Neonatal maternal separation enhances central sensitivity to noxious colorectal distention in rat

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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s

Research Report

Neonatal maternal separation enhances central sensitivity to noxious colorectal distention in rat Elaine K.Y. Chung a , XiaoJun Zhang a , Zhi Li a , Hongqi Zhang a , HongXi Xu b , ZhaoXiang Bian a,⁎ a

School of Chinese Medicine, Hong Kong Baptist University, Kowloon Tong, Hong Kong, China Hong Kong Jockey Club institute of Chinese medicine, Hong Kong SAR, China

b

A R T I C LE I N FO

AB S T R A C T

Article history:

Psychological stress experienced in early life plays an important role in the development

Accepted 18 March 2007

of visceral hyperalgesia in irritable bowel syndrome (IBS). Neonatal maternal separation

Available online 23 March 2007

has been shown to trigger a long-term alternation in stress-induced responses to visceral nociceptive stimuli in rats. The aim of the present study was to show a direct evidence of

Keywords:

stress-induced alteration in central neuronal responses to colorectal distention (CRD) in

Irritable bowel syndrome (IBS)

rats by a quantitative study of c-fos expression in relevant brain structures. Male Wistar

Neonatal maternal separation (NMS)

rat pups were subjected to 180-min daily neonatal maternal separation (NMS) for 13

Visceral hyperalgesia

consecutive days (from PND 2 to PND 14). The expression of c-fos was examined by using

Colorectal distention (CRD)

immunohistochemistry. Increased c-fos expression was observed, for the first time, in the cingulate cortex (3-fold) in NMS rats in comparison with the control group at basal condition. At noxious CRD (80 mm Hg), c-fos expression was induced in the supraspinal centers and in both the superficial (laminae I–II) and the deeper laminae (laminae V–VI and X) of the spinal cord in rats. Significantly more Fos-IR nuclei were found in the laminae I and II, and laminae V–VI of the lumbarsacral spinal cord, the paraventricular thalamic nucleus, the cingulate cortex, the amygdaloid central nucleus in NMS rats, but not in the solitary tract, the central medial thalamic nucleus, the ventromedial hypothalamic nucleus, and the periaquaductal gray. The present results indicate that NMS has sensitized the cingulate cortex and upregulated the activity of the ascending pathway at spinal level as well as the thalamo-cortico-amydala pathway to CRD. The upregulation and sensitization of these pathways may be responsible for the development of visceral hypersensitivity in IBS. © 2007 Elsevier B.V. All rights reserved.

1.

Introduction

Clinical and experimental studies have shown that visceral hyperalgesia, a major symptom of irritable bowel syndrome (IBS), is caused by stress (Mayer et al., 2001; McEwen and Stellar, 1993; Pacak and Palkovits, 2001). Psychological stress

activates and triggers functional changes in the central circuitry of the autonomic nervous system, in particular the pain modulation system and the ascending aminergic pathways (Jones et al., 2006; Mayer, 2000b; Pacak and Palkovits, 2001). Evidence suggests that stress, in particular that experienced in early period of life, triggers long-term

⁎ Corresponding author. Fax: +852 34112905. E-mail address: [email protected] (Z. Bian). 0006-8993/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2007.03.047

