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Investigative Ophthalmology & Visual Science, Vol. 33, No. 10, September 1992 Copyright © Association for Research in Vision and Ophthalmology Changes in Glucose Level Affect Rod Function More Than Cone Function in the Isolated, Perfused Cat Eye Claudio Macaluso,* Shoken Onoe,t and Gunter Niemeyer The glucose concentration (gl) in mammalian serum incorporates a normal range of variation of several millimoles. We studied the effects of such variations on light-evoked electrical signals in the in vitro arterially perfused cat eye, avoiding extraocular regulatory mechanisms that might confound data interpretation. Changes in gl from the nominal control value of 5 mmol/1 were maintained for 5-40 min. Stimuli of near rod threshold intensity were presented in full dark adaptation, and stimuli of higher intensity were presented in the presence of a white background for cone responses. We recorded the dc-electroretinogram (ERG), the scotopic threshold response (STR), the optic nerve response (ONR), and the transretinal slow P-III and transepithelial retinal pigment epithelium c-wave from the subretinal space. The ocular standing potential changed by up to ±2 mV in parallel with an increase and decrease in gl, independent of the adaptation condition. Our results show that the rod-ERG, STR, and rod-driven optic nerve response (ONR) have a marked sensitivity to small changes in gl (±1 to 3 mmol/1). The field potentials increased and decreased in parallel with changes in gl. The cone ERG and cone ONR, in contrast, failed to respond consistently to increases in gl and revealed decreases in amplitudes only with an extreme decrease in gl. Decrease in gl, down to 2 mmol/1 and less, is known to induce drastic behavioral and electrophysiologic phenomena in the central nervous system. Our results imply that the "normal" glucose level, at least in the cat, could be marginal for rod-mediated retinal function. The results also suggest a marked difference in metabolic mechanisms for cone versus rod photoreceptors. Invest Ophthalmol Vis Sci 33:2798-2808,1992 for maintenance of retina or central nervous system preparations in vitro contain 10-20 mmol/1 glucose.14 Arterial perfusion of isolated cat eyes15"19 usually is performed using oxygenated tissue culture medium containing 4.5-5.5 mmol/1 glucose, corresponding to the range of glucose concentration (or 'gl') of cat serum. The glucose serum level can change in various metabolic conditions. The supply of glucose to central nervous system tissue can be reduced because of ischemia and hypoxia. It also can be enhanced locally (eg, by adenosine-induced glycogenolysis).20"21 We sought to systematically study the effects of transient, stepwise increases and decreases in the arterial supply of glucose in the isolated perfused cat eye. The preparation is well suited for changing constituents of the perfusing medium and observing the resulting effects without the multiple regulatory responses that counteract such changes in situ (ie, insulin and glucagon secretion). We monitored the effects on light-evoked and light-independent field potentials that reflect multiple levels of retinal information processing: the standing potential, the ERG b- and cwave, the scotopic threshold response, and the optic nerve response. The signals were recorded under selective stimulation of the rod system and the cone system. We found a remarkable sensitivity to discrete Little is known about the metabolic requirements of the fully dark adapted mammalian retina. However, retinal oxygen profiles,1 glucose uptake distribution,2 intraretinal pH assessments,3"5 and biochemical data6"10 indicate there is a significant difference from the light adapted state. The supply of glucose as the major source of energy may, like that of oxygen, be marginal in the fully dark adapted state.""13 Although the serum levels of glucose vary in humans (3.9-5.6 mmol/1), oxen (2.3-4.1 mmol/1), sheep (2.4-4.5 mmol/1), horses (3.5-6.3 mmol/1), pigs (3.7-6.4 mmol/1), dogs (3.4-6.0 mmol/1), cats (3.4-6.9 mmol/ 1), and rats (5.5-11 mmol/1), several standard media From the Neurophysiology Laboratory, Department of Ophthalmology, University Hospital, Zurich, Switzerland. This work was supported in part by the Kusch-Neumann Fonds, University of Zurich. * Present address: Istituto di Oftalmologia, University of Parma, Italy. f Present address: Department of Ophthalmology, Iwate Medical University, Morioka, Japan. Submitted for publication: November 15, 1991; accepted March 10, 1992. Reprint requests: G. Niemeyer, Neurophysiology Laboratory, Dept. of Ophthalmology, University Hospital, CH 8091 Zurich, Switzerland. 