Neuroscience Letters, 91 (1988) 177 182 177

Elsevier Scientilic Publishers Ireland Ltd.

NSL 05516

Calcium-dependent pirenzepine-sensitive muscarinic response in the rat hippocampal slice

Thomas A. Pitler, Madel ine McCarren* and Bradley E. Alger

. . M D _ l _ O l Department ol' Ph vsiologv, University 0[ Maryland School ql' Medicine. Baltimore, ? ~ r (,'.,S'..4..,

(Received 7 March 1988; Revised 4 May 1988: Accepted 4 May 19881

Key wor~&" Carbachol; Oxotremorine; Muscarinic: Pirenzepine: Calciunr: Slow EPSP

Using intraccllular recording techniques in the rat hippocampal slice, we observed that muscarinic ago-

nisls produce a transient Cae+-dcpendent depolarization that may be related to the phosphatidylinositol

cycle. First, it was more readily produced by muscarinic group A agonists, which strongly enhance the

breakdown of phosphatidylinositol-4,5-bisphosphale I PIP,_), than by group B agonists, which are less clti- cacious. Second, the Cae+-dependent response was blocked by pirenzepine (PRZ), a selective muscarinic

antagonist thai blocks PIPe breakdown in fnrebrain. Both group A and group B muscarinic agonists

caused equivalent maintained levels of depolarization that were relatively insensitive to PRZ. The data

suggest that the Cae+-dependent response is fundamentally unlike other muscarinic responses that have

been described in hippocampus.

Acetylcholine is a major neurotransmitter in mammalian brain, its actions mediated largely by muscarinic receptors [15, 17]. Muscarinic cholinergic agonists have a wide variety of electrophysiologically measured effects on CNS neurons [see ref. 13 for review]. It is likely that different receptor~ffector mechanisms are involved in the various muscarinic responses, and it will be essential to determine if particular effects can be associated with each.

Some muscarinic agonists may act via the by-products of phosphatidylinositol-4,5- bisphosphate (PIP2) turnover, while others probably do not. For instance, group A muscarinic agonists (e.g. carbachol) dramatically increase the breakdown of PIP> whereas group B agents (e.g. oxotremorine) are only weak partial agonists for this effect [5, 6, 10].

In forebrain regions, the ability of muscarinic agonists to cause PIP~ breakdown is readily antagonized by the non-classical muscarinic antagonist, pirenzepine (PRZ) [4, 9], which distinguishes between classes of muscarinic receptors [12]. We report

*Present address: Department of Pharmacology, The University of Chicago, 947 East 58 Street, Chicago, Ik 60637, U.S.A.

Correspondence." B.E. Alger, Department of Physiology, University of Maryland School of Medicine, 660 West Redwood Street, Baltimore, M D 21201. U.S.A.

0304-3940,'88,'$ 03.50 @ 1988 Elsevier Scientilic Publishers Ireland Ltd.

178

that a PRZ-sensitive receptor activated by group A muscarinics mediates a transient TTX-insensitive depolarization that is dependent on extracellular calcium ([Ca: * ],,). A PRZ-insensitive receptor mediates a slow, maintained depolarization associated with an input resistance increase that is insensitive to TTX and [Ca 2~ ]o. Some of these results have appeared in abstract form [2].

Intracellular recordings were made from over 100 CAI pyramidal cells from the rat hippocampal slice using conventional techniques [1]. Animals were anesthetized with ether and decapitated. The hippocampus was removed and 400/~m thick slices were cut on a homemade tissue chopper. A single slice was maintained in the constant perfusion experimental chamber at 29- 3 l C; other slices were held in an incubation chamber at room temperature (22-24~C) until needed. Electrodes were filled with 2 M KCH3SO4. The composition of the perfusion saline was (in raM): NaCI 123, KCt 3.5, CaCI: 2.5, MgC12 3.5, Na2H2PO4 1.2, NaHCO3 26.2, glucose 10. In 'low Ca :~ / Mn 2+' saline, CaCI2 was omitted, 2 mM MnC12 was added and MgC12 was raised to 4 raM, to keep divalent cation concentration constant. Additionally, NaH2PO4 was omitted in Mn 2~-containing medium to prevent formation of a precipitate. In

A B carb carb

P R Z

CO"

( ,uM)

.... 1o mV

2" rain

2O

,I. 10 mV i

4 min Fig. 1. Dose-dependent PRZ effects on carbachol response in hippocampal CA1 cell. A: PRZ slows the rate of carbachol-induced depolarization. Bar above the traces signifies duration of the carbachol applica- tion. In the top trace, carbachol (50/~M) was applied in control saline. In a different celt shown in the lower trace, the cell was bathed in 0.1 #M PRZ containing medium during application of carbachol. Although both cells eventually reached the same maintained level of depolarization, the cell bathed in PRZ depolarized with a much slower rate of rise than the cell in control saline. B: high dose PRZ (20 gM) blocks both transient and maintained carbachol-induced changes in membrane potential and resistance. After recording the control response to carbachol (50/~M, 3 min application), the cell was extensively washed for 40 min. PRZ was then applied for t 5 min and carbachol reapplied. Notice than in 20/~M PRZ, there was no response to carbachol.

