Changes in brain structure and function following chronic exposure to inhaled vaporised cannabis during periadolescence in female and male mice: A multimodal MRI study.

BACKGROUND AND AIMS: Social norms and legality surrounding the use of medical and recreational cannabis are changing rapidly. The prevalence of cannabis use in adolescence is increasing. The aim of this study was to assess any sex-based neurobiological effects of chronically inhaled, vaporised cannabis on adolescent female and male mice. METHODS: Female and male mice were exposed daily to vaporised cannabis (10.3% Δ-9-tetrahydrocannabinol [THC] and 0.05% cannabidiol [CBD]) or placebo from postnatal day 23 to day 51. Following cessation of treatment, mice were examined for changes in brain structure and function using noninvasive multimodal magnetic resonance imaging (MRI). Data from voxel-based morphometry, diffusion weighted imaging and rest state functional connectivity were registered to and analysed with a 3D mouse atlas with 139 brain areas. Following imaging, mice were tested for their preference for a novel object. RESULTS: The effects were sexually dimorphic with females showing a unique distribution and inverse correlation between measures of fractional anisotropy and apparent diffusion coefficient localised to the forebrain and hindbrain. In contrast males displayed significant increased functional coupling with the thalamus, hypothalamus and brainstem reticular activating system as compared with controls. Cannabis males also presented with altered hippocampal coupling and deficits in cognitive function. CONCLUSION: Chronic exposure to inhaled vaporised cannabis had significant effects on brain structure and function in early adulthood corroborating much of the literature. Females presented with changes in grey matter microarchitecture, while males showed altered functional connectivity in hippocampal circuitry and deficits in object recognition.

Acute neuroradiological, behavioral, and physiological effects of nose-only exposure to vaporized cannabis in C57BL/6 mice.

Objective: The rapid increase of cannabis consumption reinforces the need to elucidate the health hazards of this practice. The presence of fine particulate matter in cannabis smoke and vapor poses a major concern, as it may contribute to cardiopulmonary disease. To facilitate the assessment of risks associated with cannabis inhalation, we developed and characterized a method for exposing mice to cannabis in a way that mimics the delivery of the drug to the airways of smokers. Materials and Methods: Cannabis (10.3% THC, 0.05% CBD) was vaporized to generate aerosols with a reproducible particle profile. Aerosols were acutely delivered to male, adult C57BL/6 mice via a nose-only exposure system. Serum THC levels were measured for increasing cannabis doses. Blood pressure and heart rate were recorded at baseline and following exposure. Behavioral response to cannabis inhalation in the open field was documented. Awake neurological activity upon cannabis exposure was monitored using BOLD fMRI.Results and Discussion: Cannabis aerosols contained particles with count median diameter of 243 ± 39 nm and geometric standard deviation of 1.56 ± 0.06. Blood serum THC levels increased linearly with aerosolized mass and peaked at 136 ± 5 ng/mL. Cannabis inhalation decreased heart rate and blood pressure but promoted anxiety-like behavior. Observed differences in BOLD activation volumes linked cannabis to increased awareness to sensory stimuli and reduced behavioral arousal.Conclusions: Quantified physiological, behavioral, and neurological responses served as validation for our mouse model of cannabis inhalation. Animal models of aerosol exposure will be instrumental for uncovering the health outcomes of chronic cannabis use.

Working memory capacity and task goals modulate error-related ERPs.

The present study investigated individual differences in information processing following errant behavior. Participants were initially classified as high or as low working memory capacity using the Operation Span Task. In a subsequent session, they then performed a high congruency version of the flanker task under both speed and accuracy stress. We recorded ERPs and behavioral measures of accuracy and response time in the flanker task with a primary focus on processing following an error. The error-related negativity was larger for the high working memory capacity group than for the low working memory capacity group. The positivity following an error (Pe) was modulated to a greater extent by speed-accuracy instruction for the high working memory capacity group than for the low working memory capacity group. These data help to explicate the neural bases of individual differences in working memory capacity and cognitive control.

The smartphone and the driver's cognitive workload: A comparison of Apple, Google, and Microsoft's intelligent personal assistants.

