Exp Brain Res (2009) 198:85–94

DOI 10.1007/s00221-009-1935-x

RESEARCH ARTICLE

The commissural transfer of the horizontal optokinetic signal in the rat: a c-Fos study

Renata Ferrari · Sergio Fonda · Matteo Corradini · Giampaolo Biral

Received: 1 March 2009 / Accepted: 30 June 2009 / Published online: 17 July 2009© Springer-Verlag 2009

Abstract We applied the Fos method in rats subjected tohorizontal optokinetic stimulation (OKS) to study whetheroptokinetic information is transferred through the commis-sural pretectal Wbres from one optic tract nucleus (NOT) tothe opposite. In binocular as well as in monocular nasal-ward OKS, the highest Fos immunoreactivity was found inthe NOT contralateral to the nasalward stimulation, asexpression of the activation either of direction-selectivecells and of commissural neurons. Even the opposite NOTshowed many Fos-positive cells activated by the oppositenucleus throughout the commissural pretectal pathway.They might be the GABA positive cells, which are thoughtto allow the activation in one nucleus to be transformed intoinhibition of the opposite side. In monocular temporalwardOKS, the inhibition on direction-selective cells and theconsequent silencing of commissural neurons caused thefaint immunoreactivity in the NOT contralateral to eyestimulated. In the opposite nucleus the few Fos-positivecells emerged as a consequence of the lack of the normaltonic commissurally mediated inhibition.

Keywords Optokinetic stimulation · NOT · c-Fos · Commissural pathway · Antero-posterior distribution

Abbreviations2DG 2-DeoxyglucoseAOS Accessory optic systemDS Direction-selectiveDTN Dorsal terminal nucleusGABA �-Aminobutyric acid

hOKN Horizontal optokinetic nystagmushOKS Horizontal optokinetic stimulationIO Inferior oliveNHP Nucleus prepositus hypoglossiNOT Nucleus of the optic tractN-T Naso-temporalOKN Optokinetic nystagmusOKS Optokinetic stimulationPBS Phosphate buVered salineSC Superior colliculusT-N Temporo-nasal

Introduction

Whole surround motion causes a reXexive ocular response,optokinetic nystagmus (OKN), elicited by a retinal slip sig-nal conveyed to a set of speciWc sub-cortical nuclei. Centralnervous structures committed to process this visual displace-ment depend on the direction of movement (see Simpson1984 for review). In mammals the nucleus of the optic tract(NOT) and the dorsal terminal nucleus (DTN) of the acces-sory optic system (AOS) are the Wrst essential relay stationsfor ocular compensatory movement in the horizontal plane(Collewijn 1975; Cazin et al. 1980; HoVmann 1982; Katoet al. 1986; HoVmann et al. 1988; SchiV et al. 1988).

In rats the NOT has a very fragmented and uneven struc-ture with a number of distinct clusters of neurons extendingthroughout the entire meso-diencephalic junction (Scalia1972; Scalia and Arango 1979). Many cell types diVerent insize, shape (Gregory 1985), electrophysiological properties(Prochnow and Schmidt 2004; Prochnow et al. 2007) andGABA immunoreactivity proWle (Giolli et al. 1985; van derWant and Nunes Cardozo 1988) have been described. Asfor GABAergic neurons, they can be interneurons devoted

R. Ferrari (&) · S. Fonda · M. Corradini · G. BiralDipartimento di Scienze Biomediche, Università di Modena e Reggio Emilia, 41125 Modena, Italye-mail: renata.ferrari@unimore.it

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to local eVects as well as projection cells exerting theirinhibitory inXuence outside the nucleus, mainly upon theAOS nuclei operating on the optokinetic vertical signal (seevan der Want et al. 1992 for review). Finally, excitatory orinhibitory projecting neurons are interspersed in such a wayas to prevent their discrimination on the basis of a segre-gated localisation (Robertson 1983). Between eVerent pop-ulations in particular, peculiar attention should be focussedon the commissural pretectal pathway, as currently a gen-eral agreement to its functional operations has not beenachieved. However, a reciprocal interplay between theNOTs for assuring appropriate optokinetic performance hasbeen proved. In fact, a unilateral lesion of the NOT in themonkey induces spontaneous nystagmus contralateral tothe lesion side (SchiV et al. 1990). Sectioning of the com-missural Wbres between NOTs re-establishes the monocularhOKN asymmetry previously reduced by monocular enu-cleation in rats (Reber et al. 1991). In lower vertebrates,where monocular OKN is elicited only by a nasalwardstimulus, injection of GABAA antagonist into one pretec-tum provokes a strong increase or the appearance of thenaso-temporal component (Bonaventure et al. 1983, 1985).