17c~

'low CaZ+/Mg 2+' saline, [Ca2+]o was lowered to 0-1.0 raM, and 10 15 mM Mg 2÷

was added. TTX, 0.3/2M, was present in all experiments. Drugs, all except PRZ, were obtained from Sigma and were administered via bath perfusion at known concentra- tions. PRZ was a gift of Boehringer-Ingelheim (Ridgefield, CT).

Values reported are means -t- S.E.M. The data illustrated in Fig. 2 and described in the text were analyzed statistically by a two-way ANOVA followed by a Student Newman-Keuls test for multiple comparisons.

A

CARB

CON LOW Ca /Mn PRZ

/ j /

q E :>

E

:> 1D

U,I

E t, O UJ I.- < IZ

10

9

8

7

0

5

4

3

2

1

B

( 3 5 ) OXO

J f J

(10 )

,1 10 mV

2 rnln

CON LOW Ca PRZ CON LOW Ca PRZ

CARBACHOL O X O T R E M O R I N E

Fig. 2. A: sample traces of muscarinic effects on membrane potential in different recording conditions.

Top row: carbachol was applied to 3 cells, either in control, low Ca -~ ~/Mn 2 b salinc (see Methods) or 1

ltM PRZ. Diagonal lines above the trace represent the slope (determined by eye) of the initial rise in depo-

larization. Notc slowed rate of rise of rcsponse in low-Ca-" ~/'Mn ~ ~ - and PRZ-containing salincs. Bottom

row: oxotremorine was applied to 3 different cells under the same conditions as in A. Note that rates of

rise of oxotrcmorine responses were comparable to those of carbachol recorded under low Ca ~ ~ :Mn ~

and PRZ conditions. B: summary data of rate of rise of muscarinic-induced depolarizations from experi-

ments as shown in A. The rates of rise (in mV/min; mean +_ S.E.M.) are shown for carbachol and oxotrc-

morine rcsponses under the indicated conditions. The number of cells studied in each condition is indicated

in parentheses near the relevant bar on the histogram. A two-way analysis of variance followed by a Stu-

dent Newman Keuls test for multiple comparisons indicated that the rate of rise of the carbachol re-

sponse in control conditions was significantly different (P<0.05) from all other groups and that therc were

no significant differences among the remaining groups.

180

When bath applied to a pyramidal cell, carbachol produces a rapid rising phase of depolarization, eventually followed by a variable reduction in depolarization to a plateau that is then maintained for as long as the perfusion is continued. In the present experiments, TTX was always present to block action potential firing and permit analysis of membrane potential changes induced by the cholinergic agonists. Application of PRZ (0.1 1 /tM) during the carbachol perfusion had no effect on the plateau of the carbachol-induced depolarization, although PRZ slowed the rapid ris- ing phase of carbachol response (Fig. 1A). At much higher concentrations (10 20 /tM), PRZ entirely blocked the depolarization due to 50 llM carbachol (Fig. I B), while even at these high doses PRZ had no effect on resting membrane properties (n-- 4). The differential effects of PRZ on the early vs the maintained muscarinic re- sponse suggested that the components of the total response might be distinguishable in other ways.

We found that perfusion of a saline with zero added Ca 2+ ([Ca2+]o approximately 10/~M), 4 mM Mg 2+ and 2 mM Mn 2~ , which abolished Ca2+-dependent responses, also eliminated the fast rising phase of the carbachol-induced depolarization without altering the plateau of the response (Fig. 2A). The same effect was achieved if the saline contained 0-1.0 mM Ca 2+ and 15 mM Mg 2+ rather than Mn 2+.

The reduction of the rapid rising phase of the carbachol response by PRZ sug- gested that this phase might be associated with P! turnover. If so, then group B mus- carinic agonists, which are only partial agonists for PI turnover, wouldproduce only slowly rising depolarizations. In addition, depolarizations caused by group B ago- nists would be largely Ca 2 t_ and PRZ-insensitive.