2798 Downloaded From: http://iovs.arvojournals.org/ on 08/03/2017 No. 10 EFFECTS OF GLUCOSE ON RETINAL FUNCTION / Mocoluso er ol changes in gl for rod-driven signals, but not for conedriven signals. Methods Details of the experimental method of arterial perfusion of the isolated mammalian eye have been published.15"1722 In the present study, eyes were enucleated from deeply anesthetized cats, in accordance with the ARVO Resolution on the Use of Animals in Research and with the regulations of the cantonal veterinary authority office of Zurich. The ophthalmociliary artery was cannulated, and the eye was perfused at 37°C at a flow rate of 1-2 ml/min. The perfusate consisted of tissue culture medium (TC 199, Earle's salts; Amimed, Basel, Switzerland) and 30% newborn calf serum, buffered with HEPES buffer and 26 mmol/1 bicarbonate, adjusted to a pH of 7.4 (at 37°C), and oxygenated by bubbling with 95% O2 and 5% CO2 for 15 min at a rate of 150 ml/min. The final PO2 was 400-500 mmHg. The gl of "standard" perfusate was 5.5 mmol/1, identical with perfusate used in previous studies from our laboratory. Glucose concentration could be increased by continuously injecting additional glucose from a stock solution into the perfusion system. To decrease the gl to below 5.5 mmol/1, perfusate containing only 1.3 mmol/1 glucose was used. In such cases, additional glucose (4.2 mmol/1) was continuously infused to keep gl at a level of 5.5 mmol/1 in control and recovery phases. The rate of this injection was reduced for the "hypoglycemic" test periods. Photic stimuli were provided by a 150 W xenon lamp (11.54 log quanta [507 nm] deg~2 sec"1) at the cornea, and attenuated by neutral density and monochromatic filters to achieve rod-matched conditions in full dark adaptation. The eye was stimulated from this source in Maxwellian view via a modified fundus camera.23 The stimulus duration was 400 msec and the interval was at least 30 sec for recordings of rodmatched signals (50 msec duration for scotopic threshold response recordings). For cone stimulating conditions, an adapting beam (white; 8.6 log quanta [507 nm] deg~2 sec"1) was used to suppress the rod contribution. The light-evoked potentials, ERG, and scotopic threshold response (STR) were recorded by salt bridge Ag-AgCl electrodes24 positioned in the vitreous and on the posterior scleral surface. A pair of Ag-AgCl electrodes were positioned on the surface and on the proximal end of the optic nerve to record the compound action potential of the optic nerve (optic nerve response, or ONR). For recordings from the subretinal space, glass microelectrodes were made using a Brown-Flaming puller (Suiter Instrument Co., Novato, CA) and werefilledwith 2 mol/1 potassium-ace- Downloaded From: http://iovs.arvojournals.org/ on 08/03/2017 2799 tate, resulting in an impedance of 15-70 mohm. By referencing the microelectrode in the subretinal space to the vitreous and to the sclera, the transretinal potential (TRP) and the transepithelial potential (TEP), respectively, were monitored. For these recordings, rod-matched stimuli, 4 sec in duration, were used. After cannulation, 20 msec light pulses (narrow band filter, Xmax 620 nm) were used to monitor the condition of the eye from the beginning of perfusion for at least 60 min of dark adaptation, adjusting the flow rate for a standard amplitude of the vitreal bwave of 700-800 fiV. The standing potential (SP) and the light-evoked ERG and ONR were recorded at one low, near threshold intensity, or over a range of intensities before, during, and after changing gl. The duration of changes in gl was 3-15 min, but was 20-60 min for recording of amplitude/intensity functions (V/log I). Results Changing the concentration of glucose in the perfusate affected the electrical activity of the pigment epithelium, retina, and optic nerve in different ways. The rod-driven light-evoked signals—ERG b-wave and cwave, ONR, and STR—increased or decreased parallel to small changes in gl. Cone-driven light-evoked signals (b-wave and ONR) failed to respond to small changes in gl, but decreased during extreme hypoglycemia. The light-independent SP changed in parallel to changes in gl, independent of dark- or light-adaptation. ERG b-Wave The ERG b-wave, recorded under rod-matched conditions, showed remarkable, immediate, and reversible responses to the changes in gl. The amplitude of the rod b-wave clearly was enhanced during elevation of the gl above the "standard." The time course of the changes in b-wave amplitude is shown in