Segmental demyelination in peripheral nerves of old cats.

This study of the fine structure of sciatic nerve branches in normal old cats provides evidence indicating that segmental demyelination may account, in part, for the significant decrease with age in the mean axonal conduction velocity in these hindlimb nerves. Fibers of different diameters exhibited focal abnormalities of their myelin sheath. Lipid-like droplets and granulo-vacuolar debris were present in distended portions of the inner adaxonal rim and in the outer cytoplasmic compartment of the Schwann cell. These inclusions extended into the cytoplasm of the paranodal myelin loops and clefts of Schmidt-Lantermann. There also occurred disruption of the axoglial junctions and separation of the myelin loops from the paranodal axolemma which widens the nodes of Ranvier. Complete disruption of one or more contiguous segments of the myelin sheath was produced by interlamellar splitting and ballooning along the major dense and intraperiod lines. Axonal degeneration occurred less frequently and was not present in all hindlimb nerves.

Quantitative morphology of medium-sized caudate spiny neurons in aged cats.

These studies were designed to assess some of the morphological alterations that occur in medium-sized spiny neurons of the caudate nucleus in aged cats. Computer assistance was used to quantify in three dimensions the extent of the dendritic trees of 164 neurons from 11 cats (5 1-3 years and 6 over 10 years of age) stained by the rapid Golgi technique. In all animals beyond 10 years of age there was a decrease in the density of spines on distal dendritic segments. This decrease was moderate (16%) in 13 year old cats and reached about 50% in 15 and 18 year old animals. In addition, there was an increase in the frequency of occurrence of spines with enlarged heads in all aged cats. In cats over 13 years there was a marked loss of portions of distal dendritic segments. All measures of dendrite length displayed statistically significant decreases of 30-40% in cats 15 and 18 years of age. There were no significant age-related alterations in numbers of dendrites, number of branches per dendrite or soma diameter. These morphological results indicate that there is a sequence of age-related changes that occurs in caudate medium-sized spiny neurons and provides a basis from which to assess functional alterations.

The postnatal development of the substantia nigra: a light and electron microscopy study.

The early postnatal development of neurons, dendrites and synaptic connectivity in kitten substantia nigra (SN) was studied by light and electron microscopy. The compact and reticular divisions of the SN are present at birth but boundaries are indistinct. Most nigral neurons stain deeply in routine histological sections and their diameters increase slightly with age. Ultrastructurally, cell bodies are characterized by eccentrically located and often invaginated nuclei surrounded by cytoplasm rich in well-formed organelles. Axosomatic synapses are infrequent and cell surfaces are enveloped by glial processes. Immature dendritic features, including growth cones and filiform processes, are commonly observed during the first 10 days. Gradually the dendritic profiles elongate and thicken and contours become smoother, retaining only scattered spinelike appendages. Clear examples of the three synaptic types described in cat are found in newborn kittens, but immature terminals contain fewer synaptic vesicles and mitochondria. Approximately 90% of synapses present at birth in both nigra subdivisions are Type I, which contain large pleomorphic vesicles and contact dendrites symmetrically. Asymmetrical contacts characterize most of the remaining definable synapses. The postnatal increase in synaptic connectivity, which was estimated from random photographs of pars reticulata neuropil, is twofold during the first 50 days of life. Initially young dendrites are enveloped by glia and then gradually become ensheathed by axon terminals. Synaptogenesis in pars reticulata reflects the postnatal increase of neostriatal inputs to this subdivision and can be correlated with functional changes in strionigral connectivity.

Hypoxic inhibition of breathing in fetal sheep: relationship to brain adenosine concentrations.

