Deborah W. Vaughan

Neuroglial cells in the cerebral cortex of rats from young adulthood to old age: An electron microscope study

SummaryNeuroglial cells were examined in the rat auditory cerebral cortex to determine the effects of aging on astrocytes, oligodendrocytes and microglia. The animals ranged in age from 3 months to 29.5 months. Over this age range of 26.5 months none of the neuroglial cells exhibit striking abnormalities in the morphology or the number of their organelles other than each of these cell types accumulates a distinct membrane-bound inclusion material. Astrocytes accumulate considerably more of this material than oligodendrocytes, and demonstrate earlier and more frequent signs of it. Microglia show the most striking alterations in regard to the inclusion material and in animals of increasing age microglial cells engorged with the heterogeneous material become increasingly common. Concurrently there is a slow transformation of the microglial population from an array of multipolar forms to larger, elongated or spherical forms which contain variable amounts of the dense membrane-bound material. These alterations in the microglial cell population do not occur at a chronologically uniform rate, and in animals 18 months and older, examples of the multipolar, the elongated, and the engorged spherical forms of microglia may be encountered.The total number of neuroglial cells increases with age. However, the numbers of the various types do not increase by similar proportions. While there is little change in the populations of astrocytes and oligodendrocytes in rats between the ages of 3 and 27 months, in the same animals the number of microglia increases by about 65%.

The Structure of Neuritic Plaques in the Cerebral Cortex of Aged Rats

Discrete patches of spongiform degeneration have been found in the cerebral cortices of three rats, 28 and 30 months of age. Many of these patches have in their midst a central nonvacuolated region containing a star-shaped homogeneous core. Using Congo red stain, there is evidence for the presence of amyloid in this core. In the electron microscope, this central region is seen to be composed of degenerating and abnormal neuronal processes, reactive neuroglia, and extracellular filamentous material with the fine structural characteristics of amyloid. Microglial cells are uniquely modified to accommodate bundles of these extracellular filaments, which are often contained in furrows that deeply invaginate the surfaces of these cells. On the basis of its three main constituents, the central region is considered to be a form of neuritic plaque. The large complex clearings in the vacuolar region of each lesion appear to be formed by the coalescence of smaller vacuoles in the surrounding neuropil.

Thalamic and callosal connections of the rat auditory cortex

This study was designed to assess the relative distributions of two extrinsic afferent fiber systems in the rat auditory cortex as indicated by the patterns of specific lesion-induced degeneration evident in Fink-Heimer preparations. The auditory cortex consists of cytoarchitectural areas 41, 20 and 36. Lesions were made in the medial geniculate body (MGB) or the corpus callosum in some rats, while in other rats, lesions were made in both the MGB and the corpus callosum. Following the thalamic lesions, degenerating terminals occur throughout the auditory region of cortex, principally in layer IV and deep layer III, but also in layer VI and in the superficial part of layer I. With the exception of the band of degeneration in layer I, the density of the thalamic degeneration is uneven, such that patches of increased density of degeneration are separated by regions with few degenerating terminals. Following lesions of the corpus callosum, degenerating callosal terminals are also evident throughout the auditory region of cortex and they occur in deep layer I through layer III, superficial layer V and in layer VI. The density of the degenerating callosal terminals is not uniform throughout most of area 41, to the extent that there are radially-oriented bands of increased density which appear within the continuous callosal projection. Following the double lesions, degenerating terminals throughout the auditory region are distributed homogeneously within all cortical layers with the exception of deep layer V which is relatively free of degeneration. The results indicate that all regions within the rat auditory cortex are subject to both thalamic and callosal influence, although the input is not completely uniform, for the zones in layers IV and VI which have decreased thalamic input appear to have increased callosal input.

The effect of aging on the neuronal population within area 17 of adult rat cerebral cortex

The brains of Sprague-Dawley rats in various age groups from 3 to 33 months were fixed by perfusion with standard aldehyde solutions in order to determine the effects of aging on neuronal numbers. Several indices of cortical volume were then measured to determine whether neuronal packing densities were affected by age-related change in cortical volume. The lengths, heights and widths of individual hemispheres for 160 animals ranging in age from 1 day to 36 months were first determined, after which blocks of tissue were removed from area 17 of some of the brains. These blocks were osmicated, embedded in Araldite and sectioned at 1 micrometer to ascertain, in the vertical plane, the thickness of area 17 and, in the tangential plane, the packing density of the clusters of apical dendrites extending from layer V pyramidal neurons. Results indicate the overall dimensions of the cerebral hemispheres increased until 3 months of age, after which there was no further increase in size. Between 3 and 33 months of age there was no age-related change in either the thickness of area 17 or in the separation between dendritic clusters, indicating the volume of area 17 did not change after 3 months of age. Within individual age groups the amount of variation present is greater than that among age groups. Since the number of nucleus-containing neuronal profiles per unit area of layers II/III, IV, V, VIa and VIb was similar in two groups of three animals at 3 and 33 months of age and the diameters of neuronal nuclei were unchanged, there seems to be no significant change in the number of neurons contained in these layers of rat visual cortex between 3 and 33 months of age. It is therefore concluded that no neurons are lost from area 17 as the mature cerebral cortex ages.

