The 5-HT1A receptor: signaling to behavior
Abstract
The 5-HT1A receptor is highly expressed both in 5-HT neurons as a presynaptic somatodendritic autoreceptor, and in many brain regions innervated by 5-HT as a post-synaptic heteroreceptor.This review examines the signaling of 5-HT1A receptors to regulate 5-HT activity and behavior. Initial findings in heterologous cell systems, neuronal cell lines, neurons, and in vivo show that the 5-HT1A receptor is a Gi/o-coupled receptor that signals to the canonical pathway of inhibition of adenylyl cyclase (AC). However, new neuron-specific pathways and their roles in neuronal function have been uncovered. 5-HT1A receptor coupling via G subunits reduces neuronal activity by opening K+ channels and closing Ca++ channels. However, the receptor coupled primarily to Gi3 in 5-HT neurons and Gi2 in hippocampal neurons, which may underlie differential signaling and desensitization in these cells. While in 5-HT neurons, the 5-HT1A receptor appears to inhibit ERK1/2 activity, it signals to activate it in developing and adult hippocampal neurons, and may play roles in synaptogenesis. Recent studies implicate 5-HT1A signaling through Gβγ and tyrosine kinase receptors to activate ACII, phospholipase C/PKC, CAMKII, and phosphatidyl inositol 3’-kinase/Akt signaling mediating synaptogenesis, cell survival, and behavioral actions of antidepressants. Thus, the 5-HT1A receptor appears to modify its signaling repertoire depending on the cell type (5-HT vs. post-synaptic neurons) and the developmental state of the neuron. Enhancement of specific signaling of the 5-HT1A receptor may provide an amplification of the antidepressant actions of 5-HT1A receptor activation. In addition, in response to prolonged 5-HT elevation upon chronic antidepressant treatment, the 5-HT1A autoreceptor appears to desensitize more extensively than the heteroreceptor. The mechanisms of 5-HT1A receptor desensitization are discussed, highlighting the potential of enhancing autoreceptor desensitization to accelerate antidepressant response.
1.Introduction: 5-HT and 5-HT1A receptors
A family of at least 14 distinct, widely distributed receptors mediates the actions of serotonin (5-HT) on a huge diversity of brain functions [1-4]. These receptor subtypes differ in their regional expression and their roles in mediating serotonergic actions on physiological function, mood and emotion. The 5-HT1A receptor subtype remains among the most studied, since it is one of the most abundant and widely expressed 5-HT receptors in the brain [5-7]. 5- HT1A receptors are sub-classified into 2 distinct populations: presynaptic autoreceptors located on the dendrites and cell body of 5-HT neurons in the raphe nuclei; and postsynaptic heteroreceptors, which exist on the dendrites and cell body of target non-5-HT neurons in 5-HT projecting areas [8-11]. The 5-HT1A autoreceptors negatively regulate the 5-HT system.Serotonin released in the raphe activates 5-HT1A autoreceptors, which inhibit the firing of 5-HT neurons [12-15]. In contrast, release of serotonin in target brain regions activates 5-HT heteroreceptors, including the 5-HT1A heteroreceptor. The 5-HT1A heteroreceptor is abundantly expressed in the hippocampus, septum, amygdala, and PFC [5, 6] where it mediates serotonin actions on fear, anxiety, stress, depression and cognitive function [2, 7, 16-18]. In the forebrain, 5-HT1A heteroreceptors are expressed on excitatory pyramidal neurons and inhibitory interneurons, and can mediate complex effects of 5-HT on the pattern of neuronal firing [10, 16, 19]. Thus, 5-HT1A receptors can both suppress global serotonin release (as autoreceptor) and mediate brain region-specific responses to released serotonin (as heteroreceptor).
Several key pharmacological ligands to the 5-HT1A receptor have been used to characterize its signaling, and in vivo function. The relatively selective 5-HT1A autoreceptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8OH-DPAT) and antagonist WAY 100635 [20] have been used as pharmacological probes of 5-HT1A receptor function. While 8OH-DPAT is selective for 5-HT1A receptors, it can also activate 5-HT7 receptors with 10-fold lower affinity [21]; while WAY100635 is highly specific to block 5-HT1A receptors [22, 23]. Acute treatment with 8OH-DPAT induces hyperphagia, hypothermia, and an anxiolytic response in rodents and these effects are blocked by pretreatment with WAY 100635 [22]. 5-HT1A partial agonists, such as buspirone, are currently approved as anxiolytics. Both 5-HT1A receptor partial agonists and antagonists can enhance the therapeutic effects of antidepressants in clinical studies [24-27]. Thus, 5-HT1A ligands are potentially useful for treatment of anxiety and depression. However, a major problem is that these ligands tend not to discriminate between 5-HT1A autoreceptors and heteroreceptors, thus having opposing actions on 5-HT effects. Recent development of 5-HT1A- selective ligands that show some discrimination between these receptors and/or their signalling pathways offers hope for improved treatments for mental illness [28, 29].In this review, we survey and evaluate the coupling of 5-HT1A receptors to a diversity of signalling pathways, in heterologous cell systems, in neurons and in vivo, comparing 5-HT1A autoreceptor/heteroreceptor signalling.
