In addition to concerns about food safety, microbial pathogens are considered to be among the leading causes of water quality impairment in California agricultural watersheds . Within a watershed, pathogenic bacteria and protozoa from humans, livestock, wildlife, and pets can be found in runoff and can contaminate surface water bodies . Non-point sources of pollution have become the main sources of microbial pollution in waterways, with agricultural activities, including manure application to fields, confined animal operations, pastures, and rangeland grazing, being the largest contributors . Constructed and restored wetlands have been among the few water management options proposed as being available to growers to filter and improve the quality of water in agricultural runoff that contains a wide range of contaminants . Specifically, constructed wetlands have been shown to be highly effective at removing pathogens from water . However, wetlands may also provide habitat for wildlife, including birds, livestock, deer, pigs, rodents, and amphibians, and they may in turn vector pathogens that cause human disease. These animals deposit feces and urine within the wetland, an effect that has the potential to negate any benefit from pathogen removal caused by wetland filtering . After past outbreaks of food borne illness caused by E. coli 0157:H7 borne on lettuce and spinach grown in California, some food safety guidelines have encouraged growers to reduce the presence of wildlife by minimizing non-crop vegetation, including wetlands, that could otherwise attract wildlife to farm fields growing fresh produce . In this situation, food safety guidelines may be at odds with water quality improvement measures. Many constructed and restored wetlands in California have been built with support from the USDA-NRCS through the Environmental Quality Incentives Program and the Wetland Reserve Program . Under these programs, 4×8 botanicare tray most wetland systems were initially developed to mitigate the loss of wetlands and improve wildlife habitat. A key element of the design of these systems is that they receive agricultural runoff as input flows intended to maintain the wetland’s saturated conditions .

In addition to increasing wildlife habitat, the observed water quality improvements linked with these types of wetlands have made them an attractive “best management practice” for irrigated agriculture . Our purpose in writing this publication is to show how wetlands may be used to improve water quality in agricultural settings where pathogens are a matter of concern. In addition, we will discuss wetland design and management considerations that have the potential to maximize pathogen removal and minimize microbial contamination. The following case study highlights the effectiveness of wetlands as a tool to improve water quality and demonstrates the importance of specific design characteristics. A water quality assessment of seven constructed or restored surface flow-through wetlands was conducted across the Central Valley of California. Wetlands differed in such parameters as size, age, catchment area, vegetation type and coverage, and hydrologic residence time . W-1 through W-4, located in the San Joaquin Valley and discharging into the San Joaquin River , were continuous flow wetlands. W-5 through W-7, situated in the Sacramento Valley and discharging into the Sacramento River , were flood-pulse wetlands with a water management regime consisting of flood pulses every 2 to 3 weeks, followed by drainage for 3 to 4 days prior to the next flood pulse. W-2 and W-3 shared the same input water source, and the same was the case for W-5, W-6, and W-7. Several water quality parameters were measured at input and output locations during the growing season to evaluate the systems’ ability to improve water quality. Both concentration and load are important considerations when assessing water quality constituents. Concentration represents the mass, weight, or volume of a constituent relative to the total volume of water. Load represents the cumulative mass, weight, or volume of a constituent delivered to some location.

