Colocalization of muscarinic acetylcholine receptors and protein kinase C γ in rat parietal cortex

152 Molecular Brain Research, 18 (1993) 152-162 ',(': 1993 Elsevier Science Publishers B.V. All rights reserved 0169-328x/93/$06.00

BRESM 70589

Colocalization of muscarinic acetylcholine receptors and protein kinase C7 in rat parietal cortex

E.A. V a n d e r Z e e ~, A .D. S t rosberg b B. Bohus ~ and P .G.M. Lu i t en ~'

" Department of Animal Physiology, Unit,ersio, of Groningen, Haren (The Netherlands) and ~' Laboratoire d 'hnmunopharmacologie Mol~;culaire, lnstitut Cochin de G~n~tique Mol~culaire, Paris (France)

(Accepted 24 November 19921

Key words: Acetylcholine; Muscarinic acetylcholine receptor; Codistribution; Carbachol; Neocortex; Protein kinase C: Rat

The present investigation analyzes the cellular distribution of muscarinic acetylcholine receptors (mAChRs) and the 3' isoform of protein kinase C (PKC) in the rat parietal cortex employing the monoclonal antibodies M35 and 36G9, respectively. Muscarinic cholinoceptive neurons were most present in layers 2, 3 and 5, whereas most PKCy-positive cells were found in layers 2, 5 and 6. Under normal, non-stimulated conditions, approximately 58% of all muscarinic cholinoceptive neurons were immunoreactive for PKCT. Conversely, nearly all PKCy-positive neurons were M35-immunoreactive. Although both pyramidal and nonpyramidal neurons express the two types of protein, the pyramidal cell type represents the vast majority. Of all cortical neurons, the large (15-25 p,m in diameter) muscarinic cholinoceptive pyramidal neurons in layer 5 express the y isoform of PKC most abundantly and most frequently. Approximately 96% of these cells are immunoreactive for PKCT. Stimulation of m A C h R s by the cholinergic agonist carbachol resulted in a pronounced increase in the intensity of 36G9 immunoreactivity, which may suggest that the mAChRs are functionally linked to the colocalized PKCy. No change was found in the number of 36Gg-immunoreactive neurons. In contrast, the number of immunocytochemically detectable muscarinic cholinoceptive neurons increased by approximately 38% after carbachol stimulation. The high degree of codistribution in cortical neurons of both transduction proteins suggests a considerable cholinergic impact upon the regulation of PKCT, a candidate key enzyme in cortical learning and memory mechanisms.

I N T R O D U C T I O N

In the last decade, the cholinergic system of the forebrain received increasing attention in relation to function and dysfunction of learning and memory mechanisms, with special focus on the cholinergic synapse in the cerebral cortex 5'6'~3. The cerebral cortex receives its topographically organized cholinergic input from the neurons of the nucleus basalis magnocellu- laris (nbm) as has been accurately established in recent years by several investigators 3L35'36'47. That lesions of

the nbm as well as cholinergic receptor blockade were shown to severely impair performance on a variety of memory tasks ~7'3°'3~'46 supports the view that the forebrain cholinergic system innervating the cerebral cortex is intimately involved in cognitive processes (see ref. 59 for review).

Acetylcholine (ACh) exerts its influence on cortical cholinoceptive target cells via two types of receptors:

muscarinic (mAChRs) and nicotinic acetylcholine receptors (nAChRs). mAChRs are G-protein-mediated receptors coupled to second messenger systems 4~5°. Part of the mAChRs are known to enhance phos- phatidyl inositol turnover, resulting in the production of inositol trisphosphate and diacylglycerol (see ref. 40 for review). Protein kinase C (PKC), a key enzyme for signal transduction and various neuronal plasticity mechanisms, is activated by diacylglycerol in the pres- ence of calcium and phospholipids 41. Activated PKC can, in turn, phosphorylate mAChRs 1~, inducing a decrease in mAChR functioning. In this way PKC exerts a physiological mechanism for feedback regulation in the strength of muscarinic responses ~5, So far, it is known that rat brain PKC consists of seven distinct isoforms, each with a characteristic distribution within the central nervous system (CNS) 211"23"24"27"45"60. Acti-

vated PKC has been implicated in long-term cellular regulation including the formation and maintenance of

Correspondence: E.A. Van der Zee, Depar tment of Animal Physiology, University of Groningen, P.O. Box 14, 9750 AA tlaren, The Netherlands. Fax: (31) 50-635205.

