INTRODUCTION

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DEVELOPMENT OF THE MAMMALIAN nervous system continues well after birth. As a result, postnatal sensory experience exerts a formative influence over the maturation of numerous components of the nervous system, including visual and auditory pathways (9, 18, 23) and the somatosensory and olfactory systems (2, 28), among others. Central nervous system regions governing vegetative functions are also susceptible to environmental influences during development. Of these, among the most thoroughly investigated is the impact of neonatal handling on regulation of the hypothalamic-pituitary-adrenal (HPA) axis. The paradigm of neonatal handling, which involves brief, daily separation of mothers and pups between birth and weaning, was shown over 40 years ago to induce a lasting reduction in the HPA responses to stress (13), an effect that is consequent to enhanced central sensitivity to glucocorticoid feedback (17, 26).

Because the sympathetic nervous system (SNS) and adrenal medulla also participate in responses to stress, the following studies were undertaken to determine whether handling of preweanling animals affected sympathoadrenal function in adulthood. Previous attempts to address this question either employed outdated experimental methods or failed to detect any difference in adult animals between those handled and not handled (10, 16). An additional motivating factor underlying these studies was the possibility that neonatal handling might affect body weight regulation. In several, although not all, studies handled animals were heavier as adults than nonhandled controls (4, 6, 13, 24). Food intake monitored over a 24-h period was also noted to be increased in adults exposed to handling as neonates (24). The following studies were undertaken, therefore, to determine whether neonatal handling altered sympathoadrenal activity when the pups reached adulthood and whether differences in food intake and body weight regulation accompanied any changes observed in sympathoadrenal function.

	METHODS

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Animals. Timed pregnant Sprague-Dawley rats were obtained from Zivic-Miller Laboratories (Zelienople, PA). Pregnant rats were housed singly in plastic cages in a room maintained at 21 ± 2°C with a 14:10-h light-dark cycle. On the day after delivery (day 0), litters were culled to 10 pups each. Neonatal handling consisted of separating mothers and pups for 15 min/day every day from day 1 until weaning at day 21; litters of nonhandled animals were left undisturbed. During handling, litters of pups were placed in plastic cages containing wood shavings within the same room as the home cage, while dams remained in the home cages. The same holding cages were used for each litter throughout the handling protocol. After weaning, all male and female rats were housed three or four per cage until 5 wk of age when they were housed two per cage before study. Animals used in this study were maintained in accordance with the guidelines and approval of the Animal Care and Use Committee of Northwestern University Medical School.

Feeding protocol. Unless otherwise specified, animals were provided free access to water and standard laboratory chow (Prolab R-M-H 3000; Agway, Syracuse, NY). In the sucrose feeding study, animals were housed in pairs and received either chow or chow plus a 10% (wt/vol) solution of sucrose. Bottles containing sucrose were replaced daily. In the lard feeding study, animals were also housed in pairs and received either a chow mash [chow:water (3:1)] or a mixture of chow and lard (ICN Nutritional Biochemicals, Cleveland, OH). In the lard-enriched diet, lard provided 50% of dietary energy. Food consumption was measured for each cage over the 4-day period from Monday to Friday, whereas sucrose consumption was measured daily during the 4-day interval. Caloric intake was calculated on the basis of digestible energy of the chow (3.7 kcal/g), sucrose (3.94 kcal/g), and lard (9 kcal/g).

Assessment of abdominal fat and body composition. At the end of each experiment, animals were killed by CO₂ inhalation. In male rats, both epididymal fat pads were removed and weighed, whereas in female rats, parametrial and retroperitoneal fat pads were removed and weighed together, as previously described (32). After removal of gastrointestinal contents, body composition was determined by chemical analysis of carcass fat, water, protein, and ash at a commercial laboratory (Convance Laboratories, Madison, WI) using standard techniques.

