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Commissural Excitation and Inhibition by the Superior Colliculus in Tectoreticular Neurons Projecting to Omnipause Neuron and Inhibitory Burst Neuron Regions

Department of Systems Neurophysiology, Graduate School of Medicine, Tokyo Medical and Dental University, Tokyo, Japan

Submitted 4 April 2005; accepted in final form 18 May 2005

	ABSTRACT

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Previous electrophysiological studies have shown that the commissural connections between the two superior colliculi are mainly inhibitory with fewer excitatory connections. However, the functional roles of the commissural connections are not well understood, so we sought to clarify the physiology of tectal commissural excitation and inhibition of tectoreticular neurons (TRNs) in the "fixation " and "saccade " zones of the superior colliculus (SC). By recording intracellular potentials, we identified TRNs by their antidromic responses to stimulation of the omnipause neuron (OPN) and inhibitory burst neuron (IBN) regions and analyzed the effects of stimulation of the contralateral SC on these TRNs in anesthetized cats. TRNs in the caudal SC (saccade neurons) projected to the IBN region, and received mono- or disynaptic inhibition from the entire rostrocaudal extent of the contralateral SC. In contrast, TRNs in the rostral SC projected to the OPN or IBN region and received monosynaptic excitation from the most rostral level of the contralateral SC, and mono- or disynaptic inhibition from its entire rostrocaudal extent. Among the rostral TRNs with commissural excitation, IBN-projecting TRNs also projected to Forel's field H (vertical gaze center), suggesting that they were most likely saccade neurons related to vertical saccades. In contrast, TRNs projecting only to the OPN region were most likely fixation neurons. Most putative inhibitory neurons in the rostral SC had multiple axon branches throughout the rostrocaudal extent of the contralateral SC, whereas excitatory commissural neurons, most of which were rostral TRNs, distributed terminals to a discrete region in the rostral SC.

	INTRODUCTION

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The superior colliculus serves to orient an animal to a target in the opposite visual hemifield. Commissural connections between the bilateral superior colliculi (SCs) are known to help maintain visual fixation and suppress saccades. Sprague (1966) showed that after transection of the commissure between the SCs, cats once again visually oriented to objects in a hemifield that had been made blind by prior ablation of the occipitotemporal cortex. This outcome, called the Sprague effect, suggests that the commissural connection of the SC may play an important role in visual-orienting behavior, and may mediate mutual suppression between the two SCs to prevent competing responses in the opposite direction. Later electrophysiological studies confirmed the inhibitory nature of the commissural tectotectal projection. Extracellular recording of local field potentials or single-cell activity showed that visual responses in the superficial layers of the SC are inhibited by stimulation of the contralateral SC (Goodale 1973; Hoffmann and Straschill 1971; Mascetti and Arriagada 1981; Robert and Cuénod 1969). Similarly, tectal output neurons related to saccadic eye movements were found to be inhibited during ipsiversive orienting movements (Infante and Leiva 1986; Peck 1990) or by electrical stimulation of the contralateral SC (Munoz and Istvan 1998). With regard to these physiological studies, the anatomical features of commissural connections between the two SCs have been examined extensively (Behan and Kime 1996a; Edwards 1977; Fish et al. 1982; Grantyn and Grantyn 1982; Magalhães-Castro et al. 1978; Moschovakis and Karabelas 1985; Olivier et al. 1998; Rhoades et al. 1986; Yamasaki et al. 1984).

The existence of a direct inhibitory connection between the two SCs was demonstrated by intracellular recordings of inhibitory postsynaptic potentials (IPSPs) from tectal cells in response to electrical stimulation of the contralateral SC. These IPSPs had a latency consistent with a monosynaptic inhibitory commissural connection (Maeda et al. 1981; Moschovakis and Karabelas 1982). Consistent with these physiological observations, Appell and Behan (1990) found that commissural cells constitute a major source of GABAergic projection to the contralateral SC in the cat. On the other hand, electrophysiological studies in tectal neurons have shown that stimulation of the contralateral SC evokes very small excitatory postsynaptic potentials (EPSPs) at a monosynaptic latency followed by larger monosynaptic IPSPs (Maeda et al. 1981; Moschovakis and Karabelas 1982), suggesting that there is an excitatory component of the tectotectal pathway. Through the use of an electron microscopic technique, tectotectal synaptic terminals were found to be heterogeneous, suggesting the existence of both excitatory and inhibitory commissural neurons (Behan 1985). Recently, using a double-labeling method, Olivier et al. (2000) clearly demonstrated that some tectotectal neurons contained glutamate, whereas others contain -aminobutyric acid (GABA), and that almost equal numbers of the two tectotectal populations are present. This finding of equal numbers of GABAergic and glutamatergic tectotectal cells somewhat contradicts previous findings because they invariably indicated that the tectotectal projection was predominantly inhibitory.

Recent studies have suggested that there are two zones in the SC: the rostral fixation zone (Guitton 1991; Munoz and Guitton 1989, 1991; Munoz and Istvan 1998; Munoz and Wurtz 1992, 1993a,b, 1995; Paré and Guitton 1994; Peck and Baro 1997) and the caudal saccade zone (Munoz and Guitton 1989, 1991; Munoz and Istvan 1998; Munoz et al. 1991). In relation to this suggestion, our recent intracellular analysis of collicular inputs to omnipause neurons (OPNs) and excitatory (EBNs) and inhibitory burst neurons (IBNs) revealed that TRNs in the caudal SC projected to the contralateral EBNs and IBNs, whereas TRNs in the rostral SC projected to the OPN region (Izawa et al. 1999; Sugiuchi et al. 2005; Takahashi et al. 2005). IBNs receive strong disynaptic inhibition from the caudal "saccade zone" of the ipsilateral SC and strong monosynaptic excitation from the caudal "saccade zone" of the contralateral SC. These reciprocal inputs supplied by the two SCs at the level of EBNs and IBNs serve to prevent the inappropriate antagonistic action of the brain stem circuit for generating horizontal saccades (Izawa et al. 1999; Sugiuchi et al. 2005). On the other hand, IBNs receive strong disynaptic inhibition from the rostral "fixation zones" of the two SCs by OPNs in the nucleus raphe interpositus (Sugiuchi et al. 2005; Takahashi et al. 2005). This inhibitory effect on IBNs and probably also EBNs by OPNs may serve to prevent unnecessary saccades to unexpected targets during visual fixation.

