Encoding of Different Aspects of Afferent Activities by Two Types of Cells in the Corpus Glomerulosum of a Teleost Brain

Hidekazu Tsutsui,1 Naoyuki Yamamoto,2 Hironobu Ito,2 and Yoshitaka Oka1

 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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 1. An overview of the corpus glomerulosum (CG) system. A: lateral view of the brain of Stephanoplepis cirrhifer showing the location of the CG. The CG is schematically illustrated. Dotted line indicates the level of the transverse brain slice used in this study. Anterior is to the left, dorsal to the top. Scale bar = 2 mm. B: schematic illustration of the major fiber connections related to the CG based on previous anatomical reports (Campbell and Ebbesson 1969; Ebbesson 1968; Murakami et al. 1986; Sakamoto and Ito 1982; Shimizu et al. 1999) and our new finding in this study (dotted line showing the projection from the nucleus corticalis to the small cell). The interrupted line indicates the midline of the brain. L and R, the left and right side of the brain, respectively. Note that projections to the right CG are not shown for clarity. C: schematic 3-dimensional illustration showing the course of the axonal fibers from the nucleus corticalis (CR) of both sides to the left CG, which correspond to the horizontal commissure (CH) fibers. Projections to the right CG are not shown for clarity. Fibers from the left CR directly enter the CG from the dorsal side (solid lines), while fibers from the right CR (dotted lines) enter the right CG first (1), run anteriorly (2), enter the left side of the brain (3; commissure), and finally run posteriorly and enter the left CG (4). Even though the axonal fibers projecting ipsilaterally from the nucleus corticalis do not enter the other side of the brain, they are also referred to as the horizontal commissure fibers since they form a thick fiber bundle together with contralaterally projecting fibers and are not distinguishable macroscopically. A, P: anterior and posterior; D, V: dorsal and ventral; L, R: left and right are indicated in the inset.

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.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 2. Schematic illustration showing the experimental configuration of the present study. Intracellular recordings were made from the tip of the large cell dendrite in the glomerular layer (GL), and whole cell recordings were made from the soma of the large cell and the small cell located in the peripheral fibrous capsule (PC) and the small cell layer (SCL). The CH fibers were stimulated using a bipolar electrode to evoke postsynaptic responses. The illustration is not drawn to the scale.

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 Omega ) 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 MOmega . 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, black-triangle). 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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Fig. 3. A: a frontal brain slice preparation viewed under a dissection microscope. B: higher magnification of the CG in A. Four concentrically arranged layers [GL, fibrous layer (FL), SCL, PC] can be identified. CH fibers from the ipsilateral CR enter the CG from the dorsal side. Note that another thinner fiber bundle enters dorsolaterally to the GR (black-triangle). This fiber bundle also originates from the corticalis neuron, but rather from a posterior population (see METHODS). Scale bars = 1 mm in A and B. C: a video photomicrograph of the CG slice under a differential interference contrast (DIC) optics. The somata of the large cell (**) and the small cell (*) were easily identified from their size, location, and morphology. Bar = 50 µm.

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 D-2-amino-5-phosphonovaleric acid (D-APV) were purchased from Tocris and gamma -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).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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) MOmega ; 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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Fig. 4. Intracellular stainings of the large cell. A, 1 and 2: intracellularly labeled large cell viewed at the focal plane of soma (S) in A1 and at the tip of the dendrite (TD) in A2. Arrows, the stalk of the dendrite (ST), which is somewhat out of focus. A3: camera lucida reconstruction of the large cell in A, 1 and 2. Dotted line, the contour of the tip of the dendrite. Orientations (dorsal, lateral and center of the CG) are shown in the inset. The axon was not visible. Scale bar in A1 = 100 µm and applies to A, 1-3. A4: high magnification of the tip of the dendrite in A, 1 and 2. B, 1 and 2: tip of the dendrite in another cell at 2 different focal planes. Scale bar in A4 = 50 µm and applies to B, 1 and 2, as well. Both cells in A and B were labeled from the somata.



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Fig. 5. A: camera lucida drawings of 2 intracellularly labeled large cells. Dotted lines, the contour of the tip of the dendrite (TD). The cell in A2 has 2 dendrites with tips (TD). Orientation (dorsal, lateral, and center of the CG) are indicated in the inset, and in A2 orientation to the center of the CG is perpendicular to the plane of the drawing. Scale bar = 100 µm. B: camera lucida drawings at low magnification of large cells with long stalks of dendrites. The cell in B2 has 2 dendrites with tips (TD). Scale bar = 500 µm. Arrowheads, the location of the electrode from which cells were labeled. Axons were not visible.


