Serotonin Receptor 5-HT2A Regulates TrkB Receptor Function in Heteroreceptor Complexes

Serotonin receptor 5-HT2A and tropomyosin receptor kinase B (TrkB) strongly contribute to neuroplasticity regulation and are implicated in numerous neuronal disorders. Here, we demonstrate a physical interaction between 5-HT2A and TrkB in vitro and in vivo using co-immunoprecipitation and biophysical and biochemical approaches. Heterodimerization decreased TrkB autophosphorylation, preventing its activation with agonist 7,8-DHF, even with low 5-HT2A receptor expression. A blockade of 5-HT2A receptor with the preferential antagonist ketanserin prevented the receptor-mediated downregulation of TrkB phosphorylation without restoring the TrkB response to its agonist 7,8-DHF in vitro. In adult mice, intraperitoneal ketanserin injection increased basal TrkB phosphorylation in the frontal cortex and hippocampus, which is in accordance with our findings demonstrating the prevalence of 5-HT2A–TrkB heteroreceptor complexes in these brain regions. An expression analysis revealed strong developmental regulation of 5-HT2A and TrkB expressions in the cortex, hippocampus, and especially the striatum, demonstrating that the balance between TrkB and 5-HT2A may shift in certain brain regions during postnatal development. Our data reveal the functional role of 5-HT2A–TrkB receptor heterodimerization and suggest that the regulated expression of 5-HT2A and TrkB is a molecular mechanism for the brain-region-specific modulation of TrkB functions during development and under pathophysiological conditions.

Serotonin receptor 2A (5-HT 2A ) belongs to the G-protein-coupled receptor (GPCR) family and is broadly distributed within the brain, showing high expression in the olfactory

The Immunofluorescence Assay of Mouse Brain Sections
Adult (P90) male and female C57BL/6J mice were subjected to this assay. The mice were bred and housed at the animal facility of the University of Hohenheim at a controlled temperature (25 • C) and photoperiod (12/12 h light/dark cycle) and were allowed unrestricted access to standard food and tap water.

Linear Unmixing FRET Analysis
These measurements were performed on live N1E-115 neuroblastoma cells as described elsewhere [40]. 5-HT 2A -mTurquoise2 and TrkB-YPet served as the donor and acceptor, respectively. As a negative control, cells were co-transfected with mTurquoise2tagged CD86 and YPet-tagged CD86. After 16 h, the cells were imaged with a Zeiss LSM 780 microscope equipped with a C-Apochromat 40×/1.2 W Korr water immersion objective For the image analysis and evaluation, custom-written MATLAB scripts were employed. We calculated the predicted apparent FRET efficiency as Ef DA = 1 2 Ef D /(1 − x D ) = 1 2 Ef A /x D , assuming a standard dimerization model [39,41].

Analysis of Ca 2+ Activity
This assay was performed on N1E-115 cells expressing TrkB-YPet, 5-HT 2A -mTurquoise2, or both. We acquired time series data under the Zeiss LSM 780 microscope for 10 min per recording (5 s per frame). Ca 2+ activity was assessed by means of GCaMP6f fluorescence signals (F) and changes calculated as F/F max . To raise Ca 2+ levels to saturated, 10 µM ionomycin was applied after 6 min. This saturated Ca 2+ signal was used as F max for the calculation of basal Ca 2+ levels.

In Situ Proximity Ligation Assay (PLA)
A PLA assay was performed with the Duolink in situ PLA Probes and Duolink in situ Detection Reagents Red Kit (cat. # DUO92008). Brain sections of 20 µm thickness were utilized in the PLA. Antibodies, the same as those used for immunohistochemistry, were diluted 1:100 with the Antibody Diluent provided with the kit. For imaging, the slides were dried and mounted with a cover slip by means of~7 µL of the Duolink in situ Mounting Medium with 4 ,6-diamidino-2-phenylindole (DAPI). The imaging and analysis of the stained brain sections were carried out under the fluorescence confocal microscope (Zeiss LSM 780) using a 40× objective and excitation wavelengths of 561 nm for PLA and 405 nm for DAPI. In each brain section, 3-4 measurements in the frontal cortex, hippocampus, and striatum were performed. Each image was analyzed in ImageJ (Fiji, RRID:SCR_002285) to count individual fluorescent spots. The data are presented as the number of spots per cell, normalized to the number of cells.