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memory 1,9'26'42. One of the most abundant PKC isoforms in the CNS is the 3' isoform24- PKCy is the major isoform present in the cerebral cortex, most frequently observed in the perikarya of pyramidal neurons 51.

In a number of recent reports it was shown by us and others that the muscarinic cholinoceptive target neurons of the nbm projection can be visualized immunocytochemically employing the monoclonal antibody M35 raised against purified mAChR protein 2'3"4'38'48'52"57'58. In addition, the cortical neurons uti-

lizing PKC3, as an intracellular key intermediate in the pathway from signal recognition to biological response has been demonstrated immunocytochemically employing the monoclonal antibody 36G9 raised against purified PKCy I°'lt'55. Since the interplay of mAChRs and PKCy may play a vital role in cortical learning and memory function, we currently examined their degree of coexistence by way of fluorescent immunocytochemical double-labeling techniques in the cerebral cortex of the rat. Analysis of the anatomical characteristics of the cortical cholinoceptive system utilizing PKCy may further substantiate our understanding of the contribution of the cortical cholinergic system in learning and memory functions. Part of this study has been presented in preliminary form elsewhere 53.

MATERIALS AND METHODS

Subjecls In this study 14 male Wistar rats (body weight 300 g) were used.

All animals were investigated for single- and fluorescent double- labeling for mAChRs and PKCy. The parietal cortex was studied in greater detail, with Zilles' atlas of the rat cerebral cortex as a standard anatomical reference 01.

Tissue preparation First, the animals were deeply anesthetized with 6% sodium

pentobarbital. Fixation of the brain of 10 rats was carried out by transcardial perfusion with 300 ml fixative composed of 2 -3% paraformaldehyde, 0.05% glutaraldehyde and 0.2% picric acid in 0.1 M phosphate buffer (PB) (pH 7.4) at a perfusion speed of 20 ml /min, which was preceded by a short prerinse of saline. The brains were removed from the skull and cryoprotected by overnight storage at 4°C in 30% sucrose in 0.1 M PB. Subsequently, the brains were coronally sectioned on a cryostat microtome at a thickness of 20 p.m.

Of 4 other rats, the brains were quickly removed after a short prerinse with ice-cold saline and put in ice-cold medium. The com- position of the medium was (mM): NaCI 119, KC1 2.5, MgSO 4 1.3, CaCI 2 2.5, NaHCO 3 26.2, NaHzPO 4 1.0, glucose 11. Brain slices containing the parietal cortex were cut at a thickness of 500/.Lm. The slices were transferred to aerated, warmed (370C) normal medium or medium containing 100 p.M carbachol, a cholinergic agonist. The slices were incubated in these media for 15-20 rain. Subsequently, the slices were fixed for 30 min at room temperature (RT) by immersion in the fixative described above. Afterwards the brain slices were cryoprotected by overnight storage at 4°C in 30% sucrose in 0.1 M PB and coronally sectioned on a cryostat microtome at a thickness of 20 /~m. Hereafter, the sections of the transcardially perfused brains and the sections of the immersion-fixed brain slices

followed a similar immunocytochemical procedure as described below.

