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Keywords:

  • Addiction;
  • anxiety;
  • corticotropin-releasing factor;
  • ethanol;
  • protein kinase C;
  • RNA interference

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgments
  8. Supporting Information

Corticotropin-releasing factor (CRF), its receptors, and signaling pathways that regulate CRF expression and responses are areas of intense investigation for new drugs to treat affective disorders. Here, we report that protein kinase C epsilon (PKCɛ) null mutant mice, which show reduced anxiety-like behavior, have reduced levels of CRF messenger RNA and peptide in the amygdala. In primary amygdala neurons, a selective PKCɛ activator, ψɛRACK, increased levels of pro-CRF, whereas reducing PKCɛ levels through RNA interference blocked phorbol ester-stimulated increases in CRF. Local knockdown of amygdala PKCɛ by RNA interference reduced anxiety-like behavior in wild-type mice. Furthermore, local infusion of CRF into the amygdala of PKCɛ−/− mice increased their anxiety-like behavior. These results are consistent with a novel mechanism of PKCɛ control over anxiety-like behavior through regulation of CRF in the amygdala.

Excessive anxiety is a prominent feature of several affective disorders. Most drugs used to treat anxiety act at gamma-aminobutyric acid A (GABAA) or serotonin receptors (Salzman et al. 1993). Despite proven clinical efficacy, GABA modulators such as benzodiazepines can produce undesirable side-effects including incoordination, sedation, amnesia, tolerance and addiction. Moreover, only some patients respond to serotonergic agents, thus limiting their use. Therefore, there is a need to identify new drugs to treat anxiety.

One strategy in the search for new anxiolytic drug targets is to identify genes that modulate anxiety in rodents. Previously, we found reduced anxiety-like behavior in mice lacking protein kinase C epsilon (PKCɛ) on the elevated plus maze and in an open field (Hodge et al. 2002). As PKCɛ−/− mice also show heightened sensitivity to several positive allosteric modulators of GABAA receptors (Hodge et al. 1999), we concluded that their reduced anxiety-like behavior might involve an increased response to endogenous neurosteroids (Hodge et al. 2002). However, because sedation is a major side-effect of anxiolytic drugs (Norman et al. 1997) such as neurosteroids that act at GABAA receptors (Rupprecht 2003), yet PKCɛ−/− mice show normal activity in several behavioral tasks, we considered whether additional mechanisms contribute to reduced anxiety-like behavior in PKCɛ−/− mice.

Corticotropin-releasing factor (CRF), which is highly abundant in the amygdala and in the paraventricular nucleus (PVN) of the hypothalamus (Merchenthaler 1984), is an important regulator of anxiety. Patients suffering from depression or anxiety disorders often have elevated CRF levels in their cerebrospinal fluid (CSF) (Koob & Heinrichs 1999; Reul & Holsboer 2002), similar to high CRF levels found in the CSF of adult nonhuman primates that show chronic anxiety from exposure to early life stress (Coplan et al. 1996). In rats and mice, intracerebral infusion of CRF (Campbell et al. 2004; Nishikawa et al. 2004) and transgenic overexpression of CRF (Stenzel-Poore et al. 1994) increase anxiety-like behavior, whereas CRF1 receptor-deficient mice show reduced anxiety-like behavior (Smith et al. 1998; Timpl et al. 1998). In particular, CRF in the amygdala contributes to anxiety because injection of CRF antagonists and CRF1-receptor antisense oligonucleotides into the amygdala reduce stress-induced anxiety-like behavior in rats (Heinrichs et al. 1992; Liebsch et al. 1995).

Protein kinase C may regulate CRF expression because phorbol esters, which activate several PKC isozymes, increase CRF promoter activity, CRF messenger RNA (mRNA) levels and CRF release in transfected COS-7 cells (Vamvakopoulos & Chrousos 1993), chicken macrophages (Van 1993) and primary fetal rat hypothalamic neurons (Emanuel et al. 1990). Although these findings suggest that PKC positively regulates CRF in vitro, it is not certain if these responses are because of activation of a PKC or another phorbol ester responsive protein (Brose & Rosenmund 2002). In addition, it is not known whether PKC regulates CRF production in vivo, which PKC isozymes are involved or whether PKC stimulation of CRF production regulates behavior. In this study, we addressed these questions by testing the hypothesis that PKCɛ regulates CRF expression in the amygdala, and thereby controls anxiety-like behavior in mice.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgments
  8. Supporting Information

Animals

Experimental animals were 8 to 10-week old, F2 generation male wild-type and PKCɛ−/− mice. The generation of PKCɛ−/− mice has been described previously (Khasar et al. 1999). The null mutation was maintained on the inbred 129S4/SvJae background and PKCɛ+/− 129S4/SvJae mice were periodically crossed with C57BL6/J mice to generate F1 generation PKCɛ+/− C57BL/6J × 129S4/SvJae mice for breeding. These were intercrossed to generate F2 wild-type and PKCɛ−/− littermates for experiments. The mice were maintained on a 12-h light/dark cycle (lights on at 0700 h) and were individually housed after cannulation or lentiviral infusion (described below). Food and water were available ad libitum. The Institutional Animal Care and Use Committee of the Ernest Gallo Clinic and Research Center approved all experimental procedures.

