52 Corticotropin-Releasing Factor and the Brain Norepinephrine System E J Van Bockstaele, Thomas Jefferson University, Philadelphia, PA, USA R J Valentino, The Children’s Hospital of Philadelphia, Philadelphia, PA, USA ß 2009 Elsevier Inc. All rights reserved.Chapter Outline52.1 Introduction 166952.2 The LC–Norepinephrine System 167052.2.1 Anatomical Attributes 167052.2.2 Physiological Attributes 167052.3 CRF, Stress, and the LC–Norepinephrine System 167152.3.1 Activation of LC Neurons by Stress 167152.3.2 Activation of LC Neurons by CRF 167252.3.3 CRF Afferents to the LC 167352.3.4 Co-Transmitters in CRF Afferents to the LC 167522.214.171.124 Glutamate 1675126.96.36.199 Opioids 1676188.8.131.52 Dynorphin 1678184.108.40.206 Gamma-amino butyric acid 167852.4 Plasticity of CRF–Norepinephrine Interactions 167852.4.1 CRF Receptors in LC 167852.4.2 Agonist and Stress-Induced Internalization of LC Neurons 167952.4.3 Structural Plasticity Following Stress 168152.5 Determinants of LC Activity 168152.6 Conclusions 1682References 168352.1 Introduction and ultimately, resulting in corticosteroid secretion (Rivier et al., 1982; Vale et al., 1981). Since thatThe mammalian stress response is a multicom- discovery, multiple lines of evidence have furtherponent response that includes an endocrine limb implicated CRF as a brain neurotransmitter that(hypothalamic–pituitary–adrenal axis activation), an may facilitate other limbs of the stress response. Forautonomic limb (cardiovascular and gastrointestinal example, a number of autonomic, behavioral, andalterations), an immunological limb (immunosup- immunological changes engaged during the stresspression), a behavioral limb (anxiogenic responses), response (Selye and Horava, 1953) were demonstra-and a cognitive limb (enhanced arousal, alterations ted to occur in hypophysectomized animals, arguingin attention, and assessment of the environment). The against a sole role for pituitary–adrenal activation inprecise coordination of these limbs of the stress these effects (Owens and Nemeroff, 1991). Likewise,response is critical in adapting to physiological and centrally administered CRF reproduced many of theemotional challenges. Evidence over the last 25 years autonomic and behavioral responses to stress even inhas suggested that this coordination is performed hypophysectomized rats (Britton et al., 1986a,b; Dunnby the neuropeptide, corticotropin-releasing factor et al., 1987; Eaves et al., 1985; Fisher et al., 1983).(CRF), acting as a neurohormone and brain neuro- Importantly, central administration of CRF anta-transmitter. CRF was initially characterized as the gonists prevented similar autonomic and behavioralhypothalamic neurohormone that initiates the endo- stress-elicited effects, emphasizing a role for cen-crine limb of the stress response, causing secretion tral CRF in these nonendocrine aspects of stress.of adrenocorticotropin from the anterior pituitary Together, these findings contributed to a more global 1669
1670 Corticotropin-Releasing Factor and the Brain Norepinephrine Systemview of CRF acting as an orchestrator of the multi- Swanson and Hartman, 1975; Waterhouse et al., 1998).component stress response. A certain topographical organization of neurons within Evidence exists for CRF neurotransmission in the LC has been described with respect to neurochem-many brain regions, for example, dorsal raphe nucleus, icals that are co-localized with norepinephrine, as wellthe lateral septum, and dorsal motor vagal nucleus as efferent targets (Loughlin et al., 1986; Mason andamong others (Bittencourt and Sawchenko, 2000; Fibiger, 1979). Nonetheless, it is relatively homogeneousBittencourt et al., 1999; Chalmers et al., 1995; in that all neurons synthesize norepinephrine and manyDautzenberg and Hauger, 2002; Lovenberg et al., have collateralized projections to functionally diverse1995; Makino et al., 1999; Perrin and Vale, 2002; targets. This feature, along with the homogeneousSauvage and Steckler, 2001; Ungless et al., 2003; Van physiological characteristics of the LC, gives the systemPett et al., 2000). However, the brain norepinephrine the potential to broadly influence neuronal activitynucleus, locus ceruleus (LC), was one of the first throughout the brain.brain regions in which a confluence of evidence sup-ported this role. It remains to be one of the most well- 52.2.2 Physiological Attributescharacterized examples of how endogenous CRF isreleased during stress to influence activity of neurons Putative functions of the LC–norepinephrine systemof a specific system to produce effects that are adaptive have been hypothesized based on neuronal dischargein response to a stressor. In addition, because of the characteristics, particularly those recorded in behav-role of this system in psychiatric disorders, our knowl- ing animals. Importantly, LC neurons are spontane-edge of the impact of CRF on the LC–norepinephrine ously active and discharge in both tonic and phasicsystem provides a window into how stress may lead to modes (see, for review, Aston-Jones and Cohenpsychiatric disturbances. (2005)). Tonic discharge is related to the state of This chapter reviews the salient anatomical and arousal such that increases in tonic activity arephysiological features of the LC that form the basis of positively correlated with and sufficient to activateits proposed functions. This is followed by a consid- forebrain electroencephalographic (EEG) activityeration of physiological and anatomical evidence (Aston-Jones and Bloom, 1981; Berridge and Foote,supporting a role for CRF neurotransmission in the 1991). Conversely, decreases in LC tonic activityLC. Emerging from these multidisciplinary studies is are associated with slow-frequency forebrain EEG.a picture of complex interactions between the stress- This was well demonstrated in studies in whichrelated neuropeptide CRF, endogenous opioids, and selective LC inactivation by clonidine was sufficientthe excitatory amino acid neurotransmitter, gluta- to change high-frequency cortical EEG activity tomate. The net effect of these interactions is posited high-amplitude, low-frequency activity, indicativeto adjust the activity and reactivity of the LC– of sedation (Berridge et al., 1993). LC neurons arenorepinephrine system such that state of arousal phasically activated by multimodal sensory stimuliand processing of sensory stimuli are modified to (Aston-Jones and Bloom, 1981; Foote et al., 1980).facilitate adaptive behavioral responses to stressors. Microdialysis studies correlating LC discharge rate and pattern to extracellular forebrain norepinephrine predict that this phasic mode of discharge results in52.2 The LC–Norepinephrine System more efficient norepinephrine release in terminal fields (Florin-Lechner et al., 1996). In the phasic mode,52.2.1 Anatomical Attributes LC neurons discharge in a synchronous manner,The LC is a primary source of norepinephrine in the that is, en masse, and this has been attributed to elec-forebrain and the sole source of norepinephrine trotonic coupling through gap junctions between den-in the cortex and hippocampus, regions that govern drites outside the nucleus, in the peri-cerulear regioncognition, memory, and complex behaviors (Foote (Ishimatsu and Williams, 1996). In contrast to phasicet al., 1983). A distinguishing feature of the LC is the discharge, the tonic mode of discharge is favored bybroad-reaching, highly collateralized projection system uncoupling and this has been verified in computationalthat arises from this compactly clustered nucleus of models (Usher et al., 1999).norepinephrine-containing neurons in the pons that Tonic and phasic LC discharge modes are inter-have been described extensively and are the subject of related in that the level of tonic LC discharge deter-several reviews (Aston-Jones et al., 1995; Foote et al., mines the magnitude of the phasic response. Thus,1983; Grzanna and Molliver, 1980; Swanson, 1976; phasic responses to sensory stimuli are optimal at
Corticotropin-Releasing Factor and the Brain Norepinephrine System 1671 Phasic, coupled, focused (excitatory amino acid) Low tonic High tonic, low phasic, low phasic uncoupled, disengaged increased arousal, scanning, sampling LC discharge rate µ-opiate CRF Stress termination StressFigure 1 Schematic depicting different modes of LC activity and the relationship between tonic and phasic activity.Representative poststimulus time histograms (PSTHs) are shown that indicate neuronal activation (ordinates) in response torepeated presentations of a brief sensory stimulus (arrowhead). The abscissae of the histograms indicate time before andafter the stimulus. When locus ceruleus tonic activity is low (left), the response to sensory stimuli is relatively low and this isassociated with drowsiness and disengagement from the environment. There is an optimal level of tonic LC discharge rate atwhich the response to sensory stimuli is high (phasic). This is associated with electrotonic coupling of LC neurons, focusedattention, and the maintenance of ongoing behavior. Increases in tonic discharge rate above this optimal level result in a lossof selective sensory responses. This is associated with uncoupling, increased arousal, scanning attention, and sampling ofbehaviors. By increasing tonic discharge, CRF shifts the mode of LC activity toward the right of this spectrum. This ispredicted to occur during stress. During stress termination, endogenous opioids acting at opiate receptors in the LC shift themode of activity toward the left. Excitatory amino acid neurotransmission in the LC favors the phasic mode. Reproduced fromValentino RJ and Van Bockstaele EJ (2008) Convergent regulation of locus coeruleus activity as an adaptive response tostress. European Journal of Pharmacology 583: 195–203, with permission from Elsevier.moderate levels of tonic activity and diminished modes of neuronal activity would be advantageouseither when tonic activity is low (as in slow wave for rapidly adjusting behavior in response to asleep) or when tonic activity exceeds a moderate stressor or after stressor termination (see below).level (as in stress) (Aston-Jones et al., 2000; Usheret al., 1999; Valentino and Foote, 1987). This supportsthe concept that the LC promotes and maintains 52.3 CRF, Stress, andan appropriate level of arousal that is necessary for the LC–Norepinephrine Systemoptimally processing sensory information in the 52.3.1 Activation of LC Neurons by Stressenvironment. During times of elevated tonic LCactivity, as in stress (see below), the loss of the phasic Activation of the LC–norepinephrine system is asensory response may signify a shift toward scanning reliable and consistent component of the stressdiverse stimuli in the environment, a response that response that has been measured using several differ-may be most adaptive in a challenging environ- ent endpoints. Early studies (using shock) demon-ment. Based upon recordings in awake behaving strated stress-induced increases in norepinephrinemonkeys and computational modeling, Aston-Jones metabolites and turnover in LC forebrain targetsand Cohen (2005) have proposed a new model of that were abolished following LC lesions (CassensLC function that is more behaviorally than sensory et al., 1980, 1981; Korf et al., 1973; Thierry et al.,based. This model posits that phasic LC discharge 1968). Importantly, unpredictable shock was morefacilitates behavioral responses to specific ongoing effective than shock that was signaled by stimulitasks (Figure 1). In contrast, a shift toward higher (Tsuda et al., 1989). Increases in norepinephrine uti-tonic activity and diminished phasic activity is pro- lization in brain regions targeted by LC projectionsposed to increase sensitivity to stimuli irrelevant following immunological challenge produced byto ongoing tasks, resulting in disengagement and to interleukin-2 injection emphasized the generalizedpromote searching for alternative tasks that may pro- nature of the activation (Lacosta et al., 2000). Micro-vide better solutions in a dynamic environment or dialysis measurements of norepinephrine efflux inwhen present behavior is not optimally adaptive. LC target regions have corroborated norepinephrineThe ability of LC neurons to readily switch between turnover studies. Thus, restraint, tail shock, auditory
1672 Corticotropin-Releasing Factor and the Brain Norepinephrine Systemstress, and hypotensive stress all increase extra- and in unanesthetized rats where auditory stimulicellular norepinephrine levels in LC terminal regions were used. The shift in LC activity toward a high(Abercrombie and Jacobs, 1988; Britton et al., 1992; tonic mode produced by CRF is consistent withSmagin et al., 1995). Many of these stimuli have interruption of ongoing behavior and a shift towardbeen demonstrated to increase LC neuronal activity, a search for alternative strategies that may be optimalincluding hypotension (Svensson, 1987) and tail for coping with the stressor, an important cognitiveshock (Abercrombie and Jacobs, 1988). Additional aspect of the stress response (Figure 1).endpoints of activation of the LC–norepinephrine The cellular mechanisms by which CRF attenu-system (i.e., expression of tyrosine hydroxylase, ates LC phasic activity may relate to its relationshipc-fos expression) have also been demonstrated indicat- with glutamate, a prominent neurotransmitter ining that activation of this system by stressors appears the LC that mediates its activation by sensory sti-to be as consistent as activation of the hypothalamic– muli (Ennis and Aston-Jones, 1988). Co-localizationpituitary–adrenal axis (Beck and Fibiger, 1995; Bonaz of CRF with glutamate in axon terminals in the LCand Tache, 1994; Campeau and Watson, 1997; Chan and convergence of individual CRF- and glutamate-and Sawchenko, 1995; Chang et al., 2000; Dun et al., containing axon terminals onto common LC den-1995; Duncan et al., 1993; Funk and Amir, 2000; drites provide a structural basis for CRF attenuationGraham et al., 1995; Ishida et al., 2002; Kollackwalker of LC phasic responses (Valentino et al., 1998) (seeet al., 1997; Makino et al., 2002; Rusnak et al., 2001; below). Importantly, differential effects of these twoSabban and Kvetnansky, 2001; Smith et al., 1991, 1992). major LC afferent inputs to the LC provide a mecha- nism for rapidly changing the mode of LC activity and thereby behavior. It is tempting to speculate that52.3.2 Activation of LC Neurons by CRF glutamate inputs engaged by sensory stimuli put theSeveral lines of evidence have demonstrated that CRF LC in a phasic mode that facilitates focused attentionadministration activates LC neurons. CRF infusions and engagement in ongoing tasks (Figure 1). In con-into the lateral ventricle increase markers of cellular trast, CRF inputs activated by stressors wouldactivation (c-fos expression) in the LC (Bittencourt and increase tonic activity, disable the influence of gluta-Sawchenko, 2000). Like stress, CRF increases tonic LC mate, disrupt selective responding to sensory stimuli,discharge and it is significantly more potent when and favor sampling of alternate behaviors in the envi-administered directly into the nucleus as compared to ronment. The net effect of this switch in neuromodu-intracerebroventricularly pointing to a site of action lator influence on the LC would be adaptive in adirectly in the nucleus (Curtis et al., 1997; Valentino threatening environment (Figure 1).and Foote, 1988; Valentino et al., 1983). In vitro studies The LC–norepinephrine system is not under basalin LC slice preparations support this view, demonstrat- regulation by endogenous CRF, because administra-ing that CRF increases LC discharge even when syn- tion of CRF receptor antagonists has no effect onaptic activity is blocked ( Jedema and Grace, 2004). As LC discharge rate or norepinephrine release in tar-with other agents that increase LC tonic activity, LC gets in unstressed rats (Curtis et al., 1994; Page andactivation by CRF is temporally correlated with cor- Abercrombie, 1999). In contrast, evidence for basaltical EEG desynchronization (Curtis et al., 1997). CRF release in the LC is apparent in adrenalecto-Moreover, cortical EEG desynchronization and induc- mized rats, which have higher LC discharge ratestion of hippocampal theta rhythm produced during compared to sham-operated rats (Pavcovich andhypotensive stress were selectively blocked by local Valentino, 1997). Administration of CRF receptoradministration of CRF antagonists into the LC (Page antagonists into the LC normalizes this difference,et al., 1993). These findings suggest that one conse- suggesting that basal levels of corticosteroids nor-quence of CRF-induced increase in LC tonic activity is mally restrain CRF release within the LC. This hasto increase or maintain arousal during stress. important implications for conditions in which In addition to increasing LC tonic discharge, CRF corticosteroid receptors may be dysfunctional (e.g.,attenuates LC phasic responses to sensory stimuli depression), in which case LC activity would be(Valentino and Foote, 1987, 1988). The net effect of predicted to be tonically elevated.CRF on LC discharge is to decrease the ratio of In contrast to the unstressed state, substantial evi-phasic-to-tonic activity (i.e., the signal-to-noise dence exists for endogenous CRF release in the LCratio). This effect was observed in anesthetized rats, during stress. The best example of this is duringusing sciatic nerve stimulation to evoke LC discharge, hypotensive challenge, which mimics the effects of
Corticotropin-Releasing Factor and the Brain Norepinephrine System 1673CRF on tonic and phasic LC discharge (Valentinoand Wehby, 1988a). As blood pressure decreases, LCdischarge rate increases and this is temporally corre-lated to forebrain EEG activation, underscoring thefunctional relevance of this effect. Phasic sensory-evoked activity is inhibited at this time, presumablykeeping the LC in an elevated tonic mode that wouldpromote heightened arousal and scanning attention.These effects are completely prevented by prioradministration of a CRF receptor antagonist intothe LC (Curtis et al., 2001; Valentino et al., 1991; (a)Valentino and Wehby, 1988a). Taken with the findingthat determinants of LC sensitivity to CRF (e.g., priorstress history, opiate administration, and sex) simi-larly affect sensitivity to hypotensive challenge, theresults support the idea that hypotensive stressengages CRF release into the LC to affect the activityof this system and thereby forebrain neuronal activity(Curtis et al., 1995, 1999, 2006; Xu et al., 2004). Theseconvergent findings provided some of the strongestevidence to date for a neurotransmitter role ofCRF in the brain. Analogous electrophysiological (b)studies support a neurotransmitter role for CRF in Figure 2 (a) Immunofluorescence photomicrographthe LC during colon distention (Lechner et al., 1997). showing CRF immunoreactivity in the nuclear core andHandling-induced increases in norepinephrine peri-erulear regions of the LC. Arrows indicate dorsal (D)efflux have also been attributed to CRF neurotrans- and lateral (L) orientation of tissue section. pLC, peri-LC;mission in the LC (Kawahara et al., 2000). Finally, cLC, nuclear core of LC; IV, fourth ventricle. (b) Electron photomicrograph showing a peroxidase-labeled axonrecent evidence for swim stress-induced CRF receptor terminal for CRF containing small and large dense-coreinternalization (Reyes et al., 2008b) that can be attenu- vesicles (dcv) that forms an asymmetric-type (curved blackated by pretreatment with a CRF antagonist indicates arrow) synapse with a dendrite containing immunogoldthat this stressor also elicits CRF release to directly silver labeling for tyrosine hydroxylase (TH). (b) Reproducedaffect LC neurons. Thus, diverse conditions that can be from Van Bockstaele EJ, Colago EEO, and Valentino RJ (1998) Amygdaloid corticotropin-releasing factor targetsdefined as stressors activate the LC via CRF. locus coeruleus dendrites: Substrate for co-ordination of emotional and cognitive limbs of the stress response. Journal of Neuroendocrinology 10: 743–757, with52.3.3 CRF Afferents to the LC permission from Blackwell