Fig 2: 5-HT2A receptors modulate synaptic plasticity at the parallel fibre–Purkinje cell synapse in 8- to 10-week-old mice. a Schematic diagram of the electrophysiological recording configuration of b–d. b Examples of recordings from Purkinje cells while electrically stimulating the parallel fibres with a double-pulse protocol, from a control slice and from a slice which is preincubated with 300 nM of the 5-HT2A selective antagonist MDL 100,907. Black traces are from recordings before inducing plasticity; red traces are from recordings 10–15 min after induction of plasticity using a 8 Hz, 30 s stimulation protocol. c Example of recordings from Purkinje cells from a control slice and from a slice which is preincubated with 300 nM of the 5-HT2A selective antagonist MDL 100,907 in which the area of the EPSC, normalized to the average before the tetanus protocol, is recorded over time. The arrow indicates the start of the tetanus to induce plasticity with a 8 Hz, 30 s stimulation protocol. d The area of EPSCs recorded 10–15 min after the plasticity stimulation protocol, normalized to the area of EPSCs before the plasticity stimulation protocol, indicates enhanced plasticity after blocking 5-HT2A receptors with MDL 100,907. The numbers in the bars of the graphs indicate the number of cells used for analysis

Fig 3: Presynaptic 5-HT2A receptors modulate stability of the parallel fibre–Purkinje cell synapse. a Schematic diagram of the electrophysiological recording configuration of b–d. b Examples of currents recorded from Purkinje cells at P11–P14 after electrically stimulating parallel fibres with a double-pulse protocol, and examples of ten consecutive currents recorded from Purkinje cells at P11–P14 after electrically stimulating parallel fibres, both in control slices and in slices preincubated with 300 nM MDL 100,907. c Pharmacological blockade of 5-HT2A receptors with 300 nM MDL 100,907 results in a reduced paired-pulse ratio at both P11–P14 and at P57–P68. d At P11–P14, the effect of pharmacological blockade of 5-HT2A receptors with 300 nM MDL 100,907 on the coefficient of variance of EPSCs at the parallel fibre–Purkinje cell synapse does not reach significance. At P57–P68, blocking 5-HT2A receptors results in a lower coefficient of variance of EPSCs at the parallel fibre–Purkinje cell synapse. The numbers in the bars of the graphs indicate the number of cells used for analysis

Fig 4: Cerebellar granule cells express functional 5-HT1A and 5-HT2A receptors during postnatal development. a Schematic diagram of the electrophysiological recording configuration. b Amplitudes of inward currents recorded from granule cells, evoked by local application of 100 nM 8-OH-DPAT or 500 nM DOI at different ages during postnatal development. c Examples of a whole-cell patch-clamp recording from a granule cell in voltage-clamp (top) and current-clamp (bottom) at P8 showing an inward current and a depolarization of the membrane potential upon local application of 100 nM of the 5-HT1A agonist 8-OH-DPAT. Also shown in red is an example trace of a recording in voltage-clamp after preincubation of the slice in 10 µM of the 5-HT1A specific antagonist WAY100,635, which blocks the effect of local application of 100 nM 8-OH-DPAT. d Examples of a whole-cell patch-clamp recording from a granule cell in voltage-clamp (top) and current-clamp (bottom) at P11, showing an inward current and depolarization of the membrane potential upon local application of 500 nM of the 5-HT2 specific agonist DOI. Also shown in red is an example trace of a recoding in voltage-clamp after preincubation of the slice in 300 nM MDL 100,907, indicating no inward current upon local application of 500 nM DOI. Arrows in b and c indicate the start of drug application which lasts for 500 ms. e Immunostaining with DAPI and antibodies against 5-HT2A receptors (obtained from Immunostar) at P5 and at P90, indicating presence of 5-HT2A receptors both on Purkinje cells and in the granule cell layer, with higher expression levels in adult mice. EGL external granule cell layer, PCL Purkinje cell layer, IGL internal granule cell layer. Scale bar indicates 155 µm

Fig 5: Presynaptic release-regulating mGlu2/3 autoreceptors and 5-HT2A heteroreceptors functionally crosstalk in synaptosomes from the prefrontal cortex of adult rats. (a) Effects of (±) DOI (1 µM) and of LY379268 (0.1–1 µM) alone or concomitantly added on the 15 mM KCl-evoked [3H]D-asp exocytosis. The release of [3H]D-asp in the first 3 min fraction collected (b1) amounted to 0.39 ± 0.02 nCi and represents the 0.88 ± 0.12% of the total tritium synaptosomal content. (b) Effects of 1 µM MDL11,939 on the release of tritium elicited by the 15 mM KCl-containing medium in the absence or in the presence of LY379268 (0.1–1 µM). The release of [3H]D-asp in the first 3 min fraction collected (b1) amounted to 0.46 ± 0.03 nCi, the 1.03 ± 0.08% of the total tritium synaptosomal content. The results are expressed as induced overflow; data are the means ± SEM of five to six experiments run in triplicate. ** p < 0.01 vs. 15 mM KCl; *** p < 0.001 vs. 15 mM KCl; ^^^ p < 0.01 vs. 15 mM KCl/1 µM LY379268; ### p < 0.001 vs. 15 mM KCl/1 µM (±) DOI; °° p < 0.01 vs. 15 mM KCl/1 µM MDL11,939; § p < 0.05 vs. 15 mM KCl/0.1 µM LY379268.