1A shows that
stimulation in the contralateral pyramid evoked monosynaptic EPSPs that
were followed by IPSPs. Monosynaptic EPSPs were found in 16 (84%) of
19 motoneurons (Fig. 1H, open bars). These observations
confirm the results from previous investigations of the monkey and
baboon by Landgren et al. (1962), Shapovalov (1975)
, Fritz et al. (1985)
, Jankowska et
al. (1976)
and Maier et al. (1998)
.
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In order to test whether the low frequency of disynaptic and oligosynaptic pyramidal EPSPs was caused by inhibition at a premotoneuronal level, we systematically gave intravenous injections of strychnine (cf. METHODS). Figure 1B shows the effect 40 s after the strychnine injection. Note that the monosynaptic pyramidal EPSPs appeared without the addition of di- and oligosynaptic EPSPs. Thus, removing glycinergic inhibition at the motoneuronal level did not reveal di- or oligosynaptic EPSPs. This finding shows that lack of di- and oligosynaptic pyramidal EPSPs was not caused by an underlying inhibition in forelimb motoneurons. However, 120 s after the strychnine injection, di- and oligosynaptic EPSPs appeared along with the monosynaptic EPSPs (Fig. 1C). The result of averaging the recordings in Fig. 1, B and C is illustrated in Fig. 1D1 and the subtracted average Fig. 1, C minus B is shown in Fig. 1D2. Note that the segmental latency was in the disynaptic range (1.6 ms; D2, arrow). Temporal facilitation was necessary to elicit di- and oligosynaptic pyramidal EPSPs as shown in the sequential recordings (Fig. 1, E-G).
Recordings from another forelimb motoneuron before and after strychnine injection are illustrated in Fig. 1, I-L. Mainly monosynaptic pyramidal EPSPs were found before the strychnine injection (Fig. 1I). However, 60 s after the injection oligosynaptic EPSPs components were evoked (Fig. 1J). The quite pronounced oligosynaptic pyramidal EPSPs compared with the monosynaptic EPSPs are shown with a slower sweep speed (Fig. 1K). We also observed a recovery from the effect of strychnine (Fig. 1L). In this case, the pyramidal EPSPs were reduced after 3 min. However, such clear recovery was only apparent after the first few injections, thereafter there was an accumulating effect of strychnine.
No oligosynaptic pyramidal EPSPs were found in the seven forelimb
motoneurons that were recorded before the strychnine injection. This
result is in agreement with that of Maier et al. (1998)
who found oligosynaptic EPSPs only in a small percentage of the
motoneurons. However, after strychnine injections oligosynaptic
pyramidal EPSPs were observed in all 15 tested cells. In nine of these
motoneurons the EPSPs were within a disynaptic range (1.2-1.8 ms) and
the remaining six EPSPs were either tri- or polysynaptic in nature (Fig. 1H, closed bars).
Note the synaptic volley (marked by arrows) that appeared after direct corticospinal volley (after 3rd and 4th stimuli; Fig. 1, A-C and I-L). This synaptic volley was enhanced after strychnine injection (Fig. 1, C and J) and declined after recovery (Fig. 1L).
Segmental location of interneurons mediating di- and oligosynaptic pyramidal EPSPs
In order to test whether the disynaptic pyramidal EPSPs can be
mediated via C3-C4 PNs as
in the cat (Illert et al. 1977), we first transected the
corticospinal tract at the
C4/C5 segmental border,
denoted as C5 lesion (cf. METHODS).
Figure 2, C-F illustrates a motoneuron recorded after the C5 lesion. Eleven strychnine injections were given before recording this motoneuron. As seen in Fig. 2C, already a single pyramidal stimulus evoked small EPSPs which were markedly facilitated by the second (Fig. 2D) and third (Fig. 2E) pyramidal stimuli. The segmental latency of this pyramidal EPSP was 1.3-1.4 ms as observed in the expanded records taken with higher sweep speed in Fig. 2F. The result suggests a strong disynaptic linkage from the corticospinal tract.
