Direct observation of liquid crystals using cryo-TEM: Specimen preparation and low-dose imaging

Liquid crystals (LCs) represent a challenging group of materials for direct transmission electron microscopy (TEM) studies due to the complications in specimen preparation and the severe radiation damage. In this paper, we summarize a series of specimen preparation methods, including thin film and cryo-sectioning approaches, as a comprehensive toolset enabling high-resolution direct cryo-TEM observation of a broad range of LCs. We also present comparative analysis using cryo-TEM and replica freeze-fracture TEM on both thermotropic and lyotropic LCs. In addition to the revisits of previous practices, some new concepts are introduced, e.g., suspended thermotropic LC thin films, combined high-pressure freezing and cryo-sectioning of lyotropic LCs, and the complementary applications of direct TEM and indirect replica TEM techniques. The significance of subnanometer resolution cryo-TEM observation is demonstrated in a few important issues in LC studies, including providing direct evidences for the existence of nanoscale smectic domains in nematic bent-core thermotropic LCs, comprehensive understanding of the twist-bend nematic phase, and probing the packing of columnar aggregates in lyotropic chromonic LCs. Direct TEM observation opens ways to a variety of TEM techniques, suggesting that TEM (replica, cryo, and in situ techniques), in general, may be a promising part of the solution to the lack of effective structural probe at the molecular scale in LC studies. Microsc. Res. Tech. 77:754-772, 2014. © 2014 Wiley Periodicals, Inc.

Lesion location predicts transient and extended risk of aspiration after supratentorial ischemic stroke

BACKGROUND AND PURPOSE To assess the association of lesion location and risk of aspiration and to establish predictors of transient versus extended risk of aspiration after supratentorial ischemic stroke. METHODS Atlas-based localization analysis was performed in consecutive patients with MRI-proven first-time acute supratentorial ischemic stroke. Standardized swallowing assessment was carried out within 8±18 hours and 7.8±1.2 days after admission. RESULTS In a prospective, longitudinal analysis, 34 of 94 patients (36%) were classified as having acute risk of aspiration, which was extended (≥7 days) or transient (<7 days) in 17 cases. There were no between-group differences in age, sex, cause of stroke, risk factors, prestroke disability, lesion side, or the degree of age-related white-matter changes. Correcting for stroke volume and National Institutes of Health Stroke Scale with a multiple logistic regression model, significant adjusted odds ratios in favor of acute risk of aspiration were demonstrated for the internal capsule (adjusted odds ratio, 6.2; P<0.002) and the insular cortex (adjusted odds ratio, 4.8; P<0.003). In a multivariate model of extended versus transient risk of aspiration, combined lesions of the frontal operculum and insular cortex was the only significant independent predictor of poor recovery (adjusted odds ratio, 33.8; P<0.008). CONCLUSIONS Lesions of the insular cortex and the internal capsule are significantly associated with acute risk of aspiration after stroke. Combined ischemic infarctions of the frontal operculum and the insular cortex are likely to cause extended risk of aspiration in stroke patients, whereas risk of aspiration tends to be transient in subcortical stroke.

Diffusion-weighted MR imaging of upper abdominal organs: field strength and intervendor variability of apparent diffusion coefficients

