Biomedical subjects
Publications and source records attributed to G M Shepherd.
An enzyme-linked immunosorbent assay (ELISA) to detect anti-idiotype (Id) antibody is described. Using this assay auto-anti-Id was detected in the serum of aged mice immunized with 2,4,6-trinitrophenylated-Ficoll (TNP-F). Hapten eluates from anti-TNP-F immune spleen cells also contained readily detectable auto-anti-Id.
An eluate prepared by brief incubation of spleen cells from 2,4,6-trinitrophenyl (TNP) lysyl-Ficoll immunized mice with TNP-epsilon-amino-n-caproic acid causes a specific inhibition of the induction of contact sensitivity by 2,4,6-trinitrochlorobenzene skin painting. The active factor in the eluate binds to an anti-mouse immunoglobulin (Ig) immunoadsorbent column but not to a TNP immunoadsorbent column and is therefore Ig but not anti-TNP antibody. The active factor does bind to an immunoadsorbent prepared from anti-TNP antibody, suggesting that the factor has anti-idiotype specificity. Evidence based upon hapten-reversible inhibition of plaque formation and an enzyme-linked immunosorbent assay (ELISA) indicates that the eluates contain auto-anti-idiotype antibody specific for anti-TNP antibody. It is suggested that auto-anti-idiotype antibody spontaneously produced during the immune response to a T-independent antigen can specifically downregulate contact sensitization to the same epitope.
Pyramidal neurons in the cerebral cortex characteristically give rise to an apical dendrite, whose distal dendritic branches in layer I are covered with spines. These spines are known to be sites of synaptic connections, but the physiological properties of the spines and the functional significance of their responses are still largely unknown. The main function attributed thus far to these synaptic responses, situated at a great distance from the neuronal cell body, is slow background modulation of impulse output in the axon. In pursuing computer simulation analysis of electrical properties of dendrites, we have obtained results suggesting interactions between distal dendritic spines. If the heads of dendritic spines have excitable membrane properties, the spread of current from one or several spines could bring adjacent spines to their thresholds for impulse generation. This could give rise to a sequence of spine head action potentials, representing a saltatory propagation, from one or more excitable spine heads to nearby excitable spine heads, in the distal dendritic branches. Both the amplification due to several spine action potentials and the possibility of propagation into more proximal branches would increase the efficacy of distal synaptic inputs. Because of nonlinear dependence upon several modifiable parameters (such as spine stem resistance and membrane excitability) and upon the spatio-temporal pattern of synaptic input, such contingent synaptic enhancement would be particularly relevant to cortical functions underlying information processing and to plasticity underlying learning and memory.
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An in vitro preparation of the salamander olfactory epithelium has been developed for electrophysiological analysis. Intracellular measurements of membrane properties of the main epithelial cell types have been carried out, combined with Lucifer Yellow injections. The most prevalent type of cell had a high resting membrane potential and relatively low input resistance. This cell never discharged impulses, either spontaneously or to injected current. Lucifer Yellow injections identified this cell type as a supporting cell. A less frequent type had a medium resting potential and a very high input resistance. This type always discharged impulses in response to injected depolarizing current. Lucifer Yellow injections identified this cell type as an olfactory receptor neuron. The least frequent type had a medium resting potential and a high input resistance. It never generated action potentials. This nonspiking type was tentatively identified as an immature receptor neuron in the process of differentiating from basal stem cells in the epithelium. These are the first results to document physiological properties for the main cell types and morphological identification of two of the types in the same preparation of the olfactory epithelium. Our results support previous suggestions regarding the glial-like properties of the supporting cells. The membrane properties of the receptor neurons appear to be well suited for mediating the olfactory sensory response of these cells.
Transection of olfactory nerves causes degeneration of receptor neurons in the olfactory epithelium, followed by generation of new receptor neurons. We have carried out intracellular recordings to document changes in epithelial cell populations during receptor neuron degeneration and regrowth at 1, 2, and 4 weeks following olfactory nerve transection in the salamander. Receptor neurons were greatly reduced in numbers at 1 week, and gradually returned to the normal percentage of intracellular penetrations by 4 weeks. They had a resting membrane potential between -30 and -50 mV and high input resistance, 100 to 600 megohms, characteristically seen in normal epithelium. However, at 1 week, the receptor neurons were able to generate only a single spike in response to injected current, and did not re-acquire their ability to respond repetitively until 4 weeks. Cells with the properties of immature receptor neurons (resting membrane potential between -30 and -50 mV and high input resistance, 100 to 600 megohms, but unable to generate spikes) increased significantly in number in the post-transection period. This correlates with the burst of mitotic activity giving rise to new receptor neurons after nerve transection. Supporting cells changed their properties in the aftermath of transection. One type (A) showed a decrease in resting membrane potential and a small increase in input resistance. A second type (B) showed a very large increase in input resistance. These results imply that the degenerating receptor neurons transmit a signal that leads to changes in the functional properties of the glial-like supporting cells. These may involve changes in the membrane properties or in electrical coupling between cells.
