Chăm Sóc Của Mẹ, Các Thụ Thể Glucocorticoid Hippocampal, và Phản Ứng Hypothalamic-Pituitary-Adrenal Đối Với Căng Thẳng
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#Chăm sóc của mẹ #thụ thể glucocorticoid hippocampal #phản ứng hypothalamic-pituitary-adrenal #hormone adrenocorticotropic #corticosterone #mRNA #điều hòa ngược glucocorticoid #hormone cortisol-releasingTài liệu tham khảo
Zarrow M. X., Campbell P. S., Denenberg V. H., Proc. Soc. Exp. Biol. Med. 356, 141 (1972).
The handling procedure involves removing the mother and then rat pups from their cage placing the pups together in a small container and returning the animals 15 min later to their cage and their mothers. The manipulation is generally performed daily for the first 21 days of life. Handling does not represent a period of maternal deprivation because over the course of the day mothers are routinely off their nests and away from pups for periods of 20 to 25 min. At the same time the artificial and nonspecific nature of the handling paradigm is unsettling [
]. Normal development in a rat pup most often occurs in the rather dark tranquil confines of a burrow where the major source of stimulation is the mother and littermates.
M. J. Meaney D. H. Aitken S. Bhatnagar Ch. Van Berkel
Sapolsky R. M., Science 238, 766 (1988);
Plotsky P. M., Otto S., Sapolsky R. M., ibid. 119, 1126 (1986);
de Kloet E. R., Front. Neuroendocrinol. 12, 95 (1991);
; S. Levine in Society Stress and Disease L. Levi Ed. (Oxford Univ. Press London 1975) pp. 43–50; W. P. Smotherman and R. W. Bell in Maternal Influences and Early Behavior R. W. Bell and W. P. Smotherman Eds. (Spectrum New York 1980) pp. 201–210.
J. R. Alberts and C.P. Cramer in. Handbook of Behavioral Neurobiology E. M. Blass Ed. (Plenum New York 1989) vol. 9 pp. 1–39.
Fleming A., Rosenblatt J. S., Behav. Neurosci. 86, 221 (1974);
B. C. Woodside M. J. Meaney J. E. Jans unpublished data.
. The animals used in all studies were Long-Evans hooded rats obtained from Charles River Labs. (St. Constant Québec Canada) and housed in 46 cm by 18 cm by 30 cm Plexiglas cages that permitted a clear view of all activity within the cage. Food and water were provided ad libidum. The colony was maintained on a 12:12 light:dark (L:D) schedule with lights on at 0800. All procedures were done in accordance with guidelines developed by the Canadian Council on Animal Care and protocol approved by the McGill University Animal Care Committee. Handling was done as described (2). Nonhandled animals were completely undisturbed until day 12 of life at which time normal cage maintenance was initiated. The behavior of each dam was observed [
Myers M. M., Brunelli S. A., Shair H. N., Squire J. M., Hofer M. A., Dev. Psychobiol. 22, 55 (1989);
] for eight 60-min observation periods daily for the first 10 days after birth with six periods during the light phase and two periods during the dark phase of the L:D cycle. The distribution of the observations was based on the finding that nursing in rats occurs more frequently during the light phase of the cycle. Handling occurred each day at 1100 and an observation was scheduled at 1130 to correspond to the reunion of the mothers and pups. Within each observation period the behavior of each mother was scored every 4 min (15 observations per period × 8 periods per day = 120 observations per mother per day) for mother off pups mother licking and grooming any pup or mother nursing pups in either an arched-back posture a “blanket” posture in which the mother lies over the pups or a passive posture in which the mother lies either on her back or side while the pups nurse. Behavioral categories are not mutually exclusive.
D. Liu et al. data not shown.
Nine Long-Evans female rats were mated in our animal facility and housed and observed as described (16). The animals underwent routine cage maintenance beginning on day 12 but were otherwise not manipulated. At the time of weaning on day 22 of life the male offspring were housed in same-sex same-litter groups. Testing of offspring occurred no earlier than 100 days of age.
We then rank-ordered the dams on licking and grooming identifying those mothers whose scores fell above the mean and as a group these dams were classified as high LG-ABN. The remaining dams were classified as low LG-ABN. The offspring were tested beginning at 100 days of age.
