Biomedical subjects
D M Lam
Publications and source records attributed to D M Lam.
Localization of classical neurotransmitters in interneurons of the larval tiger salamander retina.
Autoradiography was used to visualize the neurons in the tiger salamander retina that exhibit high-affinity uptake of 3H-dopamine, [3H]-serotonin, [3H]-glycine, and [3H]-GABA. Both [3H]-dopamine and [3H]-serotonin were accumulated by amacrine cells and by displaced amacrine cells. [3H]-glycine was taken up by amacrine cells, displaced amacrine cells, bipolar cells, and displaced bipolar cells. [3H]-GABA was accumulated by amacrine cells and by cells in the ganglion cell layer that may be displaced amacrine or ganglion cells. [3H]-GABA was also taken up by horizontal cells, bipolar cells, and displaced bipolar cells.
Localization of serotoninlike-immunoreactive amacrine cells in the larval tiger salamander retina.
Light microscopic immunocytochemistry was used to study the populations of serotoninlike-immunoreactive cells in the larval tiger salamander retina. Of 1,135 serotonin-immunostained cells observed in transverse cryosections, 87% were identified as amacrine cells, whereas 13% were tentatively designated as displaced amacrine cells. The somas of the vast majority of serotonin-amacrine cells were situated in the innermost cell row of the inner nuclear layer. Only a few serotonin-immunostained amacrine cell somas were observed in the second row of cells from the inner nuclear layer. Serotonin-immunoreactive processes generally appeared as a diffuse plexus distributed evenly throughout all levels of the inner plexiform layer. As determined in whole-mount preparations, serotonin-amacrine cells were divisible into two populations on the basis of the diameters of their somas. Large cells (45%) ranged from 16 to 19 microns in diameter with the vast majority measuring 17-18 microns. Smaller and sometimes less intensely stained cells ranged from 14 to 16 microns in diameter with the large majority measuring 15 microns. The diameters of serotonin-displaced amacrine cells ranged from 19 to 22 microns with the large majority measuring 20 microns in diameter. An examination of whole-mount retinas revealed that serotonin-immunoreactive amacrine and displaced amacrine cells were distributed throughout the center and the periphery of the retina. The density of serotonin-amacrine cells (large and small combined) was calculated to be 173 +/- 4.5 (mean +/- standard error) cells per mm2.
Expression of high molecular weight astroglial extracellular proteins is altered by growth environment.
Conditioned medium from primary rat cortical glia was analyzed with respect to the composition of the secreted high molecular weight protein species. Developmental characteristics of astroglia are affected by growth in the presence and absence of serum. These growth conditions had a pronounced effect on the extracellular protein profile, cellular morphology, and cell substratum adhesion. Cells cultured in defined serumless medium did not express certain proteins expressed in the presence of serum but rather synthesized proteins specifically stimulated by the defined serumless environment. A morphological change from flat amorphous to a contracted fibrous network having an increased affinity for self-self cellular adhesion rather than adhesion to the surface of the tissue culture dish was also stimulated by the defined serumless medium environment. A comparison of the extracellular proteins secreted by the rat C6 glioma and the rat PC12 cell demonstrated the cell-specific nature of the primary glial proteins.
A peptide histidine isoleucine/peptide histidine methionine-like peptide in the rabbit retina: colocalization with vasoactive intestinal peptide, synaptic relationships and activation of adenylate cyclase activity.
Antisera against peptide histidine isoleucine and peptide histidine methionine were found to label a subpopulation of amacrine and displaced amacrine cells in the rabbit retina with processes ramifying in sublaminas 1, 3 and 5 of the inner plexiform layer. Preadsorption controls demonstrated that this immunoreactivity was specific for a peptide histidine isoleucine- or peptide histidine methionine-like (peptide histidine isoleucine/peptide histidine methionine-like) peptide, and was not caused by cross-reactivity of the peptide histidine isoleucine or peptide histidine methionine antibodies with vasoactive intestinal peptide vasoactive intestinal peptide. In double-label studies, vasoactive intestinal peptide and peptide histidine isoleucine/peptide histidine methionine-like immunoreactivity were colocalized in the same population of retinal neurons. Electron microscopic analysis revealed that the peptide histidine isoleucine/peptide histidine methionine-labelled cells interacted with processes of bipolar cells, amacrine cells and ganglion cells. Peptide histidine methionine and peptide histidine isoleucine were slightly less potent than vasoactive intestinal peptide in stimulating adenylate cyclase activity in the rabbit retina, while the related peptides secretin, glucagon, and the C-terminal vasoactive intestinal peptide fragment, vasoactive intestinal peptide (10-28), showed little or no stimulatory activity. Stimulation of adenylate cyclase by high concentrations of vasoactive intestinal peptide and peptide histidine methionine were non-additive. These results suggest that a peptide histidine isoleucine/peptide histidine methionine-like peptide may function as a neuroactive peptide in the mammalian retina, and that this peptide appears to be cosynthesized and colocalized with vasoactive intestinal peptide and to mimic the activity of vasoactive intestinal peptide through interaction with vasoactive intestinal peptide receptor-adenylate cyclase complexes.