Ultrastructure of neurons in the auditory cortex of ageing rats: a morphometric study

SummaryThe cell bodies of pyramidal cells in layers II and V of rat auditory cortex were quantitatively examined in groups of rats 3, 6, 15, 23, 27 and 34–36 months of age. The mean diameters of cell bodies of both layer II and layer V neurons, as measured in 1 μm plastic sections, increased between 3 and 15 months of age, then decreased to a diameter that was less in the 36-month-old than in the 3-month-old rats. Morphometry of the nuclei of the cells was done by measuring nuclear area and nuclear envelope length directly on electron micrographs. In the layer II cells, neither parameter changed with advancing age. In the layer V cells, the mean nuclear area decreased significantly in the old animals and the mean envelope length increased. Point-counting techniques were applied to electron micrographs of cell bodies to determine the relative volume of selected organelles, inclusions and ground substance in the perikaryal cytoplasm. In this part of the study the chronological pattern of change in layer II and layer V pyramidal cells was similar. The relative volume of dense bodies increased linearly with advancing age, with a slightly more accelerated rate in layer II cells. The relative volume of ground substance remained essentially constant through 27 months, and then at 34–36 months decreased to 83% and 89% of the three-month level in layer V and layer II, respectively. The relative volume of the rough endoplasmic reticulum (RER) did not change significantly until after 15 months, at which time it began to occupy increasingly a larger fraction of the perikaryal cytoplasm. Finally, the relative volumes of mitochondria, multivesicular bodies and Golgi apparatus did not show clear trends of change during the 33-month period.

Chromatin Structure in Neuronal and Neuroglial Cell Nuclei as a Function of Age

Abstract: Nuclei from the cerebral cortices of animals of different ages were separated into neuronal and neuroglial populations. Nuclei from cerebellar neurons were also studied. Using the enzyme micrococcal nuclease as a probe for chromatin structure, we found that the DNA from both neuronal preparations showed a decreased susceptibility to digestion during aging, although the onset of this alteration varies. In addition, both neuronal populations showed dramatic increases in the nucleosome spacing of the chromatin. Cerebral neuronal chromatin has a repeat length (nucleosome core and linker region) of 164 base pairs at 22 days and 11 months, 186 base pairs at 24 months, and 199 base pairs at 30 months. Cerebellar neuronal chromatin has a repeat of 188 base pairs at both 22 days and 11 months, 208 base pairs at 24 months, and 243 base pairs at 30 months. Neuroglial chromatin, on the other hand, showed no change in either accessibility to nuclease or repeat length.

Rehabilitation following early malnutrition in the rat: Body weight, brain size, and cerebral cortex development

Sprague-Dawley rats were malnourished by giving their mothers an 8% casein diet starting at day 10 of gestation, while controls were fed a 24% casein diet. Starting at postnatal day 20 (P20), rehabilitation of the malnourished animals was attempted by: (1) feeding both mother and young a 24% casein diet, (2) leaving the pups with their mothers until they were 40 days old, and (3) reducing the litter size from 8 to 4 pups. Observations were made on aldehyde-perfused tissue from animals 20, 40 and 70 days old. The somatosensory cortex from one hemisphere was embedded in Araldite, and that from the other side was processed fro Golgi staining. At 20 days of age the body weight of the malnourished animals was 21% that of the controls, but at 70 days it was no longer different. The anterior-posterior length, the width, and the height of the cerebral hemispheres were also significantly reduced at P20, but the differences had disappeared by P70. The thickness of area 3 of the cerebral cortex was measured in 1 micron sections. It was significantly reduced in the malnourished animals at P20, but at P40, following rehabilitation, the difference was no longer statistically significant. In tangential 1 micron sections the fraction of the volume of tissue occupied by neuropil was measured in layers II through IV. At P20 it was significantly reduced only in the upper half of layers II/III of the malnourished animals; at P40 this difference was no longer present. The mean volume of upper layer II/III cell bodies was estimated and found to be significantly reduced in the experimental animals at P20 but not at P40. In the Golgi preparations, pyramidal cells in upper layer II/III were studied. Their estimated volume, as well as the thickness of their basal dendrites, was significantly reduced in the 20 day malnourished animals, but not in the rehabilitated animals. These results show that animals severely malnourished until 20 days of age can reach normal body weight and attain cerebral hemispheres of normal size when proper nutrition is provided. The effects of malnutrition on the cerebral cortex of these animals are most apparent in upper layer II/III which, during the time of nutritional restriction, is the least developed of the cortical layers. However, when proper nutrition is provided, the cerebral cortex may attain normal morphology.

Synaptic proliferation in the auditory cortex of the young adult rat following callosal lesions.

