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Keywords:

  • Animal model;
  • attachment;
  • infant;
  • learning;
  • maltreatment;
  • memory

Abstract

  1. Top of page
  2. Abstract
  3. Methods and materials
  4. Results
  5. Discussion
  6. References
  7. Acknowledgments

Attachment to an abusive caregiver has wide phylogenetic representation, suggesting that animal models are useful in understanding the neural basis underlying this phenomenon and subsequent behavioral outcomes. We previously developed a rat model, in which we use classical conditioning to parallel learning processes evoked during secure attachment (odor-stroke, with stroke mimicking tactile stimulation from the caregiver) or attachment despite adversity (odor-shock, with shock mimicking maltreatment). Here we extend this model to mice. We conditioned infant mice (postnatal day (PN) 7–9 or 13–14) with presentations of peppermint odor and either stroking or shock. We used 14C 2-deoxyglucose (2-DG) to assess olfactory bulb and amygdala metabolic changes following learning. PN7-9 mice learned to prefer an odor following either odor-stroke or shock conditioning, whereas odor-shock conditioning at PN13-14 resulted in aversion/fear learning. 2-DG data indicated enhanced bulbar activity in PN7-9 preference learning, whereas significant amygdala activity was present following aversion learning at PN13-14. Overall, the mouse results parallel behavioral and neural results in the rat model of attachment, and provide the foundation for the use of transgenic and knockout models to assess the impact of both genetic (biological vulnerabilities) and environmental factors (abusive) on attachment-related behaviors and behavioral development.

Attachment across mammalian species represents the processes that maintain and regulate long-term social relationships (Bowlby 1969; Hofer 2006). Infants, including rodents, display a range of adaptive behaviors to secure attachment to caregivers. Though infants exhibit evidence of prenatal predispositions for attachment, such as a preference for the odor of amniotic fluid or maternal voice (DeCasper & Fifer 1980; Marlier et al. 1998; Porter & Winberg 1999), infants readily learn associations with stimuli they encounter in the postnatal environment (Blass et al. 1984; Bushnell et al. 1989; Fifer et al. 2010; Little et al. 1984; Sullivan et al. 1991). Postnatal learning therefore has a pivotal role in facilitating infant recognition of the caregiver and ultimately ensures the infant will prefer and seek proximity of the caregiver.

Clinical data demonstrate that the lack of secure attachment to the caregiver, attributable to both environmental and biological factors, is associated with subsequent behavioral problems and psychopathologies. For example, an abused or severely neglected child forms an insecure attachment to an abusive caregiver, and lasting emotional and mental effects range from excessive anxiety and aggressive behaviors (abuse, violence), to substance abuse, mood and social disorders, and post-traumatic stress disorder (Andersen & Teicher 2009; Bos et al. 2011; Cicchetti & Toth 2005; Fisher et al. 2011; Lupien et al. 2009; Neigh et al. 2009). Furthermore, adults maltreated as children often form relationships that involve insecure attachments and abusive behavior (Riggs et al. 2011; Shah et al. 2010; Styron & Janoff-Bulman 1997).

To contribute to the understanding of the neurobiology underlying infant attachment, even in the context of aversive parenting, we previously developed a mammalian model of mother-infant attachment in rats to explore neurobehavioral processes evoked during secure attachment (odor-stroke, with stroke mimicking caregiver tactile stimulation) or attachment with abusive infant-caregiver interactions (odor-shock, with shock mimicking maltreatment). We have shown that presentations of odor and either stroking or moderate tail/foot-shock result in learned odor preferences in male and female rats younger than postnatal day (PN) 9. In older rats (PN10+) in contrast, odor-stroke conditioning fails to produce conditioned preferences and odor-shock conditioning results in learned odor aversions (Moriceau et al. 2010; Raineki et al. 2010b; Roth & Sullivan 2005; Sullivan et al. 2000). Using this model we also have shown that the paradoxical odor-shock learned preference in infancy is attributable to unique circuitry that includes cellular and physiological changes in the locus coeruleus, olfactory bulb, anterior piriform cortex, and limited amygdala participation (Moriceau et al. 2010; Raineki et al. 2010b; Roth & Sullivan 2005; Sullivan et al. 2000). As evidence of the lasting effects of early-life adversity and in a striking parallel to outcomes typically associated with abusive infant-caregiver relationships, adult rats with infant odor-shock pairings show increased depression-like behavior and altered amygdala responsivity (Sevelinges et al. 2011). These effects of early-life odor-shock treatment in infants and adults converge with odor learning data obtained using a mother maltreating the pups (Raineki et al. 2010a, 2012; Roth & Sullivan 2005).

