Brain Researctt. 512 (1990) 60-t~9 Elsevier
60
BRES 15289
Corticotropin-releasing factor receptors in the rabbit brain visualized by in vitro autoradiography S.Y. Chai 1, E. Tarjan 2, M.J. McKinley 2, G.
Paxinos 3 and F.A.O. Mendelsohn J
1University of Melbourne, Department of Medicine, Austin Hospital, Heidelberg, Vict. (Australia), 2Howard Florey Institute for Experimental Physiology and Medicine, Parkville, Vict. (Australia) and 3School of Psychology, University of New South Wales, Kensington, N.S. W (Australia) (Accepted 15 August 1989)
Key words: Corticotropin-releasing factor; Receptor; Rabbit; Pineal gland; Autoradiography
Corticotropin-releasing factor (CRF) binding sites were visualized in the rabbit brain by in vitro autoradiography using the radioligand lzsI-[Tyr°]ovineCRF. The radioligand binding to sections of rabbit cingulate cortex were competed for by ovine and rat CRF with inhibitory constants (Ki) of 26 and 37 nM, respectively, whereas sauvagine and alpha-helical CRFg_41were approximately 10-fold less potent. In the rabbit brain, the highest densities of binding sites for CRF are found in the pineal gland and the choroid plexus. The cerebral cortex is labelled throughout, with the highest concentration of binding sites in the piriform and primary olfactory divisions. In the cerebellar cortex, the granular layer is more intensely labelled than the molecular layer. The distribution of CRF binding sites in the hippocampus follows a laminar pattern; the molecular layer of the dentate gyrus is intensely labelled, the oriens, radiatum and lacunosum moleculare layers of Ammon's horn contain moderate densities of binding and no binding is observed in the granular layer of the dentate gyrus and the pyramidal cell layer. The ventral subnucleus of the lateral septum, the zonal and superficial layers of the superior colliculus contain high densities of receptors. A moderate concentration of binding sites is observed in the caudate nucleus, putamen, bed nucleus of the stria terminalis, paraventricular, anterodorsal and anteroventral thalamic nuclei and the medial nucleus of the mammillary body. The fundus striati, the dorsal subnucleus of the lateral septum, the lateral dorsal thalamic, lateral and medial geniculate nuclei, the lateral and anterior cortical amygdaloid nuclei, central and posterior interpeduncular nuclei all contain low but significant levels of CRF binding sites. No detectable binding is observed in the pons, medulla oblongata or fibre tracts. The high densities of CRF binding sites found in the cortex, hippocampus and structures associated with the limbic system supports a role for CRF in the modulation of behavioural and physiological responses associated with stress. However, the extremely high density of CRF binding sites found in the pineal gland and choroid plexus suggest a more extensive role for CRF in the rabbit central nervous system. INTRODUCTION Corticotropin-releasing factor (CRF) was first isolated from ovine hypothalami as the secretagogue of A C T H and fl-endorphinz°. The role of this peptide in the central nervous system, however, was found to be more extensive. In the rat, intracerebro-ventricular administration of C R F increased blood pressure and heart rate 16, increased grooming is and locomotor activity 18, potentiated the effects of exposure to a novel e n v i r o n m e n t7 and decreased food intake 1°. The central administration of C R F induced behavioural effects which resemble responses to acute stress. Similar effects were also observed in the rabbit after intracerebroventricular administration of CRF, namely, increased respiratory rate 1, increased exploratory activity and reduced food intake 19. In addition, infusion of C R F into the cerebral ventricles of the rabbit induced a large sodium appetite 19. CRF-like immunoreactivity is distributed extensively within the rat central nervous system where high con-
centrations of C R F - i m m u n o r e a c t i v e cell bodies were found in regions including the paraventricular and supraoptic hypothalamic nuclei, the median preoptic area, locus coeruleus and parabrachial nucleus3"15. The major site of C R F synthesis in the rat brain is the paraventricular hypothalamic nucleus21, although C R F m R N A has also been detected in the inferior olivary nucleus and nucleus of the solitary tract 12. Receptor binding sites for C R F have been demonstrated in the rat brain where they are concentrated in the cortex and structures associated with the limbic system 4'22. This study investigates the distribution of C R F binding sites in the rabbit central nervous system using quantitative in vitro autoradiography in order to elucidate possible central sites where C R F may exert its action. MATERIALS AND METHODS
Source of peptide~ The radioligand used was 125I-[Tyr°]ovineCRF, (spec. act. 2200 Ci/mmol) which had been purified by reverse-phase high pressure
Correspondence: S.Y. Chai, University of Melbourne, Department of Medicine, Austin Hospital, Heidelberg, Vict. 3084, Australia. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
61 liquid chromatography (New England Nuclear Research Products, Boston, MA). The ovine and rat CRF, the antagonist alpha-helical ERE9_41 , the C-terminal fragment CRF34~1, and the related peptide sauvagine used in the competition experiments, were all purchased from Peninsula Laboratories (Belmont, CA). ACTH~_~4 (Synacthen, Ciba-Geigy, Australia), angiotensin II, and arginine vasopressin (Peninsula Laboratories, Belmont, CA) were also used in the competition experiments.