Immunocytochemical procedure mAChR proteins and PKCy were visualized by means of the

monoclonal antibodies M35 and 36G9, respectively. The M35 monoclonal IgM antibody was raised in mice against muscarinic acetylcholine receptor protein purified from bovine forebrain homo- genates 2-4. The 36G9 monoclonal IgG antibody was raised in mice against purified bovine PKC7 protein l°'al. Extensive descriptions of their use in immunocytochemistry have been reported previously 38,48,52,55,57,58,

For single-labeling, free floating brain sections were incubated one overnight at 4°C under gentle movement with the primary antibody solution in phosphate buffered saline (PBS) containing mouse anti-mAChR IgM (M35, 1:200) or mouse anti-PKCy IgG (36G9, 1:200). After rinsing in PBS, the sections were exposed for 2 h at RT to biotinylated rabbit IgG anti-mouse IgM (/z chain-di- rected, F(ab')2 fraction, 1:200; Zymed) or biotinylated sheep anti- mouse IgG (1:200; Amersham) for M35 and 36G9, respectively. After thorough rinsing in PBS, all sections were incubated with Streptavidin-HRP (2 h at RT, 1:200; Zymed). The sections were again rinsed in PBS and Tris buffer, and reacted under visual guidance with diaminobenzidine (DAB) (30 mg DAB in 100 ml Tris buffer, pH 7.4) and 0.01% H 2 0 2. Finally, the sections were mounted and coverslipped for light microscopic inspection.

Double-labeling experiments for the study of colocalization of mAChRs and PKC3, in the sequence of 36G9/M35 were carried out with fluorescence techniques. For dual labeling, free-floating sections were sequentially exposed to each of the primary antibodies as for single labeling. The primary antibody step with 36G9 was followed by 2 h incubation at RT in Phycoerythrin-conjugated goat anti-mouse IgG (1:50; Tago). Upon completion of the 36G9 staining, the sections were incubated with M35 as described above, followed by biotinylated rabbit anti-mouse IgM (2 h at RT, 1 : 50; Zymed) and Fluorescein Isothiocyanate (FITC)-conjugated Streptavidin (2 h at RT, 1:50; Zymed). All incubations with fluorescent labels were performed in dark. After antibody processing the sections were mounted and coverslipped in a 1 : 1 mixture of PBS and glycerin. The sections were studied and photographed with a Ploemopak Leitz fluorescence microscope with the appropriate filter blocks for FITC and Phycoerythrin labels, yielding a green and red fluorescence, respectively. Standard control experiments were performed by (a) omission of either one or both primary antibodies in the incubation cycle, (b) incubation of 36G9 followed by the nonmatching secondary antibodies used for M35 and (c) replacing the primary antibody by normal mouse serum. In all cases the controls yielded negative results, i.e. absence of any detectable labeling, excluding the appear- ance of possible cross-reactivity of secondary antisera during the incubation cycle.

Degree of colocalization of M35 and 36G9 The degree of colocalization under non-stimulated and carba-

chol-stimulated conditions was quantified in greater detail in the parietal cortex (area 1; Zilles' atlas 61) of brain slices of 4 animals. Per animal, 1 brain slice of approximately 500 /.~m thickness was taken at the coronal level of Bregma - 1 . 3 or -3 .3 , according to Zilles' atlas 61. Per animal, 1 hemisphere of the brain slice was stimulated with carbachol, while the other served as a non-stimulated control. After tissue processing as described above, 2 sections per brain slice were selected on grounds of a complete cortical profile. One radially oriented cortical strip of 200/~m in tangential size per section (one per hemisphere) was analyzed from layer 1 through the border of layer 6b. In these strips, 13 frames (200 ~zm × 150/zm; 'cortical levels') covered the entire cortical profile. In these frames, the number of single- and double-labeled M35-positive cells was counted. In total, 8 strips of the carbachol-stimulated and 8 strips of the non-stimulated parietal cortex were analyzed. In the quantified cortical profiles neurons were considered to be double- labeled if the cell body a n d / o r dendritic processes revealed im-

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munoreactivity for both markers. Occasionally, single-labeled cell bodies but with double-labeled dendritic processes were encountered, or vice versa, probably caused by incomplete penetration of the respective antibodies. Nevertheless, as ment ioned before, these cases were recorded as double-labeled neurons. Of the 4 animals, all

numbers of counted neurons were summed per "cortical levels', and the percentage of colocalization with 36G9 calculated. The data of the total cells counted per ~cortical level' is presented in Fig. 5. In Table I, the data of the 'cortical levels' have been pooled per cortical layer, and the percentage of colocalization calculated.