Figure 1. The enhancing effect started within 1 min from the beginning of gl elevation. During the return to control gl, the b-wave amplitude showed a slight undershoot prior to recovery. The enhancement varied between 8 and 125% (n = 25), with a tendency to saturate at 8-10 mmol/1 glucose (Fig. 2). There was more variability between preparations than within trials on the same isolated eye. Dose-dependency was tested and found to be present in five out of eight preparations. When we exposed preparations a priori to a higher gl of 8 or 10 mmol/1, the enhancing effect of additional glucose was smaller or absent. In three out offiveexperiments of this type, an undershoot was seen after a return to 8 or 10 mmol/1 gl before recovery, much as described for the changes starting with standard perfusate.14 2800 A 35 recovered completely upon return to the control level within 5 min. An example of the V/log I function for the rod bwave near threshold is shown in Figure 3. During increased gl, the gain of the b-wave was increased in the rod range (Fig. 3, left), without revealing a clear change in threshold. Decreasing gl induced a slight reduction in sensitivity (shift of the V/log I curve along the abscissa; Fig. 3, right). Under cone-stimulating conditions, in contrast, the ERG b-wave was not or only minimally affected by increasing (n = 7) or decreasing (n = 4) gl (Fig. 2, empty symbols). Only extensive decreases in gl of about 4.2 mmol/1 induced marked depression of the cone-driven b-wave. ° 300 ^ 250 "§ 200 "5. o I 150 100 50 increased [glucose] ( + 3 mM) i -2 0 2 i i 4 6 8 10 12 min B 35 ° T 300 ampli tud Vol. 33 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / September 1992 Optic Nerve Response Changes in gl affected all parts of the ONR, the initial ON, the following plateau, and the OFF components, corresponding to the time course of the light stimulus, under rod-matched conditions. Figure 4 shows the time course of the effect on the ON-component of the ONR, revealing an increase in amplitude, similar to the effect on the b-wave. After termination of the step increase in gl, there was a slight 250 350 200 300 r decreased [glucose] (-4.2 mM) 150 •S 250 b—wa\ 3 a. E 100 200 D 50 - 150 oo 100 -5 10 15 20 25 30 CO C D _C o Fig. 1. Time course of typical changes in normalized amplitudes of the rod ERG b-wave in response to elevation (A) and decrease (B) in concentration of glucose gl. The b-wave was enhanced by additional glucose and depressed by decreased glucose. Transient increase in gl was followed by a slight undershoot after termination of the injections. The insets show corresponding original traces of rod ERGs before and during (thicker line) the changes in gl. Figure 1 shows the effect of reducing gl on the rod b-wave (n = 4). The amplitude decreased as soon as gl decreased, with the maximal effect after 2 min. The attenuation was dose dependent (Fig. 2, filled symbols). After gl was decreased for 17 min, the b-wave Downloaded From: http://iovs.arvojournals.org/ on 08/03/2017 50 -5 -3 -1 change in [glucose] (mM) Fig. 2. Changes in normalized amplitudes of the rod (filled symbols) and cone (empty symbols) ERG b-wave in response to changes in gl. The intersection of the reference lines indicates the control amplitude and control gl in the perfusate (5.5 mmol/1). Whereas the rod signals are sensitive to both increase and decrease in gl with tendency to saturation (+2.5 to +4.5 mmol/1 change), the cone b-wave was affected only by extensive decrease in gl by -4.2 mmol/1. No. 10 2801 EFFECTS OF GLUCOSE ON RETINAL FUNCTION / MQCQIUSO er ol 60+ 4.5mM glue. X> | 40 Q. Fig. 3. V/log I function for rod ERG b-wave near threshold, under standard gl (circles), under elevated gl (triangles), and under lowered gl (squares). E O 20 •o o -2.8mM glue. E610A J -6 E611D -7 -6 log relative intensity undershoot before recovery. The enhancing effect also was observed in the OFF-component, whereas the plateau changed to a lesser extent. By "loading" the preparation with 8-10 mmol/1 gl in the perfusate, the enhancement of the ONR with additional glucose was small or absent.14 The effect of decreasing gl from the "standard" concentration on the ON-component is shown in Figure 4b. Immediately, the amplitude was markedly attenuated, and 6 min after it returned to the control gl, the response had recovered. A dose-response relation was evident for changes in the range of —4.2 to +4.5 mmol/1 glucose referred to control (Fig. 5,filledsymbols). Enhancement or attenuation of the ONR amplitude was parallel to increasing (n = 18) or decreasing (n =6) gl. Figure 6 shows