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Aberrant accumulation of serotonin in dopaminergic neurons
Neuroscience Letters 401 (2006) 49–54
Aberrant accumulation of serotonin in dopaminergic neurons Rainald M¨ossner a,∗ , Rabi Simantov b , Alexander Marx c , Klaus Peter Lesch a , Isabelle Seif d a
Department of Psychiatry and Psychotherapy, University of W¨urzburg, F¨uchsleinstr. 15, 97080 W¨urzburg, Germany b Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel c Institute of Pathology, University of W¨ urzburg, 97080 W¨urzburg, Germany d Faculty of Pharmacy, University of Paris-Sud, 92296 Chatenay-Malabry, France Received 17 June 2005; received in revised form 12 February 2006; accepted 22 February 2006
Abstract Gene targeting approaches greatly facilitate insight into the functioning of monoamine transporters, the targets of potent antidepressants. The serotonin transporter (5-HTT) is the molecular target of a large number of antidepressants. To assess the clearance of serotonin (5-HT) in the absence of the 5-HTT, we have generated double knockout mice lacking both the 5-HTT and the catabolizing enzyme monoamine oxidase A (MAOA). We found aberrant 5-HT accumulation in the striatum of these MAOA/5-HTT double knockout mice. By additional ablation of the dopamine transporter (DAT), this aberrant 5-HT accumulation was abolished in MAOA/5-HTT/DAT triple knockout mice. Thus, aberrant uptake of 5-HT occurs in dopaminergic terminals under conditions of elevated 5-HT levels, and this aberrant uptake is mediated by the DAT. These findings have important consequences for antidepressant therapy, since during treatment of depression with selective serotonin reuptake inhibitors, clearance of 5-HT by dopaminergic neurons may reduce the desired therapeutic elevation of extracellular 5-HT levels. This provides a molecular rationale for improving antidepressant efficacy by additional pharmacological inhibition of the DAT. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Serotonin; Dopamine; Transporter; Monoamine oxidase A
The serotonin transporter (5-HTT), which mediates the highaffinity reuptake of serotonin (5-hydroxytryptamine, 5-HT) from the extracellular space, is the target of a wide range of antidepressant drugs. Thus, the molecular target of the selective serotonin reuptake inhibitors (SSRIs) is the 5-HTT, with blockade of the reuptake of 5-HT leading to an increase of extracellular 5-HT levels. Moreover, the 5-HTT is also inhibited by the majority of tricyclic antidepressant drugs. Further insights into the pharmacological consequences of inhibition of the 5HTT is gained by the analysis of knockout mice lacking the 5-HTT. We have previously shown that aberrant uptake and storage of 5-HT in dopaminergic neurons can occur in vivo, under conditions of elevated extracellular 5-HT concentrations. Thus, knockout mice lacking the 5-HTT display 5-HT accumulation in the dopaminergic neurons of the substantia nigra [16]. Moreover, 5-HT uptake into the dopaminergic neurons of brainstem cultures of 5-HTT knockout mice can be blocked by pharmaco-
∗
Corresponding author. Tel.: +49 931 201 76000; fax: +49 931 201 77550. E-mail address: [email protected] (R. M¨ossner).
0304-3940/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2006.02.081
logical inhibition of the dopamine transporter (DAT) [15]. This aberrant uptake and storage of 5-HT in dopaminergic neurons is driven by elevated extracellular 5-HT levels in the absence of the 5-HTT. Thus, mice lacking the 5-HTT display 6-fold elevated extracellular 5-HT levels in the substantia nigra, measured by in vivo microdialysis [12]. Similarly, mice lacking the monoamine degrading enzyme monoamine oxidase A (MAOA), an enzyme localized on the outer mitochondrial membrane, display aberrant uptake and storage of 5-HT in the substantia nigra [4]. This aberrant uptake is also mediated by the DAT, since additional ablation of the DAT in MAOA/DAT double knockout mice abolishes the aberrant 5-HT uptake in the substantia nigra. MAOA knockout mice also display elevated extracellular 5-HT levels [8]. However, aberrant uptake and storage of 5-HT was found in the dopaminergic neurons of the substantia nigra in these 5HTT knockout mice, but not in dopaminergic projection areas. To investigate whether this effect can also operate in dopaminergic projection areas, we generated double knockout mice lacking both the 5-HTT and MAOA. To further characterize the molecular physiology, we performed an additional ablation of the DAT by generating MAOA/5-HTT/DAT triple knockout mice.