Because hypoxic inhibition of fetal breathing may be caused by a rise in central adenosine levels, the effects of O2 deficiency on fetal brain adenosine concentrations were determined at levels of hypoxia that inhibited fetal breathing. Under halothane anesthesia, the brains of fetal sheep (0.8 term) were implanted with guide cannulas exteriorized through a Silastic rubber window in the uterus and flank of the ewe. At least 4 days after surgery, a microdialysis probe was inserted into a cannula with the membrane tip placed in the rostral brain stem. During 1 h of isocapnic hypoxia, mean fetal arterial PO2 was reduced from 24.0 +/- 0.9 Torr (control) to 13 +/- 0.6 Torr and arterial pH fell progressively from 7.354 +/- 0.007 to 7.273 +/- 0.023. Hypoxia decreased the incidence of fetal breathing movements from 33 +/- 5.2 to 5 +/- 2.2 min/h, with a normal incidence (29 +/- 3.5 min/h) during the hour after arterial PO2 returned to control values. Adenosine concentrations in microdialysis perfusate under control conditions averaged approximately 35 nM, increased up to 2.3-fold during the hour of O2 deficiency, and fell toward control values when normoxia was restored. We conclude that fetal brain adenosine levels are increased at levels of O2 deficiency that inhibit fetal breathing, which are results consistent with a role for adenosine in hypoxic inhibition of fetal breathing.

The postnatal development of the caudate nucleus: a Golgi and electron microscopic study of kittens.

This study used light and electron microscopy to describe changes in the synaptic organization of developing caudate nucleus and to quantitate postnatal synaptogenesis in this region. Observation at the light microscopic level focus on the perinatal period and suggest an early maturation of spiny interneurons of the caudate nucleus. Golgi impregnation of these cells at early postnatal ages (birth to 7 days) reveals 3-5 primary dendrites which radiate from the cell body and extend for distances of 8-16 micron before branching. Secondary dendritic branches contain spines and extend, with further branching, for additional distances of 60-160 micron. The dendritic fields of neighboring caudate neurons overlap and the axons which arise from these cells course and branch within the dendritic fields. Examination of perinatal caudate neuropil (birth to 5 days) by electron microscopy reveals an extensive and well-developed axodendritic connectivity. Axonal profiles form multiple synapses en passant along single dendrites and dendritic spines or on several adjacent dendritic branches. At these ages, terminals contain few synaptic vesicles and synaptic junctions are slightly asymmetrical. By the fifteenth postnatal day, boutons are filled with vesicles, junctional complexes are distinctly asymmetrical, and axondendritic connectivity has been modified by the increase of dendritic spines and branchlets. Thus, the basic pattern underlying the organization of synapses in the mature caudate nucleus, is established within the first week postpartum and subsequent changes are primarily quantitative.

An autoradiographic study of the postnatal development of pericruciate cortical projections to the neostriatum and the claustrum of the cat.

The postnatal development of corticostriatal and corticoclaustral projections was traced by light-microscopic autoradiography following injections of tritiated proline and leucine into the pericruciate cortex of kittens, 2-56 days of age, and adult cats. A medial-lateral topography of the corticostriatal pathways was present at birth, with a smaller and more restricted projection arising from the lateral pericruciate region. During postnatal development, the volume of the ipsilateral caudate nucleus filled by labelled corticostriatal fibers was reduced by 30% and these projections became constricted within the dorsolateral head and body of the caudate nucleus. During the first postnatal week, terminal fields were diffuse but lacked homogeneity since irregular patches of intensely labelled elements clustered near the internal capsule. By the end of the first month, a fenestrated organization became apparent within the caudate nucleus where elongated clusters of intensely labelled fibers radiated from the internal capsule and partially or completely surrounded islands of neuropil virtually devoid of label. Pericruciate inputs were confined to the dorsal parts of the rostral putamen and claustrum and the patchy nature of these terminal fields was limited to small labelled clusters close to the capsular margins. Contralateral terminal fields were labelled less intensely and occupied areas symmetrical to those within the ipsilateral neostriatum and claustrum.

Development of the kitten substantia nigra: a rapid Golgi study of the early postnatal period.

