Muscarinic M2 receptor mRNA expression and receptor binding in cholinergic and non-cholinergic cells...

Neuroscience Vol. 47, No. 2, pp. 361-393, 1992 Printed in Great Britain

0306-4522/92 $5.00 + 0.00 Pergamon Press plc

0 1992 IBRO

MUSCARINIC M, RECEPTOR mRNA EXPRESSION AND RECEPTOR BINDING IN CHOLINERGIC AND

NON-CHOLINERGIC CELLS IN THE RAT BRAIN: A CORRELATIVE STUDY USING IN SITU HYBRIDIZATION

HISTOCHEMISTRY AND RECEPTOR AUTORADIOGRAPHY

M. T. VILAR~, K.-H. WIEDERHOLD, J. M. PALACIOS and G. M~GOD Preelinical Research, Sandoz Pharma Ltd. 386/228, CH4002 Base& Switzerland

Abstract-The goal of the present study was to identify the cells containing mRNA coding for the m2 subtype of m mcarinic cholinergic receptors in the rat brain. In siru hybridization histochemistry was used, with ohgonucleotides as hybridixation probes. The distribution of choline@ cells was examined in consecutive sections with probes complementary to choline acetyltransferase mRNA. Furthermore, the microscopic distribution of mumarini c cholinergic binding sites was examined with a non-selective ligand ([3H]N-methylscopolamine) and with ligands proposed to be M-selective ([3H&kenmpine) or M,-selective ([aH]oxotremorine-M). The majority of choline acetyltransferase mRNA-rich (i.e. cholmergic) cell groups (medial septum-diagonal band complex, nucleus basalii, pedunculopontine and laterodorsal tegmental nuclei, nucleus parabigeminalis, several motor nuclei of the brainstem, motoneurons of the spinal cord), also contained m2 mRNA, strongly suggesting that at least a fraction of these receptors may be presynaptic autoreceptors. A few groups of cholinergic cells were an exception to this fact: the medial habemtla and some cranial nerve nuclei (principal oculomotor, trochlear, abducens, dorsal motor nucleus of the vagus). Furthermore, m2 mRNA was not restricted to cholinergic cells but was also present in many other cells throughout the rat brain. The distribution of m2 mRNA was in good, although not complete, agreement with that of binding sites for the M, preferential agonist [3~xotremorine-M, but not with ~H@emepine binding sites. Regions where the presence of [3H]oxotmmorine-M binding sites was not correlated with that of m2 mRNA are the caudatqanamen, nucleus accumbens, olfactory tubercle and islands of Calleja.

The present results strongly suggest that the M, receptor is expressed by a majority of choline& cells, where it probably plays a role as autoreceptor. However, many noncholinergic neurons also express this receptor, which would be, presumably, postsynaptically located. Finally, comparison between the distribution of m2 mRNA and that of the proposed M,-selective ligand rH]oxotremorine-M indicates that this ligand, in addition to M, receptors, may also recognize in certain brain areas other muscarinic receptor populations, particularly M,.

The actions of the neurotransmitter acetylcholine (ACh) are mediated by receptors belonging to two classes, the muscarinic (MChR) and the nicotinic cholinergic receptors. Multiplicity of MChRs has been clearly demonstrated both pharmacologically and by molecular biology. Thus, three, and recently four, different classes of MChRs have been pharmacologically defined on the basis of their differential sensitivities to several antagonist compounds. Firen- repine provided an initial classification into M, and M, MChRs having, respectively, high and low affinities for this compound.41*42 Heterogeneity of the Mr class was evidenced by the antagonist AF-DX 116,

Abbreviations: ACh, acetylchohne; AF-DX 116, lip- [(diethyl-amino)methyll_l-piperidinyl]acetyl]-5,1 l-di- hydro-6H-pyrido[2,3-b][l,4]benzodiazepine-6-one; ChAT, choline acetyltransferase; EDTA, ethylenedi- aminetetra-acetate; HEPES, N-2-hydroxyethylpipcr- azine-N’-2-ethanesulphonic acid, MChR, muscarinic cholinergic receptor; PBS, phosphate-buffered saline; PI, phosphoinositide; SDS, sodium dodecyl sulphate; SSC, sodium chlorid+sodium citrate buffer.

which presents high affinity for M2 receptors in heart and low a&&y for M, receptors in glands.43 Later, the glandular M2 receptors have been termed M,?6 MChRs with an atypical profile, which differs from those of M,, M, or M, receptors, have been reported in some cell lines6’sa and in brain5’*“” These atypical receptors probably represent an M, subtype of MChRs. Molecular biological techniques applied to the study of MChRs have resulted in the cloning, sequencing and expression of five different genes which encode proteins with the characteristics of muscarinic receptor sites (reviewed in Ref. 11). These gene products have been named ml, m2, m3, m4 and m5 MChRs.i’ The pharmacological subtypes M,, M, and M, correspond to the cloned ml, m2 and m3 receptors,‘5 whereas the putative M4 subtype most likely represents the cloned m4 receptors.67*‘00 Ex- pression studies with the five cloned MChR subtypes have shown that each subtype couples preferentially to one effector system. Thus, the ml, m3 and m5 gene products strongly stimulate phosphoinositide (PI) hydrolysis, while the m2 and m4 subtypes are

Nsc 47,2-F 361

368 M.T. VILAR~) et al.

efficiently coupled to adenylate cyclase in an inhibitory manner.‘0.34,79 However, it has also been reported that one receptor subtype can couple to both effector systems. Thus, m2 and m4 subtypes can couple to PI turnover, although weakly and only at high receptor levels and high agonist doses.6.79

In the brain, the M, class of MChRs has been identified by ligand binding and functional studies. An interesting feature of these receptors is that they appear to represent the muscarinic autoreceptors, i.e. the receptors localized presynaptically in cholinergic terminals and regulating the release of neurotransmitter.47.54.66.84,“’ Although there is abundant functional evidence supporting this presynaptic role for M2 receptors, it has been difficult to establish unambigu- ously whether these receptors are located on cholinergic neurons and whether this location is exclusive or they are also postsynaptically located.” Lesion studies combined with biochemical or autoradiographical assays have given contradictory results.56.62.74~“7

Because of this proposed role for the M, receptors, we considered it of interest to gain more insight into their anatomical localization by means of the technique of in situ hybridization histochemistry. A previous studyi has shown a relative low abundance and restricted distribution of m2 mRNA in the rat brain. However, as discussed by the authors, this could bc due to the fact that the probe used was derived from the human cDNA sequence. Therefore, we re-examined, using probes derived from the rat cDNA sequence, the distribution in the rat brain of the mRNA coding for the m2 MChR, in correlation with-

cholinergic markers and muscarinic receptor sites.

EXPERIMENTAL PROCEDURES

Male Wistar rats (BLR) (25&300 g) were killed by de- capitation, the brains were rapidly removed, frozen in isopentane at -40°C and stored at -70°C until sectioned. Sections (20 pm thick for in situ hybridization histochemistry and 1Oprn for receptor autoradiography) were obtained with a microtome cryostat (Leitz 1720). thaw mounted and kept at -20°C until used.

In situ hybridizalion histochemistry

Oligonucleotide probes were synthesized on a 380B Applied Biosystems DNA synthesizer and purified on a 20% acrylamide-8 M urea preparative sequencing gel. The oligomers were labelled at their 3’ end with [3ZP]a-dATP (3000 Ci/mmol, New England Nuclear) and terminal de- oxynucleotidyltransferase (Td’f, Boehringer Mannheim) to specific activities of 0.9-2.0 x 104 Ci/mmol. Labelled probes were purified by chromatography through a NACS PREPAC (BRL) column. For each of the mRNAs under study, two different oligomers were synthesized based on the published mRNA sequences. In the case of m2 MChR, they were complementary to bases 452-491 (m2/1) and 1806 1852 (m2/3) of the rat m2 MChR cDNA.” These regions correspond, respectively, to the ammo and carboxy termini of the protein, known to share little similarity among the five subtypes of MChRs. In the case of choline acetyltransferase (ChAT), oligonucleotides were complementary to bases 571-618 (catl) and 1321-1368 (cat2) ofthe rat ChAT cDNA sequence.% The autoradiographic signals shown in the figures were obtained with probes m2/1 and catl.