PURPOSE To determine the variability of apparent diffusion coefficient (ADC) values in various anatomic regions in the upper abdomen measured with magnetic resonance (MR) systems from different vendors and with different field strengths. MATERIALS AND METHODS Ten healthy men (mean age, 36.6 years ± 7.7 [standard deviation]) gave written informed consent to participate in this prospective ethics committee-approved study. Diffusion-weighted (DW) MR imaging was performed in each subject with 1.5- and 3.0-T MR systems from each of three vendors at two institutions. Two readers independently measured ADC values in seven upper abdominal regions (left and right liver lobe, gallbladder, pancreas, spleen, and renal cortex and medulla). ADC values were tested for interobserver differences, as well as for differences related to field strength and vendor, with repeated-measures analysis of variance; coefficients of variation (CVs) and variance components were calculated. RESULTS Interreader agreement was excellent (intraclass coefficient, 0.876). ADC values were (77.5-88.8) ×10(-5) mm(2)/sec in the spleen and (250.6-278.5) ×10(-5) mm(2)/sec in the gallbladder. There were no significant differences between ADC values measured at 1.5 T and those measured at 3.0 T in any anatomic region (P >.10 for all). In two of seven regions at 1.5 T (left and right liver lobes, P < .023) and in four of seven regions at 3.0 T (left liver lobe, pancreas, and renal cortex and medulla, P < .008), intervendor differences were significant. CVs ranged from 7.0% to 27.1% depending on the anatomic location. CONCLUSION Despite significant intervendor differences in ADC values of various anatomic regions of the upper abdomen, ADC values of the gallbladder, pancreas, spleen, and kidney may be comparable between MR systems from different vendors and between different field strengths.

Localization and production of peptide endocannabinoids in the rodent CNS and adrenal medulla.

The endocannabinoid system (ECS) comprises the cannabinoid receptors CB1 and CB2 and their endogenous arachidonic acid-derived agonists 2-arachidonoyl glycerol and anandamide, which play important neuromodulatory roles. Recently, a novel class of negative allosteric CB1 receptor peptide ligands, hemopressin-like peptides derived from alpha hemoglobin, has been described, with yet unknown origin and function in the CNS. Using monoclonal antibodies we now identified the localization of RVD-hemopressin (pepcan-12) and N-terminally extended peptide endocannabinoids (pepcans) in the CNS and determined their neuronal origin. Immunohistochemical analyses in rodents revealed distinctive and specific staining in major groups of noradrenergic neurons, including the locus coeruleus (LC), A1, A5 and A7 neurons, which appear to be major sites of production/release in the CNS. No staining was detected in dopaminergic neurons. Peptidergic axons were seen throughout the brain (notably hippocampus and cerebral cortex) and spinal cord, indicative of anterograde axonal transport of pepcans. Intriguingly, the chromaffin cells in the adrenal medulla were also strongly stained for pepcans. We found specific co-expression of pepcans with galanin, both in the LC and adrenal gland. Using LC-MS/MS, pepcan-12 was only detected in non-perfused brain (∼40 pmol/g), suggesting that in the CNS it is secreted and present in extracellular compartments. In adrenal glands, significantly more pepcan-12 (400-700 pmol/g) was measured in both non-perfused and perfused tissue. Thus, chromaffin cells may be a major production site of pepcan-12 found in blood. These data uncover important areas of peptide endocannabinoid occurrence with exclusive noradrenergic immunohistochemical staining, opening new doors to investigate their potential physiological function in the ECS. This article is part of a Special Issue entitled 'Fluorescent Neuro-Ligands'.

(Table 1) Characteristics of Klebsormidium (filamentous green algae) strains from the Antarctic, Arctic and Slovakia

The freezing and desiccation tolerance of 12 Klebsormidium strains, isolated from various habitats (aero-terrestrial, terrestrial, and hydro-terrestrial) from distinct geographical regions (Antarctic - South Shetlands, King George Island, Arctic - Ellesmere Island, Svalbard, Central Europe - Slovakia) were studied. Each strain was exposed to several freezing (-4°C, -40°C, -196°C) and desiccation (+4°C and +20°C) regimes, simulating both natural and semi-natural freeze-thaw and desiccation cycles. The level of resistance (or the survival capacity) was evaluated by chlorophyll a content, viability, and chlorophyll fluorescence evaluations. No statistical differences (Kruskal-Wallis tests) between strains originating from different regions were observed. All strains tested were highly resistant to both freezing and desiccation injuries. Freezing down to -196°C was the most harmful regime for all studied strains. Freezing at -4°C did not influence the survival of studied strains. Further, freezing down to -40°C (at a speed of 4°C/min) was not fatal for most of the strains. RDA analysis showed that certain Antarctic and Arctic strains did not survive desiccation at +4°C; however, freezing at -40°C, as well as desiccation at +20 °C was not fatal to them. On the other hand, other strains from the Antarctic, the Arctic, and Central Europe (Slovakia) survived desiccation at temperatures of +4°C, and freezing down to -40°C. It appears that species of Klebsormidium which occupy an environment where both seasonal and diurnal variations of water availability prevail, are well adapted to freezing and desiccation injuries. Freezing and desiccation tolerance is not species-specific nor is the resilience only found in polar strains as it is also a feature of temperate strains.

