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Identification and structure of neurons in the medial geniculate body projecting to primary auditory cortex (AI) in the cat.

The neuronal types in the ventral nucleus of the cat medial geniculate body projecting to the primary auditory cortex (AI) were investigated using the retrograde transport of horseradish peroxidase. These cells were compared with the morphology of neurons as revealed in Golgi and Nissl preparations, plastic-embedded tissue, and electron microscopic material. After large injections, more than 90% of the neurons in the ventral nucleus, the principal nucleus of the lemniscal auditory pathway, were labeled, and the population of labeled cells included both large and small neuronal somata. Since the ventral nucleus contains only two varieties of cells--large neurons with bushy dendrites and an unbranched axon, and smaller cells with thin dendrites and a locally projecting axon--it is concluded that at least some of the small cells, previously believed to be interneurons, may function both as local circuit and as projection neurons. These findings were confirmed in toluidine blue-stained, 1-2 micron thick sections, and in the electron microscope, where small cells with sparse cytoplasm and a deeply invaginated nuclear envelope often contained intracellular horseradish peroxidase granules, as well as the larger neurons. Besides the small, labeled neurons in the ventral nucleus, many labeled cells were seen in the interstitial nucleus of the brachium of the inferior colliculus. This hitherto poorly characterized group of cells is embedded among the fibers of the brachium of the inferior colliculus. Many of the morphologically distinct varieties of cells in the medial division of the medial geniculate body, including small neurons, were labeled. Thus, in addition to the route embodied by the large bushy neurons which project to primary auditory cortex, at least one other pathway--represented by certain of the small cells in the ventral nucleus, reaches the primary auditory cortex.

Animals↗

Neurons accumulating [3H]gamma-aminobutyric acid (GABA) in supragranular layers of cat primary auditory cortex (AI)

The classes of neurons accumulating exogenously injected, tritiated gamma-aminobutyric acid [( 3H]GABA) were studied in the supragranular layers in the primary auditory field of the adult cat. The size, laminar locus, and somatodendritic profiles of labeled neurons were studied light microscopically in frozen- or Vibratome-sectioned, 30 micron thick material, and in semithin, 1-2 micron thick, plastic-embedded high-resolution autoradiographic preparations. The chief goals of the study were to determine which types of cells could be identified as accumulating [3H]GABA in layers I, II and III, and to establish possible relationships between these cells and neurons described in Golgi studies of these layers, and the neurons found, in parallel investigations of the connections of the primary auditory field, to participate as ipsilateral corticocortical and commissural cells of origin. The principal findings are: that neurons in every layer in the primary auditory field take up tritiated gamma-aminobutyric acid; that their Nissl-counterstained somata have a smaller average area, and a smaller range of areas, than do the unlabeled cells; that more than one type of labeled neuron-as defined by somatic size and shape, height:width ratios, and nuclear membrane morphology-could be identified in each layer; that none of the labeled neurons had a soma with a pyramidal configuration; that the labeled cells are comparable in size, shape, and laminar distribution to some populations of non-pyramidal ipsilateral corticocortical cells of origin in layers II and III, and perhaps to certain classes of commissurally projecting, layer III non-pyramidal neurons; and finally, that only a rather small proportion-perhaps 10% or less, except in layer I-of the supragranular cells appear to accumulate labeled material. With regard to the identity of particular classes of neurons accumulating silver grains above background in the individual layers, in layer I, 2 of the 4 types of neurons characterized in Golgi preparations take up gamma-aminobutyric acid and the remaining 2 types may also, and the relative number of labeled cells appears to be higher than in the other layers; in layer II, 2 of the 9 varieties are labeled, and 4 other types may also be; and in layer III, 2 of the 11 types take up gamma-aminobutyric acid, and 5 other varieties may as well. Three types of non-pyramidal layer II cells that project ipsilaterally from AI to the second auditory cortical field, AII, possibly accumulate gamma-aminobutyric acid; 3 types of commissural non-pyramidal cells of origin linking AI to AI appear to be labeled by gamma-aminobutyric acid.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

Distribution of glutamate-like immunoreactivity in excitatory hippocampal pathways: a semiquantitative electron microscopic study in rats.

