Biomedical subjects

T J Millar

Publications and source records attributed to T J Millar.

At least 37 records · Page 2Linked to original sources

PubMed1993

Distribution of the slow/cardiac isoform of skeletal muscle Ca(2+)-ATPase in developing and mature tissues of chickens determined using a monoclonal antibody.

We have established that the monoclonal antibody (MAb) AA21, raised against a crude sarcolemmal fraction prepared from adult chicken anterior latissimus dorsi muscle, recognizes the slow twitch/cardiac isoform of calcium ATPase. This was done using a combination of immunohistochemistry at the light and electron microscopic level, the change in the cell distribution in skeletal muscle during development, the molecular weight of the principal protein recognized in Western transfers, and direct comparison with another MAb of known specificity. The antigen is initially expressed by all myotubes at E10 and with development is gradually lost from all presumptive fast fibers. In addition to its immunoreaction and slow extrafusal skeletal muscle fibers, AA21 displays a highly selective immunoreactivity with a number of other cell types in different tissues. The antibody stains a subset of intrafusal muscle fibers and intestinal and arterial smooth muscle, but not venous smooth muscle. In the nervous system, a subpopulation of neurons is intensely stained, most neurons are faintly stained, and glia are not stained at all.

Animals↗

PubMed1991

Selective reduction of oscillatory potentials and pattern electroretinograms after retinal ganglion cell damage by disease in humans or by kainic acid toxicity in cats.

We recorded pattern electroretinograms, scotopic threshold responses, oscillatory potentials and ganzfeld flash electroretinograms in patients with glaucoma or other optic nerve diseases and in cats with inner retinal damage caused by intravitreal injections of kainic acid. In both studies, the scotopic b-wave and the scotopic threshold responses were normal but the oscillatory potentials and pattern electroretinograms were not. The photopic b-wave was also often reduced in patients with scotopic oscillatory potential reduction, and the reduction was proportionate to the oscillatory potential change. Oscillatory potentials were as frequently reduced as pattern electroretinograms in both patient groups, and in the few cases where only one response was reduced, there was no bias toward either measure. In cats, the effects of intravitreal injection of various doses of kainic acid on the retina were evaluated electrophysiologically, and structural damage was assessed histologically. After 25 nmol of kainic acid, the pattern electroretinograms and oscillatory potentials were reduced but neither the b-waves nor the scotopic threshold responses, were affected. Histologic studies of retinas after this dose showed swollen dendrites that were restricted to the outer part (off-sublamina) of the inner plexiform layer. Serial semithin sections indicated that most, if not all, of the swelling was confined to dendrites of large ganglion cells. Our results indicate that the size and sensitivity of the oscillatory potential response may have a role in the diagnosis and management of early glaucoma and optic nerve disease, and that the photopic electroretinogram may give similar information.

Action Potentials↗

PubMed1991

Effects of excitatory amino acids and their antagonists on the light response of luminosity and color-opponent horizontal cells in the turtle (Pseudemys scripta elegans) retina.

Both kainic acid (KA) and N-methyl-d-aspartatic acid (NMDA) depolarize luminosity-type horizontal cells (L-type H cells) in normal turtle retina. The presence of both NMDA and non-NMDA receptors for excitatory amino acids (EAAs) on these cells was highlighted by an unusual effect of the noncompetitive NMDA-antagonist, MK-801. In retinas that had been exposed to MK-801, the action of NMDA was irreversibly altered to one of hyperpolarization, while the depolarizing effect of KA was unaltered. The aim of the present study was to further characterize these receptors on L-type H cells and to extend the investigation to color-opponent H cells (C-type H cells). Intracellular recording was used to study the effects of KA, NMDA, MK-801, the competitive NMDA antagonists, 2-amino-5-phosphonopentanoic acid (AP5) and 2-amino-7-phosphonoheptanoic acid (AP7), and the nonspecific EAA antagonist, kynurenic acid (KYN) on the light responses of L-type and C-type H cells in turtle retina. The effects of combinations of these drugs were also studied. In L-type H cells the agonists caused depolarization and loss of light response, KYN caused hyperpolarization and loss of light response, and MK-801, AP5 or AP7 had no direct effect. However, application of NMDA following MK-801, AP5 or AP7, but not KYN, caused hyperpolarization and loss of light response. The depolarizing effect of KA was unaltered by these antagonists. These data confirm the presence of an unusual NMDA receptor on L-type H cells. In the case of red/green C-type H cells, application of KA caused loss of responses to both red and green light, with loss of green responses preceding loss of red responses. NMDA initially removed responses to both red and green light. The most striking effect of NMDA was seen during early washout where the responses to red were reversed (hyperpolarizing). These responses eventually recovered their normal polarity. These results suggest that the depolarizing response of C-type H cells to red light is mediated by L-type H cells, but not via inhibition of the excitatory input from green cones to C-type H cells.

