Nicotinic and muscarinic acetylcholine responses in the embryo chick ciliary ganglion cells

BRAIN RESEARCH ELSEVIER

Brain Research 657 (1994) 185-190

Research report

Nicotinic and muscarinic acetylcholine responses in the embryo chick ciliary ganglion cells Katsutoshi Furukawa a,,, Yumiko Abe b, Masaru Sorimachi c, Norio Akaike d Department of Neurophysiology, Tohoku University School of Medicine, Sendai 980, Japan b Department of Neurology, Tohoku University School of Medicine, Sendai 980, Japan c Department of Physiology, Kagoshima University School of Medicine, Kagoshima 890, Japan d Department of Physiology, Kyushu University Faculty of Medicine, Fukuoka 812, Japan

Accepted 14 June 1994

Abstract Nicotinic and muscarinic acetyicholine (ACh) responses were investigated in acutely dissociated chick ciliary ganglion neurons using the nystatin perforated patch clamp technique. ACh-induced a rapid transient inward current in 100% of the neurons at a holding potential of - 6 0 mV. This rapid inward current was mimicked by nicotine but not by muscarine. The reversal potential of the rapid inward current was + 10.5 mV and the current was inhibited by d-tubocurarine and hexamethonium in a dose-dependent manner. In 57.6% of neurons, a slow inward current was also induced by ACh at a holding potential of - 2 0 mV. This slow inward current was mimicked by muscarine but not by nicotine. The slow inward current became smaller at a hyperpolarized potential but not reversed, being consistent with the fact that this current was elicited by the inhibition of M-current. p-Fluorohexa-hydrosiladifenidol (P-F-HHSiD) strongly inhibited the slow inward current, suggesting that the current was elicited by the activation of M 3 receptors. Keywords: Acetylcholine response; Muscarinic M 3 receptor; Ciliary ganglion; Chick embryo

1. Introduction P a r a s y m p a t h e t i c ciliary g a n g l i o n n e u r o n s o f chick w e r e r e p o r t e d to receive i n n e r v a t i o n f r o m t h e accessory o c u l o m o t o r n u c l e u s ( A O N ) a n d p r o j e c t to s t r i a t e d a n d s m o o t h m u s c l e s in t h e iris a n d ciliary b o d y [11,12]. C o n c e r n i n g t h e t r a n s m i s s i o n in t h e ciliary g a n g l i o n n e u r o n s , a c e t y l c h o l i n e ( A C h ) , s u b s t a n c e P, e n k e p h a l i n a n d 3,-aminobutyric acid ( G A B A ) have b e e n r e p o r t e d to e x e r t n e u r o t r a n s m i t t e r o r m o d u l a t o r functions [3,6,9,10,15]. In fact, nicotinic a n d m u s c a r i n i c A C h r e c e p t o r s a r e r e p o r t e d to b e p r e s e n t a n d p l a y an imp o r t a n t role in t h e ciliary g a n g l i o n n e u r o n s . A c a t i o n i c i n w a r d c u r r e n t i n d u c e d by t h e s t i m u l a t i o n o f nicotinic r e c e p t o r s [17] a n d an i n c r e a s e in cytosolic Ca 2+ by t h e s t i m u l a t i o n o f m u s c a r i n i c r e c e p t o r s [18] w e r e previously r e p o r t e d in t h e s e n e u r o n s . H o w e v e r , s y s t e m a t i c

* Corresponding author. Present address: 211 Sanders-Brown Research Center on Aging, 101 Sanders-Brown Building, University of Kentucky, Lexington, KY 40536-0230, USA. Fax: (1) (606) 323-2866. 0006-8993/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0006-8993(94)00746-Y

e l e c t r o p h y s i o l o g i c a l s t u d i e s on b o t h nicotinic a n d muscarinic A C h r e s p o n s e s in ciliary g a n g l i o n n e u r o n s have not yet b e e n p e r f o r m e d . In t h e p r e s e n t study, we s t u d i e d the A C h - i n d u c e d t r a n s i e n t a n d slow i n w a r d currents, which a r e c o n s i d e r e d to b e nicotinic a n d m u s c a r i n i c r e s p o n s e , respectively, using t h e nystatin p e r f o r a t e d p a t c h t e c h n i q u e [21]. T h e physiological a n d p h a r m a c o l o g i c a l p r o p e r t i e s of nicotinic a n d m u s c a r i n i c A C h r e c e p t o r s w e r e also investigated.

