The role of G protein in muscarinic depolarization near resting potential in cultured hippocampal neurons

200

Brain Reaear~'tt. ~l 2 (1993) 200- 2(!~ ~c, 1993 Elsevier Science Publishers B.V. All rights rescr,,'cd fl()06-89q3/93/$(16.(!~)

BRES 18843

The role of G protein in muscarinic depolarization near resting potential in cultured hippocampal neurons L e s l e e D. B r o w n *, K y e o n g - M a n K i m **, Y a s u k o N a k a j i m a a n d S h i g e h i r o N a k a j i m a Department of Biological Sciences, Purdue University, West Lafayette, IN 47907 (USA) and Departments of Pharmacology and of Anatomy and Cell Biology, Unit'ersity of Illinois College of Medicine at Chicago, Chicago, IL 60612 (USA) (Accepted 22 December 1992)

Key words: Acetylcholine; G protein; Muscarinic receptor; Pertussis toxin; Hippocampus; Cell culture

Cultured neurons from the CA1 and CA3 regions of the rat hippocampus were studied by using the whole-cell version of patch clamp. Application of acetylcholine (5-10 /xM) or muscarine (20 /zM) to a neuron with a holding potential of = - 7 0 mV produced a slow inward current. This inward current was inhibited by atropine (1-2 /zM). Loading the cell with GTPTS caused a change in the muscarinic response, in the control cells the muscarine-induced inward current recovered by 89%. On the other hand, in the GTPyS-loaded cells the inward current recovered by only 30%, indicating some irreversibility. Pertussis toxin treatment did not change the muscarine-induced slow inward current. Loading the cells with cyclic AMP (100/zM) plus IBMX (1 mM) (an inhibitor of phosphodiesterase) did not occlude the effect of muscarine. We conclude that the slow inward current is mediated through a pertussis toxin-insensitive G protein, and that cyclic AMP is not a part of the signal transduction cascade. The finding that the GTPyS-loaded cells did not show complete irreversibility was discussed in relation to the results of Benson et al. (J. Physiol., 404 (1988) 479-496), which showed that there are two ionic mechanisms responsible for the muscarine-induced depolarization. Occasionally cells were encountered, in which muscarine (or acetylcholine) evoked a large and rapid inward current, followed by the usual slow inward current. The time course of this rapid response was not affected by GTPTS.

(for peripheral nervous system see

INTRODUCTION

t o r s 1'2'7'9'24"26'31"38'41

Changes in neuronal excitability induced by acetylcholine (ACh) are important cellular events for brain function. The cholinergic influence on neocortical and hippocampal neurons seems to be crucial in cognition and memory and possibly in the etiology of Alzheimer's disease s. Several events are known to be responsible for the excitatory influence of ACh on brain neurons (reviewed by Nicoll et a1.36): (1) a rapid depolarization mediated through the nicotinic receptors 11A2'29, (2) muscarinic inhibition of the M-current 13'15 (for peripheral nervous system see ref. 3), (3) muscarinic inhibition of the afterhyperpolarization (AHP) 1'7 (for peripheral nervous system see ref. 37), (4) inhibition of the A-current 33 (for peripheral nervous system see ref. 5), and (5) a slow depolarization occurring near the resting potential mediated through the muscarinic recep-

ref. 30). The present paper deals with the mechanism of the muscarine-induced slow depolarization near the resting potential in cultured hippocampal neurons. In particular, we have tried to elucidate the role of GTP-binding proteins (G proteins; reviewed by refs. 14, 21) in signal transduction of the slow depolarization. Although molecular studies of the muscarine receptors suggest a G protein-coupled structure, involvement of G protein in the muscarine-induced depolarization has not yet been established. Our data indicate that the main component of the depolarization is mediated through a pertussis toxin-insensitive G protein in much the same way as in the substance P-induced depolarization in brain cholinergic neurons 32. However, the data also suggest the existence of a component that is unrelated with G protein. This is in agreement with the data of

Correspondence: S. Nakajima, Department of Pharmacology, M / C 868, University of Illinois College of Medicine at Chicago, Chicago, IL 60612, USA. Fax: (1) (312) 996-1225. * Present address: Chemical Abstracts Service, 2540 Olentangy River Road, Columbus, OH 43202, USA. ** Present address: Department of Cell Biology, Duke University Medical Center, Durham, NC 27710, USA.