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Figure 2G shows that the effects from the contralateral
pyramid were not due to current spread. The stimulating electrode was
positioned somewhat laterally in the pyramid to avoid spread to the
ipsilateral pyramid (cf. Illert et al. 1977). Disynaptic pyramidal EPSPs were evoked when the stimulating electrode was in the
contralateral pyramid (Fig. 2, G1 and G2) but
disappeared just above it (Fig. 2, G3 and G4).
Thus the disynaptic and oligosynaptic pyramidal EPSPs found in the
forelimb motoneurons were the result of stimulation of corticospinal
axons in the contralateral pyramid. However, more dorsally in the most
ventral part of the reticular formation, another oligosynaptic EPSP
appeared in records (Fig. 2, G5 and G6). These
EPSPs may be mediated via bulbospinal neurons and/or spinal interneurons.
Our findings with pyramidal stimulation after a
C5 lesion are in agreement with the findings by
Maier et al. (1998) that di- and oligosynaptic pyramidal
EPSPs could still be elicited. However, our results differ radically
from those of Maier et al. (1998)
with respect to the
frequency of occurrence. Whereas they found that such pyramidal EPSPs
could only be evoked in 18% (14% in a disynaptic range), we found
oligosynaptic pyramidal EPSPs in all 41 (100%) investigated forelimb
motoneurons after strychnine injection. In 39 (95%) of 41 motoneurons,
the pyramidal EPSPs were within a disynaptic range (Fig.
2H). We found three motoneurons with remaining monosynaptic
pyramidal EPSPs in one experiment (Fig. 2H, open bars) when
the lesion did not extend into the ventrolateral part of the lateral
funiculus (Fig. 2A). In the experiments with more
ventrolateral extension of the C5 lesion, we
found no monosynaptic pyramidal EPSPs, but di- and oligosynaptic EPSPs
could still be evoked.
The second step in localizing the intercalated neurons mediating the
di- and oligosynaptic pyramidal EPSPs was to transect the corticospinal
axons at the C2/C3
segmental border (cf. METHODS) as was done in the cat
(Illert et al. 1977). After complete transection of the
corticospinal axons in C2 as shown for the
pyramidal volley in Fig. 3B
and the histology in Fig. 3A, we observed disynaptic pyramidal EPSPs in 2 (8%) of 25 motoneurons and EPSPs with segmental latencies longer than 2.5 ms in 11 (44%) cells as shown in Fig. 3G. All motoneurons after the C2
corticospinal lesion were recorded after repeated strychnine
injections. The results from one of the recorded forelimb motoneurons,
after the C2 lesion, is illustrated in Fig. 3,
C-F taken at fast (Fig. 3, C1-F1) and slow
(Fig. 3, C2-F2) sweep speeds. Note the absence of short
latency pyramidal EPSPs and the presence of oligosynaptic EPSPs with
latencies about 4-5 ms (Fig. 3F1, arrow). These pyramidal
EPSPs were followed by a mixture of late EPSPs and IPSPs, which
probably reflects a high excitability after repeated strychnine
injections.
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From these results we conclude that disynaptic pyramidal EPSPs in forelimb motoneurons can be effectively mediated via C3-C4 PNs in the Macaca fuscata provided that inhibition of this premotoneuronal system is reduced. However, we tentatively propose that disynaptic pyramidal EPSPs can sometimes be mediated via bulbospinal neurons projecting directly to forelimb motoneurons.
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DISCUSSION |
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Our results leave no doubt that disynaptic pyramidal EPSPs in
forelimb motoneurons can be mediated via
C3-C4 PNs after the C5 corticospinal lesion and after strychnine
injection. We cannot exclude the possibility that strychnine removes
tonic inhibition of the
C3-C4 PNs, but it seems
more likely that the appearance of EPSPs via the
C3-C4 PNs is the result of
the reduction of pyramidal IPSPs in them. In cats, disynaptic IPSPs are
evoked in the C3-C4 PNs
via two different pathways: 1) the feed-forward pathway
which is excited from the same descending pathways as the
C3-C4 PNs (Alstermark et al. 1984a) and 2) the
feed-back pathway which is activated from forelimb afferents and the
corticospinal tract (Alstermark et al. 1984b
). The
effect of strychnine may be due to removal of IPSPs from either or both
of these pathways. It is tempting to suggest that the stronger
inhibition in primates compared with that in cats should be ascribed to
the feedforward pathway and that it reflects the need for a more
focused excitatory control of the C3-C4 PNs in
the primates. However, we can by no means exclude that the feedback
inhibitory pathway is also involved.