Prior to hybridization, frozen tissue sections were brought to room temperature, air dried, fixed by immersron for 20 min in 4% paraformaldehyde in phosphate-buffered saline (PBS; 2.6 mM KCl, 1.4 mM KH,PO,, 136 mM NaCI, 8 mM Na2HP0,), washed once in 3 x PBS, twice m 1 x PBS, 5 min each, and incubated in a freshly prepared solution of pre-digested pronase at a final concentration of 24 U/ml in 50 mM Tris-HCl, pH 7.5, 5 mM EDTA for 7 min at room temperature. Proteolytic activity was stopped by immersion for 30s in 2mg/ml glycine in PBS. Tissues were rinsed in PBS and dehydrated in a graded series of ethanol (60, 80, 95 and lOO%, 2 min each).

For hybridization, labelled probes were diluted to a final concentration of 2-3 x 10’ c.p.m./ml in the following buffer: 600mM NaCl; 1OmM Tris-HCI, pH 7.5; 1 mM EDTA: 1 x Denhardt’s solution (0.02% ficoll, 0.02% polyvinyl- pyrrolidone, 0.02% bovine serum albumin); 500 pg/ml yeast tRNA; 40% formamide; and 10% dextran sulphate. Tissues were covered with 60 ~1 of hybridization solution, overlaid with Nescofilm coverslips, and incubated for 17 h in a humid chamber at 42°C. Sections were then washed at 60-C in 600 mM NaCl, 20 mM Tris-HCl, pH 7.5, 1 mM EDTA. for 3 h with four changes of buffer. Tissues were dehydrated and either apposed to /?-max film (Amersham) for two (ChAT probes) or three (m2 MChR probes) weeks at -70 C with intensifying screens, or dipped into nuclear track emulsion (NTB3, Kodak) and kept at 4°C for one month. Emulsion was developed in D-19 developer (Kodak). Dark- and bright-field photomicrographs were taken, respectively, before and after counterstaining the tissue with 0.025% Toluidin Blue.

In the co-localization studies, the identification of individual cells expressing two kinds of mRNA was done as follows. Photomicrographs of consecutive sections hybridized with two different probes were taken both under dark- and bright-field illumination. Large size (20 x 25 cm) photographic prints were obtained and the cells co-existing in both sections were identified by superimposing the bright- field images. By comparison with the dark-field images, the cells containing both kinds of mRNA were then identified.

Northern analysis

Total RNA was isolated from different rat brain regions as described by Chomczynski and Sacchi.” Poly(A) + RNA was purified by chromatography through oligo (dT)-cellu- lose. Total or poly(A)+ RNA was denatured with glyoxal,5x separated by electrophoresis on 1% agarose gels, and blotted onto nylon membranes (Hybond N, Amersham). Blots were hybridized with the labelled oligomers for 18 h at 42°C in a buffer containing 600 mM NaCl; 80 mM Tris HCI, pH 7.5; 4 mM EDTA; 0.1% sodium pyrophosphate; 0.2% sodium dodecyl sulphate (SDS); 5 x Denhardt’s solution: 500 uaiml Yeast RNA: and 50% formamide. Fibers

.I, I

were washed twice for 5 min in 2 x sodium chloride-sodium citrate buffer (SSC) (1 x SSC: 150 mM NaCl-15 mM sodium citratep. 1% SDS at room temperature and twice for 15 min in 0.2 x SSC-O.l% SDS at 42°C. Filters were apposed to an X-ray film and exposed at -7OC for the appropriate period of time. The RNA content of the different lanes was normalized by hybridizing the blots with an oligonucleotide probe complementary to p-actin mRNA.

Receptor autoradiography

Muscarinic receptor sites were labelled as previously described with the non-selective antagonist [)H]N- methylscopolamine, with the putative M,-selective agonist [‘H]oxotremorine-M and with the M,-selective antagonist [‘Hlpirenzepine. For all the ligands, nonspecific binding was determined by the addition of 1 PM atropine during the incubation.

[3H]N-Mefhylscopolomine. Tissues were incubated in 0.3 M sodium potassium phosphate buffer, pH 7.4, containing 0.3 nM [3H]N-methylscopolamine (85 Ci/mmol;

Pre- and postsynaptic muscarinic m2 receptors 369

Amersham) for 1 h at room temperature. After incubation, sections were dipped in 0.17 M Tri-HCl buffer, pH 7.4, at 4”C, rinsed in this buffer for 10 min at 4°C dipped in water at 4°C and dried rapidly under a stream of cold air. Auto- radiograms were generated by apposing the labelled tissues to [3HjHypertilm (Amersham). Exposure time was six days.

[3H]Oxotremorine-M. Incubation was essentiaily as described by Spencer et al?’ Tissues were pre-incubated in 20 mM HEPES buffer for 10 min at room temperature. In- cubation was in the same buffer with 1 nM [‘HI_ oxotremorine ([3H]Oxotremorine-M; 87 Cijmmol, Du Pont NEN) for 40 min at 30°C. Tissues were washed for 2 min in HEPES buffer at 4°C and rinsed with water at 4°C. Exposure tune was two weeks.

PHJPirenzepine. Incubation was essentially as in Quirion et al.” Tissues were pm-incubated in Krebo’ buffer, pH 7.4, for 15 min at 22°C and thereafter incubated in the same buffer containing 5nM [3Nlpirenzepine (87 Ci/mmol, Du Pont NEN) for 6Omin at room temperature. Slides were then rinsed three times (4min each) in 5OmM Tris-HCl bulfer, pH 7.4, at 4°C and dipped in water at 4°C. Exposure time was 10 days.

Acetylcholinesterase hiistochembtry

Sections close to the ones used for hybridization were stained for acetylcholinesterase activity essentially as previously described.s~ Briegy, medium A was prepared by mixing 1 g a~tyl~~ho~e iodide, 1260 ml sodium acetate, 0.06 M (PH was brought to 5.5 with acetic acid), 100 ml sodium citrate, 0.1 M, 200ml CuSO,-SH,O, 30 nM, 4ml tetraisopropylpyrophosphoramide 4 mM and 16 ml H,O. Medium B was 5 mM K,Fe(Cn), . Aliquots of both media were made and stored at - 20°C. When needed, 36 ml of medium A was mixed with 4ml of medium B and tissue sections were incubated for 60min at 37°C. Tissues were rinsed with distilled water and washed for 10 min in distilled water. They were then quickly dipped in a series of ethanol (60,80,95, lOO%), then immersed in xylol and coverslipped.

Histological staining After exposure to film, hybridized tissues were stained for

20 min in 1% Giemsa solution (Fluka). washed in water for 5min, Dvorak through a‘ graded series of ethanol, immersed in xylol for 5 min and coverslipped.

msuLTs

Controls for specificity

The specificity of the different oligonucleotide probes was asses& by Northern analysis on total or poly(A)+ RNA extracted from various regions of the rat brain. A single mRNA species of approximately 6.5 kb was detected on total RNA with probe m2/1 for the m2 MChR mRNA (Fig. 1A). The hybridizing band was strongest in midbrain (lane 1), followed by thalamus (lane 8). Fainter bands were observed in hypothalamus (lane 3), brainstem (lane 4), cerebral cortex (lane 5) and cerebellum (lane 6). A very faint band was also observed in hippocampus (lane 2) and striatum (lane 7) upon prolonged exposure of the autoradiogram. Similarly, a hybridizing mRNA species of approximately 4.7 kb was detected on poly(Af + RNA with probe cat1 for ChAT mRNA (Fig. 1B). The band was most prominent in brainstem (lane 4) and spinal cord (lane S), and much fainter in midbrain (lane 3) and thalamus (lane 2). No band was detected in cerebral cortex (lane 1). The sizes of the detected transcripts are in agreement with previous

reports’4*50 and the regional distribution revealed by Northern analysis agrees well with the pattern of hybridization signaf observed in the in situ hybridization studies (see below).

