Quantitative morphological analysis of dendritic length. A , Representative photographs of Golgi-stained CA1 pyramidal neurons accompanied by typical drawing taken from adult rats that received low (left) versus high (right) amounts of LG in infancy. Apical dendrites on top (blue) and basal dendrites at the bottom (green) are delineated by a dotted line. Yellow marks represent typical locations where spine density was assessed. Drawings are based on three-dimensional stacks of Golgi-stained cells allowing accurate distinction between the cell of interest and neighboring cells. Note that the length of dendritic trees (both basal and apical dendrites) is significantly shorter in low LG ( n ϭ 6) compared with high LG ( n ϭ 7) offspring. Magnification, 40 ϫ . Scale bars, 200 ␮ m. B , Representative photographs depicting spine density for low (left) and high (right) LG offspring. Basal dendrites at the top and apical dendrites at the bottom are delineated by a dotted line. Note that spine density is lower in offspring of low LG ( n ϭ 6) compared with high LG ( n ϭ 7) mothers. Magnification, 630 ϫ . Scale bars, 2.5 ␮ m. From each cell, two 20 ␮ m segments from the apical dendrite and one 20 ␮ m segment from basal dendrite were analyzed (see yellow marks for typical locations in A ). 

Contexts in source publication

Context 1

... predicts the risk for psy- chopathology over the lifespan (Oakley Browne et al., 1995; Kendler et al., 2002; McEwen, 2003; Nemeroff, 2004a,b; Coldwell et al., 2006). Studies in the rat suggest direct effects of maternal care on the development of neural systems that regulate cognitive, emotional, and neuroendocrine responses to stress (Meaney, 2001) and thus influence vulnerability for mood disorders. In the rat, naturally occurring variations in the frequency of pup licking and grooming (LG) provided by the dam during the first week of life are associated with individual differences in stress responsiveness, emotionality, and cognitive functioning in adult offspring. As adults, offspring of low compared with high LG mothers show increased hypothalamus-pituitary-adrenal (HPA) responses to stress (Liu et al., 1997; Weaver et al., 2004, 2005), enhanced emotionality (Caldji et al., 1998; Menard and Hakvoort, 2007), and impaired performance in tests of spatial learning and object recognition (Liu et al., 2000; Bredy et al., 2003, 2004; Toki et al., 2007). These effects are essentially reversed with cross fostering, suggesting a direct effect of maternal care (Francis et al., 1999; Caldji et al., 2003). The maternal effects on behavioral and HPA responses to stress depend on, at least in part, epigenetic programming of gene expression (Meaney and Szyf, 2005; Diorio and Meaney, 2007). However, such effects, and especially those on hippocampal- dependent forms of learning and memory, may also involve differences in synaptic plasticity. Indeed, maternal care alters the expression of synaptophysin, growth factors, and selected glutamate receptor subunits (Liu et al., 2000; Bredy et al., 2003, 2004; Toki et al., 2007). However, potential effects on the structure and function of hippocampal neurons have not been examined and are of particular relevance in light of studies suggesting parental effects on hippocampal volume in humans (Vythilingam et al., 2002; Buss et al., 2007) and of consistent reports of decreased hippocampal volume in patients with mood disorders (Sheline et al., 1996; Bremner et al., 2000). In the studies reported here, we examined the effects of variation in pup LG on the morphology of CA1 hippocampal neurons and on synaptic plasticity within this region in response to corticosteroid treatments that mimic basal versus stressful conditions (i.e., examining hippocampal function under a range of environmental conditions). Specifically, we investigated effects of maternal care on the following: (1) the morphology of CA1 neurons under resting condition, (2) LTP under low and high corticosterone (CORT) conditions ( in vitro ), (3) behavioral (learning/memory) performance in a fear conditioning task (high stress, in vivo ), and (4) levels of hippocampal mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) expression. The