 |
INTRODUCTION |
Omnipause neurons (OPNs)
located near the midline of the pons discharge tonically during
fixation and pause during saccades in all directions. OPNs play a
pivotal role in saccade generation; their tonic discharge inhibits
premotor burst neurons, and the pause allows the burst neurons to fire.
Intracellular recordings from OPNs in alert cats (Yoshida et al.
1999
) have shown that a pause in spike activity is caused by
inhibitory postsynaptic potentials (IPSPs). Analyses of the timing and
profiles of IPSPs have suggested that the duration of the pause is
controlled by afferents conveying eye-velocity signals from the brain
stem burst generator and that the onset of the pause is controlled by
input other than the burst generator. One of the most likely structures providing the latter input is the superior colliculus (SC) (for review,
see Fuchs et al. 1985
). Electrical stimulation of the SC
induces monosynaptic excitation followed by suppression of OPN spikes
(Kaneko and Fuchs 1982
; Raybourn and Keller
1977
). The monosynaptic excitation is more efficiently induced
from the rostral than from the caudal SC (Paré and Guitton
1994
). It was reported that suppression of spikes after SC
stimulation had a delay of 6-8 ms (Raybourn and Keller
1977
) and that multiple shocks from the SC had a strong
additive effect on the duration of suppression (Kaneko and Fuchs
1982
). However, the synaptic mechanism of the spike suppression
of OPNs following SC stimulation and the number of synaptic linkages
between the SC and OPNs remain to be clarified. The present study
investigates postsynaptic potentials (PSPs) in OPNs with intracellular
recordings in alert cats. We show that SC stimulation elicits
disynaptic IPSPs, evidence supporting the idea that SC activity induces
the saccadic pause by active inhibition rather than by disfacilitation.
Extracellular recordings from a larger sample of OPNs were also made to
supplement the intracellular data regarding the efficiency of the
rostral versus caudal SC in production of the inhibition.
 |
METHODS |
Experiments were performed with three adult cats. All
experimental protocols complied with the guidelines of the University of Tsukuba policy on the humane care and use of laboratory animals. The
surgical procedures and methods for recording eye movements and
painless immobilization of the animal's head have been described previously (Yoshida et al. 1999
). A pair of
microelectrodes made of Elgiloy wire (Suzuki and Azuma
1976
) was chronically implanted in the SC on each side for
stimulation. One electrode was placed in the caudal part of the SC
where a short-pulse train (10-30 µA) evoked contraversive saccades
with a large horizontal component (Fig.
1Ab). The other electrode was
placed 2.5-3.5 mm rostral to the first, where stimulation evoked small
saccades (Fig. 1Aa). Intra- and extracellular recordings
were made from OPNs with glass micropipettes filled with 4 M NaCl
solution (Yoshida et al. 1999
). Responses of OPNs to
single-pulse stimulation (0.2 ms, 200 µA) of the SC were recorded. No
eye movement was evoked by this stimulation. Histological
reconstructions located the stimulation sites in the intermediate or
deep layer in the rostral and caudal
of the SC.

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Fig. 1.
A: saccades evoked by short-pulse train stimulation
(top: 30 µA, 20 pulses, 400 Hz) of the rostral
(a) and caudal SC (b). Hor and Ver,
horizontal and vertical eye position traces. B:
identification of an omnipause neuron (OPN). , artifacts of
superior colliculus (SC) stimulation. C: intracellular
records from an OPN showing hyperpolarization (top)
associated with a saccade. Radial eye velocity trace
(middle) is inverted. D: SC-induced
extracellular spike responses (a), intracellular
potentials (b), and extracellular control records
(c) for the same OPN as in B. E: spike
suppression by SC-induced inhibitory postsynaptic potentials (IPSPs).