Publishing.The circuitry by which endogenous CRF communi-cates with the LC is being revealed through ultra- synaptic specializations with LC dendrites suggeststructural and functional neuroanatomical studies. an indirect modulation of LC activity via presynapticAlthough CRF-immunoreactive fibers innervate the actions. This may be one anatomical substrate for thenuclear core of the LC, the peri-erulear regions into regulation of LC sensory responses.which LC dendrites extend are more densely inner- CRF afferents to the LC region are topographi-vated (Figure 2(a)) (Valentino et al., 1992). Electron cally organized, with neurons from certain CRF-microscopic analyses of this region identified anato- containing nuclei terminating in the nuclear coremical substrates for both direct and indirect actions of of the LC and others terminating in specific peri-CRF on LC neurons (Van Bockstaele et al., 1996). erulear fields (Figure 3). Sources of CRF afferents toThus, identified synaptic specializations between the nuclear LC included the nucleus paragigantocel-CRF-immunoreactive axon terminals and LC den- lularis (PGi) in the ventrolateral medulla, the para-drites, the majority of which are asymmetric (excit- ventricular nucleus of the hypothalamus (PVN) andatory type), indicate a direct modulation of LC Barrington’s nucleus (Reyes et al., 2005; Valentinoactivity (Figure 2(b)). In addition, CRF terminals et al., 1992, 1996; Van Bockstaele et al., 1998a, 1999).that are apposed to unlabeled terminals that form With respect to afferents from the PVN, retrograde
1674 Corticotropin-Releasing Factor and the Brain Norepinephrine System Coordination of emotional stressors PAG Hypothalamus Amygdala (autonomic) (behavior) Forebrain (cognitive) BNST PVN Barrington’s PGi NTS nucleus Thoracic spinal cord Sacral spinal cord Ventral medulla (sympathetic) (parasympathetic) Thoracic spinal cord (sympathetic) Coordination of physiological stressorsFigure 3 Schematic depicting the localization of CRF afferents to the LC and sites of termination in the LC region.CRF neurons from Barrington’s nucleus (Bar), paraventricular nucleus of the hypothalamus (PVN), and the nucleusparagigantocellularis (PGi) terminate in the nuclear core of the LC. Via projections to preganglionic parasympathetic andsympathetic neurons, these nuclei may coordinate autonomic activity with cognitive functions. The central nucleus of theamygdala and bed nucleus of the stria terminalis (BNST) terminate outside of the nuclear core in the peri-erulear region whereLC dendrites extend. These regions may provide more highly processed information and coordinate responses to emotionalstressors.transport studies indicated that a subset (30%) of the These results suggested that CRF is a major neuro-LC-projecting PVN neurons were CRF immunore- mediator in the amygdalar–LC pathway while it mayactive but few (2%) were enkephalin (ENK) immu- play less of a role in the BNST–LC circuit.noreactive (Reyes et al., 2005). This stands in contrast Different CRF afferents are engaged by distinctto medullary afferents that provide a major source stimuli to activate the LC (Figure 3). For example,of ENK innervation to the LC, but not a signifi- LC neurons are activated by non-noxious levels ofcant CRF projection (Drolet et al., 1992; Valentino colon distention and this is mediated by CRF releaseet al., 1992). from Barrington’s nucleus neurons that project to the Studies examining inputs to the peri-LC region, nuclear LC (Kosoyan et al., 2005; Lechner et al., 1997;where CRF terminals are most dense, label a distinct Rouzade-Dominguez et al., 2001). Many of the sameset of CRF afferents that include the central nucleus LC-projecting Barrington’s nucleus neurons alsoof the amygdala (CeA) and the bed nucleus of the stria project to the parasympathetic preganglionic neuronsterminalis (BNST) (Pickel et al., 1995; Van Bockstaele in the lumbosacral spinal cord that innervate theet al., 1998b). Most of these LC projections have been pelvic viscera. Thus, by engaging CRF-containingfurther examined at the ultrastructural level and neurons in Barrington’s nucleus, pelvic visceral sti-terminals arising from the CeA and BNST form syn- muli such as colon distention can initiate a coordi-aptic specializations with LC dendrites in the rostro- nated response composed of visceral and cognitivelateral peri-LC. A semi-quantitative analysis revealed components. In contrast to Barrington’s nucleus, lim-that the percentage of terminals from the CeA and bic sources of CRF that project to the rostrolateralBNST contacting LC dendrites in this region was peri-LC may underlie activation by emotional orcomparable (20%). However, a greater percentage external stressors. The CeA coordinates autonomic(35% vs. 13%) of CeA terminals were CRF immuno- and behavioral responses to emotional stimuli (e.g.,reactive in comparison to the BNST–LC pathway conditioned fear) via projections to the lateral hypo-(Pickel et al., 1995; Van Bockstaele et al., 1998b). thalamus and central gray, respectively (LeDoux
Corticotropin-Releasing Factor and the Brain Norepinephrine System 1675et al., 1988). CRF-containing projections from the and behavioral symptoms observed in many stress-CeA to LC dendrites in the rostrolateral peri-LC related disorders (e.g., functional bowel syndrome)may serve as a cognitive limb of this fear response (Valentino et al., 1999). Similar dual retrogradethat is expressed as enhanced arousal and altered tracing approaches from the LC and median emi-attention. Interestingly, distinct roles for the CeA nence revealed that CRF neurons of the PVN thatand BNST have been postulated in fear responses, project to the LC are not those that project to thewith the former being engaged during conditioned median eminence to initiate adrenocorticotropinfear and the latter during unconditioned fear ( Jasnow release in response to stress. This indicated that theet al., 2004; Liang et al., 1992). The ultrastructural neurohormone and neuromodulator actions of CRFstudies described above would suggest that both could be independently engaged (Reyes et al., 2005).nuclei (and therefore both conditions) impact onthe LC, but that the qualitative nature of the impact 52.3.4 Co-Transmitters in CRF Afferentsmay differ because of the different neurochemical to the LCnature of the projections. In addition to its role infear responses, the CeA coordinates information from Information about potential mechanisms by whichthe cardiovascular system and other viscera with CRF can interact with other neuropeptides and/orbehavior (Saha, 2005). Thus, it is not surprising that classical neurotransmitters is steadily emerging. CRF-CRF-containing neurons of the CeA are integral to containing nerve terminals that innervate the LCLC activation by hypotensive challenge (Curtis et al., often contain other nonlabeled dense-core vesicles2002). Together, the anatomical findings suggest that and smooth clear vesicles, indicating that multipledifferent stimuli may engage selective CRF afferents neurochemicals are present within individual affer-that terminate in specific subfields of the LC with ents. The identity of the co-localized neurotransmittersmore highly processed afferents terminating in the is of interest, as the impact of CRF may be augmentedperi-LC versus the nuclear core (Van Bockstaele or modulated by corelease of another transmitter.et al., 1998a,b). Pontine-medullary CRF projections A number of neuroanatomical studies have attemptedto the LC coordinate cognitive and autonomic to identify these co-localized neurotransmitters andresponses to internal physiologic challenges, while the functional implications of their-co-existence islimbic CRF projections mediate LC activation by discussed more extensively below (Figure 4).external stressors having emotional content (Koob,1999; Makino et al., 1999). Another consideration is 220.127.116.11 Glutamatethat manipulations that affect dendritic length will Consistent with the majority of CRF axon terminalshave the power to bias LC communication with affer- forming excitatory-type synaptic specializations withents terminating within the nuclear LC or in the LC dendrites, there is a substantial degree of co-pericoerulear region depending on whether they localization of CRF and glutamate in axon terminalsdecrease or increase dendritic length, respectively. in the LC region (Valentino et al., 2001). In additionAn example of this type of structural plasticity is to co-localization, individual CRF- and glutamate-discussed below. immunoreactive terminals were often apposed to Retrograde labeling from the LC combined with each other or found to converge onto common den-other targets of CRF-containing nuclei has been drites in the rostrolateral LC dendritic zone.used to identify functions that might be coordinated As discussed above, these structural interactionswith LC activation during stress. For example, this may be important mechanisms by which CRF attenu-approach demonstrated that many CRF neurons ates LC phasic activity (which is thought to bein Barrington’s nucleus diverge to simultaneously mediated by glutamate afferents).innervate the LC and the preganglionic column of Another interesting possibility is that CRF andthe lumbosacral spinal cord that provides parasym- glutamate are differentially released from the samepathetic input to the pelvic viscera (Valentino et al., afferents at different times, to produce distinct effects1996). Because it is positioned to coregulate the on LC discharge. For example, an initial stimulusLC–norepinephrine system with the lumbosacral may elicit only glutamate release and favor phasicspinal parasympathetic system, this particular CRF- LC activation. The same stimulus, if sustained, maycontaining circuit may serve to coordinate central elicit CRF release and favor tonic activation. Thewith visceral responses to pelvic visceral stimuli and mode of LC discharge may then toggle betweenmay underlie the comorbidity of pelvic visceral tonic and phasic modes depending on the type and
