10 Brain Research, 524 (1990) 10-16

Elsevier

BRES 15721

Characterization of oxytocin-binding sites in primary rat brain cell

cultures

D. Di Scala-Guenot, M.T. Strosser, M.J. Freund-Mercier and Ph. Richard

Laboratoire de Physiologie, U.R.A. 309 C.N.R.S., Strasbourg (France)

(Accepted 30 January 1990)

Key words: Oxytocin-binding site; Brain cell culture; Oxytocin analog; Vasopressin analog

Detection and characterization of oxytocin-binding sites in dissociated brain cell cultures were performed, using a highly selective iodinated
oxytocin antagonist ([125I]OTA). Dissociated cells derived from hypothalamus and extrahypothalamic forebrain of 16-day-old fetal rats were
maintained in chemically defined medium in order to enrich the cultures in neuronal cells. Specific binding of [12~I]OTA,demonstrated in both
hypothalamic and forebrain cell cultures, was temperature- and time-dependent; maximal binding was obtained by incubating the iodinated
ligand for 60 min at 37 °C. The binding parameters were shown to be identical in both cell type cultures. The Scatchard plot analysis suggested
the presence of a single class of binding sites of high affinity (Ka about 0.06 nM) and low binding capacity (Bmax about 4 fmol/dish). The
specificity of these binding sites tested in competition experiments revealed that the unlabelled OT antagonist was the most potent in inhibiting
specific [t25I]OTA binding (Ki = 0.1 nM). A lower affinity, of the nM range was demonstrated for oxytoein (OT), arginine-vasopressin (AVP)
and the V 1 antagonist, whereas the V2 AVP agonist poorly competed for [125I]OTAbinding sites (Ki about 250 nM). In conclusion, the
[125I]OTAbinding characteristics, identical in both hypothalamic and forebrain cultures, fulfil the classical conditions required for the existence
of receptor sites since the binding was reversible, saturable and specific. As these characteristics were similar to those already described in
the adult rat, at the central level in hippocampus, and at the periphery in the mammary gland, it could be postulated that [125I]OTAbinds
to an OT receptor site. Therefore, fetal rat brain cell cultures may provide a relatively simplified in vitro model for studying the OT receptor
and its regulation.

INTRODUCTION nervous system. Using the autoradiographic technique,

Beside the well known oxytocinergic and vasopressin-
ergic projections of hypothalamic magnocellular neu-
rones on the neurohypophysial capillaries where oxytocin
(OT) is related to induce peripheric hormonal effects,
fibres and endings containing O T and arginine-vaso-
pressin (AVP) have been demonstrated in various areas
in the forebrain, the brainstem and the spinal cord 26'27.
When considering the local effects of O T injections in the
hypothalamus (facilitation of the milk-ejection reflex 21)
or in various extrahypothalamic structures (behaviour
inductions, for references see ref. 7), OT, besides its
endocrine functions, could act in the brain as a neuro-
transmitter or neuromodulator. Recent data have dem-
onstrated local O T release measured by push-pull canula
in the hypothalamic supraoptic nucleus 2° and by micro-
dialysis in extrahypothalamic structures such as the
olfactory bulb and substantia nigra 15'16. Thus, such
effects imply existence of O T receptors within the central
OT-binding sites have been localized in adult rat brain
areas (for references see refs. 12, 13, 31) within the
forebrain mainly in olfactory centres, the bed nucleus of
the stria terminalis, the central amygdaloid nucleus and
the ventral subiculum. In the hypothalamus, specific
OT-binding sites were found in the ventromedial nucleus
only. The hippocampal OT-binding sites have been
further pharmacologically characterized using the mem-
brane binding assays 3'9'1° and it appears from both
autoradiographic and m e m b r a n e binding assays that
central OT-binding sites poorly discriminate between O T
and A V P 3'13. The developmental pattern of OT-binding
sites in the rat brain has been investigated only recently
by Tribollet et al. 32. The distribution of OT-binding sites
undergoes changes between the perinatal period and
adulthood. Indeed, in some areas, binding sites detected
at an early stage remain present throughout life whereas
in other areas, they disappear after the infant period or
are expressed in the adult only. These results provide

Correspondence: D. Di Scala-Guenot, Laboratoire de Physiologie, U.R.A. 309 C.N.R.S., 21 rue Ren6 Descartes, 67084 Strasbourg Cedex,
France.

