Benzodiazepine receptors in the human cerebellar cortex: a quantitative autoradiographic and pharmacological study demonstrating the predominance of type I receptors

Brain Research, 411 (1987) 379-385 Elsevier

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Benzodiazepine receptors in the human cerebellar cortex: a quantitative autoradiographic and pharmacological study demonstrating the predominance of type I receptors R.L.M.

F a u l l 1, J . W . V i l l i g e r 1 a n d N . H . G .

Holford 2

1Department of Anatomy and 2Departmentof Pharmacology and Clinical Pharmacology, School of Medicine, University of Auckland, Auckland (New Zealand) (Accepted 10 February 1987)

Key words: [3H]Flunitrazepam;Benzodiazepine receptor subtype; Human; Cerebellar cortex

The anatomical localization of benzodiazepine receptors in the human cerebellar cortex was studied using quantitative autoradiography following in vitro labelling of cryostat sections with [3HJflunitrazepam ([3H]FNZ), and the pharmacologyof these receptors has been characterized by computerized, non-linear least squares regression analysis of [3H]FNZdisplacement by FNZ, CL218,872 and ethylfl-carboline-3-carboxylate(ECC) binding to membranes. The autoradiograms demonstrated that benzodiazepine receptors were present throughout all layers of the human cerebellar cortex; high concentrationsof receptors were present in the molecular layer, moderate concentrations were present in the granular layer and a much lower density of receptors was seen in the intervening Purkinje cell layer. The pharmacologicalstudies indicated that the human cerebellar cortex contained a high concentration of homogeneous benzodiazepine receptors which have high affinity for FNZ, ECC and CL218,872, i.e. type I sites.

Benzodiazepines act at pharmacologically specific binding sites in the mammalian central nervous system 16'27. These benzodiazepine receptors have been shown to have a heterogeneous distribution in the human central nervous system using both autoradiographic 3'4'31'32 and biochemical 15A7,19 methods. Previously, using autoradiographic methods, Young and Kuhar 32 have shown benzodiazepine receptors in the human cerebellum to be localized in both the molecular and granular layers of the cerebellar cortex. Whereas initially benzodiazepines were thought to bind to a homogeneous population of binding sites, more recent work suggests the existence of two benzodiazepine binding sites (type I and type II) which can be differentiated by their affinity for the triazolopyridazine CL218,872 (refs. 8-14, 21, 26, 30, 34) or fl-carbolines (e,g. ethyl fl-carboline-3-carboxylate (ECC)1"22), In particular, Montaldo et al. 19 studied [3H]flunitrazepam ([3H]FNZ) and [3H]ECC binding to membranes prepared from various regions of the

human brain and concluded that the human cerebellum contains a mixture of both type I and type II benzodiazepine sites. In contrast, previous work in the rat brain suggests that the cerebellum contains a homogeneous population of type I sites 8"9'22"34. In the present study, we have therefore studied the detailed anatomy and pharmacology of benzodiazepine receptors in the human cerebellum in order to quantify the relative distribution of these receptors in the major layers of the cerebellar cortex and to more accurately define the pharmacology of these binding sites. The anatomical localization of the receptors has been studied using quantitative autoradiographic methods following the incubation of fresh, unfixed cryostat sections with [3H]FNZ while the pharmacology of the receptors has been investigated using computerized analysis of FNZ, CL218,872 and ECC binding to membranes. Postmortem human brains were obtained from three subjects (cases H4, H6, H8; one female and

Correspondence: R.L.M. Faull, Department of Anatomy, Schoolof Medicine, Universityof Auckland, Private Bag, Auckland, New Zealand. 0006-8993/87/$03.50 © 1987Elsevier Science Publishers B.V. (Biomedical Division)

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Fig. 1. Autoradiograms showing the distribution of benzodiazepine receptors in adjacent cryostat sections ut the human cerebella1 cortex. Section A was incubated with 1 nM [“H]F’NZ (a ligand with a high affinity for both type I and type II benzodiazepine receptors) in order to show the overall distribution of benzodiazepine receptors in the cerebellar cortex. Section B was incubated with 1 nM 13H]FNZ in the presence of 200 nM CL218,872 (a ligand with a high affinity for type I and a low affinits for type II receptc>r<” “? in (11