Ribonuclease protection assay for CRF mRNA

Wild-type and PKCɛ−/− mice were killed by CO2 inhalation and their brains were rapidly removed. The PVN and the amygdala were dissected by micropunch (ASI Instruments, Warren, MI, USA) from a 2 mm coronal slice starting at 0.8 mm posterior to bregma (Paxinos & Franklin 2000). Total RNA was isolated with RNA STAT-60 (Tel Test Inc., Friendswood, TX, USA). The CRF riboprobe was generated from a pGem-4Z plasmid (pGem4ZPst578) containing a 578 bp PstI fragment of exon II of the mouse CRF gene that was kindly provided by Dr A. Seasholtz, University of Michigan (Seasholtz et al. 1991). This plasmid was linearized with HindIII for preparation of an [α-32P]CTP-labeled, 583 bp CRF riboprobe with SP6 polymerase. The plasmid pTRI-GAPDH rat, which recognizes glyceraldehyde 3-phosphate dehydrogenase mRNA from rats and mice, was purchased from Ambion (Austin, TX, USA), and used with T7 RNA polymerase to generate a [α-32P]CTP-labeled 316 bp probe as a control. Ribonuclease protection assays (RPAs) were performed as described previously (Walter et al. 2000) with 60 000 counts per minute (c.p.m.) of the CRF complementary RNA (cRNA) probe and 10 000 c.p.m. of the GAPDH cRNA probe. CRF mRNA levels were normalized to GAPDH mRNA in each sample. GAPDH mRNA levels were not different between PKCɛ−/− and wild-type mice; direct comparison of phosphorimaging data (in arbitrary units) for GAPDH mRNA in amygdala samples from both genotypes by t-test showed no significant difference (P = 0.85) between wild-type (7084 ± 1752; n = 9) and PKCɛ−/− samples (7582 ± 1930; n = 8).

Radioimmunoassay for CRF peptide

Wild-type and PKCɛ−/− mice were killed by CO2 inhalation and brains were rapidly removed. The amygdala was dissected bilaterally from a coronal slice 0.8–1.8 mm posterior to bregma (Paxinos & Franklin 2000) by a vertical cut tangential to the external capsule and a diagonal cut along the medial border of the ipsilateral optic tract (Raber et al. 1997). The samples were boiled in 2 n acetic acid, homogenized by sonication, lyophilized and frozen at −80°C. The lyophilized samples were resuspended in radioimmunoassay (RIA) buffer. Protein concentration was determined by a Bradford assay and CRF protein was determined using a commercial RIA kit (Phoenix Pharmaceuticals, Belmont, CA, USA). Data were expressed as pg CRF/mg protein.

Western analysis

Pro-CRF was measured as described (Meloni et al. 2005) using a rabbit polyclonal anti-CRF antibody (1:1000; Chemicon, Temecula, CA, USA). Protein kinase C ɛ protein levels were determined using rabbit polyclonal anti-PKCɛ SN134 (1:1000) (Choi et al. 2002). CRF-binding protein (CBP) was measured using a rabbit polyclonal anti-CBP antibody (1:500; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and other PKC isozymes were measured using rabbit polyclonal antibodies specific for each isozyme (1:500; Santa Cruz Biotechnology), or in the case of PKC-γ a mouse monoclonal anti-PKC-γ antibody (1:1000; BD Transduction Labs, Lexington, KY, USA). Unless otherwise specified, 40 μg protein samples were analyzed. Following incubation with primary antibody, blots were incubated with either goat anti-rabbit-peroxidase (1:1000; Chemicon) or rabbit anti-mouse-peroxidase (1:500; Chemicon) and protein bands were visualized by enhanced chemiluminescence (Pierce Biotechnology, Rockford, IL, USA) according to the manufacturer’s instructions. Optical density was measured using Image J software v. 1.31 (Abramoff et al. 2004). Optical densities were normalized to the density of protein bands at 150 kDa measured using a gel of samples run concurrently and stained with Coomassie blue. In pilot studies, the optical density of this protein band did not vary between different conditions.

CRF receptor-binding autoradiography

Autoradiography was performed as described (Sanchez et al. 1999) using 16 μm sections mounted on gelatin-coated slides and incubated with 200 μl of 0.2 nm [125I]Tyr0-sauvagine (Perkin Elmer, Boston, MA, USA) in phosphate-buffered saline (PBS) with 10 mm MgCl2, 2 mm ethyleneglycoltetraacetic acid, 0.15% bovine serum albumin, 0.15 mm bacitracin and 1.5% (w/v) aprotinin to detect total CRF receptor binding. This concentration of [125I]Tyr0-sauvagine was chosen because it approximates its Kd value for binding to CRF1 receptors (Sanchez et al. 1999). Adjacent sections were incubated with 0.2 nm [125I]Tyr0-sauvagine in the presence of 2 μm of the specific CRF1 receptor antagonist NBI 29714 (Tocris, Ellisville, MO, USA) (Chen et al. 1996). To determine non-specific binding of [125I]Tyr0-sauvagine, a third series of adjacent sections was incubated with 0.2 nm [125I]Tyr0-sauvagine in the presence of 2 μm CRF, which binds to both CRF1 and CRF2 receptors. After 2-h incubation at 22°C, the slides were dipped quickly in 4°C assay buffer five times, washed in 0.01% triton in PBS for 10 min and dipped in deionized water five times. The slides were dried overnight and apposed to Kodak Biomax MR film with [125I] microscales (Amersham, Piscataway, NJ, USA) for 3 days. Binding was quantified by densitometric scanning of autoradiograms using Image J software (Abramoff et al. 2004). Specific CRF1 receptor binding was calculated by subtraction of [125I]Tyr0-sauvagine binding in the presence of the CRF1 receptor antagonist NBI 27941 (2 μm) from total [125I]Tyr0-sauvagine binding. Specific CRF2 receptor binding was calculated by subtraction of non-specific binding ([125I]Tyr0-sauvagine in the presence of 2 μm CRF) from [125I]Tyr0-sauvagine binding in the presence of 2 μm NBI 29714.

