During a cognitive task, these ongoing processes are suspended as attention is shifted to the task demands, resulting in reduced cingulate activation. Such anterior cingulate deactivation has been observed across a variety of tasks, and increases with greater working memory demands, suggesting reallocation of attentional and working memory resources . Thus, subtle attentional and working memory deficits among MAUD youths may result in the need for greater resource allocation to brain regions subserving SWM, and therefore greater deactivation in anterior cingulate cortex. MAUD teens evidenced diminished SWM activation compared to controls in the right inferior frontal and right superior temporal/supramarginal gyri, both of which have been implicated in attentional response to salient stimuli . In particular, the right temporoparietal junction, including parts of the supramarginal and superior temporal gyri, may be crucial for identifying and shifting attention to relevant stimuli . The right inferior frontal gyrus and anterior portions of the insula may be involved in evaluating stimulus relevance and inhibiting irrelevant responses , and increased activation has been associated with better performance . The lack of SWM activation in these regions among MAUD teens suggests decrements in attention orienting systems. The absence of such attentional recruitment may necessitate increased activation in other areas involved with attention and SWM,cannabis grow systems which could result in reorganization of attention and working memory circuits.
In addition to possible neural inefficiency and reorganization among MAUD teens, aberrant regional cerebral blood flow could produce the observed BOLD response differences. Diminished resting cerebral blood flow has been demonstrated among adult marijuana users during short-term abstinence, particularly in frontal and cerebellar regions . Since BOLD fMRI contrasts a resting condition and an active condition, changes in blood flow during rest alter the magnitude of the observed BOLD response . Specifically, reductions in resting blood flow produce a larger BOLD response change between resting and active conditions . Therefore, the observed greater dorsolateral prefrontal activation among MAUD teens compared to controls could be due to reduced resting frontal blood flow associated with marijuana use. However, the current study contrasted SWM with an active baseline condition , possibly limiting the impact of resting blood flow changes on the BOLD response difference between SWM and simple attention. Deactivation abnormalities among MAUD teens could also be related to resting blood flow differences between groups. One study demonstrated increased anterior cingulate blood flow among chronic marijuana users . It has been proposed that regional BOLD response changes will be evidenced as activation if resting activity is low, and deactivation if resting activity is high . Thus, high resting blood flow in the anterior cingulate associated with marijuana use could account for enhanced deactivation among MAUD teens in this region. In sum, resting blood flow changes, in addition to neural dysfunction, could underlie the observed fMRI abnormalities among MAUD teens.
MAUD youths showed similar differences relative to AUD teens as they did to controls in most regions, including reduced SWM BOLD response in right inferior frontal and superior temporal cortices, as well as greater SWM deactivation in the anterior cingulate. AUD youths did not demonstrate altered response compared to control teens in these regions. Thus, it appears that teens with marijuana and alcohol use disorders have aberrant patterns of functional response not observed in teens with AUD alone, especially in frontal systems. Heavy marijuana use during adolescence may adversely affect frontal functioning more than other brain regions , and may be related to problems with attention as well as working memory . Further, protracted recent marijuana and alcohol use during adolescence appears associated with disrupted attention and working memory networks above and beyond the abnormalities observed in teens with AUD alone. While AUD teens demonstrated greater parietal SWM response than controls , MAUD youths did not show such a pattern, although MAUD and AUD teens were equivalent on lifetime and recent drinking characteristics. One previous study indicates that adults with MAUD may perform better than those with AUD alone on working memory, visuospatial, and problem solving tasks . These findings give rise to a possibility that marijuana use may moderate some parietal abnormalities related to heavy alcohol use among MAUD youths. However, while alcohol-related parietal changes may not be apparent in MAUD youths, other abnormalities were observed, suggesting that concomitant heavy marijuana and alcohol use during adolescence presents a unique profile of functional alterations. Previous research has suggested that impaired neuropsychological functioning among marijuana using adults may largely reflect recent use .
We observed a trend for recent marijuana use to be associated with decreased activation in the right middle temporal gyrus, where MAUD teens showed reduced response relative to AUD teens. This could indicate that, at least in this region, abnormal response in MAUD teens may be due to residual drug effects from recent use. We did not find significant relationships between recency of use and brain response patterns in other regions, suggesting that some observed group differences may be unrelated to residual effects. This lack of significant relationships could be due to the limited number of participants reporting distal use, as the majority of participants had used in the month before the scan. However, whether the results in the current study are accounted for by lingering effects of recent use or long-term changes in brain functioning, important clinical implications can be drawn. Seven percent of 11th graders report using marijuana at least 10 days per month , reflecting that sizeable numbers of youths may consistently experience shorter-term after-effects of marijuana use, involving altered brain functioning during school and other activities. Several limitations of this study need to be considered. First, our sample size of 49 is relatively small for examining moderator factors, such as gender and family history. Second, although symptom severity was relatively mild, it is possible that group differences in conduct disorder prevalence could have influenced results. Third, by not including teens that heavily use marijuana alone, we were unable to delineate effects solely related to marijuana use. Fourth, our cross-sectional design does not permit the evaluation of functional differences that may have existed before the onset of substance use. Finally, as discussed above, the MAUD adolescents in the current investigation evidenced neural dysfunction after a minimum of 48 h abstinence, yet it is unclear whether the observed differences would persist with more extended sobriety . Future studies might attempt to disentangle the residual and longer term effects of adolescent marijuana use by requiring longer periods of monitored abstinence before assessment. Despite these limitations, the current investigation raises several questions to be addressed by future studies. First, we observed increased frontal activation during SWM among MAUD youths in the context of intact performance. Future studies might attempt to parametrically alter task difficulty in order to characterize neural patterns associated with changing behavioral performance . Although both SWM and attention deficits may have influenced brain response abnormalities among MAUD teens, the task utilized in this study was not designed to directly assess attention. Thus, future neuroimaging studies of heavy marijuana using youths might attempt to examine sustained and divided attention more closely, as well as other cognitive abilities. Further, as resting blood flow abnormalities may have contributed to some fMRI differences between groups, future fMRI studies should assess resting perfusion for use in covariate analyses. In addition, the current fMRI study cannot make any direct conclusions about the neural characteristics underlying functional change. Magnetic resonance spectroscopy could elucidate the metabolic and cellular underpinnings of functional abnormalities related to combined marijuana and alcohol use. Finally, as the persisting neural effects of heavy marijuana use are unclear,ebb and flow tables longitudinal investigations should characterize the neuromaturational and functional consequences of heavy alcohol and marijuana use during youth, as well as the potential for neural recovery.
