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Endocrinology Vol. 138, No. 11 4774-4782
Copyright © 1997 by The Endocrine Society

ARTICLES

Regulation of Corticotropin-Releasing Factor (CRF) Messenger Ribonucleic Acid and CRF Peptide in the Amygdala: Studies in Primary Amygdalar Cultures

J. W. Kasckow1, A. Regmi, P. S. Gill, D. G. Parkes and T. D. Geracioti2

University of Cincinnati School of Medicine (J.W.K., A.R., P.S.G., T.D.G.), Department of Psychiatry, Cincinnati, Ohio 45267-0559; Cincinnati VAMC (J.W.K., T.D.G.), Psychiatry Service, Cincinnati, Ohio 45220; University of Cincinnati Neurosciences Program (J.W.K.), Cincinnati, Ohio 45267; Amylin Pharmaceuticals, Inc. (D.G.P.), San Diego, California 92121

Address all correspondence and requests for reprints to: J. W. Kasckow, University of Cincinnati School of Medicine, Department of Psychiatry, 231 Bethesda Avenue (ML 559), Cincinnati, Ohio 45267-0559. E-mail: Kasckojw{at}email.uc.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Amygdalar CRF has been implicated in the mediation of stress behaviors. The signal transduction pathways that regulate amygdalar CRF are not well understood. In this report, we have examined the effect of protein kinase A and C activators, dexamethasone, and interleukin 6 on CRF messenger RNA (mRNA) and CRF peptide expression in dissociated amygdalar cultures. The amygdala from E19 rat pups was dissected out bilaterally and dissociated in 0.25% trypsin for 10–15 min and plated. On day 17 in culture, CRF mRNA and peptide were measured following treatment with the following agents: forskolin, the phorbol ester-phorbol 12 myristate 13-acetate (TPA), dexamethasone, and interleukin-6 (IL6). Both forskolin and IL6, but not TPA, increased CRF mRNA in a time- and dose-dependent manner. Secretion and intracellular content of the CRF peptide also increased with both forskolin and IL6 treatment but not with TPA. Dexamethasone treatment did not alter the expression of CRF message or peptide. Transfection of the primary cultures with a rat CRF promoter-luciferase reporter construct followed by treatment with all four agents produced alterations in luciferase expression that were consistent with changes observed at the level of CRF mRNA and peptide. The results suggest that CRF regulation in the amygdala differs from that known to occur in the hypothalamus, and that elevation of IL6 levels within the central nervous system may directly act to stimulate CRF production and secretion from limbic structures such as the amygdala, to promote subsequent behavioral changes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CRF IS A neuropeptide that has been isolated from various species including the sheep, rat, mouse, and human (1, 2, 3, 4). CRF is distributed heterogeneously throughout the central nervous system (CNS); it is also found in placental tissue and other peripheral tissues as well, including the adrenal medulla and testes (5). CRF coordinates the neuroendocrine, behavioral, autonomic, and immune responses to stress (5). CRF secreted from the nerve terminals of hypothalamic neurons acts on the pituitary corticotrophs to enhance secretion of ACTH and other POMC products. ACTH, in turn, stimulates glucocorticoid synthesis and secretion in the adrenal cortex.

There is a body of evidence that supports an important role for amygdalar CRF in the mediation of stress-like behaviors. Amygdalofugal pathways have been documented to participate in autonomic, endocrine, and behavioral responses to stresses (6, 7). CRF neurons densely innervate the central nucleus of the amygdala, and injection of CRF into this area has numerous behavioral effects. These include increases in locomotor activity in a familiar environment (8), reduction in exploratory behavior in an unfamiliar open field (9), as well as decreases in exploratory behavior in the open arms of an elevated plus maze (10). Administration of the CRF antagonist, {alpha}-helical CRF9–41 into the central nucleus of the amygdala reverses the decrease in exploration of the open arms of an elevated plus maze caused by exposure to a social stressor. The dose of CRF antagonist required to reverse stress-induced suppression of behavior in the plus maze is 100-fold lower with intracerebral injections into the central nucleus than those effective by the intracerebroventricular route (10). There is also evidence from microdialysis studies that restraint stress can induce increases in the release of CRF from the rat amygdala (11). This phenomenon has also been demonstrated at the level of CRF transcription. Kalin et al. (12) demonstrated that 2 h of restraint stress in rats will increase CRF messenger RNA expression in the amygdala.

