A role for potassium channels in the regulation of cortical muscarinic acetylcholine receptors in an in vitro slice preparation

71

Molecular Brain Research, 5 (1989) 71-83 Elsevier BRM 70116

A role for potassium channels in the regulation of cortical muscarinic acetylcholine receptors in an in vitro slice preparation Christopher Shaw l, Frans van Huizen 1, Max S. Cynader 1 and Michael Wilkinson 2 1Department of Ophthalmology, Universityof British Columbia, Vancouver, B.C. (Canada) and 2Departmentsof Physiology and Biophysics, and Obstetricsand Gynaecology, Dalhousie University, Halifax, N.S. (Canada) (Accepted 30 August 1988)

Key words: Acetylcholine; Muscarinic receptor; Receptor; Cortex; Regulation; Potassium channel

The rules underlying muscarinic acetylcholine receptor (mAChR) regulation in an in vitro cortical slice preparation of adult rats were examined following various alterations of bioelectric activity and following agonist stimulation. Muscarinic ACh antagonists [3H]N-methyl scopolamine ([3H]NMS) or [3H]quinuclidinylbenzylate ([3H]QNB) were used to label cell surface vs total (i.e. surface and internal) receptors, respectively. Depolarization of neural membranes for 4 h at 22-37 °C using veratridine or high external potassium (K ÷) led to a temperature-dependent down-regulation of surface mAChR of 26.2% and 11.3%. Total mAChRs decreased by 37.6% and 8.1%. Addition of picrotoxin and glutamic acid also led to decreases in mAChRs. Increases in inward chloride ion current induced by 7-aminobutyric acid (GABA) or gold chloride had no significant effect on mAChRs. Blockade of calcium channels and synaptic transmission by magnesium or cobalt and postsynaptic calcium channels with nifedipine showed a significant effect on mAChRs only in the latter case. In contrast, agonist stimulation using carbachol led to a large down-regulation for both [3H]NMS and [3H]QNB (26.1%, 35.9%). ACh decreased [3H]QNB binding by 33.9%, but had little effect on [3H]NMS binding (6.3%). For [3H]QNB binding sites the effects of carbachol appeared to summate with those of veratridine. Down-regulation of [3H]NMS labelled mAChRs by carbachol and veratridine had an estimated half-time of 30 min and 2 h, respectively. Neither the effects of veratridine nor carbachol could be antagonized by tetrodotoxin (TTX), showing that the effects were not due to an increase in sodium ion currents. However, a common thread linking the various agents which induce mAChR down-regulation appears to involve changes in potassium (K ÷) current. Potassium channel blockers tetraethylammonium chloride (TEA), 4-aminopyridine (4-AP) and apamin had little independent effect on mAChR number, but prevented veratridine-induced down-regulation, presumably through a blockade of K +- and Ca2+-dependent K+-channels. Only TEA and 4-AP diminished carbachol-induced down-regulation suggesting that this effect involves only the non Ca2+-dependent K+-channels. It thus appears that mAChR regulation in the rat cerebral cortex is linked to changes in active K÷-channel currents: activation of the K÷-channel by depolarization-induced changes in K÷ current or by agonist stimulation leading to changes in the selective K÷ currents stimulate mAChR down-regulation; blockage of the K+-channels prevents this down-regulation.

INTRODUCTION O n e characteristic feature of n e u r o n s in m a m m a lian neocortex is their ability to modify their response properties following altered input activity. This neural 'plasticity' may be confined to a 'critical period' early in postnatal life, as it is for the cat visual cortex 18. In other cortical areas it may occur in adult animals as well 3°'41. Changes in cortical properties may be induced with a time course of days to weeks 18'36'41 or in some cases minutes to hours 4"37.

A n ongoing effort in our laboratory has been the attempt to find possible molecular correlates of cortical postnatal d e v e l o p m e n t and plasticity. To this end we have examined the characteristics and distributions of various n e u r o t r a n s m i t t e r and n e u r o m o d u l a tor receptors in cat visual cortex (for a general review see ref. 44). Those studies have demonstrated that drastic alterations in receptors are a normal feature of postnatal cortical development. O u r attention has been focussed recently on the muscarinic acetylcholine receptor ( m A C h R ) which

Correspondence: C. Shaw, VGH/UBC Eye Care Centre, Department of Ophthalmology, University of British Columbia, 2550 Willow Street, Vancouver, B.C., Canada V5Z 3N9. 0169-328X/89/$03.50 © 1989 Elsevier Science Publishers B,V. (Biomedical Division)