0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)


evidence that O T - b i n d i n g site expression could be regu-
lated by mechanisms specific to a defined p e r i o d in life.
O n the o t h e r hand, based on competition experiments,
O T - b i n d i n g sites d e t e c t e d in the infant brain a p p e a r to be
p h a r m a c o l o g i c a l l y similar to those present in the adult rat
brain 3~.
So far, no O T - b i n d i n g studies have been p e r f o r m e d on
intact living brain cells. Because of the changes in
O T - b i n d i n g site distribution t h r o u g h o u t life, and of the
known effects of local injections of O T in the hypothal-
amus o r in e x t r a h y p o t h a l a m i c structures, it was t e m p t i n g
to set up a culture system to detect and characterize such
binding sites in e m b r y o n i c rat hypothalamic and fore-
brain cell cultures.
In previous studies 3A2"13'31, OT-binding sites were
studied with tritium-labelled O T the affinity of which was
of the n a n o m o l a r range and the low specific activity
i m p o s e d limitations. A recently synthesized O T antago-
nist d(CH2)s[TyrMe2,Thra,TyrNH29]OVT labelled with
125iodine ([125I]OTA), displayed a high specific activity
and has b e e n shown to bind specifically to O T receptors,
with a higher affinity than O T itself, in the rat uterus and
h i p p o c a m p a l m e m b r a n e p r e p a r a t i o n 1°. T h e r e f o r e , this
ligand has b e e n used to detect OT-binding sites and study
their p h a r m a c o l o g i c a l characteristics in p r i m a r y brain cell
cultures.
MATERIALS AND METHODS
Brain cell cultures
Wistar pregnant rats at day 16 of gestation were anesthetized with
urethane, the embryo aseptically exteriorized and their brains
removed. The hypothalamus was isolated and cells derived from this
region were called hypothalamic cells. The cells derived from the
rest of the forebrain were called forebrain cells. The tissues, kept in
ice-cold Earle's balanced salt solution without Ca 2÷ and Mg2÷
(Gibco, France), were cleaned of their meningeal membranes and
incubated in a trypsin solution for 20 rain at room temperature
(0.05% trypsin, 0.02% EDTA, Boy, France). The enzymatic
reaction was stopped by the addition of 10% fetal calf serum (FCS).
The tissues, further mechanically dissociated, were filtered through
a 48-pm nylon mesh and spun down at 400 g for 10 min. The pellet
was suspended in Dulbecco's Modified Eagle's medium (DMEM,
Gibco, France) containing 6 g/liter glucose and supplemented with
20% FCS (Gibco, France). The dissociated cells were seeded in
35-mm Costar dishes precoated with poly-L-lysine (10 mg/liter in 0.1
M borate buffer, pH 8.4) according to Yavin and Yavin34. Cells were
plated at a density of one hypothalamus (2 x 106 4- 25 × 103 cells)
or 1/2 forebrain structures (3 x 106 4- 20 × 103 cells) per dish. The
cultures were incubated at 37 °C in a humid 5% CO 2 atmosphere.
Twenty four hours after seeding the medium was replaced by a
serum-free chemically defined medium of Bottenstein and Sato4 and
thereafter changed twice a week.
Binding studies
The OT antagonist d(CH2)5[Tyr(Me)2,Thr4,Tyr-NH29]OVT was
iodinated according to a modified procedure from Elands et al.l°
and described in ref. 29. Specific radioactivity of the monoiodinated
antagonist was about 2000 Ci/mmol depending on the activity of 125I
Na (IMS 300, Amersham). The transmutation of 1251 into lZ4Tedoes
