A genetically defined morphologically and functionally unique subset of 5-HT neurons in the mouse raphe nuclei

Abstract

Heterogeneity of central serotonin (5-HT) raphe neurons is suggested by numerous lines of evidence, but its genetic basis remains elusive. The transcription factor Pet1 is required for the acquisition of serotonergic identity in a majority of neurons in the raphe nuclei. Nevertheless, a subset of 5-HT neurons differentiates in Pet1 knock-out mice. We show here that these residual 5-HT neurons outline a unique subpopulation of raphe neurons with highly selective anatomical targets and characteristic synaptic differentiations. In Pet1 knock-out mice, 5-HT innervation strikingly outlines the brain areas involved in stress responses with dense innervation to the basolateral amygdala, the paraventricular nucleus of the hypothalamus, and the intralaminar thalamic nuclei. In these regions, 5-HT terminals establish asymmetric synaptic junctions. This target selectivity could not be related to altered growth of the remaining 5-HT neurons, as indicated by axon tracing and cell culture analyses. The residual 5-HT axon terminals are functional with maintained release properties in vitro and in vivo. The functional consequence of this uneven distribution of 5-HT innervation on behavior was characterized. Pet1 knock-out mice showed decreased anxiety behavior in novelty exploration and increased fear responses to conditioned aversive cues. Overall, our findings lead us to propose the existence of Pet1-dependent and Pet1-resistant 5-HT neurons targeting different brain centers that might delineate the anatomical basis for a dual serotonergic control on stress responses.

Figure 1.

Distribution and morphology of Tph2-positive neurons in the raphe of Pet1 knock-out mice (Pet1−/−). A, B, Tph2 mRNA expression on serial coronal sections through the raphe (B1 to B9 cell groups). A subpopulation of 5-HT-synthesizing neurons remains throughout the 5-HT cell groups in Pet1−/−. C, D, Higher-magnification micrographs of the dorsal raphe (B7) show reduced level of Tph2 mRNA per cell in Pet1−/− (D), compared to controls (C). E, F, Tph immunohistochemistry, in the B2 cell group, shows normal dendrite morphology and no difference in the staining intensity. G, H, 5-HT immunocytochemistry shows a similar intensity of staining in dorsal raphe neurons of control and Pet1−/−. Illustrations are from experiments where control and Pet1−/− littermates were processed in parallel. Scale bars: C, D, 200 μm; E, F, 100 μm; G, H, 50 μm.

Figure 2.

Sparing of 5-HT innervation in selected brain regions in the Pet1 knock-out mice. 5-HT immunohistochemistry is shown on brain sections from different rostrocaudal levels in control (A, C, E) and Pet1−/− (B, D, F) littermates. The images were assembled from multiple high-magnification micrographs. In control mice, 5-HT innervation is widely and almost uniformly distributed, whereas in Pet1−/−, 5-HT axons are strikingly preserved in the indusium griseum (B), the basolateral nucleus of the amygdala (D, F), the paraventricular hypothalamic nucleus (D), and the medial thalamic nuclei (F). In contrast, 5-HT innervation is almost completely depleted in most cortical areas (B–F), in the caudate (B), the hippocampus (D, F), and the anterior hypothalamus (D, F). CeA, Central amygdala; Cga, anterior cingulate cortex; CP, caudate nucleus; dLGN, dorsal lateral geniculate nucleus; Hip, hippocampus; IG, indusium griseum; IMD, intermediodorsal nucleus of the thalamus; OT, olfactory tubercle; S1, primary somatosensory cortex; VB, ventrobasal nucleus of thalamus; ZI, zona incerta.

Figure 3.

Quantification of the regional density of 5-HT terminals. A, Axon densities were estimated as pixels/μm² in each of the 10 structures analyzed (n = 4/group). Fiber density in Pet1−/− are expressed as percentage of density in controls (ctrl). In the histogram, brain areas are ranked according to the residual density of 5-HT fibers in Pet1−/−. Data are presented as mean ± SEM, Student's t test, **p < 0.01 and ***p < 0.005. ScN, Suprachiasmatic nucleus of hypothalamus; SNR, substantia nigra pars reticulata; dLGN, dorsal lateral geniculate nucleus; Hip, hippocampus; Cga, anterior cingulate cortex; SNC, substantia nigra pars compacta; Hb, habenula; IG, indusium griseum. B, Examples of the confocal images (0.40 μm) in the hippocampus and the BLA that were used for the quantitative analyses of the densities of 5-HT innervation in controls and Pet1−/−. C, Distribution of 5-HT-immunolabeled terminals in the hypothalamus of control and Pet1−/−. Note the high density of fibers through the whole structure in controls, whereas in the Pet1−/− only the paraventricular nucleus receives a dense 5-HT innervation.

Figure 4.

Growth characteristics of 5-HT neurons in vitro and in vivo. A, B, Raphe explants (E12 + 4DIV) from control and Pet1−/− were immunolabeled with anti-5-HT antiserum. The number of 5-HT-labeled axons growing from the explants is significantly reduced in Pet1−/−, but the length of neurites is similar in both genotypes. Scale bar, 200 μm. C, D, Growth cones of raphe neurons (E12.5 + 4DIV) have similar morphologies in control and Pet1−/− after 5-HT immunolabeling with (red) and GAP 43 (green) antibodies. E, F, The morphology and general trajectory of ascending 5-HT axons in the medial forebrain bundle is unchanged in Pet1−/− (F) E15 embryos, compared to controls (E). Scale bar, 400 μm.

Figure 5.

