Biomedical subjects

K G Smithson

Publications and source records attributed to K G Smithson.

16 recordsLinked to original sources

PubMed2000

Phrenic motoneuron morphology during rapid diaphragm muscle growth.

In the adult rat, there is a general correspondence between the sizes of motoneurons, motor units, and muscle fibers that has particular functional importance in motor control. During early postnatal development, after the establishment of singular innervation, there is rapid growth of diaphragm muscle (Dia(m)) fibers. In the present study, the association between Dia(m) fiber growth and changes in phrenic motoneuron size (both somal and dendritic) was evaluated from postnatal day 21 (D21) to adulthood. Phrenic motoneurons were retrogradely labeled with fluorescent tetramethylrhodamine dextran (3,000 MW), and motoneuron somal volumes and surface areas were measured using three-dimensional confocal microscopy. In separate animals, phrenic motoneurons retrogradely labeled with choleratoxin B-fragment were visualized using immunocytochemistry, and dendritic arborization was analyzed by camera lucida. Between D21 and adulthood, Dia(m) fiber cross-sectional area increased by approximately 164% overall, with the growth of type II fibers being disproportionate to that of type I fibers. There was also substantial growth of phrenic motoneurons ( approximately 360% increase in total surface area), during this same period, that was primarily attributable to an expansion of dendritic surface area. Comparison of the distribution of phrenic motoneuron surface areas between D21 and adults suggests the establishment of a bimodal distribution that may have functional significance for motor unit recruitment in the adult rat.

Aging↗

PubMed1995

NMDA and non-NMDA receptors on rat supraoptic nucleus neurons activated monosynaptically by olfactory afferents.

The recently discovered efferent projections from the main and accessory olfactory bulbs to the supraoptic nucleus (SON) were further investigated. Intracellular electrophysiological methods were used to determine (a) if these projections are monosynaptic, (b) which excitatory amino acid (EAA) receptor subtypes mediate responses to activation of these pathways and (c) whether the same receptor subtypes mediate responses of phasically firing (vasopressin) and continuously firing (putative oxytocin) neurons. Recordings were made from SON neurons in large explants and 500 microns thick horizontal slices, containing 2-6 mm of the piriform cortex and lateral olfactory tract (LOT). This allowed recording of synaptic responses to selective stimulation of the LOT. EPSPs in SON neurons faithfully followed stimulus frequencies of 50-100 Hz, indicating that these inputs were monosynaptic. Stimulus-evoked EPSPs were blocked by the non-specific EAA antagonist, kynurenate. Perifusion of the slice with Mg(2+)-free medium revealed the presence of NMDA receptors in addition to the non-NMDA receptors on both phasically and continuously firing cells, indeed, on all cells tested. Partial blockade of these EPSPs in Mg(2+)-free medium could be achieved with either the NMDA antagonist, AP5, or the non-NMDA antagonist, CNQX or NBQX. Full blockade of the stimulus-evoked EPSPs was effected by adding both types of antagonists to the medium, although spontaneous EPSPs were still observed in several cells. These results are consistent with prior studies showing both receptor subtypes in the SON. This is the first demonstration that afferent stimulation activates both subtypes in the same SON neuron regardless of its peptide content.

2-Amino-5-phosphonovalerate↗

PubMed1995

Growth-related alterations in motor endplates of type-identified diaphragm muscle fibres.

Using a double-labelling technique, and dual-channel confocal microscopy, we examined the three-dimensional and two-dimensional morphologies of motor endplates on type I and II muscle fibres of 21-day-old and adult rat diaphragms. Motor endplates were visualized with fluorescein-conjugated alpha-bungarotoxin, and muscle fibre type was immunocytochemically determined using an anti-fast (type II) myosin antibody with a Cy5-conjugated label. Surface (three-dimensional) and planar (two-dimensional) areas were obtained from three-dimensional reconstructions of confocal optical sections of labelled endplates. Muscle fibre diameters were also measured. Total branch lengths were measured from projection images of the three dimensional reconstructions. The surface and planar areas of endplates on type I fibres at day 21 were larger than those on type II fibres, and this difference increased with maturation. In adults, the surface area of endplates was positively correlated to muscle fibre size, but such a correlation was not found at day 21. When normalized for fibre diameter, the surface areas of endplates on type I fibres were still significantly larger than those on type II fibres in both age groups. The normalized endplate surface area for type II fibres remained constant with maturation, whereas for type I fibres, the increase in endplate surface area was disproportionate to fibre growth.