The flow-through wetlands were most effective at reducing total nitrogen , total suspended solids , and E. coli loads , and were moderately effective at reducing total phosphorus loads. In many instances, the flood-pulse wetlands were actually a source of contaminants, as indicated in table 2 by the negative numbers they show for removal efficiency. E. coli load in outflows was significantly lower than the inflow load at all flow-through wetlands , while the flood-pulse wetlands showed significant increases in E. coli : decreases of 80 to 95% as opposed to increases in total E. coli loads, respectively. The differences in contaminant removal for flow-through versus flood-pulse wetlands can be attributed to two factors. First, the input water for the flood-pulse systems was very clean, so any introduced contaminants were readily detectable. The average E. coli concentration for input water was 62 cfu 100 ml−1 in the flood-pulse wetlands, compared to over 200 cfu 100 ml−1 in the flow-through wetlands. Second, the overly long hydrologic residence times of flood pulse systems can allow contaminants to become more concentrated through the processes of water evaporation, leaching of nutrients from soils and organic matter, and introduction of nutrients and contaminants from feces and urine deposited by wildlife that inhabit the wetlands. Enterococci and E. coli are standard federal- and state regulated constituents used as indicators of fecal contamination in water. In the flow-through wetlands , approximately 47 percent of water samples collected from irrigation return flows exceeded the EPA recreational contact water standard for E. coli of 126 cfu 100 ml−1 . In contrast, E. coli concentration in wetland outflows ranged from 0 to 300 cfu 100 ml−1. Following wetland treatment, 93 percent of wetland outflows met the California water quality standard for E. coli concentration . For enterococci, 100 percent of the input water samples exceeded the water quality standard of 33 cfu 100 ml−1.

Despite exceeding the water quality standard, the bacteria levels found here are very low when compared to other contaminated water sources, such as wastewater . Although enterococci removal efficiencies ranged from 86 percent to 94 percent , only 30 percent of the outflow enterococci concentrations met water quality standards . Results from this study indicate that by passing irrigation tail water through wetlands, a grower can significantly reduce the water’s pathogen concentration and load, as well as other water quality contaminants common to agricultural settings. Some water quality standards may never be met with wetland filtering alone, especially where the standards require extremely low values, as is the case for enterococci in irrigation water used on farms that grow produce that is intended to be consumed raw. Wetland design and management need to be considered prior to construction and throughout the life of the system. In many cases, the natural mechanisms that promote contaminant removal or retention can be manipulated through careful design, management of hydrology, and maintenance of appropriate vegetation. Natural mechanisms for reducing bacteria pathogens are not fully understood and have received only limited study in irrigated agriculture. Wetlands are known to act as bio-filters through a combination of physical , chemical , and biological factors , all of which contribute to the reduction of bacteria numbers . Where input water has a relatively low concentration , wetland background levels are so low that water passing through the wetland may actually end up with increased pathogen concentrations . As high-energy input flows disperse across the wetland, the water’s velocity decreases, and particles that had been suspended in the water settle to the bottom. The energy needed to support suspended particles in the water flow dissipates as the cross-sectional area of the wetland flow path increases, flood tables for greenhouse and vegetation reduces the water’s turbulence and velocity. The rate of sedimentation is governed by particle size, particle density, water velocity and turbulence, salinity, temperature, and wetland depth. Larger pathogens tend to settle more quickly than smaller ones. The actual removal of pathogens by means of sedimentation depends on whether the pathogens are free-floating or are attached to particles. Pathogens can be attached to suspended particles such as sand, silt, clay, or organic particulates. Microbial contaminants associated with particles, especially dense, inorganic soil particles, settle out in wetlands sooner than those in the free-floating form. Studies have shown that the rate of pathogen removal is greater in wetlands where the input waters have a high sediment load . Some wetland designs are more prone to encourage wave activity, which prevents sedimentation and encourages re-suspension of settled particulates . High wind velocities promote wave activity. Large, open-water designs are more prone to water turbulence because wind velocity increases over a large, smooth surface. Wetland vegetation can help minimize water turbulence and particle re-suspension. For example, trees planted as wind barriers surrounding the wetland decrease the amount of wind on the wetland. Emergent vegetation within the wetland can anchor sediment with its roots and can dampen the velocity of wind moving across the water surface. Dendritic wetland designs, which consist of a sinuous network of water-filled channels and small, vegetated uplands, can help reduce water turbulence associated with high winds .Vegetative cover has been shown to decrease sediment re-suspension. For example, Braskerud found that an increase in vegetative cover from less than 20 percent up to 50 percent reduced the rate of sediment re-suspension from 40 percent down to near zero. Wetland depth may also have an indirect effect on sediment retention.

