Doses Of Corticotropin-releasing Factor Research Articles

Corticotropin releasing factor in the nucleus basalis of Meynert impairs attentional performance and reduces levels of glutamate and taurine in male and female rats

Psychiatric disorders that are characterized by impairments in sustained attention, including attention deficit hyperactivity disorder (ADHD), post-traumatic stress disorder (PTSD), and major depression are also sensitive to exacerbation by stress. Sustained attention relies on cholinergic and non-cholinergic projections from the nucleus basalis of Meynert (NBM) in the basal forebrain to the medial prefrontal cortex (mPFC). We have previously shown that central administration of the stress neuropeptide corticotropin releasing factor (CRF) impairs performance on the sustained attention task (SAT) in adult male and female rats. The present study investigated whether this effect was mediated by CRF's action in the NBM. Rats were administered CRF in the NBM and subsequent SAT performance was measured. A high dose of CRF (100 ng) significantly impaired performance on non-signaled events across sex. Because performance on non-signaled events is believed to depend on non-cholinergic (i.e., GABA and glutamate) signaling, high performance liquid chromatography was used to quantify amino acid levels in the NBM and mPFC. We found females have higher levels of glutamate, glutamine, GABA glycine, and alanine in the NBM than males. Importantly, CRF in the NBM led to a local decrease of taurine and several amino acids involved in glutamate synthesis in males and females, changes which may mediate the CRF-induced SAT performance deficit. Together these studies suggest that CRF regulation of amino acids in the NMB contributes to stress-induced attention deficits.

Superior control of inflammatory pain by corticotropin-releasing factor receptor 1 via opioid peptides in distinct pain-relevant brain areas

BackgroundUnder inflammatory conditions, the activation of corticotropin-releasing factor (CRF) receptor has been shown to inhibit pain through opioid peptide release from immune cells or neurons. CRF’s effects on human and animal pain modulation depend, however, on the distribution of its receptor subtypes 1 and 2 (CRF-R1 and CRF-R2) along the neuraxis of pain transmission. The objective of this study is to investigate the respective role of each CRF receptor subtype on centrally administered CRF-induced antinociception during inflammatory pain.MethodsThe present study investigated the role of intracerebroventricular (i.c.v.) CRF receptor agonists on nociception and the contribution of cerebral CRF-R1 and/or CRF-R2 subtypes in an animal model of Freund’s complete adjuvant (FCA)-induced hind paw inflammation. Methods used included behavioral experiments, immunofluorescence confocal analysis, and reverse transcriptase-polymerase chain reaction.ResultsIntracerebroventricular, but systemically inactive, doses of CRF elicited potent, dose-dependent antinociceptive effects in inflammatory pain which were significantly antagonized by i.c.v. CRF-R1-selective antagonist NBI 27914 (by approximately 60%) but less by CRF-R2-selective antagonist K41498 (by only 20%). In line with these findings, i.c.v. administration of CRF-R1 agonist stressin I produced superior control of inflammatory pain over CRF-R2 agonist urocortin-2. Intriguingly, i.c.v. opioid antagonist naloxone significantly reversed the CRF as well as CRF-R1 agonist-elicited pain inhibition. Consistent with existing evidence of high CRF concentrations in brain areas such as the thalamus, hypothalamus, locus coeruleus, and periaqueductal gray following its i.c.v. administration, double-immunofluorescence confocal microscopy demonstrated primarily CRF-R1-positive neurons that expressed opioid peptides in these pain-relevant brain areas. Finally, PCR analysis confirmed the predominant expression of the CRF-R1 over CRF-R2 in representative brain areas such as the hypothalamus.ConclusionTaken together, these findings suggest that CRF-R1 in opioid-peptide-containing brain areas plays an important role in the modulation of inflammatory pain and may be a useful therapeutic target for inflammatory pain control.

