1Misaki Marine Biological Station, Graduate School of Science, The University of Tokyo, Kanagawa 238-0225; and 2Department of Anatomy, Laboratory for Comparative Neuromorphology, Nippon Medical School, Tokyo 113-8602, Japan
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ABSTRACT |
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Tsutsui, Hidekazu, Naoyuki Yamamoto, Hironobu Ito, and Yoshitaka Oka. Encoding of Different Aspects of Afferent Activities by Two Types of Cells in the Corpus Glomerulosum of a Teleost Brain. J. Neurophysiol. 85: 1167-1177, 2001. The corpus glomerulosum (CG) is an expansive nucleus in acanthopterigian teleosts that has been suggested to be involved in vision-related information processing and the control of the hypothalamic function. The CG has only two types of constituent cells, the large cell and the small cell, and well-defined afferent/efferent fiber connections. One of the three types of teleostean CG, type III has additional outstanding morphological characters: clearly laminated organization and giant (>50 µm in diameter) tips of postsynaptic dendrites. Although such histological architecture is potentially advantageous for the study of information processing in a brain nucleus based on the physiological properties of identified cells and synapses, previous studies on the CG have been limited to anatomy. In this study, we developed a slice preparation of the type III CG in a teleost, Stephanoplepis cirrhifer, and studied the morphology and physiology of individual cells and synaptic transmission by means of dendritic intracellular and somatic whole cell recordings. The characteristic morphology of the two types of cells was revealed by intracellular staining. While both of them received similar glutamatergic and GABAergic projections from the nucleus corticalis mediated by AMPA, N-methyl-D-aspartate, and GABAA receptors, they showed quite distinctive firing properties and postsynaptic responses with current injection and synaptic inputs: the large cell fired a single spike, and the small cell fired a spike train whose frequency was dependent on the stimulus intensity. Furthermore, the large cell showed low-pass temporal filtering properties with paired stimuli. These results suggest that the large cell and the small cell may encode different aspects of the corticalis activities.
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INTRODUCTION |
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Although the understanding of how information is processed in the brain nuclei of vertebrates by the physiological properties of constituent cells and synapses is one of the fundamental challenges in neuroscience, it has been hampered by the enormous complexity of the structural organization of the CNS. However, one of the three morphological types of the corpus glomerulosum (CG) among teleost fish has an exceptionally well-defined histological architecture and may serve as an excellent model preparation for the study of information processing in identified cells and networks.
The CG is an expansive nucleus located in the thalamic region of the
brain in most euteleosts except ostariophysans. The CG is a
gourd-shaped continuous structure that consists of two parts, corpus
glomerulosum pars anterior (GA), and corpus glomerulosum pars rotunda
(GR). Two types of cells, the large cell (soma diameter of 20-30 µm)
and the small cell (7-10 µm), are the projection neurons of this
nucleus (Ito and Kishida 1975). These are the only
cellular components of this nucleus, and no interneuron has been
reported thus far. In the CG there are glomeruli, which consist of the
presynaptic axon terminals from the nucleus corticalis and the tip of
the large cell dendrites. The large cell and the small cell receive
projections bilaterally from the nucleus corticalis and ipsilaterally
from the nucleus intermedius in the area pretectalis, respectively, and
the both types of cells project ipsilaterally to the inferior lobe of
the hypothalamus (Sakamoto and Ito 1982
). The results of
these and other previous anatomical reports (Campbell and
Ebesson 1969
; Ebbesson 1968
; Murakami et
al. 1986
; Shimizu et al. 1999
) and our
supplemental macroscopic and histological observations related to the
CG are summarized in Fig.
1 These anatomical
studies and electrophysiological evidence of visual input to the
nucleus corticalis (Rowe and Beauchamp 1982
) suggest that the CG may play an important role in the processing of
vision-related information and the control of the hypothalamic
function.
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Based on a comparative study of histological organization in 21 teleost
species, Ito and Kishida (1975) reported that the CG can
be morphologically classified into three groups, "nonlaminated" (type I), "incompletely laminated" (type II), and "laminated" (type III). Among these, the type III CG has the most completely laminated architecture and, furthermore, has an extreme enlargement (>50 µm in diameter) of the postsynaptic dendrite of the large cell,
which was referred to as a "star-like structure" by Ito and
Kishida (1975
, 1977
).