Pereira et al. (1994) reported that in the opossum thecommissural pathway assures the transfer of optokineticinformation from the stimulated eye to the ipsilateralnucleus, supporting the binocularity in each nucleus.Unitary responses of the ipsilateral NOT are abolished bylidocaine injection into the opposite nucleus. In an electro-physiological study in the rat, Schmidt et al. (1993) foundthat more than 50% of NOT-DTN neurons are binocular,attributing this property to the cortico-pretectal projections.However, the ablation of visual cortex does not aVect theoptokinetic performance in the rat. In fact, a roughly nor-mal OKN is performed following naso-temporal and temp-oro-nasal stimulation, both in monocular or binocularconditions (Harvey et al. 1997), showing that a directionalsignal could be built up without cortical contribution. Morerecently, recordings from direction-selective neurons in theNOT of an Australian wallaby (Ibbotson et al. 2002) sug-gested a speciWc role of the commissural route in assuringthe large binocularity of each nucleus, in agreement withthe results in opossum. Considering all these data and givena similar percent of binocular neurons and the very fewipsilateral retinal projections in the rat NOT, in our opinionalso in the rat the binocularity of NOT cells could bestrongly accounted for by the commissural pathway asdemonstrated in the opossum and wallaby.

The aim of the present study is twofold. First, to ascer-tain whether and to what degree neurons in each NOT canbe recruited by monocular or binocular horizontal optoki-netic stimulation (hOKS) and, for the monocular hOKS, inrelation to the preferred (temporo-nasal, T-N) or non-pre-ferred (naso-temporal, N-T) stimulus direction. In the case

of optokinetic transfer through the commissural Wbres, weneed to Wnd neurons activated in both pretectal nuclei, forwhatever viewing condition and stimulus direction. A pre-vious metabolic study seems to support this possibility(Biral et al. 1982). Second, to investigate the topographicsegregation of cells along the rostro-caudal extension of theNOT, mainly in relation to whether they are directly acti-vated by the retinal slip signal from the contralateral eye orindirectly stimulated via the pretectal commissure.

To achieve our purpose we used the Fos immunocyto-chemistry method, as it allows to detect activated neuronsin all the brain nuclei at one time and to map their positionwithin each cerebral structure. Furthermore, we can com-pare the present results with our previous data (Biral et al.1982, 1984, 1987) obtained by means of 2-deoxyglucosemethod (2DG). It has been claimed that the two methods, insome cases, do not give overlapping results, each detectinga speciWc neuronal population and, by consequence, diVer-entiating functional properties within a nucleus.

Preliminary results have been presented elsewhere(Ferrari et al. 1995; Biral and Ferrari 1999).

Materials and methods

Twenty male Long–Evans rats weighing 220–280 g, housedunder normal food, water and light conditions were used inthese experiments. Two weeks prior to the experiments theanimals were anaesthetised (pentobarbital, 40 mg/kg ofbody weight, i.p.) and a pair of steel nuts coated with dentalcement was secured to the skull with cyanoacrylate glue. Inthe training sessions and actual experiment they served as areceptor for a metal screw attached to a rigid support of asmall plastic box allowing a painless positioning of the headduring optokinetic stimulation (OKS).

The rats were subjected to Wve sessions of a training pro-cedure in order to habituate them to the restrained conditionand blocked head, thus avoiding or at least minimisingstress eVects. In some preliminary experiments, we testedthe animals for optokinetic performance to diVerent veloc-ity steps and for spontaneous oculomotor behaviour bymeans of a magnetic search coil gently glued on an eyewith blue cyanoacrylate (see Hess et al. 1985 for details).Only rats with regular and consistent optokinetic nystag-mus were used.

