Ethanol induced alterations of axoplasmic flow in retinal ganglion cells.

Effects of acute ethanol administration on the axoplasmic flow in retinal ganglion cells of rats were studied by intraocular injection of 3H-1-leucine. Ethanol induced a facilitation of the fast axoplasmic flow without affecting the slow axoplasmic flow. Pretreatment with colchicine almost completely eliminated the fast flow in both control and ethanol-treated rats, while cycloheximide did not significantly affect the facilitation of the fast flow induced by ethanol. The extent of ethanol-induced changes in the fast flow did not directly correlate with the level of ethanol or acet aldehyde in the blood. Neither adrenalectomy nor hypophysectomy modified sig nificantly the facilitation of the fast axoplasmic flow due to ethanol administration. Present results indicate that ethanol-induced facilitation of the fast axoplasmic flow is not due to the direct effect of ethanol or acetaldehyde or to changes in pituitary adrenal function. It has been well established that axoplasmic transport consists of fast and slow axo plasmic flow processes. It has been documented that the rates of fast and slow transport in retinal ganglion cells were 110-150 mm/day and 1.5-2.0 mm/day respectively (1, 2). Recent studies from our laboratory indicated that acute or chronic ethanol admin istration induces a facilitation of the fast phase of axoplasmic flow in the mouse cerebral cortex (3). In the present paper we report the effect of ethanol administration on the transport of protein components in the optic system of the rat. MATERIALS AND METHODS Male Wistar rats, weighing 180-220 g, were used. Under ether anesthesia, 10 ,aCi of 3H-1-leucine (1-leucine-4, 5-T, specific activity 38 Ci/mmol; The Radiochemical Center, Amersham, England) in 1-6 Id sterile aqueous solution was injected through the sclera into the vitreous body of the right eye. Four days (in the observation of slow flow) or 2.5 hr (in the observation of fast flow) after isotope injection, optic nerves and tracts were immediately dissected and cut into pieces of 2 mm length. After homogenizing in ice cold 0.32 M sucrose, the samples were analysed for protein-bound specific radioactivity as described by Sjostrand et al. (1). Forty percent ethanol solution in saline was administered i.p. immediately after the isotope injection (in fast flow experiments) or once a day in successive 4 days (in slow flow experiments). Colchicine (0.2 ,ug) or cycloheximide (50 sag) in 2 ,al of a sterile aqueous solu tion was injected into the same eye respectively at 24 hr before or 1.5 hr after the isotope injection. Pyrazole (2.8 mg/kg) dissolved in 0.5 ml saline was administered i.p. im edi ately after the ethanol treatment. Before the intraocular injection of isotope, disulfiram (0.5 g/kg/day) emulsified in 1 ml mucilage was administered orally for 3 days. Blood serum levels of ethanol and acetaldehyde were measured using gas chromato graphy. RESULTS Effect of acute ethanol administration on axoplasmic flow in retinal ganglion cells The labelled protein was found predominantly in right optic nerve on the injected side and in left optic tract on the ipsilateral side 2.5 hr after isotope injection (Fig. 1A). Although the greater part of labelled protein was present in the optic nerve, a significant amount was also transported to the left optic tract, possibly due to the fast axoplasmic flow which has a flow rate of 120-140 mm/day (1). Four days after isotope injection, the labelled protein was also found in the right optic nerve and left optic tract (Fig. 1B). At this time, however, the amount of the labelled protein in the right optic nerve was great er than that observed at 2.5 hr after the injection. In addition, the peak of labelled pro tein in the right optic nerve was found to have shifted toward the optic chiasma, suggesting that a slow axoplasmic flow which has a flow rate of 2 mm/day (1) may dominate in the right optic nerve at this stage. FiG. 1. Effect of acute ethanol administration (3 g/kg i.p.) on fast and slow axoplasmic flow in retinal ganglion cells. 