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The sinuses, a connected system of hollow cavities in the skull lined with mucosa tissue that has a thin layer of mucus, may help humidify air in the nasal cavity. In 2015, a $15-million grant by the National Science Foundation kicked off further research into how animals, including humans, locate the source of an odor, such as food . The research focuses on how odors move in the landscape and how animals use spatial and temporal cues to move toward a target. The research is just one part of the federal BRAIN Initiative that studies olfaction as a window into understanding the brain, because olfaction is considered the most primal pathway to understanding brain evolution. At present, such information is not available for e-nose development. The olfactory epithelium contains three types of cells: olfactory receptor neurons, their precursors and supportive cells. The cilia are constantly exposed to the nasal environment and are continually replaced, even their basal cells, possibly indicating frequent damage. A layer of mucus 10 to 40 µm thick coats the mucosa epithelium, and odorants must pass into this layer to interact with the sensory neurons through a series of poorly understood “perireceptor” events . Each sensory neuron, covered in cilia, trim tray projects down from the olfactory epithelium into the mucosa.These proteins thread back and forth across the outer membrane of the cilia and interact with odorants. Various theories have been put forward on how exactly odorants interact with the proteins, and this remains an area of research.

Receptor cells of the same type are randomly distributed in the nasal mucosa but converge on the same glomerulus. Each type of neuron frequently responds to more than one odorant, even from different chemical classes, so the overall odor signal must be integrated by the olfactory bulb . Integration includes both olfactory and trigeminal signals, and workers often report odor and irritation as a combined, singular perception . The olfactory bulb also receives information from other areas of the brain to filter out background odors and enhance perception. Fascinatingly, none of the physical stimuli themselves ever reach the brain. Instead, a host of proteins transduces captured molecules into a small change in voltage that can be deciphered by the brain . The unpleasant and pleasant aspects of mixtures are represented separately in the brain . Human sensitivity to odorants ranges across several orders of magnitude . Around 1 ppt appears to be a theoretical limit for sensitivity, and many odorants are not perceived until above 1,000 ppm. The major components of air are not sensed at all . Carbon dioxide is an interesting chemical because it is odorless at ambient concentrations yet selectively triggers only the trigeminal neurons and not the olfactory neurons when it reaches 200-fold above background levels . Describing multiple odor notes in mixtures is challenging. Fewer than 15% of the people tested could only identify one of the odorants present in a mixture, and identification of 3 to 4 components was the limit for trained experts . Even 90% of wine judges were unable to reproduce their scores . General variability in odor perception is high. Factors include age, sex, lifestyle, prior exposures, culture and health status . Approximately 3% of Americans have minimal or no sense of smell .Prolonged or repeated exposure to an odor can lead to a decreased response , which has the benefit of allowing a baseline reset in preparation for a new stimulus . Habituation happens as quickly as 2.5 second and is accompanied by decreased transduction by the neurons after 4 seconds . A growing field of research throughout public health is the microbiome, the microflora that contribute to gut, mouth and skin health. The nasal cavity, too, hosts microbes that contribute to normal functioning .

Some microbes themselves emit odorants and can decrease the host’s sensitivity . Attempts to reverse engineer an odor based on the molecular properties of the odorant have been successful. Algorithms were able to predict the odor note of a given odorant based on its chemoinformatic features for 8 descriptors out of 19 total . Researchers using systems biology and computational techniques mapped odors to specific proteins on olfactory receptor neurons, which was dubbed the “odorome” . Risk assessment for estimating the non-sensory health risks of airborne chemicals has a large body of guidance and case studies. The primary focus of this paper is on the sensory health effects of odors that integrate both trigeminal response and olfactory response . In general, the olfactory pathway iscapable of informing the organism about the presence of an odorant while the trigeminal pathway helps inform the organisms about the risk of health hazards and injury .Cognitive bias plays a role in odor responses . Odors trigger memories of previous experiences and are influenced by the power of suggestion. If given a prior warning that an odor is harmful, increased irritation was reported. Fewer symptoms were reported if told the odor was healthful. Even when no odor was administered, suggestion that there was a harmful odor led to symptoms. Prior experience with an odor introduces bias, too. Emotional baseline is also a factor . Sensitization to an odorant occurs when an acute exposure triggers subsequent, more-severe responses, often at lower concentrations . Desensitization can occur when chronic exposure to an odorant increases the concentration required to trigger a response. For example, workers who are habituated and desensitized to an odorant may be baffled by neighborhood complaints . Due to the availability of human data, other animal data were not considered in the hazard identification. Further, humans have a smaller area of nasal olfactory epithelium than rodents, which makes humans more vulnerable to loss, and respiration rates are quite different . Only one experimental study of a typical, complex environmental odor and health effects was found , which included both physical effects and mood . Out of the dozens of parameters evaluated, only headaches,eye irritation and nausea symptoms were elevated among those exposed for one hour as compared to controls . The epidemiology evidence, however, indicated the full range of adverse effects from odor exposure .

Such symptoms were self-reported, which means they may include bias. The distance from facility, an objective measure, contrarily did not predict the frequency of symptoms. Interestingly,ebb and flow the relationship between odor exposure and health symptoms appeared to be greatly influenced by odor hedonic tone, perhaps more so than odor intensity. The debate whether the purely odor-related symptoms are psychological or have an actual underlying physical cause is ongoing. In the same issue of Archives of Environmental Health in 1992, two opposing perspectives were presented. Shusterman concluded that the evidence of health effects was lacking beyond odors’ ability to inflict annoyance. In the editorial immediately after his article, Ziem and David off countered that odor, and chemical sensitivity in general, may well be based on underlying physiological responses, as was often found in the case of sick building syndrome. Both agreed that better ways to determine the impact of odors were needed, and well-controlled prospective case-control studies would be especially welcome. The psychological symptoms of odor exposure include tension, nervousness, anger, frustration, embarrassment, depression, fatigue, confusion, frustration, annoyance, and general stress . Odor frequency, odor intensity and feeling their concerns are not being heard all contribute to annoyance, which leads to stress. Health worries contribute as well. See Table 4.2.Changes in odor-induced frontal lobe activity has been linked to changes in mood, drowsiness, and alertness . Unfortunately, the studies of this connection were few and additional research in this area is needed. Odor-induced brain activity is complex, involving more than 30 different regions. Exposure to malodor led to inability to focus on a task . Other studies reviewed found, however, that odors have no effect on task performance, so they concluded that the impact of odors on task performance may be odorant-specific. Increased prevalence of gastrointestinal symptoms were observed as a function of proximity to a wastewater treatment plant in Poland . The symptoms were correlated with both odors and microbiological pollutants and could not be disentangled to single out odors as the primary agent. Similarly, the negative effects of traffic noise and odor on residents in Windsor, Ontario, Canada, had a strong covariance between these two parameters and could not be differentiated .Some odorants and some co-pollutants within odors are considered hazardous air pollutants because they cause other adverse effects beyond smell and irritation.

Air that contains odorants also is known to contain odorless co-pollutants such as particulate matter and endotoxins . There was a positive correlation between the presence of odors and the prevalence of self-reported health symptoms, such as headache and nausea, when communities near hazardous waste sites were compared . However, more serious health outcomes – cancers, mortality and birth defects – were not higher compared to the control sites .Dose-response relationships for odors aim to link the percentage of people experiencing adverse effects, such as odor annoyance and irritation, to the level of exposure. For toxic chemicals, adverse effects increase as exposure increases. Odors, however, can be more inconsistent. For example, hydrogen sulfide loses its characteristic “rotten egg” odor note as the concentration increases, leading to harmful levels going unnoticed .The major goal of both risk assessment and odor assessment is to verify that exposures are below the thresholds of concern . For conventional risk assessment, the thresholds are health-based, often extrapolated from animal studies, and typically incorporate large margins of safety due to crude extrapolations and uncertainties. For odor assessment, achieving odorless air is the goal, yet due to the “lack of severity” of the effect, the acceptable limit is often set well above the odor-detection threshold. Given the wide variability in human response to odor, this approach is perilous, but a point of departure is needed, nonetheless. The amount of dilution required to achieve odorless air delivers a crude point of departure but should acknowledge the large error and be presented with only one significant figure in the final results. Thresholds for odorants typically vary by several orders of magnitude, especially the ODTC50 . Further, reliable methods may not have been used, and results for odor detection and odor recognition are sometimes mixed up. Within a controlled setting, two approaches to sensory testing of dilutions by panelists are used. One is the Odor Profile Method , which uses sugar solutions for calibration, and the other uses odor disappearance upon sufficient dilution. Both rely on dilution equipment, typically a dynamic dilution olfactometer that delivers sampled air diluted with odorless air to a nose port where the dilution is smelled by the panelist. Concentrations are presented in ascending order to avoid desensitization and anticipation bias. Statistics are then used on the panelists’ results to determine the ODTC50 for the odorant or mixture tested. For a single odorant, OPM is used to assign an odor note and intensity to each dilution. The Weber-Fechner law is applied, meaning that the logarithm of the concentration is taken and then the intensity results are fit to a line through linear regression. Extrapolation to intensity score 1 yields the ODTC50 value. Each odorant can have a vastly different slope. For example, a 200-fold change in concentrations of 1-propanol and n-amyl buterate caused a 15-fold and 0.5- fold change in odor intensity, respectively . For mixtures , the ODTC50 can be determined by forced choice, typically “triangle,” methods using the same dynamic dilution equipment . While inhaling at the nose port, the panelist rotates between three choices and then selects which is the diluted sample. A point estimate is generated rather than a curve, and no odor description is gathered. ASTM Method E679-04 has been used to determine the ODTC50 values for a range of odorants . Such methods have been used in the drinking water industry to set ODTC50 values for methyl tertiary butyl ether and improvements to ASTM Method E679-04 have been suggested for drinking water . In Europe, under EN 13725, the final dataset typically only includes the data for the four or more panelists whose results are the most consistent with the overall panel’s geometric mean value. Also, panelists may be presented with 2 samples instead of 3.

The BART scores increased significantly with abstinence , whereas the IGT scores did not change during abstinence. Self-reported total and motor impulsivity decreased significantly with abstinence and the non-planning score tended to decrease . The following changes were observed when restricting our longitudinal analysis to only those 17 PSU with baseline and follow-up data: general intelligence, executive function, working memory , visuospatial skills , global cognition , and processing speed . The 19 PSU not studied longitudinally differed from our abstinent PSU restudied on lifetime years of cocaine use . PSU not restudied performed significantly worse at baseline than abstinent PSU on cognitive efficiency, processing speed, and visuospatial learning . Furthermore, they did not differ significantly on years of education, AMNART, tobacco use severity, and proportions of smokers or family members with problem drinking, or the proportion of individuals taking a prescribed psychoactive medication.In PSU, more lifetime years drinking correlated with worse performance on domains of cognitive efficiency, executive function, intelligence, processing speed, visuospatial skills, and global cognition . More cocaine consumed per month over lifetime correlated with worse performance on executive function and greater attentional impulsivity . More marijuana consumed per month over lifetime correlated with worse performance on fine motor skills and tended to correlate with higher BIS-11 motor impulsivity ; in addition, more marijuana use in the year preceding the study correlated with higher non-planning and total impulsivity. Earlier onset age of marijuana use correlated with higher non-planning impulsivity and worse visuospatial learning . Interestingly, more lifetime years of amphetamine use correlated with better performance on fine motor skills, executive function, visuospatial skills, and global cognition . Similar to the associations found in PSU, more lifetime years drinking in AUD correlated with worse performance on cognitive efficiency, visuospatial skills, cannabis grow equipment and global cognition , and worse performance on visuospatial memory correlated with greater monthly alcohol consumption averaged over the year preceding assessment and over lifetime . In addition, longer duration of alcohol use in AUD was related to worse auditory-verbal learning and memory .

Our primary aim was to compare neurocognitive functioning and inhibitory control in onemonth-abstinent PSU and AUD. Poly substance users at one month of abstinence showed decrements on a wide range of neurocognitive and inhibitory control measures compared to normed measures. The decrements in neurocognition ranged in magnitude from 0.2 to 1.4 standard deviation units below a zscore of zero, with deficits >1 standard deviation below the mean observed for visuospatial memory and visuospatial learning. In comparisons to AUD, PSU performed significantly worse on measures assessing auditory-verbal memory, and tended to perform worse on measures of auditory-verbal learning and general intelligence. Chronic cigarette smoking status did not significantly moderate cross-sectional neurocognitive group differences at baseline. In addition, PSU exhibited worse decision-making and higher self-reported impulsivity than AUD , signaling potentially greater risk of relapse for PSU than AUD . Being on a prescribed psychoactive medication related to higher self-reported impulsivity in PSU. For both PSU and AUD, more lifetime years drinking were associated with worse performance on global cognition, cognitive efficiency, general intelligence, and visuospatial skills. Within PSU only, greater substance use quantities related to worse performance on executive function and fine motor skills, as well as to higher self-reported impulsivity. Neurocognitive deficits in AUD have been described extensively. However, corresponding reports in PSU are rare and very few studies compared PSU to AUD during early abstinence on such a wide range of neurocognitive and inhibitory control measures as administered here . To our knowledge, no previous reports have specifically shown PSU to perform worse than AUD on domains of auditory-verbal learning and general intelligence at one month of abstinence. Our studies confirmed previous findings of worse auditory-verbal memory and inhibitory control in individuals with a comorbid alcohol and stimulant use disorder compared to those with an AUD, and findings of no differences between the groups on measures of cognitive efficiency .

