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Department of Pharmacology and Therapeutics (M.M.S., D.S.), and Department of Psychiatry (D.S.), Louisiana State University Medical Center, Shreveport, Louisiana 71130-3932; Department of Cellular Biology (F.A.), Faculty of Medicine, University of Barcelona, 08036 Barcelona, Spain; and Department of Cellular Biology (F.S.-T.), Faculty of Biological Sciences, Complutense University of Madrid, 28040 Madrid, Spain
Address all correspondence and requests for reprints to: M. Mar Sánchez, Ph.D., Department of Psychiatry and Behavioral Sciences, Emory University School of Medicine, 1639 Pierce Drive, Atlanta, Georgia 30322. E-mail: sanchez{at}rmy.emory.edu
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the rat, the development of this HPA system is regulated by factors of maternal origin and is also dependent on social interactions with littermates (2, 3, 4). Thus, alterations in HPA activity of rat pups have been described after acute episodes of maternal and littermate deprivation during the preweaning period (3, 5), when the PVN-stimulating pathways are in a developing stage (2, 6). Animals that are socially isolated for extended periods also show behavioral and physiological changes, including alterations in HPA activity. These alterations, however, are not consistent among studies, because increased adrenocortical activity has been reported (7, 8), whereas other groups have not found changes (9, 10), and others have found decreased HPA function, particularly when the response to stress is examined (11, 12). The duration and age at onset of isolation could have accounted for the different results. However, these studies were focused on the effects of long-term isolation on adrenocortical function, without providing information of the functionality at other levels of the HPA axis such as the pituitary or the hypothalamus.
Previous studies from our group have found synaptic changes (13) and alterations in the activity of extrahypothalamic pathways that regulate the response of PVN neurons to some stressful stimuli [in particular, mediated by the amygdala (14)] after social isolation in rats after an early weaning. These findings suggested the possibility of an altered limbic-HPA response to stressful stimuli in isolation-reared animals. Taking into account these previous studies, the aim of the present study was to characterize the neuroendocrine effects of long-term social isolation from an early weaning period on the function of the adult rat HPA system, both under basal conditions and in response to an acute stress. In addition to analyzing the activity of the axis in terms of CS secretion, we have employed quantitative immunocytochemistry to determine ACTH content in pituitary corticotrope cells and hypothalamic CRF, AVP, and OT contents and secretory activities at the level of the PVN and the ME, respectively.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Both control and isolated animals were killed at 75 days of age by decapitation, either taking them directly from their home cages (basal condition) or after 15 min of restraint stress. Rats were exposed to restraint by introducing them into plastic cones that completely restricted the animals’ movement, following the method described by Orr et al. (16). Experiments were performed between 0900–1100 h.
Measurement of plasma CS concentrations
To analyze the effects of social isolation on adrenocortical
activity, we measured the plasma CS concentrations from trunk blood
collected into ice-cold heparinized Eppendorf tubes. After
centrifugation, plasma was stored at -20 C until the CS RIA was
carried out, according to the method of Gwosdow-Cohen et al.
(17). [3H]CS was purchased from New England Nuclear
(Boston, MA), and anti-CS antibody (sheep anti-CS-3-BSA serum no. 377)
was generously provided by Dr. G. Niswender (Colorado State
University). Steroids were extracted from both plasma and standards,
using methylene chloride. Cross-reactivity with other steroids was less
than 1%, except for deoxycorticosterone and 11-ß-hydroxyprogesterone
(7%), with intraassay and interassay coefficients of variation
calculated to be less than 5%.
The relative weights of thymus and adrenal glands also were used as indices for adrenocortical function, and testicular weight was measured to investigate the possibility of any alteration in gonadal function. Daily food and water consumption also was measured in all animals.
Immunohistochemistry
To determine whether any effects on adrenocortical secretory
activity could be caused by altered function of the
hypothalamic-pituitary complex controlling the secretion of CS,
quantitative immunohistochemical studies were carried out to detect
ACTH in the pituitary and its hypothalamic secretagogues (CRF, AVP, and
OT), both at the level of their synthesizing somata in the PVN and at
the level of the releasing terminals in the ME.