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changes in visceral functions and visceral sensitivity to noxious stimuli (Barreau et al., 2004; Murray et al., 2004; Posserud et al., 2004). Studies using functional brain imaging techniques in patients with IBS have also shown evidence on the hypersensitivity of the central pain modulation circuit demonstrating the significant role that the central nuclei play in the development and maintenance of visceral hyperalgesia (Bonaz et al., 2002; Dickhaus et al., 2003; Yuan et al., 2003). The central integration of visceral nociception is mediated at both the cortical and subcortical levels by the systems involved in the processing of visceral information and circuits engaged in processing psychological stress (Coghill et al., 1999; Jones et al., 2006). Abundant of evidence suggests that chronic activation of supraspinal centers leads to maladaptation of homeostatic mechanisms and eventually lead to visceral pain (Jones et al., 2006; Urban and Gebhart, 1999). Visceral nociceptive afferent fibers project onto spinal nociceptive neurons located in the superficial laminae, the lateral neck of the dorsal horn and lamina X of spinal cord that convey information to supraspinal centers through pathways, namely the spinothalamic, spinohypothalamic, spinosolitary and the spinoreticular tracts, etc. (Al-Chaer and Traub, 2002; Clark and Treisman, 2004; Mayer, 2000a). Growing body of evidence has demonstrated an important role that the dorsal columns pathway plays in relaying visceral nociceptive information to the supraspinal center (Palecek, 2004; Willis et al., 1999). Brain regions that have been found important in generating pain perception and descending pain modulation include the cingulate cortex, medial thalamus, amygdala, hypothalamus, periaquaductal gray and the solitary tract (Clark and Treisman, 2004; Jones et al., 2006; Millan, 2002). In rodents, neonatal maternal separation (NMS) of newborns results in visceral hyperalgesia and an increased colonic motility in response to stress in adulthood, the symptoms mimicking IBS (Barreau et al., 2004; Gareau et al., 2006; Pihoker et al., 1993; Welting et al., 2005). Upregulation of visceral pain perception in rats subjected to early life stress indicates an enhanced responsiveness of central pain circuitry (Coutinho et al., 2002; Stam et al., 2002). There is a body of evidence showing that NMS results in permanent changes in the central nervous system including both the central circuits mediating autonomic responses and the pain modulatory responses (Caldji et al., 2000; Coutinho et al., 2002; Pihoker et al., 1993). However, direct evidence of the changes in the regional brain activity in the targets of the ascending visceral afferents is scarce. In the present study, we sought to determine whether neonatal stress in rats could trigger long-term changes in nociceptive processing in the supraspinal structures by using a well-established animal model of IBS (Barreau et al., 2004). The protein product of the immediate-early gene c-fos was used as a marker to indicate the activity of the spinal and supraspinal structures to noxious colorectal distention (CRD) (Monnikes et al., 2003; Sagar et al., 1988). The present study was aimed to find direct evidence of the stress-induced central alternations of visceral nociceptive processing and the association between neonatal stress and visceral hyperalgesia.

2.

Results

2.1.

Increased pain responses in NMS rats

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NMS induced visceral hyperalgesia in adult rats. The threshold pressure that evokes AWR in response to CRD in NMS rats was found to be 24.5 ± 1.6 mm Hg (n = 18). In contrast to 34.9 ± 1.7 mm Hg (n = 18) of the normal group (Fig. 1), a significant reduction (29.8 ± 5.6%, n = 18, P < 0.001) in threshold pressure was found in the NMS rats. By EMG recording, an increase in visceral nociceptive responses was found in the NMS rats. An increase in both the amplitude and intensity of EMG signal to noxious CRD (80 mm Hg) was observed in the NMS rats compared to the normal group (Fig. 2). The mean AUC values of EMG signal of NMS rats and normal rats were 575.7 ± 17.5 (n = 12) and 415.8 ± 48.3 (n = 12) respectively, thus, a significant increase of 38.5% (P < 0.001) in pain responses to noxious CRD (80 mm Hg) was found in the NMS rats.

2.2. CRD induced c-fos expression in the brain nuclei and spinal cord of normal rat Fos-immunoreactive (IR) nuclei induced by noxious CRD (80 mm Hg) were observed in the regions of limbic system. After CRD was applied to normal rats, a significant increase in the number of Fos-IR nuclei per section per side was found in the cingulate cortex, the central amygdaloid nucleus, the central medial thalamic nucleus, the paraventricular thalamic nucleus, the ventromedial hypothalamic nucleus, the periaqueductal gray, and the nucleus of the solitary tract, as well as the laminae I and II, the laminae V– VI and the deeper lamina X of lumbarsacral spinal cord by a quantitative analysis method (Table 1, P < 0.05, n = 5). The highest increase in the number of Fos-IR nuclei induced by noxious CRD in the supraspinal centers was found in the central medial thalamic nucleus (6-fold), whereas the least was found in the periaqueductal gray (2-fold) (Table 1).

Fig. 1 – Statistical comparison of the threshold pressure of CRD that elicits an observable abdominal withdrawal reflex in normal and NMS rats. The bars in each chart represent the mean of threshold pressure that elicits a distinctive abdominal muscle contraction in response to CRD of 18 rats in each group. Error bars indicate the standard errors of the mean. Asterisks represent statistical significantly different (P < 0.001) compared to the other group according to the unpaired t test.