V/log I functions for the ONR ON-component in the rod-matched range, near threshold. By raising gl 4.5 mmol/1, the sensitivity was increased (Fig. 6, left). At a gl reduced to 2.7 mmol/1, it was decreased (Fig. 6, right). Under cone-stimulating conditions, the ONR was found to be almost unresponsive to increasing (n = 7) and decreasing (n = 2) gl in the perfusate. However, a substantial decrease of about 4.2 mmol/1 greatly attenuated the signal amplitude (Fig. 5, empty symbols). Scotopic Threshold Response Much like the rod-driven ERG b-wave and optic nerve response, the STR, the most sensitive field po- Downloaded From: http://iovs.arvojournals.org/ on 08/03/2017 tential of the rod system below and at b-wave threshold, showed clear enhancement or attenuation of its amplitude parallel to increasing (n .= 5) or decreasing (n = 3) gl, as illustrated in Figure 7. Experimental series involving the effects of changes in gl on the V/ log I functions of the STR consistently showed an increase and decrease in amplitude paralleltp corresponding changes in gl. In some series (two out of five) there was evidence for an increase in sensitivity—that is, shift of the V/log I curve to the left during an increase in gl. Standing Potential The SP was consistently affected by the changing of the gl in rod- and cone-stimulating conditions. Original traces of changes in standing potential in response to changes in gl are shown in Figure 8 (top). An increase in gl (n = 45) consistently induced a small transient decrease in SP, followed by a marked and maintained increase. Decreasing gl (n = 18) induced similar changes in SP with reversed polarity compared to those seen under an increase in gl. These changes reached the maximal and maintained effect after 10 min, in contrast to the faster changes in the lightevoked b-wave, ONR, and STR. No difference in changes in SP was observed between rod- and conestimulating light conditions. The changes in SP were dose dependent (Fig. 8, bottom) as most clearly recognized within the same preparation. Recordings from the subretinal space (n = 9) showed that the changes 2802 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / September 1992 350 A because changing gl may trigger a response resulting from modified osmolarity alone. Mannitol induced a decrease in SP (50-300 ixV) that reached a plateau, representing the hyperosmolarity response.25 This response is observed consistently as an initial, transient decrease in SP during increases in gl and as an initial increase in SP during decreases in gl (Fig. 8). 300 ampli tude 250 200 ERGc-Wave 150 ONR- z o 100 50 - increased [glucose] ( + 3 m M ) 0 -2 0 2 4 6 8 10 12 min B Vol. 33 35 r ° 300 Changes in glucose level markedly affected the amplitude of the ERG c-wave. Decreasing (n - 6) or increasing (n = 11) gl consistently induced a decrease or an increase, respectively, in the amplitude of the vitreal c-wave (Fig. 9). The changes in c-wave amplitude were dose related (Fig. 9B) and closely followed the changes in SP (Fig. 9A; only the effects of decreased gl are shown). To identify which component contributing to the c-wave was affected by glucose, we recorded from the subretinal space. Referring the subretinally positioned microelectrode to the sclera permitted assessment of the transepithelial potential and the RPE cwave. Referring to the vitreous allowed the transretinal potential and slow P-III to be assessed. Analysis 250 350 "° 200 ~a. o r 300 decreased [glucose] (-4.2 mM) vS 150 0) 250 100 I 50 200 o i -5 0 5 10 15 20 25 30 min Fig. 4. Time course of typical changes in normalized amplitudes of the rod ONR-ON response during increase (A) and decrease (B) in gl. The response was enhanced by additional glucose and depressed by decreased glucose. There was a slight undershoot after termination of the injections. The insets show the corresponding original traces of the rod ONR-ON before and during (thicker line) the changes in gl. in SP induced by glucose were due solely to a change in the TEP, not to changes in the TRP. In one eye, the effect on SP of injections of hyperosmotic solutions (4.5 and 10 mmol/1 mannitol, not shown) was tested. This experiment was performed Downloaded From: http://iovs.arvojournals.org/ on 08/03/2017 0> CO c o _c o 150 O 100 _Q 50 - 5 - 3 - 1 1 3 5 7 9 change in [glucose] (mM) Fig. 5. Changes in normalized amplitudes of the rod (filled symbols) and cone (empty symbols) ONR-ON component in response to changes in gl from the standard (about 5.5 mmol/1) perfusate. Whereas the rod signals are sensitive to both increase and decrease of gl with a tendency to