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Lack of 5-HT1B receptor and of serotonin transporter have different effects on the segregation of retinal axons in the lateral geniculate nucleus compared to the superior colliculus
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Neuroscience Vol. 111, No. 3, pp. 597^610, 2002 M 2002 IBRO. Published by Elsevier Science Ltd All rights reserved. Printed in Great Britain 0306-4522 / 02 $22.00+0.00
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LACK OF 5-HT1B RECEPTOR AND OF SEROTONIN TRANSPORTER HAVE DIFFERENT EFFECTS ON THE SEGREGATION OF RETINAL AXONS IN THE LATERAL GENICULATE NUCLEUS COMPARED TO THE SUPERIOR COLLICULUS A. L. UPTON,a;1;2 A. RAVARY,a;1 N. SALICHON,b R. MOESSNER,c K.-P. LESCH,c R. HEN,d I. SEIFb and P. GASPARa * a
INSERM U106, Ho“pital de la Pitie¤-Salpe“trie're, 75651 Paris, France b
c d
CNRS UMR 146, Institut Curie, 91405 Orsay, France
Department of Psychiatry, University of Wu«rzburg, 97080 Wu«rzburg, Germany
Center for Neurobiology and Behavior, Columbia University, New York, NY 10032, USA
AbstractAWe have shown previously that raised levels of serotonin (5-hydroxytryptamine or 5-HT) during development prevent retinal ganglion cell axons from segregating into eye-speci¢c regions in their principal targets: the superior colliculus and the dorsal lateral geniculate nucleus. Possible mediators of 5-HT in this system include its plasma membrane transporter, which is transiently expressed by a sub-population of retinal ganglion cells, and the presynaptic 5-HT1B receptor carried on retinal ganglion cell axons. We analysed the retinal projections of 5-HT1B knockout (n = 15), serotonin transporter knockout (n = 14), serotonin transporter/5-HT1B double knockout (n = 4) and monoamine oxidase A/5-HT1B double knockout (n = 3) mice. In all four di¡erent knockout mice, the ipsilateral retinal projection to the superior colliculus was more di¡use and lost its characteristic patchy distribution. The alterations were most severe in the serotonin transporter knockout mice, where the ipsilateral retinal ¢bres covered the entire rostrocaudal and mediolateral extent of the superior colliculus, whereas in the 5-HT1B and double knockout mice, ¢bres retracted from the caudal and lateral superior colliculus. Abnormalities in the 5-HT1B knockout mice appeared only after postnatal day (P) 4. Treatment with parachlorophenylalanine (at P1^P12) to decrease serotonin levels caused an exuberance of the ipsilateral retinal ¢bres throughout the superior colliculus (n = 9). In the dorsal lateral geniculate nucleus in contrast, the distribution and size of the ipsilateral retinal projection was normal in all four knockout mice. In the serotonin transporter knockout mice however, the contralateral retinal ¢bres failed to retract from the mediodorsal dorsal lateral geniculate nucleus, an abnormality that was reversed by early treatment with parachlorophenylalanine and in the serotonin transporter/5-HT1B double knockout. Our observations indicate: (1) that the lack of 5-HT transporter and the associated changes in 5-HT levels impair the segregation of retinal axons in both the superior colliculus and the dorsal lateral geniculate nucleus; (2) that 5-HT and 5-HT1B receptors are necessary for the normal re¢nement of the ipsilateral retinal ¢bres in the superior colliculus, but are not essential for the establishment of eye-speci¢c segregation in the thalamus. Thus, both an excess and a lack of 5-HT a¡ect the re¢nement of the superior colliculus retinal projection, while the establishment of eye-speci¢c patterns in the dorsal lateral geniculate nucleus appears not to be sensitive to the lack of 5-HT or 5-HT1B receptors. M 2002 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: retinal projections, development, parachlorophenylalanine, knockout, mouse.
Retinal ganglion cell (RGC) ¢bres from the two eyes segregate into eye-speci¢c territories during early postna-
tal development before eye opening. Spontaneous activity in the retina during development appears to be essential for this process (Galli and Ma¡ei, 1988; Meister et al., 1991; Penn et al., 1998). An indication of the importance of serotonin (5-hydroxytryptamine or 5-HT) in normal development of retinal projection was provided by a transgenic mouse strain lacking monoamine oxidase A (MAOA), an enzyme which breaks down 5-HT (Cases et al., 1996). As a consequence, MAOA knockout (KO) mice have very high levels of 5-HT during embryonic and neonatal life. Excess 5-HT during the early postnatal period prevents segregation of retinal a¡erents in the dorsal lateral geniculate nucleus (dLGN) and the superior colliculus (SC) (Upton et al., 1999). The disruption is severe: in MAOA KO mice the contralateral projection ¢lls the entire dLGN; the ipsi-
1
These authors contributed equally to this work. Present address: University Laboratory of Physiology, University of Oxford, Oxford OX1 3PT, UK. *Corresponding author. Tel. : +33-1-53612646; fax: +33-145709990; http://mendel.u106.eu.org/. E-mail address: [email protected] (P. Gaspar). Abbreviations : 5-HT, 5-hydroxytryptamine or serotonin; 5-HTT, serotonin transporter; ANOVA, analysis of variance ; dLGN, dorsal lateral geniculate nucleus; E, embryonic day; HRP, horseradish peroxidase; KO, knockout; MAOA, monoamine oxidase A; P, postnatal day; PCPA, parachorophenylalanine; RGC, retinal ganglion cell; SC, superior colliculus ; SGS, stratum griseum super¢ciale; SO, stratum opticum. 2
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lateral projection to the dLGN is greatly enlarged and the ipsilateral projection to the SC does not form patches but is instead di¡use (Upton et al., 1999). A similar disruptive role of excess 5-HT on the segregation of retinotectal axon terminals has also been demonstrated by several experimental approaches in hamsters (Rhoades et al., 1993; Mooney et al., 1996, 1998). The role of 5-HT1B receptors in this process has been suspected because these receptors are expressed by RGCs from embryonic day 15 (E15) onwards (Upton et al., 1999). Activation of the 5-HT1B receptor on RGC terminals in the SC is known to reduce glutamate neurotransmission in adult hamsters (Mooney et al., 1994) and could thereby prevent the transmission of activity-dependent cues from the retina to the central visual targets. There are, however, other possible sites of action for excess 5-HT in this system. We found previously that during development the serotonin transporter (5-HTT) is transiently expressed in a subset of RGCs, primarily but not exclusively those RGCs that project ipsilaterally to the dLGN and the SC (Upton et al., 1999). The function of this uptake is still unknown, but possibilities include internalisation of 5-HT for use as a borrowed neurotransmitter or clearance of 5-HT to produce a local reduction in its extracellular concentration. In a previous study, focused on the retinogeniculate projection and the barrel ¢eld in somatosensory cortex, we have reported that the genetic lack of 5-HT1B receptors does not visibly a¡ect the segregation of the ipsi/ contralateral retinal ¢bres in the dLGN or the formation of barrels in the somatosensory cortex (Salichon et al., 2001). However, the disruptive e¡ects of an excess of 5-HT in these two systems appears to be entirely corrected by the additional lack of 5-HT1B receptors: a normal barrel ¢eld and a normal segregation of the retinogeniculate projection were noted in MAOA/5HT1B double KO mice and in MAOA/5-HTT/5-HT1B triple KO mice. During the course of this study however, preliminary observations suggested that some abnormalities remained in the retinotectal projections of these double and triple KO mice. To understand the nature and signi¢cance of this ¢nding, we conducted a systematic investigation of the retinotectal and retinogeniculate projection in mice carrying single gene inactivation of the 5-HT1B receptor, of the 5-HTT (Bengel et al., 1998), and of both these genes. We compared these ¢ndings to those obtained in the MAOA/5-HT1B double KO mice.
EXPERIMENTAL PROCEDURES
Animals KO mice and some of the normal mice (C3H/HeJ and C57BL/6J) were bred at the Curie Institute (Orsay, France). Additional normal mice were purchased from Janvier (France). We backcrossed the original 5-HTT KO strain (which has a mixed genetic background of 129/Sv, C57BL/6J and CD-1; Bengel et al., 1998) onto the C3H/HeJ for six generations. The 5-HT1B KO was originally generated in the 129/Sv strain (Saudou et al., 1994), but the single KO animals used in this study were backcrossed onto the C3H/HeJ background for 10 generations. MAOA/5-HT1B KO and 5-HTT/5-HT1B double
KO mice were generated in sixth generation backcrosses onto the C3H/HeJ background. Experiments were carried out in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC). Anterograde axonal tracing by intraocular horseradish peroxidase injections Adult mice were anaesthetised with 4% chloral hydrate (0.1 ml i.p. per 10 mg of body weight) and pups aged postnatal day 3 (P3) were anaesthetised by hypothermia. Horseradish peroxidase (HRP, type VI; Sigma, St. Louis, MO, USA) 60%, 1.5^4 Wl (depending on the age of the mouse), in physiological saline was injected into the vitreous chamber of the left eye with a Hamilton syringe inserted just behind the corneoscleral margin of the eye. Twenty-four hours later, mice were anaesthetised and perfused through the aorta with 5^20 ml of saline followed by 40^150 ml of ice-cold 1% paraformaldehyde, 1.25% glutaraldehyde in a 0.12 M phosphate bu¡er (pH 7.4). The brains were removed and cryoprotected in 30% sucrose/0.12 M phosphate bu¡er overnight at 4‡C. Brains were frozen and serial 40-Wm coronal sections (52-Wm for pup brains) were collected in icecold 0.12 M phosphate bu¡er and stored at 4‡C. Within 24 h, sections were rinsed for 10 min in 0.12 M acetate bu¡er (pH 3.3). Sections, maintained at 4‡C and protected from light, were then reacted for HRP using tetramethylbenzidine (0.067%) as the chromogen. The reaction was started by the addition of 0.006% H2 O2 . Further additions were made at 20-min intervals until the reaction was deemed complete (generally three additions). Sections were rinsed in 0.12 M acetate bu¡er (pH 3.3) at 4‡C to stop the reaction, immediately mounted on glass slides and left to air dry overnight. The reaction product was stabilised by a 3-min immersion in methyl salicylate before quick dehydration and mounting. Parachlorophenylalanine (PCPA) treatment Wild-type and 5-HTT KO pups were treated with PCPA from P1 until the day of the HRP injection. Daily subcutaneous injections in the neck were made (0.2^0.3 mg/g of body weight) of PCPA (PCPA methyl ester C-3635, Sigma) in physiological saline solution. If the pup’s weight increase was too low, the injection was postponed by 12^24 h. The day of injection of HRP was chosen as that of the opening of the eyes (around P11^P12) so as to compare pups at the same development stage. In wild-type mice, PCPA caused mild weight loss (mean 6 g compared to 7.5 g in the untreated controls at P12) but more severe atrophy was noted in the 5-HTT KO that were treated with PCPA (mean 4.5 g) (see also Persico et al., 2000). Another litter of 5-HTT KO mice were treated from P1 to P8 and the visual projections analysed at P20 (Fig. 3). Quanti¢cation of volume of retinal projections to the thalamus The area covered by HRP-labelled terminals was measured in complete series of coronal sections through the dLGN of normal and KO adult mice. Images were digitised and the area of the retinal projection was measured using a specially devised package from Imstar (Paris, France). The limits of the ipsilateral projection were either traced with the mouse on the computer screen or by setting an optical density threshold, in order to include all HRP-labelled elements (in this case, artefactual labelling such as blood vessels or precipitate was removed with the mouse). The volume of the labelled region was calculated by multiplying the total measured surface area by the thickness of the sections. The total volume of the dLGN was determined from the same serial sections by delimiting the external contours of HRP labelling in the contralateral dLGN and measuring the area included within each contour. This value includes the area of contralateral HRP labelling as well as any unlabelled territory in the centre. Statistical comparisons between groups were made using the analysis of variance (ANOVA) statistical test.
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Fig. 1. Wild-type, 5-HT1B KO.
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Fig. 1. 5-HTT/MAOA DKO.
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Quanti¢cation of the ipsilateral retinotectal projection Two approaches were used to quantify the retinal projection in the SC. 1. On complete serial sections of the SC of the ¢ve genotypes (C3H wild-type, 5-HTT KO, 5-HT1B KO, 5-HTT/5-HT1B double KO, and 5-HT1B /MAOA double KO), we evaluated the following parameters: (a) the existence and number of clusters and (b) the mediolateral extension of the ipsilateral projection on each section. This allowed us to identify four main qualitative groups. Group I: retinal ¢bres are distributed over the entire width of the SC and form clusters; group II: retinal ¢bres are distributed over the entire width of the SC but do not form clusters; group III: retinal ¢bres are restricted within the medial half of the SC and are di¡use; group IV: retinal ¢bres are clustered into a single medial patch. For each case, the number of sections belonging to each group was counted and normalised for the total number of SC sections. 2. The dorsoventral extent of the ipsilateral projection in rostral SC was quanti¢ed as follows. For each case, we systematically sampled the second, ¢fth and ninth section of the complete rostrocaudal series through the SC. These levels correspond to sections previously classi¢ed as belonging to groups I or II (i.e. with labelling extending over the entire mediolateral width of the SC). The sections were photographed with a digital camera (Coolsnap). Using the MetaImaging system (Princeton Instruments, France), the area containing the HRP labelling was delimited with the mouse on the screen and was measured. To quantify the dorsoventral extent of labelling the area occupied by the HRP labelling was divided by the longest mediolateral extent of the zone of HRP labelling for each section. The mean value (from three sections) for each case was used to determine the mean per group (i.e. genotype), and statistical evaluations were calculated with the ANOVA test.
