Distractibility and locomotor activity in rat following intra-collicular injection of a serotonin 1B-1D agonist

BEHAVIOURAL BRAIN RESEARCH

ELSEVIER

Behavioural Brain Research 67 (1995) 229-239

Research report

Distractibility and locomotor activity in rat following intra-collicular injection of a serotonin 1B- 1D agonist Pascale Boulenguez a, Nigel Foreman b, Jacques Chauveau c, Louis Segu a*, Marie-Christine Buhot a ~C.N.R.S., GDR Neurosciences, Equipe M~moire et r~cepteurs s~rotonine, BP 71, 31 chemin Joseph-Aiguier, 13402 Marseille Cedex 20, France b Psychology Department, Leicester University, University Road, Leicester LE1 7RH, UK "Immunotech S.A., 130 avenue de Lattre de Tassigny, B.P. 177F, 13276 Marseille Cedex 9, France

Received 21 June 1994; revised 2 August 1994; accepted 2 August 1994

Abstract The superior colliculus (SC) is thought to be the decision center for reactions to novel and/or moving stimuli in the peripheral visual field. Serotonin 1B (5-HT1B) receptors were previously demonstrated to be located on collicular axon terminals of retinal ganglion cells and their activation might depress afferent inputs from the retina. The effects of intra-collicular injections of 5-HT1 drugs on distractibility were studied in hooded rats trained to run toward illuminated targets for a food reward in a 2-choice runway. 8-hydroxy-2-(di-n-propylamino)tetraline (8-OH-DPAT), a 5-HTIA receptor agonist, RU 24969, a mixed 5-HTxA and 5-HT~B agonist, serotonin-O-carboxymethylglycyltyrosinamide (S-CM-GTNH2), a mixed 5-HT~B and 5-HT~D receptor agonist and saline (control) were alternately injected. Following the S-CM-GTNH 2 treatment alone, animals exhibited an erratic running style, involving side-toside movements of the head, without change in the overall accuracy of their locomotor trajectories, but with substantial decrease in their running speed. When distracting peripheral lights were introduced at the mid-points of the animals' run, in the weaker distracting condition (unilateral distractor) only, distraction indexes were found lower following the S-CM-GTNH2 treatment than following the other drug or saline treatments. It is concluded that serotonin, via 5-HTla_~t~ receptors, may induce an elevation of the visual distractibility threshold by modulating directly the transmission of the primary visual signal. Key words: Superior colliculus; 5-HTI~,; 5-HTIB; Neuronal modulation; Visual distractibility; Motor control; Microinjection

I. Introduction Rat retinal ganglion cells project mainly to two contralateral primary visual structures, the lateral geniculate nucleus of the thalamus and the superior colliculus [9,25]. These structures are interconnected, but they seem to participate in different aspects of vision. The lateral geniculate nucleus and its visual cortical projections are particularly concerned, especially in the rat, with the analysis of stimulus characteristics (shape, size, texture), evaluation of distance and visuomotor control [20]. The superior colliculus (SC) is important for responses associated with environmental novelty: detection and localisation of novel stimuli in the peripheral sensory field [ 15,50], organisation of orienting reactions toward such stimuli [ 11,21,47,48] or organisation of escape from them [ 12,13,14]. * Corresponding author. Fax: (33) 91.77.50.83. 0166-4328/95/$9.50 © 1995 Elsevier Science B.V. All rights reserved SSD1 0 1 6 6 - 4 3 2 8 ( 9 4 ) 0 0 1 5 2 - 9

The superficial laminae of the superior colliculus, including the superficial grey layer (stratum griseum superficiale, sgs), are largely visual, receiving two major inputs: from the contralateral retina [9,25,32] providing a retinotopic map of the visual field [2,10,14] and from the ipsilateral primary visual cortex [29,33,34]. Deeper strata (below stratum opticum) receive visual, auditory and somatic afferents and stimulation of cells in these layers may result in eye, ear, head and/or trunk movements toward contralateral locations, depending on the site of stimulation [10,35,48]. Thus, anatomical and electrophysiological evidence also point to a superior collicular role in sensorimotor integration, specifically in the organisation of overt components of selective attention, a view most strongly reinforced by the numerous studies demonstrating an absence of distractibility toward stimuli in the peripheral visual field following collicular ablation [10,12,15,21,22, 44,52].