2. Materials and methods 2.1. Preparation

The ciliary ganglion neurons were isolated as described previously [18,19,22]. Briefly, White Leghorn embryonated chick eggs were maintained at 37°C in an incubator until use. Chick ciliary ganglia were obtained from embryos killed by decapitation on embryonic day 14. Ciliary ganglia were excised and placed in a Ca 2+ and Mg2+-free standard solution. After this preparation, the ganglia were incubated with thermolysin (1 mg/ml) for 10 min at 35°C and successively with collagenase (1 mg/ml) for another 10 min at 35°C.

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KI Furukawa et al. / Brain Research 657 (1994) 185 l O0

The ganglia were rinsed with a flesh Ca 2. and Mg2+-free standard solution and then mechanically dissociated by gently pipetting. The dissociated ganglion neurons were plated on the bottom of the petri-dish filled with the external standard solution containing Ca 2+ and Mg 2+

A ACh 10 "5 M

10 "4 M

10 "3 M

VH= -60 mV

! 100

2.2. Electrical measurement

Patch pipettes were made from glass capillary tubes using a vertical puller (Narishige, MF73). The currents were measured using a patch-clamp amplifier (List Medical, EPC-7) and recorded on a penrecorder (Sanei, Recti-Horiz-8K) and video cassette recorder (Toshiba, A-750HFD) after digitalizing the signals with a digital audio processor (Sony, PCM-501ES). The series resistance reached a steady level of 10-15Mg2 within 5-10 rain after making a seal. The series resistance was compensated more than 70%. All recordings were performed at room temperature (20-22°C).

5s

B 3

¢D ACh • nicotine

2.3. Solution

The Ca 2+ and Mg2+-free external solution contained (in mM): NaCI 150, KCI 5, HEPES 10, glucose 10, and the pH was adjusted to 7.4 with Tris-base, bubbled continuously by 100% 0 2. The standard external solution contained (in raM): NaCI 150, KC1 5, CaCI 2 2, MgCI 2 1, HEPES 10 and glucose 10, and the pH was adjusted to 7.4 with Tris-base. The patch-pipette (internal) solution contained (in mM): KCI 150 and HEPES 10, and the pH was adjusted to 7.2 with Tris-base. A nystatin stock solution consisting of 10 mg/ml nystatin in methanol was prepared and stored at - 2 0 °C. The nystatin solution was diluted to the pipette solution at a final concentration of 200/zg/ml just before filling the pipette. 2.4. Drugs

Drugs used in this experiment were acetylcholine chloride (ACh), muscarine chloride, pirenzepine, nystatin d-tubocurarine, hexamethonium and thermolysin [Sigma], atropine sulfate [Merck], carbamylcholine chloride (CCh) [Tokyo Kasei], oxotremorine, McN-A343 and p-fluorohexahydrosiladifenidol(P-F-HHSiD) [RBI], and collagenase [Yakult]. AF-DX-116 was a kind gift from Dr. T. Kamiya (Fukuoka University of Japan). All drugs were dissolved in the external solution just before use. The drugs were applied using a rapid application method termed the 'Y-tube' technique, which has been reported in detail elsewhere [14]. By this technique the external solution surrounding a neuron could be exchanged completely within 10-20 ms.

