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Corticotropin-releasing factor up-regulates its own receptor mRNA in the paraventricular nucleus of the hypothalamus
MOLECULAR BRAIN RESEARCH ELSEVIER
Molecular Brain Research 38 (1996) 166-170
Short communication
Corticotropin-releasing factor up-regulates its own receptor mRNA in the paraventricular nucleus of the hypothalamus Toshihiro Imaki a,*, Mitsuhide Naruse ", Shoko Harada a, Naoko Chikada a Junko Imaki b Hidetaka Onodera b, Hiroshi Demura a, Wylie Vale c " Department of Medicine, Tokyo Women's Medical College, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162, Japan b Department of Anatomy, Nippon Medical School, Bunkyo-ku, Japan " Clayton Foundation Laboratories for Peptide Biology, The Salk Institute, La Jolla, CA 92037, USA Accepted 27 December 1995
Abstract
We investigated the role of CRF in regulating receptor expression in the paraventricular nucleus (PVN). First, to clarify the effect of exogenously administered CRF, 1 /xg of ovine CRF was injected into rat lateral ventricle and changes in concentration of the CRF type 1 receptor (CRF1-R) and CRF mRNA in the PVN were semiquantified after in situ hybridization. Second, we determined the effect of stress, as a stimulant of endogenous CRF secretion, on mRNA accumulation. While CRF1-R mRNA expression was low to be undetectable in the PVN of controls, both intracerebroventricular administration of CRF and restraint significantly increased CRF1-R and CRF signals in the parvocellular PVN. Thus CRF may modulate CRF production and release from the PVN, by regulating CRF1-R expression. Keywords: Corticotropin-releasing factor; Receptor; Paraventricular nucleus; Stress; Hybridization, in situ
Corticotropin-releasing factor (CRF), a 41-residue peptide, is the principal hypophysiotropic factor stimulating stress-induced adrenocorticotropic hormone (ACTH) secretion [1,21,22,25]. In addition to its endocrine effects, CRF acts within the central nervous system (CNS) and influences a number of behavioral, neuroendocrine, and autonomic responses to stress [2,12], thus raising the possibility that a central CRF system may act to integrate multimodal components of the organismic response to stress. Although CRF is broadly expressed throughout the CNS, the parvocellular division of the paraventricular nucleus (PVN) of the hypothalamus plays a major role in synthesizing CRF that controls stress-induced pituitary adrenal activation [23]. We have reported previously that intracerebroventricular (i.c.v.) injection of CRF induced the expression of an immediate early gene, c-fos, in the PVN [11], suggesting that CRF stimulates neuronal activity in the PVN. Centrally administered CRF also has been reported to induce expression of another immediate early gene, NGFI-B, and to increase CRF mRNA levels in the PVN [17]. Thus, CRF may have a direct short loop effect on its own release and
* Corresponding author. Fax: (81) (3) 3357-6475. 0169-328X/96/$15.00 © 1996 Elsevier Science B.V, 1-3
1911 S 0 1 6 9 - 3 2 8 X ( 9 6 ) 0 0 0 1
production within the PVN. In addition, immunohistochemical and radioligand binding studies have shown that the PVN contains a large number of both CRF-immunoreactive fibers [24] and high affinity binding sites [7], and that CRF neurons in the PVN make numerous synaptic contacts with each other in the dorsomedial zone [24]. These findings suggest that CRF regulates its own biosynthesis through CRF receptors that exist in the PVN. Recently, a receptor for CRF, designated CRF1, has been cloned from human pituitary tumor [5], rat brain [3,18], and mouse pituitary [26]. The cloned cDNA encodes a putative protein comprising seven plausible membrane-spanning domains and is structurally related to the calcitonin/vasoactive intestinal polypeptide/growth hormone-releasing factor subfamily of G protein-coupled receptors. CRF1-R mRNA has been reported to be widely distributed throughout the rat brain, but expression in the PVN was unexpectedly low [19]. However, recent studies have demonstrated that, while CRF1-R mRNA was hardly detectable in the PVN of control rats, CRF1-R was dramatically increased by several kinds of stressful manipulations of the animal, including physical-psychological stress, osmotic stimulation [14], lipopolysaccharide administration, and immobilization stress [20]. These results further sup-
