• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Biol Psychiatry. Author manuscript; available in PMC May 1, 2008.
Published in final edited form as:
PMCID: PMC2362385

Activation of raphe efferents to the medial prefrontal cortex by CRF; correlation with anxiety-like behavior



Parallel lines of research suggest that dysfunction affecting both corticotropin-releasing factor (CRF) and serotonin (5-HT) systems is involved in the pathophysiology of psychiatric illnesses such as anxiety and depression. The effect of CRF on behavior, and on the accompanying change in activity of 5-HT neurons in the dorsal and median raphe nuclei (DR and MR) that project to the ventral medial prefrontal cortex (mPFC), a brain area implicated in mood and anxiety disorders, was studied.


Male Sprague-Dawley rats with intra-mPFC deposits of fluorescent microspheres received injections of CRF (1 μg, icv) and were tested for CRF-enhanced startle, a behavioral assay believed to reflect stress- or anxiety-like states. C-Fos immunohistochemistry was used to measure CRF-induced activity in retrogradely-labeled neurons in the DR and MR and correlate this level of activity with the level of CRF-enhanced startle.


CRF-enhanced startle was accompanied by an increase c-Fos expression in retrogradely-labeled cells in the raphe. In the DR and MR, there was a clear topography of activation, with a higher percent activation in retrogradely-labeled neurons in caudal sections. In the caudal DR, this effect was positively correlated with the level of CRF-enhanced startle. Coexpression of retrogradely-labeled cells with tryptophan hydroxylase showed that the majority (> 90%) of raphe efferents to the mPFC were from serotonergic neurons.


These data indicate that CRF activates a subpopulation of cortical-projecting 5-HT raphe neurons, and suggest that increased 5-HT release in the mPFC may be an important component driving some types of anxiety-like behaviors.

Keywords: raphe, c-Fos, serotonin, startle, prefrontal cortex, stress


The neuropeptide corticotropin-releasing factor (CRF) has been widely implicated in the development and manifestation of psychiatric illnesses such as anxiety and depression [1; 2], highly comorbid disorders that may share common elements of an underlying pathophysiology [3]. The link between CRF and anxiety/mood disorders is evident in studies demonstrating that administration of CRF or CRF antagonists, or genetic manipulations that change the expression levels of CRF receptors, can dramatically alter behavior in several animal models of anxiety and depression. In general, these studies have shown that increases in CRF-neurotransmission have anxiety- and depressive-like effects whereas decreases in CRF-neurotransmission have anxiolytic- and antidepressive-like effects [4-8]. Taken together, these preclinical findings complement reports from clinical studies where an association between hyperactive CRF systems and some types of anxiety (e.g. post-traumatic stress disorder [PTSD]) and depression (e.g. major depression) have been found [9-11].

CRFergic systems are also known to interact with other neurotransmitter systems associated with anxiety and depression, such as the serotonin (5-hydroxytryptamine, 5-HT) system [12-14], and it is possible that CRF-5-HT interactions play a role in the constellation of symptoms seen in these diseases [15; 16]. In animals, administration of CRF or related peptides (e.g. urocortins) can have either excitatory or inhibitory effects on 5-HT neurons of the dorsal raphe nucleus depending on the preparation (in vitro vs. in vivo) and dose used: low doses inhibit, whereas high doses tend to excite 5-HT neurons [17-19]. In addition, CRF administration has been shown to increase, decrease, or have no effect on 5-HT release depending on the raphe projection area examined [20-23]. In the medial prefrontal cortex (mPFC), a brain area implicated in mood and anxiety disorders [24; 25] and a major target of ascending 5-HT pathways [26], studies have shown an increase in 5-HT or 5-HT metabolites after administration of CRF [27; 28]. Interestingly, the time course of CRF-induced 5-HT release in the mPFC (peak effects occurring 80 min after CRF infusion; [27]) is temporally similar to the behavioral effects of CRF on the acoustic startle response (i.e. potentiation) seen in many laboratories [29], including ours [30]. This observation suggests that time-dependent activation by CRF of raphe efferents to the mPFC may be an important component driving some types of anxiety-like behavior. To address this possibility, we used the novel approach of combining retrograde tracing with c-Fos immunohistochemistry (as a marker of neuronal activity [31; 32]) to examine the effect of CRF on raphe efferents to the mPFC in behaving rats.

Materials and Methods


The animals were male Sprague-Dawley rats (Charles River, Raleigh, NC) weighing ~350 g and were housed in group cages of four rats each until surgery. Animals were maintained on a 12-hr light/dark cycle (lights on at 07.00h) with food and water continuously available.


The equipment used to measure the effect of CRF on the acoustic startle reflex is identical to that described elsewhere [30].


FluoSphere deposits in the mPFC

Rats were anesthetized with Nembutal (50 mg/kg, intraperitoneally [ip]) and placed in a Kopf stereotaxic instrument (Model 900) with blunt ear bars. The skin was retracted and a hole was drilled in the skull above the mPFC. A stainless steel infusion cannula (30 gauge; 150 μm internal diameter) attached to a Hamilton microsyringe (10 μl) by polyethylene tubing was lowered into the brain, and the infralimbic (IL) division of the mPFC was targeted using the following coordinates: +2.8 mm rostral to bregma, − 0.6 mm lateral to the midline, −4.8 mm ventral to dura. A Harvard Apparatus (Model 22) infusion pump was used to deliver 0.2 μl of the FluoSpheres (FS, 0.04 μm diameter polystyrene beads; Molecular Probes, Eugene, OR), diluted 1:1 in 100 mM sodium citrate (pH 7.0) directly into the mPFC at a rate of .1 μl/min for 2 min. Following the injection, the infusion cannula was left in place for 10 min to avoid leakage upon removal, and the skull hole was filled with sterile Gelfoam (Pharmacia & Upjohn, Kalamazoo, MI).