Primary culture of amygdala neurons

P1 wild-type mouse pups were killed by decapitation. The brains were removed, placed ventral side up and a coronal slice was prepared by cutting anterior and posterior to the diencephalon. A diagonal cut was then made along the lateral fissure, a horizontal cut was made just below the fimbria of the hippocampus and the amygdala was dissected out by peeling away the cortex for preparation of primary amygdala cultures as described (Kasckow et al. 1997). Approximately 2 × 105 cells, obtained from two to three pups, were plated on gelatin and polyornithine-coated 60 mm plates. After 24 h, the medium (1:1 mixture of Dulbecco’s modified Eagle’s minimal essential medium and Hams F-12, supplemented with 7.5% fetal calf serum, 14 mm glucose, 15 mm NaHCO3, 5 mm HEPES and 0.05 U/ml each of penicillin and streptomycin) was replaced with fresh medium containing 20 μm cytosine arabinoside to limit proliferation of glial cells. Primary amygdala neurons were cultured at 37°C in a humidified atmosphere of 94% air:6% CO2.

Four days after preparation of the primary amygdala cultures, neurons were infected with either control or PKCɛ short hairpin RNA (shRNA) lentivirus (see below) at 1 pg p24 antigen per neuron (approximately 1 infectious unit). Four days later, the cultures were randomly assigned to one of four treatment groups: no treatment, 30 nm phorbol-12-myristate 13-acetate (PMA), 500 nm tat or 500 nm tat-ψɛRACK. Pseudo-ɛRACK (ψɛRACK) is a peptide activator of PKCɛ (Dorn et al. 1999); the tat peptide transport sequence facilitates peptide entry into cells (Inagaki et al. 2005) and was a gift from D. Mochly-Rosen (Stanford University, Stanford, CA, USA). The tat-ψɛRACK peptide was synthesized by Synpep Corp. (Dublin, CA, USA) and its purity was 83% as measured by AnaSpec (San Jose, CA, USA). All cultures were incubated in triplicate at 37°C for the indicated times and pro-CRF and PKCɛ immunoreactivities were then measured by Western blot analysis.

CRF immunofluorescence

P1 pup brains were fixed in 4% paraformaldehyde for 48 h. After overnight incubation in 12% gelatin, brains were embedded in 5% agar and 60 μm sections were cut on a Vibratome (Leica Micro-systems, Wetzlar, Germany). CRF immunoreactivity was detected using a rabbit anti-CRF antibody (1:25 000; Peninsula Laboratories, San Carlos, CA, USA), biotin-conjugated donkey anti-rabbit secondary antibody (1:300; Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA) and a TSA amplification kit (Invitrogen Corp., Carlsbad, CA, USA). Sections were mounted on gelatinized slides, air-dried, covered with Vectashield (Vector Laboratories, Burlingame, CA, USA) mounting medium and examined for fluorescein isothiocyanate (FITC) signal. Further details are provided in Appendix S1.

RNA interference

We designed three shRNAs that contain the following sequences, which are designated by the number of the first base in the mouse PKCɛ mRNA sequence (NCBI accession no. NM_011104): 1141 (GCGUCGUCGGCCACCGAUG), 1845 (GGACGACUUGUUCGAAUCC) and 2125 (GAGCCAAUACUUACACUUG). As a control, we used a sequence that did not recognize any known mammalian gene in a BLAST search (GCGCUUAGCUGUAGGAUUC). The sequences were incorporated into a LentiLox 3.7 vector [pLL3.7, L. van Parijs, MIT (Rubinson et al. 2003)]. The vector map and links to the sequence can be found at: http://www.sciencegateway.org/protocols/lentivirus/pllmap.html. Lentivirus was produced using a Virapower Kit according to the manufacturer’s manual (Invitrogen). Viral titers, determined by p24 enzyme-linked immunosorbent assay (Zeptometrix, Buffalo, NY, USA) were approximately 80 × 106 pg/ml for viruses encoding control and PKCɛ shRNAs. Knockdown efficiency was determined by infection of Neuro2A cells at 1 pg p24 antigen per neuron (approximately 1 infectious unit). Protein kinase Cɛ protein levels were determined by semiquantitative Western blot 48 h after infection. This was performed by loading 5, 10 and 20 μg of protein for each condition onto polyacrylamide gels and separating proteins by sodium dodecyl sulphate–polyacrylamide gel electrophoresis. Proteins were then transferred to nitrocellulose membranes, which were incubated with a rabbit polyclonal anti-PKCɛ SN134 (Choi et al. 2002) (1:1000 dilution) followed by goat anti-rabbit horseradish peroxidase (Chemicon; 1:1000) and detection by enhanced chemiluminescence. Immunoreactive bands were quantified by scanning densitometry and the slope of the line determined by the optical density at each concentration of sample was used as the measure of subunit immunoreactivity to determine knockdown efficiency.

Intra-amygdala infusion of lentivirus

Mice were anesthetized with xylazine (7 mg/kg i.p.) and ketamine (100 mg/kg i.p.), placed in a digital stereotaxic alignment system (Model 1900; David Kopf Instruments, Tujunga, CA, USA) and positioned with bregma at the focal point. After the skull was leveled, 0.28 mm diameter holes were drilled for microinjections. The injectors (33 gauge, 0.20 mm outside diameter) were then lowered to target the central nucleus of the amygdala using the coordinates −0.85 mm posterior from bregma, ±3.1 mm lateral to midline and −4.9 mm ventral from bregma. Lentivirus (2 μl at a concentration of 80 × 106 pg/ml p24) was infused bilaterally at a rate of 0.2 μl/min. Pilot experiments showed that this treatment caused infection throughout the central nucleus of the amygdala with minor infection in the basolateral amygdala and along the injection tract.