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Although the behavioural effects of lesions have been extensively studied, the pharmacological basis of such effects has received less attention. The superior colliculus receives a substantial serotonergic (5-hydroxytryptamine, 5-HT) input, arising mainly from the dorsal and median raphe nuclei, whose terminals are found throughout all layers of the SC, but most densely within the stratum zonale and the upper sgs [38,40]. A relatively high density of 5-HT 1 binding sites is also found in the sgs, about 70 ~o of which being of the 5-HT1B subtype, very few of the 5-HT1D subtype (<6~o) and the remaining 30~o being 5-HT~A sites [4,8,42]. A low density of 5-HTtB sites was found in other deeper layers of the SC. 5-HT~c subtype, as well as 5-HT 2 sites, are scarcely represented in the rat SC [4,36,37,43]. Previous studies have shown that optic nerve section induced a decrease by 30~o in the number of 5-HT~B binding sites in the contralateral sgs, 28 days after surgery [4,45]. The location of these 5-HT~ sites on the axon terminals of non-serotonergic retinal cells in the sgs was demonstrated via autoradiography at the electron microscopic level [6]. These 57HT~ terminal heteroreceptors could provide the anatomical substrate for the previously demonstrated inhibition of induced activity of collicular cells after in vivo or in vitro application of serotonin [23,28,31,49]. This inhibitory effect of serotonin was indeed found to be mainly the result of a direct action on primary sensory afferents by Huang et al. [28], probably by decreasing the release of neurotransmitter from fibres of retinal ganglion cells. The aim of the present study was to test whether pharmacological stimulation of 5-HT~ receptors and particularly the 5-HT1B subtype, in the superior colliculus could inhibit the transmission of visual information within the structure. The effects of intracollicular injections of 5-HTIA and 5-HTl~ agonists on visual distractibility were therefore investigated, by means of a runway paradigm used in previous studies to demonstrate distractibility deficits in colliculectomised rats [15,21,22]. The molecules tested were as follows: (1) 8-hydroxy-2-(di-n-propylamino)tetraline (8-OH-DPAT), chosen for its high specificity toward 5-HT~A receptors [24]; (2) RU 24969, a mixed 5-HT~A_~B agonist [24,27,30]; and (3) serotonin-Ocarboxymethylglycyltyrosinamide (S-CM-GTNH2; [46]), which has been shown to be selective for 5 - H T ~ and 5-HT~D receptor subtypes compared to the 5-HT~A subtype [ 5 ]. Prior to the behavioural studies, the extent of diffusion of S-CM-GTNH 2 within the SC was analysed using a radioiodinated form of the molecule (S-CMG[ a25I]TNH2; [7]) in order to determine the most appropriate injection ~parameters.

2. Study of the diffusion of S-CM-G[~Z~I]TNH2within the SC 2.1. Materials and methods

Adult male Long-Evans hooded rats were used, weighing 250-300 g. They were injected with atropine sulphate (0.3 ml, i.p.) before being anaesthesized with sodium pentobarbital (Nembutal, 55 mg/kg body weight, i.p.). They were then placed in a Kopf stereotaxic apparatus. The scalp was incised at the midline, the skin over the cranium was reflected and four holes were drilled with a standard dental drill over the SC, at the following coordinates relative to the bregmoidal suture and the midline: A-P: -6.0 or -7.5; L: + 1.5 mm (from Paxinos and Watson [41]). At each location, a 30-gauge stainless steel needle was lowered to a depth 3.2 mm below the cortical surface. The needle was connected, via a 50-cm long polyethylene tubing, to a 10 #1 Hamilton syringe which was placed into a Harward seringe pump (model # 22). Four injections of 10 nM S-CM-G[125I]TNH2 (Immunotech, Marseille, France; [7]) were performed 5, 10, 15 or 30 min before sacrifice. One rat was injected in all four locations with 1 /~1 over 1 min and others with 1/~1 over 4 min, 0.5/~1 over 1 rain or 0.5/~1 over 4 rain. Following decapitation, the brains were rapidly removed from the skull and frozen in isopentane refrigerated in liquid nitrogen. Ten #m coronal sections were taken throughout the injection sites with a cryostat at -20 °C. They were quickly dried with hot air and were exposed for 4 days in darkness to a tritiumsensitive film (Hyperfilm-3H, Amersham). The film was developed in D19 and fixed in AL4 (Kodak) to allow the examination of S-CM-G[~25I]TNH2 diffusion within the midbrain. 2.2. Resul~

The maximum diffusion size was obtained by a 30-min post-injection delay. In this condition, 1 /~1 of the drug injected over 1 min induced a diffusion over a large region of the colliculus, of diameter of approximately 3 mm around the injection site. For 1/~1 injected over 4 min, a maximal 2 mm diffusion diameter was observed, and for 0.5/~1 injected over 4 min, a diffusion of diameter 1.5 mm was obtained (Fig. 1). In order to achieve a sufficient diffusion within the SC and at the same time, a reasonable time of immobilisation for the rat and limited delay between left and right injections, a compromise was decided upon for the injections to be used in the behavioural studies. Thus, an injection procedure with 0.8 #1 of drug injected over 2 min was chosen, with a delay of 25 min before the behavioural test. Considering the retinotopic representation of the visual field in the rat SC [ 14,19], the

