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Research Report
Maternal factors and monoamine changes instress-resilient and susceptible mice: Cross-fostering effects
Priya Prakasha, Zul Meralib, Miroslava Kolajovaa,Beth M. Tannenbaumc, Hymie Anismana,⁎aInstitute of Neuroscience, Carleton University, Life Science Research Bldg, Ottawa, Ontario, Canada K1S 5B6bUniversity of Ottawa, Departments of Psychology and Cellular and Molecular Medicine,and University of Ottawa Institute of Mental Health Research, Ottawa, Ontario, Canada K1N 6N5cMcConnell Brain Imaging Centre, Montreal Neurological Institute, Montreal, Quebec, Canada H3A 2B4
Article history:Accepted 28 June 2006Available online 31 July 2006
Genetic factors influence stressor-provoked monoamine changes associated with anxietyand depression, but such effects might bemoderated by early life experiences. To assess thecontribution of maternal influences in determining adult brain monoamine responses to astressor, strains of mice that were either stressor-reactive or -resilient (BALB/cByJ andC57BL/6ByJ, respectively) were assessed as a function of whether they were raising theirbiological offspring or those of the other strain. As adults, offspring were assessed withrespect to stressor-provoked plasma corticosterone elevations and monoamine variationswithin discrete stressor-sensitive brain regions. BALB/cByJ mice demonstrated poorermaternal behaviors than C57BL/6ByJ dams, irrespective of the pups being raised. In responseto a noise stressor, BALB/cByJ mice exhibited higher plasma corticosterone levels andelevated monoamine turnover in several limbic and hypothalamic sites. The stressor-provoked corticosterone increase in BALB/cByJ mice was diminished among males (but notfemales) raised by a C57BL/6ByJ dam. Moreover, increased prefrontal cortical dopamineutilization was attenuated among BALB/cByJ mice raised by a C57BL/6ByJ dam. These effectswere asymmetrical as a C57BL/6ByJ mice raised by a BALB/cByJ dam did not exhibitincreased stressor reactivity. It appears that stressors influence multiple neurochemicalsystems that have been implicated in anxiety and affective disorders. Althoughmonoaminevariations were largely determined by genetic factors, maternal influences contributed tostressor-elicited neurochemical changes in some regions, particularly dopamine activationwithin the prefrontal cortex.
Paralleling several presumed neurochemical correlates ofanxiety and depression, stressors influence monoamine(norepinephrine, dopamine and serotonin) and neuropeptide
(H. Anisman).
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(e.g., corticotropin-releasing hormone) functioning in severalbrain regions (Anisman et al., 1992; Deutch et al., 1993; Maierand Watkins, 2005; Merali et al., 1998; Weiss and Simson,1989). These stressor-elicited neurochemical changes aresubject to appreciable inter-individual variability, differing
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markedly across strains of mice (Anisman and Matheson,2005; Anisman et al., 1998a; 2001; Belzung et al., 2001; Cabib etal., 1988; Griebel et al., 2000), and as a function of early lifeexperiences, particularly dam–pup interactions (Heim et al.,1997; Meaney, 2001). In light of the potential moderatingeffects of maternal behaviors on genetically influencedstressor responsivity, the present investigation assessed (a)the impact of stressors on monoamine and corticosteronechanges in strains of mice differentially reactive to stressors,and (b) whether these neurochemical changes were influ-enced by early life experiences, particularly the maternal carereceived.
Of various mouse strains examined, BALB/cByJ miceexhibit particularly pronounced stressor-provoked anxietyand depressive-like behaviors, whereas C57BL/6ByJ andC57BL/6J mice tend to be more stressor-resilient and anxiety-free (Belzung et al., 2001; Crawley et al., 1997; DeFries andHegmann, 1970; Griebel et al., 2000; Logue et al., 1997; Shanksand Anisman, 1988, 1989, 1993; Griffiths et al., 1992; Zacharkoet al., 1987, 1990). These strain-specific behavioral distur-bances are accompanied by increased release of medianeminence corticotropin-releasing hormone (CRH) and argi-nine vasopressin (AVP), secretion of adrenocorticotropin(ACTH) and corticosterone, as well as pronounced norepi-nephrine (NE), dopamine (DA) and serotonin (5-HT) alterationsin several limbic brain regions (Anisman et al., 1998a, 2001;Shanks et al., 1991, 1994a,b).
Although these data are consistent with a role forgenetically determined vulnerability to stressor-provokedneurochemical changes in anxiety/depression (Anisman andZacharko, 1989), early life experiences, as well as prenatalexperiences, may program neurochemical reactivity toenvironmental challenges, thereby affecting vulnerability tosubsequent behavioral disturbances (Francis et al., 2003;Meaney, 2001). In this regard, prolonged separation fromthe dam during the early postnatal period was associatedwith increased stressor-provoked behavioral and neuroendo-crine reactivity in adulthood (Matthews and Robbins, 2003;Plotsky and Meaney, 1993; Vazquez et al., 2003; Walker, 1995).Conversely, early life stimulation (handling or brief separa-tion from the dam followed by reunion) and high levels ofmaternal care (reflected by licking and grooming and archedback nursing) increased resistance to the neuroendocrinereactivity elicited by later stressors (Francis et al., 1999a,b;Meaney, 2001). This included a wide range of stress-relatedneurochemical processes, such as altered sensitivity ofhypothalamic paraventricular nucleus (PVN) NE receptors,elevated hippocampal glucocorticoid receptor (GC-R) expre-ssion, enhanced GC feedback sensitivity and decreasedhypothalamic CRH mRNA (Liu et al., 1997, 2000a,b; Meaneyet al., 1991, 1996). Early life stimulation was also associatedwith reduced median eminence CRH and AVP (Viau et al.,1993), possibly through 5-HT modulation of hippocampal GC-R (Mitchell et al., 1992; Smyth et al., 1994), as well as elevatedlevels of the GABAA receptors (Caldji et al., 2003, 2004; Hsu etal., 2003).
