Organization, development, and plasticity of the serotonergic dorsal raphe nucleus projectome

Key results

1. ShownDRN serotonergic neurons split by target into two largely non-overlapping subsets, one reaching the telencephalon and one the spinal cord, alongside wide variation from confined to brain-wide reach.

   Of 81 neurons, 11% reach both telencephalon and spinal cord and 68% only one (46% spinal cord without telencephalon, 22% the reverse). Hierarchical clustering with principal component analysis of density in five CNS subdivisions gave three clusters; cluster 1 was spinal cord-high and telencephalon-low, cluster 2 the reverse. Over 60% of neurons reach three or more subdivisions, 20% one or two. · Organizational logic of 5-HT DRN neurons’…; Figure legends

2. SuggestedStress in the first week of life raised serotonergic fiber density in telencephalic regions, whereas chronic unpredictable stress (CUS) in the second week left projection density unchanged even though it still increased anxiety-like behavior.

   Cortisol at 2-6 days post-fertilization (dpf): area × treatment p<0.0001 (n=12, 10, 10 fish); density rose in olfactory bulbs at 5 µM and in olfactory bulbs, pallium, subpallium and dorsomedial telencephalon (Dm) at 10 µM; DRN area unchanged (p=0.422). Week-1 CUS: subpallium only (one-sided p=0.027; n=18 vs 19). Week-2 CUS: area × stress p=0.597 (n=13 vs 14), though anxiety score rose (p=0.039). · Early life stress reshapes the developing 5-HT…; A critical window of sensitivity to early-life…; Statistical analysis; Figure legends

3. ShownBetween 1 and 2 weeks of age, traced neurons kept their broad reach, but every one was locally remodeled: growth exceeded loss, projections became more bilateral, and the telencephalon, diencephalon and mesencephalon changed most.

   Same 19 neurons imaged at both ages: projection score unchanged (paired t-test, n=18, p=0.331); laterality score decreased (n=19, p=0.021); new neurites 27% vs lost 18% (p=0.001). Neurite change differed across subdivisions (mixed-effects model, p=0.0002); loss was uniform (p=0.503) while growth varied (p<0.0001). · Stability and regional remodeling of the…; Figure legends

4. ShownTarget regions differ both in how many neurons reach them and in how densely they are innervated; a zero-inflated model sorted 11 CNS regions into three innervation classes.

   Cerebellum and hypothalamus received fibers from over 70% of the 81 neurons, olfactory bulbs and optic tectum from under 10%. A zero-inflated negative binomial model, chosen by Bayesian information criterion, grouped telencephalic areas and preoptic area (dense, few neurons); pons-medulla, cerebellum, spinal cord and hypothalamus (dense, many); thalamus and optic tectum (sparse, few). · 5-HT DRN neurons provide highly heterogeneous…; Statistical analysis; Figure legends

5. ShownSoma position within the DRN partly predicts targets: anterior neurons send more neurites ventrally and to the hypothalamus, dorsal neurons send more to the cerebellum, and neurons co-expressing Gad1b sit mostly in the anterior DRN.

   n=81: ventrality index vs anterior-posterior soma position rho=-0.558, p<0.0001; no trend with dorso-ventral position (rho=0.041, p=0.712). Hypothalamic density vs anterior-posterior position rho=-0.427, p=0.002; cerebellar density vs dorso-ventral position rho=0.390, p=0.005. Tph2+Gad1b+ neurons were 36% (30 vs 51 Gad1b-negative), skewed anterior (rho=-0.302, p=0.006). · Organizational logic of 5-HT DRN neurons’…; Figure legends