On the day of the experiment, lightly ether anaesthetisedanimals were housed in the plastic box positioned at thecentre of an illuminated drum (90 cm both in diameter andin height) lined with black and white alternating stripes, 1.2and 2.4 cm wide, respectively. After 1 h to eliminate anaes-thesia eVects, optokinetic stimulation was started under thebinocular or monocular condition, as speciWed below, withthe drum rotating around its vertical axis. Rats were divided

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into four groups: controls (a still illuminated pattern, 5rats); binocular horizontal optokinetic stimulation (patternrotating clockwise at 2.30°/s, 5 rats); monocular horizontaloptokinetic stimulation (pattern rotating at 2.30°/s either intemporo-nasal or in naso-temporal direction for the leftopen eye, 5 rats per group). For monocular OKS, the righteye was covered with a double thickness of black maskingtape. After stimulation for 1 h the rats were placed free inan individual cage in the dark for an additional 30 min.After injection with an overdose of nembutal, the rats wereperfused transcardially with 150 ml 0.1 M phosphatebuVered saline (PBS), pH 7.3, at room temperature, fol-lowed by 350 ml 4% paraformaldehyde in PBS. The brainswere removed, left in the same Wxative for 2 h, then placedin 30% sucrose until they sunk. Forty-micrometer coronalsections were cut on a freezing microtome and one out ofevery two sections was processed for Fos protein immuno-chemistry. BrieXy, after washing with PBS solution the sec-tions were incubated overnight at room temperature in aprimary antiserum (Cambridge Research Biochemical OA-11-823) diluted 1:4,000 with 0.3% Triton X-100 in PBS.Following a rinse with PBS, the sections were incubated for2 h with sheep anti-rabbit serum (1:200), washed, then leftin ABC solution (Vectastain Elite, Vector Labs.) for 1 h.After two more rinses, sections were treated with 0.01%diaminobenzidine in the presence of 0.07% hydrogen per-oxide. The sections were then mounted on subbed slides,dehydrated and cover slipped. Cresyl violet staining wascarried out on the remaining sections to better identify thestructures under study.

Boundaries of the studied structures were tracedaccording to the brain rat atlas (Paxinos and Watson1986), as well as to the histological sections and distribu-tion of retinal projections to NOT and DTN obtained inprevious experiments by means of an injection of alabelled aminoacid into the rat eye (Lui et al. 1992). Wearbitrarily divided the retino-recipient area occupied bythe DTN and NOT into three parts. The DTN alone ismore caudally located and extends in rostral directionfrom the middle of the superior colliculus for about500 �m. The NOT proper starts approximately 300 �mbefore the superior colliculus and spreads for about1,000 �m in the caudal direction. In between lies a transi-tion region roughly 300 �m long where DTN and NOTcoexist. Positive cells were manually counted in duplicateunder the light microscope at 100£ magniWcation overthe entire extension of the nuclei as well as in each of theabove indicated subdivisions. As a further control, allindicative Fos immunolabelled cells were recounted aftermapping by means of a camera lucida.

Statistical analysis was performed using the Student’st test (single tail, non-paired or paired groups) and P < 0.05was chosen as minimum signiWcance level.

Results

The immunoreactivity was restricted to the neuronalnucleus, showing great variability of stain intensity depend-ing on the amount of encoded protein. Fos protein wasfound in small, medium and large sized neurons.

Immuno-positive cell number

Fos-positive cells in the right and the left NOT-DTN ofcontrol and experimental animals are shown in Fig. 1. Thenumber of Fos-labelled cells in the nuclei of all experimen-tal rats is signiWcantly increased with respect to controls,except in the left NOT-DTN of monocular temporalwardstimulated animals (Fig. 1d). However, in all cases follow-ing optokinetic stimulation the right NOT-DTN shows asigniWcantly higher Fos immunoreactivity than that of theopposite nucleus.