The goal of this study is to: (1) determine the conservation of sensitive periods of conditioned responses to abusive stimuli in infant mice, which are readily amenable to genetic manipulation and (2) examine neural correlates of their learned behavior.

Methods and materials

  1. Top of page
  2. Abstract
  3. Methods and materials
  4. Results
  5. Discussion
  6. References
  7. Acknowledgments

Subjects and husbandry

Adult male and female 129S6/SvEv mice were obtained from Taconic (Germantown, NY, USA) and housed in 35 × 22 × 13 cm polypropylene cages with aspen wood shavings. We chose this strain because it is a widely used background substrain in the mouse community (i.e. knockout mice are often produced from embryonic stem cells from this substrain). The colony was kept in a temperature (20°C) and light (12 h:12d) controlled environment with food and water readily available. Males and females were paired into breeders, and males were removed from cages prior to birth. Day of parturition was termed 0 days of age, and litters were culled to a maximum of 10 infants/litter on PN1, retaining equal numbers of males and females. No more than one male and one female from a given litter were used for any training or behavioral testing condition (see figure legends for sample sizes for each experiment). All animal care and experimental procedures were approved by the Institutional Animal Care and Use Committee, which follows the guidelines from the National Institutes of Health.

Odor-stroke and -shock conditioning

Litters were assigned to receive either odor-stroke or odor-shock conditioning. Using a within litter design, one male and one female from each litter were assigned to one of three groups: Paired, Unpaired, or Odor-only. PN7-9 or PN13-14 mice were placed inside individual 400 ml beakers and given a 10 min habituation period (Fig. 1). Each conditioning period (with the three groups always run in parallel) lasted approximately 45 min with 11 conditioning trials and a 4-min intertrial interval. Subjects within the Paired group received 11 presentations of a 30 second peppermint odor that overlapped with either 30 seconds of vigorous stroking with a soft-haired paintbrush or 1 second 0.4 mA foot-shock (if feet were not accessible to manually deliver the shock, then the shock probe was applied to the tail). Subjects in the Unpaired group received stroking or shock 2 min after an odor presentation, while subjects in the Odor-only group received simply the odor presentations. For all experiments, peppermint odor was delivered with a flow-dilution olfactometer at 1 l/min and at a concentration of 1 peppermint vapor:10 clean air. Conditioning occurred in a humid, 28–33°C environment, with the lower temperature (30°C and below) used for older mice. Following conditioning, subjects were either returned to the home cage until behavioral testing the following day or brains were removed immediately for autoradiography.

image

Figure 1. Schematic representation of experimental setup and conditioning procedure. (a) Mice were placed in glass beakers with tubes for odor delivery, and tactile stimulation with a paintbrush (for odor-stroke conditioning) or moderate foot/tail-shock (for odor-shock conditioning) served as the unconditioned stimuli (US) and peppermint odor as the conditioned stimulus (CS). (b) Mice received presentations of the US and CS that either overlapped (Paired) or had a 2-min delay between their presentations (Unpaired), or received only the CS presentations (Odor-only). (c) Twenty-four hours after conditioning, the total time spent over the CS odor (out of a total of either 180 or 300 seconds) in a two-odor-choice test was calculated.

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Behavioral testing

Twenty-four hours following conditioning, mice were tested in a two-odor-choice test to assess odor preference or avoidance. The testing apparatus was a Plexiglas arena (24 × 14 cm) with a wire mesh floor. The floor was divided into two areas by a 2 cm midline: one area contained the conditioned odor (Kimwipe scented with 25 µl of peppermint extract placed in a ventilation hood for 15 min), and the other contained 100 ml of clean aspen shavings. The time spent over the conditioned odor was recorded. Subjects from odor-stroke conditioning received three, 60-second trials, with a counterbalanced starting orientation for each trial. Subjects from odor-shock conditioning were given two additional test trials for a total of five trials. The floor was cleaned between trials with distilled water. Side assignments for the odors were counterbalanced across subjects, and preference or avoidance scores were determined by summing the time spent over the conditioned odor across the trials. Prior to each testing trial, mice from the PN7-9 cohort were held in the experimenter's hand for 10 seconds to increase body temperature to aid in motor activity. The PN13-14 cohort were tested under red light, and prior to testing, were placed on a similar wire mesh floor to habituate to the substrate before entering the testing room.

