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. Author manuscript; available in PMC: 2021 Jan 10.
Published in final edited form as: Prog Neuropsychopharmacol Biol Psychiatry. 2019 Aug 12;96:109730. doi: 10.1016/j.pnpbp.2019.109730

Crh receptor priming in the bed nucleus of the stria terminalis induces tph2 gene expression in the dorsomedial dorsal raphe nucleus and chronic anxiety

Nina C Donner a, Sofia Mani a, Stephanie D Fitz b, Drake M Kienzle a, Anantha Shekhar b, Christopher A Lowry a,c,d,e
PMCID: PMC6815726  NIHMSID: NIHMS1538928  PMID: 31415826

Abstract

The bed nucleus of the stria terminalis (BNST) is a nodal structure in neural circuits controlling anxiety-related defensive behavioral responses. It contains neurons expressing the stress- and anxiety-related neuropeptide corticotropin-releasing hormone (Crh) as well as Crh receptors. Repeated daily subthreshold activation of Crh receptors in the BNST is known to induce a chronic anxiety-like state, but how this affects neurotransmitter-relevant gene expression in target regions of the BNST is still unclear. Since the BNST projects heavily to the dorsal raphe nucleus (DR), the main source of brain serotonin, we here tested the hypothesis that such repeated, anxiety-inducing activation of Crh receptors in the BNST alters the expression of serotonergic genes in the DR, including tph2, the gene encoding the rate-limiting enzyme for brain serotonin synthesis, and slc6a4, the gene encoding the serotonin transporter (SERT). For 5 days, adult male Wistar rats received daily, bilateral, intra-BNST microinjections of vehicle (1% bovine serum albumin in 0.9% saline, n = 11) or behaviorally subthreshold doses of urocortin 1 (Ucn1, n = 11), a potent Crh receptor agonist. Priming with Ucn1 increased tph2 and slc6a4 mRNA expression selectively within the anxiety-related dorsal part of the DR (DRD) and decreased social interaction (SI) time, a measure of anxiety-related defensive behavioral responses in rodents. Decreased social interaction was strongly correlated with increased tph2 mRNA expression in the DRD. Together with previous studies, our data are consistent with the hypothesis that Crh-mediated control of the BNST/DRD-serotonergic system plays a key role in the development of chronic anxiety states, possibly also contributing to stress-induced relapses in drug abuse and addiction behavior.

Keywords: anxiety, chronic anxiety, tryptophan hydroxylase 2, tph2, serotonin transporter

Graphical Abstract

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1. Introduction

Emotional disorders, such as social anxiety disorder (SAD), cause significant suffering and are a costly health care burden to society. While both stress-responsive systems and serotonin (5-hydroxytryptamine; 5-HT) systems are often dysregulated in these and other psychiatric disorders (Arango et al., 2002;Gillespie and Nemeroff, 2005), additional research is needed to determine how these systems interact to induce chronic anxiety-like states. Susceptibility to stress-related plasticity changes in the brain are determined by inherited genetic factors, as well as environmental factors, such as adverse events or chronic stressors (Kendler et al., 2004). In rodents, chronically elevated glucocorticoid stress hormones cause anxiety-like behaviors (Donner et al., 2012b) and increase the expression of crh, the gene encoding a key stress-responsive neuropeptide, corticotropin-releasing hormone (Crh), in the bed nucleus of the stria terminalis (BNST) (Makino et al., 1994;Shepard et al., 2006), and tph2, the gene encoding the rate-limiting enzyme for brain 5-HT synthesis in the serotonergic dorsal raphe nucleus (DR) (Donner et al., 2012b). Increased rat tph2 mRNA and Tph2 protein expression after stress exposure (Donner et al., 2018), or chronic, anxiogenic corticosterone treatment is predominantly observed in the dorsal part of the DR (DRD; also referred to as the dorsomedial DR) (Donner et al., 2016;Donner et al., 2012c). As the DRD is activated by a multitude of anxiogenic pharmacologic compounds (Abrams et al., 2005), as well as anxiogenic stimuli, such as exposure to an open-field arena (Hale et al., 2008), we hypothesize that much of corticosterone’s effect on tph2 expression is indirect. One likely input comes from Crh-expressing neurons in limbic forebrain regions, such as the BNST, that project directly to the brainstem DRD (Lee et al., 2008;Sink et al., 2013;Ren et al., 2018;Dabrowska et al., 2016

The BNST is part of the extended amygdala complex, which also includes the basolateral amygdala (BL) and the central nucleus of the amygdala (CE). Specific circuits (neuronal projection systems) between components of this extended amygdala complex and functional subdivisions of the DR are thought to control different types of anxiety. We hypothesize that conflict anxiety (i.e., relevant to generalized anxiety disorder (GAD) and SAD) is controlled by a BNST-DR circuit, while panic-like anxiety is controlled by a BL/CE-DR circuit (Donner et al., 2012a;Donner and Lowry, 2013). Afferents arising from the BNST are known to innervate the DR, including the anxiety-relevant DRD (Peyron et al., 1998;Dabrowska et al., 2016). However, the identity and sensitivity of these afferents to stress-related neuropeptides remains unclear, as does their effect on serotonergic gene expression.

The purpose of this study, therefore, was to investigate mRNA expression of tph2 (tryptophan hydroxylase 2, brain-specific isoform) and slc6a4 (serotonin transporter, SERT) in a circuit-driven animal model of SAD, namely repeated sub-threshold activation of Crh receptors within the BNST. Corticotropin-releasing hormone functions as a neurotransmitter peptide within the extended amygdala (Walker et al., 2009) and also in the DR (Day et al., 2004;Donner et al., 2016; Valentino et al., 2010). It plays a critical role in anxiety-like defensive behavioral responses and can be either anxiolytic or anxiogenic, depending on the brain region, receptor type, and the downstream neuronal projections that it activates (Bale et al., 2002). In the BNST of rodents, both Crh receptor subtypes (Crhr1 and Crhr2) are found (Chalmers et al., 1995), and Crh appears to exert largely anxiogenic actions (Walker et al., 2009;Sink et al., 2013), but see (Henckens et al., 2017). Repeated, sub-threshold activation of Crh receptors in the BNST likely causes long-term plasticity changes and results in a long-lasting anxiety-like state, as measured in the rat social interaction (SI) test (Lee et al., 2008).

