Department of Integrative Physiology, National Institute for
Physiological Sciences, Myodaiji, Okazaki 444-8585, Japan
 |
INTRODUCTION |
The role of the mammalian superior colliculus (SC)
in visually guided behaviors, such as saccadic eye movements and
orientation, has long been an area of intense investigation (for
review, see Sparks 1986
; Wurtz and Albano
1980
). Anatomically, the SC consists of several layers, each
with distinct neuronal organization and specific input-output
relationships. Optic fibers project to the SC through the optic layer
(the stratum opticum, SO). The stratum griseum superficiale (SGS)
receives visual input from the retina and the primary visual cortex.
The intermediate and deep layers (the stratum griseum intermediale, SGI
and the stratum griseum profundum, SGP) receive various nonvisual
sensory and cortical inputs. These layers send descending motor
commands to the brain stem reticular formation and the spinal cord and
ascending signals to the thalamus (Huerta and Harting
1982
). The distribution of neurons related to control of
saccadic eye movements extends ventrally from the border between the
optic and intermediate layers to the deep layer (Moschovakis et
al. 1988b
; Wurtz and Goldberg 1972
).
In contrast to the abundance of anatomic studies of the SC
input-output, very little is known about the organization of its local
circuits and how visual information is processed in the SC to generate
motor commands. Moreover, although the morphological properties of
individual collicular neurons have been investigated in numerous
anatomic studies, the electrophysiological properties of the
morphologically identified cells have not (Hall and Lee 1993
; Langer and Lund 1974
; Ma et al.
1990
; Norita 1980
; Sterling 1971
). Moschovakis and colleagues (Moschovakis and
Karabelas 1985
; Moschovakis et al. 1988a
,b
)
studied the morphological characteristics of saccade-related neurons
using intracellular staining with horseradish peroxidase in alert and
anesthetized squirrel monkeys. The cells stained in their studies,
however, appeared to be limited to a population of large-sized
tectofugal neurons. Lopez-Barneo and Llinás (1988)
studied membrane properties of a population of neurons in the
intermediate layer of the SC. They described a specific group of
neurons that exhibited inward rectification at hyperpolarized membrane
potentials and have divergent dendritic trees that extend into the SGS.
Their description, however, was limited to this group of neurons and
did not include information about their axonal projections.
To determine how information is processed in the local circuits of the
SC, it is essential to characterize the membrane properties and
anatomic connectivity of individual neurons composing the circuits.
Furthermore it is also important to characterize the specific
conductances that determine the membrane properties of individual
neurons (Llinás 1988
). In the present study, we
studied the electrophysiological properties of randomly selected
neurons in the movement-related layer (SGI) using whole cell
patch-clamp recording technique. At the same time we studied the
morphological characteristics of recorded cells by staining with
biocytin (Horikawa and Armstrong 1988
). The present
results indicate that there are at least five subclasses of neurons
with distinct firing properties in the local circuits of the SGI and
each subclass is differentiated further according to rectification
properties in response to hyperpolarization. The difference in their
electrophysiological property, especially the rectification property,
was reflected in their morphological characteristics. These results,
together with our studies on the signal transmission in the local
circuits of the SC (Isa et al. 1998
), may reveal the
fundamental aspects about the dynamic properties of the SC local circuits.
 |
METHODS |
Slice preparations
Thin slices of the SC were prepared from young (17-22 days old)
and adult (7-8 wk old) Wistar rats. The body weight of adult rats
ranged from 180 to 270 g. In most cases, the brains were removed
after decapitation under ether anesthesia. In some adult rats, the
procedure was performed after transcardial perfusion of ice-cold
sucrose-Ringer solution. After removal, the brains were submerged
immediately in ice-cold sucrose-Ringer solution and bubbled with 95%
O2-5% CO2 for 5-10 min.
The sucrose-Ringer solution contained (mM): 234 sucrose, 2.5 KCl, 1.25 NaH2PO4, 10 MgSO4, 0.5 CaCl2, 26 NaHCO3, and 11 glucose. Frontal slices 200- to
300-µm thick (mostly 250 µm) were cut using a Microslicer
(DTK-2000, Dosaka EM, Kyoto, Japan). They then were incubated in
standard Ringer solution at room temperature for >1 h before
recording. The standard Ringer solution contained (mM): 125 NaCl, 2.5 KCl, 2 CaCl2, 1 MgCl2, 26 NaHCO3, 1.25 NaH2PO4, and 25 glucose,
and was bubbled continuously with 95% O2-5%
CO2 (pH 7.4). After incubation, slices to be used
for recording were placed individually in a recording chamber on an
upright microscope (Axioskop FS, Zeiss, Germany) and continuously
superfused with standard Ringer solution at a rate of 3-5 ml/min using
a peristaltic pump (Minipuls 3, Gilson, Villiers, France).
