The role of the serotonin receptor subtypes 5-HT1A and 5-HT7 and its interaction in emotional learning and memory

Serotonin [5-hydroxytryptamine (5-HT)] is a multifunctional neurotransmitter innervating cortical and limbic areas involved in cognition and emotional regulation. Dysregulation of serotonergic transmission is associated with emotional and cognitive deficits in psychiatric patients and animal models. Drugs targeting the 5-HT system are widely used to treat mood disorders and anxiety-like behaviors. Among the fourteen 5-HT receptor (5-HTR) subtypes, the 5-HT1AR and 5-HT7R are associated with the development of anxiety, depression and cognitive function linked to mechanisms of emotional learning and memory. In rodents fear conditioning and passive avoidance (PA) are associative learning paradigms to study emotional memory. This review assesses the role of 5-HT1AR and 5-HT7R as well as their interplay at the molecular, neurochemical and behavioral level. Activation of postsynaptic 5-HT1ARs impairs emotional memory through attenuation of neuronal activity, whereas presynaptic 5-HT1AR activation reduces 5-HT release and exerts pro-cognitive effects on PA retention. Antagonism of the 5-HT1AR facilitates memory retention possibly via 5-HT7R activation and evidence is provided that 5HT7R can facilitate emotional memory upon reduced 5-HT1AR transmission. These findings highlight the differential role of these 5-HTRs in cognitive/emotional domains of behavior. Moreover, the results indicate that tonic and phasic 5-HT release can exert different and potentially opposing effects on emotional memory, depending on the states of 5-HT1ARs and 5-HT7Rs and their interaction. Consequently, individual differences due to genetic and/or epigenetic mechanisms play an essential role for the responsiveness to drug treatment, e.g., by SSRIs which increase intrasynaptic 5-HT levels thereby activating multiple pre- and postsynaptic 5-HTR subtypes.


Introduction
Serotonin (5-HT) is a biogenic amine acting as a neurotransmitter and neuromodulator. The distribution of serotonin-containing neurons in the CNS have been studied in different species and have been found to be localized exclusively in the brainstem (Hunt and Lovick, 1982;Takahashi et al., 1986;Ishimura et al., 1988). The majority of the serotonergic cell bodies reside in the dorsal and median raphe nuclei but send axons almost to the entire brain, including cortical, limbic, midbrain, and hindbrain regions (Charnay and Léger, 2010). As expected from the wide projection pattern of the 5-HT neurons, serotonin modulates variable physiological functions, such as sleep, arousal, feeding, temperature regulation, pain, emotions, and cognition (Bradley et al., 1986;Barnes and Sharp, 1999;Ögren et al., 2008;Berger et al., 2009;Artigas, 2015).
The pleiotropic behavioral effects of 5-HT are mediated by a family of at least 14 5-HTR subtypes (Hoyer et al., 1994). These 5-HTR subtypes are distributed in a brain-and cell-specific manner and regulate distinct physiological processes, through different and sometimes opposing signaling pathways (Hoyer and Martin, 1997;Hoyer et al., 2002).
The 5-HT 1A R is one of the best-studied 5-HTR subtypes due to its implication in anxiety-like behaviors (Heisler et al., 1998;Parks et al., 1998;Toth, 2003), in depression (Lucki, 1991) as well as in cognitive processes that are impaired in several psychiatric disorders (review by Ögren et al., 2008;Millan et al., 2012). Its potential role as a drug target has been also investigated (Tunnicliff, 1991;Den Boer et al., 2000;Blier and Ward, 2003). The most common antidepressants, the SSRIs, act by targeting the 5-HT 1A R (Hervas and Artigas, 1998;Artigas, 2015), supporting the key role of the 5-HT 1A R in the pathophysiology of mood disorders.  Rs are implicated in depression and anxiety, and evidence has been provided for their role in learning and memory (reviewed by Leopoldo et al., 2011). Interestingly, the 5-HT 7 R and 5-HT 1A R exert opposing roles in the modulation of fear learning (Eriksson et al., , 2012, pointing at the importance of both 5-HTR subtypes and their signaling interaction in the regulation of emotional learning. After a brief introduction about the characteristics of 5-HT 1A and 5-HT 7 R (distribution, signaling, and ligands), this review will focus on the role of 5-HT 1A R, 5-HT 7 R as well as its interplay in emotional learning processes. The interaction between the 5-HT 1A R and 5-HT 7 R signaling will be discussed and results of studies using different available 5-HT 1A R and 5-HT 7 R ligands on fear learning tasks are summarized. A considerable extent of this review will also be dedicated to describe the regionspecific effects of 5-HT 1A R and 5-HT 7 R, via local rather than systemic administration. Overall, the aim of this review is to draw general conclusions about the role of both 5-HT 1A R and 5-HT 7 R in fear learning, which may contribute to our better understanding of the mechanisms underlying dysregulated learning and memory in affective disorders. The focus here is on fear learning because this one-trial learning task allows for exact timing of pharmacological manipulations to discriminate between different memory phases.