SummaryThe long-term effects of partial deafferentation in the neocortex of adult rats were studied in four-month old rats in which the corpus callosum had been completely sectioned when they were one-month old. Quantitative light microscopy was used to identify morphological changes in the auditory cortex resulting from the loss of established callosal connections. Particular attention was directed at those cortical layers known to receive the heaviest callosal projection (layers II and III) and at neurons known to be postsynaptic to callosal afferents (layer V pyramidal neurons). The comparative analysis of both semithin plastic sections and Golgi-impregnated material from long-term, callosally-lesioned rats and age-matched control animals reveals no differences in the overall cortical thickness, the thickness of cortical layers, the numbers of neurons or the density of spines along apical dendrites of layer V pyramidal neurons. However, as a result of the callosal lesion, large diameter apical dendrites are significantly thinner in the callosally deafferented cortex and there is a small increase in the number of neuroglial cells in the deeper cortical layers.To determine whether another system of afferents to the auditory cortex spreads into the deafferented callosal domain, geniculate lesions were made in long-term, callosally-lesioned animals and age-matched controls. The terminal projection patterns of thalamic afferents were compared using the Fink-Heimer technique and quantitative electron microscopy. Normally in the auditory cortex there is only a small region of overlap between the terminal projection fields of callosal afferents and thalamic afferents, the latter projecting chiefly to layer IV and low layer III. However, three months after callosal lesions, thalamic axons had proliferated superficially into part of the callosal domain. Furthermore, in the normal auditory cortex after geniculate lesions, there were three rostrocaudally oriented bands of relatively dense thalamocortical terminal degeneration separated by regions of less dense degeneration. In the doubly lesioned animals these bands of degeneration were less distinct due to a proliferation of thalamic axons into the regions characterized by sparse projections.

The effects of age on enzyme activities in the rat facial nucleus following axotomy: Acetylcholinesterase and cytochrome oxidase

Advancing age affects the ability of motor neurons to regrow axons after the facial nerve is crushed. In rats, it requires 14 days after injury for 3-month-old animals to resume normal whisker activity, compared to at least 19 days in 15-month-old animals. The present study examines central enzymatic responses of facial motor neurons to axotomy. During the postoperative period from 1 day through 8 weeks, alternate frozen sections of brain stem are histochemically reacted to demonstrate activities of acetylcholinesterase (AChE) or cytochrome oxidase (COX) and the reactions are quantified using computerized image analyzing densitometry. AChE activity is evaluated separately in perikaryal cytoplasm and neuropil, while COX is assayed in the facial nucleus as a whole. Coincident with the initiation of axon outgrowth the activities of these enzymes decrease in the neurons. For AChE the decrease is greater in the older animals; for COX the decrease is equivalent in both age groups. With regard to the perikaryal AChE and the neuropil AChE, the recovery patterns are different in the two locations. In the perikarya AChE activity begins to recover after 4 days in both age groups; however, AChE activity in the neuropil remains decreased until after functional recovery of whisker activity, when it recovers rapidly in the 3-month-old animals, but more gradually in the 15-month-old animals. In both age groups, COX activity gradually decreases in response to axotomy. In the 3-month-old animals it recovers rapidly following return of whisker activity, while in the 15-month animals COX activity is maintained at the decreased level through 28 days post-crush, before it begins its gradual recovery. The study demonstrates that age differences are most apparent after the reestablishment of functional connections. This age-related deficiency may be related to deficiencies in retrogradely transported signals arising from the reinnervated target or in the older neurons ability to respond to such signals.

Proliferation of thalamic afferents in cerebral cortex altered by callosal deafferentation.

SummaryIn area 41, the auditory region of rat neocortex, callosal afferents project to layers I through III and thalamic afferents project to deep layer III through IV. Thus, these two extrinsic systems of afferents project simultaneously to only a narrow lamina in mid to low layer III. For this study, this narrow region of overlap is quantitatively examined to determine the distribution of callosal and thalamic afferents by observing degenerating terminals produced by separate callosal and thalamic lesions. The results show that of all asymmetric synapses observed in the neuropil of this narrow zone, 84% are dendritic spines and the balance are dendritic shafts. Although both callosal and thalamic afferents prefer to synapse with dendritic spines in the neuropil, 78% of the thalamic afferents synapse with dendritic spines while 93% of the callosal afferents synapse with dendritic spines.Vaughan & Foundas (1982) have shown that 3 months after callosal lesions in 1-month-old animals, additional thalamic axons have grown into, and proliferated in, this part of mid to low layer III. Quantitative analysis of the distribution of the degenerating thalamic axon terminals in these long-term callosally lesioned animals has been used to determine whether the proliferating thalamic afferents demonstrate any specificity in the pattern of synapses they make or whether the callosally deafferented neurons determine the pattern of synapses. The results indicate that thalamic axons do exhibit axon specificity, for after they have proliferated into the callosal domain, 80% of the degenerating terminals synapse with dendritic spines and 20% synapse with shafts. This distribution is most comparable to the normal distribution of thalamic axons in this region.