Some of the cross-sectional neurocognitive and inhibitory control deficits described in this PSU cohort are associated with previously described morphometric abnormalities in primarily prefrontal brain regions of a subsample of this PSU cohort with neuroimaging data . Our neurocognitive findings also further complement studies in subsamples of this PSU cohort that exhibit prefrontal cortical deficits measured by magnetic resonance spectroscopy and cortical blood flow . Our secondary aim was to explore if PSU demonstrate improvements on neurocognitive functioning and inhibitory control measures between one and four months of abstinence from all substances except tobacco. Polysubstance users showed significant improvements on the majority of cognitive domains assessed here, particularly cognitive efficiency, executive function, working memory, self-reported impulsivity, but an unexpected increase in risk-taking behavior . By contrast, no significant changes were observed for learning and memory domains, which were also worst at baseline, resulting in deficits in visuospatial learning and visuospatial memory at four months of abstinence of more than 0.9 standard deviation units below a z-score of zero. There were also indications for significant time-by-smoking status interactions for visuospatial memory and fine motor skills, however these analyses have to be interpreted with caution and considered very preliminary, considering the small sample sizes of smoking and nonsmoking PSU at followup. Nevertheless, the demonstrations of cognitive recovery in abstinent PSU, and potential effects of smoking status on such recovery, are consistent with our observations of corresponding recovery in abstinent AUD . The 19 PSU not studied at follow-up differed significantly from abstinent PSU at baseline onseveral important variables: they had more years of cocaine use over lifetime, and performed worse on cognitive efficiency, processing speed, and visuospatial learning. As such, these differences should be tested as potential predictors of relapse in future larger studies. Several factors limit the generalizability of our findings. Our cross-sectional sample size was modest and therefore our longitudinal sample of abstinent PSU was small; as not uncommon in clinical samples, about half of our PSU cohort relapsed between baseline and follow-up, a rate comparable to what has been reported elsewhere . This made us focus our longitudinal results reporting on the main effects of time and to de-emphasize the reporting of time-by-smoking status interactions. Larger studies are needed to examine the potential effects of smoking status and gender on neurocognitive recovery during abstinence from substances. The study sample was drawn from treatment centers of the Veterans Affairs system in the San Francisco Bay Area and a community based healthcare provider,vertical grow system and the ethnic breakdown of the study groups was different.

Therefore, our sample may not be entirely representative of community-based substance use populations in general. Although preliminary, the within subject statistics are meaningful as they are more informative for assessing change over time than larger cross-sectional studies at various durations of abstinence. In addition, premorbid biological factors and other behavioral factors not assessed in this study may have influenced cross-sectional and longitudinal outcome measures. Nonetheless, our study is important and of clinical relevance in that it describes deficits in neurocognition and inhibitory control of detoxified PSU that are different from those in AUD, and that appear to recover during abstinence from substances, potentially as a function of smoking status. Our cross-sectional and longitudinal findings are valuable for improving current substance use rehabilitation programs. The higher impulsivity and reduced cognitive abilities of PSU compared to AUD, likely the result of long-term comorbid substance use, and the lack of improvements in learning and memory during abstinence indicate a potentially reduced ability of PSU to acquire new cognitive skills necessary for remediating maladaptive behavioral patterns that impede successful recovery. As such, PSU may require a post-detox treatment approach that accounts for these specific deficits relative to AUD. Our results show that PSU able to maintain abstinence for 4 months had less total lifetime years of cocaine use and performed better on cognitive efficiency, processing speed and visuospatial learning than those PSU not restudied ; these variables may therefore be valuable for predicting future abstinence or relapse in PSU. Additionally, and if confirmed in larger studies, our preliminary results on differential neurocognitive change in smoking and nonsmoking PSU may inform a treatment design that addresses the specific needs of these subgroups within this largely understudied population of substance users. Potentially, concurrent treatment of cigarette smoking in treatment-seeking PSU may also help improve long-term substance use outcomes, just as recently proposed for treatment seeking individuals with AUD.

Finally, our findings on neurocognitive improvement in PSU imply that cognitive deficits are to some extent a consequence of long-term substance use , which have the potential for remediation with abstinence. This information is of clinical relevance and of psychoeducational value for treatment providers and treatment-seeking PSU alike. Patients with obstructive sleep apnea experience apneic and hypopneic events that, when untreated, have detrimental cardiovascular and neurocognitive consequences. Under normal conditions, blood pressure and heart rate decrease during non–rapid eye movement sleep and increase commensurately upon waking. This is attributed to a decrease in sympathetic nervous system activation and a subsequent increase in cardiac vagal tone during sleep . The transient episodes of hypoxemia and hypercapnia caused by apneas or hypopneas, as well as arousals, result in an increase in cardiac output and heart rate that leads to sympathetically induced peripheral vasoconstriction that causes a marked increase in blood pressure. The result of this chronic sympathetic excitation and inflammation does not resolve upon waking, and over time, together with the loss of the normal nocturnal blood pressure dip, it can lead to pathophysiologic changes such as impaired vascular function and stiffness . This impairment in the untreated patient with moderate to severe OSA has been found to increase the risk of both acute coronary syndrome and sudden cardiac death . The increased sympathetic nervous activity, inflammation, and oxidative stress seen in OSA can lead to hypertension. The prevalence of hypertension in moderate to severe OSA ranges between 13% and 60%, and OSA is considered the most common cause of secondary hypertension . Arrhythmias can be common in patients with OSA, and the prevalence of atrial fibrillation is higher in these patients than in patients without OSA. In fact, severe sleep disordered breathing is associated with twofold to fourfold higher odds of having complex arrhythmias. In addition, untreated OSA has been associated with higher rates of failure to maintain sinus rhythm after cardio version or ablation therapy . Inflammation, atrial fibrillation, and atherosclerosis are all associated with OSA and overlap with risk factors for cerebrovascular disease. OSA may be frequently diagnosed after stroke, and it can be difficult to determine whether the condition is causal or resultant. Evidence suggests that OSA is associated with an increased risk of stroke in elderly patients, and untreated OSA after stroke increases mortality risk during 10-year follow-up . Another disease state affected by sleep apnea is heart failure. Both OSA and central sleep apnea are common in patients with acute and chronic systolic and diastolic heart failure.However, screening for sleep disordered breathing can be difficult because patients with OSA and heart failure often do not report excessive daytime sleepiness. This absent symptom raises challenges in diagnosis and treatment adherence for OSA . Untreated OSA can affect many cognitive domains, including learning, memory, attention, and executive functioning. Data suggest that OSA is linked with cognitive impairment and may advance cognitive decline or dementia . In addition, intermittent hypoxemia and sleep fragmentation have been linked to structural changes in the brain that may be responsible for cognitive impairment . Given the increased prevalence of obesity and the common nature of diagnoses such as hypertension, coronary artery disease, atrial fibrillation, heart failure, and neurocognitive impairment, healthcare providers should be cognizant of the hazards of untreated OSA .Substance use, misuse, and dependence contribute immensely to the global burden of disease. Their harms extend far beyond their corrosive effects on health, safety, and well being and additionally include those associated with healthcare expenditures, productivity losses, criminal justice involvement, and other negative effects on social welfare. The incidence and harms of substance use, misuse, and dependence involve multilevel explanatory factors.

DSI can be blocked by postsynaptic Ca2 buffers or initiated by activity restricted to the postsynaptic side, and likely involves the opening of voltage-gated Ca2 channels , or release from intracellular stores. Changes in postsynaptic GABAA receptor sensitivity have been excluded, since the response to iontophoretically applied GABA did not change, and DSI had no effect on the amplitude of miniature IPSCs. Despite the clearly postsynaptic site of initiation, numerous experiments demonstrated that DSI is expressed presynaptically, i.e., as a reduction in GABA release. With the use of minimal stimulation, DSI was found to increase failure rate, multi-quantal components were also eliminated, and components of IPSCs were differentially influenced . In the cerebellum, axonal branch point conduction failure was shown to play a role . Furthermore, DSI was reduced by 4-aminopyridine and veratridine, both acting on the presynaptic terminal . Direct evidence for an inhibitory G protein-mediated presynaptic action has been provided by Pitler and Alger , as they showed that DSI was pertussis toxin sensitive. Both laboratories hypothesized from the very beginning that they were dealing with a phenomenon that involves retrograde messengers. Llano et al. stated that “Ca2 rise in the Purkinje cell leads to the production of a lipid-soluble second messenger.” This was a remarkable prediction 10 years before the discovery that, indeed, the lipid-soluble endocannabinoids are these messengers,cannabis grow system although the earlier claim of a retrograde action of arachidonic acid in the presynaptic control of LTP made this assumption rather plausible at that time.

The quest for identifying the chemical nature of this retrograde messenger began with the discovery of DSI. The slow onset , the requirement of a lasting Ca2 rise, and the Ca2 buffer effects were all consistent with a hormone or peptide rather than classical vesicular neurotransmitter. Yet the first substance suggested by direct experimental evidence was glutamate. In the cerebellum, metabotropic glutamate receptor agonists, acting on presynaptic group II mGluRs, were shown to mimic and occlude DSI, whereas antagonists reduced it . Activation of adenylate cyclase by forskolin reduced DSI, which is consistent with the proposed reduction of cAMP levels by mGluR2/3 activation that is known to lead to a reduction of GABA release . In contrast, in the hippocampus, forskolin and group II or III mGluR ligands were without effect on DSI; however, group I agonists occluded, and antagonists reduced it . Pharmacology and the anatomical distribution of the receptors suggested that mGluR5 is likely to be involved in the reduction of GABA release , but it appeared to be confined to the somadendritic compartment of the neurons perisynaptically around glutamatergic contacts , which was difficult to reconcile with the hypothesis of glutamate being the retrograde signal molecule . The long duration of DSI is not due to the dynamics of the Ca2 transient, as it was the same in EGTA and BAPTA , but probably to the slow disappearance of the retrograde messenger molecule from the site of action around the presynaptic terminal. This again is inconsistent with glutamate being the messenger , since this transmitter is known to be rapidly taken up. The fast buffer BAPTA and the slow buffer EGTA reduced DSI to a similar degree, suggesting that the site of Ca2 entry and the site of calcium’s action in DSI induction are relatively far from each other . One possibility is that the target of incoming Ca2 may be an intracellular Ca2 store that is able to produce large Ca2 transients required for the release of the signal molecule. On the other hand, the selective N-type Ca2 channel blocker -conotoxin was able to block DSI , which, according to recent evidence , turned out to be an action on the presynaptic terminals that are sensitive to DSI and selectively express the N-type Ca2 channel.

These data suggest that Ca2 plays a dual role: it is involved in the initiation phase via Ca2 -induced Ca2 release from intracellular stores in the postsynaptic side as well as in the effector phase via N-type Ca2 channels on presynaptic terminals . Obviously, DSI-like phenomena can have a functional role in neuronal signaling only if they can be induced by physiologically occurring activity patterns. In cerebellar Purkinje cells, 100-ms depolarization was required for a detectable reduction in IPSCs , which, under physiological conditions, may correspond to a few climbing fiber-induced complex spikes . Thus a short train of climbing fiber-induced spikes is expected to lead to an increased excitability of the innervated Purkinje cell for tens of seconds. Initiation by very few spikes, occasionally even two if closely spaced, has been reported in the hippocampus. With 100 M BAPTA in the pipette, detectable DSI could be evoked already by depolarization as short as 25 ms, and half-maximal effect was produced by 187 ms, or by 109-ms depolarization in the absence of BAPTA . This suggests a lower threshold, but also a smaller magnitude and shorter time course of DSI compared with the cerebellum. The behavior-dependent electrical activity patterns in the hippocampus that may lead to DSI are discussed in section VD.Recent studies by Kreitzer and Regehr provided evidence that, at least in the cerebellum, excitatory synaptic transmission is also under the control of retrogradely acting signal molecules. Both parallel fiber and climbing fiber-evoked EPSCs were suppressed for tens of seconds by a 50- to 1,000-ms depolarization of the postsynaptic Purkinje cells from 60 to 0 mV. Due to the obvious similarity to DSI, this phenomenon has been termed depolarization-induced suppression of excitation . Paired-pulse experiments, showing that short term plasticity is affected by the depolarization paradigm for both parallel and climbing fiber responses, demonstrated that the site of expression of DSE is presynaptic and involves a reduction in the probability of transmitter release. BAPTA in the recording pipette completely abolishes DSE,cannabis grow lights providing evidence for the requirement of postsynaptic Ca2 rise to trigger the event.Earlier reports are consistent with the lack of DSE in the hippocampus, but a recent study using excessive depolarization for 5–10 s argues for its existence also in this brain region.

