We then assessed lppLTP in slices from conditional β1 KO mice. For these studies, mice with floxed β1 exon 3 were crossed with CaMKIIα-Cre mice T29-firstl/J to generate KOs with β1 expression depressed in excitatory hippocampal neurons beginning in the third week of life . The LPP I/O curve in β1 KOs was slightly depressed relative to wild types but this effect did not approach statistical significance . Waveform characteristics were also comparable between KOs and wild types ; quantitatively, the mean time constants for the rising and decay phases of the fEPSP were not detectably different. Despite seemingly normal fEPSPs, the β1 KOs had severely impaired lppLPP . A major subset of β1 family integrins bind an Arg-Gly-Asp consensus sequence in their matrix ligands . Snake toxin “disintegrins,” including echistatin, contain an RGD mimetic sequence that blocks this integrin subgroup. We confirmed that infusion of echistatin blocks the stabilization of postsynaptic LTP in field CA1 , as previously reported , and then tested for effects on lppLTP.We compared the results obtained with the various integrin manipulations by normalizing the percent lppLTP per experimental slice to the mean value for yoked vehicle cases. There was a highly significant group effect for treatment with measures of potentiation from the anti-β1 group being lower than those from control, anti-αV, or echistatin groups . The anti-αV and echistatintreatment groups exhibited potentiation that did not differ from each other or from controls. Next,cannabis grow equipment we tested for integrin signaling that is activated by HFS but not by pharmacological activation of the CB1Rs.

A likely additional factor is proline-rich tyrosine kinase-2 , a second integrin-associated tyrosine kinase that produces downstream effects beyond those targeted by the homologous FAK . Physiological stimulation increased the number of terminals with high density pPyk2 immunolabeling . In contrast, WIN did not affect the percentage of LPP terminals with dense concentrations of pPyk2 . These findings point to a hypothesis in which high-frequency LPP stimulation activates β1 family integrins and their signaling to 2 associated tyrosine kinases, one of which is targeted by 2-AG signaling through the CB1R. This co-operative signaling then produces the presynaptic cytoskeletal changes required for lasting increases in transmitter release. Activation of synaptic integrin signaling by physiological activity is reported to involve ligands generated from the extracellular matrix by metalloproteinases , and by MMP9 in particular . We tested for involvement of this mechanism in the LPP and found that perfusion of the broad MMP inhibitor GM 6001 produced a significant impairment of lppLTP = 4.94, 2-tail t-test, n = 6 ea without effect on baseline responses.Lateral entorhinal cortex neurons that generate the LPP receive dense input from the adjacent olfactory cortex and neocortical associational regions. Prior studies confirmed that the LPP is critical for learning two-odor discriminations and, more recently, that AM251 blocks this encoding whereas suppression of 2-AG degradation enhances it . We accordingly used the 2-odor paradigm to test if learning elicits signaling associated with the production of lppLTP. Rats were trained on a series of ten novel discriminations over successive days until they showed a strong preference for the positive cue in trials 6–10. Past work showed that such learning results in long-term memory . A novel odor pair was presented on the test day.

Animals were euthanized immediately after the 10th training trial and sections through rostral hippocampus were processed for dual immuno staining and FDT analysis: ~400 000 SYN+ structures fitting the size and eccentricity constraints of synapses were reconstructed in 3D from image z-stacks in the LPP field within the outer molecular layer of the DG. The data were expressed as percent of all bouton-sized SYN clusters for each of a series of increasing pROCK density bins. The immunofluorescence intensity-frequency distribution for SYN co-localized pROCK was skewed to the right for rats trained in the olfactory discrimination relative to untrained controls , indicating that the learning group had greater concentrations of pROCK in LPP terminals . Increases in pROCK immunolabeling produced by odor learning were eliminated by AM251 given 1 h before training . There was a small but significant difference in the total number of SYN clusters in the AM251 group relative to the odor learning plus vehicle or control animals. To minimize any effects of this, we recalculated the above data for each pROCK density bin for each animal as a percentage of all double-labeled contacts for that animal, with the results expressed as a cumulative probability function. The mean curve for the learning plus vehicle group was clearly displaced to the right relative to that for the learning plus AM251 or for control rats ; the results for the latter 2 groups were superimposed on each other . A bootstrap analysis using randomly selected pairs of animals confirmed that the summed deviations between curves were much greater for learning versus control groups than for the learning + AM251 versus control groups .The present studies provide a first detailed description of the mechanisms underlying a novel form of LTP in a primary cortical input to hippocampus. Given that this projection conveys semantic information to hippocampus , the findings are of basic importance to the development of neurobiologically based theories of episodic memory.

The substrates for lppLTP include a repurposing of elements utilized in conventional, postsynaptic potentiation, as described for the intensively studied S–C projection to CA1, supplemented with novel features. Both forms of potentiation are induced postsynaptically via NMDARs and increased calcium levels , and both require activation of β1 integrins and their downstream effector ROCK . Integrins are a primary membrane regulator of the actin cytoskeleton and their prominent role in both types of plasticity suggests that activity-induced structural changes constitute a shared endpoint. Potentiation of S–C synapses in field CA1 entails integrin-driven assembly of stable actin networks in dendritic spines and an associated shift in spine/synapse morphology . Morphometric studies have yet to be performed for lppLTP but results obtained with the toxin latrunculin A,mobile grow system which selectively blocks actin filament assembly, confirm that in this system the cytoskeletal reorganization is located presynaptically . Thus, integrin regulation of the cytoskeleton, via signaling common to cell adhesion junctions throughout the body, underlies both forms of potentiation but in the case of lppLTP these processes are active on the presynaptic side. This specialized feature of the LPP results in a new form of LTP. Earlier work showed that LTP induction in the LPP requires stimulation of mGluR5 receptors, something that was not the case for field CA1 or the medial perforant path . The mGluR5 receptor forms a postsynaptic signalosome with the scaffolding protein Homer and the 2-AG synthesizing enzyme diacylglycerol lipase-α . Activation of this unit results in de novo 2-AG production and retrograde signaling to presynaptic CB1Rs, 2 events shown to be essential for lppLTP . The present results establish that this dependency is not due to canonical CB1R signaling, which produces depression of transmitter release at sites throughout the brain. Instead, the critical contribution of CB1Rs to lppLTP involves activation of the integrin-associated kinase FAK and its downstream effector ROCK with high-frequency afferent activity. We found that the CB1R inverse agonist AM251 prevented such activation, whereas the CB1R agonist WIN mimicked it and also lowered the threshold for induction of lppLTP. Conversely, the release depression function of the CB1R was poorly developed in the LPP and not involved in the potentiation effect. ECB-mediated release suppression is reportedly sensitive to locally synthesized pregnenolone and involves phosphorylation of the vesicular protein Munc18-1 . These effects were evident in S–C projections to CA1 but not in the LPP. Specifically, treatment with CB1R agonist WIN increased phosphorylation of Munc18-1 and caused a Munc18-1 dependent, pregnenolone-sensitive depression of synaptic responses in field CA1. In contrast, neither pregnenolone nor reductions in Munc18- 1 expression affected lppLTP. We propose that a shift in the bias of CB1R signaling away from the Munc18-1 pathway and toward facilitation of the ROCK/FAK, integrin signaling cascade constitutes a projection system-specific specialization that enables the thus far singular form of LTP found in the LPP . There is precedence for both ligand- and cell-type-specific differences in CB1R signaling .

Ligands for CB1R, including the ECBs, synthetic cannabinoids such as WIN and phytocannabinoids such as Δ9 -tetrahydrocannabinol, all bind different residues on the receptor. This has been suggested to give rise to ligand-specific conformational changes in CB1R leading to activation of different downstream signaling pathways . Moreover, the functional selectivity of a given ligand can be cell-type-specific . In line with these observations, we found cell-type-specific differences in CB1R-mediated responses to a given ligand and differences in CB1R response to different ligands for a given cell-type . The evidence for projection-specific differences in CB1R signaling gives rise to the prediction that modulation of synaptic transmission by ECBs during behaviorally relevant patterns of synaptic activity, a topic that has received surprisingly little attention, will differ between the S–C and LPP systems. Tests confirmed that stimulation in the low-frequency gamma range, selected to simulate the activity in fields CA3 and CA1 during exploration , engages CB1Rs to depress synaptic responses generated by the S–C, but not LPP, projections. Thus, the projection-specific bias in CB1R function is likely to differentially influence throughput across the nodes of the primary hippocampal network. An interesting issue for future research concerns the extent to which the contribution of ECBs to synaptic function is frequency-tuned and differsacross hippocampal rhythms associated with various behaviors. Prior work showed that blocking or enhancing the production of 2-AG produces corresponding effects on both the magnitude of lppLTP and the encoding of olfactory cues carried to hippocampus by the LPP system . The present experiments demonstrate that olfactory discrimination learning elicits evidence for potentiation in the form of increased presynaptic pROCK within the LPP. Questions thus arise about the functional significance of using a specialized form of plasticity to encode the semantic information carried by the LPP. One possibility is that the specialization helps to maintain cue identity through downstream hippocampal processing. The novel lppLTP effect changes the frequency facilitation characteristics of the LPP, as evidenced by paired-pulse measurements, something that would be expected to alter the spiking response of granule cells to patterned input. This would serve to differentiate the DG outputs produced by learned versus unlearned cues and help maintain cue identity through downstream processing. It would also further distinguish the response of granule cells to input arriving over the LPP from those elicited by the subjacent medial perforant path . Related to this, insertion of a 2-AG step in the mechanisms for encoding opens the way for modulation of lppLTP by afferents arising from sites other than entorhinal cortex. Cholinergic inputs from the medial septum and diagonal bands are of particular interest in this regard because enhancing constitutive transmission in this projection elevates 2-AG levels and related CB1R signaling in terminals. We used this effect to confirm that the Munc18-1 release suppression system is present in the LPP although not engaged by WIN or afferent stimulation. The studies also demonstrated that increasing cholinergic transmission has a strong positive effect on the production lppLTP, particularly when release suppression machinery is blocked with pregnenolone. It is, therefore, possible that particular patterns of fifiring by septal afferents or levels of pregnenolone synthesis promote the presynaptic LTP in the LPP while depressing the postsynaptic variant found in the medial perforant path. Such a mechanism could be of value when semantic information has a higher priority than spatial data. Finally, the present evidence for biased CB1R signaling has significant implications for hypotheses about how marijuana influences cognition. The Δ9 -tetrahydrocannabinol component of cannabis is an agonist for CB1Rs and stimulates the production of pregnenolone ; results presented here indicate that the combination of CB1R stimulation and high levels of pregnenolone will lower the requirements for robust lppLTP and the encoding of near threshold cues. It is interesting regarding this possibility that marijuana promotes the formation of false memories in episodic memory tests . In all, the differential effects of CB1R stimulation across the principal nodes of hippocampal circuitry are predicted to underlie a distortion of episodic memory with cannabis exposure that is due to enhanced plasticity in the LPP.

Binge drinking was assessed per NIAAA criteria for binge drinking . Binge drinking behavior was dichotomized such that participants who had any binge drinking episode in the last 30 days were classified as binge drinkers . Lifetime history of alcohol exposure, including quantity and frequency, was assessed via a semi-structured timeline follow-back interview that evaluates drinking patterns across different periods in an individual’s life. Current depressive symptoms were assessed using the Beck Depression Inventory-II, a self-report measure . The Composite International Diagnostic Interview was administered to evaluate current and lifetime mood and SUDs . Notably, the parent grants from which baseline data were drawn were funded prior to the publication of the DSM 5. Therefore, diagnoses were made in accordance with DSM-IV criteria where alcohol/substance abuse is met when participants report recurring problems as a result of continued alcohol/substance use; and alcohol/substance dependence is met when participants experience symptoms of tolerance, withdrawal, and/or compromised control over their alcohol/substance use . In order to remain consistent with the current DSM 5 criteria and nomenclature, alcohol/substance abuse and dependence criteria were combined to capture AUD and SUD. Participants were tested for HIV by enzyme-linked immunosorbent assay with Western Blot confirmation. All participants completed a comprehensive medical evaluation including self-report measures, structured neurological and medical evaluations,cannabis drainage system and blood samples to assess the presence of medical comorbidities and HIV disease characteristics. HIV viral load in plasma was measured using reverse transcriptase-polymerase chain reaction , where viral load was deemed undetectable below 50 copies/mL.

Participants were administered a comprehensive battery of neurocognitive assessments measuring global and domain-specific neurocognitive performance: global function, verbal fluency, executive function, processing speed, learning, delayed recall, working memory, and motor skills . Raw scores from each neuropsychological test were converted into demographically-corrected T-scores . Global and domain-specific continuous T-scores were derived from averaging the demographically-corrected T-scores across all tests and within each neurocognitive domain, respectively . These global and domain-specific T-scores were used as primary outcomes for comparisons of neurocognition between HIV/Binge groups. Demographic, psychiatric, medical, alcohol and substance use, and HIV disease characteristics were compared between the four HIV/Binge groups using analysis of variance or chi-square tests, as appropriate. Pair-wise comparisons were conducted to follow up on significant omnibus results using Tukey’s Honest Significant Difference tests for continuous outcomes or Bonferroni adjustments for categorical outcomes.To examine the first study aim, one-way ANOVA and Tukey’s HSD tests were used to compare mean global and domain neurocognitive T-scores between the four HIV/binge drinking groups. For any significant one-way ANOVA result, a 2 x 2 factorial ANCOVA was used to model independent and interactive effects of HIV and binge drinking status, covarying for total drinks consumed in the last 30 days and any demographic or non-alcohol-related clinical characteristics that differed between groups at p<0.05 . These demographic covariates were included to increase confidence that any observed difference in neurocognition between HIV/Binge groups would not be attributable to confounding effects of age and sex that may that may exist above and beyond the T-scores’ demographic corrections. To further support any findings indicating additive main effects, Jonckheere-Terpstra tests for ordered alternatives examined whether there was a statistically significant negative relationship between the number of risk factors and neurocognitive performance .

