Neuronal localization of the 5-HT2 receptor family in the amygdaloid complex

The amygdaloid complex (or amygdala), a heterogeneous structure located in the medial portion of the temporal lobe, is composed of deep, superficial, and “remaining” nuclei. This structure is involved in the generation of emotional behavior, in the formation of emotional memories and in the modulation of the consolidation of explicit memories for emotionally arousing events. The serotoninergic fibers originating in the dorsal and medial raphe nuclei are critically involved in amygdalar functions. Serotonin (5-hydroxytryptamine, 5-HT) regulates amygdalar activity through the activation of the 5-HT2 receptor family, which includes three receptor subtypes: 5-HT2A, 5-HT2B, and 5-HT2C. The distribution and the functional activity of the 5-HT2 receptor family has been studied more extensively than that of the 5-HT2A receptor subtypes, especially in the deep nuclei. In these nuclei, the 5-HT2A receptor is expressed on both pyramidal and non-pyramidal neurons, and could play a critical role in the formation of emotional memories. However, the exact role of the 5-HT2A receptor subtypes, as well as that of the 5-HT2B and 5-HT2C receptor subtypes, in the modulation of the amygdalar microcircuits requires additional study. The present review reports data concerning the distribution and the functional roles of the 5-HT2 receptor family in the amygdala.


INTRODUCTION
The amygdaloid complex (or amygdala), a heterogeneous structure located in the medial portion of the temporal lobe, is involved in multiple tasks, such as the generation of emotional behavior, formation of emotional memories related to fear and anxiety and modulation of the consolidation of explicit memories for emotionally arousing events (Aggleton, 2000;Whalen and Phelps, 2009). Several neuromodulators, including serotonin, are critical for amygdalar functions. Many neurological and psychiatric diseases, especially affective disorders, are characterized by a dysfunction of the amygdaloid complex and the serotoninergic system (Sanders and Shekhar, 1995;Jasnow and Huhman, 2001;Manji et al., 2001;Amaral, 2002;Braga et al., 2002;Hariri et al., 2002;Pralong et al., 2002;Rainnie et al., 2004;Canli et al., 2005;Keele, 2005;Kim et al., 2005;Rodrigues Manzanares et al., 2005;Hariri and Holmes, 2006;Shin et al., 2006;Van Nobelen and Kokkinidis, 2006). Selective serotonin reuptake inhibitors (SSRIs) are effective in the treatment of a variety of psychiatric diseases, such as anxiety disorders, in which the amygdaloid complex may play a role. Accordingly, it has been shown that the acquisition of auditory fear conditioning in the rat was enhanced by the SSRI citalopram when administered in acute cases and reduced when as administered in chronic cases; indeed, fear conditioning is known to be a model of emotional learning in which amygdaloid circuits play an important role (Burghardt et al., 2004). Selective serotonin reuptake inhibitors also reduce conditioned fear through its effect on the amygdala (Inoue et al., 2004). Moreover, coadministration of serotonin receptor agonists with paroxetine and venlafaxine could enhance the therapeutic effects of these drugs (Dhonnchadha et al., 2005). Table 1 | Nuclei and nuclear subdivisions of the rat, the monkey and the human amygdala (modified from Pitkänen and Kemppainen, 2002 (McDonald, 1992(McDonald, , 1998Sah et al., 2003;Spampanato et al., 2011). However, recent tracing studies combined with immunohistochemistry have demonstrated that some GABAergic neurons in the deep nuclei originate long projections directed to the prefrontal cortex (McDonald, 1987), basal forebrain (McDonald et al., 2012) and mediodorsal thalamic nucleus (McDonald, 1987;McDonald and Mascagni, 2007). As in the neocortex and hippocampal region, interneurons in the deep nuclei can be classified into distinct subpopulations on the basis of their content of calcium binding proteins FIGURE 1 | Photomicrographs from thionin-stained coronal sections of the rat amygdala showing the various nuclei and nuclear subdivision. Scale bar = 500 μm in (F) (applied to A-F). For abbreviations see Table 1.
