Neurochemical Research, VoL 17, No. 11, 1992, pp. 1057-1062
Autoradiography of Muscarinic Cholinergic Receptors in Cortical and Subcortical Brain Regions of C57BL/6 and DBA/2 Mice C. Schwab l, G. Briickner x,4, T. RothC, C. Castellano 2, and A. Oliverio 2,3 (Accepted February 27, 1992)
Mice of the inbred strains C57BL/6 and DBA/2 show strain-dependent behavioural differences which have been correlated with variations in brain cholinergic systems. In the present study, the density of muscarinic cholinergic receptors in both strains of mice was determined by autoradiographic methods using [3H]quinuclidinyl benzilate (QNB) and pH]pirenzepine as ligands. C57BL/6 mice showed a significantly lower [3H]QNB binding level in the frontal cortex by one third as compared to DBA/2 mice. In the striatum and the cholinergic pontomesencephalic nucleus laterodorsalis tegmenti the [3H]QNB binding was lower in C57BL/6 by 28% and 31%, respectively. The [3H]pirenzepinebinding level was found to be significantly higher in C57BL/6 temporal cortex (by 22%). These results are discussed in relation to interstrain differences in cholinergic cell density and in the activity of cholinergic enzymes. KEY WORDS: [3H]QNB;[3H]pirenzepine;autoradiography;C57BL/6mice; DBA/2mice.
brain chotinergic systems. Using acetylcholinesterase (AChE)-pharmacohistochemistry and choline acetyltransferase (ChAT)-immunohistochemistry a higher cell density of cholinergic neurons was observed in the striaturn, and in cholinergic projection systems of the basal forebrain and the pontomesencephalon of DBA mice as compared to C57 (6,7,8). DBA mice also showed higher AChE and ChAT activities in whole brain homogenates, and even more prominent, in certain brain regions, such as striatum, basal forebrain and frontal cortex (2,9,10,11). In contrast to these findings, only small interstrain differences in muscarinic acetylcholine receptor (mAChR) levels have been described in homogenates of brain tissue (5,12). In the present study, we carried out quantitative receptor autoradiography using [3H]QNB and [3H]pirenzepine as cholinergic ligands. It was the aim of our study to show in small, anatomically defined brain areas, whether ligand binding to cholinergic muscarinic receptors may be correlated with the above-mentioned
INTRODUCTION Previous studies revealed behavioural and pharmacological differences between the two inbred strains of mice, C57BL/6 (C57) and DBA/2 (DBA). C57 mice score poorly in active avoidance behavior and maze learning in comparison to the DBA strain (1,2,3). Cholinergic agonists, such as oxotremorine and nicotine, depress open field activity and body temperature in DBA mice more severely than in C57 (4,5). These strain-dependent differences have been correlated with biochemical and morphological variations in the organization of i Paul Flechsig Institute for Brain Research, Department of Neurochemistry,Universityof Leipzig, Leipzig(F.R.G.). 2 Istitutodi Psicobiologiae Psicofarmacologiadel CNR and 3 Dipartimento di Genetica e Biologia Molecolare, Universit~ "La Sapienza", Roma(Italy). 4 To whomto addressreprintrequests:Dr. G. Briickner,PaulFlechsig Institute for Brain Research, Departmentof Neurochemistry,University of Leipzig, Jahn-Allee59, 0-7010 Leipzig, F.R.G. 1057
0364-3190/92/1100-1057506~50/0 9 1992 Plenum Publishing
Corporation
1058
Schwab, Briickner, Rothe, Castellano, and Oliverio
results on strain-dependent differences in cholinergic systems.
sorption values of autoradiograms of the tissue standards with known radioactivity. Statistical Evaluation. Differences between the groups of the two mice strains were examined using a two-tailed Student's t test.