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To generate double knockout (DKO) and triple knockout mice, mice were crossed from previously characterized knockout strains lacking either MAOA [5], 5-HTT [1], or DAT [10] (for detailed genetic background see supplementary file). Animals were anaesthetized on postnatal (P) days P3–P7 with chloralhydrate and perfused through the aorta with 4% paraformaldehyde in 0.1 M sodium phosphate buffer. Brains were collected and post-fixed for several days. After cryoprotec-
tion in phosphate buffer with 30% sucrose for 2–10 days, coronal sections of the brains were frozen and cut to 40 m thick sections with a microtome. The 5-HT immunostaining method was modified from [17] (details see supplementary file). The striatum and adjacent somatosensory cortex of 5HTT knockout mice and wildtype control mice are shown in Figs. 1 and 2. The physiological transient 5-HT staining in the somatosensory cortex of wildtype mice is clearly visible
Fig. 1. 5-HT immunostaining in wildtype control mice. Rostro-to-caudal coronal sections (A–I) of a P4 C3H brain show a weak to moderate 5-HT staining of the caudate-putamen (CPu), which is coherent with 5-HTT staining (examples of 5-HTT staining of the CPu are given in Fig. 2B and supplementary Fig. 3A). Strong 5-HT staining in sensory cortices (S1, Au, and V) delineates glutamatergic thalamocortical axons that take up 5-HT via 5-HTT. Corresponding cell bodies in the visual (DLG) and somatosensory (VP) thalamic nuclei are also labeled, which is a key criteria for staining sensitivity. Progressive magnification of panel B (J–L) depicts unexpectedly dense 5-HT staining as compared with 5-HTT staining (Fig. 2B), in the mediocaudal part of the accumbens nucleus (Acb). Abbreviations: Tu (olfactory tubercle), M (motor cortex), Cg (cingulate cortex), S1 (primary somatosensory cortex), Pir (piriform cortex), cc (corpus callosum), LS (lateral septum), MS (medial septum), HDB (horizontal limb of the diagonal band), Hi (hippocampus), BST (bed nucleus of the stria terminalis), acp (anterior commissure-posterior), Hb (habenula), GP (globus pallidus), 3 V (3rd ventricle), DLG (dorsal lateral geniculate nucleus), RS (retrosplenial cortex), V (visual cortex), Au (auditory cortex), VP (ventroposterior thalamus), SCh (suprachiasmatic nucleus), aca (anterior commissure-anterior), LV (lateral ventricle). Scale bar for A–I, 1 mm.
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Fig. 2. 5-HT immunostaining in 5-HTT KO mice. Rostro-to-caudal brain sections of a P4 5-HTT KO mouse (C3H background) shows close-to-normal 5-HT staining of the CPu (A). A medial region of the Acb magnified in (C), is densely labeled, which is not congruent with 5-HTT labeling of the same region of a wildtype C3H mouse (B). Sensory cortices lack the strong 5-HT labeling observed in wildtype mice (Fig. 1A–I). A star in (A, C) indicates the magnified section. Abbreviations are as in Fig. 1.
(Fig. 1A–G). This uptake of 5-HT into thalamocortical glutamatergic fibers is mediated by the 5-HTT, since it is abolished in 5-HTT knockout mice (Fig. 2A). For a review on these glutamatergic fibers that transiently express 5-HTT but do not synthesize 5-HT, see [9]. No aberrant accumulation of 5-HT was apparent in the striatum of the 5-HTT knockout mice (Fig. 2A). In both wildtype and 5-HTT knockout mice, raphe afferents in the caudate putamen (CPu) formed thin, loose patches, in greater abundance at proximity of the accumbens nucleus and globus pallidus. After close scrutiny, 5-HTT knockout mice display 5-HT labeling in the medial part of the accumbens nucleus (Fig. 2C), which is less prominent in wildtype control mice (Fig. 1J).
We then generated MAOA/5-HTT double knockout mice and detected aberrant 5-HT accumulation in the striatum of these mice. Fig. 3 shows the striatum of MAOA/5-HTT double knockout mice, with 5-HT immunostaining demonstrating massive 5-HT accumulation in the striosomes of the CPu. The striatal phenotype of the MAOA/5-HTT double knockout mice was strikingly different from that of the MAOA single knockout mice. Thick patches of 5-HT-labeled varicosities appeared rapidly in the rostral CPu of the DKO (Fig. 3 and supplementary online Fig. 1). Moreover, intense 5-HT accumulation was apparent over the whole nucleus accumbens and most of the contiguous bed nucleus of the stria terminalis (Fig. 3 and supplementary online Fig. 2). The caudal CPu showed also high intensity of 5-
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Fig. 3. Aberrant striatal accumulation of 5-HT in MAOA/5-HTT DKO mice. Parallel processing of rostro-to-caudal brain sections of a P4 MAOA KO mouse (A, C, E, G, I, K) and a P4 MAOA/5-HTT DKO mouse (B, D, F, H, J, L) reveals genotype-related differences in the 5-HT staining pattern of the CPu and Acb, with much stronger labeling in the double KO mouse. Magnifications of the CPu and Acb regions are supplied as supplementary material (online Figs. 1–3). The somatosensory cortex of the DKO mouse (B, D, F, H, J, L) lacks the strong 5-HT labeling typically observed in MAOA KO mice (A, C, E, G, I, K) and in wildtype mice (Fig. 1A–G). The somatosensory cortex of the MAOA KO mouse typically lacks the barrel-like organization observed in wildtype mice (Fig. 1A–G). Numbers in (C) and (K) stand for CPu (1), aca (2), Acb (3), LS (4), MS (5), M (6), Cg (7), S1 (8), Pir (9), Hi (10), 11 (thalamus), and GP (12), with abbreviations as in Fig. 1.
HT labeling (Fig. 3 and supplementary online Fig. 3), with an unusual distribution reminiscent of both 5-HTT-labeled terminals and dopamine-labeled terminals in normal animals [24]. In contrast, 5-HT-labeled projections in non-catecholaminergic areas, did not appear more strongly labeled in DKO mice as compared to MAOA KO mice. Individual raphe fibers appeared either less or similarly labeled in the DKO mice, compared to MAOA KO mice. The raphe 5-HT fibers can be observed to be thicker in the MAOA single knockout mice than in the MAOA/5HTT double knockout mice. This indicates a similar revelation time for the immunohistochemical pictures.