The numbers in the lower left corner correspond to the distance from bregma according to rat brain atlas of Paxinos and Watson (1998 (Kemppainen and Pitkänen, 2000;McDonald and Mascagni, 2001). The CB-IR neurons can express PV (CB+/PV+) or CCK (CB+/CCK+) or SOM (CB+/SOM+) (McDonald and Betette, 2001;McDonald and Mascagni, 2002;Mascagni and McDonald, 2003). Similarly, the CR-IR interneurons can be immunopositive for CCK and/or VIP Muller et al., 2003). Microcircuits located in the deep nuclei are tightly regulated through the activity of the interneurons, which can innervate pyramidal cells as well as other interneurons. In particular PV-IR interneurons form symmetrical synapses with perisomatic (cell body, axon initial segment and thick proximal dendrites) and distal dendritic (small-caliber dendrites and dendritic spines) domains of pyramidal cells (Muller et al., 2006). Interestingly, pyramidal cells constitute synapse-like contacts with the perisomatic and distal dendritic domains of the PV-IR interneurons, in this way constituting a reciprocal connection . The PV-IR interneurons also form interneuronal networks interconnected by electrical (gap junctions) and chemical synapses (Muller et al., 2005;Woodruff and Sah, 2007). PV-interneurons of the basolateral amygdala can be subdivided into four functionally distinct subpopulations. Fast spiking cells are the most common PV-IR functional cell type. Interestingly, these cells innervate the perisomatic domain of pyramidal neurons (Woodruff and Sah, 2007). The CCK-IR interneurons form synapses with the somata and the proximal dendrites of the pyramidal cells. The SOM-IR neurons provide an inhibitory innervation (symmetrical synapses), especially of the distal dendritic domain (small-caliber dendrites and dendritic spines) of pyramidal cells (Muller et al., 2007a). In addition, SOM-IR axon terminals also contact SOM-, VIP-and PV-IR interneurons (Muller et al., 2007a). The VIP-IR interneurons do not innervate pyramidal cells, but form synapses with other interneurons, especially CCK-positive interneurons Muller et al., 2003). The superficial nuclei exhibit two major cell classes: spiny pyramidal cells and spine-sparse or aspiny non-pyramidal neurons (McDonald, 1992(McDonald, , 1998Sah et al., 2003). Pyramidal cells are glutamatergic projection neurons whereas non-pyramidal neurons represent local inhibitory GABAergic interneurons. These cells are not randomly organized, as in the deep nuclei, but exhibit a laminar organization (layers I, II and III) (McDonald, 1992(McDonald, , 1998Sah et al., 2003). Interestingly, GABAergic projection neurons are also distributed in the superficial nuclei (McDonald et al., 2012). The medial nucleus does not contain pyramidal and nonpyramidal neurons but small-to medium-sized ovoid neurons which possess spiny dendrites (McDonald, 1992(McDonald, , 1998Sah et al., 2003).

SEROTONINERGIC INNERVATION OF THE AMYGDALA AND SEROTONIN RECEPTOR
Serotonin (5-hydroxytryptamine, 5-HT) is a molecule located in the central nervous system which has the role of a neurotransmitter/neuromodulator. Serotoninergic somata are located along the midline of the brainstem in cell body groups designated raphe nuclei. The amygdala receives substantial serotoninergic innervation originating mainly from the dorsal raphe nucleus and, to a lesser extent, from the median raphe nucleus (Pralong et al., 2002;Hensler, 2006;Asan et al., 2013). Within the rat amygdala, serotoninergic fibers are directed especially in the lateral nucleus, basal nucleus (magnocellular division) and amygdalohippocampal area (Steinbusch, 1981). On the contrary, in the monkey amygdala, the highest density of serotoninergic fibers is located in the central nucleus, the nucleus of the lateral olfactory tract, the paralaminar nucleus, the anterior amygdaloid area, and the amygdalohippocampal area (Bauman and Amaral, 2005). In the rat basal nucleus (magnocellular and intermediate divisions), an ultrastructure study has demonstrated that serotonin terminals contact pyramidal as well as non-pyramidal (PV-IR and VIP-IR) neurons (Muller et al., 2007b).