F: reversal of SC-induced IPSPs by chloride
injection: before injection (a), during passage of
hyperpolarizing current (4 nA) (b) and after cessation
of the current (c).
|
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Analyses of excitation and inhibition were obtained from both
intracellular and extracellular records. Whereas intracellular responses could be assessed by changes in the membrane potential and
the ability to reverse responses by current injection, extracellular assessment required statistical inference. For extracellular records, inhibition was inferred by spike suppression and excitation by an
increase in activity. Responses to stimulation were summed over 50-200
trials to obtain a peristimulus spike distribution, and the number of
spikes occurring in a window of length T (0.5 ms for excitation,
1.5-3.5 ms for suppression) was counted. The decision concerning
whether these counts reflected excitation or suppression was made by
determining the deviations of these counts from 95% confidence
intervals of activity preceding stimulation (baseline). These
confidence intervals were constructed by determining the average number
of spikes during baseline, with the variance measure reflecting the sum
of squares of deviations from this average evaluated at the same window
size used for evaluating excitation or suppression following stimulation.
 |
RESULTS |
OPNs were identified by a saccade-related pause of spikes (Fig.
1B) and, after the cell was impaled, by a hyperpolarization associated with each saccade (e.g., Fig. 1C) (cf.
Yoshida et al. 1999
). The membrane potential of OPNs was
30-40 mV, at which level spike generation mechanisms were often
inactivated. Figure 1D shows extracellular spikes
(a) and intracellular responses (b) of the same
cell following stimulation of the rostral SC. The initial excitatory
postsynaptic potentials (EPSPs; Fig. 1Db) were curtailed by
a potential change in the hyperpolarizing direction, which suppressed
tonic discharges for a certain period (Fig. 1Da). Spike
suppression without preceding excitation was also associated with a
membrane hyperpolarization (Fig. 1E). The
hyperpolarization (Fig. 1Fa) was reversed to a
depolarization during passage of a hyperpolarizing current (Fig.
1Fb) and after injection of Cl
ions
into the cell (Fig. 1Fc), indicating that the
hyperpolarization was the IPSP. The IPSPs were often followed by late
EPSPs that increased firing rates (Fig. 1E; see also Fig.
2). The initial EPSPs had latencies of
0.8-1.2 ms (mean, 1.0 ms, n = 4) and were regarded as
monosynaptic. The IPSPs were induced with latencies ranging from 1.3 to
1.9 ms (mean, 1.6 ms, for 25 IPSPs in 19 OPNs). Latencies of the late
EPSPs were 3-4 ms, a rough estimate because their onset or the
transition from IPSPs was gradual.

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Fig. 2.
A: intracellular records from an OPN showing PSPs
following stimulation of the rostral and caudal sites of the bilateral
SC. B: suppression of extracellular spikes induced by SC
stimulation. Spikes in 150 superimposed trials (top) are
summed to produce the cumulative spike distribution
(middle) and peristimulus time histogram
(bottom).
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The patterns of PSPs induced by SC stimulation depended on the
stimulation site (Fig. 2). Figure 2A shows the PSPs evoked in an OPN following stimulation of the rostral and caudal parts of the
SC on both sides. Stimulation of the rostral-left SC elicited IPSPs
preceded by monosynaptic EPSPs (Fig. 2Aa), whereas
stimulation of the caudal-left SC induced IPSPs without preceding EPSPs
(Fig. 2Ab). Table
1A shows the efficiency of the
rostral and caudal SC for production of IPSPs in 19 OPNs. Notably,
IPSPs were induced from the rostral as well as caudal SC in almost all
OPNs examined (9/9 and 13/14 cells, respectively).
Since the sample of intracellular data was small and might have been
biased toward larger cells, making it unrepresentative, both excitatory
and inhibitory effects were examined in a larger sample of OPNs using
extracellular recording (Table 1B). Overall, our
extracellular observations complemented what we observed
intracellularly (cf. Table 1). Stimulation of the rostral SC was more
efficient at eliciting monosynaptic excitation (0.8- to 1.6-ms
latencies) than stimulation of caudal sites (63 vs. 31%,
2 test, P < 0.0001). Disynaptic
inhibition, exhibited by spike suppression (Fig. 2B), began
1.3-2.4 ms following stimulation and lasted 1.5-3.5 ms. Unlike
excitation, SC stimulus location had no influence on the probability of
observing spike suppression (64 vs. 67%). When early spikes were
induced, postspike refractoriness would participate in the succeeding
suppression. However, its influence on the incidence of spike
suppression was probably minor in this study because SC stimulation
elicited IPSPs in most OPNs regardless of whether there was a preceding
EPSP. Thus both intra- and extracellular data indicated that the
rostral and caudal sites were equally effective for production of OPN inhibition.