The water should be deep enough to mitigate the effect of wind velocity on the underlying soil surface, but if the water is too deep, vegetation will not be able to establish and a significant increase in re-suspension of sediment will result. Water depths between 10 and 20 inches optimize conditions for plant establishment, decreased water velocity, well-anchored soil, and a short distance for particles to fall before they can settle . An excess of vegetation can significantly reduce a wetland’s capacity to retain E. coli. Maximum removal of E. coli occurs under high solar radiation and high temperature conditions , and vegetation provides shading that can greatly reduce both UV radiation and water temperatures. While vegetation can provide favorable attachment sites for E. coli, a dense foliage canopy can hinder the free exchange of oxygen between the wetland and the atmosphere. This vegetation induced barrier to free exchange of oxygen limits dissolved oxygen levels, and that in turn reduces predaceous zooplankton, further decreasing removal of microbial pathogens from the wetland environment . The plants’ uptake of pollutants, including metals and nutrients, is an important mechanism, but is not really considered a removal mechanism unless the vegetation is harvested and physically removed from the wetland. Wetland vegetation also increases the surface area of the substrate for microbial attachment and the biofilm communities that are responsible for many contaminant transformation processes. Shading from vegetation also helps reduce algae growth. However, certain types of vegetation can attract wildlife such as migrating waterfowl, which may then become a source of additional pathogens. Vegetation that serves as a food source or as roosting or nesting habitat for waterfowl may need to be reduced in some settings. Among other important considerations for vegetation coverage in wetlands, one must include total biomass and depth features. Vegetation should provide enough biomass for nutrient uptake and adsorptive surface area purposes, but must also be managed to allow sufficient light penetration to enable natural photo degradative processes and prevent accumulation of excessive plant residues, which would prevent the export of dissolved organic carbon. One way to promote this balance is to create areas of deeper water intermixed with the shallower areas. In an agricultural setting, it may be hard to establish plantings of native species within wetlands due to the large seed bank of exotic species that may be present in input waters . You can also manage the type and amount of vegetation by manipulating the timing and duration of periods of standing water in the system. In extreme instances, you can actually harvest excess biomass. In addition to managing vegetation and water depth to maximize sedimentation and pathogen photodegradation, growers can also manipulate hydrology to maximize the removal of microbial pollutants in wetlands. The importance of hydrologic residence time is apparent when you recognize that a longer HRT increases the exposure of bacteria to any removal processes such as sedimentation, adsorption, predation, impact of toxins from microorganisms or plants, and degradation by UV radiation . E. coli concentrations have been shown to increase in runoff from irrigated pastureland when the volume of runoff is increased .

While both macrophages and cardiomyocytes were observed to align parallel to the direction of strain, other cell types have been reported to align perpendicular to cyclic strain. For example, several studies have shown perpendicular alignment of smooth muscle cells, endothelial cells, and fibroblast when exposed to cyclic stretch. Cardiomyocytes have also been reported to align perpendicular to stretch, but only if cells were cultured for longer periods of time prior to the initiation of stretch. The orientation of cells in response to cyclic stretch is thought to be attributed in part to the frequency of stimulation: it is thought that low frequency strains allow time for cells to relax, and as a result, they align parallel to the applied strain. High frequency strains, on the other hand, do not allow time for relaxation, and thus, the cells align perpendicularly to minimize the force applied on them. This theory, however, is limited to stationary mechanically active cells, such as muscle cells and fibroblasts, and has yet to be shown in other cell types. The response of cells to cyclic stretch, therefore, is dependent on a number of factors of which include cell type, time of culture, and frequency of applied of strain.The design, building, and validation of the uniaxial cell stretcher, along with mechanobiological testing of the effects of cyclic stretch on different cell types, was demonstrated to have a positive impact on the learning experience of undergraduate students. Through their involvement on this project, the students perceived that they gained valuable skills and knowledge and experiences necessary to make informed career decisions. Nonetheless,curing cannabis the learning experience can still further be improved by implementing a few changes based on suggestions from the involved students.