T. Imaki et al. /Molecular Brain Research 38 (1996) 166-170
port the notion that CRF may play a role as a direct regulator of CRF neuronal activity in the PVN during stress. The purpose of the present study was to assay for such a direct role for CRF in controlling CRF1-R gene expression, i.c.v.-Injected CRF and restraint stress were used to identify effects of exogenous and endogenous CRF, respectively. Adults male Wistar rats weighing 180-200 g were used in the experiments. Approximately 5 days before the experiment, a polyethylene guide cannula (SP-45 polyethylene tube, Natume, Tokyo, Japan) was implanted into the right lateral ventricle of each animal under sodium pentobarbital anesthesia (50 m g / k g b.wt.) as previously described [9]. The rats then were handled for 4 min daily to familiarize them with the infusion procedure and to try to minimize non-specific stress responses. On the day of the experiment, an injection cannula (SP-10 PE tube, Natume) that was connected to a 10 /xl microsyringe (Hamilton, NV) was inserted into a guide cannula. Next, we injected 1 /zg of ovine CRF in 5 /zl of normal saline, or saline alone into the lateral ventricle. They were killed before, and 2 and 4 h after the i.c.v injection. Five animals per group were examined. In the stress experiment, the rats were killed before or after 120 min in a restraint cage (Natume, Tokyo). For in situ hybridization, rats were deeply anesthetized with pentobarbital and perfused with ice-cold 4% paraformaldehyde in a pH 9.5 0.1 M borate buffer [10]. The brains were placed for 2 days at 4°C in the same fixative containing 10% sucrose. Frozen sections (25 ~m) were cut on a sliding microtome, mounted onto silane-coated slides, and air-dried. Hybridization was performed as previously described [10], with minor modifications. Prior to hybridization, sections were dried overnight under vacuum, digested with
167
proteinase K (10 /zg/ml, 37°C, 30 min), acetylated, and dehydrated. After vacuum drying, 90 /zl of the hybridization mixture (10 6 c.p.m./ml, with 10 mM dithiothreitol (DTT)) was spotted onto each slide, sealed under a coverslip, and incubated at 65°C for overnight. The coverslips then were removed and the slides were rinsed in 4 x SSC (1 X SSC: 15 mM trisodium citrate buffer, pH 7.0/0.15 M NaCl) at room temperature. The sections were digested with RNAase A ( 2 0 / z g / m l , 37°C, 30 min) and washed in 0.1 X SSC for 30 min at 65°C. These sections were exposed to X-ray film at 4°C for 7-21 days, then dipped in NTB2 nuclear emulsion (1 : 1 with water, Kodak), exposed for 7 or 42 days and developed. The slides were counterstained with thionine. Adjoining sections were stained with thionine to provide better cytoarchitectonic definition for analysis. All samples from each experiment were run in a single assay. A pGEM-4 plasmid containing the rat CRF cDNA (1.2 kb, gift from Dr. K. Mayo) was linearized with Hind III. A full-length rat CRF1-R cDNA (1.3 kb PST 1 fragment) was subcloned into p-BlueScript KS vector (Stratagene, San Diego, CA) and linearized with B a m H | . Radioactive antisense cRNA copies were synthesized by incubation of 36 mM Tris-HCl, pH 7.5, 0.1 /xg linearized plasmid in 6 mM MgCl 2, 2 mM spermidine, 8 mM D'I'T, 25 mM A T P / G T P / C T P , 5 mM unlabeled UTP, [a-35S]UTPfor CRF probe and [tx-33p]UTP for CRF1-R probe, 1 U RNAsin (Promega, Madison, WI), and SP-6 for CRF probe and T7 polymerase for CRF-R probe, for 60 min at 37°C. Sense probes for the CRF1-R cDNA were made using the T3 polymerase. All probes were purified on resin columns (Nensorb 20, NEN, Wilmington, DE). The specific activity of each probe was approximately 1.0 X 108 d.p.m.//xl. Control experiments performed using sense probes showed no hybridization signals (data not shown).
Fig. 1. Film autoradiographs showing the CRF1-R (a,b) and CRF (c,d) mRNA expression in rat PVN 4 h after i.c.v, injection of saline (a,c) or 1 p.g of ovine CRF (c,d). Exposure time was 16 (CRF1-R mRNA) and 2 (CRF mRNA) days.
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T. lmaki et al. / Molecular Brain Research 38 (1996) 166-170
Fig. 2. Effect of 2 h restraint stress on CRF1-R (a,b) and CRF mRNA (c,d) expression in rat PVN. Exposure time was 6 (CRF1-R mRNA) and 4 (CRF mRNA) days.