A role of noradrenergic receptors in anxiolytic-like effect of high CRF in the rat frontal cortex

Corticotropin releasing factor (CRF) is a neuropeptide widely distributed in the brain as a hormonal modulator and neurotransmitter. The best known behavioral function of CRF is activation of stress and anxiety via the hypothalamus and limbic structures but the role of CRF in the cortex is still poorly understood. Our previous studies have shown anxiolytic-like effects of high doses of CRF injected into the Fr2 frontal cortex and involvement of CRF1 receptors (R) in that effect. These results seemed to be controversial as most other studies suggested anxiogenic and not anxiolytic effects of CRF1R stimulation. Since stress is associated with adrenergic system, in the present study, we focused on participation of alpha1 and alpha2 or beta adrenergic receptors in the anxiolytic-like effect of CRF. Moreover, we verified whether these effects of CRF in the Fr2 were really connected with CRF1R.Male Wistar rats were bilaterally microinjected with CRF in a dose of 0.2 μg/1 μl/site or with the specific agonist of CRF1R, stressin 1 (0.2–0.0125 μg/1 μl/site) into the Fr2 area. The elevated plus maze (EPM) test was performed 30 min later to assess the anxiolysis. An involvement of noradrenergic receptors in the CRF induced anxiolytic-like effect in the Fr2 was studied by pretreatment with the alpha1 antagonist prazosin, alpha2 agonist clonidine, alpha2 antagonist RS 79948 or beta antagonist propranolol, 20–30 min before CRF. The influence on anxiety was assessed in the EPM test. The results show that anxiolytic behavior after CRF microinjection into the Fr2 area seems to be mainly connected with the CRF1R activation because a similar effect was observed after stressin 1 administration and it was blocked by CRF1R antagonist. The results observed after administration of noradrenergic ligands indicated that anxiolytic effects of CRF in the Fr2 engaged the alpha1 and alpha2 adrenergic receptors but not beta receptors.

Sex differences in corticotropin releasing factor regulation of medial septum-mediated memory formation

Stress can disrupt memory and contribute to cognitive impairments in psychiatric disorders, including schizophrenia and attention deficit hyperactivity disorder. These diseases are more common in men than in women, with men showing greater cognitive impairments. Mnemonic deficits induced by stress are mediated, in part, by corticotropin releasing factor (CRF). However, where CRF is acting to regulate memory, and sex differences therein, is understudied. Here we assessed whether CRF in the medial septum (MS), which projects to the hippocampus, affected memory formation in male and female rats. CRF in the MS did not alter hippocampal-independent object recognition memory, but impaired hippocampal-dependent object location memory in both sexes. Interestingly, males were more sensitive than females to the disruptive effect of a low dose of CRF in the MS. Female resistance was not due to circulating ovarian hormones. However, compared to males, females had higher MS expression of CRF binding protein, which reduces CRF bioavailability and thus may mitigate the effect of the low dose of CRF in females. In contrast, there was no sex difference in CRF1 expression in the MS. Consistent with this finding, CRF1 antagonism blocked the memory impairment caused by the high dose of CRF in the MS in both sexes. Collectively, these results suggest that males are more vulnerable than females to the memory impairments caused by CRF in the MS. In both sexes, CRF1 antagonists prevented MS-mediated memory deficits caused by high levels of CRF, and such levels can result from very stressful events. Thus, CRF1 antagonists may be a viable option for treating cognitive deficits in stressed individuals with psychiatric disorders.

The Effect of Peripheral CRF Peptide and Water Avoidance Stress on Colonic and Gastric Transit in Guinea Pigs.

Functional dyspepsia (FD) and irritable bowel syndrome (IBS) are common gastrointestinal (GI) diseases; however, there is frequent overlap between FD and IBS patients. Emerging evidence links the activation of corticotropin releasing factor (CRF) receptors with stress-related alterations of gastric and colonic motor function. Therefore, we investigated the effect of peripheral CRF peptide and water avoidance stress (WAS) on upper and lower GI transit in guinea pigs. Dosages 1, 3, and 10 µg/kg of CRF were injected intraperitoneally (IP) in fasted guinea pigs 30 minutes prior to the intragastric administration of charcoal mix to measure upper GI transit. Colonic transits in non-fasted guinea pigs were assessed by fecal pellet output assay after above IP CRF doses. Blockade of CRF receptors by Astressin, and its effect on GI transit was also analyzed. Guinea pigs were subjected to WAS to measure gastrocolonic transit in different sets of experiments. Dose 10 µg/kg of CRF significantly inhibited upper GI transit. In contrast, there was dose dependent acceleration of the colonic transit. Remarkably, pretreatment of astressin significantly reverses the effect of CRF peptide on GI transit. WAS significantly increase colonic transit, but failed to accelerate upper GI transit. Peripheral CRF peptide significantly suppressed upper GI transit and accelerated colon transit, while central CRF involved WAS stimulated only colonic transit. Therefore, peripheral CRF could be utilized to establish the animal model of overlap syndrome.