5-HT 1A Receptor Localization
5-HT 1A R was the first 5-HTR subtype to be cloned and is characterized by its high affinity for 5-HT (Nichols and Nichols, 2008). 5-HT 1A Rs are widely distributed throughout the CNS and are present in both pre-and postsynaptic sites. Presynaptically, 5-HT 1A Rs are exclusively located on the cell bodies and dendrites of 5-HT neurons in the dorsal and median raphe nuclei (Riad et al., 2000) and function as 5-HT 1A autoreceptors which tightly regulate 5-HT neuronal activity.
Postsynaptically, the highest level of 5-HT 1A R is found in the limbic system based on receptor autoradiography and mRNA expression. Both techniques showed the distribution of the 5-HT 1A R in the lateral septum, cingulate and entorhinal cortices, with particularly high expression in the hippocampus (reviewed by Hannon and Hoyer, 2008). At the cellular level, the postsynaptic 5-HT 1A R is expressed in cortical pyramidal neurons as well as pyramidal, GABAergic and granular cells of the hippocampus (Hannon and Hoyer, 2008). At least in the hippocampal formation, the 5-HT 1A R is located on somata and dendrites of pyramidal and granular neurons, as well as on the dendritic spines of pyramidal neurons (Riad et al., 2000). Moreover, 5-HT 1A R immunoreactivity has been demonstrated in different subgroups of neurons in the septal complex with GABAergic septohippocampal parvalbumincontaining projection neurons, GABAergic calbindin D-28containing neurons as well as cholinergic septohippocampal neurons (Lüttgen et al., 2005a). This indicates that systemic administration of 5-HT 1A R ligands can modify hippocampal function through effects on septohippocampal neurons that are responsible for the theta rhythm which plays an important role in memory functions (Elvander-Tottie et al., 2009).

5-HT 1A Receptor Signaling
Activation of 5-HT 1A R leads to neuronal hyperpolarization, an effect mediated by pertussis-toxin-sensitive Gα i/o proteins. Gα i/o proteins are negatively coupled with the signaling pathway of adenylyl cyclase and thereby decrease the cAMP formation (De Vivo and Maayani, 1986;Weiss et al., 1986). Despite their high density in the dorsal raphe nucleus, 5-HT 1A autoreceptors do not seem to inhibit AC, but mediate neuronal inhibition through different signaling pathways (Clarke et al., 1996). Both post-and presynaptic 5-HT 1A Rs inhibit neuronal firing via the activation of G protein-coupled inwardly rectifying potassium channels as well as the inhibition of Ca 2+ channels (Sodickson and Bean, 1998;Bockaert et al., 2006). A multitude of other signaling pathways and effectors has been also linked to the activation of the 5-HT 1A R (reviewed by Raymond et al., 2001;Bockaert et al., 2006).

5-HT 7 R Localization
The 5-HT 7 R was the last 5-HTR subtype to be cloned by using a targeted screening analysis of mammalian cDNA libraries and probes from already known receptors (Bard et al., 1993;Lovenberg et al., 1993;Ruat et al., 1993). Although 5-HT 7 Rs demonstrate a high interspecies homology (>90%; To et al., 1995), they share a low homology with the other 5-HTR subtypes (<50%; Bard et al., 1993). Northern blot analysis and in situ hybridization studies demonstrate high expression of 5-HT 7 R in the CNS and particularly in the hypothalamus (suprachiasmatic nucleus), thalamus, hippocampus, and cerebral cortex (Bard et al., 1993;Lovenberg et al., 1993;Ruat et al., 1993). Like 5-HT 1A R, the 5-HT 7 R is also localized in the raphe nuclei in both rodent and human brain, which has raised questions about its role in the regulation of 5-HT levels (Martin-Cora and Pazos, 2004). At the neuronal level, 5-HT 7 R is expressed in hippocampal CA pyramidal neurons with a higher density in CA3 than in CA1 (Bonaventure et al., 2004) and a differential expression, with selective localization on the cell bodies in CA1 pyramidal neurons (Bickmeyer et al., 2002). Little is known, however, about the expression patterns of 5-HT 7 R in cortical neurons, where it is suggested that 5-HT 7 R may have a role during the developing stages of cortical circuits (Béïque et al., 2007;Celada et al., 2013).

5-HT 7 Receptor Signaling
5-HT 7 R activation activates adenylyl cyclase signaling and consequently the conversion of ATP to cAMP through coupling to Gα s (Bard et al., 1993;Lovenberg et al., 1993;Ruat et al., 1993). Although cAMP activation is commonly mediated by the PKA, it has been demonstrated that Epac, a member of the cAMPregulated guanine nucleotide exchange family, has a crucial role in PKA-independent signaling (Lin et al., 2003). For instance, 5-HT 7 Rs activate the MAPK/ERK signaling pathway (Errico et al., 2001;Norum et al., 2003) via the stimulation of the Epac factor (Lin et al., 2003). Binding of cAMP to Epac leads to the activation of several other signaling pathways (reviewed by Holz et al., 2006).

Functional Roles of 5-HT 1A R and 5-HT 7 Receptors
The expression of 5-HT 1A R and 5-HT 7 R in the limbic system (Hannon and Hoyer, 2008;Berumen et al., 2012) support a role in the modulation of functions like mood, memory processing as well as emotional association with memory. The 5-HT 1A R has been proposed to modulate anxiety based on studies with 5-HT 1A R knockout mice (Heisler et al., 1998;Parks et al., 1998;Toth, 2003) and the response to antidepressant drugs (Blier and Ward, 2003;Artigas, 2015). Several partial 5-HT 1A R agonists, e.g., buspirone, have been used to treat anxiety and depression (Tunnicliff, 1991;Den Boer et al., 2000), whereas co-administration of pindolol (β-adrenergic and 5-HT 1A R antagonist) with SSRIs enhances their therapeutic efficacy and shortens their onset of action (reviewed by Artigas et al., 2001). A considerable body of literature demonstrates the 5-HT 1A R involvement in various hippocampus-dependent learning and memory tasks (reviewed by Ögren et al., 2008).
In contrast, the available data on the function of 5-HT 7 R is relatively limited, mainly due to the lack of selective agonists specific for this 5-HTR subtype (Misane and Ögren, 2000;Nichols and Nichols, 2008;Leopoldo et al., 2011). The physiological role of 5-HT 7 R has been closely linked with the regulation of sleep, circadian rhythm, pain and also mood (reviewed by Leopoldo et al., 2011). Accumulating data implicates the 5-HT 7 R in the action of antidepressant drugs, whereas the results from anxiety studies are contradictory (Leopoldo et al., 2011). Interestingly, studies using 5-HT 7 R knockout mice revealed the crucial role of this receptor in hippocampus-dependent memory (Roberts et al., 2004;Sarkisyan and Hedlund, 2009).