Animals↗

PubMed1994

Application of the Cavalieri principle in volume estimation using laser confocal microscopy.

The Cavalieri principle, a well-established stereological technique, uses interpolation between samples to estimate volume of three-dimensional (3D) objects. Serial optical sectioning with the confocal microscope resembles certain aspects of the Cavalieri principle, albeit with no interpolation. However, reconstruction and analysis of finely spaced optical sections can be cumbersome and time consuming. Application of the Cavalieri principle to confocal sections may be advantageous in reducing the size of the data set required to obtain reliable estimates of volume. In the present study, somal volumes of phrenic motoneurons were estimated by applying the Cavalieri principle to confocal images. These estimates were compared to measurements of somal volume using on interpolation of confocal sections. Phrenic motoneurons in adult rats were retrogradely labeled with a fluorescent rhodamine dye. Confocal optical sections of 0.6 micron thickness were then obtained from 150-micron-thick spinal cord slices containing labeled neurons. These image sets were reoriented to represent transverse sections. The Cavalieri principle was applied to these confocal image sets at selected sampling intervals from 1.2 to 3.0 microns. Planimetric measurements of motoneuron somal cross-sectional area in the selected sections were made using a point-counting method. At sampling intervals less than 2.4 microns, individuals motoneuron somal volume estimates were similar for the noninterpolated confocal and the interpolated Cavalieri methods. At these sampling intervals, the distributions of motoneuron somal volumes were also similar for the two methods. At a sampling interval of 2.4 microns or greater, there was a greater variability in individual motoneuron somal volume estimates, although the population mean and median were similar to the noninterpolated confocal measurements. Therefore, a satisfactory agreement between noninterpolated confocal measurements and the Cavalieri estimates suggests that less-stringent optical sectioning parameters may suffice for individual cell volume measurements when using confocal microscopy, thus making it significantly more efficient.

Animals↗

PubMed1993

Rapid synaptic changes and bundling in the supraoptic dendritic zone of the perfused rat brain.

In the dendritic region of the supraoptic nucleus of young adult rats we found ultrastructural evidence of rapid (< 30 min) morphological reorganization following transcardial perfusion of the anesthetized animals with high (325 mOsm), but not with normal, osmolality (302 mOsm) balanced salt solutions. The changes observed included significant increases in dendritic bundling and increased numbers of multiple axodendritic synapses (where one presynaptic terminal contacted two or more postsynaptic dendrites). In our initial experiments the perfusion medium contained tannic acid, but when the tannic acid was omitted in a separate set of experiments, the results were both similar and more striking. These studies indicate that the plasticity previously seen in this region under physiological conditions can occur rapidly and probably involves astrocytic process withdrawal from between adjacent dendrites. This change in the distribution of astrocytic processes allows previously single synapses access to additional postsynaptic dendritic membrane. Together, increased dendritic bundling and multiple synapses may facilitate the synchronization of cell firing and, thus, hormone release.

Animals↗

PubMed1993

Measurements of motoneuron somal volumes using laser confocal microscopy: comparisons with shape-based stereological estimations.

Previous studies on motoneuronal morphometry have assumed the geometry and orientation of neuronal soma in estimating somal volumes based on two-dimensional measurements. In this study, the optical sectioning property of the confocal microscope was used to make direct measurements of phrenic moto-neuron somal volume. These measured volumes were compared to shape-based stereological estimates of volume. Phrenic motoneuron pools in adult rats were retrogradely labeled with fluorescent dye. Labeled motoneurons, in 150-micron-thick tissue sections, were imaged using a Bio-Rad MRC 500 confocal microscope. Somal volumes were directly measured using ANALYZE, a comprehensive image processing software package. These volumes were compared to volume estimates based on five geometrical shapes previously used to study spinal motoneurons. In the adult phrenic motoneuron pool, overestimations of somal volume up to 300% were observed in cases where the Z-axis dimensions of the neurons were not simply related to the X and Y dimensions. None of the five geometrical shapes were found to be suitable for estimating either mean or individual somal volumes of the phrenic motoneuron pool. Confocal microscopy allowed accurate reconstruction along X, Y, and Z axes, and therefore provided a more direct method of measuring motoneuron somal volumes. We conclude that significant and inconsistent errors can be introduced by using shape-based stereological methods for estimating neuronal somal volumes.