For example, most of the students desired additional education in mechanobiology when first starting this project, as few were aware of the impact of mechanical forces in influencing cell function. This could be implemented through classroom learning in mechanobiology, which together with the project would promote foundational understanding, and help students formulate independent research questions and interests. Students also found that working with cells as well as the stretchers and stretchable membranes was challenging at first and wanted to have more time to practice basic techniques prior to running experiments. Again, a formal course with laboratory cell culture work may provide additional opportunities for practicing basic techniques. Finally, the students involved in this project suggested further collaboration between labs, particularly for the cell experiments, as conducting research on multiple cell types may have enhanced their learning experience in cell mechanobiology. We propose that this mechanical stretching device can serve as a platform for experiential learning for undergraduates in mechanobiology. This could be carried out as an independent research project by a group of 4–5 students, as was performed here, or could be scaled up to a laboratory course offered in conjunction with classroom learning. For the former, we believe that a few labs with long-range interest in mechanobiology of cells could collaborate to have an undergraduate student from each lab constitute a team that can mentor younger recruits. This was an extremely successful model in our experience, and provides the students with mentoring and project management experience, valued in both industry and graduate programs. Undergraduate involvement in research has previously been shown to have a positive impact on student learning and development.

Students who were involved in research perceived greater enhancement of cognitive and personal skills were more likely to pursue graduate degrees, and were more likely to have a faculty member play an important role on their career decisions, thus highlighting the impact of faculty involvement in undergraduate growth. While there are many perceived benefits of undergraduate involvement in research, there are some challenges associated with implementing the proposed project in the research environment. For example, differences in the requirements posed by each engineering curriculum could limit the time available to some students to work on this project as part of an interdisciplinary team. A potential solution to this challenge would be to provide a platform, such as through a course with both lecture and laboratory components, in which the requirements of all engineering disciplines can be satisfied. For implementing this project in a classroom setting, we anticipate at least three modules, which could take place across several semesters or quarters over the course of one year. In the first module, students would gain experience in device design and fabrication using software such as SOLIDWORKS and basic machine shop tools. The second module would encompass device validation and basic cell culture skills needed to perform biological experiments. Students would use image analysis and software such as IMAGEJ to analyze videos and measure strains applied to stretchable membranes. In addition, students would learn aseptic cell culture technique and grow cells on culture wells fabricated on membranes. Finally, the project would culminate in the third module with hypothesis-driven experimental studies. Students would develop hypotheses based on the literature and test them experimentally using their fabricated device. Through project-based learning, students have been shown to develop critical and innovative thinking and improved learning. Therefore, we propose that implementation of this project in the classroom would provide a novel experience for students in learning about cell mechanobiology.