201 mM, o-glucose 25 mM, cysteine 1 mM, and papain 20 U/ml. The tissue fragments were dissociated by trituration in culture medium. The culture medium consisted of 95% of a modified (see below) Eagle's minimum essential medium containing Earle's salt, 5% of heat-inactivated rat serum (prepared in our laboratory), L-ascorbic acid (10 ~g/ml), penicillin (50 U/ml), and streptomycin (50 #g/ml). The Eagle's minimum essential medium with Earle's salt (Gibco, Grand Island, NY, Cat. No. 330-1430) was modified by adding the following extra-ingredients: L-glutamine (0.292 mg/ml), D-glucose (5 mg/ml, in addition to the D-glucose already in the minimum essential medium), and NaHCO 3 (3.7 mg/ml). In some cultures, fibroblast growth factor 44 (1-2 ng/ml) was added (Collaborative Research, Inc.), resulting in no noticeable difference. Neurons were cultured in a 1.2 cm diameter well made at the center of a petri dish. Before plating dissociated neurons, the bottom of the well was covered by collagen and a feeder-layer consisting of astroglia cells. The cultures were kept at 37°C in an atmosphere of 10% CO 2 and 90% air at saturated humidity. Most experiments were conducted on neurons cultured for 2-4 weeks, but neurons cultured up to 8 weeks were used occasionally.

B e n s o n e t al. 2, w h o s h o w e d t h a t t w o d i f f e r e n t i o n i c m e c h a n i s m s a r e i n v o l v e d in t h e m u s c a r i n i c d e p o l a r i z a t i o n n e a r t h e r e s t i n g p o t e n t i a l in h i p p o c a m p a l n e u r o n s . P o r t i o n s o f t h e s e r e s u l t s h a v e b e e n p u b l i s h e d in a b s t r a c t f o r m 4. MATERIALS AND METHODS

Culture The culture method for hippocampus and cerebral cortex neurons was similar to that used for culturing brain nuclei, such as the locus coeruleus 27 or the nucleus basalis 34. Specific regions of the brain are visually isolated from brain slices. The specimens obtained were dissociated and cultured. The original method 27 has recently been modified slightly 33, and this modified version was employed in the present work. The methods are described briefly here. Newborn Long-Evans rats (2-8 days old, rarely up to 11 day old) were used in this study. The brain was removed aseptically under ether anesthesia and immediately immersed in an oxygenated ice-cold balanced salt solution consisting of: NaCI 130 mM, KCI 4.5 mM, CaCI 2 2 mM, D-glucose 33 mM, and PIPES buffer 5 mM (pH 7.4). The brain was embedded in 2.5% agar in the balanced salt solution. The agar block was hardened, and brain slices (coronal sections, 400-500/~m thick) were obtained using a Vibratome (Lancer 1000). The slices were examined under a dissecting microscope and tissue fragments from the CA1 or CA3 region of the hippocampus (or the parietal region of the cerebral cortex in early experiments) were cut out using a pair of hypodermic needles as small knives. The brain tissue fragments were incubated twice (15 min each) in an oxygenated papain solution with gentle stirring at 37°C. The papain solution TM contained: NaCI 116 mM, KCI 5.4 mM, NaHCO 3 26 mM, NaH2PO 4 1 mM, CaCI 2 1.5 mM, MgSO 4 1 mM, EDTA 0.5

Electrophysiology The procedures were similar to those described in Yamaguchi et al. 45. The center well of the petri dish, where cultured neurons were present, served as the experimental chamber for electrophysiology. During the experiments, the culture was superfused continuously with an oxygenated Krebs solution containing: NaC1 146 mM, KC1 5 mM, CaCI 2 2.4 mM, MgCI 2 1.3 mM, o-glucose 11 mM, tetrodotoxin 1 /*M, HEPES(NaOH) 5.0 mM (pH 7.4). The whole-cell version of the patch-clamp method 16 was used. The patch pipette was filled, unless otherwise stated, with an internal solution containing: potassium aspartate 120 mM, NaCI 40 mM EGTA(KOH) 0.5 mM, CaC1a 0.25 mM, MgCI 2 3 mM, Na2ATP 2 mM, Na2GTP 0.1 mM, HEPES(KOH) 5 mM, KOH = 6 mM (pH 7.2).

A

!!

\

i I Fig. 1. Phase-contrast micrographs of cultured hippocampal neurons. Bar = 50/zm. A: a CA1 neuron from 17-day-old rats cultured for 8 days. B: a CA3 neuron from ll-day-old rats cultured for 16 days.

