Introduction persistent inward current grew, the presumably K -me-

Brain Research 885 2000 94–101 www.elsevier.com locate bres Research report Enhancement of persistent sodium current by internal fluorescence in isolated hippocampal neurons George G. Somjen Department of Cell Biology , Box 3709, Duke University Medical Center, Durham, NC 27710, USA Accepted 12 September 2000 Abstract Following up on an earlier chance observation, voltage-dependent whole-cell currents were recorded from isolated hippocampal neurons filled with the fluorescent dyes Fluo-3 and Fura-red, that were intermittently excited by 488 nm laser light. In the absence of any ion channel blocking drugs, in most cells depolarizing voltage steps initially evoked only the ‘Hodgkin–Huxley’ type early, fast inward surge followed by sustained outward current. Over 5–20 min of intermittent electrical stimulation and laser-excited fluorescence pulses, a 1 voltage-dependent, slowly inactivating inward current also appeared and grew, while sustained outward current diminished. When K currents were blocked, a small persistent inward current was usually detectable immediately, and then it increased in amplitude. This current was blocked by tetrodotoxin TTX and it had current–voltage I –V characteristics of a persistent sodium current, I . In cells Na,P not filled with dye but illuminated by laser, and in cells with dye but not illuminated, I remained small. There was a more than 12-fold Na,P difference in the maximal amplitude of I of fluorescent compared to non-fluorescent cells. Once induced, I decreased very slowly. Na,P Na,P 1 Fluorescence increased the duration but not the amplitude of the transient Na current, I . With membrane potential clamped to a Na,T constant voltage, the laser-induced fluorescence did not evoke a membrane current. It is not certain whether fluorescence-induced I Na,P potentiation is related to photodynamic action.  2000 Elsevier Science B.V. All rights reserved. Theme : Excitable membranes and synaptic transmission Topic : Sodium channels Keywords : Persistent sodium current; Sodium channel; Photodynamic action; Fluorescence; Dissociated neuron; Whole-cell current 1

1. Introduction persistent inward current grew, the presumably K -me-

diated outward currents became reduced in amplitude. It In a previous study [19] of the effects of low sodium seemed as though the slowly inactivating opposing cur- chloride concentration and low osmolarity, whole-cell rents interfered one with the other because, as the one sodium and potassium currents were recorded in patch- waned, the other became more prominent [19]. clamped hippocampal neurons filled with the calcium- The slow inward current appeared to be similar to the sensitive indicator dyes, Fluo-3 and Fura-red. The cells persistent voltage-dependent sodium current, I , re- Na,P were stimulated by series of depolarizing voltage pulses, corded in many central neurons [6,9,23], except for its and they were intermittently excited by laser light to record growth over time and its final, unusually large amplitude. confocal images and to measure calcium-dependent fluo- Because of the possible role of I in seizures, spreading Na,P rescence. In many trials in which no ion channel blocking depression and hypoxia [7,10,13] it seemed important to drugs were used, a slowly inactivating, inward current define the conditions under which this current can attain appeared. This current was not usually detectable at first, such unusual intensity. but it increased in amplitude over 5–20 min. As the In this article I report that internal fluorescence itself causes the growth of I which, once it is induced, Na,P reverses only very slowly. An abstract of some of the Tel.: 11-919-681-8404; fax: 11-919-684-5481. E-mail address : g.somjencellbio.duke.edu G.G. Somjen. findings has been published [20]. A companion paper [21] 0006-8993 00 – see front matter  2000 Elsevier Science B.V. All rights reserved. P I I : S 0 0 0 6 - 8 9 9 3 0 0 0 2 9 4 7 - 4 G .G. Somjen Brain Research 885 2000 94 –101 95 1 reports the effect of elevated external K concentration on The current records were read with Clampfit Axon I . Instruments software. After subtraction of linear leak and Na,P holding currents, the data were further processed with the Excel Microsoft program. Junction potentials were calcu- 2. Materials and methods lated with the JPCalc program [3].