2-Amino-5-phosphonovalerate↗

PubMed1990

Distribution of [3H]MK-801 binding in the turtle retina: an autoradiographical study.

Frozen sections (15 microns) of the turtle (Pseudemys scripta elegans) retina were incubated in 1.6 nM [3H]MK-801. Autoradiograms were generated using dry autoradiographical techniques. A band of [3H]MK-801 labelling was observed in the outer plexiform layer and cells were occasionally labelled in the inner nuclear and ganglion cell layers. This pattern of labelling was enhanced by preincubation in 1.0 mM glycine but not affected by pre-incubation in 3.0 mM N-methyl-D-aspartate (NMDA). We did not observe labelling associated with somata of bipolar cells and photoreceptors, or in the inner plexiform layer. The distribution of label suggests that [3H]MK-801 was bound to horizontal cells and occasional ganglion and/or amacrine cells. We have previously reported that exposure of L-type horizontal cells to MK-801 irreversibly altered the intracellular response of the cell to exogenous NMDA. The present finding strongly suggests that these physiological effects of MK-801 were due to specific binding of MK-801 to the NMDA receptor complex on the horizontal cell membrane.

Animals↗

PubMed1990

The effect of various anaesthetics on the spatial tuning of two major wave peaks in the transient pattern electroretinogram of the cat: evidence for pattern and luminance components.

The main PERG component of the transient contrast reversal pattern electroretinogram (PERG) in cats was a negative wave (3.5 microV average, SD 1.7 microV) peaking at about 130 msec (N130) with a spatial resolution above 5.5 c/deg, close to behavioural estimates. The early positivity (P35) was more variable, smaller and had lower spatial resolution. Different anaesthetic protocols affected both the waveform and the amplitude by spatial frequency functions. Responses of urethane anaesthetised cats were like those reported previously for decerebrate cats or cats paralysed and ventilated with N2O/O2/CO2 (75%/24%/1%). P35 was evoked only by coarse stimuli and N130 amplitude decreased linearly as spatial frequency increased. When the luminance response amplitude, predicted from the optical transfer function of the eye, was subtracted, spatial tuning appeared. An anaesthetic mixture of ketamine hydrochloride and xylazine depressed both P35 and N130 at low spatial frequencies while enhancing them at high frequencies. In paralysed animals ventilated with N2O/O2 (67%/33%) P35 was larger and recordable to 1.6 c/deg. Peak times were reduced and the inter-peak time halved. Other anaesthetics depressed the ERGs. These effects suggest that cats are a good model for studying N130 in isolation or its interaction with P35 and that both PERG peaks include luminance and pattern components.

Anesthetics↗

PubMed1990

Lipid soluble antioxidants preserve rabbit corneal cell function.

The survival time of rabbit corneal endothelial cells in isolated perfused cornea is extended dramatically by the addition of oxygen free radical scavengers. Lipid soluble antioxidants were found to be more effective than water soluble non-enzymatic antioxidants in extending the survival time. The ultrastructural appearance of endothelial cells treated with vitamin E was compared with controls. Vitamin E treated endothelial cells retained normal appearance up to 12h, but without vitamin E, breakdown of the endothelial cells occurred after 6h of perfusion. In these cells the major sites of damage were the mitochondria and the endoplasmic reticulum, whereas the plasma and nuclear membranes appeared normal.

Animals↗

PubMed1989

A monoclonal antibody against the slow isoform of skeletal muscle Ca2+-ATPase selectively stains a subpopulation of neurons in the central nervous system.

