0306.4522:90 $3.00 + 0.00 Pergamon Press plc

.Yrurosciencr Vol. 36, No. I. pp. 165-174, 1990 Printed in Cireat Britain

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LAMINAR DISTRIBUTIONS OF MUSCARINIC ACETYLCHOLINE, SEROTONIN, GABA AND OPIOID RECEPTORS IN HUMAN POSTERIOR CINGULATE CORTEX B. A. VOGT,* I/ M. D. PLAGER,? P. B. CRINO:!/

and

E. D. BIRD$

Departments

of *Anatomy, tPhysiology and $Behavioral Neuroscience, Boston University School of Medicine, 80 East Concord Street, Boston, MA 02118, U.S.A. $Brain Tissue Resource Center, McLean Hospital, 115Mill Street, Belmont, MA 02178. U.S.A. j/Veterans Administration Hospital, 200 Springs Road, Bedford, MA 01730, U.S.A.

Abstract-Experimental animal studies have demonstrated a number of receptor localizations on specific cortical afferents and neurons. The present study of human posterior cingulatc cortex evaluates the laminar distributions of particular receptors and their likely association with components of the neuropil. Coverslip autoradiographic and single grain counting techniques were used followed by heterogeneity analysis in which the layer of peak binding and an index of heterogeneity were determined for each ligand. The index was calculated by determining specific binding by layer as a percentage of binding in all layers. The differences from an absolutely homogeneous distribution, i.e. 11.1% for each of nine layers. were subtracted and the absolute laminar differences summed to form the index. High indices of over 15 reflected heterogeneous binding patterns in neocortex. The binding of ligands for muscarinic acetylcholine. serotonin, opioid, GABA and beta adrenoceptors was evaluated. Pirenzepine binding peaked in layer II of area 23a but was extremely homogeneous with an index of heterogeneity of 8.9. In contrast, oxotremorine-M binding had a peak in layer IlIc and an index of 16.4, while AF-DX I16 binding peaked in layer IIIa -b and had an index of 30.6. Of the ligands for serotonin uptake and receptor binding paroxetine binding was evenly distributed in layers I-111 and had a low index of heterogeneity of 9.8. Ketanserin binding was also homogeneous and, since it had an index of 8.9, this pattern was virtually the same as that for paroxetine. In contrast, serotonin and Il-hydroxy2-(di-~-propylamino)tetraiin binding peaked in layer II and had very high indices of 20.8 and 50.3. respectively, suggesting only a limited association with that of the paroxetine distribution. Finally. there were three layers which contained peaks in binding for ligands for opioid, GABA and beta adrenoceptors. Firstly, layer Ia had peak dynorphin-A binding, the latter of which had an index of 22.6. Secondly. Tyr-o-Ala-Gly-MePhe-Gly-ol and 2-D-penicillamine-5-D-penicillamine-enkephalin binding peaked in layer II and had indices of 8.6 and 17.4, respectively. Thirdly. muscimol and (-)-cyanopindolol binding peaked in layer IiIa-b and had indices of 29.6 and 11.1, respectively. When viewed in the context of experimental animal studies, it is likely that heterogeneities in oxotremorine-M and paroxetine binding are associated with the termination of the thalamic and raphe nuclei. respectively. While serotonin> receptors are co-distributed with serotonin uptake sites. serotonin,, receptors have a significant mismatch with these sites. Finally, postsynaptic receptors preferentially peak in different layers including kappa opioid receptors in layer la, M, acetylcholine. serotonin,,, mu and delta opioid receptors in layer II, GABA, and beta adrenoceptors in layer IIIa-b and serotonin, receptors in iayer 111~. Thus, ligand binding analyses provide new insights into the structure and connections of human cerebral cortex

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is known, however, about connections brain because most tract tracing techniques are experimental in nature and cannot be conducted prior to death. In some instances neuronal degeneration following naturally occurring or monkey.

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Ahhreriation.s: AF-DX 116, 11[[2-[(diethyl-amino)methyl]I -piperidinyI]acetyl]-5, I 1-dihydro-6H-pyrido[2,3-6][ I ,4]benzodiazepine-6-ones DAGO, Tyr-D-Ala-Giy-MePh~Gly-ol; DPDPE, Z-u-penicillamine-5-o-penicillamineN-2-hydroxyethylpiperazineenkephalin; HEPES; N’-2-ethanesulfonic acid: S-HT. serotonin: S-OHDPAT. X-hydroxy-2-(di-n-propylamino)tetralin; OXOM~pirenzepine, [ 3H]oxotremorine-M coincubated with 50 nM unlabeled pirenzepine. 165