The specificity of the autoradiographic signal obtained in the in situ hybridization experiments was conlirmed by performing a series of routine controls (see Refs 77,99). For each mRNA under study, at least two different oligonucleotides complementary to different regions of the mRNA were used indepen- dently as hybridization probes in consecutive sections from the same animal and showed identical patterns of hybridization. For a given oligonucleotide probe, addition in the hybridization solution of a SO-fold excess of the same unlabelled oligonucleotide resulted in the complete abolition of the hybridization signal. The signals were not a&&& when the oligonucleotide added in excess was complementary to a different region of the same mRNA or, in the case of m2 MChR, to the equivalent region of the mRNA for a different MChR subtype. The thermal stability of the hybrids was examined by washing at increasing t~~ratu~s: the observed melting tem~~ture of the hybrids was close to the theoretical value. Thus, a sharp decrease in the intensity of the hybridization signal was observed between 70 and 75°C with the m2/1 probe, and between 70 and 80°C for the cat1 probe. The theoretical T,,, values of the hybrids3 are 73.7 and 84.6”C, respectively. The pattern of hybridization obtained with probes for the m2 MChR mRNA is clearly distinct from those obtained with probes for the other four subtypes of MChRs.‘6,98~‘W

Distribution of m2 muscarinic cholinergic receptor mRNA

The distribution of transcripts for the m2 MChR at various coronal levels of the rat brain is illustrated in Fig. 2, left-hand panels. Labelled nuclei were identified by comparison of the film autoradiographs both with Giemsa staining of the hybridized tissues and with a~tylcholinestera~ staining of close sections (shown in Fig. 2, right-hand panels).

In the olfactory system, several structures contained m2 MChR mRNA. The olfactory bulb presented a layered, very strong hybridization signal. The internal granular layer and the mitral cell layer showed a very strong labelling. The periglomerular cells of the glomerular layer were also intensely labelled (Figs 2A1, 3). In the anterior olfactory nucleus, low but significant levels of signal were observed. No signal above background levels was detected in the olfactory tubercle and very low levels of signal were observed over the primary olfactory cortex. The endopi~fo~ nucleus presented low levels of signal.

Several basal forebrain structures were rich in m2 MChR transcripts. Within the septal region, various nuclei contained cells expressing mRNA for the m2 MChR. In the lateral septal nucleus, the intermediate part showed moderate levels of signal, whereas no

370 M. ‘I‘ VILAKi) et ul.

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Fig. 1. Northern blot analysis of m2 MChR (A) and ChAT (B) transcripts. In A, each lane contained 2Opg of total RNA extracted from different rat brain regions: 1, midbrain; 2, hippocampus; 3, hvwthalamus: 4. brainstem: 5. cortex; 6, cerebellum; 7, striatum; 8, thalamus. In B, each lane contained

II , ,

approximately 8 fig of Poly(A)+ RNA extracted from: 1, cortex; 2, thalamus; 3, midbrain; 4, brainstem; 5, spinal cord. Positions of ribosomal molecular weight markers are indicated. Lower panels: RNA

concentrations were normalized by hybridizing with an actin oligonucleotide probe.

hybridization was detected in the dorsal and ventral parts. High levels of signal were observed both in the medial septal nucleus and in the nucleus of the diagonal band, including its vertical and horizonal limbs. The triangular septal nucleus also showed a strong hybridization signal. Intense labelling was observed over the cells of the basal nucleus of Meynert at all rostrocaudal levels. In the caudate-putamen nucleus, scattered cells contained high levels of m2 MChR mRNA. These cells were not

strong hybridization signal, whereas the medial habenula showed only background levels of signal (Fig. 4).

In the hypothalamus, moderate to weak hybridization signals were observed in the anterior and ventromedial hypothalamic nuclei and in the lateral hypothalamic area.

In the hippocampal formation, labelling was not homogeneously distributed. The pyramidal cells of the very rostra1 tip of the hippocampus showed

homogeneously distributed. They were particularly strong hybridization signal. Proceeding caudally, the concentrated in the lateral aspects of the nucleus and intensity of the signal decreased rapidly, and reached not detected at rostra1 levels. very low or undetectable levels over the pyramidal

Various thalamic nuclei were enriched in m2 cells of the posterior hippocampus. The pyramidal MChR mRNA. These include the reticular, cells of CA3 close to and extending into the hilar anteroventral, anterodorsal, centromedial, rhom- region showed intermediate levels of signal rostraliy, boid, paraventricular, paracentral, dorsolateral gen- whereas no signal was detected more caudally. NO iculate and ventrolateral geniculate nuclei. In the significant signal was observed over the granule epithalamus, the lateral habenula presented very cells of the dentate gyrus. Other structures in the

Pre- and p&synaptic muscarinic m2 receptors 371

hippocampal region showing intermediate or high levels of signal include the subiculum, presubiculum and parasubiculum.

Resides the hippocampal region, several other allocortical regions were enriched in m2 MChR mRNA. Intense signals were seen over the insular, anterior cingulate and retrosplenial cortices. Weak signals were observed in some amygdaloid nuclei, viz. the nucleus of the lateral olfactory tract and the basolateral amygdaloid nucleus.

In the neocortex, a bilaminar pattern was observed in most regions, with high levels of hyb~d~tion signal over layer IV and outer part of layer VI.

Transcripts for m2 MChR were prominent all along the brainstem. Several nuclei of the reticular formation presented moderate to high levels of hybridization. In a rostrocaudal progression, these include the deep mesencephalic nucleus, the oral and caudal parts of the pontine reticular nucleus, pedunculopontine, anterior and ventral tegmental nuclei, retic~o~~~l nucleus of the pons, gigantocellular, gigantocellular pars cL, paragigantocellular

reticular nuclei, and lateral reticular nucleus. In some of these nuclei, only some scattered cells showed hybridization signal. Within the central gray, the dorsal and laterodorsal @mental nuclei were strongly labelled. Other midbrain structures present- ing intermediate or high levels of hybrid&&ion signal include the anterior pretectal area, the olivary and posterior pretectal nuclei, the interstitial magnocellu- lar nucleus of the posterior commissure, the nucleus of Darkschewitsch, the superior colliculus, particularly the super&Sal layers, the inferior colliculus and the ~abi~nal nucleus. Low levels of hybridization signal were observed in the red nucleus and central part of the interpeduncular nucleus. The pontine nuclei showed the highest levels of hybridization signal.

Certain raphe nuclei showed intermediate to low levels of signal. These include the dorsomedian group of cells of the dorsal raphe nucleus, the median raphe, and raphe pontis and the raphe obscurus.

