Department of Physiology, School of Medicine, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113, Japan
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ABSTRACT |
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Izawa, Y., Y. Sugiuchi, and Y. Shinoda. Neural organization from the superior colliculus to motoneurons in the horizontal oculomotor system of the cat. The neural organization of the superior colliculus (SC) projection to horizontal ocular motoneurons was analyzed in anesthetized cats using intracellular recording and transneuronal labeling. Intracellular responses to SC stimulation were analyzed in lateral rectus (LR) and medial rectus (MR) motoneurons and internuclear neurons in the abducens nucleus (AINs). LR motoneurons and AINs received excitation from the contralateral SC and inhibition from the ipsilateral SC. The shortest excitation (0.9-1.9 ms) and inhibition (1.4-2.4 ms) were mainly disynaptic from the SC and were followed by tri- and polysynaptic responses evoked with increasing stimuli or intensity. All MR motoneurons received excitation from the ipsilateral SC, whereas none of them received any short-latency inhibition from the contralateral SC, but some received excitation. The latency of the ipsilateral excitation in MR motoneurons (1.7-2.8 ms) suggested that this excitation was trisynaptic via contralateral AINs, because conditioning SC stimulation spatially facilitated trisynaptic excitation from the ipsilateral vestibular nerve. To locate interneurons mediating the disynaptic SC inputs to LR motoneurons, last-order premotor neurons were labeled transneuronally after injecting wheat germ agglutinin-conjugated horseradish peroxidase into the abducens nerve, and tectoreticular axon terminals were labeled after injecting dextran-biotin into the ipsilateral or contralateral SC in the same preparations. Transneuronally labeled neurons were mainly distributed ipsilaterally in the paramedian pontine reticular formation (PPRF) rostral to retrogradely labeled LR motoneurons and the vestibular nuclei, and contralaterally in the paramedian pontomedullary reticular formation (PPMRF) caudomedial to the abducens nucleus and the vestibular nuclei. Among the last-order premotor neuron areas, orthogradely labeled tectoreticular axon terminals were observed only in the PPRF and the PPMRF contralateral to the injected SC and seemed to make direct contacts with many of the labeled last-order premotor neurons in the PPRF and the PPMRF. These morphological results confirmed that the main excitatory and inhibitory connections from the SC to LR motoneurons are disynaptic and that the PPRF neurons that receive tectoreticular axon terminals from the contralateral SC terminate on ipsilateral LR motoneurons, whereas the PPMRF neurons that receive tectoreticular axon terminals from the contralateral SC terminate on contralateral LR motoneurons.
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INTRODUCTION |
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The superior colliculus (SC) plays an important role
in generation of saccades (Sparks and Hartwich-Young
1989). Electrical stimulation of the SC evokes sacccades with a
contraversive horizontal component (Guitton et al. 1980
;
Robinson 1972
), and neurons in the deeper layers of the
SC show a burst of spikes before contraversive saccades
(Schiller and Körner 1971
; Wurtz and
Goldberg 1972
). The SC contains neurons that send their axons
to a region of the reticular formation (Grantyn and Grantyn
1982
; Guitton and Munoz 1991
; Moschovakis
et al. 1998
; Scudder et al. 1996
) whose
stimulation produces, and whose lesion eliminates, horizontal conjugate
eye movements (Büttner-Ennever and Büttner 1988
; Cohen et
al. 1968
). The same region contains medium-lead burst neurons (MLBNs),
which burst before and during horizontal saccades. These MLBNs include both excitatory neurons (Cohen and Henn 1972
;
Keller 1974
; Luschei and Fuchs 1972
) and
inhibitory neurons (Hikosaka and Kawakami 1977
;
Yoshida et al. 1982
), and they project to the abducens
nucleus (Strassman et al. 1986
; Yoshida et al.
1982
).
Raybourn and Keller (1977) showed that long-lead burst
neurons (LLBNs) of the paramedian pontine reticular formation (PPRF) are activated in response to single shock stimulation of the SC, whereas MLBNs located in the same area are not. Their results suggest
that the tectoabducens connections are at least trisynaptic. Supporting
this finding, the connection from the SC to MLBNs was estimated as
disynaptic by measuring the latency of saccades evoked in response to
stimulation of the SC during natural saccades (Miyashita and
Hikosaka 1996
).
Precht et al. (1974) first analyzed the synaptic linkage
between the SC and lateral rectus (LR) motoneurons, using intracellular recording, and showed that LR motoneurons received excitation from the
contralateral SC and inhibition from the ipsilateral SC, and that the
tectoabducens connections were predominantly trisynaptic with some
disynaptic components. Later, Grantyn and Grantyn (1976)
reanalyzed tectal inputs to LR motoneurons and reported that the
excitation from the contralateral SC to LR motoneurons was mainly
disynaptic, whereas the inhibition from the ipsilateral SC was mainly trisynaptic.
Details of the neural pathways from the SC to medial rectus (MR)
motoneurons have not yet been identified. Grantyn and Berthoz (1977) reported that MR motoneurons received reciprocal inputs, excitation from the ipsilateral SC, and inhibition from the
contralateral SC and suggested that the excitatory pathway from the
ipsilateral SC to MR motoneurons was disynaptic whereas the inhibitory
pathway from the contralateral SC contained at least one additional
interneuron. Neurons that might participate in the relay of signals
from the SC to MR motoneurons are abducens internuclear neurons (AINs), which were shown to convey a horizontal canal input to MR motoneurons (Baker and Highstein 1975
; Furuya and Markham
1981
; Highstein and Baker 1978
). Despite the
numerous studies to elaborate the neural organization of saccade
generating mechanisms in the brain stem, the exact tectoabducens
connections still remain controversial, and the tectooculomotor
connections remain to be identified.