Animals↗

PubMed1992

Supraoptic nucleus afferents from the accessory olfactory bulb: evidence from anterograde and retrograde tract tracing in the rat.

Our earlier electrophysiological work provided evidence of a direct input to the supraoptic nucleus (SON) from the olfactory bulbs; however, these experiments could not determine if the input originated in the main and/or accessory portions of the olfactory bulb. Here, a connection between the accessory olfactory bulb (AOB) and the SON of the rat was examined using a combination of anatomic techniques. We employed neurophysin immunocytochemistry to delineate the morphological boundaries of the SON and the proximal arborizations of supraoptic dendrites. Accessory olfactory bulb efferents to the SON were studied by injection of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) into the AOB. The distribution of retrogradely labeled cells within the AOB was also determined after injection of either rhodamine-labeled latex microspheres (rhodamine beads) or Fluoro-Gold (FG) into the SON. Neurophysin immunocytochemistry revealed that SON dendrites extended beyond the generally accepted boundaries of the nucleus, coursing ventrolaterally along the surface of the periamygdaloid cortex. Anterograde tract tracing with WGA-HRP labeled AOB efferents including a dense plexus of terminals and fibers around the ipsilateral SON along the path of the ventrally projecting dendrites. Injections of retrograde tracers into the SON resulted in rhodamine bead or FG labeling of mitral cells throughout the ipsilateral AOB. Taken together, these anatomic studies suggest a direct projection from the accessory olfactory bulb to the SON of the rat and thus a vomeronasal organ to SON pathway.

Afferent Pathways↗

PubMed1989

Neurosecretory endings in the rat neurohypophysis are en passant.

A combination of Golgi, Phaseolus vulgaris leucoagglutinin, and transmission electron microscopic (EM) techniques was used to investigate the morphology of neurosecretory axons and their endings in the neurohypophysis of the rat. Light microscopy indicated that the neurosecretory processes are very tortuous, varicose, and branched, often running in close association with blood vessels. EM, as well as reconstruction from serial thin sections, demonstrated that the combination of synaptoid membrane specializations, dense core vesicles, and accumulations of microvesicles, thought to indicate areas of preferential hormone release, can occur anywhere that the axons contact the basal lamina (BL) lining the perivascular space. Usually, but not always, this was accompanied by some degree of axonal dilation. Individual neurosecretory axons frequently entwined around, or ran adjacent to, blood vessels, a short length of axon forming multiple "endings." Thin glial (pituicyte) processes were interposed between these endings. Axonal processes were also seen to end blindly as end-bulbs suspended in the perivascular space where they were often surrounded by pituicyte processes. The morphology observed suggests that reductions in the contact length of individual nerve terminals could be mediated through increased pituicyte coverage along BL. Conversely, a reduction of pituicyte coverage along the BL could lead to more areas of axonal contact with the BL inducing or allowing the formation of more endings, such as has been reported to occur during lactation or prolonged elevation of plasma testosterone levels.

Animals↗

PubMed1989

Supraoptic nucleus afferents from the main olfactory bulb--I. Anatomical evidence from anterograde and retrograde tracers in rat.