1676 Corticotropin-Releasing Factor and the Brain Norepinephrine System (a) (b) Co-transmitters in LC afferents x/y CRF ENK Dynorphin Glutamate GABA POMC CRF - 12% [b] 35% [f] 30% [a] 5% [a] 33% [c] Glutamate 13% [a] 43% [d] 48% [d] - ne ne GABA 6% [a] nc [e] ne ne - ne ENK 16% [b] - 10% [f] 28% [d] 38% [e] ne POMC 27% [c] ne ne ne ne - nc: not counted; ne: not examined [a] Valentino et al. (2001). [b] Tjoumakaris et al. (2003). [c] Reyes et al. (2006). [d] Barr and Van Bockstaele (2005). [e] Van Bockstaele et al. (2000). [f] Reyes et al. (2008a).Figure 4 Examples of co-transmitters in CRF afferents to the LC. (a) Electron photomicrograph of an axon terminalcontaining peroxidase labeling for CRF and immunogold silver labeling for POMC (POMC + CRF-t). The dually labeledaxon terminal directly contacts a dendrite (d). (b) An axon terminal containing peroxidase labeling for ENK and immunogoldsilver labeling for CRF targets an unlabeled dendrite (uD). An unlabeled terminal (ut) that contains large dense-corevesicles (dcv) provides convergent input onto the same. Table summarizing percentages of co-transmitters in LCafferents. Percentages are shown with respect to total number of profiles for neurotransmitters in the vertical column (e.g.,ENK/total CRF sampled = 12%). (a) Reproduced from Reyes BA, Glaser JD, Magtoto R, and Van Bockstaele EJ (2006)Pro-opiomelanocortin colocalizes with corticotropin-releasing factor in axon terminals of the noradrenergic nucleus locuscoeruleus. European Journal of Neuroscience 23(8): 2074, with permission from Blackwell Publishing. (b) Reproduced fromTjoumakaris SI, Rudoy C, Peoples J, Valentino RJ, and Van Bockstaele EJ (2003) Cellular interactions between axon terminalscontaining endogenous opiod peptides or corticotropin-releasing factor in the rat locus coeruleus and surrounding dorsalpontine tegmentum. Journal of Comparative Neurology 466: 450, with permission from Wiley-Liss Inc.duration of stimulus. CRF receptors in other brain activity of the LC–norepinephrine system and thatregions such as the amygdala or septum have been interact with CRF to do so. b-Endorphin, one of thereported to decrease and increase, respectively, glu- first endogenous opioids identified in brain, derivestamate transmission via a postsynaptic mechanism from pre–proopiomelanocortin, the same precursor(Liu et al., 2004). Finally, the possibility of presynaptic as that which gives rise to adrenocorticotropinregulation of glutamate exists, based on evidence (Hughes et al., 1980). Moreover, the most consistentfor CRF1 receptors on asymmetric (excitatory-type) means for eliciting endogenous opioid release inaxon terminals in the LC (Reyes et al., 2006). brain is through stressor or pain presentation (Drolet et al., 2001). The potential for afferent regulation18.104.22.168 Opioids of the LC by endogenous opioids is supported byEndogenous opioids represent another class of stress- anatomical evidence and the effect of opiates on LCrelated neuromodulators that are poised to regulate neuronal activity. Proopiomelanocortin (POMC), the
Corticotropin-Releasing Factor and the Brain Norepinephrine System 1677precursor that yields the potent bioactive peptide, animals, a component of which involves excitatoryb-endorphin, coexists (27%) with CRF in afferents amino acid afferents to the LC (Akaoka and Aston-to the LC (Figure 4(a)). This co-localization is Jones, 1991; Rasmussen and Aghajanian, 1989). How-observed primarily in the ventromedial aspect of the ever, stress elicits release of endogenous opioids thatLC with little evidence of co-localization in the dorso- act on m-opioid receptors in the LC and this islateral aspect of the LC. This stands in contrast to the unmasked by naloxone administration. This was firstdistribution of ENK afferents to the LC. ENK densely reported by Abercrombie and Jacobs (1988) whoinnervates the nuclear core of LC neurons and is demonstrated that systemic naloxone increased LCrobust in dorsolateral peri-cerulear dendritic zones, discharge rates of opiate-naive, stressed rats, at dosesparticularly at the level of the rostral LC where its that had no effect in unstressed rats (Abercrombiedistribution overlaps that of CRF (Tjoumakaris et al., and Jacobs, 1988). Because naloxone was administered2003; Van Bockstaele et al., 1995, 2000; Van Bockstaele systemically in this study, it was not clear whether theand Chan, 1997). ENK-immunoreactive terminals effects were due to antagonism of opioids within theform synaptic specializations with TH-immunoreactive LC or elsewhere in the brain. More recent studies,dendrites (Van Bockstaele et al., 1995, 1998a). Dual using hypotensive stress, provided evidence for endo-immunoelectron microscopy studies have demon- genous opioid release in the LC and revealed that anstrated that ENK and CRF are co-localized in a important function of this is to reset the level andsubset of axon terminals (16%) in the LC region mode of neuronal activity upon stressor termination(Figure 4(b)) and these contact TH-immunoreactive (Curtis et al., 2001). With the termination of hypo-dendrites (Tjoumakaris et al., 2003). Individual ENK tensive stress, LC discharge rapidly declines to levelsand CRF-immunolabeled axon terminals also con- that are below baseline for up to 15min, prior toverge onto LC dendrites, offering another mechanism returning to prestress levels. Microinfusion of nalox-by which these two peptides can interact. one into the LC prevents poststressor inhibition In vivo, m-opioid receptor activation in the LC without affecting LC activation during the stress. Inproduces effects that are directly opposite to those naloxone-pretreated rats, LC discharge remains ele-of CRF. The predominant action is inhibition of vated for an extended time after the termination oftonic activity and at certain doses, tonic discharge is the hypotensive episode and did not decrease belowselectively decreased and phasic activity is unaf- baseline levels. These findings suggest that conditionsfected, resulting in an increased signal-to-noise of in which the opioid influence is compromised, thethe sensory-evoked response (Valentino and Wehby, brain norepinephrine system would be elevated for1988b). The net effect of m-opioid receptor activation a more prolonged period of time after stress ter-in the LC is a shift in the mode of activity in a mination. Taken with findings that CRF receptordirection opposite to CRF. If baseline tonic activity antagonists selectively prevent LC activation duringis relatively high, engaging m-opioid receptors would hypotensive stress, but not poststress inhibition, thefavor a phasic mode of activity (Figure 1). Consistent studies suggest that stress engages both CRF andwith this, multichannel recordings in LC demon- opioid afferents to the LC, which have opposingstrate that in addition to decreasing LC discharge influences on activity. CRF release during stressrate, morphine induces synchronous oscillations in should bias tonic activity and promote arousal, scan-LC neurons that are prevented by both naloxone and ning of the environment, and sampling behavioralexcitatory amino acid receptor antagonists (Zhu and strategies. Opioid release during stress terminationZhou, 2001, 2005). Thus, whereas CRF biases LC should shift LC activity away from the high tonicactivity toward a tonic mode to promote heightened mode and toward a phasically driven mode (Figure 1).arousal and behavioral sampling, m-opiates bias activ- The poststress inhibitory opioid influence may serveity toward the phasic mode to promote focused atten- to counterbalance the excitatory effects of CRF in antion and maintenance of ongoing behavior (Figure 1). effort to facilitate termination of the brain noradren- Like CRF, opioids are not tonically released to ergic stress response (Curtis et al., 2002; Tjoumakarisregulate LC activity because pure opiate receptor et al., 2003; Valentino and Van Bockstaele, 2001).antagonists have no effect on LC discharge of previ- An implication of the opposing CRF and opioidously untreated rats (Valentino and Wehby, 1989). influences on LC activity is that changes in the sen-This stands in contrast to the dramatic activation of sitivity to one neuromodulator will alter sensitivityLC neurons produced by systemic administration to the other. A test of this hypothesis revealed thatof opiate receptor antagonists to opiate-dependent chronic opiate administration selectively increased
1678 Corticotropin-Releasing Factor and the Brain Norepinephrine Systemthe sensitivity of LC neurons to CRF (Xu et al., 2004). By specifically activating neurons that give rise toThis was apparent as an increased magnitude of acti- these terminals, LC activity may be modulated via co-vation produced by microinfused CRF, as well as by release of DYN and CRF. A source of DYN/CRFstress. More importantly, sensitization of LC neurons afferents to the LC is the CeA as dual in situ hybridiza-to CRF in chronic morphine rats translated to a tion labeling showed that in CeA neurons, 31% ofchange in the behavioral repertoire-treated in DYN mRNA-positive cells co-localized with CRFresponse to a challenge (Xu et al., 2004). Thus, expo- mRNA while 53% of CRF mRNA containing cellssure of morphine-dependent rats to swim stress co-localized with DYN mRNA (Reyes et al., 2008a).resulted in excessive climbing at times when control Moreover, electrolytic lesions of the CeA result in arats would adopt a passive coping strategy of immo- significant decrease in DYN immunoreactivity in thebility. Climbing behavior in this model has been LC on the side of the lesion (Reyes et al., 2008a). Theselinked to activation of the brain norepinephrine sys- data are consistent with other reports that have showntem (Detke et al., 1995). This represents an example that about one-third of the prodynorphin neurons inof how the level and mode of LC activity is the result the CeA co-express CRF, whereas no co-expressionof a balance of neuromodulators that are engaged and with CRF is detected in other limbic regions such asactive at a particular time. The mechanism by which the BNST (Marchant et al., 2007). Interestingly, thischronic opioids induce postsynaptic sensitization to profile was not shared by DYN and ENK afferentsCRF in LC neurons is currently unknown, although which appear to function in parallel (Reyes et al.,many changes in intracellular signaling systems 2008a). This is consistent with studies showing thatwithin LC neurons have been documented to occur ENK neurons in the amygdala do not express DYNas a result of chronic opiate administration and these (Marchant et al., 2007) and that ENK afferents to thecould contribute to altered postsynaptic sensitivity to LC do not originate from the CeA (Tjoumakaris et al.,CRF (Nestler et al., 1999). 