                              
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Table 1. Summary of intracellular staining of large cells

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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Fig. 6. A1: Photomicrograph of the intracellularly labeled small cell. Arrows, some of the dendrites that are out of focus; arrowhead, the axon; dotted lines, the outline of the small cell layer (SCL). A2: a camera lucida reconstruction of the small cell in A1. Arrowhead, the axon. Scale bar in A1 = 100 µm and also applies to A2. A3: camera lucida drawings at low magnification of the same small cell as in A, 1 and 2. The axon (arrowhead) could be followed as long as 1 mm toward the inferior lobe through the peripheral fibrous capsule. Scale bar = 500 µm.

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 MOmega , 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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Table 2. Basic electrophysiological parameters

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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Fig. 7. Firing properties of the large cell (A) and the small cell (B). A1: voltage responses of the large cell soma at steps of current pulses (-0.2 to +0.35 nA, 0.05-nA increment, 150-ms duration). The current steps were applied from a negative holding current of -0.5 nA, which hyperpolarized the cell to -76 mV from somewhat depolarized resting potentials (see RESULTS). In the control traces (top), a single action potential was evoked against each suprathreshold current step (>=  +0.15 nA), which was completely blocked by 1 µM TTX (bottom). A2: voltage responses against steps of current pulses (-1.5 to +1.0 nA, 0.5-nA increment, 200-ms duration) at the tip of the large cell dendrite (TD). B1: voltage response of the small cell soma at steps of current pulses (top, 10 pA; middle, 30 pA, 150-ms duration, without holding current). The current levels are indicated at the bottom. Resting potential of this cell was -66 mV. In contrast to the large cell, the small cell fired a train of spikes. The spike train showed little frequency accommodation, and the frequency depended on the current amplitude (B2). B2: the dependence of spike frequency on the current amplitude in small cells (n = 9). B3: the spikes were completely blocked by 1 µM TTX (current pulses: -30 to +40 pA, 10-pA increment, 150-ms duration).

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, black-triangle, 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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Fig. 8. Postsynaptic responses at the large cell (A) and at the small cell (B) in response to the CH fiber stimulation. A1: postsynaptic potentials (PSPs) at the tip of the large cell dendrite (TD). Eight sweeps with different stimulus intensities are superimposed. Resting potential was -53 mV. Note that an inhibitory PSP (IPSP) component is clearly seen in response to weaker stimuli (black-triangle). A2: PSP and PSP-induced action potentials at the large cell soma. Four sweeps with different stimulus intensities are superimposed. The cell was held at -76 mV by a negative holding current (-0.7 nA). The cell never fired more than a single spike even against strong stimuli. A3: 5 superimposed sweeps evoked at 5 Hz with the same stimulus intensity showing the time course of the onset of the PSPs at tip of the large cell dendrite. Note the short and constant latency. B1, top: 10 superimposed sweeps evoked by different stimulus intensities in the small cell. The small cell fired a train of a few spikes, and the frequency depended on the stimulus intensity. The resting potential of the cell was -68 mV. Bottom: raster plots showing the timing and frequency of the spikes in the top traces and their dependence on stimulus intensity. B2: 2 superimposed seeps evoked at 5 Hz showing the shortness and constancy of the PSP latencies at the small cell. The resting potential was -67 mV.



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Fig. 9. Pharmacology of the PSPs at the tip of the large cell dendrite. A: PSPs in normal ACSF. The excitatory component was blocked by bath application of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 µM), an AMPA receptor antagonist, and inhibitory PSP (IPSP) remained after that (A, 1 and 2). The remaining IPSP was abolished by an additional application of bicuculline (Bic; 20 µM), a GABAA receptor antagonist (A3). Five IPSP responses evoked at 5 Hz in another cell in the presence of CNQX are superimposed (A4). The latencies of IPSP responses were highly constant. B: PSPs in Mg2+-free ACSF. A slow excitatory component remained in the presence of 20 µM CNQX and 20 µM bicuculline (B, 1-3), which was finally blocked by D-2-amino-5-phosphonovaleric acid (D-APV, 25 µM), an N-methyl-D-aspartate (NMDA) receptor antagonist (B, 3 and 4). The resting potentials were -59 mV in A, 1-3, -60 mV in A4, and -58 mV in B.