3H- FNZ + CL

B

5mm

382 two males) aged 33-43 years (average age 39 years) who died immediately following myocardial infarction (H4, H8) or asphyxiation (H6). All subjects had previously been in good health with no known history of neurological disease or drug treatment and all had died suddenly without the opportunity of receiving any form of medical treatment. Following postmortem delays ranging from 5 to 18 h (average delay of 10.7 h), the brain was carefully removed and blocks were immediately selected from the cerebellum and frozen on dry-ice for subsequent anatomical and pharmacological analyses as detailed below. Frozen blocks of tissue from the cerebellar cortex were frozen onto cryostat chucks using dry-ice. They were then sectioned in the frontal plane at 16 am using a cryostat and the sections thaw-mounted onto gelatine/chrome alum-coated slides. Following storage at 4 °C for up to one week. two adjacent series of slide-mounted sections were processed for the autoradiographic localization of benzodiazepine receptors. One series of sections was incubated with 1 nM [3H]FNZ (84 Ci/mmol. Amersham Radiochemicat Centre; a ligand with a high affinity for both type I and type II benzodiazepme receptors) in 50 mM TrisHCI (pH 7.4) for 1 h in order to demonstrate the overall localization of benzodiazepine receptors The second adjacent series of sections was incubated with 1 nM [3H]FNZ in the presence of 200 nM CL218.872 (a ligand with a high affinity for type I and a low affinity for type II benzodiazepine receptors ~'~4) for 1 h in order to show the localization of type II benzodiazepine receptors. The slides were then washed (2 × 1 min in Tris-HC1 buffer, dipped in distilled water) and dried under a stream of cold air. All the above steps were performed at 4°C. Non-specific control [3H]FNZ binding was determined by incubation of slides in the presence of 1/2M clonazepam. Once dry. slides were brought to room temperature and. together with slide-mounted tritium micro-scale standards (RPA.501 and RPA.505. Amersham), were taped into X-ray cassettes, apposed with 3H-sensitive Ultrofilm (LKB, Bromma. Sweden) and exposed in the dark at 4 °C for 6-10 weeks. The exposed films were developed in Kodak D19 for 4 min at 15 °C, washed and fixed. The Ultrofilm autoradiograms were subsequently printed using standard photographic procedures to yield 'darkfield' type autoradiograms where the autoradiographic labelled receptors

appear as white dots on a black background (see Fig. 1). The optical densities of the autoradiographic labelling in the various regions of the cerebellar cortex were determined directly from the Ultrofilm autoradiograms using a spot densitometer 3'4. The optical density values were then converted to nCi tritium bound/mg protein using autoradiographic standards and these values were then converted to fmol [3H]FNZ bound/mg tissue using the known specific activity of the labelled ligand 29 (Table I). In order to study the pharmacological characteristics of the cerebeUar benzodiazepine receptors, frozen blocks of tissue from the cerebellar cortex which had been stored at -20 °C were thawed, blotted and weighed, suspended in 25-30 vols. of 50 mM TrisHC1 (pH 7.4 at 20 °C), homogenized (Ultra-Turrax. setting = 50%. 15 s) and centrifuged at 30.000 g (4 °C) for 10 min. The supernatant was then discarded, and the procedure repeated two further times (i.e. 3 washes). The membranes were then suspended into 100 vols. of 4 °C Tris-HCl buffer (i.e. 10 mg/ml). [3H]FNZ binding was assayed by incubating 1 nM [3H]FNZ (84 Ci/mmol. Radiochemical Centre. Amersham), 100/A of displacer or buffer. 500 ~1 of membrane suspension and 50 mM Tris-HC1 (to give a final incubation volume of 1 ml) for I h at 4 °C. To terminate the reaction, membranes were filtered under reduced pressure through Whatman GF/B glass fiber filter discs using a Millipore Vacuum Manifold. Filters were then washed with 3 x 5 ml aliquots of ice-cold Tris-HCl. dried and counted using liquid scintillation spectrometry at a counting efficiency of 35%. Displacement experiments were performed by adding increasing concentrations of FNZ (10 -1° to 10 -7 M), ECC (10 -1° to 5 x 10 -7 M) and CL218.872 10 -~ to 10-4 M ) . TABLE I The density o f [3H]flunitrazepam binding in the human cerebellar cortex

The densities are expressed as means -~ S.D. of fmol [3H]FNZ bound/mg tissue. The values given were determined from optical densitometric readings from sections sampled from two cases and converted into fmol/mgtissue using tritium standards (see text for details).