Finally, to examine the second study aim, multiple linear regressions modeled the interaction between age and HIV/Binge status group on global and domain-specific T-scores, also covarying for total drinks consumed in the last 30 days, sex, and lifetime history of non-alcohol. Our examination of age as a predictor of demographically-corrected T-scores will allow understanding of how the effect of age in certain vulnerable groups may go above and beyond that of normal controls on whom demographic corrections were based. Parametric statistics were used because the outcome variables were continuous and had normal distributions in each HIV/Binge group. All analyses were performed using R, version 3.5.0. Demographic and clinical factors by HIV/Binge group are displayed in Table 1. The HIV-/Binge- group was younger than both HIV+ groups , and the HIV+/Binge+ group had a higher proportion of men compared to the two HIV- groups . Regarding alcohol and substance use characteristics, the two Binge+ groups had significantly higher quantity and frequency of alcohol use in the last 30 days, higher proportions of current and lifetime AUD, higher lifetime quantity and frequency of alcohol use, and a higher proportion of lifetime nonalcohol SUDs compared to those of both Binge- groups . Alcohol use characteristics, including frequency of alcohol binges in the last 30 days, did not differ between the HIV-/Binge+ and HIV+/Binge+ groups.For each of those neurocognitive outcomes with a significant omnibus result, follow-up pairwise comparisons showed significant differences between only the HIV-/Binge- and the HIV+/Binge+ groups, with HIV+/Binge+ participants exhibiting poorer performance . Results of the 2 x 2 factorial ANCOVAs are shown in Table 2. Additive main effects of HIV status and binge drinking status were detected on global function and processing speed, however none of the interactions between HIV and binge drinking status on neurocognitive outcomes reached statistical significance. Additive main effects of HIV and binge drinking were further supported by results from Jonckheere-Terpstra tests indicating significantly lower global and processing speed performance by each increase in risk factor count . Binge drinking was also a significant predictor of delayed recall and working memory. Of note, the effects of binge drinking on neurocognition were not attenuated by accounting for total drinks in the last 30 days, which did not significantly relate to any neurocognitive outcome .

Multiple linear regression revealed significant interactions between age and HIV/Binge group on neurocognitive outcomes of learning , delayed recall , and motor skills . Specifically, the association between age and each of those three neurocognitive outcomes was significantly more negative in the HIV+/Binge+ group compared to that of the HIV-/Binge- group . This interaction was not significant for any other neurocognitive outcome . Additional analyses comparing age-slopes between all groups revealed that the difference in age-slopes between the HIV+/Binge- and HIV-/Binge groups approached significance for delayed recall and motor skills , such that the HIV+/Binge- group had a stronger relationship between age and those neurocognitive domains. Total drinks in the last 30 days, sex, and lifetime non-alcohol SUD were not significant predictors of any neurocognitive outcomes.Given the rapidly growing population of older adults with and without HIV along with the increased rates of binge drinking among them,indoor cannabis grow system studying the combined effects of HIV and binge drinking across the lifespan is timely and important. Partially consistent with our first hypothesis, the HIV+/Binge+ group demonstrated the worst global neurocognitive functioning ; however, the combined effects of HIV and binge drinking on global neurocognitive functioning exhibited an additive, rather than synergistic, pattern . Consistent with our second hypothesis, we found a novel interaction with age and HIV/Binge drinking group such that the HIV+/Binge+ group displayed a stronger negative relationship between age and three domain-specific neurocognitive outcomes compared to the HIV-/Binge- group. Importantly, the alcohol-related detriments to neurocognition appeared to be specific to binge drinking, as total 30-day alcohol consumption was not a significant independent predictor of any neurocognitive outcome in our statistical models. These findings suggest that recent, discrete episodes of heavy alcohol exposure relate to poorer brain function and highlight the need for interventions to reduce binge drinking behavior among PWH, especially older PWH, in order to promote cognitive health. The findings showing additive main effects of HIV and binge drinking are consistent with several other studies demonstrating additive, but not synergistic, effects of HIV and heavy alcohol use on neurocognitive functioning . In fact, there is only one study to our knowledge that has shown an interactive effect of HIV and heavy alcohol on neurocognition, specifically in the domains of motor and visuomotor speed . Our finding of HIV/Binge group differences in neurocognitive domains of processing speed, delayed recall, and working memory is also consistent with the frontostriatal and frontolimbic neural damage that has been observed in studies of adults with HIV and heavy alcohol use.

As briefly discussed, there are a number of possible mechanisms underlying the relationship between heavy alcohol use and adverse neurocognitive outcomes in HIV, including those related to antiretroviral therapy ; possible pharmacokinetic interactions with alcohol . Recent research, however, has revealed neuroinflammatory and neuro-immunological effects as major pathways underlying the relationship between heavy alcohol use and neurobiological damage , with several of these neuroimmune pathways overlapping with effects from HIV . Although these immunobiological underpinnings are still poorly understood in the context of comorbid HIV and heavy drinking, this represents an important line of research needed to develop potential targeted interventions for reducing the incidence and/or severity of neurocognitive impairment in this population. The current study also uniquely found that the negative relationship between age and neurocognitive functioning was steepest among the PWH who reported binge drinking in the last 30 days, particularly in the domains of learning, delayed recall, and motor skills. Our findings are consistent with what is known about the greater vulnerability to brain atrophy and neurocognitive deficits in older PWH and older adults who drink heavily . Furthermore, this result showing an age by HIV/Binge group interaction is also similar to findings from a previous study in which age was found to be a significant predictor of demographically-corrected episodic memory scores only among individuals with comorbid HIV and AUD . Speculation about accelerated aging or neurocognitive decline cannot be made from our data, as they are cross-sectional; however, this result does suggest that older adults are the most susceptible to adverse neuropsychological outcomes in the context of HIV and binge drinking. Notably, this result also held when restricting the maximum age range to 60 years old for all groups, indicating that HIV/ Binge group differences in delayed recall and motor skills emerge even in the earlier stages of older adulthood. Notably, findings from this report appear to be driven specifically by our binge drinking variable, as total 30-day alcohol consumption did not significantly predict any neurocognitive outcome above and beyond binge drinking. Although there are a dearth of studies examining the specific impact of binge drinking on neuropsychological outcomes compared to that of chronic drinking over a longer span of time, some evidence suggests that the repeated periods of high level of intoxication and withdrawal from binge drinking exacerbates the detrimental neurobiological effects of alcohol . Future research is needed to examine chronicity of binge drinking and whether there may be a specific threshold associated with the greatest level of CNS risk. Still, given evidence that binge drinking may be at least as detrimental to the central nervous system as alcohol dependence, public safety measures that aim to reduce binge drinking behavior may have widespread benefits, especially among older PWH. While this study has strengths in novelty, use of a comprehensive neuropsychological battery, and clinical relevance, it also has several limitations. First, the HIV/Binge group-specific sample sizes were relatively small, particularly in Binge+ groups. Although this limited our ability to examine a full factorial three-way interaction between age, HIV status, and binge drinking status, we were still able to examine the novel age by HIV/Binge group interaction with adequate statistical power. Next, our assessment of binge drinking is based solely on self-report and may be subject to error by recall bias, memory difficulties, and/or social desirability bias; however, the majority of alcohol and substance use research relies on self-report of use. In addition, while binge drinking data specifically pertained to use within the last 30 days, we do not know exact amounts of time between participants’ last binge episode and their participation in the study, limiting our ability to comment on how recency relates to cognitive performance. Future research may benefit from the use of more objective measures of alcohol use in daily life to more accurately characterize alcohol use patterns . Finally, our exclusion of participants with current non-alcohol substance use disorders limits generalizability to others with binge drinking behavior and/or alcohol use disorder among whom polysubstance use is common. In summary, the current study demonstrated detrimental additive effects of HIV and binge drinking on neurocognitive functioning, and that older adults appear to be most vulnerable to these adverse effects particularly in the neurocognitive domains of learning, delayed recall, and motor skills.

Arguably the most significant change that came with the fourth-generation devices is the use of nicotine salts, such as benzoic acid, as opposed to free-base liquid nicotine. Freebase nicotine is alkaline, irritating, and harsh to inhale, which limits the concentration that can be inhaled. Addition of benzoic acid or other salts results in protonation of the tertiary amine on nicotine’s pyrrolidine ring, forming nicotine salts. The reduced pH of the e-liquid allows for inhalation of much higher nicotine concentrations with less of the acute respiratory irritation typical of alkaline e-liquids. Third and fourth generation devices achieve similar doses of blood nicotine as tobacco cigarettes and in some cases are being used to deliver substances other than nicotine, including cannabinoids.The Centers for Disease Control found that non-Hispanic white males are more likely to use e-cigarettes than Hispanic whites or African Americans.Lower-income smokers are also less likely to use e-cigarettes.There is concern that e-cigarettes could be a gateway to combustible cigarette use or encourage continued or enhanced nicotine addiction . Indeed, longitudinal studies have shown that young people who start using e-cigarettes are at risk of also using combustible cigarettes.However, we know little about how e-cigarette use affects other tobacco related behaviors,cannabis grow indoor and since the marketplace and demographics are changing, whether this observation holds true for fourth generation devices remains to be determined. Obtaining reliable data on e-cigarette use will depend on the development and adoption of standardized/validated self report measures of use behaviors, perceptions, and attitudes, which will allow researchers to compare across studies over time.

While there are measures currently used in surveillance studies such as Population Assessment of Tobacco and Health and CDC surveillance, the rapidly evolving landscape of e-cigarettes presents challenges including tracking the evolution of terminology and device design. Timeliness of data collection and availability for use are crucial given the rapidly evolving marketplace. The methods used for tobacco surveillance typically do not produce usable data until a year or more after data collection and rarely allow for more than annual data collection. While in-place surveillance can remain the backbone for e-cigarette investigation, novel innovative methods are required, including real-time sentinels, such as using informatics to analyze online and big data resources including Google Trends or Twitter. Thus, despite the heterogeneity in tobacco and nicotine use behaviors and trajectories, the skyrocketing popularity of e-cigarettes suggests an urgent need to better understand the implications of e-cigarette patterns of use and uptake.While cigarette smoke comes from combustion, e-cigarette vapor results from heating liquids to high temperatures, leading to a different type of exposure and dosimetry. Vaporized components of e-liquids cool and reach critical supersaturation conditions that result in a phase transition from vapor to aerosol,followed by condensational growth.Chemicals contained in the e-cigarette vapor-aerosol mixture are partitioned between the gas and particulate phases and this partitioning affects the deposition pattern in the human respiratory tract.E-cigarette aerosols have a wide particle size distribution that ranges from nanometers to micrometers, making the prediction of delivery/ deposition and dosimetry difficult.

The deposition of gas phase and nanoparticles is mostly driven by Brownian diffusion, whereas larger particles are deposited via inertial impaction, and sub-micron particles are deposited by both diffusional and gravitational forces.E-cigarette aerosols consist of hygroscopic and relatively volatile compounds, which may either quickly grow or evaporate, depending on ambient conditions. This obviously adds additional complexity to dosimetric predictions. The development of computer algorithms to predict deposition of ecigarette aerosol is an active, challenging area of investigation which includes models for multiple-path particle dosimetry, computational fluid-particle dynamics ,and thermodynamic interactions between the droplet and vapor phases.Vapers of high power devices typically use low nicotine e-liquids, while users of low power devices use high nicotine e-liquids. However, the serum cotinine levels of these groups are the same, suggesting that the users of high power devices may be exposed to more aerosol that is generated at much higher temperature, which is likely much more harmful.In addition to emission characteristics, vaping behavior is another critically important factor required to provide input parameters to the aerosol deposition models. Most studies have been conducted in the laboratory, and therefore reported puff profiles may differ from real-world scenarios.Interestingly, properties of the e-cigarette aerosol seem to affect puffing topography. For example, St Helen et al. recently demonstrated that e-liquid flavor influenced puff duration: subjects took longer puffs on a strawberry flavored e-liquid than on a tobacco flavored eliquid .The PG to VG ratio also affected puff topography. E-liquids containing more PG lead to shorter puff times, significantly greater nicotine delivery, but paradoxically, significantly less “pleasant” and/or “satisfying” feelings.Additionally, users of powerful sub-ohmic devices and nicotine salt-based e-cigarettes may display different topography profiles compared to the users of earlier generation e-cigarettes.

As devices change, more studies will be needed to determine the input parameters required to accurately determine lung deposition models.Due to this reason, studies measuring health effects of aerosols have not always been consistent. In addition to studying the complete aerosol, there has been a parallel reductionist effort to understand the health effects of separate e-cigarette constituents. These studies have primarily focused on nicotine, flavors, PG, and VG, as well as thermal decomposition products generated during the course of e-liquid vaping. However, there is a growing appreciation that information is needed on the health effects of constituents not traditionally found in nicotine-based vaping products, including -trans-D9 -tetrahydro cannabinol and cutting agents such as Vitamin E acetate .Nicotine is a water-soluble nitrogenous organic molecule that is typically extracted from tobacco leaves. It is composed of an aromatic pyridine ring bound to a pyrrolidine ring. Nicotine binds to nicotinic acetylcholine receptors , which are ligandgated cation channels.Binding of endogenous acetylcholine, or exogenous nicotine, opens the channel pore,vertical farming supplies allowing conduction of Naþ, Kþ, and Ca2þ. The channel is subsequently closed and becomes temporarily unresponsive to further ligand stimuli.nAChRs are broadly expressed throughout multiple organ systems, and in addition to the psychotropic effects on the brain, nicotine can also affect immune cells and resident, specialized cells of the heart and lungs . Despite the long history of nicotine research, understanding the role of nicotine in cardiopulmonary disease is extraordinarily difficult due to factors such as sensitization and desensitization responses; complicated dose-response relationships; the importance of exposure route; and differences in response dependent on species, age, sex, or disease status.The known effects of nicotine on the cardiovascular system are numerous and complex. Nicotine affects angiogenesis, arrhythmogenesis, endothelial cells , hemodynamics, insulin resistance, and lipids.Nicotine increases blood pressure, heart rate, myocardial contractility, and myocardial work. Nicotine also constricts coronary arteries, reduces coronary blood flow reserve and constricts blood vessels in the skin. The majority of nicotinerelated clinical studies have involved combustible cigarettes, which expose smokers to nicotine as well as multiple combustion products. To isolate the specific effects of nicotine on human cardiovascular disease, researchers have studied populations who consume nicotine without combustion, including users of nicotine medications , and smokeless tobacco users . Smokeless tobacco has been used by non-smokers in Sweden lifelong, with no evidence of accelerated atherosclerosis or most other cardiovascular harms.Nicotine medication studies have provided evidence of tolerance to the cardiovascular effects of nicotine: Low doses of nicotine increase heart rate and systemic catecholamine release, while higher doses have little additional cardio-acceleratory and catecholamine effects. These factors are reassuring when considering whether there is a risk of treating smokers with nicotine replacement medications while they are still smoking.