Here, we bilaterally infused either vehicle or sub-threshold doses of urocortin 1 (Ucn1), a Crh-related neuropeptide that binds to Crhr1 and Crhr2 receptors (Vaughan et al., 1995), once daily for five days into the BNST of adult male rats. Their anxiety-like state was assessed in the SI test before and after priming with Ucn1, and tph2 and slc6a4 gene expression were measured in nine functionally and neuroanatomically distinct subdivisions of the serotonergic DR.

2. Materials and methods

2.1. Animals

Twenty-four adult male Wistar rats (Harlan Laboratories, Indianapolis, IN, USA; 250-300 g in weight) were single-housed with ad libitum access to food (Cat. No. 8640; Teklad 22/5 Rodent Diet, Harlan Laboratories) and water at 22 °C room temperature and a reversed 12:12 h light/dark cycle (lights on at 0700 h). Rats were used for intra-BNST priming with Ucn1 (n = 12) or vehicle (n = 12), and 12 age-matched conspecifics were used as partner rats in the SI test. All animal procedures were conducted at Indiana University Purdue University at Indianapolis School of Medicine (IUPUI, Indianapolis, IN, USA). All animal experiments complied with the ARRIVE guidelines, were carried out in accordance with IUPUI IACUC committee approval, and the Guide for the Care and Use of Laboratory Animals, Eighth Edition (Institute for Laboratory Animal Research, The National Academies Press, Washington, D.C., 2011). Every effort was made to reduce the number of animals used and their suffering.

2.2. Experimental design

The experimental timeline is illustrated in Figure 1A. After acclimation for one week, rats were deeply anesthetized using isoflurane and underwent stereotaxic surgeries. During stereotaxic surgery, bilateral stainless steel guide cannulae (26 ga, Cat. No. C315GS-4, Plastics One, Roanoke, VA, USA) were implanted, and the guide cannulae were secured with stainless steel screws and dental cement. Bilateral guide cannulae were placed directly above the BNST at the following coordinates with reference to bregma (Paxinos and Watson, 1998), anterior/posterior: −1.0 mm; lateral: +/−2.5 mm; dorsoventral: −8.0 mm, 10° angle of stereotaxic arms, incisor bar at +5.5 mm (Lee et al., 2008). After surgery, guide cannulae were sealed with dummy cannulae (Plastics One). Six days later, experimental rats were habituated by placing them individually into the SI arena for 5 min, and tested in the SI test 24 h later (Day 0) to measure baseline anxiety (File, 1980). For the subsequent five days, rats received daily bilateral intra-BNST microinjections of vehicle (100 nl of 1% bovine serum albumin, Cat. No. A2153, Sigma-Aldrich, St. Louis, MO, USA, in sterile 0.9% saline, n = 12) or Ucn1 treatment (Cat. No. U6631, Sigma-Aldrich; 6 fmoles Ucn1/100 nl vehicle per side, n = 12) at 0900 h via microinjection cannulae that were inserted into the permanent guide cannulae and extended 1 mm from the guide cannula tip into the BNST. This priming paradigm is known to produce long-lasting anxiety-like behavior in the SI test through stimulation of intra-BNST Crh receptors at a dose of Ucn1 that, following acute administration, is not anxiogenic (Lee et al., 2008). Two days after the final injection, rats were tested in the SI test again to confirm development of an anxiety-like state in Ucn1-treated rats. One day later, rats were euthanized between 0900 h and 1000 h via rapid decapitation. The brains were extracted, fresh-frozen, shipped on dry ice to the University of Colorado Boulder (Boulder, CO, USA), and stored at−80 °C until tissue sectioning. Rats were euthanized 24 h after the post-priming SI test, and not earlier, because other socially interactive paradigms, such as social defeat, have been shown to increase rat tph2 mRNA expression 4 h post-exposure (Gardner et al., 2009b). In other words, we conducted the tissue collection 24 h following behavioral testing in order to avoid acute effects of the behavioral testing on tph2 and slc6a4 mRNA expression in the DR, and we assumed that mRNA changes occurred prior to behavioral testing, and otherwise remained static.

Figure 1.

Figure 1.

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(A) Experimental timeline. Adult, male rats received five daily bilateral, intra-bed nucleus of the stria terminalis (BNST) microinjections of vehicle (n = 12) or urocortin 1 (UCN1, n = 12), and anxiety-like behavior was assessed in pre- and post-priming social interaction (SI) tests. (B) Verification and mapping of cannula placements. Each black dot represents an injection site from rostral (−0.26 mm bregma) to caudal (−0.92 mm bregma). The orientation of the sections matches the orientation of the viewer (left hemisphere on the left, right hemisphere on the right). Only rats with both injection sites within the BNST were used for analysis (n = 11 per treatment group). The vertical scale indicates dorsoventral coordinates in mm from bregma. (C) Representative photomicrographs of bilateral intra-BNST cannula placements at −0.80 mm bregma. (D) Neuroanatomical analysis atlas of rat serotonergic gene expression within anatomical subdivisions of the dorsal raphe nucleus (DR) and median raphe nucleus (MnR). Shown are 14 representative coronal brain sections of slc6a4 mRNA expression from −7.580 mm to −8.672 mm bregma (designated +7 to −6). Abbreviations are applicable to each following level until indicated differently. Scale bar: 1 mm. (E) Core and shell of the DRD. Abbreviations: 3V, third ventricle; ac, anterior commissure; BSTL, bed nucleus of the stria terminalis, lateral division; BSTM, bed nucleus of the stria terminalis, medial division; cc, corpus callosum; cDRD, caudal aspect of the dorsal raphe nucleus, dorsal part; cDRV, caudal aspect of the dorsal raphe nucleus, ventral part; cMnR, caudal aspect of the median raphe nucleus; DRC, dorsal raphe nucleus, caudal part; DRD, dorsal raphe nucleus, dorsal part; DRI, dorsal raphe nucleus, interfascicular part; DRVL, dorsal raphe nucleus, ventrolateral part; f, fornix; ic, internal capsule; LV, lateral ventricle; mlf, medial longitudinal fasciculus; ox, optic chiasm; rDRD, rostral aspect of the dorsal raphe nucleus, dorsal part; rDRV, rostral aspect of the dorsal raphe nucleus, ventral part; rMnR, rostral aspect of the median raphe nucleus; sm, stria medullaris of the thalamus; st, stria terminalis; VLPAG, ventrolateral periaqueductal gray.

