Autoradiographic study of neurosteroid binding sites labelled with [35S]-TBPS in brain of different species

Bruin Research Bulletin. Vol. 32, pp. 25 l-256, Printed in the USA. All rights reserved.

0361-9230/93

1993

$6.00 + .OO

Copyright0 1993Pergamon Press Ltd.

Autoradiographic Study of Neurosteroid Binding Sites Labelled With [35S]-TBPSin Brain of Different Species MONIQUE

VINCENS,*’ EVELYNE DARTOIS,* MARC POPELIER,* CHRISTEL MARQUETTE,? EMMANUEL MOYSE,* GILLES FILLION? AND FRANCE HAOURt

*Pharmacologic Endocrinienne, Hopital Lariboisiere, 2 rue Ambroise Part?, 75010 Paris, France fPharmacologie Neuro-Immuno-Endocrinienne, Institut Pasteur, 25 rue du Docteur Roux, 75 01.5 Paris, France VINCENS, M., E. DARTOIS, M. POPELIER, C. MARQUETTE, E. MOYSE, G. FILLION AND F. HAOUR. Autoradiographic study qf neurosteroid binding sites labelled with [35s]-TBPS in brain of d@rent species. BRAIN RES BULL 32(3) 25 l-256, 1993.-Distribution of [35S]-TBPSbinding sites was studied in various structures of brain in mouse and guinea pig and in cortex of monkey and in hippocampus of postmortem human brain. As it is observed for rat brain, high densities of [35S]-TBPSbinding sites were found in layer IV of cortex in the four species, and in thalamus of mouse and guinea pig. Intermediate densities of binding sites were observed in superficial and deep layers of cortex in those four species and in hippocampus of mouse, guinea pig, and human. In all brain structures studied. 5u3aP and picrotoxin produced a dose-dependant inhibition of [35S]-TBPS binding. No significant interregion or interspecies differences could not be detected for ICsOvalues of 5n3nP or picrotoxin to inhibit [35S]-TBPSfrom its binding sites. In all regions studied, ICso values were close to I .5 X 10m6M for 5n3aP and 2.3 X IO-’ M for picrotoxin. [35S]-TBPSbinding Guinea pig brain

Picrotoxin 501-Pregnane-3aol-20-one Monkey cortex Human hippocampus

Autoradiographic technique

Mouse brain

ceptors (16). In order to further address this hypothesis, we extended the pharmacological assessment of 5a3aP interactions with [35S]-TBPS binding sites in several species distinct of the rat and including man.

SEVERAL pregnan and androstan steroid derivatives have been shown to compete with specific binding of the [35S] tbutylbicyclophosphorothionate [35S]-TBPS, a ligand that labels the barbiturate site which is linked to the GABA-A receptor complex both in cortex (15) and pituitary (39). The most potent of them, 5a-pregnane-3aol-20-one (50301P) (6), can be naturally synthetized within the central nervous system (CNS) and is likely a neurosteroid. This progesterone metabolite appears likely to account for some neural effect of in vivo progesterone. In particular. it has been shown to have hypnotic effects in women (5). In addition, we have demonstrated that 5cu3cuP inhibits prolactin release from pituitary cells in culture via an enhancement of GABA-A receptor activity (39). Modulatory effects of 5013aP upon GABA-A receptor function was demonstrated on membrane homogenates of rat cerebra1 cortex (15) and confirmed in rat pituitary (39). The interaction of 5cu3aP with [35S]-TBPS binding sites of the GABA-A receptor complex was then characterized within rat brain by quantitative autoradiography (4 1). These pharmacological data strongly suggested that the functional interactions of 5a3aP and hypnotic barbiturates with the GABA-A receptor complex were similar in most brain structures. Such homogeneity suggests that the neurosteroid binding site and the barbiturate binding site belong to the same functional entity and presumably are not depending on the demonstrated molecular heterogeneity of brain GABA-A re-

METHOD

Animals

and

Tissues

Male Wistar rats (200-220 g) were purchased from IFFACREDO (Paris). Female mice of the CH3/Hl strain (30 g) were obtained from Pasteur Institute (Paris) and male Dunkin-Harkley guinea pigs (400-450 g) from Lebeau. These animals were sacrificed by decapitation after several days housing at +2 1“C in a standard 12L: 12D cycle with food and water ad lib. Then brains were rapidly dissected out of the skull, snap frozen in dry ice, and kept at -80°C until sectioning. Frozen pieces of one postmortem human brain (hippocampus) were obtained from the group of Psychiatric Investigation (Lyon, France). This brain had been collected at autopsy of a 45-year-old male, cardiovascular deceased patient; postmortem delay before brain congelation was 24 h. One frozen male monkey cerebral cortex was obtained from the Pasteur Institute. Serial 20 pm thick sections from all samples were cut at -20°C on a cryostat (Frigocut 2800, Reichert Jung), col-