not modify the binding property of the peptide and the ligand
11
concentration in the incubation medium was calculated for each
binding experiment, taking into account the half life of 125I (Dr. J.L.
Morgat, CEA Saclay, France, personal communication).
The binding of [~25I]OTAwas studied in 12-day-old hypothalamic
and forebrain cells attached to the plastic culture dish. The culture
medium was discarded and the cells, washed twice with Tris buffer
(170 mM Tris-HCl, pH 7.4, 5 mM MgCI2, 0.1% BSA) were allowed
to equilibrate in this buffer at 37 °C for 20 min. The reaction then
proceeded for 60 min at 37 °C. The binding was stopped by rapid
removal of the medium and by addition of 1 ml of ice-cold
Tris-buffer, followed by three 10-rain washes. The cells were scraped
with a rubber policeman and the suspension counted for radioac-
tivity.
A much higher resolution in OT-binding sites was achieved with
a ligand concentration close to the K0, therefore the total binding
was determined in the presence of 0.03 nM [125I]OTA. Non-specific
binding was determined in the presence of 0.03 nM [125I]OTA plus
an excess of unlabelled OT (5/~M). Specific binding was measured
by subtracting the non-specific binding from the total binding. All
determinations were performed at least in quadruplicate on dishes
from the same culture. The technical protocols for kinetic and
saturation studies and competition experiments with OT and AVP
analogs will be given in the Results section and the data analyzed
by the linear regression analysis carried out with the EBDA and
Ligand program of McPherson. Results are expressed as mean _+
S.D.
Chemicals
d(CH2)s[Tyr(Me)Z,Thr4,Tyr-NHE9]OVT was a gift from Dr. M.
Manning (Medical College of Ohio, Toledo). OT and AVP were
purchased from Bachem (Torrance, CA). d(CH2)sTyr(Me)EAVP
(V~ AVP antagonist) was purchased from Peninsula Laboratories
(Belmont, CA) and desamino-8-o-AVP (V2 AVP agonist) from
Ferring A.B. (Maim6, Sweden).
RESULTS
Kinetic studies
Intact 12-day-old rat h y p o t h a l a m i c and forebrain cell
cultures exhibit specific binding to [125I]OTA. Because of
the larger variations o b s e r v e d at earlier stages 12-day-old
cultures were chosen. The binding was d e t e r m i n e d on
cells incubated in the presence of 0.03 n M [125I]OTA for
various times at 22 and 37 °C. F r o m the data, it results
that the binding of [125I]OTA was time- and t e m p e r a t u r e -
d e p e n d e n t , reaching a p p a r e n t steady-state at 60 min at
37 °C (Fig. 1 A , B , solid lines), whereas at 22 °C the
equilibrium did not still p r o c e e d after 90 min of incuba-
tion (Fig. 1 A , B , d o t t e d lines). T h e semilogarithmic plot
of these data (insets Fig. 1 A , B ) is a straight line giving a
first-order rate constant of association, similar in hypo-
thalamic and forebrain cells (kapp: 0.038 --I-0.0018 min -1
and 0.042 + 0.0047 min -1, respectively). A l l subsequent
incubations were therefore p e r f o r m e d for 60 rain at
37 °C. In these conditions, at equilibrium, the average
non-specific binding was a b o u t 15%; the m a x i m u m
b o u n d [125I]OTA was not m o r e than 10% of the total
labelled O T A and it is then assumed that the concentra-
tion of the free ligand and of unlabelled p e p t i d e s in the
incubation m e d i u m r e m a i n e d constant with time.
Fig. 2 shows that specific binding of [125I]OTA to
 12