RESULTS
The patterning of retinal ¢bres varies slightly according to genetic background (LaVail et al., 1978). We examined the retinal patterning of the 5-HT1B and 5-HTT mutations on several di¡erent backgrounds (C57Bl/6J and 129/Sv). The results presented in this study, however, essentially relate to the 5-HTT KO, 5-HT1B KO, 5-HTT/5-HT1B double KO, and 5-HT1B / MAOA double KO mice on a C3H/HeJ background. The wild-type C3H/HeJ strain has been analysed in
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detail in our previous study of the visual system (Upton et al., 1999). This uniform background allows reliable quantitative evaluations to be made between genotypes. 5-HT1B KO mice We examined the e¡ect of loss of 5-HT1B alone on the ipsilateral retinotectal projection in mice aged 1 month (n = 8) and mice aged over 6 months (n = 7) (total n = 15). There were no qualitative di¡erences between mice of di¡erent ages. However, for the quantitative evaluations, the measurements were done in mice aged 4^5 weeks, and, because of the instability of the HRP label, were carried out very shortly (2 weeks) after the tracing experiments. Retinotectal projections. In all the 15 5-HT1B KO mice examined we observed changes in the distribution of the ipsilateral terminals (Fig. 1B). In comparison to the controls the ipsilateral retinal ¢bres appeared to be more di¡use than in the wild-type mice, with a visible spread of the retinal axons into the ventral stratum griseum super¢ciale (SGS). Patches were less distinct. For instance, in caudal SC, a dense medial patch is normally observed in the wild-type mice. This patch was di¡use with low level of labelling in the 5-HT1B KO (Fig. 1A, B). These abnormalities were quanti¢ed in ¢ve 5-HT1B KO mice compared to ¢ve wild-type mice. To evaluate patchiness, we counted the number of sections in which patches could be seen: 10.5 U 1.5 sections through the SC contained patches in wild-type mice, whereas only 3.5 U 1.0 sections contained ¢bre aggregates in the 5-HT1B KO. These clusters were less tightly packed than in controls. Furthermore, the ipsilateral projection was more di¡use in the dorsoventral plane (Fig. 4B), with a visible spread of the retinal axons into the ventral SGS. On the other hand, the mediolateral extent of the ipsilateral projection was similar to controls (Fig. 4C). These observations indicate that 5-HT1B receptors normally have a role in the establishment of the ipsilateral projection pattern in the SC. Retinogeniculate projections. In adult wild-type mice, the ipsilateral ¢bres are con¢ned to a mediodorsal area of the dLGN whilst the contralateral projection is dis-
Fig. 1. Retinal projections to the SC in wild-type 5-HT1B KO, 5-HTT and MAOA/5-HT1B KO mice. Retinal axons were labelled with HRP injected into the left eye. Serial coronal sections through the ipsilateral SC are shown with the most rostral sections at the bottom of the ¢gure. (A) Rostrocaudal series of one in four 40-Wm sections through the SC of a wildtype C3H mouse, aged 7 months. Distinctive patches of terminals are concentrated in the stratum opticum (SO) layer of the colliculus. These extend across most of the mediolateral extent in rostral regions, but lateral patches disappear caudally leaving a single medial patch. (B) One in three/four 40-Wm sections of the SC of a 5-HT1B KO mouse (sixth generation backcross onto the C3H/HeJ strain), aged 1 month. Apart from the most rostral region where patches of label are visible (although they are not as well de¢ned as in wild-type animals), ipsilateral labelling is di¡use in the SGS. They do not segregate into a single caudal patch but neither do they cover the width of the SC in the caudal part as in the 5-HTT KO mice. (C) One in four 40-Wm sections through the SC of a 5-HTT KO mouse (sixth generation backcross onto the C3H/HeJ strain), aged 2 months. Ipsilateral terminals extend across the whole mediolateral extent, at all rostrocaudal levels and are more di¡use than in controls. No patches can be distinguished and ¢bres are present in the SGS (arrow). (D) One in four 40-Wm sections through the tectum of a MAOA/5-HT1B double KO (sixth generation backcross onto the C3H/HeJ strain), aged 11 months.
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Fig. 2. Retinal projections in the dLGN and the SC of normal and 5-HT1B KO mice aged P4. HRP was injected in the left eye of C3H and 5-HT1B KO at P3. Pups were killed at P4. Coronal sections of 52 Wm thick are presented. (A and D) Ipsilateral ¢bres in the dLGN of wild-type (A) and 5-HT1B KO (D). The density and the extent of the labelling is more di¡use than in adults but is similar in both genotypes. Ipsilateral retinal ¢bres in the SC are shown at two levels, rostral (B, E) and caudal (C, F), in wild-type (B, C) and 5-HT1B KO (E, F) mice. In both genotypes, the projection covers the entire width of the SC but is more dense medially. No patches are visible and the ¢bres extend over both the SO and the SGS. Minor di¡erences in labelling density can be attributed to a greater variability of the tracer injections in pups than in adults. Scale bar = 0.13 mm.
tributed throughout the rest of the dLGN leaving a mediodorsal gap (Fig. 3A, B and Upton et al., 1999). The volume occupied by the ipsilateral retinal projection in the 5-HT1B KO mice was non-signi¢cantly reduced in the 5-HT1B KO mice (Fig. 4D). This con¢rms our previous qualitative observations that indicated the existence of a correct segregation of eye-speci¢c terminals in the thalamus of the 5-HT1B KO mice (Salichon et al., 2001). Development of the retinal projection in the 5-HT1B KO mice. To determine when the retinotectal abnormalities occur in the 5-HT1B KO mice, we injected HRP into one eye of wild-type and 5-HT1B KO mice at P3 and analysed the retinal projections 1 day later (n = 5 for each genotype). At this age, the retinotectal projection covers both the super¢cial and deep layers of the SC and its entire rostrocaudal extent in wild-type mice. However, caudally, the ipsilateral retinal ¢bres are mainly distributed in the medial half of the SC, similarly to previous descriptions (Godement et al., 1984; Thompson and Holt, 1989). At P4, we found no di¡erence in the distribution of the retinal ¢bres between wild-type and KO
mice (Fig. 2), indicating that the abnormalities are not due to an anomalous ingrowth of retinal ¢bres, but to later re¢nement of the projection. 5-HTT KO mice We analysed the e¡ects of lack of 5-HTT on the C57Bl/6J (n = 4) and in the C3H background (n = 10) at ages ranging from 1 to 10 months. Quantitative evaluations were done on four cases at 1 month. Retinotectal projections. The distribution of the ipsilateral retinal ¢bres was severely disrupted in the SC of all the 14 5-HTT KO mice that were examined, on either the C57Bl/6J or the C3H backgrounds. We observed ipsilateral retinal ¢bres extending into the SGS and with a broad mediolateral extension in all cases. No patches or clustering of the ¢bres in any of the sections through the rostrocaudal extent of the SC were found (Fig. 1C). This was quanti¢ed in four cases. The projection was more di¡use and scattered over a greater distance in all dimensions: in the dorsoventral plane it covered over 85% more depth through the SC than in controls (Fig. 4B)
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Fig. 3. Retinal projections to the dLGN in wild-type, 5-HTT KO, 5-HTT/5-HT1B double KO, and 5-HTT KO mice treated with PCPA. Retinal axons were labelled with HRP injected into the left eye. Coronal sections of 40 Wm from the rostral third (upper) and caudal third (lower) of the ipsilateral dLGN are shown on the left for each genotype (A, C, E, G) and the contralateral projection is shown on the right (B, D, F, H). (A and B) In wild-type C3H/HeJ mice, P30, the eye-speci¢c segregation is clear : tightly packed ipsilateral retinal axons form a patch ipsilaterally. (A) The contralateral projection ¢lls the rest of the dLGN leaving a clear gap in the region occupied by ipsilateral terminals from the other eye (B). Within this gap, contralateral ¢bres corresponding to retinal axon bundles traversing the nucleus are visible. (C and D) In 5-HTT KO mice, the ipsilateral projection appears normal (C) but the contralateral projection ¢lls the entire dLGN, including the ipsilateral territory although the contralateral ¢bre terminals are not as dense within the ipsilateral area as in the rest of the dLGN (D). (E and F) 5-HTT/5-HT1B double KO mice ipsilateral terminals are concentrated in the dorsomedial region as in wild-type animals, similarly the contralateral projection also appears normal with terminals ¢lling the dLGN apart from a dorsomedial gap. (G and H) In 5-HTT KO mice aged P20, treated with PCPA from P1 to P8, ipsilateral terminals form dense patches as in wild-type animals and the contralateral projection ¢lls the dLGN apart from the territory normally ¢lled by ipsilateral ¢bres. Scale bar = 0.1 mm.
and retinal ¢bres were always found in the SGS. The ipsilateral retinal projection was also more extensive in the mediolateral dimension: in controls, it occupies the entire width of the SC in approximately half of the sections (the rostral half) and is then progressively restricted to a medial zone, whereas in the 5-HTT KO, almost all the sections (95%) from rostral to caudal have ipsilateral
¢bres that cover the mediolateral width of the SC (Fig. 4C). In the original 5-HTT KO strain with a mixed genetic background (129/Sv-C57Bl/6J), the ipsilateral retinal ¢bres were also di¡use and did not form patches, but were more biased towards the medial border in the caudal SC (data not shown).
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Fig. 4. Quanti¢cation of changes in ipsilateral retinal projections in adult 5-HT1B KO, 5-HTT KO, 5-HTT/5-HT1B double KO, and MAOA/5-HT1B double KO mice. (A) Semi-quantitative evaluation of the patchiness of the ipsilateral projection in the SC of wild-type (WT, n = 5), 5-HT1B KO (n = 5), 5-HTT KO (n = 4), 5-HTT/5-HT1B double KO (1B/5-HTT; n = 2), and MAOA/5-HT1B double KO (1B/MAOA double KO; n = 3) mice. Sections were classi¢ed into three groups as follows : I = retinal ¢bres are distributed over the entire width of the SC and clustered (white bar); II = retinal ¢bres are distributed over the entire width of the SC but are di¡use; III = retinal ¢bres are restricted within the medial half of the SC and are di¡use (II and III: stippled bar); IV = retinal ¢bres are clustered into a single medial patch (black bar). For each case the number of sections belonging to each group was normalised by the total number of sections. (B) Evaluation of the dorsoventral exuberance of the ipsilateral projection in the SC of wild-type, 5-HT1B KO, and 5-HTT KO mice aged 1 month. Mean height of the retinal projection in the SC, calculated on three sections for each case, and on four or ¢ve mice per genotype. The values were normalised relative to controls. The error bar indicates the S.E. ; * indicates a signi¢cant di¡erence (ANOVA test calculated with K = 5%, minimal signi¢cant distance = 30 in this case). The mean height of the projection is signi¢cantly increased in the 5-HT1B KO and 5-HTT KO compared to wild-type. (C) Evaluation of the mediolateral exuberance of the ipsilateral projection in the SC of wild-type, 5-HT1B KO, 5-HTT KO, and 5-HTT/5-HT1B double KO mice. The percentage of sections with a retinal projection covering the entire width of the SC is shown. This corresponds to groups I+II, normalised by the total number of sections. Bars indicate the S.E.M. There is no signi¢cant di¡erence between controls and 5-HT1B KO but 5-HTT KO di¡er signi¢cantly from the three other genotypes (ANOVA test, minimal signi¢cant distance = 18 in this case). (D) Evaluation of the volume occupied by ipsilateral terminals in the dLGN of wild-type, 5-HT1B KO, 5-HTT KO and 5-HTT/5-HT1B double KO (DKO) mice. The percentage volume of the dLGN occupied by ipsilateral retinal terminals was calculated in four to ¢ve cases in which a complete and undamaged series through both dLGNs was obtained, but only two such cases were available for the double KO mice. Bars indicate the S.E.M. No signi¢cant di¡erence was found between the genotypes.
Retinogeniculate projections. We have reported previously that the absence of the 5-HTT produces qualitative changes in the retinal projection to the dLGN (Salichon et al., 2001). In adult wild-type mice, the contralateral projection is distributed throughout the dLGN, but
leaves a clear mediodorsal gap that corresponds to the position of the ipsilateral ¢bres (Fig. 3A, B and Upton et al., 1999). In the 5-HTT KO mice, the contralateral projection does not retract from the area normally occupied by the ipsilateral ¢bres (Fig. 3C, D), although the density
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Fig. 5. E¡ects of PCPA treatment on ipsilateral retinotectal projections in wild-type and 5-HTT KO mice. For each condition, one in six 40-Wm serial coronal sections are presented from rostral (bottom) to caudal (top). (A) Untreated normal mouse (C3H/HeJ) aged P12. The patterning is similar to the adult mouse (compare with Fig. 1A), but the rostral and caudal patches are less clearly de¢ned. (B) C3H/HeJ mouse (aged P12), treated from P1 to P11 with PCPA. Clusters are still visible rostrally, but the retinal projection is very di¡use in the caudal two thirds. Retinal ¢bres extend dorsally in the SGS and across the mediolateral plane. (C) 5-HTT KO mouse aged P13, treated from P1 to P12 with PCPA. As in the wild-type pup treated with PCPA, rostral patches are detectable but the projection is exuberant caudally. Scale bar = 0.14 mm.
of contralateral ¢bre terminals is lower in this mediodorsal region than in the rest of the dLGN, a large number of contralateral ¢bres remain in this zone. We did quantitative evaluations to establish whether this was accompanied by changes in the size of the ipsilateral projection. The volume of this ipsilateral projection was quanti¢ed in cases where a complete and undamaged series of sections through the dLGN was available (Fig. 4D). The percentage volume of the dLGN occupied by ipsilateral ¢bres was not signi¢cantly di¡erent from wild-type in the 5-HTT KO.
distribution of the ipsilateral retinal ¢bres resembled that observed in the 5-HT1B KO mice. The ipsilateral ¢bres showed some tendency to cluster in medial areas of the caudal SC. However, the rostral patches were not as well de¢ned as in the wild-type mice (Fig. 4A). Ipsilateral ¢bres were more di¡use in the SGS, but the mediolateral extension of the ¢bres in the SC was normal (Fig. 4C). Thus, removing the 5-HT1B receptors in addition to the 5-HTT or to the MAOA gene appeared to produce a phenotype that resembles that of the 5-HT1B deletion alone.
