Impaired neuronal function induced by the immune mediator leukotriene B4

Brain Research, 628 (1993) 313-316

313

© 1993 Elsevier Science Publishers B.V. All rights reserved 0006-8993/93/$06.00 BRES 25887

Impaired neuronal function induced by the immune mediator leukotriene B4 Hubertus

K611er, M a r i o S i e b l e r

*

Deparment of Neurology, Heinrich-Heine University, PO Box 10 10 07, D-40001 Diisseldorf, Germany

(Accepted 2 August 1993)

Key words: Immune mediator; Cultured cortical neuron; Late potassium outward current; IK; Membrane potential; Astrocyte

Inflammatory cerebral processes, mediated by immunologically active substances or invading of macrophages are frequently associated with neuronal dysfunction. This study describes the effects of leukotriene B 4 o n membrane potential, membrane resistance and potassium currents of cultured cortical neurons from the embryonic rat. Leukotriene B 4 (1 /.~M) did not depolarize cortical neurons but induced a reversible reduction of voltage-dependent potassium outward currents (I K) in a subpopulation of these cells (35%). The results suggest that, in comparison to astrocytes, cortical neurons lack receptors for LTB 4 or its intracellular activation pathway. Immune mediators, such as leukotrienes, may contribute to neuronal dysfunction during inflammatory diseases by affecting neuronal membrane currents.

Immunological mechanisms within the CNS are involved in the pathogenesis of many neurological diseases 16. I m m u n e processes are mediated by a large n u m b e r of cytokines and arachnidonic acid derivates, such as leukotrienes 18. Increased levels of leukotrienes have been found in cerebral ischemia 5'24 and in e d e m a surrounding brain tumors 4. The effects of these leukotrienes are mediated by specific receptors being present in many t i s s u e s 22'27'29'32. Astrocytes and neurons share many electrophysiological properties. An increasing number of voltage-dependent and ligand-operated ion channels have been described in glial cells 2'3'25"3°'33'36, closely resembling their neuronal counterparts. Microglia cells and astrocytes, in contrast to neurons, are active participants in immunological processes within the CNS 11. They are able to express M H C class II antigens 8'17'37, can be activated by lymphocytes ~° and produce immunologically active substances, such as the arachnidonic acid derivates prostaglandins 9 and leukotrienes 14'15. The effects of immunological mediators on neuronal function are mostly unknown. Palmer et al. e6 described a prolonged excitation of cerebellar Purkinje ceils in response to LTC1. Kimura et al. 19 observed an increase of synaptic activity following the application of

* Corresponding author. Fax: (49) (211) 311-8469.

prostaglandins. Recently, we reported on the depolarization of cultured astrocytes induced by leukotriene B] 1, which seems to be generated by a reduced potassium conductance. In the present study, we investigated the effects of LTB 4 on neurons in comparison to astrocytes. Part of this study has been published in abstract form 2°. Neurons were prepared from cortices of embryonic Wistar rats on embryonic day 15 (El5). Cells were plated on glass coverslips coated by poly-L-lysine (0.1 m g / m l PBS) and laminine ( 1 0 / z g / m l ) at densities of 8 - 1 0 )< 10 4 c e l l s / c m z and grown in chemically defined media, which were preconditioned for at least 3 days by spatially separated astrocytes as described previously 28,3t. Electrophysiological recordings were performed when the cells have grown for at least 5 days in vitro (DIV) by means of the patch clamp technique 13, using an EPC 7 amplifier (List, Germany). The bath solution contained (in mM): NaC1 150.0, KCl 4.0, CaC12 2.8, MgCI 2 1.0, H E P E S 10.0 and sucrose 30.0; p H adjusted to 7.4 by N a O H . Recording electrodes were used with pipette tip diameters of 2 - 5 /xm and a resistance of 5 - 6 MS2. The pipette solution contained (in mM): KCI 140.0, CaC12 1.0, MgCI 2 2.0, E G T A 11.0 and H E P E S

314 TABLE I

Neurons

Astrocytes

Effects o f L T B 4 and Ba 2 + on m e m b r a n e potential o f cultured astrocytes and neurons

0

-

20-

,-4

"'~

40 -

o

60-

-

J

80-

J

Control LTB4

-i00

Z

-

B a 2÷

Fig. 1. Comparison of effects of LTB 4 and BaCl 2 on membrane potential of cultured astrocytes and neurons Astrocytes are markedly depolarized by LTB 4 (1 p.M), applied to bath solution, whereas membrane potential of neurons remains unaffected. Ba 2+ (1 raM) induced a depolarization in neurons as well as in glial cells. Data of these experiments are given in Table I.