P. Boulenguez et aL/ Behavioural Brain Research 67 (1995) 229-239

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Fig. 1. Intracerebral diffusion of the radiolabeled drug. Autoradiograms of dried coronal sections from the brains of four different rats. The injections of 10 nM S-CM-G[ 125I]TNH 2 (2000 Ci/mmol) were carried out with a needle, without a guide-cannula, at the coordinates indicated in the Materials and methods section. Injection volume was 1 #1 for A and B and 0.5/zl for C and D. Injection time was 1 min for A and C and 4 min for B and D. For each section the largest labeled region corresponds to the longer time of survival after injection (30 min). The picture in E shows the iodinated standards of Amersham International, as a reference for grey level intensities. The examples shown in this figure illustrate worst cases (greatest diffusion, greatest leakage between structures), with the exception of F which shows the typical case that was obtained using the parameters described for D. The diameter of the diffusion core diminished with decreasing injection volume, and with increasing injection time. With 1 /~1 injections, the perfused areas are very large: label is seen in both the superior colliculus (SC) and part of the cortex (curved arrow). Labeling of the cortex was less for 0.5 #1 injections and absent for the longest injection time (D and F). Leakage of radioactive drug between the SC/thalamus and the cortical-hippocampal structures, is evident in B and C (small arrows), but does not spread further. Other regions of the section are devoid of labeling. The parameters chosen for use in the behavioural experiment lie between C and D. Superficial collicular laminae were therefore well perfused as well as deeper ones. We cannot discount the possibility of minimal invasion of cortex and a small amount of diffusion between structures, though this would not have spread far from the injected zone.

stereotaxic coordinates were chosen in order to inject the drugs close to the terminals of retinal fibers stimulated by a distractor light located 15 ° above the horizon and about 80 ° laterally in respect to the sagittal plan.

3. Behavioural studies: runway path accuracy and distractibility 3.1. Materials and methods 3.1.1. Subjects Subjects were 12 male hooded rats of the Lister strain, weighing 200-300 g, housed individually in standard laboratory cages in a colony room which was illuminated between 07.00 h and 19.00 h, with 60~o humidity.They had free access to water, but were food-restricted to maintain 85 ~o of their initial body weight throughout the study. 3.1.2. Apparatus The apparatus used for behaviourai testing has been described and shown diagrammatically in earlier publica-

tions [17,18]. It consisted of a 1 m square, gray-painted arena, having sides 40 cm high. At the midpoint of one end of the arena, a 20 cm tapering recess led from the arena to a single, top-hinged translucent perspex goal door 8 cm square, its basis being at the arena-floor level. The door could be back-illuminated by a standard 12 V stimulus bulb. Pressing the goal door when it was illuminated activated a dipper mechanism that delivered a small quantity of evaporated milk to a feeding hole 1 cm behind the front face of the door. At the opposite end of the arena (hereafter, the 'discrimination' end of the arena) were two more goal doors, flush with the face of the arena, located with their centers 20 cm f r o m its midpoint. These could also be back-illuminated and delivery of a milk reward occurred on pressing the door when illuminated. Bisecting the apparatus, between the midpoints of the two side walls, was an infrared photobeam, 2 cm above the floor level. Two meters above the center of the apparatus was located a video camera, connected to a monitor and video recording equipment in an adjacent room.

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232

3.1.3. Preoperative runway training

midpoint of the apparatus. This triggered a clock which stopped when the animal pressed the illuminated goal door at the discrimination end.

Animals were trained to shuttle back and forth between the goal doors at the opposite ends of the arena. When the animal was first placed in the arena, the single goal door at the non-discrimination end was illuminated. The animal obtained milk by pressing the goal door, whose light remained on for 2 s following the door press. The light then extinguished and one of the goal doors at the discrimination end of the arena became illuminated, without delay. The animal then had to run to the illuminated door at the discrimination end to obtain a further reward. Pressing the non-illuminated door was recorded as an error and no food was delivered, though the animal could then proceed to the illuminated door and press for reward (delivered as described for the goal door at the non-discrimination end of the arena). In this way, the animal had to shuttle back and forth between the two ends of the apparatus, traversing the apparatus 60 times in a daily session (thus making 30 discrimination responses). Animals typically took 12-14 days to learn to do this competently, i.e., to achieve a criterion of three or fewer errors per day for 2 successive test days. Animals' running latencies over the second half of each discrimination run were recorded automatically. When running toward the discrimination e n d of the arena, the animal interrupted the photobeam at the