202 In most experiments drugs were applied by the puffing technique 6 using a pressure of = 7 kPa from micropipettes (tip diameter, 4 - 5 ,am) placed 30 40 ,am from the neuronal soma surface. We tested the speed of drug application by the puff method by recording inward currents generated by ejecting a high K solution (a Krebs solution containing 10 m M K ions) onto neurons superfused with a Krebs solution containing 2.5 mM K ions. The cells responded quickly with an inward current with a half-time of about 15(I ms regardless of the position (30 ,am or 45 # m ) of the drug electrode (two culture neurons from the locus coeruleus were used; for each neuron we compared the responses at the two locations). Thus, the difference between rapid and slow muscarinic responses (see Results) cannot be explained by chance differences in the location of the electrode. The glass capillaries for making the drug electrodes were thoroughly washed before use. In most experiments the pipette was placed in air until just before drug delivery., to avoid contamination of the superfusing solution. The m e m b r a n e potential was corrected for a 9 mV liquid junction potential between the external solution and the pipette solution (external solution positive). We used only large cells with average soma diameters as follows: cortical neurons, 24.6+0.65 # m ( m e a n + S . E . M . , n = 13); CAI neurons, 22.5 +//.26 # m (n 129); CA3 neurons, 24.5 + 0.40 ,am (n - 45). Fig. 1 shows such large neurons cultured from CA1 and CA3. Experiments were done at a temperature of 30-36°C (mean 33.3°C).

ments. Controls were treated in exactly the same nlanncr as t h e pertussis toxin-treated cultures, except for omission of pertussis toxin.

RESULTS Muscarinic response

Fig. 2A1 shows a voltage response recorded using a conventional intracellular microelectrode in cultured cortical neurons. Application of acetylcholine (ACh) (2 p~M) to a cell with a relatively high resting potential ( - 7 5 mV in Fig. 2A) produced a slow depolarization. This depolarization reached a peak in about 7 s, and returned to the original level in about 40 s. As shown in Fig. 2A2, simultaneous application of ACh (2 ~M) and atropine (5/xM) through a second puffing pipette, did not result in depolarization. Fig. 2B,C show records taken from hippocampal neurons at high holding potentials ( - 6 9 mV in B, and - 7 4 mV in C) using the whole-cell patch-clamp technique. In Fig. 2B1, the application of ACh (10 /xM) produced a slow inward current, which reached a peak in about 8 s, and returned to the original level in about 1 min. The time course of the recovery of the inward current varied from cell to cell, with 90% recovery time ranging from 20-80 s. As shown in Fig. 2B2, application of ACh (10 /zM) plus atropine (1 /xM) caused a

Pertussis toxin treatment

A procedure similar to Holz et al. I7 was followed. A fresh pertussis toxin solution was prepared for each experiment, and contained: pertussis toxin (List Biological Labs, Campbell, PAl 14 , a g / m l , sodium phosphate buffer (pH 7.2) 10 mM, NaCI 50 mM, and heat-inactivated bovine serum albumin (Fraction V, No. 12659, Calbiochem) 0.04%. This solution was added to the culture at a final toxin concentration of 500 n g / m l . During the pertussis toxin treatment the culture medium did not contain serum, penicillin-streptomycin or ascorbic acid. The pertussis toxin-treated cultures were incubated at 37°C for 17-32 h before use in physiological experi-

BI

Microelectrode

B2 I

CI

Whole-cell

ACh

Whole-cell

C2

!

10 sec m m

A2

ACh +Atropine

2pM ACh + 5pM Atropine

Atropine

m

Mus

M.s

roACh

' ' 10 sec

]

Fig. 2. AI,2: depolarization produced by A C h (2 ,aM) recorded from an intracellular microelectrode in a cortical neuron from 8-day-old rats cultured for 16 days (All. Application of A C h (2 ,aM) together with atropine (5 /xM) (by putting the two compounds in the same pipette) produced no response (A2). Resting potential was - 7 5 mV. B1-3: effect of A C h on a CA3 neuron from 8-day-old rats cultured for 9 days. In these and all the following figures, the whole-cell patch clamp method was used. The downward direction indicates the inward direction of current. In B1, application of A C h (10 ,aM) produced a slow inward current. In B2, application of A C h (10 ,aM) together with atropine (1 ,aM) produced only a small inward current. The atropine effect was reversible, as shown in B3 (ACh 10 #,M). The time interval for drug application was: 110 s between B1 and B2, and 122 s between B2 and B3. The holding potential was - 69 mV, and the base line current level was ~ 80 pA (outward). C1-3: effect of muscarine on CA1 neurons from 8-day-old rats cultured for 23 days. In C1, muscarine (Mus, 20 ,aM) evoked a slow inward current. In C2, atropine (2 ,aM) was applied through a second drug electrode, and subsequently muscarine (20 ,aM) was applied through the first electrode. This did not produce a response. In C3, muscarine (20 ,aM) was again applied 190 s after the application of atropine in C2; the muscarine effect partially recovered. Holding potential was - 74 mV, and the base line current level was 10-50 pA (outward).