10.0; pH adjusted to 7.4 by KOH. All recordings were made at room temperature. Leukotriene B 4, dissolved in ethanol, BaC12, tetraethylammonium chloride (TEA) and CdCI 2 (all purchased from Sigma) were added to reach final concentrations in the bath solution as indicated. The bath solution of ethanol reached 1 vol. % after addition of leukotriene B 4. Control recordings with ethanol in this concentration did not show any effect on membrane potential ( - 4 3 . 3 -t- 11 mV, n = 14) or late outward currents. Membrane resistances were measured by hyperpolarizing current pulses from a holding potential of - 7 0 to - 1 0 0 , - 9 0 and - 8 0 mV. Voltage-dependent currents were elicited by depolarizing the neurons up to + 50 mV in pulses with a duration of 400 ms increased by steps of 10 mV in the voltage clamp mode in the whole-cell configuration. Currents were evaluated as mean current of 300-350 ms after the onset of the pulse. Membrane resistances were measured as the

Cortical neurons

Control

L TB 4 (1 I~M)

Membrane potential (mV) S.D. (mV) n

-46 6 58

-47 7 38

Astrocytes

Control

L TB 4 (1 IzM)

Membrane potential (mV) S.D. (mV) n

- 96 9 54

- 39 15 45

Ba 2 + (1 raM)

- 13 4 11 Ba 2 + (1 raM)

- 32 9 22

inverse of the slope conductance of - 100 to - 70 mV. Statistical data were presented as mean values + S.D. and statistically significant differences were calculated with the Student's t-test with P < 0.001. Cells were identified as neurons by their ability to generate action potentials after depolarizing current pulse application. A total number of 149 neurons were tested after 5-20 DIV. After establishment of the gigaseal whole-cell recording, the resting membrane potential (RMP) was measured as - 4 6 _+ 6 mV (n = 58) and the membrane resistance was 1.78 + 0.7 G,Q (n = 35). When cells were incubated in a bath solution containing LTB 4 (1 p,M) for 20-60-min resting membrane potentials ( - 4 7 + 7 mV, n = 38) as well as membrane resistances did not significantly change (1.6 -I- 0.6 GJ2, n = 38, P < 0.001; Fig. 1). When BaCi 2 (1 mM), a potent inhibitor of potassium conductances, was applied to the bath solution, neurons immediately depolarized to membrane potentials of 13 + 4 mV (n = 11). Fig. 1 compares the effects of LTB 4 and Ba 2+ on membrane potentials between neurons and astrocytes. The data of the astrocyte experiments are summarized in Table I (see also ref. 21). -

C

a

pre

0

SOpAldiv

o

i8o8°° o

?00pA b

LTB4

100 ms

•

8

o

wash LTB

ie °

oOO i

,

i

10

i

,

mVId iv

! Fig. 2. Effects of LTB 4 on late potassium outward currents in cultured neurons When cultured neurons are depolarized by intracellular current pulses, a late slowly inactivating outward current is elicited (a), which resembles characteristic features of voltage-dependent delayed rectifying potassium outward current (IK)- 10 rain after application of LTB 4 (1 p.M) I K is reversibly reduced (b). In c, I - V diagram of late currents before application of LTB 4 (pre), in presence of LTB 4 (LTB) and after wash (wash) is shown.