EXPERIMENTAL PROCEDURE RESULTS Animals. Male mice of the inbred strains C57BL/6 and DBA/2 (5 for each strain) at an age of 50 postnatal days were used. Animals were bred in our laboratory from stock originally obtained from the Charles River Lab, Italy. All animals were housed in a 12/12 h light/dark cycle with free access to food and water. Tissue Preparation. The animals were killed by cervical dislocation. Brains were removed from the skull and rapidly frozen in isopentane at - 70~ Frozen brains were stored at - 70~ Sections (20 Ixm thick) were cut at - 20~ in a coronal plane with a microtomecryostat (Tissue Tek, Miles, USA), thaw-mounted onto gelatinized slides and stored at -20~ Binding Assay. Binding studies in slide-mounted tissue sections were carried out at room temperature as described recently (13). For muscarinic cholinergic receptor binding, the sections were incubated with 50 mM sodium-potassium phosphate buffer, pH 7.4, containing [3H]l-quinuclidinylbenzilate [3H]QNB; specific activity 1.11 TBq/mmol; Radiochemical Centre Amersham) at a final concentration of 2 nM. After 3 h of incubation, slides were rinsed two times (5 min each) in ice-cold buffer before drying in a light nitrogen stream. For assaying the muscarinic Mi-receptor subtype, slides were incubated with 10 mM sodium phosphate buffer, pH 7.4, containing [3H]pirenzepine (specific activity 2.05 TBq/mmol; Radiochemical Centre Amersham) at a final concentration of 9 mM for 1 h, followed by two rinses, 1 min each, in ice-cold buffer and dried in a light nitrogen stream. Unspecific binding was determined in adjacent brain sections, coineubated with 100 ~M atropine sulphate in the incubation buffer. No measurable unspecifi c binding was observed in [3H]QNB or [3H]pirenzepine incubated sections. ReceptorAutoradiography. The labeled and dried tissue sections and slides containing calibration standards from brain paste of known radioactivity level (14) were covered with tritium-sensitive film (U1trofilm, LKB). After 3 weeks of exposure in the presence of desiccator at 4~ the films were developed with Kodak 19b. Quantitative Analysis. Quantitative analysis of the antoradiograms was carried out with a computer-assisted imaging device, consisting of a CCD video camera (EEV Photon), a monitor and a PC/AT computer with EGA graphic adapter and mouse, using an autoradiographic software package written in the Institute of Pathophysiology, Ljubljana, Slovenia. Optical density reading of the autoradiograms was performed by circumscribing the area of selected brain regions on the digitized image and by measuring the mean grey level. According to an atlas of the mouse brain (15) the following brain regions were selected: frontal cortex (fro), parietal cortex (pa), piriform cortex (pir), temporal cortex (te); amygdala (amy), hippocampus (hi), striatum (str), medial septum (sin), nucleus of the diagonal band (db), nucleus basalis of Meynert (nmb), substantia innominata (si), hypothalamus (hy), colIiculus superior (cs), colliculus inferior (ci), nucleus pedunculopontinus tegmenti (pp) and nucleus laterodorsalis tegmenti (ld). Each of these brain regions was measured in each animal in quadruplicate (in two adjacent sections at both the left and the right half of the brain) with the exception of the medial septum which was measured in duplicate. The density of the receptor binding sites was calculated from the grey level determined in the corresponding tissue region using the calibration curve obtained from the densitometrieally determined ab-
[3H]QNB binding to the total mAChR varied in a wide range between the brain regions within both strains as shown in Figures 1A, 2A-C, 3A,B. The highest levels of binding were found in neoeortical regions, the hippocampus and the striatum (with the exception of striarum and frontal cortex in C57). ci, cs and amygdala showed moderate levels of binding. Cholinergic basal forebrain regions, cholinergic pontomesencephalic nuclei and the hypothalamus displayed the lowest binding level. [3H]pirenzepine binding to the M1 AChR subtype showed the highest values in the striatum and the hippocampus whereas lower levels were detected in neocortical regions and the amygdala (Figures 1B, 2D-F, 3C,D). All other subcortical areas (sin, db, si, nbm, hy) were found to have a very low level of binding, and in ci, cs, pp, and ld, the [3H]pirenzepine binding was too low to be detectable (Figure 3C,D). A lower level of [3H]QNI3 binding in C57 mice as compared to DBA was measured in three of the evaluated regions. In the frontal cortex (67%, p