NDT for concrete under accelerated freeze/thaw tests and surface scaling

Freezing of water or salt solution in concrete pores is a main cause for severe damage and significant reduction of the service life. Most of the freeze-thaw (F-T) accelerated tests measure the scaling of concrete by weighting. This paper presents complementary procedures based on the use of strain gages and ultrasonic pulse velocity (UPV) for measuring the deterioration of concrete due to freezing and thawing. These non-destructive testing (NDT) procedures are applied to two types of concretes, one susceptible to F-T damage and the other does not. The results show a good correlation between scaling and the measurements obtained with NDT. Showing NDT the advantage to detect before the damage and to perform continuous measurement

Random positions of dendritic spines in human cerebral cortex

Dendritic spines establish most excitatory synapses in the brain and are located in Purkinje cell’s dendrites along helical paths, perhaps maximizing the probability to contact different axons. To test whether spine helixes also occur in neocortex, we reconstructed >500 dendritic segments from adult human cortex obtained from autopsies. With Fourier analysis and spatial statistics, we analyzed spine position along apical and basal dendrites of layer 3 pyramidal neurons from frontal, temporal, and cingulate cortex. Although we occasionally detected helical positioning, for the great majority of dendrites we could not reject the null hypothesis of spatial randomness in spine locations, either in apical or basal dendrites, in neurons of different cortical areas or among spines of different volumes and lengths. We conclude that in adult human neocortex spine positions are mostly random. We discuss the relevance of these results for spine formation and plasticity and their functional impact for cortical circuits.

Random positioning of dendritic spines in the human cerebral cortex

Dendritic spines establish most excitatory synapses in the brain and are located in Purkinje cell?s dendrites along helical paths, perhaps maximizing the probability to contact different axons. To test whether spine helixes also occur in neocortex, we reconstructed ?500 dendritic segments from adult human cortex obtained from autopsies. With Fourier analysis and spatial statistics, we analyzed spine position along apical and basal dendrites of layer 3 pyramidal neurons from frontal, temporal, and cingulate cortex. Although we occasionally detected helical positioning, for the great majority of dendrites we could not reject the null hypothesis of spatial randomness in spine locations, either in apical or basal dendrites, in neurons of different cortical areas or among spines of different volumes and lengths. We conclude that in adult human neocortex spine positions are mostly random. We discuss the relevance of these results for spine formation and plasticity and their functional impact for cortical circuits.

Reexamination of the mechanism of hydroxyl radical adducts formed from the reaction between familial amyotrophic lateral sclerosis-associated Cu,Zn superoxide dismutase mutants and H2O2

