Reticulostimulating properties of killed vaccines of anaerobic coryneforms and other organisms.

Vaccines prepared from 115 strains of anaerobic coryneforms and other organisms were tested in mice for their reticulostimulating ability as judged by the degree of spleen hypertrophy produced after ip injection. Almost all vaccines caused a statistically significant increase in spleen weight, but the ability to produce spleen ratios (test mean wt:control mean wt) of 4 or more was confined to Propionibacterium acnes and P. avidum strains. P. acnes, type II, gave high spleen ratios more frequently than strains of any other type.

Neurosteroid modulation of GABA IPSCs is phosphorylation dependent.

The neurosteroid 3alpha-hydroxy-5alpha-pregnan-20-one (allopregnanolone) facilitates GABA(A) receptor-mediated ionic currents via allosteric modulation of the GABA(A) receptor. Accordingly, allopregnanolone caused an increase in the slow decay time constant of spontaneous GABA-mediated IPSCs in magnocellular neurons recorded in hypothalamic slices. The allopregnanolone effect on IPSCs was inhibited by a G-protein antagonist as well as by blocking protein kinase C and, to a lesser extent, cAMP-dependent protein kinase activities. G-protein and protein kinase C activation in the absence of the neurosteroid had no effect on spontaneous IPSCs but enhanced the effect of subsequent allopregnanolone application. These findings together suggest that the neurosteroid modulation of GABA-mediated IPSCs requires G-protein and protein kinase activation, although not via a separate G-protein-coupled steroid receptor.

Experimental models and their use in studies of diabetic retinal microangiopathy.

Diabetes produces dramatic changes in retinal microvasculature, triggering endothelial cell proliferation and microaneurysms. Capillaries become weakened, releasing blood into vitreal and retinal spaces. Photoreceptors become occluded and separated from the choriocapillaris, resulting in visual acuity decline, detachment and cell death. Several models have been developed that have proved useful for the study of this disease, resulting in a better understanding of the processes involved. Streptozotocin treatment affects the pancreatic beta cells, rapidly reducing them until insulin is no longer synthesized in sufficient amounts. The galactosemic model shifts metabolism away from glucose, increasing aldose reductase and retinal polyol metabolism. Finally, two weeks of cycled oxygen from high to low tension every 24 hours, followed by return to room air, triggers microangiogenesis in developing retinas. Use of these models, separately or in combination, as well as electroretinographic analysis, has begun to reveal the events taking place as diabetic retinopathy progresses. Endothelial cells become separated from pericytes as basement membranes thicken, and vascular endothelial growth factor increases, triggering their proliferation. Finally, early changes occurring within photoreceptors can now be studied.

Neuroprotection of rat retinal ganglion cells mediated through alpha7 nicotinic acetylcholine receptors.

Glutamate-induced excitotoxicity is thought to play an important role in several neurodegenerative diseases in the central nervous system (CNS). In this study, neuroprotection against glutamate-induced excitotoxicity was analyzed using acetylcholine (ACh), nicotine and the α7 specific nicotinic acetylcholine receptor (α7 nAChR) agonist, N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-chlorobenzamide hydrochloride (PNU-282987), in cultured adult rat retinal neurons. Adult Long Evans rat retinas were dissociated and retinal ganglion cells (RGCs) were isolated from all other retinal tissue using a two-step panning technique. Once isolated, RGCs were cultured under various pharmacological conditions to demonstrate excitotoxicity and neuroprotection against excitotoxicity. After 3 days, RGCs were immunostained with antibodies against the glycoprotein, Thy 1.1, counted and cell survival was assessed relative to control untreated conditions. 500 µM glutamate induced excitotoxicity in large and small RGCs in an adult rat dissociated culture. After 3 days in culture with glutamate, the cell survival of large RGCs decreased by an average of 48.16% while the cell survival of small RGCs decreased by an average of 42.03%. Using specific glutamate receptor agonists and antagonists, we provide evidence that the excitotoxic response was mediated through α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainic acid (KA) and N-methyl-d-aspartate (NMDA) glutamate receptors through an apoptotic mechanism. However, the excitotoxic effect of glutamate on all RGCs was eliminated if cells were cultured for an hour with 10 µM ACh, 100 µM nicotine or 100 nM of the α7 nAChR agonist, PNU-282987, before the glutamate insult. Inhibition studies using 10nM methyllycaconitine (MLA) or α-bungarotoxin (α-Bgt) supported the hypothesis that neuroprotection against glutamate-induced excitotoxicity on rat RGCs was mediated through α7 nAChRs. In immunocytochemical studies, double-labeled experiments using antibodies against Thy 1.1 and α7 nAChR subunits demonstrated that both large and small RGCs contained α7 nAChR subunits. The data presented in this study support the hypothesis that ACh and nicotinic acetylcholine receptor (nAChR) agonists provide neuroprotection against glutamate-induced excitotoxicity in adult rat RGCs through activation of α7 nAChR subunits. These studies lay the groundwork required for analyzing the effect of specific α7 nAChR agonists using in vivo models of excitotoxicity. Understanding the type of ACh receptors involved in neuroprotection in the rat retina could ultimately lead to therapeutic treatment for any CNS disease that involves excitotoxicity.

