Hypoglycemia leads to age-related loss of vision.

The retina is among the most metabolically active tissues in the body, requiring a constant supply of blood glucose to sustain function. We assessed the impact of low blood glucose on the vision of C57BL/6J mice rendered hypoglycemic by a null mutation of the glucagon receptor gene, Gcgr. Metabolic stress from moderate hypoglycemia led to late-onset loss of retinal function in Gcgr(-/-) mice, loss of visual acuity, and eventual death of retinal cells. Retinal function measured by the electroretinogram b-wave threshold declined >100-fold from age 9 to 13 months, whereas decreases in photoreceptor function measured by the ERG a-wave were delayed by 3 months. At 10 months of age Gcgr(-/-) mice began to lose visual acuity and exhibit changes in retinal anatomy, including an increase in cell death that was initially more pronounced in the inner retina. Decreases in retinal function and visual acuity correlated directly with the degree of hypoglycemia. This work demonstrates a metabolic-stress-induced loss of vision in mammals, which has not been described previously. Linkage between low blood glucose and loss of vision in mice may highlight the importance for glycemic control in diabetics and retinal diseases related to metabolic stress as macular degeneration.

Anesthesia can cause sustained hyperglycemia in C57/BL6J mice.

Effects of anesthesia on the blood glucose of C57/BL6J mice were evaluated under conditions commonly used for testing retinal sensitivity with electroretinographic (ERG) recordings. We evaluated the effects of four anesthetics: nembutal (50 mg/kg), pentothal (100 mg/kg), avertin (240 mg/kg), and ketamine/xylazine (100 mg/kg) using saline as control. We measured blood glucose (BG) levels from tail vein blood before and 15 and 60 min following intraperitoneal injections. Fifteen minutes postinjection, all four anesthetics and saline elevated BG with ketamine/xylazine and avertin having substantially greater effects than nembutal, pentothal, and saline. Only the effects of ketamine/xylazine and avertin persisted throughout the test period. Sixty minutes after injecting ketamine/xylazine BG remained elevated at 400 +/- 42 mg/dl, a 167% increase over preinjection levels. Sixty minutes after injecting avertin BG was 288 +/- 10 mg/dl, a 59% increase over preinjection levels. No sustained elevation in BG was detected 60 min following injection of nembutal, pentothal, or saline. Because BG can affect the amplitude of the ERG, caution should be exercised in the use of ketamine/xylazine or avertin. The choice of anesthesia may also be important in diabetes and metabolism research where changes in blood glucose could impact physiological processes.

Spermine mediates inward rectification in potassium channels of turtle retinal Müller cells.

Retinal Müller cells are highly permeable to potassium as a consequence of their intrinsic membrane properties. Therefore these cells are able to play an important role in maintaining potassium homeostasis in the vertebrate retina during light-induced neuronal activity. Polyamines and other factors present in Müller cells have the potential to modulate the rectifying properties of potassium channels and alter the Müller cells capacity to siphon potassium from the extracellular space. In this study, the properties of potassium currents in turtle Müller cells were investigated using whole cell voltage-clamp recordings from isolated cells. Overall, the currents were inwardly rectifying. Depolarization elicited an outward current characterized by a fast transient that slowly recovered to a steady level along a double exponential time course. On hyperpolarization the evoked inward current was characterized by an instantaneous onset (or step) followed by a slowly developing sustained inward current. The kinetics of the time-dependent components (block of the transient outward current and slowly developing inward current) were dependent on holding potential and changes in the intracellular levels of magnesium ions and polyamines. In contrast, the instantaneous inward and the sustained outward currents were ohmic in character and remained relatively unaltered with changes in holding potential and concentration of applied spermine (0.5--2 mM). Our data suggest that cellular regulation in vivo of polyamine levels can differentially alter specific aspects of potassium siphoning by Müller cells in the turtle retina by modulating potassium channel function.

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.

Electroretinogram of the parietal eye of lizards: photoreceptor, glial, and lens cell contributions.

Local electroretinograms (ERGs) were recorded in the parietal eye of Xantusia vigilis. The responses to monochromatic light under dark- and light-adapted conditions were studied. We found that two antagonistic chromatic mechanisms dominate the overall response. With the electrode tip in the lumen of the eye, light stimulation under dark-adapted conditions evoked responses of negative polarity with maximum sensitivity to green light. Intense green background illumination saturated the green-sensitive mechanism, and superposition of a blue stimulus then elicited responses of opposite polarity, driving the potentials back toward the dark resting level. The spectral sensitivities of the two chromatic mechanisms were determined using chromatic adaptation. The lower threshold, green-sensitive mechanism has a maximum sensitivity at 495 nm while the antagonistic mechanism, with its maximal spectral sensitivity at 430 nm, is at least 2 log units less sensitive. The polarity of the ERG recording inverts as the electrode traverses the photoreceptor layer, suggesting that the photoreceptors are the major source of the ERG. This result was confirmed with intracellular recordings from photoreceptors, glial, and lens cells. The glial and lens cells of the parietal eye respond to local changes in [K+]o. Intracellular recordings of the responses of these cells to light stimuli follow time courses similar to changes in extracellular potassium concentrations measured with K+ -specific electrodes. These results suggest that the glial and lens cell membranes are highly permeable to potassium and, therefore, the electrical responses of these cells are evoked by changes in [K+]o elicited by light stimulation of the photoreceptors. Nevertheless, the major component of the parietal eye ERG is the photoreceptor signal. A circuit model of the ERG sources is presented.