The rapid Golgi method was used to describe the morphological maturation of substantia nigra (SN) neurons and the surrounding afferent axons. Observations were made from 25 kittens grouped at 1-3, 7-10, 18-24, and 40-55 days of age. Although variability in dendritic development among neurons is seen at each age, a common maturational sequence can be defined. The dendritic shafts at 1-3 and 7-10 days exhibit prominent varicosities and thin filiform processes along their shafts, and growth cones at their tips. The dendrites at 18-24 days are longer, thicker, and have more regular contours proximally, while varicosities and filiform processes persist distally. Neurons in this age group display shorter, spine-like processes although adult cells are known to lack typical spines. By 40-55 days, most dendritic surfaces are smooth with only scattered appendages distally. Computer-assisted measurements of dendritic growth in pars compacta neurons demonstrate a 42% increase in dendritic lengths. The number of dendrites per neuron and the number of branches per dendrite do not change between the youngest and oldest age groups. The afferent connectivity in the kitten SN is en passant in character. Two axonal types are identified at all ages. Most prominent is a thin fiber with irregularly spaced varicosities and terminal expansions 1 micron in diameter. These swellings correspond, most likely, to the type I terminals described ultrastructurally and shown to be neostriatal in origin. The second axonal type is thicker, with branches that end in clusters of enlargements, 1-2 microns in diameter. The origin of this second type is unknown.

Transplantation of B16/C3 melanoma cells into the brains of rats and mice.

The B16/C3 mouse melanoma cell line produces L-DOPA, catecholamines and melanin in tissue culture. Growth and development of these cells after transplantation into the rat and mouse brain were studied by immunocytochemical and histological techniques. The implanted cells were localized by prelabelling the cell nuclei with bisbenzimide, a fluorescent marker which binds to DNA. Following transplantation into rats, B16/C3 melanoma cells were found to survive for at least 4-6 weeks. These cells initially expressed tyrosinase and tyrosine hydroxylase immunoreactivity and in some cases contained catecholamines. After 3 weeks, the cytoplasm of the transplanted cells began to accumulate melanin; catecholamines and tyrosinase immunoreactivity were no longer detected. Ultimately the cells became round in shape and densely pigmented. Growth of the tumor in the rats was restricted and the implant was encapsulated within a glial sheath. There was evidence of an immune reaction to the tumor in that cells with Ia antigen immunoreactivity were present surrounding the graft. The rat hosts were not adversely affected by the presence of the tumor, nor did the tumor cell grafts alter rotational behavior consequent to unilateral substantia nigra lesions. In mouse hosts, however, the melanoma grew rapidly, was not encapsulated by glia and led to death of all animals. These data suggest that the tumor was not rapidly destroyed in rats, even though its growth was controlled through immunological mechanisms. Both trophic and immunological mechanisms may therefore be involved in the regulation of survival and differentiation of intracerebral grafts of tumor cells.

Age-dependent changes in cat masseter nerve: an electrophysiological and morphological study.

The present study was undertaken to determine the manner in which aging affects the function and structure of the masseter nerve in old cats. Electrophysiological data demonstrated a significant decrease in the conduction velocity of the action potential in old cats compared with that observed in adult cats. Light microscopic analyses revealed an age-dependent decrease in axon diameter. Electron microscopic observations of the masseter nerve in the aged cats revealed a disruption of the myelin sheaths and a pronounced increase in collagen fibers in the endoneurium and perineurium. These morphological changes are discussed and then related to the decrease in conduction velocity which was observed in the electrophysiological portion of this study.

Quantitative developmental studies of feline neostriatal spiny neurons.

This research documents aspects of the quantitative development of the 'medium' spiny neuron in the kitten from 2 to 143 days of age. Using material derived from 244 Golgi-impregnated neurons in 15 kittens and with computer assistance the changes in somatic, dendritic and spine development were quantified. Although mean somatic diameter increased only slightly from 2--3 to 8--10 days of age, the proportion of neurons with large diameters increased significantly during the developmental period. In addition, the radius of the dendritic field of caudate spiny neurons increased significantly over the age period examined. An unexpected finding was that the number of dendrites per neuron decreased with age, probably due to a decrease in the proportion of neurons with 6 or more dendrites in animals 90--143 days of age. Growth of dendritic segments occurred throughout the age period studied. This growth was apparently caused by lengthening of all dendritic segments and this resulting increase was proportional to the initial length of the individual branches. Number of branches per dendrite and frequency of dendritic branches with different orders remained constant across age indicating that the basic dendritic branching pattern is probably set for the cat before birth. With maturation the density of spines on distal dendritic branches increased while on proximal dendritic branches spine density decreased. The time course of these quantitative changes was related to alterations in synaptogenesis and physiological changes in caudate neurons.

Postnatal differentiation and growth of cat entopeduncular neurons. A transient spiny period associated with branch elongation.

