DiOLISTIC Labeling of Neurons from Rodent and Non-human Primate Brain Slices

DiOLISTIC staining uses the gene gun to introduce fluorescent dyes, such as DiI, into neurons of brain slices (Gan et al., 2009; O'Brien and Lummis, 2007; Gan et al., 2000). Here we provide a detailed description of each step required together with exemplary images of good and bad outcomes that will help when setting up the technique. In our experience, a few steps proved critical for the successful application of DiOLISTICS. These considerations include the quality of the DiI-coated bullets, the extent of fixative exposure, and the concentration of detergent used in the incubation solutions. Tips and solutions for common problems are provided

Facilitation of Task Performance and Removal of the Effects of Sleep Deprivation by an Ampakine (CX717) in Nonhuman Primates

The deleterious effects of prolonged sleep deprivation on behavior and cognition are a concern in modern society. Persons at risk for impaired performance and health-related issues resulting from prolonged sleep loss would benefit from agents capable of reducing these detrimental effects at the time they are sleep deprived. Agents capable of improving cognition by enhancing brain activity under normal circumstances may also have the potential to reduce the harmful or unwanted effects of sleep deprivation. The significant prevalence of excitatory α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamatergic receptors in the brain provides a basis for implementing a class of drugs that could act to alter or remove the effects of sleep deprivation. The ampakine CX717 (Cortex Pharmaceuticals), a positive allosteric modulator of AMPA receptors, was tested for its ability to enhance performance of a cognitive, delayed match-to-sample task under normal circumstances in well-trained monkeys, as well as alleviate the detrimental effects of 30–36 h of sleep deprivation. CX717 produced a dose-dependent enhancement of task performance under normal alert testing conditions. Concomitant measures of regional cerebral metabolic rates for glucose (CMR(glc)) during the task, utilizing positron emission tomography, revealed increased activity in prefrontal cortex, dorsal striatum, and medial temporal lobe (including hippocampus) that was significantly enhanced over normal alert conditions following administration of CX717. A single night of sleep deprivation produced severe impairments in performance in the same monkeys, accompanied by significant alterations in task-related CMR(glc) in these same brain regions. However, CX717 administered to sleep-deprived monkeys produced a striking removal of the behavioral impairment and returned performance to above-normal levels even though animals were sleep deprived. Consistent with this recovery, CMR(glc) in all but one brain region affected by sleep deprivation was also returned to the normal alert pattern by the drug. The ampakine CX717, in addition to enhancing cognitive performance under normal alert conditions, also proved effective in alleviating impairment of performance due to sleep deprivation. Therefore, the ability to activate specific brain regions under normal alert conditions and alter the deleterious effects of sleep deprivation on activity in those same regions indicate a potential role for ampakines in sustaining performance under these types of adverse conditions

Atlas-Guided Segmentation of Vervet Monkey Brain MRI

The vervet monkey is an important nonhuman primate model that allows the study of isolated environmental factors in a controlled environment. Analysis of monkey MRI often suffers from lower quality images compared with human MRI because clinical equipment is typically used to image the smaller monkey brain and higher spatial resolution is required. This, together with the anatomical differences of the monkey brains, complicates the use of neuroimage analysis pipelines tuned for human MRI analysis. In this paper we developed an open source image analysis framework based on the tools available within the 3D Slicer software to support a biological study that investigates the effect of chronic ethanol exposure on brain morphometry in a longitudinally followed population of male vervets. We first developed a computerized atlas of vervet monkey brain MRI, which was used to encode the typical appearance of the individual brain structures in MRI and their spatial distribution. The atlas was then used as a spatial prior during automatic segmentation to process two longitudinal scans per subject. Our evaluation confirms the consistency and reliability of the automatic segmentation. The comparison of atlas construction strategies reveals that the use of a population-specific atlas leads to improved accuracy of the segmentation for subcortical brain structures. The contribution of this work is twofold. First, we describe an image processing workflow specifically tuned towards the analysis of vervet MRI that consists solely of the open source software tools. Second, we develop a digital atlas of vervet monkey brain MRIs to enable similar studies that rely on the vervet model

