Research reportDifferential role of the basolateral amygdala 5-HT3 and 5-HT4 serotonin receptors upon ACPA-induced anxiolytic-like behaviors and emotional memory deficit in mice
Introduction
Marijuana and hashish which are derived from hemp plant Cannabis sativa are amongst the most common drugs of abuse worldwide [1]. Based on molecular cloning, two cannabinoid receptors (CB1 and CB2) have been identified [2], [3], however some investigations have introduced an additional cannabinoid receptor type known as non-CB1 and CB2 (CB3) [4], [5]. CB1 cannabinoid receptors are essentially expressed in the central nervous system (such as the basal ganglia, hippocampus, cerebral cortex and amygdala) as well as in peripheral tissues, while CB2 cannabinoid receptors have a predominant peripheral presentation [6]. It seems that the main physiological, cognitive and non-cognitive effects of cannabinoids are induced by central CB1 receptors expressed in various brain regions [7], [8], [9]. Both CB1 and CB2 cannabinoid receptors belong to a seven transmembrane Gi/o-coupled receptor family which share 44% of their protein identity while showing different pharmacological properties [2], [3]. A plethora of research has demonstrated that cannabinoids induce a unique syndrome of cognitive and non-cognitive behavioral effects including the altered anxiety state, short- and long-term memory impairment, the sense of time dilation, mood alterations, enhanced body awareness, reduced ability to focus attention as well as filtering out irrelevant information, discoordination and sleepiness [10], [11], [12], [13], [14], [15], [16], [17]. Several reports have indicated that endogenous cannabinoids (i.e. 2-arachidonoylglycerol and anandamide) are produced at post synaptic level and activate the presynaptic CB1 cannabinoid receptors. This in turn reduces the release of neurotransmitters/hormones including serotonin, GABA, cholecystokinin, corticotropin-releasing factor and opioids [18], [19]. Supported by evidence, CB1 cannabinoid receptors may be considered as an alternative therapeutic target in neuropsychiatric disorders which involve serotonin receptors [20], [21]. Moreover, axon terminals from the serotonergic raphe neurons are endowed with the CB1 cannabinoid receptor in the hippocampus and amygdala [22], suggesting that CB1 cannabinoid receptors may directly control the release of serotonin in such brain regions.
One of the most important neurotransmitters in the central nervous system which involves in several cognitive and non-cognitive behaviors such as mood, sex, appetite, sleep, memory, emotion and anxiety is serotonin [5-hydroxytryptamine (5-HT)]. This neurotransmitter is mainly produced and released from the neurons having their cell bodies in the brainstem raphe nuclei. Today, at least 15 presynaptic and postsynaptic serotonin receptors are identified. Based on these receptors’ structure, their affinity for different ligands, and activation of the secondary messenger, they are classified into seven families (5-HT1-7). Among these receptors, 5-HT3 is the only ligand-gated ion channel receptor with its activation leading to Na+ and Ca2+ influx [23]. This receptor comprises two subunits known as 3A and 3B [24], [25], [26], however the actual mechanism(s) involved with the function of these subtypes are not fully understood [27], [28]. The remaining six subtypes of serotonin receptors are the rhodopsin-like G protein-coupled receptors possessing three extracellular and three intracellular domains with seven transmembrane spanning helices. The 5-HT1, 5-HT2, 5-HT4, 5-HT5, 5-HT6, and 5-HT7 receptors comprise multiple subunits [29]. Moreover, all 5-HT4 receptor isoforms are positively coupled with the adenylyl cyclase enzyme with their activation resulting in cAMP production [30].
Several investigations have strongly supported the critical role of the serotonergic system processes in memory consolidation following one-trial avoidance and conditioned-fear testing paradigms, in which experimental animals have often showed anxiogenic-like behaviors [31], [32], [33], [34]. These effects are thought to be regulated via multiple 5-HT receptor subtypes, thus the behavioral outcomes may notably vary based on the 5-HT receptor subtype involved, the site of administration (central vs. systemic), type of the drugs used, timing of the drug administration, and the behavioral tests employed [35]. 5-HT3 and 5-HT4 serotonin receptors are found in several parts of the brain such as the entorhinal cortex, amygdala and the hippocampus [36], [37], [38], [39], [40]. These receptors which extensively express in the amygdala are shown to play a distinctive role in anxiety-like behaviors, learning and memory processes [29], [37], [39]. As such, 5-HT3 receptor antagonists have demonstrated consistent effects ranging from anxiolytic to anxiogenic behaviors [39], [41], while pharmacological antagonism or knocking out the 5-HT4 gene induces anxiolytic-like behaviors [42].