Calcium preconditioning triggers neuroprotection in retinal ganglion cells.

In the mammalian retina, excitotoxicity has been shown to be involved in apoptotic retinal ganglion cell (RGC) death and is associated with certain retinal disease states including glaucoma, diabetic retinopathy and retinal ischemia. Previous studies from this lab [Wehrwein E, Thompson SA, Coulibaly SF, Linn DM, Linn CL (2004) Invest Ophthalmol Vis Sci 45:1531-1543] have demonstrated that acetylcholine (ACh) and nicotine protects against glutamate-induced excitotoxicity in isolated adult pig RGCs through nicotinic acetylcholine receptors (nAChRs). Activation of nAChRs in these RGCs triggers cell survival signaling pathways and inhibits apoptotic enzymes [Asomugha CO, Linn DM, Linn CL (2010) J Neurochem 112:214-226]. However, the link between binding of nAChRs and activation of neuroprotective pathways is unknown. In this study, we examine the hypothesis that calcium permeation through nAChR channels is required for ACh-induced neuroprotection against glutamate-induced excitotoxicity in isolated pig RGCs. RGCs were isolated from other retinal tissue using a two step panning technique and cultured for 3 days under different conditions. In some studies, calcium imaging experiments were performed using the fluorescent calcium indicator, fluo-4, and demonstrated that calcium permeates the nAChR channels located on pig RGCs. In other studies, the extracellular calcium concentration was altered to determine the effect on nicotine-induced neuroprotection. Results support the hypothesis that calcium is required for nicotine-induced neuroprotection in isolated pig RGCs. Lastly, studies were performed to analyze the effects of preconditioning on glutamate-induced excitotoxicity and neuroprotection. In these studies, a preconditioning dose of calcium was introduced to cells using a variety of mechanisms before a large glutamate insult was applied to cells. Results from these studies support the hypothesis that preconditioning cells with a relatively low level of calcium before an excitotoxic insult leads to neuroprotection. In the future, these results could provide important information concerning therapeutic agents developed to combat various diseases involved with glutamate-induced excitotoxicity.

Acetylcholine neuroprotection against glutamate-induced excitotoxicity in adult pig retinal ganglion cells is partially mediated through alpha4 nAChRs.

In the mammalian retina, excess glutamate release has been shown to be involved in retinal ganglion cell (RGC) death associated with various diseases. Recent studies have determined that activation of alpha7 nicotinic acetylcholine receptors (nAChRs) partially protect isolated RGCs from glutamate-induced excitotoxicity. In this study, we further classify the types of nAChRs involved in neuroprotection against glutamate-induced excitotoxicity using isolated adult pig RGCs. Cells were isolated with a modified two-step immunoselective panning technique designed to isolate RGCs from other retinal neurons. Once isolated, nAChR subunits were identified using a combination of pharmacological and immunocytochemical techniques. In cell culture experiments, a variety of alpha4 nAChR specific agonists were found to have a partial neuroprotective against glutamate-induced excitotoxicity. This neuroprotection was abolished in the presence of the alpha4 nAChR antagonist, dihydro-beta-erythroidine (DHbetaE). Immunocytochemical results localized several nAChR subunits on isolated adult pig RGCs; in particular alpha4, alpha7 and beta2 nAChR subunits. Large RGCs exclusively immunostained with antibodies against alpha7 nAChR subunits whereas alpha4 and beta2 subunits exclusively immunostained only small RGCs. Double label experiments provided evidence that alpha4 and beta2 subunits co-localize on small RGCs. Knowledge of the receptor subtypes responsible for neuroprotection may lead to treatments associated with glutamate-induced excitotoxicity.