qRT-PCR
Total RNA was extracted from the brain tissue of C57BL/6J mice using ExtractRNA (Evrogen, Moscow, Russia), treated with RNA-free DNase (Promega, Madison, WI, USA), and diluted to 0.125 µg/µL with diethyl pyrocarbonate-treated water. One microgram of total RNA was subjected to cDNA synthesis with a random hexanucleotide mixture [42][43][44]. The number of cDNA copies for all studied genes was evaluated by qPCR on a LightCycler 480 (Roche Applied Science, Rotkreuz, Switzerland) with specific primers (Table 1), SYBR Green I fluorescence detection (R-414 Master mix, Syntol, Moscow, Russia), and 50, 100, 200, 400, 800, 1600, 3200, or 6400 copies of genomic DNA as external standards. The calibration curve in the coordinates Ct (threshold cycle value) and minus log P (decimal logarithm of the amount of DNA standard) was plotted automatically using the LightCycler 480 System software. Gene expression is presented as the relative number of cDNA copies per 100 copies of DNA-dependent RNA polymerase 2 subunit A (Polr2a) cDNA, which served as an internal standard [42][43][44]. A melting-curve analysis was performed at the end of each run for each primer pair, allowing us to control the amplification specificity.

Statistical Analysis
The data are presented as means ± the standard error of the mean (SEM). Unless stated otherwise, the significance of pairwise differences was assessed by Student's t test after a Gaussian distribution evaluation by the D'Agostino-Pearson normality test. Groupwise comparisons were made by one-way ANOVA followed by Fisher's post hoc test. Cell groups co-expressing different amounts of 5-HT 2A receptors and treated with 7,8-DHF were compared by two-way ANOVA followed by Fisher's post hoc test. In figures, significance is displayed as p < 0.05 (*), p < 0.01 (**), and/or p < 0.001 (***).

5-HT 2A and TrkB Receptors Form Heterodimers in Neuroblastoma N1E-115 Cells
To analyze the specific interaction between 5-HT 2A and TrkB, we performed coimmunoprecipitation experiments with neuroblastoma N1E-115 cells co-expressing HAtagged 5-HT 2A and GFP-tagged TrkB. It is noteworthy that non-transfected N1E-115 cells express neither 5-HT 2A nor TrkB (data not shown). After immunoprecipitation with an antibody against the HA tag, GFP-tagged TrkB was detectable only in cells co-expressing both HA-and GFP-tagged receptors ( Figure 1A). To assay the extent of artificial protein aggregation, cells expressing only one receptor type (either HA-5-HT 2A or GFP-TrkB) were mixed prior to lysis and analyzed in parallel. While individual receptors could be detected by the same antibody, co-immunoprecipitation did not occur ( Figure 1A). This result confirmed the specificity of the 5-HT 2A -TrkB hetero-oligomerization. express neither 5-HT2A nor TrkB (data not shown). After immunoprecipitation with an antibody against the HA tag, GFP-tagged TrkB was detectable only in cells co-expressing both HA-and GFP-tagged receptors ( Figure 1A). To assay the extent of artificial protein aggregation, cells expressing only one receptor type (either HA-5-HT2A or GFP-TrkB) were mixed prior to lysis and analyzed in parallel. While individual receptors could be detected by the same antibody, co-immunoprecipitation did not occur ( Figure 1A). This result confirmed the specificity of the 5-HT2A-TrkB hetero-oligomerization.  To overcome the limitations related to protein solubilization and concentration during the co-immunoprecipitation procedure, which can cause artificial protein aggregation [45], we further analyzed the interaction between 5-HT 2A and TrkB in living cells using a Förster resonance energy transfer (FRET)-based approach. We measured the apparent FRET efficiency (Ef DA ) between mTurquoise2-labeled 5-HT 2A (5-HT 2A -mTurquoise2, donor) and YPet-labeled TrkB (TrkB-YPet, acceptor) in living N1E-115 cells using the linear unmixing FRET (lux-FRET) method combined with confocal microscopy ( Figure 1B-D). This approach can detect the physical interaction of individual molecules on the nanoscale [40]. The lux-FRET analysis revealed a high apparent FRET efficiency (Ef DA = 14.6%) for 5-HT 2A -mTurquoise2 and TrkB-YPet. In contrast, cells expressing a monomeric fluorophore-tagged CD86 protein [46] yielded significantly lower Ef DA values ( Figure 1D). These experiments demonstrated the selective heterodimerization of 5-HT 2A and TrkB.