We propose that the outstanding morphological characteristics of the
type III CG, i.e., small number of constituent cell types (only 2 types), completely laminated architecture, huge postsynaptic structure,
and simple afferent/efferent fiber connections, may be advantageous as
a preparation for the elucidation of the mechanisms of information
processing in brain nuclei. However, the previous studies on the CG
have been limited merely to anatomy. Here, we first developed an in
vitro brain slice preparation of the type III CG in the file fish,
Stephanoplepis cirrhifer (Ito 1978), and
characterized the physiological properties of the cells and synapses in
the CG as a basis for future study of intra-nuclear information processing.
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METHODS |
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Fish
Adult male and female filefish, S. cirrhifer, ranging from 15 to 22 cm in the total length, were caught in the Sagami Bay or purchased from a local dealer and kept in a tank of overflowing natural sea water system under a natural dark-light cycle until used. The water temperature ranged from 13 to 25°C throughout the year. The fish were mainly fed mussels (Mytilus edulis) or commercial food pellets, "Fukuyoka-3P" (Nihon Nohsan, Tokyo), and could be kept in good conditions for longer than a year but were usually used within 3 mo after capture.
Brain slice preparation
The fish were deeply anesthetized by immersing them in seawater
containing tricaine methane sulfonate (MS222, Sigma) at a concentration
of 150 mg/l and were decapitated. The head was fixed in a custom-made
acrylic stereotaxic device, the brain skull was opened, and frontal
cuts were made at the border of the telencephalon and the optic tectum
and at the brain stem caudal to the cerebellum. The brain block
including the optic tectum and the underlying thalamus and hypothalamus
(hence containing the CG) was then dissected out and immersed in an
ice-cold artificial cerebrospinal fluid (ACSF, see Solutions and
drugs) for ~60 s. The brain block was quickly embedded in 2.5%
agarose of low-gelling temperature (Sigma, type IV) in ACSF and was
hardened in a freezer (30°C for 3 min). The agar-embedded brain
block was trimmed and glued to the microslicer, DSK-1000 (Dosaka EM,
Kyoto, Japan). Frontal slices of 400-500 µm and 250-300 µm in
thickness containing the CG and the horizontal commissure fiber (CH
fiber) were cut in the ice-cold ACSF for intracellular and whole cell
patch-clamp recordings, respectively. Modified low-Na ACSF (see
Solutions and drugs) was used when slices of 250-300 µm
were cut to reduce excitotoxic damages (Aghajanian and Rasmussen
1989
). Because the CH fiber from the ipsilateral nucleus
corticalis enters the GR dorsally, the slice preparation of the CG used
in the present study was taken from the GR (see Fig. 1, A
and C).
Electrophysiology
The experimental configuration in the present study is schematically illustrated in Fig. 2. Intracellular recordings were made from the large cell dendrites with a microelectrode amplifier equipped with a bridge-circuit (MEZ-8300, Nihon Kohden, Tokyo). The electrode was guided to the glomerulular layer, which is clearly identifiable in the CG slice under a dissection microscope, and penetrations were done blindly while monitoring voltage responses to small current pulses (0.2 nA).
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Whole cell patch clamp recordings were made from the soma of the large cell and the small cell with a patch-clamp amplifier Axopatch 200B (Axon Instruments) in the current-clamp mode under an upright microscope (ECLIPSE E600-FN, Nikon, Tokyo) equipped with a water-immersion objective (×40, 0.80 numerical aperture), differential interference contrast (DIC) optics, and a CCD camera with infrared sensitivity (C2400, Hamamatsu Photonics, Hamamatsu, Japan). The large cell and the small cell could be easily identified by their location, size, and morphology (see RESULTS).
Intracellular microelectrodes and patch pipettes were pulled from
borosilicate glass capillaries of 1.5 mm OD (Narishige, Tokyo) by a
Flaming-Brown type microelectrode puller (P-97; Sutter Instruments).
Microelectrodes filled with 2 M KCl (DC resistance: 30-50 77 ) were
used for intracellular recordings. Patch pipettes filled with
K-gluconate-based pipette solution (see Solutions and drugs)
were used for whole cell recordings, and these electrodes showed tip
resistance of 4-6 M
. The liquid junction potential of the
K-gluconate-based pipette solution to the ACSF, which was as large as
-14 mV, was corrected for the membrane potential in the whole cell
recordings (Neher 1992
).