72 shows pronounced changes in number, affinity, and in laminar distribution during postnatal developm e n ¢ 2'44. The putative neurotransmitter, acetylcholine (ACh), appears to play a crucial role in regulating neural activity in the brain by altering potassium ion conductance 8,28'29 following activation of mAChR M 1 and M 2 receptor subtypes 51'56; it also has been implicated as playing a role in cortical plasticity mechanisms 2. The developmental changes in mAChRs which we have studied occur with a time course of days to weeks and may contribute to some plasticity mechanisms in the visual cortex. These alterations take place over too long a time, however, to account for some of the shorter term alterations in cortical response properties which have been demonstrated z" 27,37. Relatively rapid regulatory processes, the now extensively studied 'up-' and 'down-regulations' in receptor number, would seem to provide a possible mechanism for rapid alterations in neural activity (for general reviews see refs. 16, 17 and 20). Studies of ACh receptors, both nicotinic and muscarinic 34, form a large part of the literature concerning receptor regulation, and include studies using a bewildering variety of preparations, ligands and conditions. As no general review is intended, only a fraction of the literature is cited in the following synopsis. It is generally accepted that AChR down-regulation is a consequence of either cellular depolarization elicited by electrical stimulation 1"24, various chemical agents or ions 1"6'24(but see ref. 23), or agonist activation of the receptor 6"10"19"22"24"2(''46"48"52'5~.For both depolarization and agonist stimulation, the observed down-regulation reflects a loss of receptor number rather than a change in receptor affinity 22"23'2~'5s. AChR up-regulation may also occur subsequent to cellular depolarization induced by either veratri dine 23 or high external potassium ion concentrations 23'52, blocking of sodium channels with tetrodotoxin ~, or blocking AChRs with antagonists 48~5s. Down-regulation appears to involve first the loss of surface AChRs ~2"4°followed by internal degradation of the receptor 1°19"22"26"4°'46'48.The amount of downregulation of AChRs (16% (ref. 1) to 88% (ref. 22)), and the time required for this to occur varies widely. In general, significant down-regulation occurs in a matter of hours 19"22"46, although more rapid (minutes) down-regulation has been reported 26. Recov-

ery following down-regulation appears to require protein synthesis 19'22'46 whereas up-regulation may not 23. The time courses of recovery and of up-regulation also appear to be measured in hours 2223"46'4s but seem to be longer than that measured for down-regulation. In order to investigate possible m A C h R regulation in the cortex, we have employed an in vitro cortical slice preparation in which the majority of the neurons are still alive s3'ss'57 (and companion paper~4). The advantage of this preparation is that we are able to study cortical neurons in a situation in which they are maintained with relatively intact circuitry and activity43, yet one in which we can effectively control a number of experimental parameters. In the companion paper 5~ we have shown that the majority of neurons in the preparation are viable, that m A C h R binding characteristics meet normal criteria, and that the distributions and characteristics of the m A C h R are quite similar for rat neocortex and cat visual cortex. In the present article we examine the effect of various alterations of bioelectric activity and/or receptor stimulation on mAChR regulation in adult rat neocortex. Preliminary data have been presented elsewhere 53. MATERIALS AND METHODS

General For details of the slice preparation and mAChR binding methods see the companion paper by van Huizen et al. 54. As in this first paper, we used [3H]Nmethyl scopolamine (NMS) to label surface mAChRs and [3H]quinuclidinyl benzilate (QNB) to label both surface and internal mAChRs 1°-12"~9"26. Unlike the previous paper, however, all incubations with labelled ligands (typically 5 nM concentrations) were carried out at 4 °C to avoid possible receptor regulatory effects induced by the use of ligands which are themselves antagonists. Saturation binding data were analyzed by Eadie-Hofstee plots as described elsewhere 59. Bioelectric activity of the slices was altered for various periods of time (2-4 h) by various substances before addition of the labelled ligand (see concentrations in Table I). Of these KC1, CoC12, MgC12 and AuCI 2 were obtained from Fisher Scientific. The remainder were purchased from Sigma. (For a general

73

review of the effects of the various substances, see refs. 9, 14 and 33.) These substances included those added to depolarize the neurons (e.g. veratridine and high external potassium (K+out)). Glutamate and picrotoxin would be expected to increase general neural spontaneous activity, although the effects of glutamate may include activation of inhibitory circuits as well 13. y-Aminobutyric acid (GABA), a putative inhibitory neurotransmitter in the visual cortex 47 was used to depress neural activity by binding to G A B A receptors, one class of which act through a chloride

ionophore to hyperpolarize neurons 49. We used gold chloride (AuCI2) to achieve the same effect: High Cl-out should increase C1- diffusion into the cell to produce a hyperpolarization which is not linked to activation of GABA receptors. Synaptic transmission was blocked by addition of cobalt chloride (COC12) or magnesium chloride (MgCI2) which also block calcium channels both pre- and postsynaptically. Specific blockade of postsynaptic voltage-sensitive Ca 2÷ (ref~ 45) was achieved using nifedipine. Neural activity and sodium currents were suppressed by addition of