203 duced rapid response became very small (3.7 + 1.9% of the control) after superfusing with atropine-containing Krebs for 3 - 6 min (mean, 280 s). The antagonistic effect of atropine appears to be genuine, not merely reflecting desensitization. In 7 cells (using either the standard internal solution or the GTPyS-containing solution, see below), in which a rapid response was obtained, 20 /xM muscarine (or 5 IzM ACh) was applied twice in succession with an interval of = 3 min (without applying atropine). The second application produced a response with an amplitude of 70.4 + 12% (mean + S.E.M., n = 7; S.D. = 32%) of the first response, reflecting desensitization. (Desensitization varied greatly from cell to cell, as the large value of S.D. indicates.) The experimental conditions were somewhat different between the two groups, but it is very likely that atropine did inhibit the rapid response.

much smaller inward current. This inhibitory effect of atropine was reversible (Fig. 2B3). In the experiment illustrated in Fig. 2C, muscarine was applied instead of ACh. Muscarine (20 /zM) (applied through a drug electrode) produced a slow inward current (C1). In C2 atropine (2 /zM) was applied through a second electrode, and 20 s later, muscarine was applied from the first electrode. This produced no response. The suppression of muscarinic effects by atropine was partially reversible (C3). In another set of experiments (not shown) we applied atropine by switching the superfusing solution rather than by puffing. Although this produced an almost complete block of subsequent muscarine effects, we could not obtain reversibility of the atropine effect. Using the various protocols described above, we observed the antagonistic effect of atropine on the slow depolarization or the inward current in 19 cells, of which 5 showed some reversibility. These results indicate that the slow depolarization or the slow inward current evoked by ACh or muscarine in these cultured neurons is mediated through muscarinic receptors. Thus, this response corresponds to those described in cortical or hippocampal neurons in in situ or in slice preparations by various investigators 1,2,7,9,24,26,41.

Slow and rapid responses The above description of slow (dome-like) and rapid (spike-and-plateau) responses is not very quantitative. It is convenient to categorize the responses by using one quantitative variable. Thus, we have measured the maximum rate of rise of the inward current. We designated the cells with the rate of rise less than 25 p A / s as the low rate-of-rise cells (70% of the cells in GTP or G T P y S internal solution; see below about the G T P y S internal solution), and those with the rate of rise > 90 p A / s as the high rate-of-rise cells (14%), and the remainder (16%) as the intermediate. We found that in the standard internal solutions almost all (97%) of the low-rate-of-rise cells showed an inward current with a dome-like time course (slow type), and almost all (83%) the high-rate-of-rise cells revealed the spike-andplateau shape. The intermediate type showed either the dome-like or the spike-and-plateau shape, or an unclassified shape. Thus, under this new definition, the low rate-of-rise sample approximately represents the slow response (dome-shape) cells. The occurrence histogram (not shown) of the maximum rate of rise of the inward current formed a continuous spectrum without a distinct dichotomy of the two types of cells. There

Rapid response Occasionally, application of muscarine or ACh caused a rapid, large inward current. In the example shown in Fig. 3 the rapid inward current had a maximum rate of rise of 300 p A / s , peaking at = 3 s, and subsided with a half-time of 5 s, followed by a much slower inward current. Thus, the shape of this response at this time base looks like a spike followed by a small plateau. This characteristic is in contrast with a domeshaped time course of the ordinary slow response (Fig. 2). After the superfusing solution was switched to atropine (1 tzM)-containing Krebs, this rapid muscarinic response almost disappeared (right-side of Fig. 3). Due to the length of time to wash out the atropine completely, this effect was not reversible. In 3 cells (using the standard internal solution) the muscarine-in-

"+

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','r'l-~-~t~- - w ,

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] 200 p A 11) sec "

Fig. 3. A large, rapid inward current was evoked by muscarine (20 IzM) (left record) in a CA1 neuron from 3-day-old rats cultured for 24 days. After obtaining the large response, the superfusing solution was switched to the one containing atropine (1 p.M). This suppressed the muscarine (20/zM)-induced response (right record). The interval between the first and second muscarine application was ~ 6 min. The holding potential was - 74 mV. The arrows indicate zero current level.