Amygdalar CRF clearly plays a role in the mediation of behavioral responses to stress. The regulation of amygdalar CRF is not well understood. Hence, determination of the signal transduction pathways responsible for amygdalar CRF expression would help investigators better understand what role CRF regulation plays in the mediation of stress behaviors. There is evidence from in vivo studies that glucocorticoids may both positively and negatively regulate amygdalar CRF depending on the dose used (13). On the other hand, there are other studies that suggest that CRF levels in the amygdala are refractory to glucocorticoids (14). Furthermore, Raber et al. (15) demonstrated that interleukin-2, norepinephrine, and acetylcholine can increase CRF release from amygdala slices in vitro. These investigators implicated the nitric oxide signaling pathway in the IL2 effect; in addition, they implicated both {alpha} and ß-adrenergic receptors in the actions of norepinephrine and both muscarinic and nicotinic receptors in the actions of acetylcholine.

Regulation of CRF in other tissues is well characterized. This includes CRF in the hypothalamus, in the placenta, and in tumor-derived clonal cell lines that express CRF (16, 17, 18, 19, 20, 21). Glucocorticoids are known to consistently decrease hypothalamic CRF levels. On the other hand, in the placenta there is evidence that glucocorticoids consistently increase CRF levels (22). Other compounds known to increase both hypothalamic and placental CRF include forskolin, norepinephrine, serotonin, acetylcholine, and interleukin-1 (16, 17, 18, 19, 20). Protein kinase C (PKC) activators increase hypothalamic CRF but not placental CRF (23).

Interleukin-6 (IL6) levels in the brain are normally expressed at low levels; immune stressors will increase IL6 expression in the brain (24), and IL6, in turn, has been implicated as a CRF secretagogue in the hypothalamus (25, 26). There are reports that suggest that IL6 may play a role in other stress behaviors. For instance, Lacroix et al. (27) recently demonstrated that IL6 administration in rats can activate c-fos expression in the central nucleus of the amygdala.

In this report, we seek to further our understanding of the regulation of CRF expression in the amygdala by examining the effects of various stimuli on CRF messenger RNA and peptide expression in cultures of amygdalar neurons. The stimuli examined have included activators of the protein kinase A (PKA) and PKC pathway, the synthetic steroid dexamethasone, and the cytokine-IL6.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Preparation of primary amygdalar cultures
Methods were based on Brouard et al. (28) and Cratty and Birkle (29) and were also approved by the University of Cincinnati Animal Use Committee. Pregnant Sprague-Dawley rats (Zivic-Miller, Indianapolis, IN), containing embryonic day 19 pups were killed by administration of CO2. The uterus was placed in PBS (NaCl, 137 mM; Na2HPO4, 21 mM; KH2PO4, 29 mM; KCl, 1.2 mM, pH 7.3), and the embryos were removed and decapitated. Following this, the brain was removed and after slicing away the brain stem, the remainder of the brain was placed ventral side up. A coronal cut was made posterior to the optic chiasm and anterior to the diencephalon to remove the frontal cortical region. A diagonal cut was then made along the lateral fissure and the amygdaloid region was separated by gently peeling away the cortex.

Brain slices were placed in PBS. Cells were dissociated for 10–15 min at 37 C in 0.25% trypsin containing 75 U/ml of DNAse I in serum-free medium (SFM) consisting of a mixture of DMEM and Ham’s F-12 (1:1, vol/vol from GIBCO (Gaithersburg, MD) supplemented with 14 mM glucose, 15 mM NaHCO3, 5 mM Hepes and 0.05 U/ml of penicillin-streptomycin (Sigma Chemical Co., St. Louis, MO). Cells were collected by centrifugation (500 x g, 5 min), resuspended in SFM supplemented with 7.5% FCS (Atlanta Biologicals, Atlanta, GA). For analysis of CRF peptide, cells were plated at a density of 2 million cells per well using 6 well plates (Costar, Cambridge, MA). For analysis of CRF messenger RNA (mRNA), cells were plated onto 100-mm tissue culture dishes (Falcon, Lincoln Park, NJ). For microscopic analysis, cells were grown in 8-well Lab-Tek II Chamber Slide Systems (Nalge Nunc International, Naperville, IL).