Endogenous 5-HT 2A and TrkB Receptors Form a Protein Complex in the Mouse Brain
Having identified the interaction between recombinant 5-HT 2A and TrkB receptors in vitro, we next investigated whether this interaction also occurs in vivo. An immunohistochemical analysis was performed on slices of mouse hippocampus, cortex, and striatum, revealing that 5-HT 2A and TrkB receptors were highly co-localized ( Figure 2A). We then performed a co-immunoprecipitation assay using brain tissue lysates from C57BL/6J mice and identified 5-HT 2A -TrkB oligomeric complexes in samples from the frontal cortex, hippocampus, and striatum ( Figure 2B).
As an additional highly sensitive assay of hetero-oligomerization, we performed an in situ proximity ligation assay (PLA) [47]. In all analyzed brain regions (striatum, hippocampus, and cortex), we found specific PLA-positive blobs, confirming the physical interaction between 5-HT 2A and TrkB ( Figure 2C). The quantification revealed a significantly higher number of 5-HT 2A -TrkB heterodimers in hippocampal cells, indicating a greater prevalence of 5-HT 2A -TrkB heteromeric complexes in this brain region compared to in the frontal cortex and striatum ( Figure 2D).

Calcium Signaling Is Not Affected by the Heterodimerization
To evaluate possible functional consequences of TrkB-5-HT 2A heterodimerization, we first assessed Ca 2+ activity in N1E-115 cells expressing TrkB-YPet, 5-HT 2A -mTurquoise2, or both. To this end, we used the Ca 2+ indicator GCaMP6f [48] and analyzed Ca 2+ dynamics with the multi-threshold event detection approach [49]. The ratio of the GCaMP6f fluorescent signal (F) to the saturated Ca 2+ signal (F max ) indicated that basal Ca 2+ levels were significantly higher following heterodimerization compared to control cells ( Figure 3). The addition of the Ca 2+ ionophore ionomycin to N1E-115 cells elevated Ca 2+ levels under all conditions, with a significantly stronger signal in cells expressing either 5-HT 2A alone or co-expressing TrkB and 5-HT 2A (Figure 3). This finding may reflect constitutive 5-HT 2A activity [50][51][52]. On the other hand, the basal Ca 2+ levels did not differ between cells expressing 5-HT 2A and those co-expressing TrkB and 5-HT 2A (Figure 3), indicating that 5-HT 2A -TrkB hetero-oligomerization did not affect the constitutive 5-HT 2A receptor activity toward a Ca 2+ response. Based on this observation, we focused on whether heterodimerization had functional consequences for TrkB receptors.

