Ex vivo electrophysiological responses to 5-HT 2B -receptor stimulation or overexpression

To examine the ability of 5-HT 2B receptors to regulate 5-HT neuron activity, we first performed cell-attached electrophysiological recordings of identified DRN 5-HT neurons in coronal slices of wild-type mice expressing GFP in Pet1-positive neurons (Pet1-GFP mice). In vivo, DRN neurons fire with a regular pattern varying from 0.5 to 5 Hz. 5-HT neurons in ex vivo coronal brain slices are electrically quiescent, but an excitatory noradrenergic tone facilitates firing [33]. In order to reproduce noradrenergic input, subsaturating concentrations of phenylephrine (Phe, 100–300 nM) were added to the bath [25]. Low concentration (100 nM) of Phe was unable to initiate regular firing nor was the application of a preferential 5-HT 2B receptor agonist BW723C86 (1 µM). Higher concentration of Phe (300 nM) was able to initiate and maintain a regular firing. Subsequent bath application of BW723C86 (1 µM) induced a significant increase in firing frequency (1.73 ± 0.23-fold) compared to Phe alone (Fig. 1a) (one-way ANOVA RM, effect of treatment, F 1.6, 11.0 = 98; P < 0.0001, n = 8 cells). Bonferroni’s post hoc analysis showed a significant increase in firing frequency between each group (Fig. 1a). These data suggested that 5-HT 2B -receptor stimulation could increase Pet1-positive raphe 5-HT neuron firing activity.

Fig. 1 Ex vivo electrophysiological recordings of wild-type Pet1-GFP mice. a Recordings of 5-HT neuron in ex vivo wild-type slices. Cell-attached recordings made in identified Pet1-GFP neuron did not detect firing in the presence of 100 nM phenylephrine (Phe) or of 1 µM of the 5-HT 2B -receptor agonist BW723C86 (top line—5 cells). In the presence of higher concentration of phenylephrine (300 nM), Pet1-positive neuron firing was observed, and BW723C86 (1 µM) was able to increase this firing rate (8 cells). Representative traces (top) and quantification (bottom left) reveal a significant BW723C86-induced increase in firing frequency (one-way repeated measures (RM) ANOVA; bar graph and scatter plots and mean ± SEM; Bonferroni post test, *P < 0.05). b Current-clamp recordings of ex vivo raphe slices from AAVs injected Pet1-GFP mice, overexpressing 5-HT 2B -HA receptor in Pet1-positive 5-HT neuron. (Left) Sample traces of action potential for a +200 pA current step in two experimental groups. (Right) Quantification of the number of action potentials obtained in function of injected currents showed a significant increase in action potential number in Pet1-positive 5-HT neurons of mice overexpressing 5-HT 2B -HA receptor, compared to controls (n = 20 cells from three control mice and 21 cells from four 5-HT 2B -HA mice; two-way ANOVA RM, Bonferroni post test, *P < 0.05; data are mean ± SEM). c Input resistance values. Bar graph and scatter plot for input resistance values showed that input resistance was also increased in mice overexpressing HA-5-HT 2B receptor in Pet1-positive 5-HT neuron (unpaired ttest, *P < 0.05; data are mean ± SEM) Full size image

To further establish that 5-HT 2B receptors can affect 5-HT neuron activity, we overexpressed a HA-tagged 5-HT 2B receptor specifically in Pet1-positive neurons, using a DIO AAV construct that allows Cre-mediated expression of the tagged receptor. Viruses packaged into AAV2.9 serotype, AAV-DIO-5-HT 2B -HA or control AAV-DIO-TdTomato, were unilaterally injected into B7 raphe nucleus of Pet1-GFP mice (Fig. S1A). We confirmed the proper injection site by colocalization of either TdTomato expression or HA immunofluorescence with Pet1-GFP-positive neurons (Fig. S1B). Coronal raphe-containing brain slices were used to record Pet1-GFP-positive neuron excitability performed in current clamp mode. Recordings at various current steps showed that the number of action potentials obtained in function of the injected current was significantly increased in Pet1-positive neurons overexpressing 5-HT 2B receptors (two-way ANOVA RM, effect of overexpression, F 1, 39 = 4.94, P = 0.032, n = 20 and 21 cells from 3 and 4 mice) (Fig. 1b). Input resistance value from recorded neurons was also significantly increased in 5-HT 2B -HA-overexpressing neurons (unpaired ttest, t 39 = 2.05, P = 0.047) (Fig. 1c). Ex vivo recordings indicated that the 5-HT 2B -receptor overexpression increased Pet1-positive neuron excitability.

