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Muscarinic cholinergic receptors in the human spinal cord: differential localization of [3H]pirenzepine and [3H]quinuclidinylbenzilate binding sites
I N c " , i~'r
BRE 21092
Muscarinic cholinergic r ~ o r s in the human ~inal cord: differential localization of [3H]pirenzepine and [aH]quinuclidinylbenzilate binding sites JOHN W. VILIAGER1and RICHARD L. M. FAULL2
1Department of Pharmacology and Clinical Pharmacology and 2Department of Anatomy, School of Medicine. University of A uckland, Auckland (New Zealand) (Accepted May 21st, 1985)
Key words: [3H]quinuclidinylbenzilate - - [3H]pixenzepine- - muscarinic cholinergic receptor subtype - - human --spinal cord
The localization of muscarinic cholinergic receptor subtypes was studied in the human spinal cord using in vitro labelling of cryostat sections with pH]quinuclidinylbenzilate (QNB) and [3H]pirenzepine (PZ) followed by autoradiography. The highest densities of [3H]QNB binding were localized in laminae II (substantia gelatinosa) and IX (motor neurons); in contrast, the highest density of [3H]pZ binding was localized to lamina II where the binding density was 22-32% higher than in lamina IX. These results suggest that the M 1and M2 muscarinic cholinergic receptor subtypesmay be differentially localized in sensory and motor regions of the human spinal cord. Considerable evidence suggests that acetylcholine functions as a neurotransmitter in the mammalian spinal cord 9. In the human spinal cord the distribution of choline acetyltransferasel, 6 and muscarinic cholinergic receptors 3.10.12 have been studied using biochemical and histochemical techniques. These studies have consistently shown enzyme activity and receptor density to be greatest in the superficial dorsal horn and ventral horn gray matter. More specifically, in the human cord muscarinic receptors have been localized in lamina II and the lateral portion of lamine IX 10. Pirenzepine (PZ) is a non-classical muscarinic antagonist which has been shown to distinguish two subclasses of muscarinic receptors 4 termed M1 and Me receptors, Pirenzepine has high affinity for M t sites and low affinity for M 2 sites, while other muscarinic antagonists (e.g. atropine, quinuclidinylbenzilate) exhibit high affinity for both sites. Binding studies have shown both M 1 and M 2 sites to exist in rat 4,5.11 and human2 brains. Furthermore, autoradiographic studies have demonstrated a differential distribution of [3H]pZ and [3H]quinuclidinylbenzilate (QNB) binding sites in the rat spinal cord and brainstem a3. In
the rat spinal cord [3H]QNB sites were localized in both the substantia gelatinosa of the dorsal horn and in the ventral horn, whereas [3I-t]PZ sites were concentrated predominantly in the substantia gelatinosa 13. In this study we have compared the localization of [3H]QNB and [3H]pZ binding sites in sections of the human spinal cord. Postmortem human spinal cord tissue was obtained from 4 subjects with no known history of neurological disease or drug treatment. Following postmortem delays ranging from 11-32 h. the spinal cord was removed and 1-2-cm blocks were immediately selected from the cervical, lumbar and sacral regtons of the cord and frozen onto microtome chucks using dry ice. They were then sectioned transversely at 16 p m using a cryostat and mounted onto acid-cleaned gelatine/chrome alum-coated slides. Slide-mounted sections were preincubated in 150 ml 50 mM sodium phosphate buffer (pH 7.4) for 30 mm at 20 °C and then incubated in the same buffer containing 1 nM [3H]QNB (40.2 Ci/mmol, New England Nuclear) for 2 h or 20 nM [3H]pZ (82.3 Ci/ retool. New England Nuclear) for 1 h at 20 °C. The reaction was terminated by I ([3H]pZ) or 2
Correspondence: R. L. M. Faull. Department of Anatomy, School of Medicine. University of Auckland. Private Bag, Auckland New Zealand. 0006-8993/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
197
i:~H)QNB
la
(H!PZ
C7
lb
07
(:~H)QNB
2a
L4
Figs. 1-3. Autoradiograms showing the distribution of [3HIQNB (Figs. la, 2a, 3a) and [3H]pZ (Figs• lb, 2b, 3b) binding sites in transverse hemiseetions of the human spinal cord at the levels indicated. At each level, the distance between section a and section b is 64 ~tm. Note that in each autoradiogram the region of dense labelling in the dorsal horn of the gray matter is located within lamina II and the region of moderate to heavy labelling in the lateral two-thirds of the ventral horn is localized to the region of lower motor neuron nuclei which constitute lamina IX. Fig. 3 on page 198.
