Binding studies in the lurcher mutant suggest an uneven distribution of putative benzodiazepine receptor subclasses in the mouse cerebellum

Neuroscience Letters, 48 (1984) 333-338

333

Elsevier Scientific Publishers Ireland Ltd. NSL 02823

B I N D I N G S T U D I E S IN T H E L U R C H E R M U T A N T SUGGEST A N U N E V E N D I S T R I B U T I O N OF P U T A T I V E B E N Z O D I A Z E P I N E R E C E P T O R SUBCLASSES IN T H E M O U S E C E R E B E L L U M

GABRIELE SAUER1, WOLFGANGWILLE2 and WALTER E. MI:ILLER1'3'* IPharmakologisches Institut der Universitiit Mainz, D-6500 Mainz; 2lnstitut fiir Genetik der Universitiit K61n, D-5000 K61n; and 3Psychopharmakologisches Labor, Zenstralinstitut fiir Seelische Gesundheit, D-6800 Mannheim (F.R.G.)

(Received February 16th, 1984; Revised version received April 18th, 1984; Accepted April 24th, 1984)

Key words: benzodiazepinereceptors - cerebellum - receptor subclasses distribution - Lurcher mutant

Binding studies using [3H]flunitrazepamin the cerebellum of normal and Lurcher mutant miceindicate that about 90070of the total benzodiazepinereceptor population reside on the Purkinje and granule cells. Moreover, the specific binding of the BZ~ receptor subclass specific ligand [3H]propyl-/3carboline-3-carboxylatesuggests that neuronal elements of the mouse cerebellum other than the Purkinie and granule cells contain about 18070of the BZ~ receptors but only about 7070of the BZ2 receptors of the mouse cerebellum. A variety o f evidence suggests the presence o f multiple benzodiazepine receptors in the central nervous system, especially regarding the presence o f two putative receptor subclasses (BZI and BZ2 receptors) [2]. Most benzodiazepine drugs are not able to discriminate between these subclasses and bind with about equal affinity to the BZ1 and BZ2 receptors. In contrast, some non-benzodiazepines, e.g. the triazolopyridazine compound Cl 218,872, a partial agonist, and the propyl-/3carboline-3-carboxylate, an antagonist, exhibit a certain degree o f selectivity for the BZ1 receptor subclass and enable us to determine the number o f BZ1 receptors in direct binding experiments. Binding studies with both tritiated ligands have demonstrated that the two putative subclasses are differently distributed in the brain. Usually high concentrations o f the BZ~ receptors ( > 80°70) in relation to the total benzodiazepine receptor concentration occur in the cerebellum, but considerably lower levels (about 5007o) exist in most other brain areas [3, 8-10, 15]. The reason for this different distribution is not yet clear. Since benzodiazepine receptors usually reside on nerve cells it seems likely that both subclasses are differently distributed over the neuronal cell types of the cerebellum. Accordingly, we in*Author for correspondenceat: PsychopharmakologischesLabor, Zenstralinstitut ffir Seelische Gesundheit, D-6800 Mannheim, F.R.G. 0304-3940/84/$ 03.00 © 1984 Elsevier Scientific Publishers Ireland Ltd.

