Transient and steady state focal and pattern electroretinogram nerve section losses in cats with unilateral optic.

This study shows the ketamine/xylazine anaesthetised cat is a useful model for the effect of unilateral optic nerve section on pattern electroretinograms (PERGs), especially if stimuli extending to previously untested low spatial frequencies and preferably down to the focal ERG (FERG) are included. The transient reversal rate, seldom used in animals,has advantages over steady state recording. Transient PERGs had signs of true spatial tuning, a higher amplitude and signal noise ratio and showed the effect of optic atrophy at low spatial frequencies more rapidly.

Multifocal, pattern and full field electroretinograms in cats with unilateral optic nerve section.

AIM: To look for a subcomponent of the mFERG generated at the optic nerve head and increasing in latency with distance from it. To compare multifocal electroretinogram (mFERG, mPERG) changes to those in full field ERGs and transient and steady state pattern and focal ERGs (PERGs, FERGs) in cats with total unilateral optic nerve section. METHOD: We recorded multifocal flash ERGs (mFERGs) at three levels of intensity and multifocal pattern ERGs (mPERGs) within 61 equal areas after total unilateral optic nerve section in five long term (> 18 month) survival cats, as part of a long term serial study of full field flash and pattern ERG changes to many stimuli, in a larger population. Cats were anaesthetised with Ketamine/Xylazine and wore Henkes electrodes with 6mm artificial pupils. Intact retinal circulation was verified by fluorescein angiography and optic nerve section by retinal photography and histology. We compared the mean and mean summed multifocal responses, from the normal and denervated eyes. We also compared the mean interocular difference around the area centralis and as a function of distance from the optic nerve head, across the horizontal meridian for the mFERG to the most intense stimulus. The degree of change was compared to that in other types of ERG, in the larger set of cats. RESULTS: mFERGs were similar across cats. Response density was flat with no prominence at the area centralis. Average summed mFERGs were similar in the normal and denervated eye. In the interocular differences a component near OP2 was reduced in the first kernel to the most intense stimulus, near OP1 and OP3 in the second kernel and locally, there was a hint of a component near OP2, which varied in latency in a ring around the disk and at the area centralis. Nevertheless, no component could be seen, varying in latency with distance from the optic nerve head, across the horizontal meridian. No mPERG was recordable in these conditions. Full field PERGs and FERGs were very reduced. Full field flash ERG amplitude changes were small (4-20%) and slower to appear than PERG changes. Degree of ERG reduction and correlation with PERG losses was greatest for the mesopic OPs, low for the scotopic tests (STR, ERG and OPs) and near zero for the mesopic ERG. The mFERG and mesopic ERG both lost OPs without overall amplitudes. CONCLUSIONS: The cat mFERG does not have a component, varying in latency with distance from the optic nerve head. The only change was qualitiatively similar to that in the light adapted ERG. With intense stimuli there were local changes around the time of OP2 near the area centralis which might be explained by local variations in ganglion cell density.

Characterization of an inwardly rectifying potassium channel in the rabbit superior lacrimal gland.

