Sazu Yoshimoto

Hypothalamic oxytocin attenuates CRF expression via GABAA receptors in rats

Centrally released oxytocin (OXT) has anxiolytic and anti-stress effects. Delayed gastric emptying (GE) induced by acute restraint stress (ARS) for 90 min is completely restored following 5 consecutive days of chronic homotypic restraint stress (CHS), via up-regulating hypothalamic OXT expression in rats. However, the mechanism behind the restoration of delayed GE following CHS remains unclear. Gamma-aminobutyric acid (GABA)-projecting neurons in the paraventricular nucleus (PVN) have been shown to inhibit corticotropin releasing factor (CRF) synthesis via GABA(A) receptors. We hypothesized that GABA(A) receptors are involved in mediating the inhibitory effect of OXT on CRF expression in the PVN, which in turn restores delayed GE following CHS. OXT (0.5 μg) and selective GABA(A) receptor antagonist, bicuculline methiodide (BMI) (100 ng), were administered intracerebroventricularly (icv). Solid GE was measured under non-stressed (NS), ARS and CHS conditions. Expression of CRF mRNA in the PVN was evaluated by real time RT-PCR. Neither OXT nor BMI changed GE and CRF mRNA expression under NS conditions. Delayed GE and increased CRF mRNA expression induced by ARS were restored by icv-injection of OXT. The effects of OXT on delayed GE and increased CRF mRNA expression in ARS were abolished by icv-injection of BMI. Following CHS, delayed GE was completely restored in saline (icv)-injected rats, whereas daily injection of BMI (icv) attenuated the restoration of delayed GE. Daily injection of BMI (icv) significantly increased CRF mRNA expression following CHS. It is suggested that central OXT inhibits ARS-induced CRF mRNA expression via GABA(A) receptors in the PVN. GABAergic system is also involved in OXT-mediated adaptation response of delayed GE under CHS conditions.

Central and peripheral release of oxytocin following chronic homotypic stress in rats.

Accumulation of continuous life stress (chronic stress) often causes gastric symptoms. Centrally released oxytocin has anxiolytic and anti-stress effects. We have recently shown that impaired gastric and colonic motility observed in acute restraint stress was restored following repeated restraint stress for 5 consecutive days (chronic homotypic stress) in mice and rats. Chronic homotypic stress upregulates oxytocin mRNA expression and downregulates corticotrophin-releasing factor (CRF) mRNA expression at the hypothalamus. However, it still remains unclear whether stress responses induced by chronic homotypic stress are accompanied by the central or peripheral release of oxytocin. Adult male SD rats were chronically implanted with microdialysis probes at the hypothalamic paraventricular nucleus (PVN) and jugular vein catheters. Microdialysis and blood sampling were performed following 1st, 3rd and 5th of chronic homotypic stress. Oxytocin levels in the dialysates and plasma were measured by radioimmunoassay (RIA). On day 1 of chronic homotypic stress, oxytocin release was slightly, but not significantly, increased in the PVN and plasma. Oxytocin release was significantly increased in the PVN on day 3 (12.7 ± 1.3 pg/sample, n=5, P<0.05) and day 5 (28.2 ± 2.4 pg/sample, n=5, P<0.05) from basal (6.9 ± 1.8 pg/sample, n=5). In contrast, there were no significant changes observed in the plasma on day 3 and day 5. This suggests that central, but not peripheral, release of oxytocin plays an important role in response to chronic homotypic stress in rats.

Impaired adaptation of gastrointestinal motility following chronic stress in maternally separated rats

Exposure to early life stress causes increased stress responsiveness and permanent changes in the central nervous system. We recently showed that delayed gastric emptying (GE) and accelerated colonic transit (CT) in response to acute restraint stress (ARS) were completely restored following chronic homotypic stress (CHS) in rats via upregulation of hypothalamic oxytocin (OXT) expression. However, it is unknown whether early life stress affects hypothalamic OXT circuits and gastrointestinal motor function. Neonatal rats were subjected to maternal separation (MS) for 180 min/day for 2 wk. Anxiety-like behaviors were evaluated by the elevated-plus-maze test. GE and CT were measured under nonstressed (NS), ARS, and CHS conditions. Expression of corticotropin-releasing factor (CRF) and OXT in the paraventricular nucleus (PVN) of the hypothalamus was evaluated by real time RT-PCR and immunohistochemistry. MS increased anxiety-like behaviors. ARS delayed GE and accelerated CT in control and MS rats. After CHS, delayed GE and accelerated CT were restored in control, but not MS, rats. CRF mRNA expression was significantly increased in response to ARS in control and MS rats. Increased CRF mRNA expression was still observed following CHS in MS, but not control, rats. In response to CHS, OXT mRNA expression was significantly increased in control, but not MS, rats. The number of OXT-immunoreactive cells was increased following CHS in the magnocellular part of the PVN in control, but not MS, rats. MS impairs the adaptation response of gastrointestinal motility following CHS. The mechanism of the impaired adaptation involves downregulation of OXT and upregulation of CRF in the hypothalamus in MS rats.