’ To whom requests for reprints should be addressed. 251

-

5a3aP

---picrotoxin

-+

cortex

4hippocampus -

tila1amus

FIG. I. Autoradiographic distribution o~[~‘S]-TBPS binding sites in mouse midbrain (coronal section at level p2 I of Paxinos and Watson’s atlas). CO: cerebral cortex, HI: hippocampus (field CA I), DC? dentate gyrus, PU: caudate putamen, TH: thalamus. ZI: zona incerta. (A): Total of [‘%I-TBPS specific binding sites: (B): Inhibition of [3SS]-TBPSbinding by Sa3cuP 3 X 10e6 M:(C) Inhibition of [3LSJ-TBPSbinding by picrotoxin 3 X 10e6M;(D): inhibjt~on of f%]-TBPS binding with ~n~reasing~n~nt~tion ( 10m8M to lo-’ M) of 5cu3aP and picrotoxin. Each point corresponds to mean + SEM of 8-12 densitometric measurements and is expressed as percent of maximal [“SJ-TBPS specific binding in the absence of drug.

lected onto 2% gelatin-coated used.

slides, and kept at -20°C

until

Drugs

The ra~o~~~nd [35S]-tertiary butylbicyclophosphorothionate was purchased from NEN (specific activity IOO- 140 Ci/mmol).

The neurosteroid 5a-pregnane-3aoL20-one (Sa3~uP, Sigma) was dissolved at IO-* M in a mixture of 50% ethanol and 50% dimethylsulfoxide (DMSO), further diluted at IO-’ M in 65%’ DMSO, 25% incubation buffer solution, then at 10m4M in 10% DMSO, 90% incu~tion buffer. Picrotoxin (Sigma) was dissolved at 10-z M in distilled water: further dilution was done in 10%

NEUROSTEROID

BINDING

IN MAMMAL

*

BRAINS

.

-SdaQ

---picrotoxin

4

cortex

+.

hippocampus

Athalamus

F’IG. 2. Autoradiographic distribution of [35S]-TBPS binding sites in guinea pig midbrain (coronal section at level p2 I of Paxinos and Watson’s atlas). CO: cerebral cortex, HI: hippocampus (field CA]), DC: dentate gyms, PU: caudate putamen, TH: thalamus, Zl: zona incerta. (A) Total of [%ITBPS specific binding sites. (B) Inhibition of [“S]-TBPS binding by 5a3olP 3 X 10m6M. (C) Inhibition of [“S]-TBPS binding by picrotoxin 3 X 10m6 M. (D) Inhibition of [‘?5]-TBPS binding with increasing concentration (lo-@ M to 10 m5M) of 5n3uP and picrotoxin. Each point corresponds to mean t SEM of 8- 12 densitometric measurements and is expressed as percent of maximal [‘?5.]-TBPS specific binding in the absence of drug.

DMSO, 90% incubation buffer solution so as to standardize the final concentration of DMSO in the binding assays (7% DMSO for 10m4 M concentration of drugs). [35s]- TBPS A Ictovudio~raphJ~ Slides were brought to room temperature and preincubated 30 min in 50 mM Tris-HC1, ph 7.4. Sections were then rapidly dried out in ambient air and drop incubated with 3 nM [35S]-

TBPS in 50 mM Tris-HCI buffer, ph 7.4. containing 500 mM NaCl and 10m4 M ascorbic acid, 3 h at room temperature, in the absence (total binding) or in the presence of unlabeled compound (mnge concentration assayed: 1O-8 M to 10e4 M); alternate sections were incubated with 1O-5 M TBPS for nonspecific binding assesment. All slides were rinsed in three successive baths of 5 min each at 4°C in fresh buffer, dried out at room temperature, and apposed onto [3H]-hyperfilm (Amersham) in the dark. After 36 h exposure at 4°C in autoradiographic cassettes

FIG. 3. Autoradiographic distribution of [“S]-TBPS binding sites in cortex (A.B.C.1 and in hlppocampus (D.E,F) of monke!. (A,D) Total of [%]-TBPS specific binding sites (CO: cerebral cortex . Hi :hippocampus (lield CAI). DC: dentate g~rusl. (B,E) Inhibition of [35S]-TBPS binding b! 5tu3nP 3 X IOmhM. (C.F) Inhibition of [35S]-TBPS binding by picrotoxin 3 ~6 IO ’ M.

(Kodak), films were routinely developed 2 min in Kodak D19, fixed 4 min in Kodak rapid fixer, and rinsed I h under tap water.