A B
10000

5OO0

800C
4000 g
. ..""
6000

2000 4013(]

I0O0 2000
60
NINUTES NINIJ1ES

O 20 40 6 B0 100 1 0 2 40 60 100 ] 20
#]NUTES NINUTES
Fig. 1. Time-dependence of specific binding of [125I]OTAto rat brain hypothalamic (A) and forebrain cells (B) at 22 °C (---) and 37 °C ( ).
The experiment was repeated twice with similar results, Inset: the semilogarithmic plots of the time-dependent binding curves performed at
37 °C. Values of the second-order rate constant of association k÷l are given in Table I. Beq: specific binding at equilibrium. Bt: specific binding
at a given time. Data are means + S.D. of 4 determinations.

cultured cells is a reversible process. After 60-min reaction (k_l) of 0.010 + 0.0039 min -~ for hypothalamic
incubation with labe!led O T A , the medium was discarded cells and 0.013 + 0.001 min -1 for forebrain cells.
and replaced with 5 ~ M unlabelled O T and the amount Knowledge of both kapp and k_~ allowed us to calculate
of remaining bound [zesI]OTA was determined at various the second-order rat constant of association k+l: 0.93 and
time intervals. After 90 min of incubation in the presence 0.9 nM-l.min -~ for hypothalamic and forebrain cells,
of unlabelled OT, no specific radioactivity remained in respectively (Table I).
forebrain cells, whereas less than 30% of the radioactivity
bound at zero-time remained in hypothalamic cells. The Saturation studies
linear regression analysis (inset Fig. 2) allowed determi- At equilibrium, specific binding of [125I]OTA to
nation of the first-order rate constant for the dissociation cultured cells was saturable over ligand concentrations
from 0.0075 to 1.92 nM (Fig. 3). The computer analysis
of the data revealed a linear Scatchard plot and a Hill
coefficient close to the unity suggesting the presence of a
6000 9.5
single class of binding sites, with high affinity, Kd: 0.03 +
~ 9.0 0.01 nM and 0.09 + 0.011 nM and a low capacity Bmax:
2.4 + 0.13 fmol/dish and 5.3 + 0.06 fmol/dish for
hypothalamic and forebrain cells, respectively. All data
--- 4000 V ~ ?:l. . . . . ~ . are summarized in Table I. K d values calculated from
kinetic data were in good agreement with those deter-
•-,°- \ ~z o z,o 4o so oo ioo l~
mined in saturation studies arguing for a bimolecular
binding reaction, Both cell types exhibit very similar
binding constants.
=zc:z 200013000000
coc°
Competition studies
The ligand specificity of OT-binding sites was evalu-
ated from competition studies with unlabelled O T A , O T
0 I I i i i • II ,
and AVP, V 2 AVP agonist and V~ A V P antagonist at
0 20 4060 80 100 120 concentrations ranging from 0.05 nM to 5 ~M. The
NINUTES regressions were parallel and their slope close to unity
Fig. 2. Time-course of the dissociation of [I~5I]OTAspecific binding (Fig. 4A,B) indicating a c o m m o n class of binding sites for
in hypothalamic (D D) and forebrain cultures ( B - - - - I ) . Inset: the peptides tested. The IC5o values of each peptide are
semilogarithmic plots of the data. Values ol the first-order rate
constant of dissociation k~ are given in Table I. Data are means + given in Table II. All values were in the same range for
S.D. for 4 determinations. hypothalamic and forebrain cells. The affinity of [125I]-
 13

TABLE I ,6[
Binding characteristics of [1251]0TA to cultured cells .t4
k,pp and k~ are the linear regression slope values in association and .t2 ~ •
dissociation experiments, respectively. F is the free labelled iigand
141
concentration. Values are means + S.E.M. .to
Lit. • •
Hypothalamic Extrahypotha- ~-
¢-=
.08
cells lamic cells Z
= .06
o
n,.t
k,pp (min-l) 0.038 + 0.0018 0.042+ 0.0047
k_ 1(rain -1) 0.010 + 0.0039 0.013+ 0.001 .04
k~pp- k_l
k+l--- (nM-Lmin -1) 0.93 0.9 .02
F
K d from kinetic I

k_l 0 1 2 3 4 5 6
data = (nM) 0.010 0.014 BOUND fmol / dish
k+l Fig. 3. Scatcbard plots of saturation studies in hypothalamic
Kd from saturation ([] •) and forebrain cultures (~ II) using increasing con-
studies (nM) 0.03 + 0.01 0.09 + 0.011 centrations of [1zsI]OTA. Values of K d and Bmaxare given in Table
Bin,x (fmol/dish) 2.4 + 0.13 5.3 + 0.06 I. Data are means ___S.D. of 4 determinations.