198 ([3H]QNB) 5-rain washes in sodium phosphate buffer at 4 °C. The slides were then washed for 1 min ([3H]PZ) or 2-3 s ([3H]QNB) in ice-cold distilled H20 and dried under a fan in the cold room (4 °C). Once dry, the slides were brought to room temperature, taped into X-ray cassettes and exposed to [3H]sensitive Ultrofilm for 6-8 weeks at 4 °C. Films were then developed in Kodak D19 for 4 min and fixed. The slide-mounted sections were subsequently fixed and stained with cresyl violet and/or osmium tetroxide in order to demonstrate the cytoarchitecture and/or myeloarchitecture of the spinal sections. Control binding was determined by labelling sections in the presence of 1 ~M atropine sulphate; in these sections the density of labelling in both the gray and white matter was similar to that in the white matter of Figs. 1-3. At each level of the spinal cord examined, the optical density of the labelling in the gray matter laminae was determined directly from the Ultrofilm negative by projecting the image onto an accurately metered photocell which had been zero calibrated on the background labelling in the adjacent white matter. In all cases, the autoradiograms demonstrated a
TABLE I
The optical densities of [3H]QNB and {~H]PZ binding in laminae 11 and IX of the human spinal cord The optical density is given as a value on a 100 point scale ranging between the density of the adjacent non-specifically-labelled white matter (= zero) and the density of a fully exposed film (= 100). The values given are the mean ± S . D of 4 sections from C7, L4 and S1 levels of one human spinal cord.
[-+H]QNB Lamina ll Cervical(C7) Lumbar (L4) Sacral (S1)
[+~H]PZ Lamina IX
Lamina 11
Lamina IX
75.2_+1.5 72.2_. +2.8 74.2_+3.b 56.7_+5.5* 76.1_+2.0 74.8_+2+3 78.6±1.4 61.0_+2.5 + 74.5_+1,1 73.8_+1.5 74.2_+0.4 44+5±3.3"
* The values of [3HJPZ binding in lamina iX are significantly different from the corresponding [3H]QNB value at P < 0.01 according to Student's t-test.
consistent pattern of [3H]QNB (Figs. la. 2a, 3a) and [3H]pZ (Figs. lb, 2b. 3b) binding in the gray matter of the cervical (Fig. 1), lumbar (Fig. 2) and sacral (Fig. 3) regions of the human spinal cord. The highest densities of [3H]QNB binding sites were localized in lamina lI (substantia gelatinosa) of the dorsal horn and lamina IX of the ventral horn (Figs. la. 2a. 3a) as
+
+
Fig. 3. Legend on p. 197.