3. Results

3.1. Acetylcholine-induced current Two kinds of cells were identified on the basis of cell size. In the present electrophysiological study, we used only the larger cells ( > 20 mm) which were regarded as ciliary ganglion neurons [6,7]. At a holding potential ( V . ) of - 6 0 mV, the application of 10 -4 M acetylcholine (ACh) induced a rapid transient inward current in all examined neurons (Fig. 1A). This transient inward current was mimicked by the application of 1 0 - 4 M nicotine but not by 1 0 - 4 M muscarine. At a V H of - 2 0 mV, 10 -4 M A C h also elicited a slow

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Fig. 1. Acetylcholine (ACh) induced transient rapid inward current. A: the transient inward current induced by three different of concentrations of ACh at a holding potential (Vn) of - 60 mV. B: concentration-response relationships for ACh- and nicotine-induced responses. All responses were normalized to the peak response induced by 10 4 M ACh. Each point is the average from 5 cells. Vertical bars indicate mean _+S.E.M.

activated and successively inactivated inward current in 34 of the 59 neurons examined (Fig. 4A). This slow inward current was mimicked by 10 -4 M muscarine but not by 10 - 4 M nicotine. The transient inward current was accompanied by an increase in m e m b r a n e conductance, but the slow current was accompanied by a decrease in m e m b r a n e conductance. A 7-rain interval between applications of ACh did not affect the amplitude of the transient inward current, and a 5-min interval did not affect that of the slow inward current. Intervals shorter than these periods caused the desensitization of these two types of currents. I n currentclamp conditions, 10 -4 M nicotine and 10 - 4 M muscarine induced rapid and slow m e m b r a n e depolarization, respectively. The firing rate of action potentials was also increased by nicotine and muscarine. Based on the results, it was conjectured that the transient inward current was induced by the activation of nico-

K Furukawa et al./Brain Research 657 (1994) 185-190

tinic ACh receptors, whereas the slow current was evoked by the activation of muscarinic A C h receptors.

A

187

d-tubocurarine

ACh 3x10 "5 M

10 .6 M - - ]

10 -5 M --J

3.2. Nicotinic acetylcholine response Both ACh and nicotine produced a rapid transient inward current at a VH of - 6 0 mV. We used the ciliary ganglia which exhibited no response to muscarine in the experiments concerned with the nicotinic response. The inward currents produced by these cholinergic compounds increased in a sigmoidal fashion with an increase in the concentration. The half maximum ( K o) concentrations of ACh and nicotine were 1.7 x 10 - 4 M and 1.0 × 10 - 4 M , respectively. The Hill coefficients of ACh and nicotine were 1.92 and 1.77, respectively. The current-voltage ( l - V ) relationship of the nicotine-induced response was investigated with K÷-free external and internal solutions containing Cs ÷ instead of K ÷. Fig. 2A and B show 10 -4 M nicotine-activated c u r r e n t s (INic s) at various Vrts and the I - V relationship, respectively. The reversal potential of the I Nic

-60 mV

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0.8 0.6 0.4

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nicotine 10"4 M

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Concentration (M)

Fig. 3. Effects of nicotinic antagonists on the nicotinic ACh response. A: inhibitory effect of d-tubocurarine at various concentrations The pretreatment time with d-tubocurarine was 1 rain. B: concentrationinhibition curves of nicotinic antagonists on the responses induced by 3 × 10-5 M ACh. Each point is the average from five cells.

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-400 Fig. 2. Current-voltage ( I - V ) relationship of nicotinic ACh response. A: the currents induced by the application of 10 -4 M nicotine at various VHS. B: the amplitude of the nicotinic ACh response plotted as a function of VH.