Molecular layer Granularlayer

[JH]FNZ

/~H]FNZ + CL218 872

152.6 +__8.6 85.3 + 11.4

25.9 ± 1.7 16.8 ± 1.1

383 Binding of [3H]FNZ to two binding sites as a function of the total concentrations of the ligands in the system was described using the law of mass action model proposed by Feldman 5. A correction factor for the relative binding site concentration was included when data from different experiments was modelled simultaneously 2°. The equilibrium dissociation constants (Ka) for labelled and unlabelled FNZ at both binding sites were constrained to be identical. The fit was not improved by more complex models testing for isotope effects or selective binding to one of the sites. The only labelled ligand used was [3H]FNZ. The non-specific binding of each of the unlabelled ligands was constrained to be the same as that of [3H]FNZ. Binding parameters were estimated by non-linear regression using an extended least squares objective function and a homoscedastic variance model for each experiment 25. The variance of each individual experiment was estimated separately when different experiments were modelled simultaneously. Programs ( B I N D I N G library with MKMODEL 7) were written in BASIC, compiled and executed on an Apple IIe microcomputer. In all cases examined the autoradiograms demonstrated that benzodiazepine receptors were distributed throughout all layers of the human cerebellar cortex. As shown in Fig. 1A, high concentrations of receptors were present in the molecular layer, moderate concentrations were present in the granular layer and a much lower density of receptors was seen in the intervening monolayer of Purkinje cells. Quantitative analyses of the sections (Table I) confirmed the marked difference in the numbers of receptors in the molecular and granular layers; the density of receptors in the molecular layer was 78.8% greater than the receptor density in the granular layer (Table I). The resolution of our densitometer 3'4 did not allow us to reliably quantify the density of [3H]FNZ binding to the monolayered Purkinje cells. Studies on the pharmacology of the benzodiazepine receptors in the cerebellar cortex indicated that this region contains predominantly type I binding sites. This conclusion is based on two lines of evidence. Firstly, in experiments using cerebellar membranes when one-site and two-site models were fitted to FNZ, CL218,872 and ECC displacement of [3H]FNZ, a one-site model consistently resulted in a better fit than a two-site model. This is consistent

with the monophasic nature of the FNZ, CL218,872 and ECC displacement curves illustrated in Fig. 2. This computerized analysis, conducted on 3 experiments suggested a maximal number of binding sites (Bmax)of 60.91 + 12.1 pmol/g of wet tissue (mean + S.D.) with Kd's of 1.64 _ 0.4, 1.43 _ 0.3 and 60.4 + 12.2 nM for FNZ, ECC and CL218,872 respectively. Secondly, when the receptor densities in cerebellar sections incubated with [3H]FNZ in the presence of 200 nM CL218,872 (Fig. 1B; Table 1) were compared with the densities in adjacent sections incubated with [3H]FNZ alone (Fig. 1A; Table I), there was an 83% reduction in binding in the molecular layer and an 80.3% reduction in binding in the granular layer. These results show the anatomical distribution and pharmacological characteristics of bcnzodiazepine receptors in the human cerebellar cortex. The finding that benzodiazepine receptors are concentrated in both the molecular and granular layers (Fig. 1A) is in general agreement with Young and Kuhar's 32 previous autoradiographic studies on the human cerebellar cortex. However, in contrast to their findings showing equal densities of receptors in both layers, our autoradiograms (Fig. 1A) and quantitative studies (Table I) show that, as in the rat cerebellar cortex 23'28'32"33, the density of receptors in the molecular layer is substantially higher than the density in the granular layer. These autoradiographic findings therefore suggest a species similarity in the distribution of benzodiazepine receptors in the mammalian cerebellar cortex. It is now well established that the benzodiazepine

140-

ZO 10080O~ 60;~ 40U_

"1" 200 log [DISPLACER] M

Fig. 2. Displacement of [3H]FNZ binding to membranes prepared from human cerebellar cortex by FNZ (closed circles), ECC (open circles) and CL218,872 (diamonds). The figure shows the computer-fitted curves for a single representative experiment. This experiment was repeated on membranes prepared from 3 brains, with similar results in each case.