2.5-HT1A receptor signaling
2.1Signaling of cloned 5-HT1A receptors in cell lines
The 5-HT1A receptor is a heptahelical G protein coupled receptor that couples via inhibitory G proteins (Gi/Go) to inhibit adenylyl cyclase and reduce cAMP levels [30, 31] (Fig. 1). The first studies to demonstrate this canonical pertussis toxin-sensitive pathway used cloned 5-HT1A receptors transfected in heterologous non-neuronal (Hela, Cos7, CHO, Ltk) or neuroendocrine (GH4C1) cell lines [5, 32-34]. Using stable expression of antisense constructs to Gi1-3 in GH4 cells, knockdown of Gi2 or Gi3 but not Gi1 blocked 5-HT1A induced inhibition of cAMP [35]. More recent studies in CHO cells suggest that 8OH-DPAT recruits Gi3 at higher potency than Gi2, but that Gi2 signaling predominates at high receptor occupancy [36].Interestingly, in non-neuronal cells but not neuroendocrine GH4C1 cells, the receptor also coupled via Gi/Go proteins to phospholipase C (PLC), a pathway mediated by Gβγ coupling to PLCβ2/3 [5, 32-34, 37] (Fig. 1). The 5-HT1A/Gi/PLC pathway has also been described in hippocampal and raphe cell lines [37, 38] (Fig. 2, 3), but this was seen in non-differentiated cells and may not reflect signaling in differentiated neurons. Coupling to this pathway in brain cells has not been shown directly, but in prefrontal cortex (PFC) 5-HT1A receptors associated with PLCβ to increase CAMKII activation, suggesting that this pathway may be present [39]. In early postnatal hippocampus, 5-HT1A signaling to PKC induces synaptogenesis that was blocked in 5- HT1A -/- tissue and rescued by PKC activation [40], suggesting a role for this pathway in development.
However, in raphe neurons, PLC inhibitors did not affect signaling of 5-HT1A autoreceptors [41], although inhibition of PI turnover was seen in raphe membranes [42]. There was no effect of 8OH-DPAT on basal hippocampal PI turnover [42], although in rat hippocampal slices 5-HT1A receptors inhibited carbachol-stimulated PLC activation [43]. However, an indirect effect could not be ruled out in the latter studies. Thus, 5-HT1A coupling to stimulation of PLC may be restricted to neurons that express PLCβ2/3 [37], possibly during development [40].In contrast to canonical Gi/Go-mediated inhibition of cAMP, 5-HT1A receptors have been shown to constitutively stimulate adenylyl cyclase under certain conditions (Fig. 1, 3). In particular, when co-expressed with adenylyl cyclase type II (ACII) in HEK293 cells, 5-HT1A receptors stimulate cAMP via Gβγ subunits derived from Gi2 [44], and a similar pathway is seen using Xenopus oocyte expression system [45]. Furthermore, specific knockdown of Gαi1 unveiled 5-HT1A-mediated stimulation of cAMP in neuroendocrine GH4 cells, which express endogenous ACII [35]. As ACII is enriched in the hippocampus and also expressed in the dorsal raphe nucleus [46], 5-HT1A coupling to stimulate ACII may be present in these regions.
