Article Summary

Maternal Care, Hippocampal Glucocorticoid Receptors, and Hypothalamic- Pituitary- Adrenal Responses to Stress

Author(s): Dong Liu, Josie Diorio, Beth Tannenbaum, Christian Caldji, Darlene Francis, Alison Freedman, Shakti Sharma, Deborah Pearson, Paul M. Plotsky and Michael J. Meaney

Source: Science , Sep. 12, 1997, New Series, Vol. 277, No. 5332 (Sep. 12, 1997), pp. 1659- 1662

Published by: American Association for the Advancement of Science

Stable URL: https://www.jstor.org/stable/2894142

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. . .......fea G,G_,.Ga-..... S.=G< .*Ga.... WgEW EmE REPO RT S of cells after exposure to the peptide.

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mined because of the lack of success in establishing a valid binding assay with radioactively labeled eotaxin or MCP-3. However, the observation that pretreatment with vMIP-11 could inhibit the calcium

mobilizing effect of MCP-3 in cells expressing CCR3 indicates that vMIP-11 does bind to CCR3 (Fig. 3A).

23. U87/CD4 cells expressing CCR3, CCR5, and CXCR4 were kindly provided by Dan Littman. Pe- ripheral blood mononuclear cells (PBMCs) were pre- pared from buffy coat white blood cells from blood banks. After separation by density-gradient centrifu- gation on Ficoll-Paque, cells were cultured and stim- ulated with PHA. After 2 days IL-2 was added and after 3 to 5 days cells were infected. The HIV-1 strains have been described (25, 26). Virus stocks were prepared in PBMC cultures stimulated with PHA and IL-2. Although the same tissue culture ID50 (1900 for PBMC) of virus was used to challenge the cells, replication and infection were more efficient on the U87/CD4 cells expressing CXCR4 than on those expressing CCR3 or CCR5. However, lowering the amount of virus failed to substantially increase the inhibition by chemokines shown in Fig. 4 for the CXCR4-expressing cells. For determination of che- mokine inhibition of infectivity, cells were seeded into 96-well dishes at 4 x 103 cells per well. On the following day chemokines were added and incubat-. ed for 30 min at 37?C before virus was added and incubated an additional 3 hours before washing three times to remove residual virus. The cells were then incubated for 5 days at 37?C before the medi- um was harvested and p24 concentrations were es- timated (31). Initial time-course studies had deter- mined that optimal production of p24 was obtained

after 5 days with the virus strains that were used in the U87 cells before confluency was reached.

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32. Abbreviations for amino acids are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, lie; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; Y, Tyr.

33. We thank Lisbet Elbak, Helle Iversen, and Tina Ja- kobsen for technical help and Ulrik Gether for use of the spectrofluorometer. Supported by the Danish Medical Research Council (MRC), the Biotechnology Research Unit for Molecular Recognition, and the Danish AlDSFoundation (to T.W.S.) and by the UK MRC (to G.S. and P.R.C.).

26 March 1997; accepted 23 July 1997

Maternal Care, Hippocampal Glucocorticoid Receptors, and Hypothalamic-Pituitary-Adrenal

Responses to Stress

Dong Liu, Josie Diorio, Beth Tannenbaum, Christian Caldji,

'Darlene Francis, Alison Freedman, Shakti Sharma, Deborah Pearson, Paul M. Plotsky, Michael J. Meaney*

Variations in maternal care affect the development of individual differences in neuroen- docrine responses to stress in rats. As adults, the offspring of mothers that exhibited more licking and grooming of pups during the first 10 days of life showed reduced plasma adrenocorticotropic hormone and corticosterone responses to acute stress, increased hippocampal glucocorticoid receptor messenger RNA expression, enhanced glucocor- ticoid feedback sensitivity, and decreased levels of hypothalamic corticotropin-releasing hormone messenger RNA. Each measure was significantly correlated with the frequency of maternal licking and grooming (all r's > -0.6). These findings suggest that maternal behavior serves to "program" hypothalamic-pituitary-adrenal responses to stress in the offspring.