Limitations of nicotine medication studies include the fact that the subjects are all former smokers, nicotine medication use is generally of short duration , and the delivery of nicotine by gum or patch is sustained and does not simulate the spike in nicotine levels seen after smoking combustible cigarettes or e-cigarettes. In contrast, regular e-cigarette exposure likely takes place over years to decades, with vascular nicotine eliciting chronic hemodynamic changes. Nicotine has complex dose-response patterns and may exert species- and cell/tissue-specific effects. Primary rat cardiomyocytes incubated with nicotine showed increased intracellular Ca2þ concentrations, which likely contributed to the observed hypertrophy.Rats and mice that either consumed or were injected with nicotine showed weakening of the aortic walls and destruction of aortic elastin and collagen, all of which were likely a result of the upregulation of matrix metalloproteases. These findings indicate a potential risk for abdominal aortic aneurysms as a result of chronic nicotine consumption.Nicotine also promotes trans-differentiation of vascular smooth muscle cells to a calcifying phenotype by inducing a proinflammatory state, impairing endothelial function, and causing oxidative stress.The growing body of evidence in animal models has illustrated that nicotine can directly affect cardiovascular function without exposure to the byproducts of combustion-based delivery mechanisms. The contribution of specific vapor constituents to the observed endothelial dysfunction has not been fully delineated. Some studies have suggested that nicotine may be partially responsible, as nicotine exposure can damage epithelial and ECs, induce epithelialmesenchymal transition markers such as a-SMA and fibronectin, and release inflammatory mediators such as transforming growth factor-beta, all of which suggests that nicotine may promote tissue remodeling and fibrosis.While some investigators have hypothesized that the adverse effects of e-cigarettes were largely due to nicotine delivery, others have proposed that e-cigarette aerosol without nicotine might also contribute to these effects. Of note, in the study by Schweitzer et al., adverse nicotine-independent effects of e-cigarette aerosol on endothelial barrier function were also observed, which the authors attributed to other toxic components of e-cigarettes such as acrolein.The effects of nicotine on the lung have been established from studies in humans, animal models, and cultures of human cells. The use of smokeless tobacco permits the study of people who were never smokers and have used nicotine for many years. For example, snus users have a lower mortality rate than tobacco smokers. As discussed before, nicotine levels are 50-fold lower in plasma after use of e-cigarettes, snus, or tobacco than in the lung after tobacco or e-cigarette inhalation . This suggests that vaping and tobacco smoke inhalation lead to higher levels of pulmonary nicotine than snus due to their delivery route. Because of this, snus may not have as much effect on the lung as vaping due to different nicotine pharmacokinetics.Incubating isolated alveolar epithelial cells with nicotine-induced oxidative stress via activation of NADPH oxidase 1, leading to cell apoptosis.This observation was confirmed in vivo with nicotine injections to Wistar rats, which resulted in oxidative stress signaling in lung tissue.In contrast to promoting oxidative stress, a study in mice found that nicotine can prevent inflammation by inhibiting the release of pro-inflammatory cytokines in the lungs.However, a more recent study has challenged this observation by demonstrating that nicotine binding to a7 nAChR, lead to increased type I collagen deposition in lung fibroblasts: Fibroblasts from wild type and a7 nAChR knockout mice were exposed to nicotine, and U937 monocytes were cocultured on matrices derived from these fibroblasts. Nicotine exposure in WT but not a7 nAChR knockout fibroblasts resulted in monocyte activation and release of IL-1ß.Finally, nicotine has been shown to cause mucociliary dysfunction in human airway epithelial cells and animal models by reducing ion channel function via TRP channels.However, the effects of nicotine may be both cell type- and concentration-specific, which may lead to the disparity of results .Nicotine readily crosses the placenta and produces higher nicotine concentrations in the fetal circulation than in the maternal circulation.Epidemiologic studies have shown that cigarette smoking during pregnancy is associated with an increased risk of cardiovascular disease in offspring, although this may or may not be due to direct effects of nicotine.Moreover, nicotine may be a link between maternal smoking and the risk for sudden infant death syndrome.It is also well-established that maternal smoking, mediated most likely through nicotine, results in impaired fetal lung development and function, and these effects, while small, persist into childhood and beyond.Recent animal studies further indicate that pre-conception and prenatal ecigarette exposure to nicotine and PG/VG impairs embryo implantation and offspring’s metabolic health later in life.This is of particular concern, given the current rate of e-cigarettes use among young adults of childbearing age.Regardless of its source, fetal nicotine exposure during pregnancy has become a public concern and the impact of vaping nicotine-containing eliquids on fetal development remains to be determined.

Cross-sectional observational studies using Waves 2 and 3 data from the Population Assessment of Tobacco and Health Study have found an association between e-cigarette use and respiratory symptoms. One longitudinal W3-W4 PATH Study analysis found no relation between exclusive e-cigarette use and incident respiratory symptoms but suggested that dual users of cigarettes and e-cigarettes had significantly higher risk for symptom onset compared to exclusive cigarette users.Finally, one prospective study of young adults found an association between cannabis vaping and respiratory symptoms.There are many design issues that make these studies hard to compare. The clinical importance of the respiratory outcome is not clear in most cases because the multiple wheezing questions are analyzed in isolation from each other, or an endorsement of only one item is considered symptomatic. Many of the studies included adults with COPD, which is a diagnosis strongly linked to a history of cigarette smoking, and many people with COPD have chronic severe wheezing and dyspnea. Another concern is residual confounding: Most of the studies showing an association between e-cigarette use and respiratory symptoms failed to adjust for cigarette smoking history and concurrent marijuana use, both associated with respiratory problems and concurrent e-cigarette use. Finally, few studies addressed alternative tobacco product categories besides e-cigarettes. To better understand these divergent findings on how tobacco product use relates to respiratory health, we analyzed W2 and W3 data from the PATH Study.

We developed a dependent variable that incorporated all available questions on wheezing and nighttime cough and determined cut-off values associated with functional outcomes. We focused on both cross sectional and longitudinal associations between functionally-important respiratory symptoms and ten mutually exclusive tobacco product use categories,grow rack adjusting for past cigarette smoking history and concurrent marijuana use. We also examined results for two different cut-off values for a respiratory symptom index to test for sensitivity to symptom severity. Recruitment for W1 of the PATH Study employed stratified address-based, are a probability sampling with oversampling of adult tobacco users, young adults , and African-Americans. An in-person screener selected youths and adults from households at W1, and audio computer-assisted self-interviews collected data on tobacco use and health outcomes. Respiratory symptoms were assessed in W2 and W3 , including 28,362 and 28,148 adult participants, respectively . Mean time between W2 and W3 adult interviews was 53.8 weeks. Our analyses utilized the adult W2 and W3 Restricted Use Files. We selected all W2 adults without COPD or other nonasthma respiratory diseases . We report a complete case analysis excluding participants lost to follow up at W3 and those with missing data on any variables with final analytic sample of 16,295. PATH Study design and methods,interviewing procedures, questionnaires, sampling, weighting, and response rates are in the PATH Study Restricted Use Files User Guide.All respondents provided informed consent; Westat’s IRB approved the study. The PATH Study utilized the seven wheezing/cough questions from the International Study of Allergies and Asthma in Childhood core wheezing module.Responses to the ISAAC questions were used to create a respiratory symptom index . This index was validated in the PATH Study adult sample based on its internal consistency, test-retest reliability, and its strong association with self-reported physician diagnosis of asthma. Respondents over cut-off values of ≥2 and ≥3 had significantly higher risk for physical limitations, fatigue, and poorer perception of health assessed by items from the Patient Reported Outcomes Measurement Information System .

The full validation of this measure is published elsewhere.Because the validation supported cut-off values of of ≥2 and ≥3, we examined both as a test of the sensitivity of the findings to respiratory symptom severity. Covariates were derived from W1 and W2, and included variables associated with both tobacco exposure and functionally-important respiratory symptoms.Sociodemographic variables included age, sex, race/ethnicity, education, income, and urbanicity. Medical conditions that could result from tobacco use and also cause respiratory symptoms included asthma, congestive heart failure, heart attack, diabetes, cancer, being overweight, and use of antihypertensives known to cause coughing or wheezing . Smoke-related exposures included pack-years of cigarette smoking, second-hand smoke exposure, and marijuana use. Calculating pack years of smoking We were particularly concerned with adjusting results carefully for each individual’s cigarette smoking history, an important predictor of respiratory outcomes. We derived lifetime pack years to account for cigarette smoking history in this analysis. Lifetime pack years is a clinical metric calculated by multiplying the number of packs of cigarettes per day someone smokes by the number of years they have smoked cigarettes. The following text annotates the algorithm to calculate Wave 1 lifetime pack years. Data from Wave 1 lifetime pack years was used in conjunction with variables describing subsequent cigarette use to determine lifetime pack years at W2 and beyond. Never smokers were assigned a pack years value of zero. All questions used in the algorithm and response categories are listed in Supplemental Table 3. Because of routing instructions in the PATH Study interview, only those respondents who said that they have smoked cigarettes “fairly regularly” were asked about how long they have smoked or did smoke . For any respondent at Wave 1 who currently smokes regularly or formerly smoked fairly regularly, lifetime pack years was calculated by multiplying the number of cigarette packs smoked per day by the number of years they have smoked fairly regularly. Two different formulas were used for this calculation, depending on answers to the questions for variable R01_AC9004 and R01_AC9009 .

All main analyses were weighted using the W3 longitudinal full-sample and replicate weights to adjust for the complex sample design and loss to follow up. Variances were estimated using the BRR method with Fay’s adjustment set to 0.3 to increase estimate stability. Pack-years of cigarette smoking, tobacco product P30D frequency variables , and second-hand smoke exposure were Winsorized at the 95th, 95th and 99th percentiles, respectively,microgreens shelving to address outliers.We examined unadjusted associations between tobacco product use at W2 and the presence of functionally-important respiratory symptoms then used multivariable weighted Poisson regression to obtain adjusted risk ratios and 95% CIs for each dichotomous outcome.Next, we evaluated longitudinal associations between W2 tobacco product use and changes in respiratory symptoms from W2 to W3. Symptoms “worsened” if the symptom score was <3 at W2 but > 3 at W3. Symptoms “improved” if the symptom score was ≥ 3 at W2 but < 3 at W3. Finally, we tested the sensitivity of the findings to symptom severity level by rerunning all analyses at a cutoff level of ≥ 2. For each multivariable analysis, post-hoc two-group comparisons were completed to determine if the adjusted risk for each tobacco product use category was significantly different from exclusive cigarette users. All analyses used Stata survey data procedures, version 15.1; standard errors forTables 3 and 4 and estimates for all covariates for Tables 2-4 are included in Supplemental Tables 4-6.At W2, the prevalence of functionally-important respiratory symptoms was 7.2% . Table 1 shows that respiratory symptoms were more common in the four categories of tobacco use that included cigarettes , compared to never tobacco use, and among those who used marijuana. Functionally-important respiratory symptoms were much more common among those with asthma, and also more common among those with comorbid conditions, obesity, and those using medications known to cause coughing or wheezing . Figure 1 illustrates the unadjusted linear relationship between frequency of cigarette use and proportion of persons with functionally-important respiratory symptoms for the four use categories featuring cigarettes. The shape of the dose-response lowess lines were almost identical and the 95th percentile for cigarette use intensity was essentially the same for all four groups, regardless of what other tobacco products were added to cigarettes, emphasizing the importance of cigarettes in these four most prevalent categories of tobacco use. In the full, adjusted, multi-variable cross-sectional model , all four tobacco use categories that featured cigarette smoking were associated with a doubling of the risk of functionally-important respiratory symptoms vs. never tobacco users , and risk for the multiple use categories were not significantly different from exclusive cigarette use .

