Timed-pregnant Sprague Dawley female rats were housed in an uncrowded, quiet animal facility room on a 12 h light/dark cycle and were provided with food and water ad libitum. Parturition was checked daily, and the day of birth was considered P0. On P1, litters were mixed and adjusted to establish similar numbers of males and females. Complete litters were randomly assigned to one of the following early-life rearing conditions: handled daily from P2 to P8 or P9 (see below); handled once, on P5, P8, or P9; undisturbed during P2–P9; or undisturbed and deprived of maternal care for 3 h on P9. These studies used 132 male pups. All experiments were approved by the University Animal Care Committee and conformed to National Institutes of Health guidelines.
Handling and maternal observation
The daily handling procedure was modified from that originally described by Levine (1957)
, as described previously (Avishai-Eliner et al. 2001
; Fenoglio et al., 2004
; Fenoglio et al., 2005
). Cages were brought into the laboratory daily at 8:30 A.M. (Watts et al., 2004
). The dam and pups were placed into separate bedded cages (pups were kept euthermic via a heating pad located underneath the cage). After 15 min, pups were placed back into their home cage, followed by the dam, and returned to the vivarium. Undisturbed litters remained in the vivarium from P2 to P9. For all experimental groups, cage changing did not occur during this time. Every day, after handled pups were reunited with the dam and returned to the vivarium, maternal behavior (specifically licking and grooming) was observed using a protocol modified from Liu et al. (1997)
as used by Brunson et al. (2005)
. Each maternal observation session consisted of 12 3 min epochs. Within each epoch, the duration of licking and grooming of pups was recorded during the first 2 min, and the total amount of time spent licking and grooming was scored per session. Maternal behavior was also determined in undisturbed litters and in those handled once on P5 or P8 (on the day of handling).
Fos protein and phosphorylated extracellular-signal regulated kinase (pERK) were examined in rats handled daily and killed at different time points (at 5, 30, 60, 90, or 120 min) after their return to the dam on P9 compared with several control groups. These included pups handled once on P9 and killed at the same time points, the undisturbed group, and pups deprived from the mother for 3 h and not returned to the cage. Rats were anesthetized with sodium pentobarbital (100 mg/kg, i.p.) and then perfused through the ascending aorta with 0.9% saline solution, followed by freshly prepared, cold 4% paraformaldehyde in 0.1 M sodium phosphate buffer (PB), pH 7.4. Brains were removed and postfixed overnight, cryoprotected in 15 and 30% sucrose/PB solutions, and stored at −80°C. Brains were sectioned at 35 μm using a cryostat, and sections were collected in tissue-culture wells containing 0.1 M PB.
For neuroanatomic orientation, one in every six sections were stained with toluidine Nissl stain. Adjacent series at the levels of the anterodorsal (AD) BnST (4.7 mm, anterior to the interaural line), ACe (3.8–2.9 mm), and PVT (2.9–2.0 mm) were processed for Fos immunoreactivity (IR) (Sherwood and Timiras, 1970
). The coordinates for the regions examined were derived to compare with the corresponding structures in adult rats (Paxinos and Watson, 1982
): BnST (Ju et al., 1989
; Herman et al., 1994
), ACe (Prewitt and Herman et al., 1994
), PVT (Bhatnagar and Dallman, 1998
) as shown in . Series of sections at the level of the PVN (3.8–3.5 mm) were processed for pERK-IR.
Figure 4 Time course of Fos expression in PVT, ACe, and BnST of daily handled pups after their return to the dam. Quantification and bright-field photomicrographs of Fos-labeled neurons (blue arrows) in PVT (A), ACe (B), and BnST (C) of pups handled recurrently (more ...)
Free-floating sections were subjected to the standard avidin–biotin complex (ABC) methods as described previously (Chen et al., 2000
). Briefly, after three washes with 0.01 M PBS containing 0.3% Tri-ton X-100, pH 7.4 (PBS-T), sections were treated with 0.3% H2
in PBS for 30 min. Nonspecific binding was blocked with 10% normal goat serum in PBS-T for 30 min. PVT, ACe, and BnST sections were incubated with polyclonal Fos antibody (1:20,000; Oncogene Sciences, Cambridge, MA) in PBS for 24 h. PVN sections were incubated with polyclonal pERK antibody (1:400; Sigma, St. Louis, MO) in PBS for 72 h at 4°C. Subsequently, sections were washed in PBS-T (three times for 5 min) and incubated in biotinylated goat anti-rabbit IgG (1:400; Vector Laboratories, Burlingame, CA) in PBS for 2 h. After washing in PBS-T, sections were incubated in ABC solution (1:100; Vector Laboratories) for 3 h and rinsed in three 5 min PBS-T washes. The reaction product was visualized by incubating sections for 8–10 min in 0.04% 3,3′-diaminobenzidine containing 0.01% H2
. The specificity of the primary antisera was tested by preadsorbing the antiserum overnight with purified Fos or pERK blocking peptide (100 μ
g/ml). The specificity of the secondary antibody was tested by omitting the primary antiserum during the first incubation. There were no immunoreactive cells in sections subjected to these testing conditions.