Several years ago Levine, Denenberg, and others (1) showed that the development of hypothalamic-pituitary-adrenal (HPA) re- sponses to stress is modified by early en- vironmental events, including infantile stimulation [or handling (2)]. As adults, animals exposed to brief periods of han- dling daily for the first weeks of life show reduced pituitary adrenocorticotropic hor-

D. Liu, J. Diorio, B. Tannenbaum, C. Caldji, D. Francis, A. Freedman, S. Sharma, D. Pearson, M. J. Meaney, Devel- opmental Neuroendocrinology Laboratory, Douglas Hos- pital Research Center, Departments of Psychiatry, and Neurology and Neurosurgery, Faculty of Medicine, McGill University, Montreal, Canada H4H 1 R3. P. M. Plotsky, Department of Psychiatry and Behavioral Science, Emory University, Atlanta, GA 30322, USA.

*To whom correspondence should be addressed.

mone (ACTH) and adrenal corticosterone (the principal glucocorticoid in the rat) responses to stress compared with non- handled animals (3). These differences are apparent as late as 24 to 26 months of age (4), indicating that the handling effect on HPA function persists throughout life.

Glucocorticoids act at a number of neural sites to exert an inhibitory, negative-feed- back effect over the synthesis of hypotha- lamic releasing-factors for ACTH, notably corticotropin-releasing hormone (CRH) and arginine vasopressin (AVP) (5). Postnatally handled animals show enhanced glucocorti- coid negative-feedback sensitivity compared with nonhandled rats (6) and therefore decreased hypothalamic CRH and AVP

mRNA expression, as well as lower levels of both CRH and AVP immunoreactivity (7). The handling effect on feedback sensitivity is mediated by an increase in glucocorticoid receptor (GR) expression in the hippocam- pus (8, 9), a region that has been strongly implicated in glucocorticoid negative-feed- back regulation (10). The increased hip- pocampal GR gene expression is therefore a central feature of the handling effect on HPA responsivity to stress, resulting in in- creased feedback inhibition of CRH and AVP synthesis and reduced pituitary ACTH release during stress.

A number of authors ( 11) have proposed that the effects of postnatal handling are mediated by changes in mother-pup inter- actions and that the handling manipulation itself might map onto naturally occurring individual differences in maternal care. Spe- cifically, Levine proposed that handling of the pups altered the behavior of the mother and that these differences in mother-pup interactions then mediate the effect of han- dling on the development of endocrine and behavioral responses to stress. The question, then, is how this maternal mediation might occur and whether such factors might con- tribute to naturally occurring individual dif- ferences in HPA responses to stress.

In the Norway rat, mother-pup contact occurs primarily within the context of a nest-bout, which begins when the mother approaches the litter and gathers the pups under her; she then nurses her offspring, intermittently licking and grooming the pups (12, 13). Handling results in changes in mother-pup interactions (14). Mothers of handled pups spend the same amount of time with their litters as mothers of non-

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handled pups; however, mothers of handled litters had shorter, but more frequent, nest- bouts (15).

We examined the behavior of mothers of handled or nonhandled litters over the first 10 days of life, a "critical" period for the handling effect on HPA development (16). Mothers of handled pups showed increased levels of licking and grooming Qf pups and arched-back nursing (LG-ABN) compared with mothers of nonhandled pups (Table 1). The frequency of these two behaviors was highly correlated (r = +0.91); over 90% of the instances of licking and groom- ing occurred while the mother was nursing her pups in the arched-back posture. Moth- ers of nonhandled pups nursed no less fre- quently than those of handled pups (17), but tended to more frequently adopt a "blanket" or passive posture when nursing, lying over or beside the pups. These differ- ences in licking and grooming (and the accompanying arched-back nursing pos-

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Fig. 1. Mean (?SEM) plasma ACTH (top) and corticosterone (middle) responses to a 20-min pe- riod of restraint stress (solid bar) in the offspring of high- versus low-licking and grooming and arched-back nursing (LG-ABN) mothers. * Sig- nificantly different at P < 0.05. Two animals from each of the nine litters were randomly choosen for testing, that is, n = 8 to 1 0 per group. (Bottom) Scattergram for the correlation between the fre- quency of maternal licking and grooming during

thirt10 day of life and the inerae plasm

ture) were the only behaviors that served to reliably distinguish mothers of handled from those of nonhandled pups.

To determine whether the increased ma- ternal licking and grooming affects the de- velopment of HPA responses to stress, we examined the relation between naturally occurring individual differences in maternal care and HPA development (18). We de- tected pronounced and stable individual differences in maternal licking and groom- ing (which again was highly correlated with arched-back nursing; r = +0.94). The vari- ability among the dams in licking and grooming was substantial and of sufficient range to meaningfully study the relation between variations in- postnatal maternal care and the development of adult respons- es to stress (19).