Whole cell patch-clamp recording
Individual neurons in the SC were visualized with Nomarski
optics with the use of a ×40 water immersion objective. Whole cell patch-clamp recording (Edwards et al. 1989
;
Hamill et al. 1981
) was performed in randomly selected
neurons in the SGI under visual control of the patch pipettes. Patch
pipettes were prepared from borosilicate glass capillaries (GC150TF-15,
Clark Electromedical Instruments, Pangbourne, UK) with a micropipette
puller (P-97, Sutter Instrument, Novato, CA). The pipettes were filled
with an internal solution containing (mM): 140 K-gluconate, 20 KCl, 0.2 EGTA, 2 MgCl2, 2 Na2ATP, 10 HEPES, and 0.1 spermine (pH 7.3). To stain the recorded neurons,
biocytin (5 mg/ml, Sigma, St. Louis, MO) was dissolved in the solution
just before recording. The osmolarity of the internal solution was
280-290 mOsm/l. The liquid junction potential of the patch pipette
solution and standard Ringer solution was estimated to be
10 mV. The
measured membrane potentials were offset by this value to reflect the
actual membrane potential. The resistance of the electrodes was
2.5-7.0 M
in the bath solution, and the series resistance during
recording was 10-25 M
. The electrophysiological properties of the
recorded cells were investigated in current clamp mode using an EPC-7
patch-clamp amplifier (List, Darmstadt, Germany). Depolarizing and
hyperpolarizing current pulses were given routinely to the cells with a
duration of 400 ms at 20- to 80-pA steps from two different levels of
the membrane potential (
55 to
70 mV, and
75 to
90 mV) set by
varying the intensity of constantly injected current. The neurons were
classified according to their firing responses under these conditions.
The firing responses of the recorded neurons remained stable for 10 min
after establishment of the whole cell recording configuration. All the
recordings were performed at room temperature. Data were acquired and
analyzed using a pClamp hardware/software system (Axon Instruments,
Foster City, CA). The input resistance of each neuron was calculated from the voltage change induced by a hyperpolarizing current pulse (typically
40 pA) from the membrane potential of
60 to
70 mV. The
average firing frequency was calculated from the number of spikes
during the current pulse.
Histological procedure
To visualize the recorded neurons by biocytin staining
(Horikawa and Armstrong 1988
), patch pipettes were
carefully detached from the cells after recording. Slices then were
fixed with 4% paraformaldehyde in 0.12 M phosphate buffer (pH 7.4) for
2-3 days at 4°C. The slices were rinsed in 0.05 M phosphate-buffered
saline (PBS, pH 7.4) and incubated in methanol containing 0.6%
H2O2 for 30 min. After
rinsing again in PBS, the slices were incubated in the solution
containing 1% avidine-biotin peroxidase complex (Vector Laboratories,
Burlingame, CA) and 0.3% Triton-X100 for 3 h. The slices were
rinsed in PBS and 0.05 M Tris-buffered saline (TBS, pH 7.6) and then
incubated in a TBS solution containing 0.01% diaminobenzidine
tetrahydrochloride (DAB), 1% nickel ammonium sulfate, and 0.0003%
H2O2 for 30 min. All
procedures for visualization of biocytin were performed at room
temperature. The slices were mounted on gelatin-coated slides,
counterstained with cresyl violet or neutral red, dehydrated, and then coverslipped.
Only cells with intact somata and proximal dendrites were drawn using a
camera lucida attached to a light microscope. All quantitative data
were expressed as means ± SE. T-test was used for
statistical analysis.
 |
RESULTS |
In the present study, recordings were made from 223 neurons in
slices from young rats and 44 neurons in slices from adult rats. The
neurons were recorded in the SGI. Of these, 131 neurons from young rats
and 26 from adult rats were stained successfully with biocytin and used
for morphological analysis. Recording sites covered virtually all
regions of the SC, both mediolaterally and rostrocaudally. No
distribution bias was observed for any particular subclass of neurons
in the SC. Neurons with resting membrane potentials more negative than
60 mV and that exhibited action potentials more positive than 0 mV at
their peak were used for electrophysiological analysis.
Records from young rats
VOLTAGE RESPONSES TO DEPOLARIZING CURRENT PULSES.
Regular-spiking neurons. Regular-spiking neurons (Fig.
1) exhibited repetitive firing with
relatively constant interspike intervals in response to depolarizing
current pulses (Fig. 1, A and D). Mild spike
frequency adaptation was observed in some cases; however, an interspike
interval never more than doubled the preceding one. The level of the
resting membrane potential did not affect the regular firing property
as shown in Fig. 1, B and C. The relationship
between the average firing frequency and the amplitude of the injected
current was analyzed systematically in 13 cells, and linear-like
correlation was observed in all the cases according to the linear
regression analysis (0.986 < r < 0.997, mean ± SD; 0.992 ± 0.004, P < 0.001) as
shown in Fig. 1E. Fifty-one percentage (113/223) of neurons
showed this type of firing responses.