2.3. Social interaction (SI) test

The SI test was performed using a 91.5 cm × 91.5 cm wooden box with 30.5 cm-high walls and an open top under low light conditions (dark room with red light positioned directly above) as previously described (Gehlert et al., 2005). An unfamiliar age- and weight-matched male partner rat was paired with an experimental rat for 5 min between 0900 h and 1300 h. A video camera mounted above recorded the behavior for later analysis using the “The Observer” behavioral analysis software (version 5.0, Noldus Information Technologies, Wageningen, The Netherlands). Social interaction time was defined as any behavior initiated or lully participatory by the experimental rat: face-on contact, sniffing, following, grooming, crawling over or under the partner rat. No experimental rat was exposed to the same partner rat more than once. Decreased SI time is indicative of an anxiogenic treatment effect (File, 1980).

2.4. Brain tissue sectioning and verification of cannula placements

Forebrains were cryosectioned (Leica CM1900, North Central Instruments, Denver, CO, USA) coronally between approximately 0.70 mm and −1.30 mm bregma at 30 μm. Sections were thaw-mounted onto Vista-Vision HistoBond® microscope slides (VWR Scientific, West Chester, PA, USA), stained with cresyl violet (Paxinos and Watson, 1998), cover-slipped with Entellan mounting medium (Cat. No. 14802, EMS, Hatfield, PA, USA), and cannulae placements were identified with reference to a stereotaxic rat brain atlas (Paxinos and Watson, 1998). Only rats with both injection tip sites within the BNST were used for analysis (Figure 1B and 1C). Two animals were excluded from the study based on cannulae placements outside of the BNST (final n = 11 per group). Hindbrains were cryosectioned coronally at 12 μm in series of seven, thaw-mounted onto Vista-Vision HistoBond® microscope slides (VWR Scientific), and stored at −80°C.

2.5. tph2 and slc6a4 in situ hybridization histochemistry

In situ hybridization histochemistry for tph2 mRNA was performed using a synthetic 45-base oligonucleotide probe with the sequence 5’-TCC GTC CAA ATG TTG TCA GGT GGA TTC AGC GTC ACA ATG GTG GTC-3’ (Integrated DNA Technologies, Coralville, IA, USA), complementary to bases 489–533 of rat tph2 mRNA. After labeling with [35S]-deoxyadenosine-5’-trisphosphate (dATP, Cat. No. NEG034H001MC, PerkinElmer, Waltham, MA, USA) on the 3’-end using terminal deoxynucleotidyl transferase (20 U/μl, Cat. No. EP0161, Fermentas, Glen Burnie, MD, USA) for 2 h at 37 °C, the probe was cleaned using a QIAquick® nucleotide removal kit (Cat. No. 28304, Qiagen, Valencia, CA, USA) and used in the assay, as described previously (Lukkes et al., 2013). After overnight hybridization at 42 °C, slides were apposed to an autoradiography film (BioMax MR, Catalogue Number 871 5187, Carestream Health, Rochester, NY, USA) for 14 days. In situ hybridization histochemistry for slc6a4 mRNA was performed at 37 °C using a 3′-tail-[35S]-dATP-labeled synthetic 50-base anti-sense oligonucleotide probe (5′-ACT GCA GAG TAC CCA TTG GAT ATT TGG CTA GGC TCT GCC CTG TCC GCT GT-3′, Integrated DNA Technologies), complementary to bases 207–256 of rat slc6a4 mRNA, as previously described (Gardner et al., 2009a). Slides were apposed to autoradiographic film for 11 days.

2.6. Semi-quantitative analysis of gene expression

Digital autoradiography images obtained for the two genes of interest were analyzed with ImageJ (NIH, Bethesda, MD, USA) while the experimenter was blinded to the treatment groups. Gray value × area was measured using matrices in the shape of the rostral and caudal median raphe nucleus (rMnR and cMnR) and nine functionally different DR subdivisions. Area (mm2) was defined as the pixels falling above a certain gray value threshold that was determined empirically and kept consistent throughout each gene’s analysis. Fourteen rostro-caudal sections, designated levels +7 through −6 (Figure 1D), were analyzed: core and shell of the rostral aspect of the dorsal raphe nucleus, dorsal part (rDRD core and rDRD shell, Figure 1E), −7.580 to −7.916 mm bregma; rostral aspect of the dorsal raphe nucleus, ventral part (rDRV), −7.580 to −7.916 mm bregma; core and shell of the caudal aspect of the dorsal raphe nucleus, dorsal part (cDRD core and cDRD shell), −8.000 to − 8.336 mm bregma; caudal aspect of the dorsal raphe nucleus, ventral part (cDRV), −8.000 to −8.504 mm bregma; dorsal raphe nucleus, ventrolateral part/ventrolateral periaqueductal gray (DRVL/VLPAG), −7.832 to −8.504 mm bregma; dorsal raphe nucleus, caudal part (DRC), −8.420 to −8.672 mm bregma; dorsal raphe nucleus, interfascicular part (DRI), −8.420 to −8.672 mm bregma (Paxinos and Watson, 1998). The lateral wing (DRVL/VLPAG) values were averaged between the left and right hemisphere. In addition, we analyzed the rMnR, −7.832 to −8.084 mm bregma, and the cMnR, −8.168 to −8.504 mm bregma. Background gray value in a region adjacent to the DR was subtracted individually within each image. Gene expression in the entire DR was computed averaging all subdivisions.

2.7. Statistical analysis

Data were analyzed using SPSS (version 22.0, SPSS Inc., Chicago, IL, USA) after removal of extreme outliers (Grubbs, 1969). Social interaction time was analyzed using a linear mixed model (LMM) analysis with treatment (vehicle or Ucn1) as the between-subjects factor, and time (baseline versus post-priming SI test) as the within-subjects factor. Gene expression was assessed using LMM analysis, with treatment as the between-subjects factor and subdivision as the within-subjects factor. Where appropriate, post hoc pairwise comparisons were made using Fisher’s Least Significant Difference tests. Correlation analysis between post-priming SI time and tph2 or slc6a4 mRNA expression in the rDRD and cDRD core and shell were performed using SigmaPlot (Systat Software Inc, San Jose, CA, USA). Graphs were prepared using SigmaPlot, and figures were assembled in CorelDraw (Version 12.0, Corel Inc., Mountain View, CA, USA). Significance was accepted at p < 0.05. Values are shown as the mean + or ± the standard error of the mean (SEM).