5-HTT/5-HT1B and MAOA/5HT1B double KO mice In the MAOA KO mice and MAOA/5-HTT double KO mice, we have already shown that the additional lack of 5-HT1B receptor appears to correct most of the alterations in ipsi/contralateral retinal segregation in the thalamus. Thus, MAOA/5-HT1B double KO mice, and MAOA/5-HTT/5-HT1B triple KO mice have a normal distribution of the ipsi/contralateral retinal ¢bres in the dLGN (Salichon et al., 2001). In the present study, we show that a normal ipsi/contralateral segregation is present in the dLGN of 5-HTT/5-HT1B double KO mice on a C3H background (Figs. 3E, F and 4D). In the SC, on the other hand, retinal abnormalities were not entirely corrected. In the 5-HTT/5-HT1B double KO (n = 4 mice aged 1^11 months; data not shown) and the MAOA/5-HT1B double KO (n = 3) (Fig. 1D), the
E¡ects of PCPA treatments on retinal development in normal and 5-HTT KO mice To reduce 5-HT levels, we administered PCPA from P1 to the day of eye opening (P11^P12) and examined the retinal projections one day later (P12^P13), when the projections are deemed mature. This schedule was chosen because PCPA caused a high mortality in the 5-HTT KO mice. Untreated control mice were examined at P12 (n = 4). At this age, the retinal projection to the dLGN was qualitatively similar to adults but was still more di¡use: ipsilateral ¢bres occupied 26% of the dLGN at P12 compared to only 16% in adults (Fig. 6C). In the SC, the retinal ¢bres had a slightly immature pattern with less clearly de¢ned clusters (Fig. 5A). However, the proportion of sections with a projection covering
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Fig. 6. Quanti¢cation of changes in ipsilateral retinal projections after PCPA treatment. Wild-type mice (WT, n = 4), and wild-type mice treated with PCPA (n = 5), 5-HTT KO mice treated with PCPA (n = 3) were analysed at P12^P13. (A) Semiquantitative evaluation of the patchiness of the ipsilateral projection in the SC of wild-type and 5-HTT KO mice after PCPA treatment. Sections were classi¢ed into three groups as follows : I = retinal ¢bres are distributed over the entire width of the SC and clustered (white bar); II = retinal ¢bres are distributed over the entire width of the SC but are di¡use; III = retinal ¢bres are restricted within the medial half of the SC and are di¡use (II and III: stippled bar); IV = retinal ¢bres are clustered into a single medial patch (black bar). For each case the number of sections belonging to each group was normalised by the total number of sections. (B) Evaluation of the mediolateral exuberance of the ipsilateral projection in the SC of wild-type and 5-HTT KO treated with PCPA. The percentage of sections with a retinal projection covering the entire width of the SC is shown. This corresponds to groups I+II, normalised by the total number of sections. Bars indicate the S.E.M. There is no signi¢cant di¡erence between 5-HTT KO and wild-type treated with PCPA but both di¡er signi¢cantly from untreated controls. (C) Evaluation of the volume occupied by ipsilateral terminals in the dLGN of wild-type and 5-HTT KO mice treated with PCPA compared to untreated wild-type. Bars indicate the S.E. The size of the projection in the treated wild-type is signi¢cantly reduced compared to untreated wild-type (ANOVA test, the minimal signi¢cant di¡erence is 6 in this case) but not signi¢cantly reduced in the 5-HTT KO.
the entire width of the SC was similar to adults (Fig. 6B) and retinal ¢bres had retracted from the SGS and were con¢ned to the stratum opticum (SO). Normal mice treated with PCPA In wild-type mice treated with PCPA (n = 9, from two
di¡erent litters, quanti¢cation on four cases) the projection to the SC was severely altered: in the rostral part of the SC a row of patches was observed in the SO but the number of sections with such a labelling was reduced by half in comparison to untreated controls (18% compared to 35%) (Fig. 6B). Caudally, the retinal projections were exuberant in both the dorsoventral and in the mediolat-
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eral plane of the SC (Figs. 5B and 6B). In the dLGN, the presence of a gap free of retinal ¢bres was observed contralaterally as in untreated controls. However, the volume occupied by ipsilateral retinal projection appeared to be reduced relative to matched controls (Fig. 6C). 5-HTT KO mice treated with PCPA In the 5-HTT KO mice treated with PCPA (n = 3), we found abnormalities in the SC that were similar to those found in the wild-type mice treated with PCPA (Figs. 5C and 6A, B). In the dLGN, a complete and clear reversal of the retinogeniculate abnormalities was observed (n = 3+2 aged P20). The contralateral retinal projection had completely cleared from the area corresponding to the ipsilateral territory, similarly to normal mice (Figs. 3G, H and 6C).
DISCUSSION
The present report extends previous observations indicating the importance of 5-HT in the patterning of ipsi/ contralateral retinal a¡erents during development (Mooney et al., 1998; Rhoades et al., 1993; Salichon et al., 2001; Upton et al., 1999). In addition, it reveals an interesting di¡erence in the e¡ects of 5-HT in two central targets of the retinal ¢bres, the dLGN and the SC. Our analysis of the retinal projections in 5-HTT KO mice indicates that the segregation of crossed and uncrossed ¢bres is sensitive to an excess of extracellular 5-HT in both the SC and dLGN, whereas the analysis of the 5-HT1B KO mice and of PCPA-treated mice shows that the lack of the 5-HT1B receptors or the lack of 5-HT a¡ects the re¢nement of retinal ¢bre patterning only in the SC, and could also cause an atrophy of the ipsilateral retinogeniculate projection. We analysed the e¡ects of a genetic lack of 5-HTT in the retinal system because during development a subset of RGCs, predominantly those that project ipsilaterally, transiently express the 5-HTT (Upton et al., 1999). The 5-HTT is expressed along the length of the axon and could act locally to reduce the extracellular concentration of 5-HT to which adjacent 5-HT receptors are exposed. We demonstrate here that, in the absence of 5-HTT, retinal axons do not segregate normally into eye-speci¢c patches, in either the thalamus or the SC. However, the alterations di¡er in the two structures: in the dLGN, the abnormality concerns the retraction of contralateral retinal ¢bres from the territory occupied by the ipsilateral ¢bres but there is no measurable change in the extent of the ipsilateral retinal projection. In the SC, a structure where ipsilateral and contralateral retinal ¢bres normally overlap (Godement et al., 1984), there is a visible increase in the extent of the ipsilateral retinal projection. The distribution of the ipsilateral retinal ¢bres in the SC is very similar to that observed at early stages of development (Godement et al., 1984; Upton et al., 1999) suggesting that the 5-HTT phenotype results from the lack of retraction of the ipsilateral retinal ¢bres from inappro-
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priate targets, in the caudal and lateral SC, and in the super¢cial layer of the SC, the SGS. It is most likely that these abnormalities result from modi¢ed brain levels of 5-HT. Indeed, 5-HTT has been shown to be essential in the clearance of 5-HT from extracellular space, and acute blockade of the transporter increases the extracellular levels of 5-HT (Fuller, 1994; Daws et al., 1998). As a consequence, a down-regulation of 5-HT production has been found in the 5-HTT KO mice (Bengel et al., 1998). However, this down-regulation is not su⁄cient to reduce the 5-HT in the extracellular space, since in vivo microdialysis in di¡erent brain structures of the adult 5-HTT KO mice (Andrews et al., 1998; Fabre et al., 2000) show six- to eight-fold elevations of 5-HT levels. Furthermore, in 5-HTT KO pups, dopaminergic neurones abnormally capture 5-HT via the dopamine transporter indicating that 5-HT accumulates in the extracellular space soon after birth (Ravary et al., 2001). To ascertain that the phenotype of the 5-HTT KO mice is due to an increase of 5-HT, we treated the pups with PCPA. We found that the ¢bre segregation, i.e. the retraction of contralateral ¢bres, was normalised in the dLGN of the 5-HTT KO mice. In the SC, the phenotype was partly reversed, and ipsilateral patches were found in the rostral SC. The incomplete reversal of the phenotypic abnormalities in the SC are most probably related to the fact that PCPA, in itself, caused abnormalities in the re¢nement of the retinotectal projections. Until now, only increased levels of 5-HT have been shown to disrupt the retinal ¢bre segregation (Mooney et al., 1998; Upton et al., 1999; Crnko-Hoppenjans et al., 2001). Our present results, together with those of the literature, therefore indicate that both increased and decreased levels of 5-HT alter retinotectal ¢bre re¢nement and could, in addition, reduce the extent of the ipsilateral retinal projection. What targets might extracellular 5-HT act on in this system? 5-HT1B receptors are a strong candidate for the e¡ects of increased 5-HT during development. RGCs express the 5-HT1B receptor mRNA from E15 onwards (Boschert et al., 1994; Upton et al., 1999) and the protein is detected on RGC axons in the SC in adult rats (Boulenguez et al., 1993), essentially in the preterminal region (Pickard et al., 1999; Sari et al., 1999). Stimulation of the presynaptic 5-HT1B receptor on RGC terminals in the tectum of adult hamster is known to inhibit glutamatergic neurotransmission (Mooney et al., 1994) and thus excessive activation of the high-a⁄nity 5-HT1B receptors on retinal a¡erents in MAOA KO (Salichon et al., 2001) and 5-HTT KO mice could block the transmission of spontaneous activity generated in the retina to the retinorecipient targets, and thus prevent activity-dependent processes involved in the patterning of a¡erents. The present results indicate that such a mechanism could be operating in the dLGN of the 5-HTT KO mice since removing the 5-HT1B receptor (the 5-HTT/5-HT1B double KOs) appeared to normalise the patterning of the retinal projections. Contrary to the clear e¡ects of 5-HT1B overstimulation, the developmental consequences of lack of 5-HT1B receptors have not been demonstrated. In the visual sys-
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tem of the 5-HT1B KO mice there were no visible di¡erences in the distribution of the ipsilateral and contralateral projections in the dLGN. Similarly, in the primary somatosensory cortex the development of the principal barrels appeared to be normal (Boylan et al., 2000; Salichon et al., 2001), although alterations of the smaller rostral barrels were observed in 5-HT1B KO mice with a C3H/HeJ background (I. Seif, unpublished results). The present ¢nding of alterations in the uncrossed retinotectal projection in the 5-HT1B KO mice is the ¢rst report demonstrating the existence of persistent anatomical alterations due to the lack of the 5-HT1B receptor. We found that these abnormalities appear only after P4, indicating that the 5-HT1B receptors have a role in the secondary re¢nement of the retinotectal map. It will be interesting to determine whether similar developmental abnormalities exist in other parts of the brain since the 5-HT1B KO mice have a number of behavioural changes (Boutrel et al., 1999; Brunner et al., 1999), some of which may have a developmental origin. The ¢nding that 5-HT1B receptors are required for the normal segregation of retinal ¢bres in the SC explains the phenotype that we have observed in the 5-HTT/5HT1B and the MAOA/5-HT1B double KO mice. In these mice, alterations are corrected in the dLGN but persist in the SC. Based on the distribution of the ipsilateral and contralateral retinal ¢bres in these two targets, it appears that the phenotype of the double knockouts is closer to the phenotype of the 5-HT1B KO than to the phenotype of the MAOA or the 5-HTT KO mice. Thus it is probable that, double knockouts revert to the single 5-HT1B KO phenotype. How could the 5-HT1B receptors act in conjunction with the 5-HTT transporter in shaping the ipsilateral retinal map? A possible explanation could reside in the distribution of RGCs that express 5-HTT and 5-HT1B receptors in the retina. The 5-HTT positive neurones are distributed in a ventral peripheral crescent that coincides in part with the temporo-ventral crescent of ipsilaterally projecting retinal neurones, whereas 5-HT1B receptors are present in all RGCs (see Upton et al., 1999 for a full description). 5-HTT could modulate the local concentration of 5-HT around growing axon terminals. This in turn would change the activation state of the 5-HT1B receptors and modulate the level of excitatory neurotransmission. This would produce a discrepancy in the activity levels of the a¡erent retinal inputs from both eyes or from di¡erent RGCs within one eye. Retinal axons that express 5-HTT would be more likely to transmit excitatory signals than axons that do not, because they can internalise extracellular 5-HT and relieve 5-HT1B -mediated inhibition. When 5-HTT or 5-HT1B receptors are inactive this di¡erential e¡ect would be lost and result in abnormalities in the elimination of misplaced axonal branches in the SC. However, this hypothesis does not explain why similar changes do not occur in the dLGN. One possible reason for the observed di¡erence between the SC and the dLGN could be the local conditions of 5-HT innervation in both retinal targets. The SC contains a higher density of 5-HT innervation than
the dLGN (Lidov and Molliver, 1982; Dinopoulos et al., 1995) and could thus accumulate higher levels of 5-HT when the clearance of 5-HT is disturbed. These targets could also di¡er in their 5-HT receptor expression (Claeysen et al., 1996; Gerard et al., 1997; Mooney et al., 1998). Another possibility that can be considered is that the activity-dependent interactions that shape the ipsi/contralateral projection di¡ers in the dLGN and the SC. In experiments and in KO mice that interrupt activity patterns from both eyes, the ipsilateral projections to the SC and to the dLGN both remain exuberant (Upton et al., 1999; Vercelli et al., 2000; Rossi et al., 2001). However, when activity is suppressed in one eye, di¡erent patterning defects appear to occur in the dLGN and the SC. In ferrets (Penn et al., 1998) and mice (Rebsam et al., 1999) it has been reported that the ipsilateral and contralateral retinal projections from the untreated eye are exuberant in the dLGN, whereas projections from the silenced eye are normal or atrophic. An opposite e¡ect was observed in the SC of hamsters: projections from the silenced eye were di¡use whereas the retinal projections from the untreated eye showed normal clustering (Thompson and Holt, 1989). Although such studies require con¢rmation by studying the entire retinal projection in a given species, this could indicate that activity driven interactions within one eye predominate in the SC to produce clustering of ipsilateral ¢bres (Thompson and Holt, 1989), whereas competitive interactions between the eyes are more important for segregation in the dLGN. The lack of 5-HT1B receptors may thus produce a situation in which the competitive interactions within one eye are disturbed to a greater degree than those between eyes. RGC axons must interact with a large number of factors in their target nuclei to ¢nd their correct topographic location within the correct eye-speci¢c area, and then to form stable synapses. This process is believed to be directed by both spontaneous activity independently generated in the two retina (Galli and Ma¡ei, 1988) and by topographic expression of guidance molecules in the target nuclei and on RGC axons. A variety of molecules have been implicated in topographic guidance such as the Eph receptors and their ligands the ephrins which are expressed in gradients across the retina, the tectum and the thalamus, and are believed to encode positional information (Connor et al., 1998). Activity-dependent mechanisms are thought to be important in the ¢ne-tuning of the projections and are believed to involve release of trophic molecules such as brainderived neurotrophic factor (Cohen-Cory, 1999; Isenmann et al., 1999) as well as negative factors that induce growth cone collapse such as nitric oxide produced by nitric oxide synthase (Vercelli et al., 2000). The action of 5-HT, via the 5-HT1B receptor, and possibly other 5-HT receptors, could be an important modulator of these activity-dependent interactions in the ¢nal stage of retinal a¡erent patterning.