Fig. 1. Photomicrographs depicting the cellular distribution of mAChR- (A) and PKCy-positive neurons (B) in the parietal cortex through layers 1 to 6 of perfusion-fixed tissue. For both markers, the large layer 5 pyramidal neurons (large arrows) with their apical dendrites traversing the superficial layers (small arrows) display the strongest immunoreactivity. In the deeper layers, immunoreactive pyramidal-like neurons (arrowheads)

predominate. The cortical layers are indicated in the right of B. Bars (A.B) = 100 #m.

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RESULTS

Distribution patterns of M35 and 36G9 immunoreactivity A consis tent d is t r ibut ion pa t t e rn of M35-positive

neu rons was appa ren t for all animals s tudied through-

out the parietal cortex. A n overview of the parietal

cortex for the M35-positive neu rons is given in Fig. 1A.

In short, the cellular s taining with M35 in the cortex

revealed a clear l aminar distr ibution. Most M35-posi-

tive neu rons were present in layers 2 - 3 and 5. In the

@

i

Fig. 2. Immunofluorescent double-labeling for M35 (left panel) and 36G9 (right panel) in layer 5 of the parietal cortex of perfusion-fixed tissue. A high degree of colocalization becomes apparent (A,B), both in the cell bodies and processes. However, besides double-labeled neurons (small arrows in C and D), some small pyramidal and non-pyramidal neurons appear to be M35 single-labeled (large arrows in C and D). Bars

(A,B) = 30/~m; (C,D)= 15/xm.

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Fig. 3. The effects of 15-20 min 100/xM carbachol stimulation in brain slices on the immunoreactivity for 36G9 (B) and M35 (D) compared to non-stimulated brain slices (left panel). A clear increase in 36G9 immunoreactivity is found after mAChR-stimulation (A versus B). Carbachol stimulation induced a redistribution of the immunoreactivity to mAChR, most notably in the basal dendrites of the layer 5 pyramidal neurons (C

versus D; small arrows point at the dendritic arborizations). Bars (A,B) = 130 ~m; (C,D) = 20/zm.

superficial layers immunoposi t ive apical dendr i tes of

layer 5 pyramidal neurons, t raversing layer 4 (small

arrows in Fig. 1A), b ranched frequently. The basal

dendri t ic complexes of layer 5 pyramidal neurons

formed a band of immunoreac t ive processes, e i ther cut

longitudinally or transversely. Layer 4 and, to a lesser

extent, layer 6 of the parietal cortex showed lower

numbers of immunos ta ined neurons. In layer 6 pyrami-

dal-like neurons were f requent ly immunoreact ive . In

addit ion, M35-posit ive neurons were observed in sub-

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layer 6b of the parietal cortex, positioned adjacent to the white matter of the corpus callosum. Some M35- positive astrocytes were encountered, most frequently in the superficial layers and the corpus callosum. The

numerical density of M35-positive neurons through the cortical profile is depicted in Fig. 5.

An overview of the parietal cortex for the 36G9- positive neurons is given in Fig. lB. Like for M35, little

Fig. 4. Photomicrographs depicting the fluorescence double-labeling of mAChR (left panel) and PKCy (right panel) in brain slices after carbachol stimulation in the layers 2, 3 and 4 (A,B) and 5 (C,D: low power; E,F: high power). Clearly double-labeled neurons were found (arrow heads in A,B and C,D), while also numerous M35 single-labeled neurons could be discerned throughout the cortical layers (arrows in A,B and C,D). In E and F, double-labeled (arrow heads), M35 single-labeled (thin arrow) and occasionally 36G9 single-labeled neurons (thick arrow) are

present. Bars (A,B and C,D) = 30/.Lm; (E,F) = 15 p.m.