199 defined on cytoarchitectonic and myeloarchitectonic criteriaT.S; only very low levels of labelling were present in other laminae of the spinal gray. In contrast, the highest density of [3H]pZ binding was confined to lamina II, with significantly lower levels of binding detectable in lamina IX and other regions of the gray matter (Figs. l b , 2b, 3b). D e n s i t o m e t r i c analysis confirmed the visual impression o b t a i n e d from the autoradiograms (Table I). In particular the density of [3H]QNB labelling in laminae II and 1X was virtually identical at all spinal levels examined. In contrast, the optical density of [3H]pZ labelling in lamina IX was 6 8 - 7 8 % of that in lamina II. In this study we have localized subclasses of muscarinic cholinergic receptors in the h u m a n spinal cord using [3H]QNB and [3H]pZ. First, using [3H]QNB as a ligand to label both M~ and M 2 receptors, we have shown that there is a similar density of muscarinic cholinergic receptors present in laminae II and 1X. This confirms the findings of Scatton et al.10. Secondly, results o b t a i n e d with [3H]pZ which label M 1 re-
1 Aquilonius, S.-M., Eckernas, S.-A. and Gillberg, P.-G., Topographical localization of choline acetyltransferase within the human spinal cord and comparison with some other species, Brain Research, 211 (1981) 329-340. 2 Garvey, J. M., Rossor, M. and Iversen, L. L., Evidence for multiple muscarinic receptor subtypes in human brain, J. Neurochem., 43 (1984) 299-302. 3 Gillberg, P.-G., Nordberg, A. and Aquilonius, S.-M., Muscarinic binding sites in small homogenates and in autoradiographic sections of rat and human spinal cord, Brain Research, 300 (1984) 327-333. 4 Hammer, R., Berrie, C. P., Birdsall, N. J. M., Burgen, A. S. and Hulme, E. C., Pirenzepine distinguishes between different subclasses of muscarinic receptors, Nature (London), 283 (1980) 90-92. 5 Luthin, G. R. and Wolfe, B. B., Comparison of [3H]pirenzepine and [3H]quinuclidinylbenzilate binding to muscarinic cholinergic receptors in rat brain, J. Pharrnacol. Exp. Therap., 228 (1984) 648-655. 6 Nagata, Y., Okuya, M., Watanabe, R. and Honda, M., Regional distribution of cholinergic neurons in human spinal cord transections in the patients with and without motor neuron disease, Brain Research, 244 (1982) 223-229. 7 Rexed, B., A cytoarchitectonic atlas of the spinal cord in
ceptors, show a 2 2 - 3 2 % higher density of labelling in lamina II c o m p a r e d with lamina IX. T a k e n together, these results conclusively show that both Mt and M 2 receptors are present in lamina IX of the ventral horn. In addition, while it is not possible to d e t e r m i n e the relative density of M l and M 2 sites in the substantia gelatinosa from our study, the high concentration of [3H]pZ binding sites in this region suggests a high proportion of M1 receptors. In future studies, quantitation of these binding sites using standards will be required to accurately define the numbers of Ml and M 2 receptors in laminae II and IX. Nevertheless, these findings suggest that M~ and M2 cholinergic receptors may be differentially involved in sensory and m o t o r function in the h u m a n spinal cord. This research was s u p p o r t e d by the New Z e a l a n d Medical Research Council, the New Z e a l a n d Neurological F o u n d a t i o n and the A u c k l a n d Medical Research F o u n d a t i o n .
the cat, J. Comp. Neurol., 100 (1954) 297-380. 8 Rexed, B., Some aspects of the cytoarchitectonics and synaptology of the spinal cord, Prog. Brain Res., 11 (1964) 58-92. 9 Ryall, R. W., Cholinergic transmission in the spinal cord. In R. A. Davidoff (Ed.), Handbook of the Spinal Cord, Vol. 1, Marcel Dekker, New York, 1983, pp. 203-240. 10 Scatton, B., Dubois, A., Javoy-Agid, F. and Camus, A., Autoradiographic localization of muscarinic cholinergic receptors at various segmental levels of the human spinal cord, Neurosci. Lett., 49 (1984) 239-245. 11 Watson, M., Roeske, W. R. and Yamamura, H. I., [3H]Pirenzepine selectively identifies a high-affinity population of muscarinic cholinergic receptors in the rat cerebral cortex, Life Sci., 31 (1982) 2019-2023. 12 Whitehouse, P. J., Walmsley, J. K., Zarbin, M. A., Price, D. L., Tourtelone, W. W. and Kuhar, M. J., Amyotrophic lateral sclerosis: alterations in neurotransmitter receptors, Ann. Neurol., 14 (1983) 8-16. 13 Yamamura, H. I., Walmsley, J. K., Deshmulch, P. and Roeske, W. R., Differential light microscopic autoradiographic localization of muscarinic cholinergic receptors in the brainstem and spinal cord of the rat using [3H]pirenzepine, Eur. J. Pharmacol., 91 (1983) 147-149.