Sensory thalamocortical fibers in MAOA single knockout mice are labeled with 5-HT (Fig. 3), as in wildtype mice (Fig. 1), due to transient expression of the 5-HTT on these fibers during neurodevelopment. This transient labeling of thalamocortical fibers is absent in the MAOA/5-HTT double knockout mice due to the ablation of the 5-HTT in these double knockout mice [17]. To further characterize the molecular biology of aberrant 5-HT accumulation in the striatum and accumbens of MAOA knockout mice and MAOA/5-HTT double knockout mice, we generated triple knockout mice lacking MAOA, 5-HTT, and DAT. The aim of additionally ablating the DAT was to
R. M¨ossner et al. / Neuroscience Letters 401 (2006) 49–54
assess whether aberrant 5-HT uptake is mediated by the DAT. Supplementary online Fig. 4 shows that in MAOA/5-HTT/DAT triple knockout mice, additional ablation of the DAT abolishes the 5-HT accumulation seen in the striatum and accumbens of MAOA/5-HTT double knockout mice. Therefore, the DAT mediates aberrant 5-HT uptake in the MAOA/5-HTT double knockout mice and is responsible for 5-HT uptake into the striatum of MAOA/5-HTT double knockout mice. The remarkable 5-HT pattern that is obtained by ablating both 5-HTT alleles in MAOA KO mice strongly suggested the existence of compensatory 5-HT uptake by dopaminergic terminals, despite the known capacity of noradrenergic neurons to take up and store 5-HT, at least at the level of the cell bodies [4] and despite the possibility of monoaminergic fiber sprouting in the DKO. In the coronal sections corresponding to the rostral part of the CPu of the triple KOs, the septum remained strongly labeled, a region that we did not further examine as it is traversed by numerous ascending fibers. More caudally, at the level of the substantia nigra pars compacta (SN), some dopaminergic cell bodies remained labeled in the triple KOs, although much more faintly than in MAOA KO and DKO mice. In the SN of the DKO pups, the cell body of dopaminergic cells was intensely labeled as well as the whole dendritic tree, whereas the dendritic tree was only poorly labeled in the MAOA KO and 5-HTT KO mice (data not shown), as observed also in our previous studies [4,16]. Deficiency in MAOA is known to allow more 5-HT to be stored in raphe projections, which was readily observed by 5-HT immunochemistry in the present study, and also in our previous study [4]. This increased storage has for direct consequence an increase in extracellular 5-HT, which may facilitate 5-HT clearance by alternative mechanisms. Despite this facilitating aspect, MAOA KO mice did not provide strong evidence of abnormal 5-HT clearance by dopaminergic projections (Fig. 3). Indeed, the stronger 5-HT immunoreactivity in the striatum of MAOA KO mice, as compared with wildtype mice, appears largely attributable to legitimate clearance by 5-HTT and is only putatively linked to other mediators such as DAT or the norepinephrine transporter NET. Accordingly, DAT in the MAOA KO mice may transport 5-HT into a more limited subset of dopaminergic fibers than in the double knockouts, with a distribution more closely mimicking the wildtype distribution of 5-HTT fibers and 5-HT fibers. As shown by microdialysis studies, adult 5-HTT knockout mice have 6 to 14-fold elevated extracellular 5-HT levels in striatum [12,19], while adult MAOA knockout mice have approximately 2 to 3-fold elevated extracellular 5-HT levels in striatum [8]. The elevations of extracellular 5-HT levels in single knockout mice appear not to be sufficient to cause aberrant 5-HT accumulation in striatum. The striatal differences between 5-HTT KO and MAOA/5-HTT DKO mice suggest two explanations, which are not mutually exclusive: either extracellular 5-HT concentrations in 5-HTT KO mice are insufficient to allow uptake through DAT, or such uptake does occur but internalized 5-HT is largely degraded by MAOA since the MAOA gene is expressed in the midbrain dopaminergic cells that abundantly innervate the CPu and accumbens nucleus [23]. In contrast, the
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striatal differences between the MAOA KO and MAOA/5-HTT DKO suggest a single explanation, that of a powerful clearance effect of 5-HTT in MAOA KO. Within short distances of the serotonergic release sites, such as within the synaptic cleft, MAOA KO mice may possess the highest extracellular 5HT levels of all three genotypes (MAOA KO, 5-HTT KO, and MAOA/5-HTT DKO), since 5-HTT is absent from synaptic profiles. In contrast, at large distances from the serotonergic release sites, MAOA/5-HTT double knockout mice probably possess extracellular striatal 5-HT levels that are significantly higher than in either single knockout mouse. The serotonergic innervation of the cell bodies in the substantia nigra compared to the serotonergic innervation of dopaminergic terminal areas, is different. There is a very pronounced serotonergic pathway to the substantia nigra, which provides a large amount of 5-HT directly onto the cell bodies of that area. In the striatum, on the other hand, the serotonergic terminals are much less abundant and are less likely to be in close apposition to DAT sites. Thus, diffusion of 5-HT over relatively large distances, as rendered possible by ablation