The present study was performed to reexamine the synaptic connections from the SC to horizontal ocular motoneurons in the anesthetized cat using intracellular recordings from LR and MR motoneurons and AINs, and electrical stimulation of the SC. To locate last-order premotor neurons that receive direct input from the SC and terminate on LR motoneurons, we labeled last-order premotor neurons with wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP) transneuronally and tectoreticular axon terminals with dextran-biotin in the same preparations. The results show that excitation from the contralateral SC to LR motoneurons is mainly disynaptic via last-order premotor neurons in the ipsilateral PPRF rostral to the abducens nucleus, whereas inhibition from the ipsilateral SC is mainly disynaptic via last-order premotor neurons in the contralateral paramedian pontomedullary reticular formation (PPMRF) caudomedial to the abducens nucleus. In contrast, excitation from the ipsilateral SC to MR motoneurons is at least trisynaptic via AINs, and inhibition from the contralateral SC to MR motoneurons is negligible.
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METHODS |
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Experiments were performed in 15 cats weighing 2.4-4.7 kg. The
surgery and animal care conformed to "The Principles of Laboratory Animal Care" (NIH publication No. 85-23, revised in 1985) and to
"Guiding Principles for the Care and Use of Animals in the Field of
Physiological Sciences" (The Physiological Society of Japan 1988);
experimental protocols were approved by the Animal Care Committee of
Tokyo Medical and Dental University. The animals were initially
anesthetized with an intramuscular injection of ketamine hydrochloride
(Ketalar, Parke-Davis; 25 mg/kg) followed by an intravenous injection
of -chloralose (40-45 mg/kg initial dose, supplemented with an
additional dose of 10-25 mg/kg). Muscle nerves innervating the LR, MR,
inferior rectus (IR), and inferior oblique (IO) muscle were detached
from the muscles and mounted on bipolar hook electrodes for electrical
stimulation (IR nerve stimulation was monopolar against an electrode in
the orbit). Electrical stimulation of the vestibular nerve bilaterally
(single or double 0.2-ms stimuli, maximum intensity of 500 µA) was
delivered via silver ball electrodes placed on the round and oval
windows (Shinoda et al. 1992
; Shinoda and Yoshida
1974
). The bone over the parietal and occipital cortex was
removed, and the cerebral cortex overlying the SC was suctioned
unilaterally or bilaterally to introduce stimulating or recording
electrodes into the SC. Usually four, but sometimes seven, concentric
bipolar stimulating electrodes (0.1 mm ID and 0.3 mm OD; interelectrode
distance, 0.5 mm) were positioned in the intermediate or deep layer
(1.5-2.0 mm deep from the surface) of the SC on either side. The
vermis overlying the fourth ventricle was aspirated to facilitate
intracellular recording from ocular motoneurons and AINs. To examine
axonal projections of single tectoreticular neurons, a concentric
stimulating electrode was placed in the PPRF and two other electrodes
in the descending medial longitudinal fascicle (MLF) near the obex
contralateral to the SC in two animals. After these two experiments,
0.05 µl of 30% horseradish peroxidase (HRP) (Toyobo, Osaka, Japan)
was injected into the abducens nucleus ipsilateral to the stimulated PPRF for retrograde labeling of last-order interneurons terminating in
the abducens nucleus. After a survival time of 16 h, the animals were deeply anesthetized with pentobarbital sodium (45 mg/kg, Nembutal,
Abbott, Switzerland) and perfused with 2 l of 10% sucrose phosphate buffer (pH 7.4) followed by 2 l of a fixative solution containing 2% paraformaldehyde and 1% glutaraldehyde in 4% sucrose phosphate buffer. Frozen sections of 75 µm were cut from the brain stem and the midbrain and treated for HRP by the tetramethylbenzidine method (Mesulam 1978
).
Glass microelectrodes for intracellular recording were filled with 3 or
0.4 M KCl and had a resistance of 8-15 M. A tungsten electrode
insulated in a glass microelectrode was used for recording extracellular spikes in the SC. Negative pulses of 0.2 ms were delivered at 100-500 µA for stimulation of the SC, at <200 µA (usually <100 µA) for stimulation of the PPRF and at a maximum of 500 µA for stimulation of the MLF. The positions of the
stimulating electrodes were marked by passing negative currents of 20 µA for 20 s after each experiment, and stimulated sites in the
SC, PPRF, and MLF were histologically confirmed in sections stained
with thionin. During recording, the animals were paralyzed by the
intravenous administration of pancuronium bromide (Mioblock, Organon,
The Netherlands) and artificially ventilated with the end-tidal
CO2 concentration held at ~37 mmHg. The body temperature
was kept between 37.0 and 38.5°C by a heating pad. Heart rate was
constantly monitored by electrocardiogram.
To examine whether axon terminals of tectoreticular neurons terminate
on last-order premotor neurons that, in turn, terminate on LR
motoneurons, 12.5% dextran-biotin (Molecular Probes) was injected into
the SC, and WGA-HRP (Toyobo, Japan) was injected into the abducens
nerve in the same preparations in each of five animals (Sugiuchi
et al. 1995). After 4-6 days, the animals were anesthetized
with pentobarbital sodium (45 mg/kg) and perfused with 2 l of 10%
sucrose phosphate buffer (pH 7.4) followed by 2 l of a fixative
solution containing 4% paraformaldehyde and 0.05% glutaraldehyde with
0.2% picric acid in 4% sucrose phosphate buffer. Frozen sections of
50 or 75 µm were cut from the brain stem and the midbrain, incubated
in anti-WGA antibody and avidin-biotin complex, and then treated for
HRP by the heavy metal intensification method of Adams
(1981)
.