Other labelled nuclei within the brainstem include the ventral nucleus of the lateral lemniscus, the dorsal

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Abbreviations used in the figures

mbdeus aixumbens anterior cinaulate cortex anterodorsal thahunk nucleus nucleus ambiguus anterior olfactory nucleus anterior pretectal area anteroventral thaltic. nucleus cells of the basal nucleus of Meynert basolateral amygdaloid nucleus geld CA1 of Ammon’s horn tkld CA4 of Amman’s horn central gray choroid plexus ciaustrum central medial thalamic nucleus caudatiputamen cuneate nucleus cerebral cortex dentate gyms dorsal lateral geniculate nucleus dorsal parabrachial nucleus deep rn~~h~~ nucleus dorsal tegmental nucleus endopiriform nucleus entorbinal cortex external plexiform layer olfactory bulb gigantocelhtlar reticular nucleus glomerular layer olfactory bulb globus pallidus . . _

LV lateral ventricle L9.LlO lobules 9 and 10 of cerebellum Mirb Mi Mo5 MS MVe PaS PBg PC

RSpl Rt RtTg S SFi SI SPS SuG SU7 Th TS Tu VCoA VDB

medial habenular nucleus mitral cell layer, olfactory bulb motor trigeminal nucleus medial septal nucleus medial vestibular nucleus parasubiculum parabigeminal nucleus paracentral thahunic nucleus posterior cingulate cortex pontine nuclei pedunculopontine tegmental nucleus principal sensory trigeminal nucleus rhomboid thalamic nucleus retrosplenial cortex reticular thalamic nucleus ~tic~o~~~t~ nucleus of the ports subiculum septofknbrial nucleus substantia innominata nucleus of the spinal tract of the trigeminal nerve superficial gray layer of the superior colliculus suprafacial nucleus thalamic nuclei triangular septal nucleus olfactory tubercle ventral cochlear nucleus, anterior nucleus of the vertical limb of the diagonal band

@acne uweus mtcleus of the horizontal limb of the diagonal band

VDBD nucleus of the vertical limb of the diagonal bana, dorsal

inferior colhculus internal capsule internal gramslar layer olfactory bulb intermediate gray layer, superior colliculus internal plexiform layer, olfactory bulb laterodorsal tegmental nucleus lateral hypothalamic area lateral habenular nucleus lateral reticular nucleus lateral septal nucleus, dorsal lateral septal nucleus, intermediate lateral nucleus, trapezoid body

VDBV nucleus of the vertical limb of the diagonal band, ventral

VLG ventral lateral geniculate mrcleus VMH ventromedial hypothalamic nucleus VPB ventral parabrachial nucleus VTg ventral tegmental nucleus ZI zona incerta 3 principal oculomotor nucleus 4 trochlear nucleus 7 facial nucleus 10 dorsal motor nucleus of vagus 12 hypoglossal nucleus

372 MT’. VILARO et u(.

Fig. 2 (A-D).


Fig. 2 (E-H).

374 M. I. VILAR~ et at,


Fig. 2 (M-P).

376 M.T. VILAR~ rt al.

Fig. 2 (Q- S).

Fig. 2. Regional distribution of m2 MChR mRNA (A,-S,) and ChAT mRNA (A,--S2) at several coromdl levels of the rat brain. Pictures are photomicrographs from film autoradiograms. Dark areas correspond to regions rich in hybridization signal. A,-& are photomicrographs of acetylcholinesterase-stained sections. Sections shown in the left-hand and central panels are consecutive, whereas sections shown in the right-hand panels are approximately 100 pm more caudal. Abbreviations (see list) are as in Paxinos

and Watson.‘* Scale bar = 3 mm.

and ventrai parabrachiai nuclei, the iocus coeruleus, the lateral nucleus of the trapezoid body, and the gracile, cuneate and external cuneate nuclei. High levels of hybridization signal were observed in several cranial nerve nuclei: motor trigeminal, facial, suprafacial, ambiguus and hypoglossal nuclei. In addition, the principal sensory trigeminal nucleus, the nucleus of the spinal tract of the trigeminal nerve, the vestibular, and the dorsal and ventral cochlear nuclei were also labelled.

In the cerebellum, very low levels of hybridization were detected over the granule cell layer, with the

signal being much stronger in lobules 9 and IO. The Purkinje cell layer was also weakly labelled.

In the spinal cord, high levels of hybridization signal were detected in the motoneurons of the ventral horn.

The anterodorsal part of the choroid plexus within the lateral ventricles showed strong hybridization signal (Fig. 5).

Distribution of choline acetyltransferase mRNA

In order to compare the distribu~on of cells expressing the m2 MChR with that of cholinergic cells,


Fig. 3. Cellular localization of m2 MChR mRNA in the olfactory bulb. A is a dark-field illumination photomicrograph of a section hybridized with the m2/1 oligonucleotide probe. and dipped in photographic emulsion. Autoradio- graphic grains are seen as bright points. B is a bright-field photomicrograph of the same section counterstained with

Toluidine Blue. Scale bar = 200 pm.

sections consecutive to those hybridized with the probe for the m2 MChR mRNA were hybridized

with the probe for ChAT mRNA. Transcrr ‘pts for ChAT showed a more restricted distribution (Fig. 2, central panels) which partially overlapped that of transcripts for m2 MChR.

Within the olfactory system, cells expressing ChAT mRNA were detected only in the olfactory tubercle region.

Cells containing high levels of ChAT mRNA were abundant within several basal forebrain structures. In the medial septum, ChAT mRNA-rich cells were particularly concentrated along the lateral borders of this nucleus, although some cells were also more medially located. A similar situation was observed in the vertical limb of the diagonal band, where the cells containing ChAT mRNA were arranged in the borders of the nucleus, whereas the central part was practically devoid of positive cells. Within the horizontal limb of the diagonal band, cells rich in ChAT mRNA were much more abundant in the medial half of the nucleus, with only some cells present in the lateral half. The cells of the basal nucleus of Meynert were intensely labelled throughout the rostrocaudal extent of the nucleus. Many intensely labelled cells were scattered within the accumbens and caudate- putamen nuclei, whereas much fewer labelled cells were observed within the globus pallidus.

In the epithalamus, the medial habenula showed very high levels of hybridization signal which was restricted to the ventral two-thirds of the nucleus. No signal was detected in the lateral habenula (Fig. 4).

Within the midbrain, the parabigeminal nucleus was intensely labelled.

Starting at caudal mesencephalic levels and extending into the pons, cells in the pedunculopontine tegmental nucleus were heavily labelled. The laterodorsal tegmental nucleus also contained cells with high levels of ChAT mRNA.

The motoneurons of all cranial motor nuclei (the principal oculomotor, trochlear, motor trigeminal, abducens, facial, suprafacial, ambiguus and hypoglossal nuclei) showed very strong hybridization

Fig. 4. Cellular localization of m2 MChR (A) and ChAT (B) mRNAs in the habenular nuclei. A and B are dark-field photomicrographs of emulsion-dipped sections. C is the bright-field image of the section

shown in A. Scale bar = 200 pm.

378 M. T. VILAR~ et trl.

Fig. 5. Autoradiographic visualization of m2 MChR mRNA in the rat choroid plexus. Dark-field (A.Bt and bright-field (A’$‘) photomicrographs of a section hybridized with the m2jl probe and dipped in photographic emulsion. B is a higher magnification of a detail from A. Box in C (modified from Paxinw

and Watson”) ilfustrates the anatomical level. Scale bars = 400 slrn.

signal. The dorsal motor nucleus of the vagus was also intensely labelled. The iateral nucleus of the trapezoid body showed intermediate levels of signal.

In the spinal cord, the ventral horn motoneurons and the cells in the intermediolateral cell column at toracolumbar and lumbo~cral levels were strongly labelled. Some scattered labelled cells were observed in the region around the central canal.

Co-localization studies

In order to examine at the cellular level the coexistence of m2 and ChAT mRNAs in the same cell populations, consecutive sections were hybridized with each one of the probes and dipped in photographic emulsion. Individual cells ~nta~njng both kinds of mRNA were identified as described in Experimental Procedures. It should be stressed, however, that an exhaustive quantitative study of the degree of co-localization was not the aim of the present study, mainly due to the limitations of the use of consecutive sections for co-localization experiments (see Discussion).

The cellular localization of m2 and ChAT mRNAs in different structures of the basal forebrain is shown in Figs 6-9. In the medial septum-diagonal band complex (Figs 6-8). several cells were found to contain both kinds of mRNA (arrows in the figures). At all levels of the complex, ChAT mRNA- containing cells presented a more restricted distribution than m2 mRNA-containing cells. Therefore, it is possible that m2 MChRs are also expressed by GABAergic ceils present in these structures. In the cells of the basal nucbus of Meynert (Fig. 9X a much higher degree of co-localization could be established.