As adults, the offspring of high-LG- ABN mothers that showed significantly re- duced plasma ACTH and corticosterone responses to restraint stress (20) compared with the offspring of low-LG-ABN mothers (Fig. 1). There were no differences in basal hormone levels (Fig. 1). These findings par- allel those observed in handled versus non- handled rats, which differ in stress-induced, but not basal HPA, activity (3). Moreover, the frequency of maternal licking and grooming was significantly correlated with the magnitude of the plasma ACTH (r = -0.66, P < 0.01) and corticosterone (r =

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-0.65, P < 0.01) responses to stress in the adult offspring. Thus, the greater the fre- quency of maternal licking and grooming during infancy, the lower the HPA response to stress in adulthood.

We then examined glucocorticoid feed- back sensitivity in the high- and low-LG- ABN offspring by administering a bolus in- jection of corticosterone 3 hours before acute restraint stress (21). Corticosterone treatment suppressed plasma ACTH re- sponses to restraint stress to a significantly greater extent in the high-LG-ABN off- spring compared with their low-LG-ABN counterparts (75 + 5 versus 37 + 12%, respectively; P < 0.01). These findings sug- gest that the offspring of the high-LG-ABN mothers, like the handled animals, show increased sensitivity to the inhibitory ef- fects of glucocorticoids on stress-induced HPA activity.

Glucocorticoid inhibition of hypotha- lamic. CRH gene expression represents a critical feature of feedback action (5). Thus, we examined CRH mRNA expression (22) in parvocellular neurons of the paraven- tricular nucleus of the hypothalamus (PVNh), which send projections to the me- dian eminence and provide the neural sig- nal for the stimulation of ACTH release (23). CRH mRNA expression in the PVNh was significantly decreased in the offspring

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Fig. 3. (Top) Mean (?SEM) grains over individual cells (as a function of cell area) in dentate gyrus (DG), CA1, and 0A3 cell fields of the hippocam- pus in adult offspring of high- (n = 8) versus low- LG-ABN mothers (n = 6) ) from in situ hybridiza- tion studies of GR mRNA levels. *P < 0.01; *P < 0.001; **P < 0.0001. (Bottom) Scattergram of the correlation between the frequency of maternal

1660 SCIENCE * VOL. 277 * 12 SEPTEMBER 1997 * www.sciencemag.org

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of high-LG-ABN mothers compared with those of low LG-ABN mothers (Fig. 2). Moreover, CRH mRNA expression in the PVNh was significantly correlated with the frequency of maternal licking and grooming during the first 10 days of life (Fig. 2).

Considering the importance of the hip- pocampal GR system for negative-feedback regulation of HPA activity (10), we exam- ined GR mRNA expression in the hip- pocampus of the offspring of high- and low-LG-ABN mothers (24). Across each of the hippocampal cell fields there was in- creased GR mRNA expression in the off- spring of the high- compared to low-LG- ABN mothers (Fig. 3). Again, GR mRNA levels -in each cell field of the hippocampus were significantly correlated with the fre- quency of maternal licking and grooming (r = +0.76, P < 0.002 for the dentate gyrus; r = +0.64, P < 0.02 for the CAl region; r = +0.79, P < 0.001 for the CA3 region) (Fig. 3).

These findings reveal a marked similar- ity between the HPA responses to stress in the offspring of high-LG-ABN mothers and those of handled animals. The offspring of high-LG-ABN mothers, like handled ani- mals, show dampened plasma ACTH and corticosterone responses to stress, increased hippocampal GR expression, enhanced glu- cocorticoid feedback sensitivity, and de- creased.' hypothalamic CRH expression. There is considerable evidence for the im- portance of the hippocampus as a critical site for glucocorticoid feedback inhibition over hypothalamic CRH synthesis (10). In- deed, hippocampal GR levels have been directly correlated with CRH concentra- tions in the portal system of the anterior pituitary as well as with pituitary-adrenal activity (25). The offspring of the high-LG- ABN mothers showed increased glucocorti- coid feedback sensitivity coupled with de- creased hypothalamic CRH mRNA expres- sion and, as in the handled animals, the increased hippocampal GR expression ap-

Table 1. Mean (?SEM) number of observations (from a total of 1200) of licking and grooming in the mothers of handled or nonhandled litters (Han- dling study) or high- or low-LG-ABN mothers (Maternal behavior study). Differences in maternal behavior were stable over the 1 0-day period of observation. In neither study were there group differences in the frequency with which dams nursed pups or in pup contact (16). *P < 0.01.