BRE 21092
Muscarinic cholinergic r ~ o r s in the human ~inal cord: differential localization of [3H]pirenzepine and [aH]quinuclidinylbenzilate binding sites JOHN W. VILIAGER1and RICHARD L. M. FAULL2
1Department of Pharmacology and Clinical Pharmacology and 2Department of Anatomy, School of Medicine. University of A uckland, Auckland (New Zealand) (Accepted May 21st, 1985)
Key words: [3H]quinuclidinylbenzilate - - [3H]pixenzepine- - muscarinic cholinergic receptor subtype - - human --spinal cord
The localization of muscarinic cholinergic receptor subtypes was studied in the human spinal cord using in vitro labelling of cryostat sections with pH]quinuclidinylbenzilate (QNB) and [3H]pirenzepine (PZ) followed by autoradiography. The highest densities of [3H]QNB binding were localized in laminae II (substantia gelatinosa) and IX (motor neurons); in contrast, the highest density of [3H]pZ binding was localized to lamina II where the binding density was 22-32% higher than in lamina IX. These results suggest that the M 1and M2 muscarinic cholinergic receptor subtypesmay be differentially localized in sensory and motor regions of the human spinal cord. Considerable evidence suggests that acetylcholine functions as a neurotransmitter in the mammalian spinal cord 9. In the human spinal cord the distribution of choline acetyltransferasel, 6 and muscarinic cholinergic receptors 3.10.12 have been studied using biochemical and histochemical techniques. These studies have consistently shown enzyme activity and receptor density to be greatest in the superficial dorsal horn and ventral horn gray matter. More specifically, in the human cord muscarinic receptors have been localized in lamina II and the lateral portion of lamine IX 10. Pirenzepine (PZ) is a non-classical muscarinic antagonist which has been shown to distinguish two subclasses of muscarinic receptors 4 termed M1 and Me receptors, Pirenzepine has high affinity for M t sites and low affinity for M 2 sites, while other muscarinic antagonists (e.g. atropine, quinuclidinylbenzilate) exhibit high affinity for both sites. Binding studies have shown both M 1 and M 2 sites to exist in rat 4,5.11 and human2 brains. Furthermore, autoradiographic studies have demonstrated a differential distribution of [3H]pZ and [3H]quinuclidinylbenzilate (QNB) binding sites in the rat spinal cord and brainstem a3. In
the rat spinal cord [3H]QNB sites were localized in both the substantia gelatinosa of the dorsal horn and in the ventral horn, whereas [3I-t]PZ sites were concentrated predominantly in the substantia gelatinosa 13. In this study we have compared the localization of [3H]QNB and [3H]pZ binding sites in sections of the human spinal cord. Postmortem human spinal cord tissue was obtained from 4 subjects with no known history of neurological disease or drug treatment. Following postmortem delays ranging from 11-32 h. the spinal cord was removed and 1-2-cm blocks were immediately selected from the cervical, lumbar and sacral regtons of the cord and frozen onto microtome chucks using dry ice. They were then sectioned transversely at 16 p m using a cryostat and mounted onto acid-cleaned gelatine/chrome alum-coated slides. Slide-mounted sections were preincubated in 150 ml 50 mM sodium phosphate buffer (pH 7.4) for 30 mm at 20 °C and then incubated in the same buffer containing 1 nM [3H]QNB (40.2 Ci/mmol, New England Nuclear) for 2 h or 20 nM [3H]pZ (82.3 Ci/ retool. New England Nuclear) for 1 h at 20 °C. The reaction was terminated by I ([3H]pZ) or 2
Correspondence: R. L. M. Faull. Department of Anatomy, School of Medicine. University of Auckland. Private Bag, Auckland New Zealand. 0006-8993/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
197
i:~H)QNB
la
(H!PZ
C7
lb
07
(:~H)QNB
2a
L4
Figs. 1-3. Autoradiograms showing the distribution of [3HIQNB (Figs. la, 2a, 3a) and [3H]pZ (Figs• lb, 2b, 3b) binding sites in transverse hemiseetions of the human spinal cord at the levels indicated. At each level, the distance between section a and section b is 64 ~tm. Note that in each autoradiogram the region of dense labelling in the dorsal horn of the gray matter is located within lamina II and the region of moderate to heavy labelling in the lateral two-thirds of the ventral horn is localized to the region of lower motor neuron nuclei which constitute lamina IX. Fig. 3 on page 198.
