Volume 17, Issue 5, Pages 823-835 (November 1996) Transient Uptake and Storage of Serotonin in Developing Thalamic Neurons  Cécile Lebrand, Olivier Cases, Christine Adelbrecht, Anne Doye, Chantal Alvarez, Salah El Mestikawy, Isabelle Seif, Patricia Gaspar  Neuron  Volume 17, Issue 5, Pages 823-835 (November 1996) DOI: 10.1016/S0896-6273(00)80215-9

Figure 1 Confocal Microscopic Views of 5-HT-Labeled Structures in the Cortex and Thalamus of Mouse Pups In P7 pups, 5-HT levels were enhanced with clorgyline pretreatment and 5-HT was revealed with fluorescein. Confocal images represent the superimposition of two to seven images (two images with z step of 10 μm in [A] and [C], and six images with z step of 1 μm in [B] and [D]) acquired with 16 frame averagings on a high resolution monitor (1024 × 1024 pixels). (A) and (B) In the cortex, both intensely fluorescent varicose fibers (arrows) and fibers with a low level of fluorescence (arrowheads) are visible, the latter being most abundant in layer IV of the sensory cortices. (A) On a low power view of the primary somatosensory cortex, 5-HT labeling outlines the center of a barrel (arrowheads). (B) Higher power view in layer IV displays intensely fluorescent fibers with small varicosities (filled arrows) or large varicosities (open arrow) and weakly stained 5-HT immunoreactive fibers (arrowheads) that surround unlabeled cell bodies. (C) and (D) In the thalamus, 5-HT is present in cell bodies and fibers. (C) Low power view of the thalamus shows the high density of 5-HT-labeled perikarya in the visual (dlgn) and the somatosensory (vb) thalamic nuclei, and dense neuropil labeling in the reticular thalamic nucleus (rt). (D) Higher magnification of the 5-HT-labeled neurons in the vb, with few intensely fluorescent varicose fibers (arrow) coursing in between. Bar, 78 μm (A); 250 μm (C); 12 μm and 5 μm (B and D). Neuron 1996 17, 823-835DOI: (10.1016/S0896-6273(00)80215-9)

Figure 2 Transient 5-HT Immunolabeling in Thalamic Neurons and Thalamocortical Fiber Tracts Does Not Correspond to TPOH Immunostaining (A)–(D) Transient 5-HT labeling of thalamic neurons and fiber tracts in mouse pups. (E) and (F) Comparison with TPOH immunostaining. In untreated mouse pups also, 5-HT immunoreactivity labels the dorsal lateral geniculate nucleus (dlgn) (C), the ventrobasal nucleus (vb) (C and D), as well as fiber tracts in the internal capsule that arise from the lateral side of the thalamus (A). (A) and (C) On these sections, transient 5-HT labeling of the cortex is visible, outlining layer IV of the visual (V1), auditory (A1), and somatosensory (S1) cortices. (B) and (D) Higher magnifications. (B) 5-HT-labeled axons in the internal capsule are rectilinear and grouped in bundles (full arrows) contrasting with the usual varicose 5-HT fibers (open arrows). (D) The vb displays both 5-HT-labeled perikarya and staining of the neuropil. (E) Labeling with TPOH shows no immunostaining in the thalamic neurons, whereas ascending TPOH-postitive fiber tracts are visible in the medial forebrain bundle (mfb). (F) Raphe serotonergic cell bodies are clearly labeled by TPOH antibodies. Bar, 590 μm (A, C, and E); 30 μm (B); 212 μm (D); 533 μm (F). Neuron 1996 17, 823-835DOI: (10.1016/S0896-6273(00)80215-9)

Figure 3 5-HT-Immunoreactive Fibers Partially Colocalize with Thalamocortical Axons After injections of dextran–biotin in the VB, anterogradely labeled thalamocortical fibers were revealed with Cy3-coupled streptavidin (red in [B] and [E]) and 5-HT was revealed on the same sections with fluorescein-coupled antibodies (green in [A] and [D]). Pictures show the addition of five to seven confocal images with a z step of 1 μm acquired simultaneously with a two-channel excitation laser beam. (C) and (F) The superimposition of the green and red images shows the double-labeled structures as yellow-orange. (A)–(C) Low power view in the striatum (st), in which the weakly labeled 5-HT fiber tracts (A) are superimposable to the thalamocortical fiber tracts (B) (arrows). (D)–(F) High power view within layer IV of the somatosensory cortex in which some of the anterogradely labeled thalamocortical fibers (E) clearly display 5-HT immunoreactivity (D) (arrows). Bar = 124 μm (A, B, and C); 20 μm (D, E, and F). cx: cortex; v: lateral ventricle. Neuron 1996 17, 823-835DOI: (10.1016/S0896-6273(00)80215-9)