PURPOSE: To characterize the properties of an inwardly rectifying K+ (KIR) current in fresh, enzymatically isolated acinar cells from the rabbit superior lacrimal gland. METHODS: New Zealand White rabbits of both sexes were killed by injecting 45 mg/kg pentobarbital sodium, and the glands were excised. Single acinar cells were isolated enzymatically from these glands. Standard patch-clamp techniques were used to record ion currents. RESULTS: Hyperpolarizing voltages evoked KIR currents that had a conductance of 2.7 +/- 0.16 nS (n = 6) in the range -50 mV to -160 mV. The KIR current was activated with steep voltage dependence on hyperpolarization, and the conductance was approximately dependent on the square root of the external K+ concentration. Increasing the pipette Ca2+ concentration from 10(-9) M to 10(-6) M increased the conductance to 5.3 +/- 0.45 nS (n = 7). Internal substitution of K+ with various cations gave the following permeability sequence: K+ (1.0) > Rb+ (0.83) > Li+ (0.15). The KIR current was inhibited by Ba2+ (100 microns), tetraethylammonium (10 mM), and Cs+ (5 mM) but was insensitive to 4-aminopyridine (5 mM). The single-channel conductance was 43 +/- 2.7 pS (n = 11), and the relationship between between single-channel conductance (gamma) and external K+ concentration ([K]o) is given by: gamma = 7.04[K]o0.37 (pS, r2 = 0.99, P < 0.05). The relationship between [K]o and zero current potential (Erev) is given by: Erev = 35.5 log[K]o - 77.8 (mV; r2 = 0.99, P < 0.05). CONCLUSIONS: The KIR current identified in these lacrimal acini has a similar dependence on [K]o as other inward rectifiers of excitable tissues and exocrine glands. However, this study highlights that there are interspecies variations and similarities between KIR channels that could be related to their individual physiological roles. The authors' investigations suggest that one role of the KIR channel in the rabbit superior lacrimal gland acinar cells is to set and stabilize the resting membrane potential. However, this KIR channel may also be involved in secretion, as has been shown to occur in the sheep parotid gland.

Separation and characterisation of cyclic nucleotide phosphodiesterases from the lacrimal, harderian and zygomatic glands of the rabbit.

PURPOSE: To investigate the activity profile of cyclic nucleotide phosphodiesterase (PDE) isoenzymes and the effects of isoenzyme selective inhibitors in the superior and inferior lacrimal glands, Harderian gland, and zygomatic gland of the rabbit. METHODS: Protein fractions extracted from crude homogenates on an anion exchange column were examined for PDE activity using an HPLC method for detecting nucleotides. RESULTS: The superior and inferior lacrimal glands had identified PDE activity profiles. PDE I was the major type of activity and there was also a minor PDE III peak of activity. The main activity detected in the lipid secreting Harderian gland was PDE II and for the mucus secreting zygomatic gland PDE III. All glands contained PDE IV activity. The kinetics of the peak enzyme activities were examined and found similar, but not identical to the kinetics for PDE activities obtained from other tissues. Inhibitors of specific PDE classes and the general PDE inhibitor, IBMX, were tested on the peak enzyme activities. Activities designated by their substrate specificity or co-factor modification were most strongly inhibited by the corresponding class selective inhibitor. For example, PDE I activity in the lacrimal gland was most strongly inhibited by nicardipine. All activities were inhibited by IBMX. CONCLUSIONS: The superior and inferior lacrimal glands of the rabbit have the same PDE profile and this differs from the PDE subtypes detected in the mucus secreting zygomatic gland and the lipid secreting Harderian gland.

Kainic acid blocks a TTX-sensitive sodium channel in retinal horizontal cells of the turtle (Pseudemys scripta elegans).

The aim of this study was to investigate the effects of excitatory amino acids on channels found in horizontal cell membranes using patch-clamp techniques. We unexpectedly found that the excitatory amino acid receptor agonist, kainic acid, reversibly inhibited the transient tetrodotoxin (TTX)-sensitive Na+ current in isolated horizontal cell bodies and axons from the retina of the turtle (Pseudemys scripta elegans). The effect of kainic acid was antagonized by the glutamate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione. Kainic acid activated a non-selective cation current, a finding that was consistent with previous reports, and which would account for the kainate induced depolarisation of these cells. The inhibition of the transient TTX-sensitive Na+ current by kainic acid might be important in the modification of the kinetics of responses to excitatory amino acid analogues often observed during intracellular recording from these cells.

Immunohistochemical and histochemical characterisation of epithelial cells of rabbit lacrimal glands in tissue sections and cell cultures.

