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Endocannabinoids are derivatives of arachidonate-based lipids that activatea network of signaling pathways in eukaryotes...

Cannabis has been used medicinally for millennia, but the cannabinoid (CB) field exploded with the identification of its endogenous receptors and endocannabinoids (eCBs). In vitro experimentation established that eCBs alter synaptic plasticity at presynaptic nerve terminals; however, the characterization of the eCB system (ECS) in vivo remains incomplete. This study aimed to determine the mechanism of in vivo eCB-mediated hippocampal synaptic plasticity and to analyze the effects this plasticity had on spatial working memory (SWM). With in vivo recordings of field excitatory postsynaptic potentials (fEPSPs) in anesthetized mice and rats as well as pharmacological manipulation of the ECS and glutamate receptor antagonism, it was found that eCBs, both anandamide (AEA) and 2-arachnidonyl glycerol (2-AG), caused LTD at hippocampal CA3-CA1 synapses. Induction of eCB-LTD occurs via a sequential activation of cannabinoid type-1 receptor (CB1R) and NR2B-containing NMDA receptor (NR2BR) and is expressed through the endocytosis of AMPA receptors (AMPARs). Increased eCB tone also caused an impairment of SWM for over 24 hours in the Delayed Non-Match-To-Sample (DNMTS) T-maze. This study provides the first evidence that an acute administration of eCB degradative enzyme inhibitors not only produces an in vivo LTD at hippocampal CA3-CA1 synapses that requires CB1R, NR2BR, and AMPAR, but also impairs SWM, a phenomenon also caused by an acute injection of exogenous CBs.

Endocannabinoid (eCB) system is composed of endogenous CB ligands including anandamide (AEA) and 2-Arachidonyl glycerol (2-AG), enzymes involved in their biosynthesis and degradation such as diacylglycerol lipase-α (DAGL- α), and CB receptors. Primarily, there are three types of CB receptors - CB receptor 1(CB1), CB receptor 2 (CB2) and non CB1 non CB2 types of CB receptor (e.g. GPR, TRPV1) and they belong to G-protein (Gi/o) coupled receptors (GPCR) family.CB1 receptors are abundant in the brain where they modulate neuronal activities. On the other hand, CB2 receptors are predominantly expressed in the immune cells and regulate the growth and proliferation of different immune cells and modulate the activities of cytokines network and anti-oxidant machinery in stress conditions. Inflammation plays a central role in hearing loss (HL) caused by different ototoxic insults including anti-neoplastic agents such as cisplatin, aminoglycosides and acoustic trauma. These insults can trigger chronic production of reactive oxygen species (ROS) in regions of cochlea such as organ of Corti, stria vascularis (SVA), spiral ligament (SL) and spiral ganglion neurons (SG). This leads to increased synthesis of pro-inflammatory cytokines, disruption of mitochondrial membrane integrity, activation of DNA damage/repair pathways and activation of pro-apoptotic enzymes. Jeong et al. (2007) have shown that CB2 receptor specific agonist (JWH-015) protects the HEI-OC1 hair cell cultures against cisplatin-induced cytotoxicity in-vitro. The goal of the current study was to examine the distribution and function of CB receptors (mainly CB2) in the cochlea and determine whether activation of these receptors could protect the cochlea by altering the expression of ROS generating proteins, along with pro-inflammatory and pro-apoptotic proteins. This study also investigated whether inhibition of eCB synthesis can causes HL. Aim 1 of the current study investigated the expression of CB receptors in the cochlea using different in-vivo models such as male Wistar rat and knock-in mice with GFP-tagged CB2 receptors, in-vitro models such as organotypic culture of neonatal mouse (C57BL/6) cochlea and University of Bristol organ of Corti (UB/OC1) cells. We show that both CB1 and CB2 receptors are expressed in the outer and the inner hair cells (OHCs and IHCs), SV, SG and supporting cells (SCs) included outer and inner pillar cells. The distribution of DAGL- α was also examined in the male Wistar rats and we found the similar distribution pattern of this enzymes as CB2. DAGL- α catalyzes the hydrolysis of DAG to synthesize 2-AG, which acts as a chief endogenous CB2 ligand. Our initial studies suggested a role of CB2 and not CB1 in protection, leading us to focus on CB2 receptors for subsequent studies. Aim 2 examined the otoprotective role of trans-tympanic application of CB2 specific agonist (JWH-015) against cisplatin-induced hearing loss in male Wistar rats. Activation of CB2 receptors restored cisplatin-induced elevations in ABR thresholds which was significantly reversed by CB2 antagonist AM-630. Pre-treatment with JWH-015 protected against cisplatin-induced loss of hair cell and synaptic ribbons. In-vitro studies in UB/OC-1 cells demonstrated that pre-treatment of JWH-015 modulates the activities of signal transducer and activator of transcription 1 and 3 (STAT1 and STAT3), increases the expression of anti-apoptotic protein Bcl-xL, indicating its role in regulating the apoptosis Activation of CB2 also abrogated cisplatin-induced decrease in Na+/K+ATPase- α in the SV and SL fibrocytes and ameliorated the expression of different pro-inflammatory genes including TRPV1, COX2, NOX3, KIM1, iNOS and TNF- α. We also found that blocking of CB2 by AM630 itself resulted in hearing loss and loss in CB2 receptors, indicating eCB system is tonically active and could be important for physiological function of the cochlea. Indeed, we observed that inhibition of DAGL- α by RHC80267 results in HL. Aim 3 of this current study investigated whether pre-treatment of CB2 agonist will interfere with anti-cancer efficacy of cisplatin against various cancer cell lines head and neck cancer cells (UMSCC10B), and colon cancer cells (HCT116). Our data indicate that JWH-015 did not interfere with cisplatin-induced apoptosis in these cells. Overall, this study provides novel insights into the essential role eCBs plays in protection the cochlea under non-stressed conditions and following exposure to ototoxic agents. It also demonstrates that application of exogenous CB2 agonist (JWH-015) could serve as an effective protective agent against cisplatin ototoxicity These data suggest that localized delivery of CB2 agonists should be studied in human for protection against hearing loss.