Project-based classes are already offered at UCI, although none incorporate biological experimentation. For example, the Engineering seven series, which contains both a lecture and laboratory component, students design, build, and test a quadcopter, fitness tracker, or a microfluidic chip. The project described here can be implemented using this course framework but would likely require more than one academic quarter to complete design, fabrication, and cell-based experiments. Alternatively, the project can be integrated directly into UCI’s biomedical engineering curriculum, where undergraduate students are already required to learn CAD software and the basics of fabrication during their second year. An additional course would follow, where students learn about fundamentals in cellular mechanobiology and use their fabricated device in cell-based experiments. While a large-scale course may have broader impact and accommodate many students, the project may not have the same impact as an undergraduate research project where faculty-student interactions may be more predominant. Faculty involvement and direction in creating an experience similar to that of within the research environment would be critical in order to ensure student development and growth. Nonetheless, in any of the aforementioned formats, this project would provide students experiential learning in mechanical design and fabrication, testing and validation, and biological experimental design. The process involved in designing, fabricating, and validating a cell stretching platform and then using the created device to study the effects of mechanical strain on different cell types provides undergraduate students with unique experiences in learning cell mechanobiology. While experiential- and project-based learning may be prevalent in other engineering disciplines, biomedical engineering has traditionally followed a theory-based instructional model, which limits practice in applying expertise and knowledge gained to new contexts. This project not only offers undergraduate students experience in engineering design but also provides experience in cell and tissue culture and biological experimental design, both of which are difficult to obtain in a standard biomedical engineering curriculum. In addition, the process of working in interdisciplinary teams,how to dry cannabis presenting to different audiences, and interacting with graduate students and faculty helped undergraduate students to gain valuable knowledge and skills to improve learning and also help make informed career decisions. Ongoing efforts to improve the device including development of multi-well substrates for testing of many different conditions, modification of the device base to allow visualization of cells by microscopy during stretch, or addition of a three-dimensional hydrogel to the substrate to render a more physiological micro-environment for cells. The described device may be used for both research and educational purposes, as a low-cost, easy to build and maintain, hands-on experience for learning of how mechanical forces regulate cellular structure and function.Among young adults in the United States, cigarette smoking and marijuana use are strongly associated. In 2011, 36% of U.S. cigarette smokers aged 18–25 had used marijuana in the past month, almost three times the rate of the general adult population . A systematic review of studies of cigarette and marijuana co-use in adolescence and young adulthood found consistently significant associations .

There is also a reverse relationship, whereby those who use marijuana in early young adulthood are more likely to initiate cigarettes use and have a greater likelihood of developing nicotine dependence than their non-marijuana using peers . Combined smoking of cigarettes and marijuana in young adulthood has been associated with worse health outcomes than smoking either substance alone . There are multiple suggested mechanisms underlying the co-use of cigarettes and marijuana including both genetic and environmental factors . Limited research has focused directly on cognitive factors sustaining co-use, but cigarette and marijuana co-use may be perpetuated in part by similar beliefs about the two substances or that one substance supports the use of another. For example, in a study of 233 college students who smoked both cigarettes and marijuana, 65% smoked both substances in the same hour; 31% smoked cigarettes to prolong and sustain the effects of marijuana; and 55% had friends who engaged in these behaviors, suggesting that use is related both behaviorally and socially . Another explanation for perpetuation of co-use includes a phenomenon called “blunt chasing,” or the smoking of a cigarillo or cigar following a blunt , which reportedly increases the sense of euphoria from taking these drugs .The two most commonly used addictive substances among young adults, there is a need to examine whether behaviors and thoughts related to cigarettes and marijuana are similar among those who use both substances. If use and constructs associated with reducing use relate similarly across substances, it would support interventions that target both drugs simultaneously. Motivation to quit smoking cigarettes and marijuana is generally low among young adults , suggesting that the Transtheoretical Model of behavior change may be particularly appropriate to understand co-use of these substances. The TTM includes three interrelated constructs: stages of change, temptations to use, and decisional balance , that have been used to describe cigarette smoking and predict quitting . Our development and earlier analysis of a staging scale for marijuana use was found to relate to concurrent frequency of marijuana use, temptations to use, and cons of using marijuana, consistent with what has been found in the cigarette smoking literature . Among young people, relapse to cigarettes and marijuana use is also high among those who have made a quit attempt. For example, in a review of 52 studies, median rates of smoking relapse among adolescents aged 20 or young who made a cessation attempt were 34% after one week and 89% after 6 months . In a study of 385 marijuana users who had made a self-initiated quit attempt, 88% had relapsed within 5 years . Constructs of thoughts about abstinence, including desire to quit, perceived success at quitting, difficulty with staying quit, and abstinence goals, as originally described in Marlatt’s Relapse Prevention model , are also predictive of cigarette and other substance use outcomes and related to TTM constructs . Applied to marijuana, the thoughts about abstinence items assessing desire to quit, perceived success, anticipated difficulty, and abstinence goal correlated significantly with frequency of marijuana use and stage of change . Young people may think differently about their cigarette smoking and marijuana use. For example, the 2011 National Survey of Drug Use and Health showed that, 66% of youth age 12 to 17 perceived “great harm” from smoking one or more packs of cigarettes per day, compared to 45% for smoking marijuana once or twice a week . In a qualitative study of 99 adolescents who smoked cigarettes and marijuana, while most desired to quit smoking cigarettes at some point in the future, few intended to stop using marijuana . Conversely, among youth surveyed in an addictions treatment program, intention to quit smoking cigarettes was lower than intention to quit using drugs . Study of more representative samples is needed to explore cigarette and marijuana co-use patterns and cognitions. In a national, anonymous, cross-sectional survey of young adults who smoke cigarettes and use marijuana, the current investigation examined the relationship between: severity of use and quit attempts , thoughts about abstinence , and TTM constructs of stage of change, temptations, and decisional balance .

