Neuroscienee Let ters, 115 (1990) 219-225 Elsevier Scientific Publishers Ireland Ltd.

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NSL 7014

Extracellular tumor necrosis factor induces a decreased K + conductance in an identified neuron o f Aplysia kurodai M a s a s h i S a w a d a I, N o b u m a s a

Hara 2 and Takashi Maeno 1

1Department of Physiology and 2Central Research Laboratories, Shimane Medical University, Izumo (Japan)

(Received 23 February 1990; Revised version received 30 March 1990; Accepted 31 March 1990) Key words." Tumor necrosis factor; Extracellular ejection; Ionic mechanism; Inward membrane current; Aplysia

Recombinant human tumor necrosis factor (rhTNF) was pressure-applied onto the soma of identified neuron R12 in the Aplysia abdominal ganglion, rhTNF induced a slow inward current (I7NF, 80-100 s in duration, 5-10 nA in amplitude) associated with a conductance decrease. 1TN~begins 1 2 s after applying rhTNF and peaks in 5 ~ s. ITNFwas decreased by hyperpolarization and had a reversal potential of approximately - 8 7 mV (close to the K ÷ equilibrium potential). Ion substitution and pharmacological experiments suggest that IxN~ is due to a decreased K + conductance and that TNF, a product of macrophages, may form an important link in communications between nervous and immune systems.

T u m o r necrosis f a c t o r ( T N F ) , a c y t o k i n e released by m a c r o p h a g e s a n d o t h e r related cells in response to infection a n d cancer, is a m a j o r i m m u n o r e g u l a t o r y p r o t e i n [2, 4]. T h e i m m u n e a n d n e r v o u s systems are closely linked a n d m a y e m p l o y the same biologically active molecules [18]. It has been d e m o n s t r a t e d t h a t i n t r a c e r e b r o v e n t r i c u l a r m i c r o i n f u s i o n o f r e c o m b i n a n t h u m a n t u m o r necrosis factor ( r h T N F ) a n d r e c o m b i n a n t h u m a n i n t e r l e u k i n - l f l ( r h l L - l f l ) s u p p r e s s e d f o o d i n t a k e in rats, a n d e l e c t r o p h o r e t i c a l l y a p p l i e d r h T N F a n d r h l L - l f l specifically s u p p r e s s e d the activity o f glucose-sensitive n e u r o n s in the lateral h y p o t h a l a m i c a r e a [15]. Pioneering w o r k by M e t c h n i k o f f f d e m o n s t r a t e d t h a t e c h i n o d e r m s possess large m o n o n u c l e a r p h a g o c y t i c cells t h a t p a r t i c i p a t e in host defense a n d these cells have been likened to m a m m a l i a n m a c r o p h a g e s [11]. F u r t h e r m o r e , a primitive I L - l - l i k e p r o t e i n has been isolated from an i n v e r t e b r a t e , the starfish [3]. In the p r e s e n t p a p e r we have investigated the effects o f m i c r o p r e s s u r e - e j e c t e d r h T N F on the m e m b r a n e o f an identified n e u r o n R 12 in the a b d o m i n a l g a n g l i o n o f Aplysia using a v o l t a g e - c l a m p , m i c r o p r e s s u r e - e j e c t i o n a n d ion s u b s t i t u t i o n techniques. T h e results p r e s e n t e d here clearly d e m o n s t r a t e that extracelCorrespondence: M. Sawada, Department of Physiology, Shimane Medical University, Izumo 693, Japan.

0304-3940/90/$ 03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd.