5-HT 1A and 5-HT 7 Receptor Ligands General Receptor Ligand Principles
Agents that act as receptor ligands may be agonists or antagonists. Agonists initiate physiological changes by activating downstream signaling pathways, whereas antagonists bind to receptors without producing any effect (Rang et al., 2015). Ligands can be divided in three categories based on their function: (1) Full agonists produce a maximal response equivalent to the endogenous agonist (here 5-HT). These agonists have high efficacy (i.e., the ability to initiate changes which leads to effects) for the binding receptor.
(2) Partial agonists are not capable of producing the maximal functional response even when they occupy the entire receptor population. These agonists present intermediate efficacy.
Respectively, we could refer to partial antagonists that bind to the active site (competitive antagonism) but do not completely abolish the receptor-mediated effects. (3) Mixed profile ligands that (appear to) act both as agonists and as antagonist in distinct receptor populations. More likely, they have different agonist profiles at different receptor sites (e.g., pre-versus postsynaptic 5-HT 1A R) and therefore appear to exert antagonist function in the presence of a full agonist, while acting as weak (partial) agonist thereby lowering the efficacy of the full agonist.
The function of any ligand used to study the role of 5-HT 1A R and 5-HT 7 R is essential for the correct interpretation of the behavioral outcome. It is also important to mention that the intrinsic efficacy of a ligand is equally depended on the characteristics of response system; in our case the different brain populations of 5-HT 1A R and 5-HT 7 R and their downstream signaling pathways. Agonists acting on the same receptor can produce different effects depending on their physicochemical properties, brain distribution, full or partial agonism as well as the number of coupled receptors in a brain area. The specificity of the compounds used is another very important characteristic that should be always taken into consideration and is referred to the ligand's specific binding to the targeted receptor. Ligands with low specificity cannot be used to clarify the functional role of 5-HT 1A R and 5-HT 7 R, since the produced effects can be also mediated via the binding to other proteins than the receptor of interest.
The physicochemical properties of compounds play an essential role for the drug uptake and diffusion with lipophilicity, solubility and molecular mass being among the most important properties (Waterhouse, 2003). The lipophilic nature of ligands is particularly important when they are administered locally. Increasing lipophilicity leads to enhanced blood-brain barrier diffusion, prevents the drug restriction in the area of interest and consequently produces wider effects, despite local application. This is evident from dorsohippocampal infusion of the bloodbrain barrier penetrating drug 8-OH-DPAT, a full 5-HT 1A R agonist, which impairs tone-dependent memory (Stiedl et al., 2000a), whereas this does not occur when the NMDAR antagonist APV (Stiedl et al., 2000b) and the GABA A R agonist muscimol are locally applied . The latter study is one of the few demonstrating the selective drug action in the dorsal hippocampus based on fluorescently labeled muscimol as bodipy conjugate. Besides the solubility of compounds and the applied dose, it is thus of high importance to consider other physicochemical properties, such as half-life in vivo, to avoid misleading conclusions due to their wider spread (e.g., diffusion or potential active transport) in brain outside the target sites. The molecular weight of compounds can also provide valuable information about the diffusion capacity.

5-HT 1A Receptor Agonists
The prototypic 5-HT 1A R agonist 8-OH-DPAT was the first full agonist developed (Arvidsson et al., 1981;Gozlan et al., 1983) and is still the most widely used to study the functional role of 5-HT 1A R in behavioral manipulations (Barnes and Sharp, 1999). Despite its high selectivity for the 5-HT 1A R, 8-OH-DPAT also acts as a 5-HT 7 R agonist (Bickmeyer et al., 2002;Eriksson et al., 2008) and observed effects can be the result of an interplay between the two receptor subtypes (see below).
Additionally, several full and partial agonists have been synthesized (see Table 1), but only a few of them have been used in fear learning studies, such as the buspirone and tandospirone. Buspirone belongs to the arylpiperazine (partial) agonists (Hjorth  , 1982) and acts also as antagonist with high specificity for the dopamine D 2 receptor (Witkin and Barrett, 1986). Tandospirone (SM-3997) is a 5-HT 1A R partial agonist and was initially studied for its anxiolytic properties in rats and mice (Shimizu et al., 1987). Similar to buspirone, tandospirone also exhibits dopamine antagonist action with a potency that is considerably lower than the one for the 5-HT 1A R (Shimizu et al., 1987). An overview of currently available 5-HT 1A R agonists is provided in Table 1.
The agents that were initially used as 5-HT 1A R antagonist were 2-methoxyphenylpiperazine derivatives with structural similarity to buspirone, such as BMY-7378 and NAN-190 (Greuel and Glaser, 1992). However, these ligands were characterized as partial 5-HT 1A R antagonist with antagonist properties only at the postsynaptic HT 1A R and lower affinity for the α-adrenergic receptors (Greuel and Glaser, 1992).
Finally, S-15535 is reported to act as a postsynaptic 5-HT 1A R antagonist while also behaving as an agonist on presynaptic 5-HT 1A autoreceptors, and therefore, it is characterized as a mixed profile ligand (Millan et al., 1993;Carli et al., 1999). However, a more recent study indicates predominantly weaker agonist activity of S-15535 at postsynaptic 5-HT 1A Rs (Youn et al., 2009). An overview of currently available 5-HT 1A R antagonists is provided in Table 2.