2.2 Signaling to ion channels
The 5-HT1A receptor has also been shown to couple to ion channels (Fig. 1-3). In the raphe and hippocampus, 5-HT1A receptors couple via Gβγ subunits to activation G-protein inward rectifying potassium (GIRK) channels to hyperpolarize the membrane potential [47-49]. Similarly, in the PFC 5-HT1A receptors couple to outward GIRK currents, and this coupling is maximal in young adult rats, declining in adulthood and upon chronic stress [50]. Interestingly, chronic glucocorticoid treatment induced down-regulation of GIRK2 channels in raphe that correlated with 5-HT1A autoreceptor uncoupling from inhibition of 5-HT neuronal firing [51]. Heteroreceptors also couple to GIRK channels in hippocampus and cortex to inhibit neuronal activity [52, 53]. Importantly, 5-HT1A heteroreceptors in cortical pyramidal neurons are enriched at the axon hillock [54, 55], and have been shown recently to play a key role in suppressing action potential generation [56]. Although multiple GIRKs may couple the 5-HT1A autoreceptor to inhibition of neuronal firing [57], GIRK2 seems to be most prominent. Coupling of 5-HT1A receptors to outward currents was blocked in hippocampus [53] and reduced in dorsal raphe of GIRK2 -/- mice. Also, 8OH-DPAT-induced hypothermia mediated by 5-HT1A autoreceptors was blocked in GIRK2 -/- mice [58], which display a depression resistant phenotype [59] (Fig. 2). The resistance to depression in GIRK2 -/- mice could result from the uncoupling of 5-HT1A autoreceptors leading to a stress-resilient phenotype, as seen upon knockdown of 5-HT1A autoreceptors [60].
The 5-HT1A receptor also couples via Gαo and Gβγ subunits to voltage-dependent calcium channels (VDCC) (Fig. 1). Gαo was obligatory for receptor coupling to dihydropyridine (BAYK8644)-sensitive (L-type VDCC) calcium influx in GH4C1 pituitary cells [61], while Gi1- 3 were not [35]. However, 5-HT1A receptors primarily coupled to Gαi3 to inhibit TRH (Gq)- mediated hormone secretion in neuroendocrine GH4C1 cells [62]. In the raphe and target regions, 5-HT1A receptors couple via Gβγ subunits to inhibit N-type VDCCs, resulting in decreases in excitability [63-68] (Fig. 2). However, the importance of 5-HT1A-induced inhibition of VDCC in behavior has not been addressed.
The 5-HT1A heteroreceptor has also been shown to reduce ionotropic glutamate receptor function (Fig. 3). In young adult rat (3-5 week postnatal) cortical neurons, 5-HT1A-mediated inhibition of cAMP and CAMKII activity reduces AMPA currents [69]. Similarly, 5-HT1A signaling to inhibit PKA and CAMKII inhibits NR2B translocation in cortical neurons, reducing cell surface NMDA receptors [70, 71]. Conversely, in prefrontal cortical tissue, clozapine induces a complex between 5-HT1A/CAMKII, and NR2B that enhances NMDA currents [39]. However, clozapine targets multiple receptors including 5-HT2A receptors that may contribute to this NMDA-enhancing effect [71]. In the hippocampus, there is evidence that tonic 5-HT1A activity inhibits CAMKII to reduce NMDA receptor function associated with cognitive impairment and neuroprotection [72, 73]. In agreement, early post-natal blockade of 5-HT1A receptors results in hippocampal CAMKII activation associated with an anxiety phenotype [74]. Overall these studies indicate that 5-HT1A mediated inhibition of cAMP and CAMKII activity reduces ionotropic glutamate receptor signaling.
2.3 Signaling to ERK1/2 and Akt pathways
In addition to canonical 5-HT1A receptor signaling, 5-HT1A receptors activate growth factor-regulated signaling pathways, including mitogen-activated protein kinase ERK1/2 and PI3K-AKT-GSK3 signaling pathways, as first shown in 5-HT1A-transfected CHO fibroblasts (CHO-1A cells) [75] (Fig. 1). Signaling of the 5-HT1A receptor to ERK1/2 in CHO-1A cells was blocked by transfection of sequestration-deficient arrestin or dynamin mutants, implicating arrestin-induced internalization [76]. In CHO-1A cells, activation of transfected 5-HT1A receptors induced AKT signaling, leading inactivation of GSK3β [77]. 5-HT1A receptor coupling to ERK1/2 is observed in hippocampal derived cell lines with endogenous expression of 5-HT1A receptors [78], as well as in hippocampal tissue [79], but was not observed in e18 hippocampal primary cultures [80]. Signaling to Akt was found in adult hippocampal tissue [81]. However, although Gi signaling is implicated, the role of arrestin signaling in these tissues has not been as extensively studied as for other monoamine receptors.