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When trained, animals were placed on ad libitum feed for 2 days and then given surgical implantation of cannulae under deep anaesthesia. Anaesthesia was induced by placing the rat into a chamber with 5 % Halotane. It was then given midazolam (Hypnovel, Roche) by intraperitoneal injection at a dose of 4 mg/kg and fentanyl (0.315 mg/ml) / fluanisone (10 mg/ml) (Hypnorm, Janssen) by intramuscular injection (0.5 ml/kg). The depth and duration of anaesthesia was controlled by careful titration using further doses of Hypnorm The anaesthesized rat was placed in a stereotaxic frame with the skull horizontal. The head was shaved and holes were drilled in the cranium overlying the superior colliculus at the following stereotaxic coordinates with respect to bregma: A-P: -5.75, L: + 1.2 mm (from Paxinos and Watson [41]). The dura was sectioned bilaterally. A pair of parallel stainless steel, 23 gauge cannula guides, glued together and 2.4 mm apart, were lowered into position 0.7 mm above the surfaces of the colliculi, 2.3 mm below the cortical surface (Fig. 2). The guides were fitted with 30 gauge keepers, and were

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P. Boulenguez et al. / Behavioural Brain Research 67 (1995) 229-239

held in place using cranioplast dental acrylic cement, fixed to the cranium with three, 20 BS stainless steel screws. The skin was drawn around the cap with interrupted sutures. Animals were given topical and systemic antibiotic treatments postoperatively and given an opiate analgesic during postoperative day 1. Seven days following surgery, the animals were again food-restricted and retrained to run in the runway. Note that by restricting the length of cannula to 1.0 cm above the surface of the cranium, the animals were able postoperatively to push open goal doors and obtain milk rewards with no greater difficulty than during the pre-surgical training. When animals reached the criterion (three errors per day maximum on 2 successive days) after 10-15 days of retraining, a 7-day test period began, consisting of 4 days when animals were tested following intracollicular injections, each injection day being separated by a non-injection day. On non-injection days, animals were tested as previously. Cannula keepers were cleaned in alcohol daily.

3.1.5. Injection procedure On the day prior to the first treatment day, all animals were injected bilaterally with saline to familiarize them with handling and procedures. On treatment days, each animal was taken in the home cage to a quiet room where it was wrapped gently in a cloth and the keepers were removed from the guide cannulae. Into each guide was inserted a 30 gauge injection needle that extended 1.2 mm beyond the tip of the guide when fully inserted and which was connected via flexible tubing to a 10 #1 Hamilton syringe. A volume of 0.8 #1 of saline or drug was injected over a 2-rain period, first through the left cannula and then the right. The needle was always left in place for one supplementary minute after an injection was complete, before being removed from the brain. Needles were cleaned in alcohol between injections. The rats were injected with saline or the following drugs diluted in 0.9~'/o saline: 8-OH-DPAT (3 /~g//~l, Research Biological, Wayland, MA), RU 24969 (5/~g//al, RousselUclaf, Romainville, France) or S - C M - G T N H 2 (0.38 #g/ #1, Immunotech, Marseille, France), as shown in Table 1 which furthermore indicates the affinity of the drugs for their binding sites (as measured by the K d, i.e., the equilibrium dissociation constant). Table 1

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3.1.6. Postoperative distractibility testing Testing in the distraction runway began 25 min after the commencement of the first (left) injection. The animal was placed in the test arena and run as described earlier. However, on four occasions per test session, breaking the photobeam at the midpoint of the animal's run triggered distractor light flashes in the animal's peripheral visual field. Distractors consisted of 12 V standard stimulus bulbs, mounted on the side walls of the apparatus, 16 cm from the photobeam (toward the front wall) and 26 cm above the level of the apparatus floor. A distraction was either unilateral (left or right bulbs flashed) or bilateral (both bulbs flashed simultaneously), two flashes occurring in 1 s in each case. On each test day, the first and third distractors were unilateral and the second and fourth were bilateral. On test day 1, distractors occurred on trials 6, 12, 21 and 27; on day 2, on trials 8, 15, 21, 28; on day 3, on trials 5, 13, 19 and 28 and on day 4, on trials 7, 14, 21 and 27. Unilateral distractors were presented before bilaterals to maximize the probability of a head-turn response, which occurs more often to unilateral than bilateral distractors; note that the second unilateral distractor occurred on the opposite side to the first and that side of first distraction was alternated between test days. The first distractor occurred on the left side for half of the subjects and on the right side for the other half; this order was reversed for each rat on the following test day. Each rat was tested on 4 test days, each test day being separated from the next by a normal training day (without injection and without distractor lights); this procedure minimized habituation to the occurrence of distractors. Orientation behaviours of the rats were recorded on videotape and rated by three independent observers and the latency between photobeam break and goal door press was recorded automatically for each trial, providing a further measure of distractibility. Animals' accuracy in running toward goal doors was assessed by measuring the length of their run paths, traced from videotape recordings played back a frame at a time using the animal's nose as a reference. Trajectory accuracy was measured at the midpoint of the animals' run by the absolute distance in screen cm of the animal's nose from the nearest point of the line joining the centers of the single goaldoor (at the nondiscrimination end of the runway) and the illuminated door to which the animal was running on that trial. This value was obtained for the first three 'correct' trials (correct in terms of goal door presses), for each of the left and right goaldoors at the discrimination end of the arena,