In order to validate in vivo knockdown of PKCɛ, immunofluorescence for PKCɛ and green fluorescence protein (GFP) was performed on coronal sections from mice in which one amygdala was injected with the control virus, and the contralateral amygdala with PKCɛ shRNA. Briefly, coronal sections were incubated in a mixture of the goat polyclonal anti-PKCɛ antibody (1:2500; Santa Cruz Biotechnology) and mouse monoclonal anti-GFP antibody (3E6, 1:1500; Invitrogen), washed and incubated for 2 h in secondary antibodies: donkey anti-goat Texas Red and donkey anti-mouse FITC (both at 1:250; Jackson ImmunoResearch). Sections were scanned using an LSM 510 laser confocal microscope and images were analyzed using SigmaScan Pro v. 5.0 software (SPSS Inc., Chicago, IL, USA). GFP-positive cells were measured for the intensity of PKCɛ immunoreactivity. Results for each mouse are presented as PKCɛ immunoreactivity in each amygdala normalized to the mean value for immunoreactivity measured in control shRNA-infected neurons in the same mouse. Further details are provided in Appendix S1.

For subsequent behavioral experiments, mice were injected bilaterally with either control or PKCɛ shRNA expressing lentivirus. Injection sites were verified with immunoperoxidase staining for GFP using a monoclonal anti-GFP antibody (3E6, 1:1500; Invitrogen) followed by incubation with a biotin-conjugated donkey anti-mouse secondary antibody (1:300; Jackson ImmunoResearch). Peroxidase was detected using diaminobenzidine (Sigma, St Louis, MO, USA). Sections were mounted on gelatinated slides, air-dried and counterstained with cresyl-violet. Infection sites were then assessed for individual animals with the mouse brain atlas (Paxinos & Franklin 2000) as a reference for the borders of the amygdala. Only mice with bilateral infection in the amygdala were included in the analysis. Further details are provided in Appendix S1.

Anxiety-like behavior

Two weeks after bilateral infusion of lentivirus to express control or PKCɛ shRNA, anxiety-like behavior was assessed using an open field and the elevated plus maze as described in prior work (Hodge et al. 2002). For open-field tests, locomotor activity was measured in 43 × 43 cm chambers. Post hoc, the chamber floor was divided into center and periphery zones. Distance traveled and time spent in the center zone (21 × 21 cm; center equidistant from all four walls of the chamber) and the periphery zone (the remaining area of the floor) were calculated from the locomotor activity data. For the elevated plus maze task, the mice were placed on the central platform facing the open arm and allowed to explore the maze for 10 min. Ethovision software v. 3.08 (Noldus Information Technology, Leesburg, VA, USA) was used for behavioral analysis.

CRF diffusion in the amygdala

To assess the extent of diffusion of injected CRF, mice were injected with CRF that was labeled with FITC using an EZ-label kit (Pierce Biotechnology) as described (Sahuque et al. 2006). The FITC-labeled CRF solution was dried under nitrogen and resuspended in sterile saline to a final concentration of 100 ng/μl, and 100 ng of labeled CRF (Matys et al. 2004) were microinjected into the amygdala at a rate of 1 μl over 10 min. Mice were killed and perfused with 4% paraformaldehyde 10 min after microinjection. Brains were post-fixed overnight in 4% paraformaldehyde and then cut in sections, which were examined by fluorescence microscopy. Images were made using an Axioplan-2 microscope and Axiocam HR CCD camera (Carl Zeiss Micro Imaging Inc., Thornwood, NY, USA) as described above.

Intra-amygdala microinjection of CRF

For bilateral cannulation of the amygdala, 21 PKCɛ−/− mice (11 saline and 10 CRF) were anesthetized with xylazine (7 mg/kg i.p.) and ketamine (100 mg/kg i.p) and were placed in a digital stereotaxic alignment system (Model 1900; David Kopf Instruments). After the skull was exposed, the mouse was positioned with bregma at the focal point and the skull was leveled using a dual tilt measurement tool. At the desired coordinates (−0.85 mm posterior from bregma and ±3 mm lateral to midline) 0.5 mm holes were drilled for guide cannula placement. Two guide cannulae (26 gauge; Plastics One, Roanoke, VA, USA) were lowered to terminate 0.5 mm dorsal to each central nucleus of the amygdala (−4.9 mm ventral from bregma). The cannulae were secured to the skull with dental cement (Jet Repair; Henry Schein, Melville, NY, USA) and a screw for support. After surgery, dummy cannulae were inserted and the mice were allowed to recover for at least 1 week before experiments commenced.

For injection, dummy cannulae were removed and sterile 33-gauge injectors were inserted bilaterally to a depth 0.5 mm below the tip of the guide cannulae. CRF (human/rat; Tocris), dissolved in sterile saline at a concentration of 100 ng/μl, was infused at 0.1 μl/min per hemisphere over 10 min (Castane et al. 2004; Matys et al. 2004; Momose et al. 1999). Control mice received a saline injection (1 μl/hemisphere). After 5 min had elapsed to permit diffusion of the drug, the injectors were removed and the mice were immediately placed on the elevated plus maze. Post-mortem histological analysis was performed to verify cannula placement according to the mouse brain atlas (Paxinos & Franklin 2000). Only mice with bilateral amygdala cannula placement were included in the analysis.