saturation at +2.5 to +4.5 mmol/1 change, the cone ONR-ON component was affected only at extensive decreases in gl by -4.2 mmol/1. EFFECTS OF GLUCOSE ON RETINAL FUNCTION / Mocoluso er ol No. 10 2803 40 + 4.5mM glue. Q. Fig. 6. V/log I function of the rod ONR-ON component under standard (circles), elevated (triangles), and under lowered gl (squares). en -2.8mM glue. o 20 10 CL o -8 -7 -8 -7 log relative intensity of these light-evoked signals revealed that the changes in the amplitude of the c-wave during increased (n = 6) or decreased (n = 3) gl resulted from a change in the amplitude of the transepithelial c-wave rather than change in the amplitude of slow P-III (Fig. 10, only the effects of decreased gl are shown). control Discussion In this study, we found that changing the supply of glucose, even within the "normal" range, influences light-evoked signals and the standing potential, which all arise from different retinal layers. That the various retinal layers are affected differently in dark and light adaptation supports and extends the concept of their different metabolic characteristics. The external retina is recognized as being the most sensitive to the light adapting condition, probably because the photoreceptors reach their highest activity in the d a r k effect +3.6 mM glucose effect - 2.7 mM glucose control 50 msec Fig. 7. Original STR traces under increased gl (+3.6 mmol/l, top), and decreased gl (-2.7 mmol/l, bottom). The thicker line shows the effects. (Calibration bars: 2 ^V [top] and 10 M V [bottom].) Downloaded From: http://iovs.arvojournals.org/ on 08/03/2017 1-10,26-28 b-Wave Small increases and decreases in gl increased and decreased, respectively, the rod-driven b-wave, but did not or only slightly increased and decreased the cone-driven b-wave. The effect in the dark adapted condition appears to be a change in gain rather than a change in sensitivity, as derived from the V/log I curves. The b-wave is thought to be generated by the movement of K+ along the Miiller cell,29'30 as a result of the light-evoked depolarization of the ON bipolar cells.31 Our results regarding the b-wave could be due to an effect of glucose on the neuronal activity or the Miiller cell resting potential, as hypothesized by Masland and Ames.32 The finding of a clear-cut difference of effects in rod- and cone-stimulating conditions tends to exclude the latter possibility and points to a 2804 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / Seprember 1992 change in standing potential — — glucose glucose : 3 - 2/ 2 - 1 +4.5mM -4.2mM • - 1 A • -1 -2 * : # • °* o o - 5 - 3 - 1 1 3 5 the guinea pig in low glucose, suggesting a decrease in the number of activated neurons. Creutzfeldt35 reported a decrease in the firing rate in cortical recordings from the anesthetized cat under hypoglycemia. Changes in gl may directly affect the mechanisms that control firing frequency. We favor the hypothesis that in our ONR data the modulation induced by changes in glucose is simply carried along the information processing pathway within the retina, resulting in changes in the number or firing rate of activated ganglion celis. Scotopic Threshold Response o n \j Vol. 33 7 9 change in [glucose] (mM) Fig. 8. Changes in standing potential (SP) induced by changing gl. Top: original traces; the heavy line indicates the duration of the change in gl, which lasted 30 min and 25 min for the increase and decrease, respectively. Bottom: changes in amplitude (mV) of the SP induced by changes in gl in rod (filled symbols) and cone (empty symbols) adapting conditions. The SP consistently increased during increases, and decreased during reduction in gl, independently of the light-adapting condition. primary change in the neural response, mainly of rod ON bipolar cells, underlying the changes in b-wave amplitude. Optic Nerve Response The ONR reflects the light-evoked retinal ganglion cells activity. Its configuration, which makes up the ON, plateau, and OFF components, is understood to be the summation of all the on-going excitation and inhibition of the axons underlying the electrodes.17'33 The changes seen in the ONR, induced by small changes in gl, are similar to those for the ERG b-wave. They have a virtually identical time course, good correlation of change in amplitude for a given change in gl and, finally, marked effects in rod-matched, but not cone-stimulating conditions. Cox and Bachelard34 reported attenuation of the population spike in superfused hippocampal slices of Downloaded From: http://iovs.arvojournals.org/ on 08/03/2017 The STR is a newly characterized rod-driven