TABLE I

Effects of changes in bioelectric activity and agonist stimulation on mA ChRs T h e effects of preincubation at 2 2 - 3 7 °C for 4 h with the different substances on [3H]NMS and [3H]QNB binding in cortex slices (in percent difference from control specific binding). Preincubation and incubation conditions are similar in this and all following figures: all preincubations were for 4 h at 2 2 - 3 7 °C. All incubations with 3H-ligand ( - 5 nM) were for 2 h at 4 °C. In each experiment total binding was d e t e r m i n e d in 4 slices and non-specific binding, with 10 -4 M of unlabelled ligand, was determined in 2 slices. T h e n u m b e r s inside each graph represent the n u m b e r of separate experiments performed. Abbreviations in tables and figures: Ver, veratridine; K +, high potassium out; Glu, L-glutamic acid; PTX, picrotoxin; G A B A , y-aminobutyric acid; AuCI2, gold chloride; COC12, cobalt chloride; MgCI2, m a g n e s i u m chloride; T T X , tetrodotoxin; T E A , t e t r a e t h y l a m m o n i u m chloride; 4-AP, 4-aminopyridine; CsCI 2, cesiu m chloride; A C h , acetylcholine chloride; Carb, carbamylcholine chloride; Eth, ethanol (1% f.c.); + E t h , veratridine dissolved in ethanol. In Table I and subsequent figures, error bars indicate S.E.M.

Conc. (M)

Number mA ChRs: (% differencefrom control + S. E. M.) [3H]NMS

[3H]QNB - 3 7 . 6 _+ 7.9*** 8.1+4.0 - 4.5+3.9

Depolarizing agents Veratridine K+ L-Glutamic acid (HCI) Picrotoxin

10 -5 5 x 10-2 10 5 10 -5

-26.2 -11.3 - 11.6 - 10.4

Hyperpolarizing agents GABA AuCI 2

10-5 10 -5

- 5.8 + 7.1 - 8.5 + 9.7

17.6 + 7.4 - 2 0 . 4 _+ 3.4

Synaptic transmission/Ca2+-channel blockers CoCI 2 MgCI 2 Nifedipin

10 -2, 10-3 10 -2 10-5

+2.8,-11.2 - 1 0 . 7 _+ 5.6 - 8.8 + 1.0"

- 2 5 . 5 + 13.4 +22.0

Ion-channel blockers T T X (Na +) T E A ( K +) 4-Aminopyridine (K +) A p a m i n (K +) CsCI 2 (K +)

10 -5 10 3 10 -3 10-5 to 5.10 -5 10 3

+ -

- 2 7 . 6 _+ 9.0 + 6.4+4.7 - 1 6 . 2 + 11.2 +43.9 _+ 35.3 7.1

m A C h R agonists ACh-chloride Carbachol

10-5 10 -3

- 6.3 + 8.7 - 2 6 . 1 + 1.7"**

Miscellaneous Eth Veratridine + E t h

1% 10 s, 1%

+16.2 + 8.0 - 1 5 . 0 + 4.1"*

+ + + +

3.6*** 3.0** 4.9* 6.2

8.4 + 5.6 2.4+5.1 1.9 _+ 5.1 16.8 + 16.0 3.6 + 2.6

Significance was analysed by one sample t-test: *P < 0.05, **P < 0.01, ***P < 0.001 (one-tailed).

- 3 3 . 9 _+ 2.8* - 3 5 . 9 _+ 3.8***

74 t e t r o d o t o x i n (TTX), a sodium channel blocker. Different drugs were used to selectively block classes of potassium ion channels. The substances used included the quaternary a m m o n i u m ions tetraethyla m m o n i u m chloride ( T E A ) and 4-aminopyridine (4A P ) , cesium ions (CsCI2) and apamin, a neurotoxin derived from bee venom. Each of these substances was used to block certain of the 5 types of channels thought to be present in hippocampus and neocorrex 3s. (Some of these K+-channels a p p e a r to correspond to those described in other systemsS"14.) All of these substances were a d d e d 10-15 min before other substances such as veratridine, carbachol, etc. A g o n ist stimulation of m A C h R s was accomplished by addition of carbamylcholine chloride (carbachol) 6"m and A C h chloride. All preincubations were at 2 2 37 °C and the incubation buffer containing the labelled ligand did not contain the preincubation substance.

Autoradiography A f t e r incubation with the a p p r o p r i a t e radioligand, slices were rinsed, and then m o u n t e d flat onto liver paste or frozen e m b e d d i n g medium (Tissue-Tek, O.C.T. compound, Miles Labs) and rapidly frozen in liquid freon. Sixteen-/~m-thick sections were cut in a cryostat, thaw-mounted onto subbed glass slides, and used to expose LKB Ultrofilm. The Ultrofilm was conventionally d e v e l o p e d and fixed after which the original sections were stained for Nissl substance to identify the cortical laminae.