In vivo responses to 5-HT 1A and 5-HT 2B receptor agonists

Since our initial ex vivo results indicated that 5-HT 2B receptors could increase 5-HT neuron activity, we hypothesized that it could work in an opposite way as to 5-HT 1A -negative autoreceptor. We thus tested the effect of BW723C86 on 5-HT 1A receptor agonist 8-OHDPAT-induced inhibition of neuronal firing frequency in vivo. Interestingly, 8-OHDPAT was significantly less potent in suppressing putative 5-HT neuron firing activity after BW723C86 injection as shown by two-way ANOVA RM (main effect of BW723C86 treatment, F 1, 9 = 7.34; P = 0.024, n = 6–5 mice). Bonferroni’s post hoc analysis revealed a reduced effect of 8-OHDPAT at 50 and 100 µg/kg on putative 5-HT neuron firing activity (Fig. 2a). 8-OHDPAT ED50 was shifted about 3.3-fold from 45 to 148 µg/kg; BW723C86 alone did not modify firing (not illustrated).

Fig. 2 In vivo response of wild-type mice to combined 5-HT 2B and 5-HT 1A receptor agonists. a In vivo extracellular electrophysiological recordings of putative raphe neurons in anesthetized wild-type mice. (Left) BW723C86 (5 mg/kg subcutaneously (s.c.)) injected 20 min before test counteracted the 5-HT 1A agonist 8-OHDPAT (0.05 mg/kg s.c.) inhibitory cumulative effects to putative 5-HT neuron firing (n = 6–5 mice per group; two-way ANOVA RM, followed by Bonferroni’s multiple comparisons test, *P < 0.05; data are mean ± SEM). (Right) Examples of typical recordings of putative DRN 5-HT neurons obtained in each experimental group. Each arrow represents an injection of 8-OHDPAT (0.05 mg/kg s.c.). The injection of the 5-HT 1A receptor antagonist WAY100635 (0.3 mg/kg s.c.) completely reversed the inhibitory effect of 8-OHDPAT. b In vivo hypothermic effects. The 5-HT 2B -receptor agonist BW723C86 (5 mg/kg s.c.) injected 20 min before the test was able to counteract 5-HT 1A agonist 8-OHDPAT (0.3 mg/kg s.c.) hypothermic effects on wild-type mice (scattered plot, n = 4–4 mice two-way ANOVA RM, followed by Bonferroni’s multiple comparisons test, *P < 0.05) Full size image

Although the mechanism of 5-HT 1A receptor agonist-induced hypothermia is incompletely understood [34], the 8-OHDPAT-induced hypothermia in mice is known to be mediated by 5-HT 1A autoreceptor [35]. We thus used the 8-OHDPAT-induced hypothermia as another in vivo readout of the functional status of 5-HT 1A autoreceptor and tested putative effects of stimulating 5-HT 2B receptors. In agreement with the above-mentioned effect on firing, a pretreatment of wild-type mice with BW723C86 (5 mg/kg s.c,) was able to significantly reduce the ability of 8-OHDPAT (0.3 mg/kg s.c.), to induce hypothermia in wild-type mice (Fig. 2b), as shown by two-way ANOVA RM analysis (interaction between the time and treatment, F 9, 54 = 10.34; P < 0.0001, n = 4 mice per group). Bonferroni’s post hoc analysis showed a significant reduction by BW723C86 at 30 and 40 min post-injection of the 8-OHDPAT-induced hypothermia (Fig. 2b). These data indicated that 5-HT 1A autoreceptor inhibitory activities can be attenuated by concomitant activation of 5-HT 2B receptors.

Absence of response to ecstasy, MDMA, in Htr2b 5-HTKO mice

To establish putative direct actions of 5-HT 2B receptors on 5-HT neurons, we developed mice with conditional ablation of this receptor gene specifically in Pet1-positive neurons. We inserted recombination sites (loxP) flanking the second exon of Htr2b gene (Htr2blox/lox) and crossed these mice with mice expressing the Cre recombinase under Pet1 gene promoter (BAC transgenic). Pet1 gene expression in the brain is restricted to most of differentiated 5-HT neurons and their postmitotic precursors [23]. We thus generated Pet1-Cre+/0; Htr2blox/lox mice (Htr2b5-HTKO) (Fig. S2A). Restricted recombination in raphe 5-HT neurons was revealed by analysis of raphe DNA and by colocalization of a Cre-dependent GFP reporter with 5-HT staining (Fig. S2B–C) [23]. We quantified the efficiency of recombination and found that 83.6 ± 6.1% of the Pet1-Cre-dependent GFP was colocalized with 5-HT staining, 11.6 ± 3.9% was only 5-HT positive and <5% (4.8 ± 2.9%) was only GFP positive (n = 12 from 3 different mice), supporting an efficient and specific recombination in 5-HT neurons (Fig. S2C).