[³H]norepinephrine turnover procedure. L-[ring-2,5,6-³H]norepinephrine (NE; 40-60 Ci/mmol sp act; DuPont NEN Research Products, Boston, MA) was diluted with 0.9% NaCl and injected intravenously into the tail veins of unanesthetized animals in a total volume of 1.0 ml. The dose of [³H]NE used in the current study was 59 µCi/kg (~0.24 µg NE/kg). The rats were killed at preselected times by CO₂ inhalation. For each time point in the NE turnover studies, six animals were killed from each experimental group. The tissues were rapidly removed, frozen on dry ice, and stored at 20°C for later processing (usually within 2 wk). In these studies, [³H]NE turnover rates were measured in heart, interscapular brown adipose tissue (IBAT), spleen, kidney, and stomach.

Extraction and analysis of tissue catecholamines. For NE analysis, the organs were weighed and homogenized in iced 0.2 N perchloric acid in a Polytron homogenizer (Brinkmann Instruments, Westbury, NY) to extract the catecholamines. After addition of the internal standard, 3,4-dihydroxybenzylamine (DHBA; Sigma, St. Louis, MO), catecholamines were isolated from the perchloric acid extract by adsorption onto alumina (Woelm neutral; ICN Nutritional Biochemicals) in the presence of 2 M Tris buffer (pH 8.7; Sigma) containing 2% EDTA. Catecholamines were eluted from the alumina with 0.2 N perchloric acid. Aliquots of the alumina eluate were injected onto a liquid chromatographic system for catecholamine analysis following the method of Eriksson and Persson (8) with slight modification. Unless otherwise specified, all chemicals were obtained from Fisher Scientific, Fair Lawn, NJ. Aliquots of the alumina eluates were counted for [³H]NE by scintillation spectrometry in a Packard Tri-Carb 2100TR liquid scintillation analyzer (Packard Instrument, Meriden, CT). Efficiency for ³H is 58% in this system.

Urine collection. Urine was collected daily under light mineral oil in the presence of 0.5-2.0 ml 2 N HCl and 40 mg sodium metabisulfite. The internal standard, DHBA (Sigma), was added to the entire urine sample before processing, and the pH was adjusted to between 2 and 3, if necessary, before storage at 20°C. Samples were subsequently analyzed within 2-4 wk.

Determination of catecholamines in urine. Analysis of catecholamines in urine followed the method of Smedes et al. (20) as modified by Macdonald and Lake (14). The pH of the samples was adjusted to 7.5, and this mixture was then added to 30-50 mg of Analytichem Bondesil SCX (Varian Associates, Sunnyvale, CA), previously activated by exposure to 1 ml of 0.2 M NaH₂PO₄, pH 7.5, and mixed vigorously. After removal of the supernatant, the SCX was washed twice with water. Catecholamines were eluted with 1 ml of 1 M NaH₂PO₄, pH 2.9. This supernatant was transferred quantitatively into screw-topped, glass tubes. Approximately 1.0 ml of NH₄Cl-NH₄OH buffer containing 0.2% (wt/vol) diphenylboric acid ethanolamine complex (Aldrich Chemical, Milwaukee, WI) and 0.5% EDTA, 2.0 ml 2 M (NH₄)₂HPO₄, pH 9.0, and ~150 µl 5N NaOH were added to bring pH to 8.6. After addition of 2.5 ml of n-heptane containing 1% (vol/vol) octanol and 0.35% (wt/vol) tetraoctylammonium bromide (Fluka, Buchs, Switzerland), tubes were capped and shaken. The heptane supernatant was then transferred to a centrifuge tube, and 1 ml octanol and 0.2 ml of 0.4 N acetic acid containing 10-15 mg glutathione were added. After mixing and centrifugation, aliquots of the acetic acid phase were injected onto a chromatographic system for quantitation of catecholamines. In the urine assays presented here, intra-assay coefficients of variation were 2-3% and interassay coefficients of variation were 2-4% for epinephrine (Epi) and NE.