Cannabis has been used medicinally for millennia, but the cannabinoid (CB) field exploded with the identification of its endogenous receptors and endocannabinoids (eCBs). In vitro experimentation established that eCBs alter synaptic plasticity at presynaptic nerve terminals; however, the characterization of the eCB system (ECS) in vivo remains incomplete. This study aimed to determine the mechanism of in vivo eCB-mediated hippocampal synaptic plasticity and to analyze the effects this plasticity had on spatial working memory (SWM). With in vivo recordings of field excitatory postsynaptic potentials (fEPSPs) in anesthetized mice and rats as well as pharmacological manipulation of the ECS and glutamate receptor antagonism, it was found that eCBs, both anandamide (AEA) and 2-arachnidonyl glycerol (2-AG), caused LTD at hippocampal CA3-CA1 synapses. Induction of eCB-LTD occurs via a sequential activation of cannabinoid type-1 receptor (CB1R) and NR2B-containing NMDA receptor (NR2BR) and is expressed through the endocytosis of AMPA receptors (AMPARs). Increased eCB tone also caused an impairment of SWM for over 24 hours in the Delayed Non-Match-To-Sample (DNMTS) T-maze. This study provides the first evidence that an acute administration of eCB degradative enzyme inhibitors not only produces an in vivo LTD at hippocampal CA3-CA1 synapses that requires CB1R, NR2BR, and AMPAR, but also impairs SWM, a phenomenon also caused by an acute injection of exogenous CBs.