A semiquantitative electron microscopic immunocytochemical procedure was used to study the cellular and subcellular distribution of glutaraldehyde-fixed glutamate in rat hippocampal formation. Ultrathin plastic-embedded sections were incubated with a primary glutamate antiserum followed by a secondary antibody coupled to colloidal gold particles. A computer-assisted assessment of gold particle densities revealed that the axon terminals of all of the main excitatory pathways in the hippocampus were enriched with glutamate-like immunoreactivity relative to other tissue elements, including the parent cell bodies (granule and pyramidal cells). The different excitatory pathways showed slightly different labelling intensities: boutons in the termination zone of the lateral perforant path were covered by higher gold particle densities than boutons situated in the termination zones of the medial perforant path, the Schaffer collateral/commissural pathway and the hilar associational/commissural pathway. The mossy fibre terminals were significantly less enriched in immunoreactivity than terminals of the lateral perforant path and the Schaffer collateral/commissural pathway. Within the terminals, glutamate-like immunoreactivity was concentrated over synaptic vesicles and mitochondria. Terminals establishing symmetric junctions with cell bodies or dendritic stems displayed low particle densities, as did glial cell processes. These findings support the idea that glutamate is a major excitatory neurotransmitter in hippocampal excitatory synapses. Our observations are also in line with biochemical data pointing to the existence of a considerable neuronal and a smaller glial, metabolic pool of glutamate.

Animals↗

The origin and spread of human leukemia.

The human leukemias are a group of hematologic neoplasms characterized by uncontrolled proliferation of cells concerned with blood cell production. The cause(s) of human leukemia remains unknown. Bone marrow (BM) is believed to be the site of origin of human leukemias, although the specific locus(i) and/or cell(s) from which it arises have not been definitively identified. Generally, human leukemias and related proliferative diseases are thought to be clonal in nature; affecting a single hematopoietic stem cell, which then proliferates and replaces the marrow of normal hematopoietic stem cell systems. The condition is believed to be malignant in nature. Results of our current morphologic studies on well-fixed, ideally-stained thin sections of plastic-embedded bone marrow biopsies (BMB) from a large number of acute (AML, ALL) and chronic (CGL, CLL) leukemia patients suggest that human leukemias may not be clonal diseases. Instead, a large population of other resident cells--'endosteal cells'--appears to become involved in the process and it is possible that all members of this group enter the activity simultaneously. This change (transformation) in the endosteal cell population might be due to an abnormality (qualitative or quantitative) of diffusable, humoral factors (yet to be identified) that are responsible for the growth and proliferation of these hematopoietic precursor cells. In this context, the human leukemias may be considered not as malignant, but rather the result of an aberration of factor(s) that control hematopoiesis. In this respect, the human leukemias, particularly AML, ALL and CML, might be analogous to pernicious anemia (megaloblastic anemia) as it was understood 40-50 years ago.(ABSTRACT TRUNCATED AT 250 WORDS)

Bone Marrow↗

A note on the distribution of glial cells in the molecular layer of the dentate gyrus.

The distribution of the perikarya of astrocytes and other glial cells in the molecular layer of the dentate gyrus has been studied in gold chloride-sublimate preparations of rats and of normal and reeler mice, and in plastic embedded material from young adult rats. Contrary to previous reports (Rose et al. [7]), we have found no evidence for a distinct "line" or "band" of astrocyte cell bodies along the interface between the zones of termination of the entorhinal and hippocampal afferents to the dentate gyrus. Indeed, apart from a conspicuous accumulation of astrocytes immediately beneath the pial surface and hippocampal fissure, the distribution of glial cells across the extent of the molecular layer appears to be more or less random. In view of this it is difficult to ascribe a critical role to the astrocytes in either determining the normal distribution of afferent fibers to the dentate gyrus or in promoting their re-organization following its partial deafferentation.

Afferent Pathways↗

A simple device for sectioning fresh rat brain.

This article describes a simple device that enables sectioning of an unfixed unfrozen fresh rat brain corresponding in position with the de Groote's stereotaxic coordinate systems. The construction of this device allows adjustment of the thickness of the individual brain slices and, thus, facilitates quick and precise removal of whole discrete brain particles or nuclei from a minimal number of slices. Adaptation of this device for other stereotaxic brain atlases is easily possible.