Whether the mechanisms of DSE are similar in the hippocampus and cerebellum is discussed in the following section.The discovery by Wilson and Nicoll , OhnoShosaku et al. , and Kreitzer and Regehr that DSI/DSE are mediated by endocannabinoids revealed that investigations in both the cannabinoid and DSI/DSE fields have been dealing accidentally with the same subject, i.e., the mechanism of retrograde synaptic signaling via endocannabinoids. Both receptor localization data and identification of the physiological actions of cannabinoids on synaptic transmission confirmed that cannabinoids act on presynaptic axons, reducing transmitter release , whereas endocannabinoids are most likely released from the postsynaptic neuron upon strong stimuli that give rise to large Ca2 transients. Thus the signal molecules, which turned out to be endocannabinoids, travel from the post- to the presynaptic site and thus enable neurons to influence the strength of their own synaptic inputs in an activity-dependent manner. This may be considered as a short definition of retrograde synaptic signaling and perhaps, at the same time, summarizes the function of the endocannabinoid system. However, before trying to correlate the findings of cannabinoid and DSI studies, one should be aware of the major limitations. There are numerous examples of mismatch in receptor/transmitter distribution in the brain; receptors can be found in locations where they hardly ever see their endogenous ligand. Nevertheless, these receptors readily participate in mediating the effects of its exogenous ligands, e.g., during pharmacotherapy. We are facing the same problems with the relative distribution of cannabinoid receptors versus endocannabinoid release sites both at the cellular and subcellular levels. In addition, the distance to which anandamide and 2-AG are able to diffuse is also an important question from the point of identifying the degree of mismatch. Thus correlation of the sites of action of cannabinoid drugs and the sites of expression of DSI should reveal the regional, cellular, and subcellular domains where receptor and endogenous ligand distributions match, i.e., where endocannabinoids are likely to have a functional role in synaptic signaling. Several lines of evidence have been provided that endocannabinoids represent the retrograde signal molecules that mediate DSI both in the hippocampus and cerebellum, as well as DSE in the cerebellum. Antagonists of CB1 receptors fully block and agonists occlude DSI and DSE, whereas DSI is absent in CB1 receptor knock-out animals . In these experiments either single cell or paired recording has been used, and retrograde synaptic signaling has been evoked by the same procedures as described in the original work of Alger’s and Marty’s groups . In addition, Wilson and Nicoll demonstrated that uncaging of Ca2 from a photolabile chelator induces DSI that was indistinguishable from that evoked by depolarization. Thus a large intracellular Ca2 rise is a necessary and sufficient element in the induction of the release of endocannabinoids. As expected from the membrane-permeant endocannabinoids, their release does not require vesicle fusion, since botulinum toxin delivered via the intracellular recording pipette did not affect DSI.

A further crucial question concerns the range to which the released endocannabinoids are able to diffuse. Recordings at room temperature from pyramidal cells at various distances from the depolarized neuron releasing the signal molecules revealed that it is only the adjacent cell, at a maximum distance of 20 m, to which endocannabinoids are able to diffuse in a sufficient concentration to evoke detectable DSI . However, a considerably greater endocannabinoid uptake and metabolism should be expected at physiological temperatures, which likely results in a decreased spread and a more focused action. Earlier data indicating the involvement of glutamate and mGluR receptors in DSI also needed clarification . Varma et al. demonstrated that enhancement of DSI by mGluR agonists could be blocked by antagonists of both group I mGluR and CB1 receptors, whereas the same mGluR agonists were without effect in CB1 receptor knock-out animals. This provides direct evidence that any mGluR effects on DSI published earlier were mediated by endocannabinoid signaling, and glutamate served here as a trigger for the release of endocannabinoids rather than as a retrograde signal molecule as thought earlier. These data were subsequently confirmed by paired recordings from cultured hippocampal neurons . In a recent paper, Maejima et al. demonstrated that mGluR1 activation induces DSE in Purkinje cells even without changing the intracellular Ca2 concentration. This suggests that, at least in the case of cerebellar Purkinje cells, two independent mechanisms may trigger endocannabinoid synthesis ; one involves a transient elevation of intracellular [Ca2], and the other is independent of intracellular [Ca2] and involves mGluR1 signaling. This may imply that, under normal physiological conditions, different induction mechanisms may evoke the release of different endocannabinoids. With the growing number of potential endocannabinoids , the question arises whether they are involved in distinct functions, i.e., by acting at different receptors and/or at specific types of synapses. This question represents one of the hot spots of current endocannabinoid research, and direct measurements of the different endocannabinoid compounds during retrograde signaling should provide an answer. They may induce branch-point failure, decrease action potential invasion of axon terminals, reduce Ca2 influx into the synaptic varicosities via N- or P/Q-type channels, or block the release machinery somewhere downstream from the Ca2 signal. Using Ca2 imaging of single climbing fibers provided evidence that DSE involves a reduction of presynaptic Ca2 influx, which has the same time course as the reduction of the EPSC. Branch-point failure was shown not to contribute to DSE, at least in the case of climbing fibers, as stimulation of the examined single axon evoked a uniform rise of Ca2 throughout its entire arbor. These findings are supported by the fact that cannabinoids are known to block N-type Ca2 channels in neuroblastoma cells and reduce synaptic transmission by inhibiting both N- and P/Q-type channels in neurons . Inhibition of the release machinery is unlikely to play a role, particularly in GABAergic transmission, since CB1 receptor activation has little if any effect on mIPSC frequency in the presence of tetrodotoxin and cadmium . Furthermore, CB1 receptors tend to be localized away from the release sites, having a high density even on preterminal axon segments, which also argues against this possibility . In the hippocampus, evidence has been provided that DSI likely involves a direct action of G proteins on voltage-dependent calcium channels.

This clear morphological finding is also supported by work with CB_x0005_ /_x0005_ mice, which suggests that an additional receptor, pharmacologically related, but molecularly distinct from the CB1, may mediate the cannabinoid modulation of glutamatergic transmission in the hippocampus . We will return to this hypothesis in section IVB1B.CB1 receptors are also present in several subcortical nuclei of the basal forebrain. Cells expressing moderate levels of this receptor are located mainly in the teniatecta, the lateral and medial septum, and the nuclei of the vertical and horizontal limbs of the diagonal band . Colocalization experiments show that CB1 receptors may be present in somatostatin-positive neurons of the lateral septum and in cholinergic cells in the medial septum and the nucleus basalis of Meynert .In keeping with the profound impact of cannabimimetic drugs on motor activity , in situ hybridization studies have invariably reported strong expression of CB1 mRNA in the striatum . Detailed analysis at the regional and cellular level uncovered a selective expression pattern in specific components of basal ganglia networks . In rodents, the highest density of CB1 mRNA is found in the dorsolateral portion of the striatum, where the transcript is primarily localized to GABAergic medium spiny cells, which constitute  90% of striatal neurons. In contrast, CB1 mRNA expression is rather low in two key output structures of the basal ganglia in the globus pallidus and in the substantia nigra. This is also true for the human basal ganglia, which lack however the dorsoventral gradient of mRNA expression seen in rodents . Although the globus pallidus and the substantia nigra pars reticulata contain little CB1 mRNA,cannabis plant growing cannabinoid binding is remarkably dense in these structures, implying that CB1 receptors may be mainly localized to the axons of striatonigral and striatopallidal GABAergic neurons .

Indeed, two colocalization studies have now established that CB1 mRNA is expressed by neurons that also contain high levels of the enzyme GAD65 and low levels of its higher molecular mass isoform, GAD67 . In a separate study, CB1 mRNA was found to be coexpressed with both preproenkephalin and prodynorphin , indicating that striatal projection neurons express CB1 receptors irrespectively of their specific target region . Interestingly, a small fraction of CB1-positive neurons contain neither preproenkephalin nor prodynorphin and express high levels of GAD67, which is typical of striatal interneurons. Thus, in addition to medium spiny projection cells, other neurons also may express CB1 mRNA. Because colocalization experiments revealed that CB1 is found neither in somatostatin-positive nor in cholinergic interneurons , the presumptive candidates are the remaining parvalbumin-containing cells . Indeed, Marsicano and Lutz demonstrated that 15% of the CB1-expressing neurons are positive for parvalbumin, providing direct evidence that striatal local-circuit neurons express CB1 receptors. It is important to reiterate that this expression pattern is opposite to the one found in cortical and amygdaloid structures, where parvalbumin-positive interneurons do not express CB1 receptors . While the above results are based on the presence of CB1 mRNA in striatal projection neurons and local-circuit cells, the cellular expression pattern has not been confirmed yet at the protein level, although the presence of the CB1 protein in striatal neurons has already been demonstrated by immunostaining .In situ hybridization studies have reported very low levels of CB1 mRNA expression in the thalamus . Subsequent work confirmed this finding both at the mRNA and at the protein level and extended it to the human brain . Neurons expressing moderate amounts of CB1 mRNA were observed in the habenula and the anterior dorsal part of thalamus, while CB1-immunoreactive cells were found in the reticular nucleus and zona incerta . Further studies are needed, however, to unambiguously identify these cells and solve remaining inconsistencies in the literature regarding their exact location in different nuclei. This need is further underscored by the finding that anterior and dorsal nuclei of the thalamus may express high levels of monoacylglycerol lipase, an intracellular serine hydrolase implicated in terminating the biological effects of the endocannabinoid, 2-AG .There is a coherent body of evidence indicating that the endocannabinoid system participates in the hypothalamic regulation of feeding and neuroendocrine function . Likewise, anatomical investigations agree in finding moderate levels of CB1 receptor expression in the ventromedial and anterior nuclei of the hypothalamus , while pharmacological experiments suggest that these receptors may be particularly well coupled to G proteins .

Importantly, a double-labeling study showed that CB1 receptors are colocalized with calretinin, a marker for glutamatergic neurons in select hypothalamic nuclei , but not with GAD65 . This suggests that glutamatergic, but not GABAergic, cells may express CB1 receptors in these nuclei. Other hypothalamic nuclei display very low levels of CB1 expression in a population of uniformly distributed cells. These nuclei include the medial and lateral preoptic nucleus, the magnocellular preoptic and hypothalamic nucleus, the premammilary nucleus and the lateral nucleus of the mammilary body, and the lateral hypothalamus . However, as elsewhere in the brain, there is still disagreement as to the precise identity and localization of hypothalamic CB1-expressing neurons, which will undoubtedly foster further scrutiny.The finding that noxious stimuli trigger anandamide release in the PAG, as assessed by in vivo microdialysis , implies that this midbrain structure may serve as a relay in the pain-processing circuit modulated by the endocannabinoids. Yet, a coherent description of the regional and cellular expression of CB1 receptors in the midbrain is still lacking. Although current data suggest that several midbrain nuclei may have very low to moderate expression of CB1 mRNA, they are in conflict regarding the exact identity of these nuclei . Immunostaining studies have shown that the superior colliculus contains CB1-positive neuronal cell bodies, but the identity of these cells was not determined . To be able to interpret the growing body of work on the analgesic and antihyperalgesic effects of cannabinoid agents, these morphological gaps need to be filled.Detailed morphological studies of the hindbrain are also rare. A notable exception is represented by the recent immunocy to chemical demonstration of CB1 receptors in the dorsal vagal complex of the ferret, which may be relevant to the autonomic and antiemetic effects of cannabinoid agonists . The exclusive presence of these receptors in local GABAergic interneurons, but not in preganglionic motor neurons , shows how this intriguing morphological leitmotif may recurrently be found at most levels of the neuraxis.CB1 receptor mRNA is highly abundant in the cerebellum . Owing to the well-determined circuitry of the cerebellar cortex, along with its laminar structure, the identification of neuronal elements expressing CB1 receptors in this region is relatively straightforward.

Strong expression levels are found in glutamatergic granule cells, but not in the GABAergic Purkinje cells . In the molecular layer, several large cells were also reported to express CB1,vertical grow rack which might belong to the basket and stellate cells . However, it is not known whether all cerebellar interneurons express CB1 or a subtype selectivity exists among them.One of the most important aspects of cannabinoids in terms of medicinal usefulness is their analgesic and antihyperalgesic effect at multiple stages of the pain-processing pathway, from high cognitive centers of the forebrain to the midbrain and down to peripheral tissues . The spinal cord, where cells expressing CB1 receptors have been extensively characterized, is obviously an integral component of this circuit. Most in situ hybridization and immuno staining studies agree that CB1 receptors are present in select neuronal populations of the spinal dorsal horn . In lamina II, GABAergic neurons expressing CB1 also contain nitric oxide synthase , a marker for a subset of spinal interneurons called islet cells . In addition, CB1- positive cells have also been found in lamina X, which surrounds the central canal of the spinal cord ; however, by using a different antibody, these cells could only be visualized after spinal transection.Primary sensory neurons in these ganglia are classified based on the selective expression of various neuropeptides [calcitonin gene-related peptide , substance P, somatostatin], or the responsiveness to neurotrophic factors [nerve growth factor , glial-derived growth factor , present in nociceptive neurons]. These cell-specific markers are rather heterogeneously colocalized with CB1 receptors. In a small population of dorsal root ganglion cells, CB1 receptors are present in CGRP and substance P-expressing neurons, but not in somatostatin-positive cells . This suggests that CB1 receptors may be expressed only by a subset of peptidergic nociceptive neurons, which represent 25% of all CB1- positive cells, whereas the remaining CB1-expressing cells may belong to other sub-populations of nociceptive or nonnociceptive neurons. Work in dorsal root ganglion cultures suggests that CB1 receptors colocalize with another nociceptor marker, the acid- and heat-sensitive vanilloid receptor 1 . Further triple immuno labeling experiments confirmed this observation and suggested that 25% of CB1-bearing neurons are nonnociceptive and that distinct types of nociceptive neurons express the receptor as well .