Given the profound effects of early life stimulation, it ispossible that the stressor reactivity differences betweenBALB/cByJ and C57BL/6ByJ mice, typically attributed togenetic vulnerabilities, may be moderated by maternal
factors. Indeed, BALB/cByJ mice showed inferior maternalcare relative to their C57BL/6ByJ counterparts (Anisman etal., 1998b), and BALB/cByJ pups raised by a relatively stress-resilient C57BL/6ByJ dam exhibited attenuated cognitivedisturbances as adults (Zaharia et al., 1996). However,maternal behavior alone was not a sufficient condition toalter stressor reactivity, as being raised by a BALB/cByJ damdid not create behavioral or neuroendocrine disturbances inthe hardy C57BL/6ByJ pups. As such, there is reason tosuppose that both BALB/cByJ genes and BALB/cByJ damrearing were necessary for phenotypic expression of theheightened stress reactivity (Anisman et al., 1998b). Accord-ingly, in the present study, we assessed whether BALB/cByJmice would exhibit maternal styles inferior to those ofC57BL/6ByJ mice, irrespective of whether they were fosteringtheir own pups, those of another dam of the same strain orthose of a different strain. It was further hypothesized thatthe elevated monoamine changes ordinarily elicited bystressors among stress-reactive BALB/cByJ would be attenu-ated if they had been raised by a maternally attentive(stress-resilient) C57BL/6ByJ dam. However, as the effects ofstressors may stem from the conjoint effects of genetic andexperiential factors, it was expected that fostering geneti-cally resilient mice onto a reactive dam would be less likelyto result in altered neurochemical responses elicited bystressors encountered in adulthood.
2. Results
2.1. Maternal behaviors
As depicted in Fig. 1, C57BL/6ByJ dams exhibited maternalbehaviors that were considered superior to that of BALB/cByJdams. This was the case irrespective of whether they werecaring for cross-fostered pups or their own pups. Specifi-cally, the frequency of arched back nursing was consider-ably greater among the C57BL/6ByJ mice relative to theirBALB/cByJ counterparts, F(1,36)=32.69, P<0.01. Moreover,BALB/cByJ mice were away from the nest more frequentlythan were C57BL/6ByJ mice, F(1,36)=44.21, P<0.01, and inthe pup retrieval test C57BL/6ByJ mice retrieved the pupswith shorter latencies than did BALB/cByJ dams, F(1,36)=22.32, P<0.01. Finally, the nests of C57BL/6ByJ mice werescored as superior to that of BALB/cByJ mice (3.49±0.04 vs.2.54±0.05) F(1,36)=71.33, P<0.01. Importantly, in the case ofeach of the maternal behaviors, the superior C57BL/6ByJmaternal behavior occurred irrespective of whether theywere retrieving their own pups or those pups that had beencross-fostered. The pronounced difference between thestrains with respect to maternal behaviors was particularlyevident when the overlap between distributions of thebehaviors was considered. Specifically, when the scores ofeach of the measures were summed over the repeatedsamplings, normal distributions were evident for bothstrains and there was only a subject overlap in thedistributions with regard to arched back nursing and timeaway from the nest (of 20 C57BL/6ByJ and 20 BALB/cByJdams), 3-subject overlap with respect pup retrieval latencyand no overlap in nest ratings.
Fig. 2 – Mean (±SEM) levels of plasma corticosterone (μg/dl)among male and female mice that had been raised by theirbiological mother (B×B and C×C; B=BALB/cByJ,C=C57BL/6ByJ) or had been raised by a dam of the oppositestrain (C×B and B×C; the first term represents the pup and thesecond term the dam). Mice were exposed to either acompound stressor (noise plus light restraint) or notreatment 10 min before sacrifice. *P<0.05 relative tononstressed animals of the same strain and rearingcondition. †P<0.05 relative to similarly treated BALB/cByJmice that had been raised by a dam of the same strain.
Fig. 1 – Mean (±SEM) frequency of arched-back nursing (top),frequency of being away from the nest (middle) and latency(seconds) to retrieve 3 pups (bottom) among BALB/cByJ andC57BL/6ByJ mice fostering their own pups or those of theother strain. *P<0.05 relative to BALB/cByJ mice.
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In the second study assessing maternal behaviors, it wasagain observed that the C57BL/6ByJ maternal style wassuperior to that of BALB/cByJ mice. Specifically, relative tothe BALB/cByJ moms, the C57BL/6ByJ dams exhibited morefrequent licking and grooming, arched back nursing, highernest rating and less frequent time away from the nest, F(1,62)=22.62, 101.71, 150.93, 76.57, P<0.001. Neither the main effectsnor the interactions involving the relation between the pupand dam approached significance (F's<1), indicating that thestrain differences in maternal style were unaffected by whichpups were being fostered. Data are not shown as they werehighly redundant with those of the initial study in which onlythe simple cross-fostering manipulation was conducted.