The 5-HT2A receptor is coupled to G-protein and stimulates phosphoinositide-specific phospholipase C with a consequent increment of inositol triphosphate (Raymond et al., 2001;Hoyer et al., 2002;Hannon and Hoyer, 2008). This serotonin receptor also activates phospholipase D and phospholipase A2 by interacting with additional G-proteins. The 5-HT2A receptor activation also closes potassium channels, producing neuronal depolarization (Aghajanian, 1995;Barnes and Sharp, 1999). In addition, the activations of this receptor subtype increases also cGMP levels by means of a mechanism dependent on N-methyl-Daspartate (NMDA) receptor activation (Regina et al., 2003(Regina et al., , 2004. Interestingly, the 5-HT2A and 5-HT2C receptors are paradoxically regulated by agonists and antagonists (Gray and Roth, 2001;Van Oekelen et al., 2003).
Using autoradiography, in situ hybridization and immunohistochemistry, it has been demonstrated that 5-HT 2 receptor family mRNA and protein are present in the amygdala. Interestingly, the expression of the 5-HT 2A and 5-HT 2C receptors varied during postnatal development in the rat amygdaloid complex .

DEEP NUCLEI
An autoradiographic study has demonstrated a 5-HT 2 receptor binding site in rat deep nuclei, especially in the lateral nucleus (Pazos et al., 1985). In rodents, the presence of the 5-HT 2 receptor in the lateral, basal and accessory basal nuclei was also verified with in situ hybridization experiments (Wright et al., 1995). Autoradiography and in situ hybridization studies have reported that binding sites and 5-HT 2A receptor mRNA are present in the lateral (dorsomedial division) and basal (magnocellular division) nuclei (Lopez-Gimenez et al., 2001). Pompeiano et al. (1994) have reported the presence of the 5-HT 2C receptor mRNA in rat deep nuclei, with the highest levels in the lateral nucleus.
Interestingly, these Authors failed to find 5-HT 2A receptor mRNA in the same nuclei. Radioactive in situ hybridization studies on the rat (Greenwood et al., 2012) and the mouse  amygdala have shown that the 5-HT 2C receptor mRNA is located in the lateral nucleus and, to a lesser extent, in the basal nucleus. Using non-radioactive in situ hybridization procedures, the highest number of cells containing 5-HT 2C receptor mRNA in the rat amygdala has been observed in the lateral and accessory basal nuclei. On the contrary, only a few 5-HT 2C receptor mRNAreactive cells have been reported in the rat basal nucleus (Bonn et al., 2012(Bonn et al., , 2013. 5-HT 2C receptor mRNA has been reported in the deep nuclei of the human amygdala (Pasqualetti et al., 1999).
In rat deep nuclei, 5-HT 2A receptor immunoreactivity has been observed in GABAergic interneurons (in somata and dendrites) and GABAergic projection neurons (Morilak et al., 1993;McDonald and Mascagni, 2007;Bombardi, 2011). The GABAergic interneurons are present in the lateral, basal and accessory basal nuclei where the 5-HT 2A receptor is expressed by 66.3, 70.6, and 66.4% of interneurons, respectively (Bombardi, 2011). These interneurons are particularly abundant in the medial subdivision of the lateral nucleus (74.7% of interneurons) and in the parvicellular and magnocellular subdivisions of the basal nucleus (73.8 and 71.9% of interneurons, respectively) (Bombardi, 2011). In the rat amygdala, 59.8% of PV-IR neurons in the medial subdivision of the lateral nucleus, and 75.6% of PV-IR neurons in the magnocellular subdivision of the basal nucleus exhibit the 5-HT 2A receptor (McDonald and Mascagni, 2007). On the contrary, only 33.1% of SOM-IR neurons in the lateral nucleus (medial subdivision), and 32.6% of SOM-IR neurons in the basal nucleus (magnocellular subdivision), express the 5-HT 2A receptor (McDonald and Mascagni, 2007). The GABAergic/5-HT 2A receptor-IR projection neurons are especially distributed near the external and internuclear borders of the rat basolateral amygdala and project to the mediodorsal thalamus (McDonald and Mascagni, 2007). 5-HT 2C receptor-IR neurons, possibly pyramidal cells, have been observed in rat lateral and basal nuclei (Clemett et al., 2000).