The anterior cingulate has been implicated for its role in attentional control and conflict monitoring and has been shown to be involved during adult SWM performance . Diminished anterior cingulate activity during a cognitive task may be related to reorganization of attentional resources as task demands arise . Such cingulate deactivation was observed only in females in this study, suggesting that adolescent females may require greater reallocation of attentional resources than males during SWM. Males consistently perform better than females on visuospatial tasks . Further, recent fMRI studies have demonstrated gender-specific activation patterns during mental rotation, theorizing that females use more detail oriented analytic strategies, while males use more “gestalt” perceptual strategies . Thus, anterior cingulate deactivation among females in this study could represent greater attentional demand to maintain performance. In addition to gender differences in cingulate activation, males in this study evidenced greater activation in right frontopolar cortex. Further, an interaction between age and gender was observed in the frontopolar cluster, with activation in this region decreasing with age in males and increasing with age in females. The right frontopolar cortex has been associated with SWM in adults , and has also received attention for its more general role in subgoal processing and integration during working memory tasks, as well as more efficient retrieval during episodic memory . Such activation among males may indicate a more economical strategy to achieve task demands. This could preclude the need for increased attentional control, and therefore anterior cingulate deactivation, as demonstrated in females. Further, the age-related decrease in frontopolar activity among males may reflect more efficient processing as development progresses,rolling benches for greenhouse while the age-related increase among females could indicate increased ability to reallocate attention from extraneous regions to task-relevant areas, including the frontopolar cortex.

Although it remains unclear when in the course of development the male advantage on spatial tests emerges, meta-analysis has indicated that this gender difference appears some time in early adolescence and increases with age . Similarly, the observed gender differences in brain response in this study could represent the emergence of sexually dimorphic activation patterns and cognitive strategies as neural maturation progresses.It is critical to interpret fMRI results in the context of task performance. We therefore examined whether performance indices mediated the relationship between age or gender and SWM BOLD response. Although vigilance reaction time was negatively associated with age, it did not predict brain response in any region where age and BOLD response were related. This suggests that age-related differences in brain response represent changes in neural utilization and strategy, rather than behavioral alterations. Likewise, boys had faster vigilance reaction times than girls, yet vigilance reaction time was not related to brain response in either region demonstrating a gender difference in SWM BOLD response. This provides evidence that gender differences in brain response are related to gender differences in neurocognitive features other than those manifested behaviorally. The study was also limited by a sample size that may not have been sufficient to detect more subtle variations between the genders. In addition, participants in the current study came from relatively high income families, which may not accurately represent the general population. Motion during scanning is a concern for all functional neuroimaging studies, and constraints of existing motion management programs are a limitation. Further, while this SWM task was used because it has been previously reported on by our group in adolescents and young adults , the high accuracy on both conditions in this study suggests a potential ceiling effect among healthy adolescents. A more difficult task, with greater working memory load and/or more spatial locations could elicit age-related performance differences and elucidate different patterns of functional development. Moreover, the fact that teens performed somewhat faster during the spatial working memory condition than during the vigilance baseline condition warrants consideration. Although this discrepancy has been observed in our previous studies with this task , it is not clear whether reaction time differences may have contributed to fMRI findings.