334 vestigated the presence of benzodiazepine receptors ([3H]flunitrazepam binding) and of the putative BZ1 subclass ([3H]propyl-~-carboline-3-carboxylate binding) in the cerebellum of the normal and the Lurcher mutant mouse. The adult semidominant neurological mutant is characterized by a loss of more than 99.5% of the Purkinje and 90% of the granule cells [4]. The experiments should show if both subclasses are differently distributed on the Purkinje and granule cells on the one hand and on other neuronal structures of the cerebellum on the other hand. The heterozygous mutant mice (Lc/r) used in this study were raised in breeding colonies at the Institut fiir Genetik der Universit/it K61n, F.R.G., and were on a C 5 7 B L / 6 H a n background (obtained from Zenstralinstitut fiir Versuchstiere, Hannover, F.R.G.). Mutants and controls (mutant-free animals of the same background) were sacrificed (after 100 or more days postnatal) by decapitation. The brains were removed, dissected on ice and stored at - 2 0 ° C (stable for at least 4 weeks). For the receptor binding assay the frozen tissue was homogenized in about 50 vols. of ice-cold 0.1 M Tris-HCl buffer, pH 7.4, and centrifuged for l0 min at 48,000 g. The supernatant was discarded and the pellet was resuspended in the same buffer to give a final tissue concentration of about 2 mg original wet weight per ml. Nine hundred microliters of the tissue homogenate were incubated in triplicate at 4°C for 45 min together with 50/zl containing one of the two radioligands and 50 /~l buffer alone or buffer containing the blank (clonazepam 3 /~M) to determine nonspecific binding. The incubation was terminated by rapid filtration through Whatman-GF-B filters. The filters were washed three times with 3 ml ice-cotd incubation buffer, placed in minivials and dried for 30 min at 60°C. Radioactivity on the filters was determined by liquid scintillation spectrophotometry in 4 ml Quickszint 402 (Zinsser, Frankfurt, F.R.G.). Under these conditions, specific binding of both ligands was in equilibrium and linearly depending on the tissue concentration. Tritiated flunitrazepam ([3H]FNT) (spec. act. 87 Ci/mmol) and tritiated propyl-Bcarboline-3-carboxylate ([~H]PrCC) (spec. act. 96 Ci/mmol) were obtained from New England Nuclear (Dreieich, F.R.G.). Clonazepam was a gift of H o f f m a n n - L a Roche AG (Basel, Switzerland). All other chemicals were obtained from commercial suppliers. In agreement with previous findings [1] the density of the total benzodiazepine receptor population as labeled by the specific binding of [3H]FNT is reduced in the Lurcher cerebellum to about half of the density of control animals (Fig. l, Table I). This observation together with the much smaller size (about 30°70 of the controls) explains why the total number of benzodiazepine receptors in the mutant cerebellum is only about 10% of the number of receptors present in the normal cerebellum (Table I). The cerebellum of the Lurcher mutant mouse is characterized by an almost complete loss of the Purkinje cells, a 90% loss of the granule cells, and a 7 5 0 loss of the climbing fibers [4]. Since the climbing fibers do not contain benzodiazepine receptors [12] our findings indicate that about 90% of the benzodiazepine receptors in the mouse cerebellum are localized on the Purkinje and

335

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020

3H - FNT • Controls • Lurcher

k,....

0.15

.~ \

314 - PrCC

A L,.,,.cher no

010

!

005 A ~ ' - - ~ ,,

o

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d5

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113

specifically bound 3H-Ligand [pmol/mg prot] Fig. I. Scatchard analysis of the specific binding of [3H]FNT and [3H]PrCC in cerebellar homogenates of normal and Lurcher mutant mice. Data are means of triplicate determinations.

TABLE I PROPERTIES OF BENZODIAZEPINE RECEPTOR BINDING IN THE CEREBELLUM OF NORMAL AND LURCHER MUTANT MICE The data for Ka (dissociation constant) and Bmax(maximal number of binding sides) were obtained from linear Scatchard plots by regression analysis and represent means ± S.E.M. of n individual determinations, each using 4-6 different concentrations of the radioligands. * P < 0.001; nsp> 0.05 when compared with controls. Controls

Lurcher

1.14 + 0.02 (6) 4.98

0.51 + 0.04 (5)* 0.56

44 11

0.39 + 0.02 (7) 1.68

0.30 + 0.04 (3)as 0.32

77 19

Ka [nM] [3H]FNT [3H]PrCC

10.220.6 (6) 2.8 ± 0.1 (7)

15.3 + 1.1 (5)"~ 2.4 ± 0.4 (3)ns

Bm~ [~H]PrCC as percent of Bm~ [3H]FNT

33.7

57.8

Bmax [SH]FNT pmol/mg prot. pmol/cerebellum Bmax [3H]PrCC pmol/mg prot.