Acknowledgements1We thank Aude Muzerelle, Diana Haranger, and Pierre Walrafen for their help, and Constantino
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Sotelo for his support. This work was supported by the European Commission (BMH4 CT97-2412), the CNRS, the INSERM, the British Council and Ministe're des A¡aires
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Etrange'res (Alliance programme), and the Bundesministerium fu«r Bildung und Forschung (BMBF, No. 512-400101KS9603).
REFERENCES
Andrews, A.M., Wichems, C.H., Li, O., Lesch, K.P., Murphy, D.L., 1998. A microdialysis study of the e¡ects of high K+ and paroxetine on extracellular serotonin concentrations in serotonin transporter knockout mice. American Society for Neuroscience, 440.2. Bengel, D., Murphy, D.L., Andrews, A.M., Wichems, C.H., Feltner, D., Heils, A., Mossner, R., Westphal, H., Lesch, K.P., 1998. Altered brain serotonin homeostasis and locomotor insensitivity to 3,4-methylenedioxymethamphetamine (‘Ecstasy’) in serotonin transporter-de¢cient mice. Mol. Pharmacol. 53, 649^655. Boschert, U., Amara, D.A., Segu, L., Hen, R., 1994. The mouse 5-hydroxytryptamine1B receptor is localized predominantly on axon terminals. Neuroscience 58, 167^182. Boulenguez, P., Abdelke¢, J., Pinard, R., Christolomme, A., Segu, L., 1993. E¡ects of retinal dea¡erentation on serotonin receptor types in the super¢cial grey layer of the superior colliculus of the rat. J. Chem. Neuroanat. 6, 167^175. Boutrel, B., Franc, B., Hen, R., Hamon, M., Adrien, J., 1999. Key role of 5-HT1B receptors in the regulation of paradoxical sleep as evidenced in 5-HT1B knock-out mice. J. Neurosci. 19, 3204^3212. Boylan, C.B., Kesterson, K.L., Crnko-Hoppenjans, T.A., Ke, M., Rizk, T., Mooney, R.D., Rhoades, R.W., 2000. The cortical vibrissae representation is normal in transgenic mice lacking the 5-HT(1B) receptor. Dev. Brain Res. 120, 91^93. Brunner, D., Buhot, M.C., Hen, R., Hofer, M., 1999. Anxiety, motor activation, and maternal-infant interactions in 5HT1B knockout mice. Behav. Neurosci. 113, 587^601. Cases, O., Vitalis, T., Seif, I., De Maeyer, E., Sotelo, C., Gaspar, P., 1996. Lack of barrels in the somatosensory cortex of monoamine oxidase Ade¢cient mice: role of a serotonin excess during the critical period. Neuron 16, 297^307. Claeysen, S., Sebben, M., Journot, L., Bockaert, J., Dumuis, A., 1996. Cloning, expression and pharmacology of the mouse 5-HT(4L) receptor. FEBS Lett. 398, 19^25. Cohen-Cory, S., 1999. BDNF modulates, but does not mediate, activity-dependent branching and remodeling of optic axon arbors in vivo. J. Neurosci. 19, 9996^10003. Connor, R.J., Menzel, P., Pasquale, E.B., 1998. Expression and tyrosine phosphorylation of Eph receptors suggest multiple mechanisms in patterning of the visual system. Dev. Biol. 193, 21^35. Crnko-Hoppenjans, T.A., Mooney, R.D., Rhoades, R.W., 2001. Neonatally elevated serotonin levels alter terminal arbors of individual retinal ganglion cells in superior colliculus of hamsters. J. Comp. Neurol. 432, 528^536. Daws, L.C., Toney, G.M., Gerhardt, G.A., Frazer, A., 1998. In vivo chronoamperometric measures of extracellular serotonin clearance in rat dorsal hippocampus : contribution of serotonin and norepinephrine transporters. J. Pharmacol. Exp. Ther. 286, 967^976. Dinopoulos, A., Dori, I., Parnavelas, J.G., 1995. Serotonergic innervation of the lateral geniculate nucleus of the rat during postnatal development: a light and electron microscopic immunocytochemical analysis. J. Comp. Neurol. 363, 532^544. Fabre, V., Beaufour, C., Evrard, A., Rioux, A., Hanoun, N., Lesch, K.P., Murphy, D.L., Lanfumey, L., Hamon, M., Martres, M.P., 2000. Altered expression and functions of serotonin 5-HT1A and 5-HT1B receptors in knock-out mice lacking the 5-HT transporter. Eur. J. Neurosci. 12, 2299^2310. Fuller, R.W., 1994. Uptake inhibitors increase extracellular serotonin concentration measured by brain microdialysis. Life Sci. 55, 163^167. Galli, L., Ma¡ei, L., 1988. Spontaneous impulse activity of rat retinal ganglion cells in prenatal life. Science 242, 90^91. Gerard, C., Martres, M.P., Lefevre, K., Miquel, M.C., Verge, D., Lanfumey, L., Doucet, E., Hamon, M., el Mestikawy, S., 1997. Immunolocalization of serotonin 5-HT6 receptor-like material in the rat central nervous system. Brain Res. 746, 207^219. Godement, P., Salaun, J., Imbert, M., 1984. Prenatal and postnatal development of retinogeniculate and retinocollicular projections in the mouse. J. Comp. Neurol. 230, 552^575. Isenmann, S., Cellerino, A., Gravel, C., Bahr, M., 1999. Excess target-derived brain-derived neurotrophic factor preserves the transient uncrossed retinal projection to the superior colliculus. Mol. Cell. Neurosci. 14, 52^65. LaVail, J.H., Nixon, R.A., Sidman, R.L., 1978. Genetic control of retinal ganglion cell projections. J. Comp. Neurol. 182, 399^421. Lidov, H.G., Molliver, M.E., 1982. An immunohistochemical study of serotonin neuron development in the rat: ascending pathways and terminal ¢elds. Brain Res. Bull. 8, 389^430. Meister, M., Wong, R.O., Baylor, D.A., Shatz, C.J., 1991. Synchronous bursts of action potentials in ganglion cells of the developing mammalian retina. Science 252, 939^943. Mooney, R.D., Crnko-Hoppenjans, T.A., Ke, M., Bennett-Clarke, C.A., Lane, R.D., Chiaia, N.L., Rhoades, R.W., 1998. Augmentation of serotonin in the developing superior colliculus alters the normal development of the uncrossed retinotectal projection. J. Comp. Neurol. 393, 84^92. Mooney, R.D., Huang, X., Shi, M.Y., Bennett-Clarke, C.A., Rhoades, R.W., 1996. Serotonin modulates retinotectal and corticotectal convergence in the superior colliculus. Prog. Brain Res. 112, 57^69. Mooney, R.D., Shi, M.Y., Rhoades, R.W., 1994. Modulation of retinotectal transmission by presynaptic 5-HT1B receptors in the superior colliculus of the adult hamster. J. Neurophysiol. 72, 3^13. Penn, A.A., Riquelme, P.A., Feller, M.B., Shatz, C.J., 1998. Competition in retinogeniculate patterning driven by spontaneous activity. Science 279, 2108^2112. Persico, A.M., Altamura, C., Calia, E., Puglisi-Allegra, S., Ventura, R., Lucchese, F., Keller, F., 2000. Serotonin depletion and barrel cortex development : impact of growth impairment vs. serotonin e¡ects on thalamocortical endings. Cereb. Cortex 10, 181^191. Pickard, G.E., Smith, B.N., Belenky, M., Rea, M.A., Dudek, F.E., Sollars, P.J., 1999. 5-HT1B receptor-mediated presynaptic inhibition of retinal input to the suprachiasmatic nucleus. J. Neurosci. 19, 4034^4045. Ravary, A., Muzerelle, A., Darmon, M., Murphy, D.L., Moessner, R., Lesch, K.P., Gaspar, P., 2001. Abnormal tra⁄cking and subcellular localization of an N-terminally truncated serotonin transporter protein. Eur. J. Neurosci. 13, 1349^1362. Rebsam, A., Tsuchimoto, Y., Fagiolini, M., Hensch, T.K., 1999. Spontaneous retinal activity patterns drive competitive segregation of mouse retinogeniculate axons. Society for Neuroscience Meeting, 604.3. Rhoades, R.W., Bennett-Clarke, C.A., Lane, R.D., Leslie, M.J., Mooney, R.D., 1993. Increased serotoninergic innervation of the hamster’s superior colliculus alters retinotectal projections. J. Comp. Neurol. 334, 397^409. Rossi, F.M., Pizzorusso, T., Porciatti, V., Marubio, L.M., Ma¡ei, L., Changeux, J.P., 2001. Requirement of the nicotinic acetylcholine receptor L2 subunit for the anatomical and functional development of the visual system. Proc. Natl. Acad. Sci. USA 98, 6453^6458.
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A. L. Upton et al.