2.2. Startle response
Startle amplitude varied as a function of the Strain ofmouse×Noise intensity interaction, F(4,334)=6.91, p<0.001.At lower intensities (65–95 dB), where the magnitude of thestartle response was modest, a strain difference was notevident, but at the highest intensity (105 dB), the startleresponse among BALB/cByJ mice (M±SEM=965.57+55.57) wasgreater than among the C57BL/6ByJ mice (M±SEM=624.43±51.18). Startle amplitude did not differ as a function of the damstrain or with gender.
2.3. Plasma corticosterone
Corticosterone levels varied as a function of the interac-tions between Pup strain×Dam strain×Stressor treatment,F(1,188)=5.10, p<0.05, and the Stressor treatment×Gender,F(1,188)=19.57, p<0.01. As depicted in Fig. 2 and confirmedby follow-up comparisons, in the absence of a stressor,plasma corticosterone levels did not differ between thestrains or sexes. The stressor increased corticosteronelevels, but this outcome was significantly more pronouncedin BALB/cByJ raised by a same strain dam (B×B) than inC57BL/6ByJ mice that had been raised by a C57BL/6ByJ dam(C×C) and was more pronounced among females than amongmales. Furthermore, in C57BL/6ByJ mice, the post-stressorcorticosterone levels were unaffected by the strain of thedam. Likewise, in female BALB/cByJ mice raised by a C57BL/6ByJ dam, the corticosterone response was similar to thatseen when the dam was a BALB/cByJ. In contrast, however,
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among male BALB/cByJ mice, the fostering dam influencedthe corticosterone response to the stressor. Specifically, thestressor-provoked corticosterone rise was less marked amongmale BALB/cByJ mice raised by a C57BL/6ByJ dam than inBALB/cByJ that had been raised by a BALB/cByJ dam. In effect,the heightened stressor reactivity of male BALB/cByJ mice (butnot females) was less apparent when a stress-resilient C57BL/6ByJ dam raised them.
2.4. Brain monoamine levels and utilization
In general, the stressor treatment elicited marked brain-region-specific differences between the strains with respectto monoamine turnover. The cross-fostering manipulationhad limited effects, although the influence of the foster dam,as will be seen, had a clear effect with respect to DA variationselicited by the stressor in the PFC.
2.4.1. Paraventricular nucleus (PVN)The levels of NE within the PVN were unaffected by any of theexperimental variables, but the accumulation of the NEmetabolite MHPG varied as a function of the Pup strain×Dam×Stressor treatment interaction, F(1,181)=3.79, p=0.05.The follow-up comparisons indicated that, in the absence of astressor, MHPG in BALB/cByJ mice was lower than in C57BL/6ByJ mice. As depicted in Fig. 3, stressor exposure increasedMHPG accumulation in both strains, but this outcome wasgreater in BALB/cByJ mice. Moreover, the increased MHPG inBALB/cByJ occurred irrespective of the fostering dam. Incontrast, in C57BL/6ByJ mice raised by a similar strain dam,the stressor did not increase MHPG accumulation. Interest-ingly, among male C57BL/6ByJ mice that were raised by a
Fig. 3 – Mean (±SEM) MHPG accumulation within the PVNamong male and female mice that had been raised by theirbiological mother (B×B and C×C) or had been raised by a damof the opposite strain (C×B and B×C, where the first termrepresents the pup and the second term the dam). Mice wereexposed to either a compound stressor (noise plus lightrestraint) or no treatment 10 min before sacrifice. *P<0.05relative to nonstressed animals of the same strain andrearing condition.
BALB/cByJ dam, the stressor elicited elevated MHPG levels justas it did in BALB/cByJ mice. Among female C57BL/6ByJ miceraised by a BALB/cByJ dam, the increase of MHPG provoked bythe stressor was not significant. In effect, it seems that thelimited MHPG accumulation ordinarily associated with stres-sor exposure in C57BL/6ByJ mice was appreciably increased ifthey had been raised by a BALB/cByJ dam, but this was onlyapparent in males.
The levels of 5-HT within the PVN of C57BL/6ByJ mice weregreater than in BALB/cByJ mice (23.62±1.22 vs. 17.70±1.29 μg/mg protein, respectively) F(1,179)=10.92, P<0.01, but wereunaffected by either the dam that raised the pups or by thestressor treatment. Likewise, the levels of 5-HIAA within thePVN were higher in C57BL/6ByJ than in BALB/cByJ mice(22.72±1.91 and 16.54±1.37 μg/mg protein, respectively) F(1,178)=5.00, P<0.05, but again the metabolite level did notvary as a function of the stressor condition or the dam.
2.4.2. Locus coeruleusThe levels of NE within the locus coeruleus did not vary withthe strain, cross-fostering treatment, stressormanipulation oras a function of their interactions (data not shown). Theaccumulation of MHPG, however, varied as a function of thePup strain×Stressor treatment interaction, F(1,177)=3.89,P<0.05. The follow-up tests confirmed that the stressorincreased the accumulation of MHPG in BALB/cByJ, but hadlittle effect in C57BL/6ByJ mice (see Fig. 4). These effects wereapparent irrespective of the fostering dam.