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individual variation in the distribution of 36G9-positive neurons was found. 36G9 Immunoreactivity was present in the cytoplasm of the cell bodies and the dendritic arborizations. Occasionally, some pyramidal neurons showed weak nuclear staining. The distribution of 36G9-positive neurons in the parietal cortex from layer 1 to 6 partly parallelled the distribution of M35-positive cells. Most 36G9-positive neurons were present in layers 2, 5 and 6. In layer 2, multipolar and bipolar neurons with associated dendrites were strongly stained. In addition, in layers 1 and 2 the neuropil revealed dense immunoreaction. In layer 5, conspicu- ously the large pyramidal neurons (15-25 /xm in size) with associated basal and apical dendrites, the latter traversing layer 4 and branching in the superficial layers, were strongly 36G9-positive. In layer 6, different types of cells displayed 36G9 immunoreactivity. No- tably the pyramidal-like neurons and the small bipolar ceils in layer 6b were immunopositive. In contrast, layers 3 and 4 contained few 36G9-positive neurons. The sparsely immunoreactive neurons in these layers were often of the bipolar type with perpendicularly oriented dendrites. 36G9-Positive astrocytes were only rarely encountered. The numerical density of 36G9- positive neurons appeared to be identical to the numerical densisty of the M35/36G9-positive neurons shown in Fig. 5A (open circles), since nearly all 36G9- immunopositive neurons are M35-immunoreactive (see below).

Colocalization of M35 and 36G9 immunoreactivity Fluorescent double labeling revealed a characteris-

tic distribution of M35/36G9 double-labeled somata. In these double-labeled neurons, both markers were present in the cell body and the dendritic processes, most prominently in the apical dendrites of the large layer 5 pyramidal cells (Fig. 2). The cortical profile through all layers showing the total number of M35- positive cells and the number of double-labeled cells revealed that the largest proportion of colocalization was found in layers 2, 5 and 6 (Fig. 5; Table I). Both pyramidal and non-pyramidal neurons appeared to be double labeled. However, the highest degree of coexistence was found in the large pyramidal neurons of layer 5 (Fig. 2), which were 15-25/ . tm in size. Of 310 sampled large M35-positive pyramidal neurons, 297 (95.8%) appeared to be double-labeled. Pooling all layer 5 neurons, 59.7% (Table I) of the M35-positive neurons was found to be 36G9-positive. Pooling the data of the 'cortical levels' per cortical layer, 58.2% of the M35-positive neurons appeared to be 36G9-immunoreactive (Table I). Conversely, only occasionally single-labeled 36G9-positive cells were found (see Fig.

TABLE I

Percentage ~)f n~4ChR-positil'e neurons colocalized with PKCy in the different cortical layers o f the parietal cortex under non-stimulated conditions and after carbachol stimulation

The percentage of mAChR-posit ive neurons found to be colocalized with PKCy differs considerably between the cortical layers. The highest degrees of codistribution is found in layers 2 /3 , 5 and 6 under normal, non-stimulated conditions. After carbachol stimulation, the overall degree of colocalization is reduced from 58.2% to 44.6%, mainly caused by the changes in layers 2 /3 , 4 and 6. In these layers, an increase in the number of M35-positive neurons was found after carbachol stimulation, whereas no apparent change in the number of 36G9-positive somata was observed.

Layer Non-stimulated Carbachol-stimulated ('cortical let~el')

1 (1) 40.0 54.5 2 / 3 (2, 3, 4) 49.9 37.9 4 (5, 6) 30.9 8.7 5 (7, 8) 59.7 56.2 6 (9 to 13) 81.6 67.8 All (1 to 13) 58.2 44.6

4 E - F for an example of a 36G9 single-labeled neuron). Of all 36G9-positive neurons approximately 5% was found to be single-labeled.