Salichon, N., Gaspar, P., Upton, A.L., Picaud, S., Hanoun, N., Hamon, M., De Maeyer, E., Murphy, D.L., Mossner, R., Lesch, K.P., Hen, R., Seif, I., 2001. Excessive activation of serotonin (5-HT)1B receptors disrupts the formation of sensory maps in monoamine oxidase a and 5-HT transporter knock-out mice. J. Neurosci. 21, 884^896. Sari, Y., Miquel, M.C., Brisorgueil, M.J., Ruiz, G., Doucet, E., Hamon, M., Verge, D., 1999. Cellular and subcellular localization of 5-hydroxytryptamine1B receptors in the rat central nervous system : immunocytochemical, autoradiographic and lesion studies. Neuroscience 88, 899^915. Saudou, F., Amara, D.A., Dierich, A., LeMeur, M., Ramboz, S., Segu, L., Buhot, M.C., Hen, R., 1994. Enhanced aggressive behavior in mice lacking 5-HT1B receptor. Science 265, 1875^1878. Thompson, I., Holt, C., 1989. E¡ects of intraocular tetrodotoxin on the development of the retinocollicular pathway in the Syrian hamster. J. Comp. Neurol. 282, 371^388. Upton, A.L., Salichon, N., Lebrand, C., Ravary, A., Blakely, R., Seif, I., Gaspar, P., 1999. Excess of serotonin (5-HT) alters the segregation of ispilateral and contralateral retinal projections in monoamine oxidase A knock-out mice: possible role of 5-HT uptake in retinal ganglion cells during development. J. Neurosci. 19, 7007^7024. Vercelli, A., Garbossa, D., Biasiol, S., Repici, M., Jhaveri, S., 2000. NOS inhibition during postnatal development leads to increased ipsilateral retinocollicular and retinogeniculate projections in rats. Eur. J. Neurosci. 12, 473^490. (Accepted 23 November 2001)
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Neuroscience Vol. 111, No. 3, pp. 597^610, 2002 M 2002 IBRO. Published by Elsevier Science Ltd All rights reserved. Printed in Great Britain 0306-4522 / 02 $22.00+0.00
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LACK OF 5-HT1B RECEPTOR AND OF SEROTONIN TRANSPORTER HAVE DIFFERENT EFFECTS ON THE SEGREGATION OF RETINAL AXONS IN THE LATERAL GENICULATE NUCLEUS COMPARED TO THE SUPERIOR COLLICULUS A. L. UPTON,a;1;2 A. RAVARY,a;1 N. SALICHON,b R. MOESSNER,c K.-P. LESCH,c R. HEN,d I. SEIFb and P. GASPARa * a
INSERM U106, Ho“pital de la Pitie¤-Salpe“trie're, 75651 Paris, France b
c d
CNRS UMR 146, Institut Curie, 91405 Orsay, France
Department of Psychiatry, University of Wu«rzburg, 97080 Wu«rzburg, Germany
Center for Neurobiology and Behavior, Columbia University, New York, NY 10032, USA
AbstractAWe have shown previously that raised levels of serotonin (5-hydroxytryptamine or 5-HT) during development prevent retinal ganglion cell axons from segregating into eye-speci¢c regions in their principal targets: the superior colliculus and the dorsal lateral geniculate nucleus. Possible mediators of 5-HT in this system include its plasma membrane transporter, which is transiently expressed by a sub-population of retinal ganglion cells, and the presynaptic 5-HT1B receptor carried on retinal ganglion cell axons. We analysed the retinal projections of 5-HT1B knockout (n = 15), serotonin transporter knockout (n = 14), serotonin transporter/5-HT1B double knockout (n = 4) and monoamine oxidase A/5-HT1B double knockout (n = 3) mice. In all four di¡erent knockout mice, the ipsilateral retinal projection to the superior colliculus was more di¡use and lost its characteristic patchy distribution. The alterations were most severe in the serotonin transporter knockout mice, where the ipsilateral retinal ¢bres covered the entire rostrocaudal and mediolateral extent of the superior colliculus, whereas in the 5-HT1B and double knockout mice, ¢bres retracted from the caudal and lateral superior colliculus. Abnormalities in the 5-HT1B knockout mice appeared only after postnatal day (P) 4. Treatment with parachlorophenylalanine (at P1^P12) to decrease serotonin levels caused an exuberance of the ipsilateral retinal ¢bres throughout the superior colliculus (n = 9). In the dorsal lateral geniculate nucleus in contrast, the distribution and size of the ipsilateral retinal projection was normal in all four knockout mice. In the serotonin transporter knockout mice however, the contralateral retinal ¢bres failed to retract from the mediodorsal dorsal lateral geniculate nucleus, an abnormality that was reversed by early treatment with parachlorophenylalanine and in the serotonin transporter/5-HT1B double knockout. Our observations indicate: (1) that the lack of 5-HT transporter and the associated changes in 5-HT levels impair the segregation of retinal axons in both the superior colliculus and the dorsal lateral geniculate nucleus; (2) that 5-HT and 5-HT1B receptors are necessary for the normal re¢nement of the ipsilateral retinal ¢bres in the superior colliculus, but are not essential for the establishment of eye-speci¢c segregation in the thalamus. Thus, both an excess and a lack of 5-HT a¡ect the re¢nement of the superior colliculus retinal projection, while the establishment of eye-speci¢c patterns in the dorsal lateral geniculate nucleus appears not to be sensitive to the lack of 5-HT or 5-HT1B receptors. M 2002 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: retinal projections, development, parachlorophenylalanine, knockout, mouse.
Retinal ganglion cell (RGC) ¢bres from the two eyes segregate into eye-speci¢c territories during early postna-
tal development before eye opening. Spontaneous activity in the retina during development appears to be essential for this process (Galli and Ma¡ei, 1988; Meister et al., 1991; Penn et al., 1998). An indication of the importance of serotonin (5-hydroxytryptamine or 5-HT) in normal development of retinal projection was provided by a transgenic mouse strain lacking monoamine oxidase A (MAOA), an enzyme which breaks down 5-HT (Cases et al., 1996). As a consequence, MAOA knockout (KO) mice have very high levels of 5-HT during embryonic and neonatal life. Excess 5-HT during the early postnatal period prevents segregation of retinal a¡erents in the dorsal lateral geniculate nucleus (dLGN) and the superior colliculus (SC) (Upton et al., 1999). The disruption is severe: in MAOA KO mice the contralateral projection ¢lls the entire dLGN; the ipsi-
1
These authors contributed equally to this work. Present address: University Laboratory of Physiology, University of Oxford, Oxford OX1 3PT, UK. *Corresponding author. Tel. : +33-1-53612646; fax: +33-145709990; http://mendel.u106.eu.org/. E-mail address: [email protected] (P. Gaspar). Abbreviations : 5-HT, 5-hydroxytryptamine or serotonin; 5-HTT, serotonin transporter; ANOVA, analysis of variance ; dLGN, dorsal lateral geniculate nucleus; E, embryonic day; HRP, horseradish peroxidase; KO, knockout; MAOA, monoamine oxidase A; P, postnatal day; PCPA, parachorophenylalanine; RGC, retinal ganglion cell; SC, superior colliculus ; SGS, stratum griseum super¢ciale; SO, stratum opticum. 2
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lateral projection to the dLGN is greatly enlarged and the ipsilateral projection to the SC does not form patches but is instead di¡use (Upton et al., 1999). A similar disruptive role of excess 5-HT on the segregation of retinotectal axon terminals has also been demonstrated by several experimental approaches in hamsters (Rhoades et al., 1993; Mooney et al., 1996, 1998). The role of 5-HT1B receptors in this process has been suspected because these receptors are expressed by RGCs from embryonic day 15 (E15) onwards (Upton et al., 1999). Activation of the 5-HT1B receptor on RGC terminals in the SC is known to reduce glutamate neurotransmission in adult hamsters (Mooney et al., 1994) and could thereby prevent the transmission of activity-dependent cues from the retina to the central visual targets. There are, however, other possible sites of action for excess 5-HT in this system. We found previously that during development the serotonin transporter (5-HTT) is transiently expressed in a subset of RGCs, primarily but not exclusively those RGCs that project ipsilaterally to the dLGN and the SC (Upton et al., 1999). The function of this uptake is still unknown, but possibilities include internalisation of 5-HT for use as a borrowed neurotransmitter or clearance of 5-HT to produce a local reduction in its extracellular concentration. In a previous study, focused on the retinogeniculate projection and the barrel ¢eld in somatosensory cortex, we have reported that the genetic lack of 5-HT1B receptors does not visibly a¡ect the segregation of the ipsi/ contralateral retinal ¢bres in the dLGN or the formation of barrels in the somatosensory cortex (Salichon et al., 2001). However, the disruptive e¡ects of an excess of 5-HT in these two systems appears to be entirely corrected by the additional lack of 5-HT1B receptors: a normal barrel ¢eld and a normal segregation of the retinogeniculate projection were noted in MAOA/5HT1B double KO mice and in MAOA/5-HTT/5-HT1B triple KO mice. During the course of this study however, preliminary observations suggested that some abnormalities remained in the retinotectal projections of these double and triple KO mice. To understand the nature and signi¢cance of this ¢nding, we conducted a systematic investigation of the retinotectal and retinogeniculate projection in mice carrying single gene inactivation of the 5-HT1B receptor, of the 5-HTT (Bengel et al., 1998), and of both these genes. We compared these ¢ndings to those obtained in the MAOA/5-HT1B double KO mice.
EXPERIMENTAL PROCEDURES
Animals KO mice and some of the normal mice (C3H/HeJ and C57BL/6J) were bred at the Curie Institute (Orsay, France). Additional normal mice were purchased from Janvier (France). We backcrossed the original 5-HTT KO strain (which has a mixed genetic background of 129/Sv, C57BL/6J and CD-1; Bengel et al., 1998) onto the C3H/HeJ for six generations. The 5-HT1B KO was originally generated in the 129/Sv strain (Saudou et al., 1994), but the single KO animals used in this study were backcrossed onto the C3H/HeJ background for 10 generations. MAOA/5-HT1B KO and 5-HTT/5-HT1B double
KO mice were generated in sixth generation backcrosses onto the C3H/HeJ background. Experiments were carried out in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC). Anterograde axonal tracing by intraocular horseradish peroxidase injections Adult mice were anaesthetised with 4% chloral hydrate (0.1 ml i.p. per 10 mg of body weight) and pups aged postnatal day 3 (P3) were anaesthetised by hypothermia. Horseradish peroxidase (HRP, type VI; Sigma, St. Louis, MO, USA) 60%, 1.5^4 Wl (depending on the age of the mouse), in physiological saline was injected into the vitreous chamber of the left eye with a Hamilton syringe inserted just behind the corneoscleral margin of the eye. Twenty-four hours later, mice were anaesthetised and perfused through the aorta with 5^20 ml of saline followed by 40^150 ml of ice-cold 1% paraformaldehyde, 1.25% glutaraldehyde in a 0.12 M phosphate bu¡er (pH 7.4). The brains were removed and cryoprotected in 30% sucrose/0.12 M phosphate bu¡er overnight at 4‡C. Brains were frozen and serial 40-Wm coronal sections (52-Wm for pup brains) were collected in icecold 0.12 M phosphate bu¡er and stored at 4‡C. Within 24 h, sections were rinsed for 10 min in 0.12 M acetate bu¡er (pH 3.3). Sections, maintained at 4‡C and protected from light, were then reacted for HRP using tetramethylbenzidine (0.067%) as the chromogen. The reaction was started by the addition of 0.006% H2 O2 . Further additions were made at 20-min intervals until the reaction was deemed complete (generally three additions). Sections were rinsed in 0.12 M acetate bu¡er (pH 3.3) at 4‡C to stop the reaction, immediately mounted on glass slides and left to air dry overnight. The reaction product was stabilised by a 3-min immersion in methyl salicylate before quick dehydration and mounting. Parachlorophenylalanine (PCPA) treatment Wild-type and 5-HTT KO pups were treated with PCPA from P1 until the day of the HRP injection. Daily subcutaneous injections in the neck were made (0.2^0.3 mg/g of body weight) of PCPA (PCPA methyl ester C-3635, Sigma) in physiological saline solution. If the pup’s weight increase was too low, the injection was postponed by 12^24 h. The day of injection of HRP was chosen as that of the opening of the eyes (around P11^P12) so as to compare pups at the same development stage. In wild-type mice, PCPA caused mild weight loss (mean 6 g compared to 7.5 g in the untreated controls at P12) but more severe atrophy was noted in the 5-HTT KO that were treated with PCPA (mean 4.5 g) (see also Persico et al., 2000). Another litter of 5-HTT KO mice were treated from P1 to P8 and the visual projections analysed at P20 (Fig. 3). Quanti¢cation of volume of retinal projections to the thalamus The area covered by HRP-labelled terminals was measured in complete series of coronal sections through the dLGN of normal and KO adult mice. Images were digitised and the area of the retinal projection was measured using a specially devised package from Imstar (Paris, France). The limits of the ipsilateral projection were either traced with the mouse on the computer screen or by setting an optical density threshold, in order to include all HRP-labelled elements (in this case, artefactual labelling such as blood vessels or precipitate was removed with the mouse). The volume of the labelled region was calculated by multiplying the total measured surface area by the thickness of the sections. The total volume of the dLGN was determined from the same serial sections by delimiting the external contours of HRP labelling in the contralateral dLGN and measuring the area included within each contour. This value includes the area of contralateral HRP labelling as well as any unlabelled territory in the centre. Statistical comparisons between groups were made using the analysis of variance (ANOVA) statistical test.
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Fig. 1. Wild-type, 5-HT1B KO.
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Fig. 1. 5-HTT/MAOA DKO.
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Quanti¢cation of the ipsilateral retinotectal projection Two approaches were used to quantify the retinal projection in the SC. 1. On complete serial sections of the SC of the ¢ve genotypes (C3H wild-type, 5-HTT KO, 5-HT1B KO, 5-HTT/5-HT1B double KO, and 5-HT1B /MAOA double KO), we evaluated the following parameters: (a) the existence and number of clusters and (b) the mediolateral extension of the ipsilateral projection on each section. This allowed us to identify four main qualitative groups. Group I: retinal ¢bres are distributed over the entire width of the SC and form clusters; group II: retinal ¢bres are distributed over the entire width of the SC but do not form clusters; group III: retinal ¢bres are restricted within the medial half of the SC and are di¡use; group IV: retinal ¢bres are clustered into a single medial patch. For each case, the number of sections belonging to each group was counted and normalised for the total number of SC sections. 2. The dorsoventral extent of the ipsilateral projection in rostral SC was quanti¢ed as follows. For each case, we systematically sampled the second, ¢fth and ninth section of the complete rostrocaudal series through the SC. These levels correspond to sections previously classi¢ed as belonging to groups I or II (i.e. with labelling extending over the entire mediolateral width of the SC). The sections were photographed with a digital camera (Coolsnap). Using the MetaImaging system (Princeton Instruments, France), the area containing the HRP labelling was delimited with the mouse on the screen and was measured. To quantify the dorsoventral extent of labelling the area occupied by the HRP labelling was divided by the longest mediolateral extent of the zone of HRP labelling for each section. The mean value (from three sections) for each case was used to determine the mean per group (i.e. genotype), and statistical evaluations were calculated with the ANOVA test.