Therefore, future tasks should be designed in attempt to equate reaction times on experimental and control conditions. Finally, future investigations might attempt to more objectively characterize pubertal development using biological assays as opposed to retrospective self-report measures that can be influenced both by participant recollection of pubertal events and willingness to disclose information.Marijuana is the most commonly used illicit drug among teenagers: almost half of 12 th graders have used cannabinoids, 20% report past-month use, and 5% disclose daily use . During this period of increasing marijuana use, continued neuromaturation includes synaptic refinement, myelination, and improved cognitive and functional efficiency . The potential long-term consequences of marijuana use on the developing adolescentbrain have not been well delineated, but could have major implications for academic, occupational and social achievement. Neuropsychological studies in adults have indicated that within a few days of abstinence, heavy users demonstrate impairments in learning and memory, attention, visuospatial skills, processing speed, and executive functioning .Heavy marijuana users have demonstrated reduced cerebellar and frontal blood flow both at rest and during verbal learning and memory, while also showing poorer verbal learning abilities . Functional magnetic resonance imaging evidence suggests that marijuana users show increased and widespread spatial working memory activation after 6 – 36 hours of abstinence, both in anterior cingulate and prefrontal regions normally associated with SWM, as well in additionally recruited brain areas not activated among controls . During verbal working memory, marijuana users had similar fMRI response patterns as controls, yet failed to show practice-related decreases in parietal activation . However, it isunclear whether these neurocognitive abnormalities only represent effects of recent use. Pope and colleagues demonstrated deficits on verbal learning up to 7 days after use among current heavy marijuana users compared to former users and nonusing controls.

However, after 28 days of abstinence, current users performed similarly as former users and controls on all tests, suggesting that neurocognitive decrements may resolve within a month of abstinence . Importantly, fMRI evidence indicates that both abstinent users and active users show brain response abnormalities relative to controls during visual attention , suggesting lasting changes in patterns of neural activity. Together, these studies indicate that neuropsychological decrements observed after one week of use may not persist, and highlight the importance of examining neural responding after several weeks of abstinence. Few studies have examined neurocognitive functioning among adolescent marijuana users. Among poly substance using youths, marijuana use has been linked to learning and memory and attention . In a longitudinal study, Fried and colleagues assessed cognitive functioning in 9- to 12-yearolds before the initiation of marijuana use, and again when youths were ages 17 – 21. After controlling for baseline performance and demographics, current heavy marijuana users showed poorer immediate and delayed memory, processing speed,and overall IQ. Further, a longitudinal study of ten cannabis-dependent adolescents demonstrated incomplete recovery of learning and memory impairments after six weeks of monitored abstinence , indicating that adolescents may be more susceptible to long-term changes than adults . Together, these studies point to dysfunctional working memory and attention abilities among heavy marijuana using youths that may persist after several weeks of abstinence. We previously investigated fMRI response to a SWM task among adolescents with comorbid marijuana and alcohol use disorders compared to teens with alcohol use disorder alone and non-abusing teens. After an average of eight days of abstinence, adolescents with comorbid marijuana and alcohol use disorders showed brain response abnormalities not evidenced by those with alcohol use disorders alone, including increased dorsolateral prefrontal activation and reduced inferior frontal response,cannabis grow equipment suggesting compensatory working memory and attention activity associated with heavy marijuana use during youth . Yet it is unclear whether these abnormalities are solely a function of recent use or would present after more abstinence, suggesting persistent effects. A preliminary fMRI study explored verbal working memory among 7 adolescent marijuana, 7 demographically similar tobacco smokers and 7 non-users after a month of abstinence . Compared to other groups, marijuana users demonstrated increased right hippo campal activity and poorer attention and verbal working memory performance. Recently, these researchers evaluated verbal working memory among abstinent adolescent marijuana users and non-users during nicotine withdrawal . After at least two weeks of abstinence, marijuana users showed increased parietal activation during nicotine withdrawal and poorer verbal delayed recall, while non-marijuana users did not . Together, these studies suggest persisting brain response abnormalities during working memory among adolescent marijuana users. To investigate the potentially enduring neurocognitive effects of chronic marijuana use during adolescence, we examined fMRI response during a SWM task among marijuana using teens and non-abusing controls after 28 days of monitored abstinence. Blood oxygen level-dependent fMRI was collected during a SWM task that typically activates bilateral prefrontal and posterior parietal networks in adolescents , and has been associated with neural dysfunction among youths with alcohol use disorders as well as comorbid alcohol and marijuana use disorders .