Heterodimerization Reduces TrkB Phosphorylation and Blunts the Response to 7,8-DHF
The dimerization and autophosphorylation of tyrosine residues in the intracellular kinase domain of TrkB are crucial steps for the activation of TrkB-mediated intracellular signaling cascades [8]. To determine whether 5-HT 2A -TrkB heterodimerization led to changes in TrkB phosphorylation, we examined the level of the phosphorylated TrkB (pTrkB) protein in N1E-115 cells, using a phospho-specific antibody that specifically recognizes the Y515 site [53]. Notably, the basal pTrkB/TrkB ratio was significantly reduced in cells coexpressing 5-HT 2A (Figure 4). Moreover, 5-HT 2A co-expression not only decreased the amount of phosphorylated TrkB but also prevented its activation by 7,8-DHF, a well-known high-affinity TrkB agonist ( Figure 4A) [36,54]. We also treated cells co-expressing 5-HT 2A and TrkB with the selective 5-HT 2A receptor agonist 25CN-NBOH, which had no significant impact on the pTrkB/TrkB ratio ( Figure 4B). As an additional highly sensitive assay of hetero-oligomerization, we performed an in situ proximity ligation assay (PLA) [47]. In all analyzed brain regions (striatum, hippocampus, and cortex), we found specific PLA-positive blobs, confirming the physical interaction between 5-HT2A and TrkB ( Figure 2C). The quantification revealed a significantly higher number of 5-HT2A-TrkB heterodimers in hippocampal cells, indicating a greater prevalence of 5-HT2A-TrkB heteromeric complexes in this brain region compared to in the frontal cortex and striatum ( Figure 2D). levels under all conditions, with a significantly stronger signal in cells expressing either 5-HT2A alone or co-expressing TrkB and 5-HT2A (Figure 3). This finding may reflect constitutive 5-HT2A activity [50][51][52]. On the other hand, the basal Ca 2+ levels did not differ between cells expressing 5-HT2A and those co-expressing TrkB and 5-HT2A (Figure 3), indicating that 5-HT2A-TrkB hetero-oligomerization did not affect the constitutive 5-HT2A receptor activity toward a Ca 2+ response. Based on this observation, we focused on whether heterodimerization had functional consequences for TrkB receptors.

Heterodimerization Reduces TrkB Phosphorylation and Blunts the Response to 7,8-DHF
The dimerization and autophosphorylation of tyrosine residues in the intracellular kinase domain of TrkB are crucial steps for the activation of TrkB-mediated intracellular signaling cascades [8]. To determine whether 5-HT2A-TrkB heterodimerization led to changes in TrkB phosphorylation, we examined the level of the phosphorylated TrkB (pTrkB) protein in N1E-115 cells, using a phospho-specific antibody that specifically recognizes the Y515 site [53]. Notably, the basal pTrkB/TrkB ratio was significantly reduced in cells co-expressing 5-HT2A ( Figure 4). Moreover, 5-HT2A co-expression not only decreased the amount of phosphorylated TrkB but also prevented its activation by 7,8-DHF, a well-known high-affinity TrkB agonist ( Figure 4A) [36,54]. We also treated cells co-expressing 5-HT2A and TrkB with the selective 5-HT2A receptor agonist 25CN-NBOH, which had no significant impact on the pTrkB/TrkB ratio ( Figure 4B). We then tested whether the heterodimerization rate influenced TrkB phosphorylation. To this end, we analyzed cells expressing a constant amount of TrkB (1 µg), either alone or together with an increasing concentration of HA-tagged 5-HT 2A ( Figure 4C). Basal TrkB phosphorylation was significantly reduced by the expression of even a small amount (0.25 µg) of 5-HT 2A receptor. Treatment with 7,8-DHF yielded a robust enhancement of TrkB phosphorylation in cells expressing TrkB alone, and this response continuously diminished when TrkB was co-expressed with increasing amounts of 5-HT 2A ( Figure 4B).