The 5-HT1A/PI3K/Akt/GSK3β pathway appears to be important for antidepressant response. 8OH-DPAT treatment induced hippocampal Akt and GSK3β phosphorylation, actions that were blocked by a PI3K inhibitor, as was the anti-anxiety effect of 8OH-DPAT [81].Knockin of a phospho-site mutant of GSK3β in mice blocked 5-HT1A-induced anti-anxiety effects, and prevented fluoxetine-induced anti-depressant effects [81]. PI3K inhibitors also blocked intra-hippocampal 8OH-DPAT-induced Akt phosphorylation, GSK3β phosphorylation and inactivation and antidepressant effects.
In the prefrontal cortex, inhibition of 5-HT1A receptors also blocked ketamine’s antidepressant action and prevented ketamine-induced increase in Akt phosphorylation, suggesting a role for 5-HT1A/Akt signaling in ketamine actions [82], and possibly involving GSK3β inactivation [83]. Interestingly, Akt signaling may play a role in the desensitization of the 5-HT1A autoreceptor, as 5-HT1A autoreceptor-induced hypothermia was enhanced in mice with reduced Akt phosphorylation [84]. However, antidepressant induced uncoupling or reduction of GIRK channels cannot be ruled out [85]. By contrast, GSK3β knockout in 5-HT neurons diminished 5-HT1B functions, but did not affect 5- HT1A autoreceptors [86]. There is indirect evidence in embryonic rhombencephalic cultures that the pro-survival effect of 5-HT1A agonists is PI3K-dependent [87], suggesting that 5-HT1A autoreceptors may couple to this pathway.
How 5-HT1A receptors couple to the PI3K pathway in neurons remains unclear (Fig. 2, 3). In the raphe RN46A cell line, hypoxia induced PDGFβ-dependent activation of the PI3K/Akt pathway [88]. Similarly, 5-HT1A autoreceptors may couple to PDGF signaling to trigger this pathway. For example, in CHO cells dopamine D2L and D4 receptors trans-activate the PDGFβ receptor to induce ERK1/2 activation that requires PI3K [89]. In human SH-SY5Y neuroblastoma cells, 5-HT1A receptors weakly trans-activated PDGFβ receptors via Gi/Go protein signaling involving PLC activation [90].
2.4 Differences between 5-HT1A autoreceptor and heteroreceptor signaling
There is evidence that 5-HT1A autoreceptors may signal differently than heteroreceptors (Fig. 2 vs. 3). In the raphe nucleus, the 5-HT1A autoreceptor preferentially couples to Gi3, distinctly from hippocampal heteroreceptors that preferentially couple to Gαi2 or Gαo [91, 92]. However, early studies using rat raphe-midbrain membranes failed to demonstrate 8OH-DPAT- induced inhibition of forskolin-stimulated adenylyl cyclase (FSAC), while this effect was seen using hippocampal membranes [42, 93]. Subsequently, three different groups have shown 5- HT1A coupling to a modest (20-30%) inhibition of FSAC or PKA activity in rat and human dorsal raphe [92, 94, 95]. However, 5-HT1A mediated inhibition of AC in dorsal raphe depends on which agonist is used, with flibanserin ineffective, while 5-HT, 5-CT, and buspirone are effective [94, 96], while flibanserin was effective in prefrontal cortex. Using 8OH-DPAT, while one group showed efficacy in human raphe [94], this coupling was not seen in rat raphe [42, 92, 93], yet buspirone was effective [92]. These differences in agonist and species dependence may explain the lack of effect of 8OH-DPAT on FSAC, as seen in the earlier studies [42, 93]. The presence of ACII in the raphe [46] may mediate 5-HT1A-stimulated AC, and attenuate 5-HT1A action to inhibit FSAC activity. These results suggest that 5-HT1A-mediated inhibition of AC is ubiquitous, but displays different agonist and G protein specificity in raphe vs. hippocampal neurons.
Differences in 5-HT1A auto- and heteroreceptor coupling to ERK1/2 activation have also been reported. In raphe RN46A cells, endogenous 5-HT1A receptors couple to inhibit adenylyl cyclase and ERK1/2 phosphorylation, particularly in differentiated cells [37]. In contrast, 5- HT1A receptors in hippocampal-derived cells increases in ERK1/2 phosphorylation [78].
Interestingly, 5-HT1A-mediated ERK1/2 activation has also been reported in blood monocytes [97], which could provide a biomarker for 5-HT1A heteroreceptor function in humans.