Synoretin--A new protein belonging to the synuclein family.

Aoffa-Synuclein, a presynaptic nerve terminal protein, may be an important component of Lewy bodies in Parkinson's disease, dementia with Lewy bodies, and other neurodegenerative diseases. Additionally, recent genetic studies based on linkage analysis and cosegregation of A53T and A30P missense mutations demonstrated that the alpha-synuclein gene may be responsible for the development of at least some cases of familial Parkinson's disease. Despite intense interest in the members of the synuclein family, their function(s) and exact role in the diseases remained unknown. Here we describe a new member of the synuclein family, which we term synoretin, and show that it is expressed in different retinal cells, as well as in the brain, and it may affect the regulation of signal transduction through activation of the Elk1 pathway.

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.

A cGMP-gated cation channel and phototransduction in depolarizing photoreceptors of the lizard parietal eye.

Photoreceptors of the lizard parietal eye, unlike rods and cones but like most invertebrate photoreceptors, respond to light under dark-adapted conditions with a depolarization. Using excised-patch recordings, we have nonetheless found a cGMP-gated, non-selective cation channel present at high density at the presumptive light-sensitive part (the outer segment) of these cells. This channel resembles the rod cGMP-gated channel in its activation characteristics, and by showing a relative non-selectivity among alkali monovalent cations, a high permeability to Ca2+, a high sensitivity to L-cis-diltiazem, as well as a negative modulation by Ca(2+)-calmodulin. This channel appears to mediate phototransduction by opening in the light to produce the depolarizing response.

An unusual cGMP pathway underlying depolarizing light response of the vertebrate parietal-eye photoreceptor.

All cellular signaling pathways currently known to elevate cGMP involve the activation of a guanylyl cyclase to synthesize cGMP. Here we describe an exception to this rule. In the vertebrate parietal eye, the photoreceptors depolarize to light under dark-adapted conditions, unlike rods and cones but like most invertebrate photoreceptors. We report that the signaling pathway for this response involves a rise in intracellular cGMP resulting from an inhibition of the phosphodiesterase that hydrolyzes cGMP. Furthermore, this phosphodiesterase is driven by an active G protein in darkness. These results indicate an antagonistic control of the phosphodiesterase by two G proteins, analogous to the Gs/Gi control of adenylyl cyclase. Our findings demonstrate an unusual phototransduction mechanism and at the same time indicate that signaling involving cyclic nucleotides is more elaborate than previously known.

A cGMP-gated cation channel in depolarizing photoreceptors of the lizard parietal eye.

Rods and cones of the two vertebrate lateral eyes hyperpolarize when illuminated, a response generated by a cyclic GMP cascade leading to cGMP hydrolysis and consequently the closure of cGMP-gated, non-selective cation channels that are open in darkness. Lizards and other lower vertebrates also have a parietal (third) eye, which contains ciliary photoreceptors that under dark-adapted conditions depolarize to light instead. Depolarizing light responses are characteristic of most invertebrate rhabdomeric photoreceptors, and are thought to involve a phosphoinositide signalling pathway (see, for example, refs 7-9). Surprisingly, we have found in excised membrane patches a cGMP-gated channel that is selectively present at high density on the outer segment (the presumptive light-sensitive part) of the parietal eye photoreceptor. Like the light-activated channel of the cell, it is non-selective among cations. Inositol trisphosphate (InsP3) had no effect on the same membrane patches. These findings suggest that the photoreceptors of the parietal eye, like rods and cones, use a cGMP cascade and not an InsP3-mediated pathway for phototransduction, but in this case light increases cGMP. A unifying principle of evolutionary significance emerges: that phototransductions in various ciliary photoreceptors, whether hyperpolarizing or depolarizing, uniformly use a cGMP cascade and a cGMP-gated channel to generate the light response, although there are rich variations in the details.

Antagonistic chromatic mechanisms in photoreceptors of the parietal eye of lizards.

Photoreceptors are the first in the chain of neurons that process visual information. In lateral eyes of vertebrates, light hyperpolarizes rod and cone photoreceptors that synapse onto bipolar and horizontal cells in the first synaptic layer of the retina. The sign of the photoreceptor signal is either conserved or inverted in bipolar cells, resulting in chromatically dependent depolarizing and hyperpolarizing responses to visual stimuli. Visual information is then conveyed to the second synaptic layer for encoding and transmission to the brain by ganglion cells. The parietal (third) eye of lizards does not contain bipolar cells or other interneurons. Photoreceptors synapse directly onto ganglion cells and yet, even in the absence of interneurons, antagonistic chromatic mechanisms modulate the ganglion cell responses. We report here that chromatic antagonism in the third eye originates in the chromatically dependent hyperpolarizing and depolarizing response of the photoreceptors to light. We also suggest that the antagonistic nature of these photoresponses may provide lizards with a mechanism for the enhanced detection of dawn and dusk.