View larger version (26K):
[in this window]
[in a new window]
|
Fig. 1.
Electrophysiological properties of a regular-spiking neuron.
A: responses to depolarizing current pulses (values
given at right). B and C:
responses to current pulses from different membrane potentials
(B: 69 mV; C: 88 mV).
D: interspike interval between successive spikes,
recorded at different current intensities. E:
relationship between average firing frequency and the injected current
with a linear regression line (r = 0.996, P < 0.001).
|
|
Late-spiking neurons.
Late-spiking neurons were characterized by a considerably delayed first
spike after weak current pulses (Fig.
2A). When stronger current
pulses were used, a long interval occurred between the first spike and
the second. This appeared to be due to a transient hyperpolarization
that occurred after the onset of the depolarizing pulse (Fig.
2A,
). Late spiking was observed when the depolarizing pulse was applied from a hyperpolarized level (
85 mV; Fig.
2B). When the depolarizing current pulse was applied from a
more depolarized level (
62 mV), late spiking was not observed and the
firing pattern became more regular (Fig. 2C). These results
suggest that the transient hyperpolarization is due to an A-like
transient outward current, which is inactivated at depolarized membrane
potentials (Connor and Stevens 1971
). This suggestion is
supported by the observation that application of 4-aminopyridine, a
blocking agent of A channels, abolished the transient hyperpolarization
and changed the firing property of late-spiking neurons to resemble
that of regular-spiking neurons (Fig. 2D) (Gustafsson
et al. 1982
; Thompson 1977
). Twenty-two
percentage (48/223) of neurons showed this type of firing responses.

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 2.
Electrophysiological properties of a late-spiking neuron.
A: responses to depolarizing current pulses (values
given at right). , transient hyperpolarization
induced at the onset of the depolarizing current pulse.
B and C: responses to depolarizing
current pulses from 2 different membrane potentials (B:
85 mV; C: 62 mV). D: effects of 5 mM
4-aminopyridine (4-AP). Note that the characteristic delayed spike
generation was abolished by application of 4-AP.
|
|
Burst-spiking neurons.
Burst-spiking neurons (Fig. 3) generated
a cluster of more than two spikes (transient burst) at threshold level
for spike generation. The instantaneous firing frequency was
93.0-190.5 Hz at the threshold level (n = 14). This
was a quite contrast to the regular-spiking neurons, which generated
solitary spikes at the threshold level. The transient burst was
followed by an afterdepolarization (Fig. 3C,
). In
response to prolonged depolarizing current pulses, solitary spikes
followed the transient burst (Fig. 3A). The transient burst
was observed only when the depolarizing current pulse was applied at a
hyperpolarized membrane potential (
82 mV in Fig. 3, A and
B;
78 mV in Fig. 3C). When the current pulse
was applied at a more depolarized membrane potential (
66 mV in Fig.
3, D and E;
62 mV in Fig. 3F), no
transient burst or marked afterdepolarization was observed. Rebound
depolarization and spike generation was observed after termination of
the hyperpolarizing current pulse, when the resting membrane potential
was more depolarized (Fig. 3E) but not when hyperpolarized
(Fig. 3B). These results suggest that the rebound
depolarization was due to the same conductance as that underlying the
transient burst; the membrane potential in Fig. 3, A and
B, (
82 mV) was below the threshold for activation of the
conductance, whereas the membrane potential in Fig. 3, D and
E (
66 mV), was above the threshold for activation of the conductance, and the conductance therefore was inactivated by constantly holding the membrane potential at that level (Fig. 3D). Thirteen percentage (29/223) of neurons showed this
type of firing.

View larger version (29K):
[in this window]
[in a new window]
|
Fig. 3.
Electrophysiological properties of burst-spiking neurons.
A-C: responses to current pulses from hyperpolarized
membrane potentials (A and B: 82 mV;
C: 78 mV). A: responses to depolarizing
current pulses (values given at right). B,
top: responses to depolarizing and hyperpolarizing current
pulses. Bottom: injected current steps.
C: responses to current pulses near threshold for spike
generation. , afterdepolarization. D-F: responses to
current pulses from depolarized membrane potentials (D
and E: 66 mV; F: 62 mV). Same
arrangement as A-C. Note that a transient burst did not
appear at the onset of the depolarizing current pulse and the firing
pattern was regular. Also note the rebound depolarization and spike
generation after the termination of the hyperpolarizing current pulse
(E, in contrast to B). No transient burst
was observed with current pulses at threshold for spike generation in
F.
|
|
The transient burst appeared to be induced by a transient voltage hump
that occurred in response to the depolarizing current pulse. This
transient voltage hump remained in the presence of tetrodotoxin (TTX;
0.25-1.0 µM), indicating that it was Na+
independent (Fig. 4, A1 and
B1). The transient depolarization was suppressed effectively
by 1 mM Co2+ and 0.5 mM
Ni2+ (Fig. 4, A2 and B2).