Hypothalamic circuit regulating colonic transit following chronic stress in rats

Although acute stress accelerates colonic transit, the effect of chronic stress on colonic transit remains unclear. In this study, rats received repeated restraint stress (chronic homotypic stress) or various types of stress (chronic heterotypic stress) for 5 and 7 days, respectively. Vehicle saline, oxytocin (OXT), OXT receptor antagonist or corticotropin-releasing factor (CRF) receptor antagonists were administered by intracerebroventricular (ICV) injection prior to restraint stress for 90 min. Immediately after the stress exposure, the entire colon was removed and the geometric center (GC) of Na51CrO4 (a nonabsorbable radioactive marker; 0.5 μCi) distribution was calculated to measure the transit. Gene expression of OXT and CRF in the paraventricular nucleus (PVN) was evaluated by in situ hybridization. Accelerated colonic transit with the acute stressor was no longer observed following chronic homotypic stress. This restored colonic transit was reversed by ICV injection of an OXT antagonist. In contrast, chronic heterotypic stress significantly accelerated colonic transit, which was attenuated by ICV injection of OXT and by a CRF receptor 1 antagonist. OXT mRNA expression in the PVN was significantly increased following chronic homotypic stress, but not chronic heterotypic stress. However, CRF mRNA expression in the PVN was significantly increased following acute and chronic heterotypic stress, but not chronic homotypic stress. These results indicate that central OXT and CRF play a pivotal role in mediating the colonic dysmotility following chronic stress in rats.

Anti-Stress Effects of Transcutaneous Electrical Nerve Stimulation (TENS) on Colonic Motility in Rats

BackgroundDisorders of colonic motility may contribute to symptoms in patients with irritable bowel syndrome (IBS), and stress is widely believed to play a major role in developing IBS. Stress increases corticotropin releasing factor (CRF) of the hypothalamus, resulting in acceleration of colonic transit in rodents. In contrast, hypothalamic oxytocin (OXT) has an anti-stress effect via inhibiting CRF expression and hypothalamic–pituitary–adrenal axis activity. Although transcutaneous electrical nerve stimulation (TENS) and acupuncture have been shown to have anti-stress effects, the mechanism of the beneficial effects remains unknown.AimsWe tested the hypothesis that TENS upregulates hypothalamic OXT expression resulting in reduced CRF expression and restoration of colonic dysmotility in response to chronic stress.MethodsMale SD rats received different types of stressors for seven consecutive days (chronic heterotypic stress). TENS was applied to the bilateral hind limbs every other day before stress loading. Another group of rats did not receive TENS treatment.ResultsTENS significantly attenuated accelerated colonic transit induced by chronic heterotypic stress, which was antagonized by a central injection of an OXT antagonist. Immunohistochemical study showed that TENS increased OXT expression and decreased CRF expression at the paraventricular nucleus (PVN) following chronic heterotypic stress.ConclusionsIt is suggested that TENS upregulates hypothalamic OXT expression which acts as an anti-stressor agent and mediates restored colonic dysmotility following chronic stress. TENS may be useful to treat gastrointestinal symptoms associated with stress.

Social interaction attenuates stress responses following chronic stress in maternally separated rats

Early life stress has been implicated as a risk factor for functional gastrointestinal (GI) disorders. Hypothalamic oxytocin (OXT) is well known to regulate social interactions and affiliative behaviors. We have shown that maternal separation (MS) induces GI dysmotility and impair hypothalamic OXT expression in response to chronic homotypic stress (CHS). We studied whether social interaction can improve GI dysmotility and OXT expression in MS rats. Male neonatal SD rats were exposed to MS for 180 min from postnatal day (PND)-2 to PND-14. After weaning, 3MS rats were housed together (pure MS). In another group, 1MS rat was housed with 2 control rats (mixed MS). Anxiety-like behaviors were evaluated in elevated plus maze (EPM). Solid gastric emptying (GE) and colonic transit (CT) were measured following CHS loading. Expression of corticotropin releasing factor (CRF) and OXT in the paraventricular nucleus (PVN) were evaluated by real time RT-PCR and immunohistochemistry. Pure MS rats demonstrated increased anxiety-like behaviors, which were significantly reduced in mixed MS rats. Delayed GE (31.5±2.8%, n=6) and accelerated CT [Geometric center (GC) =8.9±0.8, n=6] observed in pure MS rats were restored in mixed MS rats (GE=67.8±3.8%, GC=6.7±1.2, n=6, P<0.05) following CHS. OXT mRNA expression was upregulated, while CRF mRNA expression was downregulated in mixed MS rats, compared to pure MS rats. The number of OXT-immunoreactive cells was significantly increased following CHS at the PVN in mixed MS rats. Our study may contribute to the treatment strategies for GI motility disorders associated with early life stress.

