Deformation of tissue sections in the z-axis can bias optical disector counting. When samples of particle densities are not representative for the entire tissue section, significant bias of estimated numbers can result. To assess the occurrence, prevalence, extent, sequence of events, and causes of z-axis distortion, the distribution of neuronal nucleoli in thick paraffin and vibratome sections was determined in chicken, rodent, and human brain tissues. When positions of neuronal nucleoli were measured in the z-axis, nucleoli were more frequent at the surfaces (bottom and top) of tissue sections than in the core. This nonlinear z-axis distribution was not lab-, equipment-, or investigator-specific, and was independent of age, fixation quality, coverslipping medium, or paraffin melting temperature, but in paraffin sections, was highly correlated with the tilt of the knife (cutting) angle. Manipulation of subsequent tissue processing steps revealed that two events contribute to z-axis distortion. Initially, a higher density of particles results at surfaces after sectioning, apparently due to section compression. Subsequently, particles can be lost to varying degrees from surfaces during floating or staining and dehydration, resulting in "lost caps." These results may explain different degrees of z-axis distortion between different types of sections and different labs, and reinforce the importance of checking z-axis distributions as a "quality control" prior to selection of guard zones in optical disector counting. Indirect approaches to assess section quality, such as resectioning in a perpendicular plane, yield additional artifacts, and should be replaced by a direct quantitative measurement of z-axis distribution of particles.

Multivesicular bodies (MVBs) are defined by multiple internal vesicles enclosed within an outer, limiting membrane. MVBs have previously been quantified in neuronal cell bodies and in dendrites, but their frequencies and significance in axons are controversial. Despite lack of conclusive evidence, it is widely believed that MVBs are the primary organelle that carries neurotrophic factors in axons. Reliable information about axonal MVBs under physiological and pathological conditions is needed for a realistic assessment of their functional roles in neurons. We provide a quantitative ultrastructural analysis of MVBs in the normal postnatal rat hypoglossal nerve and under a variety of experimental conditions. MVBs were about 50 times less frequent in axons than in neuronal cell bodies or dendrites. Five distinct types of MVBs were distinguished in axons, based on MVB size, electron density and size of internal vesicles. While target manipulations did not significantly change MVBs in axons, dystrophic conditions such as delayed fixation substantially increased the number of axonal MVBs. Radiolabeled brain-and glial-cell derived neurotrophic factors (BDNF and GDNF) injected into the tongue did not accumulate during retrograde axonal transport in MVBs, as determined by quantitative ultrastructural autoradiography, and confirmed by analysis of quantum dot-labeled BDNF. We conclude that for axonal transport, neurotrophic factors utilize small vesicles or endosomes that can be inconspicuous at transmission electron microscopic resolution, rather than MVBs. Previous reports of axonal MVBs may be based, in part, on artificial generation of such organelles in axons due to dystrophic conditions.

Strabismus is a misalignment of the visual axes, due to an imbalance in extraocular muscle (EOM) function. Botulinum neurotoxin (BoNT) treatment can correct the misalignment with permanent therapeutic effects in infants, possibly because the toxin causes structural alterations in developing EOM. To determine whether BoNT indeed permanently weakens developing EOMs, we examined the chicken oculomotor system. Following injections of BoNT in hatchling chicks, we quantified physiological parameters (contractile force measurements) and morphological parameters (myofiber morphometry, innervation, quantitative transmission electron microscopy of mitochondria/fiber types). Treatment of developing EOM with BoNT caused acute reductions of muscle strength and mitochondrial densities, but minimal changes in muscle fiber diameter and neuromuscular junction structures. Contrary to expectations, contractile force was fully recovered by 3-4 months after treatment. Thus, permanent therapeutic effects of BoNT most likely do not cause permanent changes at the level of the peripheral effector organ, but rather involve central (CNS) adaptive responses.

Using small tags, we tracked the pathway of tumor necrosis factor (TNF)alpha across cerebral vascular endothelial cells. In cerebral microvessel derived RBE4 cells, (125)I-TNFalpha had rapid endocytosis within the first 20 min and showed substantial exocytosis in the intact form. Biotinylated TNFalpha was detected at different time points after endocytosis by streptavidin-Quantum dots which showed its time-dependent colocalization with intracellular organelles. In mice, electron microscopic autoradiography after intravenous injection of (125)I-TNFalpha showed its transcytosis, as signals emerged on the abluminal side of the endothelial cells and reached brain parenchyma. The vesicular trafficking of TNFalpha reflects the immunomodulatory potential of peripheral cytokines for the CNS.

Precise control of contractile force of extraocular muscles is required for appropriate movements and alignment of the eyes. It is unclear how such precise regulation of contractile force is achieved during development and maturation. By using the posthatch chicken as a model, we describe and quantify critical parameters of the developing superior oblique extraocular muscle from hatching to 16 weeks of age, including contractile force, muscle mass, myofiber diameters, classification of fiber types, and distribution and quantification of mitochondria. Analysis at the light-and electron microscopic levels shows that chicken myofiber types largely correspond to their mammalian counterparts, with four fiber types in the orbital and four types in the global layer. Twitch tension muscle force and muscle mass gradually increase and stabilize at approximately 11 weeks. Tetanic tension continues to increase between 11 and 16 weeks. Myofiber diameters in both the orbital and global layer increase from hatching to six weeks, and then stabilize, whereas the myofiber number is constant after hatching. This finding suggests that muscle mass increases during late maturation due to increasing fiber length rather than fiber diameter. Quantitative ultrastructural analysis reveals continuing changes in the composition of the four muscle fiber types, suggesting ongoing fiber type conversion or differential replacement of myofiber types. Muscle fiber composition continues to change into late juvenile and adult age. Our study provides evidence for gradual, incremental, and continuing changes in avian myofiber composition and function that is similar to postnatal oculomotor maturation in visually oriented mammals such as kitten. Anat Rec, 290:1526Rec, 290: -1541Rec, 290: , 2007Rec, 290: . 2007 Wiley-Liss, Inc.

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in CD44 null mice. These results suggest that CD44 is required for the normal development of the hippocampus.

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