corticotrophin - releasing factor ( crf ) is not only a stress hormone but also acts as a neuromodulator outside the hypothalamic - pituitary - adrenocortical ( hpa ) axis , playing an important role in anxiety , depression , and pain modulation ( 1 ) . previous human studies have also found elevated levels of crf in the cerebrospinal fluid of patients with depression ( 2 , 3 ) or posttraumatic stress disorder ( ptsd ) ( 4 - 6 ) . there is accumulating evidence that the central pain circuits may be anatomically related to those that control depression and anxiety ( 7 ) . thus , a major site of extra - hypothalamic expression of crf and its receptors is the cea , which not only mediates many aspects of anxiety and fear ( 8) , but is also called the nociceptive amygdala for being an important site for central processing of nociceptive information ( 9 ) . colorectal distension ( crd ) is commonly used to model visceral pain in experimental animals and induces significant up - regulation of the immediate early gene product c - fos , as well as the phosphorylated extracellular signal - regulated kinase ( p - erk ) in the spinal cord , brainstem , limbic areas , and neocortex in conscious rats ( 10 - 13 ) . previous studies have indicated that crf and its receptors in the brain play a critical role in crd - elicited anxiogenic responses and visceral sensitivity in conscious rats ( 14 - 17 ) . indeed , intracerebral administration of crf stimulated anxiety - like behavior and hypersensitivity to crd ( 17 ) and a crf receptor 1 antagonist attenuated the crd - induced visceral pain and anxiety in rats ( 16 ) . however , few studies have investigated the differences in visceral pain - induced crf expression in the cea between conscious and unconscious rats ( 18 , 19 ) . since the cea integrates descending cortical control with ascending nociceptive input from the parabrachial nucleus , and the conscious recognition of pain attributes the behavioral or emotional response to pain ( 20 ) , we wanted to ascertain whether the suppression of cortical activation during noxious visceral stimulation affects the expression of crf in the cea . for this , we studied the expression of crf , together with that of p - erk as a marker for pain - specific activation of neurons in the cea , and of c - fos as a marker for activation of spinal neurons . all experiments were performed on adult male sprague - dawley rats ( 280 - 300 g ; orient bio inc . , gyeonggi - do , korea ) orient bio , korea ) according to the protocol 2008 - 08 approved by the institutional animal investigation committee of hanyang university . rats were divided into a colorectal distention group ( crd , n=6 ) , a colorectal distention with general anesthesia group ( crd+anesthesia , n=6 ) , and a control group with balloon insertion without distention ( con , n=6 ) . all animals were handled for 7 days prior to the experiment to minimize stress induced by manipulation . for crd , a 6 - 7 cm - long polyethylene catheter with a latex balloon at the end was inserted through the anus into the descending colon and rectum and fastened to the tail with surgical tape ( 21 ) . the length of the balloon was 4 cm and a total length of 5 cm ( with catheter ) was inserted into the anus and placed in the descending colon and the rectum . after 30 min , distension was produced by inflating the balloon to 80 mmhg three times for 5-min with a 1-min inter - trial interval . rats were allowed to survive for 30 min after crd to study the expression of p - erk and c - fos or for 3 days to study the expression of crf . for tissue fixation , rats were deeply anesthetized with a mixture of tiletamine+zolazepam ( 27.78 mg / kg , intraperitoneal [ i.p . ] ) and xylazine ( 0.647 mg / kg , i.p . ) and perfused intracardially with 100 ml of normal saline , followed by 600 ml of 4% paraformaldehyde in 0.1 m phosphate buffer ( ph 7.2 ) . the brain and spinal cord at the level of l6-s1 were removed and postfixed for 4 hr in the same fixative and then transferred to a 30% sucrose solution for a minimum of 18 hr . sections of amygdala and spinal cord were cut on a cryostat at 40 and 30 m , respectively , and processed for immunofluorescence . sections were blocked with 10% normal donkey serum in 0.01 m phosphate buffered saline ( pbs , ph 7.4 ) for 40 min at room temperature and incubated overnight at 4 with rabbit anti - crf ( 1:1,000 ; phoenix pharmaceuticals , inc . , belmont , ca , usa ) , rabbit anti - phospho - p44/42map kinase ( 1:100 ; cell signaling , beverly , ma , usa ) or goat anti - c - fos ( 1:500 ; serotec , raleigh , nc , usa ) antibodies in pbs . sections were then incubated with cy3-conjugated donkey antibodies ( 1:200 in pbs ; jackson immunolab , west grove , pa , usa ) for 3 hr , rinsed , mounted , and coverslipped with vectashield ( vector laboratories inc . , burlingame , ca , usa ) . for quantitative assessment of immunostaining ( 22 ) , the crf - positive area in the cea was measured with image analysis software ( analysis pro.v3.1 sis mnster , germany ) . the immunofluorescence images