This highly heterogeneous distribution may contribute to explain the unprecedented analgesic effectiveness of cannabinoid agents, particularly in animal models of persistent pain of neuropathic origin .Based on the selective distribution of CB1 receptors in the CNS and their pervasive association with GABAergic interneurons, one would predict that the endocannabinoid system may play important and, possibly, unique roles in the local control of neuronal network activity. A growing body of functional evidence supports this prediction. For example, microdialysis experiments have found that anandamide is released in the striatum by activation of dopamine D2 receptor, where it may act as a short range mediator to counterbalance dopamine activity . Furthermore, an endocannabinoid substance, which remains unfortunately uncharacterized, has been recently identified as a key component in two related forms of trans-synaptic communication, known as depolarization induced suppression of inhibition and depolarization-induced suppression of excitation . In section V, we discuss how the endocannabinoid system may participate in these processes. But to do that, we first need to take a further step in the localization of CB1 receptors, down to the sub-cellular level. G protein-coupled receptors, such as the CB1, are embedded within the lipid bilayer of the plasma membrane. The membrane surface of a nerve cell can be subdivided into two functionally distinct spatial domains. The dendritic tree and cell body are equipped to receive synaptic contacts at specialized structures called active zones, where receptors for fast-acting neurotransmitters such as glutamate or GABA are concentrated. G protein coupled receptors are rarely associated with these structures; rather, a significant proportion of these receptors are found outside the synapse, within the so-called perisynaptic zone or even further away on the dendritic tree , where they can influence synaptic currents and neuronal excitability by triggering the formation of diffusible intracellular second messengers. Another group of G protein-coupled receptors is situated on axon terminals, where they are exquisitely poised to regulate the release of neurotransmitters, thereby controlling the final output of a neuron. Thus the question arises, in which neuronal surface domain are CB1 receptors localized? The most direct way to approach this question consists, when a receptor-specific antibody is available, in analyzing the sub-cellular distribution of the receptor by using electron microscopy. This approach can also provide a wealth of information on the structure and function of the synapse, such as the complement of neurotransmitters and additional membrane receptors present. Evidence from anatomical studies such as these, as well as functional experiments, indicates that CB1 receptors are predominantly found in axon terminal membranes, where they may be involved in the presynaptic regulation of neurotransmitter release.Indirect anatomical evidence for the localization of CB1 receptors on axon terminals was first provided by in situ hybridization and receptor binding experiments . These studies showed that, in the basal ganglia, CB1 receptor mRNA is almost exclusively localized to neurons within the striatum , whereas cannabinoid binding is strongest in the globus pallidus and the substantia nigra pars reticulata . This mismatch implies that CB1 receptors synthesized in the cell bodies of striatal projection neurons are transported to axon terminal fields in the pallidum and substantia nigra. In keeping with this hypothesis, ibotenic acid lesion of the rat striatum produces a marked loss of cannabinoid binding in these two regions .

First, motivated by the potential therapeutic applications of cannabis-like molecules, laboratories in academia and the pharmaceutical industry began to develop families of synthetic analogs of delta-9-THC. These agents exerted pharmacological effects that were qualitatively similar to those of delta-9- THC but displayed both greater potency and stereoselectivity. The latter feature cannot be reconciled with nonspecific membrane interactions, providing the first evidence that delta-9-THC exerts its effects by combining with a selective receptor. Second, as a result of these synthetic efforts, it became possible to explore directly the existence of cannabinoid receptors by using standard radioligand binding techniques. In 1988, Howlett and her co-workers described the presence of high-affinity binding sites for cannabinoid agents in brain membranes and showed that these sites are coupled to inhibition of adenylyl cyclase activity. Conclusively supporting these findings, in 1990 Matsuda et al. serendipitously came across a complementary DNA encoding for the first G protein-coupled cannabinoid receptor, now known as CB1. In heterologous expression systems, CB1 receptors were found to be functionally coupled to multiple intracellular signaling pathways, including inhibition of adenylyl cyclase activity,cannabis growing inhibition of voltage-activated calcium channels, and activation of potassium channels . In situ hybridization and immunohistochemical studies have demonstrated that CB1 receptors are abundantly expressed in discrete regions and cell types of the central nervous system but are also present at significant densities in a variety of peripheral organs and tissues .

The selective distribution of CB1 receptors in the CNS provides a clear anatomical correlate for the cognitive, affective, and motor effects of cannabimimetic drugs. The cloning and characterization of CB1 receptors left several important problems unsolved. Since antiquity, it has been known that the actions of Cannabis and delta-9-THC are not restricted to the CNS, but include effects on nonneural tissues such as reduction of inflammation, lowering of intraocular pressure associated with glaucoma, and relief of muscle spasms. Are these peripheral effects all produced by activation of CB1 receptors? An initial answer to this question was provided by the discovery of a second cannabinoid receptor exquisitely expressed in cells of immune origin . This receptor, called CB2, only shares 44% sequence identity with its brain counterpart, implying that the two sub-types diverged long ago in evolution. The intracellular coupling of the CB2 receptor resembles, however, that of the CB1 receptor; for example, in transfected cells, CB2 receptor activation is linked to the inhibition of adenylyl cyclase activity . The experience with opioid receptors and the enkephalins has accustomed scientists to the idea that whenever a receptor is present in the body, endogenous factor that activate this receptor also exist. Not surprisingly, therefore, as soon as cannabinoid receptors were described, a search began to identify their naturally occurring ligand. One way to tackle this problem was based on the premise that, like other neurotransmitters and neuromodulators, an endogenous cannabinoid substance should be released from brain tissue in a calcium dependent manner. Taking this route, Howlett and coworkers incubated rat brain slices in the presence of a calcium ionophore and determined whether the media from these incubations contained a factor that displaced the binding of labeled CP-55940, a cannabinoid agonist, to brain membranes. These studies demonstrated that a cannabinoid-like activity was indeed released from stimulated slices, but the minute amounts of this factor did not allow the elucidation of its chemical structure .

Devane, Mechoulam, and co-workers , at the Hebrew University in Jerusalem, adopted a different strategy. Reasoning that endogenous cannabinoids may be as hydrophobic as delta-9-THC, they subjected porcine brains to organic solvent extraction and fractionated the lipid extract by chromatographic techniques while measuring cannabinoid binding activity. This approach turned out to be highly successful, and the researchers were able to isolate a lipid cannabinoid-like component, which they characterized by mass spectrometry and nuclear magnetic resonance spectroscopy as the ethanolamide of arachidonic acid. They named this novel compound “anandamide” after the sanskrit “ananda,” inner bliss. The chemical synthesis of anandamide confirmed this structural identification and allowed the characterization of its pharmacological properties . In vitro and in vivo tests showed a great similarity of actions between anandamide and cannabinoid drugs. Anandamide reduced the electrogenic contraction of mouse vas deferens and closely mimicked the behavioral responses produced by delta-9-THC in vivo; in the rat, the compound was found to produce analgesia, hypothermia, and hypomotility. However, these effects may not be exclusively due to cannabinoid receptor activation, as anandamide is readily metabolized to arachidonic acid, which can be converted in turn to a variety of biologically active eicosanoid compounds. Subsequent studies demonstrated that anandamide is released from brain neurons in an activity-dependent manner and elucidated the unique biochemical routes of anandamide formation and inactivation in the CNS . Thus anandamide fulfills all key criteria that define an endogenous cannabinoid substance. In their 1992 study, Devane, Mechoulam, and coworkers reported that several lipid fractions from the rat brain contained cannabinoid-binding activity, in addition to anandamide’s. In characterizing these fractions, they discovered that some of them were composed of polyunsaturated fatty acid ethanolamides similar to anandamide , but others were instead constituted of a distinct lipid component, sn-2-arachidonoyl-glycerol . Sugiura et al. arrived independently to the same conclusion.

That polyunsaturated fatty acid ethanolamides should mimic anandamide, to which they are structurally very similar, does not come as a great surprise. Moreover, the pharmacological properties of these fatty acid ethanolamides, essentially indistinguishable from those of anandamide, and their scarcity in brain relegate them, at least for the moment, to a position secondary to anandamide’s. We cannot say the same for 2-AG. This lipid, considered until now a mere intermediate in glycerophospholipid turnover , is present in the brain at concentrations that are 170-fold greater than those of anandamide and possesses two pharmacological properties that make it crucially different from the latter: it binds to both CB1 and CB2 cannabinoid receptors with similar affinities, and it activates CB1 receptors as a full agonist, whereas anandamide acts as a partial agonist.We have learned much over the past 10 years on the behavioral effects of these molecules, on how these lipid mediators are produced physiologically, and on the functional roles that they may serve. A major step was the discovery that depolarization-induced suppression of inhibition , a type of short-term synaptic plasticity originally discovered in the cerebellum and the hippocampus ,cannabis grow equipment is mediated by endocannabinoids . This discovery allowed the results of over a decade of research on retrograde synaptic signaling in these networks to be considered as functional characteristics of endocannabinoid signaling. The substrate of retrograde signaling and DSI is the predominantly presynaptic distribution of CB1 receptors on axon terminals in the hippocampus , as well as throughout the brain, where activation of CB1 by endocannabinoids can efficiently veto neurotransmitter release in many distinct types of synapses . The conditions of synthesis, release, distance of diffusion, duration of effect, and site of action were all extensively characterized for the mediator of DSI that turned out to be an endocannabinoid . The fact that neurons are able to control the efficacy of their own synaptic input in an activity-dependent manner is functionally very important, since this mechanism may sub-serve several functions in information processing by neuronal networks from temporal coding and oscillations to group selection and the fine tuning of signal-to-noise ratio.

The crucial involvement of endocannabinoids in these functions just began to emerge from recent studies, which are reviewed in section V. Due to the exceptionally rapid expansion of this field in recent years , we decided to focus the present review on questions related to the composition of the endocannabinoid system and its physiological roles in controlling brain activity at the regional and cellular levels as synaptic signal molecules. We did not aim to provide detailed accounts of studies dealing with other, similarly important, aspects of cannabinoid research, which have been dealt with in excellent recent reviews, e.g., about the relation of the endocannabinoid system to pain modulation , the immune system , neuroprotection , and addiction . The final message of the present review is that to understand the possible physiological functions of the endogenous cannabinoids, their roles in normal and pathological brain activity, pharmacological agents targeting the cascade of anandamide and 2-AG formation, release, uptake, and degradation will have to be developed. Such drugs, which undoubtedly will become invaluable research tools to study the potential functions listed above, may also provide novel therapeutic approaches to diseases whose clinical, biochemical, and pharmacological features suggest a link with the endogenous cannabinoid system.A basic principle that has emerged from the last two decades of research on cellular signaling is that simple phospholipids such as phosphatidylcholine or phosphatidylinositol should be regarded not only as structural components of the cell membrane, but also as precursors for transmembrane signaling molecules. Intracellular second messengers like 1,2-diacylglycerol and ceramide are familiar examples of this concept. Along with their intracellular roles, however, lipid compounds may also serve important functions in the exchange of information between cells. Indeed, biochemical mechanisms analogous to those involved in the generation of DAG or ceramide give rise to biologically active lipids that leave their cell of origin to activate G protein-coupled receptors located on the surface of neighboring cells. Traditionally overshadowed by amino acid, amine, and peptide transmitters, biologically active lipids are now emerging as essential mediators of cell-to-cell communication within the CNS, where G protein-coupled receptors for multiple families of such compounds, including lysophosphatidic acid and eicosanoids, have been identified . In this section, we discuss the biochemical properties of endogenous lipids that activate brain cannabinoid receptors. These compounds share two common structural motifs: a polyunsaturated fatty acid moiety and a polar head group consisting of ethanolamine or glycerol . Because of these features, endocannabinoid substances seemingly resemble the eicosanoids, ubiquitous bio-active lipids generated through the enzymatic oxygenation of arachidonic acid. However, the endocannabinoids are clearly distinguished from the eicosanoids by their different bio-synthetic routes, which do not involve oxidative metabolism. The two best characterized endocannabinoids, anandamide and 2-AG , may be produced instead through cleavage of phospholipid precursors present in the membranes of neurons, glia, and other cells. In the following sections, we will first focus on the biochemical pathways that lead to the formation of endocannabinoids in neurons and then turn to the mechanism by which these compounds are deactivated.Anandamide formation via energy-independent condensation of arachidonic acid and ethanolamine was described in brain tissue homogenates soon after the discovery of anandamide and was attributed to an enzymatic activity that was termed “anandamide synthase” . Subsequent work has demonstrated, however, that this reaction is in fact catalyzed by fatty acid amide hydrolase , the primary enzyme of anandamide hydrolysis, acting in reverse . Since FAAH requires high concentrations of arachidonate and ethanolamine to synthesize anandamide, higher than those normally found in cells, this enzyme is unlikely to play a role in the physiological formation of anandamide . Another model for anandamide biosynthesis is illustrated schematically in Figure 2. According to this model, anandamide may be produced via hydrolysis of the phospholipid precursor N-arachidonoyl phosphatidylethanolamine , catalyzed by a phospholipase D -type activity . The precursor consumed in this reaction may be resynthesized by a separate enzyme activity, N-acyltransferase , which may transfer an arachidonate group from the sn-1 glycerol ester position of phospholipids to the primary amino group of PE . The validity of this model was initially questioned, because previous studies had failed to detect N-arachidonoyl PE in mammalian tissues . More recent chromatographic and mass spectrometric analyses have unambiguously shown, however, that N-arachidonyl PE is present in brain and other tissues, where it may serve as a physiological precursor for anandamide . Although biochemically distinct, anandamide formation and N-arachidonoyl PE synthesis are thought to proceed in parallel. Both reactions may be initiated by intracellular Ca2 rises and/or by activation of neurotransmitter receptors . For example, administration of dopamine D2-receptor agonists to rats in vivo causes a profound stimulation of anandamide release in the striatum , which is likely mediated by de novo anandamide synthesis . Unfortunately, the two key enzyme activities responsible for these reactions, PLD and NAT, have only been partially characterized, and their molecular properties are still unknown .