M35 and 36G9 binding characteristics after carbachol stimulation

The distribution pattern of M35- and 36G9-positive neurons in immersion-fixed brain slices resembled that of perfusion-fixed brain sections. After 15-20 min of 100 /xM carbachol stimulation, however, a strong and pronounced increase in the intensity of the 36G9 immunoreactivity was observed (Fig. 3A,B). This increase was present in the cell bodies and dendritic processes of both pyramidal and nonpyramidal neurons in layers 2 /3 , 5 and 6. However, the total number of 36G9-positive neurons did not change dramatically (non-stimulated versus carbachol stimulated: 1749 versus 1818 neurons [approximately 4% increase in the number of 36G9-1abeled neurons]). In contrast to the observed changes in 36G9 immunoreactivity, the intensity of the M35 immunoreactivity did not notably change, but the number of M35-positive neurons markedly increased (non-stimulated versus carbachol stimulated: 2835 versus 3912 neurons [approximately 38% increase in the number of M35-positive neurons]). The cortical profiles of the numerical density of M35-positive neurons under non-stimulated conditions and after carbachol stimulation are depicted in Fig. 5. The increase in M35-positive neurons after carbachol stimulation was most apparent in layers 4 and 6 (Fig. 5C). Besides a numerical change of M35-positive cells, a change in M35 immunoreactivity was found predominantly in the basal dendrites of the layer 5 pyramidal neurons (arrows

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CORTICAL LEVEL Fig. 5. Cortical profiles through the parietal cortex showing the number of M35-positive and M35/36G9-posit ive neurons in brain slices. The number of cells were counted in thirteen adjacent frames ('cortical level') through the cortex from layer 1 to layer 6. The corresponding cortical layers are indicated in the bar at the top. Under non-st imulated conditions (A) the number of M35-positive neurons (closed circles; o) are highest in layers 2, 3 and 5. Double- labeled neurons (open circles; ©) were most frequently observed in layer 2, 5 and 6. After carbachol stimulation (B), an increase in the number of M35-positive neurons (closed triangles; A) was found. The distribution of M35-positive cells under normal conditions and after carbachol stimulation (closed circles (e) and closed triangles (A), respectively) found in A and B are combined in C. The carbachol induced increase is most noticeable in layers 4 and 6. However, there was no increase in the number of double-labeled

neurons (open triangles ( zx ) in B).

in Fig. 3D). The immunostaining of these dendritic processes was more pronounced and more widespread (Fig. 3C,D). Similar carbachol-induced changes in M35 and 36G9 immunoreactivity were found in other brain regions such as the hippocampus and striatum.

Colocalization of M35 and 36G9 immunoreactivity after carbachol stimulation

After carbachol stimulation similar cellular double- labeling characteristics as to those described for the non-stimulated condition were found (Fig. 4), with

both markers present in the cell body and the dendritic processes. However, due to the increase in the total number of M35-positive neurons in layers 4 and 6 in the absence of changed numbers of 36G9-positive cells, a considerable decrease in the ratio of M35-positive neurons immunoreactive for 36G9 was found in these layers (layer 4: 30.9% versus 8.7%; layer 6: 81.6% versus 67.8% for non-stimulated versus carbachol stimulated conditions, respectively; Table I). Pooling the data of the 'cortical levels' per cortical layer of the carbachol stimulated brain slices, 44.6% of the M35- positive neurons were found double-labeled (Table I).

DISCUSSION

The present study shows that more than half of all muscarinic cholinoceptive neurons in the parietal cortex express PKCy in a laminar-specific fashion. Con- versely, nearly all cortical neurons utilizing PKCy appeared to be muscarinic cholinoceptive. Carbachol stimulation suggests that the coexpressed PKCy is functionally linked to the mAChRs in the cholinoceptive cortical neurons.

