Electrophysiological and Morphological Properties of Neurons in the Rat Superior Colliculus. I. Neurons in the Intermediate Layer

Yasuhiko Saito and Tadashi Isa

Department of Integrative Physiology, National Institute for Physiological Sciences, Myodaiji, Okazaki 444-8585, Japan


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Saito, Yasuhiko and Tadashi Isa. Electrophysiological and Morphological Properties of Neurons in the Rat Superior Colliculus. I. Neurons in the Intermediate Layer. J. Neurophysiol. 82: 754-767, 1999. To begin characterizing the neural elements underlying the dynamic properties of local circuits in the mammalian superior colliculus (SC), electrophysiological and morphological properties of individual neurons in the intermediate layer [stratum griseum intermediale (SGI)] were investigated using whole cell patch-clamp recording and intracellular staining with biocytin in slice preparations from young (17-22 days old) and adult rats (7-8 wk old). Voltage responses to depolarizing current pulses of 223 neurons recorded in young rats were classified into six subclasses: regular-spiking neurons (n = 113), interspike intervals during depolarizing current pulses were constant; late-spiking neurons (n = 48), initiation of repetitive firing was delayed markedly from the onset of depolarizing pulses because of a transient hyperpolarization caused by A-like currents; burst-spiking neurons (n = 29), transient burst firing due to low-threshold Ca2+ channels were observed at the firing threshold level; fast-spiking neurons (n = 19), constant repetitive firings at frequencies >100 Hz were observed for the duration of the depolarizing pulse; neurons with marked spike frequency adaptation (n = 11), interspike intervals more than doubled due to spike frequency adaptation during depolarizing pulses; and neurons with rapid spike inactivation (n = 3), spike amplitude rapidly reduced, width increased during depolarizing pulses, and spiking was terminated after generating a few spikes. In response to hyperpolarizing current pulses, two different types of inward rectification were observed; time-dependent inward rectification by hyperpolarization-activated current (Ih; n = 29) and time-independent inward rectification (n = 111). Morphological analysis showed that neurons expressing time-dependent inward rectification by Ih had large somata, extended divergent dendrites dorsally into the superficial layers, and projected axons ventrally and sometimes dorsally, all characteristic features of wide-field vertical cells. Other neurons exhibited heterogeneous morphological properties, such as multipolar, fusiform, horizontal, or pyramidal-shaped cells. In adult rats, a total of 44 neurons showed similar electrophysiological properties except for the last type. These results indicate that the local circuits of the SC include neurons with at least five different firing properties and two different rectification properties; each with distinct electrophysiological and morphological characteristics that may be correlated with the functional output of the SC.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 MOmega in the bath solution, and the series resistance during recording was 10-25 MOmega . 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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.



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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, down-arrow ). 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.



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Fig. 2. Electrophysiological properties of a late-spiking neuron. A: responses to depolarizing current pulses (values given at right). down-arrow , 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, down-arrow ). 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.



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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. down-arrow , 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.



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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.



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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.



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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, down-arrow ). 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.



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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 (down-arrow ). 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).



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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.


                              
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Table 1. Characteristics of each subclass of neurons recorded in young rats



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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).



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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 (up-arrow  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).



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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).



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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.


                              
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Table 2. Characteristics of each subclass of neurons recorded in adult rats

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.



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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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    ACKNOWLEDGMENTS

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.


    FOOTNOTES

Address reprint requests to T. Isa.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 29 July 1997; accepted in final form 13 April 1999.


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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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0022-3077/99 $5.00 Copyright © 1999 The American Physiological Society