Expression Patterns of TrkB and 5-HT 2A during Postnatal Development
The results depicted in Figure 4 suggest that 5-HT 2A -TrkB oligomerization impacted TrkB function in a manner depending on the receptors' expression ratio. Therefore, we next determined the expression profiles of 5-HT 2A and TrkB in mouse hippocampus, striatum, and cortex at different stages of postnatal development using real-time quantitative RT-PCR. An analysis of the Ntrk2 transcript encoding TrkB revealed that the Ntrk2 mRNA level was continuously elevated from P1 to P30 in the hippocampus and frontal cortex and that this increase was sustained in the striatum until P60 ( Figure 5B).
An analysis of the Htr2a gene transcript encoding 5-HT 2A revealed that the mRNA level continuously increased from P1 to P60 in the hippocampus and cortex but not in the striatum ( Figure 5A). Moreover, the number of Ntrk2 transcripts was approximately 10 times higher than that of Htr2a transcripts ( Figure 5A). In the hippocampus and cortex, the Htr2a/Ntrk2 mRNA ratio mimicked the pattern of mRNA levels, while the Htr2a/Ntrk2 mRNA ratio in the striatum drastically increased from P1 to P60 ( Figure 5C). These findings suggest that the balance between TrkB and 5-HT 2A may shift in certain brain regions during postnatal development. In light of the above-described suppressive effects of 5-HT 2A on TrkB phosphorylation, this balance might significantly impact TrkB functions.    alone or together with an increasing concentration of HA-tagged 5-HT2A ( Figure 4C). Basal TrkB phosphorylation was significantly reduced by the expression of even a small amount (0.25 μg) of 5-HT2A receptor. Treatment with 7,8-DHF yielded a robust enhancement of TrkB phosphorylation in cells expressing TrkB alone, and this response continuously diminished when TrkB was co-expressed with increasing amounts of 5-HT2A ( Figure 4B).

Expression Patterns of TrkB and 5-HT2A during Postnatal Development
The results depicted in Figure 4 suggest that 5-HT2A-TrkB oligomerization impacted TrkB function in a manner depending on the receptors' expression ratio. Therefore, we next determined the expression profiles of 5-HT2A and TrkB in mouse hippocampus, striatum, and cortex at different stages of postnatal development using real-time quantitative RT-PCR. An analysis of the Ntrk2 transcript encoding TrkB revealed that the Ntrk2 mRNA level was continuously elevated from P1 to P30 in the hippocampus and frontal cortex and that this increase was sustained in the striatum until P60 ( Figure 5B).

Restoration of TrkB Phosphorylation by Ketanserin Treatment
We next analyzed whether a pharmacological 5-HT 2A receptor blockade could restore TrkB phosphorylation. To address this question, we pretreated N1E-115 cells expressing both receptors with the preferential 5-HT 2A antagonist ketanserin. It is noteworthy that after ketanserin treatment basal TrkB autophosphorylation was recovered to the level obtained in the cells expressing TrkB alone. In contrast, the 5-HT 2A receptor blockade by ketanserin did not restore the TrkB phosphorylation in response to 7,8-DHF treatment ( Figure 6A).
We next analyzed whether a pharmacological 5-HT2A receptor blockade could restore TrkB phosphorylation. To address this question, we pretreated N1E-115 cells expressing both receptors with the preferential 5-HT2A antagonist ketanserin. It is noteworthy that after ketanserin treatment basal TrkB autophosphorylation was recovered to the level obtained in the cells expressing TrkB alone. In contrast, the 5-HT2A receptor blockade by ketanserin did not restore the TrkB phosphorylation in response to 7,8-DHF treatment (Figure 6A).  Cell lysates were then subjected to SDS-PAGE and Western blot (WB) analysis with antibodies against phosphorylated or total TrkB. β-tubulin was used as a loading control. Representative WBs are shown. Bars show means ± SEM (n ≤ 8). * p < 0.05; *** p < 0.001 (two-way ANOVA). (B) Mice were intraperitoneally injected with ketanserin (1 mg/kg) or 25CN-NBOH (1 mg/kg), resulting in increased TrkB phosphorylation in the hippocampus and frontal cortex. At 10 min (for 25CN-NBOH) or 30 min (for ketanserin) after injection, the animals were euthanized, and brain lysates were prepared from the frontal cortex, hippocampus, and striatum. This was followed by SDS-PAGE and WB to detect phosphorylated and total TrkB. GAPDH was used as a loading control. Representative WBs are shown. Bars represent means ± SEM (5 ≤ n ≤ 8) * p < 0.05; ** p < 0.01 (two-way ANOVA).
To determine whether 5-HT 2A blockade or activation could alter TrkB autophosphorylation in vivo, we intraperitoneally injected adult (P60) C57BL/6J mice with either ketanserin (1 mg/kg) or the selective 5-HT 2A agonist 25CN-NBOH (1 mg/kg). TrkB phosphorylation was significantly increased in the hippocampus and frontal cortex of mice treated with ketanserin but not those treated with 25CN-NBOH ( Figure 6B).
These results implied that 5-HT 2A -TrkB heterodimerization specifically attenuates TrkB autophosphorylation and that the heterodimerization rate specifically regulates agonist-mediated TrkB phosphorylation. In vitro 5-HT 2A blockade with the selective antagonist ketanserin reversed the decrease of basal TrkB autophosphorylation but failed to restore the TrkB response to 7,8-DHF. At the same time, ketanserin administration considerably enhanced TrkB phosphorylation in the mouse brain in vivo.