TABLE I
Inhibitory constants (Ki) for the different analogues of CRF in competing for the binding of 1251-[Tyr°]ovine CRF to rabbit cingulate cortex sections These values were obtained by analysis with LIGAND (Munson and Rodbardll).
K, (nM)
Binding characteristics of the radioligand The specificity of 12sI-[Tyr°]ovine CRF binding to brain sections was determined by competition studies using a range of unlabelled CRF analogues and unrelated peptides. The binding of the radioligand to serial sections of cingulate cortex was evaluated in the presence of a range of concentrations (10 -11 to 10-6 M) of ovine CRF, rat CRF, alpha-helical CRF9~a, sauvagine or CRF34~1. The unrelated peptides angiotensin II, ACTH and arginine-vasopressin, in the concentration range 10-4 to 10-9 M, were also added to parallel incubations. After incubation, sections of cingulate cortex were exposed to X-ray film for 1 month together with a set of ~25I-radioactivity standards. The autoradiographs were quantitated by computerized densitometry (see below) and the binding data obtained were then exported to an iterative, model-fitting program LIGAND 1~ to determine the affinities of the different unlabelled peptides in competing for the radioligand binding sites.
Autoradiographic localization of CRF receptors
1. Rat CRF 1 41 2. Ovinel_4l 3. Sauvagine 4. a-Helical CRF9_41 5. CRF34_41 6. Vasopressin 7. Angiotensin II 8. ACTH
inactive at 1000
C R F b o t h c o m p e t e d for the r a d i o l i g a n d b i n d i n g to rabbit cingulate c o r t e x with similar affinities, with inhibition c o n s t a n t s (Ki) o f 37 and 26 n M , r e s p e c t i v e l y (Table I and Fig. 1). S a u v a g i n e and t h e a l p h a - h e l i c a l a n a l o g u e of C R F w e r e a p p r o x i m a t e l y 10-fold less p o t e n t t h a n t h e t w o C R F
Four wild rabbits were housed under laboratory conditions, given food and water ad libitum, for several weeks before they were killed with an intravenous overdose of sodium pentobarbitone. The brains were then rapidly removed, blocked and frozen in a liquid hexane/dry ice mixture. Twenty-/zm coronal sections were cut in a cryostat maintained gelatinized at -15 °C, thaw-mounted onto getalinized slides and dehydrated in a dessicator at 4 °C for 2 h. The sections were subjected to two 15-min preincubations in 50 mM Tris-HCl, pH 7.4 containing 5 mM MgClz, 2 mM EGTA, 0.1 mM bacitracin, 100 K.I.U. aprotinin/ml and 1% B.S.A., followed by a 1-h incubation in a fresh aliquot of the same buffer containing 45 pM of 125I-[Tyr°]ovine CRE Under these incubation conditions, only minimal degradation of 125I-[Tyr°]rat CRF occurred in rat olfactory bulb membranes 5. Non-specific binding was determined in parallel incubations with the addition of 1/zM ovine or rat CRE After incubation, the sections were carried through a series of four 2-min washes at 0 °C in 50 mM Tris-HCI buffer, and dried rapidly under a stream of cold air. The sections were then exposed to Agfa-Scopix CR-3B X-ray film for one month at room temperature together with a set of radioactivity standards. Preparation of these standards, consisting of brain sections with known amounts of applied 125I-radioactivity, have been described previously w. The films were developed using a Kodak RP X-Omat automatic developer. The autoradiographic images obtained were quantitated using an MCID microcomputer imaging device (Imaging Research Inc., Ont., Canada) coupled to an IBM AT computer. The radioactive standards which have been included in each X-ray cassette and subjected to the same exposure period were then fitted to calibration curves by the computer to enable conversion of optical densities on the autoradiographs to units of 1251-radioactivity (dpm/mm2). The sections were then stained with thionin for anatomical localization of the autoradiographs.
p e p t i d e s , K i o f 210 and 130 n M r e s p e c t i v e l y (Table I
100
A• o&
80
60
~
40
20
o
~ -II
RESULTS
-Z0
-9
-8
L o g [peptide]
Specificity o f the radioligand 125I-[Tyr°]ovine
37 26 210 130
CRF
displayed
specific and
high-
affinity b i n d i n g to r a b b i t brain sections. R a t and o v i n e
-7
-6
-5
(M)
Fig. 1. Binding isotherms showing the displacement of binding of 125I-[Tyr°]ovine CRF from rabbit cingulate cortex sections by ovine CRF (
60
BRES 15289
Corticotropin-releasing factor receptors in the rabbit brain visualized by in vitro autoradiography S.Y. Chai 1, E. Tarjan 2, M.J. McKinley 2, G.