displayed properties of saturability, reversibility and

O T A for OT-binding sites was 10 times higher than that
of the unlabelled peptide O T A and 100 times higher than
that of OT, making it the more selective ligand among the
known compounds. A V P and V 1 A V P antagonist were
almost as effective as O T in competing for [~25I]OTA
whereas the V 2 A V P agonist poorly competed for
OT-binding sites (K i about 250 nM). The rank order of
potency for these peptides was O T A > O T = V 1
antagonist = A V P > V 2 agonist.
DISCUSSION
Firstly, this study demonstrated that dissociated fetal
brain cell cultures exhibit OT-binding sites. The binding
specificity that satisfy a classical ligand-receptor associ-
ation. The high specific activity of the iodinated ligand
used, allowed detection of relatively low concentrations
of OT-binding sites and its high degree of specificity, a
limited non-specific binding (~< 15%). M o r e o v e r from the
experiments performed, it results that the characteristics
of OT-binding sites in hypothalamic cells are identical to
those of forebrain cells.
Secondly, it was demonstrated from the saturation and
competition experiments that the pharmacological char-
acteristics of the binding observed in the present study,
were similar in many respects to those reported for
OT-binding sites at the periphery 2s and in the brain 1°
using the same labelled ligand. Finally, the results

100 A 1oo B

:f
40[

7'0

0
'-;o -; -; -;
i"
40

20

-b
t LINLABELLED PEPTIDE ) iOC 14 ( UNLABELLED PEPTIDE ) lOG N

Fig. 4. Competition curves for [~25I]OTA binding to hypothalamic (A) and forebrain cells (B). Cultures were incubated with increasing
concentrations of unlabelled peptides (0.05 nM to 5/~M): 4F-------~, OTA; • • , OT: • • , AVP; O-----@, V t antagonist; [] r-l,
V2 agonist. Values of IC5o and K~ are given in Table II. Data are means + S.E.M. of 4 determinations.
14

