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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
J Neurosci. Author manuscript; available in PMC May 6, 2012.
Published in final edited form as:
PMCID: PMC3345266
NIHMSID: NIHMS176564

Early life stress disrupts attachment learning: The role of amygdala corticosterone, locus coeruleus CRH and olfactory bulb NE

Abstract

Infant rats require maternal odor learning to guide pups proximity-seeking of the mother and nursing. Maternal odor learning occurs using a simple learning circuit including robust olfactory bulb norepinephrine (NE) release from the locus coeruleus (LC) and amygdala suppression by low corticosterone (CORT). Early life stress increases NE but also CORT and we questioned whether early life stress disrupted attachment learning and its neural correlates (2-DG autoradiography). Neonatal rats were normally-reared or stressed-reared during the first 6-days of life by providing the mother with insufficient bedding for nest building and were odor-0.5mA shock conditioned at 7-day old. Normally-reared paired pups exhibited typical odor approach learning and associated olfactory bulb enhanced 2-DG uptake. However, stressed-reared pups showed odor avoidance learning and both olfactory bulb and amygdala 2-DG uptake enhancement. Furthermore, stressed-reared pups had elevated CORT levels and systemic CORT antagonist injection reestablished the age appropriate odor preference learning, enhanced olfactory bulb and attenuated amygdala 2-DG. We also assessed the neural mechanism for stressed-reared pups' abnormal behavior in a more controlled environment by injecting normally-reared pups with CORT. This was sufficient to produce odor aversion, as well as dual amygdala and olfactory bulb enhanced 2-DG uptake. Moreover, we assessed a unique cascade of neural events for the aberrant effects of stress rearing: the amygdala-LC-olfactory bulb pathway. Intra-amygdala CORT or intra-LC corticotropin releasing hormone (CRH) infusion supported aversion learning with intra-LC CRH infusion associated with increased olfactory bulb NE (microdialysis). These results suggest that early life stress disturbs attachment behavior via a unique cascade of events (amygdala-LC-olfactory bulb).

Keywords: amygdala, locus coeruleus, norepinephrine, corticosterone, corticotropin releasing hormone, stress, memory, learning, infant rat, sensitive period, imprinting, development

Introduction

Early life stress appears to disrupt the programming of the hypothalamus-pituitary-adrenal axis (HPA), the limbic system (hippocampus, amygdala, prefrontal cortex, etc.) and the locus coeruleus (LC), all of which are considered critical mediators of early life trauma on later life compromised mental health in humans and animal models (Caldji et al., 1998; Glaser, 2000; Dent et al., 2001; Sanchez et al., 2001; Grossman et al., 2003; Teicher et al., 2003; De Bellis, 2005; Plotsky et al., 2005; Gunnar and Quevedo, 2007; Champagne et al., 2008; McEwen, 2008; Cirulli et al., 2009; Gunnar et al., 2009; Lupien et al., 2009). Furthermore, early life prolonged stress produces an exaggerated corticosterone (CORT) response, increased corticotropin releasing hormone (CRH) expression and heightened norepinephrine (NE) levels (Smith et al., 1997; Hatalski et al., 1998; Koob, 1999; Koob and Heinrichs, 1999; Vazquez et al., 2006; Korosi and Baram, 2008).

These neural targets of early stress may have particular importance because they overlap with the infant's unique attachment learning neural circuit, which is used to support attachment to the mother via learning the maternal odor (Brunjes and Alberts, 1979; Galef and Kaner, 1980; Campbell, 1984; Sullivan et al., 1986; Sullivan et al., 1990; Sullivan et al., 2000a; Moriceau and Sullivan, 2004b; Roth and Sullivan, 2005; Moriceau et al., 2006). This circuit also enables myriad stimuli (milk, stroking, 0.5mA shock) to support this odor learning required for pup survival and is characterized by robust odor approach learning and attenuated aversion learning even when the reinforcer is painful, such as 0.5mA shock, tailpinch (Haroutunian and Campbell, 1979; Sullivan and Leon, 1986; Camp and Rudy, 1988; Sullivan et al., 2000a), or abusive mother (Roth and Sullivan, 2005). The enhanced learned odor approach responses is supported by a unique infant hyperfunctioning LC that releases copious amounts of NE into the olfactory bulb to produce the olfactory bulb's learning-induced plasticity (Shipley et al., 1985; McLean and Shipley, 1991; Sullivan and Wilson, 1991; Wilson and Sullivan, 1991; Rangel and Leon, 1995; Sullivan and Wilson, 1995; Langdon et al., 1997; Moriceau and Sullivan, 2004b). It is important to mention that moderate levels of olfactory bulb NE support preference learning, while higher NE levels support aversion learning in rat pups (Sullivan et al., 1989; Sullivan et al., 1991; Yuan et al., 2000; Harley et al., 2006; Christie-Fougere et al., 2009). The attenuated odor avoidance learning is supported by suppression of amygdala due to pups' naturally low CORT and CORT is critical for early life amygdala plasticity (Sullivan et al., 2000a; Moriceau and Sullivan, 2006; Moriceau et al., 2006). Indeed, in older animals, CORT increase produces a cascade of events beginning with CORT activation of the amygdala increasing the expression of CRH mRNA into the central amygdala (Makino et al., 1994; Hatalski et al., 1998; Hsu et al., 1998; Van Bockstaele et al., 1998; Korosi and Baram, 2008), then CRH release exciting the LC to increase the release of NE (Butler et al., 1990; Makino et al., 1994; Curtis et al., 1997; Hatalski et al., 1998; Hsu et al., 1998; Lehnert et al., 1998; Van Bockstaele et al., 1998; Page and Abercrombie, 1999; Van Bockstaele et al., 2001; Bouret et al., 2003; Dunn et al., 2004; Jedema and Grace, 2004; Korosi and Baram, 2008).