Superficial nuclei
In the rat, 5-HT 2 receptor mRNA levels are moderate in every superficial nuclei (Wright et al., 1995). In rat superficial nuclei, 5-HT 2A receptor mRNA is detectable only in the bed nucleus of the accessory olfactory tract where it is strongly expressed (Pompeiano et al., 1994). On the contrary, 5-HT 2C receptor mRNA is located in different superficial nuclei, such as the anterior cortical nucleus, the bed nucleus of the accessory olfactory tract and the medial nucleus. In particular, 5-HT 2C receptor mRNA levels are high in the bed nucleus of the accessory olfactory tract, intermediate in the medial nucleus and low in the anterior cortical nucleus (Pompeiano et al., 1994). Autoradiographic analyses of the rat brain have demonstrated the presence of the 5-HT 2 receptor binding sites especially in the anterior cortical nucleus, but also in other superficial nuclei (Pazos et al., 1985). Immunoreactivity for the 5-HT 2A receptor has been observed in every superficial nucleus of the rat amygdala. However, a high density of immunopositive neurons is present, especially in the nucleus of the lateral olfactory tract and in the bed nucleus of the accessory olfactory tract (Morilak et al., 1993;Cornea-Hébert et al., 1999;Bombardi, 2011). Using in situ hybridization procedures, high levels of 5-HT 2C receptor mRNA have been observed in the medial nucleus and in the anterior cortical nucleus of the rodent amygdala Bonn et al., 2012Bonn et al., , 2013Greenwood et al., 2012). Accordingly, many 5-HT 2C receptor-IR neurons are located in the rat medial nucleus (Clemett et al., 2000). A moderate level of 5-HT 2C receptor-IR neurons has also been observed in the posterior cortical nucleus of the rat amygdala (Clemett et al., 2000).
As in the deep nuclei, the 5-HT 2A receptor is also expressed in pyramidal and non-pyramidal neurons in the rat superficial nuclei (Bombardi, 2011). Pyramidal cells are especially distributed in the nucleus of the lateral olfactory tract (layer II), the anterior cortical nucleus (layers II and III), the periamygdaloid cortex (layers II and III) and the posterior cortical nucleus (layers II and III) (Bombardi, 2011). In these cells, the 5-HT 2A receptor is strongly expressed in the apical dendrites where it may induce excitatory synaptic currents. The 5-HT 2A receptor-IR non-pyramidal neurons are distributed in the nucleus of the lateral olfactory tract, the anterior cortical nucleus, the periamygdaloid cortex and the posterior cortical nucleus. These cells are heterogeneous in shape (multipolar and fusiform) and size (from small to large), and are particularly abundant in layers II and III (Bombardi, 2011). Since the cell types in the medial nucleus are not cortex-like as in the other superficial nuclei, 5-HT 2A receptor-IR pyramidal and non-pyramidal neurons are not present in this nucleus (McDonald, 1992(McDonald, , 1998Sah et al., 2003). Accordingly, the rat medial nucleus contains 5-HT2AR-IR principal neurons with ovoid cell bodies (Bombardi, 2011). The rat medial nucleus is the only amygdaloid area containing 5-HT 2B receptor-IR neurons. These cells are numerous and show a multipolar and bipolar morphology (Duxon et al., 1997a).

Remaining nuclei
In the rat amygdalohippocampal area, the presence of the 5-HT 2A receptor has been demonstrated only with immunohistochemical procedures which have revealed many 5-HT 2A receptor-IR neurons with angular-and ovoid-shaped cell bodies (Bombardi, 2011). The rat amygdalohippocampal area also contains a high density of 5-HT 2C receptor-IR neurons (Clemett et al., 2000). A moderate level of 5-HT 2A receptor mRNA has been revealed in the rat central nucleus (Wright et al., 1995). Accordingly, immunohistochemical procedures have demonstrated the presence of many 5-HT 2A receptor-IR ovoid somata in the different subdivisions of the rat central neurons (Cornea-Hébert et al., 1999;Bombardi, 2011). These cells could be GABAergic local neurons as well as GABAergic projecting neurons (Bombardi, 2011). Cells containing 5-HT 2C receptor mRNA have been observed in the rat central nucleus where they are particularly numerous in the lateral capsular subdivision (Bonn et al., 2012(Bonn et al., , 2013. Both 5-HT 2A and 5-HT 2C receptor mRNA are present at low density in the rat amygdalohippocampal area. Accordingly, pyramidal and non-pyramidal neurons of the rat amygdalohippocampal area contain the 5-HT 2A receptor (Bombardi, 2011). In the rat intercalated nuclei, 5-HT 2C receptor mRNA is present at a low density while 5-HT 2A receptor mRNA has not been detected (Pompeiano et al., 1994). A different distribution of the 5-HT 2A receptor has been observed using immunohistochemical procedures. In fact, Xu and Pandey (2000), and Bombardi (2011) have observed that small and large neurons in the rat intercalated nuclei express the 5-HT 2A receptor. The rat intercalated nuclei contain only weak 5-HT 2C receptor mRNA-reactive cells (Bonn et al., 2012(Bonn et al., , 2013. These data are in disagreement with immunohistochemical studies showing that intercalated nuclei contain a high density of 5-HT 2C receptor-IR neurons (Clemett et al., 2000).