After penetration of a large cell dendrite with an intracellular
microelectrode, the electrode resistance was re-canceled carefully with
the bridge-balance circuit to eliminate rapid voltage jump in response
to a current pulse (0.5 nA). The membrane resistance and capacitance
were then measured by exponential fitting of the passive voltage
response with increasing constant current steps (
1.5 to +1.5 nA,
0.5-nA increment, 100- to 200-ms duration). The current-voltage
relation was linear in this range of current amplitude, while the
linearity was lost for the current steps larger than 2 nA due to a
nonlinearity of the microelectrode impedance. The series resistance,
membrane resistance, and capacitance in the whole cell recordings were
measured from current responses with voltage steps to
100 mV (30- to
50-ms duration) from the holding potential of
80 mV in the
voltage-clamp mode. Series resistance was compensated when current
injection protocols were performed. Signals of intracellular and whole
cell recordings were filtered at 3 and 5 kHz, respectively, by the
filter circuit built in the amplifiers and digitized at 5-10 kHz using
pClamp software (Axon Instruments).
For electrical stimulation, a parallel bipolar electrode, made by
placing two tungsten electrodes (FHC) at a distance of 0.3-0.5 mm and
by removing insulation near the tips manually, was lightly inserted in
the horizontal commissure (CH), which is a clearly identifiable fiber
bundle that enters the CG dorsally (Fig. 1C). Rectangular
current pulses (100-µs duration) were delivered through an isolator
(SS-201J, Nihon Kohden, Tokyo). Another thinner fiber bundle also
entered the CG dorsolaterally (Fig.
3B, ). This fiber bundle
also originated from the corticalis neurons but rather from the
posterior part (unpublished observation). Although electrical stimulation of this fiber bundle evoked postsynaptic potentials similar
to that of the CH fibers, the main bundle was stimulated in the present
study. The intensity of the stimulus current was usually set at the
minimum value that evoked the maximum postsynaptic response in the
control condition (0.02-0.5 mA), unless otherwise noted.
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Intracellular staining
Neurobiotin or biocytin was sometimes added to the
microelectrode solution (2%) or to the pipette solution (0.5-1%) for
the intracellular stainings. Intracellularly recorded neurons were labeled by passing depolarizing current pulse (4-7 nA, 800-ms pulse at
1 Hz, for 5 min), while patch clamped neurons could be labeled only by
diffusion of the pipette solution. After survival time of 30 min, the
brain slice was fixed with 5% paraformaldehyde in 0.1 M phosphate
buffer (pH 7.4) for 30 h. The slice was treated with 0.3%
H2O2 in phosphate-buffered
saline (PBS) for 30 min to reduce endogenous peroxidase activity and
rinsed with PBS containing 0.3% Triton-X 100 (PBST). The slice was
then incubated with ABC solution of 0.5% vol/vol (Vector Laboratory)
for 3 h. The slice was rinsed again with PBST, and labeled neurons
were then visualized by 3',3'-diaminobenzidine (DAB) reaction.
Counterstaining was not performed because the macroscopic structure
could be identified without staining. Half of the slices were
dehydrated with ethanol series and xylene and then cover-slipped. It
was previously reported that morphological artifacts of bending and
curling are induced in the intracellularly labeled dendrites with DAB
products during the alcohol-dehydration process (Grace and
Llinas 1985). To avoid this potential artifact, the remaining
half of the slices were cleared by immersing in dimethyl sulfoxide
(DMSO), which has been shown to induce little artifact (Grace
and Llinas 1985
), and were directly coverslipped by sealing the
edge with silicon-based glue. However, we could not find significant
differences between the two groups of preparations.