Chronic inflammation and immune activation potentially predispose PLWH to greater risk for premature and accentuated aging, making prevention and treatment of comorbidities an important component of HIV-related care . Specifically, the three most common comorbidities for PLWH are hypertension, hyperlipidemia, and endocrine disease , which are all components of metabolic syndrome . MetS is conceptualized as a constellation of interrelated metabolic risk factors associated with an increased risk of developing cardiovascular disease : Expert Panel on Detection and Treatment of High Blood Cholesterol in Adults 2002. MetS is defined differently by various organizations, but generally includes visceral obesity, hypertension, endothelial dysfunction, atherogenic dyslipidemia, and insulin resistance . MetS is common in the USA, with approximately 34–35% of adults aged 18 years or older in the general population falling within the clinical criteria, and greater prevalence of MetS is observed with increasing age . Furthermore, MetS has also been consistently linked to neurocognitive dysfunction and brain abnormalities . Among PLWH, MetS is highly prevalent and there is evidence that its impact on neurocognitive impairment might be even more notable among PLWH than in the general population , possibly because of the impact of MetS on blood–brain barrier integrity and systemic inflammation . Aside from MetS, neurocognitive impairment is prevalent in HIV, occurring in approximately 40% of PLWH . HIV infiltrates the central nervous system early in the course of disease, resulting in neurocognitive impairment, which is often mild to moderate in severity in the era of combination ART. Another manifestation of CNS dysfunction in HIV are neurobehavioral disturbances or changes in daily behavior indicative of problems with motivation, initiating and organizing behavioral responses,cannabis drainage system and regulating emotional and behavioral responses in appropriate contexts .

These neurobehavioral disturbances have been empirically linked to frontal-subcortical dysregulation and central nervous system disease and may be present in the absence of neurocognitive impairment . Previous research has shown a consistent link between HIV serostatus and increased neurobehavioral disturbances, particularly increased apathy , disinhibition , and executive dysfunction . However, no studies have investigated the role of MetS on neurobehavioral disturbances associated with HIV. The main purpose of the present study was to examine the association between MetS and neurobehavioral disturbances in the context of HIV disease. Given prior findings by Yu et al. , we hypothesized that there would be an interaction between HIV serostatus and MetS on neurobehavioral disturbances, such that the association between MetS and neurobehavioral disturbances would be stronger among PLWH than among a HIV-uninfected comparison group, even after controlling for significant demographic and psychiatric characteristics and neurocognitive impairment.Participants included 215 adults enrolled in the baseline visit of the MultiDimensional Successful Aging among HIV-Infected Adults study conducted at the University of California San Diego HIV Neurobehavioral Research Program and the UCSD Sam and Rose Stein Institute for Research on Aging from 2013 to 2015 . Inclusion criteria included being between the ages of 36–65 years old, being fluent in English, and having the ability to provide informed consent. Exclusion criteria included the presence of a neurologic condition other than HIV known to impact cognitive functioning , diagnosis of a psychotic condition that could impact neurocognitive test performance , and having a positive urine toxicology screen on day of testing for an illicit substance other than cannabis.In order to be included in present analyses, participants had to have data available on the main variables of interest, i.e., MetS and neurobehavioral disturbances.

The UCSD Institutional Review Board approved the study, and all participants provided written informed consent and were compensated for participating. Participants self-reported demographic characteristics and completed a comprehensive neuropsychiatric and neuromedical evaluation in individual sessions conducted by trained study staff.Neurobehavioral disturbances were assessed via the 46-item self-report version of the Frontal Systems Behavior Scale . Participants provide retrospective ratings of how often they experience specific neurobehavioral symptoms “before the illness or injury” and “at the present time” using a Likert-type scale ranging from 1 to 5 . The scale yields T-scores adjusted for demographics for the entire scale and three sub-scales measuring apathy, disinhibition, and executive dysfunction. T-scores have a mean of 50 and a standard deviation of 10, and higher T-scores indicate greater reports of neurobehavioral disturbances, with a Tscore of 65 and higher suggesting clinical neurobehavioral disturbance. Previous studies have reported good internal reliability and consistent factor structure of the FrSBe sub-scales . Consistent with previous research present time FrSBe T-scores were used as the outcome of interest for the current study.All statistical analyses were conducted using SPSS 24 and JMP 11.0.0 and considered statistically significant at p < .05. Descriptive statistics were computed for FrsBe scores, MetS, demographic and psychiatric characteristics, neurocognitive impairment, and HIV disease characteristics. Group comparisons by HIV serostatus on demographic and psychiatric characteristics and neurocognitive impairment were conducted . HIV serostatus and MetS group differences on FrsBe scores were also conducted via independent sample t tests. In order to examine the potential interactive effect of HIV and MetS on neurobehavioral disturbances, we conducted a series of linear regression models with terms for HIV serostatus, MetS, and their interaction on each FrSBe subscale. In cases where the interaction term was not significant,it was removed from the model. The independent and unique effect of HIV serostatus and MetS on each FrSBe subscale was then assessed by evaluating linear regression models that included terms for HIV serostatus, MetS,indoor cannabis grow system and relevant covariates. In order to adjust for relevant covariates while also obtaining a parsimonious model for each FrSBe subscale, we followed these steps: identified sociodemographic and psychiatric variables in Table 1 that were associated with each FrSBe subscale at p < .10 via a series of univariate regression models; entered variables identified in Step 1 of the linear regression models and applied a stepwise backwards selection method based on Akaike information criterion to obtain a parsimonious model for each FrSBe subscale.

Age, sex, and education were not considered as possible covariates as FrSBe T-scores are adjusted for those demographic factors.MetS disproportionally impacts neurocognitive impairment, a marker of central nervous system dysfunction, among PLHIV compared to HIV-uninfected persons . Present findings revealed that similar interactive effects of HIV serostatus and MetS were not observed in the present sample on other behavioral manifestations associated with central nervous system dysfunction, i.e., neurobehavioral disturbances . Instead, there was an additive effect such that being HIV seropositive and having a clinical identification of MetS were independently associated with greater apathy and executive dysfunction, and there was a significant effect of HIV serostatus only on disinhibition. The presence of greater difficulties with neurobehavioral disturbances in PLWH compared to the HIV-uninfected comparison group aligns with previous research . When looking at the frontal subcortical circuits that may be affected, neuroimaging research has documented links between damage to the anterior cingulate cortex, dorsolateral prefrontal cortex, and orbital frontal cortex with difficulties with apathy, executive dysfunction, and disinhibition, respectively . Further, HIV-associated structural changes in both gray and white matter corresponding to these same areas have been documented . These cross-sectional findings inform the hypothesis that neurobehavioral disturbances seen in PLWH are associated with disease-related volume reduction and over-activation in comparison to HIV-uninfected comparison participants. These findings are of great interest not only from understanding how HIV affects the brain but also everyday functioning. For example, previous studies have shown associations between apathy and IADL decline, disability, and quality of life ; disinhibition, impulsivity, and risk-taking behaviors ; and executive dysfunction and ART medication adherence and employment status in PLWH. Further research is needed to understand association directionality and how these associations progress with age over time. While MetS and the related risk factors have been shown to be an important and unique predictor of neurocognitive change and decline in the general population , little is known on how MetS is related to neurobehavioral disturbances. Previous research has shown that the presence of MetS is associated with increased odds of presenting with higher white matter hyperintensity volumes and characterized by an anterior-posterior pattern of deterioration, such that greater deterioration is observed in the frontal lobe structures . Although not fully understood, several possible hypotheses on the mechanisms underlying the association between MetS and central nervous system dysfunction have been proposed, such as neuroinflammation, oxidative stress, abnormal brain lipid metabolism, altered cerebral hemodynamics, regional hypoperfusion, impaired cerebrovascular reactivity, and small vessel disease . While the current findings are based on self-report, greater difficulties with apathy and executive dysfunction parallels the known associations between MetS and brain structure and function. Specifically, executive function, typically measured by neuropsychology test performance, appears to be particularly sensitive to the neurologic impact of MetS .

Examination of individual components of MetS indicates that insulin resistance , diabetes mellitus , obesity , and hypertension have been linked to impaired performance on executive function tasks. Interestingly, HIV serostatus and MetS were independently associated with increased neurobehavioral disturbances within the same model, but there was no evidence of a statistical interaction. This is somewhat inconsistent with findings from Yu et al. who found evidence of an interaction on neurocognitive performance, such that association between MetS and neurocognitive impairment was stronger among PLWH than an HIV-uninfected comparison group. Yu et al. included a similar sample to ours, indicating that differences in participants’ characteristics are unlikely to explain differential findings across studies. At least part of the reason for the apparent inconsistency might be that while neurocognitive impairment and neurobehavioral disturbances are both indicative of underlying central nervous system dysfunction, there is evidence that they can exist in isolation from one another . Neurobehavioral disturbances are usually associated with dysfunction to frontal-subcortical circuitry, while our index of global neurocognitive deficits includes various neurocognitive domains which are mediated by a variety of brain structures. Furthermore, prior findings indicate that the impact of MetS on neurocognition among PLWH is most notable in the domains of learning and motor functioning, with smaller effects on executive function . Additionally, differences in HIV and MetS findings with neurobehavioral disturbances could allude to differential impacts on frontal-subcortical circuitry as there are both shared and segregated components to their structure that can only be better investigated and understood using neuroimaging and longitudinal design.Given the high prevalence of MetS and related negative health outcomes, efforts to prevent, treat, and possibly reverse the effects of MetS in middle-aged and older adults is of the greatest public health interest .Additionally, increasing daily physical exercise and reducing physical inactivity in combination with a healthy diet has also shown to be effective in reducing metabolic risk and reversing MetS components . These lifestyle modifications may be particularly important for PLWH as previous research has shown varying levels of sedentary leisure time and physical inactivity . For example, Moore et al. showed using ecological momentary assessment in a sample of middle-aged PLWH that 32% of their time was spent engaging in passive leisure activity compared to only 5% of their time which was spent engaging in physical exercise. Given the links between neurocognition and physical activity in PLWH , more research is needed to see if interventions designed to increase physical activity for an example and improve diet may further reverse MetS components as well as the related-cognitive consequences.While we have discussed findings primarily in the direction of HIV and MetS impacting neurobehavioral disturbances, it is important to consider that the directionality of the findings cannot be ascertained in the context of the present cross-sectional analyses. It might be the case that pre-existing neurobehavioral disturbances resulted in persons being more likely to acquire HIV and/or MetS. Additionally, given the strong association between certain ART regimens and lipid and glucose metabolism in PLWH and the high prevalence of these drug class types in our sample, further longitudinal investigation of lifetime and current ART regimens may provide more detail on the associations between HIV, MetS, and neurobehavioral disturbances. Another limitation included that neurobehavioral disturbances were based on self-report rather than informant report, observation, or performance-based tasks. While self-report might provide insight into daily behavior that may not be adequately captured or assessed with laboratory or clinic testing, self-report may also be confounded with bias, lack of awareness, and stigma of disease.

Vaping is a term relating to heating a liquid and inhaling the “vapor” produced. Vaping first became popular as a means of inhaling tobacco without combusting it. Electronic cigarettes consisting of a battery, heating coil, and liquid reservoir usually containing nicotine were developed and went through several “generations” of modification. The third generation consists normally of a vape pen, holder containing the battery and heating oil, and a liquid cartridge with or without nicotine. The liquid consists of glycerin and ethylene glycol typically in amounts of 30–70%. A third liquid is also included at about 1/3 of the total amount. Some terpenes for taste and odor and nicotine may also be included. The vaper heats the cartridge for a few seconds , inhales the aerosol and exhales it into the room or ambient air. Many studies of secondhand exposure to e-cigarette aerosol have been published.Recently, vaporization of marijuana has emerged as a popular method of delivering marijuana. This method heats marijuana liquid to the point of vaporization, avoiding combustion.Shortly after this method was introduced, Earley wine and Van Dam reported on four subjects who smoked marijuana and agreed to switch to vaping for a short period. All four refused to return to smoking marijuana at the end of the experiment. Eight years later, these early adopters of vaping have been joined by millions of persons worldwide. For example, 3.8%, 10.5%, and 13.8% of about 25 million US high school students reported vaping marijuana in 2016, 2017, and 2018, respectively . Abrams et al. showed that vaping marijuana produced similar levels of THC in blood as smoking marijuana cigarettes,hydro tray without the increase in carbon monoxide in exhaled breath associated with combustion.

