Experiment 1
Effects of intranasal administration of NPY on central CRF and NPY mRNA expression in response to CCS.
In the NS groups, CRF mRNA expression in the PVN showed very low level, intranasal administration of NPY (150 μg, daily, before stress loading) did not change the CRF mRNA expression significantly, compared to that of saline intranasal administered NS rats (saline 10 μl as a control, n = 6). In the CCS groups, CRF mRNA expression increased significantly compared to that of saline intranasal administered NS rats (n = 6, F (1, 20) = 26.663, p<0.001). Intranasal administration of NPY significantly decreased the CRF mRNA expression compared to that of saline intranasal administered CCS rats (n = 6, P = 0.001, saline 10 μl intranasal administered as a control; Figure. 1A).
In the NS groups, NPY mRNA expression in the PVN showed low level, intranasal administration of NPY (daily, before stress loading) did not change the NPY mRNA expression significantly, compared to that of saline IN administered NS rats (saline 10 μl as a control, n = 6). In the CCS groups, NPY mRNA expression increased significantly compared to that of saline intranasal administered NS rats (n = 6, F (1, 20) = 17.486, p=0.027). Intranasal administration of NPY further significantly increased the NPY mRNA expression compared to that of saline intranasal administered CCS rats (n = 6, p=0.011, saline10 μl intranasal administered as a control; Figure. 1B).
Effects of intranasal administration of NPY on serum corticosterone and NPY levels in response to CCS.
In the NS groups, serum corticosterone concentration showed low level. Intranasal administration of NPY (daily, before stress loading) did not change the serum corticosterone levels significantly (64.6 ± 6.3 ng/ml) compared to that of saline intranasal administered rats (67.2 ± 6.4 ng/ml, saline 10 μl intranasal administered as a control, n = 6). In the CCS groups, serum corticosterone concentration significantly increased to 146.3 ± 11.2 ng/ml compared to that of saline intranasal administered NS rats (n = 6, F (1, 20) = 18.215, p<0.001). Intranasal administration of NPY significantly decreased the corticosterone level to 93.8 ± 10.4 ng/ml, compared to that of saline intranasal administered CCS rats (n = 6, p=0.002, saline 10 μl intranasal administered as a control; Figure. 2A).
In the NS groups, serum NPY concentration showed very low level. Intranasal administration of NPY (daily, before stress loading) did not change the serum NPY levels significantly (1.83 ± 0.15 ng /ml), compared to that of saline intranasal administered rats (1.75 ± 0.13 ng/ml, saline 10 μl intranasal administered as a control, n = 6). In the CCS groups, serum NPY level decreased to 1.47 ± 0.17 ng /ml, but did not show significant change (n = 6, F (1, 20) = 1.666, p=0.206). However intranasal administration of NPY increased the NPY level to 1.92 ± 0.25 ng/ml, but also did not show significant change (n = 6, p=0.185, saline 10 μl intranasal administered as a control; Figure. 2B).
Effects of intranasal administration of NPY on colonic motility recording, MI changes and FPO in response to CCS.
In the NS groups, intranasal administration of NPY (daily, before stress loading) had no effects on the amplitude of distal colonic contraction, saline 10 μl intranasal administered as a control. In the CCS groups, restraint stress enhanced the amplitude of distal colonic contraction in the saline group (saline 10ul intranasal administered). However, Intranasal administration of NPY helped to partially restore the amplitude of distal colonic contraction groups. (n = 6, saline 10ul intranasal administered as a control; Figure. 3A).
In the NS groups, intranasal administration of NPY (daily, before stress loading) did not significantly alter the colonic MI change (95 ± 9%, n=6) compared to that of saline intranasal administered rats (100 ± 10%, saline 10 μl intranasal administered as a control, n = 6; F (1, 20) = 16.856, p<0.001, Figure. 3B). In the CCS groups, the enhanced colonic MI change in the saline group, was significantly attenuated by intranasal administration of NPY (from 196 ± 14% to 131 ± 11%, p=0.003, n=6, saline 10ul intranasal administered as a control; Figure. 3B).
In the NS groups, intranasal administration of NPY (daily, before stress loading) did not change the FPO significantly 3.7 ± 0.8 (number/90 min, n = 6) compared to that of saline intranasal administered rats (3.5 ± 0.7, saline 10 μl intranasal administered as a control, n = 6). In the CCS groups, FPO was significantly increased to 9.7 ± 0.9 (number/90 min, n = 6) compared to that of saline intranasal administered NS rats (n = 6, F (1, 20) = 13.842, p<0.001). Intranasal administration of NPY significantly decreased the FPO to 5.3 ± 0.7 (number/90 min, n = 6), compared to that of saline intranasal administered CCS rats (n = 6, p=0.004, saline 10 μl intranasal administered as a control; Figure. 3C).
Experiment 2
Effects of intranasal administration of NPY and GABAA receptor antagonist BMI on central CRF mRNA expression and NPY mRNA expression in response to CCS.
In the non-stressed (NS) groups, CRF mRNA expression showed very low level (saline 10 μl intranasal administered, and saline 5 μl ICV injected as a control), both intranasal administration of NPY (150 μg, daily) and ICV administered GABAA receptor antagonist BMI (100 ng/5 μl, for 7 consecutive days) did not change the CRF mRNA expression significantly (n = 6). In the CCS conditions, the CRF mRNA expression in the nasal saline groups was highly elevated (n = 6, F (1, 40) = 34.970, p<0.001). ICV administered BMI (100 ng/5 μl, 15 min prior to the stress loading for 7 consecutive days) did not change the CRF mRNA expression significantly (n = 6, saline 5 μl ICV injected as a control). However, in the nasal NPY groups, the decreased CRF mRNA expression was significantly increased by ICV administered BMI (n = 6, p=0.001, nasal saline and ICV saline as a control; Figure. 4A).
