Injection of Nicotine Into the Superior Colliculus Facilitates Occurrence of Express Saccades in Monkeys

Hiroshi Aizawa,1 Yasushi Kobayashi,1,3 Masaru Yamamoto,1 and Tadashi Isa1,2

 1Department of Integrative Physiology, National Institute for Physiological Sciences;  2Graduate University for Advanced Studies; and  3Core Research for the Evolutional Science and Technology Program (CREST), Myodaiji, Okazaki 444-8585, Japan


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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Aizawa, Hiroshi, Yasushi Kobayashi, Masaru Yamamoto, and Tadashi Isa. Injection of Nicotine Into the Superior Colliculus Facilitates Occurrence of Express Saccades in Monkeys. J. Neurophysiol. 82: 1642-1646, 1999. To clarify the role of cholinergic inputs to the intermediate layer of the superior colliculus (SC), we examined the effect of microinjection of nicotine into the SC on visually guided saccades in macaque monkeys. After injection of 0.4-2 µl of 1-100 mM nicotine into the SC, frequency of extremely short latency saccades (express saccades; reaction time = 70-120 ms) dramatically increased, for the saccades the direction and amplitude of which were represented at the location of the injection site on the collicular map. However, no marked change was observed for the relationship between the peak velocities and the amplitudes of saccades. These results suggested that activation of nicotinic acetylcholine receptors in the SC can facilitate initiation but causes no major change in dynamics of visually guided saccades.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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The mammalian superior colliculus (SC) is a central structure controlling visually triggered behaviors such as saccadic eye movements and orienting behaviors etc. (for review, see Sparks 1986; Wurtz and Albano 1980). The superficial layer of the SC (the stratum griseum superficiale; SGS) receives visual input from the retina (Altman 1962; Lashley 1934) and the primary visual cortex (Harting et al. 1992). The intermediate layer (the stratum griseum intermediale; SGI) and the deep layer send descending motor command to the brain stem reticular formation and the spinal cord (Huerta and Harting 1982). As to the connection between the SGS and SGI, recent studies by Lee et al. (1997) and our laboratory (Isa et al. 1998a) showed the electrophysiological evidences for existence of an excitatory connection from the SGS to the SGI in the SC slices obtained from the tree shrew (Lee et al. 1997) and rat (Isa et al. 1998a).

It is well known that the SGI receives dense acetylcholinergic innervation in various species of mammals such as rats, guinea pigs, cats, ferrets, and primates (Beninato and Spencer 1986; Graybiel 1978; Hall et al. 1989; Henderson and Sherriff 1991; Jeon et al. 1993; Ma et al. 1991; Schnurr et al. 1992). These acetylcholinergic fibers originate from the pedunculopontine and laterodorsal tegmental nuclei (Beninato and Spencer 1986; Hall et al. 1989). These observations may suggest that the acetylcholinergic input to the SGI modulates execution of visually triggered behaviors. Despite the abundance of anatomic data, however, the functional implication of the acetylcholinergic input to the SC has not been understood.

Recently in slice preparations of the rat SC, we found that application of an acetylcholinergic agonist nicotine induced inward currents and depolarization in the SGI neurons by direct activation of nicotinic acetylcholine receptors (nAChRs) (Isa et al. 1998b). We have clarified further that activation of nAChRs in the SGI neurons enhances signal transmission in the pathway from the SGS to the SGI neurons. If this mechanism holds true also in primates, the consequence of the facilitation may be direct activation of saccade-related burst neurons in the SGI via the SGS-SGI pathway, behaviorally allowing the immediate release of a reflex-like saccade toward a target.

Behavioral studies on the saccadic reaction times (SRTs) have shown that distribution of SRTs shows at least two distinct peaks: regular saccades with SRTs of 150-250 ms and express saccades with SRTs ~100 ms (Fischer and Boch 1983; Fischer and Ramsperger 1984). In the present study, we examined the effects of microinjection of nicotine into the SGI in monkeys, especially the effects on the SRTs, and found that activation of nAChRs in the SC markedly reduced the SRTs and increased the frequency of express saccades without changing their dynamics. The present results may explain some of the neuronal mechanisms related to execution of express saccades and how nAChRs are involved in control of attentive behaviors.