5-HT1A heteroreceptor signaling to ERK1/2 is inhibited by RGS19-induced uncoupling of Gi2 and enhanced by FGF2 in SH-SY5Y cells and hippocampal neurons [98]. In the presence of the mitogen FGF2, 5-HT1A autoreceptors in raphe tissues and a raphe cell line actually increase ERK1/2 activation, indicating that 5-HT1A autoreceptor signaling may depend on trophic stimulation [99]. These synergistic actions may involve FGFR1/5-HT1A receptor heterodimers [99, 100]. Modeling studies in HEK293 cells indicate that 5-HT1A-mediated ERK1/2 activation is transient when compared to 5-HT2A/Gq mediated ERK1/2 activation that is sustained and predominates over the 5-HT1A effect [101]. In vivo, systemic 8OH-DPAT injection reduces hippocampal ERK1/2 activation, which was blocked by 5-HT1A antagonist WAY 100635 and mimicked by 5-HT1A partial agonist buspirone, with no effect in striatum or prefrontal cortex [102]. However, these drug actions could be indirect, and due to 5-HT1A autoreceptor-mediated reduction of 5-HT neurotransmission. Taken together, the above findings show 5□HT1A autoreceptors and heteroreceptors can signal to diverse, sometimes opposing, intracellular signaling pathways, which are integrated in the cell to mediate the acute and sustained effects of the serotonin system of neural circuits (Fig. 2, 3).Another signaling pathway that may involve hippocampal 5-HT1A heteroreceptors is the paradoxical coupling to stimulate cAMP (Fig. 3). In HEK293 cells, 5-HT1A receptors constitutively stimulated cAMP when cotransfected with ACII and Gi2 [44]. Interestingly, while full agonists like 5-HT or 8OH-DPAT or antagonists (WAY100636 or spiperone) did not modify this pathway, anxiolytic compounds that are 5-HT1A partial agonists (e.g., buspirone, flesinoxan) displayed inverse agonism to reduce cAMP [44]. These ligands preferentially target 5-HT1A heteroreceptors and the hippocampus (dentate gyrus, CA1) and dorsal raphe express high levels of ACII RNA [46]. Thus, 5-HT1A coupling to the cAMP stimulatory pathway could account for 5-HT1A-induced increases in hippocampal cAMP [103-105], and could be involved in 5-HT1A partial agonist anti-anxiety actions [44]. However, the role of ACII in 5-HT1A receptor function in vivo has yet to be tested using knockout or knockdown approaches.
2.5 Hippocampal 5-HT1A heteroreceptor signaling to behavior
There is evidence that 5-HT1A heteroreceptors couple via Gi2 to induce antidepressant actions. Mice with knockin of an RGS-insensitive Gαi2 mutant that enhances Gi2 signaling show reduced depression- and anxiety like behavior, and increased GSK3β phosphorylation/inactivation in hippocampus and prefrontal cortex, actions that were 5-HT1A- dependent [106]. These mice had increased sensitivity to SSRI- or 8OH-DPAT-induced anti- depressant actions, but not 8OH-DPAT-induced hypothermia suggesting an effect on hetero- receptor, rather than autoreceptor signaling. These in vivo studies suggest that the hippocampal 5-HT1A/Gi2/GSK3β signaling mediates antidepressant and anti-anxiety actions of SSRI treatments. In agreement, activation of 5-HT1A receptors mediates SSRI-induced GSK3β phosphorylation/inactivation in prefrontal cortex and hippocampus [81, 107]. Similarly, in 5- HT1A -/- mice, dopamine induced GSK3β activation and synaptic remodeling in prefrontal
cortex are blocked, implicating 5-HT1A/GSK3β in cortical neuroplasticity [108]. Furthermore, TPH2 mutant mice that have 5-HT deficiency and display increased anxiety and depression-like behavior are rescued by heterozygous deletion of GSK3β [109], implicating GSK3β in 5-HT signaling to these behaviors. More recently, hippocampal 5-HT1A receptors on mature granule cells specifically have been knocked out in mice, resulting in resistance to chronic SSRI-induced hippocampal neurogenesis and anti-depressant actions [110].
Hippocampal 5-HT1A signaling via CAMKII and ERK1/2 to CREB mediates neurogenesis and neuroplasticity leading to reduced anxiety-like behavior [111, 112]. The 5-HT1A/ERK1/2 pathway has also been implicated in developmental hippocampal synaptogenesis at p15 [40]. In particular, stimulation of hippocampal 5-HT1A receptors induced PSD95 expression and spine formation via ERK1/2- PKC signaling and this action was blocked in 5-HT1A -/- tissue, but rescued by PKC activation. This developmental window (p13-34) is when 5-HT1A receptors are required for normal adult anxiety-like behavior, as shown using transient administration of a 5-HT1A blocker [74].Interestingly, hippocampal CAMKII activity was induced in young but not affected in adult 5- HT1A -/- mice [74], suggesting a development shift in 5-HT1A-CAMKII signaling. In a complementary manner, 5-HT1A gene rescue in the forebrain of 5-HT1A -/- mice during this developmental period rescues adulthood anxiety [113]. However, increasing 5-HT output by deleting 5-HT1A autoreceptors during this period also results in adulthood anxiety [114], indicating that a balance of 5-HT levels is required for normal development of anxiety behavior [7, 16].