Late excitation was observed intra- and extracellularly in a majority
of OPNs following stimulation of the rostral and caudal SC, frequently
of the SC on both sides (Table 1).
 |
DISCUSSION |
It has recently been shown that a saccade-related pause of
activity in OPNs is initiated by IPSPs (Yoshida et al.
1999
). The current study provides direct evidence that signals
originating in the SC are capable of inducing the IPSPs. The IPSPs
induced by SC stimulation had latencies as short as 1.3-1.9 ms. They
are thought to be induced disynaptically because the difference in mean
latency between monosynaptic EPSPs (1.0 ms) and IPSPs (1.6 ms) is
appropriate for the intercalation of single inhibitory interneurons. We
suggest that the disynaptic inhibitory pathway from the SC mediates a
saccadic signal that initiates an OPN pause. Such short-latency
inhibition appears favorable to rapid initiation of saccades.
The spatial pattern of inhibition was different from that of
excitation. The rostral as well as caudal SC stimulation sites in this
study were able to induce saccades, and both were equally effective for
producing inhibition. This fact is consistent with the idea that the
first element of the disynaptic pathway consists of saccade-related
burst neurons, which are distributed widely in the SC. In contrast,
monosynaptic excitation was more efficiently induced from the rostral
SC than from the caudal SC. This supports the previous finding of
Paré and Guitton (1994)
and the notion that the
rostral pole of the SC is involved with fixation (e.g., Munoz
and Guitton 1991
).
In our intracellular study, the incidence of monosynaptic EPSPs
was lower than that of extracellularly studied excitation, particularly
for caudal stimulation (Table 1). This is probably due to a fairly
depolarized state of impaled cells, which would make EPSPs smaller and
more difficult to detect. It is likely that monosynaptic EPSPs induced
by caudal stimulation are small and easily missed under such conditions.
Inhibitory interneurons mediating SC-induced IPSPs remain to be
identified. One candidate is reticular long-lead burst neurons (LLBNs),
which are monosynaptically activated from the SC (Raybourn and
Keller 1977
). Intracellular HRP techniques have shown that some
pontine LLBNs project to the OPN area (Scudder et al.
1996b
). Another candidate may be neurons at the dorsomedial
margin of the nucleus reticularis tegmenti pontis, as suggested by
Langer and Kaneko (1990)
. This region receives direct
projections from the SC and contains neurons that are labeled
retrogradely after HRP injection in the OPN area. A third possibility
is that the disynaptic inhibition is mediated by local interneurons
close to OPNs. It has been shown that axonal branches of
saccade-related SC burst neurons reach the OPN region (Scudder
et al. 1996a
). The fourth candidate, medium-lead inhibitory
burst neurons (IBNs), probably do not relay the disynaptic IPSPs, as
evidenced by the fact that single-pulse stimulation of the SC during
intersaccadic intervals did not induce spikes of IBNs (Chimoto
et al. 1996
; Raybourn and Keller 1977
) but did
induce disynaptic IPSPs in OPNs.
Since OPNs pause for saccades in all directions, we first
expected that individual OPNs might receive inhibitory effects
following stimulation of the colliculus on each side. However, nearly
half of the OPNs examined in our study were inhibited from one
colliculus and not from the other (Table 1B), possibly
implying that for a given saccade, not all OPNs are evenly inhibited by
a signal from the SC. A partial reduction in OPN population activity
could be sufficient to allow medium-lead burst neurons to begin firing, as suggested by Scudder (1988)
, and the activity of
these burst neurons could, in turn, inhibit a whole population of OPNs,
including those that have not been inhibited by SC signals. An
alternative explanation is that all OPNs receive disynaptic inhibitory
connections from the colliculi on both sides but single-pulse
stimulation is not always sufficient to activate inhibitory
interneurons. Further studies are needed to distinguish between these possibilities.
This study was supported by Grants-in-Aid for Scientific Research
(No. 11145209) from the Ministry of Education, Science and Culture of
Japan and by Core Research for Evolutional Science and Technology of
Japan Science and Technology Corporation.
Address for reprint requests: K. Yoshida, Dept. of Physiology,
Institute of Basic Medical Sciences, University of Tsukuba, Tsukuba,
Ibaraki 305-8575, Japan (E-mail:
kyoshida{at}md.tsukuba.ac.jp).