Fig. 1 Mean § SEM of Fos-positive cell numbers in the NOT andDTN of rats subjected to four experimental conditions: illuminated sta-tionary drum (a); clockwise directed binocular optokinetic stimulation(b); monocular optokinetic stimulation nasalward to the open eye (c);monocular optokinetic stimulation temporalward to the open eye (d).OKS signiWcantly increases the number of Fos-positive cells in bothnuclei apart from the left nucleus in temporalward stimulated rats;however, immuno-positive cells are signiWcantly greater in the rightthan in the left nucleus. See “Results” for signiWcance levels

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Control condition

In rats kept in front of the stationary illuminated drum, thenumber of positive cells is low in the NOT-DTN (Fig. 1a),without any signiWcant diVerence between the left(30.20 § 9.09) and the right nucleus (22.40 § 7.50).

Binocular stimulation

Fos expression in both NOT-DTN is remarkablyincreased following binocular optokinetic stimulation(Figs. 1b, 2a, b). Fos-positive cells in the right nucleuscontralateral to the nasalward stimulated eye signiW-cantly outnumber (P < 0.001) those in the left NOT-DTNreceiving temporalward stimulation: 284 § 30.20 and128 § 46.53, respectively. However, note that immuno-reactive cells are four times those found in controls in the

NOT contralateral to the eye viewing the non-preferredstimulus motion.

Monocular stimulation

Temporo-nasal stimulus motion: preferred directionStrong Fos immunoreactivity is also expressed in mon-ocularly viewing rats submitted to nasalward directedoptokinetic stimulation (Figs. 1c, 2c, d). Fos-positivecells in the right NOT-DTN (298.50 § 98.90; Fig. 1c)contralateral to the open eye reach a value similar to thatfound after binocular OKS in similarly stimulated nuclei(Fig. 1b, c). On the other hand, several stained cells arepresent in the nucleus contralateral to the closed eye: thenumber (94.00 § 48.60) is not signiWcantly diVerentfrom that found in the left NOT-DTN following binocu-lar OKS (128.00 § 46.53), but signiWcantly higher

Fig. 2 Fos expression in coronal brain sections in the left (a, c, e) and right (b, d, f) rostral NOTs of stimulated rats. a and b binocular optokinetic stimula-tion directed clockwise; c and d monocular optokinetic stimulation nasalward to the left eye; e and f monocular optoki-netic stimulation temporalward to the left eye. Fos protein is more remarkably expressed in the right nucleus contralateral to the eye stimulated by the OKS in the preferred direction (b, d). Scale bar 200 �m

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(P < 0.05) than that in the nucleus contralateral to theclosed eye in rats stimulated by a monocular naso-temporal OKS (37.25 § 10.43).

Naso-temporal stimulus motion: non-preferred directionWhen a temporalward optokinetic stimulus is provided tomonocularly viewing animals, we found Fos immunoreac-tivity to a lesser degree than either the binocular and mon-ocular nasalward OKS, as reported above (Figs. 1d, 2e, f).In the right NOT-DTN contralateral to the open eye thenumber of cells (70.25 § 24.58) is signiWcantly diVerent(P < 0.05) from that found in the left nucleus contralateralto the closed eye (37.25 § 10.43), a value similar to that ofcontrol rats. The diVerences (0.001 < P < 0.01) in Fosexpression in the NOT-DTN contralateral to the diVerentlystimulated open eye (nasalward vs. temporalward) are evenmore striking: temporo-nasal rotation of the stimulusproves to be more powerful than the stimulus moving in theopposite direction.

The left eye of naso-temporal monocular OKS (Fig. 1d)and the right eye of binocular stimulated rats (Fig. 1b) receivethe same moving stimulus. If we compare Fos expression inthe respective NOT-DTNs, i.e. the right for the monocularand the left for the binocular OKS, a signiWcant diVerence isfound, immunoreactive cells being 70.25 § 24.58 and128.00 § 46.53, respectively (0.01 < P < 0.05).