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RESULTS |
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To determine the neural pathways from the SC to horizontal ocular motoneurons, intracellular potentials were recorded from LR and MR motoneurons and AINs in cats, and the effects of bilateral electrical stimulation of the superior colliculi on these neurons were examined. All lateralities in this study are described with reference to the recording side.
Effects of stimulation of the SC on LR motoneurons
The resting membrane potentials of antidromically identified LR
motoneurons (Fig. 1A) ranged from
45 to
80 mV (
59 ± 12 mV, mean ± SD, n = 66). Single stimuli applied to the SC usually evoked no response or a
very small response (Fig. 1, Ba and Ca). Double
or triple stimuli of the contralateral SC evoked depolarization (Fig.
1Bb), and those of the ipsilateral SC evoked
hyperpolarization (Fig. 1Cb) in a LR motoneuron. Latencies
and amplitudes of these postsynaptic potentials (PSPs) fluctuated,
suggesting that these responses were induced polysynaptically. The
behavior of these PSPs on passing hyperpolarizing or depolarizing
currents through the recording microelectrode confirmed that the
depolarization evoked by contralateral SC stimulation was an excitatory
postsynaptic potential (EPSP; Fig. 1D), and the
hyperpolarization evoked by ipsilateral SC stimulation was an
inhibitory postsynaptic potential (IPSP; Fig. 1E)
(Eccles 1964
).
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As the stimulus intensity at the most effective site in the contralateral SC was increased, the size of early EPSPs increased, and their latencies decreased by 0.2-0.3 ms, whereas late EPSPs appeared on the falling phase of the early EPSPs (Fig. 2Aa). This indicates that stronger stimuli recruit a larger number of tectofugal neurons over a wider SC area. When double stimuli were used (Fig. 2Ab), multiple EPSPs of various latencies became prominent in addition to the facilitation of early EPSPs. This spatial facilitation in SC-evoked EPSPs and IPSPs was found in all of the examined LR motoneurons.
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Increasing the number of stimuli produced a significant temporal
facilitation of EPSPs (Fig. 2B). The increased number of preceding stimuli produced a greater facilitatory effect on the EPSPs
evoked by the last stimulus in each trial in Fig. 2, Bb-Be, suggesting that more last-order excitatory interneurons were recruited. Similar temporal facilitation was also remarkable in IPSPs (Fig. 2C). To avoid the saturation of hyperpolarized IPSPs, IPSPs
were reversed to depolarized IPSPs by injecting Cl into a
motoneuron (Fig. 2, Cb-Cf). With an increase of stimuli, late IPSPs with an additional latency of ~1.0 ms were superimposed on
the early IPSPs (Fig. 2Cf). The temporal facilitation in
SC-evoked EPSPs and IPSPs was observed in all of the examined LR
motoneurons. In each motoneuron examined, the number of stimuli was
changed between one and five, while keeping the stimulus intensity
constant, and the least number of effective stimuli for significant
PSPs was determined, and then the number of the stimuli was further increased. The PSP latencies were measured by comparing PSPs evoked by
the preceding stimuli with PSPs evoked by one more additional stimuli
to the preceding stimuli (see Fig. 2, B and C).
One to three additional SC stimuli to the first effective stimuli
usually decreased such PSP latencies by 0.1-0.3 ms, but further
increasing the number of the stimuli did not shorten their latencies.
Therefore these shortest latencies were regarded as latencies for the
PSPs. The latencies of contralateral SC-evoked EPSPs ranged from 0.9 to
1.9 ms (1.6 ± 0.2 ms, n = 56; Fig.
3A), and those of ipsilateral SC-evoked IPSPs ranged from 1.4 to 2.4 ms (1.8 ± 0.3 ms,
n = 57; Fig. 3B).
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The effect of the stimulation site in the SC on the size of PSPs was examined by recording PSPs from LR motoneurons while stimulating different parts of the SC with pulses of the same intensity. In the contralateral SC (Fig. 4, A1-A4), stimulation of the caudomedial site evoked the largest EPSPs at the shortest latency, and stimulation of the central site evoked smaller EPSPs at longer latencies, whereas rostral and rostrolateral stimulation evoked small or no EPSPs. In the ipsilateral SC (Fig. 4, A5-A8), stimulation of the caudomedial and caudolateral sites evoked the largest IPSPs, whereas stimulation of the rostral and rostrolateral sites evoked only small or no IPSPs. Similar tendencies were observed in all of the experiments. In three experiments, seven stimulating electrodes were arranged in the rostrocaudal direction of the SC along the horizontal meridian of the motor map (Fig. 4B). Evoked EPSPs became larger and their rising phase became sharper as the stimulation sites were shifted more caudally in the SC, but the most caudal stimulation usually evoked slightly smaller EPSPs. In general, stimulation at either the caudolateral or caudomedial site evoked large EPSPs.
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The effect of electrode depth was examined by recording EPSPs from a LR motoneuron while a single stimulating electrode was moved vertically and stimuli of the same intensity were applied at different depths in the contralateral SC (Fig. 4C). The size of evoked EPSPs was small when the electrode was located in the superficial layers and increased together with its distance from the surface of the SC.
Inputs from the SC to internuclear neurons in the abducens nucleus
Inputs from the SC to AINs were examined by recording
intracellular potentials from them while stimulating the SC. AINs
received disynaptic EPSPs from the contralateral vestibular nerve (Fig. 5C) and IPSPs from the ipsilateral
vestibular nerve (Fig. 5D) (cf. Baker and Highstein
1975). Single pulse stimulation of the SC evoked small PSPs
more often in AINs than in LR motoneurons (Fig. 5, Ea and
Fa). Increasing the number of stimuli always produced larger
PSPs, with shorter latencies, with depolarlizations from the
contralateral SC (Fig. 5, Eb and Ec) and
hyperpolarizations from the ipsilateral SC (Fig. 5, Fb and
Fc). Intracellular injection of Cl
reversed
the hyperpolarization evoked from the ipsilateral side (Fig.