As illustrated in Figure 2, we observed a significant positive dose-response relationship for current use of cigarettes . Exclusive use of non-cigarette products not associated with added risk Compared to never users, the risk of functionally-important respiratory symptoms were not significantly different for exclusive users of e-cigarette, cigar, hookah and smokeless tobacco; moreover post hoc testing indicated that risk ratios for each of these categories were significantly lower compared to exclusive cigarette use . None of these cross-sectional results changed when the analysis was repeated at a respiratory index cut-off level of ≥2. Testing sensitivity to key confounders of the e-cigarette—respiratory symptom association Cigarette smoking pack-years, second-hand smoke exposure, and marijuana use were also associated with functionally-important respiratory symptoms . Table 2 highlights the importance of cigarette smoking pack-years and past-month marijuana use as confounders of the association between tobacco product use and respiratory symptoms. Cigarette pack-years was a particularly strong confounder; adding this variable alone to the cross-sectional multi-variable model attenuated association estimates for cigarettes and cigarettes+e-cigarettes by 30% and for exclusive e-cigarettes by 25%. That was partly because all three groups had a similarly long cigarette smoking history—weighted mean 13.4 cigarette pack-years for exclusive cigarette smokers, 12.9 for the dual users, and 10.8 for exclusive e-cigarette users. Similarly, 19.2% of exclusive e-cigarette users also currently used marijuana; adding P30D marijuana use to the multi-variable model attenuated association for e-cigarettes by 9%. Adding all three confounders together attenuated the e-cigarette-respiratory symptom association RR from 1.53 to 1.05. The categorical analysis did not address whether functionally-important respiratory symptoms increased with increasing frequency of use. Figure 2 explored this for cigarettes and e-cigarettes, adjusting for cigarette smoking history. For cigarettes, there was a significant linear increase in the percent with functionally-important respiratory symptoms with higher intensity of use; prevalence of respiratory symptoms was less than 5% for never users and over 30% for those smoking a pack a day or more. There was also an increase in respiratory symptoms with higher intensity of e-cigarette use, but the trend did not reach statistical significance .Table 3 gives results for the two longitudinal models for worsening respiratory symptoms. Symptoms worsened for 5% and 8%, respectively, for cutoff levels of ≥ 3 and ≥ 2. Symptom worsening was most common in the four categories featuring cigarette use, with risk ratios for worsening symptoms for the four categories ranging from 1.64 to 2.80, and always significantly higher than for never users, regardless of threshold. Also regardless of threshold, post hoc testing indicated that risk ratios for dual use of cigarettes+e-cigarettes were never different compared to exclusive cigarette use, whereas combustible plus noncombustible use was always associated with lower risk. Cigarette pack-years, second-hand smoke exposure, and marijuana use at W2 were also associated with symptom worsening at W3, at both cutoff levels. There were no statistically significant associations between exclusive use of cigars, smokeless tobacco or hookah and worsening of respiratory symptoms compared to never users. Post hoc testing indicated that risk ratios were significantly smaller than for exclusive use of cigarettes, regardless of cutoff level for the respiratory symptom outcome . In contrast, findings for exclusive e-cigarette use were sensitive to symptom severity, showing a significant association with worsening symptoms at a threshold of ≥2 , but not at a symptom threshold of ≥3 . Table 4 gives results for the two longitudinal models for improving respiratory symptoms . Symptoms improved for 21% and 29%, respectively, for cutoff levels of ≥2 and ≥3. In contrast to symptom worsening models, tobacco use was less apt to be associated with improvement and more sensitive to cutoff threshold. Categories of use featuring cigarettes were not reliably less likely to be associated with symptom improvement compared to never users; only exclusive use of cigarettes at a threshold of ≥2 was associated with lower risk ratio for symptom improvement . At this threshold, former smokers, e-cigarette, cigar and smokeless tobacco users were all significantly more likely to show symptom improvement compared to exclusive cigarette users . This was also true for e-cigarette users at a threshold of ≥3, where e-cigarette users were also more likely show symptom improvement compared to never users . This study underscores the adverse consequences of continued cigarette smoking among people without COPD or other non-asthma respiratory disease on functionally-important respiratory symptoms.

Despite these limitations, as illustrated in Figure 1, following a thorough algorithm can aid in detecting cases of drug-induced pancreatitis that would otherwise have been difficult to diagnose . In accordance with the aforementioned limitations, evidence implicating numerous medications is inconsistent and, at times, even contradictory. Hence, although not uniform, nor universally accepted, official tier systems exist to help quantify the likelihood of a drug to be established as a culprit of acute pancreatitis. The earliest classification system was developed in 1980 and was designed to include three classes; Class I: Included drugs that were implicated to induce pancreatitis in a minimum of 20 cases of which at least one case documented drug re-exposure, Class II: Included drugs that were implicated to induce pancreatitis in 10-20 cases with or without documented drug re-exposure, and Class III: Included all drugs implicated in pancreatitis. Trivedi et al reviewed the top 100 prescription medications in the United States for their association with acute pancreatitis using this three-tier classification system. They noted that, of the top 100 most frequently prescribed medications, 44 were Class III pancreatitis medications. Additionally, 14 of these medications were Class I or II. The most recent classification system was developed by Badalov et al, in which the authors categorized implicated drugs into four classes . Class I drugs are medications in which a re-challenge was established in at least one case report. This class is further divided into whether other causes of acute pancreatitis were ruled out or not . Class II drugs are medications in which there is a latency period in 75% of at least four reported cases,seedling grow rack all with no evidence of re-challenge. Class III drugs are medications that neither a re-challenge nor a consistent latency period was established but had two or more case reports published. Class IV drugs are medications that neither a re-challenge nor a consistent latency period was established, and only 1 case report had been published.

Additionally, the Naranjo adverse drug reaction probability scale can be helpful in establishing the degree of association between a drug and an adverse reaction. This tool determines the likelihood of an adverse drug reaction based on the cumulative score on 10 questions. A score of < 1 signifies a doubtful drug reaction, 1-4 a possible drug reaction, 5-8 a probable drug reaction, and > 9 a definitive drug reaction . Finally, and most recently, our proposed specific drug-induced pancreatitis probability scale can serve as a standardized tool for determining the likelihood of drug-induced pancreatitis based on the aggregate score from a series of 10 questions. A score of < 2 suggests doubtful DIP, 3-5 possible DIP, 6-8 probable DIP, and > 9 highly probable DIP . We believe this tool, in particular, enhances one’s ability to accurately identify and implicate potential acute pancreatitis-causing drugs. While consensus has yet to be reached regarding the cause of drug-induced pancreatitis in many cases, numerous potential mechanisms have been speculated. These include, pancreatic/biliary duct constriction, cytotoxic effects, metabolic effects, accumulation of a toxic metabolite or intermediary, and idiosyncratic and/or hypersensitivity reaction, with idiosyncratic response or direct toxic effect likely accounting for the majority of cases. Studies concerning the incidence of drug-induced pancreatitis have established a range of 0.3% to 1.4% of all acute pancreatitis cases being due to drugs. Certain medications such as azathioprine/mercaptopurine and didanosine are well-known culprits of drug-induced pancreatitis with incidences of 5% and 23% respectively. As illustrated in Table 3, compiling a list of drug classes implicated in pancreatitis may yield clinical use owning to increased clinician awareness of other medications in these classes. Among the many drugs that have been associated with pancreatitis, statins have been increasingly reported as a cause of acute pancreatitis. In fact, as numerous members of this class have been implicated in acute pancreatitis, statin-induced pancreatitis may indeed be a class-effect.

Mechanisms of action of statin-induced acute pancreatitis are associated with rhabdomyolytic and cytochrome P-450 interactions leading to an immune-mediated inflammatory response, direct cellular toxicity, or perhaps a metabolic effect. As with many other drugs, its true prevalence in acute pancreatitis remains unknown, as the onset of statin-induced pancreatitis has been observed from hours to years after treatment. Interestingly, the degree of P-450 CYPA4 inhibition correlates with individual statin safety profiles. Although rare, several 5-aminosalicylic acid -induced acute pancreatitis cases have been published in the literature. Interestingly, both oral and enema mesalamine preparations have been implicated in causing pancreatitis within days. In addition, sulfasalazine has been implicated in inducing pancreatitis perhaps through an immune-mediated mechanism . In general, however, a hypersensitivity mechanism seems to be involved and pancreatitis can occur from days to years after starting mesalamine therapy.Metronidazole has been reported in association with acute pancreatitis, although the mechanism is not fully known. Free-radical production, immune-mediated, direct toxic affect, and metabolic effects have been suggested as possible pathophysiological mechanisms. Notably, a study showed that patients receiving metronidazole as part of Helicobacter pylori triple-therapy have an approximate eight-fold increased risk of acute pancreatitis. The tetracycline class has also been associated with acute pancreatitis, with the mechanism believed to be a direct toxic-effect, or hypersensitivity reaction. In addition, numerous cases of erythromycin-induced pancreatitis have been reported to date.

Although less established, other antibiotics such as ampicillin, ceftriaxone, clarithromycin, trimethoprim-sulfamethoxazole, and nitrofurantoin have been implicated in pancreatitis as well. Numerous steroids have been associated with inducing acute pancreatitis nearly all with a short latency period. As a large proportion of these cases resulted in death, it has been suggested that this drug class may be linked to a more severe disease course. The most common non-steroidal anti-inflammatory drugs that have been reported to cause pancreatitis are sulindac and salicylates, with latency ranging from weeks to years, however others have been implicated as well. A clear limitation that exists is the fact that NSAIDs may be initiated in response to early symptoms of unrecognized pancreatitis leading to erroneously attributing the pancreatitis to this class of medication. Interestingly, naproxen has been recommended as the preferred analgesic in this scenario owning to its limited risk of inducing acute pancreatitis. The mechanism being, a structural effect on the sphincter of Oddi leading to acute pancreatitis. Of note, both diclofenac and indomethacin may significantly reduce the risk of acute pancreatitis post-endoscopic retrograde cholangiopancreatography. Immunotherapy agents have long been associated with acute pancreatitis, however their increased use in recent decades has led to a concomitant increase in immunotherapy-associated pancreatitis. Interleukin-2 immunotherapy-associated pancreatitis in particular has been reported. The mechanism of injury is believed to be either immune-mediated or a direct drug toxicity. Newer programmed cell death protein 1 blockers and anti-cytotoxic T-lymphocyte-associated protein 4 agents have been associated with acute-pancreatitis as well. The exact mechanism is currently unknown,greenhouse growing racks but it is speculated to be associated with Tlymphocyte mediated inflammation. There have been many well-documented case reports of acute pancreatitis due to Angiotensin-converting-enzyme inhibitors. One case-control study suggested a dose-dependent correlation with an odds ratio of 1.5. While enalapril has been the most extensively reported culprit in this class, other agents have been described in the literature as well. Similar to ACE-Is, angiotensin receptor blockers has also been implicated in acutepancreatitis. Interestingly, the latency period between ACE-I initiation and an associated pancreatitis event may range from days to years and may be associated with severe disease. The proposed mechanism involved is due to decreased bradykinin degradation, increasing pancreatic vascularity and edema, and pancreatic enzyme trapping causing local tissue damage secondary to pancreatic duct obstruction. Although proven to be relatively safe for the management of type 2 diabetes mellitus, numerous classes of oral anti-glycemic agents including biguanides, dipeptidyl peptidase-4 inhibitors, glucagon-like peptide-1 analogues, and sodium glucose co-transporter-2 inhibitors have been associated with acute pancreatitis.

The highest incidence is reported with GLP-1 analogues, of which exenatide has the highest association with an up to 6-fold increase in the rate noted in post-marketing surveillance. The proposed mechanism is pancreatic acinar cell hypertrophy, the subsequent release of pro-inflammatory cytokines, leading to increased vascular permeability, resulting in pancreatic inflammation. Another hypothesis suggests an obstructive type phenomenon, as an increased incidence of gallstones with the use of GLP-1 analogues has been recorded.As such, many case reports suggesting the association between cannabis and pancreatitis have been published in the literature, and may even suggest a dose-dependent phenomenon. While cannabinoid receptors are found in the islet of Langerhans cells, the exact pathophysiology of cannabis-induced pancreatitis is currently not well-understood. Cocaine-induced pancreatitis has also been reported, and is believed to be due to splanchnic vasoconstriction and thrombotic micro-angiopathy leading to ductal obstruction. Similarly, codeine has also been described in the literature with regards to inducing acute pancreatitis with a mechanism believed to relate to dysfunctional sphincter of Oddi contraction as well.The highly active anti-retroviral therapy therapy drugs have long thought to be associated with the development of acute pancreatitis. The most common offenders include nucleoside reverse transcriptase inhibitors , non-NRTIs , and protease inhibitors. In a large retrospective cohort study including nearly 5000 patients who received antiretroviral therapy, 3.2% developed AP. Furthermore, 5.2% of patients who received didanosine, 4.2% of patients who received a PI plus either an NRTI or a NNRTI, and 3.5% of patients who received NRTIs combined with NNRTIs—developed acute pancreatitis. The exact mechanism by which NRTIs and NNRTIs cause pancreatitis is unidentified, but it is thought to be related to direct drug toxicity or ductal obstruction leading to mitochondrial damage resulting in cellular death and organ damage. PIs are directly related to induction of hypertriglyceridemia which is a well-established cause of pancreatitis in the literature. Diuretics [e.g. furosemide, chlorothiazide, hydrochlorothiazide , and others] have long been implicated in the development of acute pancreatitis, with the majority of cases suggesting a short latency period and a more mild disease course. Notably, HCTZ has well-established side effects of causing hypercalcemia and hyperlipidemia, both of which are well known to lead to acute pancreatitis. HCTZ can further cause hyperparathyroidism, which can also lead to hypercalcemia induced pancreatitis. It has been postulated that furosemide affects the pancreas by causing a hyper-stimulation of secretions leading to a direct toxic injury and/or ischemia. Numerous cases have been reported in which estrogen-containing products were thought to induce acute pancreatitis. Hypercoagulability and hypertriglyceridemia have been speculated as the main cause of inducing pancreatitis in patients taking hormone replacement therapy and oral contraceptives. Nonetheless, patients with existing hypertriglyceridemia and familial hyperlipoproteinemia can have an exacerbation of their underlying condition leading to pancreatitis.Although both H2-blockers and proton-pump inhibitors have been reported in the literature to cause acute pancreatitis, the evidence regarding this relationship is controversial. A retrospective study conducted by Eland et al failed to identify any association of pancreatitis with the use of ranitidine , cimetidine , and/or omeprazole . Case reports in the literature have generally linked pancreatitis in these cases to excessive consumption of antacids which likely was directly related to hypercalcemia. Many cases have linked antidepressants to acute pancreatitis; nonetheless, a population-based study by Nørgaard et al failed to demonstrate a significant association between selective serotonin reuptake inhibitors and acute pancreatitis. Only a mild increase in the risk of pancreatitis was seen with first-time users of SSRI ; however, the results are limited due to confounding variables. A recent meta-analysis demonstrated a significant association between SSRIs and acute pancreatitis. The risk was much higher in the first 2 weeks of following initiation of SSRIs. The exact mechanism by which SSRIs can lead to pancreatitis is unknown, though it is speculated that SSRIs can cause cellular apoptosis, insulin secretioninhibition, and further development of diabetes and chronic pancreatitis as well. Roberge et al additionally reported a case of a patient who developed acute pancreatitis due to an acute overdose of clomipramine. Numerous anti-seizure medications have been associated with inducing pancreatitis, especially in the pediatric population. Interestingly, this class seems to be associated with a more severe disease course that may result in pancreatic necrosis and death. The mechanism has been postulated to involve a direct toxic effect on pancreatic cells causing depletion of superoxide dismutase, catalase, and glutathione peroxidase on a biochemical level. To our knowledge, two cases of vitamin-induced acute pancreatitis have been reported, both involving vitamin D. One involved oral vitamin D, wherein the injury was seemingly related to the hypercalcemic effect of vitamin D.