Effects of Sleep Deprivation and Sleep Deprivation + CX717

<div><p>(A) Sleep deprivation condition disrupts DMS performance. Mean percent correct performance across animals for sessions following 30–36 h of sleep deprivation (nine of 11 monkeys in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030299#pbio-0030299-g002" target="_blank">Figure 2</a>A). Plus signs indicate significant mean differences (⁺<i>p</i> < 0.01, ⁺⁺<i>p</i> < 0.001) compared to the respective number of images curve in the normal vehicle condition; dashed line at 60% allows comparison with <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030299#pbio-0030299-g002" target="_blank">Figure 2</a>A.</p> <p>(B) Match response latencies in s (mean ± SEM) sorted for different trial types as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030299#pbio-0030299-g002" target="_blank">Figure 2</a>B. Asterisks indicate significant difference (*<i>p</i> < 0.01, **<i>p</i> < 0.001) at all delays compared to two-image trials. Plus signs indicate significant differences (⁺<i>p</i> < 0.01, ⁺⁺<i>p</i> < 0.001) compared to same trial types, and dashed line allows comparison with minimum latencies in normal vehicle condition (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030299#pbio-0030299-g002" target="_blank">Figure 2</a>B)<b>.</b></p> <p>(C) Sleep deprivation + CX717 condition. Effects of administration of CX717 (0.8 mg/kg, IV) 10 min prior to DMS session following 30–36 h of sleep deprivation. Mean (± SEM) percent correct DMS trials for same delay (1–30 sec, in 5-s increments) and #image (two to six) DMS conditions generated by same monkeys tested in (A). Plus signs indicate significant mean differences (⁺<i>p</i> < 0.01, ⁺⁺<i>p</i> < 0.001) compared to the respective number of images curve in the normal vehicle condition (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030299#pbio-0030299-g002" target="_blank">Figure 2</a>A); pound signs (^#<i>p</i> < 0.01,^##<i>p</i> < 0.001) indicate significant differences compared to the same trial types in sleep deprivation condition. Dashed reference line allows comparison with sleep deprivation (A) and normal vehicle conditions (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030299#pbio-0030299-g002" target="_blank">Figure 2</a>A).</p> <p>(D) Match response latencies for same sleep deprivation + CX717 condition shown in (C), sorted by delay and number of images and labeled as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030299#pbio-0030299-g002" target="_blank">Figure 2</a>B. Pound signs (^#<i>p</i> < 0.01, ^##<i>p</i> < 0.001) indicate significant differences compared to the same trial types in sleep deprivation condition (A).</p> <p>DOI: 10.1371/journal.pbio.0030299g004</p></div

Effects of Different Doses of CX717 on DMS Performance in Normal + CX717 Condition

<div><p>(A) CX717 is shown administered on consecutive sessions for nine monkeys over three dose ranges of CX717 (0.3–0.5 mg/kg, 0.8–1.0 mg/kg, and 1.5 mg/kg, IV). Each CX717 session (C, arrows) was interspersed with a single normal vehicle (V) session. Curves show mean (± SEM) percent correct performance over the entire session from each different monkey, as indicated by separate symbols. Arrows indicate CX717 sessions (also denoted by “C” on axis). Note escalating doses (0.3, 0.8, and 1.5 mg/kg) shown were in four of the nine monkeys.</p> <p>(B) Performance on split sessions (<i>n</i> = 4 monkeys) in which vehicle was administered at the start of the DMS session, and midway through same session CX717 (0.8 mg/kg, IV) was administered via remote pump without interruption. Mean (± SEM) percent correct performance over at least 50 trials was calculated separately for the first half (vehicle) and second half (CX717, 0.8 mg/kg) of the same session. Asterisks indicate a significant (** <i>p</i> < 0.001) increase in the second half of the session relative to the first (vehicle).</p> <p>(C) Overall mean dose-effect relationship of CX717 on normal alert DMS performance across monkeys (<i>n</i> = 9) in sessions in which each of the three doses (0.3, 0.8, and 1.5 mg/kg, IV) was administered. DMS sessions in which the three doses were received in an escalating order were compared with sessions in which the same doses were administered in a randomized order (F_[1,50] = 2.31, <i>p</i> = 13.5, NS). Asterisks indicate significant (*<i>p</i> < 0.01, **<i>p</i> < 0.001) difference relative to vehicle sessions.</p> <p>DOI: 10.1371/journal.pbio.0030299g003</p></div

SPM Maps of Brain Regions Showing Changes in Regional CMR_glc during Performance of DMS Task for Normal Vehicle and Normal + CX717 Conditions

<div><p>(A–E) Maps are shown for the comparison of CMR_glc during DMS performance in the normal vehicle to the baseline, no-task condition. CMR_glc was significantly increased during normal vehicle condition compared to baseline, no-task condition.</p> <p>(F–H) Maps are shown for the comparison of CMR_glc during DMS performance in the normal + CX717 condition to normal vehicle condition. CMR_glc was increased following the administration of CX717 (0.8 mg/kg, IV) as compared to normal vehicle condition (C–E).</p> <p>Regional changes are displayed on both horizontal section (A) and coronal sections (B–H) of MR images of rhesus monkey brain at the level of the motor and premotor cortex (A and B), DPFC (C and F), MTL (D and G), and parietal cortex (E and H). Statistical parametric maps were generated using SPM99 software. Colors indicate the location of clusters with a height (magnitude) threshold of <i>p</i> < 0.05 and spatial extent greater than 50 voxels. Color bar indicates <i>t</i> values for the comparisons (red, <i>t</i> = 2.0, <i>p</i> < 0.05; yellow, <i>t</i> = 5.0, <i>p</i> < 0.001). Note: left and right half of hemisphere are shown on left and right of images respectively. Area 6, premotor cortex; Cere, cerebellum; FEF, frontal eye fields; Prec, precuneus; S1, primary somatosensory cortex.</p> <p>DOI: 10.1371/journal.pbio.0030299g007</p></div