Binding sites recognized by M35 and 36G9 Currently, the exact epitope on the mAChR protein

recognized by M35 is unknown, but M35 most likely recognizes a conformational determinant only present on the native receptor 3'4. Although M35 can partly mimic the physiological effects of ACh 33, occupation of the agonist and antagonist binding sites on the mAChRs by specific ligands could not block M35 binding (Van Huizen et al.58; our own unpublished observations). This is in line with the present data, showing that carbachol binding to mAChRs in brain slices did not prevent M35 binding. Since M35 does not discriminate between the different mAChR subtypes 52'54, the epitope must be localized on a part of the receptor with high level of homology between the subtypes.

The epitope of 36G9 is well characterized and is located on the regulatory domain of PKCy on the amino acid residues between positions 164-19711. 36G9 Binding to PKCy is affected by cofactor binding of phorbol esters and phospholipids 1°, and 36G9 immunoreactivity is enhanced in behaviorally activated neurons 55.

Distribution patterns of M35 and 36G9 immunoreactivity The distribution pattern found for M35 in the rat

parietal cortex resembled previously described distribution patterns 52'57. The cortical profile of M35-positive neurons, showing the highest densities in layers 2/3, 5 and 6b, parallelled the findings of the laminar distribu-

160

tion of the responses to ACh observed in the first somatosensory cortex of unanesthetized rats s. In the latter study, the highest percentages of neurons excited by ACh were found in layers 2 /3 , 5 and 6b. We found the highest numbers of muscarinic cholinoceptive neurons in the same layers. After carbachol stimulation, we encountered an increase in the number of muscarinic cholinoceptive neurons in, particularly, layer 4. This finding appears in contrast with the observations of Bassant and coworkers s, who found a relatively low percentage of cholinoceptive neurons in this layer. However, if the cholinoceptive layer 4 neurons were excited by whisker stimulation, a response suppression was found after ACh application 12. These results indi-

cate an inhibitory action of ACh in layer 4, that can only be recorded by electrophysiological methods after induction of neuronal activity by application of somatic sensory stimuli.

The distribution pattern observed for 36G9 in the parietal cortex resembled previously reported PKCy distribution patterns, employing different antibodies raised against the y isoform 4s'sl. The PKCy-positive

cell bodies were mostly seen in layers 2, 5 and 6, and the vast majority was represented by pyramidal neurons. The large muscarinic cholinoceptive layer 5 pyramidal neurons typically expressed abundant amounts of PKCy. This observation suggests that these neurons are amongst the most active cholinoceptive neurons of the parietal cortex. Indeed, layer 5 of the parietal cortex receives a dense cholinergic innervation 14'22'37,

providing cholinergic terminals that contact apical and basal dendrites of layer 5 pyramidal neurons 22.

M35 and 36G9 binding characteristics after carbachol stimulation

The increase in 36G9 immunoreactivity in brain slices after mAChR stimulation with carbachol may reflect activation a n d / o r down regulation of PKCT and suggests that the y isoform of PKC is functionally linked to the mAChRs in double-labeled cortical neurons. Carbachol applied to cortical slices stimulates phosphoinositide hydrolysis and subsequently activates PKC Ls'~6. 36G9 recognizes the regulatory domain of both intact and trypsin-cleaved PKCy ~°. Biochemical data show that 36G9 recognizes purified PKCy protein with high affinity in cofactor-free conditions, and that binding of 36G9 is inhibited after concomitant diacylglycerol analog and phospholipid binding ~°. If these in vitro binding characteristics of 36G9 are comparable to in vivo conditions, one may expect a decrease in 36G9 immunoreactivity after PKCT activation due to diacylglycerol and phospholipid binding. The observed in-