Brain imaging of developmental learning

To assess brain areas activated by odor-stroke or odor-shock conditioning, a second cohort of mice were used. Five minutes prior to conditioning, mice of both age groups were injected (20 µCi/100 g, subcutaneous) with 14C 2-deoxyglucose (2-DG) (Sigma, St. Louis, MO, USA). Immediately following conditioning, subjects were decapitated and brains were removed and frozen in 2-methylbutane (−45°C) and stored in a −70°C freezer. For brain imaging, brains were sectioned (20 µm) in a −20°C cryostat, and every other section was collected on a coverslip (Fisher, Pittsburgh, PA, USA). Sections were then exposed for five days to X-ray film (Kodak, Rochester, NY, USA) along with 14C labeled standards (10 × 0.02 mCi; American Radiolabeled Chemicals Inc, St. Louis, MO, USA). We then measured 2-DG uptake within the glomerular layer of the olfactory bulb and basolateral complex of the amygdala using quantitative optical densitometry with NIH image software (with each region of interest drawn freehand) as previously reported (Sullivan et al. 2000; Sullivan & Wilson 1995). For the olfactory bulb, data were analyzed as the uptake within glomeruli relative to the uptake in the periventricular core. For the amygdala, data were analyzed as the uptake relative to the corpus callosum.

Statistical analysis

We used analysis of variance (anova) and post hoc Fisher tests to analyze differences between conditioning groups for both behavioral and brain imaging experiments. Because there were no differences noted between male and female behavior or imaging measures, data were collapsed across sexes for statistical analysis. Both differences in mean time spent over the conditioned odor (dependent variable for behavioral experiments) and differences in mean 2-DG uptake (dependent variable for brain imaging experiments) were considered significant when P < 0.05.

Results

  1. Top of page
  2. Abstract
  3. Methods and materials
  4. Results
  5. Discussion
  6. References
  7. Acknowledgments

Mice exhibit developmental differences in odor learning

A one-way anova revealed a significant group effect for infant behavior one day after odor-stroke conditioning (Fig. 2a; F2,12 = 13.4, P < 0.001). PN7-9 mice that received paired presentations of odor-stroke showed robust odor preferences during the testing period, as demonstrated by the significant amount of time they spent over the peppermint odor in the two-odor choice test relative to controls (P < 0.001 vs. Unpaired and P < 0.01 vs. Odor-only subjects). There was no significant difference in time spent over the odor between subjects in the two control conditions (P = 0.39). Likewise, there was a significant group effect for PN7-9 mice one day after odor-shock conditioning during both the three (Odor-only 33.91 ± 8.51 seconds, Unpaired 24.0 ± 8.18, Paired 68.91 ± 12.42; F2,29 = 5.58, P < 0.01) and five (Fig. 2b; F2,29 = 7.18, P < 0.01) test trial periods. Post-hoc analyses indicated that the Paired odor-shock subjects spent significantly more time over the peppermint odor (three or five trial, P < 0.01 vs. Unpaired and Odor-only subjects), whereas there was no significant difference in times between the controls (three trial, P = 0.49; five trial, P = 0.89).

image

Figure 2. Developing mice display a sensitive period of attachment-based learning. Both (a) odor-stroke and (b) odor-shock classical conditioning produced odor preferences in PN7-9 mice, as indicated by the significant mean (±SEM) time spent over the peppermint odor during a two-odor-choice test the day after conditioning. In contrast, (c) odor-stroke conditioning was no longer effective at producing odor preferences in PN13-14 mice, while (d) odor-shock conditioning produced an odor aversion. PN = postnatal day; Panel a n = 5/group, Panel b n = 10–11/group, Panel c n = 10–12/group, Panel d n = 11–14/group; *designates P-values significant vs. controls.

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In contrast to PN7-9 mice, odor-stroke conditioning in PN13-14 mice was ineffective at producing a conditioned preference. The three groups spent equal amounts of time over the peppermint in the two-odor choice test (Fig. 2c; F2,31 = 0.62, P = 0.54). Odor-shock conditioning however produced robust group differences in behavior during both the three (Fig. 2d; F2,36 = 8.2, P < 0.01) and five (Odor-only 144.21 ± 13.62 seconds, Unpaired 151.46 ± 7.0, Paired 76.5 ± 10.11; F2,36 = 14.16, P < 0.001) test trial periods. Post-hoc tests revealed that subjects with paired odor-shock presentations spent significantly less time over the peppermint odor during the behavior test in comparison to control subjects (three trial, P < 0.001 vs. Unpaired and P < 0.01 vs. Odor-only subjects; five trial, P < 0.001 vs. Unpaired and Odor-only subjects). There was no significant difference in time spent over the odor between pups in the Unpaired or Odor-only groups (three trial, P = 0.39; five trial, P = 0.66).