neurosurgicaily-placed lesions in human brain have been studied with cell degeneration,>“ axon demyelination ‘*z~J’ silver-stained axon degeneration’.“.” and imm;nohistochemical” techniques. Most of these approaches require the fortunate association of a restricted and well placed lesion with an approp~ate post-lesion survival time; requirements which greatly restrict their systematic application in studies of the human brain. The advent of neurotransmitter receptor subtype localization using cryomicrotome section autoradiography has provided the basis for systematic and high resolution studies of the structural organization of the human cerebral cortex. It is proposed here that this technique, in conjunction with heterogeneity analysis and comparison with experimental

localization findings. can be used to gain insights into specific connections of the human cerebral cortex. Heterogeneity analysis involves determining the layer(s) in which peak(s) in specific binding occur(s) and calculating an index of heterogeneity. This index is calculated by determining specific binding in each layer as a percentage of binding in all layers. The differences from an absolutely homogeneous distribution, i.e. 11.1% for each of nine layers in neocortex, are then subtracted from the former distribution. The absolute differences are summed to produce the index. Thus, the index of heterogeneity measures the extent to which one proportionate laminar distribution in receptor binding differs from another or from absolute homogeneity.” For example, ligands for muscarinic acetylcholine receptors have ditrerent laminar distributions in rat posterior cingulate cortex. Oxotremorine-M (0X0-M) binds preferentially to M2 acetylcholine receptors and this binding peaks in layers Ia and IV, while pirenzepine binds to M, receptors and is relativeiy holnogeneous throughout all layers. The index of heterogeneity can be calculated by subtracting the proportionate laminar distribution for pirenzepine from the same distribution for 0X0-M binding. The absolute values of these differences are summed to form the index. Higher indices reflect heterogeneous distributions of receptors, while low indices represent homogeneous distributions. Therefore. this procedure dissociates the binding in peak layers, i.e. heterogeneities. from that which is homogeneously distributed throughout all layers. There are two instances in which heterogeneities in receptor binding have been experimentally demonstrated to be associated with afferent connections and one in which they have been associated with neuronal dendrites. Firstly, ablations of rat anterior thalamic nuclei reduce binding of 0X0-M by 50% in layers Ia and IV of rat cingulate cortex. These are likely to be M, acetylcholine heteroreceptors.“’ Secondly, removal of raphe afferents to cingulate cortex in the rat with either ablation of the dorsal raphe nuclei or intraventricular 5,7-dihydroxytryptamine injections reduces peak binding of paroxetine to serotonin (5-HT) uptake sites by 70% in layer Ia.’ Thirdly, the peak in muscimol binding to GABA, receptors in layer Ia of rat cingulate cortex is reduced following neurotoxin-induced removal of cortical neurons, suggesting that these sites are on the apical dendritic tufts of pyramidal neurons.” The distribution of thalamic projections and dendrites of cortical neurons have been described in posterior cingulate cortex of non-human primates. Projections of the anterior thalamic nuclei terminate mainly in the granular layer of area 2936 and those of the medial pulvinar in layers 111~ and IV of area 23.4 The distribution of apical and basal dendrites of cortical pyramidal neurons as well as some of the features of non-pyramidal neurons have been describedJ5 and can be used in the context of

interpreting receptor binding patterns in thlh cortlcsl region. However, previous receptor binding studies of human brain have generally not provided ;L high degree of regional or laminar localization information so that it is difficult to draw conclusions about possible associations between particular components of the neuropil and specific receptors. For example, although autoradiographic studies have demonstrated the binding of paroxetine.” S-l-IT and 8-hydroxy-2-(di-~-propylamino)tetralill (&OHDPAT)‘7,‘8 and ketanserin’R,‘7 in human cerebral cortex, the laminar binding distributions of these ligdnds were not fully described in cingulate cortex and reports for other areas were only partial. The strategy for the present study was to evaluate laminar heterogeneities in receptor subtype binding as a probe for specific connections in human posterior cingulate cortex. Radiolabeled probes were used to analyse the laminar distributions of M, and M, acetylchoIine, 5-HT uptake and 5-HT,,, 5-HT,. GABA,. opioid and beta adrenoceptors. The distribution of thalamic and raphe afferents and intrinsic dendrites were then inferred from heterogeneities in the binding of ligands to these receptors. As probes for receptor subtypes are refined it may be expected that this type of analysis can be applied to a complete range of specific connections in the mammalian central nervous system including the human species.