2.4.3. Medial prefrontal cortex (mPFC)The levels of NE and 5-HT within the PFC did not vary as afunction of either the mouse strain or the cross-fosteringmanipulation, whereas the stressor treatment increased NElevels marginally, F(1,184)=3.79, P=0.05 (M±SEM=9.04±0.39and 8.00±0.35 μg/mg protein, for stressed and nonstressedmice, respectively). The accumulation of MHPG in this regionwas elevated among C57BL/6ByJ mice relative to their BALB/cByJ counterparts, F(1,176)=22.79, P<0.01, and was increasedby the stressor in both strains, F(1,176)=25.97, P<0.01. Asshown in Fig. 5 (top), this effect was not influenced by cross-fostering (F<1). The 5-HT metabolite 5-HIAA also varied as afunction of the Pup strain×Stress interaction, F(1,177)=4.41,P<0.05. The stressor produced a significant increase of 5-HIAA accumulation in BALB/cByJmice, irrespective of the damthat raised them, whereas the stressor did not affect 5-HIAAaccumulation in C57BL/6J mice (see Fig. 5, bottom).
Dopamine functioning within the PFC was sensitive to thestressor and cross-fostering manipulation as the level of DAwithin the PFC varied as a function of the Pup×Strain×Stres-sor condition interaction, F(1,179)=14.41, P<0.01. The follow-up comparisons indicated that DA levels were appreciablyhigher in BALB/cByJ than in C57BL/6ByJ mice. Levels of thisamine did not vary across stressed and nonstressed C57BL/6ByJ mice, regardless of the strain that raised them. AmongBALB/cByJ mice, in contrast, a marked reduction of DA wasapparent if they were raised by a BALB/cByJ dam, whereas nosuch reduction was evident among BALB/cByJ mice that hadbeen fostered by a C57BL/6ByJ dam (see Fig. 6, top). This profileof DA levels in response to the stressor was apparent in bothmales and females.
Fig. 5 – Mean (±SEM) MHPG (top) and 5-HIAA (bottom)accumulation within themPFC amongmale and femalemicethat had been raised by their biological mother (B×B and C×C)or had been raised by a dam of the opposite strain (C×B andB×C; the first term represents the pup and the second termthe dam). Mice were exposed to either a compound stressor(noise plus light restraint) or no treatment 10 min beforesacrifice. *P<0.05 relative to nonstressed animals of the samestrain and rearing condition.
Fig. 4 – Mean (±SEM) MHPG accumulation within the locuscoeruleus amongmale and female mice that had been raisedby their biological mother (B×B and C×C) or had been raisedby a dam of the opposite strain (C×B and B×C, where the firstterm represents the pup and the second term the dam). Micewere exposed to either a compound stressor (noise plus lightrestraint) or no treatment 10 min before sacrifice. *P<0.05relative to nonstressed animals of the same strain andrearing condition.
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The accumulation of DOPAC increased modestly, butsignificantly, in response to the Stressor treatment, F(1,179)=3.95, P<0.05. The Pup strain×Dam×Stressor treatment inter-action did not reach statistical significance. Nonetheless,given the a priori hypothesis, coupled with the DA changesthat were observed, follow-up comparisons were conducted.As shown in Fig. 6 (bottom) and confirmed by the follow-uptests, in C57BL/6ByJ mice, the accumulation of DOPAC wasmodestly increased by the stressor. In contrast, among BALB/cByJ mice raised by a same-strain dam, the accumulation ofDOPAC increased significantly in both males (54%) andfemales (66%). However, in BALB/cByJ mice raised by aC57BL/6ByJ dam, the elevated DOPAC otherwise elicited bythe stressor was absent. It is likely that the reduced DA amongBALB/cByJ mice was due to excessive DA utilization and thatthis outcome was moderated by the fostering dam.
2.4.4. Central amygdala (CeA) and hippocampusThe concentrations of NE and 5-HT within the CeA did notvary appreciably as a function of the dam. However, withinthis region, MHPG accumulation varied as a function of thePup×Stressor treatment interaction, F(1,177)=3.99, P<0.05.The follow-up comparisons confirmed, as depicted in Fig. 7,that the stressor provoked a greater increase of MHPG inBALB/cByJ (78.4%) than in C57BL/6ByJ mice (30.6%). Theaccumulation of 5-HIAA was likewise elevated in stressedmice, F(1,177)=7.39, P<0.01 (M±SEM=21.73±1.50 vs. 16.49±1.13 for stressed and nonstressed mice), but this outcomedid not interact with the strain of mouse or the gender.Within the hippocampus, the accumulation of MHPG wasgreater among stressed than nonstressed mice, F(1,177)=17.02, P<0.01 (mean±SEM–3.89± 0.22 and 2.68±0.15, respec-
tively) but did not vary with the strain or dam. Theconcentration of 5-HIAA, in contrast, was elevated inC57BL/6ByJ relative to BALB/cByJ mice, F(1,179)= 25.82,P<0 .01 (mean±SEM=6.20±0.51 and 3.99±0.42) but wasunaffected by the stressor or the dam that raised the pups.