Developmental differences in learning parallel differences in neurobiology

We next explored underlying neural correlates of group differences in behavior of PN7-9 mice. A one-way anova revealed a significant group effect for 2-DG uptake (a marker of neural activity) within the olfactory bulb glomeruli (Fig. 3a; F2,13 = 7.2, P < 0.01) but not the basolateral amygdala complex (Fig. 3b; F2,12 = 3.0, P = 0.09) of odor-stroke conditioned PN7-9 mice. Post-hoc analysis showed that PN7-9 mice that received paired presentations of odor-stroke, a condition that again produced robust odor preferences during the testing period in our other cohort of animals, showed enhanced glomerular activity within the olfactory bulb in comparison to Unpaired and Odor-only controls (both P's < 0.01). There was no significant difference in 2-DG activity between control subjects (P = 0.84). Likewise, PN7-9 mice that received paired presentations of odor-shock, a treatment that also produced a learned odor preference, showed increased glomerular activity within the olfactory bulb (Fig. 3c; F2,12 = 15.7, P < 0.001) and no group difference in activity within the basolateral amygdala complex (Fig. 3d; F2,12 = 0.03, P = 0.98). Paired presentations of odor and shock increased 2-DG activity within the olfactory bulb in comparison to control treatments (P < 0.01 vs. Unpaired and P < 0.001 vs. Odor-only subjects), and again there was no difference in activity between control subjects (P = 0.13).

image

Figure 3. Neural correlates of sensitive period attachment-based learning. Odor-stroke and odor-shock conditioned PN7-9 mice showed increased 14C 2-deoxyglucose (2-DG) uptake in (a, c) olfactory bulb glomeruli but no significant uptake in (b, d) the basolateral amygdala complex (vs. Unpaired or Odor-only controls). PN = postnatal day; mean (±SEM) 2-DG uptake relative to 2-DG uptake in periventricular core or corpus callosum; Panel a n = 5–6/group, Panel b n = 5/group, Panel c n = 5/group, Panel d n = 5/group; *designates P-values significant vs. controls.

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Finally, we examined neural correlates of group differences in odor-shock conditioned behavior in PN13-14 mice. In PN13-14 mice, who demonstrate a learned odor aversion following odor-stroke conditioning, a one-way anova revealed no group difference in glomerular activity within the olfactory bulb (Fig. 4a; F2,13 = 0.50, P = 0.62) but a significant group difference in activity within the basolateral amygdala complex (Fig. 4b; F2,14 = 5.7, P < 0.02). Post-hoc analyses showed enhanced 2-DG levels in pups that received paired presentations of odor-shock relative to controls (P < 0.01 vs. Unpaired and P < 0.05 vs. Odor-only subjects), and no difference in levels between control subjects (P = 0.86).

image

Figure 4. A transition in learning is supported by significant amygdala activity. (a) Odor-shock conditioned PN13-14 mice who learn an aversion to the odor did not show significant 14C 2-deoxyglucose (2-DG) uptake in olfactory bulb glomeruli in comparison to controls and instead (b) showed enhanced basolateral amygdala activity compared to controls. PN = postnatal day; mean (±SEM) 2-DG uptake relative to 2-DG uptake in periventricular core or corpus callosum; Panel a n = 5–6/group, Panel b n = 5–7/group; *designates P-values significant vs. controls.

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Discussion

  1. Top of page
  2. Abstract
  3. Methods and materials
  4. Results
  5. Discussion
  6. References
  7. Acknowledgments

To contribute to the understanding of the neurobiological mechanisms responsible for infant attachment despite aversive parenting, we previously developed a rat model in which we use an olfactory-based classical conditioning paradigm to parallel learning processes evoked during abusive or nurturing infant-caregiver interactions. In the current study we tested whether this controlled experimental model was applicable to infant mice of a 129 substrain. We show that odor-stroke conditioning, which mimics positive tactile stimulation from a caregiver, produces conditioned odor preferences in infant mice within the first postnatal week of development (our PN7-9 group). We also show that odor-shock conditioning, with shock mimicking maltreatment, likewise produces strong odor preferences in same-aged infant mice. Association of odor and stroke or odor and shock produces enhanced neural activity within the olfactory bulb, and these training paradigms do not differentially affect amygdala activity. This is the same neural pathway we have shown that infant rats use to learn the natural maternal odor (Raineki et al. 2010a; Roth & Sullivan 2005).