(E~i ¢) estimated from the I - V relationship was + 10.5 mV. A significant inward rectification was observed at m e m b r a n e potentials more positive than - 2 0 mV. The effects of nicotinic antagonists (d-tubocurarine and hexamethonium) on the nicotinic ACh response were examined. In this experiment, the preparations were pretreated for 1 min with each antagonist. Then, one of the antagonists and 3 × 10 -5 M ACh were applied simultaneously to the preparation, d-Tubocurarine and hexamethonium reversibly inhibited the nicotinic response in a concentration-dependent manner. The half-maximum inhibitory concentrations (ICs0) and the Hill coefficients were 1.2 × 10 -5 M and 1.13 for d-tubocurarine and 2.6 × 10 -5 M and 1.16 for hexamethonium, respectively. Atropine also inhibited the transient rapid inward current completely at the concentration of 10 -5 M (data not shown).

3.3. Muscarinic acetylcholine response At a VH of - 2 0 mV, ACh, muscarine and carbamylcholine (CCh) induced a slow inward current. This slow

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K. Furukawa et al. / Brain Research 657 (1994) 185-190

inward current was associated with a decrease in membrane conductance. However, neither McN-A-343 (a selective MI agonist) nor oxotremorine (a cardiac M e agonist) produced any currents in the concentrations up to 10 - 4 M. The slow inward current produced by ACh, muscarine and CCh increased in a sigmoidal fashion with an increase in the concentration. The half maximal concentration ( K o) of ACh, muscarine and CCh were 1.8 × 10 -5, 2.5 × 10 - 4 and 4.5 × 10 - 4 M, respectively. To clarify the ionic mechanism of the muscarinic ACh response in the ciliary ganglion neurons, the I - V relationship was examined by the ramp voltage technique. The concentrations of K + in the external and internal (pipette) solution were 5 and 150 mM, respectively. The voltage-dependent Ca 2+ and Na + channels were suppressed by adding 10 -5 M La 3+ and 10 - 7 M tetrodotoxin, respectively. Fig. 5 shows the I - V relationship to asymmetrical hyperpolarizing ramp voltage

A ACh 10-5 M

3x10 "5 M

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VH= -20mV

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Fig. 5. Current-voltage ( I - V ) relationship of muscarlnic ACh response. The inset shows recordings of voltages and currents by the asymmetrical hyperpolarizing ramp. I - V curves were obtained by ramp-commands between - 2 0 mV and - 9 6 mV in the presence and absence of 10 -5 M muscarine.

10 -4 M

l

21

muscarine 10-5M

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between - 2 0 mV and - 9 6 mV, before and during the application of muscarine. The two lines became close to each other at more hyperpolarized potentials a n d fit at around - 9 2 mV, indicating that this current does not have any reversal in the range of the voltage used. The effects of muscarinic antagonists (atropine, pirenzepine, AF-DX-116 and p-fluorohexahydrosiladifenidol (P-F-HHSiD)) on the muscarinic ACh response were investigated. In this experiment neurons were pretreated for 1 min with each antagonist. Then, one of the antagonists and 10 -5 M ACh were applied simultaneously to the preparation. Atropine (nonspecific) and P-F-HHSiD (M3-selective) inhibited the muscarinic ACh response in a concentration-dependent manner. The IC50 values of atropine and P-FH H S i D were 6.0 × 10 -9 M and 1.1 × 10 -7 M, respectively. The M~-selective antagonist, pirenzepine (PZP) and M2-selective antagonist, AF-DX-116 of 10 -6 M showed no effect on the muscarinic A C h response though P Z P inhibited the muscarinic ACh response approximately 50% at the concentration of 10 -5 MI

o[a 10.7

10.6

10.5

10.4

10.3

4. Discussion

Concentration (M) Fig. 4. Acetylcholine (ACh) induced sustained slow inward current. Brief hyperpolarizing pulses from -20 to -30 mV and 100 ms in duration were applied every 3 s. A: the slow inward currents induced by ACh of different concentrations. B: concentration-response relationships for ACh, muscarine (Mus), carbamylcholine (CCh), oxotremorine, and McN-A-343 responses. All responses were normalized to the peak response induced by 3 × 10-5 M ACh. Each point is the average from 5 cells.