The purpose of this study was to establish conditions for isolation and long term culture of acinar cells from the Harderian gland, and superior and inferior lacrimal glands of the rabbit and to compare the in vitro growth patterns of cultured cells from these glands. In order to determine the predominant cell type in the cultures, cells and tissue sections were stained using a variety of antibodies to cytokeratins, smooth muscle actin, and neuron specific enolase. Similarly, PAS and alcian blue histochemistry were used to test for the presence of mucins. The glands were excised and cells isolated using enzymatic digestion and then established in long term culture. Different media and substrata were trialed for suitability. When cultured on uncoated Costar plastic in DMEM/10%FBS, the pattern of cell growth was similar for all glands with distinct phases involving aggregation and migration out from the aggregates before cells died between 20 to 30 days. Immunohistochemical staining indicated that the cultures were of acinar cells with a small percentage of ductal cells. The acinar cells of the lacrimal glands in situ and in vitro stained with antibody MNF116 directed against cytokeratins 5, 6, 8 and 17 but did not stain for antibodies to cytokeratin 18. The reverse staining pattern was true for the Harderian gland. Sections from the white lobe of the Harderian gland showed islets of serous secreting cells which showed positive staining when MNF116 was used. In situ, PAS positive cells were found in a small number of demilunes in the superior and inferior lacrimal glands and also in cells of the intercalated ducts. Surprisingly, in culture nearly all cells, including those isolated form the Harderian gland became PAS positive. In this study we have demonstrated that acinar cells from the Harderian and lacrimal glands of rabbit can be isolated and maintained in culture for 20 to 30 days, and that despite dramatic morphological changes, these cells retain their distinctive phenotype as indicated by antibody staining to specific cellular structural proteins such as cytokeratins and actin. However, the cultured cells also begin to produce mucins as indicated by PAS staining.

A tetraethylammonium-insensitive inward rectifier K+ channel in Müller cells of the turtle (Pseudemys scripta elegans) retina.

Ion channels present in isolated glial (Müller) cells from the retina of the turtle (Pseudemys scripta elegans) were studied with the patch clamp technique. The predominant conductance in these cells was due to an inward rectifying potassium current. The whole-cell conductance of the inward rectifier was 20.2 +/- 1.9 nS (n = 7 cells) in a standard extracellular saline solution (3 mM extracellular potassium). This conductance was dependent on the extracellular potassium concentration, with a 2.88-fold change in conductance per tenfold shift in concentration. The relative permeability sequence to potassium of the inward rectifier was found to be: potassium (1.0) > rubidium (0.7) > ammonium (0.2) > lithium (0.1) = sodium (0.1), which corresponded to the Eisenman sequence IV or V for a strong-field-strength potassium binding site on the channel. The single channel conductance measured in cell-attached patches with potassium chloride (150 mM) in the pipette was 68.5 +/- 6.0 pS (n = 3 patches). The inward rectifier current was not blocked by extracellular tetraethylammonium (TEA+, 20 mM), but was blocked by extracellular barium (5 mM) or cesium (5 mM). The TEA+ insensitivity of the inward rectifier potassium channel in Müller cells is unusual, given that this type of channel in most excitable cells is sensitive to micromolar concentrations of this compound, and may be a characteristic of inward rectifier potassium channels that are primarily involved with extracellular potassium regulation.

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.

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.

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.

Modulation of cone-to-horizontal-cell signal transmission in the turtle retina by magnesium ions.

The modulation by magnesium ions of cone-to-horizontal-cell signal transmission was studied in the turtle superfused everted eye-cup preparation using solutions which contained various concentrations of divalent cations and/or chelating agents (EDTA and EGTA). Removal of magnesium ions from 'normal' solutions had no apparent effect on the horizontal cells provided that 'normal' levels of calcium ions were maintained. Solutions which actively removed calcium but contained 'normal' levels of magnesium hyperpolarized horizontal cells, reduced the amplitude of their photoresponses but also changed the character of these photoresponses; dim stimuli evoked purely depolarizing responses while bright flashes elicited triphasic responses. Active removal of both calcium and magnesium from the superfusate and from retinal stores with chelators depolarized the horizontal cell and eliminated light-evoked responses. These solutions also depolarized the cones but augmented their light-evoked responses. We conclude that magnesium ions can modulate the membrane potential of horizontal cells, but this role can be revealed only in the absence of calcium ions. Further, the depolarizing responses seen in low calcium, 'normal' magnesium superfusion indicate that the ionic mechanisms underlying the horizontal cell photoresponse are more complex than has been previously described.