2003). Finally, the potential exists for k-opioid receptor modulation of afferents to the LC as k-opioid receptors22.214.171.124 Dynorphin have been localized to CRF axon terminals in thisThe dynorphin (DYN) system has been recently region (Kreibich et al., 2006). Taken together, theseimplicated in stress-induced facilitation of drug data indicate that DYN- and CRF-labeled axon term-abuse (Shirayama et al., 2004). In addition, k-opioid inals, most likely arising from amygdalar sources, arereceptor antagonists prevent stress-elicited behaviors positioned to dually impact LC function and providethat are endpoints of depression such as immobility an anatomical basis for regulation of the stressin the forced swim test and passive behavior in responses and other CRF-related functions by thelearned helplessness (Mague et al., 2003; McLaughlin brain DYN/k-opioid receptor system.et al., 2003; Shirayama et al., 2004). Our recent analy-sis of DYN immunoreactivity in the LC (Reyes et al., 126.96.36.199 Gamma-amino butyric acid2007b) has compelled us to consider the impact of Neuroanatomical studies argue against substantialthis additional opioid peptide family in our model of co-localization with GABA in CRF terminals inco-regulation of the LC system by endogenous the rostrolateral peri-LC (Valentino et al., 2001).opioids and the stress peptide, CRF. In addition This is somewhat surprising, considering reportsto considering opposing postsynaptic effects on that the majority of amygdalar neurons containingLC neurons, the working model includes potential CRF mRNA also contain mRNA for glutamic acidk-opioid receptor-mediated presynaptic regulation, dehydroxylase. Nonetheless, this is consistent withwhich could directly impact on phasic activity of LC the findings that the majority of CRF synaptic spe-neurons by inhibiting afferent signaling. cializations is asymmetric (excitatory type). In contrast to relatively minor co-localizationof ENK and CRF, immunofluorescence microscopyrevealed prominent coexistence between DYN and 52.4 Plasticity of CRF–NorepinephrineCRF terminal fields in the LC region (Figure 4). InteractionsCoexistence of DYN and CRF in individual axon 52.4.1 CRF Receptors in LCterminals was confirmed at the ultrastructural level inthe LC core and dorsolateral peri-LC. The percentage Two major CRF receptor subtypes, CRF1 and CRF2,of DYN-immunolabeled terminals that exhibited CRF have been cloned during the past decade. They belongimmunolabeling in this study was significant (35%). to the class B1 group of the G-protein-coupled
Corticotropin-Releasing Factor and the Brain Norepinephrine System 1679receptor (GPCR) superfamily (Dautzenberg and by CRF1 receptors. Interestingly, both CRF1 bindingHauger, 2002; Hauger et al., 2003; Perrin and Vale, sites and mRNA are dense just lateral to the rostral2002). The transmembrane and intracellular domains pole of the LC, in the peri-LC region where LCof both CRF receptor subtypes are highly homolo- dendrites extend. It is possible that the binding sitesgous and both receptor subtypes couple to the and mRNA are localized to these distant dendrites.same Ga proteins and signal through similar second The dendritic localization of mRNA has been demon-messengers (Aguilera et al., 1983; Chen et al., 1993; strated for other GPCRs (Sutton and Schuman, 2005;Dautzenberg et al., 1997; Dautzenberg and Hauger, Tran et al., 2007; Zhong et al., 2006). It is also possible2002; Hauger and Dautzenberg, 1999; Hauger et al., that CRF1 mRNA is rapidly transported to LC den-1997; Hoffman et al., 1985; Olianas et al., 1995; Perrin dritic processes and because these are so extensive, theand Vale, 2002). CRF1 and CRF2 receptors are differ- signal is diluted and not sensitive to visualization byentially localized in the brain and periphery. CRF1 in situ hybridization. Indeed, there is evidence of dif-receptors are distributed throughout neocortical, lim- ferential distribution of mRNA in dendrites versusbic, and brainstem regions while distribution of CRF2 soma in other systems (Bramham and Wells, 2007;receptors is limited to discrete brain regions, includ- Sossin and DesGroseillers, 2006; Wu et al., 2007). Toing raphe nuclei, lateral septum, cortical and medial date, the inability to detect CRF1 mRNA in LC usingamygdalar nuclei, and paraventricular and ventrome- in situ hybridization has not been resolved. Althoughdial hypothalamic nuclei (Bale and Vale, 2004; mixed reports exist for the rodent LC, the rhesusDautzenberg and Hauger, 2002; Hauger et al., 2003; monkey exhibits both CRF1 receptor mRNA and bind-Perrin and Vale, 2002). ing in the LC (Sanchez et al., 1999). The receptor mediating LC activation by CRFis thought to be the CRF1 subtype because this 52.4.2 Agonist and Stress-Inducedactivation is sensitive to selective CRF1 antagonists Internalization of LC Neurons(Curtis et al., 1994). Signaling through the CRF1receptor involves GS-mediated generation of cyclic Cellular sensitivity to neurotransmitters is deter-AMP-dependent activation of protein kinase A and mined by the number of receptors on the plasmasubsequent downstream events such as cAMP response membrane, and for GPCRs this is highly regulatedelement binding (CREB) phosphorylation (Aguilera by cellular trafficking. Trafficking of CRF1 has beenet al., 1983; Bilezikjian and Vale, 1983; Boundy et al., characterized in cell culture (Childs et al., 1986;1998; Chen et al., 1993; Dautzenberg and Hauger, 2002; Perry et al., 2005; Rasmussen et al., 2004). MoreGraziani et al., 2002; Hauger and Dautzenberg, 1999; recent electron microscopic studies characterizingHoffman et al., 1985; Litvin et al., 1984; Olianas et al., CRF1 trafficking in LC neurons in vivo link these1995; Perrin and Vale, 2002; Rossant et al., 1999). While cellular events to functional correlates. ElectronCRF1 immunoreactivity and low levels of CRF1 recep- microscopic analysis revealed the presence of CRF1tor binding are reported in rodent LC (Chen et al., in LC neurons of unstressed rats that was localized2000; Sauvage and Steckler, 2001), CRF1 receptor to plasma membrane and cytoplasm. The ratio ofmRNA has not been detected in the LC using in situ plasma membrane to cytoplasmic CRFr immunola-hybridization (Aguilera et al., 2004; Bittencourt and beling was approximately 50:50. Exposure to levels ofSawchenko, 2000; Bittencourt et al., 1999; Chalmers CRF that activate LC neurons results in a shift in thiset al., 1995; Van Pett et al., 2000). An explanation for ratio to approximately 20:80 (plasma membrane:cyto-this discrepancy is that CRF may interact only presyn- plasm) (Reyes et al., 2006). This apparent agonist-aptically with receptors on terminals within the induced internalization is present at both 5min andLC region whose cell bodies are distant from the LC. 3min after injection. Internalized CRFr was associatedHowever, in vitro electrophysiological studies support with early endosomes, indicative of degradation ora direct postsynaptic action of CRF on LC neurons recycling. Agonist-induced CRFr internalization in( Jedema and Grace, 2004). In addition, CRF1 mRNA LC neurons is functionally relevant and may underlieis not abundant in regions identified as sources of LC acute desensitization to CRF that is seen in electro-afferents (Barrington’s nucleus, PGi, CeA, BNST, and physiological studies following exposure to a singlePVN). This discrepancy could also be explained dose of CRF (Conti and Foote, 1995, 1996; Curtisif CRF effects on LC neurons were not receptor et al., 1997).mediated. However, this is unlikely because pharma- CRFr internalization in LC neurons not only iscological analysis is consistent with an action mediated a pharmacological phenomenon, but is also initiated
1680 Corticotropin-Releasing Factor and the Brain Norepinephrine System (a) (c) (b) (d) Control 1h 24 h U U PM* PM* U Plasma ER ER ER membrane Golgi* sm. ves Golgi * sm. ves* Golgi*@ *@ VB VB M M sm. ves MVB*@Figure 5 (a, b) CRFr-labeled axon terminals in LC from control and stressed rats. (a) CRFr immunogold labeling is present inan axon terminal (CRFr-t) from a control rat and is associated with the plasma membrane (arrowheads). Also shown is anastrocytic process (asterisks) apposed to the dendrite. (b) CRFr immunogold-labeling (arrows) is prominently associatedwithin the intracellular compartment in a CRFr-labeled axon terminal (CRFr-t) from a case perfused 1 h following 15-minswim stress. dcv, dense core vesicles; m, mitochondria; N, nucleus. Scale bar = 0.5 mm. (c) Immunogold-silver labelingfor CRFr (arrowheads) can be seen along the plasmalemma in a dendritic profile. The dendrite receives synaptic contactsfrom axon terminals (t). (d) CRFr labeling shifts from the plasmalemma to the cytoplasm 1 h following 15-min swim stress.Arrows point to immunogold-silver labeling in the cytoplasm. Double-headed arrows point to endosome-like vesicles.Asterisks indicate astrocytic processes. Pie charts summarizing distribution of gold–silver labeling for CRFr receptorassociated with the plasma membrane (PM) small vesicles (sm.ves), multivesicular bodies (mvb), Golgi apparatuses,endoplasmic reticulum (ER), or undetermined structures (U) in control rats or rats at 1 h or 24 h following swim. (a-d)Reproduced from Reyes BA, Valentino RJ, and Van Bockstaele EJ (2008) Stress-induced intracellular trafficking ofcorticotropin-releasing factor receptors in rat locus coeruleus neurons. Endocrinology 149: 124, 126, with permissionfrom the Endocrine Society.