component of the ERG, elicited by very dim illumination below the threshold of the rod b-wave and originating in the proximal retina.36 The mechanism for its generation possibly involves an increase in K+37 induced by activation of inner retinal neurons.38 The glucose-induced changes in amplitude of the STR, parallel to those observed for b-wave and ONR, provide clear evidence for a remarkable sensitivity of the rod system to small changes in gl. Based on this view, it is not surprising that the STR reacts similarly to the optic nerve response. Standing Potential and c-Wave The increase and decrease in SP parallel to changes in gl were consistent and reversible. They differed from changes in b-wave, ONR, and STR in two ways: (1) the changes in SP were independent of light and dark conditions; and (2) they revealed a slower time course. The c-wave amplitude also increased and decreased in parallel to corresponding changes in gl and with a time course identical to that of the SP. Covariation of the two voltages39 has been described for several conditions, such as the enhancement of the cwave during the light peak.40 Our recordings from the subretinal space identified the transepithelial component of the c-wave as the source of these changes, indicating a probable change in RPE resistance, rather than a change in the light-induced decrease in [K+]o in the subretinal space.40 Therefore, glucose changes would not primarily affect the light-evoked electrical activity—ie, hyperpolarization—of photoreceptors. In preliminary experiments, the aspartate-isolated receptor potential (fast P-III) was not modified by changing gl. Similarly, Winkler7 reported that the fast P-III amplitude remained constant during changes in gl, in the range 1-10 mmol/1. These features point to a different mechanism un- No. 10 EFFECTS OF GLUCOSE ON RETINAL FUNCTION / MQCOIUSO er ol 2805 c-waves 0.5 mV I ©w > a) 100 6§. 50 e-e Glucose -1.7 mM cc Fig. 9. (A) DC recording of the standing potential (upper trace) and amplitude of the ERG c-waves (lower trace) during a decrease in gl plotted against the same time scale. The c-wave amplitude appears to covary with the standing potential. (B) Changes in normalized c-wave amplitudes in response to changes in gl from the standard (about 5 mmol/1). The c-wave increased during increases and decreased during reduction in gl. The inset shows original traces of c-waves before (thin trace) and during (heavy trace) an increase in gl. 10 20 15 B effect: + 3.5 mM glucose 300 Mi '•• 250 I control T3 I \ 1 mV 200 4 sec Q. CO 6 I 100 c CO " 50 - 5 - 3 - 1 1 3 5 7 change in [glucose] (mM) derlying the glucose-induced change in SP and c-wave compared to the mechanism of the effects on b-wave, ONR, and STR. Harik et al41 demonstrated an abundance of glucose transporters in the RPE, in addition to their presence in capillaries of retina and optic nerve—that is, at the blood-eye barriers. These glucose transporters are of the "brain-type," also referred to as "erythrocyte-type" or GLUT-1,42 They perform a facilitated transport, without cotransport with ions, as demonstrated in the RPE.43"44 Modifications of the rate of this nonelectrogenic transport of glucose across the RPE membrane cannot explain the observed glucose-induced changes in SP. Rather, these Downloaded From: http://iovs.arvojournals.org/ on 08/03/2017 might depend on metabolism of glucose in the RPE cell, leading to change in electrogenic transport mechanisms. The initial increase and decrease in SP opposite to changes in gl were due to the osmolarity change induced by injection of glucose. Mannitol injections in the same osmolarity range as glucose allowed us to distinguish between simple osmotic and glucose-specific effects. Reasons why the change induced by glucose is smaller than that induced by mannitol might be: (1) that glucose enters cells, whereas mannitol does not; thus, the effective osmotic change pressure of glucose is smaller; or (2) because of algebraic sum- 2806 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / September 1992 Vol. 33 mV 1.5 Fig. 10. Changes in amplitude of the cwave recorded from the vitreous, and of its components, transepithelial c-wave and slow P-III, recorded from the subretinal space, during a decrease in gl. Left column: amplitude vs. time changes. Right column: original traces; thicker line designates effects. The changes in the amplitude of the c-wave during decreased gl resulted from a change in the transepithelial c-wave rather than in slow P-III. 7.5 Q. a> w c 5.5 Glucose -1.7 mM 10 4 sec 15 min mation with the nonosmotic, opposite, and more prominent