Thesis advisor: John P. Christianson / Stress is a risk factor for neuropsychiatric disorders such as post-traumatic stress disorder and depression, yet not all individuals who are exposed to stress develop such disorders. Several factors influence susceptibility versus resilience to the effects of stress, including coping strategy biological sex. A growing body of research in humans has demonstrated that active coping strategies – defined as using available resources to problem solve – are positively correlated with resilience. In rodents, resilience to a potent acute stressor can be achieved through active coping, such as controlling the termination of a stressor, but only in males. During controllable stress males engage a stress mitigating pathway between the prelimbic (PL) and dorsal raphe nucleus (DRN), but this pathway isn’t engaged by control in females or when stress is uncontrollable in both sexes. Thus, neural activity within the ventromedial prefrontal cortex (vmPFC) is a critical determinant of stressor-induced anxiety. The mechanism that engage vmPFC excitability are not well understood. Therefore, the goals of the dissertation were 1) determine if eCBs in the PL promote neuronal excitability and behavioral resilience 2) test if ES and IS result in differential activation PL afferents, and will specifically test if ES results in greater activation PL-inputs from action-outcome associated regions, while IS leads to greater engagement of stress/fear inputs to the PL, and 3) identify network-wide patterns of activation and test the hypothesis that the stress and action-outcome networks are differentially activated as a function of stressor controllability and/or sex. We’ve demonstrated that augmenting eCBs in the PL increased excitability through a CB1 and GABA receptor dependent mechanism and was sufficient to block the stress induced decrease in social exploration. Regarding goal 2, PL inputs from the orbitofrontal cortex and DRN were activated in response to stress per se, but were not sensitive to stressor controllability and did not differ between males and females. PL afferents from the basolateral amygdala and mediodorsally thalamus were not sensitive to stress. Lastly, we quantified Fos expression in response to controllable and uncontrollable stress in male and female rats in 24 brain regions associated with stress, action-outcome learning, and showing sex differences in response to stress. Using interregional correlations, we found differences in functional connectivity as a function of stressor controllability and sex when considering all 24 regions and when considering only stress associated regions. Females showed greater overall functional connectivity compared with males, and IS resulted in greater overall connectivity than ES. We also reveal potentially important nodes in functional connectivity networks using centrality measures to identify network hubs. The findings of this research emphasize the need to study differences between males and females across all realms of neuroscience, particularly in relation to disorders of stress and anxiety. / Thesis (PhD) — Boston College, 2019. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Psychology.

The endocannabinoid (EC) system has a role in metabolic homeostasis. The purpose of this study was to determine the effect of ECs and the fatty acids they are derived from on lipid metabolism and mitochondrial function in adipocytes. 3T3-L1 adipocytes on day 8 of differentiation were treated with ECs and fatty acids for 48 hours in the absence or presence of insulin and various inhibitors. Lysates were analyzed via Western immunoblotting, a lipolysis assay and Seahorse XF Analyzer for changes in protein levels, phosphorylation state, lipolysis, and oxygen consumption rate. Results showed that ECs (2-arachidonoyl glycerol) stimulated lipolysis via a novel AMPK-dependent pathway, while fatty acids had varying effects on insulin signaling and mitochondrial function . These data suggest adipose tissue EC receptors may be a suitable target for anti-obesity therapy. Further research is needed to understand how the dietary fatty acid profile may influence synthesis of ECs. / May 2016

Anandamide (AEA), a well characterized endocannabinoid that has actions at multiple targets in the eye, may have potential as a novel therapeutic in the treatment of retinal disease. However, AEA is rapidly degraded by fatty acid amide hydrolase (FAAH). Therefore this study examined the drug URB597, that inhibits FAAH degradation of AEA, to assess AEA effects in experimental models of retinal damage. The objectives were to: 1) evaluate changes present in the aging retina, 2) determine whether the aging retina is more susceptible to tissue damage, and 3) investigate whether increasing AEA can provide retinal neurovascular protection in young and aged retina following damage. The results from this study showed that URB597 had protective effects on retinal ganglion cells and retinal capillaries and inhibited phagocytotic MG in models of retinal damage in young, but not the aged retina.

Diss. (sammanfattning) Umeå : Umeå universitet, 2005. / Härtill 4 uppsatser.

Thesis (Ph. D.)--Virginia Commonwealth University, 2008. / Prepared for: Dept. of Pharmacology and Toxicology. Bibliography: leaves 153 - 179. Available online via the internet.