3. Results

3.1. tph2 mRNA expression

There was a significant interaction between treatment and subdivision in the DR (Figure 2A; subdivisions × treatment, F(8, 4) = 3.22, p < 0.05). Urocortin 1-priming of the BNST elevated tph2 mRNA expression, specifically in the cDRD core, compared to vehicle-primed controls (Figure 2A and 2D; p < 0.05), but not in any other subdivision of the DR or MnR. In contrast, Ucn1 decreased tph2 expression in two rostrocaudal levels of the DRVL/VLPAG compared to controls (Figure 2A, p < 0.05 at level +3; p < 0.01 at level −2). No treatment effect of Ucn1 was detected on overall tph2 mRNA expression in the entire DR or MnR when all subdivisions were averaged (Figure 2B and 2C).

Figure 2.

Figure 2.

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Effects of five days of daily bilateral intra-bed nucleus of the stria terminalis (BNST) microinjections of vehicle (n = 11, open circles) or urocortin 1 (UCN1, n = 11, closed circles) on tph2 mRNA expression in subdivisions of (A) the dorsal raphe nucleus (DR), (B) in the entire DR (average of all subdivisions), and (C) in the median raphe nucleus (MnR). Tph2 mRNA expression is displayed throughout the entire rostrocaudal extent of each subdivision, with bregma levels (+7 through −6) on the x-axis, and gene expression on the y-axis. Borders between rostral and caudal aspects of each subdivision are indicated with dashed vertical lines. Shown is the mean ± or + the standard error of the mean (SEM). (D) Representative photomicrographs showing tph2 mRNA expression in the DR of vehicle- and Ucn1-primed rats at approximately −8.168 mm from bregma. The white arrow indicates the core of the caudal aspect of the dorsal raphe nucleus, dorsal part(cDRD core). Abbreviations: DRC, dorsal raphe nucleus, caudal part; DRD, dorsal raphe nucleus, dorsal part; DRI, dorsal raphe nucleus, interfascicular part; DRV, dorsal raphe nucleus, ventral part; DRVL/VLPAG, dorsal raphe nucleus, ventrolateral part/ventrolateral periaqueductal gray; Scale bar: 1 mm. p* < 0.05, p** <0.01, compared to vehicle controls.

3.2. slc6a4 mRNA expression

An interaction between intra-BNST Ucn1 treatment and subdivision was found for slc6a4 mRNA expression in the DR (subdivisions × treatment, F(8, 4) = 4.55, p < 0.01). Specifically, in the rDRD core, slc6a4 mRNA expression was significantly higher after Ucn1 treatment compared to controls (Figure 3A and 3F; p < 0.01), while slc6a4 mRNA expression in the DRI was decreased by Ucn1-priming (Figure 3A, 3D and 3H; p < 0.05). In the rMnR, Ucn1-treated rats displayed increased expression of slc6a4 mRNA compared to controls (Figure 3C and 3G; p < 0.01). Expression of slc6a4 mRNA in other subdivisions or the entire DR when all subdivisions were averaged did not differ between treatment groups (Figure 3B).

Figure 3.

Figure 3.

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Effects of five days of daily bilateral intra-bed nucleus of the stria terminalis (BNST) microinjections of vehicle (n = 11, open circles) or urocortin 1 (UCN1, n = 11, closed circles) on slc6a4 mRNA expression in subdivisions of (A) the dorsal raphe nucleus (DR), (B) in the entire DR (average of all subdivisions), and (C) in the median raphe nucleus (MnR). Slc6a4 mRNA expression is displayed throughout the entire rostrocaudal extent of each subdivision, with bregma levels (+7 through −6) on the x-axis, and gene expression on the y-axis. Borders between rostral and caudal aspects of each subdivision are indicated with dashed vertical lines. (D-E) Bar graphs displaying the overall expression of slc6a4 mRNA expression in the DRI and rostral MnR (rMnR). Shown is the mean ± or + the standard error of the mean (SEM). Representative photomicrographs show slc6a4 mRNA expression in the (F) rostral DRD core at approximately −7.916 mm from bregma, (G) the rMnR at approximately −7.916 mm bregma, and (H) in the dorsal raphe nucleus, interfascicular part (DRI) at approximately −8.588 mm bregma of vehicle-and Ucn1-primed rats. The white arrow indicates the core of the rostral aspect of the dorsal raphe nucleus, dorsal part (rDRD core). Abbreviations: DRC, dorsal raphe nucleus, caudal part; DRD, dorsal raphe nucleus, dorsal part; DRI, dorsal raphe nucleus, interfascicular part; DRV, dorsal raphe nucleus, ventral part; DRVL/VLPAG, dorsal raphe nucleus, ventrolateral part/ventrolateral periaqueductal gray; rMnR, rostral aspect of the median raphe nucleus; Scale bar: 1 mm. p* < 0.05, p** < 0.01, compared to vehicle controls.

3.3. Behavior in the SI test

Analysis of defensive behavioral responses in the SI test revealed a time × treatment interaction (F(1, 18) = 33.22, p < 0.001). There was no difference in baseline SI time between the vehicle and the Ucn1 group, but 48 h after the final intra-BNST priming injection, Ucn1-treated rats displayed increased anxiety-like behavior in the SI test (less SI time, p < 0.01), relative to vehicle-primed controls (Figure 4A). Also, Ucn1-primed rats spent significantly less time interacting with the partner rat than they did on Day 0 (p < 0.01), while vehicle-primed control rats displayed no difference in SI time compared to Day 0.

Figure 4.

Figure 4.

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Behavioral responses to intra-bed nucleus of the stria terminalis (BNST) priming. (A) Baseline (Day 0) and post-priming (Day 7) anxiety-like behavior of adult, male rats that received daily bilateral intra-BNST microinjections of vehicle (Veh, n = 12/11, open bars) or urocortin 1 (Ucn1, n = 12/11, black bars), as measured in the social interaction (SI) test. Shown is the mean SI time (s, seconds) + SEM. p** < 0.01 versus vehicle controls; p## < 0.01 versus baseline SI time on Day 0. (B) Correlation of SI time on Day 7 and tph2 mRNA expression in the caudal core of the dorsal raphe nucleus, dorsal part (cDRD core) of vehicle- (open circles) or Ucn1-(closed circles) primed rats.