EFFECT OF 5-HT 2 RECEPTOR FAMILY ACTIVATION ON AMYGDALAR NEURONS AND MICROCIRCUITS
Serotonin influences amygdalar information processing by activating multiple 5-HT 2 receptor subtypes. Inasmuch as the amygdaloid microcircuits are complex and the expression patterns of the 5-HT 2 receptor subtypes are not fully characterized, the mechanisms by which 5-HT 2 receptor subtypes modulate amygdalar neurotransmission remains poorly understood. This modulation is complex and has been studied especially for 5-HT 2A and 5-HT 2C receptors.
The 5-HT 2 receptor family also modulates the excitability of GABAergic interneurons in the deep nuclei. In fact, electrophysiological studies have demonstrated that the application of α-methyl-5-hydroxytryptamine (a 5-HT 2 receptor agonist) and DOI (a 5-HT 2A /5-HT 2C receptor agonist), induces the activation of GABAergic interneurons of the rat basolateral amygdala (Rainnie, 1999;Stein et al., 2000;Sokal et al., 2005). In addition, the stimulation of the 5-HT 2A receptor increases the frequency and amplitude of spontaneous inhibitory postsynaptic currents (sIPSCs) recorded from the pyramidal neurons of the juvenile rat basolateral amygdala (Jiang et al., 2008). Accordingly, the inhibition of pyramidal cell firing in the lateral nucleus of the rat amygdala obtained after the local application of serotonin is blocked by the simultaneous application of bicuculline methiodide, a GABA antagonist (Stutzmann and LeDoux, 1999). The activation of GABAergic interneurons of the corticomedial amygdala has been demonstrated by iontophoretic injections of DOI (Stein et al., 2000). Amygdala microcircuitry has not been studied as extensively as that of the neocortex and hippocampal region. However, numerous studies report that the amygdala circuit organization combines cortex-like (deep nuclei, the majority of the superficial nuclei and the amygdalohippocampal area) and striatum-like structures (central nucleus and intercalated nuclei) (McDonald, 1992(McDonald, , 1998Sah et al., 2003). Since these amygdaloid areas provide numerous intra-amygdaloid and extra-amygdaloid connections, the amygdala is considered to be the interface of the information exchange between the various functional systems of the brain (Pitkänen, 2000). Traditionally, the extra-amygdaloid afferents (all the modalities of sensory inputs and polymodal inputs) target the input side of the amygdala (deep and superficial nuclei) where they are processed locally and then directed, by intraamygdaloid connections, to the medial and central nuclei which act as an output station. The medial nucleus especially projects to the hypothalamic neuroendocrine zone whereas outputs from the central nucleus especially innervate the hypotalamic and brainstem nuclei which regulate autonomic functions (Pitkänen, 2000; Figure 2).