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sparse labeling was seen in the normal rats (Fig. 3D). In amygdala, intense c-fos expression distinctly concentrated in the central nucleus was found in the NMS rat (Fig. 4A) in comparison with the normal rats (Fig. 4B). Fos-IR nuclei in paraventricular thalamic nucleus (PV) of NMS rat concentrated predominantly in the dorsal zone of the medial parvocellular part, with considerable expression in other parvocellular region and magnocellular zone (Fig. 4C). However, only moderate c-fos expression was found in all parts of PV in normal rats (Fig. 4D). In the spinal cord, a significant increase in Fos-IR cells was observed in the superficial laminae, I and II in the dorsal horn (Fig. 4E) compared to the normal rats (Fig. 4F), as well as the neck of the dorsal horn at laminae V–VI of NMS rats (Fig. 4G) compared to the normal (Fig. 4H). Variable numbers were also found in other laminae of the spinal cord, but only those receiving the visceral afferents (Al-Chaer and Traub, 2002; Menetrey et al., 1989), were analyzed in the present study. The number and the pattern of distribution of Fos-IR nuclei in other selected regions of the central medial thalamic nucleus (Fig. 5A), the ventromedial hypothalamic nucleus (Fig. 5B), the periaquaductal gray (Fig. 5C), and the solitary tract (Fig. 5D) of the NMS rats were found to be unchanged. Fig. 2 – Representative electromyographic (EMG) recordings of the normal rat (A) and the NMS rat (B) in a 20-s noxious (80 mm Hg) colorectal distention (CRD) period. The arrows indicate the time CRD started and ended. (C) Statistical analysis of the average AUC value of the EMG amplitude of the normal and the NMS rat. Asterisks represent statistical significantly different (P < 0.001) compared to the other group according to the unpaired t test.

2.3. NMS-induced c-fos expression in the cingulate cortex of adult rats In the rats subjected to NMS, under the condition without any external stimuli, the number of Fos-IR nuclei per section per side was significantly increased (3-fold) in the cingulate cortex as compared to the normal group (Table 1, P < 0.05, n = 5). Representative micrographs of NMS and normal cingulate cortex were shown in Figs. 3A and B respectively. Slightly increases in the number of Fos-IR nuclei were also observed in all other selected brain regions and in the spinal cord of NMS rats, but the differences were not statistically significant (Table 1).

2.4. Upregulated c-fos expression in different brain nuclei and spinal cord of NMS rat in response to CRD Comparing the number of Fos-IR nuclei induced by noxious CRD in NMS and the normal rats, significant increases were found in the cingulate cortex, the central amygdaloid nucleus, the paraventricular thalamic nucleus, the laminae I–II and laminae V–VI of spinal cord of NMS rats, in comparison with those same regions of NH rats (Table 1, P < 0.05, n = 5). Dense Fos-IR cells were observed in the whole region of the cingulate cortex of NMS rats (Fig. 3C), whereas only scattered

3.

Discussion

Early life stress has long been implicated in the aetiology of IBS. Neonatal maternal separation (NMS), one form of early life stress, has been found to trigger visceral hyperalgesia and

Table 1 – The mean number of c-fos-IR nuclei per section per side from the brain nuclei and the lumbosacral spinal cord of normal and NMS rat with or without noxious (80 mm Hg) colonic distension (CRD)

Cg CeA T: CM T: PV Hp: VMH PAG NTS S: laminae I–II S: laminae V–VI S: lamina X

Normal (n = 5)

NMS (n = 5)

Normal CRD (n = 5)

NMS CRD (n = 5)

22.3 ± 3.5 21.3 ± 6.0 15.4 ± 6.2 22.1 ± 6.2 34.1 ± 6.0 38.7 ± 4.4 6.3 ± 1.2 3.0 ± 0.7

69.8 ± 13.5 ⁎ 27.3 ± 1.7 34.0 ± 8.6 30.6 ± 6.8 48.4 ± 6.4 49.7 ± 6.2 10.3 ± 1.3 6.0 ± 0.7

76.2 ± 8.7 ⁎ 54.9 ± 11.8 ⁎ 96.0 ± 37.5 ⁎ 57.3 ± 9.0 ⁎ 67.2 ± 8.2 ⁎ 70.6 ± 10.0 ⁎ 24.4 ± 5.5 ⁎ 9.1 ± 0.8 ⁎

127.6 ± 9.9 ⁎, ⁎⁎ 85.2 ± 8.1 ⁎, ⁎⁎ 104.8 ± 33.5 ⁎ 85.9 ± 11.6 ⁎, ⁎⁎ 83.0 ± 10.6 ⁎ 68.2 ± 6.9 ⁎ 29.1 ± 2.8 ⁎ 13.4 ± 2.3 ⁎, ⁎⁎