3. Discussion
As previously observed (Anisman et al., 1998a, 2001; Shanks etal., 1991, 1994a,b), BALB/cByJ exhibited elevated stressor-provoked reactivity with respect to plasma corticosteroneconcentrations and increased utilization of NE, as reflected byMHPG accumulation within the PVN, locus coeruleus andcentral amygdala as well as 5-HT andDAutilizationwithin theprefrontal cortex. It will be recognized that, although mono-amine changes within these brain regions have been
Fig. 7 – Mean (±SEM) MHPG accumulation within the centralamygdala amongmale and female mice that had been raisedby their biological mother (B×B and C×C) or had been raisedby a dam of the opposite strain (C×B and B×C; the first termrepresents the pup and the second term the dam). Mice wereexposed to either a compound stressor (noise plus lightrestraint) or no treatment 10 min before sacrifice. *P<0.05relative to nonstressed animals of the same strain andrearing condition.
Fig. 6 – Mean (±SEM) DA (top) and DOPAC (bottom) levelswithin themPFC amongmale and femalemice that had beenraised by their biological mother (B×B and C×C) or had beenraised by a dam of the opposite strain (C×B and B×C; the firstterm represents the pup and the second term designates thedam). Mice were exposed to either a compound stressor(noise plus light restraint) or no treatment 10 min beforesacrifice. *P<0.05 relative to nonstressed animals of the samestrain and rearing condition. †P<0.05 relative to similarlytreated BALB/cByJ mice that had been raised by a dam of thesame strain.
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implicated in the mediation of anxiety (Kent et al., 2002; Leschet al., 2003; Morilak and Frazer, 2004), numerous otherneurochemical processes, across a number of brain regions,have been associated with anxiety-related responses (e.g.,GABAA subunit expression, CRH activity; (Landgraf, 2001; Roy-Byrne, 2005)). Accordingly, the intent of the present study wasnot to single out any one transmitter as being particularlygermane to reactivity/anxiety or unique to one strain. Rather,the focus of the study was to determine NE, DA and 5-HTfunctioning in stress-relevant brain regions of BALB/cByJ andC57BL/6ByJ mice, with particular attention given to potentialgender differences, and to assess whether maternal factorswould influence subsequent stressor-provoked monoaminechanges.
Commensurate with the possibility that the markedstressor reactivity and anxiety characteristic of BALB/cByJmice might be related to maternal factors, in the presentinvestigation, thematernal behaviors of BALB/cByJ and C57BL/6ByJ dams could readily be distinguished from one another. Aspreviously observed (Anisman et al., 1998b; Priebe et al., 2005),arched back nursing and licking grooming were less frequentin the BALB/cByJ than in C57BL/6ByJ mice. Moreover, BALB/cByJ nests were rated as being inferior, and in a pup retrievaltest, BALB/cByJ mice exhibited longer response latencies.Importantly, the inferior maternal behavior of BALB/cByJdams was apparent irrespective of whether they were caringfor their own pups, those fostered from another dam of thesame strain or those fostered from the alternative strain.Clearly, the act of cross-fostering pups did not alter thematernal behavior of the dams, and thus any effects onneurochemical functioning (or lack thereof) associated withcross-fostering were likely related to the maternal carereceived.
In earlier cross-fostering studies involving these strains(Zaharia et al., 1996), the poor performance of male BALB/cByJmice tested in a Morris water maze (e.g., attributable toanxiety reflected by thigmotaxis) could be attenuated if aC57BL/6ByJ dam raised them. However, among C57BL/6ByJmice raised by a BALB/cByJ dam, there was no diminution ofthe otherwise proficient performance. Thus, it was suggestedthat both BALB/cByJ genes and a poor mother were needed forthe expression of poor performance. A similar profile wasobserved in a step-down anxiety test (Caldji et al., 2004).Interestingly, in the latter study, γ2 mRNA expression in thecentral amygdala of BALB/cByJ mice was lower than in C57BL/
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6ByJ mice. Moreover, levels of γ2 mRNA expression were lowerin the biological offspring of C57BL/6ByJ mothers fostered toBALB/cByJ dams relative to peers fostered to C57BL/6ByJ dams.The converse was true of BALB/cByJ mice fostered by a C57BL/6ByJ dam. Although these findings suggested that the straindifferences in the BZ/GABAA receptor system could beinfluenced by parental care, the dissociation between the γ2mRNA expression and the behavioral effects of cross-fosteringsuggests that further biological processes subserve thebehavioral effects prompted by maternal factors in thesestrains.
It had previously been reported that cross-fosteringinfluenced basal corticosterone levels in BALB/c and C57BL/6mice, but not the response to a restraint stressor (Priebe et al.,2005). In the present investigation, cross-fostering influencedthe stressor (noise/restraint) corticosterone response in astrain- and a gender-specific fashion. Specifically, the stres-sor-elicited corticosterone increase in C57BL/6ByJ mice wasunaffected by the dam, whereas in the more corticosterone-reactive BALB/cByJ mice, the pronounced effect of the stressorwas moderated if they were fostered by a C57BL/6ByJ dam.However, this only occurred inmales, and among females, theexaggerated corticosterone response was evident irrespectiveof the fostering dam.
The mechanisms responsible for the sex-dependent effectof cross-fostering on corticosterone are uncertain. It maysimply be that the corticosterone response, being appreciablygreater in female than in male mice, was also more resistantto change by early life manipulations. Alternatively, maternalfactorsmay have preferentially influenced neuronal processesamong male pups, and indeed sex- and strain-dependenteffects of stressors have been reported with respect to severalneuronal processes (Bale et al., 2002; Jones et al., 1998).Furthermore, it has been reported that prenatal stressorsdisrupted habituation of the corticosterone response torestraint in male rats, but not in females (Bhatnagar et al.,2005). Thus, the strain- and sex-specific effects of the cross-fostering manipulation in the present investigation were notentirely unique. It is uncertain what factors might havecontributed to the sex-specific effects of cross-fostering.