RESULTS
The patterning of retinal ¢bres varies slightly according to genetic background (LaVail et al., 1978). We examined the retinal patterning of the 5-HT1B and 5-HTT mutations on several di¡erent backgrounds (C57Bl/6J and 129/Sv). The results presented in this study, however, essentially relate to the 5-HTT KO, 5-HT1B KO, 5-HTT/5-HT1B double KO, and 5-HT1B / MAOA double KO mice on a C3H/HeJ background. The wild-type C3H/HeJ strain has been analysed in
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detail in our previous study of the visual system (Upton et al., 1999). This uniform background allows reliable quantitative evaluations to be made between genotypes. 5-HT1B KO mice We examined the e¡ect of loss of 5-HT1B alone on the ipsilateral retinotectal projection in mice aged 1 month (n = 8) and mice aged over 6 months (n = 7) (total n = 15). There were no qualitative di¡erences between mice of di¡erent ages. However, for the quantitative evaluations, the measurements were done in mice aged 4^5 weeks, and, because of the instability of the HRP label, were carried out very shortly (2 weeks) after the tracing experiments. Retinotectal projections. In all the 15 5-HT1B KO mice examined we observed changes in the distribution of the ipsilateral terminals (Fig. 1B). In comparison to the controls the ipsilateral retinal ¢bres appeared to be more di¡use than in the wild-type mice, with a visible spread of the retinal axons into the ventral stratum griseum super¢ciale (SGS). Patches were less distinct. For instance, in caudal SC, a dense medial patch is normally observed in the wild-type mice. This patch was di¡use with low level of labelling in the 5-HT1B KO (Fig. 1A, B). These abnormalities were quanti¢ed in ¢ve 5-HT1B KO mice compared to ¢ve wild-type mice. To evaluate patchiness, we counted the number of sections in which patches could be seen: 10.5 U 1.5 sections through the SC contained patches in wild-type mice, whereas only 3.5 U 1.0 sections contained ¢bre aggregates in the 5-HT1B KO. These clusters were less tightly packed than in controls. Furthermore, the ipsilateral projection was more di¡use in the dorsoventral plane (Fig. 4B), with a visible spread of the retinal axons into the ventral SGS. On the other hand, the mediolateral extent of the ipsilateral projection was similar to controls (Fig. 4C). These observations indicate that 5-HT1B receptors normally have a role in the establishment of the ipsilateral projection pattern in the SC. Retinogeniculate projections. In adult wild-type mice, the ipsilateral ¢bres are con¢ned to a mediodorsal area of the dLGN whilst the contralateral projection is dis-
Fig. 1. Retinal projections to the SC in wild-type 5-HT1B KO, 5-HTT and MAOA/5-HT1B KO mice. Retinal axons were labelled with HRP injected into the left eye. Serial coronal sections through the ipsilateral SC are shown with the most rostral sections at the bottom of the ¢gure. (A) Rostrocaudal series of one in four 40-Wm sections through the SC of a wildtype C3H mouse, aged 7 months. Distinctive patches of terminals are concentrated in the stratum opticum (SO) layer of the colliculus. These extend across most of the mediolateral extent in rostral regions, but lateral patches disappear caudally leaving a single medial patch. (B) One in three/four 40-Wm sections of the SC of a 5-HT1B KO mouse (sixth generation backcross onto the C3H/HeJ strain), aged 1 month. Apart from the most rostral region where patches of label are visible (although they are not as well de¢ned as in wild-type animals), ipsilateral labelling is di¡use in the SGS. They do not segregate into a single caudal patch but neither do they cover the width of the SC in the caudal part as in the 5-HTT KO mice. (C) One in four 40-Wm sections through the SC of a 5-HTT KO mouse (sixth generation backcross onto the C3H/HeJ strain), aged 2 months. Ipsilateral terminals extend across the whole mediolateral extent, at all rostrocaudal levels and are more di¡use than in controls. No patches can be distinguished and ¢bres are present in the SGS (arrow). (D) One in four 40-Wm sections through the tectum of a MAOA/5-HT1B double KO (sixth generation backcross onto the C3H/HeJ strain), aged 11 months.
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Fig. 2. Retinal projections in the dLGN and the SC of normal and 5-HT1B KO mice aged P4. HRP was injected in the left eye of C3H and 5-HT1B KO at P3. Pups were killed at P4. Coronal sections of 52 Wm thick are presented. (A and D) Ipsilateral ¢bres in the dLGN of wild-type (A) and 5-HT1B KO (D). The density and the extent of the labelling is more di¡use than in adults but is similar in both genotypes. Ipsilateral retinal ¢bres in the SC are shown at two levels, rostral (B, E) and caudal (C, F), in wild-type (B, C) and 5-HT1B KO (E, F) mice. In both genotypes, the projection covers the entire width of the SC but is more dense medially. No patches are visible and the ¢bres extend over both the SO and the SGS. Minor di¡erences in labelling density can be attributed to a greater variability of the tracer injections in pups than in adults. Scale bar = 0.13 mm.
tributed throughout the rest of the dLGN leaving a mediodorsal gap (Fig. 3A, B and Upton et al., 1999). The volume occupied by the ipsilateral retinal projection in the 5-HT1B KO mice was non-signi¢cantly reduced in the 5-HT1B KO mice (Fig. 4D). This con¢rms our previous qualitative observations that indicated the existence of a correct segregation of eye-speci¢c terminals in the thalamus of the 5-HT1B KO mice (Salichon et al., 2001). Development of the retinal projection in the 5-HT1B KO mice. To determine when the retinotectal abnormalities occur in the 5-HT1B KO mice, we injected HRP into one eye of wild-type and 5-HT1B KO mice at P3 and analysed the retinal projections 1 day later (n = 5 for each genotype). At this age, the retinotectal projection covers both the super¢cial and deep layers of the SC and its entire rostrocaudal extent in wild-type mice. However, caudally, the ipsilateral retinal ¢bres are mainly distributed in the medial half of the SC, similarly to previous descriptions (Godement et al., 1984; Thompson and Holt, 1989). At P4, we found no di¡erence in the distribution of the retinal ¢bres between wild-type and KO
mice (Fig. 2), indicating that the abnormalities are not due to an anomalous ingrowth of retinal ¢bres, but to later re¢nement of the projection. 5-HTT KO mice We analysed the e¡ects of lack of 5-HTT on the C57Bl/6J (n = 4) and in the C3H background (n = 10) at ages ranging from 1 to 10 months. Quantitative evaluations were done on four cases at 1 month. Retinotectal projections. The distribution of the ipsilateral retinal ¢bres was severely disrupted in the SC of all the 14 5-HTT KO mice that were examined, on either the C57Bl/6J or the C3H backgrounds. We observed ipsilateral retinal ¢bres extending into the SGS and with a broad mediolateral extension in all cases. No patches or clustering of the ¢bres in any of the sections through the rostrocaudal extent of the SC were found (Fig. 1C). This was quanti¢ed in four cases. The projection was more di¡use and scattered over a greater distance in all dimensions: in the dorsoventral plane it covered over 85% more depth through the SC than in controls (Fig. 4B)
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Fig. 3. Retinal projections to the dLGN in wild-type, 5-HTT KO, 5-HTT/5-HT1B double KO, and 5-HTT KO mice treated with PCPA. Retinal axons were labelled with HRP injected into the left eye. Coronal sections of 40 Wm from the rostral third (upper) and caudal third (lower) of the ipsilateral dLGN are shown on the left for each genotype (A, C, E, G) and the contralateral projection is shown on the right (B, D, F, H). (A and B) In wild-type C3H/HeJ mice, P30, the eye-speci¢c segregation is clear : tightly packed ipsilateral retinal axons form a patch ipsilaterally. (A) The contralateral projection ¢lls the rest of the dLGN leaving a clear gap in the region occupied by ipsilateral terminals from the other eye (B). Within this gap, contralateral ¢bres corresponding to retinal axon bundles traversing the nucleus are visible. (C and D) In 5-HTT KO mice, the ipsilateral projection appears normal (C) but the contralateral projection ¢lls the entire dLGN, including the ipsilateral territory although the contralateral ¢bre terminals are not as dense within the ipsilateral area as in the rest of the dLGN (D). (E and F) 5-HTT/5-HT1B double KO mice ipsilateral terminals are concentrated in the dorsomedial region as in wild-type animals, similarly the contralateral projection also appears normal with terminals ¢lling the dLGN apart from a dorsomedial gap. (G and H) In 5-HTT KO mice aged P20, treated with PCPA from P1 to P8, ipsilateral terminals form dense patches as in wild-type animals and the contralateral projection ¢lls the dLGN apart from the territory normally ¢lled by ipsilateral ¢bres. Scale bar = 0.1 mm.
and retinal ¢bres were always found in the SGS. The ipsilateral retinal projection was also more extensive in the mediolateral dimension: in controls, it occupies the entire width of the SC in approximately half of the sections (the rostral half) and is then progressively restricted to a medial zone, whereas in the 5-HTT KO, almost all the sections (95%) from rostral to caudal have ipsilateral
¢bres that cover the mediolateral width of the SC (Fig. 4C). In the original 5-HTT KO strain with a mixed genetic background (129/Sv-C57Bl/6J), the ipsilateral retinal ¢bres were also di¡use and did not form patches, but were more biased towards the medial border in the caudal SC (data not shown).
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Fig. 4. Quanti¢cation of changes in ipsilateral retinal projections in adult 5-HT1B KO, 5-HTT KO, 5-HTT/5-HT1B double KO, and MAOA/5-HT1B double KO mice. (A) Semi-quantitative evaluation of the patchiness of the ipsilateral projection in the SC of wild-type (WT, n = 5), 5-HT1B KO (n = 5), 5-HTT KO (n = 4), 5-HTT/5-HT1B double KO (1B/5-HTT; n = 2), and MAOA/5-HT1B double KO (1B/MAOA double KO; n = 3) mice. Sections were classi¢ed into three groups as follows : I = retinal ¢bres are distributed over the entire width of the SC and clustered (white bar); II = retinal ¢bres are distributed over the entire width of the SC but are di¡use; III = retinal ¢bres are restricted within the medial half of the SC and are di¡use (II and III: stippled bar); IV = retinal ¢bres are clustered into a single medial patch (black bar). For each case the number of sections belonging to each group was normalised by the total number of sections. (B) Evaluation of the dorsoventral exuberance of the ipsilateral projection in the SC of wild-type, 5-HT1B KO, and 5-HTT KO mice aged 1 month. Mean height of the retinal projection in the SC, calculated on three sections for each case, and on four or ¢ve mice per genotype. The values were normalised relative to controls. The error bar indicates the S.E. ; * indicates a signi¢cant di¡erence (ANOVA test calculated with K = 5%, minimal signi¢cant distance = 30 in this case). The mean height of the projection is signi¢cantly increased in the 5-HT1B KO and 5-HTT KO compared to wild-type. (C) Evaluation of the mediolateral exuberance of the ipsilateral projection in the SC of wild-type, 5-HT1B KO, 5-HTT KO, and 5-HTT/5-HT1B double KO mice. The percentage of sections with a retinal projection covering the entire width of the SC is shown. This corresponds to groups I+II, normalised by the total number of sections. Bars indicate the S.E.M. There is no signi¢cant di¡erence between controls and 5-HT1B KO but 5-HTT KO di¡er signi¢cantly from the three other genotypes (ANOVA test, minimal signi¢cant distance = 18 in this case). (D) Evaluation of the volume occupied by ipsilateral terminals in the dLGN of wild-type, 5-HT1B KO, 5-HTT KO and 5-HTT/5-HT1B double KO (DKO) mice. The percentage volume of the dLGN occupied by ipsilateral retinal terminals was calculated in four to ¢ve cases in which a complete and undamaged series through both dLGNs was obtained, but only two such cases were available for the double KO mice. Bars indicate the S.E.M. No signi¢cant di¡erence was found between the genotypes.
Retinogeniculate projections. We have reported previously that the absence of the 5-HTT produces qualitative changes in the retinal projection to the dLGN (Salichon et al., 2001). In adult wild-type mice, the contralateral projection is distributed throughout the dLGN, but
leaves a clear mediodorsal gap that corresponds to the position of the ipsilateral ¢bres (Fig. 3A, B and Upton et al., 1999). In the 5-HTT KO mice, the contralateral projection does not retract from the area normally occupied by the ipsilateral ¢bres (Fig. 3C, D), although the density
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Fig. 5. E¡ects of PCPA treatment on ipsilateral retinotectal projections in wild-type and 5-HTT KO mice. For each condition, one in six 40-Wm serial coronal sections are presented from rostral (bottom) to caudal (top). (A) Untreated normal mouse (C3H/HeJ) aged P12. The patterning is similar to the adult mouse (compare with Fig. 1A), but the rostral and caudal patches are less clearly de¢ned. (B) C3H/HeJ mouse (aged P12), treated from P1 to P11 with PCPA. Clusters are still visible rostrally, but the retinal projection is very di¡use in the caudal two thirds. Retinal ¢bres extend dorsally in the SGS and across the mediolateral plane. (C) 5-HTT KO mouse aged P13, treated from P1 to P12 with PCPA. As in the wild-type pup treated with PCPA, rostral patches are detectable but the projection is exuberant caudally. Scale bar = 0.14 mm.
of contralateral ¢bre terminals is lower in this mediodorsal region than in the rest of the dLGN, a large number of contralateral ¢bres remain in this zone. We did quantitative evaluations to establish whether this was accompanied by changes in the size of the ipsilateral projection. The volume of this ipsilateral projection was quanti¢ed in cases where a complete and undamaged series of sections through the dLGN was available (Fig. 4D). The percentage volume of the dLGN occupied by ipsilateral ¢bres was not signi¢cantly di¡erent from wild-type in the 5-HTT KO.
distribution of the ipsilateral retinal ¢bres resembled that observed in the 5-HT1B KO mice. The ipsilateral ¢bres showed some tendency to cluster in medial areas of the caudal SC. However, the rostral patches were not as well de¢ned as in the wild-type mice (Fig. 4A). Ipsilateral ¢bres were more di¡use in the SGS, but the mediolateral extension of the ¢bres in the SC was normal (Fig. 4C). Thus, removing the 5-HT1B receptors in addition to the 5-HTT or to the MAOA gene appeared to produce a phenotype that resembles that of the 5-HT1B deletion alone.