However, the newly acquired knowledge on the physiological roles of the endocannabinoid system has significantly enhanced these prospects. At the June 27, 2004 workshop ‘‘Future Directions in Cannabinoid Therapeutics II: From the Bench to the Clinic’’, sponsored by the University of California Center for Medicinal Cannabis Research, we on the Scientific Planning Committee were asked to identify the areas of research with the most immediate promise for the development of novel therapeutic agents. The Committee identified four broad areas involving modulation of the endocannabinoid system as particularly promising in this regard: agonists for central CB1 cannabinoid receptors and peripheral CB2 receptors, antagonists of CB1 receptors, inhibitors of endocannabinoid deactivation, and endocannabinoid-like compounds that act through mechanisms distinct from CB1 and CB2 receptors activation. Below, we summarize the data presented at the Workshop and the consensus of its participants on the most exciting opportunities for drug discovery.Two endogenous agonists of cannabinoid receptors have been well characterized and are now widely used in research: anandamide , and 2-arachidonoylglycerol . Both molecules derive chemically from the polyunsaturated fatty acid, arachidonic acid, which is used in nature as the starting material for other important signaling compounds,cannabis growing system such as the eicosanoids. Additional endocannabinoid-related compounds present in the body include virodhamine, which may act as an endogenous antagonist of CB1 receptors, and arachidonoylserine, which may engage an as-yet-uncharacterized cannabinoid-like receptor expressed in the vasculature.

As is well-known, the Cannabis plant contains more than 60 cannabinoids, which include -D9 -tetrahydrocannabinol , cannabigerol, cannabidiol, cannabinol, cannabichromene and cannabicyclol. Attention has been mostly focused on D9 -THC, because of its multiple biological properties. Nevertheless, less studied compounds such as cannabidiol may also be important, although we do not yet know at which receptors they may act to achieve their effects. D9 -THC is the only natural cannabinoid presently used in the clinic. In addition to these plant-derived cannabinoids, an extensive set of synthetic cannabinergic agonists has been developed over the last 30 years. Products of these efforts include CP-55940 , created by opening one of the rings of the tricyclic D9 -THC structure and introducing other small changes in its structure; HU-210 , a very potent cannabinoid agonist resembling some D9 -THC metabolites; and WIN55212-2 , which belongs to an altogether different class of chemicals, the aminoalkylindoles. Additionally, the metabolically stable synthetic analog of anandamideR-methanandamide is routinely used as a pharmacological probe to circumvent the short half life of the natural substance. Two important new additions to this armamentarium under discussion at the workshop include a peripherally acting cannabinoid agonist in preclinical development by Novartis for the treatment of neuropathic and inflammatory pain , andBAY-387271 , a centrally acting cannabinoid agonist in Phase II clinical studies for the treatment of stroke. The interest of the pharmaceutical industry in the application of cannabinoid agonists to the treatment of pain conditions is not recent. Indeed, most of the compounds now in experimental use derive from such an interest. Historically however cannabinoid agonist development has not proved clinically fruitful, largely because of the profound psychotropic side effects of centrally active cannabinoid agonists, hence the attention given to peripherally acting cannabinoids, which exhibit significant analgesic efficacy and low central activity in animal models. Neuroprotection is a relatively new area for cannabinoid agonists, but one that appears to be already well advanced.

Preclinical studies have made a convincing case for the efficacy of cannabinoid agents not only in experimental brain ischemia, but also in models of Parkinson’s disease and other forms of degenerative brain disorders. The results of a Phase II clinical trial with BAY-387271 are awaited with great excitement. Also highlighted during the conference were various derivatives of cannabidiol.Like D9 -THC, 7-OH-DMH-CBD is a potent inhibitor of electrically evoked contractions in the mouse vas deferens. However, 7-OH-DMH-CBD does not significantly bind to either CB1 or CB2 receptors and its inhibitory effects on muscle contractility are not blocked by CB1 or CB2 receptor antagonists, suggesting that the compound may target an as-yet-uncharacterized cannabinoid-like receptor. This hypothesis is reinforced by pharmacological experiments, which suggest that 7-OH-DMH-CBD displays anti-inflammatory and intestinal-relaxing properties, but does not exert overt psychoactive effects in mice. However, the nature of this hypothetical receptor and its relationship to other cannabinoid-like sites in the vasculature and in the brain hippocampus remains to be determined.Another way to reduce central side effects is to target peripheral CB2 receptors, which are expressed in the brain only during inflammatory states and even then are limited to microglia. Selective CB2 receptor agonists include the compounds AM1241 and HU-308 . Compounds of this type offer a great deal of promise in the treatment of pain and inflammation. Studies conducted on multiple animal models of pain have shown that the CB2-selective agonist AM1241 has robust analgesic effects and is very potent in neuropathic pain models. These effects are maintained in CB1-defificient, but not in CB2-defificient mice.CB1 cannabinoid receptors are present on the cell surface of neurons within the brain reward circuitry. Furthermore, endocannabinoids may be released from dopamine neurons in the ventral tegmental area , and from medium spiny neurons in the nucleus accumbens of the brain reward circuit. Additionally, endocannabinoids and D9 -THC activate CB1 receptors and by doing so regulate reward strength and drug craving. Though we do not know how this occurs, it is likely that these mechanisms extend to all drugs of abuse,hydroponic rack system because collectively these drugs show the propensity to increase VTA dopamine neuron activity, which might be coupled to augmented endocannabinoid production from the dopamine neurons themselves.

Finally, cannabinoid receptor antagonists block the effects of endocannabinoids in these reward circuits. Preclinical work shows that priming injections of cannabinoid agonists reinstate heroin-seeking behavior after a prolonged period of abstinence in rats trained to self-administer heroin. The cannabinoid antagonist rimonabant fully prevents heroin-induced reinstatement of heroin-seeking behavior. Additionally, rimonabant significantly attenuates cannabinoid-induced reinstatement of heroinseeking behavior. All these findings clearly support the hypothesis of a functional interaction between opioid and cannabinoid systems in the neurobiological mechanisms of relapse and might suggest a potential clinical use of cannabinoid antagonists for preventing relapse to heroin abuse. It has also been shown that cannabinoid antagonists can prevent drug reinstatement with cocaine, alcohol, and nicotine. Thus, it seems that the future of cannabinoid antagonists in substance abuse treatment is particularly promising, especially in the clinical setting, where poly drug abuse is exceedingly more common than isolated single-drug abuse.The available data suggest that CB1 antagonism produces relatively mild side effects in people. Yet several potential risks were discussed and three in particular received a great deal of attention. First, the possibility of neuropsychiatric sequelae, such as anhedonia and anxiety: preclinical studies have consistently shown such effects in animals, though they have not yet been observed in the clinic. Second, pain and hyperalgesia, because of the pervasive role played by the endocannabinoid system in the control of pain processing. Last, hypertension, as indicated by the contribution of the endocannabinoids to blood pressure regulation and the pressor effects of rimonabant in animal models of hypertension.The endocannabinoid signaling system differs from classical neurotransmitter systems, picking up where classical neurotransmitters leave off. That is, the activation of receptors initiates a series of chemical events that leads to the release of endocannabinoids from the postsynaptic spine e the final step of which is the enzymatic production and subsequent release of anandamide and/or 2-AG. Once released, the endocannabinoids are then directed to the presynaptic cell and the CB1 receptor responds by inhibiting further release of that cell’s neurotransmitters. The termination of this cascade is accomplished via a transporter that internalizes the endocannabinoids, after which intracellular enzymes such as fatty-acid amide hydrolase break them down. There is a general consensus that endocannabinoids are transported into cells via a facilitated diffusion mechanism. This process may differ both kinetically and pharmacologically from cell to cell. In brain neurons, endocannabinoid transport is blocked by certain agents, which include the compounds AM404, OMDM-8 and AM1172 . However, the pharmacological properties of these drugs in vivo are only partially understood.

Once inside cells, endocannabinoids are hydrolyzed by three principal enzyme systems.Potent and selective FAAH inhibitors have been developed and shown to exert profound antianxiety and antihypertensive effects in animals. The latter effects were discussed at length at the workshop, highlighting the important role of anandamide in two important examples of vascular allostasis e shock and hypertension. In addition to FAAH, another amide hydrolase has been recently characterized, which may participate in the degradation of anandamide and other fatty-acid ethanolamides such as oleoylethanolamine . This amidase prefers acid pH values and has a different tissue distribution than FAAH, being notably high in lung, spleen and inflammatory cells. Inhibitors of this enzyme are being developed. Finally, 2-AG is hydrolyzed by an enzymatic system separate from FAAH, which probably involvesa monoacylglycerol lipase recently cloned from the rat brain. Inhibitors of this enzyme are currently under development.College students often drink alcohol and use drugs simultaneously during parties and other social events . Dual marijuana and alcohol use is especially prevalent, with 47% of marijuana users reporting simultaneous use of alcohol . Furthermore, individuals who have a cannabis use disorder are at increased likelihood for the development of an alcohol use disorder , and rates of substance use disorders and treatment admissions are highest among individuals that use marijuana or alcohol compared to other substances . Approximately 68% of individuals with current CUD and over 86% of those with a history of CUD meet criteria for an AUD. Cannabis dependence doubles the risk for long-term persistent alcohol consequences and dual marijuana and alcohol users consume higher levels of alcohol and experience more alcohol-related consequences than only drinkers . Despite these additional risks, 60% of college students do not perceive regular marijuana use to be harmful .The combination of low perceived risk, policy changes surrounding marijuana legalization, and the rise in marijuana use over the past 10 years heightens the importance of effective interventions for alcohol and marijuana use. In the adult substance use treatment literature, it is relatively well-established that alcohol use negatively impacts treatment of other substances . In contrast, literature examining the impact of marijuana use on the treatment of other substances is mixed. With the exception of a few studies that do not show marijuana use to negatively influence alcohol or smoking cessation outcomes , many studies have demonstrated that using marijuana before or during alcohol treatment is associated with higher levels of drinking at follow-up . For example, among alcohol dependent individuals, those who used marijuana during alcohol treatment reported fewer days abstinent from alcohol one year following treatment than those who did not use marijuana . Thus, marijuana use seems to have a negative impact on alcohol treatment outcomes. A number of studies have also examined secondary changes in marijuana use following receipt of an alcohol-specific intervention. A recent integrative data analysis study indicated that alcohol BMIs may not facilitate changes in marijuana use among college students ; instead, regardless of treatment condition, college students who successfully reduced their drinking at short- and long-term follow-ups were more likely to be non-users of marijuana or reduce their marijuana use at follow-up. This complementary relationship between marijuana and alcohol use is also supported by research indicating that the risk factors for initiation and maintenance of problematic use are similar across substances . Together, these studies suggest that interventions for alcohol may lead to secondary changes in marijuana use. Consistent with this hypothesis, young adults who participated in an in-person BMIs for alcohol use in an emergency department setting reported greater decreases in marijuana use at the 6-month follow-up than those who received feedback only . Similarly, weekly marijuana users who were seeking treatment for cigarette smoking and completed a brief alcohol intervention within the context of the smoking cessation intervention, demonstrated reductions not only in heavy drinking and tobacco smoking but also in marijuana use . In the college setting, BMIs that target multiple substances have also been associated with reductions in poly-drug use . One explanation for the differential influence of alcohol interventions on marijuana use across these studies may be related to the populations examined. Thus far, alcohol interventions delivered to acute-risk populations have had an impact on marijuana use outcomes, while collectively, interventions delivered to ‘college students’ have not.

The dilution result is called “European odour unit” , which is defined as “the amount of odorants that, when evaporated into 1 m3 of neutral gas at standard conditions, elicits a physiological response from a panel equivalent to that elicited by 1 European reference odour mass [123 μg n-butanol] evaporated in 1 m3 of neutral gas at standard conditions” . Thus, the European approach accounts for the variation in detection thresholds of the panelists. Despite these strictures, proficiency tests in 2007 and 2008 found two thirds of the European laboratories claiming to work in accordance to the EN 13725 standard failed to demonstrate compliance with the required performance criteria . Dilution equipment can also be used to evaluate the odor hedonic tone, as is required in the Netherlands . Panelists express the degree of pleasantness using a 9-point scale ranging from H -4 to H +4 . The logarithm of the odor concentration is plotted versus the H-value, which approximates a straight line. Using linear regression, the level of dilution required to achieve Dutch regulatory criteria can be estimated. OPM used to determine the ODTC50 goes against the guidance upon which it was built,dry racks yet still delivers useful results . Both the FPA and APHA Method 2170 advise against conducting statistical analysis on the intensity scores. The scores are categorical rankings and not an interval scale. In other words, an intensity score of 8 is not necessarily twice as intense as a score of 4.