5G) but did not affect the polarity of the depolarization evoked from the contralateral side, thus indicating that the
depolarization was an EPSP and the hyperpolarization was an IPSP. The
latencies of contralateral SC-evoked EPSPs ranged from 0.9 to 1.7 ms
(1.4 ± 0.2 ms, n = 14; Fig. 3C),
whereas those of ipsilateral SC-evoked IPSPs ranged from 1.3 to 2.1 ms
(1.8 ± 0.3 ms, n = 12; Fig. 3D). The
effects of stimulating various sites in the SC on AINs were similar to
those on LR motoneurons.
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Inputs from the SC to MR motoneurons
Intracellular potentials were recorded from MR motoneurons to
investigate their inputs from the SC. Their resting membrane potentials
ranged from 40 to
70 mV (
56 ± 12 mV, n = 22). MR motoneurons were identified by their antidromic responses to
stimulation of the MR muscle nerve (Fig.
6A). Because the MR nerve is short from where it separates from the third nerve trunk, current sometimes spread from a stimulating electrode for the MR nerve to the IR or IO
nerve in some preparations. To confirm that intracellular potentials
were recorded from MR motoneurons, we always checked that the
motoneurons activated by MR nerve stimulation were not activated by
stimulation of the IR and the IO nerve at lower stimulus intensities.
MR motoneurons were also reliably distinguished from IR and IO
motoneurons by examining their vestibular inputs. For example,
stimulation of the ipsilateral vestibular nerve evoked small disynaptic
EPSPs (1.2-1.8 ms, 1.4 ± 0.2 ms, n = 10)
followed by larger trisynaptic EPSPs (1.9-3.1 ms, 2.4 ± 0.3 ms,
n = 10) in MR motoneurons; contralateral stimulation of
the vestibular nerve produced EPSPs (1.7-2.5 ms, 2.0 ± 0.3 ms,
n = 5). In contrast, stimulation of the vestibular
nerve evoked ipsilateral disynaptic inhibition and contralateral
disynaptic excitation in IR and IO motoneurons (Ito et al.
1976
).
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In MR motoneurons, single stimulation of the ipsilateral SC often
evoked small depolarization (Fig. 6Da), and double or triple stimuli always evoked larger depolarization at shorter latencies (Fig.
6, B, Db, and Dc). This depolarization was an
EPSP because injection of Cl into the cell had no effect
and injection of small depolarizing currents decreased the
depolarization but did not reverse it. The latencies of these EPSPs
ranged from 1.7 to 2.8 ms (2.1 ± 0.3 ms, n = 22;
Fig. 3E) and were longer by 0.7 ms on average than those of
the EPSPs in AINs (t-test, P < 0.001).
Stimulation of the contralateral SC did not evoke any potentials even
with double or triple stimuli in most MR motoneurons (Fig.
6C), although EPSPs were evoked in some neurons from
stimulation of the medial or rostral part of the contralateral SC. To
ensure that EPSPs were not canceled by IPSPs or vice versa in such
cases, either depolarizing or hyperpolarizing currents were always
passed through the recording electrode, but no hidden PSPs were
revealed. In some MR motoneurons, stimulation of the medial or rostral
part of the contralateral SC evoked EPSPs, whereas caudal stimulation never evoked potentials even in such cases.
Excitation from the ipsilateral SC to MR motoneurons may be mediated by
way of the contralateral AINs. These interneurons have been previously
shown to relay excitation from the ipsilateral vestibular nerve to MR
motoneurons (Baker and Highstein 1975), and, as the
present results have shown, they receive an excitatory input from the
SC 0.7 ms before excitation is observed in MR motoneurons. To confirm
that AINs relay excitation from the SC to MR motoneurons, the
interaction of SC-evoked EPSPs and vestibular-evoked EPSPs was examined
in a conditioning-test paradigm (Fig. 7).
Stimulation of the ipsilateral vestibular nerve (Fig. 7Eb)
and that of the ipsilateral SC (Fig. 7Ea) were adjusted to
near threshold for evoking EPSPs in the same MR motoneuron. The SC
conditioning stimulation given at 1.5 ms before the test vestibular
stimulation did not affect the early disynaptic component of the
vestibular-evoked EPSPs but increased the amplitude and rising slope of
the later trisynaptic component in the MR motoneuron (Fig.
7Ec). The time course of this facilitation was examined by
changing the intervals between the conditioning and test stimuli.
Facilitation occurred when the vestibular stimuli were given at the
same time as the SC stimuli (at 0 ms) reached its peak at 1.0 ms and
then gradually decreased after 2.0 ms (Fig. 7F). Because
conditioning SC stimulation facilitated the late trisynaptic component
of the vestibular-evoked EPSPs, but not the early disynaptic component,
these results suggest that an input from the SC and a vestibular input
converge onto common AINs terminating on MR motoneurons. Similar
facilitation was observed in all of the eight MR motoneurons tested.
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Tectoreticular neurons projecting to the PPRF
To determine the location of interneurons mediating excitation
from the SC to LR motoneurons, last-order interneurons terminating on
LR motoneurons were identified in the brain stem by transneuronal labeling after injection of WGA-HRP into the abducens nerve (Figs. 8-10).
The details of the distribution of transneuronally labeled neurons will
be reported separately (unpublished observations). Briefly,
those neurons were mainly distributed ipsilaterally in the PPRF just
rostral to the abducens nucleus (Fig. 9), the vestibular nuclei, and
contralaterally in the PPMRF just caudomedial to the abducens nucleus
(Fig. 10), the vestibular nuclei, and the prepositus hypoglossi
nucleus. Among them, the PPRF just rostral to the abducens nucleus and
contralateral to the SC most likely corresponds to the location where
stimulation produced and lesion eliminated horizontal conjugate eye
movements (Cohen et al. 1968), and where MLBNs that
burst at the onset of a horizontal saccade were found (Cohen and
Henn 1972
; Luschei and Fuchs 1972
).