5-HTT/5-HT1B and MAOA/5HT1B double KO mice In the MAOA KO mice and MAOA/5-HTT double KO mice, we have already shown that the additional lack of 5-HT1B receptor appears to correct most of the alterations in ipsi/contralateral retinal segregation in the thalamus. Thus, MAOA/5-HT1B double KO mice, and MAOA/5-HTT/5-HT1B triple KO mice have a normal distribution of the ipsi/contralateral retinal ¢bres in the dLGN (Salichon et al., 2001). In the present study, we show that a normal ipsi/contralateral segregation is present in the dLGN of 5-HTT/5-HT1B double KO mice on a C3H background (Figs. 3E, F and 4D). In the SC, on the other hand, retinal abnormalities were not entirely corrected. In the 5-HTT/5-HT1B double KO (n = 4 mice aged 1^11 months; data not shown) and the MAOA/5-HT1B double KO (n = 3) (Fig. 1D), the
E¡ects of PCPA treatments on retinal development in normal and 5-HTT KO mice To reduce 5-HT levels, we administered PCPA from P1 to the day of eye opening (P11^P12) and examined the retinal projections one day later (P12^P13), when the projections are deemed mature. This schedule was chosen because PCPA caused a high mortality in the 5-HTT KO mice. Untreated control mice were examined at P12 (n = 4). At this age, the retinal projection to the dLGN was qualitatively similar to adults but was still more di¡use: ipsilateral ¢bres occupied 26% of the dLGN at P12 compared to only 16% in adults (Fig. 6C). In the SC, the retinal ¢bres had a slightly immature pattern with less clearly de¢ned clusters (Fig. 5A). However, the proportion of sections with a projection covering
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Fig. 6. Quanti¢cation of changes in ipsilateral retinal projections after PCPA treatment. Wild-type mice (WT, n = 4), and wild-type mice treated with PCPA (n = 5), 5-HTT KO mice treated with PCPA (n = 3) were analysed at P12^P13. (A) Semiquantitative evaluation of the patchiness of the ipsilateral projection in the SC of wild-type and 5-HTT KO mice after PCPA treatment. Sections were classi¢ed into three groups as follows : I = retinal ¢bres are distributed over the entire width of the SC and clustered (white bar); II = retinal ¢bres are distributed over the entire width of the SC but are di¡use; III = retinal ¢bres are restricted within the medial half of the SC and are di¡use (II and III: stippled bar); IV = retinal ¢bres are clustered into a single medial patch (black bar). For each case the number of sections belonging to each group was normalised by the total number of sections. (B) Evaluation of the mediolateral exuberance of the ipsilateral projection in the SC of wild-type and 5-HTT KO treated with PCPA. The percentage of sections with a retinal projection covering the entire width of the SC is shown. This corresponds to groups I+II, normalised by the total number of sections. Bars indicate the S.E.M. There is no signi¢cant di¡erence between 5-HTT KO and wild-type treated with PCPA but both di¡er signi¢cantly from untreated controls. (C) Evaluation of the volume occupied by ipsilateral terminals in the dLGN of wild-type and 5-HTT KO mice treated with PCPA compared to untreated wild-type. Bars indicate the S.E. The size of the projection in the treated wild-type is signi¢cantly reduced compared to untreated wild-type (ANOVA test, the minimal signi¢cant di¡erence is 6 in this case) but not signi¢cantly reduced in the 5-HTT KO.
the entire width of the SC was similar to adults (Fig. 6B) and retinal ¢bres had retracted from the SGS and were con¢ned to the stratum opticum (SO). Normal mice treated with PCPA In wild-type mice treated with PCPA (n = 9, from two
di¡erent litters, quanti¢cation on four cases) the projection to the SC was severely altered: in the rostral part of the SC a row of patches was observed in the SO but the number of sections with such a labelling was reduced by half in comparison to untreated controls (18% compared to 35%) (Fig. 6B). Caudally, the retinal projections were exuberant in both the dorsoventral and in the mediolat-
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eral plane of the SC (Figs. 5B and 6B). In the dLGN, the presence of a gap free of retinal ¢bres was observed contralaterally as in untreated controls. However, the volume occupied by ipsilateral retinal projection appeared to be reduced relative to matched controls (Fig. 6C). 5-HTT KO mice treated with PCPA In the 5-HTT KO mice treated with PCPA (n = 3), we found abnormalities in the SC that were similar to those found in the wild-type mice treated with PCPA (Figs. 5C and 6A, B). In the dLGN, a complete and clear reversal of the retinogeniculate abnormalities was observed (n = 3+2 aged P20). The contralateral retinal projection had completely cleared from the area corresponding to the ipsilateral territory, similarly to normal mice (Figs. 3G, H and 6C).
DISCUSSION
The present report extends previous observations indicating the importance of 5-HT in the patterning of ipsi/ contralateral retinal a¡erents during development (Mooney et al., 1998; Rhoades et al., 1993; Salichon et al., 2001; Upton et al., 1999). In addition, it reveals an interesting di¡erence in the e¡ects of 5-HT in two central targets of the retinal ¢bres, the dLGN and the SC. Our analysis of the retinal projections in 5-HTT KO mice indicates that the segregation of crossed and uncrossed ¢bres is sensitive to an excess of extracellular 5-HT in both the SC and dLGN, whereas the analysis of the 5-HT1B KO mice and of PCPA-treated mice shows that the lack of the 5-HT1B receptors or the lack of 5-HT a¡ects the re¢nement of retinal ¢bre patterning only in the SC, and could also cause an atrophy of the ipsilateral retinogeniculate projection. We analysed the e¡ects of a genetic lack of 5-HTT in the retinal system because during development a subset of RGCs, predominantly those that project ipsilaterally, transiently express the 5-HTT (Upton et al., 1999). The 5-HTT is expressed along the length of the axon and could act locally to reduce the extracellular concentration of 5-HT to which adjacent 5-HT receptors are exposed. We demonstrate here that, in the absence of 5-HTT, retinal axons do not segregate normally into eye-speci¢c patches, in either the thalamus or the SC. However, the alterations di¡er in the two structures: in the dLGN, the abnormality concerns the retraction of contralateral retinal ¢bres from the territory occupied by the ipsilateral ¢bres but there is no measurable change in the extent of the ipsilateral retinal projection. In the SC, a structure where ipsilateral and contralateral retinal ¢bres normally overlap (Godement et al., 1984), there is a visible increase in the extent of the ipsilateral retinal projection. The distribution of the ipsilateral retinal ¢bres in the SC is very similar to that observed at early stages of development (Godement et al., 1984; Upton et al., 1999) suggesting that the 5-HTT phenotype results from the lack of retraction of the ipsilateral retinal ¢bres from inappro-
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priate targets, in the caudal and lateral SC, and in the super¢cial layer of the SC, the SGS. It is most likely that these abnormalities result from modi¢ed brain levels of 5-HT. Indeed, 5-HTT has been shown to be essential in the clearance of 5-HT from extracellular space, and acute blockade of the transporter increases the extracellular levels of 5-HT (Fuller, 1994; Daws et al., 1998). As a consequence, a down-regulation of 5-HT production has been found in the 5-HTT KO mice (Bengel et al., 1998). However, this down-regulation is not su⁄cient to reduce the 5-HT in the extracellular space, since in vivo microdialysis in di¡erent brain structures of the adult 5-HTT KO mice (Andrews et al., 1998; Fabre et al., 2000) show six- to eight-fold elevations of 5-HT levels. Furthermore, in 5-HTT KO pups, dopaminergic neurones abnormally capture 5-HT via the dopamine transporter indicating that 5-HT accumulates in the extracellular space soon after birth (Ravary et al., 2001). To ascertain that the phenotype of the 5-HTT KO mice is due to an increase of 5-HT, we treated the pups with PCPA. We found that the ¢bre segregation, i.e. the retraction of contralateral ¢bres, was normalised in the dLGN of the 5-HTT KO mice. In the SC, the phenotype was partly reversed, and ipsilateral patches were found in the rostral SC. The incomplete reversal of the phenotypic abnormalities in the SC are most probably related to the fact that PCPA, in itself, caused abnormalities in the re¢nement of the retinotectal projections. Until now, only increased levels of 5-HT have been shown to disrupt the retinal ¢bre segregation (Mooney et al., 1998; Upton et al., 1999; Crnko-Hoppenjans et al., 2001). Our present results, together with those of the literature, therefore indicate that both increased and decreased levels of 5-HT alter retinotectal ¢bre re¢nement and could, in addition, reduce the extent of the ipsilateral retinal projection. What targets might extracellular 5-HT act on in this system? 5-HT1B receptors are a strong candidate for the e¡ects of increased 5-HT during development. RGCs express the 5-HT1B receptor mRNA from E15 onwards (Boschert et al., 1994; Upton et al., 1999) and the protein is detected on RGC axons in the SC in adult rats (Boulenguez et al., 1993), essentially in the preterminal region (Pickard et al., 1999; Sari et al., 1999). Stimulation of the presynaptic 5-HT1B receptor on RGC terminals in the tectum of adult hamster is known to inhibit glutamatergic neurotransmission (Mooney et al., 1994) and thus excessive activation of the high-a⁄nity 5-HT1B receptors on retinal a¡erents in MAOA KO (Salichon et al., 2001) and 5-HTT KO mice could block the transmission of spontaneous activity generated in the retina to the retinorecipient targets, and thus prevent activity-dependent processes involved in the patterning of a¡erents. The present results indicate that such a mechanism could be operating in the dLGN of the 5-HTT KO mice since removing the 5-HT1B receptor (the 5-HTT/5-HT1B double KOs) appeared to normalise the patterning of the retinal projections. Contrary to the clear e¡ects of 5-HT1B overstimulation, the developmental consequences of lack of 5-HT1B receptors have not been demonstrated. In the visual sys-
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tem of the 5-HT1B KO mice there were no visible di¡erences in the distribution of the ipsilateral and contralateral projections in the dLGN. Similarly, in the primary somatosensory cortex the development of the principal barrels appeared to be normal (Boylan et al., 2000; Salichon et al., 2001), although alterations of the smaller rostral barrels were observed in 5-HT1B KO mice with a C3H/HeJ background (I. Seif, unpublished results). The present ¢nding of alterations in the uncrossed retinotectal projection in the 5-HT1B KO mice is the ¢rst report demonstrating the existence of persistent anatomical alterations due to the lack of the 5-HT1B receptor. We found that these abnormalities appear only after P4, indicating that the 5-HT1B receptors have a role in the secondary re¢nement of the retinotectal map. It will be interesting to determine whether similar developmental abnormalities exist in other parts of the brain since the 5-HT1B KO mice have a number of behavioural changes (Boutrel et al., 1999; Brunner et al., 1999), some of which may have a developmental origin. The ¢nding that 5-HT1B receptors are required for the normal segregation of retinal ¢bres in the SC explains the phenotype that we have observed in the 5-HTT/5HT1B and the MAOA/5-HT1B double KO mice. In these mice, alterations are corrected in the dLGN but persist in the SC. Based on the distribution of the ipsilateral and contralateral retinal ¢bres in these two targets, it appears that the phenotype of the double knockouts is closer to the phenotype of the 5-HT1B KO than to the phenotype of the MAOA or the 5-HTT KO mice. Thus it is probable that, double knockouts revert to the single 5-HT1B KO phenotype. How could the 5-HT1B receptors act in conjunction with the 5-HTT transporter in shaping the ipsilateral retinal map? A possible explanation could reside in the distribution of RGCs that express 5-HTT and 5-HT1B receptors in the retina. The 5-HTT positive neurones are distributed in a ventral peripheral crescent that coincides in part with the temporo-ventral crescent of ipsilaterally projecting retinal neurones, whereas 5-HT1B receptors are present in all RGCs (see Upton et al., 1999 for a full description). 5-HTT could modulate the local concentration of 5-HT around growing axon terminals. This in turn would change the activation state of the 5-HT1B receptors and modulate the level of excitatory neurotransmission. This would produce a discrepancy in the activity levels of the a¡erent retinal inputs from both eyes or from di¡erent RGCs within one eye. Retinal axons that express 5-HTT would be more likely to transmit excitatory signals than axons that do not, because they can internalise extracellular 5-HT and relieve 5-HT1B -mediated inhibition. When 5-HTT or 5-HT1B receptors are inactive this di¡erential e¡ect would be lost and result in abnormalities in the elimination of misplaced axonal branches in the SC. However, this hypothesis does not explain why similar changes do not occur in the dLGN. One possible reason for the observed di¡erence between the SC and the dLGN could be the local conditions of 5-HT innervation in both retinal targets. The SC contains a higher density of 5-HT innervation than
the dLGN (Lidov and Molliver, 1982; Dinopoulos et al., 1995) and could thus accumulate higher levels of 5-HT when the clearance of 5-HT is disturbed. These targets could also di¡er in their 5-HT receptor expression (Claeysen et al., 1996; Gerard et al., 1997; Mooney et al., 1998). Another possibility that can be considered is that the activity-dependent interactions that shape the ipsi/contralateral projection di¡ers in the dLGN and the SC. In experiments and in KO mice that interrupt activity patterns from both eyes, the ipsilateral projections to the SC and to the dLGN both remain exuberant (Upton et al., 1999; Vercelli et al., 2000; Rossi et al., 2001). However, when activity is suppressed in one eye, di¡erent patterning defects appear to occur in the dLGN and the SC. In ferrets (Penn et al., 1998) and mice (Rebsam et al., 1999) it has been reported that the ipsilateral and contralateral retinal projections from the untreated eye are exuberant in the dLGN, whereas projections from the silenced eye are normal or atrophic. An opposite e¡ect was observed in the SC of hamsters: projections from the silenced eye were di¡use whereas the retinal projections from the untreated eye showed normal clustering (Thompson and Holt, 1989). Although such studies require con¢rmation by studying the entire retinal projection in a given species, this could indicate that activity driven interactions within one eye predominate in the SC to produce clustering of ipsilateral ¢bres (Thompson and Holt, 1989), whereas competitive interactions between the eyes are more important for segregation in the dLGN. The lack of 5-HT1B receptors may thus produce a situation in which the competitive interactions within one eye are disturbed to a greater degree than those between eyes. RGC axons must interact with a large number of factors in their target nuclei to ¢nd their correct topographic location within the correct eye-speci¢c area, and then to form stable synapses. This process is believed to be directed by both spontaneous activity independently generated in the two retina (Galli and Ma¡ei, 1988) and by topographic expression of guidance molecules in the target nuclei and on RGC axons. A variety of molecules have been implicated in topographic guidance such as the Eph receptors and their ligands the ephrins which are expressed in gradients across the retina, the tectum and the thalamus, and are believed to encode positional information (Connor et al., 1998). Activity-dependent mechanisms are thought to be important in the ¢ne-tuning of the projections and are believed to involve release of trophic molecules such as brainderived neurotrophic factor (Cohen-Cory, 1999; Isenmann et al., 1999) as well as negative factors that induce growth cone collapse such as nitric oxide produced by nitric oxide synthase (Vercelli et al., 2000). The action of 5-HT, via the 5-HT1B receptor, and possibly other 5-HT receptors, could be an important modulator of these activity-dependent interactions in the ¢nal stage of retinal a¡erent patterning.