In these previous studies, stimulation of the rostral SC on each side evoked IPSPs in IBNs at latencies of 1.3–2.8 ms, which suggests that these IPSPs were disynaptic and trisynaptic (Sugiuchi et al. 2005). To explain the inhibition in an IBN evoked bilaterally by stimulation of the rostral SC, there are two possible pathways: by contacting OPNs, TRNs in both SCs may independently inhibit the same IBN, or TRNs on one side of the SC may inhibit an IBN by way of OPNs, and these TRNs may in turn be activated by the opposite SC. Experiments in our previous study, which used surgical cuts made in the brain stem, ensured the existence of the former independent pathways, but did not exclude the possibility of the latter pathway (Sugiuchi et al. 2005). There are two possible pathways whereby TRNs could be activated by stimulation of the opposite SC: commissural excitation of the TRNs and antidromic activation of commissural axon collaterals of the TRNs. It is still unclear how TRNs in the "fixation zone" and the "saccade zone" of the SC are influenced by commissural inputs from the contralateral SC.

The present study was performed to determine whether TRNs that project to different targets in the brain stem receive excitatory or inhibitory inputs from the contralateral SC, and to understand how commissural connections between the two SCs function to maintain visual fixation and to generate and suppress saccades in the cat. Using intracellular recording combined with electrical stimulation of the SC, we showed that TRNs in the caudal "saccade zone" mainly received monosynaptic inhibition from the contralateral rostral SC and mono- or disynaptic inhibition from its caudal part, whereas TRNs in the rostral SC received monosynaptic excitation from the contralateral rostral SC, and mono- or disynaptic inhibition from all rostrocaudal levels of the contralateral SC. The latter population in the rostral SC contained TRNs that projected to the OPN region and TRNs that projected to both the IBN region and the nucleus of the field of Forel (FFH). The functional roles of commissural excitation and inhibition in these different groups of TRNs and non-TRN commissural neurons will be discussed.

	METHODS

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Experiments were performed in 15 cats weighing 2.7–4.5 kg. Animal experimentation was conducted in accordance with the "Policies on the Use of Animals and Humans in Neuroscience Research" revised and approved by the Society for Neuroscience in 1995, and the "Guiding Principles for the Care and Use of Animals in the Field of Physiological Sciences" (The Physiological Society of Japan, revised in 2001). The experimental protocol was approved by the Animal Care Committee of Tokyo Medical and Dental University. The animals were initially anesthetized with ketamine hydrochloride (Ketalar, Parke-Davis; 25 mg /kg, intramuscular) followed by -chloralose (40–45 mg/kg, intravenous [iv], initial dose, supplemented with additional doses of 10–25 mg/kg, iv throughout the remainder of the experiment). During recording, the animals were paralyzed by the intravenous administration of pancuronium bromide (Mioblock, Organon, Oss, The Netherlands), and artificially ventilated with end-tidal CO₂ held at 35–40 mmHg. The heart rate was constantly monitored by an electrocardiogram. The body temperature was kept at 37.0–38.5°C by a heating pad. The abducens nerve was detached from the muscle and mounted on a bipolar hook electrode for electrical stimulation. The bone over the parietal and occipital cortex was removed, and the cerebral cortex was removed bilaterally by suction to allow the introduction of stimulating electrodes into the right SC and recording electrodes into the left SC under direct visual observation. Usually, four concentric bipolar stimulating electrodes (inner diameter and outer diameter, 0.1 and 0.3 mm; interelectrode distance along the longitudinal axis, 0.5 mm) were arranged rostrocaudally at 1.0- to 1.2-mm intervals along the presumed horizontal meridian of the motor map in the SC on the right side (McIlwain 1986). For antidromic activation of TRNs in three experiments, four concentric bipolar stimulating electrodes were placed on each side in the SC and two concentric bipolar electrodes of the same type were placed on each side in the FFH. The tips of the SC electrodes were positioned in the intermediate or deep layer (1.5–2.0 mm deep from the surface) (Izawa et al. 1999; Kawamura and Hashikawa 1978; Moschovakis and Karabelas 1985).

The vermis overlying the fourth ventricle was removed by suction to facilitate the placement of stimulating electrodes or intraaxonal recording from axons of TRNs in the OPN region and the IBN region. We first identified the location of the abducens nucleus by locating antidromic field potentials following electrical stimulation of the sixth nerve (Maeda et al. 1971; Shinoda and Yoshida 1974). Then, stimulating sites in the OPN (Büttner-Ennever et al. 1988; Curthoys et al. 1981; Evinger et al. 1982) and IBN regions (Hikosaka and Kawakami 1977; Hikosaka et al. 1978) were determined relative to the abducens nucleus (Sugiuchi et al. 2005). For antidromic identification of TRNs projecting to the OPN region and the IBN region, separate electrode arrays were placed in the right OPN and IBN regions contralateral to the recording site in the SC. These electrode arrays consisted of four monopolar electrodes (100 µm in diameter) insulated except at the tip, which were glued together around a pillar, so that the tips of the four electrodes were arranged dorsoventrally at 1-mm intervals. For stimulation of the IBN region, one electrode array was placed 0.8 mm lateral to the midline and 0.5–1.0 mm caudal to the caudal border of the abducens nucleus. For stimulation of the OPN region, the other electrode array was placed 0–0.3 mm lateral to the midline and 0.3–1.0 mm rostral to the rostral border of the abducens nucleus. Stimulus currents were delivered between two adjacent tips, and the pillar was grounded to reduce stimulus artifacts. Negative pulses of 0.2-ms duration were delivered at <500 µA for stimulation of the SC, the FFH, and the OPN and IBN regions. Ranck (1975) estimated the effective current spread of 1.0–1.5 mm around an electrode tip by monopolar stimulation at 500 µA (200-µs-duration pulse) in the mammalian CNS. Because we used bipolar stimulation, the effective current spread should be much less than the estimated values by Ranck (1975) (Shinoda et al. 1977). Sasaki et al. (1970, 1972) estimated that 500 µA could not activate fibers or cells beyond 1.0 mm from an electrode tip, when a concentric bipolar electrode was used. The positions of the stimulating electrodes in the brain stem were marked by passing negative currents of 20 µA for 20 s after each experiment, and the stimulated sites in the SC, the OPN and IBN regions, and the FFH were histologically confirmed on sections stained with thionine. Histological examination showed that the distance between a stimulating electrode in the IBN region and the presumed caudal border of the OPN region was about 2.0 mm (1.5–2.5 mm) in each experiment. Glass microelectrodes for intracellular recording were filled with 0.4 M KCl or 2 M K-citrate and had a resistance of 10–15 M.

	RESULTS

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To determine the properties of commissural inputs from the contralateral SC to TRNs that project to different sites in the brain stem, we recorded intracellular potentials from TRNs in the rostral and caudal parts of the SC and examined the effects of electrical stimulation of four sites located rostrocaudally at approximately equal intervals along the presumed horizontal meridian of the motor map (McIlwain 1986) of the contralateral SC in the cat. All lateralities in the present paper are described with reference to the recording site, if not stated otherwise.

