Fig 2: Lmx1b and Pet1 act in adult 5-HT neurons to ensure survival of pericellular baskets(A) Coronal schematic highlighting location (red box) of pericellular baskets in the dorsolateral septum (dLS).(B–D) Left, representative confocal images (63×) of TdTomato+ (anti-RFP) light pericellular baskets (B), dense baskets (C), and dense basket remnants (D) at 1 year post-TAM. Right, quantification of basket or remnant numbers relative iCON numbers; ±SEM; n = 3 mice per genotype; 2–3 sections per animal were used for counting; two-way ANOVA with Welch’s correction; unpaired t test with Welch’s correction for iCON versus iPKO. Scale bars, 10 µm.(E) Top, immunohistochemical analysis of synaptic triads in dLS at 6 months post-TAM, identified with RFP, presynaptic marker, synapsin 1 (Syn1), postsynaptic marker gephyrin (Gphn), and DAPI. Bottom, highlights overlap of pre- and post-synaptic makers. Right, relative numbers of triads; ±SEM; n = 3 mice with three sections analyzed per animal; one-way ANOVA. Scale bar, 5 µm. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not significant. See also Figure S2.

Fig 3: Decreased phosphorylation of pSer9Syn1 in excitatory but not in inhibitory synapses of BsnGT neurons ARepresentative images of 19 DIV cultured hippocampal neurons from WT and BsnGT immunolabelled for pSer9Syn1 and total Syn1 and for VGLUT1 or VGAT to identify excitatory and inhibitory synapses.BQuantification of IF intensity of Syn1 in excitatory and inhibitory synapses labelled for VGLUT1 and VGAT, respectively, reveals a similar synaptic abundance in BsnGT and WT neurons.CQuantification of IF intensity of pSer9Syn1 relative to the intensity of synaptic Syn1 IF reveals a decreased synaptic abundance of pSer9Syn1 in excitatory but not in inhibitory synapses of BsnGT neurons, indicating synapse type-specific effect of Bsn deletion on Syn1 phosphorylation. Data information: In the plots, the interquartile range and median are depicted as boxes, minimal and maximal values as whiskers, and + indicates mean. The sample size is given in brackets and corresponds to quantified independent visual fields obtained from three independent culture preparations (B, C). Statistical significance was assessed using the Student's t-test in (B) and (C) and is given as: ***P < 0.0001. Scale bar is 5 µm.

Fig 4: Increased phosphorylation of pSer551Syn1 in excitatory but not in inhibitory synapses of BsnGT neurons ARepresentative images of 20 DIV cultured hippocampal neurons from WT and BsnGT immunolabelled for pSer551Syn1, total Syn1 and for VGLUT1 or VGAT to identify excitatory and inhibitory synapses.BQuantification of synaptic Syn1 IF intensity in Vglut1-labelled excitatory and VGAT-labelled inhibitory synapses does not differ between BsnGT and WT neurons.CQuantification of IF intensity of pSer551Syn1 relative to synaptic Syn1 IF reveals an increased pSer551Syn1 abundance in excitatory but not in inhibitory synapses of BsnGT neurons, indicating a synapse type-specific effect of Bsn deletion on phosphorylation of synaptic Syn1 at Ser551. Data information: In the plots, the interquartile range and median are depicted as boxes, minimal and maximal values as whiskers, and + indicates mean. The sample size is given in brackets and corresponds to quantified independent visual fields obtained from three independent culture preparations (B, C). Statistical significance was assessed using the Student's t-test in (B) and (C) and is given as: ***P < 0.0001. Scale bar is 5 µm.

Fig 5: CDK5-dependent phosphorylation of Ser551 of Syn1 is increased in BsnGT in vivo and in vitro and not regulated by neuronal activity in vitro ARepresentative immunoblot with antibody against CDK5-dependent pSer551 of Syn1 (pSer551Syn1) and total Syn1 and total protein stain used as loading control for quantification of Syn1 on P2 fractions prepared from hippocampal tissue lysates of WT and BsnGT mice.B, CQuantification of pSer551Syn1 /Syn1 ratio (in B) and total Syn1 (in C) abundance on blots from (A).DScheme depicts the regulation of phosphorylation of Ser551 of Syn1 by CDK5/calcineurin balance that mediates recruitment of SVs from TRP to the ResP. Changes in this signalling detected in BsnGT neurons are depicted in red.ERepresentative images of 19 DIV cultured hippocampal neurons from WT and BsnGT immunostained for pSer551Syn1 and total Syn1.FQuantification of IF intensity of pSer551Syn1 in synapses from WT, BsnGT and WT cells treated with roscovitine.GRepresentative images of 21 DIV hippocampal WT and BsnGT neurons at baseline activity, upon network activity silencing (TTX, 1 µM, 72 h) or in conditions of increased activity (BIC, 30 µM, 48 h).HQuantification of experiment in (G). Data information: In the plots, the interquartile range and median are depicted as boxes, minimal and maximal values as whiskers and + indicates mean. The sample size n (in brackets) corresponds to the number of independently processed animals in (B) and (C) or to the number of independent images (E, G) quantified from two (F) and four (H) independent cultures per condition. For statistics, the Student's t-test in (B) and (C) one-way ANOVA with the Sidak's multiple comparison test in (F) and two-way ANOVA with the Tukey's multiple comparison test in (H) were used, *P = 0.05, **P < 0.01, ***P < 0.001. Scale bar is 5 µm in (E) and (G).