3.4. Correlation of behavior and gene expression

Based on the proposed role of the cDRD, but not the rDRD, in anxiety-like behaviors, as well as learned helplessness (Donner et al., 2018;Hale et al., 2012;Rozeske et al., 2011;Hammack et al., 2002), and on reports of increased TPH2 mRNA and protein expression (Bach-Mizrachi et al., 2006;Bach-Mizrachi et al., 2008;Boldrini et al., 2005), as well as increased CRH immunoreactivity (Austin et al., 2003) in the human equivalent of the cDRD in the brains of depressed suicide victims, we performed correlation analyses of SI time and serotonergic gene expression in these areas. Expression of tph2 mRNA, but not slc6a4 mRNA, within the cDRD core was negatively correlated with post-priming SI time (Figure 4B), meaning that increased tph2 mRNA expression in the cDRD core was strongly associated with decreased SI time (r = 0.55, r2 = 0.31, p < 0.01, N = 22). No other correlations of gene expression and behavior were found.

4. Discussion

Intra-BNST priming with Ucn1 induced an anxiety-like state and altered tph2 and slc6a4 mRNA expression. Specifically, Ucn1-priming increased both tph2 mRNA expression within the caudal DRD, a region associated with serotonergic responses to anxiogenic drugs, anxiety-related neuropeptides, anxiogenic behavioral stimuli, and learned helplessness. Altered tph2 mRNA expression was limited to the caudal DRD core and was not observed in the DRD shell. Reduced social interaction time was strongly correlated with tph2 mRNA expression in the DRD core. Furthermore, Ucn1-priming decreased tph2 expression in DRVL/VLPAG serotonergic neurons, a subpopulation of serotonergic neurons that has been implicated in inhibition of panic-like physiological and behavioral responses (Wscieklica et al., 2017;Donner and Lowry, 2013;Hassell Jr et al., 2017). In contrast, Ucn1-priming altered slc6a4 expression in the rostral DRD core, rostral MnR and DRI, regions that are implicated in stress resilience (Paul and Lowry, 2013).

Crh receptors in the BNST

Early studies of the distribution of Crhr1 and Crhr2 receptor mRNA in rat brain reported moderate expression of both Crhr1 and Crhr2 within the BNST (Chalmers et al., 1995). Chalmers and colleagues noted Crhr1 mRNA expression in both the medial and lateral aspects of the posterior BNST, and higher concentrations of Crhr2 mRNA expression in the medial (perifornical) aspects of the posterior BNST. Consistent with these findings, Van Pett and colleagues reported that Crhr1 mRNA expression is evident throughout the BNST, including the rostromedial, rostrolateral, posterodorsal, and posteroventral regions, whereas Crhr2 mRNA expression is restricted to the posterodorsal and posteroventral regions (Van Pett et al., 2000). Thus, both Crhr1 and Crhr2 are present in the BNST, and actions of Ucn1 on Crhr1, Crhr2, or both, could mediate the effects of BNST priming in our study.

Topographical organization of the BNST

Intra-BNST injections spanned a region of approximately 660 μm in the anterior/posterior plane. Thus, injections encompassed diverse subregions of the BNST, including anterior regions of the BNST (i.e., BSTLD, BSTLI, BSTLJ, BSTLV, BSTMA, and BSTMV according to Paxinos and Watson, (1998)), and posterior regions of the BNST (i.e., BSTLP, BSTMPI, BSTMPL, and BSTMPM according to Paxinos and Watson, (1998)); for a detailed discussion of anterior and posterior regions of the BNST, their neurochemical properties, and relevance to anxiety-like defensive behavioral responses, see (Lebow and Chen, 2016). A previous study involving reversible inactivation of the BNST using bilateral infusion of small volumes (100 nl) of the nonselective synapse blocker, CoCl2, with a similar distribution of microinjection sites as used here, found that inactivation of the BNST prevented both anxiety-like defensive behavioral responses in the Vogel conflict test and conditioned freezing responses in a model of contextual fear (Resstel et al., 2008). Furthermore, although many studies have focused on anterior regions of the BNST in control of anxiety-like defensive behavioral responses (Kim et al., 2013;Levita et al., 2004;Hammack et al., 2009), other studies have found that the posterior BNST also plays a role in control of anxiety-like defensive behavioral responses (Henckens et al., 2017). Specifically, activation of neurons in the oval nucleus of the BNST promotes an anxiety-like state, whereas activation of the adjacent anterodorsal BNST (BSTLP) decreases multiple features of anxiety-like states (Kim et al., 2013). Other studies have shown that activation of glutamatergic neurons within the ventral BNST (BSTLV) projecting to the ventral tegmental nucleus increase anxiety-like defensive behavioral responses, whereas activation of GABAergic neurons within the ventral BNST projecting to the ventral tegmental nucleus decrease anxiety-like defensive behavioral responses (Jennings et al., 2013). Meanwhile, selective activation of Crhr2 expressing GABAergic projection neurons in the posterior BNST decreases anxiety and stress-induced anxiety (Henckens et al., 2017). Taken together, the evidence for functional heterogeneity within the anterior and posterior regions of the BNST suggests that BNST priming using Ucn1 may induce a chronic anxiety-like state by sensitizing BNST neurons in the oval subnucleus of the BNST (BSTLD), inhibiting BNST neurons in the anterodorsal/posterior BNST, increasing activity of glutamatergic ventral tegmental-projecting neurons within the ventral BNST, decreasing activity of GABAergic ventral tegmental-projecting neurons within the ventral BNST, or a combination of these effects. Future studies will be required to explore the potential mechanisms involved.