In summary, this study found aberrant brain response to a spatial working memory task among adolescents with comorbid marijuana and alcohol use disorders. Compared to non-abusing controls and teens with AUD alone, MAUD youths evidenced frontal and temporal dysfunction, suggesting that heavy marijuana use could be related to attentional decrements and compensatory responses in areas subserving spatial working memory. These neural abnormalities were not observed among AUD-only teens, despite similar drinking characteristics, indicating that combined marijuana and alcohol use may have a unique influence on brain functioning. Together, these findings demonstrate that teens with comorbid marijuana and alcohol use disorders show subtle disruptions in brain functioning after a minimum 48 h of abstinence. Studies of how mechanical forces influence cell function, or mechanobiology, have been one of the major contributions of biomedical engineers to cell biology and physiology. Through the use of in vitro mechanical stimulation devices, it has become well-appreciated that physical cues influence cellular form and function, and that deviations in normal physical stimulation are often drivers of pathology. While the complexity of the in vivo environment is difficult to fully recapitulate, mechanical stimulation devices allow for the simulation of more physiologically relevant environments through application of physical stimuli to cells in culture. These devices have been instrumental in developing a better understanding of how physical forces affect a number of different cell types. Although such tools have aided in many discoveries, their use has been limited to specialized research environments, and widespread adoption remains a challenge. Furthermore, while mechanobiology has become an integral component of undergraduate curricula in biomedical engineering, education remains largely in the classroom. In this study, we describe the design, fabrication, and use of a mechanical stretching system for widespread research and educational purposes, and propose an experimental platform for mechanobiology education at the undergraduate level. Cell stretching devices are available commercially and can also be fabricated in-house, but current devices have several constraints that limit their widespread use in research or educational environments. Commercially available cell stretchers are costly to purchase and maintain, ranging in cost from thousands to tens of thousands of dollars, and also require an additional recurring cost of device-specific stretchable culture well plates. Several less expensive alternatives have been developed, but these devices often still rely on expensive equipment for fabrication or require customized stretchable membranes available exclusively from commercial sources. As such, most devices are unable to adapt to experiments that require modifying the stretchable membranes, for example, the inclusion of multiple wells for high throughput analysis. Therefore, a low-cost and reliable cell stretcher is required to increase accessibility of such devices and, ultimately, further the study of cell mechanobiology. Project-based methods have been introduced in many pedagogical environments and are generally recognized as enhancing development of critical thinking skills. In addition, undergraduate students who participate in research projects gain valuable hands-on skills and knowledge and are thought to play important roles in career decisions. Previous studies have also suggested that project-based and experiential learning techniques create better student motivation, which results in significant enhancement in learning. Although mechanobiology has become a component of many undergraduate biomedical engineering curricula in recent years, project-based and experiential learning modules in this area remain limited given the high cost of the necessary equipment. Here, we describe the design and validation of a cost-efficient uniaxial cell stretcher that can be integrated with undergraduate mechanobiology education. This project was carried out predominantly by undergraduate students with the involvement of high school students. The device is composed of durable, yet inexpensive aluminum parts, and the mechanical motions are controlled through the use of a computer-programmed servomotor, gear, and gear rack system. Stretchable membranes were made of silicone materials, which can be used in different sizes and shapes allowing for greater flexibility in performing different experimental assays. All components used to fabricate this cell stretcher are readily available and the device itself is, therefore, inexpensive to create and maintain. As proof-of-principle, the device was used to mechanically stimulate macrophages and cardiomyocytes. The changes in cell morphology and function were consistent with previous findings. We propose that this cell stretching device may be integrated with undergraduate mechanobiology education to provide students experiential learning in design and fabrication of mechanical devices that can be directly utilized in cutting edge research. The students can also get in-depth exposure to the application of such devices in the study of cell physiology applicable to many physiological systems. We demonstrate that these projects allow the students to learn both hard and soft skills, and gain experience that aids in more informed career decisions.