In the caudate-putamen nucleus, no detailed attempts were made to co-localize both mRNAs in consecutive sectians. However, the two populations of scattered cells containing each one of the mRNAs shared a number of features. Thus, the patterns of distribution and relative abundances were campar- able, although cells containing ChAT mRNA were slightly more numerous and present at rostra1 levels of the nucleus, where m2 mRNA was not detected.


Fig. 6. Distribution of cells expressing m2 MChR (A) or ChAT (B) mRNAs in the medial septal nucleus. C is the bright-field image of the section shown in A. Box in D (modified from Paxinos and Watson”) illustrates the anatomical level. Arrows in A and B point to some of the cells which contain transcripts

for both m2 MChR and ChAT. Scale bar = 200 ym.

Furthermore, both mRNAs were found in large cells which stained less intensely with Toluidine Blue than other surrounding, smaller cells (Fig. 10). These facts strongly suggest that m2 MChRs are expressed by at least a fraction of the striatal choline@ cells.

The motoneurons of all the cranial nerve motor nuclei (Fig. ll), as well as the spinal ventral horn motoneurons (Fig. 12), contained, as expected, very high levels of ChAT mRNA. In some of these nuclei (motor trigeminal, facial, ambiguus, hypoglossal) and in the ventral horn, the motoneurons also contained m2 mRNA, whereas in some others (principal oculomotor, trochlear, abducens) no hybridization with the m2 probe was detected. In those nuclei expressing both kinds of mRNA, the motoneurons expressing m2 mRNA appeared to be less numerous than the ChAT mRNA-containing neurons. This could be due to the fact that transcripts for ChAT are much more abundant than transcripts for m2 MChR. Thus, when only a small portion of the cell body is present in a given tissue section, ChAT transcripts would still be detected whereas the much lower levels of m2 transcripts could be under the detection limits. The neurons of the dorsal motor nucleus of the vagus (Fig. 11F) contained high levels of ChAT mRNA but undetectable levels of m2 mRNA.

Distribution of muscarinic binding sites : comparison with the distribution of m2 mRNA-expressing cells

A non-selective antagonist ([‘H]N-methylscopolamine), an M,-selective antagonist ([3H]pirenzepine) and a putative M,-selective agonist ([3H]- oxotremorine-M), were used in consecutive sections of the rat brain to visualize MChRs. The distribution of the binding sites for these three ligands at several coronal levels of the rat brain is illustrated in Fig. 13.

[3HJPirenzepine sites (Fig. 13, right-hand panels) were enriched in the cerebral cortex, hippocampal formation (particularly in strata oriens and radiatum of field CA1 of comu Ammonis and in the molecular layer of the dentate gyrus), and in basal ganglia regions such as the caudate-putamen, nucleus accumbens and olfactory tubercle. In contrast, the brainstem and cerebellum were practically devoid of labelled sites.

[3H]Oxotremorine-M binding sites (Fig. 13, central panels) showed a more extensive distribution. They were enriched in cortical areas, although with a different laminar distribution when compared to [3H]pirenzepine sites. Differences in the pattern of labelling were also apparent in the hippocampal formation. Densities of [3Hloxotremorine-M sites were high in two narrow bands in comu Ammonis, one in stratum oriens just above the pyramidal layer, and the other in stratum radiatum just adjacent to

380 M. iv VILARO et al.

............................ C C C.( '-~l ....'~ )\\\~

Fig. 7. Distribution of cells expressing m2 MChR (A) or ChAT (B) mRNAs in the vertical limb of the diagonal band at the level shown in the box in C. Arrows in A and B point to some of the cells which contain transcripts for both m2 MChR and CHAT. C is modified from Paxinos and

Watson. TM Scale bar = 200#m.

the stratum lacunosum--moleculare. Densities of [3H]oxotremorine-M sites were very low in the dentate gyrus. The basal ganglia (caudate-putamen, nucleus accumbens) and associated areas (olfactory tubercle, septal region) were also enriched in [3H]oxotremorine-M sites. Nuclei in the thalamus, hypothalamus, midbrain and hindbrain contained intermediate to high or very high densities of [3H]oxotremorine-M sites, contrasting with the vir- tual absence of [3H]pirenzepine sites in these regions.

Fig. 8. Distribution of cells expressing m2 MChR (A) or ChAT (B) mRNAs in the horizontal limb of the diagonal band at the level shown in the box in C. Arrows in A and B point to some of the cells which contain transcripts for both m2 MChR and ChAT. C is modified from Paxinos and

Watson. TM Scale bar = 300 pro.

The distribution of [3H]N-methylscopolamine binding sites (Fig. 13, left-hand panels) can be considered as being additive of the distributions of [3H]oxotremorine-M and [3H]pirenzepine sites. How- ever, [3H]N-methylscopolamine labels less intensely and less distinctly many of the brain areas which are labelled by [3H]oxotremorine-M but not by [3H]pirenzepine (cf. e.g. the labelling of the thalamus in Fig. 13B1, B2).

The comparison between the distribution of m2 mRNA and that of [3H]oxotremorine-M binding sites is illustrated in Fig, 14 at some selected levels of the rat brain. Regions of good correlation include the olfactory bulb, cortex, septal-diagonal band complex, thalamic nuclei, midbrain nuclei such as the


Fig. 9. Co-expression of m2 MChR (A) and ChAT (B) mRNAs in the eelIs of the basal nncleus of Meynert. Arrows point to some of the cells co-expressing both transcripts. The anatomical level is illustrated in the

acetylcholinesterase staining of a close section (C). Scale bar in A = 200 pm; in C = 1 mm.

parabigeminal nucleus and superior and inferior col- liculi, pontine nuclei, several nuclei of the medulla (particularly the cranial nerve nuclei) and the cerebellum. In the olfactory bulb, a complementary rather than overlapping distribution of both markers is observed. Thus, the very high levels of m2 mRNA in the internal granular layer are not paralleled by high densities of receptor sites in this layer. In contrast, the external plexiform layer, which is devoid of m2

mRNA, contains very high densities of receptor sites, suggesting that these receptors are located on the dendritic processes of the internal granule cells extending into the external plexiform layer. The correlation of both markers is not complete throughout the brain. Thus, there are certain brain regions where high densities of [‘H]oxotremorine-M binding sites are paralleled by much restricted distributions of transcripts for the m2 MChR. This is the case of

Fig. 10. Cells expressing m2 MChR (A,A’) or ChAT (B,B’) mRNAs in the caudattputamen nucleus. Note the similar size, intensity of staining and relative abundance of both populations of cells.

Scale bar = 80 pm.

382 M. T. VILAR~ ef al.

Fig. 11. Cellular localization of m2 MCbR mRNA (A,-F,) and ChAT mRNA (At-F& in several cranial nerve nuclei. Stained tissues (A,-F,) correspond to the sections hybridized with the ChAT probe. Note the absence of detectable hy~di~t~n with the m2 probe in the nuclei of the third (A,), fourth (B,) and sixth (DJ cranial nerves as well as in the dorsal motor nucleus of the vagus nerve (F,). 3, principal oculomotor nucleus; 4, trochlear nucleus; MO& motor trigeminal nucleus; 6, abducens nucleus; 7, facial nucleus; 10, dorsal motor nucleus of the vagus; 12, hypoglossal nucleus. Scale bar = 500 pm (260 pm for Ft and DI.


12. Mlular hcalization of m2 MChR mRNA (A-A’) and ChAT mRNA (R-B’) in the n of the spinal cord. Scale bar = 300 pm.

.ont RI1 ans

the caudate-putamen, where only some scattered cells contain n-12 mRNA, of the olfactory tubercle and islands of Calleja, where no m2 mRNA was detected, and of the hippocampus, where m2 mRNA was detected only in the most rostra1 part of this structure.