Group Licking and grooming

Handling study Handled 155 ? 21* Nonhandled 78 ? 25

Matemal behavior study High LG-ABN 136 ? 22* Low LG-ABN 72 ? 8

pears likely to mediate these effects. The magnitude of the HPA response to

stress in adult animals was strongly correlat- ed with matemal licking and grooming (Figs. 1 to 3). These findings support the hypoth- esis of Levine that the effect of postnatal handling on HPA development is mediated by effects on mother-pup interactions. Thus, handling increases the frequency of licking and grooming (Table 1) and these matemal behaviors are, in tum, associated with damp- ened HPA responsivity to stress (Figs. 1 to 3). Tactile stimulation derived from mater- nal licking and grooming regulates pup phys- iology and affects central nervous system (CNS) development (26). Variation among dams in this form of matemal behavior ap- pears also to be associated with the develop- ment of individual differences in neuroendo- crine responses to stress.

The results of the handling study suggest that the frequency of matemal licking and grooming can be regulated by stimuli associ- ated with the pup. Thus, handling pups con- sistently increased matemal licking and grooming (Table 1), effectively ensuring a consistently high level of licking and groom- ing by the dam. This is consistent with ear- lier studies showing that handling increases ultrasonic vocalizations in pups which, in tum, serve to increase matemal care, includ- ing licking and grooming (14). However, it remains possible that the differences in ma- temal behavior observed here are associated with factors intrinsic to the mother-such as emotionality-in which case the data pre- sented here may, in part, offer an example of a nongenomic mode of inheritance between parent and offspring.

We believe that the effects of early envi- ronment on the development of HPA re- sponses to stress reflect a naturally occurring plasticity whereby factors such as matemal care are able to program rudimentary, bio- logical responses to threatening stimuli. Like humans, the Norway rat inhabits a great variety of ecological niches, each with varied sets of environmental demands. Such plas- ticity could allow animals to adapt defensive systems to the unique demands of the envi- ronment. Since most mammals usually spend their adult life in an environment that is either the same as or similar to the one in which they were bom, developmental "pro- gramming" of CNS responses to stress in early life is likely to be of adaptive value to the adult (12, 27). Such programming af- fords the animal an appropriate HPA re- sponse, minimizing the need for a long and perhaps unaffordable period of adaptation in adult life. Our results suggest that this neo- natal programming occurs via the differenti- ation of the GR system in forebrain neurons that govem HPA activity in response to variations in maternal behavior.

REFERENCES AND NOTES

1. S. Levine, Science 126, 405 (1957); ibid. 135, 795 (1962); , G. C. Haltmeyer, G. G. Karas, V. H. Denenberg, Physiol. Behav. 2, 55 (1967); M. X. Zar- row, P. S. Campbell, V. H. Denenberg, Proc. Soc. Exp. Biol. Med. 356,141 (1972).

2. The handling procedure involves removing the moth- er 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 [M. Daly, Br. J.

Psychol. 64, 435 (1972)]. Normal development in a rat pup most often occurs in the rather dark, tranquil confines of a burrow where the major source of stim- ulation is the mother and littermates.

3. S. Bhatnagar, N. Shanks, M. J. Meaney, J. Neuroen- docrinol. 7,107 (1995); J. L. Hess, V. H. Denenberg, M. X. Zarrow, W. D. Pfeifer, Physiol. Behav. 4, 109 (1969); M. Vall6e et al., J. Neurosci. 17, 2626 (1997).

4. M. J. Meaney, D. H. Aitken, S. Bhatnagar, Ch. Van Berkel, R. M. Sapolsky, Science 238, 766 (1988); M. J. Meaney, D. H. Aitken, S. Bhatnagar, R. M. Sapolsky, Neurobiol. Aging 12, 31 (1991).

5. T. Imaki, J.-L. Nahan, C. Rivier, P. E. Sawchenko, W. Vale, J. Neurosci. 11, 585 (1991); P. M. Plotsky and P. E. Sawchenko, Endocrinology 120, 1361 (1987); P. M. Plotsky, S. Otto, R. M. Sapolsky, ibid. 119, 1 126 (1986); P. E. Sawchenko, J. Neurosci. 7, 1093 (1987).

6. M. J. Meaney, D. H. Aitken, S. Sharma, V. Viau, A. Sarrieau, Neuroendocrinology 50, 597 (1989); V. Viau, S. Sharma, P. M. Plotsky, M. J. Meaney, J. Neurosci. 13,1097 (1993).