We coated the plates with gelatin (250 µg/ml, 30 min, room temperature; Sigma) and polyornithine (mol wt = 40,000; 1.5 µg/ml, overnight at room temperature; Sigma) based on Brouard et al. (28). Following plating, cells were incubated at 37 C in a 95% O2/5% CO2 atmosphere. Medium was totally removed at day 5 and replaced by fresh medium containing cytosine arabinoside (AraC; 20 µM) to limit the proliferation of glial cells (30). Following this, half of the medium was replaced every day from day 6 until day 10 with medium that did not contain AraC. After this, half of the medium was changed every third day. Cells were used for analysis on day 17, based on the report of Cratty and Birkle (29).

Immunocytochemistry
The ABC method using a VectaStain kit (Vector Labs, Burlingame, CA) was used using a rabbit antibody to CRF (rC70, provided by W. Vale, Ph.D., Salk Institute, La Jolla, CA). Protocols from Vector Labs were used. Tissue was incubated initially with normal goat serum with 2% BSA for 30 min at RT to block nonspecific binding and then incubated with rC70 (1:2000) overnight at 4 C. This was followed by a 30 min incubation at 4 C with biotinylated IgG (Vector Labs, Burlingame, CA) and then with the ABC complex (Vectastain Elite ABC Kit, Vector Labs). The cultures were then incubated with the substrate-diaminobenzidine/H2O2 (DAB substrate Kit, Vector Labs) at RT for 2–10 min. Cultures were photographed with a Leitz Ortholux microscope interfaced with a Leica Wild MPS52 camera.

Stimulation experiments
Cells were washed with SFM. Test substances were then added in an incubation medium consisting of ß-Pit Julip + 0.1% BSA, based on Vale et al. (31). For CRF mRNA analysis, cultures were grown in 100-mm dishes and incubated with the following test substances at the following concentrations: forskolin (Sigma; 0, 3, 10, 30 µM), the phorbol ester-phorbol 12 myristate 13-acetate (TPA; Sigma; 0, 1, 20, 50 nM), interleukin-6 (IL6; Promega, Madison, WI; 0, 10, 50, 100 pM), and dexamethasone (Sigma; 0, 10, 50, 100 nM) for 6, 12, or 24 h. For CRF mRNA analysis, the total volume of these substances in the ß-Pit Julip + 0.1% BSA incubation medium was 5 ml.

For CRF peptide analysis, test substance was added in a total volume of 700 µl ß-Pit Julip + 0.1% BSA incubation medium, initially for 12 h of incubation. In those treatments for which significant changes were observed at 12 h of treatment, we performed time course analyses at 3, 6, and 9 h of treatment. For analysis of CRF secretion, medium was removed at the end of the incubation period and stored at -20 C for use in a CRF RIA. For determination of CRF intracellular content, cells were lysed with 0.1% NP-40 in 700 ml ß-Pit Julip + 0.1% BSA incubation medium and stored as mentioned for the secretion medium. CRF peptide levels from both secretion media and lysed cells were then measured in a RIA as described below.

RIA
RIA was performed according to the protocol provided by IgG Corporation (Nashville, TN) using their reagents, which included their CRF antibody (32, 33). Rabbit anti-hCRF serum was diluted 1:100 in RIA buffer (63 mM Na2HPO4 7H2O [pH 7.4), 13 mM Na2EDTA, 3 mM sodium azide, 0.1% Triton X-100 [vol/vol] and 250 KIU aprotinin) and incubated with 100 µl tissue samples or supernatant for 3 days at 4 C. One hundred µl [125I]-0Tyr-CRF (1000 cpm; DuPont-New England Nuclear, Boston, MA) was then added and samples were left for two additional days at 4 C. Following this, goat antirabbit gamma globulin was added and after 4 h at 4 C, samples were precipitated by centrifugation at 5000 x g at 4 C in RIA buffer, containing 2.5% BSA. Radioactivity of the pellet was measured using a Packard MultiPrias 4 gamma counter (Packard Instruments, Downers, IL). The CRF RIA exhibited an ED90 of 1.66 ± 0.13 pg/100 µl (n = 7) with a interassay coefficient of variability of 2.9%. The intraassay coefficient of variability was 2.2% (n = 5). The recovery of exogenous nonlabeled CRF added to culture wells averaged 81 ± 9.3% (n = 48) over a range of concentrations and times consistent with those used in the experimental groups.