Data analysis. Data are displayed as means ± SE, unless otherwise noted. Statistical analysis of variance and covariance (ANCOVA) were performed using Data Desk 5.0 statistical software (Data Description, Ithaca, NY) (25). Post hoc, pairwise comparisons after ANOVA used Scheffé's test. In studies of NE turnover, the data were plotted semilogarithmically. The method of least squares was used to calculate the slope (k) of decline in NE specific activity over time after tracer injection (19). In all measurements of [³H]NE turnover, no significant variation in endogenous NE was observed over the 24 h of the experiment. The statistical significance of each computed regression line was assessed by ANOVA, and ANCOVA was used in comparison of fractional turnover rates. Goodness-of-fit for each regression line was evaluated by examination of externally studentized residuals. NE turnover rates were calculated as the product of the fractional turnover rate and the endogenous NE concentration; 95% confidence limits were calculated as previously described (22).

	RESULTS

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Effect of neonatal handling on [³H]NE turnover in adult male rats. To determine if neonatal handling affects SNS activity, [³H]NE turnover was measured in heart, IBAT, spleen, kidney, and stomach of 7-wk old male rats. The results of the [³H]NE turnover measurements are presented in Table 1 and Fig. 1. The impact of neonatal handling on [³H]NE turnover varied among the peripheral tissues examined. In heart, tissue NE levels did not differ between groups; fractional NE turnover, however, was slightly diminished in the handled animals (P = 0.0580), and total cardiac NE turnover was 23% lower in the handled rats (32.8 ± 4.1 vs. 42.7 ± 4.1 ng/h). In IBAT, tissue NE levels were 12% lower in handled rats (P = 0.0022), but neither fractional nor total NE turnover rates differed between groups. By contrast, in spleen both tissue NE levels and fractional NE turnover were significantly reduced in handled animals. Because of the 26% decrease in splenic NE levels (P < 0.0001) and the 35% lower fractional NE turnover (P = 0.0415), total splenic NE turnover in handled rats was less than half that observed in the nonhandled animals (24.7 ± 6.3 vs. 52.4 ± 7.8 ng/h). [³H]NE turnover was also measured in kidney and stomach in these same animals, but neither tissue NE levels nor fractional NE turnover rates differed in handled and nonhandled rats. Thus neonatal handling led to reductions in SNS activity ([³H]NE turnover) in heart and spleen but not in IBAT, stomach, or kidney compared with nonhandled controls 4 wk after weaning.

View larger version (22K): [in this window] [in a new window]   Fig. 1.   Effects of neonatal handling on [3H]norepinephrine ([3H]NE) turnover in heart, interscapular brown adipose tissue (IBAT), spleen, kidney, and stomach of 7-wk-old male rats. Data are plotted as means ± SE for specific activity of peripheral tissues from 5 or 6 animals in each group at each time point. , Nonhandled; , handled animals. t1/2, half-life.

Effect of neonatal handling on urinary catecholamine excretion in fed and fasted animals. To assess the impact of neonatal handling on adrenal medullary function, nine handled and nine nonhandled 7-wk-old male rats were placed in metabolic cages and urine was collected for catecholamine analysis over the subsequent 16 days. The results of this experiment are depicted in Fig. 2. Shown are the animals' immediate responses to placement within the metabolic cages (Fig. 2A), after 1 wk of acclimatization to the metabolism cages (Fig 2B), and the animals' responses during a 3-day fast (Fig. 2C). Throughout the study, handled animals weighed 5-6% more than nonhandled animals (P < 0.05) and lost more weight than nonhandled animals over the 3-day fast (65.2 ± 1.5 g vs. 60.7 ± 1.3, P = 0.0381). The loss of weight in relation to prefast weight, however, did not differ between groups.

View larger version (12K): [in this window] [in a new window]   Fig. 2.   Effect of neonatal handling on urinary epinephrine (Epi) excretion in 7-wk-old male rats. Urine was collected simultaneously from 9 nonhandled and 9 handled rats. Days on the abscissa refers to the number of days since entering the metabolism cages. A: initial exposure to the novel environment; B: 1 wk of acclimatization to the metabolism cages. For the first 13 days of the experiment, all rats were fed a lab chow diet ad libitum. Beginning on day 14 (C), all animals were fasted for 3 days. Rats received distilled water to drink while fed chow and 0.45% NaCl during the 3-day fast. Data are presented as means ± SE. , Nonhandled animals; , handled rats.