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P. Boulenguez et al. / Behavioural Brain Research 67 (1995) 229-239

which occurred between distraction trials 2 and 3. Note that where insufficient suitable trials occurred in this interval, additional suitable trials were sampled after the third distraction trial. Finally, the resulting six values were averaged.

3.1.7. Histology At the end of behavioural testing, rats were sacrificed with an overdose of barbiturate anaesthetic (Sagital Forte) and intracardially perfused with a buffered solution of 10~o formol saline. Immediately prior to killing, an injection of 0.8 pl of China ink was made into each of the colliculi, following the same procedure as for experimental injections. The brains were removed, and stored for 20 days in 10~o formalin. They were then frozen and 50 #m coronal sections were taken throughout the SC at -20 °C using a cryostat (Microm). Sections were stained with Cresyl violet. The position of the cannula tracks as well as the extent of diffusion of the dye were determined by microscopic examination. 3.2. Results 3.2.1. Anatomical findings The china ink injected through the cannulae just before perfusion with formalin was resistant to the fixative and Cresyl violet staining procedures, remaining clearly evident in every case. From the positions of the ink marker, it was clear that the guide cannula tips were well placed in ten of the twelve subjects, antero-posterior and mediolateral positions being as expected and approximately 0.6 mm ventral to the most superficial collicular lamina so that the drug diffusion reached both the most superficial layers and deep layers. A representative example of the placement of the cannulae + injection needles ensemble within the colliculus is illustrated in Fig. 2. In particular, the antero-posterior and medio-lateral positioning was such that the central points of the injections (dyes) were found in the projection zone of the retinal area that would be stimulated by distractor lights positioned about 80 ° laterally with respect to the sagittal plane and 15 ° above the horizon in the rat's visual field, according to the retinotopic representation of the visual field in the rat sgs [14,19]. In two cases, cannula placements were found to be very posteriorly placed (bordering on the inferior colliculus and also too deeply located within the colliculus). Data from these two animals were omitted from statistical analyses.

their latencies in traversing the runway on non-distraction trials. As in previous studies [15], a measure was obtained that best estimated the animal's typical running latency, termed the Median Non-Distraction trial Latency (MNDL), calculated separately for each animal on each day. The M N D L is the median of all non-distraction trial latencies excluding the first trial of the day and the four latencies on trials immediately following distractor trials (since the latter can show some residual effects of distraction). When M N D L latencies were entered into a one-way Analysis of Variance (ANOVA), a significant treatment effect emerged (F3,zv = 43.44; P<0.001). Multiple F-tests confirmed that the S - C M - G T N H 2 treatment differed from all others and did so highly significantly ( P < 0.001), since the rats ran significantly more slowly than after any other treatment. There were no significant differences among the remaining treatments ( P > 0.05; Fig. 3). From. the overhead videorecordings of animals' run paths, the effects of drug treatments on general trajectory accuracy were examined. Examples of run paths are shown in Fig. 4. It was clear from observing the animals that the S - C M - G T N H 2 treatment produced a characteristic running style, consisting of side-to-side movements of the head as the animal moved across the arena. There was no evidence whatsoever of such movements when the animals were given RU 24969 or 8-OH-DPAT. This phenomenon is clear from tracings of the run paths shown in Fig. 4. However, while this disorganisation of running had consequences for the animals' latencies, overall accuracy (in terms of goal-directedness) of animals' trajectories remained good in all cases. Fig. 5 shows the trajectory accuracy measured as described in Materials and methods (subsection 3.1.6). Trials were also sampled for the nondrug day preceding the first drug treatment. Note that by sampling mid-way through the trial sequence, we obtained this accuracy data at a point when animals ought to have

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3.2.2. Behavioural findings 3.2.2.1. Runway latency and trajectory accuracy. The four drug treatment conditions differed significantly in terms of

Fig. 3. Mean value of the M N D L (Median Non-Distraction trial Latency) for arfimals in each of the four treatment conditions. The asterisk indicates that latencies were significantly ( P < 0.001) greater in rats injected with S-CM-GTNH2 than in rats injected with any of the other drugs. The latter did not differ significantly.