Acknowledgements1We thank Aude Muzerelle, Diana Haranger, and Pierre Walrafen for their help, and Constantino
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Sotelo for his support. This work was supported by the European Commission (BMH4 CT97-2412), the CNRS, the INSERM, the British Council and Ministe're des A¡aires
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Etrange'res (Alliance programme), and the Bundesministerium fu«r Bildung und Forschung (BMBF, No. 512-400101KS9603).
REFERENCES
Andrews, A.M., Wichems, C.H., Li, O., Lesch, K.P., Murphy, D.L., 1998. A microdialysis study of the e¡ects of high K+ and paroxetine on extracellular serotonin concentrations in serotonin transporter knockout mice. American Society for Neuroscience, 440.2. Bengel, D., Murphy, D.L., Andrews, A.M., Wichems, C.H., Feltner, D., Heils, A., Mossner, R., Westphal, H., Lesch, K.P., 1998. Altered brain serotonin homeostasis and locomotor insensitivity to 3,4-methylenedioxymethamphetamine (‘Ecstasy’) in serotonin transporter-de¢cient mice. Mol. Pharmacol. 53, 649^655. Boschert, U., Amara, D.A., Segu, L., Hen, R., 1994. The mouse 5-hydroxytryptamine1B receptor is localized predominantly on axon terminals. Neuroscience 58, 167^182. Boulenguez, P., Abdelke¢, J., Pinard, R., Christolomme, A., Segu, L., 1993. E¡ects of retinal dea¡erentation on serotonin receptor types in the super¢cial grey layer of the superior colliculus of the rat. J. Chem. Neuroanat. 6, 167^175. Boutrel, B., Franc, B., Hen, R., Hamon, M., Adrien, J., 1999. Key role of 5-HT1B receptors in the regulation of paradoxical sleep as evidenced in 5-HT1B knock-out mice. J. Neurosci. 19, 3204^3212. Boylan, C.B., Kesterson, K.L., Crnko-Hoppenjans, T.A., Ke, M., Rizk, T., Mooney, R.D., Rhoades, R.W., 2000. The cortical vibrissae representation is normal in transgenic mice lacking the 5-HT(1B) receptor. Dev. Brain Res. 120, 91^93. Brunner, D., Buhot, M.C., Hen, R., Hofer, M., 1999. Anxiety, motor activation, and maternal-infant interactions in 5HT1B knockout mice. Behav. Neurosci. 113, 587^601. Cases, O., Vitalis, T., Seif, I., De Maeyer, E., Sotelo, C., Gaspar, P., 1996. Lack of barrels in the somatosensory cortex of monoamine oxidase Ade¢cient mice: role of a serotonin excess during the critical period. Neuron 16, 297^307. Claeysen, S., Sebben, M., Journot, L., Bockaert, J., Dumuis, A., 1996. Cloning, expression and pharmacology of the mouse 5-HT(4L) receptor. FEBS Lett. 398, 19^25. Cohen-Cory, S., 1999. BDNF modulates, but does not mediate, activity-dependent branching and remodeling of optic axon arbors in vivo. J. Neurosci. 19, 9996^10003. Connor, R.J., Menzel, P., Pasquale, E.B., 1998. Expression and tyrosine phosphorylation of Eph receptors suggest multiple mechanisms in patterning of the visual system. Dev. Biol. 193, 21^35. Crnko-Hoppenjans, T.A., Mooney, R.D., Rhoades, R.W., 2001. Neonatally elevated serotonin levels alter terminal arbors of individual retinal ganglion cells in superior colliculus of hamsters. J. Comp. Neurol. 432, 528^536. Daws, L.C., Toney, G.M., Gerhardt, G.A., Frazer, A., 1998. In vivo chronoamperometric measures of extracellular serotonin clearance in rat dorsal hippocampus : contribution of serotonin and norepinephrine transporters. J. Pharmacol. Exp. Ther. 286, 967^976. Dinopoulos, A., Dori, I., Parnavelas, J.G., 1995. Serotonergic innervation of the lateral geniculate nucleus of the rat during postnatal development: a light and electron microscopic immunocytochemical analysis. J. Comp. Neurol. 363, 532^544. Fabre, V., Beaufour, C., Evrard, A., Rioux, A., Hanoun, N., Lesch, K.P., Murphy, D.L., Lanfumey, L., Hamon, M., Martres, M.P., 2000. Altered expression and functions of serotonin 5-HT1A and 5-HT1B receptors in knock-out mice lacking the 5-HT transporter. Eur. J. Neurosci. 12, 2299^2310. Fuller, R.W., 1994. Uptake inhibitors increase extracellular serotonin concentration measured by brain microdialysis. Life Sci. 55, 163^167. Galli, L., Ma¡ei, L., 1988. Spontaneous impulse activity of rat retinal ganglion cells in prenatal life. Science 242, 90^91. Gerard, C., Martres, M.P., Lefevre, K., Miquel, M.C., Verge, D., Lanfumey, L., Doucet, E., Hamon, M., el Mestikawy, S., 1997. Immunolocalization of serotonin 5-HT6 receptor-like material in the rat central nervous system. Brain Res. 746, 207^219. Godement, P., Salaun, J., Imbert, M., 1984. Prenatal and postnatal development of retinogeniculate and retinocollicular projections in the mouse. J. Comp. Neurol. 230, 552^575. Isenmann, S., Cellerino, A., Gravel, C., Bahr, M., 1999. Excess target-derived brain-derived neurotrophic factor preserves the transient uncrossed retinal projection to the superior colliculus. Mol. Cell. Neurosci. 14, 52^65. LaVail, J.H., Nixon, R.A., Sidman, R.L., 1978. Genetic control of retinal ganglion cell projections. J. Comp. Neurol. 182, 399^421. Lidov, H.G., Molliver, M.E., 1982. An immunohistochemical study of serotonin neuron development in the rat: ascending pathways and terminal ¢elds. Brain Res. Bull. 8, 389^430. Meister, M., Wong, R.O., Baylor, D.A., Shatz, C.J., 1991. Synchronous bursts of action potentials in ganglion cells of the developing mammalian retina. Science 252, 939^943. Mooney, R.D., Crnko-Hoppenjans, T.A., Ke, M., Bennett-Clarke, C.A., Lane, R.D., Chiaia, N.L., Rhoades, R.W., 1998. Augmentation of serotonin in the developing superior colliculus alters the normal development of the uncrossed retinotectal projection. J. Comp. Neurol. 393, 84^92. Mooney, R.D., Huang, X., Shi, M.Y., Bennett-Clarke, C.A., Rhoades, R.W., 1996. Serotonin modulates retinotectal and corticotectal convergence in the superior colliculus. Prog. Brain Res. 112, 57^69. Mooney, R.D., Shi, M.Y., Rhoades, R.W., 1994. Modulation of retinotectal transmission by presynaptic 5-HT1B receptors in the superior colliculus of the adult hamster. J. Neurophysiol. 72, 3^13. Penn, A.A., Riquelme, P.A., Feller, M.B., Shatz, C.J., 1998. Competition in retinogeniculate patterning driven by spontaneous activity. Science 279, 2108^2112. Persico, A.M., Altamura, C., Calia, E., Puglisi-Allegra, S., Ventura, R., Lucchese, F., Keller, F., 2000. Serotonin depletion and barrel cortex development : impact of growth impairment vs. serotonin e¡ects on thalamocortical endings. Cereb. Cortex 10, 181^191. Pickard, G.E., Smith, B.N., Belenky, M., Rea, M.A., Dudek, F.E., Sollars, P.J., 1999. 5-HT1B receptor-mediated presynaptic inhibition of retinal input to the suprachiasmatic nucleus. J. Neurosci. 19, 4034^4045. Ravary, A., Muzerelle, A., Darmon, M., Murphy, D.L., Moessner, R., Lesch, K.P., Gaspar, P., 2001. Abnormal tra⁄cking and subcellular localization of an N-terminally truncated serotonin transporter protein. Eur. J. Neurosci. 13, 1349^1362. Rebsam, A., Tsuchimoto, Y., Fagiolini, M., Hensch, T.K., 1999. Spontaneous retinal activity patterns drive competitive segregation of mouse retinogeniculate axons. Society for Neuroscience Meeting, 604.3. Rhoades, R.W., Bennett-Clarke, C.A., Lane, R.D., Leslie, M.J., Mooney, R.D., 1993. Increased serotoninergic innervation of the hamster’s superior colliculus alters retinotectal projections. J. Comp. Neurol. 334, 397^409. Rossi, F.M., Pizzorusso, T., Porciatti, V., Marubio, L.M., Ma¡ei, L., Changeux, J.P., 2001. Requirement of the nicotinic acetylcholine receptor L2 subunit for the anatomical and functional development of the visual system. Proc. Natl. Acad. Sci. USA 98, 6453^6458.
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A. L. Upton et al.
Salichon, N., Gaspar, P., Upton, A.L., Picaud, S., Hanoun, N., Hamon, M., De Maeyer, E., Murphy, D.L., Mossner, R., Lesch, K.P., Hen, R., Seif, I., 2001. Excessive activation of serotonin (5-HT)1B receptors disrupts the formation of sensory maps in monoamine oxidase a and 5-HT transporter knock-out mice. J. Neurosci. 21, 884^896. Sari, Y., Miquel, M.C., Brisorgueil, M.J., Ruiz, G., Doucet, E., Hamon, M., Verge, D., 1999. Cellular and subcellular localization of 5-hydroxytryptamine1B receptors in the rat central nervous system : immunocytochemical, autoradiographic and lesion studies. Neuroscience 88, 899^915. Saudou, F., Amara, D.A., Dierich, A., LeMeur, M., Ramboz, S., Segu, L., Buhot, M.C., Hen, R., 1994. Enhanced aggressive behavior in mice lacking 5-HT1B receptor. Science 265, 1875^1878. Thompson, I., Holt, C., 1989. E¡ects of intraocular tetrodotoxin on the development of the retinocollicular pathway in the Syrian hamster. J. Comp. Neurol. 282, 371^388. Upton, A.L., Salichon, N., Lebrand, C., Ravary, A., Blakely, R., Seif, I., Gaspar, P., 1999. Excess of serotonin (5-HT) alters the segregation of ispilateral and contralateral retinal projections in monoamine oxidase A knock-out mice: possible role of 5-HT uptake in retinal ganglion cells during development. J. Neurosci. 19, 7007^7024. Vercelli, A., Garbossa, D., Biasiol, S., Repici, M., Jhaveri, S., 2000. NOS inhibition during postnatal development leads to increased ipsilateral retinocollicular and retinogeniculate projections in rats. Eur. J. Neurosci. 12, 473^490. (Accepted 23 November 2001)
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