Amyotrophic lateral sclerosis (ALS) involves the progressive degeneration of motor neurons in the spinal cord and motor cortex. Mutations to Cu,Zn superoxide dismutase (SOD) linked with familial ALS are reported to increase hydroxyl radical adduct formation from hydrogen peroxide as measured by spin trapping with 5,5′-dimethyl-1-pyrrolline N-oxide (DMPO). In the present study, we have used oxygen-17-enriched water and H2O2 to reinvestigate the mechanism of DMPO/⋅OH formation from the SOD and SOD mutants. The relative ratios of DMPO/⋅17OH and DMPO/⋅16OH formed in the Fenton reaction were 90% and 10%, respectively, reflecting the ratios of H217O2 to H216O2. The reaction of the WT SOD with H217O2 in bicarbonate/CO2 buffer yielded 63% DMPO/⋅17OH and 37% DMPO/⋅16OH. Similar results were obtained from the reaction between familial ALS SOD mutants and H217O2: DMPO/⋅17OH (64%); DMPO/⋅16OH (36%) from A4V and DMPO/⋅17OH (62%); and DMPO/⋅16OH (38%) from G93A. These results were confirmed further by using 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide spin trap, a phosphorylated analog of DMPO. Contrary to earlier reports, the present results indicate that a significant fraction of DMPO/⋅OH formed during the reaction of SOD and familial ALS SOD mutants with H2O2 is derived from the incorporation of oxygen from water due to oxidation of DMPO to DMPO/⋅OH presumably via DMPO radical cation. No differences were detected between WT and mutant SODs, neither in the concentration of DMPO/⋅OH or DEPMPO/⋅OH formed nor in the relative incorporation of oxygen from H2O2 or water.

Dissociation of response conflict, attentional selection, and expectancy with functional magnetic resonance imaging

Two different attentional networks have been associated with visuospatial attention and conflict resolution. In most situations either one of the two networks is active or both are increased in activity together. By using functional magnetic resonance imaging and a flanker task, we show conditions in which one network (anterior attention system) is increased in activity whereas the other (visuospatial attention system) is reduced, showing that attentional conflict and selection are separate aspects of attention. Further, we distinguish between neural systems involved in different forms of conflict. Specifically, we dissociate patterns of activity in the basal ganglia and insula cortex during simple violations in expectancies (i.e., sudden changes in the frequency of an event) from patterns of activity in the anterior attention system specifically correlated with response conflict as evidenced by longer response latencies and more errors. These data provide a systems-level approach in understanding integrated attentional networks.

Memory's echo: Vivid remembering reactivates sensory-specific cortex

A fundamental question in human memory is how the brain represents sensory-specific information during the process of retrieval. One hypothesis is that regions of sensory cortex are reactivated during retrieval of sensory-specific information (1). Here we report findings from a study in which subjects learned a set of picture and sound items and were then given a recall test during which they vividly remembered the items while imaged by using event-related functional MRI. Regions of visual and auditory cortex were activated differentially during retrieval of pictures and sounds, respectively. Furthermore, the regions activated during the recall test comprised a subset of those activated during a separate perception task in which subjects actually viewed pictures and heard sounds. Regions activated during the recall test were found to be represented more in late than in early visual and auditory cortex. Therefore, results indicate that retrieval of vivid visual and auditory information can be associated with a reactivation of some of the same sensory regions that were activated during perception of those items.

Neuroimaging of cerebral activations and deactivations associated with hypercapnia and hunger for air

There are defined medullary, mesencephalic, hypothalamic, and thalamic functions in regulation of respiration, but knowledge of cortical control and the elements subserving the consciousness of breathlessness and air hunger is limited. In nine young adults, air hunger was produced acutely by CO2 inhalation. Comparisons were made with inhalation of a N2/O2 gas mixture with the same apparatus, and also with paced breathing, and with eyes closed rest. A network of activations in pons, midbrain (mesencephalic tegmentum, parabrachial nucleus, and periaqueductal gray), hypothalamus, limbic and paralimbic areas (amygdala and periamygdalar region) cingulate, parahippocampal and fusiform gyrus, and anterior insula were seen along with caudate nuclei and pulvinar activations. Strong deactivations were seen in dorsal cingulate, posterior cingulate, and prefrontal cortex. The striking response of limbic and paralimbic regions points to these structures having a singular role in the affective sequelae entrained by disturbance of basic respiratory control whereby a process of which we are normally unaware becomes a salient element of consciousness. These activations and deactivations include phylogenetically ancient areas of allocortex and transitional cortex that together with the amygdalar/periamygdalar region may subserve functions of emotional representation and regulation of breathing.