Intracerebroventricular (icv) cannulation

Following the FS deposit in the mPFC, a stainless steel guide cannula (23 gauge; Plastics One, Roanoke, VA) with an internal dummy stylet extending 1.5 mm beyond the guide cannula tip was lowered into the brain aimed at the lateral ventricle using the following coordinates: −0.8 mm caudal to bregma, + 1.3 mm lateral to the midline, −3.5 mm ventral to dura. Three stainless steel screws (size 0-80; Small Parts, Miami Lakes, FL) were also placed in the skull to anchor the guide cannula, and Loctite adhesive (Newington, CT ) and dental acrylic (Stoelting, Wood Dale, IL) were used to cement the cannula in place. Animals were placed under a heating lamp, and after recovery the rats were singly-housed in plastic cages (45 × 24 × 20 cm) with wood-shaving bedding.



One week after surgery, rats were given pre-test handling sessions on three consecutive days to familiarize them to the apparatus and the startle stimuli. These sessions also served to habituate c-Fos activation that occurs due to the stress of handling. To do this, rats were placed in the cages and given a 5-min acclimation period followed by the presentation of 100 startle stimuli at each of three different intensities (95, 100 and 105 dB) in a semirandom order with a 30-s interstimulus interval (ISI). We used the startle data acquired on these pre-test handling days (average across the three days) to match the animals so that the two treatment groups had nearly equivalent levels of baseline startle (Pre-CRF match; see Fig 1).

Figure 1
Effect of CRF on startle


Twenty-four hours after the last pre-test handling day, rats were returned to the startle testing room and received icv infusions of either vehicle (artificial cerebrospinal fluid [aCSF]; Harvard Bioscience, Holliston, MA) or CRF (1 μg; American Peptide Company, Sunnyvale, CA) according to previously described methods [30]. Following icv infusion, rats were immediately placed in the startle cages and given a 5-min acclimation period followed by presentation of two habituating startle stimuli (100 dB, 30-s ISI). Rats were then presented with 300 startle stimuli at each of three different intensities (95, 100 and 105 dB) in a semirandom order with a 30-s ISI for a total test session of 150 min. Immediately following the end of the test session, animals were overdosed with pentobarbital (130 mg/kg, ip) and perfused intracardially with 0.9% saline (200 ml) followed by 4% paraformaldehyde (500 ml). The brains were removed and stored (4°C) for 3-4 days in a 30% sucrose/0.1M PBS (pH 7.4) solution.

Statistical analysis

Startle amplitude data were expressed as the mean averaged across the three startle-eliciting intensities across time. The effect of CRF on startle was evaluated using analyses of variance (ANOVA) with treatment group (Vehicle and CRF) as a between-subjects factor and blocks of time (3, 50-min blocks) as a within-subjects factor. Subsequent multiple comparisons for significant differences between the Vehicle and CRF groups at each block of time were made using Dunn's test.

Anatomical Studies

We used the nomenclature, delineation of structures, and stereotaxic reference system of Paxinos and Watson (1997) [33]. In addition, we used a neuroanatomic map of the DR adapted from Abrams et al., (2004) [34] to define rostral (−7.37 to −7.64), middle (−7.73 to 8.27) and caudal levels (−8.36 to −8.54) of the DR; numbers are in millimeters caudal to bregma. Although the map of Abrams et al., (2004) comprises many more sections through the DR (from −6.92 to −9.26 mm), we limited our analysis to those sections with the greatest densities of 5-HT neurons.

Retrograde labeling and c-Fos / tryptophan hydroxylase (TrpOH) immunohistochemistry

Please see Supplementary information for immunohistochemical methods.



Figure 1 illustrates the effect of icv CRF (1 μg) on startle. A two-way ANOVA with treatment as a between-subjects factor and blocks of time (1-3) as a within-subjects factor revealed a significant main effect of treatment (F(1,13) = 14.8; p < 0.005), block of time (F(2,13) = 3.9; p < 0.05), and a significant treatment by block interaction (F(2,26) = 10.1; p < 0.005). Multiple comparisons showed no significant differences between groups during the first 50-min block of time. Further comparisons showed that startle was significantly elevated in the CRF group compared to the vehicle group across the next two blocks of time (p < 0.005), with the peak effect occurring at approximately 60 min and lasting for the duration of the test session (full time course illustrated in Fig 1B). The behavior observed in this study is similar to that observed in previous studies from our laboratory [30], indicating that FS deposits in the mPFC had no untoward effects on the ability of CRF to potentiate the startle response.