A comparison of the inhibitory actions of glycine and GABA on acetylcholine release from the rabbit retina.

The inhibition of [3H]acetylcholine (ACh) release from cholinergic amacrine cells by glycine and GABA was studied using an in vivo eyecup preparation in anesthetized rabbits. Glycine (1 mM) had no effect on basal ACh release, but completely blocked the light-evoked release of ACh. Glycine also blocked the strong potentiating effects of picrotoxin (20 microM) normally observed on basal and light-evoked release. Strychnine (20 microM) increased basal release, albeit less than picrotoxin, but partially inhibited and altered the shape of light-evoked responses. Co-perfusion of picrotoxin and strychnine after strychnine application resulted in a larger additional basal increase. However, light-evoked responses were not restored to a control shape and magnitude, or to potentiated levels as with picrotoxin alone, but remained altered and partially inhibited. These results support the concept of a sustained GABA-mediated inhibition of the cholinergic pathway in the intact retina. In contrast, glycine-mediated inhibition of the cholinergic pathway differs, with the present results indicating a significantly smaller sustained inhibition of basal release and a temporal inhibition of light-evoked release. The lack of effect of any of these compounds on kainate-evoked responses indicates that these effects are predominately indirect, possibly on the presynaptic bipolar cell.

Bacteriological quality of runoff water from pastureland.

Runoff from a cow-calf pasture in eastern Nebraska was monitored for total coliforms (TC), fecal coliforms (FC), and fecal streptococci (FS) during 1976, 1977, and 1978. Bacteriological counts in runoff from both grazed and ungrazed areas generally exceeded recommended water quality standards. The FC group was the best indicator group of the impact of grazing. Rainfall runoff from the grazed area contained 5 to 10 times more FC than runoff from the fenced, ungrazed area. There was little difference in TC counts between the two areas, but FS counts were higher in runoff from the ungrazed area and reflected the contributions from wildlife. Recommended bacteriological water quality standards, developed for point source inputs, may be inappropriate for characterizing nonpoint source pollution from pasture runoff. The FC/FS ratio in pasture runoff was useful in identifying the relative contributions of cattle and wildlife. Ratios below 0.05 were indicative of wildlife sources and ratios above 0.1 were characteristic of grazing cattle. Occasions when the FC/FS ratio of diluted cattle waste exceeded one resulted from differential aftergrowth and die-off between FC and FS. The FC/FS ratio and percentage of Streptococcus bovis in pasture runoff are useful indicators for evaluating the effectiveness of livestock management practices for minimizing bacterial contamination of surface water. The importance of choice of medium for the enumeration of FS in runoff derived from cattle wastes is discussed.

Acetylcholine release from the rabbit retina mediated by NMDA receptors.

The cholinergic amacrine cells of the rabbit retina may be labeled with 3H-choline, and the activity of the cholinergic population may be monitored by following the release of 3H-ACh. In magnesium-free medium, the glutamate analog NMDA caused massive ACh release, up to 50x the basal efflux. Magnesium blocked the NMDA-evoked release of ACh with an IC50 of 151 microM. The NMDA-evoked release of ACh was unchanged in glycine-free medium or in the presence of 500 microM glycine. However, the block of NMDA-evoked release by 7-chlorokynurenic acid (7-Cl-Kyn) was reversed by exogenous glycine. This suggests that the NMDA receptors mediating ACh release possess an allosteric glycine binding site, but under normal conditions, it is saturated by endogenous glycine. Submaximal doses of NMDA were used to determine the potency of NMDA antagonists and their specificity was established with submaximal doses of other glutamate agonists. DL-2-amino-7-phosphonoheptanoate (DL-AP-7) was a competitive NMDA antagonist, with an IC50 of 33 microM and (+)5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK-801) was a noncompetitive NMDA antagonist, with an IC50 of 10.6 nM. Neither antagonist blocked the light-evoked release of ACh from the retina. Furthermore, light stimulation did not activate the use-dependent block characteristic of MK-801, indicating that the endogenous transmitter did not open the NMDA channel. These results suggest that NMDA receptors do not mediate the physiological input to cholinergic amacrine cells in the rabbit retina.

Homocysteate-evoked release of acetylcholine from the rabbit retina.