Statistical analysis

All results are shown as mean ± SEM values. Data were tested for normality using the Kolmogorov–Smirnov test. For normally distributed data, mean values were compared by either two-tailed, unpaired t-tests or anova with post hoc Tukey tests, and differences between pairs of means were considered significant with α = 0.05. Data for downregulation of amygdala PKCɛin vivo by RNA interference were not normally distributed and therefore pairs of means were compared using Mann–Whitney rank sum tests.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgments
  8. Supporting Information

Reduced CRF levels in the amygdala of PKCɛ−/− mice

As amygdala CRF regulates anxiety-like behavior, we investigated whether CRF levels are altered in the central nervous system of PKCɛ−/− mice. CRF mRNA abundance, quantified by RPA, was reduced by 50% (P = 0.032, df = 15, two-tailed t-test) in the amygdala of PKCɛ−/− mice compared with wild-type littermates (Fig. 1a). In contrast, CRF mRNA in the PVN of the hypothalamus, the other major site of CRF expression in the brain, was not different between genotypes (Fig. 1b). CRF protein levels in the amygdala of PKCɛ−/− mice, as determined by Western blot analysis of the CRF precursor pro-CRF (Fig. 1c), were also reduced by 50% (P = 0.0097, df = 7, two-tailed t-test), while there was no difference by genotype in pro-CRF immunoreactivity in the PVN (Fig. 1d). Furthermore, RIA showed a 58% reduction (P = 0.0279, df = 24, two-tailed t-test) in CRF peptide in the amygdala of PKCɛ−/− mice (0.085 ± 0.013 pg/mg; n = 12) compared with wild-type mice (0.201 ± 0.039 pg/mg; n = 14). As availability of CRF at CRF receptors is modulated by CBP, we also measured levels of CBP in amygdala samples from wild-type and PKCɛ−/− mice. There was no difference between genotypes in CBP abundance by Western blot analysis (Fig. 1e). These data indicate that PKCɛ regulates CRF levels in the amygdala.

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Figure 1. CRF mRNA abundance and protein levels are reduced in the amygdala of PKCɛ−/− mice. In each panel, individual experiments are shown on the left and mean ± SEM values from all experiments are shown in bar graphs to the right. (a) RPA showing reduced CRF mRNA abundance in the amygdala of PKCɛ−/− mice (K; n = 8) compared with wild-type mice (W; n = 9). U, undigested probe; D, digested probe. (b) CRF mRNA levels were similar by RPA in the PVN of wild-type (n = 9) and PKCɛ−/− (n = 8) mice. (c) Western analysis showed reduced pro-CRF immunoreactivity in the amygdala of PKCɛ−/− mice (n = 4) compared with wild-type mice (n = 5). (d) Pro-CRF levels in the PVN were similar in both genotypes (n = 7). (e) Levels of CBP were similar in the amygdala of PKCɛ−/− (n = 4) and wild-type mice (n = 4). *P < 0.05 by two-tailed, unpaired t-tests.

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Amygdala CRF receptor levels are not altered in PKCɛ−/− mice

Pharmacological studies have implicated PKC in both stimulation and inhibition of CRF1 receptor expression in CATH.a and human myometrial cells (Iredale et al. 1997; Parham et al. 2004). Given the possibility that PKCɛ might regulate levels of CRF1 receptors in the brain, we quantified CRF1-receptor-binding sites in PKCɛ−/− and wild-type mice by receptor-binding autoradiography using [125I]Tyr0-sauvagine, which binds to both CRF1 and CRF2 receptors, in the absence or presence of the selective CRF1 antagonist NBI 29714. We found that the distribution of [125I]Tyr0-sauvagine binding in the brain (Fig. 2), and the amount of specific binding to CRF1 (Fig. 2a) and to CRF2 (Fig. 2b) receptors in the amygdala were similar in PKCɛ−/− and wild-type mice. Therefore, absence of PKCɛ does not alter CRF receptor binding in the amygdala.

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Figure 2. CRF1 receptor binding is not altered in the amygdala of PKCɛ−/− mice. Results are from six wild-type and six PKCɛ−/− mice. (a) Representative autoradiograms of brain sections from wild-type (+/+) and PKCɛ−/− (−/−) mice for total binding of [125I]Tyr0-sauvagine, binding in the presence of the CRF1 receptor antagonist NBI 27941 and non-specific binding in presence of CRF. (b) Specific binding to CRF1 receptors in the amygdala was similar for wild-type and PKCɛ−/− mice. (c) Specific binding to CRF2 receptors in the amygdala was also similar among the genotypes. *P > 0.05 by two-tailed, unpaired t-tests.

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Expression of PKC isozymes in amygdala of PKCɛ−/− mice

The PKCɛ−/− mice lack PKCɛ in all tissues and throughout development. Therefore, reduced levels of amygdala CRF in PKCɛ−/− mice could be because of developmental abnormalities, indirect effects of PKCɛ deficiency in other brain regions or endocrine tissues, compensatory changes in other PKC isozymes in the amygdala or absence of PKCɛ signaling in the adult amygdala. To screen for changes in other PKC isozymes, we first investigated whether the abundance of other PKC isozymes was altered in the amygdala of PKCɛ−/− mice. Using Western blot analysis, we found no differences in immunoreactivity for PKC-α, βI, βII, γ, δ, λ or ζ in wild-type and PKCɛ−/− samples (see Figure S1). Protein kinase Cη and PKCθ could not be detected in the amygdala of either genotype (not shown).

Knockdown of amygdala PKCɛ by RNA interference

We next determined if PKCɛ can regulate CRF within amygdala neurons and if the low-anxiety phenotype in PKCɛ−/− mice is because of a local deficiency of amygdala PKCɛ. For this purpose, we designed three shRNAs for specific and local reduction of PKCɛ expression in wild-type neurons by RNA interference. A sequence that did not recognize any known mammalian gene in a BLAST search was used as a control. These constructs were incorporated into a pLentiLox 3.7 vector (Rubinson et al. 2003) and lentivirus was produced to allow long-lasting PKCɛ knockdown. In vitro, two of the shRNAs reduced PKCɛ in Neuro2A cells and one did not (Fig. 3a). Semiquantitative western analysis from three separate experiments showed 45.7 ± 13% knockdown after infection with the 1845 PKCɛ shRNA expressing lentivirus compared with untreated cells, while the 2125 PKCɛ shRNA reduced PKCɛ levels by 26.0 ± 6%. In vivo, 51–62% knockdown of PKCɛ in amygdala neurons was achieved after infection with the 1845 PKCɛ shRNA expressing lentivirus and 2125 PKCɛ shRNA reduced amygdala PKCɛ immunoreactivity by 42–54% (Fig. 3b,c). Analysis of these data by Mann–Whitney rank sum tests showed significant knockdown of PKCɛ by these shRNAs in all mice (P < 0.001). The most effective PKCɛ shRNA (1845) was chosen for experiments.