CRF mRNA detection by Northern hybridization
Total RNA from cells was isolated using the PUREscript RNA isolation kit (Gentra Systems, Minneapolis, MN). Twenty micrograms of total RNA per lane was electrophoresed in an acrylamide-formaldehyde gel (1.2%/2.2 M) for 2 h at 80 V as previously described (34). RNA was transferred to a Hybond-N nylon membrane (Amersham) at 2 C overnight at 0.25 mA. The membrane with the RNA was UV cross-linked using a Stratalinker 1800 apparatus (Stratagene, La Jolla, CA) and prehybridized in ExpressHyb hybridization solution (Clonetech, Palo Alto, CA) for 30 min at 65 C. A 32P-labeled complementary DNA (cDNA) probe for CRF was generated with a PrimeIt-II oligonucleotide kit (Stratagene). The CRF probe was provided by Dr. A. Seasholtz (University of MI, Ann Arbor, MI). It consisted of a 761-bp fragment of the rat CRF exon II (+54 to +872) that was ligated to the BamHI site in a pGEM3Z vector (35). The membrane containing RNA was then hybridized at 65 C with 1 x 106 cpm/ml labeled probe in ExpressHyb hybridization solution for 1 h. After washing, the membrane was exposed to Xomatic film (Kodak, Rochester, NY) for 24–48 h and developed. A Foto/Eclipse Imager (Fotodyne Inc., Hartland, WI) interfaced with a Power Macintosh 8100/110 using NIH ImageQuant analysis software was used for densitometric quantitation of the mRNA bands.

Transfection
Cells (2 x 106 per dish) were transfected using the Lipofectamine Reagent (GIBCO-BRL). The plasmid used (pTKSL) was provided by Dr. M. Rosenfield (Howard Hughes Medical Institute, La Jolla, CA). It was based on pSV2CAT (ATCC; Bethesda, MD) with CAT replaced by luciferase at bp 4558 to 6398. The vector also contained an SV40 promoter region at bp 2929 to 4557 as well as a portion of the 5' regulatory region of the rat CRF promoter. The latter was a 360-bp BglII/HindIII fragment of the rat CRF gene (35; from -337 to +23) which, in turn, replaced the first 160 bp of the pTKSL plasmid. Five micrograms of pTKSL was diluted in OptiMem medium with the Lipofectamine reagent (GIBCO-BRL) based on the manufacturer’s recommended procedures. DNA solution was then added to the cells and incubated at 37 C for 2–3 h. Following washing and a subsequent 24 h incubation at 37 C, cells were then treated 6 h with either 30 µM forskolin, 100 pM IL6, 50 nM TPA, 100 nM dexamethasone, or vehicle in serum free ß-pit Julip medium containing 0.1% BSA. After treatment, cells were lysed using 100 µL Reporter Lysis Buffer (Promega) and rapidly frozen on dry ice. Luciferase assay was performed with 20 µl of cell extract and 100 µL luciferase substrate (Promega) using a Monolight 2010 luminometer (Analytical Luminescence Laboratory, San Diego, CA). Transfection efficiency was accounted for by cotransfecting pTKSL with 1 µg pSV-ß-galactosidase control vector (Promega) and measuring these levels based on the Promega protocol. ß-galactosidase values were then normalized to protein; protein levels had been determined based on Bradford et al. (36). Levels of luciferase expression were also normalized to ß-galactosidase and protein levels.

Statistical analysis
The quantitative data obtained were expressed as mean ± SE. Data were subjected to ANOVA followed by Student’s t test with the Bonferroni correction. A P value less than 0.05 was considered sufficient to reject the null hypothesis.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Figure 1Go illustrates a typical amygdalar culture stained with anti-CRF antibody 17 days following dispersion. By this time, cells grew as monolayers, and many cells developed long processes extending from the cell bodies to the neighboring cells. Cell viability at this stage was 97.8 ± 0.5% (n = 4) based on trypan blue exclusion. CRF immunoreactive cells were present in our cultures. These cells displayed immunoreactive cell bodies and elaborate varicose cell processes. The size of the CRF transcript in these cells was 1.3 kb, as depicted in Fig. 2Go.



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  		Figure 1. This photograph demonstrates the primary amygdalar cultures on day 17 stained with anti-CRF antibody based on methods outlined in the text. Magnification, x170.