In this study, four animals from each experimental group were used, with immunodetection of the peptides being carried out using the peroxidase-antiperoxidase (PAP) method. Briefly, pituitaries and hypothalami, obtained immediately after decapitation, were immersed in a fixative solution of paraformaldehyde 4% for 24 h and 72 h, respectively, at 4 C. After fixation of tissues, blocks were cryoprotected by immersion in a 30% sucrose solution for 48 h at 4 C. After this treatment, 30-µm-thick coronal sections of the blocks were obtained using a cryostat. After eliminating the endogenous peroxidase activity by incubation with 0.3% H2O2, pituitary sections were used for ACTH immunostaining; and adjacent serial hypothalamic sections were used for the immunodetection of CRF, AVP, and OT, following the PAP method described by Sternberger (18). CRF immunohistochemistry was performed using antiserum rC-70 (generously provided by Dr. W. Vale, The Salk Institute, La Jolla, CA) at a final dilution of 1:3,000. Antibodies to ACTH(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24), AVP, and OT also were polyclonal antibodies purchased from Chemicon International (Temecula, CA) and used in final dilutions of 1:8,000, 1:3,000, and 1:3,000, respectively.
Quantitative immunocytochemistry
For each antibody, different parameters in each area of interest
were quantified using the NIH 1.52 Image Analysis System (NIH,
Bethesda, MD). The image was acquired using an Optronics CCD video
camera LX-450A (Optronics Engineering, Goleta, CA), the camera being
mounted in an Olympus BH-2 Microscope (Olympus Optical Co., Tokyo,
Japan). In the case of ACTH, the number of ACTH-immunoreactive
(ACTH-ir) cells in five different fields (0.05 mm2 each)
were quantified per section, in five sections/animal. Only
immunoreactive cells with full nuclear profiles were counted and
subsequently studied. The total cell and immunoreactive cytoplasmic
areas (in µm2), as well as the intensity of
immunostaining, were measured in 100 ACTH-immunopositive cells/animal
within the adenohypophysis, at a magnification of 900x. The last
parameter was obtained by measuring the mean OD of the immunoreactivity
of a given cell and then corrected by subtraction of the background OD
on the same section.
To quantify the immunoreactivity for CRF, AVP, and OT in the ME, three sections/animal were studied at different levels between bregma -2.56 mm and -3.30 mm, according to the atlas of Paxinos and Watson (19). In each section, 15 measurements (spot diameter: 12.5 µm) of the OD were made in the ME, as described previously (20, 21). Six background measurements also were made in each section at the level of the arcuate nucleus (where no CRF-, AVP-, or OT-positive structures were found). After background substraction, the OD was averaged for each animal, and the mean for each experimental group was then determined. Quantitative immunocytochemistry has been demonstrated as a good approach to study relative changes in neuropeptide stores in the ME (21, 22), and these data show good correlation with absolute concentrations of the neuropeptides, as measured by RIA, in ME extracts (21, 23). The number of CRF-, AVP-, and OT-ir neurons also were counted at three different rostrocaudal levels of the PVN per animal, at a magnification of 200x (number of immunolabeled cells/section).
Statistical analysis
Plasma CS concentrations and quantitative immunocytochemistry
data were analyzed by two-way ANOVA, followed by the post
hoc Scheffé’s test to identify which groups had
significantly different mean values. Body, thymus, adrenal, and
testicular weights, as well as daily food and water consumption data,
were statistically analyzed using Student’s t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. However, when the effect of
isolation on plasma CS concentrations was analyzed, socially deprived
rats showed reduced basal plasma CS concentrations and a reduced
adrenocortical response to 15 min of restraint stress, as compared with
the control group (Fig. 1). Two-way ANOVA
revealed that these differences were statistically significant
(F1,28 = 59.92, P < 0.0001), the exposure
to stress always evoking an increase in plasma CS concentrations in
relation to basal levels (F1,28 = 125.48, P
< 0.0001). However, the interaction factor of rearing and physiologic
state was not statistically significant (F1,28 = 0.35, NS),
indicating that isolation affected both the basal and stress CS
concentrations in the same way.
