Barrel pattern formation requires serotonin uptake by thalamocortical afferents, and not vesicular monoamine release

Abstract

Thalamocortical neurons innervating the barrel cortex in neonatal rodents transiently store serotonin (5-HT) in synaptic vesicles by expressing the plasma membrane serotonin transporter (5-HTT) and the vesicular monoamine transporter (VMAT2). 5-HTT knock-out (ko) mice reveal a nearly complete absence of 5-HT in the cerebral cortex by immunohistochemistry, and of barrels, both at P7 and adulthood. Quantitative electron microscopy reveals that 5-HTT ko affects neither the density of synapses nor the length of synaptic contacts in layer IV. VMAT2 ko mice, completely lacking activity-dependent vesicular release of monoamines including 5-HT, also show a complete lack of 5-HT in the cortex but display largely normal barrel fields, despite sometimes markedly reduced postnatal growth. Transient 5-HTT expression is thus required for barrel pattern formation, whereas activity-dependent vesicular 5-HT release is not.

Fig. 1.

Somatosensory cortical barrels in coronal sections of brains of 5-HTT wt (A, D,G, J), hz (B,E, H, K), and ko (C, F, I,L) mice. A–C, 5-HT immunocytochemistry of P7 mouse brains. Notice the reduction and the nearly complete absence of 5-HT immunostaining in 5-HTT hz and ko mice, respectively. D–F, Higher magnifications of sections shown in A–C; only sparse 5-HT-stained fibers are visible in the cortex of 5-HTT ko mice (F). G–I, CO staining in coronal sections of adult mouse brains, 4–5 months old, at the level of the primary somatosensory cortex.J–L, Higher magnifications of sections shown in G–I. Barrels and septa appear normal in wt and hz animals (J,K), whereas the barrel pattern is nearly absent in 5-HTT ko mice (L). Scale bar (shown inL): A–C, 560 μm;D–F, 240 μm;G–I, 800 μm;J–L, 250 μm.

Fig. 2.

Barrel pattern in tangential sections through the primary somatosensory cortex of 5-HTT wt (A,D), hz (B, E), and ko (C, F) mice.A–C, 5-HT immunocytochemistry of P7 brains; D–F, CO staining of adult brains. In 5-HTT ko mice (F), the barrel pattern is lost, except for the more caudal PMBSF barrels (arrowheads). The anterior-posterior (a) and mediolateral (l) axes are indicated in A. Scale bar (shown inF): A, B,D–F, 740 μm; C, 1380 μm.

Fig. 3.

Nissl staining of coronal sections through the somatosensory cortex in adult 5-HTT wt (A), hz (B), and ko (C) mice.Arrows in A and B point toward two adjacent barrel septa. Notice that the septa appear to be enlarged in B and are absent inC. Scale bar (shown in C):A–C, 400 μm.

Fig. 4.

Thalamic barreloids and trigeminal barrelettes in adult 5-HTT wt (A, D), hz (B, E), and ko (C,F) mice, revealed by CO staining.A–C, Thalamic ventrobasal complex.Arrowheads in B and Cpoint to the ventromedial portion of the barrelloid pattern, which appears less distinct in 5-HTT hz (B), compared with wt animals (A), and virtually absent in ko animals (C). D–F, Spinal trigeminal nucleus. Trigeminal barrelettes appear normal in wt and hz animals (D, E), and less organized in ko animals (F). Scale bar (shown inF): A–C, 100 μm;D–F, 160 μm.

Fig. 5.

Recovery of somatosensory cortical barrel patterns in 5-HTT ko mice by systemic PCPA (300 mg/kg) injected once daily for 2 consecutive days in early postnatal life. If the injections are performed after P2, only the PMBSF barrels are rescued. The barrel pattern was revealed with CO staining. A, Significant decrease in barrel cortex area when the first PCPA injection is performed later than P1 [one-way ANOVA: F = 96.1 (df = 4, 21), p < 0.001]. Symbols represent significant differences (p < 0.05) fromP0+P1 and P1+P2 (∗), fromP2+P3 (○), and from P3+P4 (+). Sample sizes are shown in parentheses above eachcolumn. B, Schematic representation of somatosensory cortical barrel fields visible in F2 5-HTT ko mice treated with PCPA on 2 consecutive postnatal days. C, Representative examples of different degrees of rescue by PCPA, depending on time of PCPA injection. The time points of PCPA injections are indicated on the left of the photomicrographs.

Fig. 6.

GAT-1 immunostaining of the somatosensory barrel cortex of 5-HTT wt (A, B) and ko (C, D) mice. A,C, Low-power magnification of 5-HTT wt (A) and 5-HTT ko (C) mice, showing all cortical layers. Layer IV is indicated (IV). B, D, High-power magnification of layer IV of wt (B) and ko (D) mice. Scale bar (shown inD): A, C, 280 μm;B, D, 110 μm.

Fig. 7.

Electron micrographs showing 5-HT (A) and GAT-1 (B) immunostaining in layer IV of the barrel cortex. A, 5-HT-immunostained axon terminal (Ax), forming an asymmetrical contact (arrow) with a dendritic profile, in a 5-HTT wt mouse. B, GAT-1-immunostained axon terminal (Ax), forming a symmetrical synaptic contact (arrow) with a dendritic profile, from cortical layer IV of a 5-HTT ko mouse. Scale bar (shown in B):A, B, 0.2 μm.

Fig. 8.

Optic-electron microscopy correlation method used in this study. A, Semithin section through the somatosensory cortex of a P8 mouse, counterstained with toluidine blue; layer IV is always clearly visible because of its higher cell density.bv, Blood vessel. B, C, Same as in A, at a higher magnification.D, Ultrathin section of the same area as inC, obtained after resectioning the area of interest.E, Example of a neuropil-rich area within layer IV, photographed to carry out the quantitative study. Thearrow points to a synaptic contact. Scale bar (shown inE): A; 200 μm; B, 100 μm; C, 30 μm; D, 7 μm;E, 520 nm.

Fig. 9.

5-HT immunocytochemistry in brains of VMAT2 wt (A, D), hz (B,E), and ko (C, F) mice assessed at P7. A–C, 5-HT immunostaining of coronal sections reveals the barrel pattern in wt and hz mice; no 5-HT immunostaining is visible in VMAT2 ko mice (C). D–F, Higher magnification of sections shown in A–C. Scale bar (shown in F): A,B, 400 μm; C, 430 μm;D–F, 130 μm.

Fig. 10.

Cortical development and barrel pattern in VMAT2 wt (A, D), hz (B,E), and ko (C, F) mice assessed by Nissl staining and CO histochemistry at P7.A–C, Nissl staining of coronal sections show normal development of all cortical layers.Arrowheads mark the boundaries of layer IV. Barrel septa are less well defined in neonates, compared with adults (Fig. 3).D–F, CO staining of tangential sections through layer IV of the PMBSF, displaying normal barrel patterns. The anterior-posterior (a) and medial-lateral (l) axes are indicated in D. Scale bar (shown in F):A–C, 100 μm; D,E, 180 μm; F, 140 μm.