384 receptor is part of the y-aminobutyric acid ( G A B A ) receptor complex 6,24. Since most of the local-circuit neuronal cell types in the cerebellar cortex (except for the granule cells) are G A B A e r g i c 2, it is perhaps not surprising that our studies have demonstrated high densities of benzodiazepine receptors in the cerebellar cortex. In this respect it is interesting to note that autoradiographic studies on the distribution of G A B A receptors in the rat cerebellar cortex using the G A B A agonist [3H]muscimo123'28 have shown higher concentrations in the granular layer. We have observed a similar distribution of [3H]muscimol binding sites in the human cerebellar cortex, i.e. highest concentrations in the granular layer (unpublished observations). The reason for this mismatch between benzodiazepine and G A B A receptors in the cerebellar cortex is not clear since there is a generally good correlation between these two receptors in most other regions of the central nervous system ~s. It is interesting to compare the results of these various autoradiographic receptor binding studies on the b e n z o d i a z e p i n e - G A B A receptor complex to the recent immunohistochemical studies of Mohler et a1.~8'23':4 using specific monoclonal antibodies to the b e n z o d i a z e p i n e - G A B A receptor complex in the cerebellum of the rat, bovine and human brains. Their immunocytochemical studies showed a preferential staining in the granular layer with lower levels of immunoreactivity in the molecular layer of the cerebellar cortex 18'23,24. Thus, taken together, these various results demonstrating a differential distribution of [3H]FNZ binding (higher levels in the molecular layer) and of [3H]muscimol binding and benzodiazepin e - G A B A receptor immunoreactivity (higher levels in the granular layer) in the cerebellar cortex suggest that the b e n z o d i a z e p i n e - G A B A receptor complex in the molecular layer may be different from that in the granular layer (see Schoch et al. 24 for a detailed discussion).

1 Braestrup, C. and Nielsen, M., GABA reduces binding of [SH]methyl fl-carbofine-3-carboxylate to brain benzodiazepine receptors, Nature (London), 294 (1981) 472-474, 2 Chan-Palay, V., Neurotransmitters and receptors in the cerebellum: immunocytochemical localization of glutamic acid decarboxylase, GABA-transaminase, and cyclic GMP and autoradiography with 3H-muscimol. In S.L. Palay and

However. irrespective of these considerations of a possible difference between the benzodiazepineG A B A receptor complex in the molecular and granular layers, our pharmacological studies indicate that the human cerebellar cortex contains a high concentration of homogeneous benzodiazepine receptors which have high affinity for FNZ. E C C and CL218,872 (i.e. type 1 sitesl. These pharmacological results are consistent with those reported previously for the r a t 8"92234 but are at variance with those reported previously by Montaldo el at. ~9 in the human which suggested that the cerebellum contains a mixture of type I and type II sites. The reason for this discrepancy is not clear as the assay conditions used bv Montaldo et al. 19 were similar to those used in the present study However. m all our experiments we have consistently obtained monophasic ECC and CL218,872 displacement curves in cerebellar membrane experiments, and almost complete displacement of [3H]FNZ binding to cmebeltar secnons by 200 nM CL218.872. Since the cerebellum is primarily concerned with the control of muscle tone and coordination, it i~ tempting to speculate that these cerebellar benzodiazepine receptors may be involved in the mediat ion of the muscle relaxant and ataxic effects of benzodiazepines However. these motor effects of the benzodiazepines have been related lo the type II receptors while the type I receptors that we have demonstrated in the cerebellar cortex have been related to the anti-anxiety effects of the benzodiazepines (see Montaldo et al. 19 and Young el al. ~4 for refs. I. Clearly further research is required to elucidate the orgamzation and functional significance of benzodiazepine receptors in the human cerebellar cortex. This work was supported by grants from the Medical Research Council of New Zealand and the New Zealand Neurological Foundation