The effects of cross-fostering on brain monoamine func-tioning were relatively limited in scope. As previouslyobserved (Anisman et al., 1998a,b), BALB/cByJ mice exhibitedgreater variations of NE and 5-HT activity in several brainregions (e.g., prefrontal cortex, central amygdala, locuscoeruleus, PVN); however, these effects were typically notmoderated by the dam that raised the pups. In the case of NEneuronal activity, it did appear that within the PVN theelevated MHPG accumulation wasmodest in C57BL/6ByJ mice,but among males raised by a BALB/cByJ dam the stressor wasfound to increase MHPG accumulation. The processes respon-sible for this outcome are not evident. It will be recognized,however, that the effect of the stressor on NE utilizationwithin the PVN did not parallel the corticosterone changes.Thus, it is unlikely that PVN NE activity subserved thecorticosterone effects associated with cross-fostering, despitethe known contribution of PVN NE to HPA functioning.
Consistent with reports that early life experiences influ-ence the subsequent accumbal DA response to stressors(Brake et al., 2004), in the present study, DA release within
the mPFC was affected by noise exposure, and this outcomewas influenced by the maternal dam. Specifically, within themPFC, the basal levels of DA were appreciably higher in BALB/cByJ than in C57BL/6ByJ mice, but following stressor exposure,DA levels in BALB/cByJ mice raised by a BALB/cByJ dam weremarkedly reduced. As frequently observed in response tostressors (Anisman et al., 1992; Deutch et al., 1993), noise/restraint increased DOPAC accumulation in these mice,suggesting that DA utilization exceeded synthesis leading tothe DA decline in this strain. This outcomewas entirely absentin C57BL/6ByJ mice raised by a dam of the same strain. This isnot to say that stressors cannot affect DA functioning withinthe PFC of C57BL/6ByJ mice, but only that with the relativelymodest stressor (noise plus limited restraint) used in thepresent investigation, altered DA activity was not provoked inthis strain. Indeed, in the present investigation, a modeststressor severity was specifically chosen to preclude ceilingeffects from limiting detection of differences between strainsand between potential effects of the maternal manipulation.To be sure, it is possible that with relatively severe stressorssubstantial DA neuronal changes would be evident in bothstrains, irrespective of the dam.
Of particular interest was the finding that the effect of thestressor on DA activity was tied to the fostering dam. Asobserved with respect to behavior in the Morris water-mazeand in anxiety situations (Zaharia et al., 1996; Caldji et al.,2004), the altered DA variations ordinarily elicited by thestressor among BALB/cByJ mice were absent if they had beenraised by a C57BL/6ByJ dam. This cross-fostering effect wasasymmetrical as the stressor did not affect DA functioningwithin the mPFC of C57BL/6ByJ mice that had been raised by aBALB/cByJ dam. Clearly, being raised by a more attentive damhad the effect of diminishing the DA reactivity within themPFC, but being raised by a less attentive damdid not result inexcessive DA functioning in an otherwise hardy mouse strain.
Inasmuch as DA functioning within the mPFC is particu-larly sensitive to stressors (Kaneyuki et al., 1991) and has beenimplicated in both anxiety (Finlay et al., 1995) and depression(Cummings, 1992), these data provide prima facie support forthe proposition that anxiety- and depressive-like behaviors ingenetically prone mice (Belzung et al., 2001; Crawley et al.,1997; Griebel et al., 2000; Logue et al., 1997; Shanks andAnisman, 1988, 1989) may involve DA functioning and may beinfluenced bymaternal factors. Yet, as anxiety and depressionin humans are more frequent in females than inmales, but nosuch sex difference was apparent with respect to stressor-related changes of DA functioning, in modeling anxiety anddepression in BALB/cByJmice, factors other than or in additionto mPFC DA neuronal activity need to be considered. In thisregard, the propensity for greater changes among stressedfemales was evident with respect to plasma corticosteroneand MHPG accumulation within the central amygdala.Whether these represent direct vulnerability factors fordepression or reflect a bystander effect (i.e., not being causallyrelated to pathology) is uncertain.
It is thought that early life experiences, such as dam–pupinteractions, instigate a cascade of epigenetic processes (e.g.,through DNAmethylation), culminating in altered steroidal orneuronal functioning, which may influence anxiety (Szyfet al., 2005; Weaver et al., 2004). As dam–pup interactions
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affect subsequent stressor reactivity (Meaney, 2001), thefrequently reported differences of reactivity/anxiety betweenthe inbred BALB/cByJ and C57BL/6ByJ mice could reflect, atleast in part, effects related to differences of maternalbehaviors characteristic of these strains. Of course, thematernal styles in these strains may themselves have beenmediated by genetic factors, thus affecting later behavioraloutcomes among the pups tested in adulthood. Yet, it has alsobeen reported that the maternal style of the rearing dam canbe inherited by her offspring and that these behaviors can betransmitted across generations (Champagne and Meaney,2001; Champagne et al., 2001; Fleming et al., 2002). This non-genomic transmission of maternal care could potentially bemediated by oxytocin levels in several limbic regions, estrogensensitivity in brain regions that regulate maternal behavior orneuronal functioning within the medial preoptic area and theparietal and piriform cortices (Champagne et al., 2001;Gonzalez and Fleming, 2002). Alternatively, pleiotropic effectsmay exist such that the gene(s) that influence maternalbehaviors also influence anxiety, but the maternal behaviorsare not causally related to the evolution of anxiety in theoffspring. The present findings raise the possibility that DAfunctioning within the PFC may be fundamentally affected byearly life events, ultimately affecting adult stressor reactivityand vulnerability to anxiety, although it is uncertain whetherthis comes about through epigenetic processes.