Effect of neonatal handling on responses to ingestion of sucrose-enriched diet. At 50 days of age, male rats from handled and nonhandled litters were divided into two groups of approximately equal weight and provided access either to chow alone or to chow plus a 10% sucrose solution to drink. Body weights over the 7-wk feeding study are illustrated in Fig. 3, and summary data in the four handling and diet groups are presented in Table 2. Body weights did not differ between handled and nonhandled animals; weight gain also was not different between handled and nonhandled animals. Although sucrose-fed animals gained slightly more weight than chow-fed controls, the differences were of borderline statistical significance (P = 0.0974). Energy intake was also slightly greater in the sucrose-fed rats (+9%, overall effect of diet, P = 0.0723), a difference that was greatest during the first 4 wk of exposure to sucrose (diet × week interaction, P < 0.0001). Despite the lack of difference between handled and nonhandled animals in weight gain or energy intake, epididymal fat pad weights were heavier in the handled rats (overall effect of handling, P = 0.0240 and 0.0037 for fat pad weight without and with adjustment for body weight, respectively). Epididymal fat pad weights were also increased in sucrose-fed animals compared with chow-fed controls. Because body weight and epididymal fat pad weights did not differ between handled and nonhandled rats at 7 wk of age in the [³H]NE turnover experiment, the group difference in epididymal fat pad weight seen here likely arose largely over the interval from 7 to 14 wk of age.

View larger version (14K): [in this window] [in a new window]   Fig. 3.   Effect of dietary sucrose on body weight of handled and nonhandled male rats. Sucrose supplementation began at 50 days of age and continued for 7 wk. Data are presented and means ± SE. Data summary and statistical analyses are presented in Table 2.

Effect of neonatal handling on responses to ingestion of lard-enriched diet. In a subsequent experiment, 60-day old male rats from handled and nonhandled litters were divided into two groups of approximately equal weight and provided ad libitum access either to chow alone or to a lard-enriched diet containing isoenergetic amounts of chow and lard. Body weights over the 8-wk feeding study are illustrated in Fig. 4, and summary data in the four handling and diet groups are presented in Table 3. Body weights and weight gain did not differ between handled and nonhandled animals. Animals fed the lard-enriched diet gained 21% more weight than chow-fed controls (477 ± 9 vs. 394 ± 14 g, P < 0.0001). Energy intake was also 11% greater in the lard-fed rats (overall effect of diet, P = 0.0060).

View larger version (15K): [in this window] [in a new window]   Fig. 4.   Effect of dietary lard on body weight of handled and nonhandled male rats. Dietary lard began at 60 days of age and continued for 8 wk. Data are presented and means ± SE. Data summary and statistical analyses are presented in Table 3. The decline in body weight during the first week of the feeding protocol coincided with the change in food presentation from standard lab blocks to powdered chow-based diets in feeding cups.

	DISCUSSION

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The findings from the present series of experiments confirm the hypothesis that neonatal handling affects sympathoadrenal function in adult animals when examined >4 wk after weaning. SNS activity, as measured by [³H]NE turnover, was lower in heart and spleen of handled animals compared with tissues from nonhandled controls, but did not differ in IBAT, kidney, or stomach in the same animals. Because the [³H]NE turnover procedure was conducted in a laboratory outside the animal facility where the animals had been housed since birth, this measurement unavoidably compared sympathetic activity in both groups during simultaneous exposure to a novel environment. Whether the differences observed during measurement of [³H]NE turnover are also obtained in a truly basal state cannot be determined at present. In contrast to tissue-specific reductions in SNS activity, adrenal medullary secretion, reflected in urinary Epi excretion, was increased in handled rats during a 3-day fast. The adrenal medullary response to a novel environment was also slightly, although not significantly, elevated in handled animals. These data indicate that neonatal handling exerts divergent effects on sympathoadrenal function, decreasing SNS activity in some tissues but not in others, and increasing adrenal medullary responsiveness to fasting.