CRF-induced c-Fos expression in the raphe

Vehicle-treated rats showed almost no c-Fos expression in the DR (Fig 2A) or MR (data not shown), likely due to handling the rats on multiple pre-test days. These data suggest that the icv infusion itself, or the presentation of the startle-eliciting stimuli, was not sufficient to induce c-Fos activation in the raphe in these well-handled rats. As such, we did not quantify the expression of c-Fos in FS-filled raphe efferents from this group. In contrast, CRF-treated rats showed heavy c-Fos expression throughout the DR (Fig 2B), and to a lesser extent, the MR. Because CRF-enhanced startle has a delayed onset to peak effect (~ 60-80 min; see Fig 1B), and the c-Fos protein requires 60-90 minutes for maximal expression [35], sacrificing animals at 150 min after CRF infusion would reveal neuronal activation occurring during this peak behavioral effect.

Figure 2
Representative coronal brain sections through the dorsal raphe (DR) demonstrating the lack of c-Fos expression in vehicle-treated rats (A) compared with CRF-treated (B) rats. For this comparison, c-Fos immunohistochemistry was performed according to previously ...

FluoSphere deposits in the mPFC

FS deposits in the mPFC were primarily restricted to the infralimbic (IL) division; a representative image from one of these cases is shown in Fig 3A. The centers of the deposits were most often found in layer 5 of the IL with some mediolateral spread into layers 2-6 of cortex. The use of a fine gauge stainless steel cannula, to prevent clogging of the FS beads, most likely contributed to the relatively long dorsoventral extent of the deposit center. However, this had the favorable outcome of depositing the FluoSpheres in a narrow but long track throughout most of the dorsoventral extent of the IL. There was minimal spread of the tracer into the ventrally located dorsal peduncular cortex and negligible deposit artifact along the cannula track through the prelimbic (PL) and cingulate (Cg) subdivisions.

Figure 3
(A) Coronal brain section showing discrete deposit of retrograde tracer (red FluoSpheres; FS) in the infralimbic division (IL) of the mPFC. The fluorescent image was overlaid onto an image from the same section after cresyl violet staining to determine ...

Retrograde labeling of raphe efferents to the mPFC and c-Fos activation by CRF

FS deposits restricted to the IL division of the mPFC retrogradely labeled cells throughout the rostrocaudal extent of the dorsal raphe (DR) nucleus. FS-filled neurons were found almost exclusively, and in roughly equal numbers, in the dorsal and ventral subdivisions of the DR (DRD and DRV respectively); very few cells were seen in the DR lateral wings. FS-filled neurons were also found throughout the median raphe (MR), but in fewer numbers and with much less intense labeling than that seen in the DR. The distribution of FS-filled neurons and those double-labeled for c-Fos, at different levels of the DR and MR from a representative case, is illustrated in Figure 3B. We do not believe that we have labeled a specialized population of DR neurons which project to the ventricle walls and have been shown to be able to take up retrograde tracers that infiltrate the lumen of the ventricular system [see 36; 37]. If so, we would have expected to see pronounced bilateral labeling of DRD neurons [as shown in 36; 37], rather than the almost exclusively ipsilateral pattern of labeling of DRD neurons seen in the current study.

Table 1 shows the quantification of FS-filled neurons and those double-labeled for c-Fos, at different levels of the raphe, as well as the corresponding size/layer of the deposit center in the IL for each of the CRF-treated rats used in this study. Across animals, approximately 38% of all retrogradely labeled DR neurons were also positive for c-Fos immunolabel, and there was a clear topography of expression; the percent number of FS-filled neurons that were also c-Fos positive increased from the rostral (23%) to middle (38%) to caudal (53%) levels of the DR. Double-labeled neurons were also seen in the MR, but the overall percent of FS-filled cells that were also positive for c-Fos (23%) was less than that seen in the DR. Like the DR, caudal sections of the MR tended to have a higher percentage of double-labeled cells than rostral sections. A representative image of FS-filled cells, c-Fos positive cells, and double-labeling of these cell populations at mid-level through the DR is shown in Fig 4A-D. As shown in Table 2, over 90% of FS-filled neurons in the DR and MR were immunopositive for TrpOH (representative images from the DR are shown in Fig 4E-F), suggesting that the majority of raphe efferents to the IL are from 5-HT-containing neurons.

Figure 4
(A) Coronal section through the dorsal raphe (dorsal and ventral division; DRD and DRV respectively) showing FS-filled cells (marked with arrowheads) labeled after deposit of retrograde tracer (red FluoSpheres; FS) in the mPFC (see Fig 3). (B) c-Fos-positive ...
Table 1
Quantification of FluoSphere-filled cells (FS+) and those double labeled for c-Fos in the raphe after FS deposit in the mPFC
Table 2
Quantification of FluoSphere-filled cells (FS+) and those double labeled for tryptophan hydroxylase (TrpOH) in the raphe after FS deposits in the mPFC

One of the main findings of this study was a significant correlation between the level of CRF-enhanced startle (measured as % change from Pre-CRF match test) and the degree of activation (measured as % number of FS-filled cells that also expressed c-Fos) of neurons in the caudal part of the DR. As shown in Figure 5, there was a trend for a positive relationship between the level of CRF-enhanced startle and the level of overall activation in the DR (r2 = .37, p = .10). An examination of this relationship at different levels of the DR showed that this correlation was only significant for activation in the caudal DR (r2 = .5; F(1,6) = 6.07; p < .05). There was no obvious relationship between the level of CRF-enhanced startle and activation of MR efferents to the mPFC.