315 Voltage-dependent outward currents were elicited with a current amplitude between 300 and 1200 pA by depolarizing the neurons with an intracellular current application from a holding potential of - 7 0 m V to + 5 0 mV and measured as mean current of 300-350 ms after onset of depolarizing pulses. The different amplitudes of these currents in various cells is probably due to different cell size and time in culture 1. The late outward currents were activated after depolarizing the neurons from a holding potential of - 7 0 to - 4 0 mV and above. They could be reduced reversibly by application of tetraethylammonium chloride (TEA, 10 mM, n = 9) whereas CdC12 (1 mM, n = 7) had no effect on the sustained outward current (data not shown). During intracellular recordings for longer time periods up to 50 min, the amplitude of late outward currents decreased ("run down") with a time constant of 14 min and reached minimal values of 45% of the initial currents (n = 10; data not shown). After application of LTB 4 (1 /xM) to the bath solution the peak of this outward current was reduced to 25 + 12% in 19 of 54 neurons tested (35%). Reduction of the outward current appeared within a few minutes after application and was reversible after wash (n = 4; Fig. 2). No effect was observed, when a lower concentration of LTB 4 (0.5 /xM) was used (n = 12). T h e r e were no obvious differences in cell morphology or m e m b r a n e potential between those cells which responded to LTB 4 and nonresponders. As principal findings, we showed that LTB 4 reversibly reduced a voltage-dependent potassium current in a subpopulation of cultured cortical neurons. In contrast to astrocytes, however, resting m e m b r a n e potentials of neurons were not affected by LTB 4. The slowly inactivating outward currents as investigated here were activated, when neurons were depolarized from a holding potential of - 7 0 to - 4 0 m V and above. They were sensitive to T E A but insensitive to CdCI 2. These are characteristic features of the voltage-dependent delayed rectifying potassium outward current I K, as described for cultured cortical neurons 1. During recording time, a slow decrease in the amplitude of the late outward currents appeared, similar to that described in entorhinal cortex cells 7, probably due to the dialysis of the intracellular milieu by the pipette solution 23. The decrease of outward currents mediated by LTB 4, however, clearly exceeded this " r u n down". The reduction of the I K appeared within a short time after bath application of LTB 4 and was reversible after wash, indicating a direct interaction between LTB 4 and the potassium channels. Indirect effects mediated by second messengers or by newly synthesised proteins as

discussed for inducing the depolarization of astrocytes 21 are unlikely. In our cultures, the I K of only a subpopulation of neurons was reduced by LTB 4. All cells were harvested on E l 5 and should be in an early postmitotic stage at the time of preparation 6, resulting in only a small number of different neuronal cell types in culture. However, in these cultures, different subtypes of neurons could be classified by immunohistochemical staining of neurotransmitter contents 31. Sensitivity to the immune mediator LTB 4 seems to be a property of a particular neuronal subpopulation, which so far could not be further classified. It is conceivable that the portion of LTB4-responding neurons further increases with maturation. The m e m b r a n e potential of the neurons was not affected by LTB 4, which is in contrast to our findings in cultured glial cells 21. The LTB4-induced depolarization in astrocytes was probably generated by a general reduction of potassium conductances, as it could be mimicked by BaC1 e, a potent unselective potassium channel blocker 12. The m e m b r a n e potential of neurons 6 in culture is less negative than that of astrocytes 35'36. The m e m b r a n e potential of glial cells mainly depends on the potassium conductance 36 whereas Na ÷, Ca z+ and C1- conductances contribute to the neuronal m e m b r a n e potential and explain the great differences of both m e m b r a n e potentials. The depolarizing effect of BaC12, however, was very similar in neurons and in astrocytes, indicating a similar sensitivity of potassium conductances to channel blockade. Therefore, the lack of LTB4-induced depolarization in neurons may be due to a lack of LTB 4 receptors or a different response to receptor activation. Functionally, LTB 4 may increase excitability in neurons because of: (i) a slowing of repolarization after action potentials in a subpopulation of neurons due to reduced voltage-dependent potassium outward currents; and (ii) a reduced capacity of astrocytes to maintain ionic homeostasis 34 and, especially, to clear a high extracellular potassium. Further studies are needed to clarify these mechanisms. The results may help to understand clinical symptoms in the course of inflammatory or autoimmunological CNS diseases. We gratefully acknowledge H.-J. Freund, Di.isseldorL for continuous support, H.W. Miiller, Diisseldorf, for helpful discussions and G. Stoll, Diisseldorf, for critical reading of the manuscript. This study was supported by grants from the Deutsche Forschungsgemeinschaft (SFB 194/B7). 1 Ahmed, Z., Expression of membrane currents in rat neocortical neurons in serum-free culture. II Outward currents, Det', Brain Res., 40 (1988) 297-305. 2 Barres, B., New roles for gila, J. Neurosci., 11 (1991) 3685-3694.

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