T A B L E II embryonic neuronal cell cultures becomes relevant when

Potency of OTA, OT and AVP analogs to compete with [1251]0TA on considering the study of Tribollet et al. 32 on OT receptor
0 T binding sites ontogeny. In the central nervous system, many regions
IC5o, concentration which displaced 50% of the [tzsI]OTA binding. expressed OT receptors between embryonic day 20 (E20)
Values are m e a n s _+ S.D. of 4 determinations. K~, inhibitory constant and postnatal day 5 (P5), except in the brainstem where
calculated from C h e n g and PrusoffS: they could be detected as soon as El4. This raises the
IC5o × Kd question of the nature of the endogenous ligand for such
Ki - binding sites; indeed the fully processed immunoreactive
K~+F
form of OT was only detectable after birth, as demon-
where K d is the dissociation constant at equilibrium for the labelled strated with immunocytochemistry and radioimmuno-
ligand. F i s the free labelled ligand concentration. assays 1'2'33. Moreover, a previous report from our labo-
ratory demonstrated that immunoreactive-OT (IR-OT)
Ligand 1C5o(nM) Ki (nM)
could not be detected in embryonic hypothalamic cells
Hypotha- Extrahypo- Hypotha- Extrahypo- prepared from 16-day-old embryos, and cultured with s or
lamic thalamic lamie thalamic
cells cells cells cells without fetal calf serum (unpublished observation). How-
ever, 1R-AVP could be measured and the cellular content
AOT 0.23+0.09 0.16+0.09 0.11 0.08
OT 5 + 0.17 1.84 + 0.4 2.6 0.9
increased with time in culture. Therefore, it could be
AVP 13 + 1.6 16 + 1.9 6.5 9 postulated that, in our cultures, precursor forms of OT or
V l antagonist 11.5 + 0.21 20 + 0.81 5.5 10 fully processed AVP could act as the endogenous ligand
V z agonist 510 + 85 500 + 101 250 240
since central OT receptors poorly discriminate between
OT and A V P 3"13. These results also raise the question
about appearance of the OT-binding sites during time in
culture. Thus, local interactions between cultured cells or
presented in this paper demonstrate a pharmacological differentiation during the first days in culture and
similarity between embryonic and adult rat brain OT- onwards, could trigger OT-binding site expression via
binding sites; this has already been described by mechanisms yet unknown.
Tribollet32 who reported, in competition studies with In the adult rat brain, autoradiographic studies re-
unlabelled OT, that the OT-binding sites detected in the vealed a dense labelling for OT-binding sites in the
infant and adult rat brain displayed similar pharmaco- ventromedial nucleus of the hypothalamus 12'31 whereas
logical characteristics. only a faint and diffuse labelling was visible in the young
Taken together, our results suggest that the binding rat hypothalamus at P532 (corresponding more or less to
sites detected in our embryonic cultured cells may 10 days in culture). At this stage, the most intensively
represent a single class of OT-binding sites which affinity labelled structures were observed outside the hypothal-
for OT and AVP was of the nanomolar range. amus. Therefore, we could expect a lower binding
All binding studies performed in this work, were capacity in hypothalamic than in forebrain cells, whereas
realized on enriched neuronal cultures (by means of a they are of the same range (about 4 fmol/dish). It is likely
defined culture medium). Neuronal cultures have already that we failed to detect all OT-binding sites in forebrain
been used for the detection and subsequent character- cultures because of the higher cell density. Indeed,
ization of specific binding sites for peptides6'14'17"lS; Nilsson et al. 22 demonstrated that the growth hormone-
however it cannot be excluded that binding occurred binding rate in cultured chondrocytes was higher in cells
partially on glial cells. Indeed, non-neuronal binding has seeded at low densities. However, the reduced binding
been clearly demonstrated for other peptides such as rate with high cell densities is not a general feature since
insulin in brain cell cultures 24 and opioid peptides in rat 25 Foord et ai. H and Lewis et al. TM reported that specific
or chicken forebrain glial cultures 19'23. The hypothesis binding was linear and increased significantly with the
that astroglial cells could be OT target cells is supported number of cells. Binding studies performed on cultures
by the ability of OT to induce glial plasticity in the seeded with the same density should clarify this problem.
magnocellular system of the lactating rat 3°. Indeed, glial In conclusion, dissociated brain cell cultures containing
coverage diminishes during lactation and OT could be mainly neuronal cells, exhibited OT-binding sites, the
responsible for the structural reorganization of the pharmacological characteristics of which are similar to
supraoptic and paraventricular nuclei. It might be there- those previously described in the brain and peripheral
fore valuable to detect OT-binding sites in pure astroglial tissues. It may provide a useful model for studying the
cell cultures. regulation of OT receptors and their ontogeny in culture
The physiological significance of OT binding studies in conditions.