2.6 5-HT1A signaling in human depression
While the above studies in animal models are instructive, it remains difficult to address whether similar 5-HT1A signaling alterations occur in humans. Both post-mortem studies of depressed suicide brain and imaging studies of depressed subjects have shown that 5-HT1A receptor binding is reduced widely in the cortex and hippocampus [18, 115, 116], while in the raphe increased binding of 5-HT1A autoreceptors has been reported [117, 118]. In one study of membranes prepared from post-mortem occipital cortex, 5-HT1A coupling to Gi2 and Gi/Go proteins was reduced in depressed suicide brain with no change in Gi/Go protein levels, and 5- HT1A-mediated inhibition of cAMP was reduced [119]. However this effect could be due to reduced levels of occipital cortex 5-HT1A receptors in depressed suicide [120]. Interestingly, the depressed suicide samples showed reduced Akt and GSK3β phosphorylation, and reduced ERK1/2 phosphorylation, suggesting reduced overall activity of these signaling pathways [119], although not necessarily involving 5-HT1A signaling.
3.5-HT1A receptor desensitization
3.1 Acute desensitization mechanisms
The mechanisms of acute agonist-induced desensitization of the 5-HT1A receptor remain unclear (Fig. 1). Protein kinases PKC, PKA, and GRK2 may each contribute to 5-HT1A receptor desensitization. Early studies using purified 5-HT1A receptors from baculovirus infected insect Sf9 cells showed that 5-HT induces rapid uncoupling of the receptor from G proteins and inhibition of cAMP, and induces receptor hyper-phosphorylation [121]. Agonist- induced phosphorylation of the receptor was not blocked by PKA or PKC inhibitors, suggesting a role for GRK2 in homologous desensitization of the 5-HT1A receptor. Consistent with this, co-expression of GRK2 and arrestin enhanced agonist-mediated 5-HT1A receptor internalization in HEK-293 cells [122]. In HEK-293 and CHO-1A cells, 8OH-DPAT induced arrestin2 recruitment to the 5-HT1A receptor, as detected by bioluminescence resonance energy transfer (BRET) [100, 123]. In Ltk- cells transfected with 5-HT1A receptor and arrestin2-GFP, 8OH-DPAT induced redistribution of arrestin2-GFP from diffuse cytosol localization to plasma membrane and intracellular puncta (Fig. 4). In cells transfected with arrestin2-GFP alone, no redistribution was observed, suggesting 5-HT1A-dependent recruitment of arrestin2. Taken together, these studies to argue for a role of arrestin2 recruitment in agonist-induced 5-HT1A receptor desensitization. However it should be noted that these experiments were carried out in transformed cell lines with arrestin overexpression.
On the other hand, PKC and PKA have been implicated in heterologous desensitization of 5-HT1A receptors. In non-neuronal cells, PKC and PKA have been shown to phosphorylate the 5-HT1A receptor and PKA augments the PKC-dependent desensitization of the receptor [124, 125]. Activation of PKC preferentially uncouples Gβγ over Gαi signaling [33, 126]. The effect of PKC on 5-HT1A-mediated Gβγ signaling was blocked by mutation of 3 PKC sites in the i3 loop, and one site in the i2 loop that was obligatory for Gβγ signaling [37, 126-128].
Thus, PKC could mediate 5-HT-induced homologous desensitization in cells where the 5-HT1A receptor couples to PLC activation [129]. In agreement, in dorsal raphe neurons and F11 neuronal cells, PKC uncouples 5-HT1A-induced inhibition of N-type VDCC, but not GIRK activation [130-132]. However, in the dorsal raphe neurons, the inhibition of PKA but not PKC reduced agonist-induced uncoupling from N-type VDCC inhibition [95]. Hence, depending on the cell type and signaling pathway, PKA, PKC or both can uncouple 5-HT1A receptor signaling.