 


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  		Figure 2. This photograph displays the size of the CRF transcript from the primary amygdalar cultures. The molecular weight of the transcript was 1.3 kb.

 
CRF response to forskolin
Figure 3Go, A and B, depicts changes in CRF messenger RNA (mRNA) following various doses and times of forskolin incubation. Figure 3AGo depicts the changes from autoradiograms at 12 h with concentrations of 0, 3, 10, and 30 µM. As depicted in Fig. 3AGo, absolute levels of CRF mRNA increase with treatment, whereas those for ß-actin remain constant. Figure 3BGo depicts changes in CRF mRNA normalized to ß-actin mRNA based on densitometric analyses, at 6, 12, and 24 h with the same concentrations of forskolin. CRF mRNA started to increase at 6 h. Levels of CRF mRNA in amygdalar cultures were significantly elevated at concentrations of 30 µM forskolin with 6 h relative to control (P < 0.05). At 12 h of incubation, CRF mRNA levels were also significantly elevated relative to control with 10 µM and 30 µM forskolin treatment (P < 0.05). The peak response to forskolin occurred at 12 h. With 30 µM forskolin treatment, the mRNA levels at 12 h were significantly higher from those observed at 6 h. By 24 h, levels of CRF mRNA at all concentrations of forskolin returned to levels not statistically different from control.



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  		Figure 3. CRF messenger RNA changes in primary cultures at concentrations of 3, 10, and 30 µM forskolin following 6, 12, and 24 h of incubation. A, Representative autoradiogram of the Northern blots probed for ß-actin or CRF expression at 6 h, with increasing concentrations of forskolin. B, Densitometric values of each concentration at various time points normalized to values obtained from a ß-actin probe. Bars represent mean ± SE of the mean (n = 3). Statistical significance for each concentration in comparison to control, as determined by ANOVA, followed by Student’s t test with Bonferroni’s correction is represented by * for P < 0.05. {dagger} indicates that 30 µM forskolin treatment at 12 h resulted in significantly higher mRNA levels than that at 6 h. The data were pooled from three independent experiments.

 
Figure 4AGo depicts changes in CRF peptide secretion at 9 and 12 h of incubation. Basal rates of CRF secretion in these cells were below the limits of detection at 3 and 6 h, obviating comparisons to control. At 9 and 12 h, basal levels of secretion were readily detectable and ranged from 10–15 pg/2 x 106 cells (17–25 pg/ml). The highest concentration of forskolin, i.e. 30 µM, was examined on the basis of the CRF mRNA data. Levels of CRF secretion were significantly elevated with 30 µM forskolin treatment following 9 and 12 h of incubation (P < 0.05).



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  		Figure 4. Secreted (A) and intracellular content (B) of CRF-like immunoreactivity (CRF-LI) from primary amygdalar cultures in response to 30 µM forskolin. The abscissa represents time course of incubation for 30 µM forskolin vs. control. The ordinate represents pg per 2 x 106 cells of CRF-LI. Bars represent mean ± SEs. Statistical significance for each concentration in comparison to control, as determined by ANOVA, followed by Student’s t test with Bonferroni’s correction is represented by * for P < 0.05. This figure is representative of three independent experiments.

 
Basal levels of CRF intracellular content in these cultures varied from 30–40 pg/2 x 106 cells (50–67 pg/ml) at the given time courses. Figure 4BGo reveals respective changes in CRF intracellular content following 3, 6, 9, and 12 h of incubation with 30 µM forskolin. At all time points, basal levels of CRF intracellular content were above the detection limits of the CRF RIA. At 6, 9, and 12 h of incubation, intracellular content of CRF was significantly elevated relative to control with 30 µM forskolin treatment (P < 0.05).

Response to interleukin-6 (IL6)
Figure 5Go, A and B, depicts changes in CRF mRNA following various concentrations and times of IL6 incubation. Figure 5AGo depicts the changes from autoradiograms at 12 h and concentrations of 0, 10, 50, and 100 pM. Absolute levels of CRF mRNA increased with treatment while those for ß-actin did not. Figure 5BGo depicts changes based on densitometric analyses at 6, 12, and 24 h at the same concentrations of IL6. Levels of CRF mRNA started to increase at 6 h. As was demonstrated for forskolin treatment, levels of CRF mRNA in amygdalar cultures were significantly elevated with 100 pM IL6 following 6 and 12 h of incubation (P < 0.05). The peak response to IL6 occurred at 6 h; with 100 pM IL6, the mRNA levels at 6 h were significantly higher than those observed at 12 h. By 24 h, levels of CRF mRNA expression at all concentrations were not significantly different from controls.