Anxiety-promoting CRF neurons in the oval nucleus of the BNST selectively target the caudal DRD

Recent studies by Ren and colleagues (2018) have demonstrated that BNST projection neurons preferentially innervate DR serotonergic neurons projecting to the central nucleus of the amygdala (DRSERT→CeA neurons) that in turn increase anxiety-like defensive behavioral responses, a finding consistent with previous retrograde tracing from the dorsomedial DR (Peyron et al., 1998). Although not discussed by Ren and colleagues (2018), the BNST neurons projecting to DRSERT→CeA neurons (located in the dorsomedial DR) are almost exclusively located in the oval nucleus of the BNST (BSTLD), the location of a dense population of Crh-expressing neurons in rats and mice (Hu et al., 2019;Nguyen et al., 2016;Dabrowska et al., 2016). Furthermore, using injections of a‘floxed’ anterograde tracer (rAAV5/EF1a-DIO-mCherry) into the oval nucleus of the BNST of CRFp3.0CreGFP transgenic mice and analogous methods in rats, Dabrowska and colleagues showed that CRF neurons in the oval BNST selectively target the DRD, also referred to as the dorsomedial DR (Dabrowska et al., 2016), i.e., the same region where we observed changes in Tph2 mRNA expression in our study. Overexpression of Crh selectively in BNST neurons decreases Crhr2 receptor expression selectively within the dorsomedial DR and increases expression of conditioned emotional memory (Sink et al., 2013). Conversely, serotonergic projections from the DR to BNST Crh-expressing neurons, through activation of 5-HT2C receptors, promote development of an anxiety-like state (Marcinkiewcz et al., 2016). Finally, direct activation of Crh neurons in the oval BNST nucleus induces an anxiety-like state that mimics that observed following chronic stress (Hu et al., 2019). Thus, Crh-expressing neurons in the oval subnucleus of the BNST and serotonergic neurons within the dorsomedial DR appear to have reciprocal connections that play a role in control of emotional behavior, including anxiety-like defensive behavioral responses. Intra-BNST Ucn1 priming may sensitize or dysregulate these reciprocal circuits, leading to development of a chronic anxiety-like state. Nevertheless, as noted above, the BNST is complex in terms of its topographical organization and neurochemical properties (Lebow and Chen, 2016), and further research is needed to determine how intra-BNST priming alters BNST function and downstream signaling.

Intra-BNST priming induces a chronic anxiety-like state

Intra-BNST priming with Ucn1 decreased social interaction time, an anxiogenic effect, measured 48 h following the final Ucn1 injection. Previous studies have shown that Ucn1-induced priming of the BNST increases social anxiety, but not panic-like physiological responses (Lee et al., 2008), suggesting that the BNST plays an important role in anxiety-like defensive behavioral responses that are anticipatory in nature, or involve a conflict between approach and avoidance. In contrast, Ucn1-induced priming of the BL increases both social anxiety and panic-like anxiety (Hale et al., 2012). Ucn1 priming may activate an anxiety-promoting DR-amygdala circuit identified using viral-genetic approaches by Ren and colleagues (2018). DR-amygdala serotonergic neurons are preferentially located in the DRD (Ren et al., 2018). DR-amygdala serotonergic neurons, relative to serotonergic neurons projecting to the orbitofrontal cortex, preferentially receive input from the stress-, anxiety- and fear-related brain regions, including the central nucleus of the amygdala, the paraventricular nucleus of the hypothalamus, the nucleus of the solitary tract, and, importantly, as discussed in detail above, the BNST (Ren et al., 2018). DR-amygdala serotonergic neurons give rise to collateral projections to forebrain systems controlling anxiety- and fear-related defensive behavioral responses and anxiety-like states, including the central nucleus of the amygdala, lateral amygdala, intercalated nucleus of the amygdala, basolateral nucleus of the amygdala, paraventricular nucleus of the hypothalamus, substantia nigra, and the ventral bed nucleus of the stria terminalis (Ren et al., 2018). Activation of a DR-BNST serotonergic circuit, acting on 5-HT2C receptors to stimulate Crh neurons within the BNST, has also been implicated in anxiety-like defensive behavioral responses (Marcinkiewcz et al., 2016). Together with these findings, our data elucidate how serotonergic circuits downstream of the BNST may control chronic conflict anxiety-like states, suggesting that serotonergic neurons within the DRD core play an important role.

Evidence that dorsomedial DR serotonergic neurons promote acute- and chronic anxiety-like states

Ucn1-induced priming of the BNST increased tph2 mRNA expression within serotonergic neurons located in the caudal DRD, also referred to as the dorsomedial DR. Serotonergic neurons within the caudal DRD are activated by anxiogenic drugs, Crh-related neuropeptides, including Ucn2, and anxiogenic stimuli, including acoustic startle, anxiety due to intimate partner violence, the avoidance task on the elevated T-maze, open-field exposure, social defeat, and inescapable shock in a model of learned helplessness (Abrams et al., 2005;Bouwknecht et al., 2007;Rozeske et al., 2011;Staub et al., 2005;Spiacci, Jr. et al., 2012;Cordero et al., 2012) (for review, see Hassell Jr et al., 2017). Activation of caudal DRD serotonergic neurons likely depends on specific neuronal afferents (Hassell Jr et al., 2017;Ren et al., 2018). In support of the assumption that a key source of these afferents is the oval subnucleus of the BNST (Ren et al., 2018;Dabrowska et al., 2016), Sink et al (2013) reported that crh overexpression in the BNST decreases Crhr2 binding selectively in the caudal DRD. Based on the published autoradiograms, this decrease was clearly located in the DRD core, the only subregion of the DR that contains serotonergic neurons that co-express Crh (Commons et al., 2003). The fact that tph2 mRNA expression was elevated in the caudal DRD core in our study reinforces the hypothesis that the BNST selectively innervates and controls serotonergic function in this subregion. Indeed, previous anterograde and retrograde tracing studies have confirmed direct projections from the oval subnucleus of the BNST to the DRD core (Peyron et al., 1998;Dabrowska et al., 2016).

Serotonergic neurons in the DRD core have unique morphological properties, relative to serotonergic neurons in the DRD shell (Steinbusch, 1984). Serotonergic neurons in the DRD core also have unique neurochemical properties; as mentioned above, Commons et al (2003) have described a population of Crh-expressing serotonergic neurons that are restricted to the DRD core. Crh is a potent anxiogenic neuropeptide, and serotonin is a potent anxiogenic neurotransmitter via actions on 5-HT2C receptors located in forebrain target regions of DRD-efferents, including the BL, the BNST, and the dorsal hypothalamic area (Christianson et al., 2010;Strong et al., 2011;Commons et al., 2003;Marcinkiewcz et al., 2016). Activation of this Crh-expressing subset of serotonergic neurons may have particularly potent anxiogenic effects. As mentioned above, a recent study by Marcinkiewcz et al (2016) describes projections from the DR to the BNST that act on 5-HT2C receptors to silence anxiolytic output from the BNST. It is possible that activation of Crh-expressing serotonergic neurons in the DRD core leads to a downstream increase in Crh release within the BL. Acute anxiogenic actions of Crh (Cipriano et al., 2016), as well as development of chronic anxiety-like states following intra-BL Crh priming within the BL (Gehlert et al., 2005;Rainnie et al., 2004), are known to be mediated via Crhr1.