Animals↗

Endolymphatic hydrops in the rabbit: auditory brainstem responses and cochlear morphology.

A rabbit model of endolymphatic hydrops was studied using detailed functional and cytohistologic methods. Immediately following surgical destruction of the endolymphatic sac and the distal portion of the duct, measures of the evoked auditory brainstem response (ABR) revealed mild to profound losses specific to low- and high-frequency test stimuli while responses to mid-frequency signals remained unchanged for the majority of animals. Rabbits exhibited varying degrees of vestibular upset involving both overt behavior and reduced responses to caloric stimulation. Histologic processing of the plastic embedded cochleae demonstrated distended Reissner's membranes along with extensive damage to apical and basal turn sensory cells and myelinated afferent nerve fibers while the middle portion of the cochlear duct remained relatively unaltered. An atypical pattern of hair cell lesions involving a greater loss for inner than for outer hair cells was identified at the interface between damaged apical sensorineural elements and the normal appearing organ of Corti of the middle turns.

Animals↗

Determination of the regional cochlear blood flow in the rat cochlea using non-radioactive microspheres and serially sectioned cochleas.

The regional blood flow to the rat cochlea has been studied using a method which combines the microsphere method with observation of serially-sectioned, plastic-embedded cochleas. Direct quantitation of the microspheres in a reference blood sample and in the different vascular areas of the cochlea allows the analysis of blood flow patterns with respect to the different capillary beds.

Animals↗

Single-neuron labeling and chronic cochlear pathology. III. Stereocilia damage and alterations of threshold tuning curves.

Tuning curves were obtained from 100 to 150 auditory-nerve fibers spanning the range of characteristic frequencies (CFs) in each of eight cases of permanent noise-induced and three cases of permanent kanamycin-induced threshold shift. In each ear, from one to six neurons were intracellularly labeled with horseradish peroxidase. Locating the labeled terminals in plastic-embedded surface preparations of the cochlea enabled us to accurately correlate particular tuning-curve abnormalities with the condition of the sensory cells generating them. The correlations between structural and functional changes suggest that a normal tuning-curve tip requires that the stereocilia on both the IHCs and OHCs (especially those from the first row) be normal. Selective damage to the OHCs is associated with elevation of the tips and hypersensitivity of the tuning-curve tails. This tuning-curve pattern also originates from cochlear regions at the basal border of hair cell lesions where the local hair cells (and their stereocilia) appear completely normal at the light-microscopic level. Total destruction of the OHCs in a region in which the IHCs appear normal (as can happen in cases of kanamycin poisoning) is associated with bowl-shaped tuning curves which appear to lack a tip. Combined damage to the IHCs and OHCs (as typically happens in cases of acoustic trauma) is invariably associated with elevation of both tips and tails on the tuning curve. A framework for the interpretation of the results is suggested in which the activity of the OHCs is transmitted via the tectorial membrane to the tall row of stereocilia on the IHCs.

Animals↗

Computerized reconstruction of the regional blood flow in the rodent cochlea.

The regional blood flow in the cochlea has been studied by mapping the distribution of microspheres in 22 micron thick serial sections of plastic embedded rat and rabbit cochleas. Using a computer-assisted reconstruction technique, a graphic display of the microspheres in each cochlea is obtained. From this display, microspheres may be counted and their distribution may be observed both in relation to the different vascular areas and with respect to the length of the cochlea. This technique, coupled with quantitation of the microspheres in a reference blood sample, offers a method for calculating total and regional cochlear blood flow using non-radioactive microspheres. In addition, this technique allows analysis of blood flow patterns with respect to the different capillary beds along the length of the cochlea. Thus it may be possible to determine if regional blood flow displays a longitudinal gradient, as do other functional, morphologic and biochemical characteristics of the cochlea.

Animals↗

Colored microspheres for blood flow determinations twice in the same animal.

The regional cochlear blood flow has been studied in the rabbit cochlea by mapping the distribution of non-radioactive microspheres in serial sections of plastic embedded cochleas. By using microspheres of two different colors injected at two different times, it is now possible to measure the 'regional' cochlear blood flow twice in the same animal.

Animals↗

Morphological correlates of aging in the chinchilla cochlea.