effect of gl on the SP. The Photoreceptor Synapse as a Possible Site for the High Sensitivity of the Retina to "Mild Hypoglycemia" in the Dark The effect of changing gl on b-wave and ONR under cone stimulating conditions, showing a decrease only for extremely low arterial gl, is consistent with data obtained in other parts of the central nervous system under hypoglycemia. Only severe hypoglycemia (1.2 mmol/1 and less) induces convulsions, coma, and, finally, an isoelectric electroencephalogram in association with a significantly decreased level of adenosine triphosphate and creatine phosphate.45"48 Central nervous system functions (monitored as electrical or as behavioral activity) are affected when the arterial gl falls below approximately 2 mmol/1, sometimes referred to as "mild hypoglycemia."34'46"50 The metabolic correlate of this condition is very interesting. Different from what might be expected, the energy state of the tissue—eg, the concentration of high energy phosphates—is not lowered.34-45"49 Rather, failure of neurotransmission is thought to be the underlying mechanism, because glucose is metabolically coupled to molecules involved in the release of transmitters. Levels of gluta- Downloaded From: http://iovs.arvojournals.org/ on 08/03/2017 mate, glutamine, alanine, 7 aminobutyric acid, and acetylcholine have been found to be decreased, and levels of aspartate, lysine, and NH4+ have been found to be increased in mild hypoglycemia.45"51 The damage to and death of cells occurring in severe hypoglycemia have been demonstrated to be a result of an increase in excitotoxins—the aspartate/glutamate proportion reaches a new steady-state in hypoglycemia.51 In the present study, thefindingthat the rod-driven b-wave, the ONR, and the STR respond to decreases in gl to about 3 mmol/1 reveals an exquisite sensitivity to hypoglycemia of the dark adapted retina. Parallels to the high sensitivity to mild hypoxia of the dark adapted cat retina in vivo are evident in the work of Linsenmeier1 and Steinberg.52 This is attributed to the high Na+/K+ ATPase activity of photoreceptors, necessary for keeping the light-sensitive cation channels open.52 Because "mild hypoglycemia" does not inhibit energy metabolism, the highly energy-dependent Na+/K+ ATPase activity in photoreceptors should not be influenced by lowering gl. Our finding that the light-evoked electrical activity of photoreceptors is not affected by changing gl would confirm this view. The possibility arises that the neurotransmission between photoreceptors and second order neurons is the site primarily affected by change in gl. This hypothesis is consistent with three facts. First, in dark adaptation, not only does the Na+/K+ ATPase pump No. 10 EFFECTS OF GLUCOSE ON RETINAL FUNCTION / Mocoluso er ol exert maximal activity, but neurotransmission to second order neurons reaches its highest rate. Second, glutamate is considered most likely to be the neurotransmitter at those synapses.53'54 Third, glutamate levels are significantly lowered in hypoglycemia.45"51 Conclusion 11. 12. 13. These observations, particularly that small changes in gl induced marked changes in the amplitudes of rod-driven responses, raise the question whether the supply of glucose in fully dark-adapted conditions is marginal or suboptimal for rod-driven retinal function. From recording field potentials and from single cell responses in this preparation, which are comparable to and exhibit the same sensitivity as in the in vivo cat retina, we consider the isolated eye to be nourished with an adequate amount of oxygen. It remains to be tested whether the light-evoked electrical signals of the rod system in vivo also are sensitive to small changes in gl. 14. 15. 16. 17. 18. 19. Key words: glucose, retina, optic nerve, electrophysiology, perfused eye 20. Acknowledgments We thank Dr. Urs Gerber, Zurich, for valuable comments on the manuscript, and Ms. F. Uldry and Ms. F. Werren for excellent technical assistance. 21. References 22. 1. Linsenmeier RA: Effects of light and darkness on oxygen distribution and consumption in the cat retina. J Gen Physiol 88:521, 1986. 2. Bill A and Sperber GO: Aspects of oxygen and glucose consumption in the retina: Effects of high intraocular pressure and light. Graefes Arch Clin Exp Ophthalmol 228:124, 1990. 3. Borgula GA, Karwoski CJ, and Steinberg RH: Light-evoked changes in extracellular pH in frog retina. Vision Res 29:1069, 1989. 4. Oakley Bll and Wen R: Extracellular pH in the isolated retina of the toad in darkness and during illumination. 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