Repeated restraint stress lowers the threshold for response to third ventricle CRF administration

Rats and mice exposed to repeated stress or a single severe stress exhibit a sustained increase in energetic, endocrine, and behavioral response to subsequent novel mild stress. This study tested whether the hyper-responsiveness was due to a lowered threshold of response to corticotropin releasing factor (CRF) or an exaggerated response to a standard dose of CRF. Male Sprague–Dawley rats were subjected to 3h of restraint on each of 3 consecutive days (RRS) or were non-restrained controls. RRS caused a temporary hypophagia but a sustained reduction in body weight. Eight days after the end of restraint, rats received increasing third ventricle doses of CRF (0–3.0μg). The lowest dose of CRF (0.25μg) increased corticosterone release in RRS, but not control rats. Higher doses caused the same stimulation of corticosterone in the two groups of rats. Fifteen days after the end of restraint, rats were food deprived during the light period and received increasing third ventricle doses of CRF at the start of the dark period. The lowest dose of CRF inhibited food intake during the first hour following infusion in RRS, but not control rats. All other doses of CRF inhibited food intake to the same degree in both RRS and control rats. The lowered threshold of response to central CRF is consistent with the chronic hyper-responsiveness to CRF and mild stress in RRS rats during the post-restraint period.

Sex differences in corticotropin releasing factor-evoked behavior and activated networks

Hypersecretion of corticotropin releasing factor (CRF) is linked to the pathophysiology of major depression and post-traumatic stress disorder, disorders that are more common in women than men. Notably, preclinical studies have identified sex differences in CRF receptors that can increase neuronal sensitivity to CRF in female compared to male rodents. These cellular sex differences suggest that CRF may regulate brain circuits and behavior differently in males and females. To test this idea, we first evaluated whether there were sex differences in anxiety-related behaviors induced by the central infusion of CRF. High doses of CRF increased self-grooming more in female than in male rats, and the magnitude of this effect in females was greater when they were in the proestrous phase of their estrous cycle (higher ovarian hormones) compared to the diestrous phase (lower ovarian hormones), which suggests that ovarian hormones potentiate this anxiogenic effect of CRF. Brain regions associated with CRF-evoked self-grooming were identified by correlating a marker of neuronal activation, cFOS, with time spent grooming. In the infralimbic region, which is implicated in regulating anxiety, the correlation for CRF-induced neuronal activation and grooming was positive in proestrous females, but negative for males and diestrous females, indicating that ovarian hormones altered this relationship between neuronal activation and behavior. Because CRF regulates a number of regions that work together to coordinate different aspects of responding to stress, we then examined more broadly whether CRF-activated functional connectivity networks differed between males and cycling females. Interestingly, hormonal status altered correlations for CRF-induced neuronal activation between a variety of brain regions, but the most striking differences were found when comparing proestrous females to males, particularly when comparing neuronal activation between prefrontal cortical and other forebrain regions. These results suggest that ovarian hormones alter the way brain regions work together in response to CRF, which could drive different strategies for coping with stress in males versus females. These sex differences in stress responses could also help explain female vulnerability to psychiatric disorders characterized by CRF hypersecretion.

Corticotropin releasing factor impairs sustained attention in male and female rats

Stressful life events and stress-related psychiatric disorders impair sustained attention, the ability to monitor rare and unpredictable stimulus events over prolonged periods of time. Despite the link between stress and attentional disruptions, the neurobiological basis for stress regulation of attention systems remains underexplored. Here we examined whether corticotropin releasing factor (CRF), which orchestrates stress responses and is hypersecreted in patients with stress-related psychiatric disorders, impairs sustained attention. To this end, male and female rats received central infusions of CRF prior to testing on an operant sustained attention task (SAT), where rats were trained to discriminate signaled from non-signaled events. CRF caused a dose-dependent decrease in SAT performance in both male and female rats. Females were more impaired than males following a moderate dose of CRF, particularly during the middle part of the session. This sex difference was moderated by ovarian hormones. Females in the estrous cycle stage characterized by lower ovarian hormones had a greater CRF-induced attentional impairment than males and females in other cycle stages. Collectively, these studies highlight CRF as a critical stress-related factor that can regulate attentional performance. As sustained attention subserves other cognitive processes, these studies suggest that mitigating high levels of CRF in patients with stress-related psychiatric disorders may ameliorate their cognitive deficits.