Statistical analysis Differences in m A C h R number following alterations of bioelectric activity and agonist stimulation were analysed using a one sample t-test (one-tailed). The paired t-test was used for comparing the effects of an agent alone versus the effects of combinations of agents. R e c e p t o r binding values following various manipulations are usually expressed as percent difference from control m A C h R n u m b e r +__S . E . M . RESULTS Table 1 summarizes the various agents, the concentrations used to alter bioelectrical activity and/or to stimulate m A C h R s , and the effect on m A C h R number. The various agents in each group had little

or no effect on receptor n u m b e r at 4 °C (Fig. 1) under our conditions. In other studies some effects have been noted 25. This point is quite important since we show in what follows that some of these substances can alter m A C h R Bmax at 2 2 - 3 7 °C. A substantial decrease in m A C h R n u m b e r at 4 °C would imply a direct, competitive action on the receptor itself, and m a k e an i n d e p e n d e n t in vitro evaluation of true regulatory p h e n o m e n a impossible. Note that this problem can occur for both agonists and antagonists ( A C h and carbachol; atropine sulfate, NMS and Q N B , respectively; Fig. 1). In the first case, since the preincubation substance was not included in the incubation buffer and since agonists are removed reasonably rapidly, the p r o b l e m should not be severe. For carbachol, which does show some displacement of ['~H]N M S binding at a concentration of 10 -3 M, extensive washes (3 × 5 min) gave the same result as seen when simply replacing the preincubation with the incubation medium. These results (not shown) strongly suggest that little of the original preincubation buffer re-

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Fig. 1. Competition studies with [3H]NMS in the in vitro cortex slice preparation. A: the effects of veratridine, atropine sulphate, and carbachol on [3H]NMS binding (2 h at 4 °C) were examined. Veratridine had essentially no effect on [3H]NMS binding. Atropine displaced [3H]NMS with an IC50 of 1.3 × 10 s M. Carbachol showed effects on [3H]NMS binding only at the highest concentrations used (--28% at 10-3 M: - 5 % at 10-5 M). B: the effects of various ion channel blockers on [3H]NMS binding (2 h at 4 °C) were examined: TTX (Na+-channel), TEA, and 4-AP (K+-channel) had no significant effect on [3H]NMS binding.

75 mains in the well following replacement with the incubation medium. With antagonists, especially almost irreversible ones like those employed here, the problem is more complex. Our estimate of mAChR Bm~x at 30 °C (ref. 54) after a 2 h incubation in [3H]NMS or [3H]QNB may, in part, be a consequence of regulatory effects initiated by the antagonist ligands used. (As a further caveat we have observed that one commonly used depolarizing agent, veratrine, also displaces ligand binding to mAChRs. These latter results, and their interpretation, are presented more fully elsewhere 55. Similar effects were noted by other investigators31,32.)

Effects of depolarization on A ChRs Increases in bioelectric activity induced by veratridine (10 -5 M) led to a decrease in both [3H]NMS and [3H]QNB binding sites of 26.2% and 37.6%, respectively (Table I). The effects of veratridine, whose stock solution was first dissolved in 100% ethanol

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Fig. 2. Saturation binding isotherms (at 4 °C for 2 h) of [3H]NMS (A) and [3H]QNB (B) after a 4 h preincubation at 30 °C in the presence (+, ×) or absence (O, 0) of veratridine. The right side of the figure shows Eadie-Hofstee plots of the data. B~ax and Kd values for [3H]NMS were 1042 + 136 fmol/mg protein, and 6.2 + 1.1 riM, respectively in the control condition and 604 + 118 fmol/mg protein and 5.2 + 1.8 nM following veratridine treatment. Bmax and Kd values for [3H]QNB were 1432 _+301 fmol/mg protein and 8.7 + 2.8 nM, respectively in the control condition and 576 + 182 fmol/mg protein and 7.2 + 4,2 nM followingveratridine treatment.

(Eth), depended on the final well concentration of Eth. Larger decreases with veratridine were seen for lower (0.01%) Eth concentrations. The observation that higher concentrations of Eth diminished the effects of veratridine provides evidence that veratridine's effects are not due solely to cellular damage induced by this substance. A 1% Eth well concentration alone increased m A C h R number by 16.2% (Table I). E a d i e - H o f s t e e analysis 59 of saturation binding isotherms (Fig. 2) indicates that the loss of mAChRs reflects a lowered Bmax, without a significant change in Kd for both [3H]NMS (Fig. 2A) and [3H]QNB (Fig. 2B). In these experiments it was not possible to determine if the mAChRs lost from the internal pool were first removed from the surface of the cell, although in the Discussion we will attempt some estimate of relative surface and internal ratios for m A C h R down-regulation. The autoradiograms of sections from slices pre-incubated with veratridine (Fig. 3) show a loss of mAChRs ([3H]NMS binding) across all laminae in which mAChRs are normally present 54, although the loss in the middle and deep laminae is more pronounced. In a separate series of experiments various influences on veratridine-induced down-regulation and the rate of down-regulation were examined. The amount of veratridine-induced down-regulation increased with increasing temperature to 37 °C (Fig. 4A). At 37 °C, m A C h R down-regulation in response to veratridine treatment showed an initial plateau followed by a decline to 27.4% of the control value (Fig. 4B). A similar loss following a 2 h carbachol treatment was followed by an increase towards control values at longer preincubation times (Fig. 4C). Time to half maximal (at 4 h) down-regulation was about 30 min for carbachol and about 2 h for veratridine. Muscarinic down-regulation was also observed following preincubation in high K+out, glutamate, or picrotoxin (Table I). Of these, the effects of high K+out and glutamate on external m A C h R labelled with [3H]NMS were significant, showing a decrease in mAChRs of 11.3% and 11.6%, respectively.