Immuno-positive cell distribution

The substantial increase of Fos-positive neurons followinga hOKS is unevenly distributed and mainly restricted to therostral portions of the NOT. This segregation appears evenmore striking when comparing the labelled cell distributionwith the curve representing the area of the NOT and DTNcovered by the retinal aVerents. For all experimental groups(Fig. 3) more than 95% of the total positive cells are locatedin the NOT proper region which, on the other hand,accounts for about 80% of the total retinal aVerent area. Theremaining c-Fos positive neurons are restricted to the DTNalone region covered by 7–8% of retinal aVerents. The tran-sition zone of coexistence of NOT and DTN appears nearlydevoid of Fos expressing cells. In addition, the NOT properpresents a further uneven distribution with more than 70%of total immunoreactivity conWned to the anterior half ofthe nucleus.

Furthermore, we looked into a diVerential distribution ofFos-positive neurons along the NOT proper extension, byconsidering the right and the left nucleus in both monocularOKSs (Fig. 4a, b). It is well ascertained that in the monocu-lar viewing condition, clockwise or anticlockwise rotationof the drum elicits speciWc responses, respectively excit-atory or inhibitory contralateral to the open eye NOT neu-rons. So, Fos immunoreactivity represents for each nucleus

diVerent classes of cells. As for the NOT ipsilateral to theopen eye, its activation has to be indirect, via a commis-sural transfer or whatever other route, and the immunoreac-tive cells have to belong to neuron classes very diVerentfrom that of the contralateral NOT (see “Discussion” fordetails). In our experiments, following the T-N monocularOKS (Fig. 4a), the distribution of labelled neurons diVersonly slightly in the two NOTs. In the nucleus ipsilateral tothe stimulated eye c-Fos immunoreactivity is more locatedin the rostral sections, whereas in the NOT receiving retinalinput immuno-positive neurons are slightly more caudal.Even if not so evident, the same distribution is produced byN-T monocular stimulation (Fig. 4b). Hence, despite theresponse pattern elicited (excitatory or inhibitory) and theactivation pathway (direct or indirect), the absence of anysigniWcantly diVerent spatial distribution of Fos-positivecells in two nuclei can be considered an implicit, but nottrivial, validation of the large intermixture of distinctiveclasses of neurons classically described in the NOT.

Discussion

Present data reveal a consistent expression of Fos protein inneurons of the NOT and DTN following whole-Weldmotion in the horizontal plane, conWrming the role of thesenuclei as Wrst relay stations in processing and transferringthe retinal slip signal along the horizontal optokinetic route.

Fig. 3 Percent distribution of Fos-labelled cells along the antero-pos-terior extension of the right NOT and DTN following OKS. At eachlevel, the percentage of Fos-positive neurons is compared to the per-centage of the area covered by retinal aVerents obtained by antero-grade transport of labelled aminoacids. It is striking that about 95% ofFos immunoreactivity is conWned on the rostralmost part of the NOTthat accounts for only 80% of the whole nucleus area. In the caudal-most region of the NOT and in the DTN few cells express Fos protein

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Depending on visual motion direction, similar heavy Foslabelling has been detected in the optokinetic nuclei of thebirds stimulated with a striped drum rotating about a hori-zontal or vertical axis (Rojas et al. 2009).

Our results show that the distribution of Fos-labelledcells is far from uniform.

On the basis of the very uneven Fos labelling elicited byhOKS, the NOT can be subdivided into a lateral (posterior)and a medial (anterior) part. The lateral part of the NOT,and the DTN as well, has very few Fos-labelled cells,whereas the anterior part of the NOT shows an impressiveincrease of Fos expressing neurons, indicating a possibledistinction of operations within the nucleus. A diVerentialinvolvement of the lateral and medial part of the NOTcould have been supposed from a series of metabolic stud-ies in the rat and guinea pig (Biral et al. 1982; Lui 1987)using the deoxyglucose method. In fact, in those previous