V. Chan-Palay reds.), l'he Cerebellum New Vistas. Springer. Berlin. 1982, pp. 552-584 3 Faull. R.L.M. and Villiger, J.W.. Benzodiazepine receptors in the human spinal cord: a detailed anatomical and pharmacological study, Neuroscience. 17 ~t986 b791-802. 4 Faull. R.L.M. and Villiger. J.W.. Heterogeneous distribunon of benzodiazepme receptors in the human stnatum: a

385

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9

10

11

i2

13

14

15

16

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quantitative autoradiographic study comparing the pattern of receptor labelling with the distribution of acetylcholinesterase staining. Brain Research, 381 (1986) 153-158. Feldman, H.A., Mathematical theory of complex ligand binding systems at equilibrium, Anal. Biochem., 48 (1972) 317-338. Haring. P., Stahli. C., Schoch, P., Takacs, B., Staehelin, T. and Mohler, H , Monoclonal antibodies reveal structural homogeneity ot' 7-aminobutyric acid/benzodiazepine receptors in different brain areas, Proc. Natl. Acad. Sci. U.S.A.. 82 (1985) 4837 4841. Holford, N.tt.G.. MKMODEL: a modelling tool for microcomputers - - pharmacokinetic evaluation and comparison with standard computer programs, Clin. Exp. Pharmacol. Physiol., Suppl. 9 (1985) 95. Klepner, C.A., Lippa, A.S., Benson, D.I., Sano, M.C. and Beer, B., Resolution of two biochemically and pharmacologically distinct benzodiazepine receptors, Pharmacol. Biochem. Behav., 11 (1979) 457-462. Lippa, A.S., Beer, B., Sano, M.C., Vogel, R.A. and Mcyerson, L.R., Molecular substrates of anxiety: clues from the heterogeneity of benzodiazepine receptors, Life Sci.. 28 (1981) 2343-2347. Lippa, A.S., Coupet, J., Greenblatt, E.N., Klepner, C.A. and Beer, B., A synthetic non-benzodiazepine ligand for benzodiazepine receptors: a probe for investigating neuronal substrates of anxiety, Pharmacol. Biochem. Behav., II (1979) 99-106. Lo, M.M.S., Niehoff, D.L., Kuhar, M.J. and Snyder, S.H., Autoradiographic differentiation of multiple benzodiazepine receptors by detergent solubilization and pharmacologic specificity, Neurosci. Lett., 39 (1983) 37-44. Lo, M.M.S., Niehoff, D.L., Kuhar, M.J. and Snyder, S.H., Differential localization of type I and type II benzodiazepine binding sites in substantia nigra, Nature (London), 306 (1983) 57-611. Lo, M.M.S. and Snyder, S.H., Two distinct solubilized benzodiazepine receptors: differential regulation by ions, J. Neurosci.. 3 (1983) 2270-2279. Lo, M.M.S.. Strittmatter, S.M. and Snyder, S.H., Physical separation and characterization of two types of benzodiazepine receptors, Proc. Natl. Acad. Sci. U.S.A., 79 (1982) 68(/-684. l_uabeya, M.K., Maloteaux, J.-M. and Laduron, P.M., Regional and cortical laminar distributions of serotonin S~, benzodiazepine, muscarinic, and dopamine D 2 receptors in human brain, J. Neurochem., 43 (1984) 1068-1071. Mohler, H. and Okada, T., Benzodiazepine receptor: demonstration in central nervous system, Science, 198 (1977) 848-851. Mohler, H. and Okada, T., The benzodiazepine receptor in normal and pathological human brain, Br, J. Psychiatr., 133 (1978) 261-268. Mohler. H., Schoch, P., Richards, J.G., Haring, P., Takacs, B. and Stahli, C., Monoclonal antibodies: probes for structure and location of the GABA receptor/benzodiazepine receptor/chloride channel complex. In R.W. Olsen and J.C. Venter (Eds.), Benzodiazepine/GABA Receptors and Chloride Channels: Structural and Functional Properties', Liss, New York, 1986, pp. 285-297. Montaldo, S., Serra, M., Concas, A., Corda, M.G., Mele, S. and Biggio, G.. Evidence for the presence of benzo-

