Plasma membrane transporters of serotonin, dopamine, and norepinephrine mediate serotonin accumulation in atypical locations in the developing brain of monoamine oxidase A knock-outs

Abstract

Genetic loss or pharmacological inhibition of monoamine oxidase A (MAOA) in mice leads to a large increase in whole-brain levels of serotonin (5-HT). Excess 5-HT in mouse neonates prevents the normal barrel-like clustering of thalamic axons in the somatosensory cortex. Projection fields of other neuron populations may develop abnormally. In the present study, we have analyzed the localization of 5-HT immunolabeling in the developing brain of MAOA knock-out mice. We show numerous atypical locations of 5-HT during embryonic and postnatal development. Catecholaminergic cells of the substantia nigra, ventral tegmental area, hypothalamus, and locus ceruleus display transient 5-HT immunoreactivity. Pharmacological treatments inhibiting specific monoamine plasma membrane transporters and genetic crosses with mice lacking the dopamine plasma membrane transporter show that the accumulation of 5-HT in these catecholaminergic cells is attributable to 5-HT uptake via the dopamine or the norepinephrine plasma membrane transporter. In the telencephalon, transient 5-HT immunolabeling is observed in neurons in the CA1 and CA3 fields of the hippocampus, the central amygdala, the indusium griseum, and the deep layers of the anterior cingulate and retrosplenial cortices. In the diencephalon, primary sensory nuclei, as well as the mediodorsal, centrolateral, oval paracentral, submedial, posterior, and lateral posterior thalamic nuclei, are transiently 5-HT immunolabeled. The cortical projections of these thalamic nuclei are also labeled. In the brainstem, neurons in the lateral superior olivary nucleus and the anteroventral cochlear nucleus are transiently 5-HT immunolabeled. None of these structures appear to express the monoamine biosynthetic enzyme L-aromatic amino acid decarboxylase. The administration of monoamine plasma membrane transporter inhibitors indicates that the 5-HT immunolabeling in these structures is attributable to an uptake of 5-HT by the 5-HT plasma membrane transporter. This points to neuron populations that form highly precise projection maps that could be affected by 5-HT during specific developmental stages.

Fig. 1.

Catecholaminergic neurons accumulate 5-HT in P7 MAOA knock-outs. Coronal section through the SN–VTA complex (A, C) and the LC (B,D) were double immunostained with antibodies to 5-HT (A, B) and TH (C,D). As indicated by the arrowheads, almost all TH-positive neurons contain 5-HT immunolabeling. The varicose 5-HT-positive terminal network contains no TH immunolabeling (arrow). Scale bar (in D):A, C, 140 μm; B,D, 50 μm.

Fig. 2.

Atypical locations of 5-HT accumulation in the telencephalon of E18 MAOA knock-outs. Coronal brain sections are shown for controls (A, C) and MAOA knock-outs (B, D–G). A, In controls, 5-HT-immunostained fibers are primarily observed in the medial forebrain bundle (MFB). B, In MAOA knock-outs, 5-HT immunostaining of the MFB is increased, and a dense 5-HT immunolabeling is visible in the nucleus reticularis (RT), the thalamocortical fibers in the internal capsule (IC), the hippocampus (HI), and the amygdala (AMG). A higher magnification of the medial cortical area is shown inC and D at a more rostral level through the corpus callosum (CC), anterior cingulate cortex (ACG), and indusium griseum (IG).C, In controls, 5-HT immunoreactivity is only observed in terminal fibers or fiber tracts in the septum and ACG; the 5-HT-positive fibers in ACG form a bilaminar pattern in layer I and in the deep cortical layers. D, In MAOA knock-outs, 5-HT-positive fibers are more intensely stained, and additional labeling is visible in the fornix (FX) and in neuronal cell bodies in ACG and IG. E, A closer view of the 5-HT-immunolabeled cell bodies in the hippocampus reveals that these neurons have the morphological aspect of the principal pyramidal cells. Arrow indicates a neuron with a clear labeling of the dendritic tree. F, A closer view of the 5-HT immunolabeled neurons in the central nucleus of the amygdala.Arrows indicate neurons having a typical ovoid shape.G, Higher magnification of the 5-HT-positive thalamocortical fibers as they reach the cortical primordium. A dense network of fibers (fiber tracts and varicose fibers) is observed in the subplate (SP), with some fibers (open arrows) starting to penetrate in the cortical plate (CP). In contrast, a few long varicose fibers (arrow), probably representing afferents from the raphe, run in the intermediate zone (IZ). Only varicose fibers in SP and IZ were 5-HT immunoreactive in control mice, and this staining was much less intense than in MAOA knock-outs.VZ, Ventricular zone. Scale bar (in G):A, B, 625 μm; C,D, 150 μm; E, 27 μm;F, 40 μm; G, 33 μm.

Fig. 3.