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).
Immunohistochemical study: ACTH, CRF, AVP, and OT
Most of the ACTH-ir cells in the pars distalis of the
adenohypophysis showed a stellate morphology under basal conditions,
with the immunoreactive granules distributed peripherally in the
cytoplasm, although some polygonal cells were also found (Fig. 2, A and B). No differences were observed
comparing ACTH immunostaining in the adenohypophysis of control (Fig. 2A) and socially deprived rats (Fig. 2B).
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, C and D).
Moreover, a change in the cellular shape and size was also observed,
the cells seeming larger and more rounded.
After carrying out the corresponding quantitative immunocytochemical
study, a two-way ANOVA revealed an overall significant difference
between the density of ACTH-ir cells in the pituitary of control and
isolated groups (F1,12 = 8.54, P < 0.05),
the stress factor producing a significant increase in the density of
immunopositive cells (F1,12 = 25.26, P <
0.001), with a significant interaction between factors also being
present (F1,12 = 5.95, P < 0.05).
Scheffé’s post hoc analysis revealed that isolated
animals showed a smaller increase in the density of ACTH-ir cells in
response to stress, in comparison with control animals (S = 3.79,
P < 0.01); however, no differences were found in the
basal density of ACTH-ir pituitary cells comparing control and isolated
animals (Fig. 3). In fact, the increase
in ACTH-ir cellular density, provoked by stress in control animals, did
not reach statistical significance in the isolated group.
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). However, the cellular corticotroph
area in isolated animals was not significantly different from that of
the control animals under basal conditions.
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). Two-way ANOVA revealed that
differences caused by isolation were statistically significant
(F1,12 = 16.90, P < 0.01), the stress
factor causing a significant increase in the ACTH-ir area of pituitary
cells, in comparison with the basal condition (F1,12 =
27.07, P < 0.001). However, no significant interaction
between factors was found (F1,12 = 4.43, NS).
Scheffé’s post hoc analysis revealed that differences
caused by isolation appeared only after exposure to stress, the
isolated animals showing a smaller increase in the ACTH-ir area than
the control group (S = 4.39, P < 0.01), whereas
no differences were found under basal conditions.
No significant differences were found between groups when the ACTH-ir
intensity was quantified in the pituitary (rearing factor:
F1,12 = 2.73, NS; physiologic state factor:
F1,12 = 3.04, NS; interaction between factors:
F1,12 = 1.61, NS; two-way ANOVA), as shown in Fig. 4C.
To test the hypothesis of an altered synthesis and/or release of
ACTH secretagogues from the hypothalamus, we performed a quantitative
immunohistochemical analysis of CRF, AVP, and OT (both in the PVN and
in the ME). Social isolation did not affect the distribution or the
number of CRF, AVP, or OT immunopositive cells within the PVN (see
Table 2), nor did it produce any changes
in the content of AVP- or OT-ir fibers in the ME (data not shown).
However, as shown in Fig. 5, isolated
animals showed some changes, both in the pattern of distribution and in
the content of CRF-ir fibers in the ME. The immunopositive fibers for
this neuropeptide were mainly located in the ZEME, perpendicularly
oriented to its surface. In this region, isolated animals showed an
increased content of CRF-ir fibers, mainly in the lateral regions, both
under basal conditions and after exposure to restraint stress.
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). The exposure to restraint stress
evoked a significant decrease in the CRF-ir in the ME of control
animals (S = 2.32, P < 0.05), reflecting a
decrease in the stores of this neuropeptide in this structure,
presumably caused by release into the hypophyseal portal system
(21, 22, 23). The reduction in the content of CRF-ir fibers was much lower
in isolated animals, and this was not significantly different from that
found under basal (unstressed) conditions (S = 0.75, NS).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All our measurements were done at the zenith of the diurnal cycle, the plasma ACTH and CS levels being highly dependent on the activity of hypothalamic CRF neurons (24). Other authors have previously demonstrated increased plasma CS and ACTH concentrations 10–60 min after the onset of restraint stress, both in the AM and the PM components of the circadian rhythm (25, 26, 27). Under these conditions, CRF and AVP stores in ME decrease by 66%, as measured by RIA, indicating release of these ACTH secretagogues into the portal vasculature (26).