of 5-HTT, seems an important requirement for 5-HT uptake by dopaminergic projections, whereas this is not a requirement in the substantia nigra, as shown in a previous report, since 5-HT accumulation was observed in nigral cell bodies in MAOA KO mouse pups but not in MAOA/DAT double KO littermates [4]. Finally, the density and variety of storage organelles may cause differences in accumulation between cell bodies and axonal afferents. In the substantia nigra and ventral tegmental area, dopaminergic cell bodies and dendrites are filled with extensive networks of VMAT2-labeled tubulovesicular organelles that are believed to store dopamine and serotonin [13]. Aberrant 5-HT uptake in catecholaminergic terminals has previously been observed in autoradiographic investigations of uptake of tritiated 5-HT in brain slices, and in electrophysiological studies [2,7,11,18,20]. Here we show that this can also be visualized in vivo, which is consistent with the finding that the DAT is localized not only to dendritic, but also to axonal plasma membranes [6,14]. Very recently, it was shown by Zhou et al. that 5-HT may be released by striatal dopaminergic neurons as a false neurotransmitter [25]. Our investigations further support and extend these findings by demonstrating the DAT- and MAOA-dependent aberrant accumulation of 5-HT in the striatum of double knockout mice. Moreover, we demonstrate that striatal dopaminergic terminals display aberrant 5-HT accumulation less readily than dopaminergic cell bodies. Furthermore, our findings show that double and triple knockout mice represent valuable models to assess the effect of blockade of neurotransmitter transporters and metabolizing enzymes on the distribution and disposition of neurotransmitters. Another clinical condition in which the aberrant uptake of 5-HT into dopaminergic neurons is expected to be important is Brunner syndrome. Patients with this syndrome lack MAOA activity due to a point mutation in the MAOA gene [3]. These patients exhibit marked aggression and are occasionally violent. Given that in MAOA knockout mice, the shell of the nucleus accumbens shows relatively intense 5-HT labeling, we suggest that MAOA-deficient men might have 5-HT in their
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catecholaminergic neurons in this area. After treatment with SSRIs, it is possible that extensive aberrant uptake and storage of 5-HT in dopaminergic neurons would take place. However, Brunner syndrome patients must not be treated with 5-HT reuptake inhibitors and other 5-HT agonists due to the high risk of developing adverse reactions, as was noted with buspirone [22]. In conclusion, elevated extracellular 5-HT levels drive aberrant uptake of 5-HT in the somatodendritic compartment of dopaminergic cell groups, but also in dopaminergic terminals. During antidepressant therapy with selective serotonin reuptake inhibitors, clearance of 5-HT by the DAT may lead to a lesser degree of elevation of extracellular 5-HT levels than desired. Thus, 5-HT accumulates in small amounts in dopaminergic terminals when wildtype mice are treated with fluoxetine [25]. To achieve the desired elevation of extracellular 5-HT levels during antidepressant therapy, it may therefore be useful to block not only the 5-HTT, but also the DAT. Hence, antidepressants that inhibit both the 5-HTT and the DAT [21], such as sertraline, may be advantageous in antidepressant therapy. Acknowledgements The IZKF W¨urzburg (N-2; 01KS9603), the CNRS, the Deutsche Forschungsgemeinschaft (SFB 581; KFO 125/11), and the European Commission (NEWMOOD LSHM-CT2003–503474) supported this work. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.neulet.2006.02.081. References [1] D. Bengel, D.L. Murphy, A.M. Andrews, C.H. Wichems, D. Feltner, A. Heils, R. M¨ossner, H. Westphal, K.P. Lesch, Altered brain serotonin homeostasis and locomotor insensitivity to 3,4-methylenedioxymethamphetamine (“Ecstasy”) in serotonin transporter-deficient mice, Mol. Pharmacol. 53 (1998) 649–655. [2] B. Berger, J. Glowinski, Dopamine uptake in serotoninergic terminals in vitro: a valuable tool for the histochemical differentiation of catecholaminergic and serotoninergic terminals in rat cerebral structures, Brain Res. 147 (1978) 29–45. [3] H.G. Brunner, M. Nelen, X.O. Breakefield, H.H. Ropers, B.A. van Oost, Abnormal behavior associated with a point mutation in the structural gene for monoamine oxidase A, Science 262 (1993) 578– 580. [4] O. Cases, C. Lebrand, B. Giros, T. Vitalis, E. De Maeyer, M.G. Caron, D.J. Price, P. Gaspar, I. Seif, Plasma membrane transporters of serotonin, dopamine, and norepinephrine mediate serotonin accumulation in atypical locations in the developing brain of monoamine oxidase knock-outs, J. Neurosci. 18 (1998) 6914–6927. [5] O. Cases, I. Seif, J. Grimsby, P. Gaspar, K. Chen, S. Pournin, U. M¨uller, M. Aguet, C. Babinet, J.C. Shih, E. De Maeyer, Aggressive behavior and altered amounts of brain serotonin and norepinephrine in mice lacking MAOA, Science 268 (1995) 1763–1766. [6] B.J. Ciliax, C. Heilman, L.L. Demchyshyn, Z.B. Pristupa, E. Ince, S.M. Hersch, H.M. Niznik, A.L. Levey, The dopamine transporter: immunochemical characterization and localization in the brain, J. Neurosci. 15 (1995) 1714–1723.