6.0 ± 1.0

9.9 ± 0.9

24.7 ± 2.5 ⁎

32.4 ± 3.5 ⁎, ⁎⁎

7.8 ± 0.8

7.8 ± 0.9

13.7 ± 1.6 ⁎

15.1 ± 1.2 ⁎

Data Data are are expressed expressed as as mean mean ± ± SEM SEM as as determined determined by by the the average average number of c-fos-IR c-fos-IR nuclei nuclei per per section section per per side. side. number of Abbreviations: Cg, cingulate cortex; CeA, central amygdaloid Abbreviations: Cg, cingulate cortex; CeA, central amygdaloid nucleus; nucleus; T, thalamus; CM, central medial thalamic nucleus; PV, T, thalamus; CM, central medial thalamic nucleus; PV, paravenparaventricular thalamic nucleus; VMH, ventromedial hypothalatricular thalamic VMH, ventromedial hypothalamic mic nucleus; PAG,nucleus; periaqueductal gray; NTS, solitary tract; S,nucleus; spinal PAG, periaqueductal gray; NTS, solitary tract; S, spinal cord. cord. <0.05 0.05 vs. vs. Normal. Normal. ⁎⁎ PP< <0.05 0.05vs. vs.Normal NormalCRD. CRD. ⁎⁎⁎⁎ PP<

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Fig. 3 – Light micrographs of representative sections in the region showing the cingulate cortex of normal (in right column) and NMS rat (in left column) without CRD (A and B) or with CRD (C and D). Scare bar in A: 200 μm (for A–D).

colonic dysfunction in several rodent models (Barreau et al., 2004; Gareau et al., 2006; Rosztoczy et al., 2003). The present study further confirmed that NMS-induced upregulation of visceral nociception in Wistar rats was manifested as a reduction in threshold pressure to elicit AWR in response to CRD and an increased pain response to noxious CRD. Increased activity in the cingulate cortex of NMS rats at basal condition was observed for the first time in our study. Furthermore, at noxious CRD (80 mm Hg), the neural activity in the spinal cord and supraspinal centers, involving the laminae I and II, and the neck of dorsal horn at laminae V and VI of the lumbarsacral spinal cord, the paraventricular thalamic nucleus, the cingulate cortex, the amygdaloid central nucleus were significantly upregulated in NMS rats, but not in the solitary tract, the central medial thalamic nucleus and the ventromedial hypothalamic nucleus, and the periaquaductal gray in comparison with the normal rats. These data indicate that NMS has significantly upregulated the activity of the cingulate cortex and sensitized the ascending pathway at spinal level as well as the thalamo-cortico-amydala pathway. Pain perception is a dynamic and plastic process that is affected by sensory, emotional and cognitive experiences. Visceral pain processing is complicated starting from the nociceptors in viscera, proceeding to the spinal cord, then to the brain. However, the mechanisms involved in the ascending pain signal transmission and perception pathways are still unclear. Previous studies in IBS have shown that visceral afferent neurons remained sensitized after the initial inflammatory insult leading to persistent visceral hypersensitivity and intestinal dysmotility (Bercik et al., 2004). The data of the present study showed that the number of Fos-IR nuclei induced by noxious CRD in the superficial laminae (I and II)

in the dorsal horn of the NMS rats was significantly increased in comparison to the normal rats. Previous study has shown that only those neurons in the superficial laminae of spinal cord are the projecting neurons that convey visceral information to the supraspinal centers (Menetrey et al., 1989). The elevated neural activities in laminae I and II to CRD indicated that NMS triggered sensitization of spinal cord neurons in response to peripheral stimulation that may have resulted in an increase in ascending pathway. Apart from the superficial laminae, laminae V–VI located in the neck of dorsal horn were also found to have a higher neuronal activity to CRD in the NMS rats. Lamina V contains wide dynamic range (WDR) neurons which respond to both the innocuous and noxious stimulus. Evidence have point out that the plasticity in the dorsal horn circuits involving the WDR neurons plays a critical role for the mechanisms of chronic pain (Benarroch, 2001). Therefore, the present results indicated that NMS-induced hyperalgesia may at least take place at the spinal level. In the supraspinal centers, the present study demonstrated for the first time an elevated neuronal responsiveness to noxious CRD in NMS rats manifested as an increase in c-fos expression in brain nuclei including the cingulate cortex, the paraventricular thalamic nucleus and the central amygdaloid nucleus. We also demonstrated that NMS induced a significant c-fos expression in the cingulate cortex in adult rats at basal condition. As the expression of c-fos reflects activity of neurons, the present results indicates that NMS not only trigger long-term sensitization of cingulate cortex, but also significantly elevates its activity level in response to noxious visceral stimuli. Many studies on pain processing in human as well as in animals have demonstrated that the anterior cingulate cortex