Blocks of posterior cingulate cortex were obtained posf mortem from nine individuals without a clinical history of dementia and who died from non-neurological causes. In some cases blocks were taken from previously frozen, coronal, 1-2-cm-thick slabs and in other cases blocks were removed from brains in an ice slurry and then frozen to - 70°C. In all instances the brain was frozen to - 70°C and not rewarmed for dissection. The post mortem interval was 14 + 1.5 h (mean t: S.E.M.). There were seven male and two female cases whose age at death was 66 & 4.2 years and whose brains weighed 1336 + 52 g. Sections were cut on a cryomicrotome at a 16 hrn thickness for receptor binding or were cut at three times this thickness and counterstained with Thionin for cytoarchitectural analysis. The sections were mounted on chrome-alum subbed slides. Materials

Unlabeled pirenzepine was kindly provided by Boehringer Ingelheim, Ltd. Radiolabeled ligands were purchased from New England Nuclear and included the foIlowing: 13H]pirenzipine (specific activity 84 Ci/mM), [3HfoXO-M; (specific activity 85.1 CijmM), (3H]I 1[~2-(d~ethyl-amino)methyl]-lpi~ridinyl]a~tyl]-5,l l-dihydr~6H-py~do[2,3-6]~1,4]~nzodiazepine-&one ([)H]AF-DX I 16; specific activity 59.8 Ci/mM), 13HJparoxetine (specific activity 23.1 CijmM), [ ‘HIS-HT (specific activity 30 Ci/mM), [ 3H]8-OH*DPAT (specific activity 142.9 Ci/mM), [ ‘HI-ketanserin (specific activity 61 Ci/mM), [3H]muscimol (specific activity 23.2 Ci/mM), [3H]Tyr-o-Ala-Gly-MePhe-Gly-ol (DAGQ; specific activity 30.3 Ci/mM), [“H]2-D-penicillamine-!&Dpenicillamine-enkephalin (DPDPE; specific activity 43 Ci/mM), [3H]dynorphin-A (l-8) (specific activity

Receptor 27.6 CLmM) 2200 WmM).

and

[ “‘I]( - )-cyanopindolol

localization

(specific

in human

activity

During the course of these studies a total of nine cases were available for analysis. All nine cases were evaluated for pirenzepine. 0X0-M, 5-HT and muscimol binding. Six cases were used for &OH-DPAT, ketanserin. dynorphin-A and (-)-cyanopindolol binding. Four cases were employed for the analysis of AF-DX 116, DAGO, DPDPE and parcltoxine binding. The conditions for ligand binding experiments have been reported previously for both human and experimental animal brainsY,‘X.“.29.“.‘X.“Uand so will only be briefly stated here. All buffers were at pH 7.4. Pircpn:rpinr. Sections were incubated in I2 nM [ ‘Hjpircnzepine in Krebs-Henseleit buffer for 70 min at 25 C followed by two buffer washes at 4 C for 2 min each. Non-specific binding was assessed in a parallel series with I HIM atropine. O.wtremorinr-M. Sections were incubated in 0.1 or 5 nM [?H]OXO-M in 20mM HEPES Tris buffer with 10 mM Mg’ ’ and 50 nM pirenzepinc for 30 min at 25 C followed by two buffer washes at 4 ‘C for 2 min each and one 2-min water wash. Non-specific binding was determined with 1I’M atropine in a parallel series of sections. Purosetinc~. Sections were incubated in 100 pM ~~~]~ir~xetine in 50 mM Tris buffer with 120 mM NaCl and 5mM KC1 for 120min at 25 ‘C followed by two washes at 15 C for I h each and a I-min water wash at 4’C. Non-specific binding was assessed in a parallel series which inclu~~~d 100 ~5M Auoxetine. Erotonin. Sections were preincubated in 5OmM Tris buffer with 5.7 mM ascorbatc, IO,L~M pargyline and 4 mM CaCI, for 15 min at 25 C. They were then incubated in the Same buffer with 3 nM [‘HJS-HT at 25‘C for 40 min followed by three buffer washes at 4 C for 1min each. Non-specific binding was determined in a parallel series with I /lM 5-HT. K~~rcmsc~rin.Sections were preincubated in 50 mM Tris at 25 C for IOmin and then incubated in 2 nM [:H]ketanserin at 30 C for 30 min followed by three buffer washes at 4 C for I min each. 8-ff~~lrt)x~-2-(rli-n-~rc)~~luminn)terru~in. Sections were preiilcubated in I70 mM Tris with 4 mM CaCI, and 0.01% ascorbate at 25 C for 3Omin. The sections were then incubated in buffer with 2 nM I’HWOH-DPAT at 25 C for 60 min followed by two buf& ha&es at 4 C for 5 min each. Non-specific binding was evaluated in a parallel series of sections with 100 11M unlabeled 8-OH-DPAT. ~~z~~s~,~t?~~~~. Sections were preincubat~ in 50 mM Tris buffer at 20 C for 40min. They were then incubated in the same buffer with 2OnM [ ‘H]muscimol for 15 min at 20’C followed by one buffer wash at 4 C for 2 min and a Water wash at 4 C for 2min. 2-l~-Pf~ni~illumin~-S-u-~~mi~ilk~minr-clnke~halin. Sections were preincubated in 50mM Tris with 5mM MgCL, 2 my ml bovine serum albumin. ZO~g/ml bacitracin, IOOmM NaCl and 50pM GTP at 25 C for IS min. The sections were then washed twice in a similar buffer to remove the GTP at 25 C for S min each. Incubati(~n of [ ‘H]DPDPE was conducted in 50 mM Tris with 2 mg/ml bovine serum albumin at 25 C for 60 min followed by three butTer washes at 4 C for IO min each. Non-specific binding was assessed in a parallel series with 1JIM DPDPE. T,,r-u-Aku-Cam-M~Pllr-GI,-oi. Sections were incubated in 50 mM Tris with I nM [jH]DAGO at 25 C for 45 min followed by three buffer washes at 4 C for 1 min each. Non-specific binding was evaluated in a parallel series of sections with 1!tM levdllorphan which was kindly provided by Ho~tnanu-~ Roche. Inc. Dynorphm-A. Sections were incubated in 50 mM Tris with 5 nM [?H]dynorphin-A at 4 C for 90 min followed by four buffer washes at 4 C for 2 min each. Non-specific