The density of CRF and CRF1-R mRNAs on autoradiograms was semiquantified using an MCID image analysis system (Imaging Research, Inc., St. Catherines, Ontario, Canada) [15]. This system digitizes the continuous range of image gray shades into 256 discrete gray levels, in which the lower values are given to the darker group. The levels obtained were converted to relative optical densities (ROD) using the formula: ROD = loglo (256/levels). The optical densities of the PVN were measured bilaterally on at least two sections from each subject. The ROD within the window was measured and the background was assessed by measuring ROD with the window placed over another area of the brain where no specific CRF or CRF1-R hybridization was detected. Values are presented as means + S.E.M. Statistical analysis was performed by analysis of variance followed by Fisher's protected least-significance
difference. A single comparison was made by Student's t-test. P < 0.05 was considered significant. In control rats, CRF1-R m R N A was expressed in the neocortex, piriform cortex and the medial nucleus of the amygdala (Fig. la), as recently reported by Potter et al.[19] A low to moderate signal was also detected in the supraoptic nucleus (SON). Control rats displayed virtually undetectable signals for CRF1-R m R N A in the PVN (Fig. la). However, CRF signals in the PVN were much increased after i.c.v, injection of CRF (Fig. lb). CRF1-R gene expression in the SON appeared to be slightly increased after i.c.v, administration of CRF as shown in the Fig. lb. Centrally administered CRF did not appear to modulate the expression of CRF1-R m R N A in other brain regions studied. Similarly, i.c.v.-administered CRF induced an increase in CRF m R N A signals in the PVN (Fig. la,b). Restraint
Fig. 3. Dark-field photomicrographsdemonstrating the distribution of silver grains over the PVN. Hybridizationsignals positive for CRF1-R cRNA probes were distributed mainly in the parvoce!lulardivision of the PVN. Exposure time was 19 days. Bar = 100 /zm. 3V, third ventricle.
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T. lmaki et al. / Molecular Brain Research 38 (1996) 166-170
stress induced strong CRF1-R mRNA signals in the PVN (Fig. 2a,b). In brain regions other than the PVN, CRF1-R mRNA accumulations appeared to be unchanged in response to stress. CRF mRNA also appeared increased in the PVN following stress (Fig. 2c,d). Emulsion-coated sections were prepared for in situ hybridization autoradiography to identify the cellular localization of the CRF1-R mRNA and demonstrated that CRF1-R mRNA was mainly distributed within the parvocellular division of the PVN in CRF-treated rats (Fig. 3). Densitometric analysis showed that both i.c.v.-injected CRF and restraint stress significantly increased CRF1-R (Figs. 4 and 5a) as well as CRF signals (Fig. 4b and 5b) in the PVN. Densitometry showed no significant increase in CRF1-R mRNA levels in the SON (data not shown). This study demonstrated that i.c.v.-injected, exogenous, CRF increased both CRF1-R and CRF mRNA expression in the PVN, and that restraint stress, which produced an increase in CRF mRNA accumulations, also stimulated CRF1-R gene expression in the PVN. This suggested that CRF up-regulates CRF1-R mRNA expression in the PVN, and thereby up-regulates its own biosynthesis. Neuronal activation of the PVN by CRF previously has been proposed, since central CRF administration activates the immediate early genes, c-los and NGFI-B [11,17], both useful markers for delineating the functional circuitry involved in hormonal activation of the PVN [4]. The present finding of increased CRF mRNA accumulations after i.c.v.-injected CRF is consistent with a previous report.
a
i
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Fig. 4. Effect of i.c.v, injection of CRF on CRF1-R (a) and CRF (b) hybridization signals in the PVN. Results, expressed as ROD, are the mean 5: S.E.M. of 5 animals for each group. * P < 0.05, * * P < 0.005 vs. saline-injected control.
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Fig. 5. Effect of restraint stress on CRF1-R (a) and CRF (b) hybridization signals in the PVN. Results, expressed as ROD, are the mean :1:S.E.M. of 5 animals for each group. * P < 0.01, ° * P < 0.0005 vs. control (0 h) rats.