of the bilateral cea in each of 5 sections per rat 400 m apart between coronal levels -0.80 mm and -2.80 mm ( paxinos ) were acquired by a cooled ccd camera attached to the microscope under a constant exposure and digital gain , and a frame size of 1,7601,321 m . in each image , the crf - positive area was segmented by setting a value for a given threshold , and the number of segmented pixels was counted . p - erk - positive neurons were counted in 12 images of the bilateral cea from 6 sections per rat 400 m apart ( magnification 200 , frame size=439330 m ) . c - fos positive nuclei were counted in 12 images of the bilateral dorsal horn from 6 sections per rat 300 m apart ( magnification 100 , frame size=898660 m ) . all results were analyzed with spss 17.0 ( spss , chicago , il , usa ) . differences between groups were analyzed by one - way analysis of variance ( anova ) , followed by a tukey posttest in which all groups were compared for crf expression and number of p - erk and c - fos positive - neurons . all experiments were performed on adult male sprague - dawley rats ( 280 - 300 g ; orient bio inc . , gyeonggi - do , korea ) orient bio , korea ) according to the protocol 2008 - 08 approved by the institutional animal investigation committee of hanyang university . rats were divided into a colorectal distention group ( crd , n=6 ) , a colorectal distention with general anesthesia group ( crd+anesthesia , n=6 ) , and a control group with balloon insertion without distention ( con , n=6 ) . all animals were handled for 7 days prior to the experiment to minimize stress induced by manipulation . for crd , a 6 - 7 cm - long polyethylene catheter with a latex balloon at the end was inserted through the anus into the descending colon and rectum and fastened to the tail with surgical tape ( 21 ) . the length of the balloon was 4 cm and a total length of 5 cm ( with catheter ) was inserted into the anus and placed in the descending colon and the rectum . after 30 min , distension was produced by inflating the balloon to 80 mmhg three times for 5-min with a 1-min inter - trial interval . rats were allowed to survive for 30 min after crd to study the expression of p - erk and c - fos or for 3 days to study the expression of crf . for tissue fixation , rats were deeply anesthetized with a mixture of tiletamine+zolazepam ( 27.78 mg / kg , intraperitoneal [ i.p . ] ) and xylazine ( 0.647 mg / kg , i.p . ) and perfused intracardially with 100 ml of normal saline , followed by 600 ml of 4% paraformaldehyde in 0.1 m phosphate buffer ( ph 7.2 ) . the brain and spinal cord at the level of l6-s1 were removed and postfixed for 4 hr in the same fixative and then transferred to a 30% sucrose solution for a minimum of 18 hr . sections of amygdala and spinal cord were cut on a cryostat at 40 and 30 m , respectively , and processed for immunofluorescence . sections were blocked with 10% normal donkey serum in 0.01 m phosphate buffered saline ( pbs , ph 7.4 ) for 40 min at room temperature and incubated overnight at 4 with rabbit anti - crf ( 1:1,000 ; phoenix pharmaceuticals , inc . , belmont , ca , usa ) , rabbit anti - phospho - p44/42map kinase ( 1:100 ; cell signaling , beverly , ma , usa ) or goat anti - c - fos ( 1:500 ; serotec , raleigh , nc , usa ) antibodies in pbs . sections were then incubated with cy3-conjugated donkey antibodies ( 1:200 in pbs ; jackson immunolab , west grove , pa , usa ) for 3 hr , rinsed , mounted , and coverslipped with vectashield ( vector laboratories inc . , burlingame , ca , usa ) . for quantitative assessment of immunostaining ( 22 ) , the crf - positive area in the cea was measured with image analysis software ( analysis pro.v3.1 sis mnster , germany ) . the immunofluorescence images of the bilateral cea in each of 5 sections per rat 400 m apart between coronal levels -0.80 mm and -2.80 mm ( paxinos ) were acquired by a cooled ccd camera attached to the microscope under a constant exposure and digital gain , and a frame size of 1,7601,321 m . in each image , the crf - positive area was segmented by setting a value for a given threshold , and the number of segmented pixels was counted . p - erk - positive neurons were counted in 12 images of the bilateral cea from 6 sections per rat 400 m apart ( magnification 200 , frame size=439330 m ) . c - fos positive nuclei were counted in 12 images of the bilateral dorsal horn from 6 sections per rat 300 m apart ( magnification 100 , frame size=898660 m ) . all results were analyzed with spss 17.0 ( spss , chicago , il , usa ) . differences between groups were analyzed by one - way analysis of variance ( anova ) , followed by a tukey posttest in which all groups were compared for crf expression and number of p - erk and c - fos positive - neurons . an immunoreaction for crf was observed in the neuronal perikarya and neuropil throughout the rostrocaudal extent of the cea . after crd , the crf - immunopositive area had increased significantly in both conscious ( 72.753.48 m ) and unconscious ( 23.105.24 m ) rats when compared to the control animals . however , the