The goal of this research was to examine the impact of voice-based interactions using 3 different intelligent personal assistants (Apple's Siri, Google's Google Now for Android phones, and Microsoft's Cortana) on the cognitive workload of the driver. In 2 experiments using an instrumented vehicle on suburban roadways, we measured the cognitive workload of drivers when they used the voice-based features of each smartphone to place a call, select music, or send text messages. Cognitive workload was derived from primary task performance through video analysis, secondary-task performance using the Detection Response Task (DRT), and subjective mental workload. We found that workload was significantly higher than that measured in the single-task drive. There were also systematic differences between the smartphones: The Google system placed lower cognitive demands on the driver than the Apple and Microsoft systems, which did not differ. Video analysis revealed that the difference in mental workload between the smartphones was associated with the number of system errors, the time to complete an action, and the complexity and intuitiveness of the devices. Finally, surprisingly high levels of cognitive workload were observed when drivers were interacting with the devices: "on-task" workload measures did not systematically differ from that associated with a mentally demanding Operation Span (OSPAN) task. The analysis also found residual costs associated using each of the smartphones that took a significant time to dissipate. The data suggest that caution is warranted in the use of smartphone voice-based technology in the vehicle because of the high levels of cognitive workload associated with these interactions. (PsycINFO Database Record

Audiogenic seizure activity following HSV-1 GAD65 sense or antisense injection into inferior colliculus of Long-Evans rat.

Herpes virus technology involving manipulation of GAD65 was used to study effects on audiogenic seizures (AGS). Audiogenic seizure behaviors were examined following injections of replication-defective herpes simplex virus (HSV-1) vectors incorporating sense or antisense toward GAD65 along with 10% lac-Z into the central nucleus of inferior colliculus (CNIC) of Long-Evans rats. In seizure-sensitive animals developmentally primed by intense sound exposure, injection of GAD65 in the sense orientation increased wild running latencies and reduced incidence of clonus compared with lac-Z only, unoperated, and vehicle seizure groups. In contrast, infection of CNIC with GAD65 antisense virus resulted in 100% incidence of wild running and clonus behaviors in AGS animals. Unprimed animals not operated continued to show uniform absence of seizure activity. Administration of GAD65 antisense virus into CNIC produced novel wild running and clonus behaviors in some unprimed animals. Staining for ß-galactosidase in all vector animals revealed no differences in pattern or numbers of immunoreactive cells at injection sites. Qualitatively, typical small and medium multipolar/stellate and medium fusiform neurons appeared in the CNIC of vector animals. These results demonstrate that HSV-1 vector constructs implanted into the CNIC can predictably influence incidence and severity of AGS and suggest that viral vectors can be useful in studying GABA mechanisms with potential for therapeutic application in epilepsy. This article is part of a Special Issue entitled "Genetic and Reflex Epilepsies, Audiogenic Seizures and Strains: From Experimental Models to the Clinic".

Talking to your car can drive you to distraction.

This research examined the impact of in-vehicle information system (IVIS) interactions on the driver's cognitive workload; 257 subjects participated in a weeklong evaluation of the IVIS interaction in one of ten different model-year 2015 automobiles. After an initial assessment of the cognitive workload associated with using the IVIS, participants took the vehicle home for 5 days and practiced using the system. At the end of the 5 days of practice, participants returned and the workload of these IVIS interactions was reassessed. The cognitive workload was found to be moderate to high, averaging 3.34 on a 5-point scale and ranged from 2.37 to 4.57. The workload was associated with the intuitiveness and complexity of the system and the time it took participants to complete the interaction. The workload experienced by older drivers was significantly greater than that experienced by younger drivers performing the same operations. Practice did not eliminate the interference from IVIS interactions. In fact, IVIS interactions that were difficult on the first day were still relatively difficult to perform after a week of practice. Finally, there were long-lasting residual costs after the IVIS interactions had terminated. The higher levels of workload should serve as a caution that these voice-based interactions can be cognitively demanding and ought not to be used indiscriminately while operating a motor vehicle.

Assessing Cognitive Distraction in the Automobile.