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Aberrant accumulation of serotonin in dopaminergic neurons Rainald M¨ossner a,∗ , Rabi Simantov b , Alexander Marx c , Klaus Peter Lesch a , Isabelle Seif d a
Department of Psychiatry and Psychotherapy, University of W¨urzburg, F¨uchsleinstr. 15, 97080 W¨urzburg, Germany b Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel c Institute of Pathology, University of W¨ urzburg, 97080 W¨urzburg, Germany d Faculty of Pharmacy, University of Paris-Sud, 92296 Chatenay-Malabry, France Received 17 June 2005; received in revised form 12 February 2006; accepted 22 February 2006
Abstract Gene targeting approaches greatly facilitate insight into the functioning of monoamine transporters, the targets of potent antidepressants. The serotonin transporter (5-HTT) is the molecular target of a large number of antidepressants. To assess the clearance of serotonin (5-HT) in the absence of the 5-HTT, we have generated double knockout mice lacking both the 5-HTT and the catabolizing enzyme monoamine oxidase A (MAOA). We found aberrant 5-HT accumulation in the striatum of these MAOA/5-HTT double knockout mice. By additional ablation of the dopamine transporter (DAT), this aberrant 5-HT accumulation was abolished in MAOA/5-HTT/DAT triple knockout mice. Thus, aberrant uptake of 5-HT occurs in dopaminergic terminals under conditions of elevated 5-HT levels, and this aberrant uptake is mediated by the DAT. These findings have important consequences for antidepressant therapy, since during treatment of depression with selective serotonin reuptake inhibitors, clearance of 5-HT by dopaminergic neurons may reduce the desired therapeutic elevation of extracellular 5-HT levels. This provides a molecular rationale for improving antidepressant efficacy by additional pharmacological inhibition of the DAT. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Serotonin; Dopamine; Transporter; Monoamine oxidase A
The serotonin transporter (5-HTT), which mediates the highaffinity reuptake of serotonin (5-hydroxytryptamine, 5-HT) from the extracellular space, is the target of a wide range of antidepressant drugs. Thus, the molecular target of the selective serotonin reuptake inhibitors (SSRIs) is the 5-HTT, with blockade of the reuptake of 5-HT leading to an increase of extracellular 5-HT levels. Moreover, the 5-HTT is also inhibited by the majority of tricyclic antidepressant drugs. Further insights into the pharmacological consequences of inhibition of the 5HTT is gained by the analysis of knockout mice lacking the 5-HTT. We have previously shown that aberrant uptake and storage of 5-HT in dopaminergic neurons can occur in vivo, under conditions of elevated extracellular 5-HT concentrations. Thus, knockout mice lacking the 5-HTT display 5-HT accumulation in the dopaminergic neurons of the substantia nigra [16]. Moreover, 5-HT uptake into the dopaminergic neurons of brainstem cultures of 5-HTT knockout mice can be blocked by pharmaco-
∗
Corresponding author. Tel.: +49 931 201 76000; fax: +49 931 201 77550. E-mail address: [email protected] (R. M¨ossner).
0304-3940/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2006.02.081
logical inhibition of the dopamine transporter (DAT) [15]. This aberrant uptake and storage of 5-HT in dopaminergic neurons is driven by elevated extracellular 5-HT levels in the absence of the 5-HTT. Thus, mice lacking the 5-HTT display 6-fold elevated extracellular 5-HT levels in the substantia nigra, measured by in vivo microdialysis [12]. Similarly, mice lacking the monoamine degrading enzyme monoamine oxidase A (MAOA), an enzyme localized on the outer mitochondrial membrane, display aberrant uptake and storage of 5-HT in the substantia nigra [4]. This aberrant uptake is also mediated by the DAT, since additional ablation of the DAT in MAOA/DAT double knockout mice abolishes the aberrant 5-HT uptake in the substantia nigra. MAOA knockout mice also display elevated extracellular 5-HT levels [8]. However, aberrant uptake and storage of 5-HT was found in the dopaminergic neurons of the substantia nigra in these 5HTT knockout mice, but not in dopaminergic projection areas. To investigate whether this effect can also operate in dopaminergic projection areas, we generated double knockout mice lacking both the 5-HTT and MAOA. To further characterize the molecular physiology, we performed an additional ablation of the DAT by generating MAOA/5-HTT/DAT triple knockout mice.