cingulate

cortex

167

binding was evaluated in a parallel series with I JIM levaliorphan. (- )-C~anopindoW Sections were incubated in i 70 mM Tris buffer with 135 mM NaCl, I ,uM 5-HT and 65 pM [ “‘I]( -)-cyanopindoloi at 25 C for 120 min foilowed by three buffer washes at 4:C for 20min each and a 5-s water wash. Non-specific binding was determined in a parallel series with 30 it M isoproterenol. 11[[2-[(i3i~~f~~~-unlina)m~fh~i]l-pjperjdj~~l ]WPf~~l]-L,l I The dih.ydro- 6H -p.~rin’o[2,3 - 6][ I ,4]ben-_udiazupinP-hww

protocol for [jH]AF-DX I I6 binding included the following steps: pr~in~ub~tion in 50mM NajKPO, buffer (pH 7.4, 30 min, 25’ C); incubation in the same buffer with 5 nM [?H]AF-DX 1I6 (30 min, 25’ C); a wash in buffer (3 min. 4’ C); a wash in distilled water (1 min, 4’ C); rapid drying. Autoradiographs were prepared according to the method of Young and Kuhar.4’ Coverslips were acid cleaned and dipped in Kodak NTB-2 nuclear tract emulsion. The dried coverslips were attached to slides with cyanoacryiatc and exposed in the dark at -20 C for three weeks to eight months. All autoradiographs were developed in Kodak D-19 without hardener, fixed in Kodak Rapid Fixer and then counterstained with Thionin. Q~u~ti~~,at~on

of u~t~)rf~dj~~rup~l.~

The cytoarchitecture of posterior cingulate cortex in and human brains has been thoroughly monkey analvsed.0,2’,“4 For the present study the exact boundaries of areas in posterior cin&ate cortex”are those of Vogt’h and Vogt et ul.‘OThe mediclateral distribution of areas in the posterior cingulate cortex is shown in Fig. I. The five areas that were analysed in this study included areas 29 I, 29m, 30, 23a and 23b. Each cortical area and its layers and sublayers. i.e. layers la, Ic, If. II&b, 111~. IV. Va, Vb and VI were identified on bright-field illumination and then dark-field illumination was used so that a computerized image analysis system (Image Technology Model 1000, DonSanto Corp. were quantified per 2500pm’ of a cortical layer in three

i

Fig. I. Medial surface of the human brain. Sections were cut coronally through posterior cingulate cortex. The borders of each area are marked with arrows in the trdW#erSe section. Grain densities were quantified in each area at levels marked by the dotted lines. CC. corpus callosum: CS. cingulate sulcus; Sub, subiculum.

168

H A. Lo

Laminar distributions of muscarinic acetylcholine, serotonin, GABA and opioid receptors in human posterior cingulate cortex.

Experimental animal studies have demonstrated a number of receptor localizations on specific cortical afferents and neurons. The present study of huma...
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