198 ([3H]QNB) 5-rain washes in sodium phosphate buffer at 4 °C. The slides were then washed for 1 min ([3H]PZ) or 2-3 s ([3H]QNB) in ice-cold distilled H20 and dried under a fan in the cold room (4 °C). Once dry, the slides were brought to room temperature, taped into X-ray cassettes and exposed to [3H]sensitive Ultrofilm for 6-8 weeks at 4 °C. Films were then developed in Kodak D19 for 4 min and fixed. The slide-mounted sections were subsequently fixed and stained with cresyl violet and/or osmium tetroxide in order to demonstrate the cytoarchitecture and/or myeloarchitecture of the spinal sections. Control binding was determined by labelling sections in the presence of 1 ~M atropine sulphate; in these sections the density of labelling in both the gray and white matter was similar to that in the white matter of Figs. 1-3. At each level of the spinal cord examined, the optical density of the labelling in the gray matter laminae was determined directly from the Ultrofilm negative by projecting the image onto an accurately metered photocell which had been zero calibrated on the background labelling in the adjacent white matter. In all cases, the autoradiograms demonstrated a
TABLE I
The optical densities of [3H]QNB and {~H]PZ binding in laminae 11 and IX of the human spinal cord The optical density is given as a value on a 100 point scale ranging between the density of the adjacent non-specifically-labelled white matter (= zero) and the density of a fully exposed film (= 100). The values given are the mean ± S . D of 4 sections from C7, L4 and S1 levels of one human spinal cord.
[-+H]QNB Lamina ll Cervical(C7) Lumbar (L4) Sacral (S1)
[+~H]PZ Lamina IX
Lamina 11
Lamina IX
75.2_+1.5 72.2_. +2.8 74.2_+3.b 56.7_+5.5* 76.1_+2.0 74.8_+2+3 78.6±1.4 61.0_+2.5 + 74.5_+1,1 73.8_+1.5 74.2_+0.4 44+5±3.3"
* The values of [3HJPZ binding in lamina iX are significantly different from the corresponding [3H]QNB value at P < 0.01 according to Student's t-test.
consistent pattern of [3H]QNB (Figs. la. 2a, 3a) and [3H]pZ (Figs. lb, 2b. 3b) binding in the gray matter of the cervical (Fig. 1), lumbar (Fig. 2) and sacral (Fig. 3) regions of the human spinal cord. The highest densities of [3H]QNB binding sites were localized in lamina lI (substantia gelatinosa) of the dorsal horn and lamina IX of the ventral horn (Figs. la. 2a. 3a) as
+
+
Fig. 3. Legend on p. 197.