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Fig. 4 – Light micrographs of representative sections in different regions of normal (in right column) and NMS rat (in left column) with noxious CRD. (A and B) Central amygdaloid nucleus (CeA); (C and D) paraventricular thalamic nucleus (PVT); (E and F) the superficial laminae; (G and H) the neck of dorsal horn of the lumbarsacral spinal cord. I: lamina I; II: lamina II; V: lamina V; VI: lamina VI of dorsal horn; BLA: bassolateral amygdaloid nucleus; MD: mediodorsal thalamic nucleus. Scare bars in panels A, C and E: 200 μm (for B, D and F).

is an important area for processing sensory information related to pain (Buchel et al., 2002; Coghill et al., 1999; Posserud et al., 2004; Rainville et al., 1997). A previous study has identified neurons in the cingulate cortex that were selectively responsive to painful stimuli, supporting a role for the cingulate cortex in pain perception (Hutchison et al., 1999). A recently published study in patients has showed that early life

stress triggers morphological changes in the cingulate cortex, but not in the hippocampus and amygdala (Cohen et al., 2006). In another study, inhibiting neural activation in the limbic anterior cingulate cortex and parietal association cortex with intervention resulted in inhibition of pain and other symptoms exacerbated by stress in IBS patients (Morgan et al., 2005). These compelling data suggest the important role of the

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Fig. 5 – Light micrographs of representative sections in different regions of normal (in right column) and NMS rat (in left column) with noxious CRD. Row A: central medial thalamic nucleus (CM); row B: ventromedial hypothalamic nucleus (VMH); row C: periaquaductal gray (PAG); row D: solitary tract (NTS). Arc: arcuate hypothalamic nucleus; DLPAG: dorsolateral periaqueductal gray; DMN: dorsal motor nucleus of the vagus; LPAG: lateral periaqueductal gray; VLPAG: ventrolateral periaqueductal gray. Scare bars in panels A–D: 200 μm.

cingulate cortex in the visceral pain processing. Therefore, the activation and sensitization of the cingulate cortex may play a crucial role in NMS-induced visceral hypersensitivity. The thalamus and amygdala which are the regions receiving cortical projections as well as afferent inputs from the viscera may also play a critical role in the development of visceral hyperalgesia in NMS rats. The thalamus and amyg-

dala are involved in generating emotional and autonomic responses to stimuli, as well as part of the central pain matrix (Almeida et al., 2004; Delvaux, 1999; Mayer, 2000a). Recent studies have found that the thalamus plays an important role in visceral nociceptive processing through the dorsal columns pathway (Palecek, 2004; Willis et al., 1999). The amygdala is also involved in the central processing of visceral pain