Solutions and drugs
The electrolyte composition and osmolarity of the serum of two
marine teleosts, filefish (Navodon modestus) and labrid
(Halichoeres poecilopterus), were measured (data not shown),
and the composition of normal ACSF for marine teleost was determined as
follows (in mM): 126 NaCl, 4.0 KCl, 1.0 MgSO4,
1.7 CaCl2, 26 NaHCO3, 1.0 NaH2PO4, and 10 glucose.
The composition of sucrose based low-Na ACSF was (in mM) 0 NaCl, 4.0 KCl, 8.5 MgSO4, 0.17 CaCl2,
26 NaHCO3, 1.0 NaH2PO4, 10 glucose, and
210 sucrose. Ca2+-free ACSF was prepared by
substituting CaCl2 with equimolar
MgCl2, and Mg2+-free ACSF
was prepared by just omitting MgSO4. ACSF was
continuously bubbled with 95% O2-5%
CO2 (pH ~ 7.4). Compositions of the
K-gluconate based patch pipette solution was (in mM) 147 KOH, 3 KCl,
0.2 EGTA, 2 MgCl2, 2 NaATP, and 10 HEPES,
adjusted to pH 7.3 with gluconic acid. Glutamate receptor antagonists,
6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and
D2-amino-5-phosphonovaleric acid (D-APV) were
purchased from Tocris and
-aminobutyric acid (GABA) receptor
antagonist bicuculline from Sigma. Tetorodotoxin (TTX) was from Wako
pure chemicals (Kyoto, Japan), and the other salts and popular
chemicals were from Sigma and Nacalai tesque (Kyoto, Japan).
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RESULTS |
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Brain slice preparation and cell identification
The concentrically layered structure of the type III CG previously
described in histological preparations (Ito and Kishida 1975) could be clearly identified in nonfixed brain slices
under a dissection microscope (Fig. 3, A and B).
Furthermore soma of the large cell and the small cell were visually
identified and distinguished using DIC optics under an upright
microscopes (Fig. 3C), which could be further verified by
whole cell patch-clamp and subsequent intracellular staining (see the
next section). The structures that correspond to the glomerulus
comprising the tip of the large cell dendrite were sometimes clearly
identifiable in the glomerular layer at the surface of a slice under a
dissection microscope (Fig. 3B), but no successful
penetration with intracellular microelectrode could be done from these
structures probably because these dendrites were severely damaged
during the slicing process. In contrast, stable intracellular
recordings could be performed rather blindly from the deeper part of
the slice. These recordings were regarded to be from the tip of the
large cell dendrite, because all of the subsequent successful
intracellular staining revealed the characteristic morphology of the
large cells (n = 34 cells, see the next section), the
penetrations were usually stable against substantial movement
(10-20 µm) of the microelectrode along the long axis of the
electrode, and the passive membrane properties revealed low input
resistance [3.8 ± 1.4 (SD) M
; n = 16] and large membrane capacitance (1.2 ± 0.6 nF;
n = 16; see the later section), and there is no
candidate structure that shows such unusual electrical properties other
than the tip of the large cell dendrite.
Morphology
LARGE CELL.
The intracellularly labeled large cells revealed their characteristic
morphology (Figs. 4 and
5). Most cells had a single dendritic
stalk extending from the soma, which is located in the peripheral
fibrous capsule, toward the center of the GR and had an extremely
enlarged dendritic tip with complex arborizations (Fig. 4, A
and B). This tip structure is the postsynaptic component in
the glomerulus and corresponds to the "star-like structure" previously described by Ito and Kishida (1975, 1977
).
There was no significant difference among the morphology of the large
cells labeled using "dendritic" intracellular microelectrodes and
"somatic" patch pipettes. However, no soma was observed in 47%
(16/34 cells) of the dendritic stainings, likely because their soma was
cut out of the brain slice, leaving the dendritic tips in the slice. It
was also revealed that 18% (10/56 cells) of the large cells (dendritic
as well as somatic stainings) had two or more dendritic tips (Fig. 5,
A2 and 2; TD). In that case, the cell had two
dendritic stalks arising directly from the soma or had a dendritic
branch point in the GL. Most of the stalks of the dendrites of the
large cells were radially oriented toward the center of the GR (Figs. 4
and 5A), while some large cells had an extremely long stalk and nonradial orientations (Fig. 5B). The results of the
dendritic and somatic stainings of the large cell are summarized in
Table 1.
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SMALL CELL.
A typical example of a labeled small cell is shown in Fig.
6. The small cell had several dendrites
extending toward the center of the GR from the soma located in the
small cell layer. Because the dendrites were usually as long as
150-300 µm and sometimes exceeded 500 µm, many of them were long
enough to reach the glomerular layer. The previous anatomical study on
fiber connections of the CG (Sakamoto and Ito 1982) did
not describe direct synaptic connections from the nucleus corticalis to
the small cell. However, such synaptic inputs could be demonstrated
electrophysiologically as described in a later section (see
Postsynaptic responses evoked by the CH fiber stimulation).