Figure 5
Correlations between CRF-enhanced startle (collapsed across blocks of time and expressed as percent difference from Pre-CRF match) and the level of c-Fos activation in raphe efferents to the mPFC. There was a trend for a positive relationship between ...

The data from Table 1 show a positive relationship between the size of the deposit in the IL and the overall number of FS-filled cells in the DR, but not the MR, indicating that a bigger deposit in the IL produced a higher number of retrogradely labeled cells in the DR (see Supplementary Data Fig 1A). Despite this relationship, there was no significant correlation between the size of the deposit (and the accompanying increase in the number of retrogradely-labeled cells) and the percentage of FS-filled cells in the DR that are activated by CRF (Supplementary Data Fig 1B). Thus, we believe that the significant correlation between activation of caudal DR efferents to the mPFC and CRF-enhanced startle is not confounded by an inflated/deflated retrograde cell count due to the size of the deposit. To address this further, we examined the relationship between activation of caudal DR neurons and CRF-enhanced startle in a subset of CRF-treated rats (n=5) with roughly equivalent deposit sizes and levels of total retrograde tracing. These animals had between 134 and 160 total FS-filled cells, and between 26 and 40 FS-filled cells in the caudal DR, as indicated in Table 1 (rat id#s: C2-5, C2-7, C2-2, C2-9 and C2-4). In this subset of animals, there was still a strong positive relationship between the percent activation of caudal DR neurons and CRF-enhanced startle (r2 = .631; F(1,3) = 5.1; p < .11; see Supplementary Fig. 2) although the correlation was not significant likely owing to the reduced power of the analysis (i.e. fewer observations).


Using c-Fos immunohistochemistry as a marker of neuronal activity, the results of the present study show that icv CRF activates a population of raphe neurons that project to the mPFC. Based on our double labeling studies of FS-filled neurons with TrpOH, the enzyme involved in the production of 5-HT, our data suggest that the majority of CRF-activated raphe efferents to the IL division of the mPFC are serotonergic. FS-filled neurons were found at all levels of the DR, and in roughly equal numbers in two of the major subdivisions of the DR (i.e. the DRD and DRV). However, CRF-induced activation of DR neurons showed a clear rostral-caudal topography, with a progressive increase in the percent activation of raphe-mPFC projecting neurons from rostral to middle to caudal levels of the DR. There was also a significant correlation between the level of CRF-enhanced startle and the degree of activation of DR efferents to the mPFC (caudal division only). These data suggest that the level of anxiety-like behavior induced by CRF may be controlled by specific 5-HT circuits originating in the caudal parts of the DR, and projecting to the ventral mPFC. Interestingly, this CRF effect on topographically organized subpopulations of 5-HT neurons closely resembles the pattern of c-Fos activation seen in the DR after administration of other anxiogenic drugs such as m-chlorophenyl piperazine (mCPP), yohimbine, and FG-7142 [34]. Furthermore, our data overlap with the finding that neurons in these mid-to-caudal levels of the DR also have projections to other brain areas known to regulate anxiety-like behaviors, such as the basolateral amygdala, and support the hypothesis that subpopulations of 5-HT neurons are involved in functional anxiety circuits [38].

The simplest explanation to account for the effect of exogenously applied CRF on raphe neurons is through a direct action on CRF receptors located on DR and MR neurons. While immunohistochemical studies have shown that CRFergic innervation of the DR is high [39], and has been shown to make direct synaptic contact with both 5-HT and GABA-containing neurons in the DR [40], it is somewhat surprising that levels of CRF-1 receptors (the primary receptor through which CRF acts), and mRNA for this receptor, are quite low in the DR [41; 42]. Instead, mRNA for the CRF-2 receptor is found in relatively high levels in the DR, with moderate levels of both CRF-1 and CRF-2 receptor mRNA found in the MR [43]. Although CRF can act at CRF-2 receptors, these observations suggest that CRF-related peptides, such as the urocortins (Ucn, including Ucn1, Ucn2 and Ucn3), which bind with high affinity to the CRF-2 receptor [44; 45], may play a prominent role in modulating the activity of raphe neurons. This idea is supported by the finding that intra-DR administration of a sufficiently high dose of Ucn2 can increase the activity of DR 5-HT neurons [18], increase c-Fos expression in these neurons, and increase 5-HT efflux in DR projection areas [46]. Likewise, icv administration of Ucn2 has been shown to increase c-Fos expression in DR 5-HT neurons with a topography remarkably similar to that seen in the present study using CRF [47]. Thus, while direct activation of DR neurons by CRF would be the most straightforward mechanism to account for our data, indirect activation through other CRF-modulated neurotransmitter systems, such as the urocortin system, is also a possibility [48].