199 defined on cytoarchitectonic and myeloarchitectonic criteriaT.S; only very low levels of labelling were present in other laminae of the spinal gray. In contrast, the highest density of [3H]pZ binding was confined to lamina II, with significantly lower levels of binding detectable in lamina IX and other regions of the gray matter (Figs. l b , 2b, 3b). D e n s i t o m e t r i c analysis confirmed the visual impression o b t a i n e d from the autoradiograms (Table I). In particular the density of [3H]QNB labelling in laminae II and 1X was virtually identical at all spinal levels examined. In contrast, the optical density of [3H]pZ labelling in lamina IX was 6 8 - 7 8 % of that in lamina II. In this study we have localized subclasses of muscarinic cholinergic receptors in the h u m a n spinal cord using [3H]QNB and [3H]pZ. First, using [3H]QNB as a ligand to label both M~ and M 2 receptors, we have shown that there is a similar density of muscarinic cholinergic receptors present in laminae II and 1X. This confirms the findings of Scatton et al.10. Secondly, results o b t a i n e d with [3H]pZ which label M 1 re-
1 Aquilonius, S.-M., Eckernas, S.-A. and Gillberg, P.-G., Topographical localization of choline acetyltransferase within the human spinal cord and comparison with some other species, Brain Research, 211 (1981) 329-340. 2 Garvey, J. M., Rossor, M. and Iversen, L. L., Evidence for multiple muscarinic receptor subtypes in human brain, J. Neurochem., 43 (1984) 299-302. 3 Gillberg, P.-G., Nordberg, A. and Aquilonius, S.-M., Muscarinic binding sites in small homogenates and in autoradiographic sections of rat and human spinal cord, Brain Research, 300 (1984) 327-333. 4 Hammer, R., Berrie, C. P., Birdsall, N. J. M., Burgen, A. S. and Hulme, E. C., Pirenzepine distinguishes between different subclasses of muscarinic receptors, Nature (London), 283 (1980) 90-92. 5 Luthin, G. R. and Wolfe, B. B., Comparison of [3H]pirenzepine and [3H]quinuclidinylbenzilate binding to muscarinic cholinergic receptors in rat brain, J. Pharrnacol. Exp. Therap., 228 (1984) 648-655. 6 Nagata, Y., Okuya, M., Watanabe, R. and Honda, M., Regional distribution of cholinergic neurons in human spinal cord transections in the patients with and without motor neuron disease, Brain Research, 244 (1982) 223-229. 7 Rexed, B., A cytoarchitectonic atlas of the spinal cord in
ceptors, show a 2 2 - 3 2 % higher density of labelling in lamina II c o m p a r e d with lamina IX. T a k e n together, these results conclusively show that both Mt and M 2 receptors are present in lamina IX of the ventral horn. In addition, while it is not possible to d e t e r m i n e the relative density of M l and M 2 sites in the substantia gelatinosa from our study, the high concentration of [3H]pZ binding sites in this region suggests a high proportion of M1 receptors. In future studies, quantitation of these binding sites using standards will be required to accurately define the numbers of Ml and M 2 receptors in laminae II and IX. Nevertheless, these findings suggest that M~ and M2 cholinergic receptors may be differentially involved in sensory and m o t o r function in the h u m a n spinal cord. This research was s u p p o r t e d by the New Z e a l a n d Medical Research Council, the New Z e a l a n d Neurological F o u n d a t i o n and the A u c k l a n d Medical Research F o u n d a t i o n .
the cat, J. Comp. Neurol., 100 (1954) 297-380. 8 Rexed, B., Some aspects of the cytoarchitectonics and synaptology of the spinal cord, Prog. Brain Res., 11 (1964) 58-92. 9 Ryall, R. W., Cholinergic transmission in the spinal cord. In R. A. Davidoff (Ed.), Handbook of the Spinal Cord, Vol. 1, Marcel Dekker, New York, 1983, pp. 203-240. 10 Scatton, B., Dubois, A., Javoy-Agid, F. and Camus, A., Autoradiographic localization of muscarinic cholinergic receptors at various segmental levels of the human spinal cord, Neurosci. Lett., 49 (1984) 239-245. 11 Watson, M., Roeske, W. R. and Yamamura, H. I., [3H]Pirenzepine selectively identifies a high-affinity population of muscarinic cholinergic receptors in the rat cerebral cortex, Life Sci., 31 (1982) 2019-2023. 12 Whitehouse, P. J., Walmsley, J. K., Zarbin, M. A., Price, D. L., Tourtelone, W. W. and Kuhar, M. J., Amyotrophic lateral sclerosis: alterations in neurotransmitter receptors, Ann. Neurol., 14 (1983) 8-16. 13 Yamamura, H. I., Walmsley, J. K., Deshmulch, P. and Roeske, W. R., Differential light microscopic autoradiographic localization of muscarinic cholinergic receptors in the brainstem and spinal cord of the rat using [3H]pirenzepine, Eur. J. Pharmacol., 91 (1983) 147-149.