Antidromic identification of TRNs projecting to the OPN and IBN regions

TRNs in the caudal part of the SC are known to project to EBNs in the contralateral paramedian pontine reticular formation (PPRF) (Grantyn and Grantyn 1982; Izawa et al. 1999; Moschovakis et al. 1988; Scudder et al. 1996; Sugiuchi et al. 2005) and IBNs in the paramedian pontomedullary reticular formation (PPMRF) (Grantyn and Grantyn 1982; Grantyn et al. 1987; Olivier et al. 1993; Scudder et al. 1996), and these TRNs are mainly responsible for the generation of saccades (Gandhi and Keller 1999; Munoz and Istvan 1998). On the other hand, TRNs in the rostral part of the SC are known to be involved in visual fixation (Guitton 1991; Munoz and Guitton 1989, 1991; Munoz and Wurtz 1993a,b, 1995; Peck 1989) and project to OPNs (Büttner-Ennever et al. 1988; Gandhi and Keller 1997; Langer and Kaneko 1990; Paré and Guitton 1994; Raybourn and Keller 1977; Sugiuchi et al. 2005; Takahashi et al. 2005). Therefore we tried to analyze the properties of commissural inputs onto these two groups of TRNs. To determine the projection sites of TRNs in the brain stem, we recorded intracellular potentials from cell bodies of TRNs in the left SC and identified TRNs that projected to different sites in the brain stem by their antidromic responses to stimulation of the contralateral OPN and IBN regions (Fig. 1A). For this purpose, we stimulated three dorsoventral sites in the OPN region (Fig. 1D) and three dorsoventral sites in the IBN region (Fig. 1E) on the right side. Intracellular recordings were made from 95 TRNs, but 23 of them could not be used for analysis of synaptic potentials because of quick spontaneous diffusion of Cl^– from the recording electrode into the cells. Therefore the remaining 72 TRNs were used for the present analysis. Their resting membrane potentials ranged from –45 to –70 mV (mean ± SD, –54 ± 18 mV, n = 72).

View larger version (39K): [in this window] [in a new window]   FIG. 1. Experimental arrangement for the intracellular analysis of commissural effects of the superior colliculus (SC) on tectoreticular neurons (TRNs). A: dorsal view of the brain stem showing an experimental setup. Intracellular potentials were recorded in the left SC. To examine the effects of the contralateral SC on TRNs, 4 stimulating electrodes (sites 1–4) were placed in the right SC rostrocaudally along the horizontal meridian of the motor map of the SC (McIlwain 1986). For antidromic identification of TRNs, one array of 3 stimulating electrodes was placed in the right omnipause neuron (OPN) region (sites 5–7) and the other array in the right inhibitory burst neuron (IBN) region (sites 8–10). VI, abducens nucleus. B: montage of the lateral view of the brain stem showing stimulating electrode positions in the right SC (sites 1–4), the right OPN (sites 5–7), and the right IBN regions (sites 8–10). Rost, rostral; Caud, caudal; BC, brachium conjunctivum; IC, inferior colliculus; IO, inferior olive; NRTP, nucleus reticularis tegmenti pontis; PN, pontine nucleus; RN, red nucleus; IIIn, third nerve. C–E: photomicrographs showing locations of stimulating electrodes (arrowheads) in the right SC (parasagittal section including 4 electrode tracks) (C), the right OPN region (sites 5–7) (transverse section parallel to the electrode track) (D), and the right IBN region (transverse section parallel to the electrode track) (sites 8–10) (E). Locations of 3 cathodal electrode tips are indicated by arrowheads in the OPN (D) and IBN regions (E). Arrows indicate the midline. Lt, left; Rt, right; CG, central gray; VN, vestibular nucleus; F, facial nucleus; SO, superior olive; Tmo, trigeminal motor nucleus. F: electrophysiological identification of a TRN projecting to the contralateral IBN region. Antidromic spikes were evoked by stimulation of the right OPN region (sites 5–7) and the right IBN region (sites 8–10). Stimulus intensity is indicated at the top right of each panel. Note that this tectal neuron was antidromically activated at similar short latencies (0.3–0.4 ms) from 3 OPN sites (5–7), but at different latencies from 3 IBN sites (0.5, 0.7, and 1.1 ms) (8–10), indicating that the stem axon of this TRN passed through the OPN region and ramified to terminate in the IBN region.

In support of this interpretation, the latencies of the antidromic spikes in a TRN were very similar at three sites in the OPN region (within 0.1-ms difference) (Fig. 1F, 5–7) because the passing stem axon was activated there. Furthermore, this neuron was antidromically activated at different latencies (0.5, 0.7, and 1.1 ms) by stimulation of the three different sites (Fig. 1, B, 8–10 and E, 8–10) in the IBN region (Fig. 1F, 8–10), suggesting that the stimulus activated thin TRN axon collaterals ramifying in the IBN region, and most probably terminating there. Therefore TRNs that were antidromically activated from the OPN and IBN regions were considered to project to the IBN region. Such IBN-projecting TRNs as in Fig. 1F were found at all rostrocaudal levels of the SC, and the latencies of the antidromic spikes in TRNs of the caudal SC that were activated from the IBN region ranged from 0.5 to 1.2 ms (0.8 ± 0.2 ms, n = 47) (see Fig. 5Ab).

View larger version (26K): [in this window] [in a new window]   FIG. 5. Latency histograms in different groups of TRNs of antidromic spikes evoked by stimulation of the OPN (a) and IBN regions (b) (A, D, and G), and commissural EPSPs (B, E, and H) and IPSPs (C, F, and I) evoked by stimulation of the contralateral SC. A–C, IBN-projecting TRNs in the caudal SC (n = 28 tested); D–F, OPN-projecting TRNs in the rostral SC (n = 13 tested); G–I, IBN-projecting TRNs in the rostral SC (n = 31 tested). Same arrangement of stimulation sites in the contralateral SC, site 1 (a), site 2 (b), site 3 (c), and site 4 (d) in the rostrocaudal order as shown in Fig. 1A. Total number of antidromic latencies exceeds the total number of TRNs tested because all latencies were included when a TRN was activated from more than one site in the OPN or IBN region. Total number of PSP latencies falls below the total number of TRNs tested because some latencies could not be determined as a result of the appearance of spikes, masking IPSPs due to Cl– leak from the pipette or the difficulty in reversing IPSPs by Cl– injection.