The cholinergic amacrine cells of the rabbit retina can be labeled with [3H]choline and the activity of the cholinergic population monitored by following the release of [3H]acetylcholine. It has been proposed that L-homocysteate may be the main endogenous transmitter released onto cholinergic amacrine cells by bipolar cells. Therefore, we have examined the effects of the isomers of homocysteate on the release of [3H]acetylcholine. In magnesium-free medium, D-homocysteate was slightly more potent than the L-isomer. The addition of magnesium, which blocks responses mediated by NMDA receptors, preferentially reduced but did not eliminate, the response to L-homocysteate. 2-Amino-7-phosphonoheptanoate, a potent NMDA antagonist, preferentially blocked L-homocysteate evoked responses. 6,7-Dinitroquinoxaline-2,3-dione, a potent kainate antagonist, preferentially blocked D-homocysteate-evoked responses. Therefore, in the rabbit retina, L-homocysteate is an NMDA-preferring agonist, whereas D-homocysteate is a kainate-preferring agonist. In addition, we found that L-homocysteate can activate the physiologically activated kainate receptor but only when used in millimolar concentrations and under conditions that minimize NMDA-receptor activation. However, the low potency of L-homocysteate combined with low affinity for the glutamate transporter, lack of immunocytochemical localization in bipolar cells, and low retinal content place serious limitations on the role of L-homocysteate at the bipolar-to-cholinergic amacrine cell synapse.

GABA inhibits ACh release from the rabbit retina: a direct effect or feedback to bipolar cells?

The cholinergic amacrine cells of the rabbit retina may be labeled with [3H]-Ch and the activity of the cholinergic population monitored by following the release of [3H]-ACh. We have tested the effect of muscimol, a potent GABAA agonist, on (1) the light-evoked release of ACh, presumably mediated via bipolar cells, which are known to have a direct input to the cholinergic amacrine cells and (2) ACh release produced by exogenous glutamate analogs that probably have a direct effect on cholinergic amacrine cells. Muscimol blocked the light-evoked release of ACh with an IC50 of 1.0 microM. In contrast, ACh release produced by nonsaturating doses of kainate or NMDA was not reduced even by 100 microM muscimol. Thus, we have been unable to demonstrate a direct effect of GABA on the cholinergic amacrine cells. GABA antagonists, such as picrotoxin, caused a large increase in the base release and potentiated the light-evoked release of ACh. Both these effects were abolished by DNQX, a kainate antagonist that blocks the input to cholinergic amacine cells from bipolar cells. DNQX blocked the effects of picrotoxin even when controls showed that the mechanism of ACh release was still functional. Together, these results imply that the dominant site for the GABA-mediated inhibition of ACh release is on the bipolar cell input to the cholinergic amacrine cells. This is consistent with previous anatomical and physiological evidence that bipolar cells receive negative feedback from GABA amacrine cells.

Contributions of GABAA receptors and GABAC receptors to acetylcholine release and directional selectivity in the rabbit retina.

GABA is a major inhibitory neurotransmitter in the mammalian retina and it acts at many different sites via a variety of postsynaptic receptors. These include GABAA receptors and bicuculline-resistant GABAC receptors. The release of acetylcholine (ACh) is inhibited by GABA and strongly potentiated by GABA antagonists. In addition, GABA appears to mediate the null inhibition which is responsible for the mechanism of directional selectivity in certain ganglion cells. We have used these two well-known examples of GABA inhibition to compare three GABA antagonists and assess the contributions of GABAA and GABAC receptors. All three GABA antagonists stimulated ACh release by as much as ten-fold. By this measure, the ED50s for SR-95531, bicuculline, and picrotoxin were 0.8, 7.0, and 14 microM, respectively. Muscimol, a potent GABAA agonist, blocked the effects of SR-95531 and bicuculline, but not picrotoxin. This indicates that SR-95531 and bicuculline are competitive antagonists at the GABAA receptor, while picrotoxin blocks GABAA responses by acting at a different, nonreceptor site such as the chloride channel. In the presence of a saturating dose of SR-95531 to completely block GABAA receptors, picrotoxin caused a further increase in the release of ACh. This indicates that picrotoxin potentiates ACh release by a mechanism in addition to the block of GABAA responses, possibly by also blocking GABAC receptors, which have been associated with bipolar cells. All three GABA antagonists abolished directionally selective responses from ON/OFF directional-selective (DS) ganglion cells. In this system, the ED50S for SR-95531, bicuculline, and picrotoxin were 0.7 microM, 8 microM, and 94.6 microM, respectively. The results with SR-95531 and bicuculline indicate that GABAA receptors mediate the inhibition responsible for directional selectivity. The addition of picrotoxin to a high dose of SR-95531 caused no further increase in firing rate. The comparatively high dose required for picrotoxin also suggests that GABAC receptors do not contribute to directional selectivity. This in turn suggests that feedforward GABAA inhibition, as opposed to feedback at bipolar terminals, is responsible for the null inhibition underlying directional selectivity.