The morphological features of a putative connection between the main olfactory bulb and the supraoptic nucleus of the rat was studied using a combination of anatomical techniques. Immunocytochemistry of neurophysin-containing processes were employed to delineate morphological features of supraoptic dendrites. Main olfactory bulb efferents to the supraoptic nucleus were studied by injection of the anterogradely transported substances, wheatgerm agglutinin conjugated horseradish peroxidase or Phaseolus vulgaris leucoagglutinin, into the main olfactory bulb. To confirm the results of these studies, the distribution of retrogradely labeled cells within the main olfactory bulb was determined after injection of rhodamine-labeled latex microspheres or Fluoro-Gold into the supraoptic nucleus. Neurophysin immunocytochemistry revealed the supraoptic nucleus dendritic plexus which coursed anteroposteriorly beneath supraoptic somata. Additionally, a portion of this plexus also projected ventrolaterally into periamygdaloid areas, a feature of supraoptic architecture which is not generally appreciated. The anterograde tracers labeled main olfactory bulb efferents including a dense plexus of terminals and fibers ventrolateral to the ipsilateral supraoptic nucleus. The pattern of anterogradely labeled fibers and terminals appeared to overlap with the distribution of ventrolaterally projecting neurophysin-containing processes. Since the latter consists of dendritic processes of supraoptic origin, this suggests that the main olfactory bulb projects to the supraoptic nucleus. Injections of rhodamine-labeled latex microspheres or Fluoro-Gold resulted in retrogradely labeled mitral cells throughout the ipsilateral main olfactory bulb. Taken together, these anatomical studies demonstrate a direct projection from the main olfactory bulb to the supraoptic nucleus of the rat. A comparison electrophysiological study confirmed these results.

Animals↗

PubMed1988

Synaptic inputs and electrical coupling among magnocellular neuroendocrine cells.

This paper first briefly reviews the evidence for synaptic and nonsynaptic plasticity among the neurons and glia of the magnocellular hypothalamo-neurohypophysial system. Emphasis is placed upon the importance of the roles played by astrocytes in the remodeling of the magnocellular nuclei under various conditions of increased hormone demand. Evidence is then reviewed from more recent studies showing that there is electrical coupling among magnocellular neurons, and that this coupling shows plasticity similar to that shown for other characteristics of the system (e.g., chemical synapses, dendritic bundling etc.). Further, evidence is presented that extent of electrical coupling can be modified not only by manipulating the physiological state of the animal (such as lactation), but also by electrical stimulation of newly described olfactory afferent inputs to the cells of the supraoptic nucleus. The possible functional significance of these findings is discussed in relation to the behavior of nursing rats.

Animals↗

PubMed1986

Immunoreactivity to vasopressin- but not oxytocin-associated neurophysin antiserum in phasic neurons of rat hypothalamic paraventricular nucleus.

Bursts of action potentials were recorded intracellularly from 11 phasically firing magnocellular neurons in the paraventricular nucleus in slices of rat hypothalamus. The bursts of overshooting, often broadening action potentials (63-87 mV peak-to-peak) were superimposed on depolarizing plateau potentials. Phasic activity was recorded before and/or after the neurons were injected with the fluorescent dye Lucifer Yellow CH. Injected neurons were first examined in whole slices, and subsequently, in sectioned material, characterized immunocytochemically using antisera to vasopressin- and oxytocin-associated neurophysins (VP-NP and OT-NP respectively). The 11 injections produced 8 single dye filled neurons and 3 pairs of dye-coupled neurons, 14 dye-filled cells in all. Six of the single cells and all the dye coupled pairs were immunoreactive with VP-NP antiserum and not reactive with OT-NP antiserum. Most of these neurons were in areas of the nucleus in which VP-NP reactive cells predominated, but two were surrounded by OT-NP reactive cells. Two single, dye-filled, phasically active, magnocellular neurons failed to show immunoreactivity to either antiserum.

Animals↗

PubMed1985

Lactation-associated redistribution of the glial fibrillary acidic protein within the supraoptic nucleus. An immunocytochemical study.

Electron-microscopic evidence indicates that during conditions of high hormone demand such as lactation there is a dramatic reduction in the number of fine glial processes which are normally interposed between magnocellular neuroendocrine cell somata in the supraoptic nucleus (SON). The purpose of this study was to corroborate these data at the light-microscopic level and to gain some insight into what underlying events might accompany these apparent morphological changes. The distribution of the glial fibrillary acidic protein (GFAP), an intermediate filament component of the astrocytic cytoskeleton, was visualized in lactating or estrous rats using peroxidase-antiperoxidae immunocytochemistry. Computerized image analysis was employed to determine and compare the staining distributions of this protein for the two groups of rats. Statistical analysis revealed a redistribution of GFAP immunostaining in the SONs of lactating animals as compared to controls. No differences in staining were found in a control area dorsolateral to SON. The pattern of change was to a less dense, more homogeneous distribution of GFAP in lactating rats, a change which could be interpreted as reflective of a reduction in the number of densely staining glial processes.