The effects of prolonged superfusions with acidic amino acids and their agonists on field potentials and horizontal cell photoresponses in the turtle retina.

The effects of prolonged superfusions with acidic amino acids and their agonists, kainic acid (KA) and N-methyl-D-aspartate (NMDA), on horizontal cells, and extracellular field potentials were studied in the turtle everted eyecup preparation using simultaneous intracellular and extracellular recordings. In a fresh preparation initial superfusions with each of the above agents usually induced a large (up to 60 mV) transient negative extracellular field potential recorded adjacent to horizontal cells, followed by a sustained negative potential of lesser amplitude (up to 10 mV). The amplitude of the sustained potential did not vary with subsequent superfusions, whereas that of the transient phase was reduced. KA and NMDA were much more potent (at least 300 times) in evoking these field potentials than either acidic amino acid. The horizontal cell transmembrane potential was monitored as the difference between the intra- and extracellular potentials. Superfusion with KA and NMDA produced a triphasic time course of the drug effect consisting of an initial depolarization with reduced photoresponses, a rehyperpolarization of the membrane accompanied by a growth of the light responses followed by a gradual depolarization and loss of photoresponses. Superfusion with the acidic amino acids usually produced a biphasic response that resembled qualitatively the first two phases of the response to KA and NMDA. This biphasic response was occasionally followed by a gradual depolarization and loss of the light response. The kinetics of the transient component of the field potential and the rapid reduction and regrowth of the photoresponses recorded during superfusion with these agents suggests an initial action of these drugs, which is of a nonsynaptic origin and which may be an expression of a drug-induced spreading depression. The kinetics of the photoresponses recorded during superfusion with KA, L-aspartate, and L-glutamate were similar but differed from those recorded during superfusion with NMDA. The difference in the effects of NMDA and KA on photoresponse kinetics suggests that two types of acidic amino acid receptors may be present in the outer plexiform layer of the turtle retina.

Measles-virus-persistent infection in BGM cells. Modification of the incorporation of [3H]arachidonic acid and [14C]stearic acid into lipids.

In BGM cells chronically infected with measles virus, although the composition of the phospholipids is unaltered, the fatty acid composition is modified. Uninfected, lytic and persistently infected cells were labelled with [3H]arachidonic acid and [14C]stearic acid and their metabolic fate analysed. No difference in the total incorporation was observed in the different systems. However, the [14C]stearic acid and [3H]arachidonic acid were incorporated up to 2-fold and 13-fold respectively greater into the neutral lipid of persistently infected compared with that of uninfected cells. Both radioactive fatty acids were specifically accumulated in the triacylglycerol and non-esterified fatty acids fractions. Lytically infected cells were similar to uninfected cells. Although there was no significant difference in the incorporation of radioactivity into the total phospholipid in either system, there was a large decrease in [3H]arachidonic acid incorporated into phosphatidylethanolamine and to a lesser extent phosphatidylcholine and phosphatidylinositol in persistently infected cells. [14C]Stearic acid incorporation was also reduced in phosphatidylcholine and phosphatidylethanolamine fractions of persistently infected cells.

Accumulation of radiolabelled fatty acids in the neutral lipid fraction of measles virus persistently infected BGM cells.

The effect of measles virus infection (acute and persistent) on fatty acid metabolism has been studied in BGM (African green monkey kidney) cells. In persistently infected cells, there was an increase in the incorporation of radiolabelled fatty acids into the neutral lipid fraction. Compared to uninfected cells, the increase was up to 2-fold for palmitic, stearic and oleic acids and 8-14 fold for arachidonic acid. The lipid metabolism in measles virus acutely infected cells was unmodified. The radiolabelled fatty acids incorporated into the neutral lipids in persistently infected cells were principally associated with the triglyceride fraction. The implications of a virus- induced modification of lipid metabolism are discussed.