The voltage hump that occurred as a rebound depolarization after the
hyperpolarizing current pulse also was blocked by 0.5 mM
Ni2+ (Fig. 4C). Furthermore transient
burst to depolarizing current pulses was abolished by 0.5 mM
Ni2+ (Fig. 4, D and E).
These observations suggest that low-threshold Ca2+ channels mediate the transient voltage hump
and transient burst in burst-spiking neurons.

View larger version (28K):
[in this window]
[in a new window]
|
Fig. 4.
Effect of Co2+ and Ni2+ on the transient
voltage hump and transient burst. A and
B: effects of Co2+ and Ni2+ on
the transient voltage hump. C: effects of
Ni2+ on the transient voltage hump observed as a rebound
depolarization. 1: responses to depolarizing
(A and B) and hyperpolarizing
(C) current pulses in standard Ringer solution
containing 0.25 µM TTX. Bottom: current steps.
2: responses in the presence of 1 mM Co2+
(A) or 0.5 mM Ni2+ (B and
C). D and E: effects of
Ni2+ on spike firings. Control (D) and
during application of 0.5 mM Ni2+ (E). In
these experiments, the control solution was composed of 140 NaCl, 2.5 KCl, 2 CaCl2, 1 MgCl2, 5 HEPES, and 10 glucose
(pH 7.4) and bubbled with 100% O2.
|
|
Fast-spiking neurons.
Fast-spiking neurons (Fig. 5) sustained
high-frequency repetitive firing throughout the depolarizing pulse with
virtually no spike frequency adaptation (Fig. 5, A and
C). There appeared to be a threshold for induction of stable
high-frequency firing. For example, injection of a 76-pA depolarizing
pulse in the neuron shown in Fig. 5 caused fluctuating low-frequency
firing, whereas currents >152 pA induced stable high-frequency firing.
Further increases in the current amplitude did not much increase the
firing frequency (Fig. 5, B and C). The firing
frequency exceeded 100 Hz when strong current pulse was applied. Nine
percentage (19/223) of neurons showed this type of firing responses.

View larger version (38K):
[in this window]
[in a new window]
|
Fig. 5.
Electrophysiological properties of a fast-spiking neuron.
A: responses to depolarizing current pulses (values
given at right). B: relationship between
average firing frequency and amplitude of injected current.
C: interspike intervals between successive spikes. Note
that stable high-frequency firing occurred with current pulses > 152 pA.
|
|
Neurons with marked spike frequency adaptation.
In response to a depolarizing current pulse, the interspike interval of
full-sized action potentials recorded from neurons with marked spike
frequency adaptation increased during the spike train, and an
interspike interval could become more than double the preceding one
(Fig. 6). Generation of the spike train
often was terminated during the current pulse (Fig. 6A,
bottom). Burst-spiking neurons often exhibited spike frequency
adaptation (Fig. 3B), however, these neurons exhibited
marked spike frequency adaptation without a transient burst of spikes
even from a hyperpolarized level (
89 mV; Fig. 6A). Five
percentage (11/223) of neurons showed this type of firing responses.

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 6.
Electrophysiological properties of a neuron with marked spike frequency
adaptation. A: responses to depolarizing current pulses
(values given at right). B: interspike
intervals between each pair of successive spikes.
|
|
Neurons with rapid spike inactivation.
In three neurons, the amplitude of action potentials in the spike train
decreased, their width increased, and the spike train was terminated
after the generation of two to three spikes although the membrane
potential was still above threshold (Fig.
7A). Two of these neurons
showed long tail of depolarization after termination of the
depolarizing pulses (Fig. 7B,
). Cessation of firing may have been due to inactivation of Na+ channels,
because spike generation recovered after a brief pause (10-20 ms) in
the depolarizing pulse (Fig. 7C). Furthermore although the
number of the recorded cells was too small (n = 3) for
statistical analysis, the input resistance in neurons with rapid spike
inactivation was higher and whole cell membrane capacitance was smaller
than those in other type neurons.

View larger version (12K):
[in this window]
[in a new window]
|
Fig. 7.
Electrophysiological properties of a neuron with rapid spike
inactivation. A: responses to depolarizing current
pulses (values given at right). B, top:
responses to depolarizing current pulses. Note the slowly inactivating
depolarization after termination of the current injection ( ).
Bottom: injected current steps. C:
recovery of the spike generation. Depolarizing current step (400 pA)
was paused briefly (20 ms).
|
|
VOLTAGE RESPONSES TO HYPERPOLARIZING CURRENT PULSES.
Time-dependent inward rectification. In response to
hyperpolarizing current pulses, a group of neurons showed
time-dependent inward rectification as shown in Fig.