20

21

22

23

24

25

26

27 28

29

30

31

32

33 34

diazepine receptor subclasses in different areas of the human brain, Neurosci. Lett., 52 (1984) 263-268. Munson, P.J. and Rodbard, D., LIGAND: a versatile computerized approach for characterization of ligand-binding systems, Anal. Biochem., 107 (1980) 220-239. Niefhoff, D.C., Mashal, R.D., Horst, W.D.~ O'Brien, R.A., Palacios, J.M. and Kuhar, M.J., Binding of a radiolabelled triazolopyridazine to a subtype of benzodiazepine receptor in the rat cerebellum, J. Pharmacol. Exp. Ther., 221 (1982) 670-675. Nielsen, M. and Braestrup, C,, Ethyl fl-carboline-3-carboxylate shows differential benzodiazepine receptor interaction, Nature (London), 286 (1980) 606-6(17. Richards, G., Mohler, H. and Haefely, W., Mapping benzodiazepine receptors in the CNS by radiohistochemistry and immunohistochemistry. In P. Panula, H. Paivarinta and S. Soinila (Eds.), Neurohistochemistrv: Modern Methods and Applications, Liss, New York, 1986, pp. 629-677. Schoch, P., Richards, J.G., Haring, P., Takacs, B., Stahli, C., Staehelin, T., Haefely, W. and Mohler, H., Co-localization of GABA a receptors and benzodiazepine receptors in the brain shown by monoclonal antibodies, Nature (London), 314 (1985) 168-171. Sheiner, L.B., Analysis of pharmacokinetic data using parametric models - - 1: regression models, J. Pharmacokin. Biopharmacol., 12 (1984) 93-117. Squires, R., Benson, D.I., Braestrup, C., Coupet, J., Klepner, C.A., Meyer, V. and Beer, B., Some properties of brain benzodiazepine receptors: new evidence for multiple receptors, Pharmacol. Biochem. Behav., 10 (1979) 825-830. Squires, R.F. and Braestrup, C., Benzodiazepine receptors in rat brain, Nature (London), 266 (1977) 732-734. Unnerstall, J.R., Kuhar, M.J., Niehoff, D.L. and Palacios, J.M., Benzodiazepine receptors are coupled to a subpopulation of 7-aminobutyric acid (GABA) receptors: evidence from a quantitative autoradiographic study, J. Pharmacol. Exp. Ther., 218 (1981) 797-804. Unnerstall, J.R., Niehoff, D.L., Kuhar, M.J. and Palacios, J.M., Quantitative receptor autoradiography using [3H]UItrofilm: application to multiple benzodiazepine receptors, J. Neurosci. Meth., 6 (1982) 59-73. Villiger, J.W., CL218,872 binding to benzodiazepine receptors in rat spinal cord: modulation by 7-aminobutyric acid and evidence for receptor heterogeneity, J. Neurochem., 43 (19841 903-905. Whitehouse, P.J., Walmsley, J.K., Zarbin, M.A., Price, D.L., Tourtellotte, W.W. and Kuhar, M.J., Amyotrophic lateral sclerosis: alterations in neurotransmitter receptors, Ann. Neurol., 14 (1983) 8-16. Young, W.S. and Kuhar, M.J., Autoradiographic localization of benzodiazepine receptors in the brains of humans and animals, Nature (London), 280 (1979) 393-395. Young, W.S. and Kuhar, M.J., Radiohistocbemical localization of benzodiazepine receptors in rat brain, J. Pharmacol. Exp. Ther., 212 (1980) 337-346. Young, W.S., Niehoff, D.L., Kuhar, M.J., Beer, B. and Lippa, A.S., Multiple benzodiazepine receptor localization by light microscopic radiohistochemistry. J. Pharmacol. Exp. Ther., 216 (1981) 425-430.