Qualitative and computer-assisted analyses were performed on Golgi-impregnated neurons which were serially reconstructed in 3 dimensions. Analysis of the temporal pattern of growth indicated that the initial outgrowth, formation of the adult number of dendrites and virtually all dendritic branching occurred in the prenatal period. About 40% of the total growth of the dendrites occurred in the postnatal period. Maturation was completed by 90-120 days. Analyses of the mode of dendritic growth and of the morphological changes associated with growth revealed two significant findings. First, the outward expansion of the dendritic tree was not due to the addition of new branches but resulted from the elongation of terminal and non-terminal branches. Thus, growth occurred between branch points as well as on terminal portions of dendrites. Second, a transient population of spines was found during the period of postnatal growth. These spines may play an integral role in synaptogenesis and dendritic branch elongation. We suggest that developing afferent fibers initially contact spines. As spines retract, axon terminals are brought to the shaft of the dendrites. Further, the dendrites elongated because membrane associated with spines is incorporated into the shafts of dendrites. Striopallidal projections and other afferents may provide an important trophic influence for the normal dendritic differentiation of pallidal neurons by inducing the elaboration or retraction of spines.

The morphogenesis of glutamic acid decarboxylase in the neostriatum of the cat: neuronal and ultrastructural localization.

Correlative light and electron microscopic immunohistochemical methods were adapted for a descriptive analysis of the normal time course and pattern of expression and intraneuronal localization of the enzyme glutamic acid decarboxylase (GAD) in the neostriatum (Ns) of fetal, postnatal and adult cats. The differentiation of this synthesizing enzyme demonstrated the establishment of gamma-aminobutyric acid (GABA) transmitter identity in these neurons and their connections. The structural modifications of these GABAergic profiles revealed the morphogenesis of important inhibitory synaptic inputs in the Ns. The expression of GAD began during late fetal development and proceeded in a diagonal gradient from the first-formed ventrolateral putamen to the last-formed dorsomedial caudate nucleus. The frequency of GAD-positive elements increased with age particularly during the early postnatal period. After the initial expression of GAD, 3 interrelated processes contributed to its differentiation: (1) enzyme accumulation; (2) enzyme association with membranous organelles and (3) progressive elaboration of neuronal infrastructure. Synaptogenesis was both coincident and subsequent to GAD differentiation. Two principal types of GABAergic structures, cell bodies and axonal 'terminals', were evident from the initiation of GAD expression. The GABAergic cell bodies were polymorphic by and after the day of birth and consisted of ubiquitous medium sized cells (often having somatic and/or dendritic spines) and rare large sized cells (apparently aspiny and confined to a limited region of the Ns). The GABAergic axonal terminals changed from growth cone and prototerminal forms to mature bouton en passage and bouton terminaux forms establishing axosomatic and axodendritic contracts, having symmetric synaptic specializations and providing inputs to both medium- and large-sized GABAergic target neurons.

Ultrastructural alterations in caudate nucleus in aged cats.

These studies provide information on the changes in the ultrastructure in the caudate nucleus of aged cats. The major finding was that there was a decrease in the density of synapses in caudate neuropil. This decrease occurred in animals after 3 years of age and remained relatively constant in older animals. In conjunction with this change a population of unusually long synapses also occurred. These larger synaptic appositions were associated with enlarged spine heads. The caudate also showed a number of qualitative ultrastructural alterations. Many neurons contained accumulations of lipofuscin or lipopigment granules in aged animals. These inclusions occurred in both soma and dendrites of neurons and all types of glial cells. A unique configuration of collapsed agranular cisterns also was observed in aged animals. The present results indicate that decreases in synaptic density may be one morphological event underlying functional alterations observed in caudate neurons in aged cats.

The entopeduncular nucleus: Golgi morphometrics of serially reconstructed neurons in adult cats.

Computer-assisted morphometrics were used to characterize mature somatodendritic architecture in Golgi-stained neurons of the entopeduncular nucleus (EN) of the adult cat. Only one form of adult EN neuron was apparent and characterized by common features including: relatively large conical somata, long aspiny and moderately branched dendrites and discoid to spherical dendritic fields oriented randomly within the EN. These results indicate that feline EN neurons have some properties in common with large neurons of the primate medial pallidal segment.