The intensity scale was not designed for 1 to be the ODTC50 value, and historically used the symbol “. Surprisingly, such “against the guidance” extrapolation was found to be not off-the-mark from ODTC50 values from other studies , which may be confirmation of the enormous range of such values . Day-to-day variation by panels is typically within an order of magnitude . Faint environmental odors are sensed but cannot be measured by dynamic dilution olfactometry . Typically, dynamic dilution values are never given for levels lower than 10 OUE/m3 , and generally the lower values start in a range of 50–100 OUE/m3 . The use of human subjects as panelists is strictly controlled . International codes of conduct apply as do reviews by a human subjects review boards to protect participants.Exposure limits are intended to protect the health of populations and arrive at quite different results for workers versus the general public. Workers are considered healthier and less diverse in susceptibility than the general population. They also only spend a portion of their day and week on-the-job. Political pressures, too, may influence worker limits to prevent onerous restrictions to industry . The morbid or lethal outcomes of occupation exposures likely overshadowed concerns about impact on olfactory function. The end result is that worker exposure limits are usually many times higher than those for residential exposures. A survey by National Geographic revealed that factory workers reported poorer senses of smell , although sensory loss is typically gradual and may be confused with aging . Sensory-impaired workers can miss out on warning signals that impact their nutrition and quality of life. Monitoring of workplace air for hazardous chemicals cannot be replaced by sensory cues, due to a portion of the workforce having little or no sense of smell , odors can create a workplace nuisance for most employees. Workers, too, are subject to the psychological strain of odor exposure, and setting and occupational exposure limit above the odor threshold may lead to perceived risk and well as physical response . For these reasons, occupational exposure limits consider odor.

OELs have long considered irritation . In the United States and Europe, about 40% of the OELs are set to avoid sensory irritation . Sensitization is especially a concern, and OELs should be set low enough to avoid such initial, triggering exposures . Only three chemicals are regulated by OSHA based on “obnoxious odor” and worker complaints: isopropyl ether, phenyl ether, and vinyl toluene . Critical reviews suggest setting OELs by taking into account both irritation and odor thresholds . For residential exposures, sensory effects are considered in emergency situations but rarely for ongoing, long-term exposures . The latter protect from cancer and non-cancer effects by focusing on system organ toxicity. Whether odor is a health-protective signal varies from odorant to odorant. Carcinogens have especially low exposure limits, so the ODTC50 tends to be well above the residential exposure limit. Benzene and formaldehyde are prime examples of such carcinogens . Because ODTC50 values range over several orders of magnitude, they commonly overlap with exposure limits. Re-visiting the work by Rosenfeld et al. and adding the ODTC50 ranges from AIHA , this overlap became apparent for hydrogen sulfide and ammonia .Exposure assessment involves the measurement of odorant concentrations in the hope the subset measured is responsible for the odor. It also involves the use of human panels to identify odor characteristics . Such categorical measures cannot be used in a quantitative risk assessment; however, they do provide qualitative information. The measurement of the number of dilutions required to remove an odor is plagued by inconsistent results and driven by the final remaining odorant in the dilution . When exposure measurements have been determined for a subset of odorants, the readings can be compared to the exposure limits presented in Section 4.4. This is the risk characterization. For residential exposure, the comparison is with the ODTC50 by a ratio known as the “Odor Activity Value” .

Such a comparison, however, carries with it the inherent weakness in the numerator and denominator values in the ratio, undermining some of its usefulness. Specifically, ODTC50 values usually span odors of magnitude, and measured concentrations are typically a snapshot in time. Some studies find the inputs to OAVs too variable and are now pursuing other methods instead . In addition, for ongoing rather than intermittent odor exposures, comparisons with health based exposure limits for residents, such as the USEPA Reference Concentration or California EPA Reference Exposure Level , can be performed for non-cancer effects. For carcinogens, the cancer risk estimate involves the inhalation USEPA Unit Risk Factor or equivalent. Note that exposure assessment usually has less uncertainty than other portions of risk assessment . Mixtures are challenging for risk assessment. Combined effects of sensory irritants can be considered additive as a first approximation . The interplay of odorants, however, is often unknown . Hydrogen-sulfide-equivalents are a proposed approach ,cannabis drying similar to the dioxin equivalents for the 17 dioxin-like congeners and the carbon dioxide equivalents for various greenhouse gases.As a literature review, where no exposure data were generated, the case-study approach is used to illustrate how odorant concentration data can be used in conjunction with odor threshold information to characterize the health risks. Case studies were selected that addressed both odor and health risks within the same study. Although the most significant risks are due to chemical exposures in the workplace , the focus of this paper is on residential exposures to environmental odors, so worker exposure studies were excluded .Not only have OAVs been evaluated for on-site worker exposures , they also have been evaluated for odorants from sewers across Australia over a period of 3.5 years . Both efforts were to prioritize the subset of odorants monitored within the mixture to identify “high priority” odorants for further analysis. For the sewer study, the upper bound concentrations were compared to the ODTC50 values from Nagata and Takeuchi . The ranking by OAV reduced the number of compounds from 31 to 8 “high-priority” odorants. Hydrogen sulfide and methyl mercaptan dominated across all sites , and other volatile sulfur compounds also ranked high for most sites. Diethyl sulfide, limonene, toluene and m,p-xylenes each ranked high for fewer sites. The limitations of such ranking are substantial. The choice of ODTC50 value is paramount and typically ranges over several orders of magnitude across studies, complicating OAV calculations and their utility as a ranking scheme. Although Sivret et al. acknowledged the enormous ranges of ODTC50 values available in the compilation by van Gemert in several graphs, the final ranking was performed using the single values found in Nagata and Takeuchi for simplicity.

Presenting the final OAVs as ranges would have muddied the results. Finally, those compounds without ODTC50 values available in the literature were dropped from the ranking, essentially rewarding lack of data for potentially odorous compounds. As with any study of mixtures, the analyzed odorants are only a subset and may not reflect the overall, total odor experienced by residents.Risk assessment is often predictive rather than retrospective. The impacts of a proposed oil well in Hermosa Beach, California, were forecast . Except for upset conditions, the anticipated negative health outcomes were largely nuisance-related . The evaluation concluded that the oil well would have no substantial effect on community health, which was based in part on a risk assessment of hydrogen sulfide as the odorant of primary concern. The results of the modeling indicated that fugitive emissions from normal operations could produce concentrations greater than the odor threshold without mitigation, which would reach nearby residences. Concentrations could be as high as 6 times the odor threshold, primarily driven by hydrogen sulfide. The acute REL for hydrogen sulfide would only be exceeded, according to the model, during accidental or unplanned release. Odor impacts from normal operations were, therefore, considered potentially significant without mitigation, so an Odor Minimization Plan was required.Over years, the residents of communities north of Denver have complained of intermittent, unpredictable “tar” and “asphalt” odors. The symptoms in Globeville, Colorado, included burning eyes and throat, headaches, skin irritation and sleep problems. A USEPA funded environmental justice study was conducted there in 2012 . Air samples were collected from locations near potential sources and within the community over a period of 7 months. Out of a list of 23 analytes, the most prevalent were hexane, heptane, benzene, toluene, m,p-xylenes and naphthalene. The maximum concentrations were below odor and toxicity thresholds , except for the carcinogens , which were considered within normal ranges for urban air. To see how the risk characterization might change if different thresholds were selected, a comparison with the ODTC50 ranges from AIHA and the RELs from California was conducted .The comparison thresholds did not change most of the risk characterization performed by Morgan et al. . Only the maximum measurement of naphthalene enters within the comparison range of ODTC50 values , while the low ends of other comparison ranges for toluene and m,p-xylenes are now within a factor of 3. The health risk characterization remains the same, with the carcinogen benzene exceeding the threshold that accounts for cancer. Urban levels of benzene are typically around 1.8 ppb , which are also above the cancer threshold.An emerging area for environmental odors is cannabis cultivation. In the states that have legalized cannabis use medicinally and by adults, the cultivation in warehouses has led to nuisance odor complaints. The grow cycle includes a budding stage with “skunk” odor notes. Most cultivation warehouses stagger the growth cycles, so budding is an ongoing occurrence. As a best practice, exhaust ventilation is passed through an activated carbon filter; however, the efficiency is typically 50-98% , so odorous compounds are still emitted. Colorado has required odor management plans certified by an industrial hygienist to ensure best practices are implemented. In 2003, California was the first state to allow medicinal cannabis use . In 2018, cultivation for adult use became legal . Today, regions throughout the state – such as Sonoma County, Sacramento County and Greater Palm Springs – are managing the side-by-side growth of the indoor-cultivation industry and nearby residences. The first study to report Odor Activity Values for odorants emitted from cannabis identified a list of over 20 substances, which were different than those traditionally associated with cannabis odor .Samples were not living cannabis plants, but rather fresh, old and desiccated cannabis, and some monitoring was outside storage bags to simulate a person being pulled over by police while transporting cannabis. The most odorous compounds based on OAVs are listed in Table 4.8 and provide a starting point for future odor research.

Interventions to reduce their individual- and population-level harms can often be hampered by biological, psychological, and social complexity, especially because substance use is often syndemic with other health and social problems such as HIV infection, hepatitis B and C, mental health disorders, and violence. It is in this context that PLOS Medicine devotes its November 2019 Special Issue to research on substance use, misuse, and dependence. The contributions to the issue cover a wide range of topics, including social determinants of substance use, health harms resulting from substance use, and interventions to prevent or reduce the harms associated with substance use. There are also a number of gaps in terms of the topics covered, the robustness of the evidence, and its global scope.For the first time in several decades—and concomitant with the rise in opioid use, misuse, and dependence—life expectancy has declined in the United States, and life expectancy gains have stalled in Canada. Consistent with these global estimates,cannabis grow equipment in an accompanying paper for the Special Issue, Astrid Guttmann and colleagues analyzed 2002–2016 national data from the United Kingdom and Canada to identify women who likely used opioids during pregnancy and demonstrated markedly elevated mortality rates over up to 10 years of follow-up. The elevated rates were particularly striking for mortality due to avoidable causes like unintentional and intentional injuries. Using 1998–2014 data from a large sample of primary care practices in the UK, John MacLeod and colleagues show that coprescription with benzodiazepines was highly prevalent among patients receiving opioid agonist and partial agonist treatment and that coprescription was strongly associated with drug-related poisonings.

This study adds to the relatively thin evidence base about the potential hazards of benzodiazepine coprescription in the setting of opioid agonist treatment. Although opioid agonist treatment should not be with held from patients concurrently taking benzodiazepines or other central nervous system depressants, these studies suggest a need for vigilance by healthcare professionals providing care for such patients to minimize the risk of overdose or death. Coprescription of alprazolam may warrant particularly heightened scrutiny, however, given that it is the short-acting benzodiazepine most frequently involved in drug overdose deaths.The elevated mortality risks facing people with opioid use disorders are attributable to a complex web of interrelated structural and psychological causes. The concept of the “risk environment”may be useful to reference here, given its focus on the interplay between various structural factors that increase vulnerability to morbidity and mortality. The study by Zehang Li and colleagues provides an example of the use of spatiotemporal data to characterize one aspect of the risk environment. Applying a Bayesian space–time model to emergency medical services dispatch data on suspected heroin-related overdose incidents from Cincinnati in 2015–2019, the investigators identified significant spatial heterogeneity in the distribution of these calls, with strong associations with features of the built environment and temporal spikes corresponding to local media reporting. Analyzing 2005–2016 claims data, Yu-Jung Wei and colleagues identified more than 200,000 adults with new claims related to opioid use disorder or overdose. They found that, by the end of the study period, nearly one-half had filled no opioid prescriptions in the 12 months prior to an incident opioid use disorder diagnosis or overdose. Among those who had filled opioid prescriptions, nearly three-quarters were prescribed a mean daily dose lower than the threshold needed to trigger most risk stratification algorithms.

Also noteworthy is the analysis of 2015–2016 data from the US National Survey on Drug Use and Health by Joel Hudgins and colleagues. These authors found that approximately 1 in 20 adolescents and young adults reported either past-year opioid use disorder or past-year non-medical use of prescription opioids and that three-quarters of those reporting non-medical use of prescription opioids had obtained them from outside the healthcare system. These estimates are generally consistent with trends identified in similar, previously published analyses of NSDUH data. Thus, although opioid prescribing patterns undoubtedly played a significant role in how opioid use disorders came to be so highly prevalent and asymmetrically distributed in the US, a public health response that focuses solely on prescribing behavior is likely to be ineffective in reducing the number of fatal and nonfatal opioid overdoses.For people with existing opioid use disorders, opioid agonist treatment is known to reduce mortality. Monica Malta and colleagues add to this evidence base with a systematic review showing a wide range of health and prosocial benefits of opioid agonist treatment for people with opioid use disorders who are incarcerated or have recently been released. Opioid agonist treatment may have important collateral health effects as well. Analyzing data from a 3-country cohort of people who inject drugs, Charles Marks and colleagues found that people who inject drugs and who receive opioid agonist treatment are approximately half as likely to assist others in initiating injection drug use. They then developed a deterministic, dynamic transmission model of initiation into injection drug use, ongoing drug use, and cessation of drug use. Currently about 1 in 5 people with opioid use disorders receive any kind of treatment; if treatment coverage were doubled to 40%, Marks and colleagues’ model suggests that the number of initiations into injection drug use could fall significantly. This is an underappreciated benefit of opioid agonist treatment and underscores its potential public health impact, extending beyond its benefit to the individual.