4. Experimental procedures
4.1. Subjects
The breeding colony comprised of 18–20 female and 6–8 maleBALB/cByJ and C57BL/6ByJ mice at any given time. Miceobtained from the Jackson Laboratory, Bar Harbor, Mainewere housed in standard polypropylene cages in same-sexgroups of 3–4 for 10 to 14 days after arrival at the laboratory.Mice were maintained on a 12-h light–dark cycle (lightphase: 07:00–19:00 h), with temperature (22 °C) and humidity(63%) kept constant and free access to food and water.Following this acclimatization period, 3 female mice and 1male of the same strain were housed in standard, clearplastic cages. Experimental procedures were approved by theCarleton University Animal Care Committee and wereconsistent with the guidelines set out by the CanadianCouncil on Animal Care.
4.2. Procedure
Once females were visibly pregnant, they were housedindividually (approximately Day 15 of gestation). Pups ofeach strain were raised either by their biological mother orwere cross-fostered to the other strain. Cross-fostering, whichentailed transferring whole litters, was conducted if a BALB/cByJ and C57BL/6ByJ dam gave birth within ±6 h of oneanother. An experimenter removed the dams from their homecages and placed them into temporary individual holdingcages, while the pupswere removed from the original nest andplaced into the foster dam's nest. The dams were then placedback into their original home cage, which now contained the
foster pups. Cross-fostered litters were culled so that eachcross-fostered pair had an equal number of pups. Litters bornmore than ±6 h apart were raised by their biological parent. Alllitters were culled so that notmore than 10 pups remained in alitter, and litters of less than 3 (e.g., due to cannibalism) werenot used.
Pups remained with their biological or foster mother untilthey were 22 days old, after which they were housed in groupsof 2–5 (depending on the male/female breakdown) with same-sex littermates. If only a single male or female came from agiven litter, then this pup was not subsequently used asindividual housing could potentially influence outcomes inresponse to stressors.
4.3. Maternal behaviors in BALB/cByJ and C57BL/6ByJmice
A total of 40 litters were used in the principle experiment,comprising of 7–12 litters of each of the four conditions (i.e.,pups raised by the parent strains or cross-fostered to thealternative strain), to assess maternal behaviors. As maternalbehaviors became progressively less notable with age, dam–pup interactions were limited to postnatal day (PD) 2–6. Oneach day, assessments of dam maternal behaviors wererecorded twice (between 09:00 and 10:00, and again at 13:00–14:00). During each 1-h period, a 3-min time samplingprocedure was used so that the dam–pup interaction wasmonitored and recorded for 15 s every 3 min. Maternalbehaviors, based on those previously described (Liu et al.,1997), consisted primarily of: arched back nursing (ABN),blanket nursing, passive nursing (lying on the side with noactive effort to nurse), licking and grooming, and time awayfrom nest. In a separate set of animals in which two ratersrecorded behaviors independently, better than 90% agreementwas obtained.
In addition tomonitoring the dam–pup interaction, on PD-2through PD-7, nest ratings were taken to determine whetherthe BALB/cByJ and C57BL/6ByJ mice could be distinguishedin this regard. The procedure for nest ratings, modifiedslightly from that described by Oxley and Fleming (Oxleyand Fleming, 2000), assessed nest characteristics using a4-point scale: 1=nesting material untouched or the nesthad no form; 2=nesting material in one section, flat, but nestcould be distinguished; 3=nesting material loosely gatheredinto a definite nest shape of moderate height; 4=nest wassymmetrical, tightly packed and high walls or in a sphericalshape.
Finally, a pup retrieval test was conducted on PD-6 todetermine whether dams would retrieve opposite strain pupsas readily as their own pups. To this end, 3 pupswere removedfrom the nest and placed in the three furthest corners of thecage. The latency for the dam to retrieve each pup and place itback in the nest was recorded.
In the preceding study, mice that were not cross-fosteredstayed with their biological parent. Thus, differences asso-ciated with cross-fostering vs. not-cross-fostering mice couldbe attributable to the influence of the cross-fostering proce-dure itself rather than to characteristics of the fostering dam(e.g., (Bartolomucci et al., 2004)). Accordingly, a second studywas conducted in which BALB/cByJ and C57BL/6ByJ pups
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either stayed with their own birth parent (N=8 and 17 litters,respectively), were cross-fostered to a dam of the same strain(N=11 and 13 litters for C57BL/6ByJ and BALB/6ByJ mice) orfostered to a dam of the opposite strain (N=9 and 10 litters forthe C57BL/6ByJ and BALB/cByJ dams, respectively). Maternalbehaviors were recorded throughout PD 1–6, as described inthe initial experiment.