Corticotropin-releasing Factor in the Rat Dorsal Raphe Nucleus Promotes Different Forms of Behavioral Flexibility Depending on Social Stress History.

The stress-related neuropeptide, corticotropin-releasing factor (CRF) regulates the dorsal raphe nucleus-serotonin (DRN-5-HT) system during stress and this may underlie affective and cognitive dysfunctions that characterize stress-related psychiatric disorders. CRF acts on both CRF1 and CRF2 receptor subtypes in the DRN that exert opposing inhibitory and excitatory effects on DRN-5-HT neuronal activity and 5-HT forebrain release, respectively. The current study first assessed the cognitive effects of intra-DRN microinfusion of CRF or the selective CRF2 agonist, urocortin II in stress-naive rats on performance of an operant strategy set-shifting task that is mediated by the medial prefrontal cortex (mPFC). CRF (30 ng) facilitated strategy set-shifting performance, whereas higher doses of CRF and urocortin II that would interact with CRF2 were without effect, consistent with a CRF1-mediated action. This dose decreased 5-HT extracellular levels in the mPFC, further supporting a role for CRF1. The effects of CRF were then assessed in rats exposed to repeated social stress using the resident-intruder model. Repeated social stress shifted the CRF effect from facilitation of strategy set shifting to facilitation of reversal learning and this was most prominent in a subpopulation of rats that resist defeat. Notably, in this subpopulation of rats 5-HT neuronal responses to CRF have been demonstrated to shift from CRF1-mediated inhibition to CRF2-mediated excitation. Because 5-HT facilitates reversal learning, the present results suggest that stress-induced changes in the cellular effects of CRF in the DRN translate to changes in cognitive effects of CRF. Together, the results underscore the potential for stress history to shift cognitive processing through changes in CRF neurotransmission in the DRN and the association of this effect with coping strategy.

Effects of intracerebroventricular corticotropin releasing factor on sensory-evoked responses in the rat visual thalamus

Corticotropin releasing factor (CRF) coordinates the brain׳s responses to stress. Recent evidence suggests that CRF-mediated activation of the locus coeruleus-norepinephrine (LC-NE) system contributes to alterations in sensory signal processing during stress. However, it remains unclear whether these actions are dependent upon the degree of CRF release. Using intracerebroventricular (ICV) infusions, we examine the dose-dependent actions of CRF on sensory-evoked discharges of neurons in the dorsal lateral geniculate nucleus of the thalamus (dLGN). The LGN is the primary relay for visual signals from retina to cortex, receiving noradrenergic modulation from the LC. In vivo extracellular recording in anesthetized rats was used to monitor single dLGN neuron responses to light flashes at three different stimulus intensities before and after administration of CRF (0.1, 0.3, 1.0, 3.0 or 10.0μg). CRF produced three main effects on dLGN stimulus evoked activity: (1) increased magnitude of sensory evoked discharges at moderate doses, (2) decreased response latency, and (3) dose-dependent increases in the number of cells responding to a previously sub-threshold (low intensity) stimulus. These modulatory actions were blocked or attenuated by intra-LC infusion of a CRF antagonist prior to ICV CRF administration. Moreover, intra-LC administration of CRF (10ng) mimicked the facilitating effects of moderate doses of ICV CRF on dLGN neuron responsiveness to light stimuli. These findings suggest that stressor-induced changes in sensory signal processing cannot be defined in terms of a singular modulatory effect, but rather are multi-dimensional and dictated by variable degrees of activation of the CRF-LC-NE system.