View larger version (27K): [in this window] [in a new window]   FIG. 4. Commissural inputs from the contralateral SC to 2 TRNs terminating in the OPN region. A: experimental setup. Intracellular potentials were recorded in the rostral part of the left SC. B–D: typical commissural inputs to a TRN projecting to the OPN region. Stimulus intensities, 500 µA for all traces in B–D. B: antidromic spikes evoked by stimulation of the contralateral OPN region at site 5 but not at sites 6 and 7. C: no spikes evoked by stimulation of the contralateral IBN region at sites 8–10. D: effects of the contralateral SC on the TRN. Injection of Cl– into the cell reversed the hyperpolarizations (middle traces) to depolarizing potentials (top traces), but did not change the polarity of the early depolarizations. E–G: typical commissural inputs to another TRN projecting to the OPN region. E and F: antidromic spikes evoked at different latencies (1.2 and 1.0 ms) by stimulation of the 2 sites in the OPN region (5, 7), respectively, but not of the 3 sites in the IBN region (8–10). Note that a comparison of the top (after Cl– injection into the cell) and middle traces (before Cl– injection) in E indicates deterioration of the partial antidromic spikes evoked at sites 5 and 7 and reversed IPSPs after Cl– injection. Similarly, this TRN was also antidromically activated from the rostral site (site 2) of the contralateral SC (G2, middle traces). G: properties of synaptic inputs from the contralateral SC (1–4) to the same TRN as in E and F. Stimulus intensities, 500 µA for all traces, but for the top traces in G1 and G2 where weak stimulation (200 µA) was applied.

To analyze the properties of commissural inputs from the contralateral SC to TRNs in the caudal SC, we recorded intracellular potentials from the caudal part of the left SC and stimulated the right OPN region, the right IBN region, and four rostrocaudal sites in the right SC, as shown in Fig. 1A. Figure 2, A–C shows a typical example of commissural input to a caudal TRN. In this caudal TRN, antidromic spikes were evoked by stimulation of the right OPN region (Fig. 2A, 5) and also the right IBN region (Fig. 2A, 8). Therefore this neuron was considered to be a TRN that projected to the contralateral IBN region. Stimulation of all four rostrocaudal sites in the contralateral SC evoked large hyperpolarizations at 500 µA (Fig. 2B). To determine whether these hyperpolarizations were IPSPs or disfacilitation attributed to a decrease in EPSPs, we iontophoretically injected Cl^– into the cell. After the injection of Cl^–, hyperpolarizing potentials were changed to large depolarizing potentials, indicating that these hyperpolarizations were inhibitory postsynaptic potentials (IPSPs) (Coombs et al. 1955; Eccles 1964). This input pattern from the contralateral SC was usually observed in most TRNs in the caudal SC. With respect to the IPSPs, generally their amplitudes were larger, their latencies were shorter, and the slopes of their falling phase were steeper, when more rostral sites were stimulated (Fig. 2B). We determined the onsets of IPSPs by superimposing IPSPs on their corresponding field potentials recorded just outside the penetrated cells or by superimposing the IPSPs on reversed depolarizing potentials after Cl^– injection into the cells. The latencies of the IPSPs from sites 1–4 in the contralateral SC were 1.0, 1.1, 1.4, and 1.7 ms, respectively (Fig. 2B, 1–4). The latencies of antidromic spikes of putative inhibitory commissural neurons (see Tectal neurons mediating commissural excitation or inhibition) evoked by stimulation of the contralateral SC were 0.3–1.2 ms (see Fig. 10, C–F). Therefore the IPSPs evoked at sites 1 and 2 were safely considered to be monosynaptic, and those evoked at site 4 appeared to be disynaptic (Fig. 2B).

View larger version (26K): [in this window] [in a new window]   FIG. 2. Commissural inhibition of 2 TRNs in the caudal SC evoked by stimulation of the contralateral SC. Same experimental setup as in Fig. 1A. A–C: commissural inhibition from the SC to a TRN in the caudal SC. A: antidromic spikes of a TRN in the caudal SC evoked in an all-or-none manner at threshold by stimulation of the contralateral OPN region at 100 µA (site 5) and IBN region at 45 µA (site 8). B: properties of inhibitory postsynaptic potentials (IPSPs) evoked by stimulation of the contralateral SC at 500 µA in the same TRN as in A. Number attached to each panel corresponds to each stimulation site in the contralateral SC (1–4), OPN (5–7), and IBN regions (8–10) as shown in Fig. 1A. Same arrangement of stimulating sites and their corresponding intracellular records as used in this figure is used in the following figures. Top, middle, and bottom traces indicate intracellular potentials after and before Cl– injection into the cell, and juxtacellular field potentials recorded just outside the penetrated cell, respectively. Hyperpolarizing potentials were changed to depolarizing potentials after Cl– injection. C: temporal facilitation of monosynaptic commissural inhibition. a: single-pulse stimulation of site 2 at 90 µA. b: double-pulse stimulation of site 2 at 90 µA. Note the presence of clear temporal facilitation of the reversed IPSPs whose onset is indicated by an arrow. D–F: effects of the stimulus intensity in the contralateral SC on postsynaptic potentials (PSPs) in another TRN in the caudal SC. D: antidromic spikes evoked by stimulation of the OPN (site 6) and the IBN region (site 8). E and F: properties of synaptic inputs from the contralateral SC to the same TRN as in D. IPSPs evoked by stimulation of the 4 rostrocaudal sites in the contralateral SC (1–4) at 500 µA (E) and 150 µA (F) before (middle traces) and after injection of Cl– into the cell (top traces). Bottom traces indicate juxtacellular field potentials. Latencies of the IPSPs were 1.0 (1), 1.1 (2), 1.2 (3), and 1.1 ms (4) for E, and 1.3 (1), 1.4 (2), 1.2 (3), and 1.2 ms (4) for F. Note that the amplitudes, latencies, and slopes of the falling phases of individual IPSPs evoked from the 4 sites at the same intensities were similar in this TRN. Scales in E also apply to F.

View larger version (27K): [in this window] [in a new window]   FIG. 10. Putative inhibitory intratectal commissural neuron in the rostral SC. A and B: a commissural neuron with multiple axonal branches in the contralateral SC that did not project to either the OPN or IBN region. A: schematic diagram of the experimental setup and the presumed axonal projection of a commissural neuron shown in B. B: antidromic spikes evoked by stimulation of 4 rostrocaudal sites in the contralateral SC (1–4). Each stimulus intensity shown at the top right in each panel was fixed at 1.5 times threshold for spike activation. a: single-pulse stimuli, b, c: double-pulse stimuli at different intervals to examine refractory periods of the cell. Note that spikes followed double-pulse stimuli at intervals of <1 ms (B1c, B2c, B3b, and B4b), indicating that these spikes were activated antidromically from these 4 stimulating sites. C–F: latency histograms of antidromic spikes in 11 putative inhibitory commissural neurons evoked by stimulation of the contralateral SC. Eight neurons were activated from all 4 rostrocaudal sites (site 1–4) and 3 neurons were activated from the 3 rostral sites (sites 1–3). G: latency histogram of antidromic spikes in rostral OPN-projecting and IBN-projecting TRNs with commissural axons evoked by stimulation of the contralateral rostral SC (sites 1 and/or 2). Total number of latencies exceeds the total number of commissural neurons tested (n = 28) because a single neuron was activated from more than one site in the SC. Mean ± SD values are shown for each histogram.