Considering the results of the intracellular staining of the small cell
in the present study and the previous report that the lesion of the
nucleus corticalis produced degenerating nerve terminals in the
glomeruli and the glomerular layer (Sakamoto and Ito
1982
), it is probable that the long dendrites of the small cell
in the glomerular layer receive synaptic inputs from the nucleus
corticalis.
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Membrane parameters and firing properties
Resting potentials of the large cell were -60 ± 5.0 mV
(n = 45) and -55 ± 5.5 mV (n = 74) in the dendritic and the somatic recordings, respectively, and
those of the small cell were 65 ± 3.3 mV (n = 50). As expected from its characteristic morphology, the tip of the
large cell dendrite had very low input resistance, 3.8 ± 1.4 M
, and large membrane capacitance, 1.2 ± 0.6 nF, which reflects the extremely abundant arborizations (Fig. 4). If we assume
that the membrane of the large cell dendrite has a specific capacitance
of 1.0 µF/cm2 (De Bruinje 1984),
the mean membrane capacitance of 1.2 nF corresponds to 1.2 × 105 µm2, which is as
large as the surface area of a spherical membrane of 100 µm in
diameter. These and other physiological parameters for the large cell
and the small cell soma are shown in Table 2.
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To examine the firing properties of the large cell soma, current pulses
(0.2 to +0.35 nA, 0.05-nA increment, 150-ms duration) were
applied in the current-clamp mode of the somatic whole cell recordings
(Fig. 7). In a preliminary study, most of
the voltage-dependent channels that are involved in the generation of
action potentials seemed to be inactivated at the resting potentials
(
55 ± 5.5 mV) because the active voltage responses with
depolarizing current steps were very small, while anode break spikes
were generated after the hyperpolarizing current steps (
0.2 to
0.1 nA). Therefore current steps were applied to the cell held at
around
75 mV by injecting a hyperpolarizing DC holding current
(
0.1 to
0.7 nA), realizing that the resting potentials of the
large cell in the slice preparations may be more depolarized than those
in the in vivo condition. The large cell soma fired a single action
potential with suprathreshold current pulses (0.1 ± 0.07 nA,
n = 13). The large cell soma always fired a single
spike in response to much longer or stronger current stimuli. Because
the spikes were blocked by 1 µM TTX, a fast voltage-dependent
Na+ channel blocker, they were regarded to be
fast Na+ spikes (Fig. 7A1). On the
other hand, no dendritic action potential could be evoked at the
dendritic tip of the large cell by depolarizing current pulses as large
as 2.5 nA (Fig. 7A2) or by depolarization up to
20 mV
induced by bath application of L-glutamate (4 mM; the tips
have ionotropic glutamate receptors, see later section).
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In contrast, the small cell had completely different firing properties from that of the large cell (Fig. 7B). The small cell generated regular spike trains in response to depolarizing current pulse injections of much smaller amplitude (without hyperpolarizing holding current) and showed almost no spike frequency accommodation (Fig. 7B1). The spike frequency depended on the amplitude of the injected current and easily exceeded 100 Hz by current pulses 30 pA (Fig. 7B2). The spikes were also TTX sensitive (n = 5) and could be regarded as conventional fast Na+ spikes (Fig. 7B3).
Postsynaptic response evoked by the CH fiber stimulation
PHYSIOLOGY.
It has been described anatomically that the large cell receives
synaptic inputs mainly at the tip of the dendrite in the glomerulus from the CH fiber which originates in the nucleus corticalis
(Sakamoto and Ito 1982). To study the physiology of the
synaptic transmissions from the nucleus corticalis to the large cell,
postsynaptic potentials (PSPs) evoked by stimulation of the CH fiber
were recorded at the tip of the large cell dendrite (intracellular
recording) and at the soma (whole cell recording). The major component
of the evoked PSP at the tip of the large cell dendrite was excitatory. Strong CH fiber stimulation did not evoke postsynaptic dendritic spikes
(Fig. 8A1, see also Fig.
7A2). The PSPs sometimes contained a small inhibitory
component (Fig. 8A1,
, see also Fig.
9). As expected for monosynaptic
transmissions, latencies from the CH fiber stimulation to the onset of
the PSP were highly constant among the responses against repeated
stimulation at 5 Hz (Fig. 8A3). At the soma, a single spike
was induced by the PSP evoked by a suprathreshold stimulation. Similar
to the voltage response with increasing current injections (previous
section), the large cell always fired a single spike with stimulation
of a stronger intensity. (Fig. 8A2).