Corticotropin-releasing factor in the nucleus accumbens shell induces swim depression, anxiety, and anhedonia along with changes in local dopamine/acetylcholine balance

The nucleus accumbens shell (NAcS) has been implicated in controlling stress responses through corticotropin-releasing factor (CRF). In addition to studies indicating that CRF in the NAcS increases appetitive motivation, there is indirect evidence suggesting that NAcS CRF may also cause aversive responses and that these behaviors may be mediated through local dopamine (DA) and acetylcholine (ACh) systems. To provide a direct test of this hypothesis, we used male Sprague–Dawley rats with implanted cannulas aimed at the NAcS. Experiment 1 showed local CRF injection (10 or 50 ng/side) to increase immobility in the forced swim test and a CRF antagonist D-Phe-CRF (12-41) to attenuate this depressive-like behavior. In Experiment 2, injection of CRF (250 ng/side) also decreased the rats' preference for sucrose, while in Experiment 3, CRF (50 or 250 ng/side) induced anxiety-like behaviors in an elevated plus maze and open field. These same doses of CRF in Experiment 4 failed to alter the rats' locomotor activity, indicating that these behavioral changes were not caused by deficits in activity. In Experiment 5, results from in vivo microdialysis revealed that CRF in the NAcS markedly increased local extracellular ACh, while also producing a small increase in DA. These results show that NAcS CRF can generate a variety of aversive behaviors, including swim depression, anhedonia, and anxiety, in addition to approach behavior. They suggest that these behaviors may occur, in part, through enhanced activation of ACh and DA in the NAcS, respectively, supporting a role for this brain area in mediating the dual effects of stress.

Corticotropin-Releasing Factor in the Norepinephrine Nucleus, Locus Coeruleus, Facilitates Behavioral Flexibility

Corticotropin-releasing factor (CRF), the stress-related neuropeptide, acts as a neurotransmitter in the brain norepinephrine nucleus, locus coeruleus (LC), to activate this system during stress. CRF shifts the mode of LC discharge from a phasic to a high tonic state that is thought to promote behavioral flexibility. To investigate this, the effects of CRF administered either intracerebroventricularly (30-300 ng, i.c.v.) or directly into the LC (intra-LC; 2-20 ng) were examined in a rat model of attentional set shifting. CRF differentially affected components of the task depending on dose and route of administration. Intracerebroventricular CRF impaired intradimensional set shifting, reversal learning, and extradimensional set shifting (EDS) at different doses. In contrast, intra-LC CRF did not impair any aspect of the task. The highest dose of CRF (20 ng) facilitated reversal learning and the lowest dose (2 ng) improved EDS. The dose-response relationship for CRF on EDS performance resembled an inverted U-shaped curve with the highest dose having no effect. Intra-LC CRF also elicited c-fos expression in prefrontal cortical neurons with an inverted U-shaped dose-response relationship. The number of c-fos profiles was positively correlated with EDS performance. Given that CRF excites LC neurons, the ability of intra-LC CRF to activate prefrontal cortical neurons and facilitate EDS is consistent with findings implicating LC-norepinephrine projections to medial prefrontal cortex in this process. Importantly, the results suggest that CRF release in the LC during stress facilitates shifting of attention between diverse stimuli in a dynamic environment so that the organism can adapt an optimal strategy for coping with the challenge.

Interactions between corticotropin-releasing factor and the serotonin 1A receptor system on acoustic startle amplitude and prepulse inhibition of the startle response in two rat strains

Both the neuropeptide, corticotropin-releasing factor (CRF) and the serotonin 1A (5-HT1A) receptor systems have been implicated in anxiety disorders and there is evidence that the two systems interact with each other to affect behavior. Both systems have individually been shown to affect prepulse inhibition (PPI) of the acoustic startle response. PPI is a form of sensorimotor gating that is reduced in patients with anxiety disorders including post-traumatic stress and panic disorder. Here, we examined whether the two systems interact or counteract each other to affect acoustic startle amplitude, PPI and habituation of the startle response. In experiment 1, Brown Norway (BN) and Wistar-Kyoto (WKY) rats were administered ether an intraperitoneal (IP) injection of saline or the 5-HT1A receptor agonist, 8-OH-DPAT 10 min prior to receiving an intracerebroventricular (ICV) infusion of either saline or CRF (0.3 μg). In a second experiment, rats were administered either an IP injection of saline or the 5-HT1A receptor antagonist, WAY 100,635 10 min prior to receiving an ICV infusion of saline or CRF. Thirty min after the ICV infusion, the startle response and PPI were assessed. As we have previously shown, the dose of CRF used in these experiments reduced PPI in BN rats and had no effect on PPI in WKY rats. Administration of 8-OH-DPAT alone had no effect on PPI in either rat strain when the data from the two strains were examined separately. Administration of 8-OH-DPAT added to the effect of CRF in BN rats, and the combination of 8-OH-DPAT and CRF significantly reduced PPI in WKY rats. CRF alone had no effect on baseline startle amplitude in either rat strain, but CRF enhanced the 8-OH-DPAT-induced increase in startle in both strains. Administration of WAY 100,635 did not affect the CRF-induced change in PPI and there were no interactions between CRF and WAY 100,635 on baseline startle. The results suggest that activation of the 5-HT1A receptor can potentiate the effect of CRF on endophenotypes of anxiety disorders in animal models.This article is part of a Special Issue entitled ‘Anxiety and Depression’.