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To examine the projection of tectofugal neurons to this PPRF area,
extracellular spikes were recorded from neurons in the SC. One group of
tectoreticular neurons (n = 19) was antidromically activated from the contralateral PPRF, but not from the descending MLF
(Fig. 8B), and the other group (n = 27) was
activated from both the contralateral PPRF and the contralateral
descending MLF (Fig. 8C). In the neuron in Fig.
8B, spikes activated by PPRF stimulation were regarded as
antidromic, because they had a fixed latency of 1.9 ms even at
threshold and followed double shock stimuli at a 0.9-ms interval. This
neuron could not be activated by MLF stimulation at 500 µA (Fig.
8Bc). In the neuron in Fig. 8C, spikes were
evoked by PPRF stimulation at a fixed latency of 0.8 ms at threshold
and followed double shock stimuli at a 0.5-ms interval (Fig.
8Cb). This neuron was also activated by MLF stimulation at a
latency of 0.8 ms (Fig. 8Cc). To examine the possibility of
current spread from the PPRF stimulus to the nearby MLF, a spike
collision test was carried out between spikes activated from the
contralateral PPRF and MLF (Fig. 8, Ce-Ch) (Shinoda
et al. 1976, 1986
). Spikes evoked by the
preceding PPRF stimulation had no effects on spikes evoked by the
following MLF stimulation at latencies longer than 1.1 ms, but blocked
MLF-evoked spikes at an interval of 1.0 ms (Fig.
8Cf). On the other hand, spikes evoked by the preceding MLF
stimulation had no effect on PPRF-evoked spikes at intervals of
1.0 ms, but blocked PPRF-evoked spikes at intervals of
0.9 ms (Fig. 8Ch). By using the values (spike
latencies, refractory periods, and maximal intervals for spike
collision) obtained in this spike collision test, the additional conduction time along the axon collateral in the PPRF was calculated (Shinoda et al. 1976
) to be 0.3 ms (Fig. 8F).
If the stem axon had been activated by current spread from the PPRF,
this conduction time would have been zero. Conduction times along
presumed axon collaterals in the PPRF were calculated in all of the
neurons examined, and neurons were discarded from the sample when
calculated conduction times were <0.1 ms (see the details of the
calculation of axonal conduction time and associated problems in
Shinoda et al. 1976
, 1986
).
At the conclusion of these experiments, the PPRF was identified by injecting HRP into the ipsilateral abducens nucleus. We confirmed that the electrolytic lesion marking the location of the stimulating electrode was located in the PPRF where retrogradely labeled neurons were abundant (Fig. 8, G and H) in both animals. Latencies of antidromic spikes from the PPRF were 0.5-2.8 ms (1.6 ± 0.6 ms, n = 19) for tectoreticular neurons (Fig. 8D) and 0.5-2.3 ms (1.0 ± 0.4 ms, n = 27) for tectoreticulospinal neurons (Fig. 8E). Tectoreticular neurons tended to have slower conducting axons, which is consistent with the finding that smaller spikes tended to be recorded from them. These tectofugal neurons were recorded at depths of 1.5-3.0 mm from the SC surface.
By comparing the latencies of PSPs evoked by SC stimulation (Fig. 3, A-E) and the antidromic latencies of tectoreticular neurons projecting to the PPRF, SC-evoked EPSPs and IPSPs in LR motoneurons, and EPSPs and IPSPs in AINs were most likely disynaptic. SC-evoked EPSPs in MR motoneurons were most likely trisynaptic, because their latencies were ~0.7 ms longer than those of disynaptic EPSPs in AINs.
Morphological evidence of disynaptic excitatory and inhibitory pathways from the SC to LR motoneurons
To confirm that tectoabducens connections are mainly disynaptic and to identify last-order premotor neurons in these connections, we anatomically examined the relationship between tectoreticular axons (identified by injecting dextran-biotin into the SC) and last-order premotor neurons terminating on LR motoneurons (identified by transneuronal transport of WGA-HRP injected in the abducens nerve). Areas showing both the presence of tectoreticular axon terminals and transneuronally labeled neurons were restricted to the PPRF, PPMRF, and to some extent in the abducens nucleus. Terminal axons in these areas were traced proximally, from serial sections, and they were seen to join the predorsal bundle after crossing the midline. Labeled tectoreticular axon terminals were distributed in the same PPRF area as transneuronally labeled neurons were located, but they spread more widely in the ventromedial direction (Fig. 9). Many of these labeled neurons seemed to have the labeled tectoreticular axon terminals on their cell bodies and proximal dendrites. Figure 11A shows a photomicrograph of such synaptic contacts of labeled tectoreticular axon terminals with a transneuronally labeled neuron in the PPRF ipsilateral to the abducens nucleus. Figure 12 shows a reconstruction of labeled tectoreticular axons terminating on transneuronally labeled neurons in the PPRF. In this example, 0.59 µl of dextran-biotin was relatively widely injected into the right SC, and 91 of 285 transneuronally labeled neurons in the PPRF ipsilateral to the injected abducens nerve had labeled tectoreticular axon terminals on them. In the other two experiments (0.1 µl and 3.3 µl injected), 24 of 177 and 104 of 262 transneuronally labeled neurons in the PPRF had labeled tectoreticular axon terminals on them, respectively. These morphological data show that the shortest main pathway from the contralateral SC to LR motoneurons is disynaptic via the ipsilateral last-order premotor neurons in the PPRF rostral to the abducens nucleus. Synaptic contacts of a small number of labeled terminals observed in the abducens nucleus with retrogradely labeled LR motoneurons were scarcely observed in each preparation.