As discussed above, functional differences between pre- and postsynaptic 5-HT1A receptor signaling, desensitization and transcriptional regulation have been reported in animal models of depression. This has led researchers to propose a key role for transcriptional regulators of the 5-HT1A receptor in successful antidepressant treatment. For example, prolonged stimulation of 5-HT1A autoreceptors by fluoxetine (SSRI) results in desensitization and downregulation of the 5-HT1A autoreceptor that requires at least 1-2 weeks of treatment, as shown in several depression models and animal species [133-138].
Interestingly, postsynaptic 5- HT1A heteroreceptors do not desensitize as easily, although the mechanism by which this difference occurs is not yet known [138-141]. 8OH-DPAT-induced desensitization of the 5- HT1A autoreceptor in vivo involves a rapid internalization of the receptor, followed by recycling of the inactivated receptor to the membrane [142]. This recycling event could involve receptor dephosphorylation, as suggested by the effect of protein phosphatase 2A inhibition on 5-HT1A signaling [101, 143]. By contrast, hippocampal 5-HT1A heteroreceptors did not internalize. The exact mechanisms involved remain unclear, but agonist-induced recruitment of βarrestin2 to 5- HT1A receptors has been shown using BRET in 5-HT1A-transfected HEK-293 and CHO-1A cells [100, 123], and could mediate this internalization. Agonist-induced 5-HT1A receptor internalization has been shown in 5-HT1A-transfected HEK293 cells for a variety of 5-HT1A agonists, but requires co-transfection of GRK2 and βarrestin2 [122]. Furthermore, in 5-HT1A- transfected Ltk- fibroblast cells, but not vector-transfected cells, 8OH-DPAT treatment induces redistribution of transfected arrestin2-GFP to puncta at the membrane and within the cells, suggesting that arrestin2 co-internalizes with 5-HT1A receptors upon agonist stimulation (Fig. 4). In CHO-1A cells, blockers of arrestin- or dynamin-dependent internalization prevent 5- HT1A coupling to ERK1/2, suggesting a role for endogenous arrestin in 5-HT1A internalization-dependent signaling to ERK1/2 [76]. However, to date, no studies have specifically examined the role of βarrestin in 5-HT1A receptor desensitization in vivo nor in vitro without βarrestin overexpression.
The regulator of G protein signaling (RGS) family of proteins inactivates G protein signaling [144, 145]. Signaling to G proteins by the 5-HT1A receptor is also regulated by RGS proteins, as shown by the effect of knock-in of RGS-insensitive Gi2 on 5-HT1A signaling in vivo [106]. There is evidence that RGS4, RGS6, RGS10, RGS19, and RGS20 suppress 5-HT1A signaling [98, 146-149], but other RGS proteins may also play a role. While most RGS proteins were analyzed in cell culture, the requirement for RGS6 was examined in vivo. 5-HT1A-induced antidepressant actions were enhanced in RGS6 +/- mice, while RGS6 -/- mice displayed reduced baseline anxiety and depression phenotypes [149]. The extent of RGS regulation could depend on the tissue expression of different RGS subtypes [150]. For example, knockdown of RGS4 in nucleus accumbens conferred resistance to SSRI actions, while its global knockout enhanced ketamine antidepressant action [151].
3.2 5-HT1A autoreceptor down-regulation mechanisms
A key difference noted between 5-HT1A autoreceptors and heteroreceptors on hippocampal pyramidal neurons was that the former desensitize upon chronic treatment with SSRI or 5-HT1A partial agonists, while the latter are resistant to desensitization [152-154]. The SSRI’s rapidly block 5-HT transporters, leading to an increase in 5-HT levels, both in target regions and in the raphe. These initial changes occur with 1-3 hours, as detected by PET imaging studies in monkeys [141]. The increase in 5-HT in the raphe activates 5-HT1A autoreceptors, which inhibit the firing of 5-HT neurons via GIRK activation, and prevent the SSRI-induced increase in 5-HT in target areas. However, after several weeks of treatment, the 5-HT1A autoreceptors desensitize, and 5-HT firing recovers, leading to increased 5-HT and clinical responses [136, 154]. This prolonged time required for desensitization is much longer than for uncoupling (sec), internalization (1 hr), or even receptor degradation (1-2 day) [155].Furthermore, differences in G protein coupling and pharmacological responses of these receptors have been described [156-158]. Molecular cloning of the human, rat and mouse 5-HT1A genes revealed that they lack introns in the coding sequence, indicating only a single protein can be made [5, 159-161]. Because there are no introns in the promoter and coding portions of the 5- HT1A receptor gene, alternate splicing of these regions cannot explain this difference in long- term desensitization. Hence, differential transcriptional regulation of the 5-HT1A promoter in pre- and post-synaptic neurons could mediate this difference in long-term desensitization.
