318 BRES 17048 Brain Research, 561 (1991) 318-323 © 1991 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/91/$03.50 ADONIS 0006899391170482 Transferrin can alter physiological properties of retinal neurons Arnold G. Hyndman 1, Philip E. Hockberger 2'*, Gail D. Zeevalk 1'** and John A. Connor 3 t Department of Biological Sciences, Rutgers University, Piscataway, NJ 08855 (U.S.A.), 2Department of Molecular Biophysics, Bell Laboratories, Murray Hill, NJ 07974 (U.S.A.) and 3Department of Neuroscience, Roche Institute of Molecular Biology, Nutley, NJ 07110 (U.S.A.) (Accepted 21 May 1991) Key words: Transferrin; Calcium; Retina; Glutamate; Neuron; Culture The role of transferrin as a possible neurotransmitter was examined in cultured chick retinal cells. Brief exposure to transferrin caused a dramatic and transient increase in intracellular calcium levels in approximately 20% of the total population of cultured retinal neurons. The increase in intracellular calcium was observed in cell bodies and neuronal processes. Electrophysiological analysis of a subset of the popu- lation, bipolar-like neurons, demonstrated that more than half of these cells responded to the application of transferrin with a transient membrane depolarization. Under voltage clamp conditions, the currents evoked by transferrin were similar to glutamate in that they both displayed non-linearvoltage dependence. Furthermore, acute transferrin exposure resulted in a 200% increase in the amount of Na ÷ indepen- dent [3H]glutamate binding observed in these cultures. These results suggest that transferrin may function as a neurotransmitter or neuro- modulator in the developing vertebrate nervous system. INTRODUCTION The serum glycoprotein, transferrin, has long been known as a major iron transport protein and is widely used as a growth factor for cells maintained in vitro 3'z°. There is evidence suggesting that transferrin has a role in CNS growth, differentiation and function 2'11'17'27"35. Among the more novel and interesting suggestions is that transferrin may serve as a neua'omodulator, or in- fluence the maintenance and regulation of synaptic con- tacts in the developing CNS 28'30'34. Using immunohistochemical techniques, we have shown that transferrin is present in neurites during for- mation of the inner and outer plexiform layers, as well as the optic nerve fiber layer of the embryonic chick ret- ina 34. The relative concentration of transferrin in the neural retina increases 5-fold between embryonic day (E) 6 and 10, and then rapidly declines by El4 35. This time is a critical period in retinal maturation, including the formation of the retinal synaptic layers. The coincident timing between elevated transferrin and retinal maturation suggests that transferrin may be important for certain cellular activities associated with neurite growth and connectivity. In this study, we exam- ined the effects of transferrin on intracellular calcium levels, electrical activity and transmitter binding. Other neurotrophic factors such as epidermal growth factor, insulin and nerve growth factor have been shown to be capable of modulating these types of neuronal activities 5" 29,33 Our monolayer tissue culture system of purified chick retinal neurons has proven useful in the examination of transferrin's effects on the developing CNS. These neu- rons can be maintained in vitro without transferrin and yet are capable of binding and internalizing transferrin in a manner consistent with their developmental stage 2°' 35. In this study, retinal cultures were used to examine the effects of transferrin on neuronal physiology. MATERIALS AND METHODS Purified neuronal cultures The preparation and characterization of purified retinal neurons from chick embryo has previously been described 1"22'33. Briefly, neural retinas from 8-day chick embryos were dissected free from other ocular tissue, incubated for 10 min in Ca2+-Mg2+-free Hank's balanced salt solution (CMF) and further incubated for 20 min in 0.25% trypsin (1:250 Nutritional Biochemical, Cleveland, OH) in CMF. Cells were mechanically dissociated and suspended in 2 ml of modified Eagle's basal medium (Gibco, Grand Island, NY) sup- plemented with eatalase (5.6 U/ml) and insulin (8.3 x 10-TM). All media supplements were purchased from Sigma Chemical Corp. Cells were seeded (982 cell/mm 2) on 15-mm coverslips coated with 0.1% polyornithine in 60-mm tissue culture dishes. All cultures were incubated at 37 °C in a humidified atmosphere of 5% CO2 in air. * Current address: Department of Physiology, Northwestern University Medical School, 303 E. Chicago Ave., Chicago, IL 60611, U.S.A. ** Current address: UMDNJ-Robert Wood Johnson Medical School, Department of Neurology, Piscataway, NJ 08855, U.S.A. Correspondence: A.G. Hyndman, Department of Biological Sciences, Rutgers University, Piscataway, NJ 08855, U.S,A.