Experiments designed to examine the energy requirements of neurophysiological function were performed on isolated rabbit retina. Function was altered by photic stimulation or by function-specific drugs, and the response of energy metabolism was assessed by simultaneous measurements of O2 consumption and lactate production. In other experiments, the supply of O2 or glucose was reduced and the effect on energy metabolism and electrophysiological function was observed. Energy requirements under control conditions in darkness were high, with O2 consumption (per gm dry wt) at 11.3 mumol min-1, with lactate production at 14.8 mumol min-1, and with the derived value for glucose consumption at 9.3 mumol min-1 and for high-energy phosphate (approximately P) generation at 82.6 mumol min-1. Energy reserves were small. Removing glucose abolished the b-wave of the electroretinogram (ERG) with a t1/2 of 1 min, but did not immediately affect O2 consumption or the PIII of the ERG. Removing O2 caused increases of up to 2.7-fold in glycolysis (Pasteur effect) and caused both PIII and b-wave to fail, with a t1/2 of about 5 min. Neurotransmission through the inner retina was supported almost entirely by glycolysis, as evidenced by large increases in lactate production in response to flashing light and decreases in response to transmitter blockers (2.3-fold overall change), with no change in O2 consumption. Phototransduction, on the other hand, was normally supported by oxidative metabolism. The dark current accounted for 41% of the retina's O2 consumption. With O2 reduced, the dark current was partially supported by glycolysis, which accounts (at least in part) for the large Pasteur effect. Na+ transport by NaK ATPase accounted for about half of all energy used, as evidenced by the response to strophanthidin, that is, for 49% of the oxidative energy and 58% of the glycolytic energy. The t1/2 for the turnover of intracellular Na+ was calculated from these data to be less than 1 min. Changes in temperature caused changes in the amplitude of light-evoked electrical responses of 6.5% per degree and caused changes in both O2 consumption and glycolysis of 6.8% per degree (Q10 = 1.9). A surprisingly large fraction of oxidative energy, corresponding to about 40% of the total energy generated, could not be assigned to phototransduction, to neurotransmission, to Na+ transport for other purposes, or to vegetative metabolism. We cannot account for its usage, but it may be related to the (previously reported) rapid turnover of the gamma-phosphate of retinal GTP, the function of which also remains unknown.(ABSTRACT TRUNCATED AT 400 WORDS)
1. Rabbit retinas were isolated and superfused with a physiological medium. Ganglion cell activity was recorded during stimulation with focused light, and receptive fields were mapped. Receptive fields were identical to those found in vivo and did not change during a 6-h incubation. After the receptive field of a ganglion cell had been identified, acetylcholine or related agents were introduced singly or in combination into the medium, and their effect on the cell's spontaneous and light-evoked activity was observed. 2. Ganglion cells with on-center or directionally selective receptive fields were excited when ACh was added to the medium. The response to exogenous ACh was prevented by cholinergic antagonists. 3. These cells' spontaneous activity and response to light were enhanced by anticholinesterase and depressed by cholinergic antagonists. Antagonists varied in their ability to block the light-evoked response, with dihydro-beta-erythroidine the most effective. 4. Thresholds for ACh or the related agents were low, ranging from 1 to 40 muM; their effects were rapidly and completely reversed when the retina was returned to control medium. 5. In retinas incubated in medium containing 20 mM Mg2+ and 0.2 mM Ca2+, ganglion cells lost completely both their spontaneous and light-evoked activity, but retained their ability to generate action potentials in response to elevated K+. Ganglion cell activity rapidly returned to normal when the retina was returned to medium containing normal electrolytes. On-center and directionally selective cells were excited by ACh in retinas where synaptic transmission had been inhibited by 20 mM Mg2+ and 0.2 mM Ca2+. 6. The responses of on-center and directionally selective cells to ACh, to anticholinesterase, and to cholinergic antagonists in control medium indicate that the retina contains one or more synapses using ACh as a neurotransmitter. The response to ACh in retinas exposed to 20 mM Mg2+ and 0.2 mM Ca2+ suggests that at least one such synapse in on the ganglion cell itself. 7. Off-center cells were inhomogenous in their response to ACh. Although some responded just as the other classes of cell, the majority responded quite weakly and a subgroup was encountered which was entirely unaffected by even 1 mM ACh, by levels of physostigmine which inactivate virtually all retinal acetyl-cholinesterase, or by high concentrations of cholinergic antagonists. Only 2 of 20 off-cells tested in the presence of 20 mM Mg2+ and 0.2 mM Ca2+ were excited by ACh. Apparently ACh is not a primary transmitter for most off-cells.
Methods are described for isolating adult rabbit retinal and maintaining it in a medium designed to resemble CSF. Morphologic, metabolic, nd electrophysiologic measurements obtained on the in vitro retinas showed that they remained in a nearly physiological state for at least 8 h, and even after 2 days in vitro they still exhibited a high level of metabolic activity and electrical responsiveness to light. Physiological activity was modified by photic stimulation, and data are presented to document changes in metabolism in response to the changes in function. The isolated retina appears to offer a number of unusual advantages for studying relationships between function and metabolism in organized mammalian central nervous tissue.
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