We did not find differences between groups in the density of pituitary ACTH-ir cells under resting conditions, which would predict a similar basal release of ACTH and CS for both groups of animals. However, the basal CS plasma levels were lower in the isolated animals. This could be caused, for example, by an altered sensitivity of the adrenal cortex to ACTH [caused by a down-regulation of ACTH receptors or an altered steroidogenesis in the gland, mechanisms that are still in development by postnatal day (PND) 16 (6)]. The fact that social isolation did not alter basal ACTH content in the anterior pituitary is in agreement with previous studies indicating that chronic social, or nonsocial, stress of low intensity does not necessarily affect the morphology or proportion of pituitary cellular types (28) or the basal pituitary content of ACTH (29). There is some evidence, however, for a decreased content of pituitary POMC-derived peptides, such as ß-endorphin, in animals isolated from weaning until adulthood (30). The discrepancy between results could be caused by the different age at onset of isolation. In this regard, Vázquez and Akil (6) showed that intermittent chronic isolation produced a decrease in the basal content of ACTH and POMC messenger RNA (mRNA) in the anterior pituitary of PND 21 rats, whereas it did not produce alterations in PND14 animals.
In response to restraint stress, however, isolation caused a reduced activation of pituitary corticotroph cells, as demonstrated by the blunted increase in the density of ACTH-ir cells, these showing a smaller increase in immunoreactive area than that found in the control group. Previous studies have demonstrated that anterior pituitary corticotrophs enlarge and the percentage of ACTH-ir cells increases after exposure to stress (31) or after stimulation by CRF (32). Apart from a likely decrease in the release of CRF in the ZEME to the hypophyseal portal system (as indicated by the smaller decrease in CRF-ir caused by acute stress in the ZEME of isolated, as compared with control animals), this result also could be caused by:
1) A decreased proteolytic cleavage of POMC to render ACTH. POMC and ACTH levels increase in the anterior pituitary between the second and fifth week of the rat life because of: an increase in the proteolytic processing of POMC and a change in the nature of its final products (33, 34); and an increase in the number of corticotroph cells in the anterior pituitary by a process of selection (34). During postnatal development there are pituitary corticotrophs with multihormonal content that differentiate either into corticotrophs or other phenotypes (35, 36). This process of differentiation could have been affected by the social isolation. However, we did not find differences in basal density of pituitary corticotrophs after isolation-rearing, but a blunted increase in their density in response to restraint stress. Fifteen to 30 min of stress provokes an activation of more pituitary corticotrophs (in addition to those showing basal ACTH content), which show a higher threshold in the concentration of ACTH secretagogues required for activation (36). Therefore, a blunted release of CRF from the ZEME terminals into the portal vasculature of isolated animals could explain the fact that fewer of those high-threshold corticotrophs were activated in response to restraint stress. Moreover, a change in the ratio of AVP to CRF in portal blood also could affect the activation of different corticotroph populations secreting ACTH in the isolated animals; this would include cells responsive to CRF but not AVP, to CRF or AVP, or to CRF and AVP (37).
2) A down-regulation of CRF and/or AVP pituitary receptors and/or a reduction in their affinities, which has been previously reported in adult rats after brief periods of maternal deprivation on PND 2–20 (38), or in PND18 rat pups maternally deprived for 24 h (39). However, these authors relate the down-regulation of CRF receptors to increased CRF secretion into the hypophysial portal vasculature and found increased CS plasma levels in the maternally-deprived animals.
3) An up-regulation of glucocorticoid receptors (GR) or/and a reduced corticosteroid-binding globulin content, or an increase in CRF-binding protein within the pituitary corticotrophs (2). An increase in GR occupancy/translocation, together with an increase in corticosteroid-binding globulin, has been shown previously in PND15 rats after 24 h of maternal separation, resulting in an increased CS negative feedback at the level of ACTH-containing cells (40). Again, these authors found increased basal and stress plasma CS and ACTH concentrations in the deprived animals. The low basal and stress plasma CS concentrations found in our isolation-reared animals make the possibility of increased negative feedback of glucocorticoids at the level of pituitary corticotrophs an unlikely one.