7. P. M. Plotsky and M. J. Meaney, Mol. Brain Res. 18, 195 (1993); D. Francis, S. Sharma, P. M. Plotsky, M. J. Meaney, Ann. N. Y Acad. Sci. 794,136 (1996).

8. M. J. Meaney etal., Behav. Neurosci. 99, 760 (1985); A. Sarrieau, S. Sharma, M. J. Meaney, Dev. Brain Res. 43,158 (1988).

9. D. O'Donnell, S. Larocque, J. R. Seckl, M. J. Meaney, Mol. Brain Res. 26, 242 (1994).

10. E. R. de Kloet, Front. NeuroendocrinoL 12, 95 (1991); L. Jacobson and R. M. Sapolsky, Endocr. Rev. 12, 118 (1991).

11. S. A. Barnett and J. Burn, Nature 213, 150 (1967); 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 Influ- ences and Early Behavior, R. W. Bell and W. P. Smotherman, Eds. (Spectrurf, New York, 1980), pp. 201-21 0.

12. J. R. Alberts and C.P. Cramer, in. Handbook of Be- havioral Neurobiology, E. M. Blass, Ed. (Plenum, New York, 1989), vol. 9, pp. 1-39.

13. A. Fleming and J. S. Rosenblatt, Behav. Neurosci. 86, 221 (1974); J. E. Jans and B.C. Woodside, Dev. Psychobiol. 23, 519 (1990); M. Leon, P. G. Croskerry, G. K. Smith, Physiol. Behav. 21, 793 (1978).

14. R. W. Bell, W. Nitschke, T. H. Gorry, T. Zachma, Dev. Psychobiol. 4, 181 (1971); M. H. S. Lee and D. I. Williams, Anim. Behav. 22, 679 (1974).

15. B. C. Woodside, M. J. Meaney, J. E. Jans, unpub- lished data.

16. S. Levine and G. W. Lewis, Science 129, 42 (1959); M. J. Meaney and D. H. Aitken, Dev. Brain Res. 22, 301 (1985). The animals used in all studies were Long-Evans hooded rats obtained from Charles Riv- er Labs. (St. Constant, Qu6bec, 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 Conm- mittee. Handling was done as described (2).

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Nonhandled animals were completely undisturbed until day 12 of life at which time normal cage main- tenance was initiated. The behavior of each dam was observed [M. M. Myers, S. A. Brunelli, H. N. Shair, J. M. Squire, M. A. Hofer, 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 1 100, and an obser- vation was scheduled at 1 130 to correspond to the reunion of the mothers and pups. Within each obser- vation period the behavior of each mother was scored every 4 min (15 observations per period x 8 periods per day = 120 observations per mother per day) for mother off pups, mother licking and groom- ing 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 mutu- ally exclusive.

17. D. Liu et al., data not shown.

18. Nine Long-Evans female rats were mated in our an- imal facility and housed and observed as described (16). The animals underwent routine cage mainte- nance 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.

19. 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.

20. 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 in- dwelling 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 [L. C. Krey et al., Endocrinology 96, 1088 (1975)]. Plasma (25 pI) ACTH was measured by radioimmunoassay as de- scribed [C.-D. Walker, S. F. Akana, C. S. Cascio, M. F. Dallman, 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 1 1 % for ACTH. The data were analyzed by two-way anal- yses of variance (ANOVA) with one between (group) and one within (sample) measure. Post hoc analysis was performed by Tukey test.

21. We used a delayed negative-feedback paradigm [M. Keller-Wood and M. F. Dallman, Endocr. Rev. 5, 1 (1984)] 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 feed- back 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 pe- riod of restraint, a time point that corresponds to the peak plasma ACTH level (6). The percentage sup- pression of plasma ACTH responses to stress for the high- versus low-LG-ABN groups was derived by comparing A(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). Per- centage suppression scores were used to accommo- date for the groups differences in plasma ACTH re- sponses to acute stress. The results were examined statistically by Mann-Whitney U test on the basis of percentage scores.

22. CRH mRNA in situ hybridization was done with a

48-base pair (bp) oligonucleotide sequence

(CAGTTTCCTGTTGCTGTGAGCTTGCTGAGCTA-

ACTGCTCTGCCCTGGC) (Perkin-Elmer,Warring- ton, UK) and a modified version of the procedure

previously described [N. Shanks, S. Larocque, M. J.