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Figure 3. Knockdown of amygdala PKCɛ by RNA interference. (a) Decreased PKCɛ immunoreactivity 48 h after infection of Neuro2A cells with lentiviruses encoding 2125 or 1845 shRNA; control and 1141 shRNAs had no effect. (b) In control shRNA-infected amygdala, GFP (infected neuron) and PKCɛ immunoreactivity were often observed in the same neuron (arrowheads). By contrast, PKCɛ immunoreactivity was absent in most 1845 shRNA-infected neurons in the contralateral amygdala (arrows). Non-infected neurons expressed PKCɛ in both the control-infused and 1845 PKCɛ-infused amygdala (asterisks). Scale bar = 50 μm. (c) Two weeks after amygdala infection, analysis of PKCɛ immunoreactivity showed knockdown of PKCɛ in 1845 PKCɛ-infected neurons (n = 54 in mouse 1; 69 in mouse 2) and 2125 PKCɛ-infected neurons (n = 82 in mouse 3; 94 in mouse 4). Results are expressed relative to PKCɛ immunoreactivity in approximately 75 control shRNA-infected neurons in the contralateral amygdala of each individual mouse. *P < 0.001 by Mann–Whitney rank sum tests.

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PKCɛ regulates CRF levels in primary amygdala neurons

As PKCɛ−/− mice show reduced levels of CRF in the amygdala and PKCɛ is expressed throughout the amygdala (Choi et al. 2002), we predicted that PKCɛ regulates levels of CRF within amygdala neurons. To test this hypothesis, we measured levels of pro-CRF in amygdala neurons cultured from P1 mouse pups. Immunofluorescence staining of brain slices from these pups showed CRF-like immunoreactivity in the amygdala and paraventricular nucleus of the hypothalamus (Fig. 4a). Treatment with 30 nm PMA increased pro-CRF immunoreactivity in cultured amygdala neurons with a maximal 1.6-fold increase observed after 60 min (Fig. 4b). Infection of the cultures with lentivirus to express 1845 PKCɛ shRNA against PKCɛ prevented PMA-induced increases in pro-CRF immunoreactivity, whereas a control shRNA had no effect (Fig. 4c). One-way anova showed an effect of shRNA treatment (F2,8 = 8.8, P = 0.0095). Tukey post hoc analysis showed that pro-CRF levels were significantly (P < 0.05) lower in the cells infected with the 1845 PKCɛ shRNA lentivirus compared with untreated neurons or those infected with the control lentivirus. Western analysis showed that the 1845 PKCɛ shRNA decreased PKCɛ levels by 55.5 ± 3.2% in the cultures (Fig. 4d). To confirm that CRF levels are regulated by PKCɛ, we also treated neurons with a peptide activator of PKCɛ, the pseudo-ɛRACK (ψɛRACK) peptide conjugated to the tat47–57 peptide transport sequence, which facilitates peptide entry into cells. One-way anova indicated an effect of peptide treatment (F2,23 = 11.87, P = 0.0004). Tukey post hoc analysis showed that treatment with 500 nm tat-ψɛRACK for 60 min increased pro-CRF by 1.5-fold when compared with untreated control neurons (P < 0.05), while the tat47–57 peptide alone did not alter levels of pro-CRF (Fig. 4e). Taken together, these experiments show that endogenous PKCɛ regulates CRF levels within amygdala neurons.

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Figure 4. PKCɛ regulates CRF in primary amygdala neurons. (a) CRF-like immunoreactivity was detected in the central amygdala (arrows) and the paraventricular nucleus of the hypothalamus (arrowheads) in brain slices from P1 mouse pups. Scale bar = 1 mm. (b) Western blot showing increased pro-CRF immunoreactivity in amygdala neurons after treatment with 30 nm PMA with a maximal increase observed after 60 min. (c) PMA induced a 1.6-fold increase in pro-CRF in neurons that were not infected (n = 5) or were infected with control shRNA lentivirus (n = 3), but not in 1845 PKCɛ shRNA-infected neurons (n = 3). *P < 0.05 compared with PMA or PMA + control shRNA by Tukey test. (d) Representative Western blot showing reduced PKCɛ immunoreactivity in primary amygdala neurons after infection with 1845 PKCɛ shRNA lentivirus when compared with control shRNA-infected neurons. (e) Tat-ψɛRACK increased pro-CRF in primary amygdala neurons, while the tat peptide alone had no effect on pro-CRF immunoreactivity (n = 8 for each condition). *P < 0.01 compared with untreated control or tat by Tukey test.

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Knockdown of amygdala PKCɛ reduces anxiety-like behavior

In addition to reduced levels of amygdala CRF, PKCɛ−/− mice show reduced anxiety-like behavior (Hodge et al. 2002). If this behavior is also because of absence of PKCɛ in the adult amygdala, then local knockdown of PKCɛ in the amygdala of adult wild-type mice should reduce their anxiety-like behavior and mimic the PKCɛ−/− phenotype. To test this hypothesis, we injected virus encoding control or 1845 PKCɛ shRNA bilaterally into the amygdala of male wild-type mice (Fig. 5a). Two weeks after infection, mice were tested for anxiety-like behavior. The total distance traveled in the open field was similar in both treatment groups (Fig. 5b). However, mice treated with 1845 PKCɛ shRNA traveled a greater distance in the center of the open field (Fig. 5c; P < 0.05, df = 27, two-tailed t-test) and spent more time in the center zone of the open field (Fig. 5d; P < 0.05, df = 27, two-tailed t-test) when compared with control shRNA-treated animals. In an elevated plus maze task, mice treated with the 1845 PKCɛ shRNA spent more time on the open arms (Fig. 5e; P < 0.05, df = 39, two-tailed t-test) and entered the open arms more frequently (Fig. 5f; P < 0.05, df = 39, two-tailed t-test) than mice treated with the control shRNA. Treatment with the 1845 PKCɛ shRNA did not alter locomotor activity, as shown by the similar number of closed arm entries in mice treated with the control shRNA and mice treated with the 1845 PKCɛ shRNA (Fig. 5g). Bilateral infection in the amygdala was confirmed by post-mortem histological examination. These results indicate that PKCɛ signaling in the adult amygdala regulates anxiety-like behavior in mice.