Effects of hyperpolarization on mA ChRs Neither G A B A nor AuC12 treatment had a signifi-

76 cant effect on mAChR number labelled by [3H]NMS or [SH]QNB (Table I).

Effects of blocking synaptic transmission or selected ion channels Table I shows the effects on mAChRs of blocking Ca 2+-, Na +- and K+-channels. C o C l 2 and MgC12 produced small, but insignificant, decreases of mAChRs. Nifedepine had a significant effect (-8.8%) on surface mAChRs only. Preincubation with TTX at 2237 °C led to a small decrease in mAChRs, but had no effect on mAChRs when added during incubation at 4 °C (Fig. 1B). The decrease in mAChRs following blockade of sodium channels by TTX led us to examine the blockade of other ion channels. Since ACh is believed to alter K+-channel conductance, these channels seemed an obvious choice. In a series of experiments we examined the effects of TEA, 4-AP,

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lmm Fig. 3. Effects of veratridine on mAChR distribution. Autoradiograms are from 16-/~m-thick sections cut from a cortical slice incubated with [3H]NMS at 4 °C for 2 h after a 2 h preincubation at 22 °C in (A) control medium or (B) 10-s M veratridine. Sections were apposed to LKB Ultrofilm for 21 days to generate autoradiograms. Following the development of the autoradiograms, the sections were stained with Cresyl violet for Nissl substance. Both autoradiograms and stained sections were photographed individually for highest contrast, but to the same magnification. Sections of each point were aligned to identify the cortical laminae. Note the greater qualitative loss of [3H]NMS binding sites in laminae IV-VI than in laminae I-III after veratridine preincubation. See text for quantitative effects of veratridine treatment on [3H]NMS binding. Dorsal is up, medial is to the left for both slices. Note that [3H]NMS binding is less dense in the cingulate cortex (medial portion; see also 54; Fig. 1) than in the neocortex lateral.

201c Pre-incubation time (hr) 0 I -20-.

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Fig. 4. A: effects of temperature on veratridine-induced downregulation for [3HINMS (left) and [3H]ONB (right) in the cortex slice. All conditions as given in Table I, except for the variations in veratridine preincubation temperature. B,C: time course of (B) veratridine-induced and (C) carbachol-induced down regulation of [3H]NMS binding sites at 37 °C. Other conditions as in Table I, except for the variations in veratridine preincubation time.

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ber following a 4 h preincubation period at 22-37 °C for receptors labelled with [3H]NMS. The carbacholinduced down-regulation was even larger for [3H]QNB (33.9%). [3H]NMS binding sites were not significantly altered by ACh, unlike those labelled by [3H]QNB which showed a 35.9% decrease. Some of the loss of m A C h R may have involved a direct competitive interaction between carbachol and the mAChR, although this seems unlikely. The concentration in the well after replacing the preincubation medium with the incubation medium should not have been more than 10 -5 M carbachol, although this would not take into account possible residual, bound,

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apamin, and Cs + on m A C h R number. T E A had no effect on [3H]NMS binding to a concentration of 10 -3 M at 4 °C (Fig. 1B). After preincubation at 30-37 °C with 10 -3 M TEA, m A C h R number was also near control values. Similarly, preincubation with 4-AP and CsCI 2 had little effect on [3H]NMS binding unlike apamin which showed a substantial increase. Similar results were obtained using these K+-channel blockers with [3H]QNB.

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Effects of agonist stimulation on mA ChRs Table I shows that the addition of a 1 mM concentration of the muscarinic agonist carbachol, led to a highly significant decrease (26.1%) in m A C h R num-

Fig. 6. Effects of veratridine and carbachol alone and combined with "Iq'X for 4 h at 22-37 °C on [3H]NMS (A) and [3H]QNB (B) binding in cortex slices. Incubation condition as in Table I. Note that in this figure the numbers shown reflect data from these experiments only.

78 carbachol. At these presumed concentrations the amount of displacement was extremely low ( - 5 % , Fig. 1).

to maximal values for this incubation time (data not shown).