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lularly ejected rhTNF can induce a slow inward current associated with a decrease in K + conductance causing membrane depolarization. The methods used in this study are similar to those described previously [16]. Briefly, isolated desheathed abdominal ganglia of Aplysia kurodai were pinned to a plexiglass chamber. The ganglia were superfused with buffered saline (normal seawater (NSW): 587 mM Na +, 12 mM K +, 671 mM CI-, 14 mM Ca 2+, and 52 mM Mg 2+) at room temperature (19-20 ~'C). The pH was adjusted to 7.6 with Tris and HC1. An identified neuron, R12 (regularly firing cell, nomenclature of ref. 7) was implied with two microelectrodes, filled with 4 M potassium acetate for conventional two-electrode voltage-clamp. A third double-barreled microelectrode, one barrel filled with rhTNF (1050 nM in NSW), the other with heat-inactivated (90°C for 20 rain) rhTNF, was positioned near the soma of the neuron, rhTNF was applied by micropressure ejection (using a PPM-2 pneumatic pump, Medical System Corp.). Recombinant human TNF-~ (rhTNF) was purchased from Genzyme Corp. Drugs applied extracellularly were added to NSW. The following drugs were used: tetraethylammonium (TEA, Sigma): 4-aminopyridine (4-AP, Nakarai); and tetrodotoxin (TTX, Sankyo Co.). Micropressure ejection of rhTNF onto the soma of identified neuron v,12 voltageclamped at - 6 0 mV (its resting potential) induced a slow inward current (ITNF) associated with a conductance decrease. At the peak of ITN~ there was a 44% decrease in input-membrane conductance (Fig. I A). ITNV reached a maximum amplitude (5 10 nA) within 5-6 s and lasted at least 80-100 s (Fig. IB). The slow inward current

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Fig. 1. Micropressure ejection of rhTNF onto the soma of identified neuron RI2 voltage-clamped al a holding potential of 60 mV (its resting potential), r h T N F was ejected by a constant pressure pulse (100 ms duration, 2 kg/cm 2 intensity in A C). Hyperpolarizing command pulses (2 mV) with a duration of 500 ms were applied every 5 s to measure the membrane conductance (A and subsequent figures). Numbers on the lowest trace of A C represent the duration of the pressure pulse (ms), A: rhTNF-induced slow inward current (,/I'Nb) associated with a decrease in the membrane conductance. B: the same IIN~ as in A illustrated on a faster time base. C: IrNv at different holding potentials. Note that current direction reverses between - 8 0 and - 9 0 inV. D: voltage sensitivity of [rNv plotted with current on ordinate (negative values indicate inward current) and clamped membrane potential on abscissa, The reversal potential is intercalated as - 87,5 mV.

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response to ejected r h T N F was observed in 48 of 49 identified neurons R12. Control ejections of heat-inactivated r h T N F were without effects (n = 3). The voltage dependence of ITNF was studied by holding the neuron at a series of different voltages from - 1 0 0 to - 6 0 mV and ejecting r h T N F with a constant pressure pulse (Fig. 1C,D). ITNF decreased with hyperpolarizing holding potentials and reversed at hyperpolarized holding potentials more negative than - 8 7 mV (close to the K + equilibrium potential). The mean reversal potential for IVNVwas -- 79.3 _+ 6.3 mV (mean -I- S.D., n = 6). T h e f a c t t h a t ITNF r e v e r s e d a t ca. - - 8 0 m V a n d w a s a s s o c i a t e d w i t h a d e c r e a s e i n conductance

s u g g e s t e d t h a t ITNF is d u e t o a d e c r e a s e in a r e s t i n g K + c o n d u c t a n c e .

To test this hypothesis, the external ionic compositions

were altered. Increasing

e x t r a c e l l u l a r K + f r o m t h e c o n t r o l level o f 12 t o 24 m M c a u s e d a m a r k e d r e d u c t i o n i n ITNF (23 _+ 3.4% o f t h e c o n t r o l , m e a n _+ S . D . , n = 3, Fig. 2 A ) . F u r t h e r m o r e , recorded at a holding potential of -60