Autoradiography of Muscarinic Cholinergic Receptors in Cortical and Subcortical Brain Regions of C57BL/6 and DBA/2 Mice C. Schwab l, G. Briickner x,4, T. RothC, C. Castellano 2, and A. Oliverio 2,3 (Accepted February 27, 1992)
Mice of the inbred strains C57BL/6 and DBA/2 show strain-dependent behavioural differences which have been correlated with variations in brain cholinergic systems. In the present study, the density of muscarinic cholinergic receptors in both strains of mice was determined by autoradiographic methods using [3H]quinuclidinyl benzilate (QNB) and pH]pirenzepine as ligands. C57BL/6 mice showed a significantly lower [3H]QNB binding level in the frontal cortex by one third as compared to DBA/2 mice. In the striatum and the cholinergic pontomesencephalic nucleus laterodorsalis tegmenti the [3H]QNB binding was lower in C57BL/6 by 28% and 31%, respectively. The [3H]pirenzepinebinding level was found to be significantly higher in C57BL/6 temporal cortex (by 22%). These results are discussed in relation to interstrain differences in cholinergic cell density and in the activity of cholinergic enzymes. KEY WORDS: [3H]QNB;[3H]pirenzepine;autoradiography;C57BL/6mice; DBA/2mice.
brain chotinergic systems. Using acetylcholinesterase (AChE)-pharmacohistochemistry and choline acetyltransferase (ChAT)-immunohistochemistry a higher cell density of cholinergic neurons was observed in the striaturn, and in cholinergic projection systems of the basal forebrain and the pontomesencephalon of DBA mice as compared to C57 (6,7,8). DBA mice also showed higher AChE and ChAT activities in whole brain homogenates, and even more prominent, in certain brain regions, such as striatum, basal forebrain and frontal cortex (2,9,10,11). In contrast to these findings, only small interstrain differences in muscarinic acetylcholine receptor (mAChR) levels have been described in homogenates of brain tissue (5,12). In the present study, we carried out quantitative receptor autoradiography using [3H]QNB and [3H]pirenzepine as cholinergic ligands. It was the aim of our study to show in small, anatomically defined brain areas, whether ligand binding to cholinergic muscarinic receptors may be correlated with the above-mentioned
INTRODUCTION Previous studies revealed behavioural and pharmacological differences between the two inbred strains of mice, C57BL/6 (C57) and DBA/2 (DBA). C57 mice score poorly in active avoidance behavior and maze learning in comparison to the DBA strain (1,2,3). Cholinergic agonists, such as oxotremorine and nicotine, depress open field activity and body temperature in DBA mice more severely than in C57 (4,5). These strain-dependent differences have been correlated with biochemical and morphological variations in the organization of i Paul Flechsig Institute for Brain Research, Department of Neurochemistry,Universityof Leipzig, Leipzig(F.R.G.). 2 Istitutodi Psicobiologiae Psicofarmacologiadel CNR and 3 Dipartimento di Genetica e Biologia Molecolare, Universit~ "La Sapienza", Roma(Italy). 4 To whomto addressreprintrequests:Dr. G. Briickner,PaulFlechsig Institute for Brain Research, Departmentof Neurochemistry,University of Leipzig, Jahn-Allee59, 0-7010 Leipzig, F.R.G. 1057
0364-3190/92/1100-1057506~50/0 9 1992 Plenum Publishing
Corporation
1058
Schwab, Briickner, Rothe, Castellano, and Oliverio
results on strain-dependent differences in cholinergic systems.
sorption values of autoradiograms of the tissue standards with known radioactivity. Statistical Evaluation. Differences between the groups of the two mice strains were examined using a two-tailed Student's t test.