204 was no d i f f e r e n c e in the size of n e u r o n s b e t w e e n the low rate-of-rise cells and the high rate-of-rise cells (23.7 vs. 24.3 ~tm; P > 0.5).

Role of G protein T h e m a i n objective o f the following e x p e r i m e n t s is to e x a m i n e the role of G p r o t e i n s in signal t r a n s d u c tion involved in the m u s c a r i n i c effects. O n e way of testing for the i n v o l v e m e n t of G p r o t e i n is i n t r a c e l l u l a r a p p l i c a t i o n of a n o n - h y d r o l y z a b l e a n a l o g u e of G T P , such as G T P T S ( g u a n o s i n e 5 ' - O - ( 3 - t h i o t r i p h o s p h a t e ) ) (see ref. 14). If a G p r o t e i n is involved in the m u s c a r i n e effect, the p r e s e n c e of a n o n - h y d r o l y z a b l e a n a l o g u e would cause the m u s c a r i n i c effect to persist w i t h o u t recovery, since the G p r o t e i n will r e m a i n activated. Fig. 4 A (control) shows a c u r r e n t r e c o r d u n d e r whole-cell voltage c l a m p from a c u l t u r e d h i p p o c a m p a l neuron. T h e p a t c h p i p e t t e c o n t a i n e d 200 /~M G T P .

A

Control

_ MHS

-"

A f t e r b r e a k i n g the patch, we w a i t e d for 5 min to allow for diffusion of G T P into the i n t e r i o r of the neuron. T h e a p p l i c a t i o n of m u s c a r i n e p r o d u c e d the usual slow inward current, which s u b s i d e d almost c o m p l e t e l y in ---60 s. M u s c a r i n e was e j e c t e d again 3 min after the first a p p l i c a t i o n , p r o d u c i n g a similar response. As shown in Fig. 4B, d i f f e r e n t results were o b t a i n e d w h e n the p a t c h p i p e t t e c o n t a i n e d G T P y S , i n s t e a d of G T P . A f t e r the r u p t u r e of the g i g a - s e a l e d patch, 5 min was allowed to pass. ( W e o b s e r v e d previously that 5 min was long e n o u g h for G T P y S to diffuse into the cell i n t e r i o r a n d p r o d u c e its effects on the s o m a t o s t a t i n r e s p o n s e in locus c o e r u l e u s n e u r o n s ~', and on thc s u b s t a n c e P r e s p o n s e in nucleus basalis neurons32.) D u r i n g the waiting p e r i o d , the d r u g ejecting p i p e t t e was p l a c e d in the air to p r e v e n t c o n t a m i n a t i o n of the s u p e r f u s i n g solution with the drug. S p o n t a n e o u s activation of the inward c u r r e n t was not o b s e r v e d without

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0

pA Mus

B

10 sec

GTP'IS - loaded

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Mus C

GTP~S - loaded

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Mus

D

Fig. 4. Effect of loading cells with GTPyS. A: a control CA3 neuron from 3-day-old rats cultured for 33 days loaded with GTP (200 rtM). Application of muscarine (20/zM) produced an inward current, which returned virtually to base line. A second application produced a similar response. In all the records in this figure, 5 min passed after the rupture of the patch before the first muscarine application. B,C: two examples of GTP3,S-loaded cells. Both are CA1 neurons from 4-day-old rats cultured for 21 days. Muscarine (20/xM) produced a slow inward current, which did not return to base line. The patch pipette solution contained GTPyS (instead of GTP), and other ingredients were the same as in the standard patch pipette solution. The concentration of GTPyS was 350/~M in B and 200 ~M in C. In all the records, the holding potential was - 74 inV. Arrows indicate the zero current levels. D: a diagram to illustrate the notations in Table 1.