Meaney, J. Neurosci. 15, 376 (1995)] 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 35S-labeled standards pre- pared with known amounts of radiolabeled 35S in a brain paste. The hybridization signal within the par- vocellular subregion of the PVNh was quantified by densitometry with an MCID image analysis system (Imaging Research, St. Catherine's, Ontario, Cana- da). The data are presented as arbitrary absorbance units after correction for background. These data were analyzed by t test for unpaired groups.

23. P. M. Plotsky, J. Neuroendocrinol. 3, 1 (1991); W. H. Whitnall, Prog. NeurobioL 40, 573 (1993).

24. GR in situ hybridization was done as described [(9); J. R. Seckl, K. L. Dickson, G. Fink, J. Neuroendocri- nol. 2, 911 (1990)] with [35S]UTP-labeled cRNA an- tisense 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, sec- tions were dehydrated, dried, and dipped in photo- graphic emulsion (NTB-2, Kodak), and then stored at 4?C for 21 'days before development and counter- staining wlth Cresyl Violet. The hybridization signal within dorsal hippocampal subregions was quanti- fied by grain counting within high-power microscop- ic 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 [J. T. McCabe, R. A. Desharnais, D. W. Pfaff, Meth- ods Enzymol. 168, 822 (1989)]. These data were analyzed by two-way ANOVA with one between measures (group) and one repeated measure (hip- pocampal sub-field) by Tukey post hoc test.

25. R. M. Sapolsky, L. C. Krey, B. S. McEwen, Proc. Natl. Acad. Sci. U.S.A. 81, 6174 (1984); R. M. Sapol- sky, M. P. Armanini, D. R. Packan, S. W. Sutton, P. M. Plotsky, Neuroendocrinology 51, 328 (1990); J. L. W. Yau, T. Olsson, R. G. M. Morris, M. J. Meaney, J. R. Seckl, Neuroscience 66, 571 (1995).

26. C. M. Kuhn, G. E. Evoniuk, S. M. Schanberg, Science 204,1034 (1978); S. R. Butler, M. R. Suskind, S. M.

Schanberg, ibid. 199,445 (1978); S. Levine, Ann. N. Y Acad Sci. 746, 260 (1994); C. L. Moore, Dev. Psy- chobiol. 17, 347 (1984); M. M. Myers, H. N. Shair, M. A. Hofer, Experentia 48, 322 (1992); S. M. Schan- berg and T. M. Field, Child Dev. 58,1431 (1987).

27. M. A. Hofer, in L. A. Rosenblum and H. Moltz, Eds., Symbiosis in Parent-Offspring Interactions (Plenum, New York, 1983), pp. 61-75.

28. 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 Can- ada (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 grad- uate fellow of the MRCC.

2 May 1997; accepted 31 July 1997

Structure of a Murine Leukemia Virus Receptor-Binding Glycoprotein at

2.0 Angstrom Resolution

Deborah Fass, Robert A. Davey, Christian A. Hamson, Peter S. Kim,* James M. Cunningham,* James M. Berger

An essential step in retrovirus infection is the binding of the virus to its receptor on a target cell. The structure of the receptor-binding domain of the envelope glycoprotein from Friend murine leukemia virus was determined to 2.0 angstrom resolution by x-ray crys- tallography. The core of the domain is an antiparallel 3 sandwich, with two interstrand loops forming a helical subdomain atop the sandwich. The residues in the helical region, but not in the sandwich, are highly variable among mammalian C-type retroviruses with distinct tropisms, indicating that the helical subdomain determines the receptor spec- ificity of the virus.

Retroviruses are simultaneously a profound human medical problem and a potential med- ical solution. They can be pathogenic, causing immunodeficiency, leukemia, and neurologi- cal disease, but they are also actively studied for their proposed utility as gene therapy vec- tors. Essential to both roles is the targeting of the virus to the host cell through interactions between viral envelope proteins and cell sur- face proteins.

Retrovirus envelope glycoproteins (1) are

synthesized as single chain precursors that are subsequently cleaved into two subunits, the surface (SU) and the transmembrane (TM) (Fig. 1). The SU glycoprotein binds the receptor. The TM subunit contains the hydrophobic fusion peptide and transmem- brane segments and is likely to participate directly in the fusion of the viral and cellular membranes after receptor binding.

Efforts to understand the structural basis of retroviral binding and entry and to devel-

1662 SCIENCE * VOL. 277 * 12 SEPTEMBER 1997 * www.sciencemag.org

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