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(Almeida et al., 2004; Clark and Treisman, 2004; Han and Neugebauer, 2004). The latero-capsular part of the amygdaloid central nucleus has been defined as the ‘nociceptive amygdala’ because of its high content of nociceptive neurons (Neugebauer and Li, 2003). Both thalamus and amygdala receive excitatory inputs from the anterior cingulate cortex forming the thalamo-cortico-amygdala pathway which is part of the emotional motor system being activated by psychosocial stressors (Boatman and Kim, 2006; Clark and Treisman, 2004). The major outputs of this system may affect autonomic, pain modulatory and endocrine responses. Therefore, the elevated neuronal activation to CRD in the thalamus and amygdala that were found in NMS rat in the present study could be a result of the increased inputs of ascending visceral afferents from spinal cord and/or the increased inputs from the sensitized cingulate cortex. The present results demonstrate that NMS activates and triggers functional changes and outcome of the thalamo-cortico-amygdala pathway that may lead to pain facilitation and dysfunction of pain modulation responses. However, in the present study, increase in c-fos expression was found only in the spinal cord level and brain nuclei that are involved in the thalamo-cortico-amygdala pathway but not in all the regions of supraspinal centers in NMS rats. For instance, the activity in the solitary tract which is also a target of the visceral ascending projection to noxious visceral stimuli was found to be unchanged in NMS rats in our study. Although the solitary tract also receives intestinal afferents through the spinosolitary tract, and provides ascending projections that convey this visceral sensory information to the rostral brainstem and forebrain (Almeida et al., 2004; Traub et al., 1996; Urban and Gebhart, 1999), our data suggest that its activity is not involved in NMS-induced visceral hyperalgesia. Further investigation is required to ascertain whether NMS triggers sensitization of specific ascending pain pathways and to elucidate the possible circuits as well as the underlying mechanisms. Apart from the ascending pathway of visceral nociceptive information, there are powerful inhibitory influences arising from the brain (Jones et al., 2006; Millan, 2002), that also contributes to the development of visceral hyperalgesia (Millan, 2002). The cingulate cortex that receives ascending activation as demonstrated in this study is also a major site sending inhibitory efferent signals directly or indirectly through the PAG and the amygdala to the dorsal horn of the spinal cord (Rainville et al., 1997; Zhang et al., 2005). Evidence also suggested that the midbrain PAG plays a major role in descending nociceptive inhibition by means of opioids (Burdin et al., 1992; Hosobuchi et al., 1977). Apart from PAG, the hypothalamus is also involved in descending inhibition (Dafny et al., 1996; Millan, 2002). However, our study showed that the c-fos expression induced by noxious CRD in the hypothalamic nucleus and PAG was not significantly changed in NMS rats, suggesting that the activity of these nuclei to visceral stimuli was unaffected by neonatal stress. In conclusion, the present study demonstrates direct evidence of stress-induced central alternation of visceral sensitivity in rat, and provides further support for the association between neonatal stress and visceral hyperalgesia. NMS-induced visceral hyperalgesia is associated with the

activation of the limbic circuits particularly the thalamocortico-amygdala pathway, in which sensitization may contribute to pain facilitation. The altered brain responses to noxious visceral stimuli may possibly represent a cascade of molecular events that are triggered by the stress. The expression of c-fos is just one of the indicators of early changes in the pathways; other possibilities include the changes in neurotransmitters and receptors expression. More permanent changes may also occur including changes in synaptic plasticity by means of nerve sprouting and formation of new synapses. In conclusion, NMS-induced central sensitization to noxious visceral stimuli is a manifestation of complex plasticity changes in the nervous system. Investigations on the change of plasticity in the thalamo-cortico-amygdala pathway and the underlying mechanisms in stress-induced visceral hyperalgesia may provide a promising future therapeutic strategy for treating IBS.

4.

Experimental procedure

4.1.

Animals

Primiparous timed-pregnant Wistar female rats were obtained from the Laboratory Animal Services Centre, the Chinese University of Hong Kong, on gestational day 15. Dams were housed individually in plastic cages and maintained on a 12:12-h light–dark cycle with free access to food and water. The handling of rats and all procedures performed were approved in accordance with the Animals (Control of Experiments) Ordinance, Hong Kong, China.

4.2.

Maternal separation

On postnatal days (PND) 2 to 14, pups were separated from their maternity cages to adjacent cages of identical type for 180 min daily as described previously (Barreau et al., 2004). Briefly, NMS pups were placed in isolated cages lined with bedding materials, under a lamp to keep them warm. After the separation period, pups were returned to their maternity cages. Apart from the designated daily separation period and the weekly changing of bedding material, pups were left undisturbed. Control groups were not exposed to handling and were maintained in their maternity cages with the dams. On PND 22, sex of pups was determined. Female pups were culled, and male pups were weaned and litter housed in individual cages. After weaning, pups were weighted weekly. Only male rats weighted 250–300 g were used in the present study to avoid variations due to hormonal cycles.

4.3.

Behavioral testing

4.3.1.

Abdominal withdrawal reflex (AWR)

Colorectal distention was performed as previously described (Al-Chaer et al., 2000). The rats undergoing CRD experiments were lightly anesthetized with ether, and then a 6-cm-long flexible latex balloon was inserted in the distal colon with the distal tip 1 cm from the anal verge and secured to the base of the tail. Animals were allowed to recover for at least 15 min. CRD was then applied in increments of 5 mm Hg until an

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observable identifiable contraction of the abdominal wall. Threshold intensity of CRD that elicited an observable AWR of all groups (n = 18 for each group) was recorded and repeated three times with at least 4-min intervals for recovery. Data of each group were averaged and compared by unpaired t test.