frequency of the maternal behaviors is listed in Table 1. In addition to differing in the percentage of LG averaged over the first week of life, dams used in the current study also differed in the percentage of flat-back nursing (a form of passive maternal care) with low LG dams displaying significantly more flat-back nursing than high LG dams ( t ϭ 2.78; df (30) , p Ͻ 0.01, unpaired t test). High LG dams displayed significantly more LG than low LG dams across all days of the observation period ( F (1,21) ϭ 145.11; p Ͻ 0.0001, two-way ANOVA, repeated measures) (Fig. 1 B ). Furthermore, the frequency of maternal LG decreased over days across both groups (uncorrected, F (5,105) ϭ 3.10, p Ͻ 0.05; H-F corrected, F (3.8,79.7) ϭ 3.10; ␧ ϭ 0.759; p Ͻ 0.05). These findings are in agreement with previous reports and support the view that, in general, low LG dams display a more passive maternal care style than high LG dams (Champagne et al., 2003; Menard and Hakvoort, 2007). Enduring effects of variations in maternal care on hippocampal development have been suggested to occur via mechanisms similar to those associated with experience-dependent neural development (for review, see Meaney, 2001). This raises the question of whether early sensory experiences such as maternal LG (a form of tactile stimulation) could stably alter dendritic morphology and/or spine density during a critical period of development. To address this question, we used Golgi staining, a sensitive method allowing accurate assessment of morphological aspects of hippocampal neurons. We report morphological alterations in the length of both basal ( t ϭ 3.056; df ϭ 11; p Ͻ 0.01, unpaired t test) and apical ( t ϭ 2.38; df ϭ 11; p Ͻ 0.05, unpaired t test) CA1 pyramidal cell dendrites that were significantly shorter in adult offspring of low versus high LG mothers (Fig. 2 A , Table 2). Spine density (as determined in 20 ␮ m segments) of apical ( t ϭ 2.88; df ϭ 11; p Ͻ 0.05, unpaired t test) and basal ( t ϭ 2.57; df ϭ 11; p Ͻ 0.05, unpaired t test) dendrites was also significantly lower in low versus high LG offspring (Fig. 2 B , Table 2). Although no significant differences were observed in the number of dendritic branches (data not shown) as a function of maternal care, a trend toward statistical significance for the number of branching points was observed between offspring of low and high LG mothers, with low LG offspring displaying a trend toward fewer branching points on apical ( t ϭ 1.97; df ϭ 11, p Ͼ 0.05, unpaired t test) but not basal ( t ϭ 0.91; df ϭ 11; p Ͼ 0.05, n.s. unpaired t test) dendrites than high LG offspring (Table 2). Previous reports showed that as early as P8, there is decreased expression of genes encoding NMDA receptor (NR) subunits (i.e., NR2A and NR2B, particularly in the CA1 subfield) in the offspring of low compared with high LG mothers (Liu et al., 2000; Bredy et al., 2004). These characteristics endure into adulthood and correlate with impaired cognitive performance in hippocampal-dependent tasks (e.g., Morris water maze, object recognition) (Liu et al., 2000; Bredy et al., 2003). Therefore, we induced tetanic stimulation of Schaffer collaterals in hippocampal slices taken from low and high LG rats, which were bathed with either VEH or CORT and recorded synaptic activity for the next 60 min afterward. We report a significant interaction between maternal care (low versus high LG) and treatment (VEH versus CORT; F (1,8) ϭ 60.31; p Ͻ 0.001, two-way ANOVA, repeated measure). Sidak post hoc comparisons revealed dramatic differences after tetanic stimulation as a function of maternal care ( p Ͻ 0.001). Although synaptic potentiation was very prominent in rats from high LG mothers, no significant synaptic potentiation was seen 60 min after tetanic stimulation (10 Hz; 900 pulses) in offspring from low LG mothers (Fig. 3). It is noteworthy that the maximal responses evoked by low-frequency stimulation were significantly lower in offspring of low LG versus high LG mothers ( t ϭ 3.59; df ϭ 17; p Ͻ 0.01, unpaired t test), whereas half-maximum stimulus intensity values were comparable between both groups (Table 3). These findings suggest alterations in pre- and post-LTP