8. The neurons in Fig. 8, A-C, showed regular-, late-, and burst-spiking properties,
respectively. In all the cases, hyperpolarizing current pulses elicited
a rapid hyperpolarization followed by a slow redepolarization or a
"voltage sag" (arrow in Fig. 8). Thus the voltage sag was
observed in a subpopulation of regular-, late-, and burst-spiking
neurons (see Table 1). Previous studies
have shown that a voltage sag is caused by a
hyperpolarization-activated current
(Ih,
If, or
Iq) that is suppressed by
Cs+ but is resistant to
Ba2+ (DiFrancesco and Ojeda 1980
;
Halliwell and Adams 1982
; Mayer and Westbrook
1983
; Takahashi 1990
; Yanagihara and
Irisawa 1980
). We tested the effects of
Cs+ and Ba2+ on the
time-dependent inward rectification in 13 cells. Application of 3 mM
Cs+ in the present study had little effect on the
voltage response during the early phase of the response (50 ms) but
eliminated the voltage sag (Fig. 9,
A-E). In contrast, Ba2+ did not
affect either the early or late phase of the response (Fig. 9,
F-J). An increase in membrane conductance due to the activation of Ih at hyperpolarized
membrane potentials also accounts for the significantly low input
resistance of neurons with Ih, which
have a membrane capacitance comparable with those without Ih (t-test,
P < 0.01, Table 1). After the end of current pulses, the membrane potential exhibited a rebound depolarization (Fig. 8,
double arrows).

View larger version (27K):
[in this window]
[in a new window]
|
Fig. 8.
Neurons with time-dependent inward rectification. Voltage responses of
a regular-spiking neuron (A), a late-spiking neuron
(B), and a burst-spiking neuron (C) with
time-dependent inward rectification, respectively. Note that
hyperpolarizing current pulses elicited a rapid hyperpolarization
followed by a slow redepolarization (an arrow). Rebound depolarization
after the hyperpolarizing current pulses is indicated by a double
arrow.
|
|

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 9.
Effect of Cs+ and Ba2+ on the time-dependent
inward rectification. A-E: effect of bath application
of Cs+. Membrane hyperpolarization in response to
hyperpolarizing current pulses in the control solution
(A), during application of 3 mM Cs+
(B), and after washing out of Cs+ (10 min
later) (C). D and E:
current-voltage plots for traces shown in A-C. Membrane
potential at the peak (D; 50 ms from onset of current
pulse) and steady-state level (E; 300 to 350 ms from onset of current
pulse). F-J: effect of extracellular application of 1 mM Ba2+. Details as in A-E. In these
experiments, the control solution was composed of 140 NaCl, 2.5 KCl,
2.8 MgCl2, 0.2 CdCl2, 5 HEPES, and 10 glucose
(pH 7.4) and bubbled with 100% O2 to avoid the
contamination of Ca2+ currents.
|
|
Time-independent inward rectification.
In another group of neurons, hyperpolarizing current pulses elicited
membrane hyperpolarization with a marked decrease in input resistance
at more hyperpolarized levels in the regular-spiking neuron shown in
Fig. 10A. The inward
rectification in this case was time-independent and such inward
rectification was observed in a large proportion of neurons without
time-dependent inward rectification by
Ih [82% (60/73) of the
regular-spiking, 82% (27/33) of late-spiking, 74% (14/19) of
burst-spiking, 62% (8/13) of fast-spiking neurons, and 40% (2/5) of
neurons with marked spike frequency adaptation and no neurons with
rapid spike inactivation]. Application of 3 mM
Cs+ (Fig. 10, B-E) or 1 mM
Ba2+ (Fig. 10, F-I) to the
extracellular solution abolished the inward rectification with
virtually complete recovery after wash out (n = 9).
These results suggested that the time-independent inward rectification
was caused by inward rectifier potassium channels (Hagiwara and
Takahashi 1974
; Standen and Stanfield 1978
) (see also DISCUSSION).

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 10.
Effect of extracellular Cs+ and Ba2+ on
time-independent inward rectification. A, top: response
of a regular-spiking neuron with time-independent inward rectification
to depolarizing and hyperpolarizing current pulses.
Bottom: injected current steps. B-E:
effect of extracellular application of Cs+. Membrane
hyperpolarization in response to hyperpolarizing current pulses in
standard Ringer solution (B), during application of
solution containing 3 mM Cs+ (C), and after
washing out of Cs+ (D). E:
current-voltage plots for traces shown in B-D. Membrane
potentials at steady-state level [380-390 ms from onset of the
current pulse ( in B)], were measured and plotted.
F-I: effect of extracellular application of 1 mM
Ba2+. Details as in B-E.
|
|
MORPHOLOGICAL CHARACTERISTICS.