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  		Figure 5. CRF messenger RNA changes in primary cultures at concentrations of 4, 20, and 100 pM interleukin-6 (IL6) following 6, 12, and 24 h of incubation. A, Representative autoradiogram of the Northern blots at 6 h with increasing concentrations of forskolin. B, Densitometric values of each concentration at various time points normalized to values obtained from a ß-actin probe. Bars represent mean ± SE of the mean (n = 3). Statistical significance for each concentration in comparison to control, as determined by ANOVA, followed by Student’s t test with Bonferroni’s correction is represented by * for P < 0.05. {dagger} indicates that 100 pM IL6 treatment at 6 h resulted in significantly higher mRNA levels than that at 12 h. The data were pooled from three independent experiments.

 
Figure 6AGo depicts the effects of IL6 on CRF secretion at 9 and 12 h, at a concentration of 100 pM. Basal levels of secreted CRF at 3 and 6 h were below the detection limits of the CRF RIA. At 9 and 12 h, levels of CRF secretion with 100 pM IL6 treatment were significantly elevated relative to control. Figure 6BGo depicts at 3, 6, 9, and 12 h, levels of CRF intracellular content. Basal levels of CRF content were above the limits of detection at all time points tested. At 9 and 12 h, 100 pM IL6 produced elevations in levels of CRF intracellular content that were significantly higher relative to controls (P < 0.05).



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  		Figure 6. Secreted (A) and intracellular content (B) of CRF-like immunoreactivity (CRF-LI) from primary amygdalar cultures in response to 100 pM interleukin 6 (IL6). The abscissa represents time course of incubation for 100 pM IL6 vs. control. The ordinate represents pg per 2 x 106 cells of CRF-LI. Bars represent mean ± SEs. Statistical significance for each concentration in comparison to control, as determined by ANOVA, followed by Student’s t test with Bonferroni’s correction is represented by * for P < 0.05. This figure is representative of three independent experiments.

 
We also studied the effect of 500 pM IL6 on levels of CRF secretion and intracellular content for the same time courses. At 9 and 12 h, this concentration of IL6 increased levels of CRF secretion and content to levels that were significantly higher than controls (P < 0.05) but not significantly different from that achieved with 100 pM IL6 (data not shown).

Response to dexamethasone
CRF peptide and CRF mRNA levels did not change with dexamethasone treatment at concentrations of 1, 10 and 100 nM following 6, 12, and 24 h of incubation (data not shown). Figure 7Go, A and B, reveals that there was no effect of 100 nM dexamethasone on levels of CRF secretion (Fig. 7AGo) nor CRF intracellular content (Fig. 7BGo) after 12 h of incubation. Furthermore, coincubation of 100 nM dexamethasone with either 30 µM forskolin or 100 pM IL6 for 12 h was not able to significantly alter the stimulation achieved at the level of CRF secretion (Fig. 7AGo) nor at the level of CRF intracellular content (Fig. 7BGo).



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  		Figure 7. Secreted (A) and intracellular content (B) of CRF-like immunoreactivity (CRF-LI) from primary amygdalar cultures in response to 100 nM dexamethasone (DEX) as well as coincubation of 100 nM DEX with either 30 µM forskolin (FOR) or 100 pM interleukin 6 (IL6). The abscissa represents treatment and the ordinate represents pg per 2 x 106 cells of CRF-LI. Bars represent mean ± SEs. Statistical significance for each concentration in comparison to control, as determined by ANOVA, followed by Student’s t test with Bonferroni’s correction is represented by * for P < 0.05. Furthermore, there was no significant difference between levels of CRF-LI obtained with 30 µM FOR vs. 30 µM FOR + DEX as well as with 100 pM IL6 vs. 100 pM IL6 + DEX. This is true for levels of secretion (A) and for levels of intracellular content (B). This figure is representative of three independent experiments.

 
Response to TPA
CRF mRNA levels did not change with TPA treatment at doses of 1, 10, and 50 nM following 6, 12, or 24 h of incubation (data not shown). In addition, there was no effect of 50 nM TPA on CRF secretion nor CRF intracellular content after 12 h of incubation (data not shown).