Microinjection of Different Doses of Corticotropin-releasing Factor into the Medial Prefrontal Cortex Produces Effects Opposing Anxiety-related Behavior in Rats

Corticotropin-releasing factor (CRF) in the medial prefrontal cortex (mPFC) is suggested to play an important role in mediating fear, anxiety, and depression. The results of the studies of the actions of CRF in the mPFC regarding anxiety-related behavior, however, seem contradictory. In one study, microinjection of CRF into the mPFC produced an increase in anxiety-related behavior on the elevated plus maze, whereas in another study CRF produced an anxiolytic-like effect. To test whether the different doses of CRF used in these experiments are responsible for the differing results, we examined the dose-dependent effects of CRF (0.015, 0.05, 0.15, 0.5, and 1.0 µg/0.5 µL/site) microinjected into the bilateral mPFC of male Wistar rats on anxiety-related behavior in the elevated plus maze. We found that microinjection of 0.05 µg CRF significantly decreased the number of open-arm entries, whereas 1.0 µg CRF significantly increased the time spent on the open arms. The results indicate that CRF has effects opposing anxiety-related behavior in the elevated plus maze: anxiety-related behavior at a lower dose and an anxiolytic-like effect at a higher dose.

Interactions of Stress and CRF in Ethanol-Withdrawal Induced Anxiety in Adolescent and Adult Rats

Repeated stress or administration of corticotropin-releasing factor (CRF) prior to ethanol exposure sensitizes anxiety-like behavior in adult rats. Current experiments determined whether adolescent rats were more sensitive to these challenges in sensitizing ethanol withdrawal-induced anxiety and altering CRF levels in brain during withdrawal. Male adult and adolescent Sprague-Dawley rats were restraint stressed (1 hour) twice 1 week apart prior to a single 5-day cycle of ethanol diet (ED; stress/withdrawal paradigm). Other rats received control diet (CD) and three 1-hour restraint stress sessions. Rats were then tested 5, 24, or 48 hours after the final withdrawal for anxiety-like behavior in the social interaction (SI) test. In other experiments, adolescent rats were given two microinjections of CRF icv 1 week apart followed by 5 days of either CD or ED and tested in social interaction 5 hours into withdrawal. Finally, CRF immunoreactivity was measured in the central nucleus of the amygdala (CeA) and paraventricular nucleus (PVN) after rats experienced control diet, repeated ethanol withdrawals, or stress/withdrawal. Rats of both ages had reduced SI following the stress/withdrawal paradigm, and this effect recovered within 24 hours. Higher CRF doses were required to reduce SI in adolescents than previously reported in adults. CRF immunohistochemical levels were higher in the PVN and CeA of CD-exposed adolescents. In adolescent rats, repeated ethanol withdrawals decreased CRF in the CeA but was not associated with decreased CRF cell number. There was no change in CRF from adult treatments. In the production of anxiety-like behavior, adolescent rats have equal sensitivity with stress and lower sensitivity with CRF compared to adults. Further, adolescents had higher basal levels of CRF within the PVN and CeA and reduced CRF levels following repeated ethanol withdrawals. This reduced CRF within the CeA could indicate increased release of CRF, and future work will determine how this change relates to behavior.