In situ hybridization histochemistry studies
Basal levels of CRH mRNA were investigated in PVN of P9 pups that were handled daily from P2 to P8, handled once on P5 or P8, or remained undisturbed. [Note that previous studies have shown that handling from P2 to P8 is sufficient to induce long-term neuroendocrine changes (Avishai-Eliner et al., 2001
; Fenoglio et al., 2005
). Here, handling-induced changes of basal CRH mRNA expression were examined in pups handled from P2 to P8 and killed 24 h after the last day of handling, on P9.]
Levels of unedited, heteronuclear CRH RNA were determined as a measure of direct activation of CRH gene expression. Differential activation of the CRH gene on P9 was compared between pups that were handled (from P2 to P8) and those that were undisturbed during these days. On P9, both groups were subjected to the handling procedure (i.e., were separated from the dam for 15 min and then returned to the dam). CRH hnRNA was measured at the onset of this separation (basal), at the moment of the pups’ return (15 min from separation onset), as well as at 45 and 75 min after the onset of separation from the mother (i.e., 30 and 60 min, respectively, after the pups’ return to the dam). For all in situ hybridization histochemistry (ISH) studies, rats were killed by rapid decapitation, and brains were quickly removed and frozen on dry ice.
Expression levels of CRH mRNA and heteronuclear RNA (hnRNA) in PVN were determined using previously described ISH methods (Yi and Baram, 1994
; Avishai-Eliner et al., 2001
; Chen et al., 2001b
). Coronal sections (20 μ
m) were collected on gelatin-coated slides and stored at −80°C. Sections were air dried and postfixed in 4% paraformaldehyde in PB for 20 min. Sections underwent a series of dehydration and rehydration steps through graded ethanol washes and were exposed to 0.25% acetic anhydridein 0.1M triethanolamine, pH8, for 8min. After dehydration through graded ethanol washes, sections were incubated with prehybridization buffer for 1 h in a humidified chamber (at 42°C for mRNA and 55°C for hetero-nuclear RNA). Levels of mature CRH mRNA were examined using 0.25 × 106
cpm of 35
S-labeled deoxyribonucleotide probe. Sections were hybridized with probe overnight at 42°C. Sections were washed at 55°C in 2×and 1× SSC for 5 min each and then successively in four fresh solutions of 0.3×SSC for 15 min each (note that 1× SSC denotes 0.15 M NaCl, 15 mM trisodium citrate buffer, pH 7.0). In the second set of experiments, levels of unedited CRH heteronuclear RNA were determined using 1 × 106
cpm of 35
S-labeled ribonucleotide probe [courtesy of Drs. S. J. Watson (University of Michigan, Ann Arbor, MI) and S. Rivest (CHUL Research Center, Quebec, Canada)]. Sections were hybridized with probe overnight at 55°C, then rinsed in 4× and 2× SSC, subjected to a 30 min RNase wash at 37°C, followed by a series of washes at 55°C: 2× SSC (5 min), 1× SSC (30 min), 0.25× SSC (1 h), and 0.03× SSC for 30 min. All sections were dehydrated in graded ethanol solutions containing 0.3 M ammonium acetate, followed by 100% ethanol. Sections were air dried and apposed to film (Kodak BioMax MR Film, MR-1; Eastman Kodak, Rochester, NY) for ~10 d. Selected sections were dipped in emulsion (NTB-2; Eastman Kodak) and developed after 3 weeks.
Semiquantitative analyses and statistical considerations
Initial experiments evaluated neuronal activation throughout a series of sections spanning the brain from the anteriormost border of the diagonal band of Broca (6.2 mm, anterior to the interaural line) to the bottom of the brainstem (−4 mm, posterior to the interaural line). Little constitutive or handling-induced expression of the cellular activation markers used here was noted in regions other than those discussed.
Section sampling and analyses were conducted without knowledge of treatment (Eghbal-Ahmadi et al., 1999
; Brunson et al., 2001
; Fenoglio et al., 2004
). For an unbiased determination of cells labeled for Fos or pERK, a systematic random series of sections (one in two for BnST and PVN, one in three for ACe, and one in four for PVT) was selected for each animal. Cell nuclei were counted as described previously (Chen et al., 2001a
; Bender et al., 2001
) under 40× magnification in matching sections. The boundaries of each region of interest were delineated in adjacent sections labeled with methyl green Nissl stain. The average number of Fos-immunopositive cells was determined from three to five sections for PVT, three sections for ACe, and two sections for BnST. Cells positive for pERK were counted in two to four PVN sections, generating a mean number of immunoreactive cells per region for each brain. For the ISH studies, signal was analyzed on digitized films using the Image-Tool software program (University of Texas Health Science Center, San Antonio, TX). The signal was analyzed in the linear range of optical densities as evaluated using 14
C standards. Means and SEs were calculated for each brain from two to four anatomically matched sections. For all studies, statistical significance (p
< 0.05) among experimental groups was determined using one-factor ANOVA with Bonferroni’s post hoc
test (Prism; GraphPad, San Diego, CA) unless otherwise noted.