The activation of serotonergic neurons in the DR by CRF, as indicated by c-Fos expression, would presumably lead to an increase in 5-HT release in mPFC. Although the majority of raphe efferents to the mPFC were not c-Fos positive, indicating a lack of activation (or inhibition) by CRF in this population of raphe neurons, we cannot determine what the net effect on 5-HT release in the mPFC would be, given the ratios of activated vs. non-activated neurons seen in the present study. However, microdialysis studies have shown that both direct application of CRF into the DR, as well as icv CRF, can significantly increase 5-HT and 5-HT metabolite levels in the mPFC [27; 28]. Interestingly, the results of Forster et al. (2006) show that CRF-induced 5-HT release in the mPFC has a gradual onset, with a peak effect occurring approximately 80 min after CRF administration. Although the mechanisms underlying this delayed effect of CRF on 5-HT release in the mPFC are unknown, it is relevant to note that the time-course of this physiological response closely mirrors the behavioral effect of CRF on startle. As shown consistently in a number of reports, the effects of CRF on startle are not immediate, rather, the startle-enhancing effects of CRF (1 μg) begin approximately 30 min after infusion, grow steadily to a peak effect at 60-80 min, and remain elevated for 2-3 hrs at which time they begin to return to baseline [30; 49]. Because CRF in these behavioral studies was usually given icv, it is unknown if these delayed behavioral effects were due to the amount of time required for CRF to infiltrate the brain parenchyma from the ventricular system, or was a function of time-dependent recruitment by CRF of other neurotransmitter systems that were directly responsible for the potentiated response. The results of the present study, in association with the results of Forster et al., (2006), raise the intriguing possibility that CRF-induced activation of specific 5-HT inputs to the mPFC is a rate-limiting step in the expression of anxiety-like behaviors such as CRF-enhanced startle.

What then might be the effect of an increase in 5-HT on mPFC neurons, and how might that be translated into the generation of anxiety-like behaviors? The results from several studies have shown that electrical stimulation of either the DR or MR inhibits a majority of pyramidal neurons in the mPFC [50-52], an effect likely mediated by 5-HT, as the inhibition was reduced by destruction of ascending 5-HT pathways and by systemic administration of 5-HT1a receptor antagonists. Other electrophysiological studies have also demonstrated inhibition of mPFC pyramidal neurons after direct application of 5-HT in vivo [53-55], although excitatory effects of 5-HT on pyramidal neurons have also been seen in vitro [56]. Possible mechanisms for the inhibitory effects of 5-HT on mPFC pyramidal neurons include direct effects, mediated by 5-HT1a receptors, or indirect effects, mediated through activation of GABAergic interneurons via 5-HT2a and/or 5HT-3 receptors [57-59]. Interestingly, in other studies from our laboratory, we have noticed a conspicuous paucity of c-Fos positive cells in the IL and PL subdivisions of the mPFC 150 min after icv CRF administration (Meloni and Carlezon, unpublished results). These observations suggest that the majority of cells in the ventral mPFC, of which pyramidal neurons are the major cell type, are either not involved in CRF-mediated effects (i.e. their cellular activity is not changed), or are actively inhibited as a result of icv CRF administration and putative 5-HT release from raphe afferents. Unfortunately, the use of c-Fos alone cannot distinguish between these two possibilities and future studies are planned to examine the effect of direct intra-mPFC injections of 5-HT agonists/antagonists on CRF-enhanced startle. Although our data show a significant correlation between caudal DR activation and CRF-enhanced startle, it is entirely possible that enhanced 5-HT release in the mPFC may be playing an adaptive role involved in the recovery from stress and anxiety [60; 61]. Hence, these future studies will allow us to better determine if 5-HT release in the mPFC is a mechanism by which CRF elicits its anxiogenic-like behavioral effects, or is involved in the recovery from this heighten anxiety-like state.

5-HT-mediated alterations in the descending excitatory drive from pyramidal neurons in the mPFC to limbic areas such as the extended amygdala (e.g. the central nucleus of the amygdala [CeA] and bed nucleus of the stria terminalis [BNST]) would have profound effects on emotionality. For example, there is strong evidence to suggest that the input from the ventral mPFC to the CeA is an important axis for controlling the extinction of conditioned fear [62]. Furthermore, dysfunction within this particular anxiety-control circuit may underlie the development and/or expression of PTSD [63], a disorder in which CRF has been implicated [64; 65]. Although not yet fully explored, dysregulation of the strong descending input from the IL to the BNST [66], an area known to play a role in the expression of unconditioned anxiety-like behaviors [67], including CRF-enhanced startle [29], could represent another axis of pathology for anxiety disorders. Further studies are clearly needed to help elucidate the functional interactions between CRF and 5-HT occurring with raphe-cortical-limbic circuits. Such studies have the potential to identify new targets and new therapies in the treatment of mood and anxiety disorders.

Supplementary Material




This work was supported by The Stanley Medical Research Institute (to BMC), MH63266 (to WAC) and MH076230 (to EGM).


Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Financial Disclosures

Dr. Meloni reported no biomedical financial interests or potential conflicts of interest. Ms. Reedy reported no biomedical financial interests or potential conflicts of interest. Dr. Cohen reported no biomedical financial interests or potential conflicts of interest. Dr. Carlezon reported no biomedical financial interests or potential conflicts of interest.