Topography of raphe projections to the amygdala and the hippocampus in Pet1 knock-out mice. A, B, Confocal images show back-labeled raphe neurons after fluorogold injections into the amygdala in a control mouse. Arrowheads show double-labeled dorsal raphe neurons. 5-HT neurons with no retrograde labeling are indicated by arrows. C, Micrograph showing a typical fluorogold injection site into the amygdala; the principal subnuclei are delimited by dashed lines: BLAp, basolateral amygdala, posterior part; BMA, basomedial amygdala; CoA, cortical amygdalar area; LA, lateral amygdala. D, Dorsal raphe neurons projecting to the amygdala are shown at the level where the back-labeled neurons were most abundant. Green dots indicate neurons containing only the retrograde tracer and red dots indicate the retrogradely labeled neurons that were colocalized with 5-HT. E, Typical fluorogold injection into the hippocampus is shown. F, Retrogradely labeled neurons from the hippocampus are essentially concentrated in the median raphe in both control and Pet1−/−. Half of these neurons are 5-HT-positive in controls and none in Pet1−/−.

Figure 6.

Electron micrographs of 5-HTT-labeled axon terminals in control and Pet1 knock-out mice. A–F, Micrographs of 5-HTT labeling in the BLA (A–D) and in the PVH (E, F). Control and Pet1−/− 5-HT terminal boutons likewise contained numerous small vesicles and a few large dense vesicles (red arrows). Asymmetric synaptic specializations are marked by arrowheads. In the BLA of control mice, 45% of the boutons showed synaptic contact (A), while the rest did not (B). In contrast, a majority of the reconstructed varicosities from Pet1 knock-out mice show asymmetric synapses, as illustrated in the BLA (C, D) and in PVH (E, F). High-magnification of postsynaptic densities are shown in C and D as insets (arrowheads). Scale bars: A–F, 500 nm; C, D, insets, 50 nm.

Figure 7.

Functional characterization of 5-HT release in vivo and ex vivo. A, Specific binding of [³H]citalopram (selective ligand for 5-HTT) to whole-brain synaptosome membranes from Pet1−/− and control mice. Maximal number of binding sites is reduced in Pet1−/− preparations, consistent with reduced levels of 5-HTT (B_max Pet1−/− = 24.6 ± 6.0 and control = 293.7 ± 7.4 fmol/mg). B, Saturation isotherms of 5-HT uptake by synaptosomes prepared from Pet1−/− and control whole brains. Maximal uptake capacity is reduced in the Pet1−/− preparation (V_max Pet1−/− = 29.9 ± 2.3 and control = 218.7 ± 3.4 fmol/min/mg). C, Release of 5-HT from preloaded synaptosomes by MDMA. Though the loading capacity of both preparations differed in Pet1 knock-out and control preparations, MDMA released 5-HT from both synaptosome preparations in a similar manner (log EC₅₀ Pet1 knock-out = −6.26 ± 0.09 and control = −6.10 ± 0.11). D, A single injection of d-fenfluramine (10 mg/kg, i.p.) activates neurons in the PVH compared to saline. Representative images of c-Fos immunohistochemistry at 10× magnification. Squares depict the area used for quantification in E. E, The number of c-Fos-positive cells after d-fenfluramine injection was similar in Pet1−/− and control mice (*p < 0.05 vs corresponding saline treatment). F, d-Fenfluramine induces a significant increment in plasma corticosterone in Pet1 knock-out mice and control littermates (F_(1,20) = 13.27, p = 0.0045 and 0.0096, respectively, Bonferroni post hoc test after two-way ANOVA, n = 8/group). In addition, a significant genotypic difference was found in response to saline injection (F_(1,20) = 9.028, p = 0.0070, Bonferroni post hoc test after two-way ANOVA). **p < 0.01 versus corresponding saline treatment. ^##p < 0.01 saline treatment between genotypes. In all figures, data are expressed as mean ± SEM.

Figure 8.

Exploratory behavior of Pet1 knock-out mice and controls in novel environments. A, Pet1−/− mice explore the open arms of the maze more than their control littermates as indicated by a higher percentage time spent in open arms and percentage of open arm entries (***p < 0.001, Student's t test, n = 12/group). B, The percentage of time spent exploring the center of the open field was not significantly different between groups (Student's t test, n = 12/group). C, Latency to feed in a novel brightly illuminated arena was significantly lower for Pet1−/− compared to controls (**p < 0.01, Student's t test, n = 11/group). D, No differences were found in ambulatory activity between groups. Numbers of light beam breaks are reported in 10 min blocks for a total of 2 h, no significant differences were found between genotypes (two-way ANOVA, F_(1,25) = 0.8385, n = 13/group). In all figures, data are expressed as mean ± SEM.

Figure 9.

Behavioral responses to learned fear in Pet1 knock-out and control mice. A, Conditioned fear responses to context, measured as percentage of time freezing, were exaggerated in Pet1−/− compared to controls (*p < 0.05, Bonferroni post hoc test, after two-way ANOVA, n = 12/group). B, Delay fear conditioning in Pet1−/− revealed enhanced fear responses to tone after conditioning with three tone/footshock pairings (*p < 0.05, Bonferroni post hoc test, after two-way ANOVA, n = 9/group). The break in the x-axis marks the beginning of the tone, and fear responses to the new context were similar for both groups. C, For the trace fear conditioning protocol a 3 s interval was added between the tone and the shock. Fear responses were measured in a new context 24 h after the training and were enhanced in Pet1−/− (*p < 0.05, Bonferroni post hoc test, after two-way ANOVA, n = 8/group). The break in the x-axis marks the beginning of the tone, and fear responses to the new context were also enhanced in Pet1−/−. In all figures, data are expressed as mean ± SEM.