Acknowledgements. This work was supported by grants from the
C.N.R.S. (URA 309) and I.N.S.E.R.M. (CRE 88-6012). We are
deeply indebted to Dr. M. Manning for generous supply of OT
REFERENCES
1 Altstein, M. and Gainer, H., Differential biosynthesis and
posttranslational processing of vasopressin and oxytocin in rat
brain during embryonic and postnatal development, J. Neuro-
sci., 8 (1988) 3967-3977.
2 Altstein, M., Whitnall, M.H., House, S., Key, S. and Gainer,
H., An immunocytochemical analysis of oxytocin and vasopres-
sin prohormone processing in vivo, Peptides, 9 (1988) 87-105.
3 Audigier, S. and Barberis, C., Pharmacological characterization
of two specific binding sites for neurohypophyseal hormones in
hippocampal synaptic plasma membranes of the rat, EMBO J.,
4 (1985) 1407-1412.
4 Bottenstein, J. and Sato, G.H., Growth of a rat neuroblastoma
cell line in serum-free supplemented medium, Proc. Natl. Acad.
Sci. U.S.A., 76 (1979) 514-517.
5 Chen, Y.-C. and Prusoff, W.H., Relationship between the
inhibition constant (Ki) and the concentration of inhibitor which
causes 50 per cent inhibition (15o) of an enzymatic reaction,
Biochem. Pharmacol., 22 (1973) 3099-3108.
6 Ciaraldi, T., Robbins, R., Leidy, J.W., Thamm, P. and Berhanu,
P., Insulin receptors on cultured hypothalamic cells; functional
and structural differences from receptors on peripheral target
cells, Endocrinology, 116 (1985) 2179-2185.
7 De Wied, D., Gispen, W.H. and van Wimersma Greidanus,
Tj.B., Neuropeptides and Behavior: vol. 2, The Neurohypophy-
seal Hormones, Pergamon, Oxford, 1989, 188 pp.
8 Di Scala-Guenot, D., Strosser, M.T., Sarlieve, L.L., Legros, J.J.
and Richard, Ph., The development of neurophysin-containing
neurons in primary cultures of rat hypothalami is related to the
age of the embryo. Morphological study and comparison of in
vivo and in vitro neurophysins, oxytocin and vasopressin
content, J. Neurosci. Res., 25 (1990) 94-103.
9 Elands, J., Barberis, C. and Jard, S., [3H]-[Thra,GIyT]OT: a
highly selective ligand for central and peripheral OT receptors,
Am. J. Physiol., 254 (Endocrinol. Metab. 17) (1988) E31-E38.
10 Elands, J., Barberis, C., Jard, S., Tribollet, E., Dreifuss, J.J.,
Bankowski, K., Manning, M. and Sawyer, W.H., [12Sl]-labelled
d(CH2)s[Tyr(Me)2),Thr4,Tyr-NH29]OVT: a selective OT recep-
tor ligand, Eur. J. Pharmacol., 147 (1988) 197-207.
11 Foord, S.M., Peters, J.R., Dieguez, C., Scanlon, M.E and Hall,
R., Dopamine on intact anterior pituitary cells in culture:
functional association with the inhibition of prolactin and
thyrotropin, Endocrinology, 112 (1983) 1567-1583.
12 Freund-Mercier, M.J., Stoeckel, M.E., Palacios, J.M., Pazos,
A., Reichhart, J.M., Porte, A. and Richard, Ph., Pharmaco-
logical characteristics and anatomical distribution of [3H]-
oxytocin-binding sites in the Wistar rat brain studied by
autoradiography, Neuroscience, 20 (1987) 599-614.
13 Freund-Mercier, M.J., Stoeckel, M.E., Dietl, M.M., Palacios,
J.M. and Richard, Ph., Quantitative autoradiographic mapping
of neurohypophysial hormone binding sites in the rat forebrain
and pituitary gland. I. Characterization of different types of
binding sites and their distribution in the Long-Evans strain,
Neuroscience, 26 (1988) 261-272.
14 Kapcala, L.P. and De Souza, E.B., Characterization of corti-
cotropin-releasing factor receptors in dissociated brain cultures,
Brain Research, 456 (1988) 159-167.
15 Kendrick, K.M., Keverne, E.B., Chapman, C. and Baldwin,
B.A., Intracranial dialysis measurement of oxytocin, mono-
amine and uric acid release from the olfactory bulb and
substantia nigra of sheep during parturition, suckling, separation
from lambs and eating, Brain Research, 439 (1988) 1-10.
16 Kendrick, K.M., Keverne, E.B., Chapman, C. and Baldwin,
B.A., Microdialysis measurement of oxytocin, aspartate, y-
15
antagonist. The authors also thank Mrs. M. Horrenberger, B.
Waltisperger and E. Wendling for their excellent technical assis-