3H-1-leucine was injected into the vitreous body of the right eye . Abscissae indicates the distance (mm) from the right eye ball. Usually the optic chiasma was located between 9 and 11 mm from the right eye ball. x ...... x Control (right optic nerve-left optic tract) 0 Ethanol-treated (right optic nerve-left optic tract) A...... A Control (left optic nerve-right optic tract) Each point represents the mean±S.E. obtained from 6 different experi ments. * Differs from each control , P<0.05. Following acute i.p. administration of ethanol (3 g/kg), specific activities of the pro tein in the left optic tract were increased in the 2.5 hr observation (Fig. IA), indicating that the fast phase of axoplasmic flow in retinal ganglion cells is facilitated by ethanol ad ministration. The blood serum levels of ethanol were altered 150-370 mg/dl by changing the ethanol dose (2.5-3.5 g/kg i.p.) or pyrazole (2.8 mg/kg) treatment. The facilitated pattern of the fast axoplasmic flow due to ethanol remained however unchanged. These results suggest that the observed changes in the fast axoplasmic flow may be relatively independent from blood levels of ethanol at these concentration ranges. On the other hand, the rate of slow axoplasmic flow, estimated 4 days after isotope injection, did not change significantly after ethanol administration (Fig. 1B). Effect of colchicine on ethanol induced changes in axoplasmic flow In order to determine whether or not the observed facilitation of fast axoplasmic flow in retinal ganglion cells following ethanol administration is entirely due to intra-axonal transport, the effect of colchicine, which is known as a potent inhibitor of axoplasmic flow (1, 5, 6), was examined. As shown in Table I, pretreatment with colchicine remarkably reduced fast axoplasmic flows in both control and ethanol-treated animals and eliminated the ethanol-induced facilitation of the fast axoplasmic flow. The results clearly indicate that the labelled protein observed in optic nerves and tracts was transported intra-axonally and that the ethanol facilitated portion was also transported by the same process. TABLE 1. Effects of colchicine and cycloheximide on ethanol induced facilitation of fast axoplasmic flow in retinal ganglion cells. Mean S.E. obtained from 6 different experiments. 3H-1-leucine was injected into the vitreous body of right eye at 2.5 hr before sacrifice. a, Colchicine (0.2 pg) was administered intraocularly 24 hr before isotope injection. b, Cycloheximide (50 fig) was injected intraocularly 1.5 hr after isotope injection. c, Three g/kg of ethanol was administered i.p. immediately after isotope injection . d, Indicates the distance from the right eye ball in the right optic nerve and left optic tract preparations. * Difference from each control , p<0.05. Effect of cycloheximide on ethanol induced changes in axoplasinic flow Increase of the amount of labelled protein in the optic nerve and tract by acute ethanol administration can be explained in the following two ways; namely, the increase of uptake and incorporation of injected radioactive 1-leucine into the retina and/or acceleration of the fast axoplasmic flow in retinal ganglion cells. In the former possibility, the effect of intraocular injection of 501-q-:1 cycloheximide, a dose which inhibits almost completely protein synthesis in the retina (K. Kuriyama, unpublished observation) was studied. Since preliminary experiments indicated that the half time of protein synthesis in the retina was 1.5-3 hr, cycloheximide was injected 1.5 hr after the labelled 1-leucine injection. Cyclo heximide induced a decrease of the amount of radioactive protein in the right optic nerve in both control and ethanol-treated rats as shown in Table I. However, specific activities FIG. 2. Effect of pretreatment with disulfiram, adrenalectomy and hypophysectomy on ethanol (3 g/kg i.p.) induced facilitation of fast axoplasmic flow in retinal ganglion cells. Disulfiram (0.5 g/kg/day, orally) was administered for 3 days before initiating the intraocular injection of isotope. Adrenalectomized and hypophysectomized rats were employed 14 days after operation. Other experimental conditions and the expression of results are the same as in Fig. 1. of protein in the left optic tract were not significantly affected and the ethanol induced increase of labelled protein in the optic tract remained unchanged following the cyelo heximide injection. These