A simple, computerized densitometer is described which can be used to measure densities of photographic prints by means of a fiber-optic reflectance densitometric probe coupled to the movable cursor of a digitizing tablet. The cursor, with its attached probe, is moved by hand along a scan trajectory determined by the operator. In its configuration for use with 2-deoxyglucose autoradiographs, the histological section from which the autoradiograph was derived provides architectonic landmarks for guiding the path of the scan. The X, Y and density values taken along a scan line are sequentially stored in the computer memory. Algorithms are presented for plotting densities along unfolded scan lines within layers of structures with complicated shapes, for normalizing non-linearities introduced during photographic processing, for standardizing the data sets with reference to the density of average gray matter in different animals, for calculating total integrated density within defined boundaries along the scan line, for generating averages of multiple scans, and for stacking sequential scans to form pseudo-3-dimensional plots. This system allows densitometric measures to be made from autoradiographs in anatomically defined regions, thereby permitting precise correlation between isotope concentration and histological structure.
A critical gap exists in our knowledge of the topographical relationship between the olfactory epithelium and olfactory bulb. The present report describes the application to this problem of a method involving horseradish peroxidase conjugated to wheat germ agglutinin. This material was iontophoretically delivered to circumscribed glomeruli in the olfactory bulb and the characteristics and distribution of retrogradely labeled receptor cells were assessed. After discrete injections into small glomerular groups in the caudomedial bulb, topographically defined populations of receptor cells were labeled. Labeled receptor cell somata appeared at several levels within the epithelium. The receptor cell apical dendrites followed a tight helical course towards the surface of the epithelium. The data thus far demonstrate that functional units within the olfactory system may include not only glomeruli as previously suggested but, in addition, a corresponding matrix of receptor cells possessing functional and topographical specificity.
The olfactory nerves terminate exclusively on the distal dendritic tufts of mitral cells in the olfactory bulb, which makes this a favorable model for analysis of synaptic responses in distal dendrites. Intracellular recordings of responses to olfactory nerve volleys have been obtained in the isolated turtle olfactory bulb. Single mitral cells usually responded with excitatory postsynaptic potentials (EPSPs) to volleys in two different bundles, indicating convergence from separate receptor neuron populations. Paired volleys revealed long-lasting inhibition of a test EPSP by a conditioning volley. This could be shown to be independent of the inhibition of mitral cells by granule cell interneurons in the deeper layers. The results suggest that excitatory and inhibitory synaptic interactions in the glomerular layer are important in the processing of olfactory inputs. The results also provide an exception to the classical doctrine that synaptic inhibition is preferentially sited near the cell body and axon hillock in order to control impulse generation there. Our findings of inhibitory actions on distal dendrites of mitral cells may provide a model for similar actions on distal dendrites of other central neurons.
Muscarinic cholinergic receptors in the olfactory epithelium of the salamander, Ambystoma tigrinum, were studied via binding of 3-[3H]quinuclidinyl benzilate. The receptors are present on the olfactory receptor cells in the epithelium to an amount of 0.08 pmol/mg homogenate protein. Both choline acetyltransferase and acetylcholine esterase are present in the salamander olfactory epithelium.
Pregnant rats received 2-[14C]deoxy-D-glucose (2DG) intravenously on the last day of gestation, and their fetuses were delivered 1 hour later by cesarean section. Fetal brains showed high 2DG uptake spread throughout the accessory olfactory bulb and little or no differential uptake in the main olfactory bulb. These findings demonstrate that functional activity occurs in the accessory olfactory bulb in utero and suggest that the accessory olfactory system may be the pathway by which fetal rats detect the odor quality of their intrauterine milieu.
(1) Evidence has been presented that the detection of hapten-augmentable plaques indicates cells whose secretion of antibody had been blocked by the binding of auto-anti-id to cell surface idiotypes. Because of the dependence of the assay on the affinities of the various species for one another, the number of hapten-augmentable plaques detected should be regarded as a minimal estimate of the number of cells whose secretion of antibody is inhibited by auto-anti-id. For confirmation that hapten-augmentable PFC are due to auto-anti-id 2 principal controls are important: (a) incubation of the spleen cell population with hapten prior to plaquing should remove the hapten-augmentable PFC; (b) the dialyzed supernate from hapten incubated cells should inhibit plaque formation in a hapten-reversible manner. (2) Evidence has been presented that hapten-reversible inhibition of plaque formation can serve as an assay for anti-id. Apparent false positive assays can result from the presence of anti-hapten antibody or antigen-antibody complexes; however, these apparent false positives are rarely reversed by hapten. Removal of anti-hapten antibody, by passage over an antigen immunoadsorbent, will eliminate this source of false positives and the procedure is recommended. False negative results can arise from mismatching of the anti-ids in the sample to be assayed and the idiotypes of the target cells used in the assay. This can result from shifts in idiotype expression related to age and time after antigen injection. False negatives can also result from the presence of idiotype-anti-id complexes in the sample to be assayed. This source of false negatives can sometimes be eliminated by passage of the sample through an antigen immunoadsorbent.