The observation that tph2 expression in the DRD core subregion was strongly correlated with decreased SI time raises the possibility that sensitization of neurons in the DRD core contributes to the persistent anxiety-like state following Ucn1-priming. In separate studies, we have shown that chronic glucocorticoid administration, which also induces a chronic anxiety-like state, increases tph2 mRNA, Tph2 protein expression, and Tph enzyme activity in the DRD, an effect that was prevented by local blockade of Crhr2 receptors (Donner et al., 2016). This hypothesis is consistent with studies of learned helplessness by Maier and colleagues, in which a functional desensitization of 5-HT1A receptors in the DRD accounts for the chronic anxiety-like state associated with learned helplessness (Rozeske et al., 2011). Furthermore, exaggerated fear conditioning and escape deficits measured 24 h following inescapable stress in this model of learned helplessness are prevented by lesions of the BNST, suggesting that the same BNST/caudal DRD circuitry is involved in the behavioral manifestation of learned helplessness (Hammack et al., 2004). Consistent with these observations, learned helplessness can be precipitated in the absence of inescapable stress by microinjections of Crhr2 receptor agonists directly into the region of the caudal DRD, while the exaggerated fear conditioning and escape deficits 24 h following inescapable stress are prevented by blockade of Crhr2 receptors within the region of the DRD (Hammack et al., 2002). Downstream of the DRD, exaggerated anxietylike responses in the juvenile social interaction test 24 h following inescapable stress are prevented by blockade of 5-HT2C receptors in the BL (Christianson et al., 2010), while escape deficits are prevented by blockade of 5-HT2C receptors within the dorsal striatum (Strong et al., 2011). Both the BL (Abrams et al., 2005) and the dorsal striatum (Steinbusch, 1984) are innervated by the DRD core. Figure 5 presents a hypothetical model illustrating how the BNST/DRD-core circuit may contribute to the development of chronic anxiety-like states, including learned helplessness.

Figure 5.

Figure 5.

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Hypothetical model of how urocortin 1 (Ucn1) priming within the bed nucleus of the stria terminalis (BNST) may enhance corticotropin-releasing hormone (Crh) expression, Crh release, and Crh actions on Crh receptor type 2 (Crhr2) in serotonergic neurons (Lukkes et al., 2011) of the caudal core of the dorsal raphe nucleus, dorsal part (cDRD core) (Sink et al., 2013 ;Dabrowska et al., 2016), ultimately leading to increased activity of serotonergic projections innervating, for example, the conflict anxiety-related basolateral amygdaloid nucleus (BL) (Hale et al., 2008;Christianson et al., 2010;Ren et al., 2018) and the caudate putamen (CPu), where serotonin acts on 5-hydroxytryptamine receptor type 2C (5-HT2C), contributing to the manifestation of learned helplessness (Christianson et al., 2010;Strong et al., 2011). In addition, increased Crh release within the BL, possibly arising from Crh-expressing serotonergic neurons in the cDRD (Commons et al., 2003), may contribute to enhanced anxiety via Crh receptor type 1 (Crhr1) activation in the BL. Other abbreviations: aca, anterior commissure, anterior part; Aq, mesencephalic aqueduct; f, fornix; LV, lateral ventricle; mlf medial longitudinal fasciculus; scp, superior cerebellar peduncle.

BNST priming decreases tph2 mRNA expression in the lateral wings of the DR

Ucn1-induced priming decreased tph2, but not slc6a4, mRNA expression in DRVL/VLPAG serotonergic neurons. An increase in tph2 expression in the DRD core associated with a decrease in tph2 mRNA expression in the DRVL/VLPAG serotonergic neurons is consistent with a number of studies showing that whenever DRD serotonergic neurons are activated, DRVL/VLPAG serotonergic neurons are not, and vice versa (Hassell Jr et al., 2017). Thus, the chronic anxiety-like state following BNST-priming may involve dysregulation of gene expression in multiple functional subdivisions of the brainstem raphe complex.

BNST priming alters slc6a4 mRNA expression

Slc6a4, but not tph2, expression was altered in the rostral DRD, rostral MnR, and in the DRI following Ucn1-induced priming. The rhombomeric derivation of the MnR and DRI is developmentally distinct from other subdivisions of the brainstem raphe complex (Jensen et al., 2008), and the rostral DRD, MnR, and the DRI are functionally implicated in stress resilience (Paul and Lowry, 2013). For example, direct microinjection of Crh into the DRD region results in a Crhr2-dependent, immediate increase in serotonin release in the central nucleus of the amygdala, which lasts approximately 60 minutes and is associated with freezing behavior. At the termination of freezing behavior, there is a delayed increase in serotonin release in the medial prefrontal cortex, which is dependent on the MnR; if the MnR is inactivated, the serotonin release in the medial prefrontal cortex does not occur (Forster et al., 2008). The medial prefrontal cortex is a key structure for behavioral control and stress resilience (Maier et al., 2006). Thus, changes in slc6a4 expression in these regions may be secondary or temporally delayed relative to the changes observed in the DRD. While the primary anxiogenic effects of BNST priming are likely mediated by direct projections to the DRD core, internal raphe-raphe projections may modulate other subpopulations of serotonergic neurons, e.g. in the MnR and DRI, leading to altered stress resilience.

5. Limitations

One limitation of the current study is that we limited our assessment to analysis of tph2 and slc6a4 mRNA expression, and did not extend this finding to confirm effects of intra-BNST Ucn1 priming on Tph2 and Slc6a4 protein. Although, in previous studies, we have observed parallel effects of treatment, i.e., chronic administration of the glucocorticoid hormone, corticosterone, on tph2 mRNA expression, Tph2 protein expression, and Tph2 activity in the dorsomedial DR (Donner et al., 2012a;Donner et al., 2016), future studies should also evaluate Tph2 protein expression and Tph2 activity within the dorsomedial DR following intra-BNST priming.