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PHARMACOLOGY.
To study the neurotransmitters and the receptor subtypes involved in
the synaptic transmissions from the CH fibers, a series of
pharmacological experiments were performed. Bath application of CNQX
(10 µM), an -amino-3-hydroxy-5-methyl-4-isoxazole-proprionate (AMPA) type glutamate receptor antagonist, selectively blocked the
excitatory component of the PSP at the tip of the large cell dendrite
(Fig. 9A, 1 and 2; n = 20), and
at the small cell soma (Fig.
10A, 1 and
2; n = 10) in normal ACSF. In most cells (LC
dendrite: n = 17/20; SC: n = 6/10),
only the inhibitory response (IPSP) remained (Figs. 9A2 and
10A2), while the PSPs were totally blocked in the other
cells. The remaining IPSP was abolished by additional bath application
of bicuculline (20 µM), a GABAA receptor
antagonist (Figs. 9A3 and 10A3). Latencies of the
IPSP isolated by CNQX were also short and constant against repeated
stimuli, and could be regarded to be monosynaptic responses as well
(Fig. 9A4). In the presence of both CNQX and bicuculline,
PSPs were almost completely blocked (Fig. 9A3). However, in
the absence of external Mg 2+, which is known to
release N-methyl-D-aspartate (NMDA) type
glutamate receptors from a voltage-dependent blockade (Mayer et
al. 1984
; Nowak et al. 1984
), a substantial slow
excitatory PSP (EPSP) appeared (Fig. 9B, 1-3). This slow
EPSP was fully unmasked in the combined blockade of the fast EPSP by
CNQX (20 µM) and the IPSP by bicuculline (Figs. 9B3 and
10B1). Because this component was completely blocked by
further application of D-APV (25 µM; Figs. 9B4
and 102; n = 13 at the LC dendrite,
n = 7 at the SC), a selective NMDA receptor antagonist,
it can be regarded as an NMDA receptor-mediated EPSP. Thus the synaptic
transmissions from the corticalis neurons to both the large cell and
the small cell are suggested to consist of a combination of a fast AMPA
receptor-mediated EPSP, a slow NMDA receptor-mediated EPSP, and a
GABAA receptor-mediated IPSP. According to an
ultrastructural study (Ito and Kishida 1977
), S-type
axon terminals are the major terminals that synapse onto the tip of the
large cell dendrite, and F-type terminals are the minor ones, while it
has been suggested that the S and F terminals are excitatory and
inhibitory synapses, respectively (Komuro 1981
). Therefore it is very likely that the major S and the minor F terminals mediate excitatory glutamatergic and inhibitory GABAergic synaptic transmissions, respectively.
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Temporal filtering properties of postsynaptic responses against paired inputs
Finally, to examine the properties of the evoked postsynaptic response in relation to the temporal aspect of the corticalis activities, a pair of stimuli with different inter-stimulus intervals (25-500 ms) were delivered to the CH fibers, and the dependence of the amplitude of the postsynaptic responses (i.e., PSPs at the tip of the large cell dendrite, and spikes at the soma of the large cell and the small cell) on the inter-stimulus interval was studied. PSPs at the tip of the large cell dendrite were highly constant and showed no paired-pulse facilitation or depression at any inter-stimulus intervals examined in the present study (Fig. 11A, TD), which suggests that both neurotransmitter release from the CH fiber terminal and sensitivities of the postsynaptic receptor are constant for the two successive stimuli. However, at the soma, spike amplitudes of the second responses did not show all-or-none properties and were obviously depressed at intervals of 200 ms (Fig. 11A, LC soma). The relative amplitude of the second response to the first response (2nd/1st) at the 25-ms intervals was 0.17 ± 0.09 (n = 10), and the mean inter-stimulus interval at which 50% of the amplitude depressions occur was estimated to be ~75 ms (Fig. 11B, top). A stimulation intensity that evoked a single postsynaptic spike was used for the small cell. In contrast to the large cell, the small cell soma showed postsynaptic spike responses of constant amplitudes at any inter-stimulus interval (25-500 ms; Fig. 11, A, SC soma, and B, bottom; n = 9). When a short spike train was evoked by stimulation with stronger intensity, the profile of the second spike trains was almost identical to that of the first ones (Fig. 11C). Thus it is suggested that the large cell functions as a low-pass temporal filter for corticalis inputs with a cutoff frequency of 5-40 Hz (corresponding to the inter-stimulus interval of 25-200 ms), while the responses of the small cell retains fidelity to the temporal input patterns from corticalis, at least to the inputs with frequency lower than 40 Hz.