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Fig. 2. Effects of high K ÷ seawater (A), low K + seawater (B), and Na+-deficient seawater (C) on ITNF recorded from neuron R 12 voltage-clamped at - 6 0 mV. rhTNF was ejected by a constant pressure pulse (100 ms, 2 kg/cm2). A: left, control; center, 6 min after exposure to high K + (2 x normal K ÷) seawater; right, 7 min after washout. There was an inward shift (9.1 nA) of the base-line holding current in high K ÷ seawater. B: left, control; center, 6 min after exposure to low K + (0.1 x normal K +) seawater; right, 8 min after washout. There was an outward shift (7.6 nA) of the base-line holding current in low K + seawater. C: left control; center, 6 min after exposure to Na+-deficient (0.2 x normal Na ÷) seawater; right, 8 min after washout. There was a slight outward shift (1.2 nA) of the base-line holding current in N a +deficient seawater. Numberson the lowest traces represent the duration of the pressure pulse (ms). Middle traces show constant voltage pulses to test membrane input conductance.

222 NSW

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Fig. 3. Effects of high K ~ seawater (A) and 10 mM Co2+ seawater (B) o n /INI~ recorded from neuron RI2. The neuron R12 in B was obtained from another preparation, rhTNF was ejected by a constant pressure pulse (1 s, 2 kg/cm2 in A, 2 s, 2 kg/cm2 in B). Holding potentials were -60 mV in A, -52 mV in B. A: left, control; center, 6 min after exposure to high K ~ (5 x normal K +) seawater; right, 8 rain alter washout. There was an inward shift (14 nA) of the base-line holding current in high K + seawater. Note that tjN~ recorded at -60 mV in high K + seawater was reversed in polarity to outward. B: left, control; right, 9 min after exposure to [0 mM Co2+ seawater.

in high K + external solution (5 x n o r m a l K + , n = 2, Fig. 3A). O n the other h a n d , decreasing extracellular K ~ from 12 to 1.2 m M caused a n increase in ITNF (160 _ 7.5% of the control, m e a n _+ S.D., n = 3, Fig. 2B). Neither perfusion for 10 m i n with 50/~M T T X n o r perfusion with Na+-deficient (0.2 x n o r m a l N a +, replaced with N - m e t h y l - D - g l u c a m i n e ) solution c h a n g e d the a m p l i t u d e of ITNF recorded at a h o l d i n g potential of - 6 0 m V (Figs. 2C a n d 4C, n = 3). Decreasing extracellular CI from the control level o f 671 to 67.1 m M (replaced with gluconate) did n o t induce a n y changes in ITNF (n = 2, data n o t shown). Perfusion for 9 m i n with 10 m M Co 2+ seawater (calcium c h a n n e l blocking cation) also did n o t cause any changes in ITNF (Fig. 3B, n = 3). These results taken together with the o b s e r v a t i o n that the reversal potential of ITNF was close to -- 80 m V suggest that ITNF is due to decreased K + c o n d u c t a n c e b u t n o t due to a n increased N a + cond u c t a n c e or a n increased Ca 2+ conductance. We next e x a m i n e d the effects o f the K ÷ c h a n n e l blockers o n ITNF. Neither perfusion with 5 m M T E A n o r b a t h - a p p l i e d 5 m M 4-AP affected o n ITNF recorded at a holding potential of - 6 0 mV (Fig. 4A, B, n = 3). These results indicate that ITNF is separated from the Ca2+-activated, fast, a n d delayed K + currents. The present results clearly indicate that extracellularly applied r h T N F induces a

223

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20 sec Fig. 4. Effects of 5 m M TEA (A), 5 m M 4-AP (B), and 50/~M T T X (C) on ITNF recorded from neuron R12. The neuron R12 in C was obtained from another preparation, rhTNF was ejected by a constant pressure pulse (1 s, 2 kg/cm 2 in A and B; 100 ms, 2 kg/cm 2 in C). Holding potentials were - 6 0 mV in A and B, - 5 5 mV in C. Aa, control; Ab, 9 min after exposure to 5 mM TEA. Ba, control; Bb, 9 min after exposure to 5 mM 4-AP seawater. Ca, control; Cb, 10 min after exposure to 50 ltM TTX seawater.