1. Owens MJ, Nemeroff CB. The role of corticotropin-releasing factor in the pathophysiology of affective and anxiety disorders: laboratory and clinical studies. Ciba Foundation Symposium. 1993;172:296–308. [PubMed]
2. Heim C, Nemeroff CB. The role of childhood trauma in the neurobiology of mood and anxiety disorders: preclinical and clinical studies. Biological Psychiatry. 2001;49:1023–1039. [PubMed]
3. Boyer P. Do anxiety and depression have a common pathophysiological mechanism? Acta Psychiatr Scanf. 2000;102:24–29. [PubMed]
4. Bakshi VP, Kalin NH. Corticotropin-releasing hormone and animal models of anxiety: gene-environment interactions. Biological Psychiatry. 2000;48:1175–1198. [PubMed]
5. Steckler T, Holsboer F. Corticotropin-releasing hormone receptor subtypes and emotion. Biological Psychiatry. 1999;46:1480–1508. [PubMed]
6. Takahashi LK. Role of CRF1 and CRF2 receptors in fear and anxiety. Neuroscience and Biobehavioral Reviews. 2001;25:627–636. [PubMed]
7. Weiss JM, Stout JC, Aaron MF, Quan N, Owens MJ, Butler PD, et al. Depression and anxiety: role of the locus coeruleus and corticotropin-releasing factor. Brain Research Bulletin. 1994;35:561–572. [PubMed]
8. Koob GF. Corticotropin-releasing factor, norepinephrine, and stress. Biological Psychiatry. 1999;46:1167–1180. [PubMed]
9. Ströhle A, Holsboer F. Stress responsive neurohormones in depression and anxiety. Pharmacopsychiatry. 2003;36(Suppl 3):S207–S214. [PubMed]
10. Mitchell AJ. The role of corticotropin releasing factor in depressive illness: a critical review. Neuroscience and Biobehavioral Reviews. 1998;22:635–651. [PubMed]
11. Arborelius L, Owens MJ, Plotsky PM, Nemeroff CB. The role of corticotropin-releasing factor in depression and anxiety disorders. Journal of Endocrinology. 1999;160:1–12. [PubMed]
12. Ressler KJ, Nemeroff CB. Role of serotonergic and noradrenergic systems in the pathophysiology of depression and anxiety disorders. Depression and Anxiety. 2000;12:2–19. [PubMed]
13. Lowry CA, Moore FL. Regulation of behavioral responses by corticotropin-releasing factor. General and Comparative Endocrinology. 2006;146:19–27. [PubMed]
14. Linthorst ACE. Interactions between corticotropin-releasing hormone and serotonin: implications for the aetiology and treatment of anxiety disorders. Handbook of Experimental Pharmacology. 2005;169:181–204. [PubMed]
15. Ruggiero DA, Underwood MD, Rice PM, Mann JJ, Arango V. Corticotropin-releasing hormone and serotonin interact in the human brainstem: behavioral implications. Neuroscience. 1999;4:1343–1354. [PubMed]
16. Austin MC, Janosky JE, Murphy HA. Increased corticotropin-releasing hormone immunoreactivity in monoamine-containing pontine nuclei of depressed suicide men. Molecular Psychiatry. 2003;8:324–332. [PubMed]
17. Lowry CA, Rodda JE, Lightman SL, Ingram CD. Corticotropin-releasing factor increases in vitro firing rates of serotonergic neurons in the rat dorsal raphe nucleus: evidence for activation of a topographically organized mesolimbic serotonergic system. Journal of Neuroscience. 2000;20:7728–7736. [PubMed]
18. Pernar L, Curtis AL, Vale WW, Rivier JE, Valentino RJ. Selective activation of corticotropin-releasing factor-2 receptors on neurochemically identified neurons in the rat dorsal raphe nucleus reveals dual action. Journal of Neuroscience. 2004;24:1305–1311. [PubMed]
19. Kirby LG, Rice KC, Valentino RJ. Effects of corticotropin-releasing factor on neuronal activity in the serotonergic dorsal raphe nucleus. Neuropsychopharmacology. 2000;22:148–162. [PubMed]
20. de Groote L, Penalva RG, Flachskamm C, Reul JMHM, Linthorst ACE. Differential monoaminergic, neuroendocrine and behavioural responses after central administration of corticotropin-releasing factor receptor type 1 and type 2 agonists. Journal of Neurochemistry. 2005;94:45–56. [PubMed]
21. Kagamiishi Y, Yamamoto T, Watanabe S. Hippocampal serotonergic system is involved in anxiety-like behavior induced by corticotropin-releasing factor. Brain Research. 2003;991:212–221. [PubMed]
22. Price ML, Lucki I. Regulation of serotonin release in the lateral septum and striatum by corticotropin-releasing factor. Journal of Neuroscience. 2001;21:2833–2841. [PubMed]
23. Dunn AJ, Berridge CW. Corticotropin-releasing factor administration elicits a stress-like activation of cerebral catecholaminergic systems. Pharmacology, Biochemistry and Behavior. 1987;27:685–691. [PubMed]
24. Drevets WC, Price JL, Simpson JR, Todd RD, Reich T, Vannier M, et al. Subgenual prefrontal cortex abnormalities in mood disorders. Nature. 1997;386:824–827. [PubMed]
25. Shin LM, Rauch SL, Pitman RK. Amygdala, medial prefrontal cortex, and hippocampal function in PTSD. Annals of the New York Academy of Sciences. 2006;1071:67–79. [PubMed]