As shown in this example, the commissural inhibition was usually stronger from the most rostral SC site. However, in other TRNs examined, the strength of this inhibition was almost equal from individual rostrocaudal sites of the contralateral SC. Such an example is shown in Fig. 2, D–F. In this caudal TRN, stimulation of four rostrocaudal SC sites evoked IPSPs of similar large amplitudes and with short latencies at 500 µA (Fig. 2E). The latencies of the IPSPs were 1.1–1.2 ms, and therefore the IPSPs evoked at all four sites were considered to be monosynaptic. These IPSPs were still evoked at 150 µA (Fig. 2F), but their amplitudes were smaller and the slopes of the falling phase were slower than at 500 µA. With a decrease in the stimulus intensity, monosynaptic inhibition was still evoked at site 4 in this TRN (Fig. 2F, 4), indicating that at least some inhibitory commissural neurons must exist in the caudal SC. In brief, we examined commissural input to 28 TRNs in the caudal SC. All of them projected to the contralateral IBN region, and received commissural inhibition from all four rostrocaudal sites of the contralateral SC. In nearly 60% of TRNs, the commissural inhibition tended to be stronger and mainly monosynaptic from the most rostral SC stimulating site, and disynaptic from the most caudal SC stimulating site. This inhibition was almost equally strong and monosynaptic from both the rostral and caudal SC in the remaining 40% of the TRNs tested even at lower stimulation strengths (100–200 µA).

To compare the nature of commissural inhibition in TRNs located in different rostrocaudal sites in the SC, we recorded intracellular potentials from cells along the presumed horizontal meridian of the motor map (McIlwain 1986). Figure 3 shows a typical example of commissural inhibition on four TRNs located in different rostrocaudal sites of the SC in one cat. These TRNs were antidromically activated from the contralateral IBN region (not illustrated). All of the TRNs received commissural inhibition from the four rostrocaudally distributed sites in the contralateral SC. In each TRN, the slopes of the falling phase of IPSPs were steeper and the amplitudes of the IPSPs were usually larger, when more rostral sites were stimulated. In the most rostral TRN (Fig. 3Ba), small EPSPs preceded the IPSPs evoked from rostral stimulation sites (Fig. 3Ba, 1 and 2), but in the other TRNs, only IPSPs were apparent. For each TRN, the IPSPs were largest and their latencies, which ranged from 0.7 to 1.2 ms, were shortest when the most rostral site was stimulated in the contralateral SC. This indicates that all four of these TRNs received monosynaptic inhibition from the most rostral SC. This finding also suggested that inhibitory commissural neurons might be distributed with higher density in the rostral part of the SC. However, because many TRNs received monosynaptic inhibition from the more caudal SC, a few inhibitory commissural neurons must also be located in the caudal SC. In addition, TRNs tended to receive longer-latency inhibition from more caudal SC levels that was often disynaptic. Therefore it is highly likely that this late inhibition was mediated by rostral inhibitory commissural neurons that were orthodromically activated from the more caudal SC.

View larger version (23K): [in this window] [in a new window]   FIG. 3. Commissural inputs from the contralateral SC to 4 TRNs located in different rostrocaudal sites in the SC of the same cat. A: experimental setup. Intracellular potentials were recorded from cell bodies indicated as a–d along the horizontal meridian (McIlwain 1986). All of these TRNs were antidromically activated from both the OPN and IBN regions (not illustrated). B: properties of commissural inputs from the contralateral SC to TRNs in different rostrocaudal locations in the SC (a–d). Stimulus intensities were 500 µA for all traces. In a TRN in the most rostral part of the SC (a), small depolarizations preceded hyperpolarizations, when stimulation was applied to the rostral parts of the contralateral SC (1, 2), whereas only IPSPs were evoked by stimulation of its caudal parts (3, 4). Note that these 4 TRNs received stronger inhibition from the more rostral part of the contralateral SC because the amplitudes and the slopes of the falling phases of the IPSPs were greater.

To specifically examine commissural input patterns in TRNs of the rostral SC, we recorded intracellular potentials from TRNs in the left rostral SC and examined the effects of stimulation of four rostrocaudal sites of the contralateral SC on these TRNs in the same way as shown in Fig. 2. In the rostral SC, TRNs were classified into two types with regard to their projection to the brain stem: TRNs that projected to the OPN region and those that projected to the IBN region. Among 44 TRNs examined in the rostral one-fourth of the SC, 31 TRNs were antidromically activated from both the OPN and IBN regions, whereas the other 13 TRNs were activated only from the OPN region, but not from the IBN region. We will describe commissural inputs to these two types of rostral TRNs separately.

The effects of stimulation of the contralateral SC in TRNs that projected to the OPN region are illustrated in Fig. 4. In the example shown in Fig. 4, B–D, antidromic spikes were evoked at a latency of 0.7 ms by stimulation of the OPN region (site 5) at 50 µA (Fig. 4B, 5), but not by stimulation of the IBN region (Fig. 4C, 8–10). In this TRN, stimulation of the contralateral rostral SC evoked a depolarization followed by a hyperpolarization (Fig. 4D, 1, middle traces). To determine whether this depolarization was an EPSP or disinhibition resulting from a decrease in IPSPs, we passed a hyperpolarizing current through a recording electrode and injected Cl^– into the cell. This reversed the later hyperpolarization to a depolarizing potential without affecting the early depolarization (Fig. 4D, 1, top traces). Therefore this depolarization was an EPSP and the later hyperpolarization was an IPSP (Coombs et al. 1955; Eccles 1964). As the stimulation sites moved caudally, EPSPs and IPSPs decreased in amplitude (Fig. 4D, 2–3) and only IPSPs were evoked by stimulus site 4 (Fig. 4D, 4). The EPSPs were considered to be monosynaptic because their latencies were 0.8–1.1 ms. The latencies of the IPSPs were 1.2 (site 1), 1.7 (site 2), 1.9 (site 3), and 1.8 ms (site 4). Judging from the latencies of antidromic spikes in presumed inhibitory commissural neurons (see Tectal neurons mediating commissural excitation or inhibition and Fig. 10, C–F), the IPSPs evoked at site 1 were considered to be monosynaptic and those at site 4 were considered to be disynaptic.