Our findings support the hypothesis of an ultrashort positive feedback for the regulation of CRF production in the PVN. The existence of ultrashort loop positive feedback control of CRF secretion during stress has been implied by Ono et al. [16]. Such a mechanism would further increase stress-induced CRF expression and amplify the stress response. When injected i.c.v, in rats, CRF has been shown to produce a dose-dependent, behavioral, and autonomic activation in rats akin to the stress response [2,12]. To initiate these biological effects within the CNS, binding to plasma membrane receptors specific for CRF is essential. This assumption is supported by experiments showing that central administration of CRF results in a variety of physiological and behavioral stress-related phenomena, which can be blocked by application of the CRF-R antagonist a-helical CRF9.41 [8]. In contrast with the loss of receptors and the desensitization of target cells induced by many hormones, our results show that CRF produces an increase of its own receptor's mRNA in the PVN. The HPA axis is a system that markedly changes its properties with changing conditions [6]. This axis is activated by stress. In the face of stimulation by chronic stress, it changes its response characteristics so that further responsiveness is maintained [6]. The increased expression of CRF1-R mRNA by direct application of CRF would facilitate the positive feedback effects of CRF on the expression of the gene encoding CRF itself a n d / o r other genes including immediate early genes. Similarly, it has been reported that both binding sites and ACTH receptor mRNA are regulated positively by AC-'TH in cultured human adrenal cells
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11 lmaki et al. / Molecular Brain Research 38 (1996) 166-170
[13]. T h i s s u g g e s t s the e n h a n c e d g l u c o c o r t i c o i d s e c r e t i o n u n d e r c o n d i t i o n s o f i n c r e a s e d A C T H secretion, for instance d u r i n g stress. S u c h p o s i t i v e effects o f the h o r m o n e on its r e c e p t o r s m a y a c c o u n t for p r e s e r v a t i o n o f the function in the H P A axis by p r o v i d i n g a m e c h a n i s m for facilitation o f s u b s e q u e n t responses. T h u s , the a n i m a l c o u l d m a i n t a i n c o n t i n u e d activity in the H P A axis d u r i n g stress.
Acknowledgements W e t h a n k Dr. K. M a y o ( N o r t h w e s t e r n U n i v e r s i t y , C h i c a g o , IL) for the k i n d gift o f rat C R F c D N A . W e are grateful to Drs. S. M i n a m i a n d H. S u g i h a r a ( N i p p o n M e d i c a l S c h o o l ) for p e r f o r m i n g the d e n s i t o m e t r i c analysis.
References [1] Antoni, F.A., Hypothalamic control of adrenocorticotropin secretion: advances since the discovery of 41-residue corticotropin-releasing factor. Endocrine Reu., 7 (1986) 351-370. [2] Brown, M.R. and Fisher, L.A., Corticotropin-releasing factor: effects on the autonomic nervous system and visceral systems. Fed. Proc. 44 (1985) 243-248. [3] Chang, C.-P., Pearsc, R.V. I1, O'Connel, S. and Rosenfeld, MG., Identification of a seven transmenbrane helix receptor for conicotropin-releasing factor and sauvagine in mammalian brain. Neuron 11 (1993) 1187-1195. [4] Chan, R.K.W., Brown, E.R., Ericsson, A., Kovacs, K.J. and Sawchenko, P.E., A comparison of two immediate-early genes, c-los and NGFI-B, as markers for functional activation in stress-related neuroendocrine circuitry. J. Neurosci. 13 (19931 5126-5138. [5] Chen, R., Lewis, K.A., Perrin, M. and Vale, W.W., Expression cloning of a human corticotropin-releasing factor receptors. Proc. Natl. Acad. Sci. USA, 90 (19931 8967-8971. [6] Dallman, M.F., Stress update: adaptation of the hypothalamic-pituitary-adrenal axis to chronic stress. Trends EndocrinoL Metab., 4 (1993) 62-69. [7] De Souza, E.B., Insel, T.R., Perrin, M.H., Rivier, J. and Vale, W.W., Corticotropin-releasing factors are widely distributed within the rat central nervous system: an autoradiographic study. J. Neurosci., 5 (1985) 3189-3203. [8] Dunn, A. and Berridge, C.W., Physiological and behavioral responses to corticotropin-releasing factor administration: is CRF a mediator of anxiety or stress responses? Brain Res. Rev., 15 (19901 71-100. [9] lmaki, T., Shibasaki, T., Hotta, M., Masuda, A., Demura, H.. Shizume, K. and Ling, N., The satiety effect of growth hormone-releasing factor in rats. Brain Res., 340 (1985) 186-188. [10] lmaki, T., Nahon, J.-L., Sawchenko, P.E. and Vale, W., Widespread expression of corticotropin-releasing factor messenger RNA and immunoreactivity in the rat olfactory bulb. Brain Res.. 496 (1989) 35-44. [11] lmaki, T., Shibasaki, T., Hotta, M. and Demura, H., lntracerebroventricular administration of corticotropin-releasing factor induces c-los