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To generate double knockout (DKO) and triple knockout mice, mice were crossed from previously characterized knockout strains lacking either MAOA [5], 5-HTT [1], or DAT [10] (for detailed genetic background see supplementary file). Animals were anaesthetized on postnatal (P) days P3–P7 with chloralhydrate and perfused through the aorta with 4% paraformaldehyde in 0.1 M sodium phosphate buffer. Brains were collected and post-fixed for several days. After cryoprotec-
tion in phosphate buffer with 30% sucrose for 2–10 days, coronal sections of the brains were frozen and cut to 40 m thick sections with a microtome. The 5-HT immunostaining method was modified from [17] (details see supplementary file). The striatum and adjacent somatosensory cortex of 5HTT knockout mice and wildtype control mice are shown in Figs. 1 and 2. The physiological transient 5-HT staining in the somatosensory cortex of wildtype mice is clearly visible
Fig. 1. 5-HT immunostaining in wildtype control mice. Rostro-to-caudal coronal sections (A–I) of a P4 C3H brain show a weak to moderate 5-HT staining of the caudate-putamen (CPu), which is coherent with 5-HTT staining (examples of 5-HTT staining of the CPu are given in Fig. 2B and supplementary Fig. 3A). Strong 5-HT staining in sensory cortices (S1, Au, and V) delineates glutamatergic thalamocortical axons that take up 5-HT via 5-HTT. Corresponding cell bodies in the visual (DLG) and somatosensory (VP) thalamic nuclei are also labeled, which is a key criteria for staining sensitivity. Progressive magnification of panel B (J–L) depicts unexpectedly dense 5-HT staining as compared with 5-HTT staining (Fig. 2B), in the mediocaudal part of the accumbens nucleus (Acb). Abbreviations: Tu (olfactory tubercle), M (motor cortex), Cg (cingulate cortex), S1 (primary somatosensory cortex), Pir (piriform cortex), cc (corpus callosum), LS (lateral septum), MS (medial septum), HDB (horizontal limb of the diagonal band), Hi (hippocampus), BST (bed nucleus of the stria terminalis), acp (anterior commissure-posterior), Hb (habenula), GP (globus pallidus), 3 V (3rd ventricle), DLG (dorsal lateral geniculate nucleus), RS (retrosplenial cortex), V (visual cortex), Au (auditory cortex), VP (ventroposterior thalamus), SCh (suprachiasmatic nucleus), aca (anterior commissure-anterior), LV (lateral ventricle). Scale bar for A–I, 1 mm.
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Fig. 2. 5-HT immunostaining in 5-HTT KO mice. Rostro-to-caudal brain sections of a P4 5-HTT KO mouse (C3H background) shows close-to-normal 5-HT staining of the CPu (A). A medial region of the Acb magnified in (C), is densely labeled, which is not congruent with 5-HTT labeling of the same region of a wildtype C3H mouse (B). Sensory cortices lack the strong 5-HT labeling observed in wildtype mice (Fig. 1A–I). A star in (A, C) indicates the magnified section. Abbreviations are as in Fig. 1.
(Fig. 1A–G). This uptake of 5-HT into thalamocortical glutamatergic fibers is mediated by the 5-HTT, since it is abolished in 5-HTT knockout mice (Fig. 2A). For a review on these glutamatergic fibers that transiently express 5-HTT but do not synthesize 5-HT, see [9]. No aberrant accumulation of 5-HT was apparent in the striatum of the 5-HTT knockout mice (Fig. 2A). In both wildtype and 5-HTT knockout mice, raphe afferents in the caudate putamen (CPu) formed thin, loose patches, in greater abundance at proximity of the accumbens nucleus and globus pallidus. After close scrutiny, 5-HTT knockout mice display 5-HT labeling in the medial part of the accumbens nucleus (Fig. 2C), which is less prominent in wildtype control mice (Fig. 1J).
We then generated MAOA/5-HTT double knockout mice and detected aberrant 5-HT accumulation in the striatum of these mice. Fig. 3 shows the striatum of MAOA/5-HTT double knockout mice, with 5-HT immunostaining demonstrating massive 5-HT accumulation in the striosomes of the CPu. The striatal phenotype of the MAOA/5-HTT double knockout mice was strikingly different from that of the MAOA single knockout mice. Thick patches of 5-HT-labeled varicosities appeared rapidly in the rostral CPu of the DKO (Fig. 3 and supplementary online Fig. 1). Moreover, intense 5-HT accumulation was apparent over the whole nucleus accumbens and most of the contiguous bed nucleus of the stria terminalis (Fig. 3 and supplementary online Fig. 2). The caudal CPu showed also high intensity of 5-
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Fig. 3. Aberrant striatal accumulation of 5-HT in MAOA/5-HTT DKO mice. Parallel processing of rostro-to-caudal brain sections of a P4 MAOA KO mouse (A, C, E, G, I, K) and a P4 MAOA/5-HTT DKO mouse (B, D, F, H, J, L) reveals genotype-related differences in the 5-HT staining pattern of the CPu and Acb, with much stronger labeling in the double KO mouse. Magnifications of the CPu and Acb regions are supplied as supplementary material (online Figs. 1–3). The somatosensory cortex of the DKO mouse (B, D, F, H, J, L) lacks the strong 5-HT labeling typically observed in MAOA KO mice (A, C, E, G, I, K) and in wildtype mice (Fig. 1A–G). The somatosensory cortex of the MAOA KO mouse typically lacks the barrel-like organization observed in wildtype mice (Fig. 1A–G). Numbers in (C) and (K) stand for CPu (1), aca (2), Acb (3), LS (4), MS (5), M (6), Cg (7), S1 (8), Pir (9), Hi (10), 11 (thalamus), and GP (12), with abbreviations as in Fig. 1.
HT labeling (Fig. 3 and supplementary online Fig. 3), with an unusual distribution reminiscent of both 5-HTT-labeled terminals and dopamine-labeled terminals in normal animals [24]. In contrast, 5-HT-labeled projections in non-catecholaminergic areas, did not appear more strongly labeled in DKO mice as compared to MAOA KO mice. Individual raphe fibers appeared either less or similarly labeled in the DKO mice, compared to MAOA KO mice. The raphe 5-HT fibers can be observed to be thicker in the MAOA single knockout mice than in the MAOA/5HTT double knockout mice. This indicates a similar revelation time for the immunohistochemical pictures.