Lurcher as percent of controls

172

336

granule cells. This is in agreement with other findings [12, 14]. Using the BZt receptor specific ligand [3H]PrCC, considerably less binding sites were found in the cerebellum of the normal animals than have been detected by the specific [3H]FNT binding (Fig. 1) suggesting that only about one-third of the total receptor population belongs to the BZ1 subclass (Table I). This disagrees with observations in the CD-1 mouse (78o70 BZ1) [9] but goes parallel with findings in the CF-1 mouse (51°70 BZ1) [7] and our own findings in the NMRI mouse where a similar distribution was found for the same experimental conditions (38o70 BZ0 (data not shown). Obviously, profound strain differences exist in the mouse regarding this aspect. Very interestingly, pronounced strain differences in the mouse have also been reported in respect to some benzodiazepine-sensitive pharmacological tests [6, 11]. In contrast to the total receptor population, the density of the [3H]PrCC sensitive binding sites (BZ1 receptor) seems to be not or only slightly reduced in the Lurcher cerebellum as indicated by the intercepts of the extrapolated Scatchard plots with the abscissa (Fig. 1). These findings suggest that about 60o7o of the benzodiazepine receptors in the mutant cerebellum belong to the putative BZ~ subclass which is nearly twice the percentage found for the controls (Table I). In addition to the changes seen for the benzodiazepine receptors in the cerebellum we found an about 20°7o reduction of the total receptor population in the frontal cortex of the Lurcher mutant with no apparent specificity in respect to the BZ~ subclass (Table II). Presently, we cannot explain these observations, especially since

TABLE II PROPERTIES OF BENZODIAZEPINE RECEPTOR BINDING IN THE FRONTAL CORTEX OF NORMAL AND LURCHER MUTANT MICE The data for Ka (dissociation constant) and Bmax(maximal number of binding sites) were obtained from linear Scatchard plots by regression analysis and represent means _+ S.E.M. of n individual determinations, each using 4-6 different concentrations of radioligands. * P < 0.05; nsp> 0.05 when compared with controls. Controls

Lurcher

Lurcher as percent of controls

Bmax [3HIFNT pmol/mg prot.

1.50 ± 0.05 (9)

1.22 ± 0.05 (9)*

82

Bmax [3H]PrCC pmol/mg prot.

1.04 _+0.04 (9)

0.88±0.03 (10) "~

85

Kd [nM] [3H]FNT 13HIPrCC Bmax [3H]PrCC as percent of Bm~ [3H]FNT

12.7 _+0.5 (9) 5.7 _+0.3 (9)

10.5 _+0.3 (9) ns 4.5 +_0.2 (10) "~

69.1

71.9

104

337

histological lesions are not known for the frontal cortex of the Lurcher mutant. To our knowledge, other neurochemical lesions have not yet been reported for the Lurcher cortex. The most important findings of the present communication are the observations that the relative distribution of putative benzodiazepine receptor subclasses might differ between the cerebella of control animals and the Lurcher mutants. Based on these data it can be calculated that about 93°/0 of all BZ2 but only 82°70 of all B Z I receptors of the cerebellum reside on the Purkinje and granule cells. In other words, neuronal structures of the cerebellum except the Purkinje and granule cells contain about 18% of the BZ1 but only about 7°/0 of the B Z 2 receptors of the mouse cerebellum. The functional significance of the two putative subclasses of the benzodiazepine receptor is not yet known and speculations that the BZ1 subclass is more important for anxiolytic and anticonvulsant than for sedative and muscle relaxant properties [13] have not yet been substantiated. Moreover, it is still under dispute whether the two subclasses represent two structurally and functionally different receptors or only two conformational states of the same receptor [5]. We think that our findings fit better into the first hypothesis. Moreover, since the cerebellum represents a relatively well understood model system of the brain, we think that our findings are helpful for evaluating possible functional differences of the two benzodiazepine receptor subclasses. This study was supported by grants from the Deutsche Forschungsgemeinschaft (Wi 563/3-2 and Mu 467/3-4).

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