Principles of two-photon excitation microscopy and its applications to neuroscience

The brain is complex and dynamic. The spatial scales of interest to the neurobiologist range from individual synapses (approximately 1 microm) to neural circuits (centimeters); the timescales range from the flickering of channels (less than a millisecond) to long-term memory (years). Remarkably, fluorescence microscopy has the potential to revolutionize research on all of these spatial and temporal scales. Two-photon excitation (2PE) laser scanning microscopy allows high-resolution and high-sensitivity fluorescence microscopy in intact neural tissue, which is hostile to traditional forms of microscopy. Over the last 10 years, applications of 2PE, including microscopy and photostimulation, have contributed to our understanding of a broad array of neurobiological phenomena, including the dynamics of single channels in individual synapses and the functional organization of cortical maps. Here we review the principles of 2PE microscopy, highlight recent applications, discuss its limitations, and point to areas for future research and development.     

Might cardiac catheterization influence diurnal rhythm of the steroid stress hormones secretion?

Glucocorticoids (GCs) are steroid hormones produced by the adrenal cortex in reaction to stress stimuli. GCs production is not stable over a 24-hour period; the plasma concentration peaks in the morning (approximately upon awakening) and then the plasma levels decrease, reaching the nadir in the evening. In our experiments, the levels of cortisol, cortisone, DHEA and DHEAS were tested in young female pigs (n=23) during heart catheterization at two different day times (in the morning and in the afternoon). The non-parametric Mann-Whitney test for statistical analysis was used. We found only minimal statistical differences in studied markers between the morning and afternoon group (p>0.05). The absence of circadian variation in GCs levels could originate either at an early age of our experimental pigs, or in stressful conditions on the experiment day, or most likely the day before (e.g. social isolation, fasting, transport, and catheterization), respectively. We can conclude there is no difference in the stress load between morning and afternoon experiments, and therefore we can assume the stress load is not a limiting factor for the timing when catheterization should be preferably performed.     

Automated tracking of whiskers in videos of head fixed rodents

We have developed software for fully automated tracking of vibrissae (whiskers) in high-speed videos (>500 Hz) of head-fixed, behaving rodents trimmed to a single row of whiskers. Performance was assessed against a manually curated dataset consisting of 1.32 million video frames comprising 4.5 million whisker traces. The current implementation detects whiskers with a recall of 99.998% and identifies individual whiskers with 99.997% accuracy. The average processing rate for these images was 8 Mpx/s/cpu (2.6 GHz Intel Core2, 2 GB RAM). This translates to 35 processed frames per second for a 640 px×352 px video of 4 whiskers. The speed and accuracy achieved enables quantitative behavioral studies where the analysis of millions of video frames is required. We used the software to analyze the evolving whisking strategies as mice learned a whisker-based detection task over the course of 6 days (8148 trials, 25 million frames) and measure the forces at the sensory follicle that most underlie haptic perception.     

The upper paleolithic burial area at Predmostí: ritual and taphonomy

Paleoanthropological materials from Predmostí, recovered by J. Wankel in 1884, K.J. Maska in 1894, M. Kríz in 1895, and K. Absolon in 1928 (and probably 1930), represent one of the largest collections of early modern human remains. Unfortunately, most of these fossils were destroyed in 1945. The aim of this paper is to create a list of finds in accordance with the discovery dates, to place them into the spatial and chronological context of the site, and to compare them with the evidence from recent excavation in 2006. Two competing hypotheses are raised in the literature suggesting that the Predmostí individuals represent either a contemporary burial as a consequence of one catastrophic event, or a gradual accumulation of human bodies at one place. Whereas the first hypothesis is supported by the demographic structure of the buried group, including adults and children, the second interpretation is based on stratigraphic and taphonomic analysis of the burial area itself. Using the original documentation of Maska and other early researchers, and my own experience from recent excavation in the remaining part of the site, I attempt to reconstruct the plan of the site, with a focus on spatial distribution of the human fossils, especially in the main burial area. I suggest that the burial place was not the settlement center, but rather a peripheral and task-specific area. The determining factor for location of the burial area was likely the remarkable Skalka rock, a cliff that rose directly above the site. A long-term tendency to place the dead "below the rock" may have given rise to the accumulation of human remains at a single place, with a scatter of dispersed fragments in the vicinity. At this location, the human bodies were partly protected by soil coverage, limestone debris, and mammoth scapulae, but were also affected by postdepositional processes such as redeposition of sediments, predator activities, and later human activities, including the burial of additional corpses.     

Hematologic Values of Free-ranging Cebus cay and Cebus nigritus in Southern Brazil

We evaluated blood samples obtained from 80 free-ranging healthy capuchins (Cebus cay and C. nigritus) to establish hematological reference values and to assess the influence of sex and age on them. We caught the monkeys in the Paraná River region of Southern Brazil via manual or automatic traps. We anesthetized them intramuscularly with 3.6 mg/kg tiletamine/zolazepam hydrochlorides. After physical examinations, we divided the sample according to sex and age: 26 females (13 adults and 13 juveniles) and 54 males (27 adults and 27 juveniles). We collected blood and determined hematological values via traditional published methods. We analyzed data via 2-way ANOVA to test the effect of sex, age, and interactions between the 2 factors. The packed-cell volume was higher in adult males and the numbers of white blood cells and lymphocytes were higher in juveniles. There is no other significant difference.     

Antifungal Activity of the Essential Oil of Hyssop (Hyssopus officinalis)

The antifungal and fungicidal effects of hyssop ( Hyssopus officinalis ) oil and its individual components were studied in a series of in vitro and in vivo experiments. Mycelial growth of the plant pathogenic fungi Pyrenophora avenae and Pyricularia oryzae was completely inhibited by 0.4% hyssop oil. Volatile components diffusing from agar medium containing 0.4% hyssop oil also completely inhibited the growth of these two fungi. Various components of hyssop oil ( L -bornyl acetate, isopinocampheol and pinocamphone), used individually, reduced growth of P. avenae and, where combinations of individual components were used, any mixture containing isopinocampheol completely inhibited fungal growth. Growth of P. oryzae was less affected by individual components of the oil. Hyssop oil reduced germination of Botrytis fabae conidia and uredospores of Uromyces viciae-fabae , but in contrast to the data from in vitro experiments, its effects on pathogen infection were less clear cut. Thus, although 0.05% hyssop oil reduced rust infection of broad bean when applied 1, 2 or 3 days before, or 1 or 2 days after inoculation, its effects against barley powdery mildew and apple powdery mildew were variable. It is suggested that this variability might be the result of the volatile components of the oil diffusing away from leaf surfaces, thus reducing the concentration of active components on the leaf surface.     

Microbody proliferation in liver induced by nafenopin, a new hypolipidemic drug: comparison with CPIB

Nafenopin (2-methyl-2[P-(1,2,3,4-tetrahydro-1 naphthyl) phenoxy]- propionic acid, a phenolic ether with hypolipidemic properties, when administered by gavage at 100 mg/kg b wt daily for 1 to 2 weeks, caused a significant increase in the number of microbody profiles and simultaneous increase in catalase activity in livers of male rats. The concentration of catalase protein and the rate of incorporation of H³-δ-aminolevulinic acid into catalase fraction, as determined by immunochemical methods were approximately twice that of controls. The microbody proliferation resulting from nafenopin treatment was comparable to that induced by CPIB.