Possible inhibitory roles of endogenous 2-arachidonoylglycerol during corticotropin-releasing factor-induced activation of central sympatho-adrenomedullary outflow in anesthetized rats

We previously reported that intracerebroventricularly (i.c.v.) administered corticotropin-releasing factor (CRF) (0.5–3.0 nmol/animal) dose-dependently elevates plasma noradrenaline and adrenaline through brain phospholipase C-, diacylglycerol lipase- and prostanoids-mediated mechanisms in rats. Diacylglycerol produced by phospholipase C from phospholipids can be hydrolyzed by diacylglycerol lipase into 2-arachidonoylglycerol, which may be further hydrolyzed by monoacylglycerol lipase into arachidonic acid, a precursor of prostanoids. Recently, 2-arachidonoylglycerol has been recognized as a major brain endocannabinoid, which can modulate synaptic transmission through presynaptic cannabinoid CB 1 receptors. Released 2-arachidonoylglycerol is rapidly deactivated by uptake into cells and enzymatic hydrolysis. In the present study, therefore, we examined (1) the involvement of brain 2-arachidonoylglycerol, (2) the regulatory role of 2-arachidonoylglycerol as a brain endocannabinoid, and (3) the effect of exogenous cannabinoid receptor agonist, on the CRF-induced elevation of plasma noradrenaline and adrenaline using anesthetized rats. The elevation of both catecholamines induced by a submaximal dose of CRF (1.5 nmol/animal, i.c.v.) was reduced by i.c.v. administered MAFP (monoacylglycerol lipase inhibitor) (0.7 and 1.4 µmol/animal), AM 404 (endocannabinoid uptake-inhibitor) (80 and 250 nmol/animal) and ACEA (cannabinoid CB 1 receptor agonist) (0.7 and 1.4 µmol/animal), while AM 251 (cannabinoid CB 1 receptor antagonist) (90 and 180 nmol/animal, i.c.v.) potentiated the response induced by a small dose of CRF (0.5 nmol/animal, i.c.v.). These results suggest a possibility that 2-arachidonoylglycerol is endogenously generated in the brain during CRF-induced activation of central sympatho-adrenomedullary outflow, thereby inhibiting the peptide-induced response by activation of brain cannabinoid CB 1 receptors in anesthetized rats.

Brainstem reticulospinal neurons are targets for corticotropin-releasing factor-Induced locomotion in roughskin newts

Stress-induced release or central administration of corticotropin-releasing factor (CRF) enhances locomotion in a wide range of vertebrates, including the roughskin newt, Taricha granulosa. Although CRF's stimulatory actions on locomotor behavior are well established, the target neurons through which CRF exerts this effect remain unknown. To identify these target neurons, we utilized a fluorescent conjugate of CRF (CRF-TAMRA 1) to track this peptide's internalization into reticulospinal and other neurons in the medullary reticular formation (MRF), a region critically involved in regulating locomotion. Epifluorescent and confocal microscopy revealed that CRF-TAMRA 1 was internalized by diverse MRF neurons, including reticulospinal neurons retrogradely labeled with Cascade Blue dextran. In addition, we immunohistochemically identified a distinct subset of serotonin-containing neurons, located throughout the medullary raphé, that also internalized the fluorescent CRF-TAMRA 1 conjugate. Chronic single-unit recordings obtained from microwire electrodes in behaving newts revealed that intracerebroventricular (icv) administration of CRF-TAMRA 1 increased medullary neuronal firing and that appearance of this firing was associated with, and strongly predictive of, episodes of CRF-induced locomotion. Furthermore, icv administered CRF-TAMRA 1 produced behavioral and neurophysiological effects identical to equimolar doses of unlabeled CRF. Collectively, these findings provide the first evidence that CRF directly targets reticulospinal and serotonergic neurons in the MRF and indicate that CRF may enhance locomotion via direct effects on the hindbrain, including the reticulospinal system.

Corticotropin-Releasing Factor (CRF) Sensitization of Ethanol Withdrawal-Induced Anxiety-Like Behavior is Brain Site Specific and Mediated by CRF-1 Receptors: Relation to Stress-Induced Sensitization