studies, following horizontal optokinetic stimulation, atleast for the low velocities tested, the highest radiolabellingwas measured in the lateral portion of the NOT and in theDTN in the groove between the edge of the superior col-liculus and the medial geniculate body. As 2DG uptake hasmainly been considered in relation to the activity of theouabain-sensitive sodium pump (Mata et al. 1980), itshould be prevalent at the level of dendritic branches(Sharp 1976; Schwartz et al. 1979; Kadekaro et al. 1985).Consequently, 2DG uptake has been regarded as a pre-syn-aptic marker and, in our case, it has been attributed to theregion of the NOT where retinal optokinetic input is moreintensely processed. Some other Wndings further suggest apossible functional distinction between subnuclei in theNOT. For example, Nunes Cardozo and van der Want(1990) describe in the rabbit GABA-negative neurons pro-jecting to the inferior olive mainly located in the rostral partof the nucleus and GABAergic interneurons with a preva-lent caudal location. Also Vargas et al. (1997) Wnd therostralmost concentration of olivary projecting neurons anda more medial localisation of GABAergic and commissuralcells in the opossum. Therefore, these reports and the pres-ent data taken together suggest an operational dissimilaritybetween the posterior part of the NOT, which could be con-sidered as a further nucleus of AOS and a strict ally of DTN(Simpson 1984), and the other rostralmost regions of thenucleus. Furthermore, a series of anatomo-functional obser-vations give evidence that the NOT and the DTN could notbe entirely equivalent (see Büttner-Ennever and Horn 1997for review): they receive a diVerent retinal input and,besides retinal slip neurons, several additional classes ofneurons are found only in the NOT. In addition, the DTN aswell as the other AOS nuclei lack a striate cortical input, atleast in the guinea pig (Lui et al. 1994), and perhaps in allother lateral-eyed animals. Conversely, calcitonin gene-related protein is widely distributed in the AOS nuclei butnot in the NOT (Ribak et al. 1997). More recently, in theirexhaustive review Giolli et al. (2005) have introduced rea-sonable doubts concerning the NOT and the DTN, as a uni-tary functional complex.

In contrast with 2DG technique, the Fos method is ableto resolve single neuron activation and represents a power-ful tool for mapping the pattern of post-synaptic stimulation(Morgan and Curran 1991). Therefore, the number of Fos-positive cells found in the rostral part of the nucleus shouldbelong to neurons projecting out of the NOT, as neuronstransferring retinal slip signal to the inferior olive (IO) andto the nucleus prepositus hypoglossi (NPH), as well as toneurons originating in the pretectal commissural pathway,as discussed below. However, eVerent neurons towardsdiVerent stations are largely intermingled within the NOT(Robertson 1983). Neurons originating in the descendingpathways towards the IO and the NPH are not topographically

Fig. 4 Distribution of Fos-labelled cells along the antero-posterioraxis of both NOTs proper following monocular T-N (a) and N-T (b)optokinetic stimulation. The right NOT is directly receiving the retinalslip signal, whereas the left NOT is activated via indirect pathway. Noobvious segregation is observed between direct and indirect activatedneurons

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conWned with respect to cells projecting to the thalamus andcontralateral NOT. Furthermore, besides distribution nei-ther size or shape are distinctive for a particular class ofthese cells (Schmidt et al. 1995). Recently, Prochnow et al.(2007) found that within the NOT neurons projecting toipsilateral IO, ipsilateral SC and contralateral NOT wereintermingled at similar topographical locations. Moreover,two classes of GABAergic cells have been described alongthe NOT–DTN extension: small to medium sized and largecells interpreted, respectively, as local interneurons andprojection neurons mainly directed to AOS nuclei (van derWant et al. 1992). Thus, we can also infer that a part ofFos-labelled cells can be also GABAergic. Unfortunately,although Fos labelling may be interpreted as a substantialindex of neuronal activation, we still do not understand therelationship between Fos expression and the immunoreac-tivity proWle of neurons. Thus, the Fos method does notallow the diVerent classes of activated neurons to be pre-cisely diVerentiated. In our case, it cannot distinguishGABAergic from non-GABAergic cells.