4.3.2.

Electromyography (EMG)

The surgical procedures for EMG were done as previously described (Tammpere et al., 2005). Rats were deeply anesthetized by injecting 3–4 mg/kg of midazolam hydrochloride intraperitoneally. Electrodes were stitched into the external oblique musculature, just superior to the inguinal ligament, and tunneled subcutaneously exposing the recording end of the electrode. Rats were allowed to recover for at least 5 days. Wounds were tested for tenderness to ensure complete recovery from surgery before testing. After a series of three 20-s distentions of 80 mm Hg was applied to establish a stable response, EMG data were obtained as previously described (Bercik et al., 2004). In short, the EMG signal was amplified and filtered (50–5000 Hz) by the Power Lab System (AD Instruments International). The EMG data of the 20 s 80 mm Hg CRD period and the preceding 20-s baseline recording of each rat were used for the calculation of AUC (the area under the curve of EMG amplitude over baseline) values by using the commercial program (Chart 5, AD Instruments International). Triplicate EMG data of each rat were obtained with 2-min intervals between each distention periods. The AUC value of each group of rats (n = 12 for each group) was averaged and compared by unpaired t test.

4.4.

Fos induction

NMS rats and NH rats (n = 5 for each group) was randomly chosen for the CRD experiments. A series of 20 20-s distentions of 80 mm Hg was performed as previously described (Stam et al., 2002). Intervals between balloon stimulations were 2 min. One hour after the last stimulation, rats were deeply anesthetized with an overdose of midazolam hydrochloride and transcardially perfused. The remaining group of NMS and NH rats (n = 5 for each group) were lightly anesthetized with ether, without CRD stimulus, then deeply anesthetized and perfused.

4.5.

Immunohistochemistry

Rats were deeply anesthetized with midazolam hydrochloride and perfused transcardially with saline followed by 3% paraformaldehyde (pH 7.4). The brain and the lumbarsacral spinal cord segments were removed and post-fixed in fixative overnight followed by post-fixing in 30% sucrose in 0.1 M PBS at 4 °C for about 3 days, and then stored at −80 °C with embedding matrix (Shandon Cryomatrix, Thermo electron corporation). Coronal sections (30 μm) were cut on a freezing cryostat. The first of every four consecutive sections was obtained and stored in PBS at 4 °C prior to use. Immunohistochemistry was performed as previously described (Kobelt et al., 2004). Sections were incubated in polyclonal antibody solutions against c-fos (rabbit, 1:10,000 in 0.3% triton X-100, 10% BSA in PBS; Calbiochem) 48 h at 4 °C. Following primary antibody incubation, the sections were rinsed for 5 min each in PBS for three

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times and then incubated for 2 h in a biotinylated secondary antibody (1:200 in PBS; Vector Laboratories, USA) at RT. Following incubation in the solution containing avidin–biotin complex (Elite ABC kit; Vector Laboratories) for 45 min at RT and subsequently reacted with diamino-benzidine (DAB), sections were mounted on gelatin-coated slides, dehydrated in a series of graded alcohol and coverslipped.

4.6.

Quantification

The number of c-fos-IR nuclei was counted bilaterally in four non-consecutive coronal sections at an interval of at least 90 μm from each animal at 100× magnification. Measurements of c-fos-IR cell in different supraspinal centers were performed at the coordinates according to the atlas of Paxinos and Watson as given: +1.56 for cingulate cortex area and agranular insular cortex; −2.64, mediodorsal and paraventricular thalamic nucleus, dorsalmedial and ventromedial nucleus of hypothalamus, central nucleus of amygdala; −5.88, periaqueductal gray; −12.36, nucleus of the solitary tract. In lumbarsacral spinal segments, c-fos-IR nuclei were counted in the laminae receiving visceral projection (Al-Chaer and Traub, 2002; Fitzgerald, 2005). The total number of c-fos-IR nuclei of each area obtained from 5 animals in each group was averaged and compared by one-way ANOVA. Data were considered significantly different when P < 0.05.

Acknowledgments The present work was supported by the Hong Kong Jockey Club Institute of Chinese Medicine (JCICM-16-02), and Faculty Research Grant, Hong Kong Baptist University (FRG/05-06/II-54). REFERENCES

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