In abstinent alcoholics, stress induces negative affect-a response linked to craving and relapse. In rats, repeated stresses at weekly intervals before 5-day ethanol diet sensitize withdrawal-induced anxiety-like behavior ("anxiety") that is blocked by a corticotrophin-releasing factor 1 (CRF-1)-receptor antagonist. Current experiments were performed to identify brain sites that support CRF involvement in stress sensitization of ethanol withdrawal-induced anxiety-like behavior. First, different doses of CRF microinjected weekly into the central amygdala (CeA) before ethanol exposure produced a dose-related sensitization of anxiety during ethanol withdrawal. Subsequently, CRF microinjection into the basolateral amygdala, dorsal raphe nucleus (DRN), or dorsal bed nucleus of the stria terminalis (d-BNST) also sensitized ethanol withdrawal-induced anxiety. In contrast, sensitization of ethanol withdrawal-induced anxiety was not observed after weekly CRF administration into the ventral-BNST, CA1-hippocampal region, or hypothalamic-paraventricular nucleus. Then, experiments documented the CRF receptor subtype responsible for CRF and stress sensitization of withdrawal-induced anxiety. Systemic administration of a CRF-1 receptor antagonist before CRF microinjection into the CeA, DRN, or d-BNST prevented CRF-induced sensitization of anxiety during ethanol withdrawal. Furthermore, repeated microinjections of urocortin-3, a CRF-2 receptor agonist, into the CRF-positive sites did not sensitize anxiety after withdrawal from ethanol. Finally, microinjection of a CRF-1 receptor antagonist into the CeA, DRN, or d-BNST before stress blocked sensitization of anxiety-like behavior induced by the repeated stress/ethanol withdrawal protocol. These results indicate that CRF released by stress acts on CRF-1 receptors within specific brain regions to produce a cumulative adaptation that sensitizes anxiety-like behavior during withdrawal from chronic ethanol exposure.

Fluoxetine potentiates the effects of corticotropin-releasing factor on locomotor activity and serotonergic systems in the roughskin newt, Taricha granulosa

The anxiety- and stress-related neuropeptide corticotropin-releasing factor (CRF) elicits behavioral changes in vertebrates including increases in behavioral arousal and locomotor activity. Intracerebroventricular injections of CRF in an amphibian, the roughskin newt (Taricha granulosa), induces rapid increases in locomotor activity in both intact and hypophysectomized animals. We hypothesized that this CRF-induced increase in locomotor activity involves a central effect of CRF on serotonergic neurons, based on known stimulatory actions of serotonin (5-hydroxytryptamine, 5-HT) on spinal motor neurons and the central pattern generator for locomotor activity in vertebrates. In Experiment 1, we found that neither intracerebroventricular injections of low doses of CRF (25 ng) nor the selective serotonin reuptake inhibitor fluoxetine (10, 100 ng), by themselves, altered locomotor activity. In contrast, newts treated concurrently with CRF and fluoxetine responded with marked increases in locomotor activity. In Experiment 2, we found that increases in locomotor activity following co-administration of CRF (25 ng) and fluoxetine (100 ng) were associated with decreased 5-HT concentrations in a number of forebrain structures involved in regulation of emotional behavior and emotional states, including the ventral striatum, amygdala pars lateralis, and dorsal hypothalamus, measured 37 min after treatment. These results are consistent with the hypothesis that CRF stimulates locomotor activity through activation of serotonergic systems.

Corticotropin releasing factor enhances attentional function as assessed by the five-choice serial reaction time task in rats

Previous studies have shown that psychological stress affects attentional function, and corticotropin releasing factor (CRF) is closely related to stress responses. In the present study, we examined the effect of CRF on attentional function using a five-choice serial reaction time task (5-CSRTT) in rats. Accuracy in the 5-CSRTT was used as the index of attentional function. Human/rat CRF was intracerebroventricularly injected (0, 0.01, 0.1, or 1 μg/5 μl saline) 20 min before the beginning of the test trial in male Lister hooded rats that generally show high performance in the 5-CSRTT. As a result, 0.1 μg of CRF, but not other doses of CRF, increased accuracy in the 5-CSRTT. However, 0.1 μg of CRF did not affect impulsivity, motivation/appetite, perseverative tendency, or motor function. Even when Wistar rats, which generally show poor performance in the 5-CSRTT were used, 0.1 μg of CRF increased accuracy in the 5-CSRTT. However, the effect of CRF was not significantly attenuated by intracerebroventricular injection of the CRF 1 receptor antagonist antalarmin (5 μg). These results showed that CRF selectively enhances attentional function regardless of baseline attentional performance and rat strain, but this effect may be due to the pathways other than CRF 1 receptors. The present results suggest that CRF is involved in stress-related changes of attention and indicate that moderate stress, but not severe stress, may enhance attentional performance. Furthermore, these findings also indicate that the CRF-related substance could be a target for the development of an agent to improve attentional function.