slow inward current (ITNF) and this current is due to a decrease in K + conductance. Our conclusion that extracellular r h T N F can produce a decreased K + conductance in an identified neuron (R12) of Aplysia kurodai is based on the following observations: (1) ITNF is sensitive to changes in the extracellular K + concentration but not to the extracellular Na +, Ca 2+ and C1- concentrations. (2) The reversal potential of IXNF is approximately --80 mV (close to the K ÷ equilibrium potential estimated for the Aplysia neurons with K+-sensitive electrodes [10]). (3) At the peak of ITNF there is a 44% decrease in input membrane conductance. In previously studied Aplysia neurons, serotonin has been reported to induce a decreased K + conductance (S-K current [17] in sensory neurons. In addition, serotonin also has been reported to produce a decreased K ÷ conductance in ink m o t o r neuron L14 distinct from S-K currents [19]. Furthermore, histamine has been shown to induce a decreased K + conductance in the metacerebral cell of Aplysia [20]. In neurons of the vertebrate myenteric plexus, it has been demonstrated that histamine [8] and serotonin [12] also decrease K + conductance. This decreased conductance is the Ca2+-dependent K + conductance. In addition, the epinephrine-induced depolarization in neurons of sympathetic ganglia has been shown to be due to a decreased K + conductance [1], probably the M-channel [5]. The TNF-induced inward current in neuron R I 2 appears to be distinct from S-K

224 current, M - c u r r e n t , Ca 2+ current, a n d C a 2 + - a c t i v a t e d K + c u r r e n t [5, 14, 17]. Ia-NF in n e u r o n R 12 m o r e closely resembles the s e r o t o n i n - i n d u c e d response in L I 4 n e u r o n s [19] on the basis o f v o l t a g e - d e p e n d e n c e , ionic selectivity a n d p h a r m a c o l o g y . In recent years, a t t e n t i o n h a d focused on the possible role o f i m m u n e m e d i a t o r s (including T N F , I L - l a n d interferon) in influencing the firing rate o f the n e u r o n s in the central n e r v o u s system o f v e r t e b r a t e [6, 18]. L o c a l a p p l i c a t i o n o f these c y t o k i n e s has been r e p o r t e d to decrease o r increase the firing rate o f the n e u r o n s in v e r t e b r a t e b r a i n [6, 9, 13, 15). H o w e v e r , the ionic m e c h a n i s m o f the o b s e r v e d effects o f these cytokines on the n e u r o n a l activity in b r a i n u n d e r v o l t a g e - c l a m p c o n d i t i o n s has not been d e m o n s t r a t e d . A l t h o u g h m u c h a d d i t i o n a l evidence is needed to establish that a T N F - l i k e molecule is a n a t u r a l m e d i a t o r for m e m b r a n e d e p o l a r i z a t i o n o f the Aplysia n e u r o n s o r that the n e u r o n has surface receptors for T N F - l i k e proteins, o u r results p r o v i d e new a n d interesting insights into the u n d e r l y i n g m e c h a n i s m s for the g e n e r a t i o n o f a slow i n w a r d c u r r e n t by r h T N F in an identified n e u r o n R I 2 o f Aplysia. The results suggest that T N F , a p r o d u c t o f m a c r o p h a g e s , m a y form an i m p o r t a n t link in c o m m u n i c a t i o n s between n e r v o u s a n d i m m u n e systems. This w o r k was e n t r u s t e d to the S h i m a n e M e d i c a l U n i v e r s i t y by the Science a n d T e c h n o l o g y Agency, using the Special C o o r d i n a t i o n F u n d s for P r o m o t i n g Science a n d T e c h n o l o g y . W e t h a n k Dr. J.E. B l a n k e n s h i p for critical r e a d i n g o f the m a n u s cript. W e also t h a n k Mrs. Y. T a k e d a for typing the m a n u s c r i p t , a n d Mr. S. Saito for a c o n s t a n t supply o f Aplysia kurodai. 1 Akasu, T. and Koketsu, K., Evidence for epinephrine-induced depolarization in neurons of bullfrog sympathetic ganglia, Brain Res., 405 (1987) 375 379. 2 Balkwill, F., Burke, F., Talbot, D., Tavernier, J., Osbourne, R., Naylor, S., Durbin, H. and Fiers, N., Evidence for tumor necrosis factor/cachectin production in cancer, Lancet, II (1987) 1229-1232. 3 Beck, G. and Habicht, G.S., Isolation and characterization of a primitive interleukin-l-like protein from an invertebrate, Asteriasforbesi, Proc. Natl. Acad. Sci. U.S.A., 83 (1986) 7429-7433. 4 Beutler, B. and Cerami, A., Cachectin and tumor necrosis factor as two sides of the same biological coin, Nature, 320 (1986) 584-588. 5 Brown, D.A. and Adams, P.R., Muscarinic suppression of a novel voltage-sensitive K ~ current in a vertebrate neurone, Nature, 283 (1980) 673 676. 6 Dafny, N., Prieto-Gomez, B. and Reyes-Vazquez, C., Does the immune system communicate with the central nervous system?, J. Neuroimmunol., 9 (1985) 1 12. 7 Frazier, W.T., Kandel, E.R., Kupfermann, I., Waziri, R. and Coggeshall, R.E., Morphological and functional properties of identified neurons in the abdominal ganglion of Aplysia ealifornica, J. Neurophysiol., 30 (1967) 1288-1351. 8 Grafe, P., Mayer, C.J. and Wood, J.D., Synaptic modulation of calcium-dependent potassium conductance in myenteric neurones in the guinea-pig. J. Physiol., 305 (1980) 235-248. 9 Hori, T., Shibata, M., Nakashima, T., Yamasaki, M., Asami, A., Asami, T. and Koga, H., Effects of interleukin-1 and arachidonate on the preoptic and anterior hypothalamic neurons, Brain Res. Bull., 20 (1988) 75-82. 10 Kunze, D.L. and Brown, A.M., Internal potassium and chloride activities and the effects of acetylcholine on identified Aplysia neurones, Nature, 229 (1971) 229-231. I1 Metchnikoff, E., Lectures on the Comparative Pathology of Inflammation, English Translation, Dover, New York, 1893. 12 Nemeth, P.R., Ort, C.A. and Wood, J.D., Intracellular study of effects of histamine on electrical behav-