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8O - HD -PA ~T RU 24969~ SC -MG -TN ~~ H2 Fig. 4. Six typical run paths (three correct runs to each goal door) recorded for animals on the day preceding the first drug injection and on each of the drug treatment days. Recordings were made via an overhead video camera, displayed on a monitor screen and traced using slow speed playback using the animal's nose as a reference point. Selection of trials for this analysis is described in the text. Numerals indicate the order of drug treatments.

been running efficiently and thus a high value of the resulting index would indicate that an animal was running indirectly and showing poor visual-locomotor guidance toward the target goal door. The values generated were entered into a one way ANOVA, which revealed no significant drug treatment effect (F4,36 = 0.45; n.s.). In addition, groups failed to differ in terms of discrimination accuracy. During drug treatment days, all treatment groups performed well, achieving an error rate of between 1.5 and 5~o. There were no significant treatment effects on this measure when individual animals' error rates were entered into a one-way non-parametric ANOVA (Friedman-test: Zr2 =4.21; d f = 3 ; n.s.).

3.2.2.2. Distractibility. Two types of measures were taken of animals' distractibility to unexpected peripheral light presentations: latency elevation and behavioural observations. Since the M N D L represents an animal's typi-

Fig. 5. Mean locomotor accuracy scores for animals on the day immediately preceding the first drug day and on each of the drug treatment days. The vertical scale represents the mean absolute distance (in cm) of the animal, at the mid-point of its run across the arena, from the straight line joining goal door centers (see text). The greater the value, the less accurate the animal's trajectory. There were no statistically significant differences between groups on this measure.

cal runway latency on a particular day, it is an appropriate measure against which to compare latencies on distraction trials; it also corrects for individual differences in animals' run speeds [ 15]. Therefore, for each of the four distraction trials occurring on a drug treatment day, the animal's latencies were expressed as a multiple of its M N D L for that day. Separate analyses of variance were carried out on the latency indexes (multiples of M N D L ) for unilateral and bilateral distractor trials (Fig. 6). When bilateral distractor results were analysed using a 4 (drug conditions) x 2 (distractor presentations 1 and 2) ANOVA, there were no significant effects (P>0.05). However, a similar ANOVA applied to the unilateral distractor data revealed a significant group difference

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Fig. 6. Distraction indexes. Latencies expressed as multiples of the M N D L of each rat on the four distractor trials after the different drug treatments. The analysis was performed separately for the unilateral distractors (left-hand histograms) and for the bilateral distractors (righthand histograms). The asterisk shows that the index of latencies measured in S-CM-GTNHz-treated rats was significantly (P<0.05) lower compared to that measured in other drug- and saline-treated rats, in the unilateral distraction condition.

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(F3,27 = 3.16; P < 0.05). There was no significant interaction between drug condition and distractors (F3,~7 = 1.61; n.s.). Post-hoc Tukey-tests revealed that the S-CMG T N H 2 treatment group's latency indexes were lower than for the RU 24969 condition ( P < 0.05) and both the 8-OHD P A T and saline control conditions (P<0.01). A more direct measure is the observation of distraction behaviour from overhead video recordings, which has been used in the past to supplement latency measures [15]. In the present study, three independent observers rated from videorecordings the animals' distractibility on each of the four distraction trials in each drug treatment condition, scoring animals as having (a) slowed or stopped, and (b) turned their head toward the distractor source(s). When animals are distracted by a peripheral light or lights, both slowing and head movement are typically seen. In extreme cases, an animal will run to a distractor light and investigate it. Raters marked trials as 'clearly stopped/slowed', 'clearly head-turned', 'possible slowing' and 'possible head-movement'. Raters agreed in all but a very few cases; they reviewed the videotape several times where they were uncertain about a particular trial. Data were analysed in two ways: distraction trials were analysed first using only those for which raters agreed that a clear distraction (slowed/stopped/turned) had occurred. A second analysis was then carried out in which were included all those trials for which raters had recorded a 'possible distraction'. In fact, in most respects, both the conservative and liberal criteria produced the same overall outcome. Animals could score between 0 (no distractor effective) and 4 (all distractors effective) on any one drug treatment day. The analysis was conducted by taking first the conservative criterion and considering whether raters thought that animals had stopped/slowed/hesitated when the distractor occurred. A non parametric ANOVA revealed significant differences between treatments (Friedman-test, corrected for ties: Zr2 = 12.14; dr= 3; P < 0.01). Wilcoxontests (corrected for ties) further revealed that animals in the S-CM-GTNH~ treatment condition showed visible slowing less often than in the saline control (T= 0; df= 9; P<0.01), RU 24969 ( T = 0 ; d f = 7 ; P < 0 . 0 5 ) and 8-OHDPAT ( T = 3.0; df= 9; P < 0 . 0 5 ) conditions. There were no significant differences among the latter three groups. When the more liberal criterion was applied, again treatment differences emerged clearly (Zr~= 13.48; d f = 3 ; P<0.01), the S - C M - G T N H 2 treatment producing less visible slowing than saline ( T = 0; df= 9; P<0.01), RU 24969 (T= 0; dr= 7; P < 0 . 0 5 ) or 8-OH-DPAT (T= 6.0; df= 10; P<0.05). Differences between treatments were also seen when raters judged whether slowing in conjonction with head movement toward the distractor light had occurred. The