These expectations were confirmed. Depolarizations produced by oxotremorine, a typical group B muscarinic agonist, had significantly slower rates of rise than those induced by carbachol. Moreover, oxotremorine responses were nearly insensitive to low Ca2+/Mn 2+ saline or 1/~M PRZ (Fig. 2A). These experiments indicated that the rate of rise of the carbachol response in control conditions was significantly faster than other responses, whether to carbachol in low Ca 2+ or in PRZ (l /iM), or to oxotremorine in all conditions. Moreover, apart from the responses to carbachol in control saline, there were no differences among any of the other groups. The differ- ence between carbachol in control saline and all other conditions could not be explained by differences in level of depolarization or in input resistance increase [cf. 20]. Carbachol and oxotremorine caused identical depolarizations, and both drugs caused a slightly lower level of depolarization in low Ca 2+ saline. The input resis- tance increases caused by carbachol or oxotremorine did not vary among the differ- ent experimental conditions tested, although the absolute increases caused by oxotre- morine were somewhat less than those due to carbachol.

In 5 cells, short (10-15 s) applications of carbachol were administered, resulting in a transient depolarization that recovered completely within approximately 10 rain. If the cell was then washed into 'low Ca 2+' medium for a minimum of 20 min, the response was nearly completely abolished (see Fig. 3). Responses separated by 20 min in normal medium showed no decrement (Fig. 3A), suggesting that desensitization [cf. 16] could not explain the response reduction caused by the 'low Ca 2+' perfusion.

A B C O N 20 MIN CON LOW C a / M n

10 mV 250 pA

2"' mln

Fig. 3. Example of depolarizing response to short (15 s) applications of 50 ILM carbachol. A: both re-

sponses are m control medium and were recorded sequentially in the same cell separated bv 20 rain. In

this time frame and with these applications of carbachol there was no desensitization of the second re~

sponse. B: the first trace shows the response in control medium, the second trace shows the responses

recorded in low Ca2+/Mn ~+ medium after washing for 20 rain. Low Ca 2~ medium nearly completely

blocks the depolarization due to brief applications ofcarbachol.

In conclusion, we have discovered that an initial Ca2+-dependent component of the carbachol-induced depolarization is PRZ sensitive. A possible association of this Ca 2 ~ -dependent component with PIP2 breakdown was strengthened by our observa- tion that oxotremorine, a typical weak partial agonist in causing PIP2 breakdown, produced only slowly rising depolarizations that were comparatively Ca 2~- and PRZ-insensitive. We suggest that this Ca2+-dependent depolarizing phase is mediated by Pl-related muscarinic action.

Occasional reports have suggested that low Ca2+/high Mg 2+ saline abolishes cho- linergic responses [3, 7, 8, 18], whereas, in other cases, no evidence of Ca 2 ~ depen- dence was found [e.g. 11]. Our data suggest that the Ca2+-dependent phase is a tran- sient component, and that indeed the maintained plateau of the cholinergic depohtri- zation is not Ca2+-dependent. Hence the differences among earlier reports may be due to the different methods used to elicit the response and different phases of the response studied.

At low doses, PRZ reportedly blocks the muscarinic slow EPSP in hippoeampus [16]. At these doses, PRZ, however, has very little effect on the maintained muscarinic depolarization (Fig. I). Since the maintained depolarization is due to block of the M-current [11] and the K+-dependent leak current [14], a component of the slow EPSP may be due to the Ca 2 +-dependent depolarizing conductance. It should be not- ed that the present experiments do not establish that the Ca 2 ~ -dependent depolariza- tion is a postsynaptic effect. While the presynaptic muscarinic effects reported thus far are inhibitory [19, 21], it is conceivable that bath application of muscarinic ago- nists could liberate a depolarizing agent, perhaps a neurotransmitter, into the extra- cellular space in a Cae+-dependent manner. Further work will be necessary to inves- tigate the possible role of the Ca2+-dependent conductance in the slow EPSP. Nevertheless, the Ca2+-dependent effects we have described will be present when pharmacological techniques are used to study cholinergic actions in hippocampus and perhaps other brain regions as well.

182

This w o r k was s u p p o r t e d by N I H g r a n t NS22010 and a w a r d s f r o m the Epi leps)

F o u n d a t i o n o f A m e r i c a and the M c K n i g h t F o u n d a t i o n , and f r o m N I H g ran t

NS08330 (T.P. ) a n d N a t i o n a l Ins t i tu te o f N e u r o l o g i c a l a n d C o m m u n i c a t i v e Dis-

o r d e r s a n d S t r o k e G r a n t N S - 0 7 5 4 7 ( M . M . ) . W e t h a n k B o e h r i n g e r - l n g e l h e i m for the

d o n a t i o n o f p i r e n z e p i n e ( G a s t r o z e p i n ) .

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