Animals↗

PubMed1985

Dye-coupled magnocellular peptidergic neurons of the rat paraventricular nucleus show homotypic immunoreactivity.

Magnocellular neurons in rat hypothalamic slices are known to exhibit dye coupling: the transfer of the fluorescent dye, Lucifer Yellow, from an intracellularly-injected neuron to one or more nearby neurons. The question of the hormonal identity of coupled cells and the possibility of dye coupling as an artefact led us to determine the immunoreactivity of dye-coupled magnocellular neurons in the paraventricular nucleus of the rat hypothalamus using antisera to oxytocin- and vasopressin-associated neurophysins. In 23 pairs, one triplet, and one quadruplet, immunoreactivity to one or the other antiserum was always exclusive, and dye coupling was always homotypic, that is, coupled neurons in each instance were reactive to the same antiserum. The quadruplet, triplet and 17 pairs were immunoreactive to vasopressin-associated neurophysin, and oxytoxin-associated neurophysin immunoreactivity was observed in the remaining pairs. Immunoreactivity to each antiserum was found for somasomatic and non somasomatic modes of coupling and for coupled neurons in the three magnocellular areas of the nucleus. A relationship between mode of coupling and hormone content was not detected. The data support the hypothesis that coupling is a real, functionally significant mechanism for coordinating neuronal activity in this nucleus, particularly under conditions of high hormone demand. They do not support the idea that coupling is artefact. The possibility of a relationship between hormone content and mode of coupling, and the projection pathway(s) of the coupled neurons of each type require further study.

Animals↗

PubMed1984

A reliable method for immunocytochemical identification of Lucifer Yellow injected, peptide-containing mammalian central neurons.

An immunocytochemical procedure is described for reliably determining the hormone content of magnocellular neuroendocrine neurons that have been injected with Lucifer Yellow in slices of rat hypothalamus. The chief advantages of this procedure over others currently available are: (a) it permits whole mount observation of the tissue, and thus, of the morphology of filled cell(s) as well as of such phenomena as dye-coupling; (b) the reliability of tissue preparation and peptide determination has been optimized so that about 85% of injected cells are identified immunocytochemically; and (c) the final immunostained product is permanent, permitting bright-field examination of the injected cell. Relative advantages and limitations of this and other recently published methods are discussed.

Animals↗

PubMed1984

An incubation chamber for the simultaneous, on-slide treatment of brain sections with different reagents.

The design and use of a new microscope slide incubation chamber is described. The chief advantage of this device is that it allows the simultaneous incubation of tissue sections which are mounted on glass slides, either together with the same reagent, or individually with different reagents. Simple construction makes this chamber durable, reliable, and inexpensive. Advantages and disadvantages of this device, as well as, other published devices are discussed.

Animals↗

PubMed1983

Polyethylene glycol embedding: a technique compatible with immunocytochemistry, enzyme histochemistry, histofluorescence and intracellular staining.

A technique is described which permits rapid processing of neural tissue for light microscopic analysis of sections of 1-40 microns thickness. This technique was developed as an alternative to paraffin embedding. When compared to paraffin, polyethylene glycol (PEG) offers the following advantages: 10-15 degrees C lower embedding temperatures, net tissue shrinkage of less than 5% vs approximately 50% in paraffin, and approximately one-half the embedding time. Tissue orientation during embedding and sectioning is particularly easy to control, e.g. 500 microns brain slices can be routinely flat-embedded and sectioned at 5 microns to form excellent ribbons. Since PEG is water-soluble, tissue may be dehydrated with a series of aqueous PEG solutions; the embedding matrix is easily removed by washing with a variety of aqueous buffers. These procedures allow subsequent electron microscopic analysis of material with generally well preserved ultrastructure. However, PEG is hygroscopic, thus tissue blocks become soft and difficult to section in high (greater than 90%) relative humidity. PEG was found to be compatible with intracellular staining with Lucifer yellow, horseradish peroxidase enzyme histochemistry, aqueous histofluorescence and immunocytochemical demonstration of neuronal peptides and glial fibrillary acidic protein.

Animals↗