Two electrophysiologically distinct cell types were found with intracellular recordings for the first time in an in vitro preparation of the olfactory epithelium of the salamander, Ambystoma tigrinum. Intracellular recordings showed that Type I cells did not discharge action potentials but had high resting membrane potentials (-50 to -104 mV) and relatively low input resistances. Type II cells had resting membrane potentials of -24 to -52 mV, high input resistances, and discharged upon penetration and to depolarizing current steps. The discharge pattern of Type II cells showed the following characteristics: (1) decreased spike latency and increased discharge frequency with increasing current step intensity; (2) relatively slowly adapting spike trains; and (3) varying spike amplitude during repetitive discharges. The superficial location in the epithelium of the Type I cells implies that they may be sustentacular cells with glial-like electrophysiological properties. The Type II cells are presumably olfactory receptor cells, based on the characteristics of their spike discharge to depolarizing current and their intermediate location within the olfactory epithelium.
Uptake and retention of exogenous tritiated dopamine and L-dopa was observed within turtle olfactory bulb slices. In the more superficial layers, periglomerular and superficial tufted cells, as well as their processes, and intraglomerular dendrites were recognized as labeled. Within the deeper part of the bulb, some labeled cells between the tanycytes, as well as nerve fibers and terminals within the granule cell layer, are reported. The results confirm the presence of specific intrinsic dopaminergic cells within the reptilian olfactory bulb.
Dopaminergic mechanisms were analyzed in an isolated preparation of the turtle olfactory bulb. Field potentials were evoked by antidromic or orthodromic stimulation, and the effects determined of pharmacological manipulations of the bathing medium. In the presence of dopamine or a dopamine agonist, apomorphine, there was a reduction of amplitude and delay of onset of the component of the field potentials due to granule cell responses; fluphenazine, a dopamine antagonist, had generally opposite effects. Using paired volleys, it was found that the suppression of the response to the second test volley was reduced in the presence of dopamine or apomorphine, but enhanced in the presence of fluphenazine. The most likely explanation of these results is that exogenous dopamine depresses the response of mitral cells, which in turn decreases the dendrodendritic synaptic excitation of granule cells and reduces their inhibitory feedback onto mitral cells. This suggests that the dopaminergic cells in the bulb may suppress mitral cells by modulating excitable mechanisms in the mitral dendritic membrane, or modulating long-lasting synaptic potentials.
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Selective degeneration of mitral cells, the principal relay neuron in the central olfactory pathway, is described in the mutant mouse Purkinje cell degeneration (PCD). Concomitant effects are described in adjacent layers of the olfactory bulb which suggest a reorganization of local neuronal circuits. The 2-deoxyglucose technique produced results which suggested that the topographical distribution of sensory afferent input to the bulb was not affected.
The spatial distribution of odor-induced neuronal activity in the olfactory bulb, the first relay station of the olfactory pathway, is believed to reflect important aspects of chemosensory coding. We report here the application of high-resolution 2-deoxyglucose autoradiography to the mapping of spatial patterns of metabolic activity at the level of single neurons in the olfactory bulb. It was found that glomeruli, which are synaptic complexes containing the first synaptic relay, tend to be uniformly active or inactive during odor exposure. Differential 2-deoxyglucose uptake was also observed in the somata of projection neurons (mitral cells) and interneurons (periglomerular and granule cells). This confirms and extends our previous studies in which odor-specific laminar and focal uptake patterns were revealed by the conventional x-ray film 2-deoxyglucose method due to Sokoloff and colleagues [Sokoloff, L., Reivich, M., Kennedy, C., DesRosiers, M. H., Patlak, C. S., Pettigrew, K. D., Sakurada, O. & Shinohara, M. (1977) J. Neurochem. 28, 897--916]. Based on results obtained by the two methods, it is suggested that the glomerulus as a whole serves as a functional unit of activity. The high-resolution results are interpreted in terms of the well-characterized synaptic organization of the olfactory bulb and also serve to illustrate the capability of the 2-deoxyglucose autoradiographic technique to map metabolic activity in single neurons of the vertebrate central nervous system.