Discussion
Several research articles have reported the modulation of BDNF expression by the 5-HT 2A receptor within the limbic neurocircuits [27][28][29], but the exact underlying mechanisms remain unclear. Moreover, the TrkB receptor can be involved in 5-HT 2A -mediated neuritoand spinogenesis in cortical neuronal cultures [55]. We recently found that the chronic treatment of mice with the selective 5-HT 2A receptor agonists TCB-2 and 25CN-NBOH reduced the levels of total and membrane-associated TrkB protein in the mouse brain [56], indicating TrkB downregulation. Among several possible explanations for the functional interplay between 5-HT 2A and TrkB, heterodimerization is the most intriguing. Previously, we have reported that the 5-HT 4 or 5-HT 7 receptors, the classical GPCRs, can form heterodimers with adhesion molecule L1 [33], CDK5 [34], or CD44 [35]. The existence of heteroreceptor complexes between GPCRs and receptor tyrosine kinases (RTKs) was demonstrated previously for the adenosine receptor A2AR [57,58]. Moreover, the 5-HT 1A receptor has previously been shown to form heteroreceptor complexes with fibroblast growth factor receptor 1 (FGFR1) in the mouse hippocampus [59]. In the present study, we used a combination of multiple approaches (including lux-FRET, PLA, and co-immunoprecipitation) to demonstrate the existence of 5-HT 2A -TrkB heteroreceptor complexes both in vitro and in vivo.
From a functional perspective, this heterodimerization suppressed basal TrkB autophosphorylation and prevented agonist-mediated TrkB activation without affecting 5-HT 2A receptor functions. Importantly, our data demonstrated that the degree of heterodimerization played a pivotal role in this process. This suggests that changes in the relative amounts of both receptors and the corresponding changes in heterodimerization rates can provide an intriguing mechanism for the differential regulation of TrkB functions in health and disease. Several studies have described substantially decreased TrkB expression and lower TrkB phosphorylation in the hippocampus of depressive suicidal victims [60][61][62][63]. There is also evidence of increased 5-HT 2A levels in some brain structures, including the frontal cortex and hippocampus, in post-mortem samples from patients with major depressive disorder and suicide victims [64][65][66]. Moreover, 5-HT 2A expression is reportedly sensitive to basal 5-HT concentration, with the 5-HT 2A receptor becoming more abundant in response to a diminished synaptic 5-HT concentration and vice versa [67][68][69]. In this regard, the general tendency of upregulated 5-HT 2A expression in the brain of suicide victims may be explained by an adaptive response to deficient 5-HT 2A signaling due to a reduced 5-HT level in depression. Based on the above-mentioned data, it might be hypothesized that under pathological conditions (e.g., major depressive disorder, addiction, and suicidal behavior), the balance between 5-HT 2A and TrkB becomes shifted toward increased levels of 5-HT 2A -TrkB heteroreceptor complexes, with a subsequent loss of TrkB function. The prevalence of 5-HT 2A -TrkB heteroreceptor complexes in the hippocampus, even under the basal conditions obtained in this study, suggests that this brain structure is among the most vulnerable to the above scenario.
Notably, our findings may also explain why the ability of BDNF to activate TrkB gradually declines during early postnatal development in mice [70]. Starting from approximately 2 weeks of age, BDNF application has only a weak influence on TrkB phosphorylation, while the systemic administration of selective serotonin reuptake inhibitors begins to affect TrkB signaling. Here, we showed that at P14 5-HT 2A expression was drastically increased (up to five-fold) in all analyzed brain regions, while TrkB expression exhibited only a moderate change (up to a two-fold increase). Considering that even a very low relative amount of 5-HT 2A receptor elicited inhibitory effects on TrkB, such a shift in the receptor ratio could result in the decreased ability of BDNF to activate TrkB.