Response of the rat CRF promoter in primary amygdalar cultures to stimuli
The results from the transfection studies are depicted in Table 1Go. At baseline, the CRF luciferase promoter in primary amygdalar cultures yielded 0.8 ± 0.02 luciferase counts/ß-galactosidase/µg protein after 6 h of treatment. Following 6 h of 30 µM forskolin treatment, CRF luciferase expression increased to 6.31 ± 0.31 luciferase counts/ß-galactosidase/µg protein, an increase to 184% relative of control. Six hours of IL6 treatment resulted in 6.97 ± 0.81 luciferase counts/ß-galactosidase/µg protein, an increase to 203% of control. Both of these increases were statistically significant. Treatment with 100 nM dexamethasone and 20 nM TPA did not yield significant increases in CRF luciferase expression.


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  		Table 1. Transfection of amygdalar primary cultures with pTKSL

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Primary amygdalar cultures have been used in the past to analyze the actions of stimuli on CRF expression. It has been demonstrated by others with dissociated amygdalar cultures, that CRF peptide levels are detectable by RIA and that depolarization induces CRF release (29). Furthermore, we have confirmed that our amygdalar cultures contain sufficient numbers of CRF-producing cells that are detectable by immunocytochemistry, RIA, and Northern blot analysis. In addition, we have demonstrated that various stimuli such as forskolin and interleukin-6 (IL6) can increase expression of CRF. The results observed in our experiments are consistent with the notion that CRF exhibits differential regulation in hypothalamic, amygdalar, and placental tissue. Not only does this appear to be true for physiologic systems but also in clonal cell line models that express CRF. For instance, CRF in the NPLC-KC hepatoma cell line increases in response to interleukin-1 as well as to PKA and PKC activators but decreases in response to dexamethasone (37). In the BE(2)-M17 and BE(2)-C cell line, CRF increases in the presence of PKA and PKC activators but not interleukin-1. Only CRF in the BE(2)-M17 is positively regulated by steroids, whereas in the BE(2)-C cell this is not the case (21).

Here we have provided evidence for a positive regulation of CRF expression in the amygdala by PKA and interleukin-6 (IL6) and a lack of regulation with TPA and dexamethasone. This has been verified with Northern blot analysis of CRF messenger RNA (mRNA) and with RIAs that have quantified CRF peptide expression at the level of secretion and intracellular content. In addition, treatment of the primary cultures with all these agents for 6 h following transient transfection with a CRF promoter-luciferase construct yielded data that were consistent with the Northern blot and RIA data analysis. These results suggest that measurement of CRF promoter-driven luciferase expression represent a valid and facile tool for measuring alterations in CRF expression in this model system.

The peak expression of CRF mRNA occurred at 12 h with forskolin treatment and at 6 h with IL6 treatment. With both agents, peak CRF mRNA levels increased by approximately 3-fold. At the level of CRF secretion, both forskolin and IL6 treatment lead to increases just under 2-fold. Maximal responses in levels of CRF secretion and intracellular content were observable with 30 µM forskolin at 12 h of treatment. Maximal levels of secretion at this time point were approximately 15 times greater than the ED90 of our RIA; in addition, maximal levels of intracellular content at 12 h were approximately 30 times higher than the ED90. Changes in CRF demonstrable in vivo are within the same order of magnitude as that observed with our in vitro experiments. For instance, Plotsky et al. (38) demonstrated that electrical stimulation of the ventral noradrenergic bundle in vivo results in a 3-fold elevation in portal CRF levels.

Despite continuous activation of our amygdalar cultures by various chemical stimuli, CRF mRNA levels declined by 24 h after exhibiting initial increases at 6 and 12 h. Similar declines by 24 h, with other endocrine genes in primary culture systems, have been observed (39, 40). There appear to be other cellular mechanisms that regulate amygdalar CRF gene expression, which contribute to its instability. These decrements in gene expression by 24 h could reflect local feedback or a cellular mechanism of desensitization in response to continuous stimuli.

