Wiley

A stereological method for obtaining estimates of the total number of neurons in five major subdivisions of the rat hippocampus is described. The new method, the optical fractionator, combines two recent developments in stereology: a three‐dimensional probe for counting neuronal nuclei, the optical disector, and a systematic uniform sampling scheme, the fractionator. The optical disector results in unbiased estimates of neuron number, i.e., estimates that are free of assumptions about neuron size and shape, are unaffected by lost caps and over‐projection, and approach the true number of neurons in an unlimited manner as the number of samples is increased. The fractionator involves sampling a known fraction of a structural component. In the case of neuron number, a zero dimensional quantity, it provides estimates that are unaffected by shrinkage before, during, and after processing of the tissue. Because the fractionator involves systematic sampling, it also results in highly efficient estimates. Typically only 100–200 neurons must be counted in an animal to obtain a precision that is compatible with experimental studies. The methodology is compared with those used in earlier works involving estimates of neuron number in the rat hippocampus and a number of new stereological methods that have particular relevance to the quantitative study of the structure of the nervous system are briefly described in an appendix.

Cross‐sectional internal diameter measurements were made of right and left common carotid and right femoral arteries and right and left internal jugular, superficial femoral, and common femoral veins in 32 normal human subjects utilizing duplex ultrasonography. The relationships of these vessel sizes to the subject's sex, age, height, weight, and body surface area were analyzed statistically; and graphs were constructed, indicating the relationship of blood vessel diameters to the various body size parameters. Findings indicate that (1) for the femoral veins, body surface area had the best correlation with the internal diameter of the vein; (2) for the right internal jugular vein, body weight had the best correlation with the internal diameter of the vein; (3) correlation between vein diameter and body size of the subject is better for the femoral veins than for the internal jugular veins; (4) internal diameter of the femoral and internal jugular veins increases about 20% when they are distended by 15% of positional inclination of the subject's body; (5) neither age nor sex of the subject influences the positional distensibility of the veins examined; (6) the cross‐sectional internal diameter of the femoral and internal jugular veins, as determined by duplex ultrasonography, closely relates to the external diameter of these vessels as measured by direct in vivo application of calipers and to the maximum outside diameter of cannula the vessel will accept.

Whether neural crest cells from the avian embryo are determined for chondrogenesis before they begin their migration away from the neural tube (i.e., before H. H. stages 8.5‐9) was investigated by establishing neural folds from embryos of H. H. stages 5‐11 either in organ culture, or as grafts to the chorioallantoic membranes of host embryos. Cartilage differentiated from neural folds taken from embryos of H. H. stages 5‐7 but not from those taken from older embryos. This stage specific pattern was reversed when the tissue adjacent to the neural tube was grafted to the chorioallantoic membrane. Cartilage only formed from tissues isolated later than H. H. stage 8; i.e., when these adjacent tissues contain neural crest cells. We concluded that neural crest cells are determined for chondrogenesis while still in the neural tube and before their migration to the face and head. This is in contrast to the situation in the only other group which has been examined, the urodele amphibians.

Each of the bilateral nasal glands of Dipsosaurus is surrounded by a thin cartilagenous capsule. A short excretory duct leads to the vestibule of the nasal cavity. This duct connects with the branched principal secretory tubules that end in small terminal segments. Tall columnar cells line the principal secretory tubules, but mucous and tuft cells form the terminal elements. In salt‐stressed animals the spaces between dark and light principal secretory cells are dilated. Potassium‐dependent, ouabain sensitive, adenosine triphosphatase (Ernst, '72a) was localized within the lateral plications of the principal secretory cells and in the apical microvilli of the tuft cells. These observations are consistent with current concepts of ion transport in salt‐secreting epithelia, and they suggest that the tuft cells, not found in avian salt glands, play a role in the unusual physiology (Templeton, '66) of the nasal glands in this reptile.

Germ cell degeneration in 14 normal and 14 microwave‐irradiated, adult (400–500 gm), Sprague‐Dawley rats was compared by evaluating potential sperm production rates at different developmental steps in spermatogenesis. Following 9 days of irradiation at 1.3 GHz (6 hours/day at 6.3 mW/gm using 1‐μsec pulsewidth at 600 pulses/second) or sham treatment, rats were killed at 6.5, 13.0, 26.0, or 52.0 days following treatment. Testes were perfused with 2% glutaraldehyde, embedded in Epon, and sectioned at 0.5 μm for morphometric analyses. Plasma LH and FSH concentrations were determined by radioimmunoassay from blood collected on the day of death. Considering nuclear size, percentage of nuclei in the parenchyma, and life span of different cells, potential daily sperm production was determined for type B spermatogonia, preleptotene or pachytene primary spermatocytes, or spermatids with round nuclei. No differences (P > .05) in parameters tested were found among time periods following irradiation. With the possible exception of sperm production per testis (P < .05) based on pachytene spermatocytes, microwave irradiation had no effect on the parameters evaluated. No degeneration was detected in spermatogenesis when potential sperm production rates were determined either from type B spermatogonia to spermatids or from type B spermatogonia to a posttesticular approximation of sperm production rate. Thus, it appears that regulation of sperm production rates must take place during spermatogonial mitoses, since once the number of type B spermatogonia is determined, there is essentially no subsequent alteration in sperm production potential in normal or irradiated adult rats.