PSPs evoked by the CH fiber stimulation were then recorded at the small cell soma (Fig. 8B). The latencies of the PSPs evoked by repeated stimuli at the small cell were also short and highly constant, and the synaptic transmission to the small cell could be regarded to be monosynaptic (Fig. 8B2). The small cell, in contrast to the large cell, fired a short spike train, the number of which depended on the stimulus intensity (Fig. 8B1). It is therefore suggested that the small cell can encode the number of simultaneously excited corticalis neurons at a given time in the frequency of the spike train, while the large cell cannot do so and can only respond with a single spike against the suprathreshold synaptic inputs.

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 alpha -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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Fig. 10. Pharmacology of the postsynaptic responses at the small cell in the normal ACSF (A) and in the Mg2+-free ACSF (B). PSP-induced spike train in the normal ACSF (A1). The excitatory component of the PSP was blocked by 10 µM CNQX, and a small IPSP remained (A2). A trace in slower time scale is shown for clarity in the inset. The IPSP was blocked by additional application of 20 µM bicuculine (Bic; A3). In the Mg2+-free ACSF, excitatory PSP in the presence of 20 µM CNQX and bicuculine was large enough to induce a spike train (B1). The EPSP was finally blocked by 25 µM APV (B2). The resting potentials were -66 mV in A and -67 mV in B.

It has been claimed in histochemical studies (Wullimann and Meyer 1990; Wullimann and Roth 1992) that nucleus corticalis may be cholinergic. However, high-dose application of the cholinergic agonists, acethylcholine (5 mM) and carbamylcholine (1 mM), and an antagonist, D-tubocurarine (20 µM), had no effect on the resting potentials and evoked PSPs at the tip of the large cell dendrites (not shown). Although there still remains a possibility that acethylcholine may function as a neurotransmitter via metabotropic receptors, it is clear that the neurotransmitters that mediate the "fast" synaptic transmissions via ionotropic receptors here are glutamate and GABA.

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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Fig. 11. Postsynaptic response at the tip of the large cell dendrite (TD; left), the large cell soma (middle), and the small cell soma (right) evoked by paired-stimulation with different inter-stimulus intervals (25-500 ms; delivered at 0.2 Hz). A: traces for inter-stimulus interval of 500, 100, and 25 ms are shown. At the tip of the dendrite (left), the PSP amplitudes were highly constant. In contrast, at the large cell soma (middle) the 2nd spikes were obviously depressed in amplitude at the intervals 100 ms. The small cell showed highly constant spike responses at any inter-stimulus intervals examined (right). The resting potential of the tip of the large cell dendrite and the small cell were -56 and -64 mV, respectively. The large cell soma was held at -76 mV. B: averaged ratio of the amplitude of the 2nd response normalized to the 1st (mean ± SD) as a function of inter-stimulus intervals (ms) at the tip of the large cell dendrite (; n = 10), the large cell soma (black-diamond ; n = 10) in the top graph and at the small cell (black-diamond ; n = 9) in the bottom. C: postsynaptic spike responses of the small cell with paired stimulation at a stronger intensity with 50-ms interval, which evoked 2 (top) or 3 spikes (bottom). The 2 postsynaptic responses were exactly the same in shape and amplitude. The resting potentials of the cell were -67 and -62 mV for the top and the bottom traces, respectively.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 12. Schematic summary diagram illustrating the function of the large cell and the small cell suggested in the present study. open circle  and  in the left column symbolize spatial and temporal patterns of the corticalis cell activities. open circle  and , resting and excited corticalis cells, respectively. Expected postsynaptic response corresponding to each input pattern at the large cell soma and the small cell soma are shown to the right. In response to the 1st corticalis input, the large cell soma only fires a single full spike whenever it receives suprathreshold input (middle and bottom rows). It only fires a full spike after a period long enough from the previous firing (e.g., 3rd response), whereas it fires an attenuated spike in response to inputs with shorter intervals (e.g., 2nd response). Thus the large cell extracts the temporal feature of the corticalis activities (encoding of temporal aspect) but not the number of excited corticalis cells at a given time. In contrast, the small cell encodes that numbers into the frequency of the spike train, while the responses follow the temporal patterns of the inputs.

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).


    ACKNOWLEDGMENTS

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.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

0022-3077/01 $5.00 Copyright © 2001 The American Physiological Society