225 iour of myenteric neurones in guinea-pig small intestine, J. Physiol., 355 (1984) 411-425. 13 Oomura, Y., Chemical and neuronal control of feeding motivation, Physiol. Behav., 44 (1988) 555-560. 14 Pellmar, T.C. and Carpenter, D.O., Serotonin induces a voltage-sensitive calcium current in neurons of Aplysia californica, J. Neurophysiol., 44 (1980) 423-439. 15 Plata-Salaman, C.R., Oomura, Y. and Kai, Y., Tumor necrosis factor and interleukin-lfl: suppression of food intake by direct action in the central nervous system, Brain Res., 448 (1988) 106-114. 16 Sawada, M. and Maeno, T., Forskolin mimics the dopamine induced K + conductance increase in identified neurons of Aplysia kurodai, Jpn. J. Physiol., 37 (1987) 459-478. 17 Siegelbaum, S.A., Camardo, J.S. and Kandel, E.R., Serotonin and cyclic AMP close single K ÷ channels in Aplysia sensory neurones, Nature, 299 (1982) 413-417. 18 Solomon, G.F., Psychoneuroimmunology: Interactions between central nervous system and immune system, J. Neurosci. Res., 18 (1987) 1 9. 19 Walsh, J.P. and Byrne, J.H., Analysis of decreased conductance serotonergic response in Aplysia ink motor neurons, J. Neurophysiol., 53 (1985) 590-602. 20 Weiss, K.R., Shapiri, E. and Kupfermann, I., Modulatory synaptic actions of an identified histaminergic neuron on the serotonergic metacerebral cell of Aplysia, J. Neurosci., 6 (1986) 2393-2402.