Neurons with time-dependent inward rectification by
Ih. Of the 29 neurons with time-dependent inward
rectification by Ih, 16 neurons were
successfully stained with biocytin. All extended divergent dendritic
trees dorsally (Fig. 11) that often
reached the SGS (Fig. 11, A, B, and D). These
morphological characteristics corresponded to those of wide-field
vertical cells (Langer and Lund 1974
). Some of them, in
addition to extensive dorsal projection of dendrites, extended
dendrites ventrally and/or horizontally, sharing the characteristics of
multipolar cells (Fig. 11E). The axons were mostly projected
ventrally with few terminal-like structures nearby the somata (Fig. 11,
A, C, and D); however, some neurons showed
terminal-like fine collateralization and swelling of axons ventral to
the somata and/or projection of axons dorsally (Fig. 11B).

View larger version (23K):
[in this window]
[in a new window]
|
Fig. 11.
Morphological characteristics of neurons with time-dependent inward
rectification by Ih. A-E:
camera lucida drawings of neurons with time-dependent inward
rectification by Ih. Somata and dendrites
are painted in black. Axons are drawn in half tone. Dashed lines are
the boundaries between layers. Low magnification views of the locations
of each neuron in the superior colliculus (SC) are shown in
A2-E2.
|
|
Neurons without time-dependent inward rectification by
Ih.
Neurons that did not show time-dependent inward rectification had
widely varying heterogeneous dendritic projection pattern and
heterogeneous morphological properties, including fusiform (Fig.
12, A and F),
multipolar (Fig. 12, B and C), horizontal (Fig. 12D), and pyramidal-shaped (Fig. 12E) neurons.
Some of these neurons had axon collaterals and terminal-like swellings
nearby the somata such as those shown in Fig. 12, A, B, C,
and E. Among these groups of neurons, differences in firing
responses to depolarizing current pulses did not correspond to a
particular morphological property. The only one exception so far
revealed was that neurons with rapid spike inactivation were
characterized by round somata with sparse dendrites and had the
appearance of immature cells (figure not shown).

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 12.
Morphological characteristics of neurons without time-dependent inward
rectification by Ih. A-F:
camera lucida drawings of neurons without time-dependent inward
rectification by Ih. A and
F: fusiform cells. B and
C: multipolar cells. D: horizontal cell.
E: pyramidal-shaped cell. A and
B were regular-spiking neurons, C was a
late-spiking neuron, D was a burst-spiking neuron,
E was a fast-spiking neuron, and F was a
neuron with marked spike frequency adaptation. Somata and dendrites are
painted in black and axons are drawn in half tone. Dashed lines
indicate the boundaries between layers. Low magnification views of the
locations of each neuron in the SC are shown in
A2-F2.
|
|
Records from adult rats
We recorded a total of 44 neurons in slices obtained from adult
rats (Table 2). Five of the six firing
properties observed in young rats were found in adult rats; neurons
with rapid spike inactivation were not recorded in adults.
Among the nine neurons with time-dependent inward rectification by
Ih, three neurons exhibited repetitive
spike doublets in response to depolarizing pulses (Fig.
13, A and B).
Such rhythmic spike doublets were never observed in the SGI in young
rats.

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 13.
Electrophysiological and morphological properties of a regular-spiking
neuron with time-dependent inward rectification by
Ih recorded in adult rat. A
and B: responses to depolarizing and hyperpolarizing
current pulses. Duration of the current pulse was 800 ms. Intensities
of the current pulses are indicated by the values at
right (A) and in the bottom
traces (B). C and
D: morphology of a neuron with repetitive doublets of
spikes and time-dependent inward rectification by
Ih located close to the border between the
stratum opticum (SO) and stratum griseum intermediale (SGI) of an adult
rat. This cell was a wide-field vertical cell.
|
|
Twenty-six neurons from adult rats were stained successfully with
biocytin. Among the nine neurons with
Ih, six were stained, and all of them
were wide-field vertical cells (Fig. 13, C and D). The other 20 stained neurons belonging to other
subclasses consisted of 13 multipolar, 4 pyramidal-shaped, 2 fusiform,
and 1 horizontal cells. Neurons recorded in adult rats tended to have higher input resistance and lower capacitance than those recorded in
young rats except for neurons with Ih
(Tables 1 and 2). It is, however, likely that there was a sampling bias
for smaller neurons in slices because it was difficult for large cells
to survive close to the surface of the slices made from adult rats.
 |
DISCUSSION |
The results of the present study indicate that when classified
according to electrophysiological properties, there are at least five
major types of firing responses to depolarizing pulses and two
different inward rectification properties in the SGI neurons of rat SC.
Thus there appears to be a wider variety of neurons in the local
circuit of the SC of rat than indicated by previous studies. In
addition, neurons described in previous studies can be categorized
within the context of the subclasses described in the present study.