synaptic transmission properties between the adult offspring of high and low LG mothers and reinforce the suggestion that differential synaptic transmission might be re- lated to differences in morphological aspects of CA1 neurons reported above. To ascertain that the effects reported above were not attributable to different levels of circulating basal CORT and ACTH, trunk blood samples were taken at rest when animals were killed by decapitation for either Golgi analysis (cohort 1: low LG, n ϭ 6; high LG, n ϭ 7), glucocorticoid receptor system analysis (cohort 2: low LG, n ϭ 5; high LG, n ϭ 5), or electrophysiology (cohort 3: low LG, n ϭ 10; high LG, n ϭ 11). For all cohorts, we report that CORT and ACTH levels did not differ under resting condition between low and high LG offspring. All values for CORT and ACTH levels are reported in Table 4 for each individual cohort. Stress and high CORT are known modulators of hippocampal functioning during learning and memory. CORT exerts its action via binding to MR and/or GR, which are both abundantly ex- pressed in CA1 pyramidal neurons (de Kloet et al., 1998). Although GR activation mediates the inhibitory effects of high CORT on LTP, conditions of predominant MR activation (resting state) are associated with efficient induction of LTP (Diamond et al., 1992; Pavlides et al., 1996; Joels and Krugers, 2007). Differences in MR and GR protein levels between low and high LG offspring may thus affect LTP, both under resting and stress- like conditions. We measured protein levels for both MR and GR in the hippocampus of offspring of low and high LG mothers. In agreement with previous reports (Liu et al., 1997; Weaver et al., 2004, 2005), we observed lower GR protein levels in low LG [relative optical density (ROD), 32.85 Ϯ 1.64] compared with high LG adult offspring (ROD, 58.77 Ϯ 1.75; t ϭ 10.34; df ϭ 10; p Ͻ 0.001, unpaired t test) (Fig. 4 A ). We extended this finding by showing that MR levels are also lower in low LG (ROD, 17.8 Ϯ 1.48) compared with high LG adult offspring (ROD, 33.37 Ϯ 3.31; t ϭ 3.68; df ϭ 10; p Ͻ 0.01, unpaired t test) (Fig. 4 B ). However, calculation of the MR/GR ratio revealed no differences between low LG and high LG offspring (low LG, 0.54 Ϯ 0.05, vs high LG, 0.53 Ϯ 0.05). The MR/GR ratio was obtained by dividing the ROD value of MR by that of GR for each phenotype. Next, we tested whether the differential levels of GR and MR expression reported above are associated with functional effects of high CORT on CA1 neuronal excitability. We pretreated hippocampal slices from adult offspring of low and high LG mothers with stress-like levels of CORT (100 n M ) and induced LTP 1– 4 h later to allow gene-mediated GR effects to develop (Karst et al., 2000). We report a significant interaction between maternal care (low vs high LG) and treatment (VEH vs CORT; F (1,8) ϭ 60.31; p Ͻ ...

Context 2

... quality of parent– child interactions predicts the risk for psy- chopathology over the lifespan (Oakley Browne et al., 1995; Kendler et al., 2002; McEwen, 2003; Nemeroff, 2004a,b; Coldwell et al., 2006). Studies in the rat suggest direct effects of maternal care on the development of neural systems that regulate cognitive, emotional, and neuroendocrine responses to stress (Meaney, 2001) and thus influence vulnerability for mood disorders. In the rat, naturally occurring variations in the frequency of pup licking and grooming (LG) provided by the dam during the first week of life are associated with individual differences in stress responsiveness, emotionality, and cognitive functioning in adult offspring. As adults, offspring of low compared with high LG mothers show increased hypothalamus-pituitary-adrenal (HPA) responses to stress (Liu et al., 1997; Weaver et al., 2004, 2005), enhanced emotionality (Caldji et al., 1998; Menard and Hakvoort, 2007), and impaired performance in tests of spatial learning and object recognition (Liu et al., 2000; Bredy et al., 2003, 2004; Toki et al., 2007). These effects are essentially reversed with cross fostering, suggesting a direct effect of maternal care (Francis et al., 1999; Caldji et al., 2003). The maternal effects on behavioral and HPA responses to stress