A monoclonal antibody against the slow isoform of chicken skeletal muscle Ca2+-ATPase recognises, in nervous tissue, analogous membrane proteins which are most concentrated in the microsomal fraction. Histochemically, the immunoreactivity of the antibody is restricted to neurones where the staining is most intense in cell bodies and dendrites, weak in axons and absent from cell nuclei. The expression of the antigen varies greatly between different neuronal populations and is developmentally regulated. The antigen is also axonally transported.

Animals↗

PubMed1989

Co-lamination of cholinergic amacrine cell and displaced ganglion cell dendrites in the chicken retina.

Displaced ganglion cells in the chicken retina were back-labelled with Fast blue injected into the nucleus of the basal optic root, then filled under visual control with Lucifer yellow to reveal the dendritic fields of the cells. In transverse sections, the dendrites of the displaced ganglion cells formed a narrow band in the outer part of the inner plexiform layer. Counter-staining for acetylcholinesterase (AChE) activity showed that the dendrites of the displaced ganglion cells and the type I cholinergic amacrine cells co-laminated in the outer part of the inner plexiform layer.

Acetylcholinesterase↗

PubMed1989

Urethane as a sole general anaesthetic in cats used for electroretinogram studies.

Cats are very sensitive to induction of anaesthesia by urethane. To anaesthetize cats with urethane, 1.0-1.3 g/kg, of freshly prepared urethane is administered intravenously at a rate of 1.92 g/h, while anaesthesia is maintained with 0.5% halothane in a 66%/33% nitrous oxide/carbogen gas mixture. Cats can then be maintained for up to 3 days by intravenous infusion at a rate of 4 ml/h of a 100 ml solution containing 50 IU heparin, 2.4 mg atropine, 4.7 g anhydrous D-glucose, and 240 mg urethane/kg. Using this anaesthetic, excellent electroretinograms can be recorded with no interfering eye movements.

Anesthesia↗

PubMed1989

MK801-induced antagonism of NMDA-preferring excitatory amino acid receptors in horizontal cells of the turtle retina.

Intracellular recordings were made from axon terminals of L-type horizontal cells in the turtle (Pseudemys scripta elegans) retina. Superfusion with Ringer's solution containing 3.0 mM N-methyl-D-aspartate (NMDA) or 0.2 mM kainic acid (KA) induced depolarization and reduction in the hyperpolarizing light responses of horizontal cells, consistent with an agonist effect of these excitatory amino acid (EAA) analogs on postsynaptic receptors. Delivery of 0.1 mM MK801, a selective blocker of NMDA-type EAA receptors, had no apparent effect on membrane potential or photoresponses, nor did it change the KA depolarization. Exposure of the retina to 3.0 mM NMDA following 0.1 mM MK801 always caused hyperpolarization of the horizontal cell and loss of light responses. Because MK801 is specific for NMDA-preferring receptors, we suggest that the reversal of the NMDA response to one of antagonism following MK801 is strong evidence for the presence of NMDA-preferring EAA receptors in turtle horizontal cells.

Animals↗

PubMed1988

Putative serotonergic bipolar and amacrine cells in the chicken retina.

Four populations of putative serotonergic cells could be detected in the chicken retina by histofluorescence and immunohistochemistry. Numerous (10,000/mm2) small (6 micron diameter) bipolar cells were located towards the middle of the inner nuclear layer, as were sparser (1000/mm2) larger (12 micron diameter) amacrine cells. Very sparse large (greater than 30 micron diameter) and more numerous small (12 micron diameter) ganglion cells were also detected. Prominent fibre plexuses were detected in the inner plexiform layer, close to the inner nuclear and ganglion cell layers, and appeared to be formed by the processes of the bipolar cells, amacrine cells and at least the large ganglion cells. Exogenous serotonin (5-HT) was detected in the chicken retina. From the effects of neurotoxins on 5-HT levels and 5-HT-like immunoreactivity (5-HTLI), most of this appeared to be associated with the amacrine cells. 5-HTLI bipolar cells were selectively destroyed by intravitreal injections of 5-10 nmol of kainic acid, while 5-HTLI amacrine cells were destroyed by N-methyl-D,L-aspartic acid and 5,7-dihydroxytryptamine. The sensitivity of the bipolar cells to kainic acid indicates that they are OFF-cells.

5,7-Dihydroxytryptamine↗

PubMed1988

The sensitivity of gas-phase models of dense interstellar clouds to changes in dissociative recombination branching ratios.