Although the isolation-reared animals did not show an increase in the density of pituitary ACTH-ir cells after restraint stress, there was a significant increase in their cytoplasmic immunoreactive area, in comparison with the basal state. A positive correlation has been found between the increase in area of anterior pituitary corticotrophs and the increase in serum ACTH after stress (31), which would have caused an increase in CS plasma levels after restraint stress. However, such an increase was smaller than the one observed in the control group.
The decline in CRF-ir in the ZEME in control animals, after restraint stress, indicates secretion of the neuropeptide from its neuronal stores in that area into the hypophysial portal system, in agreement with previous studies (21, 22, 26, 29).
Isolated animals showed a smaller reduction in the ZEME CRF-ir than control animals after restraint, indicating a blunted release of this neuropeptide to the portal circulation in response to stress, which could be responsible for the reduced activation of pituitary corticotrophs found in isolated animals and discussed above. Pituitary ACTH responses to restraint stress seem to be mediated by dynamic variations in both CRF and AVP (1). However, no differences in CRF release to portal system seem to occur under basal conditions, because the ZEME content of CRF-ir was similar in control and isolated animals. ME AVP- and OT-ir were also unaltered in the isolated animals. In accordance with our results, repeated or chronic exposure to a psychosocial stressor has previously been shown to differentially affect AVP- and CRF-ir contents in the ZEME (26), reflecting an adaptive response of ACTH-controlling neuronal pathways to a specific repeated or chronic activation, and suggesting that CRF and AVP release to the portal system during acute restraint stress are controlled by independent mechanisms. A strong positive correlation has been demonstrated between long-lasting increases of AVP stores in the ZEME and the initial ACTH response to an acute stressor (41), suggesting that isolation may not have been initially an intense stressor for the infant rats and, therefore, did not affect AVP release but did affect CRF release into the portal circulation.
An increased negative feedback of glucocorticoids can result in decreased CRF release to the portal blood (42, 43). This seems an unlikely mechanism to explain our finding, because baseline plasma CS concentrations are lower in isolation-reared rats than in controls. However, this possibility cannot be completely ignored until GR or mineralocorticoid receptor up-regulation in hippocampus, PVN, or pituitary is ruled out. As previously suggested under conditions such as posttraumatic stress disorder, there may exist abnormalities in HPA function, after the stressful event, that seem to be characterized by a blunted baseline glucocorticoid secretion caused by an augmented negative feedback inhibition of the axis (44).
CRF, AVP, and OT synthesis in the PVN seemed to be similar in both groups of animals, as shown by quantitative immunocytochemistry of immunoreactive somata within the different PVN subnuclei. Therefore, the blunted CRF released to the portal circulation in response to restraint does not seem to be caused by a reduced CRF content in the PVN in the isolation-reared animals, but rather caused by alterations in CRF axonal transport and/or release. Cell activation often is correlated with increases in neuropeptide synthesis and perikaryal content and immunocytochemistry, although not a good quantitative technique in terms of peptide concentrations, is useful for the detection of long-term qualitative changes in expression of neuropeptides, such as after chronic stress (45). However, it would be interesting to analyze whether social isolation altered the expression of CRF, AVP, OT, and POMC by means of in situ hybridization techniques. The changes in mRNA levels, though, would not necessarily be correlated with changes in peptide content and release (45), whereas depletion of peptide can be used as an indicator of secretion, as discussed above.