Sensory thalamocortical fibers in MAOA single knockout mice are labeled with 5-HT (Fig. 3), as in wildtype mice (Fig. 1), due to transient expression of the 5-HTT on these fibers during neurodevelopment. This transient labeling of thalamocortical fibers is absent in the MAOA/5-HTT double knockout mice due to the ablation of the 5-HTT in these double knockout mice [17]. To further characterize the molecular biology of aberrant 5-HT accumulation in the striatum and accumbens of MAOA knockout mice and MAOA/5-HTT double knockout mice, we generated triple knockout mice lacking MAOA, 5-HTT, and DAT. The aim of additionally ablating the DAT was to
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assess whether aberrant 5-HT uptake is mediated by the DAT. Supplementary online Fig. 4 shows that in MAOA/5-HTT/DAT triple knockout mice, additional ablation of the DAT abolishes the 5-HT accumulation seen in the striatum and accumbens of MAOA/5-HTT double knockout mice. Therefore, the DAT mediates aberrant 5-HT uptake in the MAOA/5-HTT double knockout mice and is responsible for 5-HT uptake into the striatum of MAOA/5-HTT double knockout mice. The remarkable 5-HT pattern that is obtained by ablating both 5-HTT alleles in MAOA KO mice strongly suggested the existence of compensatory 5-HT uptake by dopaminergic terminals, despite the known capacity of noradrenergic neurons to take up and store 5-HT, at least at the level of the cell bodies [4] and despite the possibility of monoaminergic fiber sprouting in the DKO. In the coronal sections corresponding to the rostral part of the CPu of the triple KOs, the septum remained strongly labeled, a region that we did not further examine as it is traversed by numerous ascending fibers. More caudally, at the level of the substantia nigra pars compacta (SN), some dopaminergic cell bodies remained labeled in the triple KOs, although much more faintly than in MAOA KO and DKO mice. In the SN of the DKO pups, the cell body of dopaminergic cells was intensely labeled as well as the whole dendritic tree, whereas the dendritic tree was only poorly labeled in the MAOA KO and 5-HTT KO mice (data not shown), as observed also in our previous studies [4,16]. Deficiency in MAOA is known to allow more 5-HT to be stored in raphe projections, which was readily observed by 5-HT immunochemistry in the present study, and also in our previous study [4]. This increased storage has for direct consequence an increase in extracellular 5-HT, which may facilitate 5-HT clearance by alternative mechanisms. Despite this facilitating aspect, MAOA KO mice did not provide strong evidence of abnormal 5-HT clearance by dopaminergic projections (Fig. 3). Indeed, the stronger 5-HT immunoreactivity in the striatum of MAOA KO mice, as compared with wildtype mice, appears largely attributable to legitimate clearance by 5-HTT and is only putatively linked to other mediators such as DAT or the norepinephrine transporter NET. Accordingly, DAT in the MAOA KO mice may transport 5-HT into a more limited subset of dopaminergic fibers than in the double knockouts, with a distribution more closely mimicking the wildtype distribution of 5-HTT fibers and 5-HT fibers. As shown by microdialysis studies, adult 5-HTT knockout mice have 6 to 14-fold elevated extracellular 5-HT levels in striatum [12,19], while adult MAOA knockout mice have approximately 2 to 3-fold elevated extracellular 5-HT levels in striatum [8]. The elevations of extracellular 5-HT levels in single knockout mice appear not to be sufficient to cause aberrant 5-HT accumulation in striatum. The striatal differences between 5-HTT KO and MAOA/5-HTT DKO mice suggest two explanations, which are not mutually exclusive: either extracellular 5-HT concentrations in 5-HTT KO mice are insufficient to allow uptake through DAT, or such uptake does occur but internalized 5-HT is largely degraded by MAOA since the MAOA gene is expressed in the midbrain dopaminergic cells that abundantly innervate the CPu and accumbens nucleus [23]. In contrast, the
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striatal differences between the MAOA KO and MAOA/5-HTT DKO suggest a single explanation, that of a powerful clearance effect of 5-HTT in MAOA KO. Within short distances of the serotonergic release sites, such as within the synaptic cleft, MAOA KO mice may possess the highest extracellular 5HT levels of all three genotypes (MAOA KO, 5-HTT KO, and MAOA/5-HTT DKO), since 5-HTT is absent from synaptic profiles. In contrast, at large distances from the serotonergic release sites, MAOA/5-HTT double knockout mice probably possess extracellular striatal 5-HT levels that are significantly higher than in either single knockout mouse. The serotonergic innervation of the cell bodies in the substantia nigra compared to the serotonergic innervation of dopaminergic terminal areas, is different. There is a very pronounced serotonergic pathway to the substantia nigra, which provides a large amount of 5-HT directly onto the cell bodies of that area. In the striatum, on the other hand, the serotonergic terminals are much less abundant and are less likely to be in close apposition to DAT sites. Thus, diffusion of 5-HT over relatively large distances, as rendered possible by ablation of 5-HTT, seems an important