4.4. Stressor effects on plasma corticosterone and brainmonoamine activity
The effects of the stressor treatments were assessed only inthe initial cross-fostering study. To minimize effects asso-ciated with diurnal rhythms, all procedures were conductedbetween 08:00 and 12:00 h. A total of 204 mice (N=9–15 ofeach sex in each of the fostering conditions) served assubjects. When pups reached 60–70 days of age, they wereused for the analyses of stressor effects on corticosterone andcentral monoamine turnover. Mice were randomly assignedto a stressor condition (noise while partially restrained) orwere not stressed. Ordinarily, noise, including the brief noisepulses used to elicit an acoustic startle response, potentlystimulates HPA activity (Lightman et al., 2000), includingstrain-specific elevations of plasma corticosterone (Glowaet al., 1992). Thus, in the present investigation, brief noisepulses in loosely restrained mice were used both as a stressorto determine the effects onHPA and central amine functioningand as an index of behavioral reactivity in BALB/cByJ andC57BL/6ByJ mice.
Mice were individually placed in a clear, cylindricalacrylic holder (4.4 cm×5.7 cm), which was attached to arectangular base fastened to the startle platform that washoused in a ventilated, sound-attenuating chamber(30cm×55 cm×50 cm). Owing to the small size of theacrylic holder, motor activity that might have interferedwith startle was limited. Once the animal had been securedin the holder using two removable panels placed atopposite ends of the tube, the holder was secured to thestartle platform 20 cm from the audio source. A 3-watt redfiltered light generated low levels of ambient light, andbackground white noise (45 dB) was maintained.
After a 1-min acclimation period, mice were exposed to aseries of 30 startle stimuli (from 65 to 105 dB; 50 ms induration) presented at 30 s intervals in a predeterminedrandom sequence. Given the limited movement permitted inthe acrylic holder used during noise delivery, the stressormight best be considered as a compound stressor comprisingboth noise and restraint. Nonstressed animals were handledand placed in a holding cage containing new bedding but werenot placed in the startle chamber. The presentations of stimuliwere controlled by startle reflex software (MED Associates, St.Albans, Vermont) as previously described (Anisman et al.,2001).
4.5. Blood collection, brain dissection and HPLC procedurefor analysis of brain amine and metabolite levels
Following the noise/restraint treatment, mice were returnedto their home cage and then decapitated 10 min later. Trunkblood was collected in tubes containing 10 μl of EDTA,
centrifuged and the supernatant stored at −80 °C. Plasmacorticosterone was determined, in duplicate, using a com-mercially available radioimmunoassay kit (ICN BiomedicalsInc., CA). The inter- and intra-assay variability was less than10%.
Brains were placed on a stainless steel block (immersedin ice) with slots (spaced approximately 500 μm apart) thatserved as guides for razor blades to provide a series ofcoronal brain sections. Brain sections were placed on glassslides resting on a bed of crushed ice and, following themouse brain atlas of Franklin and Paxinos (1997), micro-punches were taken from the PVN, the locus coeruleus,medial prefrontal cortex (mPFC), central amygdala (CeA)and dorsal hippocampus using hollow 16- and 20-gaugeneedles with a beveled tip. Tissue punches were placed in0.3 M monochloroacetic acid containing 10% methanol andinternal standards and stored at −80 °C for monoamineanalyses conducted by HPLC. Levels of NE and 5-HT, andtheir metabolites MHPG and 5-HIAA, were assessed in eachof the brain regions, and DA and DOPAC were alsodetermined within the mPFC. Tissue punches were soni-cated in a homogenizing solution comprising of 14.17 gmonochloroacetic acid, 0.0186 g EDTA, 5.0 ml methanol and500 ml HPLC grade water. Following centrifugation, super-natants were used for the HPLC analysis. Using an Agilent(Mississauga, Ont) pump, guard column, radial compressioncolumn (5 m, C18 reverse phase, 8 mm×10 cm) andcoulometric electrochemical detector (ESA), 40 μl of thesupernatant was passed through the system at a flow rateof 1.5 ml/min (1400–1600 PSI). Each liter of mobile phaseconsisted of sodium dihydrogen phosphate (90 mM), 1-octase sulfonic acid (sodium salt) (1.7 mM), disodiumethylenediamine tetraacetate (EDTA) (50 mM), citric acid(50 mM), potassium chloride (5 mM) and 10% acetonitrile.The mobile phase was filtered (0.22 mm filter paper) anddegassed. The area and height of the peaks were deter-mined using an Agilent integrator. Protein content of eachsample was determined using bicinchoninic acid with aprotein analysis kit (Pierce Scientific, Brockville, Ontario)and a Fluorostar colorimeter (BMG, Durham, NC). The lowerlimit of detection for the monoamines and metabolites wasapproximately 1.0 ρg.
4.6. Statistical analyses
Analyses of monoamine and metabolite levels, as well asplasma corticosterone were conducted by a 2 (Pup strain)×2(Dam)×2 (Sex)×2 (stress or no stress) analysis of variance(ANOVA). Analyses of maternal behaviors involved a mixedmeasure ANOVA comprising of Pup strain and Dam strain asbetween group factors and Days as the within group measure.Follow-up tests for significant interactions were conductedusing Bonferonni corrected t tests controlling for family-wiseerror (α=0.05).
Acknowledgments
The research was supported by the Canadian Institute ofHealth Research. HA holds a Canada Research Chair in
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Neuroscience. The assistance of Jerzy Kulczycki is gratefullyacknowledged.
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