Paxinos 3 and F.A.O. Mendelsohn J
1University of Melbourne, Department of Medicine, Austin Hospital, Heidelberg, Vict. (Australia), 2Howard Florey Institute for Experimental Physiology and Medicine, Parkville, Vict. (Australia) and 3School of Psychology, University of New South Wales, Kensington, N.S. W (Australia) (Accepted 15 August 1989)
Key words: Corticotropin-releasing factor; Receptor; Rabbit; Pineal gland; Autoradiography
Corticotropin-releasing factor (CRF) binding sites were visualized in the rabbit brain by in vitro autoradiography using the radioligand lzsI-[Tyr°]ovineCRF. The radioligand binding to sections of rabbit cingulate cortex were competed for by ovine and rat CRF with inhibitory constants (Ki) of 26 and 37 nM, respectively, whereas sauvagine and alpha-helical CRFg_41were approximately 10-fold less potent. In the rabbit brain, the highest densities of binding sites for CRF are found in the pineal gland and the choroid plexus. The cerebral cortex is labelled throughout, with the highest concentration of binding sites in the piriform and primary olfactory divisions. In the cerebellar cortex, the granular layer is more intensely labelled than the molecular layer. The distribution of CRF binding sites in the hippocampus follows a laminar pattern; the molecular layer of the dentate gyrus is intensely labelled, the oriens, radiatum and lacunosum moleculare layers of Ammon's horn contain moderate densities of binding and no binding is observed in the granular layer of the dentate gyrus and the pyramidal cell layer. The ventral subnucleus of the lateral septum, the zonal and superficial layers of the superior colliculus contain high densities of receptors. A moderate concentration of binding sites is observed in the caudate nucleus, putamen, bed nucleus of the stria terminalis, paraventricular, anterodorsal and anteroventral thalamic nuclei and the medial nucleus of the mammillary body. The fundus striati, the dorsal subnucleus of the lateral septum, the lateral dorsal thalamic, lateral and medial geniculate nuclei, the lateral and anterior cortical amygdaloid nuclei, central and posterior interpeduncular nuclei all contain low but significant levels of CRF binding sites. No detectable binding is observed in the pons, medulla oblongata or fibre tracts. The high densities of CRF binding sites found in the cortex, hippocampus and structures associated with the limbic system supports a role for CRF in the modulation of behavioural and physiological responses associated with stress. However, the extremely high density of CRF binding sites found in the pineal gland and choroid plexus suggest a more extensive role for CRF in the rabbit central nervous system. INTRODUCTION Corticotropin-releasing factor (CRF) was first isolated from ovine hypothalami as the secretagogue of A C T H and fl-endorphinz°. The role of this peptide in the central nervous system, however, was found to be more extensive. In the rat, intracerebro-ventricular administration of C R F increased blood pressure and heart rate 16, increased grooming is and locomotor activity 18, potentiated the effects of exposure to a novel e n v i r o n m e n t7 and decreased food intake 1°. The central administration of C R F induced behavioural effects which resemble responses to acute stress. Similar effects were also observed in the rabbit after intracerebroventricular administration of CRF, namely, increased respiratory rate 1, increased exploratory activity and reduced food intake 19. In addition, infusion of C R F into the cerebral ventricles of the rabbit induced a large sodium appetite 19. CRF-like immunoreactivity is distributed extensively within the rat central nervous system where high con-
centrations of C R F - i m m u n o r e a c t i v e cell bodies were found in regions including the paraventricular and supraoptic hypothalamic nuclei, the median preoptic area, locus coeruleus and parabrachial nucleus3"15. The major site of C R F synthesis in the rat brain is the paraventricular hypothalamic nucleus21, although C R F m R N A has also been detected in the inferior olivary nucleus and nucleus of the solitary tract 12. Receptor binding sites for C R F have been demonstrated in the rat brain where they are concentrated in the cortex and structures associated with the limbic system 4'22. This study investigates the distribution of C R F binding sites in the rabbit central nervous system using quantitative in vitro autoradiography in order to elucidate possible central sites where C R F may exert its action. MATERIALS AND METHODS
Source of peptide~ The radioligand used was 125I-[Tyr°]ovineCRF, (spec. act. 2200 Ci/mmol) which had been purified by reverse-phase high pressure
Correspondence: S.Y. Chai, University of Melbourne, Department of Medicine, Austin Hospital, Heidelberg, Vict. 3084, Australia. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
61 liquid chromatography (New England Nuclear Research Products, Boston, MA). The ovine and rat CRF, the antagonist alpha-helical ERE9_41 , the C-terminal fragment CRF34~1, and the related peptide sauvagine used in the competition experiments, were all purchased from Peninsula Laboratories (Belmont, CA). ACTH~_~4 (Synacthen, Ciba-Geigy, Australia), angiotensin II, and arginine vasopressin (Peninsula Laboratories, Belmont, CA) were also used in the competition experiments.