MDMA, the active compound of ecstasy, is a substrate of SERT leading to massive 5-HT release from synaptic vesicle stores, which is partially calcium-dependent [36]. The Htr2b5-HTKO mice injected with MDMA (20 mg/kg) did not display increased locomotor responses in contrast to control Htr2blox/lox littermate mice (Fig. 3a, b). Two-way ANOVA RM analysis showed a main effect of genotype (F 1, 30 = 4.22; P = 0.049, n = 16 mice per group). Bonferroni’s post hoc analysis showed a significant difference between genotypes from t = 25 to t = 45 min post-injection (Fig. 3a). The analysis of the total locomotor activity over the first 60 min after MDMA injection confirmed the significant effect of genotypes (two-way ANOVA RM, F 1, 60 = 4.61; P = 0.036, n = 16 mice per group). Bonferroni’s post hoc analysis showed a significant increase in locomotion in control Htr2blox/lox mice, but not in Htr2b5-HTKO mice (Fig. 3b). We further assessed the contribution in Pet1-positive neurons of 5-HT 2B receptors to MDMA-induced locomotor sensitization using a two-injection protocol [37]. A significant increase in locomotor activity was observed in Htr2blox/lox control mice as shown by two-way ANOVA RM analysis (main effect of genotype, F 1, 14 = 5.12; P = 0.04). Bonferroni’s post hoc analysis showed a significant increase in locomotion in control Htr2blox/lox mice at the second injection of MDMA (20 mg/kg) (first injection 639 ± 222 vs. second injection 1,291 ± 295 1/4 of turns, n = 8), but not in Htr2b5-HTKO (first injection 254 ± 132 vs. second injection 353 ± 174, n = 8) (Fig. 3c). These results indicated a lack of behavioral and sensitizing effects of MDMA in Htr2b5-HTKO mice as found in full KO, Htr2b−/− mice, confirming the need for this receptor in Pet1-positive 5-HT neurons.

Fig. 3 Behavioral response to the 5-HT releaser MDMA in Htr2b5-HTKO mice. a MDMA-induced locomotion. Mice were injected with MDMA (20 mg/kg i.p.) (arrow) after 30 min habituation. A lack of MDMA-induced locomotion was observed in Htr2b5-HTKO mice, while control Htr2blox/lox mice showed a clear increase in locomotion. Data between −30 to +60 min were analyzed using two-way ANOVA RM (means ± SEM, n = 16 mice per group) and a Bonferroni post test was applied on each graph, *P < 0.05. b Cumulative MDMA-induced locomotion. Cumulative locomotion during the first hour following MDMA injection showed a significant difference between the two genotypes. Data were analyzed using two-way ANOVA (n = 16–16 mice, scattered plot, mean ± SEM). A Bonferroni post test was also applied on each graph (*P < 0.05 Htr2b5-HTKO vs. Htr2blox/lox; #P < 0.05 MDMA vs. Veh). c Locomotor sensitization by two MDMA injection protocol. The stimulant effect of a challenge dose of MDMA (20 mg/kg i.p.) 7 days after the first (2nd) was significantly enhanced compared to the first injection in control Htr2blox/lox mice, while it had no effect in Htr2b5-HTKO mice. Data were analyzed using two-way ANOVA RM (n = 8–8 mice, scattered plot, mean ± SEM). Bonferroni post test was also applied on each graph (*P < 0.05 Htr2b5-HTKO vs. Htr2blox/lox; #P < 0.05 1st vs. 2nd injection) Full size image

Absence of response to the SSRI fluoxetine in Htr2b 5-HTKO mice

SSRIs are known to block 5-HT reuptake by SERT, leading to extracellular 5-HT accumulation following vesicular 5-HT release. A classical outcome for acute response to SSRIs is the reduced immobility time observed in FST. We tested the effect of acute fluoxetine injection (3 mg/kg intraperitoneally (i.p.)), the optimal dose determined for 129S2 strain in FST (Diaz et al., 2011). Fluoxetine injection did not affect immobility time in Htr2b5-HTKO mice (Fig. 4a) as shown by two-way ANOVA analysis (time and genotype interactions, F 1, 24 = 10.67; P < 0.003, n = 7–7). However, a significant reduction of immobility time was observed in control Htr2blox/lox littermates as Bonferroni’s post hoc analysis showed the only significant difference between vehicle and fluoxetine-treated Htr2blox/lox control mice (Fig. 4a).