5-HT 7 Receptor Agonists
The lack of selective and potent 5-HT 7 R agonists (Misane and Ögren, 2000;Leopoldo, 2004;Leopoldo et al., 2011) is one of the major limitations to study the role of 5-HT 7 R in learning and memory. Currently, only a few selective 5-HT 7 R agonists exist and even less has been used in learning and memory studies. AS-19 and LP-44 are highly selective but low efficacy (partial) HT 7 R agonists whose functional role in fear learning was recently assessed (Eriksson et al., 2012). LP-211 is a novel highly selective 5-HT 7 R agonists  but it has so far only been tested in an autoshaping Pavlovian/instrumental learning task (Meneses et al., 2015). An overview of currently available 5-HT 7 R agonists is provided in Table 3.

5-HT 7 Receptor Antagonists
SB-258719 is the first selective 5-HT 7 R antagonist described (Forbes et al., 1998) but has not yet been used to investigate the role of 5-HT 7 R in the modulation of emotional learning. Both SB-656104-A and SB-269970 possess high potency and selectivity for 5-HT 7 R (Lovell et al., 2000;Thomas et al., 2002Thomas et al., , 2003. These are the most commonly used 5-HT 7 R antagonists in behavior studies. An overview of currently available 5-HT 7 R antagonists is provided in Table 3.

Behavioral Tasks for the Assessment of Emotional Learning and Memory
The experimental studies on emotional learning and memory in animals are based originally on psychological analysis of conflict behavior involving approach and avoidance of conditioned stimuli. Traditionally, the assays used to investigate animal behavior are based on the association of pleasant (i.e., motivationally related reward like food) or aversive stimuli (i.e., conditions related to negative feelings like pain and danger) to environmental cues involving classical (Pavlovian) or instrumental conditioning (Ögren and Stiedl, 2015).
The FC and the PA tasks are the most commonly used associative learning paradigms based on contextual fear learning. This type of learning is dependent on the operation of neuronal circuits in the limbic system, such as hippocampus and amygdala (Cahill and McGaugh, 1998;LeDoux, 2000) as demonstrated by us in mice (e.g., Stiedl et al., 2000a,b;Baarendse et al., 2008). Unlike FC, PA also includes instrumental learning. In the step-through PA test, the animal needs to suppress its innate preference for the dark compartment (where it previously received a foot shock) and remain in the bright compartment. In the step-down PA paradigm, however, the retention is examined in the dark compartment, where the animal received the foot shock (unconditioned stimulus) after stepping down from an elevated platform. The PA test procedure can be modified to examine any facilitating effect of the treatment on PA retention (Madjid et al., 2006). More specific information on the PA task is provided elsewhere (Ögren and Stiedl, 2015). A refined version of this task may provide for better translational aspects to assess pathological fear states such as post-traumatic-like responses based on deliberate choice of mice (Hager et al., 2014).
The single-trial learning design of FC and PA, which is sufficient to establish long-term and remote memory, allows the exact timing of the drug treatment in relation to training and retention test. Thereby, unlike multi-session tasks, onetrial tasks provide a unique advantage to study learning mechanisms as well as drug effects (here 5-HT 1A R and 5-HT 7 R ligands) on the different phases of learning and memory, i.e., the acquisition phase that consists of encoding and early consolidation, consolidation, the recall (retrieval and expression) phase as well as the extinction phase and reconsolidation.

Effects of 5-HT 1A Receptor Ligands in Emotional Learning and Memory
An overview of the behavioral effects of various 5-HT 1A R ligands is provided in Table 4.

Systemic 5-HT 1A Receptor Ligand Effects
Despite the differences among the 5-HT 1A R ligands in their chemical and pharmacological features (e.g., receptor selectivity and partial or full agonist properties; see Tables 1 and 2), there is strong evidence for the impairing effect of postsynaptic 5-HT 1A R activation on fear memory. Systemic, pretraining administration of the full 5-HT 1A R agonist 8-OH-DPAT shows a biphasic effect on PA performance, with the low dose range (0.01, 0.03 mg/kg) facilitating and the high dose range (0.1-1 mg/kg) impairing PA retention 24 h after training in both rats (Misane and Ögren, 2000;Lüttgen et al., 2005b) and mice (Madjid et al., 2006). The impairing dose of 8-OH-DPAT (0.2 and 0.3 mg/kg) also induces signs of the serotonin syndrome (Carli et al., 1992;Lüttgen et al., 2005b) linking the postsynaptic 5-HT 1A R to the learning deficits. In line with these results, FC studies demonstrated that pretraining systemic injections of high doses (0.1-0.5 mg/kg) of 8-OH-DPAT impair fear learning (Stiedl et al., 2000a;Youn et al., 2009). Pretreatment with the selective 5-HT 1A R antagonist WAY-100635 (0.03-1 mg/kg) blocked the impairment in freezing (FC) and transfer latency (PA), confirming and extending the detrimental role of the postsynaptic 5-HT 1A R activation on memory acquisition. The observed memory deficit was already present in shortterm memory tests performed 1 h after training for FC retention (Stiedl et al., 2000a) and 5 min after PA training (Misane and Ögren, 2000). Thus, postsynaptic 5-HT 1A R activation specifically impairs memory encoding of the aversive experience and not memory consolidation. In agreement to that observation, immediate 8-OH-DPAT post-training administration did not alter PA or FC retention (Misane and Ögren, 2000;Madjid et al., 2006).
but not post-training intra-hippocampal infusion of 8-OH-DPAT impairs contextual FC (Stiedl et al., 2000a), pointing at the important role of the postsynaptic 5-HT 1A R in acquisition processes as observed after systemic administration.