Characterization of a genetically reconstituted high-affinity system for serotonin transport.
By transfecting mouse fibroblast L-M cells with human genomic DNA, we have established and identified several clonal cell lines that stably express a high-affinity serotonin (5-HT)-uptake mechanism absent in untransfected host cells. One such cell line, L-S1, possesses features of 5-[3H]HT uptake similar to those previously characterized in the central nervous system and blood platelets: (i) specificity for 5-HT; (ii) antagonism by imipramine, a known inhibitor of high-affinity 5-HT uptake; (iii) both Na+ and temperature dependences; (iv) kinetic saturability; and (v) high affinity for 5-HT (Km = 0.39 +/- 0.10 microM; Vmax = 2.14 +/- 0.55 pmol/min per mg of protein). This cell line can be used to compare the relative efficacies of known blockers of 5-HT uptake and thereby offers a rapid and reliable assay system for testing novel inhibitors of this system. Since L-S1 contains stably integrated human DNA in its genome, we postulate that the observed 5-HT-uptake system resulted from the expression of human gene(s) coding for the 5-HT transporter. Thus, cell lines such as L-S1 may represent novel means for screening and developing therapeutic agents specific for neurotransmitter-uptake systems as well as substrates for the cloning and elucidation of the genes encoding the various neurotransmitter transporters.
The coexistence of three neuroactive substances in amacrine cells of the chicken retina.
For the first time in the vertebrate retina, we have demonstrated that 3 neuroactive substances coexist in a single neuron. Using immunofluorescence, immunoperoxidase, and autoradiographic techniques, we found a subpopulation of amacrine cells in the chicken retina exhibited enkephalin- and neurotensin-like immunoreactivity and high-affinity uptake of glycine. Biochemical evidence showed that glycine release in this retina is inhibited by enkephalin and enhanced by neurotensin1.14. It is therefore possible that the two peptides form a push-pull system in self-regulating the glycine release from retinal amacrine cells.
Localization of tyrosine-hydroxylase-like-immunoreactive amacrine cells in the larval tiger salamander retina.
Immunocytochemistry was used to localize the populations of tyrosine-hydroxylase-like (TH)-immunoreactive cells in the tiger salamander retina. Ninety percent of these cells possessed somas that were situated in the innermost cell row of the inner nuclear layer and were classified as amacrine cells. Ten percent of TH-immunoreactive somas were located in the ganglion cell layer and were tentatively designated as those of displaced amacrine cells. The processes of TH-immunoreactive cells ramified most heavily in sublayer 1 of the inner plexiform layer, while a relatively small number of TH-labelled processes distributed in sublayers 3 and 5. Less than 1% of TH-immunoreactive cells in the amacrine cell layer exhibited a short process of somal origin that extended distally toward the outer plexiform layer. However, these processes did not cross the whole of the inner nuclear layer, and no immunolabelling was observed in the outer plexiform layer. An examination of retinal whole-mounts revealed that TH-immunoreactive amacrine and displaced amacrine cells were distributed throughout the center and periphery of the retina. The density of TH-immunolabelled amacrine cells was calculated to be 49 +/- 13 (mean +/- standard error) cells per mm2. The vast majority of TH-immunoreactive amacrine and displaced amacrine cells exhibited a stellate appearance and gave rise to three or more primary dendrites. A few TH-amacrine and displaced amacrine cells possessed two primary dendrites that emerged from opposite sides of their somas. The processes of TH-immunoreactive cells were generally poorly branched and varicose with terminal branches sometimes appearing thin and beaded. Because some TH-immunolabelled processes were very long, there was considerable overlap between the dendritic fields of neighboring TH-cells. Lastly, individual TH-immunoreactive amacrine and displaced amacrine cells were often observed in whole-mounts to provide processes that ramified at more than one level of the inner plexiform layer.