To assist with prevention and treatment efforts, Jesse Yedinak and colleagues aggregated data from 2015–2016 state and national data sources to estimate the proportions of Rhode Island residents in various states: at risk of, in treatment for, and in recovery from opioid use disorders. The authors’ cross-sectional approach requires assumptions about exchange ability in the transition probabilities of individuals at each stage and the absence of biases due to selective mortality, but their addition of the “recovery” stage is a novel modification to the existing framework. This body of work echoes the familiar “voltage drops” analogy pioneered by John Eisenberg and Elaine Power, who used this model to illustrate how the potential for high quality care is lost at various stages of access, enrollment,mobile grow system and treatment. So-called treatment cascade models have been used to identify gaps in the access and treatment continuum for a wide range of health conditions, including HIV treatment, prevention of mother-to-child transmission of HIV, depression, and, most recently, opioid use disorders. Finally, for people with opioid use disorders who either cannot or do not choose to achieve sustained remission, alternative approaches might be considered to reduce the harms associated with ongoing use. Stephanie Lake and colleagues analyzed 2014–2017 data from Vancouver, Canada, on people who use drugs and who experience chronic persisting pain and found that daily cannabis use was associated with significant reductions in high-frequency non-medical opioid use. This finding echoes previously published studies showing that expansions in access to marijuana in the US have been associated with reductions in opioid overdose mortality. Among those for whom opioids remain the drugs of choice, use of supervised consumption facilities can reduce the risk of overdose mortality, and the potential for either individual-level adverse health effects or neighborhood-level adverse social effects appears to be minimal. Mary Clare Kennedy and colleagues contribute to this literature by showing that, among clients of the first supervised consumption facility in North America , frequent utilization was associated with a reduction in all-cause mortality over 2006–2017. Although “deaths of despair” ranked highly among the causes of death observed in this study, other non-accidental causes of death were also prominent. In the US, only 1 supervised injection facility currently exists, although 13 cities have sought approval to support their implementation. Ongoing misalignment between state and federal laws governing use of recreational marijuana and availability of supervised injection facilities seriously undermines harm-reduction efforts in the US.

These and other interventions across multiple sectors involving healthcare, economic, and social welfare systems need to be scaled up, dramatically and immediately, in order to substantively reduce the number of opioid overdose deaths. However, as discussed by Alexander Tsai and colleagues, the single most all-consuming force that restrains an effective policy and programmatic response to the opioid overdose crisis—through multilevel pathways that have nimbly adapted to the contours of the crisis over time—is the stigma attached to opioid use. Women whose children are affected by neonatal abstinence syndrome carry a stigma for the rest of their lives. Current media attention devoted to the “mommy drinking” myth is driven by the stigma resulting from the intersecting levels of scrutiny targeted toward women who parent and toward those who consume alcohol. Moreover, the disparate geospatial burden of opioid-related incidents, such as those studied by Zehang Li and colleagues, generates a stigma that attaches to entire neighborhoods. Indeed, as a class, harm-reduction interventions have been tainted by stigma, leading to their chronic under funding and under utilization. These and other forms of stigma must be eliminated before the overdose crisis can be successfully overcome. Tsai and colleagues provide suggestions for anti-stigma interventions at multiple levels to achieve this goal.This issue of PLOS Medicine is notable for several gaps. First, the majority of contributions to the Special Issue concern the North American opioid overdose crisis, but the global burden of disease attributable to alcohol use disorders greatly exceeds that attributable to opioid use disorders . Touching on harms owing to alcohol use, McKetta and Keyes used data from the 2006–2018 US National Health Interview Survey to examine national trends in binge drinking and heavy drinking. Consistent with concurrently published findings from the NSDUH [59], they found that heavy drinking has declined or stabilized for most age/sex subgroups but that binge drinking has increased, particularly among women and older men. A second evidence gap has to do with the global reach of the evidence. Although PLOS Medicine publishes research findings of general interest to the medical and public health communities, we received manuscript submissions describing research conducted in only a limited number of countries. In this Special Issue, the sole paper representing research conducted outside the US, Canada, and the UK is the report by Samantha Harris and colleagues, who analyzed Swedish register data from 1984–2016 to show that both refugee and non-refugee migrants had lower rates of substance use disorders compared with Swedish-born individuals but that over time, the rates among migrants converged to that of Swedish-born individuals. The issue regrettably features no articles from Africa, Asia, or South America, and no articles focused on indigenous populations or on racial, ethnic, or sexual minority groups. A third evidence gap has to do with the portfolio of methods underlying the evidence base. Causal methods have an important role to play in characterizing the relationships between exposures and outcomes when experimental data are difficult to come by . Among the articles included in this Special Issue, only Kennedy and colleagues used the “E-value”to estimate the minimum strength of association on the risk ratio scale that an unobserved variable would need to have with both the exposure and outcome to fully explain away their observed association. More studies using causal mediation analysis, instrumental variables, marginal structural models, natural experiments, regression discontinuity designs, and synthetic control methods are needed. This Special Issue also lacks articles based on qualitative data. Qualitative methods can be used to study complex phenomena like substance use disorders in greater depth, probe for mechanistic pathways linking the phenomena of interest, and generate new insights that can be tested in future studies. Collectively, the articles published in this issue highlight the scope of discovery and implementation needed to reduce the global burden of disease attributable to substance use, misuse, and dependence. Challenges—some related to science, others related to politics—are apparent. Multiple lines of evidence have already charted a road map that can be followed with respect to immediate policy making and program deployment. But there is a yawning chasm between what we know works to reduce the burden of disease from substance use disorders and what, at a societal level, is actually done about this burden. Eliminating this gap is not beyond our reach, given the available scientific evidence and substantial burden of ill health and suffering calling for prompt action, making it a public health imperative.

These included the demonstration that modulation of kinase and phosphatase activities or cAMP levels has no effect on DSI, while the relatively rapid onset of IPSC suppression makes a phosphorylation-mediated change in channel activity less likely, since that would typically require several seconds. They confirmed the findings of Lenz et al. that  -conotoxin, but not  -aga-toxin is able to block DSI, which means that the G protein-mediated endocannabinoid actions target only the N-type but not the P/Q-type Ca2 channels in the hippocampus . DSI or the selective Ca2 channel inhibitors never block IPSCs completely, which may be due to a partial reduction of release from all terminals, or to the selective expression of CB1 receptors together with the N-type channels only on a particular subset of interneurons. Wilson et al. provided an elegant resolution to this dilemma using paired recordings, which revealed that interneurons producing IPSCs with distinct kinetics express different presynaptic Ca2 channels, and those that show DSI possess only N-type channels . This finding correlates well with the anatomical observations that CCK-containing basket cells selectively express CB1 receptors, whereas another basket cell type lacks CB1 receptors . The differences in IPSC kinetics observed by Wilson et al. may be due to CCK cells forming synapses that are enriched in 2-subunit-containing GABAA receptors,cannabis grow tent whereas parvalbumin-containing basket cells synapse onto GABAA receptors with five times less 2-subunits .

Taken together, these data suggest that CCK-containing basketcell terminals selectively express N-type Ca2 channels together with CB1 receptors predisposing them to DSI, whereas parvalbumin-containing interneurons may express only the P/Q-type Ca2 channels, lack CB1 receptors, and are therefore unaffected by DSI. This conclusion also suggests that the success of DSI induction in any hippocampal slice preparation depends on the relative contribution of the two basket cell types to the examined spontaneous or evoked IPSCs samples. Carbachol is known to enhance DSI, but the mechanism hasnot been revealed to date . One possibility is that carbachol activates the inositol 1,4,5-trisphosphate system via muscarinic receptors, thereby contributing to the large Ca2 transient required for endocannabinoid release . This sounds unlikely as in most experimental paradigms massive depolarizations or uncaging of calcium has been used; thus it would be difficult to further enhance calcium levels by activation of IP3 receptors on intracellular stores. Furthermore, a recent study showed in sympathetic neuronal cultures that muscarinic receptor-mediated activation of PLC- results in limited if any IP3-mediated intracellular Ca2 release ; thus the major signaling pathway there is the production of DAG. However, under physiological conditions in the hippocampus, a cholinergic activation of PLC may well contribute to endocannabinoid release via both the IP3 cascade and the DAG limb . Another likely explanation for the experimental results with carbachol is that it may suppress IPSCs produced by parvalbumincontaining basket cells via presynaptic m2 receptors, which are selectively expressed by this interneuron type , whereas the spontaneous activity of CCK-containing interneurons may be increased via m1 muscarinic, or perhaps even nicotinic actions of carbachol. The mutually exclusive distribution of CB1 and m2 receptors on two subsets of basket cell terminals is shown in Figure 16 . If this reasoning is correct, DSI could be facilitated via other receptors as well that are selectively present on CCK cells but not on parvalbumin cells, e.g., substance P receptors or 5-HT3 receptors.

Indeed, Ha´jos et al. demonstrated that the increase in the amplitude and frequency of spontaneous IPSCs after bath application of substance P fragment was brought back to near control levels by the coapplication of the CB1 receptor agonist WIN55212–2. Although endocannabinoid-mediated DSE has been convincingly demonstrated in the cerebellum, the existence of this phenomenon in the hippocampus could not be established with the same paradigm used for DSI or DSE in the cerebellum . Cannabinoids do reduce glutamatergic EPSCs in the hippocampus , but the receptor involved is unlikely to be CB1 , since the effect was found to be the same in CB1 knock-out and wild-type animals . However, in a recent study, prolonged depolarization was found to readily induce DSE in hippocampal slices, which was absent in CB1 knock-out mice . This is in conflict with the data of Ha´jos et al. and may be due to age or strain differences. Retrograde endocannabinoid signaling was shown to be responsible for another type of synaptic plasticity of glutamatergic transmission in the striatum. Long-term depression of EPSCs induced by high-frequency stimulation of afferent fibers disappeared in CB1 receptor knock-out animals.Interestingly, recent experiments uncovered that activation of postsynaptic type I mGluR receptors induce LTD in the hippocampus by decreasing glutamate release presynaptically . The striking similarity of induction parameters, as well as the potential role of type I mGluRs inendocannabinoid synthesis , suggests that retrograde signaling via postsynaptic release of endocannabinoids is likely to account for this phenomenon. Thus an important question for future research is to determine how DSE and mGluR-dependent LTD are related, along with the identification of how postsynaptic release of endocannabinoids may contribute to these phenomena. The paragraphs above dealt with cannabinoid signaling phenomena that are, or could be, brought about by endogenously released cannabinoids.

Some thought should be given also to those cannabinoid actions that are unlikely to be reproduced by endogenously released cannabinoids but may still be important for the interpretation of the mechanisms of action of delta-9-THC or synthetic ligands. For example, endogenously released cannabinoids are unlikely to act on LTP in the hippocampus, since 1) DSE could be evoked in this region only by prolonged depolarization , 2) cannabinoids had no effect on LTP or LTD when Mg2 -free solution or pairing with strong postsynaptic depolarization was used , and 3) LTP induction under quasi-physiological conditions may be insufficient stimulation for a detectable endocannabinoid release . Single postsynaptic spikes are able to induce LTP if paired with presynaptic spikes or bursts , and excess endocannabinoid release that would be capable of inhibiting glutamate release is unlikely to occur under these conditions. Thus whether endogenously released cannabinoids are able to influence the efficacy or plasticity of glutamatergic transmission in the hippocampus via a direct action on glutamate release is still to be shown. However, Carlson et al. showed that a weak train of stimuli that normally does not induce LTP will induce NMDA-dependent LTP if given during the DSI period. The simultaneously recorded field EPSPs do not undergo LTP,grow lights for cannabis showing that the weak stimulus train was indeed sub-threshold for LTP induction except in dis-inhibited cells. The single-cell LTP was prevented by pretreatment with AM251, suggesting that locally released endocannabinoids can enhance LTP by causing dis-inhibition of a pyramidal cell.As discussed above, several lines of experimental evidence suggest that rather large increases in intracellular [Ca2] are required for the induction of DSI and DSE via the release of endocannabinoids , and this elevation of Ca2 is essential for the synthesis rather than the release of endocannabinoids . Such profound Ca2 transients may occur only under special physiological conditions, e.g., upon the release of Ca2 from IP3- or ryanodine-sensitive intracellular stores via simultaneous activation of metabotropic receptors and voltage-gated Ca2 channels . Back-propagating action potentials are most likely responsible for the voltage-gated Ca2 influx both in the proximal dendritic and distal dendritic regions , although in small cellular compartments like a spine head, a single NMDA-mediated synaptic event may be sufficient to release Ca2 from the local intracellular stores . In the perisomatic region , type I mGluRs appear to supply IP3 both in pyramidal and Purkinje cells , which may partly explain the apparent involvement of this receptor type in DSI . Indeed, recent papers provide evidence that metabotropic glutamate effects on DSI are mediated by endocannabinoids, as described above. Pairing back-propagating action potentials with mGluR activation increases Ca2 release severalfold compared with spiking alone . The largest amplitude Ca2 transient was observed in the most proximal segment of the apical dendrite, an ideal location for endocannabinergic modulation of GABAergic axon terminals that innervate this region. Electron microscopic studies demonstrate the lack of glutamatergic synapses on the cell bodies and proximal apical shafts of pyramidal cells , which suggests that intracellular Ca2 release in this region has to have a role other than conveying plasticity to glutamatergic synapses.