205 TABLE I

Effect of GTPy S loading on muscarinic response Values are mean±S.E.M. Referring to the diagram in Fig. 4D, 'lst response' is the size of a; 'Max. rate of rise' is the maximum rate of rise of the 1st response; 'Recovery' is (b/a); and '(2nd/lst)' is (c/a). In controls, the patch pipette contained GTP (100-200/zM): in the GTPyS group, the patch pipette contained GTPyS (100-300/~M). Responses were produced by 20/xM muscarine. The first muscarine application was performed --- 5 min after rupture of the patch membrane, and the second muscarine application was done = 3 rain after the first application. Data from cells with a very small response (less than 30 pA) in the first agonist application were excluded. Holding potential was - 74 mV. Table I A: only the data from the cells exhibiting a response with a maximum rate of rise of the first response less than 25 p A / s are included. Table I B: data from all cell types were included. In both Table I A and I B, the differences between the test and control groups were significant both in 'Recovery' (P < 0.001) and in '(2nd/lst)' (P < 0.01).

A. Low rate-of-rise Control GTPy S-loaded B. All types Control GTPyS-loaded

Number of cells

Resting potential (mV)

6 17

-66+3.7 -73_+1.2

9 26

-69_+2.8 -72_+0.9

1st response(pA) 63+ 11 73+ 8.0 109_+28 114_+21

the presence of agonists. After loading the cell with GTPyS, application of muscarine resulted in an inward current, which showed little tendency to subside, and a second application of muscarine caused only a small additional response (Fig. 4B). Fig. 4C shows another example of a GTPyS-loaded cell. In this cell, application of muscarine evoked an inward current, which, after reaching a peak, showed a small tendency to return towards base line. The second application produced a small but definite increase in the inward current. Table I summarizes this data. As already discussed in relation with Fig. 3, muscarine occasionally produced a very rapid initial inward current. Table IA excludes these ceils, and lists only cells with the low

Max. rate of rise (pA / s)

Recovery (%)

(2nd / lst) (%)

9.8_+ 3.2 8.5+ 1.4

89_+8.9 30-+5.4

75 + 10 35+ 6.4

35 +15 29 _+ 9.5

87-+7.0 42_+5.5

76+ 7.6 36_+ 4.6

rate-of-rise response (see the legend for the sampling procedures). The sixth column of Table I, 'Recovery', represents the degree of recovery from the first muscarine response ( b / a in Fig. 4D). In the control cells recovery was 89%, whereas in the GTPyS-loaded cells recovery was only 30%. Column 7 of Table I is '(2nd/lst)'. This is the ratio of the magnitude of the second response to the first response, corresponding to (e/a) in Fig. 4D. This ratio is again larger in the control (75%) than in GTPySloaded cells (35%). (In the control cells, this ratio, 75%, approximately represents the degree of desensitization under these conditions. There was a large cell to cell variation in the degree of desensitization: S.D. = 24%. In the GTPyS-loaded cells, this ratio would

A _.1..

GTP~S-loaded '

4-

2 1

-Mus B

Mus

GTP~S-loaded

pA

-ACh

10 s e c

ACh

]

Fig. 5. Example of a rapid, large inward current evoked by muscarine (20 p.M) (A) or by ACh (5/zM) (B) in neurons loaded with GTPyS (100 p.M). A: CA3 neuron from 3-day-old rats cultured for 37 days. B: CA3 neuron from 2-day-old rats cultured for 58 days. In both records, the holding potential was - 74 mV, and arrows indicate the zero current levels.

206 A TABLE 11

B

Control

PTX

Rapid re.sponse Values are mean _+S.E.M. Only cells which showed a rapid response to either muscarine (20 /zM) or ACh (5 /xM) application with the maximum rate of the inward current > 90 p A / s were included. In the control group, GTP (100-200 /xM) was loaded, and the experiments were started 1-5 rain after rupture. In the GTPyS-loaded group, 1110-350 ~M of the nucleotide was loaded, and at least 5 min were allowed for GTPyS to diffuse into the cell. Holding potential was - 7 4 mV (except for one control cell, in which it was - 6 9 mV).

Control (n=6) GTPyS-loaded ( n = 6 )

Resting potential (mV)

Peak current (pA)

Max. rate Hall" of rise recouery (pA / s ) time (s)