Essentially all passive exposure to marijuana smoke from vaping is from exhaled breath since there is no side stream smoke. The aerosol emerging from exhaled breath will be different in many respects from the inhaled aerosol, due to lung deposition, humidification, growth, and coagulation, so it is important to test exposure under real-world conditions using human vapers. One recent study has focused on indoor air concentrations of particles due to various indoor sources, including both marijuana and tobacco smokers . This study included 193 persons, of whom about 22% and 15% smoked tobacco and marijuana, respectively. It may be the first study to look at passive exposure to marijuana from human smokers in their own homes. The authors found that nonsmokers exposed to persons smoking either tobacco or marijuana cigarettes had roughly twice the exposure to fine particles as non-exposed nonsmokers. The Dylos monitors used in this study were not calibrated by comparison to gravimetric levels, so the investigators could not estimate PM2.5 exposures or source strengths. Exposure models depend on the vaping “topography” including frequency of use, depth or time of inhalation, and time retained in the lungs. McClure et al. studied 20 heavy users who were allowed to smoke marijuana cigarettes freely over 4 days. On average, they smoked 12 cigarettes per 9-h day, taking 13 puffs from each cigarette. The volume per puff ranged from 51 to 61 ml. A second study of 98 Dutch adolescents found that they smoked an average of 2.5 joints per day of use, and 21 days of use per month . Another required parameter in an exposure model is the air exchange rate. Chan et al. used leakage information to show that rates in the US vary in log-normal fashion from about 0.1 to 2 h-1. The rate is affected by the indoor-outdoor temperature difference and by wind direction and speed .

One of the strongest effects on air exchange rates is window-opening behavior . The volume of the home is another necessary parameter, statistics for which can be obtained for the US from the US Census Bureau . Although these previous studies are useful in developing exposure models, we believe that no studies have sufficiently characterized the two crucial ingredients of such models: source strengths and decay rates of real-world aerosols from vaping marijuana liquids by human subjects. In this study, we use more than 100 controlled experiments involving human vapers in rooms within inhabited homes to provide information on source strengths and decay rates, from which models of exposure can be built. The main objective of the study was to measure, under real-world conditions, the two main parameters affecting secondhand PM2.5 exposure to marijuana aerosol from vaping: the source strength and removal rate from the air. The source strength is the mass of PM2.5 emitted and is generalizable to other locations and situations. The removal rate for nonvolatile particles not subject to coagulation is the deposition rate k . In the case of vaping marijuana, the deposition rate may be augmented by evaporation, so the removal rate = k + evaporation rate . In carrying out this objective, we evaluated the calibration factors for the 3 p.m. instruments measuring vaping aerosol. For the Side Pak and Piezo balance monitors, the CFs were determined directly using gravimetric techniques . Since the Purple Air monitors in this study were collocated with the Side Pak monitors, the CF for them was determined by direct comparison with the Side Pak readings. A secondary objective was to compare the performance of a low-cost monitor to research-grade instruments . If the low-cost monitor performed sufficiently well, it could be adopted in future studies of exposure. It offers the opportunity of a much broader sample of homes and participants. It is able to monitor continuously with no maintenance. In the case of Purple Air, there is also an existing network on the internet that can be sampled at will by a researcher.

Three main particle monitor types were used in the study: an optical monitor with a PM2.5 impactor ; a monitor employing a piezoelectric crystal , also with a PM2.5 impactor; and a low-cost optical monitor providing estimates of PM1, PM2.5 and PM10 . The Piezobalance is manufactured by Kanomax, Inc. Japan, and has previously been licensed for sale in the US by TSI Inc., Shoreview, MN. Piezobalances used in this study included models from both Kanomax USA Inc. , and TSI, Model 8510. For the Piezobalances used in this study, a special connector has been added by the factory, which allows the 1-min average frequency counts to be output to a computer where they can be logged. The instrument employs a vibrating quartz crystal exposed to a steady flow rate that has passed through a PM2.5 impactor. As the exposed crystal collects particles, its frequency changes due to the piezoelectric principle,planting table and within a certain frequency range the change in frequency is proportional to the amount of material collected on the exposed crystal. The frequency change during each measured time interval is then multiplied by the factory-set calibration factor to give an estimate of the amount of mass collected during the time interval. The SidePak is an optical particle monitor. It uses a laser to sense particles as they pass through a chamber. The scattered light is collected and calculated as a volume determined by applying Mie scattering formulae. The SidePak is calibrated at the factory using ISO 12103-1 Test Dust . As with all optical monitors, it is recommended that the particular aerosol mixture being studied be analyzed using gravimetric methods, so that a calibration factor can be determined for that aerosol. For example, the calibration factor for the SidePak has been found to be about 0.32 for tobacco smoke . We have been able to determine a calibration factor for the marijuana aerosol produced by vaping . The PurpleAir instruments use a laser of ~650 nm wavelength to sort particles into one of six size categories . There are two lasers in each monitor, providing the opportunity to detect departures from normal operation and calculate internal precision. The monitors have a small inaudible fan to counter “starvation” of air at the face of the monitor. They operate off line current and have no battery. Every 80 s they upload observations directly to the Web.

The monitors provide the number per deciliter of particles in each of the six categories. Also, they provide two data series, identified as CF1 and CF ATM for PM1, PM2.5, and PM10. The manufacturer of the sensor is a Chinese company . The company does not provide details on the calibration aerosol used, such as the density, or any correction factors employed in calculating PM1, PM2.5 or PM10. Therefore, we chose not to use the CF1 or CF ATM data series provided by Plan tower. Instead we adopted a standard method for determining PM2.5 from the particle numbers provided for the three size categories up to 2.5 μm. We chose an intermediate particle diameter within each category to represent all particles in the category, calculated the resulting particle volume, and determined a reference mass by adopting a density of 1 g cm-3. This approach succeeded in improving the limit of detection from about 2 μg/m3 using the CF1 or ATM to ~1 μg/m3 . The calibration factors for the Piezobalance and SidePak response to vaping marijuana were studied using gravimetric procedures by Zhao et al. . The Piezobalance CF was 0.97 . The SidePak CF was 0.44 . A carbon monoxide monitor was employed to estimate the air exchange rate of each of the two experimental rooms. A known amount of carbon monoxide was released using a flow rate regulator attached to a 107-liter cylinder containing 10% CO . The air exchange rate was determined as the negative slope of the background-corrected logarithm of the CO concentration. A correction was made for the temperature based on observations of the variation of the Langan monitor readings with temperature. Tests were carried out between May 21, 2018 and May 25, 2019. Each test was conducted in a room in a home. Two rooms were used, one in Santa Rosa and one in Redwood City . Rooms were sealed off from the remainder of the home. The HVAC system was off and floor registers sealed. Usually no fan was employed. In some experiments, one or two table fans were employed to test the effect of a fan on measured decay rates. Two Piezobalances, two PurpleAir monitors, and 2–3 SidePaks were employed for each experiment. They were situated at two well-separated locations at heights between 0.9 and 1 m. A single Langan electrochemical device was set at a central location in the room to monitor CO. CO was released using the cylinder discussed above. A target peak concentration above 5 ppm was set. Background PM2.5 concentrations were collected for 5–10 min before the experimenter took one or more puffs of the heated marijuana oil. A battery operated device was used to vaporize marijuana oil from a 500 ml cartridge . Oils with different CBD/THC ratios were tested: 18:1 ; 8:1 ; 7:1 ; and 2:1 . The amount of CBD and THC was listed for each cartridge. For example, the 8:1 CBD/THC ratio included 336 mg CBD and 40 mg THC. The missing 124 ml liquid was not identified. The other formulations also included about 2/3 marijuana liquid and 1/3 unidentified liquid. Only one person at each location was the vaper. He sat or stood at a location roughly equidistant from the two locations for the instruments. After completing the protocol for heating the cartridge , the experimenter left the room. Two protocols for heating the cartridges were adopted to study the effect of different heating times on the amount of vapor produced. Protocol I consisted of heating the oil for 3 s by pressing the power button on the vaping pen and then inhaling for three additional seconds while still pressing the power button. Protocol II involved heating for 12 s before the 3-s inhale, a total of 15 s of heating compared to 6 s in Protocol I. Source strengths were calculated as follows. Since the initial peaks registered by the monitors occurred under conditions of poorly-mixed air, the true estimated peak concentration assuming perfect mixing was determined by calculating decay rates after good mixing was attained . The line of best fit could then be extended “backward” in time to the time of the puff.

Such risk assessment approaches can and have been applied to environmental odors. Unlike risk assessment of toxic chemicals, odor studies can include human panels. Risk assessment is directly applicable to the investigation of health risks from odors .The objective of this thesis is to conduct a critical literature evaluation, supplemented with interviews of odor experts and investigators, of scientific methods to investigate environmental odors and assess their health impacts. The overall aim is to improve the state of knowledge of a complex, multidisciplinary topic. In an effort to further advance the field of odor investigation, advice and concepts from the overlapping field of risk assessment will be applied, when appropriate. A review of the health impacts of environmental odors, which is information frequently requested by the public during odor investigations, concludes the review. The focus of this thesis is on nuisance odor investigations of existing facilities rather than predictive modeling used for permits of new or modified facilities. Much has already been written and reviewed on such predictive dispersion models and odor mapping . While such modeling is the most common approach, other approaches include maximum separation distance, maximum emission, plant grow table maximum annoyance and best technology requirements . It is likely that, in large part, the poor track record of these approaches has led to the need for nuisance odor investigations.

Indoor sources of odors are also beyond the scope of this thesis, as are occupational exposures to odors, except for the final chapter on health effects where the information is highly valuable for public health. Odors from emergencies and accidents are also not central to the thesis. The thesis is organized around increasing use of risk assessment to inform nuisance odor investigation. Chapter 2 on regulations and techniques used around the world to investigate nuisance odor complaints only employs some of the basic risk assessment concepts. Chapter 3 on odor exposure assessment falls within the risk assessment framework. Chapter 4 on the health risks from environmental odors is entirely consistent with and reliant upon risk assessment. Each chapter also identifies best practices, gaps in current knowledge and provides suggestions on how such gaps could be filled. Chapters 2 to 4 are presented as journal-ready publications. Chapter 5 ends the thesis with concluding remarks. Fugitive emissions from such industries can lead to odor complaints due to the extremely low sensory thresholds. Further, upsets and their associated larger releases are especially problematic at oil refineries , where it can take a long time to fix the problem. Historically, odors have been regulated under public nuisance law or when permitting a new or modified facility by conducting dispersion modeling within a theoretical odor impact assessment. The latter approach is outside the scope of this paper because it has been reviewed elsewhere . The use of dispersion modeling within a theoretical odor impact assessment has only achieved partial success as evidenced by the ongoing volume of odor complaints. Odor as regulated under public nuisance law for existing facilities, rather than proposed new or modified facilities, is the focus of this paper. Public nuisance law originated in British Common Law. Such law distinguishes between “private nuisance” and “public nuisance” . Almost always, nuisance odor complaints fall under the latter category rather than individual rights .

To address nuisance odor complaints, regulators may set subjective or objective standards. Based on population-based health studies of toxic air pollutants, objective standards have been set for pollutants. Odors, however, are multidimensional and require difficult-to-enforce standards. Unsurprisingly, the setting of odor standards varies greatly throughout the world and depends heavily on the industrial sector, type of activity generating the odor and level of public outcry. Applying public nuisance law to odors has been criticized : “A direct measure of annoyance is typically made by an inspector or authorized officer of the state, and if there are no complaints there is by definition, no problem. The inherent subjectivity built into the approach is a problem, as is its susceptibility to political influence or community pressure. It lacks continuity of regulation for both the community and the industry concerned and does not offer a ‘target’ for the design and management of odor control systems.” As an alternative, odor intensity limits have been set in some jurisdictions. Such limits remain sensory-based, bringing with them the inherent variability in human responses to odors, and typically are set at a certain number of volumes of odorless air required to dilute one volume of sample air until the odor is no longer detected by 50% of panelists . Dynamic dilution olfactometry, which requires a dilution instrument and evaluation by a trained panel, is typically used to set this limit.A team of Brazilian and Australian researchers published a recent, comprehensive review of odor regulations from around the world . Their survey of 28 countries primarily focused on the dispersion modeling and criteria applied to new and modified facilities emitting odorous gases. Such regulations typically set limits to emissions and setback distances based on the theoretical calculations of odor impact. They often also require that “best available technology” be implemented.