The effect of immobilization on endplate morphology of the rat soleus muscles was studied qualitatively and quantitatively. The endplate was visualized by light microscopic zinc iodide osmium (ZIO) staining and by electron microscopy. The soleus muscle was immobilized by pinning of ankle and knee joints at right angles for 5 days. Immobilized muscles were then compared to the contralateral side and to normal litter mates. After 5 days of partial disuse, muscle fibers atrophied and nerve terminal area increased in ZIO‐determined measurements. Neuromuscular junctions (NMJs) of disuse muscle fibers visualized by electron microscopy exhibited greater amounts of degeneration than either contralateral or control NMJs. Degeneration consisted of nerve terminal disruption, exposed junctional folds, and postsynaptic areas which contained little or no postjunctional folds. Regeneration also occurred in the same NMJs, consisting of small terminals associated with large expansion of junctional folds, several small terminals occurring within the same primary synaptic cleft, and several axons wrapped by the same Schwann cell. These observations demonstrate, for the first time, that partial disuse for only 5 days produces muscle atrophy as well as denervation‐like changes at the NMJ, which leads to terminal sprouting within the endplate area and remodelling.

Transmission electron microscopy of Thai leaf frog testis revealed a unique pattern of spermatid nuclear morphogenesis. Chromatin condenses into a continuous cylindrical coil within a roughly spherical nucleus. Later the nuclear membrane conforms to the contours of the uncoiling nuclear contents. In the mature sperm, the long, tapering nuclesus is helically shaped. This developmental sequence occurs in the absence of a microtubular manchette, raising questions about the role of this structure in nuclear shaping in spermatozoa of other species.

With environmental factors rigidly standardized, Sprague‐Dawley rats were maintained under the following lighting schedules: (1) artificial light 0600 to 1800 alternating with 12 hours of darkness‐LD, (2) reversal of the above‐DL, (3) constant darkness‐DD, and (4) constant illumination‐LL.

During each regimen, both total and differential white blood counts of tail blood were done in a hemocytometer and then were compared to differential counts done by the smear technique on groups of 16 animals at bi‐hourly intervals over a 24‐hour period. Rhythms in lymphocytes, eosinophils and neutrophils were found under all lighting conditions by plotting the bi‐hourly mean values of the absolute counts along the 24‐hour time scale; rhythms were not found when the means of the differential counts were plotted. The DL rhythms always were the reverse of the ones seen in LD.

In LD, DL, and DD, but not in LL, the rhythms of the three cell types were synchronized, that is, their peaks and troughs occur at about the same time each day.

Some evidence, based on desynchronization from LD rhythms, suggests that all three cells types in DD and the lymphoctyes in LL were or had been at one time free‐running.

Expressed as an overall increase in magnitude, the greatest response in the three cell types to abnormal lighting conditions (DL, DD, and LL) was seen in the neutrophils.

Similar determinations made on a second colony of hypophysectomized animals maintained under LD conditions demonstrated that hypophysectomy did not abolish the rhythm characteristic of lymphocytes, since the timing was identical to the rhythm seen in normal LD animals. There was, however, a lymphocytosis in the hypophysectomized group. Hypophysectomy greatly modified, but did not abolish the eosinophil and neutrophil rhythms.

The significance of periodicity analysis in relation to bioassay is discussed.

The observed fit of bone mass to a healthy animal's typical mechanical usage indicates some mechanism or mechanisms monitor that usage and control the three longitudinal growth, bone modeling, and BMU‐based remodeling activities that directly determine bone mass. That mechanism could be named a mechanostat. Accumulated evidence suggests it includes the bone itself, plus mechanisms that transform its mechanical usage into appropriate signals, plus other mechanisms that detect those signals and then direct the above three biologic activities. In vivo studies have shown that bone strains in or above the 1500–3000 microstrain range cause bone modelling to increase cortical bone mass, while strains below the 100–300 microstrain range release BMU‐based remodeling which then removes existing cortical‐endosteal and trabecular bone. That arrangement provides a dual system in which bone modeling would adapt bone mass to gross overloading, while BMU‐based remodeling would adapt bone mass to gross underloading, and the above strain ranges would be the approximate “setpoints” of those responses.

The anatomical distribution of those mechanical usage effects are well known. If circulating agents or disease changed the effective setpoints of those responses their bone mass effects should copy the anatomical distribution of the mechanical usage effects. That seems to be the case for many agents and diseases, and several examples are discussed, including postmenopausal osteoporosis, fluoride effects, bone loss in orbit, and osteogenesis imperfecta.

The mechanostat proposal is a seminal idea which fits diverse evidence but it requires critique and experimental study.

A method for rapid freezing is described in which use is made of the good heat conducting properties of silver. The freezing was accomplished by bringing the tissue in contact with a polished silver surface at the temperature of liquid nitrogen either at atmospheric or reduced pressure. Helium gas flowing over this surface prevented the condensation of water or air on the silver. After freezing the tissue was placed in a substituting solvent. The best results were obtained with 2% osmium tetroxide in acetone at −85°C. The ultrastructure of the tissue was well preserved in a narrow surface layer only.