Effects of ion channel blockers on mA ChR down-regulation induced by veratridine, high K+o,t or carbachol

Combination of veratridine and carbachol effects on m A ChRs Experiments in which both carbachol and veratridine were added simultaneously showed a nearly linear summation (Fig. 5) for [3H]QNB, i.e., the combined effects of veratridine and carbachol were 88% of the total effects of separate veratridine and carbachol experiments added together. In contrast, the combined effects on [3H]NMS labelled m A C h R s were only 58% of separate determinations. These effects cannot be attributed to insufficient drug concentrations since at the normal experimental concentrations of veratridine and carbachoi (10 -5 and 10 -3 M, respectively) the down-regulation was already close

Addition of TTX (10 -5 M) totally failed to block veratridine- or carbachol-induced down-regulation of m A C h R s (Fig. 6). In contrast, addition of T E A , 4AP and apamin (Fig. 7) largely blocked down-regulation induced by veratridine while T E A and 4-AP but not apamin diminished that induced by carbachol. The effects of high K+out were not significantly affected by T E A in preliminary experiments. DISCUSSION The results of the present report support the no-

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1 2

Fig. 7. Effects of veratridine (left), carbachol (middle) and high K+out(right) alone and combined with TEA, 4-AP or apamin for 4 h at 22-37 °C on [3H]NMS (A) and [3H]QNB (B) binding in cortex slices. Incubation condition as in Table I. Note that in this figure, the numbers shown reflect data from these experiments only. v Significantly different from veratridine alone, P < 0.05.

79 tion that regulation of mAChRs occurs by a process involving alterations in the n u m b e r of receptors, both those on the external surface of neurons and those in the internal milieu. Down-regulation of both surface and total mAChRs in response to veratridine involved a change in B m a x , n o t Kd. Similar results have been reported following veratridine 23 and carbacholinduced 22'2658 down-regulation in other preparations. Depolarization induced in three different ways (with veratridine, high K+out and carbachol) all led to down-regulation for surface mAChRs. (Carbachol appears to depolarize some cortical neurons by blocking an outward K + current 28. Note that for some neurons carbachol may increase K + inward currentS,29.) Depolarization of cortical slices by veratridine led to a reasonably rapid (tl/2 ---2 h), temperature-dependent loss of 26.2% of surface mAChRs. Carbachol-induced down-regulation (26.1%) showed a faster rate of loss of surface mAChRs (tv2 = 30 min) than that found using veratridine. The amount and rate of m A C h R down-regulation is similar to that reported by other investigators 1°'a2'19'22'26'4°'46"48. The time course of down-regulation resulting from either veratridine or carbachol treatment was, however, complex, suggesting a more complex receptor elimination process than is often found 35,5°, but one that is similar in many respects to processes reported by others 26"46'4s. Replotting the data on a semi-log plot did not suggest a first order m A C h R down-regulation process. A non-first-order process of the type found here may be accounted for by postulating different populations of mAChRs. Indeed, M 1and M 2 mAChR subtypes have been characterized in neural tissue TM 56. Our own preliminary data 54 indicate that both of these m A C h R subtypes are present in rat neocortex in a M1:M 2 ratio of about 2:1. Additional complexities in the m A C h R down-regulation rate could be caused by the differential distribution of one (or more) receptor subtypes on various cellular loci, e.g., neurons vs glia, or more specifically, somata vs dendrites, etc. Quinolinic acid lesion studies in our laboratory have shown that both Ms and M 2 mAChR subtypes are neuronal, thus eliminating the first of these two possibilities 39 (and Shaw et al., manuscript in preparation). The differential loss of surface mAChRs illustrated in the autoradiograms of Fig. 3, however,

may lend support to the second possibility. While internalization of surface mAChRs is a likely explanation for the loss of [3H]NMS labelled mAChRs 12"4°, inactivation via a phosphorylation step with or without internalization is also possible TM. Using [3H]QNB, the loss of total mAChR was 37.6% and 35.9% for veratridine and carbachol, respectively. Since [3H]QNB binds to both internal and surface mAChRs (see ref. 54 for literature citations)' a loss of mAChRs thus labelled may reflect the loss of receptors from both the cell surface and the internal receptor pool. A secondary loss of internal mAChRs would indicate a possibly degradative (or phosphorylating) process and has been reported by other workers 7'52. Whether the loss of internal mAChRs in the present experiments is restricted to those mAChRs already inside the cell, or to those first removed from the cell surface awaits clarification. In spite of this uncertainty, it would seem to us valuable to be able to estimate the relative proportions of surface and internal mAChR down-regulation, and in the following discussion we will attempt such an estimate. We begin with our previous estimate of surface to internal m A C h R distribution based on the differential penetration of the ligands [3H]NMS and [3H]QNB54. Since the former binds to surface sites only, while the latter binds all mAChRs, calculating internal mAChRs can be determined by a single relation: R t - R s = R i , where R t -- total mACh'Rs labelled, R~ = surface mAChRs and R i = internal mAChRs. Our previous measurements had suggested that at 30 °C approximately 61% of mAChRs