26. Vertes RP. a PHA-L analysis of ascending projections of the dorsal raphe nucleus in the rat. Journal of Comparative Neurology. 1991;313:643–668. [PubMed]
27. Forster GL, Feng N, Watt MJ, Korzan WJ, Mouw NJ, Summers CH, et al. Corticotropin-releasing factor in the dorsal raphe elicits temporally distinct serotonergic responses in the limbic system in relation to fear behavior. Neuroscience. 2006;141:1047–1055. [PubMed]
28. Lavicky J, Dunn AJ. Corticotropin-releasing factor stimulates catecholamine release in hypothalamus and prefrontal cortex in freely moving rats as assessed by microdialysis. Journal of Neurochemistry. 1993;60:602–612. [PubMed]
29. Lee Y, Davis M. Role of the hippocampus, bed nucleus of the stria terminalis and amygdala in the excitatory effect of corticotropin releasing hormone (CRH) on the acoustic startle reflex. Journal of Neuroscience. 1997;17:6434–6446. [PubMed]
30. Meloni EG, Gerety LP, Knoll AT, Cohen BM, Carlezon WA. Behavioral and anatomical interactions between dopamine and corticotropin-releasing factor in the rat. Journal of Neuroscience. 2006;26:3855–3863. [PubMed]
31. Hoffman GE, Smith MS, Verbalis JG. c-Fos and related immediate early gene products as markers of activity in neuroendocrine systems. Frontiers in Neuroendocrinology. 1993;14:173–213. [PubMed]
32. Hoffman GE, Lyo D. Anatomical markers of activity in neuroendocrine systems: are we all ‘Fos-ed out’? Journal of Neuroendocrinology. 2002;14:259–268. [PubMed]
33. Paxinos G, Watson C. The rat brain in stereotaxic coordinates. 3rd ed. Academic Press; New York: 1997.
34. Abrams JK, Johnson PL, Hollis JH, Lowry CA. Anatomic and functional topography of the dorsal raphe nucleus. Annals of the New York Academy of Sciences. 2004;1018:46–57. [PubMed]
35. Kovacs KJ. c-Fos as a transcription factor: a stressful (re)view from a functional map. Neurochemistry International. 1998;33:287–297. [PubMed]
36. Mikkelsen JD, Hay-Schmidt A, Larsen PJ. Central innervation of the rat ependyma and subcommissural organ with special reference to ascending serotoninergic projections from the raphe nuclei. Journal of Comparative Neurology. 1997;384:556–568. [PubMed]
37. Simpson KL, Fisher TM, Waterhouse BD, Lin RCS. Projection patterns from the raphe nuclear complex to the ependymal wall of the ventricular system in the rat. Journal of Comparative Neurology. 1998;399:61–72. [PubMed]
38. Lowry CA, Johnson PL, Hay-Schmidt A, Mikkelsen J, Shekhar A. Modulation of anxiety circuits by serotonergic systems. Stress. 2005;8:233–246. [PubMed]
39. Valentino RJ, Liouterman L, Van Bockstaele EJ. Evidence for regional heterogeneity in corticotropin-releasing factor interactions in the dorsal raphe. Journal of Comparative Neurology. 2001;435:450–463. [PubMed]
40. Waselus M, Valentino RJ, Van Bockstaele EJ. Ultrastructural evidence for a role of γ-aminobutyric acid in mediating the effects of corticotropin-releasing factor on the rat dorsal raphe serotonin system. Journal of Comparative Neurology. 2005;482:155–165. [PubMed]
41. DeSouza EB, Insel TR, Perrin MH, Rivier J, Vale WW, Kuhar MJ. Corticotropin-releasing factor receptors are widely distributed within the rat central nervous system: an autoradiographic study. Journal of Neuroscience. 1985;5:3189–3203. [PubMed]
42. Day HEW, Greenwood BN, Hammack SE, Watkins LR, Fleshner M, Maier SF, et al. Differential expression of 5-HT-1A, a1b adrenergic, CRF-R1, and CRF-R2 receptor mRNA in serotonergic, γ-aminobutyric acidergic, and catecholaminergic cells of the rat dorsal raphe nucleus. Journal of Comparative Neurology. 2004;474:364–378. [PMC free article] [PubMed]
43. Van Pett K, Viau V, Bittencourt JC, Chan RKW, Li H-Y, Arias C, et al. Distribution of mRNAs encoding CRF receptors in brain and pituitary of rat and mouse. Journal of Comparative Neurology. 2000;428:191–212. [PubMed]
44. Reyes TM, Lewis MH, Perrin MH, Kunitake KS, Vaughan J, Arias CA, et al. Urocortin II: a member of the corticotropin-releasing factor (CRF) neuropeptide family that is selectively bound by type 2 CRF receptors. Proceedings of the National Academy of Sciences USA. 2001;98:2843–2848. [PMC free article] [PubMed]
45. Lewis K, Li C, Perrin MH, Blount A, Kunitake K, Donaldson C, et al. Identification of urocortin III, an additional member of the corticotropin-releasing factor (CRF) family with high affinity for the CRF2 receptor. Proceedings of the National Academy of Sciences USA. 2001;98:7570–7575. [PMC free article] [PubMed]
46. Amat J, Tamblyn JP, Paul ED, Bland ST, Amat P, Foster AC, et al. Microinjections of urocortin 2 into the dorsal raphe nucleus activates serotonergic neurons and increases extracellular serotonin in the basolateral amygdala. Neuroscience. 2004;129:509–519. [PubMed]