Neurons in the intermediate layer of slices of guinea pig SC have been
found to exhibit oval somata and extend divergent dendritic trees that
reached the stratum zonale (Lopez-Barneo and Llinás 1988
). These neurons exhibit voltage sag in response to
hyperpolarizing current pulses, and some exhibit late-spiking property
due to an A-like current. Thus these neurons appear to correspond to wide-field vertical cells (Langer and Lund 1974
) and the
regular-spiking neurons with time-dependent inward rectification by
Ih described in the present study or
the few cells that also exhibited late-spiking. Tecto-bulbo-spinal
tract neurons in cats have been found to be large multipolar cells that
exhibit marked inward rectification (Grantyn et al.
1983
). These neurons appear to correspond to the regular-spiking neurons with time-independent inward rectification. The
present results showed that the time-independent inward rectification was suppressed by both Cs+ and
Ba2+. We performed voltage-clamp analyses and
observed a shift in the reversal potential of the inward rectifier
current (Cs+-sensitive current component) in
parallel to the predicted equilibrium potential for potassium when the
extracellular potassium concentration was changed (data not shown).
Previous studies have shown that inward rectifier potassium (IRK)
channels are blocked by both Cs+ and
Ba2+ (Hagiwara and Takahashi 1974
;
Standen and Stanfield 1978
). Thus the possibility is
raised that IRK channels contribute to the time-independent inward
rectification observed in the present study. Details of the
voltage-clamp analyses of IRK channels will be reported elsewhere
(unpublished data).
The results of the present study also indicate that the
morphological properties of the neurons, particularly the dendritic arborization, correlate with the presence of
Ih in the neuron. Wide-field vertical
cells expressed Ih and exhibited
time-dependent inward rectification in response to hyperpolarizing
current pulses. In response to depolarizing pulses, these neurons
primarily responded as regular-spiking cells; however, some of these
neurons exhibited late or burst spiking. The morphology of the
wide-field vertical cells in the SGI suggests that they receive direct
or indirect visual information from the optic tract in the SGS and SO
on their dendrites and transmit it to deeper layers, that is, the SGI
and SGP. If so, these neurons are involved in the signal transmission in the direct visuomotor pathway in the SC (the optic
tract-SGS/SO
SGI) (Isa et al. 1998
). In contrast,
multipolar, pyramidal, fusiform, and horizontal cells did not exhibit
Ih, but many of them exhibited time-independent inward rectification due to activation of presumably IRK channels. In response to depolarizing pulses, these neurons showed
heterogeneous firing properties; they responded either as regular-,
late-, burst-, or fast-spiking neurons or neurons with marked spike
frequency adaptation. Further analysis of these neurons focusing on
additional characteristics, such as their projection pattern (output
neurons or interneurons) or postsynaptic effects (excitatory or
inhibitory), may give rise to a more distinct classification.
Neurons with firing properties similar to those described in the
present study also have been described in other regions of the CNS.
Regular-spiking neurons have been described primarily in the neocortex
(Connors and Gutnick 1990
; McCormick et al.
1985
), although the regular-spiking neurons recorded in the
present study exhibited milder spike frequency adaptation than
neocortical neurons. Those in the neocortex appeared to be more similar
to the neurons with marked spike frequency adaptation described in the
present study. Regular-spiking neurons with time-dependent inward
rectification by Ih have been
described in the thalamus (McCormick and Pape 1990
) and
striatum (Jiang and North 1991
; Kawaguchi
1993
). Late-spiking neurons have been described in vagal motor
nucleus (Yarom et al. 1985
), pedunculopontine tegmental
nucleus (Kang and Kitai 1990
), nucleus tractus
solitarius in the medulla (Dekin et al. 1987
), neocortex
(Kawaguchi 1995
), and cochlear nucleus (Fujino et
al. 1997
). Fast-spiking neurons have been described in the
neocortex (Connors and Gutnick 1990
; McCormick et
al. 1985
) and hippocampus (Han et al. 1993
;
Kawaguchi and Hama 1988
; Kawaguchi et al.
1987
; Schwartzkroin and Mathers 1978
).
Burst-spiking neurons with low-threshold Ca2+
channels have been described in the neocortex (Connors and
Gutnick 1990
; McCormick et al. 1985
), thalamus
(Jahnsen and Llinás 1984
), and pedunculopontine
tegmental nucleus (Kang and Kitai 1990
). The properties
of the neurons described in the present study illustrate that the
diversity of electrophysiologically distinct neurons present in the SGI
of the SC is comparable with other regions of the CNS.
Development of the SC local circuits
In the present study, 17- to 22-postnatal-day-old rats were mainly
used, because in general visually controlled patch-clamp experiments
become extremely difficult in slice preparations from older animals.