Figure 4 Depletion of the Dense Transient Cortical 5-HT Innervation after a Thalamic Lesion (A) and (B) 5-HT immunoreactivity in the barrelfield of a P7 pup ipsilateral (A) and contralateral (B) to a thalamic lesion effected at P3. (A) The dense 5-HT innervation of the barrels in layer IV as well the low-grade diffuse labeling in cortical layer VI and in the subcortical layer have disappeared on the lesioned side, whereas the well delimited 5-HT-positive varicose fibers are still visible. (C) Cytochrome oxydase–stained section shows the thalamic electrolytic lesion (asterisk) involving the dLGN, part of the VB as well as the thalamocortical efferent tracts. Bar, 125 μm (A and B); 0.8 mm (C). Neuron 1996 17, 823-835DOI: (10.1016/S0896-6273(00)80215-9)

Figure 5 Transient 5-HT Labeling in Thalamus and Cortex Depends on 5-HT Uptake At 6 hr after a single administration of paroxetine (A) or fluoxetine (B and D), two selective blockers of 5-HT uptake, the pattern of 5-HT labeling in P7 mouse pups is profoundly modified. Dense 5-HT immunostaining is abolished in the thalamocortical tracts (compare 5A with Figure 2A) and in layer IV of the sensory areas (compare 5B with Figure 4B). In the same way, 5-HT labeling in the sensory thalamic nuclei, shown here in the dlgn (C), is eliminated after 5-HT uptake inhibition (D). (B) and (D) In all these areas, the “adult type” varicose convoluted 5-HT fibers were still present in normal densities. Bar, 640 μm (A); 125 μm (B, C, and D). Neuron 1996 17, 823-835DOI: (10.1016/S0896-6273(00)80215-9)

Figure 6 High Affinity 5-HT Uptake in Brain Slices: Different Density and Distribution in Adults and Pups High affinity uptake of 3H–5-HT, revealed on autoradiographs from P7 (A, B, C, and D) and adult (E and F) mice shows striking differences. (A)–(C) In P7 thalamus, 5-HT uptake occurs in efferent fiber tracts (A) and in terminal fibers that densely accumulate in the reticular thalamic nucleus (rt), with uptake to a lesser extent in the vb and dlgn (C). At a higher magnification (arrow in [A] designates the field shown in [B]), uptake in fiber tracts of the internal capsule form linear strands. Such uptake is visible in tissue fragments that contain only the internal capsule. (D) In the primary somatosensory cortex of mouse pups, numerous terminal varicosities are visible in layer IV and layer VI, outlining the barrels. (E) In adult thalamus, 5-HT uptake occurs only in varicose fibers with a particularly low abundance of fibers in the rt. (F) This laminar distribution of 5-HT uptake is different in adults with spaced varicose fibers mainly in layer I, upper V, and VI. Bar, 159 μm (A); 50 μm (B); 212 μm (C, D, E, and F). Neuron 1996 17, 823-835DOI: (10.1016/S0896-6273(00)80215-9)

Figure 7 Retrograde Transport of 5-HT from Cortex to Thalamus (A) At the site of the 3H–5-HT injection, shown here in a P8 rat pup, local cortical fibers take up the amine. (B) Retrograde transport into the thalamocortical axonal tract is already observed 1 hr after the injection of 5-HT. (C and D) Accumulation of radioactivity is seen in fibers of the reticular thalamic nucleus (rt) and in neurons of the ventrobasal nucleus (vb) 3 hr after the cortical injection. (D) Note that in vb, not all neurons have accumulated 3H–5-HT: some neurons display silver grain accumulation (full arrows), whereas others do not (open arrows). Bar, 635 μm (A); 125 μm (B); 78 μm (C); 25 μm (D). Neuron 1996 17, 823-835DOI: (10.1016/S0896-6273(00)80215-9)

Figure 8 In Situ Hybridization of the Serotonergic and Monoaminergic Vesicular Transporters Antisense cRNA 35S-labeled probes to SERT (A and B) and to VMAT2 (C, D, E, F, and G) were hybridized to 15 μm thick coronal brain sections from P7 (A, B, C, D, F, and G) and adult (E) mice. As shown on autoradiograms, an intense hybridization signal is observed in the median and dorsal raphe nuclei (r, arrow) with the SERT-cRNA probe (A) and in the substantia nigra (sn) with the VMAT2-cRNA probe (C), confirming the specificity of these two antisense riboprobes. High levels of gene expression are also detected in the sensory thalamic nuclei, the dlgn, vb, and mgn with both SERT- (B) and VMAT2- (C, D, F, and G) cRNA probes. (E) As shown here only for the VMAT2 probe, this expression has completely disappeared in adult brains. (F) With higher magnification of emulsion-coated sections, the high level of VMAT2 gene expression is seen to overlie neurons, with higher levels in the dlgn than in the vb. (G) Using Nissl-counterstained sections, VMAT2 mRNA expression is observed over the majority of the vb neurons (full arrows); the open arrow indicates an unlabeled neuron. The limits of the vb have been outlined with a dotted line. Bar, 1.25 mm (A, B, C, and D), 1.42 mm (E), 256 μm (F), and 20 μm (G). Neuron 1996 17, 823-835DOI: (10.1016/S0896-6273(00)80215-9)