Cannabinoids activate the insulin pathway to modulate mobilization of cholesterol in C. elegans.

The nematode Caenorhabditis elegans requires exogenous cholesterol to survive and its depletion leads to early developmental arrest. Thus, tight regulation of cholesterol storage and distribution within the organism is critical. Previously, we demonstrated that the endocannabinoid (eCB) 2-arachidonoylglycerol (2-AG) plays a key role in C. elegans since it modulates sterol mobilization. However, the mechanism remains unknown. Here we show that mutations in the ocr-2 and osm-9 genes, coding for transient receptors potential V (TRPV) ion channels, dramatically reduce the effect of 2-AG in cholesterol mobilization. Through genetic analysis in combination with the rescue of larval arrest induced by sterol starvation, we found that the insulin/IGF-1signaling (IIS) pathway and UNC-31/CAPS, a calcium-activated regulator of neural dense-core vesicles release, are essential for 2-AG-mediated stimulation of cholesterol mobilization. These findings indicate that 2-AG-dependent cholesterol trafficking requires the release of insulin peptides and signaling through the DAF-2 insulin receptor. These results suggest that 2-AG acts as an endogenous modulator of TRPV signal transduction to control intracellular sterol trafficking through modulation of the IGF-1 signaling pathway.

Loss of Choline Agonism in the Inner Ear Hair Cell Nicotinic Acetylcholine Receptor Linked to the α10 Subunit.

The α9α10 nicotinic acetylcholine receptor (nAChR) plays a fundamental role in inner ear physiology. It mediates synaptic transmission between efferent olivocochlear fibers that descend from the brainstem and hair cells of the auditory sensory epithelium. The α9 and α10 subunits have undergone a distinct evolutionary history within the family of nAChRs. Predominantly in mammalian vertebrates, the α9α10 receptor has accumulated changes at the protein level that may ultimately relate to the evolutionary history of the mammalian hearing organ. In the present work, we investigated the responses of α9α10 nAChRs to choline, the metabolite of acetylcholine degradation at the synaptic cleft. Whereas choline is a full agonist of chicken α9α10 receptors it is a partial agonist of the rat receptor. Making use of the expression of α9α10 heterologous receptors, encompassing wild-type, heteromeric, homomeric, mutant, chimeric, and hybrid receptors, and in silico molecular docking, we establish that the mammalian (rat) α10 nAChR subunit underscores the reduced efficacy of choline. Moreover, we show that whereas the complementary face of the α10 subunit does not play an important role in the activation of the receptor by ACh, it is strictly required for choline responses. Thus, we propose that the evolutionary changes acquired in the mammalian α9α10 nAChR resulted in the loss of choline acting as a full agonist at the efferent synapse, without affecting the triggering of ACh responses. This may have accompanied the fine-tuning of hair cell post-synaptic responses to the high-frequency activity of efferent medial olivocochlear fibers that modulate the cochlear amplifier.

Unraveling the Molecular Players at the Cholinergic Efferent Synapse of the Zebrafish Lateral Line.

The lateral line (LL) is a sensory system that allows fish and amphibians to detect water currents. LL responsiveness is modulated by efferent neurons that aid in distinguishing between external and self-generated stimuli, maintaining sensitivity to relevant cues. One component of the efferent system is cholinergic, the activation of which inhibits afferent activity. LL hair cells (HCs) share structural, functional, and molecular similarities with those of the cochlea, making them a popular model for studying human hearing and balance disorders. Because of these commonalities, one could propose that the receptor at the LL efferent synapse is a α9α10 nicotinic acetylcholine receptor (nAChR). However, the identities of the molecular players underlying ACh-mediated inhibition in the LL remain unknown. Surprisingly, through the analysis of single-cell expression studies and in situ hybridization, we describe that α9, but not the α10, subunits are enriched in zebrafish HCs. Moreover, the heterologous expression of zebrafish α9 subunits indicates that homomeric receptors are functional and exhibit robust ACh-gated currents blocked by α-bungarotoxin and strychnine. In addition, in vivo Ca2+ imaging on mechanically stimulated zebrafish LL HCs show that ACh elicits a decrease in evoked Ca2+ signals, regardless of HC polarity. This effect is blocked by both α-bungarotoxin and apamin, indicating coupling of ACh-mediated effects to small-conductance Ca2+-activated potassium (SKs) channels. Our results indicate that an α9-containing (α9*) nAChR operates at the zebrafish LL efferent synapse. Moreover, the activation of α9* nAChRs most likely leads to LL HC hyperpolarization served by SK channels.SIGNIFICANCE STATEMENT The fish lateral line (LL) mechanosensory system shares structural, functional, and molecular similarities with those of the mammalian cochlea. Thus, it has become an accessible model for studying human hearing and balance disorders. However, the molecular players serving efferent control of LL hair cell (HC) activity have not been identified. Here we demonstrate that, different from the hearing organ of vertebrate species, a nicotinic acetylcholine receptor composed only of α9 subunits operates at the LL efferent synapse. Activation of α9-containing receptors leads to LL HC hyperpolarization because of the opening of small-conductance Ca2+-activated potassium channels. These results will further aid in the interpretation of data obtained from LL HCs as a model for cochlear HCs.

Selectivity of (±)-citalopram at nicotinic acetylcholine receptors and different inhibitory mechanisms between habenular α3ß4* and α9α10 subtypes.