The axonal location of the
C3-C4 PNs in the macaque
monkey is not known. We have assumed that they at least partly have a similar location in the ventral part of the lateral funiculus as has
been shown in the cat (Illert et al. 1978). However,
because we observed a reduction of the synaptic pyramidal volley (Fig. 2B2), presumably mediated via the
C3-C4 PNs, it cannot be
excluded that some of the propriospinal axons in the macaque monkey
have a somewhat more dorsal location than the cat. Our findings are in
contradiction with those of Maier et al. (1998)
who did
not observe a propriospinal volley after a C5
corticospinal transection.
One possible difference is that the
C3-C4 PNs were strongly
inhibited in the study by Maier et al. (1998) and thus
could not mediate the synaptic pyramidal volley. Further investigation
is necessary to find the detailed axonal location of the
C3-C4 PNs with projection
to forelimb motoneurons.
Let us consider the general hypothesis regarding a phylogenetical
replacement of the C3-C4
propriospinal system by monosynaptic cortico-motoneuronal excitation
(Lemon 1999; Maier et al. 1996 and 1998
).
Their common theme is that as the monosynaptic cortico-motoneuronal excitation gradually becomes stronger through phylogenesis because of
increased need for control of dextrous finger movements, the phylogenetically older
C3-C4 propriospinal system
should become weakened and vanish in higher monkeys and man. The
evidence is not only the findings by Maier et al.
(1998)
, which we now have explained, but also that of
Maier et al. (1997)
and Nakajima et al.
(1999)
who found that disynaptic pyramidal EPSPs are more frequent in the squirrel monkey, which has less advanced finger movements compared with the macaque monkey. Maier et al.
(1998)
conclude that these EPSPs are mediated via
C3-C4 PNs even if they have not made the necessary C2 lesion.
Furthermore, Lemon (1999) suggested that
"the positive correlation across species between more
advanced hand function and the strength of the cortico-motoneuronal
system is accompanied by a negative correlation between this function
and the strength of the PN system." This is a surprising
hypothesis because the behavioral work on the
C3-C4 propriospinal system
has shown that it can mediate the command for target reaching and is
not conveying the command for food-taking which involves digit
movements, wrist flexion, and supination. Therefore it is not logical
that this particular system would need to be replaced with the
monosynaptic cortico-motoneuronal connections, which, as is generally
accepted, are of special importance for dextrous finger movements.
Recently, Lemon (1999) suggested that the studies by
Pierrot-Deseilligny (1996) on a propriospinal system in
man "may need to be reevaluated to obtain a better understanding of
corticospinal control in man." Because our results
demonstrate the existence of C3-C4 PNs in
primates, the objective by Lemon (1999)
regarding Pierrot-Deseilligny's conclusion is removed. Thus, it now seems that
the need for such a reevaluation is not so urgent.
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ACKNOWLEDGMENTS |
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We thank Prof. Anders Lundberg and Dr. Lars-Gunnar Pettersson for constructive criticism on the first version of this paper. Furthermore, we thank Dr. H. Aizawa for valuable discussion and M. Seo, C. Suzuki, Y. Takeshima, and J. Yamamoto for technical assistance.
This work was supported by Grants 08458266, 08279207, and 09268238 from the Ministry of Education, Science, Sports and Culture of Japan, as well as grants from CREST of the Japan Science and Technology Corporation, the Daiko Foundation, the Naito Memorial Foundation, and the Mitsubishi Foundation to T. Isa.
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FOOTNOTES |
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Address reprint requests to B. Alstermark.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 13 July 1999; accepted in final form 7 September 1999.
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REFERENCES |
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