The 5-HT 2A -TrkB interaction could also play a role in the action of antidepressants. It is generally accepted that BDNF-TrkB signaling is a critical component of an antidepressant response [11,[71][72][73]. On the other hand, accumulating evidence from animal and clinical studies suggests that 5-HT 2A receptor inactivation may also facilitate antidepressant action [23,[74][75][76][77]. In one study, chronic treatment with a relatively high dosage (5 mg/kg) of the 5-HT 2A receptor antagonist ketanserin increased neurogenesis in the adult rat hippocampus [78]. However, this effect might be unspecific to the 5-HT 2A receptors because a combined treatment with fluoxetine and ketanserin applied at lower dosage (i.e., 0.1 mg/kg) failed to produce a neurogenic effect while it boosted the expression of Bdnf mRNA in the rat hippocampus [79]. Moreover, atypical antipsychotics exerting 5-HT 2A antagonism can stimulate BDNF expression and/or secretion [80][81][82][83][84][85][86][87][88][89][90]. On the other hand, 5-HT 2A receptor activation can suppress Bdnf transcription in the hippocampus [26]. Our present data showing the physical interaction of 5-HT 2A and BDNF could thus explain the functional crosstalk between these receptors at the molecular level. Indeed, we found that an acute blockade of 5-HT 2A with ketanserin reversed basal TrkB phosphorylation in vitro. We also demonstrated that acute treatment with ketanserin led to increased TrkB phosphorylation in the mouse hippocampus and frontal cortex in vivo, which is in accordance with our findings regarding the prevalence of 5-HT 2A -TrkB heteroreceptor complexes in these brain regions.
Overall, our present results demonstrated that the 5-HT 2A receptor, a member of the GPCR family, could form heterodimers with the non-GPCR receptor TrkB, both in vitro and in vivo. Heterodimerization considerably decreased TrkB autophosphorylation and prevented TrkB activation by its ligand, and these effects could be reversed by a pharmacological blockade of 5-HT 2A with ketanserin. Importantly, our findings suggest that the regulated and balanced ratio of heterodimerization in different brain structures may be crucially involved in both the onset and treatment responsiveness of psychiatric diseases such as depression and anxiety.

Conclusions
In the present study, we provide a multilevel analysis demonstrating, for the first time, the physical interaction between the 5-HT 2A receptor, a member of the GPCR family, and the non-GPCR receptor TrkB, both in vitro and in vivo.
From a functional perspective, heterodimerization suppressed basal TrkB autophosphorylation and prevented agonist-mediated TrkB activation without affecting 5-HT 2A receptor functions. Importantly, these detrimental effects on TrkB functions were reversed by a pharmacological blockade of the 5-HT 2A receptor with ketanserin, a drug widely used to treat hypertension. Moreover, our data demonstrated that the stoichiometry of heterodimerization played a pivotal role in this process. This suggests that changes in the relative expression of both receptors and the corresponding changes in heterodimerization rates can provide an intriguing mechanism for the brain-region-specific regulation of TrkB functions in health and disease.

Informed Consent Statement: Not applicable.
Data Availability Statement: Data supporting the findings of this study are available from the corresponding authors on reasonable request.