The role of potassium conductance in the generation of light responses in Müller cells of the turtle retina.

Müller cells are highly permeable to potassium ions and play a major role in maintaining potassium homeostasis in the vertebrate retina during light-evoked neuronal activity. Potassium fluxes across the Müller cell's membrane are believed to underlie the light-evoked responses of these cells. We studied the potassium currents of turtle Müller cells in the retinal slice and in dissociated cell preparations and their role in the genesis of the light-evoked responses of these cells. In either preparation, the I-V curve, measured under voltage-clamp conditions, consisted of inward and outward currents. A mixture of cesium ions, TEA, and 4-AP blocked the inward current but had no effect on the outward current. Extracellular cesium ions alone blocked the inward current but exerted no effect on the photoresponses. Extracellular barium ions blocked both inward and outward currents, induced substantial depolarization, and augmented the light-evoked responses, especially the OFF component. Exposing isolated Müller cells to a high potassium concentration did not cause any current or voltage responses when barium ions were present. In contrast, application of glutamate in the presence of barium ions induced a small inward current that was associated with a substantially augmented depolarizing wave relative to that observed under control conditions. This observation suggests a role for an electrogenic glutamate transporter in generating the OFF component of the turtle Müller cell photoresponse.

Acetylcholine release from the rabbit retina mediated by kainate receptors.

The cholinergic amacrine cells of the rabbit retina may be labeled with 3H-choline (3H-Ch), and the activity of the cholinergic population may be monitored by following the release of 3H-ACh. Glutamate analogs caused massive ACh release, up to 50 times the basal efflux, with the following rank order of potency: alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) greater than quisqualate (QQ) = kainate (KA) much greater than NMDA (in magnesium-free medium) much greater than glutamate greater than aspartate. In contrast, the release of 3H-Ch was unchanged. Submaximal doses of each agonist were used to establish the specifity of glutamate antagonists. Kynurenic acid was selective for KA much greater than QQ, and 6,7-dinitroquinoxaline-2,3-dione (DNQX) was selective for KA greater than QQ much greater than NMDA. At low doses, which selectively blocked the response to KA, both antagonists blocked the light-evoked release of ACh. These results suggest that ACh release may be produced via several glutamate receptors, but the physiological input to the cholinergic amacrine cells is mediated by KA receptors. Because these cells receive direct input from cone bipolar cells, this work supports previous evidence that the bipolar cell transmitter is glutamate.

Characterization with barium of potassium currents in turtle retinal Müller cells.

Müller cells are highly permeable to potassium ions and play a crucial role in maintaining potassium homeostasis in the vertebrate retina. The potassium current found in turtle Müller cells consists of two components: an inwardly rectifying component and a linear, passive component. These currents are insensitive to broadband potassium channel blockers, tetraethylammonium (TEA) and 4-aminopyridine (4-AP) and well blocked by barium. Differential block by the polyamine spermine suggests that these currents flow through different channels. In this study, we used barium ions as a probe to investigate the properties of these currents by whole cell, voltage-clamp recordings from isolated cells. Current-voltage (I-V) relationships generated from current responses to short (35 ms) and long (3.5 s) voltage pulses were fit with the Hill equation. With extracellular barium, the time course of block and unblock was voltage and concentration dependent and could be fit with single exponential functions and time constants larger than 100 ms. Blocking effects by extracellular barium on the two types of currents were indistinguishable. The decrease of the outward current originates in part due to charge effects. We also found that intracellular barium was an effective blocker of the potassium currents. The relative block of the inward rectifier by intracellular barium suggests the existence of two "apparent" binding sites available for barium within the channel. Under depolarizing conditions favoring the block by internal polyamines, the Hill coefficient for barium binding was 1, indicating a single apparent binding site for barium within the pore of the passive linear conductance. The difference in the steepness of the blocking functions suggests that the potassium currents flow through two different types of channels, an inward rectifier and a linear passive conductance. Last, we consider the use of barium as an intracellular K(+) channel blocker for voltage-clamp experiments.