Effects of 5-HT 1A Receptor Agonists and Antagonists on Memory Recall Systemic 5-HT 1A Receptor Ligand Effects
Unlike the unambiguous implication of the postsynaptic 5-HT 1A R in memory acquisition, its role in fear retrieval and expression is less clear. The systemic 5-HT 1A R agonist NDO-008 (0.5 mg/kg) administered before the retention test to rats impairs slightly PA performance (Misane et al., 1998). In contrast, systemic administration of buspirone at the dose of 1 and 3 mg/kg had no effect on fear expression in mice . These different effects may partly depend on the readouts and the side effects elicited by higher 5-HT 1A R dosages, such as the hypolocomotion induced together with the serotonin syndrome (Stiedl et al., 2000a). The hypolocomotion confounds the interpretation of fear expression results in mice when based on freezing. Moreover, it also possible that differences exists between rats and mice, although our own data shows high similarity of results in these two species. Therefore, a recent study tried to clarify the role of the 5-HT 1A R in fear recall, by assessing the effect of 8-OH-DPAT on fear-conditioned HR responses (reviewed by Stiedl et al., 2009) upon training and 24 h after training, in mice (Youn et al., 2013). Systemic pretest administration reduced the conditioned maximum HR as a consequence of the significantly reduced baseline HR before the presentation of the conditioned stimulus (tone). However, the tone-induced HR increase was preserved during the retention of auditory fear in mice with similar magnitude as compared to that in controls. Additionally, 8-OH-DPAT reduced the unconditioned tachycardia elicited by novelty exposure as a consequence of altered HR dynamics indicating autonomic dysregulation with enhanced parasympathetic function through postsynaptic 5-HT 1A R activation (Youn et al., 2013). Thus, the claims of anxiolytic actions of pretest injection of 5-HT 1A R agonists as initially reported in human studies and partly in animal models cannot be supported unambiguously at least in learned fear experiments.  DD, dose-dependent, FC, fear conditioning; i.h., intrahippocampal; i.p., intraperitoneal, i.v., intravenous; M, mice; n.a., not available; PA, passive avoidance; post-tr., post-training; p.o., per os; pretr, before training; R, rats; s.c., subcutaneous.

Local 5-HT 1A Receptor Ligand Effects
Local administration approaches tried to distinguish the role of the post-versus the presynaptic 5-HT 1A R in the different aspects of fear expression. Bilateral microinjections of a selective 5-HT 1A R agonist flesinoxan decreased the expression of conditioned contextual freezing when injected into the hippocampus or amygdala but not in the medial prefrontal cortex (Li et al., 2006), as well as the fear-potentiated startle responses when infused into the central amygdala (Groenink et al., 2000). The role of 5-HT 1A autoreceptors in fear expression was also studied by pretest infusion of 8-OH-DPAT into the median raphe nuclei. This resulted in impaired contextual freezing responses (Borelli et al., 2005;Almada et al., 2009), but not fear-potentiated startle (Groenink et al., 2000;Almada et al., 2009) suggesting the existence of raphe-dependent serotonergic regulation that appears to modulate the freezing response to the aversive context. In contrast, hippocampal 8-OH-DPAT impaired the expression of both contextual freezing and fear-potentiated startle (Almada et al., 2009). However, 8-OH-DPAT mediates hyperlocomotion in rats (but hypolocomotion in mice) leading to a similar problem of potentially confounded interpretation of freezing performance during the drug state as mentioned before for mice.

Effects of 5-HT 1A Receptor Agonists and Antagonists on Memory Extinction
In contrast to the well-studied implication of 5-HT 1A Rs on memory acquisition and recall, there is only one study with 5-HT 1A R ligands on fear extinction. The systemic 5-HT 1A R agonist buspirone abolishes the fear extinction in mice . Similarly, the systemic 5-HT 1A R antagonist WAY-100635 before a second sampling trial impaired the extinction of object recognition memory in rats (Pitsikas et al., 2003). Further studies are needed to determine the precise role of 5-HT 1A Rs in memory extinction and/or reconsolidation in emotional learning tasks. Furthermore, local rather than systemic approaches are necessary to identify the neurocircuitry involved in these processes. The roles of other 5-HTRs in fear learning and the consequences of altered 5-HT neurotransmission on fear extinction are reviewed by Homberg (2012).