Conditioning experiments in PN13-14 mice illustrate that, just like in rats, there is a sensitive period of learning. We show that odor-stroke conditioning becomes ineffective at producing learned odor preferences in mice within the second postnatal week of development, a phenomenon we and others have shown in post-sensitive period rats that coincides with the lack of significant bulbar activity (Sullivan & Wilson 1994; Woo & Leon 1987). We also show here that odor-shock conditioning switches from producing an odor preference to producing a strong aversion to the odor and that association of odor and shock in PN13-14 mice produces enhanced neural activity within the amygdala (in comparison to controls). Though our handling procedures during testing did differ between the two age groups, it is unlikely that this was a factor in the behavioral and neural differences, as the results presented here parallel those we and others have previously reported for rats where the handling procedures prior to testing did not differ for the two age groups (Camp & Rudy 1988; Raineki et al. 2010a; Sullivan et al. 2000).

Though previous work has shown that infant mice display preferences for maternal and homecage odors (Logan et al. 2012; Thomas et al. 2010) and are able to form conditioned memories (Akers et al. 2012; Alleva & Calamandrei 1986; Bollen et al. 2012; Bouslama et al. 2005; Drake et al. 2011; Durand et al. 2003; Nagy et al. 1972; Wiedenmayer et al. 2000), to our knowledge, our data here are the first to show preference learning in infant mice despite an aversive conditioning paradigm, and thus are first to illustrate a sensitive period for attachment-based learning. Similar to developing rats, infant mice display a stress hyporesponsive period (Cirulli et al. 1994; D'Amato et al. 1992; Schmidt et al. 2003). Specifically, from birth until around PN12 mice exhibit a blunted stress response to a mild stressor, while after PN12 mice display an adult-typical response with significant elevations of ACTH hormone and corticosterone. Our prior work in developing rats has shown that the transition in learning (from a preference to an aversion) is corticosterone and amygdala dependent (Barr et al. 2009; Moriceau et al. 2006). The 2-DG data here suggest that a similar mechanism underlies the transition in learning in this substrain of mice.

In rats, although the learned odor preference is beneficial in enhancing interaction with the mother, there are detrimental effects later in life at both the neural and behavioral levels. For example, adolescents exposed to paired presentations of odor and shock in infancy show deficits in social behavior and enhanced amygdala activation in response to a stress challenge (Raineki et al. 2012). Adults who had been exposed to paired presentations of odor-shock during infancy show increased depression-like behavior in the Forced Swim Test and a sucrose preference test and show a deficit in paired-pulse inhibition in the amygdala and piriform (olfactory) cortex (Sevelinges et al. 2011). Together, behavioral outcomes inducible with this model are in striking parallel to those of early-life stress and disorganized attachment reported in the clinical literature.

Reports continue to characterize underlying biological vulnerabilities that appear to contribute to disrupted attachment, and highlight the exquisite interplay between genotype and environment in determining subsequent mental health. For example, functional polymorphisms in the mu-opioid receptor influence the development of attachment behavior in rhesus macaques (Barr et al. 2008), and mouse pups lacking the mu-opioid receptor show deficits in attachment behavior (Moles et al. 2004). Exposure to early-life stress and increased vulnerability to post-traumatic stress disorder and other mood and anxiety disorders have been linked to polymorphisms within the gene encoding for the corticotropin-releasing hormone receptor, CRHR1, and a gene important in the regulation of glucocorticoid sensitivity, FKBP5 (Gillespie et al. 2009). Emotional problems are more likely in individuals who experienced stressful life-events and have one or two copies of the short allele of the serotonin transporter gene, 5-HTT (Caspi et al. 2003; Kumsta et al. 2010). Finally, more recent work indicates that there is an interaction between early-life stress, brain-derived neurotrophic factor (bdnf) genotype, and neurobehavioral outcome, as individuals that were institutionalized and carriers of the risk Met allele were found to have alterations in amygdala and hippocampal volume, anxiety, and depression (Casey et al. 2009; Gatt et al. 2009). As reports continue to characterize underlying genetic vulnerabilities that contribute to outcomes associated with early-life adversity, it stands to reason that use of additional background strains and genetically modified mice with the model we introduce here can yield substantial information regarding heritable factors that influence secure vs. disorganized attachment and resilience vs. vulnerability to psychopathology. Our model is also ideally suited to study neural characteristics of attachment learning under varied conditions.

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  1. Top of page
  2. Abstract
  3. Methods and materials
  4. Results
  5. Discussion
  6. References
  7. Acknowledgments
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Acknowledgments

  1. Top of page
  2. Abstract
  3. Methods and materials
  4. Results
  5. Discussion
  6. References
  7. Acknowledgments

This work was funded by grant NSF IOF-0850527 to RMS, CAPES to CR, startup funds from the University of Delaware to TLR, and RP was supported by T32MH096331.