Corticotropin-Releasing Factor and the Brain Norepinephrine System 1681by stress. Thus, 1 and 24h after a 15-min swim stress, associated with multivesicular bodies, suggesting thatthe ratio of plasma membrane to cytoplasmic CRFr is some of the internalized receptor is targeted forapproximately 20:80, whereas it is 50:50 in handled degradation. Importantly, this cellular activity israts (Figure 5). Stress-induced CRFr internalization expressed functionally as a decreased maximal effectis significantly attenuated by administration of a of CRF 24h after swim stress (Curtis et al., 1999) (seeselective CRF1 receptor antagonists prior to swim also below). In perikarya, more internalized CRFrstress (Reyes et al., 2008b). The sensitivity of stress- was associated with Golgi apparatuses 24h versusinduced CRFr internalization in LC neurons to a 1h post-stress. This is suggestive of changes inCRF1 antagonist provides further evidence for the CRFr synthesis. Alternatively, this may indicateexistence of the receptor in LC neurons and that communication between multivesicular bodies andCRF acts as a neurotransmitter here that is released Golgi apparatuses in the process of recycling. Eluci-to impact on this system during stress. The endpoint dating specific molecules involved in CRFr cellularof CRFr internalization may be useful for determin- trafficking may offer targets for manipulating theing whether endogenous CRF impacts on the LC cellular response to CRF.system during a particular challenge in studies thatpreclude neuronal recordings. 52.4.3 Structural Plasticity Following Identifying cellular compartments with which StressCRFr is associated following internalization at differ-ent times after agonist or stress can reveal informa- A variety of studies have shown that CRF can altertion about the fate of the receptor and processes dendritic morphology in a pattern similar to thatthat regulate cell sensitivity (Figure 5). For example, found after stress (Chen et al., 2004; Swinny et al.,at 1h post-stress, CRFr was often associated with 2004). Recent evidence suggests that CRF alsoearly endosomes in dendrites and perikarya (Reyes impacts LC dendritic structure, an effect with poten-et al., 2008b). By 24h, significantly more CRFr was tially long-term consequences (Swinny and Valentino, 2006). Exposure of LC explants to CRF increased dendritic length into the surrounding peri-cerulear region. This effect was mediated by CRF1 receptors Limbic afferents and involved activation of protein kinase A and Emotional mitogen-activated protein kinase. Importantly, the arousal Rho GTPases that regulate dendritic growth were identified as molecules linking CRF receptor signal- CRF ing and actin cytoskeletal dynamics. The significance of these morphological effects of CRF on LC neurons relates to the differential termination patterns of LC afferents. Because different afferents terminate in Autonomic the LC core versus the peri-cerulear region, the afferents degree of dendritic extension outside the core is rele- vant to which afferents will have predominant influ-Figure 6 Schematic depicting potential consequences of ence over this brain norepinephrine system. DendriticCRF morphological effects on locus ceruleus dendrites.In contrast to autonomically related CRF afferents to the extension into the peri-cerulear region would increaselocus ceruleus that terminate in the nuclear core, limbic the probability of communicating with limbic afferentssources of CRF terminate outside of the core in the peri- that relay emotionally related information to the LCcerulear region where locus ceruleus dendrites extend. By (Figure 6). Thus, by governing the extension of LCpromoting the extension of locus ceruleus dendrites into dendrites into the peri-cerulear region, CRF maythis region, CRF potentially increases the probability thatthese dendrites will synapse with limbic afferents that determine the level of emotional arousal.convey affective information and enhance the influence ofthese afferents on locus ceruleus activity. Through thismechanism, CRF can determine the structural basis for 52.5 Determinants of LC Activityemotional arousal. Reproduced from Valentino RJ and VanBockstaele EJ (2008) Convergent regulation of locuscoeruleus activity as an adaptive response to stress. Sensitivity of LC neurons to CRF is not static, butEuropean Journal of Pharmacology 583: 199, with can be influenced by a number of factors includingpermission from Elsevier. exposure to opiates (see above) as well as a history of
1682 Corticotropin-Releasing Factor and the Brain Norepinephrine Systemstress exposure. Prior stress can either decrease or be important in stress-related bowel disorders, suchincrease the magnitude of CRF activation of LC as irritable bowel syndrome (Valentino et al., 1999).neurons depending on the severity of the stressor, Given that colonic distention activates the LC via awhether it is acute or chronic, and the challenge CRF-dependent mechanism, LC sensitization todose of CRF. Like an acute dose of CRF, certain CRF would predictably translate to sensitization toacute stressors desensitize LC neurons to CRF. colonic stimuli, a characteristic feature of irritableFor example, immediately following a session of bowel syndrome. In addition to repeated sessions offootshocks, the CRF dose–response curve for LC footshock and a single exposure to swim stress, sensi-activation is shifted to the right (Conti and Foote, tization of LC neurons to CRF has also been reported1996; Curtis et al., 1995). The physiological impact of in rats subjected to 2 weeks of daily cold exposurethis change in sensitivity to CRF is apparent as a loss ( Jedema et al., 2001).of LC responses to hypotensive challenge (Curtis Sex is also an important determinant of LCet al., 1995) and also to colon distention (Lechner sensitivity to CRF and stress. Regardless of hormonalet al., 1997). This change in LC sensitivity is selective status, LC neurons of female rats are more sensitiveto CRF, because LC spontaneous discharge and sen- to CRF as indicated by a shift to the left in the CRFsitivity to other inputs are not altered. It is speculated dose–response curve (Curtis et al., 2006). This isthat the electrophysiological desensitization, like an intriguing finding, given the higher incidenceagonist-induced desensitization, is related to acute of depression and other stress-related disorders inreceptor internalization. females. Moreover, whereas a history of prior stress In contrast to a single session of shock, repeated regulates LC sensitivity to CRF in male rats, it doessessions over 7 days result in a complex shift in the not do so for female rats (Curtis et al., 2006). ThisCRF dose–response curve to the left with a decrease suggests that there may be sex differences in CRFin the maximum response, such that LC neurons are receptor structure or signaling that may be verysensitized to low doses of CRF, although the maximal relevant to female vulnerability to stress-relatedmagnitude of activation is decreased (Curtis et al., disorders. Given that the CRF receptor has become1995). A similar shift is seen 24h after exposure to a pharmacological target for the treatment of stress-swim stress (Curtis et al., 1999). As described above, releated disorders, which are more prevalent inthe decrease in the maximum magnitude of LC acti- females, potential sex differences in CRF receptorvation by CRF that is seen 24h after swim stress structure and/or signaling need to be taken intois consistent with cellular evidence for increased account in this line of therapeutic development.associations of the receptor with multivesicularbodies, which often indicate receptor degradationand downregulation. (Reyes et al., 2006, 2007b, 2008a,b; 52.6 ConclusionsFigure 4). Mechanisms underlying the sensitizationto low doses of CRF have not been elucidated, As an adaptive response to a perceived life-threateningalthough the type of change in the CRF dose– challenge, arousal should be elevated and maintainedresponse curve suggests changes in receptor transduc- for the duration of the challenge and attentiontion mechanisms. Stress-induced sensitization to low should be directed toward diverse stimuli in thedoses of CRF is functionally relevant because it environment, rather than being focused on specificallows low levels of CRF, that would typically have stimuli. This favors sampling of behavioral strategiesno effect, to activate the brain norepinephrine sys- that can be adopted, given the environmental con-tem by a magnitude that would impact on forebrain straints, so that the organism has the highest proba-activity. This stress-related sensitization of LC neu- bility for survival. Moreover, these changes shouldrons to CRF may play a role in the hypervigilance subside when the stressor is no longer present. LCseen in post-traumatic stress disorder, a condition in neurons with their widely distributed network ofwhich subjects exposed to a severe trauma become axons promote these aspects of the cognitive andselectively sensitized to stimuli previously associated behavioral limbs of the stress response throughwith the trauma. Subjects exhibit hyperarousal, changes in their discharge rate and pattern. Inhypervigilance, and sleep disturbances, symptoms turn, the mode of LC activity is finely tuned bythat are associated with a hyperactive LC system the convergence of afferents, particularly excitatory(Charney et al., 1993). This phenomenon may also amino acids, CRF, and endogenous opioids onto LC
