High serotonin levels during brain development alter the structural input-output connectivity of neural networks in the rat somatosensory layer IV

Abstract

Homeostatic regulation of serotonin (5-HT) concentration is critical for "normal" topographical organization and development of thalamocortical (TC) afferent circuits. Down-regulation of the serotonin transporter (SERT) and the consequent impaired reuptake of 5-HT at the synapse, results in a reduced terminal branching of developing TC afferents within the primary somatosensory cortex (S1). Despite the presence of multiple genetic models, the effect of high extracellular 5-HT levels on the structure and function of developing intracortical neural networks is far from being understood. Here, using juvenile SERT knockout (SERT(-/-)) rats we investigated, in vitro, the effect of increased 5-HT levels on the structural organization of (i) the TC projections of the ventroposteromedial thalamic nucleus toward S1, (ii) the general barrel-field pattern, and (iii) the electrophysiological and morphological properties of the excitatory cell population in layer IV of S1 [spiny stellate (SpSt) and pyramidal cells]. Our results confirmed previous findings that high levels of 5-HT during development lead to a reduction of the topographical precision of TCA projections toward the barrel cortex. Also, the barrel pattern was altered but not abolished in SERT(-/-) rats. In layer IV, both excitatory SpSt and pyramidal cells showed a significantly reduced intracolumnar organization of their axonal projections. In addition, the layer IV SpSt cells gave rise to a prominent projection toward the infragranular layer Vb. Our findings point to a structural and functional reorganization of TCAs, as well as early stage intracortical microcircuitry, following the disruption of 5-HT reuptake during critical developmental periods. The increased projection pattern of the layer IV neurons suggests that the intracortical network changes are not limited to the main entry layer IV but may also affect the subsequent stages of the canonical circuits of the barrel cortex.

Keywords: SERT; barrel cortex; columnar circuitry; morphology; pyramidal cell; serotonin; somatosensory; spiny stellate cell.

Figure 1

Thalamocortical afferent projection pattern in the layer IV. Overlay of 5 reconstructions for the SERT^+/+ (A1) and SERT^−/− (A2) rats, respectively, in reference to the barrel borders stained by cytochrome oxidase. Percentages are given for the total axon length (B1), number of nodes (B2), and number of boutons (B3) innervating the HB, S, and NB (SERT^+/+ n = 11; SERT^−/− n = 8). Gray shaded areas indicate the position of the respective home and adjacent barrels. Data are mean ± SEM, asterisks mark significant differences between genotypes. ^*P < 0.05; ^**P < 0.01; ^***P < 0.001.

Figure 2

Representative cytochrome oxidase stained tangential sections of layer IV SERT^+/+ (A1) and SERT^−/− (A2) P21 rats (SERT^+/+n = 6; SERT^−/−n = 6). The sections were photographed and barrel areas (C and E) and septal distances (D and E) were quantitatively analyzed (B). Barrel areas of SERT^−/− rats were determined corresponding to rows A-B: 1–4; C-D-E: 1–5, septa were divided into rows A–E and Arcs 1–5. The edges of the barrel borders were evaluated in the gray level pixel values (Δ GL) over distance from the barrel to the septa (D) and showed a reduced steepness in SERT^−/− compared to SERT^+/+ (F). Scale bars = 1 mm. ^*P < 0.05; ^**P < 0.01; ^***P < 0.001.

Figure 3

Action potential firing pattern and discriminant analysis of electrophysiological and morphological properties of excitatory layer IV cells. (A) Representative whole cell current clamp recordings showing regular spiking (RS) and intrinsically bursting (IB) firing patterns in SERT^−/− excitatory layer IV cells. Both firing patterns were observed in both genotypes (SERT^+/+ and SERT^−/−) as well as both morphological classes (spiny stellate cells and pyramidal cells). (B) Canonical score plots based on discriminant analysis of the genotype specific electrophysiological (upper panel) and morphological classes (lower panel) as a-priory groups. Plots were based on two functions which combined the best characteristics defining either the firing patterns (B1; function 1: high and low current 1st ISI; function 2: firing threshold, 2nd AP amplitude) and morphological classes (B2; function 1: Vrmp, high current 2nd ISI; function 2: high current 1st ISI, 2nd AP amplitude). Both analysis properties show no segregation of genotype specific populations.

Figure 4

Somatodendritic organization of excitatory spiny stellate (A,C) and pyramidal (B,D) cells in SERT^+/+ and SERT^−/− layer IV barrel cortex. Micrographs of a biocytin stained spiny stellate (A) and pyramidal (B) cells in acute slice and representative morphological reconstructions; spiny stellate (C) and pyramidal (D) of both genotypes. Gray shaded areas indicate the position of the respective home barrel.

Figure 5

Intracortical axonal projection pattern of excitatory cells in SERT^+/+ and SERT^−/− layer IV of the barrel cortex. Representative morphological reconstruction(s) of the somatodendritic structure (blue) and axonal projections (red) of spiny stellate cells (A1,B1) and pyramidal cells (A2,B2) in SERT^+/+ and SERT^−/− cortex. Overlay of 5 reconstructed neurons aligned to their position within their home barrels which illustrates the main axonal projection patterns on the population level. Gray shaded areas indicate the position of the respective home barrel (center) and adjacent barrels.

Figure 6

Quantitative analysis of the axonal projection pattern of excitatory spiny stellate (A) and pyramidal cells (B) in SERT^+/+ and SERT^−/− layer IV barrel cortex. Schematic drawings (left panels) illustrate the significant changes in relative bouton distribution within the individual layers (I–VI), highlighting layers specific shifts from home column (HC) to septal or neighboring column (NC) and vice versa. Histograms (right panels) represent the most relevant layer specific axonal properties (length, nodes, and boutons number) per genotype. Data are mean ± SEM, asterisks mark significant differences between genotypes. ^*P < 0.05; ^**P < 0.01.