-71+1.3 -74+2.8

490_+ 98 328_+ 83 3.4_+0.54 443_+124 380_+212 3.0_+0.17

represent a complicated quantity, since it would be dependent not only on desensitization but also on the degree of recovery.) To summarize, loading the cells with G T P y S caused an incomplete recovery of the low rate-of-rise muscarine response, and the second response was much smaller than the first one. These results suggest that the slow response is mediated through a G protein. Table IB lists the data for all types of neurons. Rapid response in GTPyS-loaded cells As was the case for cells under standard conditions (Fig. 3), application of muscarine or ACh occasionally produced a rapid initial inward current in cells loaded with G T P T S (Fig. 5). The time course of this rapid phase was similar to that in the control cells (compare Fig. 5 with Fig. 3). Table II summarizes the time course of this rapid phase for the high rate-of-rise cells. As shown in the last column, the half-recovery time of the rapid response was 3.0 s in the G T P y S group, essentially the same as that in the control group (3.4 s; P > 0.3). Thus, the recovery from the rapid response was not affected by the presence of G T P y S , suggesting that a G protein is not involved with the rapid response. If the rapid phase were G protein-gated, we would have observed in G T P y S loaded cells a large and rapid inward current phase, which does not recover (irreversible rapid response) or at least one in which the main recovery phase is substantially retarded. We never observed cells with such a response. Pertussis toxin sensitiuity Some G proteins, such as G i or G O, are ADPribosylated by pertussis toxin (islet-activating protein) and lose their capability to interact with the receptors 2°'43. As shown in Fig. 6, the muscarine response in our hippocampal neurons was pertussis toxin-insensitive. Table III summarizes the data. There

Fig. 6. Pertussis toxin (PTX) treatment of cultured CA1 neurons. Muscarine (20 p,M) produced the usual inward current in both control and PTX-treated cells. Both cells were from 4-day-old rats cultured for 21 days. Holding potential was - 7 4 inV. The base line current level was 40 pA (inward) in A, and 80 pA (inward) in B.

was no significant difference in the amplitude of the muscarine-induced inward current between the control and the pertussis toxin-treated cells. It is unlikely that insensitivity to pertussis toxin was due to faulty pertussis toxin treatment: the same procedure for pertussis toxin treatment completely abolished the somatostatin-induced response in cultured noradrenergic neurons from the locus coeruleus ~9 and the response to the D 2 dopaminergic agonist in cultured substantia nigra neurons 22. Insensitivity of the muscarine-induced inward current to pertussis toxin was reported in slice preparations made from the toxin-treated rats ~°. Cyclic A M P as a second messenger Adenylate cyclase and cyclic A M P (adenosine 3',5'monophosphate) have been shown to act as second messengers in many biological effects initiated by hormones or transmitters. Adenylate cyclase is activated by a G protein Gs, which is pertussis toxin-insensitive. Thus, we tested whether our muscarine-induced response was mediated through the cyclic A M P system.

TABLE 1II

Pertussis toxin treatment effect on muscarinic response Values are mean_+ S.E.M. Muscarinic response is the amplitude of the inward current elicited by the first application of muscarine (20 /zM) at a holding potential of - 7 4 mV. Experiments were done using 3 series of culture batches. Only cells with the low rate-of-rise response were sampled. (We did not encounter cells with the rapid response in these batches. One cell with the intermediate type was excluded.) There was considerable inter-batch variation in the properties of muscarine-induced inward current. However, the intra-batch variation was smaller. Therefore, for each batch, the same number of control and pertussis toxin-treated cells were sampled. (By batch, we mean several dishes of cultured cells, derived from the same brains, dissociated together, incubated together, and used for physiological experiments on the same day). The current amplitude of each cell was corrected for the cell size variation by normalizing it to a soma size of 21 /zm, assuming that the current is proportional to (diameter)< There was no significant difference between the control and the pertussis toxin-treated cells ( P > 0.5).

Control Pertussis toxin-treated

(n = 8) (n = 8)

Resting potential (mV)

Muscarinic response (pA)

