Cannabinoid-serotonin interactions in alcohol-preferring vs. drug craving, we investigated whether cannabinoid 1 (CB1) and 5-HT(1A) receptor ligands could. Total RNA was isolated and qRT-PCR for 5-HT2A, CB1, and CB2 was . and 5- HT (F1,, pinteraction between . Keywords: Endocannabinoid, serotonin, dorsal raphe, stress, synaptic So far, two cannabinoid receptors (CB1 and CB2) have been cloned and . studies have begun to examine the functional interaction between these two.
Prolonged activation and disturbances of the HPA axis contribute to depressive and anxiety symptoms seen in many psychopathological conditions. Stimulation of 5-HT 2C receptors leads to increase of corticotropin releasing hormone CRH and vasopressin mRNA in the paraventricular nucleus and proopiomelanocortin in the anterior pituitary lobe.
In rats, restraint stress which can produce depressive symptoms if being chronic induces secretion of prolactin, ACTH, vasopressin and oxytocin which is partially mediated via 5-HT 2C receptor.
Responses during such conditions as dehydration or haemorrhage causes the release oxytocin via serotonergic response that is partly mediated via 5-HT 2C. In addition, peripheral release of vasopressin involves serotonergic response which is partially mediated via 5-HT 2C.
Combination of the hormones decrease the receptor concentration in the ventral hippocampus in rats and could thus affect mood. Many human polymorphisms have been identified influencing the expression of 5-HT 2C.
Significant correlations are suggested, specifically in relation to psychiatric disorders such as depression, OCD, and anxiety-related conditions. Polymorphisms also correlate with susceptibility to a number of conditions including drug abuse and obesity. As women have two copies of the gene, but only one allele is expressed in each cell, they are a mosaic for polymorphisms, meaning that one genetic variant may be prevalent in one tissue and another variant will be prevalent in a different tissue as with all other x-linked genetic variations.
First allosteric modulators were developed in Different levels of editing result in a variety of effects on receptor function. Inosines are recognised as guanosine by the cells translational machinery.
ADAR3 is thought to have a regulatory role in the brain. The double stranded regions of RNA are formed by base-pairing between residues in the close to region of the editing site with residues usually in a neighboring intron but can be an exonic sequence.
If an editing site occurs within a coding sequence, it can result in a codon change. This can lead to translation of a protein isoform due to a change in its primary protein structure. Therefore, editing can also alter protein function. Editing occurs in 5 different closely located sites within exon 5, which corresponds to the second intracellular loop of the final protein. Several codon changes can occur due to A-to-I editing at these sites.
Thirty-two different mRNA variants can occur leading to 24 different protein isoforms. These codon changes which can occur due to A to I editing at these sites can lead to a maximum of 32 different mRNA variants leading to 24 different protein isoforms.
The number of protein isoforms is less than 32 since some amino acids are encoded by more than one codon. RNA editing of this receptor occurs at 4 locations in the rat.
The 5-HT 2c receptor is the only serotonin receptor edited despite its close sequence similarities to other family members. Disruption of this inverted repeat was demonstrated to cease all editing.
Some examples of differences in frequency of editing and site edited in different parts of the human brain of 5HT2CR include low frequency of editing in cerebellum and nearly all editing is at site D while in the hippocampus editing frequency is higher with site A being the main editing site.
Site C' is only found edited in the thalamus. The most common isoform in human brain is the VSV isoform. Mice knock out and other studies have been used to determine which ADAR enzyme are involved in editing.
Also such interactions and tissue specific expression of ADARs interaction may explain the variety in editing patterns in different regions of the brain. Second, the editing pattern controls the amount of the 5-HT2CR mRNA that leads to the expression of full-length protein through the modulation of alternative splice site selection 76, Unedited pre-mRNAs tend to be spliced at the GU1 site, resulting in the truncated, non-functional protein if translated 76, Thus, when editing is inefficient, increased splicing at GU1 may act as a control mechanism to decrease biosynthesis of the 5-HT2CR-INI and thereby limit serotonin response.