    METHODS
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INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Two Japanese monkeys (Macaca fuscata) were fitted with an acrylic skullcap and scleral search coils (Fuchs and Robinson 1966) asceptically under halothane anesthesia. The surgical procedures have been described in a preceding paper (Aizawa and Wurtz 1998). Animals were trained to perform a visually guided saccade paradigm sitting in a primate chair with their heads fixed. Visual stimuli were given as small light spots (0.8 × 0.8° square) back-projected on a tangential screen at a distance of 28 cm from the eye. Three different tasks were shuffled randomly; NO GAP paradigm (the saccade target appeared at peripheral visual field after central fixation), GAP paradigm (the target was lit after 170 ms gap after the fixation offset), and catch trials (after the gap, the target was emitted at the fixation point again). GAP and NO GAP paradigms in opposite directions were presented with equal probability (22%, respectively). Catch trials consisted 12% of the whole session. In all the paradigms, the duration of central fixation was varied randomly between 300 and 1,000 ms. Behavioral paradigms, visual displays, and collection and storage of eye-movement data were under the control of a real-time data-acquisition system (REX) (Hays et al. 1982). Location of the tip of the microsyringe was identified by the vector and threshold of saccadic eye movements evoked by electrical stimulation (<20 µA, 500 Hz, 50 biphasic pulses of 200 µs width) via the electrode attached to the microsyringe (Crist, MRM-S02) (see Aizawa and Wurtz 1998). For the injection, the areas in the SC that represent 5-20° saccades were selected. The locations of the visual targets were determined to induce the saccades with the same vector as those evoked at the injection site ("contraversive saccades"), and those of the same amplitude but on the opposite side of the horizontal and vertical meridians ("ipsiversive saccades"). Contraversive and ipsiversive saccades were shuffled randomly. The timing of saccade onset was determined at the time when the eye velocity exceeded 30°/s. Saccades with reaction time <70 ms were regarded as "anticipatory saccades," which were not rewarded and discarded from data (see RESULTS). Saccades with reaction time >350 ms were also not rewarded and discarded from data. Frequency of anticipatory saccades did not exceed 3% of the trials in the present experiments. Either solution of nicotine (1-100 mM in saline) or vehicle (saline) was injected at a rate of 0.2 µl/min until the total volume reached 0.4-2 µl, and reaction times, amplitude, and velocities of saccades were analyzed. Experiments were performed in accordance with the NIH Guidelines for the Care and Use of Laboratory Animals.


    RESULTS
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Effects of nicotine injection (0.4-2 µl, 1-100 mM) into the SC on SRTs were examined in a total of 13 experiments in two monkeys (experiments N1-N13, see Table 1). As control, injection of vehicle (saline) was tested in four experiments (experiment S1-S4 in Table 1). Figure 1, A-D, shows the result of the experiment N10, in which 2 µl of 10 mM nicotine solution was injected. As shown in Fig. 1, A and B (NO GAP paradigm), the SRTs were distributed primarily between 120 and 200 ms (mean ± SD; 163.8 ± 11.4 ms, n = 22) before the nicotine injection (Fig. 1B, ). After nicotine injection, the SRTs were shortened (146.8 ± 23.8 ms, n = 107 observed during 30 min after injection) with a statistically significant difference from those before injection (P < 0.05, Student's t-test). As clearly illustrated in Fig. 1A, the SRTs stepped down from the range of 150-200 ms to extremely short range (<120 ms). The distribution of SRTs in the present experiments thus were divided into two different subgroups at 120 ms (Fig. 1B) as in the preceding study (Dorris et al. 1997). Saccades with SRTs shorter than 70 ms were regarded as anticipatory saccades and discarded from data. Therefore we classified the saccades with SRTs between 70 and 120 ms as "express saccades" and those with SRTs longer than 120 ms as "regular saccades" (Fischer and Boch 1983). The increase in frequency of express saccades continued for longer duration in the GAP paradigm (Fig. 1, C and D) as compared with the NO GAP paradigm (Fig. 1, A and B). The average SRT of express saccades in the GAP paradigm (100.1 ± 6.5 ms, n = 51) was slightly shorter than that in the NO GAP paradigm (104.8 ± 6.9 ms, n = 22). The difference was statistically significant (P < 0.05, Student's t-test) but minor. Thus the major change caused by introducing GAP was the prolonged duration of the nicotine effect.