DISCUSSION

The main findings of these investigations could be summarized as follows, (1) m2 MChRs are widely dist~but~ dugout the neuroaxis. (2) The distribution of m2 mRNA overlaps with that of cholinergic cell bodies as visualized with probes for ChAT mRNA. (3) This overlapping is, however, limited, in the sense that not all the cholinergic cell bodies appear to express m2 receptors and, conversely, many non-choliner~c cells do express m2 receptors. (4) In

relation to the muscarinic ligands used in the present study, a good correlation between the distribution of m2 mRNA could be established with that of the subset of MChRs labelled by [3~oxo~o~n~M but not with the receptors labelled by [jH]pi~~ine. (5) However, high densities of [3H]oxotremorine-M binding sites were present in regions such as the caudate-putamen, olfactory tubercle and islands of Calleja, where m2 mRNA was scarce or not detected. In contrast, these regions are highly enriched in mRNA coding for the m4 MChR subtype and contain MChRs with an atypical pharmacological profile.‘@’

The specificity of the autoradiographic signals obtained in the in situ hybridization studies has been assessed by performing a number of control experiments which include Northern analysis, separate hyb~d~tion with two probes for the same mRNA,

M. I, VILAR~ ef d.

Fig. 13. Comparison of the regional distributions of MChRs labelled with different muscarinic ligands in consecutive coronal sections of the rat brain. Pictures am photom~cro~aphs from film autoradiograms. Dark areas correspond to regions with high densities of receptors. (A,-E,) Distribution of [3H]N- methylscopolamine binding sites. (AZ-ES) Distribution of [3H]oxotremorine-M binding sites. (A3- E,)

Distribution of [‘H]ptrenzepine binding sites. Scale bar = 3 mm.


Fig. 14. Comparison between the distribution of m2 MChR mRNA (A-F) and the distribution of MChRs labelled with [3H]oxotremorine-M (A’-F’) in the rat brain. Sections correspond to approximately

equivalent levels of the brain of different animals. Scale bar = 3 mm.

co-hybridization with excess unlabelled oligonucleotides and estimation of the melting temperature of the hybrids. In the case of the m2 MChR, the oligonucleotides used as hybridization probes were designed to hybridize with regions of the mRNA sharing very little homology among the different subtypes of MChRs, thus excluding the possibility of cross-hybridization with other subtybs. Previous in situ hybridization studiesI in rat brain with human cDNA probes have shown a more restricted distribution of transcripts for the m2 MChR. In the present study, the use of probes derived from the rat m2 MChR mRNA sequence3’ has enabled us to detect a more extensive distribution of m2 mRNA.

The use of consecutive sections to analyse the co-localization of two different markers is hampered

by the size of the cells under study. As shown by Gaspar et a1.,35 estimates of the degree of co-localization were much lower when studied on consecutive sections as thin as 5 pm than when studied by double labelling on the same section. In the present study, while we could establish a good degree of co-localization in cell populations where the size of the cell bodies is big (nucleus basalis, ventral horn of the spinal cord, cranial nerve nuclei), in other cell populations where the cells are smaller (medial septum, diagonal band, striatum) it was more difficult to find a good overlapping of cells. Thus, the unequivocal establishment of the degree of co-localization of m2 and ChAT mRNAs in the cell populations examined in this study would require the use of double labelling techniques such as the combination of isotopic and

386 M. T VILAR~ ef ol.

non-isotopic probes on the same tissue sections. Nevertheless, the high degree of co-distribution found in several cell populations strongly suggests an important level of co-localization.

m 2 mRNA expression and cholinergic cell bodies in the rat brain

The distribution of cholinergic cell bodies and terminals in the rat brain has been studied in detail using histochemical and immunohistochemical techniques (see Refs 32, 87 and 104 for reviews). These studies have shown the existence of several groups of cholinergic projection neurons with long axons as well as other cholinergic neuronal populations which are short-axon, local circuit interneurons. The projection groups include cells in the medial septum-- diagonal band complex, nucleus basalis, pedunculopontine and laterodorsal tegmental nuclei of the pontomesencephalic reticular formation, medial habenula, parabigeminal nucleus, motor neurons of cranial nerve nuclei, and somatic motor neurons and preganglionic projection neurons in the spinal cord. Cholinergic interneurons have been described in caudate-putamen, ventral striatum, cerebral cortex, hippocampal formation, amygdaloid complex, olfactory bulb and hypothalamus.”

Our in situ hybridization studies with probes for ChAT mRNA are in general in good agreement with the previous immunohistochemical studies. Thus, we detect all the Chl-Ch8 cholinergic groups described by Mesulam et al.,” and Mufson et al.,” or the four major cell groups proposed by Satoh et al.” How- ever, we also find notable discrepancies. The main difference is our failure to identify cells containing ChAT mRNA in cortical areas, including the neocortex and the hippocampus. The existence of cholinergic cell bodies in the neocortex has been controversial, with contradictory results reported by several groups (see Refs 29,32,49,51,87,104). Simi- larly, the presence of intrinsic cholinergic neurons in the hippocampus has been reported only in some recent immunohistochemical studies.“~63~‘05 The hypothalamus is another region where ChAT-immunoreactive neurons have been described82.96 and where we did not detect any ChAT mRNA-containing cells. Finally, Tago et aL9’ have reported the existence within the rat brainstem of several ChAT-immunoreactive cell groups which were not detected in the present study. A common characteristic of all these neurons for which discrepancies have been found, is their small size and weak immunostaining when compared to other larger, heavily stained cholinergic neurons of, for example, the basal forebrain complex or the striatal interneurons, which we readily detected with our probes. Several explanations could be proposed to account for these discrepancies: Firstly, the possible limited sensitivity of the technique used in this study. This is, however, unlikely because we do detect cells in the medial habenula and parabigeminal nucleus, which are also relatively small and which

have been detected in several but not all the immunohistochemical studies (see Refs 82,104). Nevertheless, one could hypothesize that there exists, in the cells undetected in this study, a low turnover rate for ChAT that results in very low levels of ChAT mRNA, which could be below our detection limits, An alternative explanation is that the antibodies used

in the immunohistochemical studies recognize, in those controversial cells, a form of ChAT which is different from the one in the major cholinergic cell groups and which could be encoded by ;t different mRNA, therefore escaping detection by our oligonucleotide probes. The existence of multiple forms of ChAT has also been controversial. Several forms ot the enzyme with different subcellular localization and physical properties have been reported in the rat and other species. *,30,59 However, some of these findings have been questioned as being artifactual results due to long conventional purification procedures.“’ It should be noted, however, that in a later study by this same group,13 involving a rapid immuno-affinity purification procedure to purify human placental and brain ChAT, heterogeneity of the brain preparation was obtained in the same conditions that gave a homogeneous preparation of placental ChAT. Some of the additional minor proteins in the brain ChAT preparation reacted with the antibody used for the purification. I3 Furthermore, recent preliminary com- munications have indicated that two species of ChAT mRNA exist in human brain which may encode isoforms of ChAT with a unique amino acid sequence.92 Finally, preliminary results in our laboratory indicate that the rat phaeocromocytoma cell line PC12 contains transcripts for ChAT which are con- siderably shorter than those detected in brain and spinal cord, thus suggesting that heterogeneity of ChAT may also exist in rat.

mRNA coding for m2 MChRs was seen in all the major cholinergic projection cell groups of the basal forebrain, including the medial septum, vertical and horizontal limbs of the diagonal band and nucleus basalis, which provide innervation to cortical and allocortical regions. 9.b5~“b The pedunculopontine and laterodorsal tegmental nuclei, which mainly provide cholinergic innervation to the thalamus,b”.“s and the parabigeminal nucleus, a major source of cholinergic innervation to the superior colliculus,70 also contain m2 mRNA. In contrast, m2 mRNA was not detected in the cells of the medial habenula, which contain high levels of transcripts for ChAT and which project to the interpeduncular nucleuszO

Another group of cholinergic nuclei which also presented high levels of m2 mRNA were the cranial nerve nuclei. However, notable exceptions were observed. Thus, both ChAT and m2 MChR mRNAs were present in the motor nucleus of the trigeminal nerve, the facial, suprafacial, ambiguus and hypoglossal nuclei. In contrast, high levels of ChAT mRNA but undetectable levels of m2 mRNA were present in the nuclei related with eye movements, i.e.


principal oculomotor, trochlear and abducens nuclei, as well as in the dorsal motor nucleus of the vagus.