crease in 36G9 immunoreactivity after carbachol application, therefore, may indicate that (a) 36G9 binding characteristics to PKCy are different for in situ localized PKCT and purified PKCy, or (b) PKCy activation is followed by proteolytic cleavage, which results in regulatory fragments 2s recognized by 36(}9. The activated form of PKC appears to be a target of proteolysis by calpain 2°. The ' formed regulatory fragments may play some role in the control of cellular function 2s. There is ample evidence demonstrating PKC activation through mAChR stimulation ~5'"'. However, carbachol acts on both mAChRs and nAChRs. These two types of cholinergic receptors are coexpressed with a high incidence in cortical neurons 4s57. Since Messing and

coworkers ~'~ showed that both muscarinic and nicotinic agonists in an additive way stimulate rapid activation of PKC in PC12 cells, part of the presently observed increase in PKCT immunoreactivity might have been induced through nAChR stimulation.

Activation of mAChRs through carbachol stimulation resulted in an increase of detectable mAChRs (notably in the dendritic part of the neurons) employing the monoclonal antibody M35. This is in agreement with biochemical data suggesting that M35 most likely recognizes a conformational determinant present on the active receptor ~4. The increase in number of M35- immunoreactive neurons after 15-20 min carbachol stimulation of brain slices is most likely due to unmask- ing of the M35 epitopc of membrane incorporated mAChRs, predominantly in the dendritic parts of the neurons. This might be induced by rapid internaliza- tion of the activated mAChRs, since in rat brain ho- mogenates mAChRs are translocated to the cytoplasm 5-10 rain after carbachol stimulation ~. Furthermore, short carbachol exposure (100 #M, 15 rain to 1 h) to cultured human fibroblasts induced sequestration, in- ternalization and subsequent redistribution of mAChRs as visualized by M35 a~.

Functional consequences of tile colocalization of m/tCht(~ and PKCy

The present results showed that 44.6% (after carbachol stimulation) to 58.2% (under non-stimulated conditions) of all M35-positive neurons express PKCy, suggesting that approximately half of all muscarinic cholinoceptive neurons in the parietal cortex use the y isoform of PKC for signal transduction. The PKCy- negative neurons most likely utilize cAMP as the pre- dominant second messenger system 4°, or contain other PKC isoforms. Colocalization of mAChRs with the non-PKCy isoforms, however, does not frequently oc- cur in rat neocortex 5~.

The cortical cell population containing PKCy appeared to be almost entirely muscarinic cholinoceptive. This high degree of colocalization of PKCy and mAChRs found in the present study suggests a pre- dominant impact of the cholinergic basal forebrain system upon the activation of this PKC isoform. In turn, the activated PKC may exert a physiological feedback upon the mAChRs, often leading to a decrease in mAChR function ~5"2~'25. Understanding the role of PKCy in muscarinic signal transduction may shed more light on the cholinergic contribution to cortical learning and memory mechanisms.

PKC is known to play an important role in the stabilization of long-term potentiation (LTP) 7, a phe- nomenon of a long-lasting enhancement of neuronal activity after repetitive synaptic stimulation related to learning and memory processes 49. LTP can be induced by cholinergic agonists in concord with glutamate in the parietal cortex 34. Therefore, the cholinoceptive neuronal cell group in the cortex should be considered as an important substrate for learning and memory related processes, with emphasis on those muscarinic cholinoceptive neurons expressing PKCy. Cortical cholinergic activity is, for example, crucial for proper passive shock avoidance performance 44. Preliminary findings revealed that the behaviorally activated cholinoceptive neurons in the rat neocortex displayed increased immunoreactivity for mAChRs, PKCy as well as microtubule-associated protein 2 (MAP2) 56. The description of the cell type and distribution of the cortical cholinoceptive neurons utilizing PKCy may further substantiate our understanding of the role of the cholinergic system in (cortical) learning and memory functions in general, and particularly in aging and Alzheimer's disease.

Acknowledgements. We would like to thank J. Gast for technical assistance and C. Streefland for helpful contribution to the paper. The 36G9 was kindly provided by S. Cazaubon.

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Colocalization of muscarinic acetylcholine receptors and protein kinase C γ in rat parietal cortex