Affiliative behavior attenuates stress responses of GI tract via up-regulating hypothalamic oxytocin expression

Hypothalamic oxytocin (OXT) has stress-attenuating effects. Social interaction in a positive environment continuously activates OXT release system. We have recently shown that pair housing restores delayed gastric emptying following chronic heterotypic stress, via up-regulation of OXT mRNA expression in rats. We tested the hypothesis that affiliative behavior attenuates stress responses via upregulating OXT expression. Adult male SD rats were divided into two groups: the rat with a stressed partner (RSP) and the rat with a non-stressed partner (RNSP). RSPs were pair housed with a partner that received different types of stress for 7 consecutive days (chronic heterotypic stress). RNSPs were pair housed with a partner who did not receive any stress. After each stress loading, the rats were returned to their home cages and the behaviors of RSPs and RNSPs toward their partners were videotaped. After the study completion, RSPs and RNSPs were loaded with acute restraint stress. Then, gastric emptying and colonic transit were measured. Corticotropin releasing factor (CRF) and OXT expression in the paraventricular nucleus (PVN) were evaluated by real time RT-PCR and immunohistochemistry. The time of affiliative behaviors toward their partners was increased in RSPs, compared to that of RNSPs. Delayed gastric emptying and accelerated colonic transit induced by acute restraint stress were significantly attenuated in RSPs, compared to RNSPs. CRF expression was reduced, while OXT expression was increased in RSPs in response to acute stress, compared to controls. It is suggested that affiliative behaviors may upregulate hypothalamic OXT expression, which in turn attenuates stress responses.

Central Oxytocin is Involved in Mediating the Adaptation Mechanism of Colonic Transit Following Chronic Homotypic Stress in Rats

Background: Central oxytocin has anti-stress effects via inhibiting the gene expression of corticotropin releasing factor (CRF) in the paraventricular nucleus (PVN).We have previously shown that accelerated colonic transit in response to acute restraint stress was no longer observed following 5 consecutive days of restraint stress loading (chronic homotypic stress) (Neurosci Lett 453, 147-150, 2009). Our recent study showed that central oxytocin is involved in mediating restored gastric emptying following chronic homotypic stress in rats (Am J Physiol 299, G946-53, 2010). We studied whether central oxytocin is involved in mediating the adaptation mechanism of colonic transit following chronic homotypic and heterotypic stress in rats. Methods: A catheter was inserted into the proximal colon to measure colonic transit. A cannula was implanted into the right lateral ventricle for intracerebroventricular (icv) access. Five days after the surgery, the rats were exposed to chronic homotypic stress or heterotypic stress. For chronic homotypic stress, the rats were given restraint stress for 90 min for 5 consecutive days. For chronic heterotypic stress, the rats were given different types of stress (water avoidance stress, force swimming stress, cold restraint stress, and restraint stress) for 7 consecutive days. Ninety min after the injection of 51Cr (0.2 μCi) into the proximal colon on the 5th or 7th day of chronic stress, the entire colon was removed to measure geometric center (GC; the distribution of 51Cr). Oxytocin (0.5 μg), oxytocin receptor antagonists {[d(CH2)5,Try(Me),Orn]-oxytocin}(100 ng), CRF1 receptor antagonists (NBI-27914; 100 μg) and CRF2 receptor antagonists (astressin2-B; 10 μg) were administered (icv) 30 min before the stress loading. Saline-injected rats served as controls. Expression of oxytocin and CRF mRNA in the PVN was evaluated by in situ hybridization. Results: Colonic transit was no longer accelerated following chronic homotypic stress (GC; 5.8 ± 0.2, n=7), which was antagonized by icv-injection of oxytocin antagonists (GC; 7.9 ± 0.1, n=6, P<0.05). The increased oxytocin and decreased CRF mRNA expression at the PVNwere observed following chronic homotypic stress. In contrast, chronic heterotypic stress significantly accelerated colonic transit (GC; 8.6 ± 0.2; n=7, P<0.01). Accelerated colonic transit following chronic heterotypic stress was attenuated by icv-injection of oxytocin (GC; 6.7 ± 0.2, n=7, P<0.05) and NBI-27914 (GC; 6.42 ± 0.1; n=7), but not astressin2-B (GC; 8.0 ± 0.2, n=7). Oxytocin mRNA expression was no longer increased following chronic heterotypic stress. Conclusion: It is suggested that central oxytocin plays a pivotal role in mediating the adaptation mechanism of colonic transit following chronic homotypic, but not heterotypic, stress in rats.