Andrey V. Dmitriev

Dendritic compartmentalization of chloride cotransporters underlies directional responses of starburst amacrine cells in retina

The mechanisms in the retina that generate light responses selective for the direction of image motion remain unresolved. Recent evidence indicates that directionally selective light responses occur first in the retina in the dendrites of an interneuron, i.e., the starburst amacrine cell, and that these responses are highly sensitive to the activity of Na-K-2Cl (NKCC) and K-Cl (KCC), two types of chloride cotransporter that determine whether the neurotransmitter GABA depolarizes or hyperpolarizes neurons, respectively. We show here that selective blockade of the NKCC2 and KCC2 cotransporters located on starburst dendrites consistently hyperpolarized and depolarized the starburst cells, respectively, and greatly reduced or eliminated their directionally selective light responses. By mapping NKCC2 and KCC2 antibody staining on these dendrites, we further show that NKCC2 and KCC2 are preferentially located in the proximal and distal dendritic compartments, respectively. Finally, measurements of the GABA reversal potential in different starburst dendritic compartments indicate that the GABA reversal potential at the distal dendrite is more hyperpolarized than at the proximal dendrite due to KCC2 activity. These results thus demonstrate that the differential distribution of NKCC2 on the proximal dendrites and KCC2 on the distal dendrites of starburst cells results in a GABA-evoked depolarization and hyperpolarization at the NKCC2 and KCC2 compartments, respectively, and underlies the directionally selective light responses of the dendrites. The functional compartmentalization of interneuron dendrites may be an important means by which the nervous system encodes complex information at the subcellular level.

A circadian clock regulates the pH of the fish retina

Although it is generally accepted that the acid/base ratio of tissue, as represented by the pH, is strictly regulated to maintain normal function, recent studies in the nervous system have shown that neuronal activity can result in significant shifts in pH. In the vertebrate retina, many cellular phenomena, including neuronal activity, are regulated by a circadian clock. We thus investigated whether a circadian clock regulates the pH of the retina. pH‐sensitive microelectrodes were used to measure the extracellular pH of the in vitro goldfish retina superfused with a bicarbonate‐based Ringer solution in the subjective day and night; that is, under conditions of constant darkness. These measurements demonstrated that a circadian clock regulates the pH of the vertebrate retina so that the pH is lower at night compared to the day. This day‐night difference in retinal pH was observed at two different values of Ringer solution pH, indicating that the circadian phenomenon is independent of the superfusion conditions. The circadian‐induced shift in pH was several times greater than light‐induced pH changes and large enough to influence synaptic transmission between retinal neurons. These findings indicate that a circadian clock regulates the pH of the vertebrate retina. Thus, an intrinsic oscillator in neural tissue may modulate metabolic activity and pH as part of normal daily function.

Retinal pH Reflects Retinal Energy Metabolism in the Day and Night

The extracellular pH of living tissue in the retina and elsewhere in the brain is lower than the pH of the surrounding milieu. We have shown that the pH gradient between the in vitro retina and the superfusion solution is regulated by a circadian (24-h) clock so that it is smaller in the subjective day than in the subjective night. We show here that the circadian changes in retinal pH result from a clock-mediated change in the generation of H+ that accompanies energy production. To demonstrate this, we suppressed energy metabolism and recorded the resultant reduction in the pH difference between the retina and superfusate. The magnitude of the reduction in the pH gradient correlated with the extent of energy metabolism suppression. We also examined whether the circadian-induced increase in acid production during the subjective night results from an increase in energy metabolism or from the selective activation of glycolysis compared with oxidative phosphorylation. We found that the selective suppression of either oxidative phosphorylation or glycolysis had almost identical effects on the dynamics and extent of H+ production during the subjective day and night. Thus the proportion of glycolysis and oxidative phosphorylation is maintained the same regardless of circadian time, and the pH difference between the tissue and superfusion solution can therefore be used to evaluate total energy production. We conclude that circadian clock regulation of retinal pH reflects circadian regulation of retinal energy metabolism.