image

Figure 5. Knockdown of amygdala PKCɛ reduces anxiety-like behavior. (a) Nissl-stained section showing lentiviral infection (GFP-immunoreactivity, brown) in the central amygdala and to a lesser extent in the basolateral amygdala and along the needle track in striatum. Scale bar = 500 μm. (b) The total distance moved in an open-field chamber was similar in mice treated with the 1845 PKCɛ (n = 15) lentivirus and the control shRNA lentivirus (n = 14). (c,d) Mice treated with 1845 PKCɛ lentivirus traveled a greater distance in the center zone of the open field. (e) Mice treated with 1845 PKCɛ shRNA lentivirus (n = 20) spent a greater percentage of total time in the open arms compared with mice treated with the control lentivirus (n = 21). (f) Mice treated with 1845 PKCɛ shRNA lentivirus also made a greater percentage of arm entries into the open arms compared with control shRNA-treated mice. (g) Local PKCɛ knockdown did not alter the number of closed arm entries. *P < 0.05 by two-tailed, unpaired t-test.

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Amygdala CRF infusion increases anxiety-like behavior in PKCɛ−/− mice

Having shown that amygdala PKCɛ controls anxiety-like behavior and that PKCɛ regulates CRF within amygdala neurons, we next investigated whether decreased levels of amygdala CRF contribute to the reduced anxiety-like behavior in PKCɛ−/− mice. If true, then infusion of CRF into the amygdala of PKCɛ−/− mice should increase their anxiety-like behavior. Using 1 μl FITC-labeled CRF, we determined that infusion of 100 ng CRF in 1 μl at 0.1 μl/min deposited CRF in the amygdala without diffusion outside this region (Fig. 6a). We then examined the effect of CRF infusion on anxiety-like behavior using the elevated plus maze and afterwards confirmed bilateral placement of the infusion cannulae into the amygdala by post-mortem histological examination (Fig. 6b). CRF infusion increased anxiety-like behavior in PKCɛ−/− mice as shown by a reduction in the percentage of time spent in the open arms (P < 0.05, df = 19, two-tailed t-test) and a decrease in the percentage of total arm entries (P < 0.05, df = 19, two-tailed t-test) into the open arms of the maze (Fig. 6c). CRF did not alter locomotor activity in PKCɛ−/− mice because the number of closed arm entries was similar in saline-treated and CRF-treated groups (Fig. 6c). These findings show that infusion of CRF into the amygdala can increase anxiety-like behavior in PKCɛ−/− mice, which supports our hypothesis that reduced levels of amygdala CRF contribute to the low-anxiety phenotype of PKCɛ−/− mice.

image

Figure 6. Bilateral infusion of CRF in the amygdala increases anxiety-like behavior in PKCɛ−/− mice. The results shown are from 21 PKCɛ−/− mice (11 treated with saline and 10 treated with 100 ng CRF). (a) Representative fluorescence micrograph of a coronal section showing the extent of CRF diffusion in the amygdala after injection of FITC-labeled CRF at 0.1 μl/min for 10 min. Scale bar = 1 mm. CeA, central amygdala; BLA, basolateral amygdala. (b) Schematic of guide cannulae placements in the amygdala for local injections of saline (▵) or CRF (▴), mapped in Nissl-stained sections in reference to the mouse Brain atlas (Paxinos & Franklin 2000). The injectors extended 0.5 mm below the tips of the guide cannulae. (c) Intra-amygdala infusion of CRF in PKCɛ−/− mice decreased the percentage of open-arm entries and the percentage of time spent in the open arms, without altering the number of closed arm entries. *P < 0.05 compared with saline-treated mice by two-tailed, t-tests.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgments
  8. Supporting Information

In previous work, we found that knockout mice lacking PKCɛ show reduced anxiety-like behavior (Hodge et al. 2002). Although this suggested that PKCɛ inhibitors might be anxiolytic in adult animals, there remained the strong possibility that the effects of the PKCɛ null mutation on anxiety were developmental and not because of loss of PKCɛ signaling in adult neurons. Such a situation has been shown for mice that lack serotonin 5HT1A receptors; these mice show increased anxiety-like behavior only when the gene is silent during fetal and early postnatal development, and this phenotype persists even after gene expression is restored during adulthood (Gross et al. 2002). By using RNA interference in our present study, we induced knockdown of PKCɛ in the amygdala of adult wild-type mice and were able to phenocopy the low-anxiety phenotype of PKCɛ−/− mice. This result confirms that PKCɛ signaling in the adult brain regulates anxiety-like behavior. Furthermore, because the expression of the shRNA was restricted to the amygdala, our results indicate that PKCɛ signaling specifically in the amygdala regulates anxiety-like behavior in adult mice.