Finally, the motoneurons of the spinal cord, which constitute another well-characterized cholinergic projection cell group, were also enriched in m2 MChR mRNA.

In addition to the projection neurons mentioned above, the cholinergic interneurons of the caudate- putamen nucleus also appear to be enriched in m2 MChR transcripts.

The expression of m2 MChR mRNA in choline@ cells, together with the presence of muscarinic binding sites in some of the nuclei or brain regions containing cholinergic cell bodies, is compatible with the presence of those binding sites in (i) presynaptic terminals of these cells where those receptors could play a role as autoreceptors, and (ii) the soma or dendrites of the cholinergic cells, where they could be innervated either by recurrent axon collaterals or by cholinergic afferents from other areas. Evidence in the literature supports the existence of both these types of muscarinic receptors in cholinergic neurons or terminals (see below).

Functional evidence for a muscarinic control of ACh release in brain is abundant.‘6~*‘~94 These biochemical studies have been focused particularly in the neocortex, hippocampus and striatum.39@‘,6’*7’,*’ In the cases where the pharmacological nature of these receptors has been established, they have been shown to belong to the M, class, 47s4*66~&1~“’ although in other regions a different subtype of MChR has also been proposed.93

Lesion studies to determine the presence of presynaptic MChRs have not always been conclusive. In some cases, reductions of high-affinity agonist62*“o or non-selective antagonist binding sites’9~25~56 have been observed in the projection areas. However, in other cases, no reduction in projection areas has been found%4% 17 using non-selective antagonists. It has been suggested 53,95 that these antagonists have a lower affinity for presynaptic than for postsynaptic receptors, and therefore the former may have gone undetected at the concentration of antagonists used in these studies. It has also been proposed53,74 that presynaptic decrease could be masked by the develop- ment of postsynaptic supersensitivity. Indeed, several studies7*23~“~52~“4 have shown up-regulation of MChRs in cortex or hippocampus after cholinergic denervation of these structures. Another piece of evidence supporting the presence of MChRs in the terminals of the septohippocampal pathway comes from the demonstration that high-affinity agonist binding sites (putative M, sites) undergo axonal transport along the fimbria and towards the hippocampus.‘W How- ever, kainic acid lesions in the hippocampus result in an almost complete loss of hippocampal muscarinic binding sites. ‘9,75 It is interesting to note that in general the reported densities of putative presynaptic receptors are relatively low. In this sense it is also worth indicating that relatively low densities of

muscarinic binding sites have been found, in the present and previous studies,22,*3*85~‘” in the basal forebrain regions where the proposed choline+ cell bodies are located. This could suggest that this particular subpopulation of receptors has a rapid turnover rate.

In contrast to the forebrain cholinergic nuclei, several cranial nerve motor nuclei innervating striated muscles (motor trigeminal, facial, ambiguus, hypoglossal) were characterized by a high abundance of both m2 mRNA and receptor binding sites labelled by [3H]oxotremorine-M. This fact indicates that a large proportion of the receptors could be located in the cell bodies or proximal dendrites of the motoneurons. An early study in the hypoglossal nucleus provides electrophysiological evidence for this location of the MChRs.69 Furthermore, ChAT-positive presumptive axon terminals have been detected in the cranial nerve motor nuclei,4s indicating that these choline& nuclei may also be cholinoceptive.*’ In addition to the mentioned somadendritic localization of M, MChRs in the motoneurons, it is also possible that a fraction of the receptors are transported to presynaptic terminals where they could be involved in the modulation of ACh release. Although there are, to our knowledge, no reports on the presence of such presynaptic M, MChRs in the terminals of these particular nerves, other motor nerve terminals have been shown to contain presynaptic MChRs modulating the release of ACh (see below). Furthermore, as will be discussed below, MChRs flowing in the vagus nerve might in fact correspond to receptors synthesized in the nucleus ambiguus.

Contrasting with the cranial nerve nuclei mentioned above, the three motor nuclei which innervate the striatal muscles responsible for eye movements (principal oculomotor, trochlear and abducens nuclei) contained undetectable levels of transcripts for m2 MChRs. Preliminary results in our laboratory indicate that these nuclei contain intermediate levels of transcripts for the m3 MChR subtype, which could account for the low or intermediate densities of MChRs (when compared to nuclei such as the facial or the hypoglossal) described in these nuclei in autoradiographic studies.2’@,‘07 Therefore, it seems that other than m2, receptors are also expressed by cholinergic neurons and could also have a role as autoreceptors. In this sense, it should be mentioned that regions such as the medial septum-diagonal band complex also contain cells expressing m3 mRNA (our own unpublished observations), but we have made no attempts yet to identify the neurotransmitter nature of these cells. The principal oculomotor, trochlear and abducens nuclei contain transcripts for both a

and B subunits of the nicotinic choline& receptor,‘03 but they also show a peculiarity in that they express the a4 subunit, whereas the motor trigeminal, facial and ambiguus nuclei express the a3 subunit.‘03 It could be hypothesized that in these three nuclei, presynaptic autoreceptors are of the nicotinic type, as

388 M. T. VILAR~ et ul.

has been suggested for the habenulo-interpeduncular pathway (see below).

The dorsal motor nucleus of the vagus nerve (which contains the cholinergic cell bodies of the preganglionic parasympathetic fibres innervating most of the viscera) was, as expected, enriched in transcripts for ChAT but no mRNA for m2 MChR was detected. We have not detected transcripts for any of the other four cloned MChR subtypes in this nucleus (unpublished observations). However, low or very low densities of muscarinic binding sites have been described in this nucleus.E6,‘07 Consequently, the levels of transcripts would also be expected to be very low, and might have gone undetected by our probes. Alternatively, it could be that the receptors present in this nucleus are encoded by a not yet cloned member of the muscarinic receptor family.” Still a further alternative would be the location of these receptors in axon terminals of afferents to the nucleus.

MChRs have been shown to flow anterogradely in the vagus nerve’08,“8 and it has been suggested that these receptors are associated with the parasympathetic motor fibres arising in the dorsal motor nucleus of the vagus. However, in both these studies’08~“8 it appears from the description of the site of the ligature that the vagus nerve was ligated at a level where it still carries motor fibres arising from the nucleus ambiguus and innervating striated muscle in the larynx and oesophagus. Given the high levels of m2 mRNA present in the nucleus ambiguus, it could well be that the MChRs flowing in the vagus nerve are synthesized in this motor nucleus and are being transported to the terminals in the striated muscle.

Finally, the medial habenula is another group of brain cholinergic cells where m2 mRNA was not detected. Despite initial controversy (see Ref. 104), the existence of a medial habenulo-interpeduncular nucleus cholinergic projection seems now well established.48.‘0’ The presence of presynaptic nicotinic receptors on the habenular terminals in the interpeduncular nucleus has been suggested by electrophysiological” and lesion” studies. The cholinergic cell bodies in the medial habenula are restricted to the ventral two-thirds of the nucleus (Ref. 20 and present results). This part of the nucleus also contains mRNA for several different subunits of nicotinic receptors 27.28~103 some of which (a 4- 1, 1x4-2 and 82) are also expiessed by the principal oculomotor, trochlear and abducens nuclei,“’ which also lack transcripts for m2 MChR. It could be postulated that these particular subunits participate in the formation of presynaptic nicotinic receptors.