These methods for assessing sympathoadrenal activity were chosen for several reasons. First, techniques of NE turnover assess the dynamic behavior of neuronal NE stores and provide a generally accepted index of sympathetic function in peripheral tissues of unanesthetized, unrestrained experimental animals. More rapid disappearance of tracer NE signifies increased SNS activity, whereas slower disappearance indicates decreased SNS activity. Extensive experience with these techniques in this laboratory has demonstrated the common occurrence of tissue-specific differences in NE turnover between experimental groups (3, 5, 29) and, moreover, indicated the relative independence of tissue NE turnover from alterations in adrenal medullary function (12). Second, although less sensitive than plasma catecholamine determinations in detecting acute changes in adrenal medullary secretion, catecholamine levels in urine provide an integrated assessment of adrenal medullary function when measured over an extended time period and lower the risk of stress associated with testing, as evidenced by the reduction in urinary Epi in both handled and nonhandled animals with acclimatization between days 1-3 and days 8-10.

The current results differ from the few previous attempts to assess sympathoadrenal responses in handled animals. Hucklebridge and Nowell (10) noted enhanced plasma catecholamine responses to acute stressors in mice, although sample collection after stress occurred under ether anesthesia and catecholamines in plasma were measured by bioassay. Although these limitations in experimental design were avoided by McCarty et al. (16), their assessment of sympathoadrenal activation was based solely on changes in plasma catecholamine levels before and immediately after footshock. Catecholamine levels that were high at the start rose similarly in the handled and nonhandled animals. In contrast to these earlier reports, the present studies used methods that are capable of discriminating between SNS and adrenal medullary responses and provide information regarding sympathetic outflow to individual peripheral tissues.

Although the effect of neonatal handling on the regulation of HPA activity has been characterized as reducing HPA responsiveness to stress (13), the changes noted here do not necessarily reflect diminished sympathoadrenal responses to stress. Because the pattern of SNS and adrenal medullary responses is dependent on both the character and duration of the stressor (31), sympathoadrenal responses to stress are inherently more complex than those of the HPA. In some circumstances, sympathetic outflow may be depressed while adrenal medullary secretion is increased (27, 31). Such divergence in SNS and adrenal medullary responses to stress provides a precedent for the reciprocal effects of neonatal handling on activity of sympathetic nerves and the adrenal medulla.

The implications of the observed changes in SNS activity associated with neonatal handling are potentially twofold. First, the difference in cardiac NE turnover in heart between handled and nonhandled animals may contribute to the diminished heart rate responses to handling and to novel stimulation noted previously in mice (1). The impact of handling during infancy on the subsequent development of hypertension may also reflect a handling-induced reduction in cardiac SNS activity (21). Second, because the innervation of lymphoid tissue, including spleen, plays a potentially important role in the acquisition of immune functions (15), the marked reduction in splenic SNS activity in the handled animals may be associated with a corresponding reduction in immune responses. Thus the differences noted in SNS activity between handled and nonhandled animals are likely to be associated with alterations in cardiovascular and immune function, although the nature of these effects remains to be determined.

The effects of neonatal handling on the regulation of body weight and body composition are especially noteworthy. First, these studies did not confirm the hypothesis that neonatal handling increased an animal's tendency to gain weight. Although adult body weights were, in general, slightly greater in handled rats, the handled animals did not gain more weight than nonhandled rats when fed diets enriched in either sucrose or lard. These data are in contrast to those obtained in animals exposed to a cool environmental temperature (18°C) during infancy, which gain more weight in response to dietary sucrose than animals reared at a warm temperature (30°C) (32). Second, handled rats exhibited heavier epididymal fat pads in males (Table 2) and abdominal fat depots in females than nonhandled animals, yet did not differ with respect to body fat content (Table 4). These data imply a difference between groups in body fat distribution, rather than a difference in body fat content.

Perspectives