However, an attempt to discriminate between diVerentcell classes compares Fos expression appearing in eachNOT following either binocular or monocular hOKN and,for the monocular condition, with respect to the preferred(nasalward) or non-preferred stimulus (temporalward)direction. All parametric studies of hOKS performance inlateral-eyed mammals demonstrated an asymmetricresponse during monocular stimulation. The most consis-tent response is elicited by a nasalward directed stimulus(Collewijn 1969; Cazin et al. 1980; Hess et al. 1985;Volchan et al. 1990) and the NOT activated is the nucleuscontralateral to the eye viewing that stimulus motion.Accordingly, following hOKS the highest Fos expression isinduced in the NOT contralateral to the temporo-nasalstimulated eye both under binocular or monocular viewingconditions. Unpredictability, a consistent Fos immunoreac-tivity also appeared in the homologous regions of the oppo-site NOT, namely the nucleus receiving the temporalwardmotion signal (non-preferred direction) in binocular OKS,as well as the nucleus contralateral to the closed eye inmonocular OKS. These results are surprising, as temporal-ward stimulus motion is known to be largely inhibitory forthe DS neurons of the contralateral NOT, and it is com-pletely unexpected to Wnd a large sign of activation in thenucleus related to the closed eye as only a poor ipsilateralretinal input, functionally ineVective, impinges upon it(Cowey and Franzini 1979).

How can the high Fos immunoreactivity in the NOTcontralateral to the closed eye be interpreted? And how canthe contrasting results obtained in NOTs receiving naso-temporal stimulation be interpreted in relation to binocularor monocular vision? One may speculate a role played bythe commissural projection through which the NOTs are

connected to each other. This commissural pathway hasbeen anatomically documented in the rat (Terasawa et al.1979; Korp et al. 1989; Reber et al. 1991; Schmidt et al.1995; Prochnow et al. 2007), whereas its functional signiW-cance has been neglected or misinterpreted. Collewijn andHolstege (1984) did not consider involvement of the pretec-tal commissural pathway when discussing improvement ofthe naso-temporal OKN in adult rabbits following unilate-ral enucleation. On the other hand, Reber et al. (1991) sug-gested that a release from the reciprocal inhibition betweenthe two NOTs running along the commissural Wbres couldcause the increased gain of the naso-temporal responses inmonoenucleated rats: in fact, subsequent commissurotomyabolished this improvement. Moreover, it has been sup-posed that the commissure between the NOTs might beinvolved in synchronizing the two hemispheres for control-ling the fast phases of the OKN (Schmidt et al. 1995).

A diVerent and more convincing opinion suggests thatthe commissural NOT projections allow the activity evokedin one nucleus to be transferred to the opposite nucleus(Pereira et al. 1994; Ibbotson et al. 2002). This transfer issupposed to be the basis of neuron binocularity in eachNOT and assures that the activation of one nucleus is trans-formed into inhibition of the other: GABA-negative com-missural cells driven by retinal slip activate GABAergicinterneurons of opposite nucleus which, in turn, inhibit DScells of the same side. In fact, it has been found that a classof GABAergic neurons inhibit the DS cells projecting tothe IO (Horn and HoVmann 1987; van der Want and NunesCardozo 1988; van der Want et al. 1992). Now, our dataseem to be validated by the experimental Wndings of Pereiraand Ibbotson. Fos-labelled cells following the nasalwardOKS in the NOT contralateral either to temporalward stim-ulated (binocular condition) and to the closed eye (monocu-lar condition) might represent the population of GABAinterneurons activated by the opposite NOT through thecommissural pathway. In conclusion, following an hOKSthe Fos-positive cells in the NOT receiving the preferredmotion signal could mainly represent two classes of excit-atory projection neurons: DS cells and commissural neu-rons crowded into a very restricted rostral region of thenucleus and largely intermixed. As far as the facing NOT isconcerned, Fos-labelled cells must be ascribed only toGABAergic interneurons which do inhibit DS cells of thesame side. Even the distribution of this inhibitory popula-tion is highly concentrated in a limited part of the NOTproper. Moreover, by comparing the distribution curves oflabelled cells in both NOTs, it seems evident that GABApositive cells occupy the same area where direction selec-tive cells and commissural neurons are located. Thisarrangement proves to be very eVective in the operation ofthe NOT, given the close proximity and interspersion of theinhibitory and projecting neurons. A further support for

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commissural transfer of optokinetic information betweenthe two pretectal nuclei is oVered by a 2DG study (Biralet al. 1982). In monocularly stimulated rats following a uni-directional OKS both NOTs were labelled. More impor-tantly, since 2DG uptake increase is related to enhancedexcitatory input, the nervous activity running along thecommissural Wbres cannot be inhibitory, as has beeninvoked (Reber et al. 1991).