OBJECTIVE: The objective was to establish a systematic framework for measuring and understanding cognitive distraction in the automobile. BACKGROUND: Driver distraction from secondary in-vehicle activities is increasingly recognized as a significant source of injuries and fatalities on the roadway. METHOD: Across three studies, participants completed eight in-vehicle tasks commonly performed by the driver of an automobile. Primary, secondary, subjective, and physiological measures were collected and integrated into a cognitive distraction scale. RESULTS: In-vehicle activities, such as listening to the radio or an audio book, were associated with a low level of cognitive workload; the conversation activities of talking to a passenger in the vehicle or conversing with a friend on a handheld or hands-free cell phone were associated with a moderate level of cognitive workload; and using a speech-to-text interfaced e-mail system involved a high level of cognitive workload. CONCLUSION: The research established that there are significant impairments to driving that stem from the diversion of attention from the task of operating a motor vehicle and that the impairments to driving are directly related to the cognitive workload of these in-vehicle activities. Moreover, the adoption of voice-based systems in the vehicle may have unintended consequences that adversely affect traffic safety. APPLICATION: These findings can be used to help inform scientifically based policies on driver distraction, particularly as they relate to cognitive distraction stemming from the diversion of attention to other concurrent activities in the vehicle.

Working memory's workload capacity.

We examined the role of dual-task interference in working memory using a novel dual two-back task that requires a redundant-target response (i.e., a response that neither the auditory nor the visual stimulus occurred two back versus a response that one or both occurred two back) on every trial. Comparisons with performance on single two-back trials (i.e., with only auditory or only visual stimuli) showed that dual-task demands reduced both speed and accuracy. Our task design enabled a novel application of Townsend and Nozawa's (Journal of Mathematical Psychology 39: 321-359, 1995) workload capacity measure, which revealed that the decrement in dual two-back performance was mediated by the sharing of a limited amount of processing capacity. Relative to most other single and dual n-back tasks, performance measures for our task were more reliable, due to the use of a small stimulus set that induced a high and constant level of proactive interference. For a version of our dual two-back task that minimized response bias, accuracy was also more strongly correlated with complex span than has been found for most other single and dual n-back tasks.

mRNA-targeted fluorescent in situ hybridization (FISH) of Gram-negative bacteria without template amplification or tyramide signal amplification.

Technologies are needed to study gene expression at the level of individual cells within a population or microbial community. Fluorescent in situ hybridization (FISH) supplies high-resolution spatial information and has been widely applied to study microbial communities at the rRNA level. While mRNA-targeted FISH has been popular for studying gene expression in eukaryotic cells, very little success has been achieved with prokaryotes. At present, detection of specific mRNAs in individual prokaryotic cells requires the use of in situ RT-PCR or tyramide signal amplification (TSA). In this study we used DNA oligonucleotide probes labeled with a single near-infrared dye in FISH assays to detect multi-copy plasmid-based and endogenous mRNA molecules in Escherichia coli and Shewanella oneidensis MR-1. We took advantage of the fact that there is much less background signal produced by biological materials and support matrices in the near-infrared spectrum and thus long camera exposure times could be used. In addition, we demonstrate that a combination of probes targeting both rRNA and mRNA could be successfully employed within the same FISH assay. These results, as well as ongoing R&D improvements in NIR and infrared dyes, indicate that the FISH approach we demonstrated could be applied in certain environmental settings to monitor gene expression in mixed populations.

Proteomic analysis of Salmonella enterica serovar typhimurium isolated from RAW 264.7 macrophages: identification of a novel protein that contributes to the replication of serovar typhimurium inside macrophages.

