The aldehydes introduced in this paper and the more appropriate concentrations for their general use as fixatives are: 4 to 6.5 per cent glutaraldehyde, 4 per cent glyoxal, 12.5 per cent hydroxyadipaldehyde, 10 per cent crotonaldehyde, 5 per cent pyruvic aldehyde, 10 per cent acetaldehyde, and 5 per cent methacrolein. These were prepared as cacodylate- or phosphate-buffered solutions (0.1 to 0.2 M, pH 6.5 to 7.6) that, with the exception of glutaraldehyde, contained sucrose (0.22 to 0.55 M). After fixation of from 0.5 hour to 24 hours, the blocks were stored in cold (4°C) buffer (0.1 M) plus sucrose (0.22 M). This material was used for enzyme histochemistry, for electron microscopy (both with and without a second fixation with 1 or 2 per cent osmium tetroxide) after Epon embedding, and for the combination of the two techniques. After fixation in aldehyde, membranous differentiations of the cell were not apparent and the nuclear structure differed from that commonly observed with osmium tetroxide. A postfixation in osmium tetroxide, even after long periods of storage, developed an image that—notable in the case of glutaraldehyde—was largely indistinguishable from that of tissues fixed under optimal conditions with osmium tetroxide alone. Aliesterase, acetylcholinesterase, alkaline phosphatase, acid phosphatase, 5-nucleotidase, adenosine triphosphatase, and DPNH and TPNH diaphorase activities were demonstrable histochemically after most of the fixatives. Cytochrome oxidase, succinic dehydrogenase, and glucose-6-phosphatase were retained after hydroxyaldipaldehyde and, to a lesser extent, after glyoxal fixation. The final product of the activity of several of the above-mentioned enzymes was localized in relation to the fine structure. For this purpose the double fixation procedure was used, selecting in each case the appropriate aldehyde.

/pdf/cytochemistry-and-electron-microscopy-the-preservation-of-kpmoxmynkl.pdf

Cytochemistry and electron microscopy. The preservation of cellular ultrastructure and enzymatic activity by aldehyde fixation.

1. A variety of physiological properties of single motor units have been studied in the gastrocnemius muscle (primarily in the medial head) of pentobarbitone-anaesthetized cats. Intracellular stimulation of individual motoneurones ensured functional isolation of the muscle units innervated by them.

2. A system for muscle unit classification was developed using a combination of two physiological properties. Almost all of the units studied could be classified into one of three major types, including two groups with relatively short twitch contraction times (types FF and FR, which were differentiable from one another on the basis of sensitivity to fatigue) and one group with relatively long contraction times (type S, which were extremely resistant to fatigue and were differentiable from FF and FR units on the basis of the shape of unfused tetani). Post-tetanic potentiation of twitch responses was observed in all three muscle unit types. The distributions of axonal conduction velocities for motoneurones innervating FF and FR muscle units were essentially the same, while conduction velocities for motoneurones innervating type S units were, in general, slower.

3. Histochemical profiles of muscle units representative of each of the physiological classes present in the gastrocnemius pool were determined using a method of glycogen depletion for muscle unit identification. Each of the physiological categories of muscle units exhibited a corresponding unique set of muscle fibre staining reactions, or histochemical profile. Within each physiological type, all of the units examined had the same histochemical profile. The results generally support the hypothesis that the histochemical characteristics of muscle fibres are meaningfully related to the physiological properties of the same fibres. However, certain limitations in the detailed application of the hypothesis were also apparent.

4. Systematic assessment of the histochemical profiles of relatively large numbers of fibres belonging to single muscle units provided strong support for the hypothesis that all of the muscle fibres innervated by a single α-motoneurone are histochemically identical.

https://physoc.onlinelibrary.wiley.com/doi/pdf/10.1113/jphysiol.1973.sp010369

Physiological types and histochemical profiles in motor units of the cat gastrocnemius.