At PND16 (when the social isolation started in our study) CRF, AVP, and OT systems and the extrahypothalamic pathways that converge on PVN CRF-containing neurons are still in a developing state (2, 6). Electrophysiological findings from our group demonstrated a hypofunctionality of extrahypothalamic pathways controlling HPA axis activity after isolation-rearing (14). Electrical stimulation of the medial amygdala was used in that study to mimic the activation evoked by stressful neurogenic stimuli (such as restraint) on pathways that converge in the PVN, and the subsequent increase in CS secretion (46, 47, 48). Isolated rats showed reduced excitation (and increased inhibition) of PVN putative CRF-containing cells in response to medial amygdala stimulation, whereas their basal activity did not show remarkable alterations (14). This reduced activation of specific limbic-hypothalamic pathways that mediate HPA responses to neurogenic stress could be responsible for (or at least, related to) a blunted release of CRF into the portal vasculature and the consequent lesser activation of pituitary corticotrophs and lower CS response to restraint stress observed in isolated animals in this study.
The effects of isolation-rearing on HPA axis function in the adult rat are not consistent: increased adrenocortical activity has been reported (7, 8), whereas other groups observe no changes (9, 10) or even find decreased HPA function (11, 12). The discrepancies between results seem to be caused by differences in the age at onset of isolation (pre- vs. postweaning), the duration of isolation, and the sensory stimuli in the environment. The effects of isolation-rearing in our model could be caused by different variables, including: 1) the maternal deprivation from PND16–21 that regulate the development of different components of the HPA axis (4); 2) separation from the peers, which deprives the animals of any social contact (e.g. play); and 3) the possibility of isolation being a chronic stress. In relation to this, 24 h of maternal deprivation on PND 5–18 increases pup plasma CS concentrations (3, 5).
The effects of isolation-rearing seem to be different from the effects of repeated brief periods of maternal separation on the HPA adult function. For example, PND2–14 rats, maternally separated for 3 h per day, showed (as adults) an increased CS and a greater depletion of CRF in the ME in response to restraint stress, which indicated an increase in CRF released into the hypophysial portal system during stress (49). Under basal, resting conditions, CRF content in the ME also was elevated in maternally separated animals, although basal CS plasma levels were similar in all the groups. However, Ladd et al. (38) found that maternal deprivation, a few hours per day, from PND 2–20, increased basal and stress plasma ACTH levels in adult animals without changing CS plasma levels; deprived animals showed a similar increase in ME CRF content under both basal and stress conditions, this in contrast with the data reported by Plotsky and Meaney (49). It seems that pup age is a factor determining the effect of maternal separation on later HPA function. Thus, 24 h of maternal separation at PND3–4 increased ACTH responses to stress at PND20 and mineralocorticoid receptor content in hippocampus, whereas separation at PND11–12 decreased the ACTH response to stress at PND20, with a down-regulation of GR mRNA in hippocampus (50).
Paradoxically, handling during the early postnatal life in the rat also results in reduced CS response to different stressors, which has been related to a blunted release of ME CRF and AVP to the portal system (27). Under resting conditions, however, plasma levels of CS remain unchanged, whereas handled rats show decreased CRH and AVP hypothalamic synthesis and ME content (responsible for the blunted release of the neuropeptides in response to stress). In contrast to handling, long-term social isolation does not affect the basal PVN or ME content of CRH or AVP, whereas the basal levels of plasma CS are reduced. As discussed above, a blunted excitatory signal in response to stress (14) could be responsible for a blunted secretion of CRH in the ME after isolation, because the readily releasable storage pools of this neuropeptide remain unchanged under resting conditions.
Therefore, different social manipulations during the early life can evoke different effects on HPA axis function of the adult rat, depending on the developmental state of the HPA system, the duration of social isolation, the number of separation periods, and the level of social and sensorial stimuli deprivation. In any case, all the above findings indicate the importance of the social environment as a factor regulating the development of the limbic-HPA system and possibly determining individual differences in the sensitivity to stress.
In summary, isolation-rearing after early weaning caused a hypofunction of the HPA axis in the adult rat, particularly evident in the response to acute stress. These results, together with previous electrophysiological and stereological results from our group, indicate a hypofunctionality of limbic-hypothalamic pathways that control the adrenocortical response to neurogenic stress in the isolated animals, whereas basal activity of the HPA axis remains less affected.
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Acknowledgments |
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Footnotes |
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Received July 8, 1997.
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