We predicted that after 28 days of monitored abstinence, marijuana using teens would demonstrate intact performance on the SWM task yet increased brain response in frontal and parietal regions, based on prior findings in recently using and abstinent adolescent marijuana users.Flyers were distributed at high schools in San Diego County to recruit adolescent participants ages 16 – 18. Interested teens and a parent provided informed assent and consent , approved by the University of California San Diego Human Research Protection Program. Each adolescent and a parent were separately administered detailed screening interviews . The computerized NIMH Diagnostic Interview Schedule for Children Predictive Scales  excluded adolescents with a psychiatric disorder based on youth or parent report. Teens in the marijuana group who met DSM-IV criteria for alcohol use disorder were not excluded due to high comorbidity with marijuana use disorder . Additional exclusionary criteria included prenatal substance exposure, psychotropic medication use, neurological dysfunction, head injury, family history of bipolar I or psychotic disorder as ascertained by the Family History Assessment Module screener , left-handedness, learning disorder, MRI contraindications, or substance use in the 28 days before scanning. Eligible teens were 15 heavy marijuana users and 17 demographically similar non-using controls . While most MJ teens were current users, five reported no use in the month before the monitored abstinence period. Groups were similar in gender and ethnic composition. Importantly, both MJ and control teens demonstrated similar levels of estimated premorbid IQ, as assessed by the Wechsler Abbreviated Scale of Intelligence Vocabulary subtest and socioeconomic status . MJ teens showed higher levels of depressive symptoms on the Hamilton Depression Rating Scale and Beck Depression Inventory , and higher levels of anxiety on the Hamilton Anxiety Rating Scale . MJ teens had more lifetime and recent experience with alcohol than controls , yet five MJ teens reported no alcohol use in the month before the abstinence period. Among current alcohol users, most were weekend binge drinkers. Both groups had low rates of nicotine use, but, MJ teens had used cigarettes more recently than controls, and four MJ teens smoked cigarettes on the day of the scan. Although MJ teens divulged more use of other drugs than controls , such use was limited to 25 lifetime experiences, most commonly oral opiates or hallucinogens.Substance use was characterized with the Customary Drinking and Drug Use Record , which collected lifetime and past 3-month information on marijuana, alcohol, nicotine, and other drug use, withdrawal symptoms, and DSM-IV abuse and dependence criteria. Based on CDDR reports, typical blood alcohol concentration achieved during drinking episodes was calculated using the Widmark method, based on amount and duration of drinking, height, weight, and gender . The Fagerstrom Test for Nicotine Dependence assessed degree of nicotine dependence on a scale of 0 – 10. The Timeline Follow back assessed substance use for 28 days before starting monitored abstinence, and for the 28 days of the abstinence period. Teens were asked to indicate for each day whether they used or drank, and if so, how many hits of marijuana, standard drinks of alcohol, or amount of other substances were used. The Hamilton Depression Rating Scale , Beck Depression Inventory , Hamilton Anxiety Rating Scale , and state scale of the Spielberger State Trait Anxiety Inventory assessed mood at the time of scanning.