Corticotropin-Releasing Factor and the Brain Norepinephrine System 1683neurons. By biasing the activity of LC neurons Referencestoward a particular mode of discharge, these affer-ents can govern predominant behavioral strategies Abercrombie ED and Jacobs BL (1988) Systemic naloxonein specific situations or environments. In addition to administration potentiates locus coeruleus noradrenergic neuronal activity under stressful but not non-stressfulits immediate role in governing ongoing LC activity, conditions. Brain Research 441: 362–366.CRF impacts the morphology of LC neurons. By Aguilera G, Harwood JP, Wilson JX, Morell J, Brown JH, anddetermining the degree to which certain afferents Catt KJ (1983) Mechanisms of action of corticotropin- releasing factor and other regulators of corticotropin releasecommunicate with LC neurons, this can have in rat pituitary cells. Journal of Biological Chemistry 258:enduring consequences on behavior or emotional 8039–8045.expression. The emerging knowledge of how the Aguilera G, Nikodemova M, Wynn PC, and Catt KJ (2004) Corticotropin releasing hormone receptors: Two decadesLC–norepinephrine system is regulated by stressors later. Peptides 25: 319–329.and determinants of sensitivity of LC neurons to Akaoka H and Aston-Jones G (1991) Opiate withdrawal-stress-related neurotransmitters is an important step induced hyperactivity of locus coeruleus neurons is substantially mediated by augmented excitatory amino acidin understanding and alleviating diverse stress-related input. Journal of Neuroscience 11: 3830–3839.psychiatric disorders. Aston-Jones G and Bloom FE (1981) Activity of norepinephrine- Evidence has accumulated over the past two containing locus coeruleus neurons in behaving rats anticipates fluctuations in the sleep-waking cycle. Journal ofdecades supporting the notion that CRF exerts Neuroscience 1: 876–886.important actions in the brain beyond stimulation of Aston-Jones G and Cohen JD (2005) An integrative theory ofthe hypothalamic–pituitary–adrenal axis. Consistent locus coeruleus-norepinephrine function: Adaptive gain and optimal performance. Annual Review of Neuroscience 28:with the dual role of CRF in acting both as a sec- 403–450.retagog for anterior pituitary hormones and as Aston-Jones G, Rajkowski J, and Cohen J (2000) Locusextra-pituitary peptide neurotransmitter, CRF may coeruleus and regulation of behavioral flexibility and attention. Progress in Brain Research 126: 165–182.coordinate coping responses to stress in the fashion Aston-Jones G, Shipley M, and Grzanna R (1995)of a final common effector pathway (Heinrichs Chemoanatomy of the locus coeruleus, A5 and A7and Koob, 2004). Since the original identification noradrenergic cell groups. In: Paxinos G (ed.) The Rat Nervous System, pp. 183–214. Orlando, FL: Academicand characterization of CRF, many advances have Press.been made that have increased our understanding Bale TL and Vale WW (2004) CRF and CRF receptors: Role inof how this peptide functions during stress and non- stress responsivity and other behaviors. Annual Review of Pharmacology and Toxicology 44: 525–557.stressful physiological conditions. Importantly, the Barr J and Van Bockstaele EJ (2005) Vesicular glutamateconditions that regulate long-term plasticity of CRF transporter-1 colocalizes with endogenous opioid peptidessystems are being identified, as are the pathological in axon terminals of the rat locus coeruleus. Anatomical Record, A: Discoveries in Molecular, Cellular, andconsequences of this plasticity. Preclinical data Evolutionary Biology 284: 466–474.show promise for the potential of small molecule Beck CH and Fibiger HC (1995) Chronic desipramine altersCRF1 receptor antagonists as novel treatments for stress-induced behaviors and regional expression of the immediate early gene, c-fos. Pharmacology, Biochemistry,depression based on the concept that abnormally and Behavior 51: 331–338.enhanced central CRF1 receptor signaling contri- Berridge CW and Foote SL (1991) Effects of locus coeruleusbutes to the pathophysiology of major depression. activation on electroencephalographic activity in neocortex and hippocampus. Journal of Neuroscience 11: 3135–3145.Continued investigations into the neurobiology of Berridge CW, Page ME, Valentino RJ, and Foote SL (1993)CRF receptor signaling and its influence on broadly Effects of locus coeruleus inactivation onprojecting neural networks such as the LC will electroencephalographic activity in neocortex and hippocampus. Neuroscience 55: 381–393.provide important insights into the complex cellular Bilezikjian LM and Vale WW (1983) Glucocorticoids inhibitand molecular mechanisms underlying normal and corticotropin-releasing factor-induced production ofpathological defense mechanisms, and their contri- adenosine 30 ,50 -monophosphate in cultured anterior pituitary cells. Endocrinology 113: 657–662.bution to onset and relapse of anxiety, stress, and Bittencourt JC and Sawchenko PE (2000) Do centrallydepression. administered neuropeptides access cognate receptors?: An analysis in the central corticotropin-releasing factor system. Journal of Neuroscience 20: 1142–1156. Bittencourt JC, Vaughan J, Arias C, Rissman RA, Vale WW, andAcknowledgments Sawchenko PE (1999) Urocortin expression in rat brain: Evidence against a pervasive relationship of urocortin-containing projections with targets bearing typeThis work was supported by PHS Grants MH40008 2 CRF receptors. Journal of Comparative Neurology 415:and DA09082. 285–312.
1684 Corticotropin-Releasing Factor and the Brain Norepinephrine SystemBonaz B and Tache Y (1994) Water-avoidance stress-induced Childs GV, Morell JL, Niendorf A, and Aguilera G (1986) c-fos expression in the rat brain and stimulation of fecal Cytochemical studies of corticotropin-releasing factor (CRF) output: Role of corticotropin-releasing factor. Brain receptors in anterior lobe corticotropes: Binding, Research 641: 21–28. glucocorticoid regulation, and endocytosis of [biotinyl-Ser1]Boundy VA, Chen J, and Nestler EJ (1998) Regulation of cAMP- CRF. Endocrinology 119: 2129–2142. dependent protein kinase subunit expression in CATH.a and Conti LH and Foote SL (1995) Effects of pretreatment with SH-SY5Y cells. Journal of Pharmacology and Experimental corticotropin-releasing factor on the electrophysiological Therapeutics 286: 1058–1065. responsivity of the locus coeruleus to subsequentBramham CR and Wells DG (2007) Dendritic mRNA: Transport, corticotropin-releasing factor challenge. Neuroscience translation and function. Nature Reviews Neuroscience 69: 209–219. 8: 776–789. Conti LH and Foote SL (1996) Reciprocal cross-desensitizationBritton DR, Varela M, Garcia A, and Rosenthal M (1986b) of locus coeruleus electrophysiological responsivity to Dexamethasone suppresses pituitary–adrenal but not corticotropin-releasing factor and stress. Brain Research behavioral effects of centrally administered CRF. Life 722: 19–29. Sciences 38: 211–216. Curtis AL, Bello NT, Connolly KR, and Valentino RJ (2002)Britton KT, Lee G, Dana R, Risch SC, and Koob GF (1986a) Corticotropin-releasing factor neurones of the central Activating and ’anxiogenic’ effects of corticotropin releasing nucleus of the amygdala mediate locus coeruleus activation factor are not inhibited by blockade of the pituitary–adrenal by cardiovascular stress. Journal of Neuroendocrinology 14: system with dexamethasone. Life Sciences 39: 1281–1286. 667–682.Britton KT, McLeod S, Koob GF, and Hauger R (1992) Pregnane Curtis AL, Bello NT, and Valentino RJ (2001) Evidence for steroid alphaxalone attenuates anxiogenic behavioral effects functional release of endogenous opioids in the locus of corticotropin releasing factor and stress. Pharmacology, coeruleus during stress termination. Journal of Neuroscience Biochemistry, and Behavior 41: 399–403. 21: RC152(1–5).Campeau S and Watson SJ (1997) Neuroendocrine and Curtis AL, Bethea T, and Valentino RJ (2006) Sexually dimorphic behavioral responses and brain pattern of c-fos induction responses of the brain norepinephrine system to stress and associated with audiogenic stress. Journal of corticotropin-releasing factor. Neuropsychopharmacology Neuroendocrinology 9: 577–588. 31: 544–554.Cassens G, Kuruc A, Roffman M, Orsulak PJ, and Curtis AL, Grigoriadis DE, Page ME, Rivier J, and Valentino RJ Schildkraut JJ (1981) Alterations in brain norepinephrine (1994) Pharmacological comparison of two corticotropin- metabolism and behavior induced by environmental stimuli releasing factor antagonists: In vivo and in vitro studies. previously paired with inescapable shock. Behavioural Brain Journal of Comparative and Physiological Psychology 268: Research 2: 387–407. 359–365.Cassens G, Roffman M, Kuruc A, Orsulak PJ, and Curtis AL, Lechner SM, Pavcovich LA, and Valentino RJ (1997) Schildkraut JJ (1980) Alterations in brain norepinephrine Activation of the locus coeruleus noradrenergic system by metabolism induced by environmental stimuli previously intracoerulear microinfusion of corticotropin-releasing factor: paired with inescapable shock. Science 209: 1138–1140. Effects on discharge rate, cortical norepinephrine levels andChalmers DT, Lovenberg TW, and De Souza EB (1995) cortical electroencephalographic activity. Journal of Localization of novel corticotropin-releasing factor receptor Comparative and Physiological Psychology 281: 163–172. (CRF2) mRNA expression to specific subcortical nuclei in rat Curtis AL, Pavcovich LA, Grigoriadis DE, and Valentino RJ brain: Comparison with CRF1 receptor mRNA expression. (1995) Previous stress alters corticotropin-releasing factor Journal of Neuroscience 15: 6340–6350. neurotransmission in the locus coeruleus. Neuroscience 65:Chan RK and Sawchenko PE (1995) Hemodynamic regulation 541–550. of tyrosine hydroxylase messenger RNA in medullary Curtis AL, Pavcovich LA, and Valentino RJ (1999) Long-term catecholamine neurons: A c-fos-guided hybridization regulation of locus ceruleus sensitivity to corticotropin- histochemical study. Neuroscience 66: 377–390. releasing factor by swim stress. Journal of PharmacologyChang MS, Sved AF, Zigmond MJ, and Austin MC (2000) and Experimental Therapeutics 289: 1211–1219. Increased transcription of the tyrosine hydroxylase gene in Dautzenberg FM, Dietrich K, Palchaudhuri MR, and Spiess J individual locus coeruleus neurons following footshock (1997) Identification of two corticotropin-releasing factor stress. Neuroscience 101: 131–139. receptors from Xenopus laevis with high ligand selectivity:Charney DS, Deutch AY, Krystal JH, Southwick SM, and Unusual pharmacology of the type 1 receptor. Journal of Davis M (1993) Psychobiologic mechanisms of Neurochemistry 69: 1640–1649. posttraumatic stress disorder. Archives of General Dautzenberg FM and Hauger RL (2002) The CRF peptide family Psychiatry 50: 295–305. and their receptors: Yet more partners discovered. Trends inChen Y, Bender RA, Brunson KL, Pomper JK, Grigoriadis DE, Pharmacological Sciences 23: 71–77. Wurst W, and Baram TZ (2004) Modulation of dendritic Detke MJ, Rickels M, and Lucki I (1995) Active behaviors in the differentiation by corticotropin-releasing factor in the rat forced swimming test differentially produced by developing hippocampus. Proceedings of the National serotonergic and noradrenergic antidepressants. Academy of Sciences of the United States of America 101: Psychopharmacology (Berl) 121: 66–72. 15782–15787. Drolet G, Dumont EC, Gosselin I, Kinkead R, Laforest S, andChen Y, Brunson KL, Muller MB, Cariaga W, and Baram TZ Trottier JF (2001) Role of endogenous opioid system in the (2000) Immunocytochemical distribution of corticotropin- regulation of the stress response. Progress in releasing hormone receptor type-1 (CRF(1))-like Neuropsychopharmacology and Biological Psychiatry immunoreactivity in the mouse brain: Light microscopy 25: 729–741. analysis using an antibody directed against the C-terminus. Drolet G, Van Bockstaele EJ, and Aston-Jones G (1992) Robust Journal of Comparative Neurology 420: 305–323. enkephalin innervation of the locus coeruleus from the rostralChen R, Lewis KA, Perrin MH, and Vale WW (1993) Expression medulla. Journal of Neuroscience 12: 3162–3174. cloning of a human corticotropin-releasing-factor receptor. Dun NJ, Dun SL, Shen E, Tang H, Huang R, and Chiu TH (1995) Proceedings of the National Academy of Sciences of the c-fos Expression as a marker of central cardiovascular United States of America 90: 8967–8671. neurons. Biological Signals 4: 117–123.