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DISCUSSION |
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In this study, we developed an in vitro brain slice preparation of the type III CG and, taking advantage of its clearly laminated architecture, analyzed the single cell morphology and physiology of the two types of the principal projection neurons. We find that the two types of cells, the large and the small cell, may encode different aspects of the afferent neuronal activities.
Large cell
The large cell of the type III CG has an outstanding morphological feature, a huge enlargement of the dendritic tip, which has not been found in the brains of other vertebrates. The larger the area of the postsynaptic membrane, the larger the number of presynaptic terminals the postsynaptic cell can receive. But the larger postsynaptic membrane is expected to have lower input resistance, which decreases the amplitude of the PSP contributed by each synaptic terminal. Therefore it may be possible that the huge dendritic tip of the large cell is specialized for "averaging" synaptic inputs from a large number of corticalis axons.
The large cell received excitatory glutamatergic and inhibitory
GABAergic synaptic inputs just like the central neurons of other
vertebrate brains. Besides AMPA and GABAA
receptors, substantial NMDA receptor-mediated postsynaptic potentials
were identified in the absence of external Mg2+,
which blocks NMDA receptors voltage dependently (Mayer et al. 1984). In the present study, it was difficult to stably
depolarize the membrane potential large enough for removing
voltage-dependent Mg2+ block of NMDA receptors in
the presence of external Mg2+ (1.7 mM) by
injecting current through the microelectrode because the membrane
resistance was too low and it required injection of too large current
to be controlled. However, PSPs evoked by stimulations of the CH fibers
had a mean amplitude of 25 mV, and the membrane potentials were
depolarized to around
35 mV, which is enough for activating some
portion of the NMDA receptors in the presence of
Mg2+ (Nowak et al. 1984
).
Furthermore, considering that only some of the CH fibers are stimulated
in the present in vitro study, there may be a chance in vivo that a
larger depolarization occurs in response to the activation of more of
the CH fibers. Thus it is concluded that the NMDA receptors can
potentially contribute to the synaptic transmission in vivo.
The large cell fired a single Na+ spike and never
fired spike trains even in response to large current injections or
strong synaptic stimuli. Furthermore the amplitudes of the PSP-induced spikes in response to paired synaptic stimuli were not all-or-none but
were gradually attenuated dependent on the inter-stimulus interval
(low-pass filtering). The half recovery time of the spike amplitude,
which was estimated to be ~75 ms, seems to be unusually long,
considering that nearly 90% of the conventional fast sodium channels
recover from the inactivated state in 10 ms (Hille
1992). These results may imply that the
Na+ channels involved in the generation of the
action potential in the large cell recover more slowly from the
inactivated state. This suggestion was supported by our preliminary
voltage-clamp experiments, which revealed an unusually long time
constant (>100 ms) of the recovery process. Thus the analysis of the
kinetics of the Na+ current may be a future key
to understanding the mechanisms underlying the low-pass filtering
property in the large cell.
The low-pass filtering property revealed by the paired CH fiber
stimulations suggests that the large cell fires a full spike only when
a substantial number of corticalis neurons are activated after a long
enough period (~200 ms) since the previous firing, while it fires an
attenuated spike in response to inputs with shorter intervals (Fig.
12, large cell). It has been reported
that in vivo corticalis neurons show burst firings with a spike
frequency of ~5-20 Hz (Rowe and Beauchamp 1982),
which is within the dynamic (sensitive) range of the temporal filtering
property of the large cell revealed in the present study. Therefore we
suggest that, in vivo, the large cells process and modify the temporal
pattern of afferent inputs from corticalis neurons.
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Small cell
Both the morphology and the physiology of the small cell
contrasted to that of the large cell. In response to current injections or synaptic stimuli, the small cell fired spike trains whose frequency depended on the current amplitude or the stimulus intensity. It was
clearly shown in the present study that spikes of the small cell are
associated with large afterhyperpolarizations that promote recovery of
voltage-dependent sodium channels from the inactivated state, while the
afterhyperpolarization of the large cell spikes are much smaller. It
remains to be determined whether the differences in the firing
properties between large cells and small cells comes from differences
in the kinetics, densities, distribution of ion channels, and/or
neuronal morphology, which have been suggested to be critical for the
determination of neuronal firing properties (Mainen and
Sejnowski 1996; Migliore et al. 1995
).