crf - positive area in the rats with crd under general anesthesia was significantly lower than that of the conscious animals ( fig . after crd , the number of perk - positive neurons significantly increased compared to control animals . in parallel with the changes in crf expression , the number of perk - positive neurons was significantly reduced in anesthetized ( 95.1722.47 m ) vs conscious ( 264.5065.98 m ) rats with crd ( fig . the number of c - fos positive nuclei in the dorsal horn was significantly increased by crd over the control animals ( 308.015.95 m ) , but the difference in expression between anesthetized ( 567.67129.35 m ) and conscious ( 505.6750.70 m ) rats was insignificant ( fig . the main result of this study is that the expression of crf and perk in the cea was significantly increased by crd - induced visceral pain , and that this effect was attenuated by general anesthesia , whereas the expression of c - fos in the spinal cord was unaffected by general anesthesia . few studies have investigated the differences between conscious and unconscious animals in regional brain activity induced by visceral pain . functional magnetic resonance imaging in anesthetized rats revealed that the amygdala , thalamus , cerebellum , and hippocampus were significantly activated during crd - induced visceral pain ( 19 ) . similarly , crd elicited a significant increase in regional blood flow in the cerebral cortex and amygdala in conscious rats by the autoradiographic cerebral perfusion method ( 23 ) . the greatest increase in evoked cortical activity induced by somatic pain was observed in the contralateral somatosensory cortex and that was significantly lower in anesthetized rats than in conscious rats ( 18 ) . our results demonstrate that the level of consciousness may affect the expression of crf and perk in the cea induced by visceral pain . polymodal sensory information of thalamic and cortical origin is related to the cea through connections with the lateral and basolateral amygdaloid nuclei ( 24 , 25 ) . the amygdala also receives nociceptive information via the spinoparabrachial and spinohypothalamic tracts ( 20 ) . we speculate that the decreased crf expression in the cea of anesthetized rats is related to the reduction of cortical activity caused by general anesthesia , which disrupts the relay of pain - related information from the neocortex to the amygdala . in our results , crd caused immediate neuronal activation in the cea as indicated by the increased expression of p - erk . activation of erk and the related signaling pathways in the cerebral cortex and the amygdala plays an important role in the enhancement of memory - related neuronal activity ( 26 ) and in the development of anxiety ( 27 , 28 ) . our results also indicate a sustained increased expression of crf in the amygdala at 72 hr after crd . previous animal studies have suggested that exposures to psychological and physiological stresses immediately increase crf expression in the cea ( 29 , 30 ) . however , to our knowledge , there has been no study examining prolonged effect of stresses on amygdala crf system in the rat . since ptsd and major depression occur frequently following traumatic events , and there is usually a temporal gap between traumatic events and their related symptoms later in life , to understand the prolonged effect of stress at a specific period on the neuroendocrine system and its related symptoms is clinically important . in conclusion , suppression of cortical activation by general anesthesia during visceral pain attenuated the expression of crf in the cea , suggesting that the conscious recognition of pain at a cortical level is an important determinant of the involvement of crf in the mechanisms of subcortical pain control .
corticotrophin - releasing factor ( crf ) , a key regulator of the hypothalamic - pituitary axis , is expressed in the central nucleus of the amygdala ( cea ) and its expression is upregulated in stress - related disorders . 
 we investigated here the effect of noxious colorectal distension ( crd ) on the expression of crf in the cea of conscious and unconscious rats . 
 adult male rats with or without general anesthesia were exposed to visceral pain induced by crd for 5 min ; this procedure was repeated 3 times with 1 min resting after each distension . 
 the rats were sacrificed and sections of the cea were immunostained for crf as an indicator for anxiety response , and for phosphorylated extracellular signal - regulated kinase ( p - erk ) as a marker for pain - specific activation of neurons ; sections of lumbosacral spinal cord were immunostained for c - fos as a marker for activation of spinal neurons . 
 crd elicited a significant increase in the expression of crf and p - erk in the cea and of c - fos in the spinal cord . 
 general anesthesia attenuated the increase in crf and p - erk in the cea , but did not affect the expression of spinal c - fos . 
 these results suggest that conscious recognition of pain at higher brain centers is an important determinant of crf expression in the cea .