Third, RNA editing controls the ultimate physiological output of constitutively active receptors by affecting the cell surface expression of the 5-HT2CR. The 5-HT2CR-VGV, which displays the lowest level of constitutive activity, is fully expressed at the cell surface under basal conditions and is rapidly internalized in the presence of agonist As mentioned editing results in several codon changes.
The editing sites are found in the second intracellular domain of the protein which is also the receptors G protein coupling domain. Therefore, editing of these sites can affect the affinity of the receptor for G protein binding. Editing results in reduced affinity for specific G proteins which in turn affects internal signalling via second messengers Phospholipase C signalling system. Most evidence for the effect of editing on function comes from downstream measurements of receptor activity, radio ligand binding and functional studies.
Samples were shaken for 90 s and centrifuged at 21, g for 90 s at room temperature. Proteins isolated from cultured cells and PFCx were used in these experiments.
Expression of membrane-associated 5-HT 2A receptors were determined by Western blot as previously described Carrasco et al. The antiHT 2A antibody was a generous gift from Dr. Muma and has been previously validated in the literature Singh et al. Negative controls included either omission of primary antibody or addition of pre-immune rabbit immunoglobulins. Each sample was measured on three independent gels.
All samples were standardized to controls and normalized to their respective actin levels. The primers used in this manuscript were: These primers have been previously validated in the literature Atkinson et al.
In all real-time PCR experiments, measurements were made from the number of cycles required to reach the threshold fluorescence intensity [cycle threshold Ct ]. We utilized two cells line in our work in order to examine whether our findings could be replicated in two independent neuronal cells lines.
CLU cells were incubated with either vehicle ethanol 0. Samples were run in triplicate. Cells were washed with PBS pH 7. Expression of membrane-associated 5-HT 2A receptors was determined by Western blot as described above. On day 2, cells were placed in serum-free medium and incubated with CP 55, 1nM for 3 days.
CLU cells were pretreated with either vehicle ethanol 0. Twenty min later cells were incubated with either vehicle ethanol 0.
In a different experiment, CLU cells were incubated with either vehicle ethanol 0. CLU or A1A1v cells were incubated with either vehicle ethanol 0. Cells were then treated with either vehicle ethanol 0. Expression of nuclear-associated pERK levels were determined by Western blot as previously described. This procedure was repeated over three days. Cells were collected 72 h after the initial treatment. Cells were treated with either vehicle ethanol 0. The next day, cells were rinsed with PBS 3x and incubated with either vehicle ethanol 0.
In a different experiment, CLU cells were treated with either vehicle ethanol 0. Here, 5-HT 2A receptors were identified as a single and prominent band with a molecular mass of approximately 42—43 kDa as previously described Singh et al.
CLU cells were used to better study the mechanisms involved in the upregulation of 5-HT 2A receptors. No significant effect on the protein levels of 5-HT 2A receptors were detected after 24 hours of exposure to CP 55, data not shown.
This experiment was designed to study whether these cannabinoid agonists can mediate similar effects in two different and independent neuronal cell lines that endogenously express 5-HT 2A receptors. Our previous data suggest that selective CB2 receptor agonists induce a strong regulation of 5-HT 2A receptors.
JTE and AM are two specific CB2 receptor antagonists used in the literature to study the specific effects of CB2 receptors in different animal or cell culture models Iwamura et al. In these experiments we studied the effect of these two antagonists on the GP 1a-induced upregulation of 5-HT 2A receptors Fig. No main interaction was detected between either pretreatment F 1,0. A main interaction was detected between pretreatment and treatment F 1, No main interaction was detected between these three factors F 1,2.