mRNA expression in brain regions related to stress responses: comparison with pattern of c-los mRNA induction after stress. Brain Res., 616 (1993) 114-125. [12] Koob, G.F. and Bloom, F.E., Corticotropin-releasing factor and behavior. Fed. Proc., 44 (1985) 259-263. [13] Lebrethon, M.-C., Naville, D., Begeot, M. and Saez, J.M., Regulation of corticotropin receptor number and messenger RNA in cultured human adrenocortical cells by corticotropin and angiotensin II. J. Clin. Invest., 93 (1994) 1828-1833. [14] Luo, X., Kiss, A., Makara, G.B., Lolait, S.J. and Aguilera, G., Stress-specific regulation of corticotropin releasing hormone receptor expression in the paraventricular and supraoptic nuclei of the hypothalamus in the rat. J. Neuroendocrinol., 6 (1994) 689-696. [15] Minami, S., Kamegai, K., Sugihara, H., Hasegawa, O. and Wakabayashi, I., Systemic administration of recombinant human growth hormone induces expression of c-los gene in the hypothalamic arcuate and periventricular nuclei in hypophysectomized rats. Endocrinology, 131 (1992) 247-253. [16] Ono, N., Samson, W., McDonald, J., Lumpkin, M., Bedran deCastro, J., McCann, S., Effects of intravenous and intraventricular injection of antisera directed against corticotropin-releasing factor on the secretion of anterior pituitary hormones. Proc. NatL Acad. Sci. USA, 82 (1985) 7787-7790. [17] Parkes, D., Rivest, S., Lee, S., Rivier, C. and Vale, W., Corticotropin-releasing factor activates c-los, NGFI-B and corticotropinreleasing factor gene expression within the paraventricular nucleus of the rat hypothalamus. Mol. Endocrinol., 7 (1993) 1357-1367. [18] Perrin, M.H., Haas, Y., Rivier, J.E. and Vale, W., Cloning and functional expression of a rat brain corticotropin-releasing factor (CRF) receptor. Endocrinology, 118 (1993) 3058-3061. [19] Potter, E., Sutton, S., Donaldson, C., Chen, R., Perrin, M., Lewis, K., Sawchenko, P.E. and Vale, W., Distribution of corticotropin-releasing factor receptor mRNA expression in the rat brain and pituitary. Proc. Natl. Acad. Sci. USA, 91 (1994) 8777-8781. [20] Rivest, S., Laflamme, N. and Nappi R.E., Immune challenge and immobilization stress induce transcription of the gene encoding the CRF receptor in selective nuclei of the rat hypothalamus. J. Neurosci., (1995) 2680-2695. [21] Rivier, C., Rivier, J. and Vale, W., Inhibition of adrenocorticotropic hormone secretion in the rat by immunoneutralization of corticotropin-releasing factor. Science, 218 (1982) 377-379. [22] Rivier, C.L. and Plotsky, P.M., Mediation by corticotropin-releasing factor (CRF) of adenohypophyseal hormone secretion. Annu. Ret,. Physiol., 48 (1986) 475-494. [23] Sawchenko, P.E. and Swanson, L.W., Organization of CRF immunoreactive cells and fibers in the rat brain: immunohistochemical studies. In: E.B. DeSouza, and C.B. Nemeroff (Eds.), Corticotropin-releasing Factor: Basic and Clinical Studies of a Neuropeptide, CRC Uniscience, Boca Raton, FL, 1991, pp. 29-51.
[24] Silvermann, A.J., Hou-You, A. and Chen, W., Corticotropin-releasing synapses within the paraventricular nucleus of the hypothalamus. Neuroendocrinology, 49 (1989) 292-299. [25] Vale, W., Rivier, C., Brown, M.R., Spiess, J., Koob, G., Swanson, L., Bilezikjian, L., Bloom, F. and Rivier, J., Chemical and biological characterization of corticotropin-releasing factor. Rec. Prog. Hormone Res., 39 (1983) 227-45. [26] Vita, N.B., Laurent, P., Lefort, S., Chalon, P., Leloas, J.M., Kaghad, M., Le Fur, G., Caput, D., Ferrara, P., Primary structure and functional expression of mouse pituitary and human brain corticotropin releasing factor receptor. FEBS Lett., 335 (1993) 1-5.