This regulatory approach is not the focus of this paper; nonetheless, the authors also covered, to a lesser degree, regulations and methods to investigate odor complaints from existing facilities, which have been tabulated .The U.S. federal government, under the Clean Air Act, does not regulate odors as airborne pollutants. That leaves odor regulation to the states. Odor regulations in 42 of the 50 states are addressed by nuisance law . The federal government, however, has provided general guidance regarding nuisance odors and their impacts . The National Research Council and the National Academies of Sciences, Engineering, and Medicine provide scientific guidance to the federal government. In the 1970s, the NRC pioneered guidance on odors . The conclusion was that odors would be difficult to regulate due to their variable adverse effects on people, standards would not be widely accepted and the two sets of data are difficult to relate toeach other, costly and time consuming. If enacted, odor standards would need to be related to a measurement of odor perception that is compared with the intensity of a specified concentration of a standard reference odorant. Duration and frequency of exposure, as well as offensiveness, should be considered. In 2014, the Agency for Toxic Substances and Disease Registry launched a web page to provide a series of documents and videos dedicated to environmental odor effects and management,hydroponic table including a reference list of 684 odorants and their sensory descriptors . For this list, ATSDR initially compiled a list of chemicals that the food and beverage industries use in production. ATSDR then added other chemicals that trained professionals or large numbers of people agree have a distinctive odor. Similar odor information has also been compiled by researchers into odor wheels . A distinctive odor can indicate the potential source and even the specific key odorants. This has helped solve odor complaint problems from landfills, trash transfer stations , composting facilities and wastewater treatment plants .Based on the information gathered from around the world, a targeted literature search and phone interviews of odor experts and air inspectors were conducted to provide current perspectives on odor investigations. Literature searches were conducted online and at the University of California, Los Angeles, and California Air Resources Board physical libraries. The focus was post-2010, and when relevant articles or books were found the “cited by” function was used to find even more up-to-date information. A California-specific literature search was conducted as well. Reviews of the latest approaches to odor investigation were sought. Finally, relevant websites and posted materials not typically available in scientific journals were searched.

To collect the latest information on nuisance odor investigations from California’s air districts and international experts, phone interviews were conducted. The interviewees’ insights were gathered and any available guidance documents and relevant case studies were requested.CARB has a long history of addressing nuisance odor complaints. In the 1970s, questionnaires were used to collect information from residents near pulp mills on exposure to the odor and on health and annoyance reactions . In general, the frequency with which odor was noticed and the frequency and intensity with which respondents were bothered by the odor were correlated with perceived odor intensity and frequency as measured by dynamic dilution olfactometry within each community. More recently, an agreement was formed between CARB and the local air districts regarding the handling of odor complaints . It stated that CARB handles mobile source odor complaints and, for stationary sources, the local air district is phoned as soon as possible, but at least within one business day. It then gives local air districts 15 working days after receipt of the CARB letter to provide a written complaint resolution or summary progress report. Finally, CARB agreed to subscribe to an over-the-phone verbal translation service that it would make available to the local air districts. Several of the local air districts were contacted to gather information on how they currently investigate nuisance odor complaints. The information gathered is summarized next. One observation during the interviews was that CARB trains the air district staff on smokestack opacity measurements using their own eyes . Training regarding environmental odor evaluation could be developed as well. A key phrase is “considerable number of persons,” which is evaluated case-by-case but generally falls into 6 to 10 complaints within a 24-hour period for a notice of violation. Rule 402 does not apply to agricultural operations. In 2008, SCAQMD Rule 410 took effect to address odors from trash transfer stations and material recovery facilities. Sufficiently large facilities were required to have an approved odor management plan with extra requirements for even larger facilities. In 2009, a panel of 10 air inspectors used odor profiling to evaluate a trash transfer station . Based on the observed odors, specific odorants were suggested and then confirmed analytically. In 2017, SCAQMD adopted Rule 415 to address odors from rendering facilities . The rule requires building enclosures with ventilation to odor control systems for odorous operations and best management practices, such as covers for trucks and trailers and time limits for moving materials during the receiving and rendering process. Also in 2017, Rule 1430 was adopted to reduce odors from grinding operations at metal forging facilities . Four confirmed odor complaints over six months trigger additional odor management controls, such as enclosures. A conversation with air inspectors covered how nuisance odor complaints are currently investigated. Air inspectors use their own noses to confirm the complaint, which does not necessarily need to match the complainant’s description of the odor. Each person’s ability to describe odors varies too greatly to require consistent descriptions. Rather, sensing that “something” is there is sufficient. The next step is to identify the source and move upwind to verify that the odor is not coming from elsewhere.They are trained on how to respond by pairing junior staff with more senior mentors. For more complex odors, monitoring and meteorological data are used. Hydrogen sulfide monitors have been used for direct measurements and SUMMA evacuated canisters and Tedlar™ bags have been used to collected air samples. Sometimes a SUMMA canister is given to a complainant to collect a sample over time. Wind direction, speed and temperature are measured using a hand-held Kestrel 1000 meter, and the cloudiness and weather are noted. More sophistical meteorological data has been used to calculate the back-trajectory of odor plumes using data from the National Weather Service in Meso West plus the HYSPLIT dispersion model. Odor investigation data is captured in an in-house database that is rather old and does not allow for easy queries. Since 2010, the number of odor complaints per year has risen from around 3,400 to 7,500, decreasing in 2017 to 6,000 . Odor complaints are received by phone or via their website, which is also available through their app.No electronic nose technology is currently used although a report surveyed dozens of emerging technologies . The authors noted that e-noses were primarily used for quality control for food and other products, and that a large effort would be required for field tests for environmental odor detection.

The identified increase in variance explained by PTSD emphasizes that PTSD is a significant predictor of later addiction severity among individuals who face early substance use problems. These results are consistent with previous research, which associated early childhood experience of trauma with early substance use onset and transition to poly drug use . Additionally, previous handful evidence has consistently established associations between SUD and PTSD and provided explanatory hypotheses underlying these associations. Given that at SSA populations such Rwandans had experienced horrific events , these results may be interpreted through well-documented risky use of psychoactive substances for coping with post disaster distress . However, it is challenging to delineate which of the two conditions occurs first because SUD and PTSD affect the stress processing system. Chronic SUD, such as alcohol use disorders, increased individual vulnerability to PTSD due to alcohol related defects of endocrinal response to distress events and reduced cortisol release . On the other hand, PTSD influences neurotransmitters changes, such as serotonin, in the hypothalamic-pituitary-adrenaline axis, which have been linked to risks for worsened SUD . The identified positive association between level of education and addiction severity may be partially explained by the Rwandan cultural and conception of mental illness. Cultural expectations are strict on the use of alcohol that interferes with social and professional functioning . Thus,vertical hydroponic garden educated people may find it challenging to seek early help for their SUD due to fear of being subject to attached stigma and use psychoactive substances as self-medication.

The present study, to our knowledge, is the first to investigate the contributions of age, motives for first substance use, and post traumatic distress to later addiction problems using a clinical sample in sub-Saharan Africa. The study used a compelling alternative to the random sampling strategy, recruiting every participant presenting for inpatient addiction care in two existing settings over 8 months. This study has a few limitations, including relying on self-reported data that may be prone to recall and social desirability biases. However, we attempted to minimize these biases by collecting data through face-to-face semi-structured interviews conducted by trained and qualified mental health professionals who were not part of a healthcare circle .Underlying its clinical symptomatology, schizophrenia is characterized by a disturbance in the regulated processing of environmental information . Especially notable are impairments in identifying and responding to stimuli that are either salient or novel. The P300 event-related potential is a physiological index of the cognitive processes elicited by such task-relevant or novel deviant stimuli.However, the scalp-recorded P300 response to an auditory target stimulus – also referred to as P3b to differentiate it from the P3a orienting response to novelty – arises primarily from inferior parietal and supramarginal cortical regions . Reduced amplitude of this response is one of the most robust physiological abnormalities associated with schizophrenia, being replicated across hundreds of studies with a relatively large effect size . Although P300 amplitude varies to a certain extent with fluctuations in clinical symptomatology , it also exhibits characteristics of a trait-like disease marker. The measure has relatively high test-retest reliability , and the patient deficit persists regardless of the level of acute symptomatology or psychotropic medication . There is also strong evidence implicating a genetic contribution to P300 amplitude in both healthy subjects and patients . Thus, P300 amplitude meets the essential criteria for a schizophrenia endophenotype .

Historically, an impediment to the utility of the P300 as a schizophrenia biomarker has been its lack of specificity. P300 is disrupted in a variety of neuropsychiatric disorders associated with cognitive impairment. These include, among others, Alzheimer’s disease , alcoholism and affective illness . Additionally, the P300 response is influenced by a variety of factors, independent of clinical diagnosis. Most notable among these are age and sex; P300 amplitude is smaller, overall, in men and exhibits both reduced amplitude and prolonged latency in the elderly . It is also altered by both the acute and chronic administration of various psychoactive substances. P300 amplitude is smaller in active smokers compared to nonsmokers, and this decrement reflects both the current number of cigarettes smoked per day and the number of years of continuous smoking . It is similarly reduced following acute administration of marijuana , alcohol , and cocaine , and among chronic users of these substances . In contrast, the effect of antipsychotic medications, among schizophrenia patients, appears to be relatively inconsequential . Another potentially important factor that has only rarely been considered is race or ethnicity, and the little evidence that exists concerning this has been inconclusive . Given the frequent co-occurrence of schizophrenia with other co-morbid neuropsychiatric and substance use disorders, as well as the differences in smoking prevalence and racial stratification that is often found in schizophrenia patient vs. healthy control samples, a comprehensive understanding of the impact of such modulating factors is critical to enhancing the utility of P300 as a disease-specific schizophrenia biomarker. Recently, with the advent of studies of the schizophrenia prodrome, the importance of the P300 as a putative biomarker has taken on added significance. There is now substantial evidence that P300 amplitude is reduced in “high-risk” individuals with prodromal symptoms, prior to illness onset . Importantly, within a high-risk cohort, P300 appears to be a sensitive predictor of which individuals will, in fact, transition to frank psychosis. Moreover, the degree of impairment indicates the proximity of illness onset . The greater the magnitude of the amplitude reduction, the more likely that psychosis onset is imminent. P300 amplitude assessment may, therefore,plant bench indoor play an important part in the clinical evaluation of at-risk individuals.

However P300 studies have thus far been confined primarily to academic neurophysiology laboratories, and data analyses have been limited primarily to between-group comparisons of measures acquired in one laboratory under identical conditions. It is thus unclear whether specific values obtained in one experimental setting can be compared or co-mingled with values obtained in another setting under less-than identical conditions. The ability to aggregate quantitative data across multiple sites is critical to the strategy employed by the Consortium on the Genetics of Schizophrenia to identify the genetic substrates of disease endophenotypes. It is also critical to the utility of this measure as a specific predictive biomarker of impending psychosis. The purpose of the current analysis was therefore, first, to determine if a standardized P300 data acquisition system could be successfully deployed to multiple settings without on-site electrophysiology expertise. We assessed the overall usability of the ERP data and the ability to detect known schizophrenia deficits. We also considered the consistency of measurements across five COGS-2 sites, and examined various socio-demographic modulating factors that can contribute to measurement variability. As noted, a careful understanding of the quantitative impact of these modulating factors is an important prerequisite to any interpretation of a specific set of measurements. Healthy control subjects and schizophrenia patients were enrolled in the COGS-2 endophenotype study at 5 sites, as detailed in the introductory article of this Special Issue . Briefly, all participants were assessed using a modified version of the Structured Clinical Interview for DSM-IV-TR , Modules A-E . All patients met criteria for either SZ or schizo affective disorder, depressed subtype. HCS were excluded for any history of a psychotic disorder in either themselves or a 1 st-degree relative, a current Axis I mood disorder, a Cluster A Axis II disorder, or current psychoactive medication use. Subjects were also excluded for any medical or neurological condition that could interfere with endophenotype assessment, history of substance abuse in the past month or substance dependence in the past 6 months, or a positive toxicology screen at the time of testing. Clinical and demographic characteristics of the P300 sample are presented, by site, in Table 1. Specific past substance related diagnoses are detailed in Table 2. Adjunctive psychoactive medications used by patients are listed in Table 3. In addition to the structured clinical interview and the various endophenotype measures, all subjects were assessed with the Mini-Mental Status Examination and the Global Assessment of Functioning Scale . Patients were further assessed with two measures of functional capacity: the 15-item clinician rated Scale of Functioning, and the UCSD Performance-based Skills Assessment-Brief , which directly assesses an individual’s capacity in multiple domains of everyday functioning through the use of props and standardized skill performance tests.