The distribution of the 5-HT 2 receptor previously reported indicates that this receptor could modulate amygdala activity acting on projection neurons (pyramidal neurons of cortex-like structures and GABAergic projection neurons of the basolateral amygdala and striatum-like structures) as well as on inhibitory interneurons (in particular, GABAergic interneurons of the cortex-like structures). The distribution of the 5-HT 2 receptor family has been studied more extensively for 5-HT 2A receptor subtype, especially in the rat basolateral amygdala (Figure 3). In the microcircuits of the rat deep nuclei, the 5-HT 2A receptor is located on both pyramidal and non-pyramidal neurons. The nonpyramidal neurons containing this receptor express PV and SOM (McDonald and Mascagni, 2007). As previously reported, the PV-IR interneurons innervate the perisomatic domain (cell body and proximal dendrites) of pyramidal cells (Muller et al., 2006). Moreover, these interneurons are connected by gap junctions and constitute an inhibitory network which synchronizes the firing of pyramidal cells (Woodruff and Sah, 2007). Interestingly, most of the pyramidal neurons form intimate synapse-like contacts with the somata and dendrites (especially proximal dendrites) of the PV-IR interneurons , in this way, constituting a reciprocal perisomatic connection which may be important in modulating the synchronized rhythmic activity associated with the formation of emotional memories (Paré and Collins, 2000;Paré et al., 2002;Rainnie et al., 2006). In the rat basolateral amygdala, SOM-IR interneurons innervate the distal dendritic domain of pyramidal cells and could modulate synaptic mechanisms related to emotional learning, including fear conditioning (Paré et al., 2002;Muller et al., 2007a). Since the 5-HT 2A receptor is located on PV-IR and SOM-IR interneurons, this receptor subtype could play an important role in the formation of emotional memories.

5-HT 2 RECEPTOR FAMILY AND AMYGDALA-MEDIATED BEHAVIOR
The involvement of the 5-HT 2 receptor family in numerous amygdala-mediated behavioral and physiological effects has been described in several reports. This receptor family plays a crucial role, especially in fear and anxiety. Local infusion of ketanserin (a 5-HT 2 receptor family antagonist) induces an anxiolytic effect in the conflict test (Hodges et al., 1987). Microinjections of nefazodone (a 5-HT 2 receptors antagonist) into the basolateral nucleus of the rat amygdala enhances the aversive responses induced by NMDA activation of the neural substrates of aversion in the inferior colliculus (Maisonnette et al., 2000). In different mouse models of anxiety, the 5-HT 2A receptor mediates different anxiolytic-like effects (Dhonnchadha et al., 2003a,b). Moreover, bilateral injections of ketanserin (a 5-HT 2A and 5-HT 2C receptors antagonist) into the rat basolateral/medial amygdala produces an anxiogenic profile in an elevated plus-maze (Zangrossi and Graeff, 1994).
The 5-HT 2A receptor is also implicated in kindling development from the rat amygdala since the subcutaneous injection of DOI, an agonist of 5-HT 2A/2C receptors, facilitates kindling development and reduces the number of amygdaloid stimulations necessary to obtain generalized seizures (Wada et al., 1997).
It is known that direct or indirect projections from the central nucleus of the amygdala to the paraventricular nucleus of the hypothalamus mediate a stress response. In vivo microdialysis studies have demonstrated that there is an increase in serotonin release in the amygdala during stress (Kawahara et al., 1993). Accordingly, the 5-HT 2A receptor located in the central nucleus of the rat amygdala is able to activate the hypothalamo-pituitaryadrenocortical axis (Feldman et al., 1998). Finally, in the rat basolateral amygdala, the 5-HT 2A receptor-mediated serotoninergic facilitation of GABAergic synaptic transmission is impaired by stress (Jiang et al., 2008).
The 5-HT 2B receptor is also involved in amygdala-mediated behavior. In fact, the activation of this receptor subtype causes anxiolysis in social interaction tests in the rat (Duxon et al., 1997b).

CONCLUSIONS
The present review reported that the 5-HT 2 receptor family plays a crucial role in regulating the activity of amygdalar microcircuits and projections. In fact, as in the cerebral cortex and the hippocampal regions (Willins et al., 1997;Hamada et al., 1998;Goldman-Rakic, 1998, 2000;Cornea-Hébert et al., 1999;Clemett et al., 2000;Xu and Pandey, 2000;Jansson et al., 2001;Miner et al., 2003;Lüttgen et al., 2004;Bombardi, 2012), excitatory as well as inhibitory neurons in the rat amygdala express the 5-HT 2 receptor family. Nevertheless, the exact role of the 5-HT 2 receptor family in the modulation of amygdala activity is still poorly understood and requires additional study. In this way, detailed knowledge of the cellular mechanism underlying the modulation of amygdalar activity mediated by the 5-HT 2 receptor family could provide valuable information for better understanding the pathogenesis of affective disorders and for utilizing a more specific pharmacological treatment.