This process was repeated for 3 days and cells were collected 24 h after the last incubation. Here, cells were treated with either vehicle or GP 1a 1nM for 24h. After rinsing the cells, they were treated with either vehicle or GP 1a 1nM for 15 min and nuclear and cytosolic proteins were isolated as previously described. Several behavioral reports suggest that cannabinoids can regulate 5-HT neurotransmission, and more specifically 5-HT 2A receptors Cheer et al.
This could explain how chronic exposure to CP 55, is associated with downregulation and a significant loss of CB1 and CB2 receptors in cortex as measured by receptor binding experiments Rubino et al.
We used CLU cells as cellular model to investigate the molecular mechanism by which cannabinoid receptors might mediate the upregulation and enhanced activity of 5-HT 2A receptor signaling in a neuronal cell line. This neuronal cell line endogenously expresses 5-HT 2A receptors coupled to the stimulation of PI hydrolysis. The two-fold increase in both 5-HT 2A receptor mRNA and protein levels suggest that an increase in the activity of 5-HT 2A receptors might be associated with the exposure to the non-selective cannabinoid agonist, CP 55, This suggests that selective CB2 receptor agonists can upregulate 5-HT 2A receptors in two independent neuronal cells that endogenously express 5-HT 2A receptors.
Both CB2 receptor antagonists completely prevented the GP 1a-induced upregulation of 5-HT 2A receptors in this neuronal cell line, supporting the role of the CB2 receptor agonist in this phenomenon. The CB2 receptor is a seven transmembrane receptors that was initially identified in the periphery but not in the brain Abood and Martin, ; Demuth and Molleman, Indeed, brain expression of CB2 receptors was much less well established and characterized in comparison to the expression of brain CB1 receptors.
However, more recent reports have established the expression of CB2 receptors in normal neurons in PFCx, amygdala, hypothalamus, hippocampus, etc. Furthermore, recent studies indicate that CB2 receptors are mainly localized in post synaptic neurons Brusco et al.
While a single administration of GP 1a triggered an increase in the nuclear-associated levels of pERK, we observed a greater response in cells that were exposed to GP 1a 24 hours earlier Fig. However, our results would suggest that repeat exposure to cannabinoid agonists might mediate enhanced responses of the CB2 receptor-mediated regulation of ERK activation.
Interestingly, studies by Lefkowitz et al. Taken together, the results of these studies indicate that eCBs reduce serotonin release in the CNS via the activation of CB1 receptors. In addition to the modulation of serotonin release, eCB signaling has also been shown to control the function and expression of various 5-HT receptors in the CNS.
The reduction in the function of 5-HT 1A and 5-HT 2A receptors appears to be associated with no significant changes in their expression levels Aso et al.
The results of these studies show an increased expression and function of 5-HT 1A receptors in the hippocampus Moranta et al. Together these studies provide strong evidence that eCBs via activation of central CB1 receptors modulate the release of 5-HT as well as the function of 5-HT receptors. Such a regulatory control may represent a mechanism that underlies the functional cross talk between the eCB and 5-HT systems.
Results from early anatomical studies have shown that both CB1 receptors and the enzymes involved in the synthesis and catabolism of eCBs are expressed in the DR Egertova et al. Consistent with this notion, in vivo and in vitro electrophysiological studies have reported that eCBs as well as exogenous cannabinoids regulate the excitability of DR 5-HT neurons Bambico et al.
For example, results from an in vivo extracellular recording study show that systemic administration of FAAH inhibitors, which presumably increases anandamide levels in the CNS, enhances the firing activity of DR 5-HT neurons via the activation of CB1 receptors Gobbi et al. Similarly, in vivo administration of the selective CB1 receptor agonist Win 55, has been shown to increase the firing rate of DR 5-HT neurons Bambico et al. The eCB-induced excitation of DR 5-HT neurons reported in vivo is blocked after lesion of the prefrontal cortex, suggesting that it is an indirect effect mediated by activation of CB1 receptors presumably located in the prefrontal cortex Bambico et al.