In the spinal cord, the cholinergic motoneurons of the ventral horn express m2 MChRs. The existence of presynaptic MChRs modulating the release of ACh on the terminals of motor nerves is still a matter of controversy.“’ Whereas some studies do not support the existence of such muscarinic autoreceptors,40.46 others indicate the presence of inhibitory2,‘.“’ or both inhibitory and facilitatory”2,‘13 muscarinic autorecep-

tors on the motor nerve terminals. Our present results indicate that these reported muscarinic autoreceptors could belong to the M2 subtype. It remains to bc established if the proposed inhibitory and facilitatory muscarinic autoreceptors belong to the same subtype. In this sense, it would be of interest to examine whether the motoneurons express in addition other subtypes of MChR mRNAs. Most of the studies mentioned above have been performed with the rat phrenic nerve-hemidiaphragm preparation. Ad- ditional evidence supporting the presence of MChRs on motor nerve terminals comes from the demonstration of anterograde transport of MChRs in the motoric axons of the sciatic nerve.78

m2 mRNA in non-choknergic cell bodies

The presence of m2 mRNA in several regions of the rat brain (olfactory bulb, cerebral cortex, thalamic nuclei, amygdala, superior colliculus, cerebellum) which receive cholinergic inputs,32,65,73,87 suggests a postsynaptic location for this MChR subtype in these brain areas. Furthermore, since in these regions the distribution of m2 mRNA shows a high degree of overlap with that of [3H]oxotremorine-M binding sites, it could be proposed that these receptors are localized either in the cell body or in the proximal processes of the cells which synthesize them. For several of these regions, cholinergic terminal fields have been described in the areas which present high densities of m2 mRNA and [3H]oxotremorine-M binding sites. In the olfactory bulb, the external plexiform layer appears to be the site of termination of cholinergic fibres arising in the horizontal limb of the diagonal band. 32 This layer contains high densities of [3H]oxotremorine-M binding sites, which are most likely located on the dendrites of the internal granule cells whose cell bodies contain high levels of m2 mRNA. In the neocortex, dense ChAT-positive fibres and terminal-like structures as well as ChAT-positive synapses have been detected throughout all cortical layers.49 Given the presence of cells expressing m2 mRNA in certain cortical layers, it is conceivable that these cells constitute the target of a fraction of those cortical cholinergic terminals. Therefore, cortical Mz receptors may be localized both to postsynaptic cortical neurons receiving cholinergic innervation and to presynaptic cholinergic terminals arising pre- dominantly from the cells of the basal nucleus of Meynert,9.65 which are also rich in m2 mRNA.

Several other structures enriched both in m2 mRNA and [3H]oxotremorine-M binding sites have been shown to receive cholinergic innervation. This is the case of, for example, certain amygdaloid nuclei (basolateral nucleus and nucleus of the lateral olfactory tract),65.*7.“4a several thalamic nuclei,hJ~X’~“5 the lateral habenula,32~48~78a the superior colliculus70 and the pontine nuclei.‘,97

In the choroid plexus, MChRs have been described*’ with an identical distribution to the one observed for m2 MChR mRNA in the present study.


It is interesting to note that m2 mRNA was detected only in the choroid plexus within the lateral ventricles, in contrast to other neurotransmitter receptor mRNAs (e.g. serotonin-1C mRNAU), which are expressed by the choroid plexus of all ventricles. Furthermore, even within the lateral ventricles, only the subset of cells of the choroid plexus located more dorsally and rostrally contain m2 mRNA. Cholin- ergic autonomic innervation of the choroid plexus has been described in several species including rat.s5 A sympathetic adrenergic innervation has also been reported and a role for autonomic nerves in the formation of the cerebrospinal fluid has been suggested.)’ The present results suggest that this cholinergic action upon the choroid plexus would be mediated by MChRs of the M, subtype.

m2 mRNA and muscarinic binding sites

The distribution of m2 MChR mRNA was compared with that of several muscat-uric ligands: [3H]N-methylscopolamine, a classical non-selective antagonist, [3H]pirenzepine, an M,-selective antagonist,42 and [‘Hjoxotremorine-M, a proposed M,-selective agonist. 36*w The autoradiographic distributions obtained in the present study are in good agreement with previous reports. 22,w The distribution of m2 mRNA correlated best with the distribution of [‘H]oxotremorine-M binding sites, although notable differences were observed. Thus, whereas high densities of [‘Hjoxotremorine-M binding sites were observed in the caudatt+putamen, accumbens nucleus and olfactory tubercle, m2 mRNA in these regions was restricted to some scattered cells in the striatum or not detected in the olfactory tubercle. The possibility that these high levels of [3H]oxotremorine-M sites are of extrinsic origin is ruled out by the fact that most of the MChRs present in the striatum are localized to cells intrinsic to the nucleus (see Ref. 100). Moreover, the small proportion of striatal MChRs of extrinsic origin could belong to the m5 subtype.98 In a previous study,‘” we have shown that the predominant MChR population in the striatum and olfactory tubercle belongs to the M, subtype, based on the high levels of m4 mRNA and on the pharmacological profile of the muscarinic sites in these nuclei.

This fact, taken together with the high affinity displayed by oxotremorine for cloned m2 and m4 receptors expressed in mammalian cell lines,” strongly suggests that [3H]oxotremorine-M is also labelling M, receptors in the striatum and olfactory

tubercle. In the hippocampus, (3H]oxotremorine-M binding sites are present at all rostrocaudal levels of the comu Ammonis, whereas m2 mRNA is detected only at rostra1 levels. In contrast, m4 mRNA is expressed by the pyramidal cells of comu Ammonis in all its rostrocaudal extent. Therefore, [3H]oxotremorine-M labelling in the hippocampus may reflect, at least in part, binding to m4 receptors. The same considerations also apply to [‘H]ACh, another proposed M,-selective agonist, which also labels high densities of MChRs in caudate-putamen, olfactory tubercle and hippocampus@’ and which displays high affinities for cloned m2 and m4 receptors.‘* [3H]AF-DX 116, a proposed M2 antagonist, binds with high affinity to two classes of MChRs in the striatum: one with higher affinity and low capacity, and the other with lower affinity and higher capacity.4 In experiments with cloned receptors, this antagonist shows the highest affinity for m2 receptors followed by m4 receptors.is These facts, taken together with the low abundance of m2 mRNA (present results) and the high levels of m4 mRNA’OO present in the striatum, suggest that the high affinity-low capacity subclass of receptors detected by [‘H]AF-DX 116 in the striatum4 corresponds to M, MChRs, whereas the low affinity-high capacity subclass represents M, MChRs.

CONCLUSIONS

We have shown that M, MChRs are expressed by choline& cells, where they most likely act as presynaptic autoreceptors regulating the release of ACh, and also by non-choline@, putative cholinoceptive cells. Therefore, the possibility arises that in certain cholinergic synapses, M2 receptors are localized to both pre- and postsynaptic elements of the synapse. It has been suggested that M,-selective antagonists could prove useful to counteract the presynaptic cholinergic deficit observed in Alzheimer’s disease.62 In view of the present results, the possible action of such antagonists on postsynaptic M, receptors should also be taken into account. If differences exist between pre- and postsynaptic M2 receptors, they are more likely to be at the level of signal transduction than at the level of the receptor protein itself. In this sense, primary cultures or cell lines derived from cholinergic neurons44,89 and from cholinoceptive regions would be a valuable tool to establish whether such differences exist between pre- and postsynaptic M, MChRs.

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2. Abbs E. T. and Joseph D. N. (1981) The effects of atropine and oxotremorine on acetylcholine release in rat phrenic nervediaphragm preparations. Br. .I. Pharmac. 13, 481483.

3. Albretsen C., Haukanes B. I., Aasland R. and Kleppe K. (1988) Optimal conditions for hybridization with oligonucleotides: a study with myc-oncogene DNA probes. Anaiyf. Biochem. 170, 193-202.

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(Accepted 9 September 1991)

Muscarinic M2 receptor mRNA expression and receptor binding in cholinergic and non-cholinergic cells...

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