Quantitative studies of enkephalin's coexistence with gamma-aminobutyric acid, glycine and neurotensin in amacrine cells of the chicken retina.
Previous double-label studies demonstrate that enkephalin coexists with gamma-aminobutyric acid, glycine or neurotensin in amacrine cells of the chicken retina. The present study utilizes double- and triple-label paradigms to quantitatively analyze these coexisting relationships. Twenty-eight percent of enkephalin-like immunoreactive amacrine cells were found to exhibit high-affinity uptake of [3H]GABA, while 53% of enkephalin-amacrine cells specifically accumulate [3H]glycine. Moreover, the present study predicts that at least 26% of enkephalin-amacrine cells which accumulate [3H]glycine should also be immunoreactive for neurotensin.
GABAergic ganglion cells in the rabbit retina.
The ganglion cells are the output neurons of the retina. There is, however, relatively little known about the neurotransmitters used by these cells. In the present study, ganglion cells identified with a ganglion cell-specific monoclonal antibody (AB5) are shown in separate double-label experiments to be gamma-aminobutyric acid (GABA)-like immunoreactive and to possess a high-affinity uptake mechanism for [3H]GABA accumulation. The localization of these markers of GABA activity to AB5-labelled ganglion cells provides the first definitive evidence for the presence of a classical transmitter in retinal ganglion cells and suggests that GABA may perform a role as a neurotransmitter in these cells.
Localization of neurotensin-like immunoreactive amacrine cells in the larval tiger salamander retina.
Light microscopic immunocytochemistry was used to localize the populations of NT-like immunoreactive amacrine cells in the larval tiger salamander retina. Seventy-nine percent of NT-immunostained cells observed in transverse cryo-prepared sections were classified as Type 1 amacrine cells. Another 6% were classified as Type 2 amacrine cells, while 15% of the NT-cells had their cell bodies situated in the ganglion cell layer and were tentatively designated as displaced amacrine cells. Each type of NT-like immunoreactive cell was observed in the central and peripheral retina. NT-immunostained processes were observed to ramify in sublayers 3 and 5 of the inner plexiform layer. An examination of retinal whole mounts revealed that NT-amacrine cells were distributed throughout the center and periphery of the retina at a density of 82 +/- 24 cells/mm2. The dendritic fields of NT-immunostained amacrine and displayed amacrine cells were observed to be either symmetrically or asymmetrically distributed about their somas. Symmetrical dendritic fields were generally oval-shaped and ranged in diameter from 250 to 500 micron (major axis) by 150 to 250 micron (minor axis). Asymmetrical dendritic fields were observed to encompass one-half or less of an imaginary circle surrounding their soma of origin and were orientated in all directions. The processes forming asymmetrical dendritic fields ranged from 75 to 260 micron in length. Furthermore, partial overlap was often observed between the dendritic fields of adjacent NT-amacrine cells.
To pop a balloon: aeromedical evacuation in the 1870 siege of Paris.
H. G. Armstrong, in the 3rd edition of his Principles and Practice of Aviation Medicine stated: "The first use of air transportation in support of medical activities occurred during the siege of Paris in 1870 when a total of 160 patients were removed from that city by means of an observation balloon." Careful review of the available literature and of reports during and subsequent to that time have offered no corroboration of this statement, whereas the names and missions of some 167 balloonists and passengers are amply documented. An extensive bibliography and footnotes, available upon request from the author, attest to the multiple uses to which this new form of transportation was put; unfortunately, aeromedical evacuation was not one of them.
Ganglion cell density in albino and pigmented rabbit retinas labeled with a ganglion cell-specific monoclonal antibody.