In the NS groups, NPY mRNA expression showed low level (saline 10 μl intranasal administered, and saline 5 μl ICV injected as a control), both intranasal administration of NPY (150 μg, daily) and ICV administered GABAA receptor antagonist BMI (100 ng/5 μl, for 7 consecutive days) did not change the NPY mRNA expression significantly (n = 6).
In the CCS conditions, the NPY mRNA expression increased significantly in the nasal saline groups (n = 6, F (1, 40) = 9.569, p=0.030). ICV administered of BMI did not change the NPY mRNA expression significantly (n = 6, p=1.000). However, in the nasal NPY groups, the further elevated NPY mRNA expression was significantly inhibited by ICV administered BMI (n = 6, p=0.021, nasal saline and ICV saline as a control; Figure. 4B).
Effects of intranasal administration of NPY and GABAA receptor antagonist BMI on serum corticosterone and NPY levels in response to CCS.
In the NS groups, the serum corticosterone concentration showed low level (67.4 ± 6.8 ng/ml, saline 10 μl intranasal and saline 5 μl ICV as a control), ICV administered BMI (100 ng/5 μl) increased the corticosterone level to 74.2 ± 6.4 ng/ml, but did not show significant change (n = 6). In the nasal NPY groups, the serum corticosterone level also did not show significant change by ICV administered saline or BMI (66.9 ± 6.6 ng/ml and 69.6 ±5.7 ng/ml, respectively, n = 6). In the CCS conditions, the serum corticosterone concentration in the nasal saline groups was significantly elevated (142.4 ± 9.5 ng/ml, saline 5 μl ICV injected, n = 6, F (1, 40) = 20.716, p<0.001, compared with NS groups). ICV administered BMI did not change the corticosterone level significantly (147.8 ± 10.4 ng/ml, n = 6, p=1.000). However, in the nasal NPY groups, the decreased serum corticosterone level was significantly increased by ICV administered BMI (from 97.1 ± 7.8 ng/ml to 140.3 ± 9.9 ng/ml, n = 6, p=0.011, nasal saline and ICV saline as a control; Figure. 5A).
In the NS groups, the serum NPY concentration showed very low level (1.76 ± 0.13 ng/ml, saline 10 μl intranasal and saline 5 μl ICV as a control), both intranasal administration of NPY (150 μg, daily) and ICV administered BMI (100 ng/5 μl) did not change the corticosterone level significantly (1.80 ± 0.16 ng/ml, 1.81 ± 0.14 ng/ml and 1.85 ± 0.15 ng/ml, respectively, n = 6). In the CCS conditions, the serum NPY level in the nasal saline groups decreased to 1.62 ± 0.14 ng /ml, but did not show significant change (n = 6, F (1, 40) = 0.670, p=0.996). ICV administered BMI also did not change the serum NPY level significantly (1.65 ± 0.12 ng/ml, n = 6). In the nasal NPY groups, the serum NPY level also did not show significant change by ICV administered saline or BMI (1.91 ± 0.11 ng/ml and 1.95 ± 0.15 ng/ml, respectively, n = 6, p=1.000, nasal saline and ICV saline as a control; Figure. 5B).
Effects of the GABAA receptor antagonist on colonic motility recording, motility index (MI) changes and FPO in response to CCS.
In the non-stressed (NS) groups, both intranasal administration of NPY (150 μg, daily) and ICV administered GABAA receptor antagonist BMI (100 ng/5 μl) had no effects on the distal colonic contractions (data not shown). In the CCS condition, restraint stress enhanced the amplitude of distal colonic contraction in the nasal saline group. ICV administered GABAA receptor antagonist BMI (100 ng/5 μl, 15 min prior to the stress loading for 7 consecutive days) did not change the distal colonic contraction obviously. However, in the nasal NPY groups, the attenuated distal colonic contraction was enhanced by ICV administered BMI.
(n = 6, saline 5 μl ICV injected as a control; Figure. 6A).
In the NS groups, the colonic MI change is 100 ± 10% (nasal saline and saline 5 μl ICV administered as a control, n=6). The combination of nasal saline or nasal NPY with ICV administered saline or BMI (100 ng/5 μl) did not alter the colonic MI change significantly (102± 10%, 94 ± 9%, and 97 ± 11%, respectively, n = 6). In the CCS condition, the colonic MI change was highly elevated (198 ± 12%, n=6, F (1, 40) = 17.760, p<0.001). ICV administered GABAA receptor antagonist BMI did not change colonic MI change significantly (195 ± 14%, n=6, p=1.000). However, in the nasal NPY groups, the decreased colonic MI change was significantly increased by ICV administered BMI (from 130 ± 11% to 201 ± 15%, nasal saline and ICV saline as a control; n = 6, p=0.003, Figure. 6B).
In the NS groups, the FPO is 3.5± 0.4 (number/90 min, nasal saline and saline 5 μl ICV administered as a control, n=6). The combination of nasal saline or nasal NPY with ICV administered saline or BMI (100 ng/5 μl) did not alter the FPO significantly (3.7 ± 0.6, 3.5 ± 0.6, and 3.8 ± 0.6, number/90 min, respectively, n = 6). In the CCS condition, the FPO was highly elevated 9.7 ± 0.7 (number/90 min, n = 6, F (1, 40) = 24.525, p<0.001). ICV administered GABAA receptor antagonist BMI did not change the FPO significantly 9.6 ± 0.6 (number/90 min, n = 6, p=1.000). However, in the nasal NPY groups, the FPO was significantly increased by ICV administered BMI (from 5.8 ± 0.6 to 9.5 ± 0.6 number/90 min, nasal saline and ICV saline as a control; n = 6, p=0.002, Figure. 6C).