Effects of 5-HT 7 Receptor Agonists and Antagonists on Emotional Learning Systemic 5-HT 7 Receptor Ligand Effects
The paucity of studies 5-HT 7 R functions on emotional learning is mainly due to the lack of selective ligands, especially agonists (Misane and Ögren, 2000;Leopoldo, 2004;Leopoldo et al., 2011; see Table 5 and text above). Recent data from an autoshaping task showing that the 5-HT 7 R agonist, LP-211, when administered systematically after the training session, reversed scolopamineinduced amnesia, in rats (Meneses et al., 2015). The same group also shows a facilitating effect on memory formation by the 5-HT 7 R agonist AS-19 administered after an autoshaping training session (Perez-García and Meneses, 2005). The enhancing effect of 5-HT 7 Rs on memory consolidation was blocked by preinjection of the 5-HT 7 R antagonist SB-269970 (Perez-García and Meneses, 2005;Meneses et al., 2015) indicating the specific involvement of the 5-HT 7 R. Eriksson et al. (2008) investigated the role of 5-HT 7 R on emotional learning in mice using a step-through PA paradigm. Pretraining systemic administration of the 5-HT 7 R antagonist SB-269970 enhanced the impairing effect of low doses of 8-OH-DPAT . This result supports the notion that 5-HT 7 R activation has a beneficial modulatory role in learning opposing the function of 5-HT 1A R activation. Accordingly, pretraining 5-HT 7 R activation by the combined use of the 5-HT 1A R antagonist NAD-299 with the 5-HT 1A R and 5-HT 7 R agonist 8-OH-DPAT facilitated PA retention (Eriksson et al., 2012). This PA facilitation by NAD-299 together with 8-OH-DPAT was again blocked by the 5-HT 7 R antagonist SB-269970 indicating a procognitive effect of 5-HT 7 R activation by this drug combination. However, the 5-HT 7 R agonists LP-44 and AS-19 failed to mediate this PA facilitation, despite dose-dependent tests. Despite their high in vitro potency to stimulate intracellular signaling cascades (Eriksson et al., 2012), the 5-HT 7 R agonists LP-44 and AS-19 have moderate agonist efficacy in vivo. This finding is in agreement with previous pharmacological characterization ( Monti et al., 2008;Bosker et al., 2009;Brenchat et al., 2009) in vivo and may explain why the facilitatory effect of NAD-299 with 8-OH-DPAT could not be mimicked by the putative agonists LP-44 and AS-19.

Local 5-HT 7 Receptor Ligand Effects
To further address the role of 5-HT 7 Rs on emotional learning, Eriksson et al. (2012) performed hippocampal infusions with the 5-HT 7 R agonist AS-19 in mice. Since they failed to find clear facilitatory effects, as observed after systemic treatment, they concluded that "5-HT 7 Rs appear to facilitate memory processes in a broader cortico-limbic network and not the hippocampus alone." The failure of the SB-269970 to enhance emotional memory, upon hippocampal infusions, may be the consequence of the low dose that can be locally infused due to the relatively poor solubility of SB-269970. However, systemic administration of this 5-HT 7 R antagonist fully blocked the PA facilitation observed after 5-HT 1A R blockade. Hence, the hippocampus-dependent involvement of the 5-HT 7 Rs needs to be re-investigated with selective highly potent 5-HT 7 R agonists, because also the low potency of AS-19 (Eriksson et al., 2012) may have contributed to the lack of effects by dorsohippocampal 5-HT 7 R agonist application on PA. Finally, although the role of 5-HT 7 R in memory consolidation has been suggested, there are currently insufficient data supporting this view. More work is also required to clarify the role of 5-HT 7 R in memory extinction and reconsolidation, which are both essentially unexplored.
The Interplay of the 5-HT 1A and 5-HT 7 for Emotional Learning The interaction of the two 5-HTR subtypes in emotional learning has been studied by using 8-OH-DPAT, which exerts agonistic effects for both 5-HT 1A Rs and 5-HT 7 Rs. To dissect the function of these 5-HTRs, pre-treatment with selective 5-HT 1A R antagonists is used to exclusively activate 5-HT 7 R. Eriksson et al. (2008) were the first to suggest the functional interplay between the two 5-HTRs on the behavioral level as the activation of 5-HT 7 R counteracted the 5-HT 1A R-mediated impairments in PA performance. The interaction between the two 5-HTRs and their functional antagonism was then extended by experiments in mice, demonstrating that 5-HT 7 R activation and concomitant 5-HT 1A R blockade leads to PA facilitation (Eriksson et al., 2012). The facilitatory effect on emotional memory by the 5-HT 1A antagonist NAD-299 was related to stimulation of 5-HT 7 Rs under conditions with reduced 5-HT 1A R transmission. These findings suggest that the states of 5-HT 1A Rs and 5-HT 7 Rs play a critical role for 5-HT effects on emotional memory. Consequently, the elevation of endogenous 5-HT via SSRIs will most likely result in differential cognitive/emotional effects depending on genetic and/or epigenetic regulation and occupancy of these two 5-HTRs in health and disease. This condition will affect the expression of the 5-HT 1A R and change the relative balance between 5-HTR subtypes, which together will A, anxiety tests; DD, dose dependent, FC, fear conditioning; i.h., intrahippocampal; i.p., intraperitoneal, i.v., intravenous; M, mice, MSRAP, ; post-tr., post-training; p.o., per os; pretr, before training; R, rats; s.c., subcutaneous. eventually determine the physiological actions of 5-HT and the clinical efficacy of SSRI treatment.
Mechanisms Underlying the Functional Interaction of 5-HT 1A R and 5-HT 7 R As described above, 5-HT 1A Rs and 5-HT 7 Rs mediate opposing effects regarding the neuronal excitability. 5-HT 1A R activation reduces the activity of adenyl cyclase, whereas 5-HT 7 R activation stimulates adenyl cyclase activity and thereby increases intracellular cAMP thereby increasing neuronal excitability (Bockaert et al., 2006;Nichols and Nichols, 2008;Berumen et al., 2012). Accordingly, 5-HT 7 R stimulation in the hippocampus was found to activate pyramidal neurons, unlike 5-HT 1A R activation which inhibited pyramidal neurons (Bickmeyer et al., 2002). Both 5-HTRs are expressed in glutamatergic hippocampal pyramidal neurons (Bockaert et al., 2006;Nichols and Nichols, 2008;Berumen et al., 2012). Therefore, it is likely that 5-HT 1A R and 5-HT 7 R stimulation decreases and increases glutamate release in the hippocampus, respectively. In line with these results, 5-HT 7 R activation enhances the AMPA receptormediated synaptic currents on CA1 pyramidal neurons, whereas 5-HT 1A R activation inhibits the AMPA receptor-mediated transmission between CA3 and CA1 pyramidal neurons in both pre-and postsynaptic sites (Costa et al., 2012). However, the 5-HT 1A R-mediated inhibitory effect on glutamatergic neurotransmission was stronger than the 5-HT 7 R-mediated facilitatory effect (Costa et al., 2012). One explanation for the increased effectiveness of 5-HT 1A R in controlling the input from the Schaffer collaterals may stem from the different localization of the two receptors on the CA1 pyramidal neurons: 5-HT 7 Rs are found on the cell bodies (Bickmeyer et al., 2002), whereas the 5-HT 1A Rs appear to be mainly localized on dendrites (Kia et al., 1996). Differences in the expression of the receptors could also play an essential role in their distinct activation pattern from the endogenous 5-HT. The progressive reduction of post-synaptic 5-HT 7 R levels during postnatal development, together with the maintenance of the expression level of 5-HT 1A R (Kobe et al., 2012;Renner et al., 2012), could increase the ratio of membrane 5-HT 1A Rs over  Rs. Consequently, a model has been proposed regarding the molecular mechanisms that underlie the regulation of the 5-HT 1A Rs and 5-HT 7 Rs. 5-HT 1A R and 5-HT 7 R form heterodimers both in vitro and in vivo . This heterodimerization plays a functional role by decreasing G i protein coupling of the 5-HT 1A R and by reducing the ability of 5-HT 1A R to activate potassium channels, without affecting the G s protein coupling of the 5-HT 7 R. The heterodimerization additionally contributes to the desensitization of the 5-HT 1A R through facilitated internalization . 5-HT 1A R and 5-HT 7 R are co-localized in the cell membrane of hippocampal neurons, where their heterodimerization induces an inhibitory effect on the 5-HT 1A R-mediated activation of potassium channels in hippocampal neurons . As mentioned above the post-synaptic levels of 5-HT 7 R are lower compared to the expression levels of post-synaptic 5-HT 1A R, whereas this is not the case for the pre-synaptic 5-HT 7 R ). These regional differences in the 5-HT 7 R levels and therefore in the concentration of the heterodimers, can explain the preferential desensitization of 5-HT 1A autoreceptors by SSRIs and more generally the regionand cell-specific differences in the signaling pathway mediated by the 5-HT 1A R activation (see Naumenko et al., 2014). In summary, the above data suggest that the positive or negative consequences of a drug on emotional memory and cognition depend on the relative level of 5-HTR expression and, its efficacy in activating different receptors with their downstream signaling pathways.