requirement for 5-HT uptake by dopaminergic projections, whereas this is not a requirement in the substantia nigra, as shown in a previous report, since 5-HT accumulation was observed in nigral cell bodies in MAOA KO mouse pups but not in MAOA/DAT double KO littermates [4]. Finally, the density and variety of storage organelles may cause differences in accumulation between cell bodies and axonal afferents. In the substantia nigra and ventral tegmental area, dopaminergic cell bodies and dendrites are filled with extensive networks of VMAT2-labeled tubulovesicular organelles that are believed to store dopamine and serotonin [13]. Aberrant 5-HT uptake in catecholaminergic terminals has previously been observed in autoradiographic investigations of uptake of tritiated 5-HT in brain slices, and in electrophysiological studies [2,7,11,18,20]. Here we show that this can also be visualized in vivo, which is consistent with the finding that the DAT is localized not only to dendritic, but also to axonal plasma membranes [6,14]. Very recently, it was shown by Zhou et al. that 5-HT may be released by striatal dopaminergic neurons as a false neurotransmitter [25]. Our investigations further support and extend these findings by demonstrating the DAT- and MAOA-dependent aberrant accumulation of 5-HT in the striatum of double knockout mice. Moreover, we demonstrate that striatal dopaminergic terminals display aberrant 5-HT accumulation less readily than dopaminergic cell bodies. Furthermore, our findings show that double and triple knockout mice represent valuable models to assess the effect of blockade of neurotransmitter transporters and metabolizing enzymes on the distribution and disposition of neurotransmitters. Another clinical condition in which the aberrant uptake of 5-HT into dopaminergic neurons is expected to be important is Brunner syndrome. Patients with this syndrome lack MAOA activity due to a point mutation in the MAOA gene [3]. These patients exhibit marked aggression and are occasionally violent. Given that in MAOA knockout mice, the shell of the nucleus accumbens shows relatively intense 5-HT labeling, we suggest that MAOA-deficient men might have 5-HT in their
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catecholaminergic neurons in this area. After treatment with SSRIs, it is possible that extensive aberrant uptake and storage of 5-HT in dopaminergic neurons would take place. However, Brunner syndrome patients must not be treated with 5-HT reuptake inhibitors and other 5-HT agonists due to the high risk of developing adverse reactions, as was noted with buspirone [22]. In conclusion, elevated extracellular 5-HT levels drive aberrant uptake of 5-HT in the somatodendritic compartment of dopaminergic cell groups, but also in dopaminergic terminals. During antidepressant therapy with selective serotonin reuptake inhibitors, clearance of 5-HT by the DAT may lead to a lesser degree of elevation of extracellular 5-HT levels than desired. Thus, 5-HT accumulates in small amounts in dopaminergic terminals when wildtype mice are treated with fluoxetine [25]. To achieve the desired elevation of extracellular 5-HT levels during antidepressant therapy, it may therefore be useful to block not only the 5-HTT, but also the DAT. Hence, antidepressants that inhibit both the 5-HTT and the DAT [21], such as sertraline, may be advantageous in antidepressant therapy. Acknowledgements The IZKF W¨urzburg (N-2; 01KS9603), the CNRS, the Deutsche Forschungsgemeinschaft (SFB 581; KFO 125/11), and the European Commission (NEWMOOD LSHM-CT2003–503474) supported this work. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.neulet.2006.02.081. References [1] D. Bengel, D.L. Murphy, A.M. Andrews, C.H. Wichems, D. Feltner, A. Heils, R. M¨ossner, H. Westphal, K.P. Lesch, Altered brain serotonin homeostasis and locomotor insensitivity to 3,4-methylenedioxymethamphetamine (“Ecstasy”) in serotonin transporter-deficient mice, Mol. Pharmacol. 53 (1998) 649–655. [2] B. Berger, J. Glowinski, Dopamine uptake in serotoninergic terminals in vitro: a valuable tool for the histochemical differentiation of catecholaminergic and serotoninergic terminals in rat cerebral structures, Brain Res. 147 (1978) 29–45. [3] H.G. Brunner, M. Nelen, X.O. Breakefield, H.H. Ropers, B.A. van Oost, Abnormal behavior associated with a point mutation in the structural gene for monoamine oxidase A, Science 262 (1993) 578– 580. [4] O. Cases, C. Lebrand, B. Giros, T. Vitalis, E. De Maeyer, M.G. Caron, D.J. Price, P. Gaspar, I. Seif, Plasma membrane transporters of serotonin, dopamine, and norepinephrine mediate serotonin accumulation in atypical locations in the developing brain of monoamine oxidase knock-outs, J. Neurosci. 18 (1998) 6914–6927. [5] O. Cases, I. Seif, J. Grimsby, P. Gaspar, K. Chen, S. Pournin, U. M¨uller, M. Aguet, C. Babinet, J.C. Shih, E. De Maeyer, Aggressive behavior and altered amounts of brain serotonin and norepinephrine in mice lacking MAOA, Science 268 (1995) 1763–1766. [6] B.J. Ciliax, C. Heilman, L.L. Demchyshyn, Z.B. Pristupa, E. Ince, S.M. Hersch, H.M. Niznik, A.L. Levey, The dopamine transporter: immunochemical characterization and localization in the brain, J. Neurosci. 15 (1995) 1714–1723.
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