Protein kinase C control of CRF release has been studied previously (Emanuel et al. 1990; Vamvakopoulos & Chrousos 1993; Van 1993) but little is known about PKC isozyme-specific effects. Here we provide the first evidence for PKCɛ-specific regulation of CRF in the amygdala. First, we showed that CRF mRNA and peptide are reduced in the amygdala of PKCɛ−/− mice, without changes in CRF receptor binding or levels of CBP. Second, we found that the abundance of other PKC isozymes is not altered in the amygdala of PKCɛ−/− mice (Figure S1), suggesting that the observed change in amygdala CRF in PKCɛ−/− mice is not related to altered expression of a different PKC isozyme. Third, we used primary cultures of amygdala neurons to show that PKCɛ regulates CRF within these neurons. Activation of PKCɛ using a selective activator, ψɛ-RACK (Dorn et al. 1999), increased pro-CRF levels in amygdala neurons, whereas phorbol-ester-induced increases in pro-CRF were completely blocked after PKCɛ knockdown by RNA interference. These results indicate that PKCɛ is the major, if not the sole PKC isozyme that regulates CRF levels in amygdala neurons.

It is not yet known how PKCɛ regulates CRF levels. The phorbol ester PMA increases CRF promoter activity in a luciferase assay (Kasckow et al. 1997), suggesting that a conventional or novel PKC can regulate CRF expression through activation of a phorbol ester responsive element in the CRF promoter. Li et al. (2000) reported that PKCɛ activates nuclear factor kappa B and (activator protein) AP-1 in cardiomyocytes. Because phorbol esters can activate gene expression through the AP-1 transcription factor and there is an AP-1 site in the CRF promoter, PKC regulation of AP-1-mediated transcription provides a potential mechanism by which PKCɛ could stimulate CRF expression. More detailed studies are required to resolve the molecular mechanisms that underlie PKCɛ regulation of amygdala CRF.

Although PKCɛ is expressed in both the amygdala (Saito et al. 1993) and the hypothalamus (De et al. 2002), we found that PKCɛ regulates CRF mRNA and protein only in the amygdala. Reduced CRF in the PVN of PKCɛ−/− mice might have been anticipated because we previously observed reduced morning plasma corticosterone and adrenocorticotrophic hormone levels in PKCɛ−/− mice (Hodge et al. 2002). However, PKCɛ−/− mice show a normal increase in plasma corticosterone in response to stress or in response to systemic injection of CRF or metyrapone, indicating that the hypothalamic–pituitary–adrenal (HPA) axis functions normally at the level of the PVN in these mice. These prior results are therefore consistent with our present findings showing that levels of CRF mRNA and pro-CRF in the PVN are normal in PKCɛ−/− mice.

Our findings in the present study indicate tissue specificity in CRF regulation by PKCɛ. Although not previously reported for PKC, tissue-specific CRF regulation is well described for glucocorticoids. In the PVN, glucocorticoids inhibit CRF gene expression, while they increase or have no effect on CRF expression in the amygdala, and stimulate CRF gene expression in the placenta and in the bed nucleus of the stria terminalis (King et al. 2002). These differential effects of glucocorticoids are thought to involve tissue-specific expression or regulation of transcription factors (Burbach 2002; King et al. 2002). Analogous mechanisms may explain amygdala-specific regulation of CRF by PKCɛ indicated by our results. Further study of the mechanisms involved in PKCɛ regulation of CRF expression may provide important information about tissue-specific regulation of CRF.

There is little information available about brain region-specific effects of CRF on behavior. Apart from reduced exploratory behavior after local infusion of CRF into the amygdala (Liang & Lee 1988), little is known about the effect of exogenous CRF infused into the amygdala on anxiety-like behavior. Infusion of CRF antagonists or CRF1-receptor antisense oligonucleotides into the amygdala reduces stress-induced, anxiety-like behavior in rats (Heinrichs et al. 1992; Liebsch et al. 1995). In addition, induction of CRF1 receptor deficiency in limbic brain regions through conditional gene targeting reduces anxiety-like behavior (Muller et al. 2003). Thus, CRF1 receptor activity in the amygdala can modulate anxiety-like behavior. Our findings in PKCɛ−/− mice show that, in the setting of amygdala CRF deficiency, local infusion of CRF into the amygdala can increase anxiety-like behavior. This result is consistent with our hypothesis that PKCɛ regulation of amygdala CRF contributes to PKCɛ modulation of anxiety.

Currently, there is widespread interest in the development of CRF1 receptor antagonists for the treatment of anxiety disorders (Holmes et al. 2003). CRF1 antagonists do not appear to suppress baseline functioning of the HPA axis, but they can reduce its activation in response to stress (Habib et al. 2000). While the effect of CRF1 antagonists on the stress response may not be detrimental to healthy individuals, it could be harmful under conditions of severe physical stress, for example, in critically ill patients where an inadequate HPA response contributes to hypotension (Marik & Zaloga 2002). Protein kinase Cɛ regulates anxiety-like behavior, possibly through regulation of amygdala CRF, but without altering responsiveness of the HPA axis to physical stress (Hodge et al. 2002). Therefore, selective PKCɛ inhibitors may prove effective for the treatment of anxiety disorders without risks associated with suppression of the HPA response to stress.

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  3. Materials and methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgments
  8. Supporting Information
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Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgments
  8. Supporting Information

The authors thank H. Walter and P. Paredes for technical assistance and D. Mochly-Rosen for the tat peptide. This work was supported by E.U. Marie Curie Fellowship MOIF-CT-2004-002812 to H.M.B.L. and by funds provided by the State of California for medical research on alcohol and substance abuse through the University of California at San Francisco, grant AA013588 from the U.S. National Institute on Alcohol Abuse and Alcoholism, and U.S. Army contract DAMD17-03-1-0058 to R.O.M.

Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgments
  8. Supporting Information

The following supplementary material is available for this article online from http://www.blackwell-synergy.com/doi/full/10.1111/j.1601-183X.2007.00356.x

Figure S1: The abundance of other PKC isozymes is not altered in the amygdale of PKC&epsi;−/− mice.

Appendix S1: Supplementary methods.

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