[ ] SALINE

T

~ 8-OH-DPAT

IT.T E

[ ] RU 24969 I]] S-CM-GTNH2

fiiiiiiii

TREATMENTS

Fig. 7. Median number ofdistractions (slowing in conjonction withhead turns) evaluated through the analysis of video recordings (see text), after the different drug treatment conditions. The asterisk indicates that this index of distraction was significantly (P < 0.05) lower following treatment with S-CM-GTNH 2 than following any other treatments.

same pattern of results was obtained for both the conservative and liberal criteria. Considering the conservative criterion, a significant treatment effect occurred (Friedman-test, corrected for ties: l:r2= 9.13; df= 3; P<0.05; Fig. 7), reflecting a highly significant difference between S-CM-GTNH2 and saline treatments ( T = 0 ; d f = 9 ; P < 0.01), though the RU 24969 and 8-OH-DPAT treatments were intermediately placed and failed to differ from either the S-CM-GTNH2 condition (T= 9.0; df= 9; n.s.; T= 7.5; df= 8; n.s., respectively) or the saline control condition (T= 9.0; df= 8; n.s.; T= 11.0; dr= 8; n.s., respectively).

4. Discussion It is clear from the results that the injection of S-CMGTNH2 into the SC had an effect on visual distractibility and locomotor activity in the runway, whereas RU 24969 and 8-OH-DPAT had no significant effects. As stated in the introduction, 8-OH-DPAT selectively stimulates 5-HT~A receptors, whereas RU 24969 stimulates both the 5-HT1A and 5-HTtB receptors and S - C M - G T N H 2 stimulates more selectively the 5-HTI~ and 5-HTtD receptors [5,24]. It is therefore clear that 5-HT~A receptors in the SC are involved neither in the control of visual distractibility nor in locomotion behaviours. RU 24969 and S - C M - G T N H 2 both stimulate 5-HT m receptors, but only the effects of S - C M - G T N H 2 reached a significant level. This discrepancy might be due to the activation of 5-HTtD receptors by the S-CM-GTNH2, but, since a low level of 5-HT~o binding sites was found in the rat SC [8], we would rather explain this discrepancy by differences in diffusion properties and/or inactivation or dissociation rates between the two drugs. However, the effects seen in the S-CM-GTNHz condition are very specific and are

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likely to reflect the behavioural functions of 5-HT~B_~D receptors in the SC. The impact of distracting visual stimuli is less for animals in the S-CM-GTNH2 treatment condition, supporting the idea that the 5-HT~_~t~ receptor subtypes are likely to be involved in the inhibition of retinal afference to the superior colliculus by serotonin. This type of neuromodulatory control by serotonin could be generalized to other primary and secondary sensory inputs, as shown by several authors [ 1,3,26,51 ]. The spread of S-CM-GTNH 2 cannot be precisely ascertained, in particular, since the extent of diffusion of the iodinated form, used in the anatomical study reported here, may be different from the non-iodinated molecule. While it was clear that the bulk of the injected material was placed in the colliculus, we cannot rule out the possibility that some might have invaded ventricular space and affected the periventricular regions of adjacent neural structures. Nevertheless, we think this an unlikely explanation for the present findings, since the autoradiographic study (which was undertaken specifically to identify optimal cannula placements) suggested that S-CM-GTNH 2 remained well localised in SC, moreover, distractibility changes of the kind seen here in the S-CM-GTNH2 condition, are particularly associated with superior collicular function [ 15,21,22,39]. It is thus reasonable to assume that the dorsal spread of drug largely affected the more superficial regions of the SC, which are likely to be the most implicated in the detection and localisation of novel stimuli in the peripheral visual field [15]. However, ventral spread into deeper laminae are likely to be responsible for the motor effects. This indicates the physiological importance of the (even) low density of 5-HT~n receptors in deeper collicular laminae involved in the organisation of coordinated orienting movements (cf. [ 15]). The most likely output for such cells is via the predorsal bundle, which has inhibitory influences on brainstem nuclei involved in motor control and locomotor movement [ 16]. Indeed, the two animals tested in the present study but whose cannula placements were both posteriorily placed and located in very deep strata of the SC, suffered the motor effects consequent upon the injection of the 5-HT~_~t~ agonist (locomotor slowing and side-to-side head movements as other animals in this treatment condition). Yet, these animals showed normal distractibility, which may be explained by the fact that their superficial collicular laminae and more particularly the projection area of retinal cells activated by the distractor lights in the sgs, were likely to have remained unaffected. It is difficult to determine the precise nature of the deficits seen in the present study. First, the side-to-side movements made by animals treated with the 5-HT~_~t) agonist are likely to reflect a disorganisation of some aspect