Figure 4, E–G shows another example of a TRN projecting to the OPN region. In this tectal neuron, antidromic spikes were not evoked by stimulation of the IBN region at 500 µA (Fig. 4F). However, partial spikes evoked at latencies of 1.2 and 1.0 ms by stimulation at sites 5 and 7 in the OPN region, respectively (Fig. 4E), were judged to be antidromic because they were evoked at constant latencies in an all-or-none manner at threshold. Similar antidromic spikes were also evoked by stimulation of site 2 in the contralateral SC (Fig. 4G, 2). Therefore we considered this neuron to be a TRN terminating in the OPN region with a commissural axon. Stimulation of the three rostral sites of the contralateral SC evoked small depolarizations followed by large IPSPs (Fig. 4G, 1–3) and stimulation of its most caudal part evoked only large IPSPs (Fig. 4G, 4) at 500 µA. Sharp spikelike depolarizations such as those in Fig. 4E, 5 and 7 also appeared in Fig. 4G, 1 and 2. Sharp spikes in Fig. 4G, 2 were judged to be antidromic because they were evoked at constant latencies in an all-or-none manner at threshold. However, sharp depolarizations in Fig. 4G, 1 were not antidromic spikes for the following reason. As stimulus intensities were decreased at sites 1 and 2 (top traces in Fig. 4G, 1 and 2), the IPSPs became smaller, and as a result, it became clear that these sharp potentials were attributed to the curtailment of EPSPs by the large IPSPs. The latencies of the EPSPs were very short (<1.0 ms) and therefore monosynaptic (Coombs et al. 1955; Eccles 1964). The latencies of the IPSPs ranged from 1.0 to 1.4 ms and were most probably monosynaptic (see DISCUSSION). As the SC stimulation sites moved more caudally, the EPSP amplitude decreased, but the IPSPs did not change so much. These results indicate that a TRN terminating in the OPN region received excitation from the rostral part of the contralateral SC and inhibition from all rostrocaudal sites within the contralateral SC. In this TRN, inhibition was also caused by stimulation of the contralateral IBN region at latencies of 1.6–1.8 ms (Fig. 4F, 8–10). This inhibition was most likely disynaptic because latencies of antidromic spikes in TRNs projecting to the IBN region were 0.5–1.7 ms (see Fig. 5, Ab and Gb).

In summary, we analyzed 13 TRNs projecting to the contralateral OPN region. The latencies of EPSPs and IPSPs evoked by stimulation of the contralateral SC ranged from 0.7 to 1.3 ms (mean ± SD, 0.9 ± 0.2 ms, n = 22) (Fig. 5E) and 0.9 to 2.0 ms (1.4 ± 0.3 ms, n = 37) (Fig. 5F), respectively. All of these TRNs received monosynaptic excitation from the rostral sites and mono- or disynaptic inhibition from all rostrocaudal levels of the contralateral SC. Both the excitation and the inhibition were generally stronger from the more rostral SC. Furthermore, most of these TRNs were disynaptically inhibited by stimulation of the contralateral IBN region. The latencies of antidromic spikes evoked by stimulation of the OPN region were often longer in most OPN-projecting TRNs (Fig. 5Da) than in IBN-projecting TRNs (Fig. 5Ga), suggesting that tectoreticular axons terminating in the OPN region, and probably their cell bodies, are smaller than those terminating in the IBN region. This suggestion was partly supported by the experience that intracellular recording from OPN-projecting TRNs was more difficult than recording from IBN-projecting TRNs.

While analyzing commissural inputs to TRNs projecting to the IBN region, we found that many TRNs in the rostral SC received commissural excitation from the contralateral SC making them different from TRNs in the caudal SC. Figure 6 shows an example of a rostral TRN projecting to the IBN region. This tectal neuron penetrated in the rostral one-fourth of the SC was antidromically activated from the OPN (Fig. 6B, 5) and IBN regions (Fig. 6B, 10). Stimulation of the most rostral part of the contralateral SC evoked depolarizations with occasional spikes, which were followed by large hyperpolarizations (Fig. 6C, 1). Stimulation of the more caudal SC evoked only hyperpolarizations (Fig. 6C, 2 and 3) but stimulation of the most caudal SC evoked very small depolarizations followed by hyperpolarizations (Fig. 6C, 4). At a weaker stimulus intensity (Fig. 6D, middle traces), a similar response pattern was observed in the same TRN. To determine the synaptic nature of the depolarizations and hyperpolarizations, Cl^– was injected into the cell (Fig. 6D, top traces). With this procedure, the hyperpolarizations were reversed to depolarizing potentials, but the depolarizations were not affected so much, indicating that the hyperpolarizations were IPSPs and the depolarizations were EPSPs (Coombs et al. 1955; Eccles 1964). These temporal and spatial features of synaptic inputs from the different rostrocaudal levels of the contralateral SC to a rostral TRN projecting to the IBN region were very similar to those observed in rostral TRNs projecting to the OPN region, but were different from those observed in caudal TRNs projecting to the IBN region in that caudal TRNs mainly received commissural inhibition without commissural excitation.

View larger version (26K): [in this window] [in a new window]   FIG. 6. Commissural excitation and inhibition of a TRN in the rostral SC projecting to the IBN region. A: experimental setup. B: antidromic activation of a tectal neuron by stimulation of the OPN (5) and IBN regions (10) at 500 µA. C: commissural effects of the contralateral SC on the TRN. Stimulus intensity, 500 µA for all stimulation sites. Orthodromic spikes were evoked on top of EPSPs by stimulation of the most rostral SC (1). D: identification of EPSPs and IPSPs evoked by contralateral SC stimulation at 150 µA. Top and middle traces: PSPs after and before injection of Cl– into the same TRN as in B and C, respectively. Bottom traces: juxtacellular field potentials.

View larger version (19K): [in this window] [in a new window]   FIG. 7. Effects of stimulus intensity applied in the contralateral SC on commissural excitation and inhibition of a TRN in the rostral SC that projected to the IBN region. A: antidromic spikes evoked by stimulation of the OPN (site 7) and IBN regions (site 8) at 500 µA. B: properties of synaptic inputs to the same rostral TRN as in A evoked by stimulation of the contralateral SC at different stimulus intensities. Effects of stimulus intensity on commissural PSPs were examined by changing stimulus intensities from 500 (a) to 300 (b), 200 (c), 100 (d), and 50 µA (e). Note that strong, monosynaptic excitation was evoked by stimulation of the rostral sites of the contralateral SC, whereas commissural inhibition occurred from all rostrocaudal sites in the contralateral SC.