                              
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Table 1. Average SRTs for all the experiments



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Fig. 1. A-D: effects of nicotine injection (10 mM, 2 µl, experiment N10) into the monkey superior colliculus (SC) on saccadic reaction time (SRT) during NO GAP and GAP (170 ms) visually guided saccade paradigms. Amplitude of the saccades was 7° in horizontal and 7° in vertical directions. Horizontal bars in A and C indicate period of nicotine injection (10 min). A and C: SRTs are plotted against time. Open circles, trials before injection; asterisks, trials during and after nicotine injection. A and C, NO GAP and GAP paradigms, respectively. B and C: histograms of the normalized distribution of SRTs before (lower open columns) and after application of nicotine (upper closed columns, 30-min recording) in NO GAP (B) and GAP (D) paradigms. E-H: effect of saline injection (experiment S4). Amplitude of saccades was 10 deg horizontal. Same arrangement as A-D.

As shown in Fig. 2, the average SRTs (A and C) decreased [plots fell below the equality line (slope = 1)], and the percentage of express saccades (E and G) increased (the plots fell above the equality line) for contraversive saccades both in NO GAP (A and E) and GAP (C and G) paradigms in a majority of nicotine injections (). Statistically significant reduction in average SRTs (P < 0.05, Student's t-test) was observed in 12 of 13 experiments for GAP paradigm and 9 of 12 experiments in NO GAP paradigm as indicated by asterisks in Table 1. The occurrence probability of express saccades showed statistically significant increase after nicotine injection both in NO GAP and GAP paradigms for the population data (n = 13, P < 0.05, Student's t-test). As to the ipsiversive saccades, on the other hand, no statistical change was observed in a majority of the cases. Statistically significant change (P < 0.05, Student's t-test) was observed in average SRTs in experiments N2, N8, N9 (NO GAP) and N11, N12 (GAP; Table 1). However, no consistent tendency was observed as to whether there was significant increase or decrease in average SRTs and in occurrence probability of express saccades (see also Fig. 2, B, D, F, and H). Thus the effect of nicotine was spatially selective. Injection of saline caused no significant effect in a majority of the cases as shown in Fig. 1, E-H (see the population data in Fig. 2) except for the experiment S3 in which the SRTs decreased for contraversive saccades in both GAP and NO GAP paradigms and experiment S4 in which the average SRTs increased for ipsiversive saccades in GAP paradigm (P < 0.05, Student's t-test; Table 1).



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Fig. 2. Comparison of average SRTs (A-D) and occurrence probability of express saccades (E-H) before and after nicotine injection. Data from 13 experiments for nicotine injection and 4 control experiments (saline injection) are plotted.  and open circle , injections of nicotine and saline, respectively. Horizontal axes indicate the values before injection and vertical axes indicate those after injection (for 30 min). A, B, E, and F show the data for NO GAP paradigm. C, D, G, and H show the data for GAP paradigm. A, C, E, and G show data for contraversive saccades. B, D, F, and H show the data for ipsiversive saccades. · · ·, equality line (slope = 1).

In addition, the effect of nicotine was dose dependent. When the effects of two different doses of nicotine into the same location of the SC were compared [0.4 µl of 50 mM (experiment N11) and 0.4 µl of 100 mM (N13)], the effect of the larger dose was longer in duration; however, there was no significant difference in the SRTs of the express saccades (104.6 ± 6.9 ms in N11 and 101.6 ± 11.0 ms in N13, P = 0.12, Student's t-test).

Figure 3 shows the relationship between the amplitude and peak velocity of eye movements during express saccades before (A and B) and after nicotine injection (C and D) in NO GAP and GAP paradigms (data from 7 experiments in 1 monkey). Linear regression lines between the amplitude and peak velocity virtually superimposed before and after the nicotine injection for both NO GAP and GAP paradigms (Fig. 3, B and D). The difference was not statistically significant for NO GAP paradigm (P = 0.10, Student's t-test) but significant for GAP paradigm (P = 0.04, Student's t-test). In the latter case, however, the difference was apparently minor (change in slope of the regression line was 3%, Fig. 3D). Thus nicotine injection increased the frequency of express saccades but caused no major change in their dynamics.