The inner ears from 80 chinchillas ranging in age from premature to 19.2 years were examined as plastic-embedded flat preparations to determine the morphological changes associated with aging. Three of the four forms of human presbycusis defined by Schuknecht were found in the chinchillas. All animals had losses of sensory cells or sensory presbycusis. Inner (IHCs) and outer hair cells (OHCs) degenerated at a rate of about 0.29% and 1.0% per year, respectively. Age-related degeneration of inner (IPs) and outer pillars (OPs) occurred at a much slower rate. In four animals (5%) the dendritic processes of some of the spiral ganglion cells had degenerated in areas where the loss of sensory cells was minimal. This pathological change is likely equivalent to neural presbycusis. Six animals (7.5%) had regions of degeneration of the stria vascularis or strial presbycusis. The other common finding in the aging cochleas was the presence of lipofuscin or age pigment. Lipofuscin deposits were found to accumulate in the subcuticular region of OHCs, IPs and OPs, near the endolymphatic surfaces of many of the supporting cells and in the epithelial cells of Reissner's membrane. The IHCs accumulated much less lipofuscin. The morphological changes seen in the ears of aging chinchillas were qualitatively similar to those seen in the temporal bones of aging humans although the magnitude of the changes was considerably less. These results suggest that some of the damage found in aging human cochleas may be due to aging plus exposure to one or more ototraumatic agents.

Aging↗

Ionic coupling among cells in the organ of Corti.

Gap junctions have been demonstrated morphologically among the supporting cells of the mammalian organ of Corti but, in contradistinction to reptiles, evidence for their existence between the supporting cells and hair cells is equivocal. The literature is ambiguous with respect to electrical coupling and dye coupling among the supporting cells, and no coupling of either kind has been demonstrated for the hair cells. We found strong coupling of both kinds among the supporting cells in the cochleas of live Mongolian gerbils and a less stable coupling between the supporting cells and the outer hair cells. The electrical coupling was established by recording alternating receptor potentials in the hair cells and following their decrement in the population of Hensen's cells; the dye coupling, by injecting Lucifer yellow electrophoretically into the hair cells or the supporting cells and investigating its spread to the neighboring cells. The electrical recordings were made by means of microelectrodes filled with either 1.5 or 3 M KCl or 1 M LiCl with 6% Lucifer yellow, the latter used for dye injection. The electrode resistances ranged from about 20 to 60 M omega in the first instance, and from about 50 to 110 M omega, in the second. The electrodes were inserted into the organ of Corti through scala media according to the method of Dallos, Santos-Sacchi and Flock (1982) modified by us. The alternating potential in Hensen's cells was usually larger than in the outer tunnel of Corti and remained practically constant up to the outer margin of the Hensen's-cell population. Its phase was the same as in the outer hair cells. When the dye was injected into a Hensen's cell, it always spread to neighboring Hensen's cells and often to Deiter's cells. Dye injected into outer hair cells (identified according to anatomical and physiological criteria) also spread to Deiter's and Hensen's cells and, usually, to other outer hair cells. Stained cells were identified in surface preparations and, on two occasions, in serial sections from plastic embedded cochleas.

Animals↗

Chronic cochlear de-efferentation and susceptibility to permanent acoustic injury.

The question of whether olivocochlear (OC) efferent feedback can decrease permanent damage from acoustic overexposure was investigated by comparing the chronic threshold shifts and cochlear histopathology in guinea pigs either surgically de-efferented or sham-operated and then exposed (awake and unrestrained) to a 109- or 112-dB narrow-band noise centered at 10 kHz for 2 h. Threshold shifts were estimated using compound action potentials; hair cell loss and stereocilia condition were evaluated via light-microscopic examination of plastic-embedded surface preparations, and the degree of de-efferentation was assessed by measuring OC fascicles in the tunnel of Corti. Among animals exposed to 109-dB noise, the mean permanent threshold shift (PTS) was less than 25 dB, and there were no significant differences between normal and de-efferented animals with respect to either physiological or histological measures of acoustic injury. Among animals exposed to 112 dB, the mean peak PTS was roughly 50 dB. There was a small (but statistically significant) increase in PTS for de-efferented animals, especially at frequencies above the region of peak threshold shift; however, the patterns of hair cell loss and stereocilia damage were statistically indistinguishable. Thus, for these particular exposure conditions, sound-evoked activity in the OC system does not play a major protective role in the auditory periphery, except perhaps for the extreme basal regions of the cochlea.