Commissural excitation of TRNs that projected to the FFH

To better understand the functional role of commissural excitation in rostral TRNs projecting to the OPN or IBN region, we examined whether these TRNs also projected to the FFH. In this experiment, a stimulating electrode was placed in the FFH on each side and four stimulating electrodes were placed in the SC on each side. Intraaxonal recordings were made from axons of TRNs in either the OPN or IBN region (Fig. 8). The records in Fig. 8, A and B show an example of intraaxonal spikes recorded in the left IBN region. Stimulation of the right rostral SC (sites 5 and 6) evoked direct spikes at 500 µA (Fig. 8B, 5 and 6, top traces) and indirect or synaptically activated spikes at lower stimulus intensities (Fig. 8B, 5 and 6, bottom traces). Stimulation of the right caudal SC (sites 7 and 8) evoked only indirect spikes (Fig. 8B, 7 and 8) in this TRN. Therefore this axon was considered to arise from a TRN in the rostral SC. The same TRN was antidromically activated from the right FFH (Fig. 8B, i-FFH), but not from the left FFH (Fig. 8B, c-FFH). The effects of stimulation of the contralateral SC on this TRN were examined by stimulating four rostrocaudal sites in the left SC (Fig. 8B, 1–4). This TRN was directly activated from site 2 and synaptically from site 1, but not activated from sites 3 or 4. Accordingly, this TRN with commissural excitation was considered to be located in the right rostral SC and to project to the right FFH and the left SC and IBN region, as shown schematically in Fig. 8A. Figure 8, C and D shows another example of intraaxonal spikes recorded in the left OPN region. Stimulation of the rostral sites (sites 5 and 6) of the right SC evoked both direct and/or indirect spikes at near thresholds for the direct spikes (Fig. 8D, 5 and 6), and stimulation of the caudal SC (site 7) evoked only indirect spikes (Fig. 8D, 7). This neuron was not activated from either FFH (Fig. 8D, c-FFH and i-FFH), but was synaptically activated from the rostral part of the contralateral SC (Fig. 8D, 1 and 2). Therefore this rostral TRN was considered to receive commissural excitation and was most likely to project to the contralateral OPN region (see DISCUSSION and Fig. 9Ba). It should also be noted that both TRNs in Fig. 8 were excited orthodromically from sites adjacent to the presumed locations of their cell bodies in the ipsilateral SC.

View larger version (22K): [in this window] [in a new window]   FIG. 8. Two types of TRNs that received commissural excitation evoked by stimulation of the contralateral SC. A and B: commissural input to a TRN in the rostral SC that sent its axons to both the contralateral IBN region and the ipsilateral Forel's field H (FFH). A: experimental setup and summary diagram of the neural circuit of the TRN shown in B. Four stimulating electrodes were arranged rostrocaudally in each SC, and a stimulating electrode was placed in each FFH. Intraaxonal spikes were recorded in the left IBN region. B: responses of the TRN evoked by stimulation of the left FFH (c-FFH) and the right FFH (i-FFH), and the left (1–4) and the right SC (5–8). Stimulus intensity for each stimulation site is indicated at the top right in each panel. Double-pulse stimuli at an interval of 1.5 ms were used at stimulation sites of c-FFH, 3, 4, 7, and 8. This TRN that was assumed to be located in the rostral SC on the right was antidromically activated from both the ipsilateral FFH and contralateral SC (site 2). Note that this TRN with commissural excitation from site 1 also sent its axon collateral to the contralateral SC (site 2). This TRN was synaptically activated from all 4 sites in the ipsilateral SC because spike latencies fluctuated near thresholds. C and D: commissural input to a TRN in the rostral SC that projected to the contralateral OPN region. C: experimental setup and summary diagram of the neural circuit about the TRN shown in D. Same experimental setup as in A, but intraaxonal spikes were recorded in the left OPN region. D: responses of the TRN evoked by stimulation of c-FFH, i-FFH, the left SC (1–4), and the right SC (5–8). Double-pulse stimuli were used for stimulation of c-FFH, i-FFH, sites 1–4 and 8. Note that this TRN projecting to the contralateral OPN region was assumed to be in the rostral SC on the right and received commissural excitation from the contralateral rostral SC (sites 1 and 2). This TRN was synaptically activated from adjacent sites in the ipsilateral SC (sites 5–7).

View larger version (25K): [in this window] [in a new window]   FIG. 9. Commissural influences from the contralateral SC on TRNs projecting to the FFH, OPN, or IBN region. Lateralities are described with reference to the location of cell bodies on the right. A: schematic diagram of the experimental setup. Intraaxonal recording was made either in the left OPN region (B) or in the left IBN region (C). Four stimulating electrodes were placed rostrocaudally in the left SC (1–4) and the right SC (5–8). Two stimulating electrodes were placed in the left FFH [contra (c) FFH] and the right FFH [ipsi (i) FFH]. B and C: summary of commissural influences on TRNs with axons that were penetrated in the OPN (B) or IBN region (C). Numbers 1–8 and c FFH and i FFH at the top indicate stimulation sites in the SC and the FFH shown in A, respectively. Each row shows the effects of stimulation of individual stimulation sites indicated at the top on a single TRN. Different symbols indicate directly activated spikes (black dots), synaptically activated spikes (open circles), and both directly and synaptically activated spikes (black dots with open circles) evoked by stimulation of individual stimulation sites. a, b: TRNs without axon collaterals to the FFH; c, d: TRNs with axon collaterals to the FFH; a, c: TRNs with commissural excitation; b, d: TRNs without commissural excitation. It is most likely that TRNs in Ba are related to visual fixation, TRNs in Bb and Cb to horizontal saccades, TRNs in Bc and Cc to vertical saccades, and TRNs in Bd and Cd to oblique saccades (see the text for more details).

Tectal neurons mediating commissural excitation or inhibition

The experimental results that have been described so far indicate that commissural excitation is exerted on rostral TRNs from the most rostral part of the contralateral SC, whereas commissural inhibition is exerted on TRNs throughout the rostrocaudal extent of the SC from the entire rostrocaudal extent of the contralateral SC. Furthermore, the shortest latency inhibition originates from the most rostral part of the contralateral SC. Based on these findings, we tried to find candidate commissural neurons that mediated either excitation or inhibition from the contralateral rostral SC to TRNs.

We searched for neurons in the left rostral SC that were activated antidromically from the contralateral SC, while stimulating four rostrocaudal sites in the right SC at 500 µA (Figs. 10 and 11). Two groups of tectal commissural neurons were identified: neurons with multiple axon collaterals in the contralateral SC that lacked projections to either the OPN or IBN region (Fig. 10, A and B), and neurons with an axon collateral limited to the rostral, contralateral SC that also projected to the OPN or IBN region (Fig. 11, A–D). Figure 10, A and B shows a typical example of a commissural neuron with multiple axon collaterals in the contralateral SC. This commissural neuron was located in the most rostral SC because extracellular spikes were recorded from a cell body in the most rostral SC. It was activated from all of the four rostrocaudal stimulating sites in the contralateral SC (Fig. 10Ba, 1–4). Spikes were evoked at short, fixed latencies of <1.0 ms, and they followed double-pulse stimulation at intervals of <1.0 ms (Fig. 10B, 1c and 2c, and 3b and 4b). Therefore these spikes were considered to be antidromically activated from each stimulation site in the contralateral SC. Spike latencies were 0.9 (site 1), 1.0 (site 2), 1.1 (site 3), and 1.3 ms (site 4), suggesting that the commissural axon ran in the rostrocaudal direction in the contralateral SC after crossing the midline at the rostral level of the SC. This neuron was not activated by stimulation of three dorsoventral sites in each of the contralateral OPN and IBN regions at 500 µA (not illustrated). Therefore this commissural neuron was considered to be a purely intratectal commissural neuron.