Fig. 4 Antidepressant action in Htr2b5-HTKO mice. a Forced swimming test (FST). The time spent immobile in the FST was significantly reduced in control Htr2blox/lox mine but not in Htr2b5-HTKO mice 30 min after SSRI antidepressant fluoxetine (Flx 3 mg/kg i.p.) injection (two-way ANOVA, Bonferroni post tests, *P < 0.05; n = 7–7 mice, scattered plots with mean ± SEM). b Neurogenesis in subgranular zone (SGZ) of the hippocampus. Fluoxetine (3 mg/kg/day i.p.), daily injected for 4 weeks, induced a significant increase in BrdU incorporation in neuron of the SGZ of control Htr2blox/lox mice, but had no effect in Htr2b5-HTKO mice (two-way ANOVA, Bonferroni post test; *P < 0.05; n = 7–8 mice, scattered plots with mean ± SEM). c SERT expression and function. (Left) SERT expression in conditional Htr2b5-HTKO and Htr2blox/lox control mice was evaluated using heterologous competition binding assays of [3H]citalopram on synaptosome membranes prepared from whole brain. No differences in the affinity (K i ) or expression (B max ) between Htr2b5-HTKO and Htr2blox/lox genotypes were observed. (Right) Saturation isotherms for [3H]5-HT uptake of this synaptosomal preparation were similar in conditional Htr2b5-HTKO and Htr2blox/lox control mice. Nonlinear regression analysis did not reveal differences in the Km or Vmax. Shown are representative curves of at least two independent experiments performed in duplicates. Individual values are presented Full size image

Long-term effects of SSRI are known to be associated with hippocampus subgranular zone (SGZ) neuronal proliferation. We performed a daily i.p. injection for 4 weeks of fluoxetine (3 mg/kg), bromodeoxyuridine (BrdU) injections the 27th day, and quantified BrdU incorporation in SGZ neurons of the hippocampus at the 28th day. Chronic injection of fluoxetine did not produce changes in BrdU incorporation in Htr2b5-HTKO mice (Fig. 4b) (two-way ANOVA analysis showed a trend for treatment and genotype interactions, F 1, 26 = 3.88; P = 0.059, n = 7–8). An increase was detected in control Htr2blox/lox littermates as Bonferroni’s post hoc analysis showed the only significant difference between vehicle and fluoxetine-treated Htr2blox/lox controls (Fig. 4b). These results indicated that the lack of both acute behavioral and chronic neurogenic effects of SSRIs found in Htr2b−/− mice was due to 5-HT 2B -receptor elimination from Pet1-positive neurons.

We recently reported that Htr2b−/− mice displayed novelty-induced hyperlocomotion and a global deficit in sensorimotor gating [31]. However, locomotor activity in a new environment was not different between Htr2b5-HTKO and Htr2blox/lox littermate control mice over the first 60 min (Fig. S3A). Similarly, prepulse inhibition (PPI) of startle reflex and startle amplitude were not different between Htr2b5-HTKO and Htr2blox/lox littermate control mice (Fig. S3B). Although present in Htr2b−/− mice, these deficits were not found in mice lacking the 5-HT 2B receptor selectively in 5-HT neurons, supporting the specificity of these conditional mice.

Since SERT-targeting drug (MDMA and fluoxetine) action is affected by the lack of 5-HT 2B receptors in Pet1-positive neurons, we next determined possible alterations of SERT expression or function in brain synaptosome preparations from Htr2b5-HTKO mice. Heterologous competition binding experiments showed no difference in the density of citalopram binding sites (B max 305 ± 18 vs. 389 ± 26 fmol/mg of protein) or affinity (K i 0.4 ± 0.1 vs. 0.17 ± 0.06 nM) between Htr2b5-HTKO mice and their Htr2blox/lox littermate control mice (Fig. 4c). Additionally, 5-HT uptake experiments on brain synaptosomes showed no difference in 5-HT transport maximum velocity (V max ; 5.57 ± 0.32 vs. 6.49 ± 0.38 fmol/sample/min) or apparent affinity (K m 40.7 ± 2.6 vs. 27.7 ± 2.5 nM) between Htr2b5-HTKO mice and their Htr2blox/lox littermate controls (Fig. 4c). Together, these results demonstrated that selective ablation of 5-HT 2B receptors in raphe Pet1-positive neurons eliminates MDMA and fluoxetine actions but does not affect SERT expression and activity.