Finally, we have to consider the Fos NOT immunoreac-tivity observed after monocular temporalward OKS, knownto elicit a weak and irregular OKN in all lateral-eyed ani-mals. Experimental data support that each nucleus is exclu-sively committed to building-up one direction of the slowphases of the optokinetic nystagmus: the right NOT and theleft NOT govern clockwise and anticlockwise slow eyemotion, respectively. In fact, electrical stimulation of eitherNOT elicits ipsiversive slow eye movements and thedestruction of either nucleus abolishes the OKN in responseto nasalward stimulation of the contralateral eye (Collewijn1975; Cazin et al. 1980; Precht and Strata 1980; Kato et al.1988; SchiV et al. 1988, 1990). Therefore, for a monocularnaso-temporal OKS the NOT initiating the OKN will bethat ipsilateral to the open eye. In our rats, OKS was givento the left eye and the slow eye phases were directed anti-clockwise: consequently, the left NOT is responsible for theoculomotor response. Are the Fos-positive cell numbers wefound in each NOT consistent with the above statements?In comparison with the other experimental groups, N-Tstimulated rats have the lowest Fos immunoreactivity;moreover, in the left NOT, working for the OKN, Fos cellsare signiWcantly fewer than in the right NOT receiving theN-T motion. Following the hypothesis of Pereira et al.(1994), silencing the commissural neurons, as occurs in theNOT receiving N-T retinal slip, releases the facing nucleusfrom tonic inhibition and allows a mild excitation toemerge. Accordingly, the few Fos cells found in the leftNOT distributed in a minimal area of the rostral nucleus,could be neurons projecting to premotor structures, consis-tent with the low gain of the optokinetic response. On theother hand, Fos expressing cells in the right NOT, locatedand concentrated in the rostral region, could be assigned toa population of inhibitory interneurons activated in someway by the temporalward OKS. DS in the NOT are largelyinhibited by naso-temporal motion, but the inhibition doesnot arise from the DS retinal ganglion cells: retinal input isindeed glutamergic, that is excitatory (Nunes Cardozo et al.1991; Schmidt 1991). Therefore, this inhibition must beintrinsically created within local circuits of NOT neuronalpopulations or imported from extraretinal sources and man-aged within the nucleus (Schmidt et al. 1994). The resultinginhibition is conveyed towards the DS as well as towardsthe commissural neurons which, inactivated, can release theinhibitory interneurons of the contralateral NOT from tonic

activation. As reported above, the Wnal eVect is a mild exci-tation of the NOT ipsilateral to the open eye and the occur-rence of weak nystagmus.

An alternative hypothesis to that proposed by Pereiraand Ibbotson could account either for NOT neuron binocu-larity and hence for the Fos immunoreactivity we observed.In fact, besides a strong contralateral input, the NOTreceives in the rat a consistent projection from the ipsilat-eral visual cortex (Schmidt et al. 1993; Shintani et al.1999). However, experimental evidence seems to rule outany consistent contribution of the cortico-pretectal pathwayin the NOT operation related to optokinetic reXex. First, noc-Fos immunoreactivity is found neither in the dorsalgeniculate nucleus nor in the visual cortex in present exper-iments. Second, it has been already demonstrated that thevisual cortical ablation is for the most part ineVective onoptokinetic performance of binocularly as well as monocu-larly viewing rats (Harvey et al. 1997). Similarly, corticallesions performed in other lower mammals do not impairoptokinetic responses (Hobbelen and Collewijn 1971; Pereiraet al. 2000).

In conclusion, following a horizontal optokinetic stimu-lation in whatever direction and viewing condition, Fos-positive cells are found in both NOTs. DiVerent subclassesof cells express Fos protein depending on whether the NOTis directly or indirectly stimulated. It seems likely thatinformation running through commissural Wbres is excit-atory even in the rat, hence accounting for the binocularityof more than 50% of NOT cells.

Acknowledgments This work was partially supported by EU-Bio-med 2 grant no. BMH4-CT96-0976

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