For restraint stress (20 min) testing (6) two animals from each of the nine litters were randomly selected for testing. Blood samples were collected from indwelling right jugular vein catheters (6) implanted 4 days before restraint stress testing and replaced with an equal volume of normal saline (0.9%) via the same route. We have found that by 72 hours after surgery basal ACTH and corticosterone levels have returned to normal (6). Plasma corticosterone was measured by radioimmunoassay [
]. Plasma (25 μl) ACTH was measured by radioimmunoassay as described [
Walker C.-D., Akana S. F., Cascio C. S., Dallman M. F., ibid. 127, 832 (1990);
; V. Viau and M. J. Meaney ibid. 129 2503 (1991)]. All samples were run within a single assay. In our lab the intra- and interassay coefficients of variation are 7 and 10% respectively for corticosterone and 8 and 11% for ACTH. The data were analyzed by two-way analyses of variance (ANOVA) with one between (group) and one within (sample) measure. Post hoc analysis was performed by Tukey test.
We used a delayed negative-feedback paradigm [
] in which animals are steroid-treated 2 to 4 hours before acute stress. The animals used in this study were the same animals prepared with jugular catheters for acute restraint testing. The animals were tested 4 days after restraint stress and all but one of the catheters remained patent during this interval. The critical measure here is the ability of the steroid to inhibit subsequent HPA responses to stress. Animals were injected subcutaneously with either vehicle alone or a low to moderate dose of corticosterone (1 mg/kg in ethanol:saline/1:9) on the basis of earlier studies (6) showing that this dose discriminates feedback sensitivity in handled versus nonhandled rats. Restraint stress was done as described above and plasma samples were obtained from jugular catheters immediately before and at the end of the 20-min period of restraint a time point that corresponds to the peak plasma ACTH level (6). The percentage suppression of plasma ACTH responses to stress for the high– versus low–LG-ABN groups was derived by comparing Δ(peak stress level − basal level) for each of the corticosterone-treated animals in both groups with that of the mean for the respective control groups (vehicle-treated high– or low–LG-ABN animals). Percentage suppression scores were used to accommodate for the groups differences in plasma ACTH responses to acute stress. The results were examined statistically by Mann-Whitney U test on the basis of percentage scores.
CRH mRNA in situ hybridization was done with a 48–base pair (bp) oligonucleotide sequence (CAGTTTCCTGTTGCTGTGAGCTTGCTGAGCTAACTGCTCTGCCCTGGC) (Perkin-Elmer Warrington UK) and a modified version of the procedure previously described [
] with brain sections obtained from animals rapidly killed under resting-state conditions. After hybridization sections were apposed to Hyperfilm (Amersham) for 21 days along with sections of 35 S-labeled standards prepared with known amounts of radiolabeled 35 S in a brain paste. The hybridization signal within the parvocellular subregion of the PVNh was quantified by densitometry with an MCID image analysis system (Imaging Research St. Catherine's Ontario Canada). The data are presented as arbitrary absorbance units after correction for background. These data were analyzed by t test for unpaired groups.
GR in situ hybridization was done as described [(9);
] with [ 35 S]UTP-labeled cRNA antisense probes transcribed with T7 RNA polymerase from a 674-bp Pst I–Eco RI fragment of the rat GR cDNA linearized with Ava I. After hybridization sections were dehydrated dried and dipped in photographic emulsion (NTB-2 Kodak) and then stored at 4°C for 21 days before development and counterstaining with Cresyl Violet. The hybridization signal within dorsal hippocampal subregions was quantified by grain counting within high-power microscopic fields under brightfield illumination. Grain counting was performed by an individual unaware of the group from which the slide was derived. For each cell field grains over ∼40 to 50 individual neurons per section were counted on three sections per animal (9). After subtraction of background (grains over neuropil) mean values were derived for each hippocampal cell field for each animal. Background ranged between 10 and 15% of values found over hippocampal cells. Grain counts are presented as a function of cell area to account for possible morphological differences [
]. These data were analyzed by two-way ANOVA with one between measures (group) and one repeated measure (hippocampal sub-field) by Tukey post hoc test.
Sapolsky R. M., Armanini M. P., Packan D. R., Sutton S. W., Plotsky P. M., Neuroendocrinology 51, 328 (1990);
Butler S. R., Suskind M. R., Schanberg S. M., ibid. 199, 445 (1978);
; S. Levine Ann. N.Y. Acad. Sci. 746 260 (1994);
M. A. Hofer in L. A. Rosenblum and H. Moltz Eds. Symbiosis in Parent-Offspring Interactions (Plenum New York 1983) pp. 61–75.
We thank R. Meisfield (Univ. of Arizona) for rat GR cDNA and H. Anisman and M. Hofer for comments on an earlier version of this manuscript. Supported by grants from the Medical Research Council of Canada (MRCC) (M.J.M.) and the National Institute of Mental Health (P.M.P. and M.J.M.). M.J.M. is the recipient of an MRCC Scientist award. D.L. is a graduate fellow of the MRCC.