Evidence for a direct role of eCB signaling and CB1 receptors located in the DR in regulating the excitability of 5-HT neurons comes from in vitro intracellular electrophysiological studies Haj-Dahmane and Shen, ; Results from these studies show that, while eCBs as well as synthetic CB1 receptor agonists have no significant effects on the intrinsic excitability of DR 5-HT neurons, they profoundly reduce the strength of glutamate synapses impinging on these neurons.
Specifically, administration of anandamide strongly reduces the amplitude of glutamate-mediated excitatory postsynaptic currents EPSCs. The inhibitory effect of eCBs as well as the synthetic CB1 receptor agonists is readily blocked by the selective CB1 receptor antagonist AM , indicating that the eCB-induced modulation of glutamtergic synaptic transmission in DR 5-HT neurons is mediated by activation of CB1 receptors.
The results of these studies also show that the CB1 receptor-induced suppression of glutamatergic transmission is consistently associated with a significant decrease in the probability of glutamate release.
Such finding indicates that the inhibition of glutamatergic synaptic transmission to DR 5-HT neurons is mainly caused by a presynaptic suppression of glutamate release.
The outcome of these studies also suggests that CB1 receptors are most likely located on glutamatergic terminals impinging on DR 5-HT neurons.
In addition to the inhibition of glutamate release, a recent in vitro extracellular recording study reports that eCB tone in the DR can regulate the firing activity of 5-HT neurons by regulating GABAergic transmission Mendiguren and Pineda, However, the detailed cellular mechanism of such regulation remains to be determined.
The general view that emerges from the above studies is that eCBs regulate the activity of DR 5-HT neurons primarily, if not exclusively, via the regulation of their excitatory and inhibitory inputs see, Fig.
By modulating the function of both the excitatory and inhibitory synapses, it is possible that eCBs exert a bidirectional modulation of the activity of DR 5-HT neurons. Clearly, additional anatomical studies are required to determine the colocalization of these receptors with GABA and glutamate and their density on synaptic terminals impinging on DR 5-HT neurons.
The results of these studies are necessary to formulate a comprehensive model of eCB modulation of 5-HT neurotransmission. CB1 receptors are expressed by local glutamate neurons and by glutamatergic inputs from forebrain areas such as, the prefrontal cortex that target 5-HT neurons and GABA interneurons of the DR. CB1 receptors are also located on 5-HT terminals, activation of these receptors reduces 5-HT release and overall 5-HT neurotransmission.
A major finding from our electrophysiological study is that DR 5-HT neurons synthesize and release eCBs in an activity-dependent manner Haj-Dahmane and Shen, ; The activity driven-eCB synthesis and release from DR 5-HT neurons can be initiated by postsynaptic membrane depolarization and the subsequent increase in intracellular calcium or in response to the activation of postsynaptic G-protein coupled receptors.
The DSE requires an increase in postsynaptic intracellular calcium, and is caused by a presynaptic decrease in glutamate release. Activation of these receptors with orexin B, also called hypocretin 2, increases the excitability of DR 5-HT neurons Liu et al.
The orexin-induced depression of synaptic transmission is mediated by the activation of postsynaptic OXR and caused by presynaptic decrease in glutamate release, indicating the involvement of retrograde messengers. Examination of the retrograde messengers involved reveals that the OXR-induced suppression of glutamate release is blocked by the CB1 receptor antagonist AM and occluded by the CB1 receptor activation.
Such findings demonstrate that the OXR-induced inhibition of glutamatergic synaptic transmission is mediated by retrograde eCB signaling Haj-Dahmane and Shen, However, additional studies are required to test the involvement of anandamide and others eCB species. Future studies are required to investigate the role of these receptors in the regulation of eCB signaling in DR 5-HT neurons. Results from the above studies indicate that eCBs through activation of CB1 receptors provide an important regulatory control of 5-HT system functions.