Htr2b 5-HTKO mice display a hyposerotonergic phenotype

To determine whether the 5-HT 2B receptor had an overall effect on 5-HT neurons, we measured firing rates of putative 5-HT DRN neurons in vivo in Htr2b5-HTKO mice. Neurons were included in this analysis based on characteristics and averaged traces of their action potentials [25]. Htr2b5-HTKO mice display a higher percentage of putative 5-HT neurons discharging with a low firing mode (16.1% vs. 9.3% < 1 Hz) and a lower percentage of neurons with a high firing mode (5.9% vs. 13.9% > 4 Hz) relative to Htr2blox/lox mice. This observation was reflected by a significant shift in cumulative distribution of neurons with lower firing rate in Htr2b5−HTKO mice compared to control Htr2blox/lox mice (n = 108 and 118 neurons, respectively; Kolmogorov–Smirnov test; P = 0.0008) (Fig. 5a). These results revealed that ablation of 5-HT 2B receptors in raphe Pet1-positive neurons is sufficient to modify 5-HT neuron firing rate.

Fig. 5 Hyposerotonergic phenotype of Htr2b5−HTKO mice. a In vivo extracellular recordings of putative DRN 5-HT neurons in anesthetized mice. (Left) The firing frequency of individual putative DRN 5-HT neuron was shifted from high to low firing rates in Htr2b5-HTKO mice. (Right) A shift in cumulative distribution was observed with significantly lower firing rate in Htr2b5−HTKO mice than in control Htr2blox/lox mice (n = 108 and 118 neurons, respectively; Kolmogorov–Smirnov test; *P < 0.05). b Head-twitch dose–response to DOI. Control Htr2blox/lox and Htr2b5−HTKO mice were i.p. injected with ±DOI (1, 3, and 5 mg/kg i.p.). Ten minutes later, the head-twitch response was scored for 10 min. DOI-induced head-twitch response was significantly increased in conditional Htr2b5-HTKO (*P < 0.05; multiple t test) compared to littermate control mice at 5 mg/kg of DOI (data are presented as scattered plot, n = 3–5 mice per group, and means ± SEM). c In vivo hypothermic effects of 8-OHDPAT. The hypothermic response to 8-OHDPAT (0.1 mg/kg s.c.) was significantly stronger in Htr2b5-HTKO mice compared to control Htr2blox/lox mice (n = 3–8 mice, scattered plot, two-way ANOVA RM, followed by Bonferroni’s multiple comparisons test, *P < 0.05) Full size image

Head-twitch response is a rhythmic paroxysmal rotational head movement that occurs in mice and rats treated by a variety of serotonergic hallucinogens, including LSD and DOI [38]. This behavior is specifically linked to 5-HT 2A receptor activation, since selective 5-HT 2A receptor antagonists block head-twitch response induced by DOI and other hallucinogens, and it is absent in Htr2a−/− mice [38]. Here, we tested ±DOI at 1, 3, and 5 mg/kg i.p. We found that a dose of 5 mg/kg induced a larger increase in head-twitch in Htr2b5-HTKO mice, as compared to Htr2blox/lox littermate control (+109%, t 13 = 2.68, n = 3–5, P = 0.016, multiple t test) (Fig. 5b). Similarly, a greater head-twitch response was observed in Htr2b−/− compared with control mice at the same dose (Fig. S4A). Nevertheless, no significant change in 5-HT 2A or 5-HT 1A receptor expression or in 5-HT content and turnover was found in PFC from Htr2b−/− compared to control mice (Fig. S4B–D). Since a drastic increase in the number of DOI-induced head-twitch was observed in the Tph2-R439H knock-in mouse [34], a mouse model with 60–80% reduction in TPH2 activity and thus with low serotonergic tone, the increased behavioral response to DOI confirmed a lower serotonergic tone in Htr2b5-HTKO mice.