Molecular Brain Research 38 (1996) 166-170
Short communication
Corticotropin-releasing factor up-regulates its own receptor mRNA in the paraventricular nucleus of the hypothalamus Toshihiro Imaki a,*, Mitsuhide Naruse ", Shoko Harada a, Naoko Chikada a Junko Imaki b Hidetaka Onodera b, Hiroshi Demura a, Wylie Vale c " Department of Medicine, Tokyo Women's Medical College, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162, Japan b Department of Anatomy, Nippon Medical School, Bunkyo-ku, Japan " Clayton Foundation Laboratories for Peptide Biology, The Salk Institute, La Jolla, CA 92037, USA Accepted 27 December 1995
Abstract
We investigated the role of CRF in regulating receptor expression in the paraventricular nucleus (PVN). First, to clarify the effect of exogenously administered CRF, 1 /xg of ovine CRF was injected into rat lateral ventricle and changes in concentration of the CRF type 1 receptor (CRF1-R) and CRF mRNA in the PVN were semiquantified after in situ hybridization. Second, we determined the effect of stress, as a stimulant of endogenous CRF secretion, on mRNA accumulation. While CRF1-R mRNA expression was low to be undetectable in the PVN of controls, both intracerebroventricular administration of CRF and restraint significantly increased CRF1-R and CRF signals in the parvocellular PVN. Thus CRF may modulate CRF production and release from the PVN, by regulating CRF1-R expression. Keywords: Corticotropin-releasing factor; Receptor; Paraventricular nucleus; Stress; Hybridization, in situ
Corticotropin-releasing factor (CRF), a 41-residue peptide, is the principal hypophysiotropic factor stimulating stress-induced adrenocorticotropic hormone (ACTH) secretion [1,21,22,25]. In addition to its endocrine effects, CRF acts within the central nervous system (CNS) and influences a number of behavioral, neuroendocrine, and autonomic responses to stress [2,12], thus raising the possibility that a central CRF system may act to integrate multimodal components of the organismic response to stress. Although CRF is broadly expressed throughout the CNS, the parvocellular division of the paraventricular nucleus (PVN) of the hypothalamus plays a major role in synthesizing CRF that controls stress-induced pituitary adrenal activation [23]. We have reported previously that intracerebroventricular (i.c.v.) injection of CRF induced the expression of an immediate early gene, c-fos, in the PVN [11], suggesting that CRF stimulates neuronal activity in the PVN. Centrally administered CRF also has been reported to induce expression of another immediate early gene, NGFI-B, and to increase CRF mRNA levels in the PVN [17]. Thus, CRF may have a direct short loop effect on its own release and
* Corresponding author. Fax: (81) (3) 3357-6475. 0169-328X/96/$15.00 © 1996 Elsevier Science B.V, 1-3
1911 S 0 1 6 9 - 3 2 8 X ( 9 6 ) 0 0 0 1
production within the PVN. In addition, immunohistochemical and radioligand binding studies have shown that the PVN contains a large number of both CRF-immunoreactive fibers [24] and high affinity binding sites [7], and that CRF neurons in the PVN make numerous synaptic contacts with each other in the dorsomedial zone [24]. These findings suggest that CRF regulates its own biosynthesis through CRF receptors that exist in the PVN. Recently, a receptor for CRF, designated CRF1, has been cloned from human pituitary tumor [5], rat brain [3,18], and mouse pituitary [26]. The cloned cDNA encodes a putative protein comprising seven plausible membrane-spanning domains and is structurally related to the calcitonin/vasoactive intestinal polypeptide/growth hormone-releasing factor subfamily of G protein-coupled receptors. CRF1-R mRNA has been reported to be widely distributed throughout the rat brain, but expression in the PVN was unexpectedly low [19]. However, recent studies have demonstrated that, while CRF1-R mRNA was hardly detectable in the PVN of control rats, CRF1-R was dramatically increased by several kinds of stressful manipulations of the animal, including physical-psychological stress, osmotic stimulation [14], lipopolysaccharide administration, and immobilization stress [20]. These results further sup-
T. Imaki et al. /Molecular Brain Research 38 (1996) 166-170
port the notion that CRF may play a role as a direct regulator of CRF neuronal activity in the PVN during stress. The purpose of the present study was to assay for such a direct role for CRF in controlling CRF1-R gene expression, i.c.v.