The approach of Bates to the determination of neutral product branching ratios in ion-electron dissociative recombination reactions has been utilised in conjunction with quantum chemical techniques to redetermine branching ratios for a wide variety of important reactions of this class in dense interstellar clouds. The branching ratios have then been used in a pseudo time-dependent model calculation of the gas phase chemistry of a dark cloud resembling TMC-1 and the results compared with an analogous model containing previously used branching ratios. In general, the changes in branching ratios lead to stronger effects on calculated molecular abundances at steady state than at earlier times and often lead to reductions in the calculated abundances of complex molecules. However, at the so-called "early time" when complex molecule synthesis is most efficient, the abundances of complex molecules are hardly affected by the newly used branching ratios.

Ammonia↗

PubMed1987

The ultrastructural localization of acetylcholinesterase-like immunoreactivity in the chicken retina.

The localization of acetylcholinesterase (AChE) in the chicken retina was studied using histochemical and immunohistochemical techniques. Using histochemistry, reaction end product was found in amacrine cells, ganglion cells, horizontal cells and in 4 bands in the inner plexiform layer. Ultrastructurally, the reaction end product was located between membranes of the endoplasmic reticulum, between the membranes of the nuclear envelope, surrounding neurites in the inner plexiform layer and filling synaptic clefts. Immunohistochemical techniques using a monoclonal antibody against AChE showed a similar staining pattern to that obtained with histochemistry. Ultrastructurally, AChE-like immunoreactivity was located on, not between, the membranes of the endoplasmic reticulum and nuclear envelope of amacrine cells, ganglion cells and horizontal cells. In the inner plexiform layer, immunoreactivity was on both pre- and postsynaptic membranes, and there was no immunoreactivity in non-terminal regions of the dendritic membranes and none within the synaptic clefts.

Acetylcholinesterase↗

PubMed1987

Localization of choline acetyltransferase-like immunoreactivity in the embryonic chick retina.

Putative sites of acetylcholine synthesis in the retina of the embryonic and posthatched chick were localized immunohistochemically with antisera to choline acetyltransferase; the resultant choline acetyltransferase-like immunoreactivity (ChAT-IR) was compared to demonstrated sites of acetyltransferase (AChE) activity, and changes were followed in localization during development. The results confirmed the early and rapid course of development of the chick's retinal cholinergic system described in previous biochemical and morphological studies. Immunoreactivity was first detected at embryonic day 6.5 in cells close to the retina's vitreal surface. By 8 days it was present in cells in two juxtaposed rows; by the ninth day the two rows were separated and immunoreactivity was evident in two subliminae of the inner plexiform layer. On the tenth day distribution was like that in the posthatched chicken, in type I cholinergic cells in the inner nuclear layer and in type II cells in the ganglion cell layer (Millar et al.: Neurosci. Lett. 61:311-316, '85), and similar to that of most vertebrates. Three days before hatching, a third population of weakly immunoreactive cells (type III cells) appeared within the inner nuclear layer. The onset of localizable ChAT-IR occurred in amacrine cells and in their processes, before the period of synaptogenesis. Acetylcholinesterase activity was localized at an earlier age than ChAT-IR, and at all ages was present in more cells. The results obtained support the view that "displaced" cholinergic amacrine cells begin to differentiate at the same time and in the same retinal region as type I cholinergic cells. Separation of the two groups is a consequence of the ramification of processes of amacrine and ganglion cells rather than a result of the secondary migration of cells between layers.

Acetylcholinesterase↗

PubMed1987

Cholinergic amacrine cells in the rabbit retina synapse onto other cholinergic amacrine cells.

Cholinergic amacrine cells in the rabbit retina have been detected by localizing choline acetyltransferase-like immunoreactivity. The characteristic array of normally placed and displaced amacrine cells was detected, as were two major synaptic plexuses in the inner plexiform layer. At the ultrastructural level, the most striking feature was the existence of contacts between the processes of cholinergic amacrine cells. Since this feature has also been detected in chicken and goldfish retina, it needs to be taken into account in any model which seeks to explain how the cholinergic amacrine cells are involved in generating directional selectivity at the ganglion cell level.

Animals↗

PubMed1987

Cholinergic amacrine cells of the chicken retina: a light and electron microscope immunocytochemical study.