The inhibitory activity of (±)-citalopram on human (h) α3ß4, α4ß2, and α7 nicotinic acetylcholine receptors (AChRs) was determined by Ca2+ influx assays, whereas its effect on rat α9α10 and mouse habenular α3ß4* AChRs by electrophysiological recordings. The Ca2+ influx results clearly establish that (±)-citalopram inhibits (IC50's in µM) hα3ß4 AChRs (5.1 ±â€¯1.3) with higher potency than that for hα7 (18.8 ±â€¯1.1) and hα4ß2 (19.1 ±â€¯4.2) AChRs. This is in agreement with the [3H]imipramine competition binding results indicating that (±)-citalopram binds to imipramine sites at desensitized hα3ß4 with >2-fold higher affinity than that for hα4ß2. The electrophysiological, molecular docking, and in silico mutation results indicate that (±)-citalopram competitively inhibits rα9α10 AChRs (7.5 ± 0.9) in a voltage-independent manner by interacting mainly with orthosteric sites, whereas it inhibits a homogeneous population of α3ß4* AChRs at MHb (VI) neurons (7.6 ± 1.0) in a voltage-dependent manner by interacting mainly with a luminal site located in the middle of the ion channel, overlapping the imipramine site, which suggests an ion channel blocking mechanism. In conclusion, (±)-citalopram inhibits α3ß4 and α9α10 AChRs with higher potency compared to other AChRs but by different mechanisms. (±)-Citalopram also inhibits habenular α3ß4*AChRs, supporting the notion that these receptors are important endogenous targets related to their anti-addictive activities.

Tricyclic antidepressants inhibit hippocampal α7* and α9α10 nicotinic acetylcholine receptors by different mechanisms.

The activity of tricyclic antidepressants (TCAs) at α7 and α9α10 nicotinic acetylcholine receptors (AChRs) as well as at hippocampal α7-containing (i.e., α7*) AChRs is determined by using Ca2+ influx and electrophysiological recordings. To determine the inhibitory mechanisms, additional functional tests and molecular docking experiments are performed. The results established that TCAs (a) inhibit Ca2+ influx in GH3-α7 cells with the following potency (IC50 in µM) rank: amitriptyline (2.7 ±â€¯0.3) > doxepin (5.9 ±â€¯1.1) ∼ imipramine (6.6 ±â€¯1.0). Interestingly, imipramine inhibits hippocampal α7* AChRs (42.2 ±â€¯8.5 µM) in a noncompetitive and voltage-dependent manner, whereas it inhibits α9α10 AChRs (0.53 ±â€¯0.05 µM) in a competitive and voltage-independent manner, and (b) inhibit [3H]imipramine binding to resting α7 AChRs with the following affinity rank (IC50 in µM): imipramine (1.6 ±â€¯0.2) > amitriptyline (2.4 ±â€¯0.3) > doxepin (4.9 ±â€¯0.6), whereas imipramine's affinity was no significantly different to that for the desensitized state. The molecular docking and functional results support the notion that imipramine noncompetitively inhibits α7 AChRs by interacting with two overlapping luminal sites, whereas it competitively inhibits α9α10 AChRs by interacting with the orthosteric sites. Collectively our data indicate that TCAs inhibit α7, α9α10, and hippocampal α7* AChRs at clinically relevant concentrations and by different mechanisms of action.

Pharmacological augmentation of endocannabinoid signaling reduces the neuroendocrine response to stress.

Activation of the hypothalamic-pituitary-adrenal axis (HPA) is critical for survival when the organism is exposed to a stressful stimulus. The endocannabinoid system (ECS) is currently considered an important neuromodulator involved in numerous pathophysiological processes and whose primary function is to maintain homeostasis. In the tissues constituting the HPA axis, all the components of the ECS are present and the activation of this system acts in parallel with changes in the activity of numerous neurotransmitters, including nitric oxide (NO). NO is widely distributed in the brain and adrenal glands and recent studies have shown that free radicals, and in particular NO, may play a crucial role in the regulation of stress response. Our objective was to determine the participation of the endocannabinoid and NOergic systems as probable mediators of the neuroendocrine HPA axis response to a psychophysical acute stress model in the adult male rat. Animals were pre-treated with cannabinoid receptors agonists and antagonists at central and systemic level prior to acute restraint exposure. We also performed in vitro studies incubating adrenal glands in the presence of ACTH and pharmacological compounds that modifies ECS components. Our results showed that the increase in corticosterone observed after acute restraint stress is blocked by anandamide administered at both central and peripheral level. At hypothalamic level both cannabinoid receptors (CB1 and CB2) are involved, while in the adrenal gland, anandamide has a very potent effect in suppressing ACTH-induced corticosterone release that is mainly mediated by vanilloid TRPV1 receptors. We also observed that stress significantly increased hypothalamic mRNA levels of CB1 as well as adrenal mRNA levels of TRPV1 receptor. In addition, anandamide reduced the activity of the nitric oxide synthase enzyme during stress, indicating that the anti-stress action of endocannabinoids may involve a reduction in NO production at hypothalamic and adrenal levels. In conclusion, an endogenous cannabinoid tone maintains the HPA axis in a stable basal state, which is lost with a noxious stimulus. In this case, the ECS dampens the response to stress allowing the recovery of homeostasis. Moreover, our work further contributes to in vitro evidence for a participation of the endocannabinoid system by inhibiting corticosterone release directly at the adrenal gland level.