Retinas from two rabbits, one normally pigmented and one albino, were labeled with monoclonal antibody AB5, which has been shown to be a specific marker for ganglion cells. This method obviates criteria for distinguishing among ganglion cells, displaced amacrine cells, and glia. Labeled cells were counted within small fields at some 2000 regularly spaced points on each retina. These counts were transformed to maps of ganglion cell density. In general, the density map for the pigmented retina was similar to those obtained by earlier studies with non-specific stains, thereby confirming the basic validity of most previous studies and demonstrating the applicability of AB5 labeling to work of this type. The ganglion cell density map of the albino retina was abnormal, showing a clear deficit of ganglion cells in the nasal portion of the visual streak. This result not only indicates that the albino anomaly has retinal effects, but also suggests a major impact on ganglion cells whose projections (in normal animals) are contralateral.
Interactions between enkephalin and gamma-aminobutyric acid in the larval tiger salamander retina.
Both double-label and intracellular electrophysiological recording techniques were utilized to investigate the interactions between enkephalin and gamma-aminobutyric acid in the larval tiger salamander retina. Double-label studies revealed that the vast majority (greater than 96%) of enkephalin-immunostained amacrine cells also exhibit high affinity uptake of [3H]gamma-aminobutyric acid. Electrophysiological evidence demonstrated that morphine and gamma-aminobutyric acid exert opposite effects on a population of On-Off ganglion cells. gamma-Aminobutyric acid decreased the activity of these cells, while enkephalin increased their activity. These findings support the idea that opiate-mediated pathways inhibit GABAergic pathways in the vertebrate retina.
Accumulation of gamma-aminobutyric acid by horizontal cells isolated from the goldfish retina.
In the goldfish retina, H1 horizontal cells, which receive input predominantly from red sensitive cone photoreceptors, possess a single high-affinity uptake mechanism for gamma-aminobutyric acid (GABA). This GABA uptake is enhanced by light stimulation, which hyperpolarizes the H1 cells. The regulation of this uptake mechanism was examined in isolated horizontal cells by measuring the accumulation of exogenously supplied 3H-GABA. Solutions containing elevated external K+ or glutamate were used to quantitatively depolarize the cells to reveal that the potential-sensitive GABA uptake is maximal under hyperpolarizing conditions and minimal with depolarization. The driving force for GABA uptake is derived from the Na+ electrochemical gradient, with approximately 2 Na+ ions being cotransported with each molecule of GABA. The results presented suggest that the uptake mechanism permits the synaptic concentration of GABA to be regulated by the membrane potential of the H1 horizontal cells. This, then permits the presynaptic horizontal cell to modulate the synaptic concentration of transmitter in this tonically active synapse.
Expression of single calcium channels in Xenopus oocytes after injection of mRNA from rat heart.
Oocytes of Xenopus laevis, after microinjection with mRNA from rat heart, display typical high-threshold calcium (Ca) whole cell currents. To prepare to study structure-function relationships of the cardiac Ca channel molecule, we examined the fidelity of expression of biophysical and pharmacological properties at the molecular level. Cell-attached gigaseal recordings in five K-depolarized oocytes injected with adult rat heart mRNA showed single channel Ba currents with mean amplitude 1.3-1.5 pA at 0 mV, slope conductance 18-25 pS, and extrapolated reversal potential 57-68 mV. Openings were predominantly brief (mean 1.2 ms) but longer openings (mean 9 ms) were greatly enhanced in 10(-6) M BAY-K 8644, increasing the ensemble average current at 0 mV by more than fivefold. These features are typical of high-threshold cardiac Ca channels. In two patches from one injected oocyte, we saw multiple Ca channel conductances, as recently observed in other preparations. We conclude that X. laevis oocytes injected with adult rat heart mRNA produce high-threshold cardiac Ca channels with molecular properties identical to native cells.
Real-time hexose monophosphate shunt activity in light- and dark-adapted rabbit retinas.
Correlation proton nuclear magnetic resonance spectroscopy was performed on light- and dark-adapted rabbit retinas to elucidate real-time hexose monophosphate shunt (HMPS) activity. Light significantly stimulated retinal HMPS initially, which consumed 17% more glucose than that in dark-adapted retinas. The glucose consumption eventually declined to 16% of total, the baseline level, after 60 min of light exposure. In contrast, dark-adapted retinas showed an initial HMPS activity of 29% total glucose consumption, which declined to the basal level after 40 min. Lactate production appeared stable in both sets of retinas. Tert-butyl hydroperoxide (1 mM) also stimulated the shunt; however, a combination of light-adaptation and tert-butyl hydroperoxide did not stimulate the shunt additively. These data indicate that the retina has limited HMPS capacity.