4.1. Characteristics of nicotinic ACh response The ACh-induced transient inward current seems to be mediated by the activation of nicotinic receptors because the transient inward current was mimicked by nicotine but not by muscarine. In the concentration-response relationships, the potency was in the order of

K. Furukawa et al./ Brain Research 657 (1994) 185-190

A

following two reasons. One possibility is that the transient inward current includes a muscarinic component. The other is that atropine has a non-selective blocking action on ion channels. However, a more detailed investigation is necessary to clarify this inhibitory action of the muscarinic antagonist. In the I - V relationship of the nicotinic ACh response, the inward rectification was observed at potentials more positive than - 2 0 inV. Similar characteristics including inward rectification were also observed in rat sympathetic ganglion neurons [13], paratracheal ganglion neurons [1], and PC12 ceils [8]. In the rat sympathetic ganglion neurons, it appeared that the inward rectification was not due to a potential sensitive change in the ACh receptor but to the number of channels that became inactive at positive potentials [13]. The non-linear I - V relationship in the present preparation also might be attributed to the same mechanism.

atropine 10-9M

ACh 10"5 M

10-8M

,

m

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--] m

VH=-20 mV

m

100p~ 1 min

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189

4.2. Characteristics of muscarinic ACh response

-9

10

-8

10

-7

10

-6

10

10.5

Concentration (M) Fig. 6. Effects of muscarinic antagonists on the nicotinic ACh response. A: inhibitory effect of atropine at various concentrations The pretreatment time with atropine was 1 rain. B: concentration-inhibition curves of muscarinic antagonists on the 10 -5 M ACh response. Each point is the average from five cells.

nicotine > ACh. The results are in agreement with those concerning the mammalian CNS neuron [20] (nucleus tractus soritarii neurons) and paratracheal ganglion cells [1]. In the present study, the Hill coefficient of ACh in the concentration-response relationship for the nicotinic response was 1.92. This result suggests that the nicotinic ACh receptor of ciliary ganglion neurons may have two binding sites. Nicotinic antagonists (d-tubocurarine and hexamethonium) inhibited the nicotinic ACh response in the ciliary ganglion neurons. In the present study, the Kt) values were 1 . 2 × 1 0 -5 M and 2 . 6 x 1 0 -5 M for dtubocurarine and hexamethonium, respectively. These values are not much different from those of rat CNS [20] and paratracheal ganglion neurons [1]. Besides the nicotinic antagonists, atropine, a nonspecific muscarinic antagonist, also inhibited the nicotinic response. This inhibitory effect of atropine on the nicotinic ACh response has been previously reported in chick ciliary ganglion neurons [17], frog isolated sympathetic neurons [16], and PC12 ceils [8]. This result might be considered to be caused by the one of the

ACh, muscarine, and CCh induced a long-lasting slow inward current in chick ciliary ganglion neurons, resulting in a slow membrane depolarization. Similar excitatory actions of ACh were reported in the rat hippocampus [2], the olfactory cortex [5] and the sympathetic ganglion [4]. In these neurons, ACh diminished a voltage- and time-dependent K + current known as the M-current. In the present study, the ACh-induced slow inward current was accompanied by a decrease in membrane conductance. Furthermore, the I - V curves in the absence and presence of muscarine became close to each other at hyperpolarized potentials and did not show any reversal. These results do not conflict with the speculation that this slow inward current was elicited by the inhibition of M-current. On the basis of the selectivity of both the muscarinic agonists and antagonists, the pharmacological heterogeneity of four (M1, M 2, M 3 and M 4) o u t of five genetically-identified muscarinic ACh receptor subtypes ( m l - m 5 ) was proposed. From the different sensitivity of ACh receptors to antagonists such as pirenzepine, AF-DX-116 and P-F-HHSiD, the M~, M 2 and M 3 muscarinic receptor subtypes were proposed. The present study indicates that McN-A-343, a selective M 1 agonist, and oxotremorine, a selective M z agonist, showed low efficacy and that, except for atropine, P-F-HHSiD was the most potent antagonist. Therefore, the subtype of muscarinic ACh receptor that elicits a slow inward current in chick ciliary ganglion neurons is considered to be M 3. By the measurement of the cytosolic Ca z+ concentration by fura-2, the M 3 muscarinic receptor subtype was proposed to mobilize intra-cellular Ca z+ in chick ciliary ganglion neurons [181.