depend on, at least in part, epigenetic programming of gene expression (Meaney and Szyf, 2005; Diorio and Meaney, 2007). However, such effects, and especially those on hippocampal- dependent forms of learning and memory, may also involve differences in synaptic plasticity. Indeed, maternal care alters the expression of synaptophysin, growth factors, and selected glutamate receptor subunits (Liu et al., 2000; Bredy et al., 2003, 2004; Toki et al., 2007). However, potential effects on the structure and function of hippocampal neurons have not been examined and are of particular relevance in light of studies suggesting parental effects on hippocampal volume in humans (Vythilingam et al., 2002; Buss et al., 2007) and of consistent reports of decreased hippocampal volume in patients with mood disorders (Sheline et al., 1996; Bremner et al., 2000). In the studies reported here, we examined the effects of variation in pup LG on the morphology of CA1 hippocampal neurons and on synaptic plasticity within this region in response to corticosteroid treatments that mimic basal versus stressful conditions (i.e., examining hippocampal function under a range of environmental conditions). Specifically, we investigated effects of maternal care on the following: (1) the morphology of CA1 neurons under resting condition, (2) LTP under low and high corticosterone (CORT) conditions ( in vitro ), (3) behavioral (learning/memory) performance in a fear conditioning task (high stress, in vivo ), and (4) levels of hippocampal mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) expression. The frequency of the maternal behaviors is listed in Table 1. In addition to differing in the percentage of LG averaged over the first week of life, dams used in the current study also differed in the percentage of flat-back nursing (a form of passive maternal care) with low LG dams displaying significantly more flat-back nursing than high LG dams ( t ϭ 2.78; df (30) , p Ͻ 0.01, unpaired t test). High LG dams displayed significantly more LG than low LG dams across all days of the observation period ( F (1,21) ϭ 145.11; p Ͻ 0.0001, two-way ANOVA, repeated measures) (Fig. 1 B ). Furthermore, the frequency of maternal LG decreased over days across both groups (uncorrected, F (5,105) ϭ 3.10, p Ͻ 0.05; H-F corrected, F (3.8,79.7) ϭ 3.10; ␧ ϭ 0.759; p Ͻ 0.05). These findings are in agreement with previous reports and support the view that, in general, low LG dams display a more passive maternal care style than high LG dams (Champagne et al., 2003; Menard and Hakvoort, 2007). Enduring effects of variations in maternal care on hippocampal development have been suggested to occur via mechanisms similar to those associated with experience-dependent neural development (for review, see Meaney, 2001). This raises the question of whether early sensory experiences such as maternal LG (a form of tactile stimulation) could stably alter dendritic morphology and/or spine density during a critical period of development. To address this question, we used Golgi staining, a sensitive method allowing accurate assessment of morphological aspects of hippocampal neurons. We report morphological alterations in the length of both basal ( t ϭ 3.056; df ϭ 11; p Ͻ 0.01, unpaired t test) and apical ( t ϭ 2.38; df ϭ 11; p Ͻ 0.05, unpaired t test) CA1 pyramidal cell dendrites that were significantly shorter in adult offspring of low versus high LG mothers (Fig. 2 A , Table 2). Spine density (as determined in 20 ␮ m segments) of apical ( t ϭ 2.88; df ϭ 11; p Ͻ 0.05, unpaired t test) and basal ( t ϭ 2.57; df ϭ 11; p Ͻ 0.05, unpaired t test) dendrites was also significantly lower in low versus high LG offspring (Fig. 2 B , Table 2). Although no significant differences were observed in the number of dendritic branches (data not shown) as a function of maternal care, a trend toward statistical significance for the number of branching points was observed between offspring of low and high LG mothers, with low LG offspring displaying a trend toward fewer branching points on apical ( t ϭ 1.97; df ϭ 11, p Ͼ 0.05, unpaired t test) but not basal ( t ϭ 0.91; df ϭ 11; p Ͼ 0.05, n.s. unpaired t test) dendrites than high LG offspring (Table 2). Previous reports showed that as early as P8, there is decreased expression of genes encoding NMDA receptor (NR) subunits (i.e., NR2A and NR2B, particularly in the CA1 subfield) in the offspring of low compared with high LG mothers (Liu et al., 2000; Bredy et al., 2004). These characteristics endure into adulthood and correlate with impaired cognitive performance in hippocampal-dependent tasks (e.g., Morris water maze, object recognition) (Liu et al., 2000; Bredy et al., 2003). Therefore, we induced tetanic stimulation of Schaffer collaterals in hippocampal slices taken from low and high LG rats, which were bathed with either VEH or CORT and recorded synaptic activity for the next 60 min afterward. We report a significant interaction between maternal care (low versus high LG) and treatment (VEH versus CORT; F (1,8) ϭ 60.31; p Ͻ 0.001, two-way ANOVA, repeated measure). Sidak post hoc comparisons revealed dramatic differences after tetanic stimulation as a function of maternal care ( p Ͻ 0.001). Although synaptic potentiation was very prominent in rats from high LG mothers, no significant synaptic potentiation was seen 60 min after tetanic stimulation (10 Hz; 900 pulses) in offspring from low LG mothers (Fig. 3). It is noteworthy that the maximal responses evoked by low-frequency stimulation were significantly lower in offspring of low LG versus high LG mothers ( t ϭ 3.59; df ϭ 17; p Ͻ 0.01, unpaired t test), whereas half-maximum stimulus intensity values were comparable between both groups (Table 3). These findings suggest alterations in pre- and post-LTP synaptic transmission properties between the adult offspring of high and low LG mothers and reinforce the suggestion that differential synaptic transmission might be re- lated to differences in morphological aspects of CA1 neurons reported above. To ascertain that the effects reported above were not attributable to different levels of circulating basal CORT and ACTH, trunk blood samples were taken at rest when animals were killed by decapitation for either Golgi analysis (cohort 1: low LG, n ϭ 6; high LG, n ϭ 7), glucocorticoid receptor system analysis (cohort 2: low LG, n ϭ 5; high LG, n ϭ 5), or electrophysiology (cohort 3: low LG, n ϭ 10; high LG, n ϭ 11). For all cohorts, we report that CORT and ACTH levels did not differ under resting condition between low and high LG offspring. All values for CORT and ACTH levels are reported in Table 4 for each individual cohort. Stress and high CORT are known modulators of hippocampal functioning during learning and memory. CORT exerts its action via binding to MR and/or GR, which are both abundantly ex- pressed in CA1 pyramidal neurons (de Kloet et al., 1998). Although GR activation mediates the inhibitory effects of high CORT on LTP, conditions of predominant MR activation (resting state) are associated with efficient induction of LTP (Diamond et al., 1992; Pavlides et al., 1996; Joels and Krugers, 2007). Differences in MR and GR protein levels between low and high LG offspring may thus affect LTP, both under resting and stress- like conditions. We measured protein levels for both MR and GR in the hippocampus of offspring of low and high LG mothers. In agreement with previous reports (Liu et al., 1997; Weaver et al., 2004, 2005), we observed lower GR protein levels in low LG [relative optical density (ROD), 32.85 Ϯ 1.64] compared with high LG adult offspring (ROD, 58.77 Ϯ 1.75; t ϭ 10.34; df ϭ 10; p Ͻ 0.001, unpaired t test) (Fig. 4 A ). We extended this finding by showing that MR levels are also lower in low LG (ROD, 17.8 Ϯ 1.48) compared with high LG adult offspring (ROD, 33.37 Ϯ 3.31; t ϭ 3.68; df ϭ 10; p Ͻ 0.01, unpaired t test) (Fig. 4 B ). However, calculation of the MR/GR ratio revealed no differences between low LG and high LG offspring (low LG, 0.54 Ϯ 0.05, vs high LG, 0.53 Ϯ 0.05). The MR/GR ratio was obtained by dividing the ROD value of MR by that of GR for each phenotype. Next, we tested whether the differential levels of GR and MR expression reported above are associated with functional effects of high CORT on CA1 neuronal excitability. We pretreated hippocampal slices from adult offspring of low and high LG mothers with ...