Atypical locations of 5-HT accumulation in the forebrain of P7 MAOA knock-outs. A, The coronal section through the frontal cortex shown in the inset indicates the position of unusual 5-HT immunolabeling in the orbital cortex (ORB) ventrally (curved arrow) and in the medial prefrontal and pregenual anterior cingulate cortex (ACG) medially; the primary somatosensory cortex (SI) was shown previously to be 5-HT-labeled in normal pups. B, At higher magnification, 5-HT immunolabeling in the orbital cortex is seen to be localized in a plexus of fine fibers in layer III. C, Higher magnification shows the presence of 5-HT-containing neurons in ACG.Arrow points to a neuron with a typical pyramidal shape.D, Coronal section through the thalamus showing strong 5-HT-immunolabeling in different thalamic nuclei. MDc, Central part of the mediodorsal nucleus; SUB, submedial nucleus; OPC, oval paracentral nucleus;VPM, ventroposteromedial nucleus. E, Higher magnification shows the presence of 5-HT-immunolabeled cell bodies in MDc and OPC. F, Higher magnification shows the presence of 5-HT-immunolabeled cell bodies in SUB. Scale bar (in E): inset, 820 μm;A, B, D, 150 μm;C, 19 μm; E, F, 24 μm.

Fig. 4.

5-HT uptake in neurons of the LSO. Coronal sections through the brainstem of P7 MAOA knock-outs (A,B, D) and control (C). A, 5-HT immunoreactivity is normally localized in 5-HT-producing neurons of the dorsal raphe (DR), median raphe (MnR), and raphe magnus (RMg) and is abnormally localized in auditory neurons of LSO. B, 5-HT immunolabeling is found in the projection area of LSO neurons in the central nucleus of the inferior colliculus (CeIC). See higher magnification in D. 5-HT immunolabeling is also visible in the stratum zonale and stratum griseum of the superior colliculus (SC) and is primarily contained in retinal afferents (A. L. Upton, N. Salichon, I. Seif, and P. Gaspar, personal communication). C, In situhybridization with a radiolabeled SERT riboprobe shows the presence of SERT RNA in LSO neurons and in the raphe nuclei. D, At higher magnification, 5-HT immunolabeling in CeIC is not seen in cell bodies but in auditory afferents, presumably from LSO, as suggested by the trajectory of corresponding fiber bundles in adjacent sections. Scale bar (in D): A–C, 200 μm; D, 80 μm.

Fig. 5.

Immunocytochemical localization of AADC in E18 MAOA knock-outs. A, B, As viewed on coronal brain sections from rostral (A) to caudal (B), AADC immunoreactivity is localized in terminal fibers in the striatum, cortex, and hippocampus and in fiber tracts in the MFB or stria terminalis. AADC immunostaining of perikarya appears to be limited to catecholaminergic and D-group (Jaeger et al., 1984) neurons in the hypothalamus (Hyp).C, D, Coronal sections at comparable levels of the diencephalon are shown with 5-HT (C) and AADC immunostaining (D); both antisera label fibers in the MFB. On the other hand, the dense 5-HT immunolabeling of the ventroposterior complex (VP), dorsal lateral geniculate nucleus (DLG), thalamocortical fibers (Ta), and optic tract (Op) has no visible counterpart with AADC immunostaining. Scale bar (in D): A–D, 625 μm.

Fig. 6.

Changes of 5-HT immunoreactivity in P7 MAOA knock-outs after administration of selective inhibitors of monoaminergic transporters. Comparable coronal brain sections are shown in the metencephalon (A, D,G), mesencephalon (B, E,H), and diencephalon (C,F, I), after repeated administration of fluoxetine (A–C), nisoxetine (D–F), or GBR12783 (G–I) at P6 and P7. Control brain sections obtained from untreated MAOA knock-outs are not shown. 5-HT immunolabeling of the raphe nuclei is not visibly affected by any pharmacological treatment, although the staining of the fine varicose afferents from the raphe is reduced by the fluoxetine treatment. A–C, Fluoxetine, a selective inhibitor of SERT, causes the disappearance of 5-HT immunolabeling in the SC (B) and thalamus at the level of DLGn and VP (C) but increases staining of dopaminergic neurons in the SN and VTA (B), with no visible change in the LC (A). D–F, Nisoxetine, a selective inhibitor of NET, greatly reduces 5-HT immunolabeling in the LC (D) but does not cause changes of staining in the SN, VTA, SC (E), or thalamus (F). G–I, GBR12783, a selective inhibitor of DAT, abolishes 5-HT immunolabeling in the SN and VTA (H) but not in the LC (G), SC (H), or thalamus (I). Scale bar (inI): A–I, 625 μm.

Fig. 7.

Increase in the number of 5-HT-containing neurons in the hypothalamus of P7 MAOA knock-outs after fluoxetine treatments. Control brain sections obtained from untreated MAOA knock-outs are not shown. A, The number of 5-HT-containing neurons is increased in the Arc. Arrow indicates a dorsal periventricular neuron. B, The number of 5-HT-containing neurons is increased in the SCN. 3V, Third ventricle. Scale bar (in B): A, 24 μm;B, 90 μm.

Fig. 8.

Lack of 5-HT accumulation in dopaminergic cell bodies of P7 MAOA–DAT double knock-outs. Comparable coronal brain sections are shown in the metencephalon (A,C, E) and the pons (B,D, F) of mice knock-outs for MAOA (A, B), DAT (C,D), or both MAOA and DAT (E,F). A, B, In the MAOA single knock-out, 5-HT-containing neurons are observed in the MGV, SN, VTA, and LC. C, D, In contrast, in the DAT single knock-out, no 5-HT-containing neurons are observed in the MGV, SN, VTA, and LC. E, F, In the MAOA–DAT double knock-out, 5-HT-containing neurons are still observed in the MGV and LC but are no longer observed in the SN and VTA. Scale bar: A–F, 265 μm.