However, we have recently identified novel splice variants in the 3’-UTR of the human 5-HT1A receptor gene that stabilizes 5-HT1A RNA stability by deleting a miRNA135 site [162], and could contribute to regulation of 5-HT1A receptor levels in humans [163].
The 5-HT1A promoter sequence contains a non-selective CG-rich housekeeping promoter sequence, including multiple strong Sp1/MAZ enhancers that drive expression in all cells [154, 164, 165]. The promoter also contains an NFkB-response element that may mediate 5-HT1A induction in immune cells [166]. Furthermore, the 5-HT1A receptor gene is regulated by a number of identified transcription factors (TF), some of which show cell-specificity. For example, specifically in 5-HT neurons, PET-1/FEV (an ETS transcription factor) acts as an enhancer at the 5-HT1A receptor promoter, and is required for raphe 5-HT1A receptor expression due to its exclusive expression in these neurons [167]. Pet-1 acts developmentally to orchestrate expression of 5-HT genes including 5-HT1A receptors, and is required in adulthood to maintain their expression, except for the 5-HT1A gene, which become Pet-1-independent [168]. Thus, inactivation of Pet-1 in adulthood did not reduce 5-HT1A receptors, but reduced TPH2 levels and induced anxiety and depression phenotypes.
Among transcriptional regulators of the 5-HT1A gene, NUDR/Deaf1 is of particular interest. Deaf1 was identified as a repressor that binds to the -1019C- but the G-allele of the human 5-HT1A promoter polymorphism (rs6295). The G-allele has been associated with depression, anxiety and resistance to SSRI treatment [169, 170]. However, Deaf1 serves a dual role, acting as a transcriptional repressor in raphe 5-HT neuronal cells [171], and as an enhancer non-5-HT neuronal cells [172]. In mice lacking Deaf1, 5-HT1A autoreceptors are up-regulated, while the expression of 5-HT1A heteroreceptors in the prefrontal cortex, but not hippocampus, is reduced [173]. Similarly, depressed patients with the G-allele show increased binding for raphe 5-HT1A autoreceptors [118, 174], and reduced 5-HT1A binding and RNA levels in the prefrontal cortex [174, 175]. Therefore, Deaf1 may play a distinct role in desensitization of 5- HT1A auto- vs. heteroreceptors after chronic SSRI treatment. However, the signaling mechanisms that lead to Deaf1 activation remain unclear.Another transcriptional regulator of the 5-HT1A receptor is Freud-1/CC2D1A, which functions as a strong repressor of the 5-HT1A gene [176]. Interestingly, in human raphe, Freud- 1, but not Freud-2/CC2D1B, is strongly expressed [177, 178] and thus could also play a key role in down-regulation of the 5-HT1A autoreceptors. Freud-1 is inactivated by Ca2+-CAMK signaling leading to an up-regulation of 5-HT1A transcription [176]. Thus, a reduction in Ca2+ levels by 5-HT1A autoreceptor activation upon treatment with SSRI’s would increase Freud-1 repression, reducing 5-HT1A gene transcription and down-regulating the 5-HT1A autoreceptor. Consistent with a role of Freud-1 in 5-HT1A autoreceptor regulation, adulthood knockout of Freud-1 in 5-HT neurons results in up-regulation of 5-HT1A autoreceptors, reduced raphe 5-HT levels, and an anxiety and depression phenotype that is resistant to SSRI treatment [179]. Thus, agents that inhibit CAMKII activation, particularly in raphe cells, could correct the 5-HT1A autoreceptor over-expression and promote more rapid and effective response to SSRI treatment.
4.Conclusion
The above findings indicate that while the 5-HT1A receptor is coupled to canonical Gi/Go- mediated inhibition of AC, it can also couple to a diversity of other signaling pathways. These additional pathways differ for 5-HT1A autoreceptors vs. heteroreceptors, and by the specific cell type, brain region (hippocampus vs. prefrontal cortex), and developmental time point (adult vs. adolescent, childhood, post-natal or embryonic). Furthermore, 5-HT1A autoreceptors appear more sensitive to 5-HT induced desensitization than heteroreceptors. Results from gene knockout and transgenic approaches indicate that specific signaling Darovasertib pathways in specific cell types may be critical for antidepressant responses, and developing new ligands that target these processes shows promise for the generation of new, more effective antidepressant treatments.