Genetic and Epigenetic Effects on 5-HT Transmission and Receptor Expression
Genetic and/or epigenetic effects regulate the receptor's state and eventually define the physiological actions of endogenous 5-HT. A characteristic example is the Ala50Val variant of the 5-HT 1A R, located in the transmembrane region 1, that leads to loss of response to 5-HT and consequently to the interruption of 5-HT signaling (Del Tredici et al., 2004). Moreover, the human polymorphism Gly22Ser attenuates the downregulating effect induced by long-term 8-OH-DPAT stimulation in comparison to the Val28 variant and wild-type without effect on the ligand binding capacity (Rotondo et al., 1997). It is suggested that individuals with the Ser22 variant have higher sensitivity to SSRIs treatment since its serotonergic effect depends on the efficiency of 5-HT 1A R transmission (Rotondo et al., 1997). Furthermore, carriers of the short (s) allele of the 5-HT transporter promotor region possess behavioral abnormalities, such as increased levels of anxiety and FC as well as stronger fear potentiated startle (Bauer, 2014) in comparison to long (l) allele carriers. Accordingly, the therapeutic efficacy of SSRIs is reduced in patients homozygous for the s-allele when compared with heterozygous or l-allele carriers (Tomita et al., 2014).
The epigenetic regulation of 5-HTR subtypes is also implicated in the differential emotional and cognitive modulation induced by the serotonergic signaling. It is widely accepted that 5-HT 1A R binding is reduced in the brain of depressed humans (e.g., Savitz et al., 2009) as well as in stressed rats (e.g., Choi et al., 2014) as indication of epigenetic modulation. 5-HT 1A R activation in the basolateral amygdala and the prelimbic area of the prefrontal cortex in low-anxious rats reduced fear potentiated startle, whereas 5-HT 1A R activation in the periaqueductal gray of high-anxious rats had the opposite effect (Ferreira and Nobre, 2014). These findings highlight how environmental conditions can contribute to individual differences in 5-HT 1A R-mediated response differences. In line with this, single-housed mice display a stronger hypothermic effect upon 5-HT 1A R activation by 8-OH-DPAT, which is associated with an increased depressivelike state, in comparison to their group-housed counterparts (Kalliokoski et al., 2014). However, the mechanisms underlying the inter-individual differences in serotonergic signaling and consequently in cognitive and emotional modulation are not clear yet.
A linkage disequilibrium study identified two polymorphisms (rs3808932 and rs12412496) in the human HTR7 suggesting that it is a schizophrenia susceptibility gene (Ikeda et al., 2006). However, to the best of our knowledge, there is no evidence for the effect of 5-HT 7 R polymorphisms on serotonergic signaling or the interaction between polymorphisms of 5-HT 7 and 5-HT 1A Rs. Therefore, to elucidate the functional interaction between 5HT 1A R and 5-HT 7 R, it is of high importance to understand which polymorphisms influence the expression of those 5-HTRs and how these changes affect emotional and cognitive functions. This knowledge could potentially reveal the polymorphisms that modulate the endophenotypes of different affective disorders, closely linked with the function of 5-HT 1A R and 5-HT 7 R, such as anxiety and depression.