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The distributions of contralateral last-order premotor neurons terminating on LR motoneurons and tectoreticular axon terminals were examined in a similar way (Fig. 10). Labeled premotor neurons were fairly localized in a vertical column in the PPMRF caudomedial to the caudal part of the abducens nucleus and at a depth of 0.5-2.5 mm from the surface of the fourth ventricle. Labeled tectoreticular axon terminals were distributed in this PPMRF area, although they spread more widely than the location of the premotor neurons. Many of the labeled premotor neurons in this area were contacted by labeled tectoreticular terminals (Fig. 11B). Figure 13 shows a reconstruction of labeled tectoreticular axon terminals terminating on transneuronally labeled neurons in the PPMRF contralateral to the injected abducens nerve. In this example, 3.1 µl of dextran-biotin was widely injected into the SC including its caudal half, and 42 of 112 transneuronally labeled neurons in the PPMRF contralateral to the injected abducens nerve had labeled tectoreticular terminals on their cell bodies and proximal dendrites. In another experiment, 2.5 µl of dextran-biotin was injected into the left SC widely including its caudal half, and 190 of 490 transneuronally labeled neurons in the PPMRF were contacted by labeled tectoreticular axon terminals on their cell bodies and proximal dendrites. These morphological data show that the shortest main pathway from the ipsilateral SC to LR motoneurons is disynaptic via the last-order premotor neurons located in the contralateral PPMRF caudomedial to the abducens nucleus. By comparing the electrophysiologically identified disynaptic excitatory and inhibitory connections and the above morphologically identified disynaptic connections between the SC and LR motoneurons, we conclude that the main connections between the SC and LR motoneurons are disynaptic. The excitatory pathway from the contralateral SC is relayed through PPRF neurons ipsilateral to the LR motoneurons, whereas the inhibitory pathway from the ipsilateral SC is relayed through PPMRF neurons contralateral to the LR motoneurons.
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DISCUSSION |
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The present study has revealed that the main excitatory pathway from the contralateral SC to LR motoneurons is disynaptic via the ipsilateral PPRF, whereas the main inhibitory pathway from the ipsilateral SC to LR motoneurons is disynaptic via the contralateral PPMRF. In contrast to these reciprocal inputs to LR motoneurons, only excitation was observed in MR motoneurons from the ipsilateral SC; inhibition from the contralateral SC was not observed.
Precht et al. (1974) first reported that the average
latency of EPSPs from the contralateral SC to LR motoneurons was 1.6 ms
(1.2-2.2 ms) and concluded that the main connection from the contralateral SC to LR motoneurons was at least trisynaptic, although some disynaptic connections were present. However, Grantyn and Grantyn (1976)
reanalyzed these pathways and concluded that
EPSP latencies in the range of 0.8-1.0 ms were monosynaptic, whereas the most frequently observed latencies of 1.4-2.0 ms were disynaptic. In the present study, the latencies of the EPSPs ranged from 0.9 to 1.9 ms (1.6 ± 0.2 ms) and were very similar to the latency ranges
reported by Precht et al. (1974)
and Grantyn and
Grantyn (1976)
. Multiple SC stimulation induced temporal
facilitation and decreased latencies of EPSPs by 0.1-0.3 ms in
virtually all LR motoneurons examined, indicating that these EPSPs were
at least disynaptic. Comparison of conduction times of tectoreticular
fibers from the SC to the PPRF with the EPSP latencies indicate that most of the EPSPs are likely to be disynaptic, although some of the
longer latency responses could involve an additional synapse. This
electrophysiological conclusion is supported by our morphological findings that axon terminals of tectofugal fibers made direct contact
with many transneuronally labeled neurons in the PPRF terminating on LR motoneurons.
The latencies of SC-evoked IPSPs in ipsilateral LR motoneurons in the
present study (1.4-2.4 ms, 1.8 ± 0.3 ms) are similar to those
reported by Precht et al. (1974) and Grantyn and
Grantyn (1976)
. The latter group reasoned that because IPSP
latencies were usually in the range of 1.8-2.4 ms, which is
substantially longer than the EPSP latencies, the shortest inhibitory
pathway from the ipsilateral SC to LR motoneurons had essentially one more synapse than the excitatory pathway (Grantyn and Grantyn 1976
). Our results confirm that the IPSP latency is, on
average, 0.2 ms longer than that of the contralaterally evoked EPSPs,
but this seems insufficient time for an additional synapse. Instead, we
ascribe this latency difference to the longer conduction distance for
the inhibitory pathway and conclude that the inhibitory connection from
the ipsilateral SC to LR motoneurons is predominantly disynaptic. Our
anatomic data support the conclusion because axon terminals of
tectofugal fibers made direct contact with transneuronally labeled
neurons in the contralateral PPMRF terminating directly on injected LR motoneurons.
With increased stimulus intensity or number of stimuli, SC-evoked
disynaptic excitation and inhibition in LR motoneurons were greatly
enhanced, and late polysynaptic components of PSPs were extensively
recruited. The former phenomenon indicated the existence of spatial
and/or temporal facilitation at the level of interneurons in the
tectoabducens pathways. Recruitment might be due to either of at least
two underlying neural mechanisms: 1) intervening pontine reticular interneurons mediating disynaptic excitation to LR
motoneurons fire repetitively, and 2) additional
interneurons involved between the above interneurons and LR motoneurons
are recruited. The latter mechanism has been discussed (see
Fuchs et al. 1985 for references), but reliable
experimental evidence has not been provided yet. Burst neurons in the
PPRF do not continue to burst for a long time after cessation of
repetitive stimulation of the SC (Grantyn and Grantyn
1976
). Therefore long-lasting EPSPs are most likely mediated by
additional interneurons such as vestibular nucleus neurons or
prepositus hypoglossi neurons. Repetitive stimulation of the
ipsilateral SC evoked late components of IPSPs in LR motoneurons, suggesting that excitatory reticular neurons in the PPRF may exert their influence on inhibitory reticular neurons in the PPMRF. This
possible pathway needs to be further investigated, although some burst
neurons in the PPRF have a collateral near the abducens nucleus
(Strassman et al. 1986
).