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Fig. 3. Relationship between the amplitude and peak velocity of express saccades in NO GAP (A and C) and GAP (B and D) paradigms, before (A and B), and after (C and D) nicotine injection into the SC. Data are from 7 experiments in 1 monkey. - - - and ---, regression lines for data obtained before and after the injection, respectively. Note that the regression line before the injection also was indicated in the bottom (- - -). In C, the 2 regression lines are completely superimposed.


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Schiller et al. (1987) showed that express saccades never occurred after ablation of the SC and concluded that the SC is an essential structure for execution of express saccades. Recent electrophysiological studies showed that express saccades are generated when the visual response of saccade-related burst neurons in the SGI triggers the motor burst (Dorris et al. 1997; Edelman and Keller 1996). Dorris et al. (1997) further showed that gradual increase in activity in a population of SGI neurons (buildup neurons) before the target appearance was correlated significantly with the occurrence of express saccades. Thus advanced increase in excitability level of the burst neurons may lead to lowering the threshold for trigerring the motor burst activity by a visual response, which leads to execution of express saccades. In contrast, when the advanced increase in activity is not sufficient, the visual response cannot trigger the motor burst; this results in regular saccades. Recently we showed that activation of nAChRs can induce inward currents and depolarization in a majority of the SGI neurons in slice preparations of the rat SC (Isa et al. 1998b). Furthermore, depolarization of the SGI neurons induced by nicotine lowered the threshold for bursting response of the SGI neurons to electrical stimulation of the SGS. Thus activation of nAChRs could facilitate signal transmission in the direct visuomotor pathway in the SC, the existence of which we proved in the rat SC slices (Isa et al. 1998a). The origin of the visual response in the SGI neurons of monkeys is not yet clear; however, these studies have suggested that express saccades can be generated by gating of direct signal transmission from the SGS to the SGI. In the present study, we observed that both introduction of GAP and increase in nicotine dose did not cause further reduction in the SRTs of express saccades. A slight increase in eye velocities was observed after nicotine injection for the GAP paradigm (Fig. 3D); however, the increase was much smaller than the effect caused by injection of GABAA antagonist bicuculline into the SC, which also increases frequency of express saccades (Hikosaka and Wurtz 1985). Thus no major change was observed in the dynamics of express saccades after nicotine injection. These results in part support the Fischer's three-loop model in which he proposed that that the loop with the shortest transmission time was used for express saccades (Fischer 1987).

Another interesting observation in the present study is that effect of nicotine injection was more prominent in the GAP paradigm than in the NO GAP paradigm even though they were randomly shuffled in the same block of trials. Thus the threshold for induction of express saccades by nicotine appeared to be lower in the GAP paradigm. This "GAP effect" may be partly caused by decrease in activity of fixation cells in the rostral pole of the SC, which can suppress initiation of saccades, during the gap period (Dorris and Munoz 1995). If so, the cholinergic system can facilitate induction of express saccades independently of release from inhibition by the fixation cells.

Besides the decrease in activity of fixation cells, advanced increase in activity of buildup cells (buildup activity) is correlated with occurrence of express saccades (Dorris et al. 1997). However, the origin of the buildup activity during the GAP period is still unknown. Furthermore it has been shown that frequency of express saccades increases with daily practice (Fischer and Ramsperger 1986). A question worth further investigation is whether the cholinergic input to the SGI originated from the pedunculopontine and laterodorsal tegmental nuclei is involved in the factors that facilitate occurrence of express saccades, such as buildup activity of the SGI neurons and daily practice.


    ACKNOWLEDGMENTS

We thank Y. Takeshima, C. Suzuki, J. Yamamoto, and C. Kamada for technical assistance.

This study was supported by grants from the Ministry of Education, Science, Sports, and Culture of Japan to T. Isa (08458266, 08279207, and 09268238), CREST of the Japan Science and Technology Corporation, the Daiko Foundation, Naito Memorial Foundation, Uehara Memorial Foundation, and Mitsubishi Foundation.

Present address of H. Aizawa: Dept. of Physiology, School of Medicine, Hirosaki University, Zaifu-Cho 5, Hirosaki 036-8562, Japan.


    FOOTNOTES

Address for reprint requests: T. Isa, Dept. of Integrative Physiology, National Institute for Physiological Sciences, Myodaiji, Okazaki 444-8585, Japan.E-mail: tisa{at}nips.ac.jp

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 13 January 1999; accepted in final form 3 June 1999.


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