6. Conclusions

In summary, our findings are relevant for clinical conditions including anxiety-disorders, stressor- and trauma-related disorders like posttraumatic stress disorder, and affective disorders. Analyses of post mortem tissue from depressed suicide patients have revealed increased CRH immunoreactivity (Austin et al., 2003) and increased TPH2 mRNA and TPH2 protein expression within the caudal DR (Bach-Mizrachi et al., 2006;Bach-Mizrachi et al., 2008). A subregional analysis revealed that TPH2 expression in the DRD of depressed suicides was 136% that of controls (Bach-Mizrachi et al., 2006). Another study of alcohol-addicted depressed suicides indicates that TPH-immunostaining was selectively increased in the DRD, with a striking elevation of TPH-immunostaining in the presumed human equivalent of the DRD core (Bonkale et al., 2006). Our data suggest that increased TPH2 mRNA and TPH2 protein expression in the DRD may be based on overactivity of a BNST/DRD core circuit, resulting in chronic anxiety or a ‘learned helplessness’ state. Based on human post mortem studies, this circuit also may play a role in vulnerability to suicidal self-directed violence

HIGHLIGHTS.

BNST priming induces a chronic anxiety-like state

BNST priming increases tph2 mRNA expression in the dorsomedial dorsal raphe nucleus

Increased tph2 mRNA expression is strongly correlated with anxiety-like behavior

Acknowledgements

We gratefully acknowledge Zachary D. Barger for proof reading the manuscript.

Funding

This study was supported by NIH grant R01MH065702 (AS, CAL) and R01MH086539 (CAL). The sponsor(s) had no role in the collection, analysis, and interpretation of data; in the writing of the report; and in the decision to submit the article for publication. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of Mental Health or the National Institutes of Health. CAL is supported by the National Institute of Mental Health (grant number 1R21MH116263), Department of the Navy, Office of Naval Research Multidisciplinary University Research Initiative Award (grant N00014-15-1-2809), Department of Veterans Affairs Office of Research and Development RR&D Small Projects in Rehabilitation Research (grant 1-I21-RX002232-01), the Colorado Department of Public Health and Environment (grant DCEED-3510), and the Alfred P. Sloan Foundation (grant G-2016-7077). D.M.K. was supported by a University of Colorado Boulder Undergraduate Research Opportunities Program (UROP) Individual grant (AY 18-19).

Abbreviations

3V

third ventricle

5-HT

5-hydroxytryptamine; serotonin

ac

anterior commissure

BL

basolateral amygdala

BNST

bed nucleus of the stria terminalis

BSTL

bed nucleus of the stria terminalis, lateral division

BSTLD

bed nucleus of the stria terminalis, lateral division, dorsal part

BSTLI

bed nucleus of the stria terminalis, lateral division, intermediate part

BSTLJ

bed nucleus of the stria terminalis, lateral division, juxtacapsular part

BSTLP

bed nucleus of the stria terminalis, lateral division, posterior part

BSTLV

bed nucleus of the stria terminalis, lateral division, ventral part

BSTM

bed nucleus of the stria terminalis, medial division

BSTMA

bed nucleus of the stria terminalis, medial division, anterior part

BSTMPI

bed nucleus of the stria terminalis, medial division, posterointermediate part

BSTMPL

bed nucleus of the stria terminalis, medial division, posterolateral part

BSTMPM

bed nucleus of the stria terminalis, medial division, posteromedial part

BSTMV

bed nucleus of the stria terminalis, medial division, ventral part

cc

corpus callosum

cDRD

caudal aspect of the dorsal raphe nucleus, dorsal part

cDRD

core, core of the caudal aspect of the dorsal raphe nucleus, dorsal part

cDRD

shell, shell of the caudal aspect of the dorsal raphe nucleus, dorsal part

cDRV

caudal aspect of the dorsal raphe nucleus, ventral part

CE

central nucleus of the amygdala

cMnR

caudal aspect of the median raphe nucleus

Crh

corticotropin-releasing hormone

DR

dorsal raphe nucleus

DRC

dorsal raphe nucleus, caudal part

DRD

dorsal raphe nucleus, dorsal part (also referred to as the dorsomedial DR)

DRI

dorsal raphe nucleus, interfascicular part

DRVL

dorsal raphe nucleus, ventrolateral part

DRVL/VLPAG

dorsal raphe nucleus, ventrolateral part/ventrolateral periaqueductal gray, also called lateral wing of the DR

f

fornix

ic

internal capsule

LMM

linear mixed model

LV

lateral ventricle

mlf

medial longitudinal fasciculus

MnR

median raphe nucleus

ox

optic chiasm

rDRD

rostral aspect of the dorsal raphe nucleus, dorsal part

rDRD core

core of the rostral aspect of the dorsal raphe nucleus, dorsal part

rDRD shell

shell of the rostral aspect of the dorsal raphe nucleus, dorsal part

rDRV

rostral aspect of the dorsal raphe nucleus, ventral part

rMnR

rostral aspect of the median raphe nucleus

SERT

serotonin transporter

SAD

social anxiety disorder

SEM

standard error of the mean

SI

social interaction

slc6a4

solute carrier family 6 (neurotransmitter transporter, serotonin), member 4, serotonin transporter, SERT

sm

stria medullaris of the thalamus

st

stria terminalis

Tph2

tryptophan hydroxylase 2, brain-specific isoform

tph2

tph2 gene

Ucn1

urocortin 1

VLPAG

ventrolateral periaqueductal gray

Footnotes

Conflict of interest

CAL serves on the Scientific Advisory Board of Immodulon Therapeutics Ltd. All other authors declare no financial conflicts of interest related to this work.

Declarations of interest: CAL serves on the Scientific Advisory Board of Immodulon Therapeutics, Ltd. The other authors report no conflicts of interest related to the submitted manuscript.

Ethical statement

This manuscript is in accordance with the Authorship statement of ethical standards for manuscripts submitted to Progress in Neuro-Psychopharmacology & Biological Psychiatry. All authors have read and approved the submission of the manuscript; the manuscript has not been published and is not being considered for publication elsewhere, in whole or in part, in any language, except as an abstract.

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