Developmental studies of the SC indicate that although calbindin-D28k-containing neurons exhibit adult-like distribution by
the end of the first postnatal week (Dreher et al. 1996
)
and SC neurons exhibit adult-like dendritic trees 15 days postnatal, dendritic growth continues beyond 30 days (Warton and Jones
1985
). Thus it is likely that the SC circuits were still in the
course of development in the rats used in the present study. Data from adult rats (7-8 wk old) demonstrated the presence of five subclasses of the firing responses in young rats; only neurons with rapid spike
inactivation were not observed. Although the sample size was limited,
the morphological characteristics of immature cell-like appearance
suggested that these neurons might be in the course of development or
cell death that is normally occurring in the developing SC
(Warton and Jones 1984
). In adult rats, neurons exhibiting time-dependent inward rectification caused by
Ih were exclusively wide-field
vertical cells, and thus correlation between morphological and
electrophysiological properties was preserved. In adult rats, there
appeared to be a larger proportion of burst- and fast-spiking neurons
than in young rats. This may reflect developmental processes; however,
it is also possible that this result was due to a sampling bias. In
addition, some of neurons with time-dependent inward rectification by
Ih in adult rats exhibited a unique
electrophysiological property not observed in young rats; repetitive
spike doublets were observed. Such doublets of spikes also have been
observed in SO neurons by Lo et al. (1998)
, who showed
that SO neurons express voltage sag by
Ih and repetitive doublets of spikes
by intracellular recordings in adult rat SC slices. These neurons were
mostly wide-field vertical cells and looked quite analogous to the SGI
neurons with Ih recorded in the
present study. This firing property may contribute to the oscillatory
activity in SC circuits (Anderson and O'Steen 1975
; Mandl 1993
).
In conclusion, the comparison of data obtained from rats of different
ages suggests that developmental changes may still be occurring 17- to
22-days postnatal; however, most of the properties observed in neurons
from young rats were stable and observed in adult rats.
Functional significance of firing characteristics in
relation to dynamic properties of the SC local circuits
Among the subclasses of SGI neurons, the regular-spiking neurons
composed the largest population (Tables 1 and 2). Regular-spiking neurons recorded in the present study exhibited more regular firings and milder spike frequency adaptation than those described in the
neocortex (Connors and Gutnick 1990
; McCormick et
al. 1985
). Such firing property may have significance in the
generation of discrete motor commands to control precise movements. In
addition, the SC circuits contain other subclasses of neurons that
exhibited marked nonlinear input-output relationships. Among these
subclasses, burst-spiking neurons may be suited for the detection of
changes in sensory events because they are strongly activated,
particularly when the neuron is excited at more hyperpolarized membrane
potentials, due to the activation and inactivation properties of
low-threshold Ca2+ channels. Late-spiking neurons
exhibited voltage-dependent change in firing property; repetitive
firing was suppressed at hyperpolarized membrane potentials but release
from the suppression occurred in a depolarization-dependent manner.
These properties may enable these neurons to discharge a large number
of spikes in response to excitatory input only when the cell is
concurrently depolarized.
The present study also provides evidence for the presence of specific
ionic conductances in each subclass of neurons. Inward rectification in
several subclasses of neurons appears to be due either to IRK channels
or Ih. The characteristic firing of
late-spiking neurons appears to be due to A-like transient outward
currents. Although not specifically investigated in this study,
Ca2+-activated K+ channels
may have a significant role in the firing property of neurons with
marked spike frequency adaptation (Blatz and Magleby 1987
; Sah 1996
). The channels mediating the
characteristic electrophysiological properties of each subclass of
neurons are known to be significantly modulated by several
neurotransmitter systems, such as acetylcholine and enkephalin (see
Nicoll et al. 1990
). Then a question may arise as to
whether the classification of neurons made in the present study is
exclusive or not. We recently found that regular-spiking neurons in the
SGI express fast inactivating transient outward currents (A channels),
the amplitude of which is smaller than those in late-spiking neurons
(Saito and Isa 1998
). Thus some aspects of the
classification made in the present study may be a matter of quantity of
a particular ionic conductance. Therefore the firing properties in some
cases can be modulated significantly by change in amplitude of a
particular ionic conductance that determines the firing properties
under physiological conditions, e.g., by action of neurotransmitters.
Further studies of the transmitter systems innervating the SC are
needed to determine their role in modulating the signal transmission in
the local circuits and possibly give rise to interesting hypotheses as
to how SC modulation may effect changes in the behavioral response of
the animal.
The authors thank Prof. Seiji Ozawa and Drs. Wen-Jie Song, Hiroshi
Aizawa, and Yasushi Kobayashi for comments on the manuscript and
helpful discussions, and M. Seo for technical assistance.
This study was supported by grants from the Ministry of Education,
Science, Sports, and Culture of Japan (Grants 08279207, 08458266, and
09268238) and by grants from the Japan Science and Technology
Corporation and Uehara Memorial Foundation.
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.