Corticotropin-Releasing Factor and the Brain Norepinephrine System 1685Duncan GE, Johnson KB, and Breese GR (1993) Topographic Ishida Y, Hashiguchi H, Takeda R, et al. (2002) Conditioned-fear patterns of brain activity in response to swim stress: stress increases Fos expression in monoaminergic and Assessment by 2-deoxyglucose uptake and expression GABAergic neurons of the locus coeruleus and dorsal raphe of Fos-like immunoreactivity. Journal of Neuroscience nuclei. Synapse 45: 46–51. 13: 3932–3943. Ishimatsu M and Williams JT (1996) Synchronous activity inDunn AJ, Berridge CW, Lai YI, and Yachabach TL (1987) locus coeruleus results from dendritic interactions in CRF-induced excessive grooming behavior in rats and mice. pericoerulear regions. Journal of Neuroscience 16: Peptides 8: 841–844. 5196–5204.Eaves M, Thatcher-Britton K, Rivier J, Vale W, and Koob GF Jasnow AM, Davis M, and Huhman KL (2004) Involvement of (1985) Effects of corticotropin releasing factor on central amygdalar and bed nucleus of the stria terminalis locomotor activity in hypophysectomized rats. Peptides 6: corticotropin-releasing factor in behavioral responses to 923–926. social defeat. Behavioral Neuroscience 118: 1052–1061.Ennis M and Aston-Jones G (1988) Activation of locus coeruleus Jedema HP, Finlay JM, Sved AF, and Grace AA (2001) Chronic from nucleus paragigantocellularis: A new excitatory cold exposure potentiates CRH-evoked increases in amino acid pathway in brain. Journal of Neuroscience electrophysiologic activity of locus coeruleus neurons. 8: 3644–3657. Biological Psychiatry 49: 351–359.Fisher LA, Jessen G, and Brown MR (1983) Corticotropin-releasing Jedema HP and Grace AA (2004) Corticotropin-releasing factor (CRF): Mechanism to elevate mean arterial pressure hormone directly activates noradrenergic neurons of the and heart rate. Regulatory Peptides 5: 153–161. locus ceruleus recorded in vitro. Journal of NeuroscienceFlorin-Lechner SM, Druhan JP, Aston-Jones G, and 24: 9703–9713. Valentino RJ (1996) Enhanced norepinephrine release in Kawahara H, Kawahara Y, and Westerink BH (2000) The role of prefrontal cortex with burst stimulation of the locus afferents to the locus coeruleus in the handling stress- coeruleus. Brain Research 742: 89–97. induced increase in the release of noradrenaline in theFoote SL, Aston-Jones G, and Bloom FE (1980) Impulse activity medial prefrontal cortex: A dual-probe microdialysis study in of locus coeruleus neurons in awake rats and monkeys is a the rat brain. European Journal of Pharmacology 387: function of sensory stimulation and arousal. Proceedings of 279–286. the National Academy of Sciences of the United States of Kollackwalker S, Watson SJ, and Akil H (1997) Social stress in America 77: 3033–3037. hamsters – defeat activates specific neurocircuits within theFoote SL, Bloom FE, and Aston-Jones G (1983) Nucleus locus brain. Journal of Neuroscience 17: 8842–8855. ceruleus: New evidence of anatomical and physiological Koob GF (1999) Corticotropin-releasing factor, norepinephrine, specificity. Physiological Reviews 63: 844–914. and stress. Biological Psychiatry 46: 1167–1180.Funk D and Amir S (2000) Enhanced fos expression within the Korf J, Aghajanian GK, and Roth RH (1973) Increased turnover primary olfactory and limbic pathways induced by an of norepinephrine in the rat cerebral cortex during stress: aversive conditioned odor stimulus. Neuroscience 98: Role of the locus coeruleus. Neuropharmacology 12: 403–406. 933–938.Graham JC, Hoffman GE, and Sved AF (1995) c-Fos expression Kosoyan HP, Grigoriadis DE, and Tache Y (2005) The CRF(1) in brain in response to hypotension and hypertension in receptor antagonist, NBI-35965, abolished the activation conscious rats. Journal of the Autonomic Nervous System of locus coeruleus neurons induced by colorectal 55: 92–104. distension and intracisternal CRF in rats. Brain ResearchGraziani G, Tentori L, Portarena I, et al. (2002) CRH inhibits cell 1056: 85–96. growth of human endometrial adenocarcinoma cells via Kreibich A, Glaser J, Curtis A, Chavkin C, Valentino RJ, and CRH-receptor 1-mediated activation of cAMP-PKA Van Bockstaele EJ (2006) Evidence for presynaptic pathway. Endocrinology 143: 807–813. regulation of excitatory afferents to the locus ceruleus (LC)Grzanna R and Molliver ME (1980) The locus coeruleus in the by kappa 1 opiate receptors (k-OR). Society for rat: An immunohistochemical delineation. Neuroscience Neuroscience Abstract 32: 724.5. 5: 21–40. Lacosta S, Merali Z, and Anisman H (2000) Central monoamineHauger RL and Dautzenberg FM (1999) Neuroendocrinology in activity following acute and repeated systemic interleukin-2 Physiology and Medicine. Totowa, NJ: Humana Press. administration. Neuroimmunomodulation 8: 83–90.Hauger RL, Dautzenberg FM, Flaccus A, Liepold T, and Spiess J Lechner SM, Curtis AL, Brons R, and Valentino RJ (1997) Locus (1997) Regulation of corticotropin-releasing factor receptor coeruleus activation by colon distention: Role of function in human Y-79 retinoblastoma cells: Rapid and corticotropin-releasing factor and excitatory amino acids. reversible homologous desensitization but prolonged Brain Research 756: 114–124. recovery. Journal of Neurochemistry 68: 2308–2316. LeDoux JE, Iwata J, Cicchetti P, and Reis DJ (1988) DifferentHauger RL, Grigoriadis DE, Dallman MF, Plotsky PM, Vale WW, projections of the central amygdaloid nucleus mediate and Dautzenberg FM (2003) International Union of autonomic and behavioral correlates of conditioned fear. Pharmacology. XXXVI. Current status of the nomenclature Journal of Neuroscience 8: 2517–2529. for receptors for corticotropin-releasing factor and their Liang KC, Melia KR, Campeau S, Falls WA, Miserendino MJ, ligands. Physiological Reviews 55: 21–26. and Davis M (1992) Lesions of the central nucleus ofHeinrichs SC and Koob GF (2004) Corticotropin-releasing factor the amygdala, but not the paraventricular nucleus of in brain: A role in activation, arousal, and affect regulation. the hypothalamus, block the excitatory effects of Journal of Pharmacology and Experimental Therapeutics corticotropin-releasing factor on the acoustic startle reflex. 311: 427–440. Journal of Neuroscience 12: 2313–2320.Hoffman AR, Ceda G, and Reisine TD (1985) Corticotropin- Litvin Y, PasMantier R, Fleischer N, and Erlichman J (1984) releasing factor desensitization of adrenocorticotropic Hormonal activation of the cAMP-dependent protein kinases hormone release is augmented by arginine vasopressin. in AtT20 cells. Preferential activation of protein kinase I by Journal of Neuroscience 5: 234–242. corticotropin releasing factor, isoproterenol, and forskolin.Hughes J, Beaumont A, Fuentes JA, Malfroy B, and Unsworth C Journal of Biological Chemistry 259: 10296–10302. (1980) Opioid peptides: Aspects of their origin, release and Liu J, Yu B, Neugebauer V, Grigoriadis DE, et al. (2004) metabolism. Journal of Experimental Biology 89: 239–255. Corticotropin-releasing factor and Urocortin I modulate