In contrast to the large cell, the small cell showed no temporal
filtering with paired stimuli of intervals of 25-500 ms. Because the
spike frequency in the previously reported corticalis activities in
vivo rarely exceeded 20 Hz (Rowe and Beauchamp 1982), it
is concluded that the response of the small cell can follow the
temporal firing patterns of the corticalis neurons (Fig. 12, small
cell). Thus the present results suggest that the small cell encodes the
number of excited corticalis neurons at a given time into the frequency
of a spike train.
Functional perspectives of the CG
In the classical view of neural coding, information in neural
systems is carried by changes in the spike discharge rate of neurons
(spike rate code). More recently, however, it has been proposed that
information may be coded by the timing of spike discharges among
numbers of neurons (timing code; Mainen and Sejnowski 1995; Riehle et al. 1997
). In particular,
synchronous firing has been shown to be related to various neural
representations in the CNS of primates (Steinmetz et al.
2000
; Vaadia et al. 1995
). If we assume that the
corticalis neurons employ a timing code, the present study suggests
that the large cell in the CG may extract a slow component of synchrony
in the firing of corticalis neurons as a timing code, while the small
cell may translate the number of corticalis inputs into a spike rate code.
From several tract tracing studies (Campbell and Ebbesson
1969; Ebbesson 1968
; Ito et al.
1980
; Sakamoto and Ito 1982
; Vanegas and
Ebbesson 1976
), it has been proposed that the CG receives multi-modal telencephalic input, somatosensory and most probably visual, and then projects to the inferior lobe of the hypothalamus. While the function of the inferior lobe is not well understood in
teleosts, it has been suggested that it too receives multimodal sensory
inputs (including gustatory) (Shimizu et al. 1999
), and it is involved in mediating feeding and aggressive behaviors
(Demski 1983
). Therefore although it is still difficult
to suggest the behavioral significance of the CG at this time, encoding
of the different aspects of corticalis activity by the large and the small cells in the CG may be critical for the control of such behaviors
by first processing visual and other sensory inputs and then relaying
such information to the inferior lobe.
Type III CG as a model system for the study of "neuronal circuit"
Because it has been hypothesized that information processing in
the CNS occurs in a parallel and a distributed way through large
numbers of cells, measuring neuronal activities in large numbers of
identified cells with high spatial and temporal resolution has been one
of the most fundamental challenges in the modern neuroscience. The
morphology of the tip of the large cell dendrite in the type III CG may
be advantageous for multiple recordings of postsynaptic activities by
means of various imaging techniques because large numbers of dendritic
tips could be retrogradely labeled with fluorescent dyes (FITC-dextran
or Ca2+-Green dextran) injected into the inferior
lobe, with each tip then clearly identified under conventional optics
with a low-magnification objective (Tsutsui and Oka, unpublished
data). Thus taking advantage of its well-defined architecture, the type
III CG may provide an excellent preparation for a "simple neuronal
circuit," in which each constituent cell can be
electrophysiologically characterized as in the present study and a
large number of postsynaptic activities may be simultaneously monitored
with the resolution of a single postsynaptic cell using various kinds
of environment-sensitive fluorescent probes, which is still difficult
to accomplish in other vertebrate neuronal systems (Tsutsui et
al. 1998).
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ACKNOWLEDGMENTS |
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We thank the technical staffs of Misaki Marine Biological Station for help in catching and maintaining fish, Drs. Y. Takei and S. Hasegaea for the analysis of electrolyte compositions of fish serum, and Dr. T. Manabe for helpful discussion and a generous gift of glutamate receptor antagonists.
This work was supported by Grants-in-Aid from the Ministry of Education, Science, Culture, and Sports of Japan to Y. Oka and by Sasagawa Scientific Grant and Research Fellowships of the Japan Society for Promotion of Science for Young Scientists to H. Tsutsui.
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FOOTNOTES |
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Address for reprint requests: Y. Oka (E-mail: okay{at}mmbs.s.u-tokyo.ac.jp).
Received 16 August 2000; accepted in final form 27 November 2000.
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REFERENCES |
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