This regulatory control occurs at the level of DR 5-HT neuron cell body as well as at their projection areas. At the level of the cell body area, eCBs, via the activation CB1 receptors, control the excitability of DR 5-HT neurons by modulating the strength and plasticity of synaptic transmission. An important finding of these studies is that DR 5-HT neurons can synthesize and release eCBs in an activity-dependent manner. The eCBs released from 5-HT neurons play a central role in mediating retrograde modulation of synaptic transmission in the DR.
Thus, it will be interesting to examine whether the inhibitory effect induced by these pharmacological agents is accompanied by an alteration of eCB signaling in the DR. More importantly, because 5-HT 1A receptor agonists are widely used for the treatment of stress-related mood disorders, elucidating their effects on the eCB signaling will determine the potential contribution of the eCB system in the therapeutic effects of 5-HT 1A agonists.
Finally, although a number of studies have shown that exposure to various stressors alters the function of DR 5-HT neurons Baratta et al. Future studies are needed to address these issues and enhance the current understanding of the impact of stress on the functional cross talk between eCB and 5-HT systems. This work was supported in part by research grants from the National Institutes of Health research grant MH to S.
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Abstract The cannabinoid CB 1 receptors and their endogenous agonists, endocannabinoids eCBs , are ubiquitously distributed throughout the central nervous system CNS , where they play a key role in the regulation of neuronal excitability.
Endocannabinoid, serotonin, dorsal raphe, stress, synaptic transmission. Endocannabinoid signaling in the mammalian brain Endocannabinoids are lipid signaling molecules with potent actions at cannabinoid receptors.
Modulation of the stress responses by eCB signaling and the 5-HT system The notion that the eCB system may be involved in the regulation of the stress responses is rooted in the observation that the recreational use of cannabis in humans has profound effects on mood and stress-related behaviors Williamson and Evans, Regulation of the 5-HT system by eCB signaling Early evidence for a role of eCB signaling in the regulation of the 5-HT system is suggested by behavioral studies showing a high level of functional overlap between the 5-HT and eCB systems.
Open in a separate window. Concluding remarks Results from the above studies indicate that eCBs through activation of CB1 receptors provide an important regulatory control of 5-HT system functions.
Acknowledgments This work was supported in part by research grants from the National Institutes of Health research grant MH to S. Cannabinoid effects on anxiety-related behaviors and hypothalamic neurotransmitters.
Lack of CB1 receptor activity impairs serotoninergic negative feedback. Cannabinoids modulate synaptic strength and plasticity at glutamatergic synapses of rat prefrontal cortex. Activation of cannabinoid receptor type 1 decreases glutamatergic and GABAergic synaptic transmission in the lateral amygdala of the mouse. Long-lasting self-inhibition of neocortical interneurons mediated by endocannabinoids.
Genetic deletion of fatty acid amide hydrolase alters emotional behavior and serotoninergic transmission in the dorsal raphe, prefrontal cortex, and hippocampus. Cannabinoids elicit antidepressant-like behavior and activate serotoninergic neurons through the medial prefrontal cortex. Selective activation of dorsal raphe nucleus-projecting neurons in the ventral medial prefrontal cortex by controllable stress.
Effects of sustained administration of the serotonin and norepinephrine reuptake inhibitor venlafaxine: Functional role of high-affinity anandamide transport, as revealed by selective inhibition. Serotonin evokes endocannabinoid release and retrogradely suppresses excitatory synapses.
Cannabinoid-serotonin interactions in alcohol-preferring vs. alcohol-avoiding mice.
information on the nature of CBHT interactions. The specific objective of this study was to examine the state of 5-HT functionality in CB1 receptor knockout. Paracetamol and unwanted 5‐hydroxytryptamine interactions COX‐2 inhibition 3 and cannabinoid receptor (CB1) agonism 4 but does not. Abstract. Cross-talk between cannabinoid CB1 and serotonin 5-HT receptors in rat on the 5-HT2 receptor and on G protein-receptor interactions, respectively.