TABLE I
Inhibitory constants (Ki) for the different analogues of CRF in competing for the binding of 1251-[Tyr°]ovine CRF to rabbit cingulate cortex sections These values were obtained by analysis with LIGAND (Munson and Rodbardll).
K, (nM)
Binding characteristics of the radioligand The specificity of 12sI-[Tyr°]ovine CRF binding to brain sections was determined by competition studies using a range of unlabelled CRF analogues and unrelated peptides. The binding of the radioligand to serial sections of cingulate cortex was evaluated in the presence of a range of concentrations (10 -11 to 10-6 M) of ovine CRF, rat CRF, alpha-helical CRF9~a, sauvagine or CRF34~1. The unrelated peptides angiotensin II, ACTH and arginine-vasopressin, in the concentration range 10-4 to 10-9 M, were also added to parallel incubations. After incubation, sections of cingulate cortex were exposed to X-ray film for 1 month together with a set of ~25I-radioactivity standards. The autoradiographs were quantitated by computerized densitometry (see below) and the binding data obtained were then exported to an iterative, model-fitting program LIGAND 1~ to determine the affinities of the different unlabelled peptides in competing for the radioligand binding sites.
Autoradiographic localization of CRF receptors
1. Rat CRF 1 41 2. Ovinel_4l 3. Sauvagine 4. a-Helical CRF9_41 5. CRF34_41 6. Vasopressin 7. Angiotensin II 8. ACTH
inactive at 1000
C R F b o t h c o m p e t e d for the r a d i o l i g a n d b i n d i n g to rabbit cingulate c o r t e x with similar affinities, with inhibition c o n s t a n t s (Ki) o f 37 and 26 n M , r e s p e c t i v e l y (Table I and Fig. 1). S a u v a g i n e and t h e a l p h a - h e l i c a l a n a l o g u e of C R F w e r e a p p r o x i m a t e l y 10-fold less p o t e n t t h a n t h e t w o C R F
Four wild rabbits were housed under laboratory conditions, given food and water ad libitum, for several weeks before they were killed with an intravenous overdose of sodium pentobarbitone. The brains were then rapidly removed, blocked and frozen in a liquid hexane/dry ice mixture. Twenty-/zm coronal sections were cut in a cryostat maintained gelatinized at -15 °C, thaw-mounted onto getalinized slides and dehydrated in a dessicator at 4 °C for 2 h. The sections were subjected to two 15-min preincubations in 50 mM Tris-HCl, pH 7.4 containing 5 mM MgClz, 2 mM EGTA, 0.1 mM bacitracin, 100 K.I.U. aprotinin/ml and 1% B.S.A., followed by a 1-h incubation in a fresh aliquot of the same buffer containing 45 pM of 125I-[Tyr°]ovine CRE Under these incubation conditions, only minimal degradation of 125I-[Tyr°]rat CRF occurred in rat olfactory bulb membranes 5. Non-specific binding was determined in parallel incubations with the addition of 1/zM ovine or rat CRE After incubation, the sections were carried through a series of four 2-min washes at 0 °C in 50 mM Tris-HCI buffer, and dried rapidly under a stream of cold air. The sections were then exposed to Agfa-Scopix CR-3B X-ray film for one month at room temperature together with a set of radioactivity standards. Preparation of these standards, consisting of brain sections with known amounts of applied 125I-radioactivity, have been described previously w. The films were developed using a Kodak RP X-Omat automatic developer. The autoradiographic images obtained were quantitated using an MCID microcomputer imaging device (Imaging Research Inc., Ont., Canada) coupled to an IBM AT computer. The radioactive standards which have been included in each X-ray cassette and subjected to the same exposure period were then fitted to calibration curves by the computer to enable conversion of optical densities on the autoradiographs to units of 1251-radioactivity (dpm/mm2). The sections were then stained with thionin for anatomical localization of the autoradiographs.
p e p t i d e s , K i o f 210 and 130 n M r e s p e c t i v e l y (Table I
100
A• o&
80
60
~
40
20
o
~ -II
RESULTS
-Z0
-9
-8
L o g [peptide]
Specificity o f the radioligand 125I-[Tyr°]ovine
37 26 210 130
CRF
displayed
specific and
high-
affinity b i n d i n g to r a b b i t brain sections. R a t and o v i n e
-7
-6
-5
(M)
Fig. 1. Binding isotherms showing the displacement of binding of 125I-[Tyr°]ovine CRF from rabbit cingulate cortex sections by ovine CRF (