Cholinergic amacrine cells of the chicken retina were detected by immunohistochemistry using an antiserum against affinity-purified chicken choline acetyltransferase. Three populations of cells were detected: type I cholinergic amacrine cells had cell bodies on the border of the inner nuclear and inner plexiform layers and formed a prominent laminar band in sublamina 2 of the inner plexiform layer, while type II cholinergic amacrine cells had cell bodies in the ganglion cell layer, and formed a prominent laminar band in sublamina 4 of the inner plexiform layer. Type III cholinergic amacrine cell bodies were located towards the middle of the inner nuclear layer, and their processes were more diffusely distributed in sublaminas 1 and 3-5 of the inner plexiform layer. Type I and type II cells were present at densities of over 7000 cells/mm2 in central areas declining to less than 2000 cells/mm2 in the temporal retinal periphery. The cells were organized locally in a non-random mosaic, with regularity indices ranging from 3 peripherally to over 5 centrally. Neither at the light nor electron microscopic levels was a lattice of cholinergic dendrites of the kind reported by Tauchi and Masland [J. Neurosci. 5, 2494-2501 (1985)] detectable. Within the two prominent dendritic plexuses, a major feature of the synaptic interactions of the type I and type II cholinergic cells was extensive synaptic interaction between cholinergic processes. Apart from this, there was little, if any, input to cholinergic processes from non-cholinergic amacrine cells, but there was input from bipolar cells. Output from the cholinergic amacrine cell processes was directed towards non-cholinergic amacrine cells as well as other cholinergic amacrine cells, and ganglion cells.

Animals↗

PubMed1987

The toxic effects of ethylcholine mustard aziridinium ion on cholinergic cells in the chicken retina.

The chicken retina has been used to examine the toxicity of a highly reactive chemical analog of choline, ethylcholine mustard aziridinium ion (ECMA). Following a single intravitreal injection, retinas were analyzed biochemically for CAT and AChE activities, and GABA, glycine, and dopamine levels. Retinas were also examined using histofluorescence for dopamine histochemistry, for AChE, and immunohistochemistry with antibodies to CAT, tyrosine hydroxylase, GABA, 5-HT, Leu-enkephalin, and somatostatin. A dose of 50 nmol ECMA caused a prolonged 70% depletion of CAT activity and a 40% depletion of AChE activity. The other biochemical parameters were unchanged. This result corresponds to the morphological finding that 2 populations of cholinergic cells were destroyed and that the AChE activity associated with their terminal arbors was lost. A third population of cholinergic cells, located towards the middle of the inner nuclear layer, was resistant to the toxic effects of ECMA. The other cell types, except for somatostatin-immunoreactive cells and photoreceptors, which showed transient effects, were unaffected. ECMA therefore appears to be a highly specific toxin for cholinergic cells in the retina.

Acetylcholine↗

PubMed1986

Acetylcholinesterase generates enkephalin-like immunoreactivity when it degrades the soluble proteins (chromogranins) from adrenal chromaffin granules.

Acetylcholinesterase was purified by passage through 3 affinity columns. The enzyme so purified was found to be homogeneous by electrophoresis and the peptidase and AChE activities co-eluted from a high pressure liquid chromatography column. The purified AChE degraded the chromogranins, the soluble proteins from the adrenal chromaffin granules, at a rate of nearly 8 micrograms/microgram AChE/h. The rate was fastest with the largest chromogranins, but proteins across the whole molecular weight spectrum were hydrolyzed. Immunoassay of extracts after incubation with AChE showed that enkephalin-like material had been produced. Incubations were also done with chromogranins that had been fractionated by size exclusion chromatography. The AChE degraded protein in all fractions and generated enkephalin-like immunoreactive material in fractions where it was produced by sequential treatment with trypsin and carboxypeptidase B. It seems likely, therefore, that AChE can hydrolyze some of the enkephalin precursors that are sensitive to trypsin and carboxypeptidase B, but the one-step nature of its action suggests a mode of action with fewer restrictions. It is concluded that AChE can hydrolyze proteins of widely differing sizes and the data add to the evidence that AChE is able to hydrolyze enkephalin precursors resulting in the generation of immunoreactive peptide.

Acetylcholinesterase↗