-Injected CRF and restraint stress were used to identify effects of exogenous and endogenous CRF, respectively. Adults male Wistar rats weighing 180-200 g were used in the experiments. Approximately 5 days before the experiment, a polyethylene guide cannula (SP-45 polyethylene tube, Natume, Tokyo, Japan) was implanted into the right lateral ventricle of each animal under sodium pentobarbital anesthesia (50 m g / k g b.wt.) as previously described [9]. The rats then were handled for 4 min daily to familiarize them with the infusion procedure and to try to minimize non-specific stress responses. On the day of the experiment, an injection cannula (SP-10 PE tube, Natume) that was connected to a 10 /xl microsyringe (Hamilton, NV) was inserted into a guide cannula. Next, we injected 1 /zg of ovine CRF in 5 /zl of normal saline, or saline alone into the lateral ventricle. They were killed before, and 2 and 4 h after the i.c.v injection. Five animals per group were examined. In the stress experiment, the rats were killed before or after 120 min in a restraint cage (Natume, Tokyo). For in situ hybridization, rats were deeply anesthetized with pentobarbital and perfused with ice-cold 4% paraformaldehyde in a pH 9.5 0.1 M borate buffer [10]. The brains were placed for 2 days at 4°C in the same fixative containing 10% sucrose. Frozen sections (25 ~m) were cut on a sliding microtome, mounted onto silane-coated slides, and air-dried. Hybridization was performed as previously described [10], with minor modifications. Prior to hybridization, sections were dried overnight under vacuum, digested with
167
proteinase K (10 /zg/ml, 37°C, 30 min), acetylated, and dehydrated. After vacuum drying, 90 /zl of the hybridization mixture (10 6 c.p.m./ml, with 10 mM dithiothreitol (DTT)) was spotted onto each slide, sealed under a coverslip, and incubated at 65°C for overnight. The coverslips then were removed and the slides were rinsed in 4 x SSC (1 X SSC: 15 mM trisodium citrate buffer, pH 7.0/0.15 M NaCl) at room temperature. The sections were digested with RNAase A ( 2 0 / z g / m l , 37°C, 30 min) and washed in 0.1 X SSC for 30 min at 65°C. These sections were exposed to X-ray film at 4°C for 7-21 days, then dipped in NTB2 nuclear emulsion (1 : 1 with water, Kodak), exposed for 7 or 42 days and developed. The slides were counterstained with thionine. Adjoining sections were stained with thionine to provide better cytoarchitectonic definition for analysis. All samples from each experiment were run in a single assay. A pGEM-4 plasmid containing the rat CRF cDNA (1.2 kb, gift from Dr. K. Mayo) was linearized with Hind III. A full-length rat CRF1-R cDNA (1.3 kb PST 1 fragment) was subcloned into p-BlueScript KS vector (Stratagene, San Diego, CA) and linearized with B a m H | . Radioactive antisense cRNA copies were synthesized by incubation of 36 mM Tris-HCl, pH 7.5, 0.1 /xg linearized plasmid in 6 mM MgCl 2, 2 mM spermidine, 8 mM D'I'T, 25 mM A T P / G T P / C T P , 5 mM unlabeled UTP, [a-35S]UTPfor CRF probe and [tx-33p]UTP for CRF1-R probe, 1 U RNAsin (Promega, Madison, WI), and SP-6 for CRF probe and T7 polymerase for CRF-R probe, for 60 min at 37°C. Sense probes for the CRF1-R cDNA were made using the T3 polymerase. All probes were purified on resin columns (Nensorb 20, NEN, Wilmington, DE). The specific activity of each probe was approximately 1.0 X 108 d.p.m.//xl. Control experiments performed using sense probes showed no hybridization signals (data not shown).
Fig. 1. Film autoradiographs showing the CRF1-R (a,b) and CRF (c,d) mRNA expression in rat PVN 4 h after i.c.v, injection of saline (a,c) or 1 p.g of ovine CRF (c,d). Exposure time was 16 (CRF1-R mRNA) and 2 (CRF mRNA) days.
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Fig. 2. Effect of 2 h restraint stress on CRF1-R (a,b) and CRF mRNA (c,d) expression in rat PVN. Exposure time was 6 (CRF1-R mRNA) and 4 (CRF mRNA) days.
The density of CRF and CRF1-R mRNAs on autoradiograms was semiquantified using an MCID image analysis system (Imaging Research, Inc., St. Catherines, Ontario, Canada) [15]. This system digitizes the continuous range of image gray shades into 256 discrete gray levels, in which the lower values are given to the darker group. The levels obtained were converted to relative optical densities (ROD) using the formula: ROD = loglo (256/levels). The optical densities of the PVN were measured bilaterally on at least two sections from each subject. The ROD within the window was measured and the background was assessed by measuring ROD with the window placed over another area of the brain where no specific CRF or CRF1-R hybridization was detected. Values are presented as means + S.E.M. Statistical analysis was performed by analysis of variance followed by Fisher's protected least-significance
difference. A single comparison was made by Student's t-test. P < 0.05 was considered significant. In control rats, CRF1-R m R N A was expressed in the neocortex, piriform cortex and the medial nucleus of the amygdala (Fig. la), as recently reported by Potter et al.[19] A low to moderate signal was also detected in the supraoptic nucleus (SON). Control rats displayed virtually undetectable signals for CRF1-R m R N A in the PVN (Fig. la). However, CRF signals in the PVN were much increased after i.c.v, injection of CRF (Fig. lb). CRF1-R gene expression in the SON appeared to be slightly increased after i.c.v, administration of CRF as shown in the Fig. lb. Centrally administered CRF did not appear to modulate the expression of CRF1-R m R N A in other brain regions studied. Similarly, i.c.v.-administered CRF induced an increase in CRF m R N A signals in the PVN (Fig. la,b). Restraint
Fig. 3. Dark-field photomicrographsdemonstrating the distribution of silver grains over the PVN. Hybridizationsignals positive for CRF1-R cRNA probes were distributed mainly in the parvoce!lulardivision of the PVN. Exposure time was 19 days. Bar = 100 /zm. 3V, third ventricle.
