Neuroscience Letters, 1 I I (1990) 246-251 Elsevier Scientific Publishers Ireland Ltd.

246

NSL 06768

Isolation-induced changes in radioligand binding to benzodiazepine binding sites S. M i a c h o n , M. M a n c h o n , J.R. F r o m e n t i n a n d M. B u d a INSERM U 171-CNRS UA 95, Centre Hospitalier Lyon Sud, Pavillon 4H, Pierre Benite (France) (Received 18 July 1989: Revised version received 5 December 1989; Accepted 5 December 1989)

Key words:

Benzodiazepine binding; 7-Aminobutyric acid; Isolated male Wistar rat

Binding of [3H]flunitrazepam was studied in brain tissues of isolated Wistar rats and compared to groupreared animals. Modifications were observed in hippocampus and cortex (Kd increased) and in cerebellum (Bm,x decreased) and when brain sections of control rats were incubated in the bath fluid that had served to incubate sections from isolated rats, a flattening of the saturation curve was observed. Results are discussed in terms of possible modulators of benzodiazepine binding sites, mainly tissue GABA concentrations.

We studied benzodiazepine (BZD) binding sites in the model of the isolated male Wistar rats ( T ) which has been described in Petkov and Yanev's homogenate studies [12] to induce an increase in their Kd, in order to study neurochemical correlates (i.e. stress index: to be published) and a possible relation to emotional states [8, 14]. Binding parameters were determined in the hippocampus, cortex and cerebellum, on account of their richness in BZD binding sites of the two subtypes and the involvement of the hippocampus in anxiety. We used male Wistar rats (170 +_ 20 g) (IFFA CREDO) which were either housed for 3 months in individual cages (30 x 20 cm) with non-transparent walls, or were group-reared ('GR') (5 animals in the same cage). The animals had free access to food and water, and were kept under standard laboratory conditions: T = 22 + 2°C, controlled light-dark cycles (light from 08.00 h to 20.00 h). After 3 months, the animals were tested for muricidal behavior 24 h before sacrifice. The animals were decapitated (always at 14.00 h) and the hippocampus, frontal cortex and cerebellum were immediately dissected out, frozen in isopentane at - 5 0 ° C and kept frozen at - 8 0 ° C until use. Frozen sections of each structure (20 /~m) were cut at - 1 5 ° C using a Reichert Jung cryostat, mounted on gelatincoated slides (4 - 6 sections were placed on the same slide, and their weight was evaluated) and kept I-7 days at - 2 0 ° C . Preliminary dissection of the brain structures

Correspondence: S. Miachon, INSERM U 171-CNRS UA 95, Centre Hospitalier Lyon Sud, Pavilion 4H, Chemin du Grand Revoyet, 693 l0 Pierre Benite, France. 0304-3940'90/$ 03.50 4!: 1990 Elsevier Scientific Publishers Ireland Ltd,

247

allows autoradiographic and biochemical evaluations of binding parameters on the same tissues and with the same incubation conditions, as first described by Wamsley et al. [19]. On the day of receptor study, the slides were removed 3 h before the experiment; whenever preincubation in the buffer (Tris-HC1 pH 7.4) was performed before the binding assay it was done 15 min at ambient temperature. Incubation (at 4°C) was carried out following a protocol previously described [8] using [3H]flunitrazepam (FLU) (Amersham) as ligand and clonazepam (CLO) l0 -6 M as displacing agent. The incubation baths (12 ml) were poured out after each series (15 - 20 sections for one concentration) except when sections from GR animals were incubated in baths that had served to incubate sections from I ('crossed incubation') and, as a control, sections from GR animals in baths that had previously incubated other sections from GR. In some experiments, ~-aminobutyric acid (GABA) (Sigma), or (+)-bicuculline (Sigma) or the putative anxiogenic 'anxiety peptide' octadecaneuropeptide (ODN: Peninsula Lab., fragment of 'Diazepam Binding Inhibitor', DBI, having the same pharmacological activity [6]) were added to the incubation bath of preincubated sections at concentrations of respectively 10- 5 M, 10 - 4 M and 10- 5 M. The results were analyzed either on the whole structure after dissolution of the tissue in a protein solubilizer (Soluene Packard) and the radioactivity bound to the tissue was determined by means of a scintillation counter, or on individual layers of each of the structures having the highest density of BZD binding sites (pyramidal layer of hippocampus, frontal cortex and molecular layer of cerebellum) by means of autoradiography on [3H]LKB ultrofilms (exposure time was 3 weeks at 4°C).

Fig. 1. Visualization of [3H]FLU binding sites on [3H]ultrofilms LKB in cerebellum of isolated (I) (left) and G R (right). Shadows of adjacent sections placed below result from incubation in the presence of 10 _6 M non labelled clonazepam.

248 Quantitative densitometric measurements were carried out using an image analyser ( I M S T A R ) and a standard plastic scale (3H-microscale Amersham RPA 501). The affinity constant (Kd) and the maximal quantity of [3H]FLU bound per mg of dry tissue (Bmax) were defined by analysis of the saturation curve with and without CLO; 8 increasing concentrations of the radioligand were used (0.5 - 12 nM), pooling tbr each concentration 16 20 sections from the same incubation bath of 5 brains of I or G R corresponding to 1 series (3 series of 5 ! and 5 G R rats were evaluated). The affinity and density of [3H]FLU binding sites were determined using an iterative computer program, 'Binding Wizard' [4]. In the same tissues, we evaluated quantitatively G A B A and the group glutamine + glutamic acid, using an amino acid analyser ( ' C h r o m a k o n 500: Kontron') with detection limits of 570 and 440 nM. Statistical comparison between I and G R was carried out using Student's "t'-test. After the 3-month isolation period, the male Wistar rats were generally very aggressive against the experimenter, and 40% of them exhibited muricidal behaviour. In the hippocampus and cortex of I, a 70% and 35% increase in the Kd of [3H]FLU (biochemical results, Fig. 1) were respectively observed, as compared to GR; densito-

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Fig. 2. Koand Bm,~values (mean of 3 series + S.D., each of them resulting from pooled tissues of 5 rats), in cortex, hippocampus and cerebellum of male Wistar rats. GR, horizontal stripe; I, stipples; GR sections incubated in baths that had served previously to incubate sections from I, hatched; *0.025 < P