tance.
aminobutyric acid and glutamate release from the olfactory bulb
of the sheep during vaginocervical stimulation, Brain Research,
442 (1988) 171-174.
17 Laribi, C., Legendre, P., Dupouy, B., Vincent, J.D. and
Simonnet, G., Characterization of two angiotensin II binding
sites in cultured mouse spinal cord neurones, Brain Research,
347 (1985) 94-103.
18 Lewis, R.E., Childers, S.R. and Phillips, M.I., [125I]Tyr-
Bradykinin binding in primary rat brain cultures, Brain Re-
search, 346 (1985) 263-272.
19 Maderspach, K. and Soiomonia, R., Glial and neuronal opioid
receptors: apparent positive cooperativity observed in intact
cultured cells, Brain Research, 441 (1988) 41-47.
20 Moos, E, Poulain, D.A., Rodriguez, E, Guern6, Y., Vincent,
J.D. and Richard, Ph., Release of oxytocin within the supraoptic
nucleus during the milk ejection reflex in rats, Exp. Brain Res.,
76 (1989) 593-602.
21 Moos, E and Richard, Ph., Paraventricular and supraoptic
bursting oxytocin cells in rat are locally regulated by oxytocin
and functionally related, J. Physiol., 408 (1989) 1-18.
22 Nilsson, A., Lindahl, A., Eden, S. and Isaksson O.G.P.,
Demonstration of growth hormone receptors in cultured rat
epiphyseal chondrocytes by specific binding of growth hormone
and immunohistochemistry, J. Endocrinol., 122 (1989) 69-77.
23 Pearce, B., Cambray-Deakin, M. and Murphy, S., Astrocyte
opioid receptors: activation modifies the noradrenaline-evoked
increase in [2-14C]deoxyglucose incorporation into glycogen,
Neurosci. Lett., 55 (1985) 157-160.
24 Raizada, M.K., Stamler, J.E, Quinlan, J., Landas, S. and
Philips, M.I., Identification of insulin receptor-containing cells
in primary cultures of rat brain, Cell. Mol. Neurobiol., 2 (1982)
47-52.
25 Rougon, G., Noble, M. and Munge, A.W., Neuropeptides
modulate the fl-adrenergic response of purified astrocytes in
vitro, Nature, 305 (1983) 715-717.
26 Silverman, A.J. and Zimmerman, E.A., Magnocellular neuro-
secretory system, Annu. Rev. Neurosci., 6 (1983) 357-380.
27 Sofroniew, M.V., Vasopressin, oxytocin and their related neu-
rophysins. In A. Bj6rklund and T. H6kfelt (Eds.), Handbook of
Chemical Neuroanatomy, Vol. 4, Elsevier, Amsterdam, pp.
93-165.
28 Soloff, M.S., Fernstrom, M.A. and Fernstrom, M.J., Vasopres-
sin and oxytocin receptors on plasma membranes from rat
mammary gland. Demonstration of vasopressin receptors by
stimulation of inositol phosphate formation, and oxytocin
receptors by binding of a specific 125I-labeledoxytocin antago-
nist, d(CH2)51[Tyr(Me)2,Thra,Tyr-NH29]OVTBiochem. Cell Biol.,
67 (1989) 152-162.
29 Stoeckel, M.E. and Freund-Mercier, M.J., Autoradiographic
demonstration of oxytocin-binding sites in the macula densa,
Am. J. Physiol., 257 (1989) F310-F314.
30 Theodosis, D.T., Montagnese, C., Rodriguez, F., Vincent, J.D.
and Poulain, D.A., Oxytocin induces morphological plasticity in
the adult hypothalamo-neurohypophysial system, Nature, 322
(1986) 738-740.
31 Tribollet, E., Barberis, C., Jard, S., Dubois-Dauphin, M. and
Dreifuss, J.J., Localization and pharmacological characteriza-
tion of high affinity binding sites for vasopressin and oxytocin in
the rat brain by light microscopic autoradiography, Brain
Research, 442 (1988) 105-118.
32 Tribollet, E., Charpak, S., Schmidt, A., Dubois-Dauphin, M.
and Dreifuss, J.J., Appearance and transient expression of
oxytocin receptors in fetal, infant, and peripubertal rat brain
studied by autoradiography and electrophysiology, J. Neurosci.,
9 (1989) 1764-1773.
16

33 WhitnaU, M.H., Key, S., Ben-Barak, Y., Ozato, K. and Gainer, 34 Yavin, E. and Yavin, Z., Attachment and culture of dissociated
H., Neurophysin in the hypothalamo-neurohypophysial system. cells from rat embryo cerebral hemispheres on poly-lysine-
II. Immunocytochemical studies of the ontogeny of oxytociner- coated surface, J. Cell Biol., 62 (1974) 540-546.
gic and vasopressinergic neurons, J. Neurosci., 5 (1985) 98-109.