- 72 _+2 - 71+ 1

47 -4-_11 54-+ 5.8

207 TABLE IV

Signal transduction of the slow response

Effect of cyclic AMP loading on the muscarinic response

The primary conclusion from the present experiments is that a G protein acts as a transducer in the signal transduction leading to the slow muscarinic inward current. This conclusion is based on the finding that the intracellular application of G T P y S caused an incomplete recovery from the muscarine-induced response. The response was not affected by pertussis toxin, indicating that the G protein is pertussis toxininsensitive. Cyclic AMP does not seem to be a messenger for the slow electrical signal obtained under our experimental conditions. These results indicate that on the whole, the signal transduction of the muscarinic slow inward current resembles the substance P-induced suppression of the inwardly rectifying K-current in brain cholinergic neurons 32'42 or the muscarine- or the LHRH-induced suppression of M-current in sympathetic neurons 39. Nevertheless, there is a quantitative difference. The present data on the G T P y S application were not as clear-cut as those of the substance P-induced response in the nucleus basalis neurons 32. In the nucleus basalis neurons, which were loaded with G T P y S (100-350/xM), application of substance P produced a virtually irreversible conductance decrease, with a recovery of only 2 - 4 % 32. In contrast, the present experiments show that in the presence of G T P y S (100-300 ~ M ) the muscarine-induced slow response recovered by as much as 30%. Recently, Benson et al. 2 investigated the ionic mechanism of the muscarinic inward current near resting potential in hippocampal neurons in slices. They observed that the reversal potential of the muscarinic response was either absent or more negative than the potassium equilibrium potential ( E K) (see also the earlier work by Dodd et al. 9 which showed a very negative value ( - 1 0 5 mV) for the reversal potential). Thus Benson et al. 2 concluded, in agreement with the earlier result on the muscarinic excitation by Kuba and Koketsu 25 in sympathetic neurons, that the muscarinic inward current originates from two different ionic mechanisms: a decrease in a K conductance and an increase in a non-specific cation conductance. (Note that the increase in the non-specific cation conductance reported by Benson et al. 2 is a small a n d / o r slow process, unlike our rapid response.) If we consider the data from our experiments and those of Benson et al. z, the following hypothesis can be proposed. The mechanism of the muscarinic depolarization in CA1 or CA3 consists of two components: (1) a decrease in K conductance, which is mediated by a pertussis toxin-insensitive G protein in analogy with the substance P-induced response, and (2) an increase

In cyclic AMP-loaded cells, 100 p,M cyclic AMP plus 1 mM IBMX was used. Values are mean _+S.E.M. 'Muscarinic response' is the amplitude of the inward current elicited by the first application of muscarine (20 ~M), which occurred 5 min after the rupture. The current amplitude of each cell was corrected for the cell size variation by normalizing it to a soma size of 23 p.m, assuming that the current is proportional to (diameter) 2. Experiments were done on 3 series of culture batches. Two rapidly responding cells (the high rate-of-rise cells) (in the cyclic AMP group) were eliminated. One cell of intermediate type was included in the control. For each culture batch, the same number of control and cyclic AMP-loaded cells were sampled. There was no significant difference between the two groups ( P > 0.3). In these experiments the cell resting potentials was lower compared to the cells in other tables, reflecting below optimal culture conditions.

Control cAMP-loaded

(n = 5) (n = 5)

Resting potential (mV)

Muscarinic response(pA)

-64_+4 -50_+6

63_+21 95_+22

A patch pipette solution containing the standard internal solution plus 100 /zM cyclic AMP and 1 mM IBMX (3-isobutyl-l-methylxanthine) (an inhibitor of phosphodiesterase) was used. This concentration of cyclic AMP (100 ~zM) far exceeds the value of K m (0.01-0.15 ~M;, see ref. 35) of cyclic AMP for the protein kinases. Therefore, if cyclic AMP is a second messenger for the muscarinic effect, this concentration of cyclic AMP would occlude the muscarinic response. After breaking the patch, we waited for 5 min, and the muscarine-induced inward current was recorded. As shown in Table IV, no difference was found in the muscarine-induced response between the cyclic AMPloaded cells and the control cells. The results suggest that it is unlikely that the muscarine-induced inward current is caused by changes in the intracellular concentration of cyclic AMP. DISCUSSION

Rapid response Occasionally, we encountered hippocampal neurons in which ACh or muscarine elicited a very rapid inward current. Our results show that the signal transduction of this response is not related to a G protein. The cellular origin of this response is obscure. But it is unlikely that this response represents a 'culture effect', since in slice preparations McCormick and Prince z8 and Reece and Schwartzkroin 4° observed rapid muscarinic responses in non-pyramidal cells in cortical and hippocampal neurons. Likewise, our rapid response may also be of a non-pyramidal origin. Immunocytochemical investigation of cultured cells may resolve this problem.

208 in the non-specific conductance, which is unrelated to a G protein. This hypothesis is in agreement with the recent report by Koyano et al. 23, which showed that the substance P response has two components in locus coeruleus neurons: one, a decrease in an inwardly rectifying K conductance mediated by a G protein, and the other an increase in a non-selective ion conductance, unrelated to a G protein. Acknowledgements~ We thank Mrs. Carolyn Partee and Mr. Charles McShane for their clerical help. This work was supported by NIH Grant NS24711.

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