Mammalian skeletal muscles consist of three main fibre types, type 1, 2A and 2B fibres, with different myosin heavy chain (MHC) composition. We have now identified another fibre type, called type 2X fibre, characterized by a specific MHC isoform. Type 2X fibres, which are widely distributed in rat skeletal muscles, can be distinguished from 2A and 2B fibres by histochemical ATPase activity and by their unique staining pattern with seven anti-MHC monoclonal antibodies. The existence of the 2X-MHC isoform was confirmed by immunoblotting analysis using muscles containing 2X fibres as a major component, such as the normal and hyperthyroid diaphragm, and the soleus muscle after high frequency chronic stimulation. 2X-MHC contains one determinant common to 2B-MHC and another common to all type 2-MHCs, but lacks epitopes specific for 2A- and 2B-MHCs, as well as an epitope present on all other MHCs. By SDS-polyacrylamide gel electrophoresis 2X-MHC shows a lower mobility compared to 2B-MHC and appears to comigrate with 2A-MHC. Muscles containing predominantly 2X-MHC display a velocity of shortening intermediate between that of slow muscles and that of fast muscles composed predominantly of 2B fibres.

Three myosin heavy chain isoforms in type 2 skeletal muscle fibres.

The multiplication of cells in the proliferative zone of the external germinal layer and the early steps in the differentiation of basket, stellate and granule cells were studied in the cerebellar cortex of rats aged 0, 3, 5, 7, 10, 12, 15, 21 and 30 days with histological, histochemical, autoradiographic and electron microscopic techniques. Between 0–9 days the proliferative zone has a constant depth of four to five cells; the bipolar cells in the underlying premigratory zone increase in depth during this period from 0–6 cells. Thereafter, there is a decline in the cell depth of both zones. In the premigratory zone there is a gradient in the length of the extruded processes of the bipolar cells (concentration of such profiles), the future parallel fibers. Presumably when the latter reach their final length, the cell body migrates downward and the parallel fiber becomes part of the upper zone of the molecular layer.

Thus, parallel fibers are progessively formed on the surface of others from the bottom upward by a stacking process and the external germinal layer, as a consequence, is continually pushed upward. This design makes possible the assembly of a matrix of very long, thin and straight beams of horizontally oriented parallel fibers which pile up vertically according to their age. When synaptogenesis starts during the second week in the molecular layer the enlarging junctional processes produce a spurt in the growth of this layer.

Basket cells which are formed in the pyramis on days 6–7 are arrested in the formative molecular layer because their processes are oriented at a right angle to the underlying bed of parallel fibers. Therefore, there is also a stacking of the cells of the molecular layer from the bottom upward as a function of time of onset of their differentiation. Parallel fiber synapses may be seen on differentiating basket cells as early as the seventh day, forming connections with these inhibitory interneurons before they synapse with spines of Purkinje cells.

Postnatal development of the cerebellar cortex in the rat. I. The external germinal layer and the transitional molecular layer.

The correlation among a variety of physiological properties and the histochemical characteristics of muscle fibers belonging to single motor units in a mixed mammalian muscle is directly demonstrated. The population of motor units making up the cat gastrocnemius was classified into three nonoverlapping groups on the basis of a combination of physiological parameters. The muscle fibers belonging to motor units of each physiological type exhibited a distinctive histochemical profile, such that the three basic histochemical "fiber types" exactly matched the three physiologically defined groups. Within each individual motor unit, the muscle fibers were histochemically uniform.

https://science.sciencemag.org/content/sci/174/4010/709.full.pdf

Mammalian Motor Units: Physiological-Histochemical Correlation in Three Types in Cat Gastrocnemius

1. A new p-nitrophenyl substituted ditetrazole, 2,2'-di-p-nitrophenyl-5,5'-diphenyl-3,3'-(3,3'-dimethoxy-4,4'-biphenylene) ditetrazolium chloride (Nitro-BT), is introduced, which possesses most of the properties of an ideal reagent for histochemical purposes. It permits the cytochemical visualization of the sites of enzymic activity in tissue sections from 2-8 µ thick, under aerobic conditions, after a relatively short incubation period.2. A comparison was made of the various tetrazoles in common use with several new p-nitro substituted analogues of these agents.3. A survey was presented of the enzyme distribution in the major tissues of the rat and dog. Evidence for the reliability of cytochemical localization is provided by illustrative material in selected tissues.4. The new tetrazole can be employed for the diphosphopyridine nucleotide and triphosphopyridine nucleotide diaphorase systems as well as for the succinic dehydrogenase system by substituting Nitro-BT for blue tetrazolium (BT) in the incubati...

M M Nachlas

Papers

Cytochemical demonstration of succinic dehydrogenase by the use of a new p-nitrophenyl substituted ditetrazole.