Acoustic Stimulation↗

How to avoid compression: a model study of latex sphere grid sections.

The parameters which might play a role in the compression of plastic-embedded objects are studied. The compression is measured on spherical polystyrene latex particles, used as markers in the grid sectioning technique. By changing independently the hardness of the latex particles through a controlled electron irradiation and the hardness of the embedding medium, it is shown that compression is a local event depending only on the mechanical properties of the sectioned object and not on the properties of its surrounding materials. It is demonstrated on one hand that the intrinsic resin compression diminishes as resin hardness increases, and on the other hand that the latex compression can be completely eliminated after a preliminary irradiation by electrons (the electron-induced vulcanization is equivalent to a hardening). It is thus concluded that compression could be greatly reduced or eliminated if objects were sufficiently hardened during their preparation. Several preparation procedures for biological specimens are suggested. For comparison latex has also been irradiated 31/2 days near a 25 000-Ci 60Co gamma source and near the core of an 8-MW nuclear reactor. Neither of these irradiations was sufficient to produce a hardening equivalent to that of the electron irraidation.

Electrons↗

Detection of yellow fever virus nucleic acid in infected mosquitoes by RNA:RNA in situ hybridization.

An in situ hybridization technique was developed for the strand-specific detection of yellow fever virus (YFV) RNA. An 35S-labeled, transcribed RNA probe was used to detect positive-sense polarity YFV genomic RNA in infected C6/36 (Aedes albopictus) cells, dissected mosquito tissues, and sections of plastic-embedded, YFV-infected Aedes aegypti mosquitoes. Mosquito tissues fixed in buffered Formalin retained morphological integrity. The low concentrations of probe used yielded high specific signal on infected specimens and low background signal on uninfected specimens.

Animals↗

No influence of an aldose reductase inhibitor on glycogen deposition in tubules from streptozotocin diabetic rats.

Streptozotocin diabetic rats were divided into two groups. One diabetic group was given an aldose reductase inhibitor (Statil) throughout the study and the other group was left untreated. An additional group of nondiabetic rats was included in the study. After 7 months, the kidneys were perfusion fixed and blocks of tissue were sampled systematically and uniformly throughout the kidney cortex. Plastic embedded blocks were sectioned and used to determine the volume fraction of tubular glycogen by light microscopy. No difference was found between the two diabetic groups in glycogen deposits in the cortical thick ascending limb of Henle's loop, expressed as volume fraction per cortex. Tubules from control animals contained no visible glycogen, as opposed to a content of about 1% in both diabetic groups. We concluded that an aldose reductase inhibitor does not aggravate this tubular lesion (i.e., the Armanni-Ebstein lesion) in rats with experimental diabetes.

Aldehyde Reductase↗

Autologous transplantation of expanded neural precursor cells into the demyelinated monkey spinal cord.

The objective of this study was to establish if neural precursor cells could safely be developed from biopsy of the subventricular zone (SVZ) in the non-human primate (marmoset), and to determine their myelinating potential after autologous transplantation into a demyelinated lesion. Small amounts of tissue were safely collected from the subventricular-subependymal zone of the adult primate brain under ultrasonography without any neurological deficit. Neural precursor cells were isolated and expanded in the presence of mitogen in vitro. The dorsal columns of the adult marmoset spinal cord were demyelinated by X-irradiation and intraspinal injections of ethidium bromide in the center of the radiation field. Cell suspensions of the neural precursors were microinjected through a micropipette into the demyelinated lesion site in the spinal cord. Lesions were histologically examined 3 weeks after transplantation. Light and electron microscopic examination of plastic embedded sections revealed a significant number of myelinating profiles in the transplantation zone; no myelination was observed in control lesions. The myelinated axons had predominantly peripheral patterns of myelination. These results demonstrate that autologous transplantation of neural precursor cells in the adult nonhuman primate can remyelinate demyelinated central nervous system (CNS) axons, thus suggesting the potential utility of such an approach in demyelinating lesions in humans.

Animals↗