Amygdala is one of the main brain sites regulating the anxiety state as well as emotional and fear-related memory acquisition [43], [44], [45], [46], [47]. Based on the anatomical studies, amygdala is shown to comprise several distinct nuclei, such as the basal and lateral nuclei (together called basolateral amygdala; BLA) and the central nucleus [48]. BLA is known to modulate anxiety and memory consolidation in a wide variety of the emotionally arousing tasks, irrespective of their positive or negative affective valence [49], [50], [51]. For instance, some investigations have demonstrated that BLA activation and inhibition increases and decreases the anxiety-related physiological and behavioral responses, respectively [52], [53], [54]. Nonetheless, the actual mechanism(s) involved in these phenomena within the BLA remain(s) unclear. It has been postulated that in emotional events, the amygdala regulates the activity of the hippocampal formation. For instance, the activation of amygdala and especially its basolateral region (BLA) in emotionally passive situations potentiates the episodic memory for visual details [55], [56], [57]. Therefore, the integration of amygdala and the hippocampal system seems to play a critical role in emotional memory enhancements [58]. Moreover, in emotional instances such as aversion and fear, this structure may enhance or impair memory formation [59].
Some investigations (i.e. behavioral, physiological, pharmacological, and genetic) have described a close memory-anxiety relationship [60], [61], [62] although this relationship does not follow a consistent synergistic rule. The above suggests that quite a complex nonlinear relation exists between these behavioral functions. Moreover, the fact that altered anxiety is usually seen together with altered memory and vice versa does not oppose with the idea that under certain circumstances both domains may be affected independently. Likewise, memory or anxiety must not be considered as a single entity as each of them clearly represents a complex multidimensional domain on its own [63]. Emotional states (i.e. fear and aversion) can be modulated by enhanced or impaired memory formation [59]. The currently available animal models of learning and memory not only lack the ability to fully detect the effect of drugs on anxiety, fear and memory but also subject to misinterpretation. Using the elevated plus-maze (EPM) test-retest paradigm in rodents is thus an attempt to concomitantly assess the effects of drugs on anxiety, learning and memory [64].
The use of EPM in testing anxiety is based on the natural tendency of animals to avoid dangerous situation when exposed to height and open spaces [65]. In general, animals acquire information with regard to safe and dangerous areas in the maze upon test. Repeated testing in the EPM (usually in 24 h) induces some experience-dependent behavioral changes which possibly represent an index for memory acquisition, consolidation and retention. During re-test, the decreased open arms exploration time may translate to rodents’ acquired emotional learning and memory [66], [67], [68], [69], [70], [71].
Within the BLA, both cannabinoid and serotonin receptors are highly expressed on GABAergic interneurons [72], [73]. Given the involvement of both cannabinoid and serotonin receptors in memory and anxiety function [74], [75], and the fact that cannabinoids directly inhibit the release of serotonin [76], this study was designed to examine the functional interaction between BLA serotonin (5-HT3 and 5-HT4) receptors upon ACPA (selective CB1 cannabinoid receptor agonist)-induced emotional amnesia in EPM test-retest protocol in mice.
Section snippets
Animals
Adult (8–9 weeks of age) male white mice (Institute for Cognitive Science Studies; Tehran, Iran) weighting 28–33 g at the time of surgery were used. Animals were housed in a colony, maintained at 22 ± 2 °C with a 12-h light/12-h dark cycle (lights on at 07:00 h) and allowed free access to food and water inside the standard polypropylene cages. Eight animals were used in each experimental group. The experiments were carried out during the light phase of the cycle. Animals handling was limited to the
Effects of the pretest intra-BLA microinjection of M-Chl on the open-arms exploratory behaviors
Repeated measure and post hoc analysis between test and retest days showed that intra-BLA injection of M-Chl at applied doses did not alter the transfer latency [Intra-groups: F(3, 24) = 1.1, P > 0.05, Inter-groups: F(1, 24) = 35.6, P < 0.001, Inter-Intra groups interaction: F(3, 24) = 0.99, P > 0.05; Fig. 1, panels 1A and 2A], %OAT [Intra-groups: F(3, 24) = 1.75, P > 0.05, Inter-groups: F(1, 24) = 48.9, P < 0.001, Inter-Intra groups interaction: F(3, 24) = 1.56, P > 0.05; Fig. 1, panels 1B and 2B], %OAE
The effects of ACPA on open-arms exploratory behaviors in naive mice subjected to EPM
Present results indicate that the intra-peritoneal infusion of the selective CB1 cannabinoid receptor agonist, ACPA, elicits the anxiolytic-like effects. Furthermore, anxiolytic-like effects of ACPA emerge into the retest session. These results confirmed the impairment of aversive memory acquisition in ACPA-treated animals upon test. Meanwhile, the drug did not alter the locomotor activity on test and retest days.
In terms of anxiety-like behaviors, the effects of cannabinoid agonists on this
Acknowledgements
The authors thank the Iran National Science Foundation (INSF) for providing financial support to this project.
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