One possibility is that this Ca2 rise is sufficiently close to the nucleus to trigger transcriptional changes. Alternatively, it may be critically involved in the induction of endocannabinoid release, which results in the down regulation of perisomatic inhibition. Thereby action potentials could better back-propagate into the distal dendrites allowing associative LTP of distal glutamatergic synapses, or would enable the neuron to dissociate itself from the population oscillation maintained by basket cell-mediated inhibition . One problem with this hypothesis, and with the interpretation of the mGluR studies , is the source of glutamate required to activate mGluRs in the somatic/proximal dendritic region, since these parts of pyramidal cells do not receive glutamatergic synapses . Thus, if mGluRs get activated at all in this region under physiological conditions, it either has to involve extrasynaptic mGluRs reached by diffusion of glutamate from distant synaptic sites, or mGluRs may be activated further away from the proximal apical dendrite , and IP3 would have to be able to diffuse very fast to its receptors located on the perisomatic or proximal dendritic endoplasmic reticulum. The latter alternative is possible, since IP3 was calculated to be able to diffuse 50 m in 0.5 s, which is faster than Ca2 diffusion in the cytosol containing Ca2 buffers . Diffusion of synaptically released glutamate, however, is unlikely, since it is limited by the efficient glial and neuronal uptake machinery; a spillover even to the adjacentsynapse is limited . An alternative trigger for IP3 synthesis is muscarinic activation. Indeed, Martin and Alger demonstrated that DSI is enhanced by muscarinic m1 or m3 receptor stimulation. Varicose cholinergic fibers are abundant in all layers of the hippocampus and particularly enriched in stratum pyramidale and near the granule cell layer . Furthermore, principal cells are known to express muscarinic receptors on their perisomatic membrane . Activation of muscarinic receptors induces a profound Ca2 rise in the soma, or Ca2 waves that propagate into the soma, and increases the Ca2 transients evoked by trains of action potentials . Thus it is important to emphasize that, in addition to group I mGluRs, cholinergic transmission may also contribute to the generation of sufficient IP3 levels to trigger large Ca2 transients followed by endocannabinoid release when coinciding with trains of action potentials. However, muscarinic receptor-mediated activation of PLC-_x0007_ in sympathetic neuronal cultures results in limited if any IP3-mediated intracellular Ca2 release; thus the major signaling pathway there is the production of DAG , which, on the other hand, is the precursor of 2-AG synthesis . Whether muscarinic activation uses primarily the DAG limb in hippocampal endocannabinoid signaling remains to be established, although the lack of an antagonist effect on DSI suggests that resting levels of acetylcholine are not involved in the generation of the required DAG pool . The same question arises also for the mechanism of mGluR-mediated endocannabinoid release, since in a recent study in hippo campal cultures, group I mGluR activation was shown to enhance DSI without increasing intracellular calcium signals . This raises the possibility that under some conditions, group I mGluR activation uses the alternative root; it may increase 2-AG synthesis via the DAG limb , and thereby could cooperate with depolarization induced Ca2 transients to enhance endocannabinoid release. The physiologically most relevant question here is which are the behavior-dependent activity patterns that could ensure the coincidence of metabotropic receptor activation and bursts of action potentials that are able to induce sufficiently large Ca2 transients to release endocannabinoids in the hippocampus . Spontaneous or low magnesium-evoked burst potentials that resemble physiological bursts were shown to induce DSI . Hippocampal pyramidal cells typically produce bursts of two to six action potentials at 6-ms intraburst intervals . These bursts were shown to invade parts of the dendritic tree quite efficiently, and therefore their pairing with presynaptic activity readily induces LTP .

A similar presynaptic localization also has been suggested for CB1 receptors in dorsal root ganglion neurons, because resection of the dorsal root significantly decreases cannabinoid binding in the dorsal horn of the spinal cord . An important achievement in cannabinoid research was the development of specific antibodies recognizing CB1 receptors, which have become indispensable research tools . Antibodies raised against either the NH2 terminus or the COOH terminus of the CB1 protein provided crucial information about the precise localization of CB1 receptors at the regional, cellular, and sub-cellular levels. However, immunohistochemical studies require careful investigation and well-designed controls, since it is rare that an antibody is absolutely specific for the desired target protein. Thus reports claiming immunore activity for CB1 receptors in cells , which do not produce the mRNA of CB1, or immunolabeling of glial cells due to the antibody recognizing the antigen carrier protein should be viewed with caution . Essential in this regard was the generation of mutant CB1 /_x0005_ mice , which were instrumental to demonstrate antibody specificity and limit the confusion resulting from staining artifacts . Initial light microscopy studies revealed the existence of numerous CB1-immunoreactive fibers throughout the brain . Based on their distinctive morphological appearance, thin and rich in varicosities, these fibers were tentatively identified as axons. This identification received its first subcellular confirmation from work conducted in the rat hippocampus . The varicosities observed at the light microscopy level were found to correspond to axon terminals packed with synaptic vesicles and to be densely covered by CB1 receptors . Notably,cannabis grow racks when an antibody against the extracellular NH2 terminus of the CB1 receptor was used in combination with silver-impregnated gold particles, the particles were exclusively found at the outer surface of the axonal plasma membrane .

On the other hand, when the staining was carried out with a different antibody, specific for the COOH terminus, the gold particles only labeled the intracellular surface of the boutons . CB1-positive axons have a scattered pattern of distribution, which largely parallels that obtained with radioli-gand binding . An especially dense fiber mesh work is observed in the globus pallidus, the substantia nigra pars reticulata, and the entopeduncular nucleus, probably on axons deriving from the striatum. In many cortical areas, as well as in olfactory systems, CB1-immunoreactive axons are abundant and form pericellular baskets around CB1-negative cell bodies. Likewise, CB1-positive axons equipped with numerous boutons cover the somata of Purkinje cells in the cerebellum and shape the characteristic pinceaux structures around the axon initial segments . In addition, the stratum moleculare of the cerebellum also exhibits strong CB1 immunoreactivity, while leaving blank the dendritic tree of Purkinje cells . The cell origin of these fifibers can sometimes be inferred from the combination of cellular CB1 expression pattern and the distribution of CB1-positive axons. For example, in the cerebellum, the dense staining seen in the stratum moleculare likely results from axons of CB1-expressing granule cells, which constitute the so-called parallel fibers. In most cases, however, the cell origin and phenotype of CB1- carrying axons is still uncertain. Recent efforts have helped determine the precise subcellular distribution of CB1 receptors in the rodent somatosensory cortex, the hippocampus, and the amygdala, as well as in the human hippocampus . In these areas, CB1 receptors localize to specific types of axon terminals, and as a rule, boutons engaged in asymmetrical synapses do not carry CB1 receptors, whereas boutons engaged in symmetrical synapses do . This indicates that GABAergic, but not glutamatergic, axon terminals contain the receptors. GABAergic interneurons are extremely heterogeneous, however, and not all of them express CB1 receptors.

Indeed, only a sub-population of GABAergic interneurons, those that utilize CCK as a peptide cotransmitter, was found to be CB1 positive , whereas those marked by parvalbumin were not . Because CCK- and parvalbumin-positive interneurons have distinct roles in the regulation of cortical activity, it is likely that endocannabinoid substances also have specific functions in the modulation of cortical network properties. This notion is strongly supported by the retrograde messenger role of endocannabinoids in DSI, which is clearly restricted to select inhibitory synapses within the hippocampus .Outside the cortex, detailed information on the subcellular distribution of CB1 receptors is only available for the peripheral nervous system, where CB1 receptors also appear to be concentrated at nerve endings. In the rat and guinea pig lung, sparse nerve fibers bearing CB1 receptors are found among bronchial smooth muscle cells . Although such fibers rarely form true synapses, immunogold labeling reveals that CB1 receptors are located close to vesicle accumulations, where they may act to modulate neurotransmitter release. Importantly, neuropeptide Y, a neurochemical marker for noradrenergic sympathetic nerve fibers , was found to colocalize with CB1 in these axon terminals . Accordingly, cannabinoids potently inhibit norepinephrine release in peripheral tissues and organs through a presynaptic mechanism .Although anatomical studies may reveal the precise localization site of a particular receptor type, they may only provide predictions about its functional importance. In the last decade, two major approaches, electrophysiological recordings and neurochemical release studies, contributed fundamentally to our understanding of the physiological role of endocannabinoids and the consequences of cannabinoid receptor activation. Most of these studies point to the same conclusion as anatomical studies, i.e., CB1 receptors presynaptically regulate the release of certain types of neurotransmitters from axon terminals. The major goal of these studies is to establish which of the numerous types of neurotransmitters are influenced by cannabinoids at certain brain areas.

Not surprisingly, the release of nearly all major neurotransmitter types was shown to be affected by cannabinoid agents. Similarly to CB1-specific antibodies in anatomical experiments, the development of pharmacological probes, such as selective CB1 receptor agonists and antagonists, was indispensable to advance the field . However, as is the case with immunohistochemical experiments, the establishment of the role of CB1 receptors in many of the described processes requires careful evaluation. Recent studies using CB1 /_x0005_ mice provided evidence that conventional cannabinoid receptor ligands, as well as the endocannabinoids, are not exclusively selective for CB1 receptors . In the following sections, we survey the various lines of pharmacological evidence for the existence of presynaptic cannabinoid receptors on many different types of axons in several brain areas and aim to evaluate in the light of anatomical data whether CB1 or another molecular target may underlie certain effects of cannabinoids.In the hippocampus,cannabis grow system electrophysiological and neurotransmitter release experiments concord in indicating that cannabimimetic agents modulate GABA release via a presynaptic mechanism. Whole cell patch-clamp experiments show that cannabinoid agonists decrease amplitude and frequency of GABAA receptor-mediated inhibitory postsynaptic currents elicited by action potentials . These effects are mediated by CB1 receptors, because they are blocked by the CB1 antagonist SR141716A and are completely absent in CB_x0005_ /_x0005_ mice . The presynaptic action of cannabinoids was suggested by the lack of effect on the amplitude of miniature IPSCs , as well as by a reduction in vesicle release probability . These data are in striking agreement with the anatomical studies showing the presynaptic localization of CB1 receptors on GABAergic axon terminals. In the basolateral amygdala, which has a morphological architecture in many respects similar to the hippocampus, cannabinoid agonists produce comparable responses. The compounds inhibit synaptic GABAA-mediated currents in principal neurons of this region, but cause no such effect in the central nucleus, which does not contain CB1 receptors . The significance of these findings was also recently confirmed in vivo in the prefrontal cortex . In accordance with the exclusive expression of CB1 by GABAergic neurons in the neocortex , the cannabinoid receptor agonist WIN 55,212–2 reduced cortical GABA levels, which was prevented by the cannabinoid receptor antagonist SR 141716A . Moreover, neurochemical release experiments extended the validity of this finding from the rat to the human hippocampus . Taken together, these results indicate that GABAergic axon terminals are one of the major targets of cannabinoids in cortical networks, where they reduce the release of GABA in a CB1 receptor-mediated manner.Results from a variety of cortical tissue preparations are consistent in indicating that cannabinoid agonists can reduce excitatorysynaptic neurotransmission.

These actions are probably exerted at a presynaptic locus, for three reasons: 1) cannabinoid agonists increase paired-pulse facilitation, 2) they do not change postsynaptic responses to glutamate or kainate applications, and 3) they cause a characteristic increase in response failures and coefficient of variation of excitatory postsynaptic currents . The ability of the CB1 antagonist SR141716A to prevent these inhibitory responses suggested early on that CB1 receptors might be involved. Nevertheless, the fact that careful anatomical analyses negated this hypothesis sent the field up an apparent cul-de-sac: how could cannabinoid agonists inhibit glutamate release if CB1 receptors are only weakly, if at all, expressed by glutamatergic neurons and are absent from glutamatergic terminals ? The use of CB1 /_x0005_ mice offered a solution to this conundrum. Cannabimimetic agents reduce glutamatergic EPSCs in CB1 /_x0005_ mice to the same degree as they do in wild-type ones, although they no longer affect GABAergic IPSCs . The most economical hypothesis compatible with this result is that glutamatergic axon terminals contain a novel cannabinoid-sensitive site, which is blocked by SR141716A, but is molecularly distinct from the cloned CB1 receptor. Further pharmacological characterization revealed that the new cannabinoid-sensitive receptor has an order of magnitude lower affinity for WIN55,212–2 compared with CB1 , as the EC50 for the suppression of EPSCs was 2.01 M, whereas for IPSCs 0.24 M . In addition, cannabinoid effects on EPSCs could be antagonized by the vanilloid antagonist capsazepine, and mimicked by the agonist capsaicin, whereas vanilloid compounds were without effect on GABAergic IPSCs . These data clearly indicate that cannabinoid receptors controlling IPSCs versus EPSCs are pharmacologically distinct. The latter type is unlikely to be the vanilloid receptor VR1, since WIN55,212–2 does not bind to VR1 on sensory nerves . Moreover, VR1 forms a nonselective cation channel , whereas cannabinoid effects on glutamatergic EPSCs are mediated via a pertussis toxin-sensitive G protein-coupled process , which is in accordance with the ability of WIN 55,212–2 to stimulate [ 35S]GTP S binding in several brain regions of CB1 knockout mice . It is reasonable therefore to conclude that a cannabinoid-sensitive receptor other than CB1 or VR1 is located on glutamatergic, but not on GABAergic, axons in the hippocampus and possibly other brain areas . C) CANNABINOID EFFECTS ON ACETYLCHOLINE RELEASE IN CORTICAL AREAS. The cannabinoid receptor agonist WIN 55,212–2 decreases acetylcholine release from electrically stimulated rat hippocampal slices . This effect is mimicked by other synthetic cannabinoid agonists, as well as by the endocannabinoid anandamide, and is prevented by the CB1 antagonists SR141716A and AM281.The role of CB1 receptors in these responses, suggested by the effects of CB1 antagonists, is further supported by anatomical and genetic data. CB1 receptors are expressed by neurons in the medial septum and ventral diagonal band, where cholinergic innervation of the hippocampus originates . In the monkey forebrain, septal CB1-immunoreactive cells, along with other CB1- positive neurons in the nucleus basalis of Meynert , express choline acetyltransferase , the synthetic enzyme for acetylcholine . Furthermore, the cannabinoid modulation of acetylcholine release was reduced in “knockdown” experiments with antisense oligonucleotides and abolished in the hippocampus and the neocortex of CB1 knock-out mice . Although unequivocal anatomical demonstration of CB1 receptors on cholinergic axon terminals is still needed, physiological evidence also supports their existence. In hippo campal slices perfused with a Ca2-free, K-rich medium containing the Na channel blocker tetrodotoxin, cannabinoid agonists attenuate Ca2 -evoked acetylcholine release, probably by inhibition of voltage-gated Ca2 channels . Importantly, a parallel result was obtained in cortical and hippo campal synaptosomes, again implying a presynaptic site of action . What is the functional significance of these in vitro findings? Cholinergic innervation of cortical brain regions is thought to play an important role in cognitive processes, many of which are strongly impaired by cannabinoid treatment . An appealing causal link between these observations is strengthened by the finding that cannabinoid agonists reduce acetylcholine levels in rat cortical and hippocampal microdialysates, when administered at relatively high doses . However, recent experiments uncovered that lower doses of these drugs cause an opposite effect, elevating acetylcholine level in the prefrontal cortex and the hippocampus .