237

of SC function. Dean et Redgrave [12,13] and Mort et al [39] have reported that side-to-side scanning movements of the head disappear after SC ablation, in rat and in hamster, and thus the movements seen here could reflect excitability in motor systems that normally control such scanning and attentional behaviours as a normal animal is investigating a novel environment or traversing an unfamiliar runway. These movements are not the result of spatial disorientation as indicated by the fact that in this condition, rats were able to take normal overall trajectories toward goal doors. Although animals in the S-CM-GTNH 2 condition were moving their heads from side to side during running, by the time animals reached the mid-point of the runway (at which the distractor sequence was triggered), their heads were usually oriented in a forward direction, toward the goal doors (Fig. 4). Head position always varies to some degree in this type of study. There were many notable instances recorded from the videotape in which S-CM-GTNH 2 animals, with forward-oriented heads, failed to respond to unilateral distractors that provoked reliable orienting in control and other drug conditions. In fact, although lateral head displacements in S-CM-GTNH 2 treated animals were of low amplitude compared with the head-turns typically made to distractors, they might occasionnally have been mistaken for orienting responses, leading to false positive 'apparent' distractions. Yet, according to both the liberal and conservative criteria used to assess distraction behavior, animals showed significantly fewer discernible orienting head turns toward distractors in the S-CMG T N H 2 condition than in any other. That they failed to detect and/or orient to distractors is further reinforced by the corresponding latency data which showed less locomotor slowing on unilateral distraction trials in this treatment condition. The deficit in distractibility can have sensorial as well as attentional components [ 12]. The rats' ability to discriminate lighted from non-lighted goal doors was unaffected, since treatment groups notably failed to differ in terms of goal door press errors; all animals maintained a very efficient level of discrimination performance despite drug treatments. The lack of distractibility observed following S-CM-GTNH 2 treatment could be interpreted as an inability of the injected rats to stop the ongoing task and completely shift their attention to an unexpected peripheral stimulus, as has been hypothesized after collicular lesions [12,15,21]. However, when treated with S-CMG T N H 2, rats were found to be more distracted by the stronger (bilateral) distractors than by the unilateral ones, which might be hypothesized as the specific ability the 5-HT~B agonist to induce the elevation of distractibility threshold by modulating the transmission of the visual signal. By this way, the serotonergic system would func-

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tion in the c o n t r o l o f s i g n a l - t o - n o i s e ratio, w h i c h m a y be u n d e r s t o o d , in the p r e s e n t study, at a p s y c h o p h y s i o l o g i c a l level. T h i s m e c h a n i s m c o u l d be i n v o l v e d in p r o c e s s e s underlying a d a p t a t i o n o f the reactivity a c c o r d i n g to the state o f a r o u s a l o f the a n i m a l a n d / o r to the salience o f the stimulus. F u r t h e r studies w o u l d be w e l c o m e t h a t i n v e s t i g a t e the effects o f 5 - H T l a a g o n i s t s in s t r u c t u r e s s u c h as s u b s t a n tia n i g r a a n d inferior colliculus to identify the i n v o l v e m e n t o f n o n - c o l l i c u l a r s t r u c t u r e s in b e h a v i o u r s e x a m i n e d in this study.

Acknowledgements T h e a u t h o r s w o u l d like to e x p r e s s t h a n k s to R o u s s e l U c l a f ( R o m a i n v i l l e , F r a n c e ) for p r o v i d i n g the R U 24969, the B i o l o g i c a l S c i e n c e s R e s e a r c h C o m m i t t e e o f the U n i versity o f L e i c e s t e r a n d the British C o u n c i l for financial s u p p o r t , also the D e p a r t m e n t o f P h a r m a c o l o g y o f the U n i versity o f L e i c e s t e r a n d D r . R o b i n S t e v e n s o f the U n i v e r sity o f N o t t i n g h a m , for t e c h n i c a l a d v i c e a n d a s s i s t a n c e . T h i s w o r k w a s also s u p p o r t e d by the E u r o p e a n C o m m i s sion ( B i o t e c h n o l o g y P r o g r a m , B i o 2 C T - C T 9 3 - 0 1 7 9 ) .

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