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K. Furukawa el al. / Brain Research 657 (1994) 185-190

I n c o n c l u s i o n , c h i c k ciliary g a n g l i o n n e u r o n s h a v e n i c o t i n i c a n d m u s c a r i n i c ( M 3) A C h r e c e p t o r s . T h e n i c o t i n i c r e c e p t o r s h a v e a r o l e in t h e r a p i d e x c i t a t i o n a n d t h e m u s c a r i n i c r e c e p t o r s h a v e a r o l e in t h e slow sustained excitation.

Acknowledgements T h e a u t h o r s t h a n k Mr. B. Bell a n d M r . J a m e s G. Begley for helpful comments.

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[9] McEachern, A.E,, Margiona, J.F. and Berg, I).K, GABA receptors on chick ciliary ganglion neurons in vivo and in cell culture, ,L Neurosci., 5 (1985) 2690-2695. [10] Margiotta, J.F. and Berg, D.K., Enkephalin and substance P modulate synaptic properties of chick ciliary ganglion neurons in cell culture, Neuroscience, 18 (1989) 175-182. [11] Martin, A.R. and Pilar, G., Dual mode of synaptic transmission in the avian ciliary ganglion, J. PhysioL, 168 (1963) 443-463. [12] Martin, A.R. and Pilar, G., Transmission through the ciliary ganglion of the chick, J. PhysioL, 168 (1963) 464-475. [13] Mathie, A., Colquhoun, D. and Cull-Candy, S.G.. Rectification currents activated by nicotinic acetylcholine receptors in rat sympathetic ganglion neurons, J. Physiol., 427 (1990) 625-655, [14] Murase, K., Randic, M,, Shirasaki, T., Nakagawa, T. and Akaike, N., Serotonin suppresses N-methyI-D-aspartate response in acutely isolated spinal dorsal horn neurones of the rat, Brain Res., 525 (1990) 84-91. [15] Role, L.W., Substance P modulation of acetylcholine-induced currents in embryonic chicken sympathetic and ciliary ganglion, Proc. NatL Acad. Sci. USA, 81 (1988) 2924-2928. [16] Sadoshima, J., Oyama, Y. and Akaike, N., Inhibition of nicotinic acetylcholine response by atropine in frog isolated sympathetic neurons, Brain Res., 508 (1990) 147-151. [17] Schmid, H.A. and Vijayaraghavan, S., Inhibition of the nicotinic acetylcholine response by serotonergic and muscarinic agents in chick ciliary ganglion neurones, Neuropharmacology, 31 (1992) 1001-1008. [18] Sorimachi, M., Caffeine- and muscarinic receptor agonist-sensirive Ca 2+ stores in ciliary ganglion cells, Brain Res., 627 (1993) 34-40. [19] Stanley E.F., Calcium currents in a vertebrate presynaptic nerve terminal: the chick ciliary ganglion calyx, Brain Res., 505 (1989) 341-345. [20] Ueno, S., Kakehata, S. and Akaike, N., Nicotinic acetylchotine receptor in dissociated rat nucleus solitarii neurons, Neurosci. lett., 149 (1993) 15-18, [21] Wakamori M., Hidaka H. and Akaike N., Hyperpolarizing muscarinic responses of freshly dissociated rat hippocampal CAI neurons, J. Physiol. 463 (1993) 585-604. [22] Wisgirda M.E. and Dryer S.E., Characteristics of multiple voltage-activated K + currents in acutely dissociated chick ciliary ganglion neurons, J. Physiol. 470 (1993) 171-189.