The present study has shown that the patterns and latencies of inputs
from the SC to LR motoneurons and AINs are similar; the main connection
is disynaptic excitation from the contralateral SC and disynaptic
inhibition from the ipsilateral SC, although the latencies of EPSPs
recorded from AINs were slightly shorter. Grantyn and Grantyn
(1976) reported EPSPs with latencies of 0.8-1.0 ms in a very
small fraction of LR motoneurons, suggesting the existence of a
monosynaptic connection. Axon terminals of tectofugal axons were
scattered in the contralateral abducens nucleus as reported by
Grantyn and Grantyn (1982)
and Olivier et al.
(1993)
. Direct contact of these terminals on retrogradely
labeled LR motoneurons was rarely observed, suggesting that they might
contact AINs, distal dendrites of LR motoneurons, or distal dendrites
of other neurons.
SC stimulation evoked EPSPs in ipsilateral MR motoneurons at latencies
of 1.3-2.6 ms, described as disynaptic (Grantyn and Berthoz
1977). In the present study, the latencies of EPSPs ranged from
1.7 to 2.8 ms (2.1 ± 0.3 ms), which were ~0.7 ms longer than in
AINs. This suggests that the main pathway from the SC to ipsilateral MR
motoneurons is trisynaptic. We have shown that the pathway from the
ipsilateral SC to MR motoneurons is mediated via contralateral AINs,
because conditioning stimulation of the ipsilateral SC facilitated the
late component but not the early component of EPSPs evoked by
ipsilateral vestibular stimulation in MR motoneurons. The discrepancy of the presence or absence of the short-latency EPSPs (<1.7 ms) in the
previous and present studies remains to be explained. One possible
explanation is that their SC stimulation might activate a part of the
SC or an adjacent tegmental structure that is not related to saccades,
because their stimulation depths were deeper than ours. The latencies
of SC-evoked EPSPs in MR and LR motoneurons slightly overlapped each
other, despite the fact that MR motoneurons receive excitation via one
more intervening interneuron than LR motoneurons. This is partly
because AINs have slightly stronger and shorter-latency inputs from the
SC. Because MR motoneurons on one side and LR motoneurons on the other
side must be synchronously activated for conjugate horizontal saccades,
this neural mechanism is functionally significant and may explain why
AINs start discharging earlier and have a higher velocity sensitivity
than LR motoneurons (Delgado-Garcia et al. 1986
). MR
motoneurons received reciprocal inputs from the two superior colliculi,
excitation from the ipsilateral SC and inhibition from the
contralateral SC, and the latencies of contralateral SC-evoked IPSPs
were 2.0-3.5 ms (Grantyn and Berthoz 1977
). The
inhibitory SC action on MR motoneurons appeared to be weaker under
pentobarbital anesthesia than in "encéphale isolé"
preparations (Grantyn et al. 1979
). The ipsilateral
dorsomedial reticular formation between the abducens and the trochlear
nuclei was suggested as a source for monosynaptic inhibition of MR
motoneurons (Grantyn et al. 1980
). In the present study
under chloralose anesthesia, contralateral SC stimulation usually did
not evoke any short-latency IPSPs in MR motoneurons, but sometimes
evoked EPSPs. The pathway responsible for these EPSPs remains
undetermined but may be related to a vergence eye movement.
Precht et al. (1974) first reported that the excitatory
and inhibitory tectoabducens pathways ran from the tectum through the
ipsilateral midbrain reticular formation to LR motoneurons. Grantyn et al. (1979)
showed that the pathways
underlying the transmission of oligosynaptic excitatory and inhibitory
effects from the SC to LR motoneurons decussate in the mesencephalic
tegmentum, and the second decussation of the inhibitory tectoabducens
pathway occurs at the level of the abducens nucleus. Grantyn and
Berthoz (1977)
first analyzed the anatomic pathways from the SC
to MR motoneurons by comparing responses in MR motoneurons before and after transecting the pontobulbar structures and speculated that reticular neurons in the paramedian pontine tegmentum receiving monosynaptic input from the contralateral SC would exert their monosynaptic excitation on MR motoneurons. Later, the "abducens region" involving interneurons within or adjacent to the abducens nucleus was thought to be responsible for disynaptic excitatory connection between the SC and MR motoneurons (Grantyn et al.
1979
), but Grantyn and co-workers had difficulty in explaining
disynaptic connections between the SC and MR motoneurons without
assuming that tectofugal fibers establish monosynaptic connections with AINs. The present analysis revealed that AINs mainly received disynaptic excitation from the contralateral SC and disynaptic inhibition from the ipsilateral SC. Conditioning stimulation of the
ipsilateral SC did not exert any effect on the disynaptic vestibulooculomotor response but had a facilitatory effect on the
trisynaptic vestibulooculomotor response. Accordingly, AINs were
regarded as last-order excitatory premotor neurons from the ipsilateral
SC to MR motoneurons, whereas vestibular nucleus neurons do not appear
to be part of this pathway.