To evade host resistance mechanisms, Salmonella enterica serovar Typhimurium (STM), a facultative intracellular pathogen, must alter its proteome following macrophage infection. To identify new colonization and virulence factors that mediate STM pathogenesis, we have isolated STM cells from RAW 264.7 macrophages at various time points following infection and used a liquid chromatography-mass spectrometry-based proteomic approach to detect the changes in STM protein abundance. Because host resistance to STM infection is strongly modulated by the expression of a functional host-resistant regulator, i.e. natural resistance-associated macrophage protein 1 (Nramp1, also called Slc11a1), we have also examined the effects of Nramp1 activity on the changes of STM protein abundances. A total of 315 STM proteins have been identified from isolated STM cells, which are largely housekeeping proteins whose abundances remain relatively constant during the time course of infection. However, 39 STM proteins are strongly induced after infection, suggesting their involvement in modulating colonization and infection. Of the 39 induced proteins, 6 proteins are specifically modulated by Nramp1 activity, including STM3117, as well as STM3118-3119 whose time-dependent abundance changes were confirmed using Western blot analysis. Deletion of the gene encoding STM3117 resulted in a dramatic reduction in the ability of STM to colonize wild-type RAW 264.7 macrophages, demonstrating a critical involvement of STM3117 in promoting the replication of STM inside macrophages. The predicted function common for STM3117-3119 is biosynthesis and modification of the peptidoglycan layer of the STM cell wall.

High efficiency recovery and epitope-specific sorting of an scFv yeast display library.

In order to more productively utilize the rich source of antigen-specific reagents present in the previously described non-immune single chain fragment variable (scFv) yeast display library, one must be able to efficiently isolate and characterize clones within the library. To this end, we have developed and validated a magnetic bead sorting technique utilizing the Miltenyi Macs system to recover greater than 90% of the antigen-specific clones present in the library. In combination with flow cytometry, we rapidly reduced diversity and enriched for antigen-specific clones in three rounds of selection. Furthermore, we demonstrate the use of pre-existing monoclonal antibodies (mAbs) for antigen labeling and subsequent flow cytometric sorting and characterization of epitope-specific scFv. Combining these two improvements in library screening allowed isolation and characterization of three epitope-specific scFv, including a previously uncharacterized epitope to a 6-kDa protein, epidermal growth factor.

Yeast mating for combinatorial Fab library generation and surface display.

Yeast display of antibody fragments has proven to be an efficient and productive means for directed evolution of single chain Fv antibodies for increased affinity and thermal stability, and more recently for the display and screening of a non-immune library. In this paper, we describe an elegant and simple method for constructing large combinatorial Fab libraries for display on the surface of Saccharomyces cerevisiae, from modestly sized, and easily constructed, heavy and light chain libraries. To this end, we have constructed a set of yeast strains and a two vector system for heavy chain and light chain surface display of Fab fragments with free native amino termini. Through yeast mating of the haploid libraries, a very large heterodimeric immune Fab library was displayed on the diploids and high affinity antigen specific Fabs were isolated from the library.

Flow-cytometric isolation of human antibodies from a nonimmune Saccharomyces cerevisiae surface display library.

A nonimmune library of 10(9) human antibody scFv fragments has been cloned and expressed on the surface of yeast, and nanomolar-affinity scFvs routinely obtained by magnetic bead screening and flow-cytometric sorting. The yeast library can be amplified 10(10)-fold without measurable loss of clonal diversity, allowing its effectively indefinite expansion. The expression, stability, and antigen-binding properties of >50 isolated scFv clones were assessed directly on the yeast cell surface by immunofluorescent labeling and flow cytometry, obviating separate subcloning, expression, and purification steps and thereby expediting the isolation of novel affinity reagents. The ability to use multiplex library screening demonstrates the usefulness of this approach for high-throughput antibody isolation for proteomics applications.

Transplantation of M213-2O cells with enhanced GAD67 expression into the inferior colliculus alters audiogenic seizures.

The purpose of the present study was to examine the effects of GABA-producing cell transplants on audiogenic seizures (AGS). The M213-2O cell line was derived from fetal rat striatum and has GABAergic properties. This cell line was further modified to express human GAD(67) and produce elevated levels of GABA. The present study compares the effects of parent M213-2O cell transplants with those of GAD(67)-modified M213-2O cells in AGS-prone Long-Evans rats. Two weeks following implantation of engineered cells, latency to AGS-typical wild running was increased compared to nonimplanted subjects. Survival of the transplanted cells was confirmed by immunochemical labeling of GAD(67) and Epstein-Barr virus nuclear antigen. These findings support the use of GABA-producing cell lines to modify seizure activity.