47. Staub DR, Evans AK, Lowry CA. Evidence supporting a role for corticotropin-releasing factor type 2 (CRF2) receptors in the regulation of subpopulations of serotonergic neurons. Brain Research. 2006;1070:77–89. [PubMed]
48. Risbrough VB, Hauger RL, Pelleymounter MA, Geyer MA. Role of corticotropin releasing factor (CRF) receptors 1 and 2 in CRF-potentiated acoustic startle in mice. Psychopharmacology. 2003;170:178–187. [PubMed]
49. Liang KC, Melia KR, Miserendino MJD, Falls WA, Campeau S, Davis M. Corticotropin-releasing factor: long-lasting facilitation of the acoustic startle reflex. Journal of Neuroscience. 1992;12:2303–2312. [PubMed]
50. Hajos M, Gartside SE, Varga V, Sharp T. In vivo inhibition of neuronal activity in the rat ventromedial prefrontal cortex by midbrain-raphe nuclei: role of 5-HT1A receptors. Neuropharmacology. 2003;45:72–81. [PubMed]
51. Puig MV, Artigas F, Celada P. Modulation of the activity of pyramidal neurons in the rat prefrontal cortex by raphe stimulation in vivo: involvement of serotonin and GABA. Cerebral Cortex. 2005;15:1–14. [PubMed]
52. Mantz J, Godbout R, Tassin J-P, Glowinski J, Thierry A-M. Inhibition of spontaneous and evoked unit activity in the rat medial prefrontal cortex by mesencephalic raphe nuclei. Brain Research. 1990;524:22–30. [PubMed]
53. Zhou F-M, Hablitz JJ. Activation of serotonin receptors modulates synaptic transmission in rat cerebral cortex. Journal of Neurophysiology. 1999;82:2989–2999. [PubMed]
54. Tan H, Zhong P, Yan Z. Corticotropin-releasing factor and acute stress prolongs serotonergic regulation of GABA transmission in prefrontal cortical pyramidal neurons. Journal of Neuroscience. 2004;24:5000–5008. [PubMed]
55. Ashby CR, Edwards E, Wang RY. Action of serotonin in the medial prefrontal cortex: mediation by serotonin2-like receptors. Synapse. 1992;10:7–15. [PubMed]
56. Aghajanian GK, Marek GJ. Serotonin induces excitatory postsynaptic potentials in apical dendrites of neocortical pyramidal cells. Neuropharmacology. 1997;36:589–599. [PubMed]
57. Yan Z. Regulation of GABAergic inhibition by serotonin signaling in prefrontal cortex. Molecular Neurobiology. 2002;26:1–14. [PubMed]
58. Puig MV, Santana N, Celada P, Mengod G, Artigas F. In vivo excitation of GABA interneurons in the medial prefrontal cortex through 5-HT3 receptors. Cerebral Cortex. 2004;14:1365–1375. [PubMed]
59. Santana N, Bortolozzi A, Serrats J, Mengod G, Artigas F. Expression of serotonin1A and serotonin2A receptors in pyramidal and GABAergic neurons of the rat prefrontal cortex. Cerebral Cortex. 2004;14:1100–1109. [PubMed]
60. Amat J, Baratta MV, Paul E, Bland ST, Watkins LR, Maier SF. Medial prefrontal cortex determines how stressor controllability affects behavior and dorsal raphe nucleus. Nature Neuroscience. 2005;8:365–371. [PubMed]
61. Deakin JWF, Graeff FG. 5-HT and mechanisms of defence. Journal of Psychopharmacology. 1991;5:305–315. [PubMed]
62. Sierra-Mercado DJ, Corcoran KA, Lebron-Milad K, Quirk GJ. Inactivation of the ventromedial prefrontal cortex reduces expression of conditioned fear and impairs subsequent recall of extinction. European Journal of Neuroscience. 2006;24:1751–1758. [PubMed]
63. Rauch SL, Shin LM, Whalen PJ, Pitman RK. Neuroimaging and the neuroanatomy of PTSD. CNS Spectrums. 1998;3(suppl 2):30–41.
64. Kasckow JW, Baker D, Geracioti TD., Jr. Corticotropin-releasing hormone in depression and post-traumatic stress disorder. Peptides. 2001;22:845–851. [PubMed]
65. Baker DG, West SA, Nicholson WE, Ekhator NN, Kasckow JW, Hill KK, et al. Serial CSF corticotropin-releasing hormone levels and adrenocortical activity in combat veterans with posttraumatic stress disorder. American Journal of Psychiatry. 1999;156:585–588. [PubMed]
66. Vertes RP. Differential projections of the infralimbic and prelimbic cortex in the rat. Synapse. 2004;51:32–58. [PubMed]
67. Walker DL, Toufexis DJ, Davis M. Role of the bed nucleus of the stria terminalis versus the amygdala in fear, stress, and anxiety. European Journal of Neuroscience. 2003;463:199–216. [PubMed]
68. Meloni EG, Davis M. Synergistic enhancement of the acoustic startle reflex by dopamine D1 and 5-HT1A agonists and corresponding changes in c-Fos expression in the dorsal raphe of rats. Psychopharmacology. 2000;151:359–367. [PubMed]
PubReader format: click here to try


Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...


  • Compound
    PubChem Compound links
  • MedGen
    Related information in MedGen
  • PubMed
    PubMed citations for these articles
  • Substance
    PubChem Substance links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...