TABLE II Percent loss mAChRs from surface or internal sites after different preincubation conditions

Surface mAChRs (Rs) were measured using [3H]NMS. Internal mAChRs were determined from the equation R i = Rt - R~, where R~is the total number of mAChRs labelled by [3H]QNB. Percent loss of surface or internal mAChRs following experimental preincubation were determined by the equations: for Ri: 100 × (Ri(exp) - Ri(comrol))/Ri(control); for Rs: 1 0 0 × (Rs(exp) -

Rs(control))/Rs(comrol)" Treatment

Surface (s)

Internal (i)

s:i Ratio

Veratridine High K+out Carbachol ACh

-26.2 -11.3 -26.1 - 6.3

-55.3 - 3.1 -54.8 -77.2

1:2.1 1:0.3 1:2.1 1:12.2

80 were on the surface while 39% were internal• Estimating the number of m A C h R s left after the various preincubation treatments ('experimental') is of the same form: Rt(exp)-Rs(cxp) = Ri(exp). Given the surface to internal ratios of 61:39 we can then calculate the percent loss of surface or internal m A C h R s by the following equations: for Ri: 100N (Ri(exp)- Ri(control))/

Ri(control) ; for Rs: 100x (Rs(exp)- Rs(control))/Rs(comrol ). The results of these calculations are given in Table II and show the percent loss of m A C h R s from the surface or internal pools and their proportions after veratridine, high K+out, carbachol, and ACh preincubations. From Table II it is apparent that the different preincubation treatments have quite different effects on surface and internal m A C h R s . For example, inwardly directed potassium current (high K+out) affects only surface mAChRs. In contrast, veratridine and carbachol affect both sites although the main effects are on the internal sites while A C h ' s actions are exclusively on internal m A C h R s following preincubations of the concentrations and lengths used here. Other investigators have also noted differences in the effect of ACh and carbachol when both drugs were used at the same high concentrations 22. One reason why the ACh and carbachol effects differ may be that they bind to different membrane sites. In our preparation carbachol does not appear to have a high affinity for the m A C h R (Fig. 1 and see ref. 54 for additional citations) since we have observed little displacement with this agonist except at very high concentrations (--28% at 10-3 M, --5% at 10 -5 M) either in the present study or in cat visual cortex 42. These results may suggest that carbachol exerts much of its influence on m A C h R regulation indirectly, perhaps at a site linked to, but not identical with, the m A C h R . The actual amount of down regulation of m A C h R s may indeed be higher than we report here due to the fact that some 15-30% of neurons in a slice are not viable s4 and hence incapable of active receptor regulation. Taking this fact into account, the down-regulation seen after veratridine and carbachol treatments are probably underestimates of the true values, perhaps by as much as a third. Additionally, the manipulation of the tissue prior to the preincubation may itself induce depolarization via spreading depression. This depolarization might, in turn, induce a m A C h R down-regulation. If true, then the down-regulation seen with the usual preincubation

treatments may be superimposed on an already present down-regulation and thus give values which are apparently smaller than the actual regulatory capacity of the system. One of the largest effects on m A C h R down-regulation observed in the present study followed exposure to veratridine, a drug which decreases sodium channel inactivation leaving these channels open for more than 50 times longer than normal 9'1433. TTX failed to block veratridine's effect. While veratridine exerts its primary effect on Na+-channels, an increased K+-flux may be expected to occur as well if not all Na+-channels have been blocked by the TTX, or if other ion channels, e.g., Ca 2+ are still active. Additionally, veratridine increases the relative K +permeability 14. Depolarization induced in other ways, notably by increased K+out, also led to a downregulation of m A C h R s as observed in other studies 3 (some reports 23, however, have shown the opposite effect). Our results suggest that the observed downregulation is not strictly related to depolarization and that it is not Na+-dependent. Furthermore, m A C h R regulation in the cortex slice does not appear to be directly Ca2+-dependent, unlike in other preparations--, since a general block of Ca2+-channels with either Mg 2+ or Co 2+ had little effect. More specific blocking of postsynaptic voltage-dependent Ca 2+channels with nifedipine, however, gave a small decrease in m A C h R s . Hyperpolarization of neurons by either G A B A (which acts to increase inward CI- conductance) or AuCI 2 had little effect on m A C h R regulation. Carbachol-induced down-regulation also appears to act through a Na +-, Ca z+- or Cl--independent process. Like ACh, carbachol has been shown to alter a K + current ~'2s and its effects on the m A C h R are clearly not blocked by TTX. Little effect in m A C h R number was noted following the addition of K+-channel blockers. However, the blocking effect of these substances on down-regulation elicited with veratridine, carbachol and, to a smaller extent, high K+,,ut, leads us to suggest that changes in K + current, in whatever direction (the inward K + current being unlikely to occur in nature), might result in m A C h R down-regulation. The relative failure of alterations in Na +, Ca 2+ or C1- ion channels to alter m A C h R number in this preparation lends further support to this notion. The relative po-