The autoradiographic labeling was quantified in selected brain structures by computerized densitometry using the RAG-200 software (BIOCOM, les Ulis, France). Data were measured in relative optical density (OD) units. Nonspecific labeling density averaged less than 5% of total 35S-TBPS total binding in the present conditions and was systematically substracted from OD readings. Drug-inhibited [‘%I-TBPS binding was then plotted as percent of the specific binding obtained in the absence of

drug. Dose-inhibition curves were established by means of a nonlinear regression analysis with the software GRAPH-PAD. The concentration of drug ensuring 50% inhibition of specific binding (ICY,,) was systematically determined with the same package. RESULTS .1 utorudiogruphic

in Bruin

of

Distribution Fhrious Spwies

~/‘[“~s]- TBPS 5)ccific

Binding

In the midbrain of mouse and guinea pig, as in rat, the highest densities of [35S]-TBPS binding were found in layer IV ofcerebral cortex, globus pallidus. and some thalamic nuclei (Figs. 1A. 2A).

NEUROSTEROID

BINDING

IN MAMMAL

255

BRAINS

Monkey cerebral cortex displayed a strong [35S]-TBPS specific binding, the highest density of which occured in deep layers (Fig. 3A). The hippocampus allowed also a high density of [)‘S]TBPS specific binding, present principally in the dentate gyrus and CA 1 field (Fig. 3D). In the human hippocampus, [35S]-TBPS specific binding sites were mainly concentrated in strata oriens and radiatum (Fig. 4A). In all tissue sample, assayed, nonspecific [35S]-TBPS binding was low and homogeneously spread (Figs. IC, 2C, 3C. 3F. 4C). @XY 0/‘5~3~P

and picrotmin

,fiwn

site in brain

its bindinK

on displacement ~?f[‘~s]- TBPS sttxclures cjf various species

In the mouse and guinea pig, 5a3cuP produced a dose-dependant inhibition of [35S]-TBPS binding according to the same sigmoid kinetics as picrotoxin. Within the midbrain of mouse as of guinea pig, significant interregion differences could not be detected, neither for 5n3otP nor for picrotoxin inhibition of [35S]TBPS binding (Figs. ID. 2D). In all regions investigated, IC50 values were IO-’ M for 5~3crP and lo-’ M for picrotoxin. DISCUSSION

FIG. 4. Autoradiographic distribution of [%I-TBPS binding sites in human hippocampus. (A) Total of [I’S]-TBPS specific binding sites (Hi: hippocampus field CA I. DC: dentate gyrus). (B) Inhibition of [‘5S]-TBPS binding by Soi3~uP3 X 10e6 M. (C) Inhibition of IX5S]-TBPS binding by picrotoxin 3 x 10m6 M.

Superficial and deep layers of cerebral cortex, strata oriens, and radiatum of the hippocampal CA 1 field, molecular layer of the dentate gyrus, and caudate putamen were less densely, though significantly, labelled (Figs. IA. 2A).

[‘%I-TBPS has been demonstrated as a highly valuable tool for autoradiographic labeling of the barbiturate binding sites in the conditions used in the present study (20,41). In rat brain preparations, the progesterone metabolite 5n3cuP, was shown to interact with [35S]-TBPS, the specific radioligand-labelled barbiturate binding sites according to Michaelian kinetic (10,20,40.4 1). The existence of distinct and interactive binding sites for barbiturates and for neurosteroids. versus a common one, has appeared from physiological discrepancies and from interregion interaction heterogeneities (7,13,23,37,42). Autoradiographic assesment of 5a3nP has a dose-dependant effect on [‘%I-TBPS specific binding within the rat brain and revealed regionally homogeneous kinetics of 5cz3aP interaction with the barbiturate binding sites (41). In the present work, the regional distributions of [35S]-TBPS binding sites in the midbrains of mouse and of guinea pig were similar to the distribution previously reported in rat brain (41). Consistently, picrotoxin inhibition of [35S]-TBPS specific binding displayed with the same ICsO value in all four species studied as inrat brain (41). This result contrasts with previously reported differences between rat and mouse regarding inhibition of [35S]TBPS binding by pentobarbital and GABA (19). The affinity of 5n3nP towards [35S]-TBPS binding sites were also remarkably similar in all four species tested. Moreover, within the brains of mouse and of guinea pig as previously reported in rat brain (41), no interregion difference of 50i301P affinity for barbiturate sites was detected. The present work demonstrated also the inhibition of [35S]TBPS by 5tu3aP and by picrotoxin in primate forebrain. These data suggest that the pattern of neurosteroid-GABA-A receptor interactions in humans is comparable to that of other species. Experimentation in rat, mouse. guinea pig, and monkey become, then, particularly relevant for this topic. Animal models allow, indeed, to couple autoradiographic investigations with behavioral tests, which began recently to improve understanding of neurosteroid mechanisms of action. The induction of an antiaggressive behavior by administration of 4 mg progesterone to a female rat was, thus, correlated in vivo with inhibition of [35S]TBPS binding in limbic structures of the animal (4).

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