results suggest that the observed increase of labelled protein in the optic tract following acute ethanol administration may be due to an actual facilita tion of axoplasmic flow with no direct relation to changes in protein synthesizing processes in the retina. Effect of pretreatment with disulfiram, adrenalectomy and hypophysectomy on ethanol in duced changes in axoplasmic flow Following pretreatment with disulfiram (0.5 g/kg/day, for 3 days), a potent inhibitor of acetaldehyde dehydrogenase, ethanol administration induced a significant increase of acetaldehyde level in the blood (0.66-1.11 mg/dl in disulfiram pretreated animals VS 0.34 0.35 mg/dl in untreated animals). Significant effects of disulfiram pretreatment on the ethanol induced facilitation were not found to exist (Fig. 2B), suggesting that acetaldehyde may not be the primary factor responsible for the facilitation of fast axoplasmic flow. In both adrenal ectomized and hypophysectomized rats, the general patterns of the axoplasmic flow were similar to those found in non-operated rats respectively (Fig. 2C and 2D). In addition, ethanol-induced facilitation of the fast axoplasmic flow was basic ally unchanged by adrenalectomy or hypophysectomy as shown in Figs. 2C and 2D re spectively. DISCUSSION The axoplasmic flow has been extensively investigated neurochemically and autoradio graphically because of its importance in the metabolic economy and physiological functions of the neuron. It is well established that in the retinal ganglion cells there are at least two kinds of axoplasmic flow, one of which has a rate of 110 to 150 mm/day and the other a rate of 1.5 to 2 mm/day (1, 2). It is generally considered that the fast flow is mainly responsible for the transport of particulate proteins, while the slow flow is 5 to 10 times greater than the fast flow in pool size, and transports mostly soluble proteins (1, 2, 4, 7). The present study demonstrates that acute ethanol administration accelerates the fast flow without affecting the slow one. Similar results were reported in studies of the intracerebral axoplasmic flow in mice following acute and chronic ethanol administration (3). A possible argument may be made that these findings are a simple reflection of the changes in local protein synthesis in the axon and/or extra-axonal flow. The present study, however, strongly argues that the migrating labelled protein in the control and ethanol treated rats was transported in the interior of axons, since colchicine (2 ,ug), a potent in hibitor of axoplasmic flow, inhibited the transport of radioactive protein in the optic nerve and tract, and clearly eliminated the ethanol-induced facilitation in the fast phase of axo plasm:c transport. Intraocular injection of eolchicine is reported to inhibit protein syn thesis in the retina, but in the present study this was not the case as the dosage employed was too low (1). Moreover, cycloheximide, a potent inhibitor of protein synthesis, did not affect the ethanol induced increase of labelled protein in the optic tract. Actually, acute ethanol administration does not change significantly protein synthesis in the retina (Y. Yamasaki and K. Kuriyama, unpublished observation). To clarify mechanisms Underlying the facilitation of the fast axoplasmic flow induced by acute ethanol administration, three possible factors have been considered. At first, the relationship between the extent of the acceleration of fast flow and blood level of etha no] was studied but a correlative relationship was absent. Secondly, a possible involve ment of acetaldehyde, the first metabolite of ethanol, on the facilitation of the fast axoplasmic flow was examined following disulfiram administration. Despite the significant increase of acetaldehyde, there were the same results as seen with ethanol treatment alone. Finally, effects of acute ethanol administration on the axoplasmic flow were examined in adrenalectomized and hypophysectomized rats. It has been reported that ethanol has a stimulatory effect on the hypophyseal-adrenal axis and induces the elevation of plasma corticosterone level (8). In this study, however, alteration in ethanol induced facilitation of the axoplasmic flow was not found to exist in adrenalectomized or hypophysectomized