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stress induced strong CRF1-R mRNA signals in the PVN (Fig. 2a,b). In brain regions other than the PVN, CRF1-R mRNA accumulations appeared to be unchanged in response to stress. CRF mRNA also appeared increased in the PVN following stress (Fig. 2c,d). Emulsion-coated sections were prepared for in situ hybridization autoradiography to identify the cellular localization of the CRF1-R mRNA and demonstrated that CRF1-R mRNA was mainly distributed within the parvocellular division of the PVN in CRF-treated rats (Fig. 3). Densitometric analysis showed that both i.c.v.-injected CRF and restraint stress significantly increased CRF1-R (Figs. 4 and 5a) as well as CRF signals (Fig. 4b and 5b) in the PVN. Densitometry showed no significant increase in CRF1-R mRNA levels in the SON (data not shown). This study demonstrated that i.c.v.-injected, exogenous, CRF increased both CRF1-R and CRF mRNA expression in the PVN, and that restraint stress, which produced an increase in CRF mRNA accumulations, also stimulated CRF1-R gene expression in the PVN. This suggested that CRF up-regulates CRF1-R mRNA expression in the PVN, and thereby up-regulates its own biosynthesis. Neuronal activation of the PVN by CRF previously has been proposed, since central CRF administration activates the immediate early genes, c-los and NGFI-B [11,17], both useful markers for delineating the functional circuitry involved in hormonal activation of the PVN [4]. The present finding of increased CRF mRNA accumulations after i.c.v.-injected CRF is consistent with a previous report.
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Fig. 4. Effect of i.c.v, injection of CRF on CRF1-R (a) and CRF (b) hybridization signals in the PVN. Results, expressed as ROD, are the mean 5: S.E.M. of 5 animals for each group. * P < 0.05, * * P < 0.005 vs. saline-injected control.
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Fig. 5. Effect of restraint stress on CRF1-R (a) and CRF (b) hybridization signals in the PVN. Results, expressed as ROD, are the mean :1:S.E.M. of 5 animals for each group. * P < 0.01, ° * P < 0.0005 vs. control (0 h) rats.
Our findings support the hypothesis of an ultrashort positive feedback for the regulation of CRF production in the PVN. The existence of ultrashort loop positive feedback control of CRF secretion during stress has been implied by Ono et al. [16]. Such a mechanism would further increase stress-induced CRF expression and amplify the stress response. When injected i.c.v, in rats, CRF has been shown to produce a dose-dependent, behavioral, and autonomic activation in rats akin to the stress response [2,12]. To initiate these biological effects within the CNS, binding to plasma membrane receptors specific for CRF is essential. This assumption is supported by experiments showing that central administration of CRF results in a variety of physiological and behavioral stress-related phenomena, which can be blocked by application of the CRF-R antagonist a-helical CRF9.41 [8]. In contrast with the loss of receptors and the desensitization of target cells induced by many hormones, our results show that CRF produces an increase of its own receptor's mRNA in the PVN. The HPA axis is a system that markedly changes its properties with changing conditions [6]. This axis is activated by stress. In the face of stimulation by chronic stress, it changes its response characteristics so that further responsiveness is maintained [6]. The increased expression of CRF1-R mRNA by direct application of CRF would facilitate the positive feedback effects of CRF on the expression of the gene encoding CRF itself a n d / o r other genes including immediate early genes. Similarly, it has been reported that both binding sites and ACTH receptor mRNA are regulated positively by AC-'TH in cultured human adrenal cells
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[13]. T h i s s u g g e s t s the e n h a n c e d g l u c o c o r t i c o i d s e c r e t i o n u n d e r c o n d i t i o n s o f i n c r e a s e d A C T H secretion, for instance d u r i n g stress. S u c h p o s i t i v e effects o f the h o r m o n e on its r e c e p t o r s m a y a c c o u n t for p r e s e r v a t i o n o f the function in the H P A axis by p r o v i d i n g a m e c h a n i s m for facilitation o f s u b s e q u e n t responses. T h u s , the a n i m a l c o u l d m a i n t a i n c o n t i n u e d activity in the H P A axis d u r i n g stress.
Acknowledgements W e t h a n k Dr. K. M a y o ( N o r t h w e s t e r n U n i v e r s i t y , C h i c a g o , IL) for the k i n d gift o f rat C R F c D N A . W e are grateful to Drs. S. M i n a m i a n d H. S u g i h a r a ( N i p p o n M e d i c a l S c h o o l ) for p e r f o r m i n g the d e n s i t o m e t r i c analysis.
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