The lack of response of CRF mRNA in these cultures to multiple doses of dexamethasone is consistent with the preliminary findings reported previously by Kasckow et al. (41); this report indicated that levels of both CRF peptide secretion and intracellular content in primary amygdalar cultures do not respond to doses of 0, 4, 20, and 100 nM dexamethasone. Furthermore, in the current set of experiments, dexamethasone was not able to alter the stimulated response of the CRF peptide achieved with forskolin or IL6. The lack of a CRF response to dexamethasone in these amygdalar cultures contrasts with the consistent decreases of CRF levels demonstrable in the hypothalamus following glucocorticoid treatment. Makino et al. (13) reported both positive and negative regulation of rat amygdalar CRF mRNA by glucocorticoids in vivo. They revealed that the direction of the regulation depends on the doses used and the time course of administration. It has been demonstrated by Honkaniemi et al. (42) that the CRF amygdalar cells possess glucocorticoid receptors. Despite this, in our experiments no effect of dexamethasone was discernable. It is possible that the results can be accounted for by the limitations of our experimental system. The use of dissociated cultures may lack the relevant neuronal connections that may be required for steroidal regulation of the amygdalar CRF cells. Our results are, however, consistent with those of Beyer et al. (14), who demonstrated that adrenalectomy does not alter the expression of CRF in the amygdala. Of interest is the fact that the CRF promoter lacks a glucocorticoid responsive element (43) yet in the hypothalamus, CRF can be regulated by steroids.

The lack of an effect on CRF with TPA in our cultures contrasts with the increases in CRF messenger RNA and peptide demonstrable in hypothalamic dissociated primary cultures following treatment with activators of the PKC pathway (44, 45). CRF in the placenta, like that demonstrable in the amygdala, also appears not to be regulated with PKC activators (23). It has been demonstrated with transient transfection protocols that phorbol esters can stimulate CRF gene expression in clonal cell lines through the cAMP responsive element (CRE; 46). As with glucocorticoid regulation, it is not clear which tissue specific transcription factors mediate the TPA effect in the hypothalamus.

Like that seen in the placenta and the hypothalamus, the PKA activator forskolin increases CRF expression. This is to be expected given the presence of an 8-bp palindromic CRE in the CRF 5' regulatory region (43). Spengler et al. (47) has provided evidence implicating the CRE in PKA stimulated CRF gene expression.

We and others have investigated the effects of cytokines on neuronal expression of CRF (15, 16). We now extend these studies by demonstrating positive regulation of amygdalar CRF by IL6. It is known that IL6 is able to stimulate the HPA axis at the level of the hypothalamus, pituitary gland, and adrenal gland (24, 25, 26). In the hippocampus and prefrontal cortex, IL6 increases serotonin activity (48), and it has been speculated that this may play a role in the stress response. Furthermore, Raber et al. (11) demonstrated that interleukin 2 (IL2) can regulate secretion of CRF from the amygdala. The IL6 receptor mRNA by in situ hybridization has been localized in the basal state to the hippocampal formation, striatum, medial habenular nucleus, hypothalamus, cortex and olfactory bulb, but not the amygdala (49).

There are several possible explanations that may account for the ability of IL6 to stimulate CRF. It is possible that IL6 may act through a subtype of the receptor that was not detected in the localization studies. It is also possible that the amygdala may not express IL6 receptors in the basal state. However, with inflammation, the receptor may be induced in the amygdala. In fact, there is evidence that tissue injury and inflammation can induce expression of the IL6 receptor in neural tissue (49, 50). To what extent the cell dispersion procedure promotes a biological situation similar to inflammation or injury is unclear.

Increased understanding of the mechanisms regulating CRF expression in the amygdala is relevant because of the implied link between CRF expression there and the behavioral manifestations of stress. The studies in this report demonstrate that amygdalar CRF appears to be positively regulated by the cytokine IL6 as well as activators of the PKA intracellular pathway. Neither PKC activation nor dexamethasone were shown to alter CRF levels in this system. The elucidation of signal transduction mechanisms in the present experiments supports the hypothesis that amygdalar CRF is regulated differently than CRF within the hypothalamus. Furthermore, physiological or pathological stimuli that promote release of IL6 within the central nervous system may promote direct activation of the CRF system within the amygdala, leading to stress related behavioral alterations.


   Acknowledgments
 
We are grateful to Wendel Nicholson and Dr. Jeffrey Mulchahey for their technical help and suggestions.


   Footnotes
 
1 Supported by a National Alliance for Research on Schizophrenia and Depression Young Investigator’s award. Back

2 Recipient of a Veteran’s Administration Merit Review. Back

Received December 9, 1996.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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