View larger version (30K): [in this window] [in a new window]   FIG. 11. Putative excitatory commissural neuron in the rostral SC. A–D: extracellular-spike recordings from a commissural neuron that had a simple axonal branch in the contralateral SC and also projected to the contralateral OPN region but not to the IBN region. A: schematic diagram of the experimental setup and the presumed axonal projection of the commissural neuron shown in B–D. B: stimulation of 4 rostrocaudal sites in the contralateral SC (1–4) and the contralateral OPN region (5) at 500 µA. Note that this neuron was activated antidromically from site 2 in the contralateral SC and the contralateral OPN region (5) but not from 3 dorsoventral stimulation sites in the contralateral IBN region at 500 µA (not illustrated). C: antidromic activation of the commissural neuron in an all-or-none manner at thresholds (a and c) from the contralateral SC (site 2) and the OPN region (site 5), and double-shock stimulation of the same sites to determine refractory periods of the cell (b and d). Refractory periods were 0.6 ms for site 2 and 1.0 ms for site 5. D: a spike collision test between SC-evoked and OPN-evoked antidromic spikes. Stimuli for the contralateral SC (site 2) were applied 3.0 ms (a) and 2.9 ms (b) after stimuli for the OPN region (site 5), and 3.0 ms (c) and 2.9 ms (d) before stimuli for the OPN region (site 5). E–H: summary of different types of tectal neurons with a commissural axon projecting to the contralateral SC. Stimulating electrodes were arranged in the same way as in Fig. 1A. E: TRNs projecting to the OPN region. F: TRNs projecting to the IBN region. G: intratectal neurons projecting only to the rostral part of the contralateral SC. These neurons were not antidromically activated from the contralateral OPN and IBN regions. H: intratectal neurons projecting to the rostral and caudal parts of the contralateral SC. Numbers indicate neurons that belong to each type. Note that the commissural neurons in E and F are excitatory and the intratectal commissural neurons in H are most likely inhibitory (see the text for more details).

Figure 11, A–D shows an example of extracellular records from a commissural neuron of the second type. This neuron was also located in the most rostral part of the left SC, and was activated from the contralateral SC (Fig. 11B, 2) and the contralateral OPN (Fig. 11B, 5), but not from the contralateral IBN region (not illustrated). Spikes were evoked in an all-or-none manner at threshold (30 µA) (Fig. 11Ca) by stimulation of the contralateral rostral SC (site 2) and followed double-pulse stimuli at an interval of 0.6 ms (Fig. 11Cb), indicating that these were antidromic spikes. In a similar way, spikes evoked by stimulation of the OPN region were regarded as antidromic (Fig. 11C, c and d). A spike collision test (Fig. 11D) confirmed that this TRN sent its axons to both the contralateral SC and the contralateral OPN region (Shinoda et al. 1976). As in this example, commissural neurons of this second type projected to the most rostral sites of the contralateral SC and also projected to the OPN or the IBN region. This second type of commissural neuron that projects to the OPN or IBN region was considered to be excitatory because TRNs that terminated on OPNs (Takahashi et al. 2005) and IBNs (Sugiuchi et al. 2005) were excitatory. That is, excitatory commissural neurons were most likely TRNs that had commissural axons.

Commissural neurons of different types are summarized in Fig. 11, E–H. In this analysis, spikes were recorded intracellularly or extracellularly from cell bodies in the rostral SC. Among 44 rostral commissural neurons that were activated antidromically from the contralateral SC, nine were TRNs that were also activated antidromically from the OPN region but not from the IBN region (Fig. 11E) and 19 were TRNs that were antidromically activated from both the OPN and IBN regions (Fig. 11F). The other 16 neurons were not activated from either the OPN or the IBN region (Fig. 11, G and H). Among them, 11 were antidromically activated from three to four rostrocaudal sites in the contralateral SC (Fig. 11H) and the other five were activated only from the most rostral one or two sites (Fig. 11G). The first three groups of commissural neurons (Fig. 11, E–G) were most likely excitatory because they projected to only the rostral SC, whereas the last group (Fig. 11H) was most likely inhibitory because they projected to the entire SC and did not project to either the OPN or the IBN region. For intratectal commissural neurons with multiple axon collaterals, the latencies of antidromic spikes evoked by contralateral SC stimulation were longer from the caudal SC (Fig. 10F) than from the rostral SC (Fig. 10C). For rostral TRNs with commissural axons, the latencies of antidromic spikes evoked by contralateral SC stimulation ranged from 0.3 to 1.2 ms (0.5 ± 0.2 ms, n = 39) (Fig. 10G).

Inputs to intratectal commissural neurons with multiple axon collaterals in the contralateral SC

To understand the functional role of commissural inhibition in TRNs, we examined inputs to presumed inhibitory commissural neurons in two ways. First, we searched for commissural axons in the caudal half of the SC, while stimulating the contralateral rostral SC (Fig. 12A). Stimulation of the most rostral part of the contralateral SC evoked direct spikes at a threshold of 50 µA (Fig. 12B, 1, right), and direct and indirect spikes at 500 µA (Fig. 12B, 1, left). The cell body of this axon was considered to be located near site 1 in the SC because such activation of direct and indirect spikes usually occurs when a cell body and excitatory presynaptic fibers on it are activated near a stimulating electrode (see Sugiuchi et al. 2005 for a more detailed identification method) (Jankowska et al. 1975; Shinoda et al. 1976, 1982, 1987). This commissural axon did not arise from a TRN because this axon was not antidromically activated from either the OPN or IBN region at 500 µA (Fig. 12C, 5–10). As shown in Fig. 11H, commissural axons projecting to the caudal SC belonged to intratectal neurons (non-TRNs) with multiple axon collaterals. Therefore this commissural axon was most likely an axon of a non-TRN with multiple axon collaterals whose cell body was located in the contralateral rostral SC. Stimulation of the more caudal SC (sites 2 and 3) also evoked direct and indirect spikes (Fig. 12B, 2 and 3), but stimulation of the most caudal SC (site 4) evoked only indirect spikes at fluctuating latencies with double-pulse stimuli (Fig. 12B, 4, right). In short, this neuron located in the rostral SC projected to the caudal part of the contralateral SC and received excitatory inputs from all four rostrocaudal sites of the ipsilateral SC. Similar results were obtained in four other commissural axons recorded in the caudal SC.