The combination of electrophysiological and morphological methods we employed allowed us to overcome some of the drawbacks of previous lesion and microstimulation studies and to specify last-order interneurons that directly receive SC outputs and terminate on LR motoneurons. In the present study, ~30% of the transneuronally labeled reticular neurons in the PPRF or the PPMRF were directly contacted by tectoreticular axon terminals. This value is probably an underestimation, because the small amounts of dextran-biotin we injected into the SC could label only a small portion of tectofugal neurons. With this reservation in mind, it seems safe to conclude that tectoreticular axons contact the somata and proximal dendrites of the vast majority of PPRF neurons that terminate directly on ipsilateral LR motoneurons and of PPMRF neurons that terminate directly on contralateral LR motoneurons, and that disynaptic connections between the SC and LR motoneurons are substantial. Together with the electrophysiological findings, it is safe to conclude that the reticuloabducens neurons with direct tectoreticular contacts in the ipsilateral PPRF and in the contralateral PPMRF are excitatory and inhibitory, respectively. The question arises as to whether all PPRF neurons contacted by contralateral tectofugal neurons and terminating on ipsilateral LR motoneurons are excitatory, and all PPMRF neurons contacted by contralateral tectofugal neurons and terminating on contralateral LR motoneurons are inhibitory. Because tectal stimulation evoked only disynaptic EPSPs in contralateral LR motoneurons, we conclude that all transneuronally labeled PPRF neurons contacted by tectofugal axons exert an excitatory influence on ipsilateral LR motoneurons. On similar grounds, we conclude that all transneuronally labeled PPMRF neurons contacted by tectofugal axons exert an inhibitory influence on contralateral LR motoneurons.
The PPRF contains MLBNs that exhibit a high-frequency burst of spikes
before and during saccades in monkeys and cats (Cohen and Henn
1972; Hikosaka and Kawakami 1977
; Keller
1974
; Luschei and Fuchs 1972
; Yoshida et
al. 1982
). These MLBNs are considered to be premotor neurons
that directly terminate on LR motoneurons (Hikosaka and Kawakami
1977
; Hikosaka et al. 1978
; Yoshida et al. 1982
). Raybourn and Keller (1977)
examined
the effect of SC stimulation on MLBNs to determine the pathway from the
SC to LR motoneurons and showed that MLBNs could not be activated at a short latency by SC stimulation in alert monkeys, whereas long-lead burst neurons (LLBNs) received short-latency excitatory input from the
SC with about one-third of the responses being in the monosynaptic
range. In contrast, Chimoto et al. (1996)
showed that
MLBNs in the paramedian reticular formation rostral and caudal to the
abducens nucleus were activated monosynaptically from the contralateral
SC in alert cats. It seems likely that, in the cat, these MLBNs in the
ipsilateral PPRF are the relay neurons that are responsible for
disynaptic excitation from the contralateral SC to LR motoneurons and
those in the contralateral PPMRF are responsible for disynaptic
inhibition from the ipsilateral SC to LR motoneurons.
The effects of stimulation at various depths in the SC on single LR
motoneurons showed that deeper stimulation induced steeper-rising and
larger EPSPs, whereas superficial-layer stimulation induced small
EPSPs. Tectoreticular neurons activated antidromically from the PPRF
were located at 1.5-3.0 mm from the SC surface, depths that
corresponded to the intermediate and deep layers. Stimulation of this
intermediate or deep layer (2.5-3.0 mm deep) resulted in marked
temporal facilitation of the EPSPs. This is consistent with the
anatomic findings that tectoreticular or tectospinal neurons are
located in the intermediate and deep layers of the SC (Kawamura
and Hashikawa 1978; Moschovakis and Karabelas
1985
). Stimulation of different tectal sites evokes
characteristic saccadic eye movements in the awake cat (Guitton
et al. 1980
; McIlwain 1986
), and the spatial
distribution of effective SC stimulating sites for evoking PSPs in
horizontal ocular motoneurons is generally similar to the "motor
map" obtained for horizontal saccadic components in awake animals.
The longer the distance from the rostral pole of the SC, the larger the
PSP evoked in LR motoneurons. Anatomic data show that neurons in the SC
extensively project to the contralateral nucleus reticularis pontis
caudalis and the rostral part of the nucleus reticularis
gigantocellularis (Grantyn and Grantyn 1982
; Kawamura et al. 1974
; Scudder et al.
1996
). In agreement with previous results (Grantyn and
Grantyn 1982
), there are two groups of tectoreticular and
tectoreticulospinal neurons. In the present study, ~60% (27/46) of
tectoreticular neurons projecting to the contralateral PPRF were found
to send their axons to the spinal cord. Our recent morphological study
showed that tectospinal neurons emit multiple axon collaterals in the
cervical cord, and spinal interneurons receiving monosynaptic
excitation from the contralateral SC directly terminate on neck
motoneurons (Muto et al. 1996
). Therefore the SC could
influence neck muscles through such tectoreticulospinal neurons as well
as reticulospinal neurons that receive monosynaptic input from the SC
(Kakei et al. 1994
; Sasaki 1992
). The SC
of the cat can be divided into three anteroposterior zones from a functional point of view; the anterior, intermediate, and posterior zones (Guitton et al. 1980
). Tectal output neurons
projecting to the spinal cord predominantly exist in the intermediate
zone (Muto et al. 1996
). Therefore tectoreticulospinal
neurons in this zone contribute to gaze control by a large-amplitude
eye movement and synchronous neck movement to fixate a visual target in
the periphery of the visual field (Guitton et al. 1980
).
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ACKNOWLEDGMENTS |
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We thank R. H. Schor for reading the manuscript, M. Takada for invaluable technical assistance, and Y. Tamai and I. Sugihara for computer programming.
This research was supported by a grant from Core Research for Evolutional Science and Technology of the Japan Science and Technology Corporation.
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FOOTNOTES |
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Address reprint requests to Y. Shinoda.
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 7 December 1998; accepted in final form 2 February 1999.
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REFERENCES |
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