Forebrain mechanisms of nociception and pain: Analysis through imaging

Pain is a unified experience composed of interacting discriminative, affective-motivational, and cognitive components, each of which is mediated and modulated through forebrain mechanisms acting at spinal, brainstem, and cerebral levels. The size of the human forebrain in relation to the spinal cord gives anatomical emphasis to forebrain control over nociceptive processing. Human forebrain pathology can cause pain without the activation of nociceptors. Functional imaging of the normal human brain with positron emission tomography (PET) shows synaptically induced increases in regional cerebral blood flow (rCBF) in several regions specifically during pain. We have examined the variables of gender, type of noxious stimulus, and the origin of nociceptive input as potential determinants of the pattern and intensity of rCBF responses. The structures most consistently activated across genders and during contact heat pain, cold pain, cutaneous laser pain or intramuscular pain were the contralateral insula and anterior cingulate cortex, the bilateral thalamus and premotor cortex, and the cerebellar vermis. These regions are commonly activated in PET studies of pain conducted by other investigators, and the intensity of the brain rCBF response correlates parametrically with perceived pain intensity. To complement the human studies, we developed an animal model for investigating stimulus-induced rCBF responses in the rat. In accord with behavioral measures and the results of human PET, there is a progressive and selective activation of somatosensory and limbic system structures in the brain and brainstem following the subcutaneous injection of formalin. The animal model and human PET studies should be mutually reinforcing and thus facilitate progress in understanding forebrain mechanisms of normal and pathological pain.

Frontoparietal cortical networks for directing attention and the eye to visual locations: Identical, independent, or overlapping neural systems?

Functional anatomical and single-unit recording studies indicate that a set of neural signals in parietal and frontal cortex mediates the covert allocation of attention to visual locations, as originally proposed by psychological studies. This frontoparietal network is the source of a location bias that interacts with extrastriate regions of the ventral visual system during object analysis to enhance visual processing. The frontoparietal network is not exclusively related to visual attention, but may coincide or overlap with regions involved in oculomotor processing. The relationship between attention and eye movement processes is discussed at the psychological, functional anatomical, and cellular level of analysis.

Effects of anesthesia on functional activation of cerebral blood flow and metabolism

Functional brain mapping based on changes in local cerebral blood flow (lCBF) or glucose utilization (lCMRglc) induced by functional activation is generally carried out in animals under anesthesia, usually α-chloralose because of its lesser effects on cardiovascular, respiratory, and reflex functions. Results of studies on the role of nitric oxide (NO) in the mechanism of functional activation of lCBF have differed in unanesthetized and anesthetized animals. NO synthase inhibition markedly attenuates or eliminates the lCBF responses in anesthetized animals but not in unanesthetized animals. The present study examines in conscious rats and rats anesthetized with α-chloralose the effects of vibrissal stimulation on lCMRglc and lCBF in the whisker-to-barrel cortex pathway and on the effects of NO synthase inhibition with NG-nitro-l-arginine methyl ester (l-NAME) on the magnitude of the responses. Anesthesia markedly reduced the lCBF and lCMRglc responses in the ventral posteromedial thalamic nucleus and barrel cortex but not in the spinal and principal trigeminal nuclei. l-NAME did not alter the lCBF responses in any of the structures of the pathway in the unanesthetized rats and also not in the trigeminal nuclei of the anesthetized rats. In the thalamus and sensory cortex of the anesthetized rats, where the lCBF responses to stimulation had already been drastically diminished by the anesthesia, l-NAME treatment resulted in loss of statistically significant activation of lCBF by vibrissal stimulation. These results indicate that NO does not mediate functional activation of lCBF under physiological conditions.