81 tencies of the 3 main K÷-channel blockers employed in these experiments (TEA, 4-AP and apamin) may suggest the specific K÷-channel through which veratridine and carbachol exert their effects (Table I and Fig. 7). An increasing order of effectiveness in veratridine-induced down-regulation was found such that apamin > T E A > 4-AP. Since T E A is known to block both Ca 2÷- and non-Ca2÷-dependent K÷-chan nels, while apamin is only effective in Ca2÷-depen dent K÷-channels, our results suggest that both types of K÷-channel normally participate in m A C h R downregulation, although to differing degrees. The less effective, but still substantial, effects of 4-AP on mAChR regulation support this view. The differential effectiveness of T E A from apamin may derive in part from T E A ' s known action on all Ca2÷-dependent K ÷channels (i.e. large conductance Ca2+-dependent K÷-channels 5) versus apamin's effects on only a subclass of the channels. In contrast to the effects of K +channels blockers on veratridine, only T E A and 4AP were able to block carbachol-induced m A C h R down-regulation, while apamin was without effect. These results clearly show that the Ca2+-dependent K÷-channels are not involved in this type of agonistinduced down-regulation. Preliminary experiments from our laboratory have shown a phorbol ester-induced m A C h R down-regulation 21, which can be blocked by TEA, but not by apamin (Jia et al., manuscript in preparation). (Phorbol esters stimulate PKC.) These results, taken together with the present effects of K÷-channel blockers on carbachol-induced m A C h R regulation, may suggest a common protein kinase C (PKC) pathway for agonist induced receptor regulation. Studies in progress are directed at clarifying this issue. The down-regulation observed following the various experimental manipulations may result from effects not only on the different K +channels, but also on surface or internal receptors. High K+outappear to act mostly on surface mAChR. Carbachol and veratridine show an approximate 1:2 effect on surface vs internal receptors while ACh appears to act mainly on internal receptors (see Table II). Based on these results we argue for separate mechanisms of regulation of surface and internal mAChR; all, however, are based on changes in K ÷ currents. Given the above data, we speculate that specific

receptor regulations might occur in response to changes in ionic current in specific ion channels induced by the relevant neurotransmitters. Thus, in the present study changes in K÷-channel currents led to decreases in mAChRs, while in muscle myotubes, in which ACh stimulation leads to increased Na ÷ current, down-regulation of AChRs appears to be linked to alterations in Na 2÷ current 6. It this principle is a general one, we may speculate that the regulation of other receptor populations in the neocortex, e.g., the G A B A A and quisqualate follow changes in G A B A or glutamate-induced CI- or Na ÷ currents, respectively. Studies in progress are directed at this issue. It is of interest that no manipulation in the present study reliably gives m A C h R up-regulation. Since depolarization leads, however indirectly, to m A C h R down-regulation, it was surprising to find that TTX which blocks Na÷-induced depolarization, and in other preparations gives up-regulation 3, had an insignificant effect on m A C h R number, yet one similar in direction to that observed with veratridine. How can we account for this? One possibility is that up-regulation occurs only after longer periods of incubation than we employed here as been suggested by other studies 23. If this is a correct interpretation, then upregulation may be expected to occur at longer times following T r X and K÷-channel blockers. Another possibility is that m A C h R up-regulation following a loss of neural activity occurs indirectly and is mediated through heterospecific receptor regulatory mechanisms ~7. Further experiments are needed to clarify this issue. The relatively rapid alterations of mAChRs which we have observed may underlie some of the rapid alterations of neural activity observed following various forms of synaptic stimulation 4.37. Similar regulatory effects on other receptor populations may contribute further to dynamic alterations of cortical chemical circuitry. These regulatory effects on mAChRs and other receptor populations may further be dependent on a number of variables, including the regions of neocortex studied (we have shown elsewhere 42 that mAChRs binding differs in primary sensory from association cortex in the cat), the postnatal age of the animal, and the species under study 44. The latter are questions of ongoing investigations.

82 ACKNOWLEDGEMENTS

Prof. J. S i m m o n s for c o m m e n t s o n t h e m a n u s c r i p t . T h i s w o r k was s u p p o r t e d b y a K i l l a m P o s t d o c t o r a l

T h e a u t h o r s t h a n k D. W h i t e f i e l d , G . T r o o p , D .

F e l l o w s h i p to F . v . H . a n d a M e d i c a l R e s e a r c h C o u n -

M a r c h , G . P r u s k y a n d F. S t e f a n i f o r a s s i s t a n c e a n d

cil o f C a n a d a g r a n t ( t o M . W . , M . S . C . a n d C . S . ) .

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