EXPERIMENTAL PROCEDURE RESULTS Animals. Male mice of the inbred strains C57BL/6 and DBA/2 (5 for each strain) at an age of 50 postnatal days were used. Animals were bred in our laboratory from stock originally obtained from the Charles River Lab, Italy. All animals were housed in a 12/12 h light/dark cycle with free access to food and water. Tissue Preparation. The animals were killed by cervical dislocation. Brains were removed from the skull and rapidly frozen in isopentane at - 70~ Frozen brains were stored at - 70~ Sections (20 Ixm thick) were cut at - 20~ in a coronal plane with a microtomecryostat (Tissue Tek, Miles, USA), thaw-mounted onto gelatinized slides and stored at -20~ Binding Assay. Binding studies in slide-mounted tissue sections were carried out at room temperature as described recently (13). For muscarinic cholinergic receptor binding, the sections were incubated with 50 mM sodium-potassium phosphate buffer, pH 7.4, containing [3H]l-quinuclidinylbenzilate [3H]QNB; specific activity 1.11 TBq/mmol; Radiochemical Centre Amersham) at a final concentration of 2 nM. After 3 h of incubation, slides were rinsed two times (5 min each) in ice-cold buffer before drying in a light nitrogen stream. For assaying the muscarinic Mi-receptor subtype, slides were incubated with 10 mM sodium phosphate buffer, pH 7.4, containing [3H]pirenzepine (specific activity 2.05 TBq/mmol; Radiochemical Centre Amersham) at a final concentration of 9 mM for 1 h, followed by two rinses, 1 min each, in ice-cold buffer and dried in a light nitrogen stream. Unspecific binding was determined in adjacent brain sections, coineubated with 100 ~M atropine sulphate in the incubation buffer. No measurable unspecifi c binding was observed in [3H]QNB or [3H]pirenzepine incubated sections. ReceptorAutoradiography. The labeled and dried tissue sections and slides containing calibration standards from brain paste of known radioactivity level (14) were covered with tritium-sensitive film (U1trofilm, LKB). After 3 weeks of exposure in the presence of desiccator at 4~ the films were developed with Kodak 19b. Quantitative Analysis. Quantitative analysis of the antoradiograms was carried out with a computer-assisted imaging device, consisting of a CCD video camera (EEV Photon), a monitor and a PC/AT computer with EGA graphic adapter and mouse, using an autoradiographic software package written in the Institute of Pathophysiology, Ljubljana, Slovenia. Optical density reading of the autoradiograms was performed by circumscribing the area of selected brain regions on the digitized image and by measuring the mean grey level. According to an atlas of the mouse brain (15) the following brain regions were selected: frontal cortex (fro), parietal cortex (pa), piriform cortex (pir), temporal cortex (te); amygdala (amy), hippocampus (hi), striatum (str), medial septum (sin), nucleus of the diagonal band (db), nucleus basalis of Meynert (nmb), substantia innominata (si), hypothalamus (hy), colIiculus superior (cs), colliculus inferior (ci), nucleus pedunculopontinus tegmenti (pp) and nucleus laterodorsalis tegmenti (ld). Each of these brain regions was measured in each animal in quadruplicate (in two adjacent sections at both the left and the right half of the brain) with the exception of the medial septum which was measured in duplicate. The density of the receptor binding sites was calculated from the grey level determined in the corresponding tissue region using the calibration curve obtained from the densitometrieally determined ab-
[3H]QNB binding to the total mAChR varied in a wide range between the brain regions within both strains as shown in Figures 1A, 2A-C, 3A,B. The highest levels of binding were found in neoeortical regions, the hippocampus and the striatum (with the exception of striarum and frontal cortex in C57). ci, cs and amygdala showed moderate levels of binding. Cholinergic basal forebrain regions, cholinergic pontomesencephalic nuclei and the hypothalamus displayed the lowest binding level. [3H]pirenzepine binding to the M1 AChR subtype showed the highest values in the striatum and the hippocampus whereas lower levels were detected in neocortical regions and the amygdala (Figures 1B, 2D-F, 3C,D). All other subcortical areas (sin, db, si, nbm, hy) were found to have a very low level of binding, and in ci, cs, pp, and ld, the [3H]pirenzepine binding was too low to be detectable (Figure 3C,D). A lower level of [3H]QNI3 binding in C57 mice as compared to DBA was measured in three of the evaluated regions. In the frontal cortex (67%, p