There were significant group differences in age , sex and racial composition . There were also significant site differences for each of these measures, and group × site interactions for age and sex. As expected, patients and controls differed on education, GAF, and MMSE scores. There were also significant main effects of site and group × site interactions for both GAF and MMSE. Site differences were also evident for rates of past substance use, major depressive disorder, and nicotine use. Among patients, site differences were observed for duration of illness, age of onset, and current symptomatology . This variability indicates that different sites drew their samples from different socio-demographic and clinical pools. The auditory P300 was conducted as the last task of the COGS-2 battery, following completion of the Mismatch Negativity experiment described by Light et al. . A San Diego Instruments 2-channel ERP-LAB system with a pre-set P300 module was used for stimulus presentation and EEG recording. One channel recorded EEG activity at the vertex referenced to the left mastoid process . A second channel recorded eye movement activity from electrodes placed mid superior and lateral to the right orbit . A ground electrode was placed on the right mastoid. All electrode impedances were below 5kΩ. The Cz electrode location represented a compromise between the preferred electrode placements for MMN and P300 recordings, given the limitations of the 2-channel recording system and the need to dedicate one of these to the EOG. Subjects were seated in front of a computer monitor and directed to fixate their gaze on the center screen. A hearing test was conducted to ensure >40 dB hearing threshold bilaterally at 1000Hz. Subjects were instructed to press the button on a hand-held counter whenever they heard a 1500 Hz target tone amid a stream of 1000 Hz tones. They were given a brief practice period to ensure initial task comprehension and compliance. Unfortunately, the experimental hardware did not allow further real-time monitoring of button-press responses over the course of the experiment. Subjects were then presented a series of 400 tones, including 62 random targets. The ERP system digitally sampled the EEG at 1000 Hz and wrote 1400 ms of data for each stimulus trial, beginning 100 ms prior to stimulus onset. At the conclusion of the experiment, the number of button presses was recorded from the counter. Raw EEG data from all 5 COGS-2 sites were uploaded to a centralized database. Quality assurance data review and analysis was then conducted by a single investigator who was blind to all demographic and diagnostic information. EEG data were processed using Brain Vision Analyzer 1.5 . Data were digitally filtered between 0.1 and 30 Hz and eye movement artifact was removed using an automated algorithm . Intervals with additional EEG artifact were excluded from further analysis. Remaining trials were then sorted and combined to form separate average ERP wave forms for the target and frequent tone conditions. These were baseline corrected relative to the 100ms pre-stim interval and visually inspected to determine the presence or absence of reliably identifiable ERP components. A highly conservative, stringent, approach to data inclusion was employed. Data without an unambiguous N100/P200 response to the frequent tone, or a reliably identifiable P300 response to the target were excluded. Subjects were also excluded if their target count deviated by more than 20 from the actual number of 62, regardless of the quality of EEG data, since appropriate task engagement could not be documented. For the remaining subjects, P300 amplitude and latency were measured from the peak target response between 250 and 400 ms. . P300 amplitude and latency differences were analyzed in two separate general linear models , with diagnosis, sex and test site as categorical factors and age as a continuous predictor. Significant main effects and interactions were parsed with post-hoc paired contrasts. The effects of various modulating factors were then initially assessed by adding these individually as separate additional factors to the original model.

The increasing use of more discreet forms of vaping, particularly JUUL , may also have had an impact on social media engagement about vaping behavior, though more research is needed. Also, the associated health risks of vaping were relatively unknown during the study period, though the outbreak of EVALI in 2019 may have generated more attention and possible concern among users about potential health risks of vaping, though these conversations were not detected in this study . Importantly, most tweets that included conversations about tobacco products and behavior expressed positive sentiment. Though unclear from these preliminary results, the influence of “party culture” on college campuses, the opportunities to experiment and initiate with forms of substance abuse behavior, and the immediacy of pleasure from substance use may outweigh concerns, including those relating to long-term health risks, among college students in the United States as observed in this user sentiment . Interestingly, though marijuana tweets exhibited the highest proportion of positive tweets, they also exhibited the highest proportion of neutral tweets and the lowest proportion of tweets with negative sentiment. This finding may suggest relative homogeneity regarding marijuana attitudes, possibly as a consequence of debate regarding marijuana legalization during this time period. As the majority of all sentiment-containing tweets were positive, results from this study may suggest that outreach efforts to raise awareness about the health risks of tobacco and ATPs on college campuses may have limited resonance.

However, these preliminary data also suggest discrepancies in sentiment between tobacco products,vertical agriculture as well as differences in sentiment toward smoking across California universities. Therefore, policymakers and health promotion advocates should consider tailoring policy implementation and health communication for specific college students in California based upon evidence of latent receptivity toward anti-tobacco approaches and existing community sentiment toward smoking behaviors as detected in this study. Furthermore, future studies should more explicitly assess user reaction and sentiment to debate, communication and implementation of state-level policies that both legalize and restrict use of tobacco and smoking products, as well as how these macro policies interact with campus-specific smoke free policy perceptions for different tobacco, marijuana, and e-cigarette product categories. For example, actionable insights based on preliminary findings from this study indicate that users generally express more positive sentiment about tobacco use and smoking behavior. This may necessitate the use of campus-based health promotion and education activities that focus on reducing appeal of these products, such as restricting any form of marketing and promotion in or near campus communities. This should be coupled with broader state legislation to further restrict marketing and promotion that targets young adults and college communities. Further, perceived penalties for violating smoke- and tobacco-free campus policies may also impact compliance based on socioeconomic factors. For example, one user from UC Riverside tweeted, “other places might be more lenient, but UCs have a shitty tobacco and smoking policy and I got caught and now it’s over” [emphasis added to denote correction of misspelling].

Hence, data-driven approaches to assess receptivity and the impact enforcement has on smoking behavior should be built into smoke free program implementation iteratively. Importantly, the breakdown of smoking-related tweets between numerous college campuses as detected in this study presents challenges with respect to whether the distribution of tweet characteristics accurately reflects distributions in the underlying college populations. Nevertheless, similar work has been conducted which presents correlational evidence between characteristics of geospatially-specific social media posts and characteristics of populations in those areas . Furthermore, as over half of college students in California are between the ages of 18 and 24 , academic and demographic distributions of tobacco consumption within colleges may be the consequence of socioeconomic disparities in childhood and potential effects of these disparities on attitudes about smoking among parents, high schools, and/or neighborhoods that warrant further study . Results from our study are limited in generalizability, though complement work by others on examining the impact of tobacco free policies on US college campuses. This includes a recent study from 2020 of small colleges in Massachusetts that found that a college with a smoke-free policy had significantly more anti smoking attitude than a control campus, but did not have lower rates of smoking itself . Relatedly, a separate earlier study from 2005 that analyzed undergraduates in Texas found that campuses with preventive education programs had lower odds of smoking, whereas designated smoking areas and cessations programs were associated with higher odds of smoking . Collectively, these prior studies and our own work helps to better characterize knowledge, attitudes and behaviors of college campus communities toward smoking, as well as the smoke-free policies attempting to discourage smoking, which in turn should aid in the development of more targeted approaches to educate college-aged populations about the health harms of tobacco and also enable better implementation of anti-tobacco policies in these critical populations.

This study was exploratory in nature and collected social media messages for which latitude and longitude coordinates could be collected from the Twitter API, but this data collection methodology is limited to collecting messages from Twitter users that enabled geolocation, a specific limitation to generating a more generalizable dataset on Twitter as it is estimated that only 1% of all tweets are geocoded . Hence, the dataset used in this study after filtering for keywords was small and likely biased, limiting the generalizability of results. This method of data collection may have introduced bias in the types of tweets collected, thereby limiting the generalizability of findings as the majority of Twitter users do not geolocate their posts. Potential sampling biases for Twitter include oversampling for certain geographic areas,hydroponics flood table filtering for specific features , and the limitations of the Twitter public streaming API in lieu of other data collection approaches . Future studies should examine the use of multiple Twitter APIs to generate a more representative Twitter dataset and compliment findings with other traditional sources of data to generate findings that are more robust and generalizable, as well as use complementary Twitter and social media datasets made publicly available by other researchers. specific to identification of Twitter users and conversations associated with colleges and universities, using keyword searches, and selecting accounts affiliated with higher education should be explored in future studies. Also, inclusion criteria required tweets to be posted from college campuses, which would not have accounted for variability in smoking related tweets from off-campus housing or areas/neighborhoods at the borders of campus properties where college students may reside. Furthermore, though the study design permitted searches of the Twitter API to return different volume of tweets for different keywords, there was a smaller number of original keywords for substances containing marijuana/cannabis than those for e-cigarettes or products containing tobacco due to our purposeful filtering for tobacco and alternative tobacco product keywords . Additionally, the majority of tweets analyzed for this study were from 2015, a period prior to major public scrutiny about default privacy settings for location sharing on Twitter . Finally, this study is an ecological study and should therefore be considered hypothesis generating and not generalizable to individuals on college campuses until further studies among individuals confirm these correlational findings. Post traumatic stress disorder is an anxiety disorder that can develop following exposure to traumatic life events. Central clinical features of PTSD include a persistent, heightened experience of alarm and distress, as well as a failure of extinction processes to diminish the emotional impact of traumatic memories. Investigation of the neural mechanisms that underlie fear acquisition, consolidation, and extinction may thus enhance our understanding of the neurobiological basis of PTSD, and open opportunities for mechanism-based drug discovery and development of the next-generation pharmacotherapies for this disabling disorder. The process by which emotionally-aversive memories become consolidated is recognized to be an interaction between glucocorticoid hormones and norepinephrine, both of which are released in response to stress. The primary component of this response appears to be a noradrenergic signal that is necessary for encoding emotionally salient information.

The hyperconsolidation of traumatic memories in PTSD is driven by a glucocorticoid-hormone facilitated potentiation of norepinephrine inputs to the basolateral amygdala. Recent work has revealed that this glucocorticoid action is mediated by cannabinoid type-1 receptors, a mechanism that is critical for the consolidation of aversive memories and thus implicates CB1 receptors in the etiology of PTSD. Moreover, there is an emerging body of evidence demonstrating an important role for CB1 receptor-mediated endocannabinoid signaling in the extinction of aversive memories. Augmenting levels of anandamide in the amygdala modulates short-term fear extinction, thereby resulting in long-term reduction in fear and highlighting the endocannabinoid system as a candidate system for developing novel pharmacotherapies for PTSD. CB1 receptors are the most abundant G-protein-coupled receptors in the central nervous system, and are found in high concentrations within an amygdala-hippocampal-cortico striatal circuit responsible for processing and storing fear-related memories and coordinating fear-related behaviors. Animal studies have shown that chronic stress is associated with decreased brain levels of the endocannabinoid anandamide and CB1 receptor adaptations, which in turn give rise to an anxious/depressive phenotype. However, it is not clear whether these animal findings apply to PTSD in humans. The development of a CB1 receptor selective radio tracer—[11C]OMAR22—now makes it possible for the first time to conduct a quantitative assessment of in vivo CB1 receptor availability using positron emission tomography . In the current study, we hypothesized that, relative to healthy non-trauma-exposed and trauma-exposed controls , individuals with PTSD would have increased CB1 receptor availability. In light of data from animal studies, we further predicted more pronounced CB1 receptor elevations in women than men with PTSD. A TC group free of lifetime PTSD or other psychiatric illness was recruited in order to assess the relation between trauma exposure alone and CB1 receptor availability. We also assessed peripheral levels of the endocannabinoids anandamide and 2-arachidonoylglycerol ; levels of the fatty acid ethanolamides oleoylethanolamide and palmitoylethanolamide ; and cortisol. We expected to find lower anandamide and cortisol levels in the PTSD group relative to the HC and TC groups. Finally, psychiatrically relevant biomarkers for PTSD are important yet elusive contributors towards accurate diagnosis and improved clinical care for trauma survivors. We predicted that measures of CB1 receptor availability, anandamide and cortisol would accurately categorize a majority of participants with regard to PTSD diagnostic status relative to healthy and trauma-exposed controls. Participants were recruited via public advertisements seeking individuals with non-combat trauma histories and healthy control participants with and without lifetime histories of trauma.In addition, none were receiving psychotherapy at the time of scanning. The protocol was approved by the New York University Institutional Review Board, the Yale University School of Medicine Human Investigation Committee, the Yale University Magnetic Resonance Research Center, and the Yale New Haven Hospital Radiation Safety Committee. After providing written informed consent, participants underwent a thorough medical and psychiatric evaluation that included physical examination, electrocardiogram, standard blood chemistry and hematology laboratory tests, urine analysis and toxicology, followed by a magnetic resonance imaging scan and a resting PET scan with the CB1 receptor antagonist radiotracer [11C]OMAR. Psychiatric diagnoses were made using Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition – Text Revision criteria and the Structured Clinical Interview for DSM-IV , which was administered by an experienced psychiatric clinician. PTSD symptom severity was assessed using the Clinician-Administered PTSD Scale for DSM-IV and trauma history was assessed using the Traumatic Life Events Questionnaire. Only traumatic events meeting DSM-IV-TR PTSD criterion A1 for severe trauma exposure, as well as criterion A2, which confirms the emotional response to the trauma , were counted towards participants’ trauma history in this study. Additional assessments included the Hamilton Rating Scale for Anxiety, the Montgomery-Åsberg Depression Rating Scale, the Alcohol Module of the Addiction Severity Index and the Fagerström Test for Nicotine Dependence. To meet TC inclusion criteria, individuals must have been exposed to at least one potentially traumatic event that met DSM-IV-TR PTSD Criteria A1 and A2, but have no lifetime PTSD or other Axis I diagnosis. Participants with significant medical or neurological conditions, substance abuse within 12 months of the PET scan, a lifetime history of substance dependence , or history of head injury with loss of consciousness were excluded from the study.