Neurochemical Effects in the Hippocampus
In contrast to the above electrophysiological results, in vivo microdialysis in awake rats showed that the local blockade of 5-HT 1A R increased extracellular acetylcholine (ACh) levels (Madjid et al., 2006;Hirst et al., 2008;Kehr et al., 2010) but failed to show changes in hippocampal glutamate release in the ventral hippocampus and the prefrontal cortex (Kehr et al., 2010). The result with ACh is consistent with the pro-cognitive effect of (postsynaptic) 5-HT 1A R blockade in PA (Madjid et al., 2006). However, the expected glutamate increase may not be detectable because of the limited capacity of microdialysis to detect small transmitter changes restricted to the synaptic cleft. More sensitive techniques are required such as enzyme-based microelectrode amperometry, which is selective for the detection of extracellular glutamate with (1) spatial resolution in the μm level, (2) sub-second temporal resolution and (3) sensitivity in the μm range of glutamate (Day et al., 2006;Konradsson-Geuken et al., 2009;Mishra et al., 2015). This novel technology is suited to provide evidence for the expected enhancement of glutamatergic transmission in the hippocampus by both 5-HT 1A R inhibition and 5-HT 7 R activation.
It is clear that the impairing effects of low dose NMDA receptor antagonists (e.g., MK-801) and cholinergic antagonist (e.g., scopolamine) can be prevented by serotonergic manipulations (Ögren et al., 2008). Thus, these two pharmacological models of cognitive impairment relevant for Alzheimer's disease are both alleviated by 5-HT 1A R inhibition FIGURE 1 | Simplified overview of 5-HT 1A R-and 5-HT 7 R-mediated modulation of fear learning in pre-and postsynaptic neurons under conditions of high (A) and low presynaptic 5-HT 1A R activation (B), resulting in low and high postsynaptic 5-HT release, respectively. This in turn causes increased and decreased acetylcholine (ACh) release in the hippocampus (and also the medial septum). A similar effect on hippocampal glutamate (Glu) levels is hypothesized (as shown in the medial septum). When high postsynaptic 5-HT levels are biased to 5-HT 7 R activation (C), e.g., by 8-OH-DPAT at the postsynaptic dose of 1 mg/kg in combination with the 5-HT 1A R antagonist NAD-299 at 0.3 mg/kg, a pro-cognitive effect in fear learning is observed. Thus, emotional learning and memory depend on intrasynaptic 5-HT levels, receptor availability and occupancy, genetic and epigenetic factors for 5-HTR regulation and its short-and long-term mechanisms underlying altered synaptic transmission via ACh and glutamate (Glu) release. Under conditions of higher (postsynaptic) 5-HT release, the cognitive consequences depend on the availability and occupancy of 5-HT 1A R and 5-HT 7 Rs with so far unknown conditions that bias toward impaired (B) or facilitated fear memory (C). The specific functions of GABAergic interneurons in 5-HT 1A R and 5-HT 7 R-mediated fear memory modulation are currently not understood.
demonstrating a role for both enhanced glutamatergic and cholinergic transmission for improved cognitive function (e.g., Schechter et al., 2005;Madjid et al., 2006). An overview of these modulatory effects is provided in Figure 1.

Conclusion and Future Perspectives
During the last three decades many studies have indicated important regulatory functions of 5-HT signaling for emotional and cognitive functions. However, the complexity of the serotonergic signaling due to the existence of at least 14 pre-and postsynaptic 5-HTRs subtypes with multiple transduction mechanisms makes it exceedingly difficult to assign unambiguously the physiological and behavioral role of a single 5-HTR subtype. However, the use of specific ligands in combination with systemic and intrahippocampal administration, receptor autoradiography and in vivo neurochemical measurements are powerful tools in identifying the action of specific ligands in local networks of the brain including subareas of the hippocampus. This approach, combined with in vivo electrophysiology and genetic tools, can also better define the functional role of 5-HT in the neuronal circuitry underlying cognitive function.
Overall a number of open questions need to be answered to further improve our understanding of the role of serotonergic signaling via the different 5-HTRs in health and disease: (1) How do 5-HTRs modulate hippocampal and cortical glutamatergic transmission with a focus on activation and inhibition of 5-HT 1A Rs and 5-HT 7 Rs? This needs to be determined with newly developed amperometry methods in in vivo recordings.
(2) What are the roles of 5-HT 1A Rs and 5-HT 7 Rs in defined hippocampal subregions for emotional and cognitive functions? This requires the development of new ligands with low lipophilicity for local actions tested in vivo. Alternatively, is should be possible to shut down the second messenger coupling of neurons selectively expressing 5-HT 1A Rs and 5-HT 7 Rs by Designer Receptors Exclusively Activated by Designer Drugs (DREADD) technology. (3) What are the roles of 5-HT 1A Rs and 5-HT 7 Rs in different memory phases? As indicated there are considerable inconsistencies about the role of 5-HT 1A Rs and 5-HT 7 Rs in the consolidation process. In addition, extinction and reconsolidation are so far poorly explored. (4) The regulation of 5-HTR expression has so far focused on the 5-HT 1A R. This needs to be extended to other 5-HTRs including the 5-HT 7 R. Besides the use of radio-ligands in imaging studies, the subcellular immunohistochemical analyses of 5-HTR protein levels requires the development of specific antibodies. (5) Finally, despite the evidence of the beneficial effects of 5-HT 1A R antagonists in preclinical models, the therapeutic potential to facilitate cholingergic and/or glutamatergic neurotransmission for improved cognitive function in human neuropathology (e.g., Alzheimer's disease) or in aging is so far not explored.