Thus, there is overlap in the attachment learning circuit and the targets of early life prolonged stress and here we test whether this is important for the unique effects of stress in infancy. Specifically, we describe a functionally compromised attachment neural circuit produced by early life stress that may contribute to the enduring effects of early life stress via disrupted social behavior of pups to the mother.

Methods

Subjects

Subjects were both male and female Long Evans rat pups born and bred in our colony (originally from Harlan Lab Animals). Mothers and pups were housed in polypropylene cages (34cm width×29cm length×17cm height) lined with pine shavings and were kept in a temperature (23°C) and light (0700-1900 hr) controlled room. Food and water were available ad libitum. The day of parturition was considered postnatal day (PN) 0 and litters were culled to 12 on PN1. No more than one male and one female from a litter were used in each experimental condition and no difference in behaviors or neural activity between male and female was found. All procedures were approved by the Institutional Animal Care and Use Committee and followed NIH guidelines.

Stressed rearing

From PN1 to PN7, the dam and her pups were housed in a cage with limited nesting/bedding material, modified version of the Baram procedure (1000ml, 0.5″ layer compared to the normal 4500 ml, 2″ layer (Gilles et al., 1996; Brunson et al., 2005)). This limited bedding environment decreased the dams' ability to construct a nest, which results in frequent nest building, transporting/rough handling of pups, less licking and nursing (Table 1). No significant difference in weight between conditioning group and rearing condition was found (F(3.46) = 0.236, p < 0.8707) which replicates previous research (Gilles et al., 1996; Avishai-Eliner et al., 2001).

Table 1
Frequency of maternal behaviors observed during mother-infant interactions within the normally-reared and stressed-reared paradigm.

Maternal and pups behaviors were observed 2 times a day (day and night) each session lasting 30 min. The maternal behaviors observed were: (1) stepping/jumping on: the mother steps or jumps on the pup; (2) rough handling: the mother aggressively grooms a pup or transports a pup by a limb; (3) chase tail: the mother was running after its own tail; (4) nest building: the mother was building a nest; (5) licking: the mother was licking its pups; and (6) nursing: the mother was nursing the pups.

Systemic drug injection

PN7 pups were injected with either CORT (Corticosterone HBC complex, Sigma; 3.0 mg/kg dissolved in saline, ip), CORT antagonist (mifepristone RU 38486, Sigma; 5.0 mg/kg dissolved in saline, ip) or saline (Takahashi, 1994; Moriceau and Sullivan, 2004a) 24 hours and 30 minutes before conditioning.

Radioimmunoassay (RIA)

The levels of circulating CORT were determined from heart blood of PN7 pups following normally-rearing and stressed-rearing. Pups were anesthetized with pentobarbital and blood taken from the heart's ventricle via a thoracotomy through the diaphragm using a 23 gauge needle. All blood was collected between 12 and 2pm immediately following conditioning. Blood samples were centrifuged at 14,000 rpm for 6 min. Plasma was stored at -70°C until radioimmunoassay was performed. Duplicate plasma samples were analyzed for CORT using the Rat Corticosterone Coat-a-Countkit (Radioassay Systems Labs, In., Carson, CA). The sensitivity of the assay was 5 ng/ml. The intraassay coefficient of variation was 1-9%.

Surgery

On PN5, pups were anesthetized by inhalation (with isoflurane) and placed in an adult stereotaxic apparatus modified for use with infants. Stainless steel cannulas (30-gauge tubing) were implanted bilaterally in the LC or the amygdaloid complex through holes drilled in the overlying skull. Stereotaxic coordinates derived from the atlas of Paxinos, were used as reference and adapted through pilot work (Paxinos et al., 1991; Sullivan et al., 2000b) for implanting cannulas into the LC (caudal -1.40 mm; lateral ±0.60 mm from lambda) or into the amygdaloid complex (caudal -0.80 mm; lateral ±3.00 mm from bregma). The cannulas were lowered 5.5 mm (LC) or 5.0 mm (amygdala) from the surface of the skull. The cannulas were fixed to the skull with dental cement. To ensure patency of the cannulas, guide wires were placed in the lumen of the tubing until conditioning. Following recovery from surgery (generally within 30 min), pups were returned to the dam and littermates for a 2 days recovery period until conditioning. The survival rate of the surgery was 91%, including recovery from anesthesia and cannibalism from the mother.

Pharmacological treatment

On the day of the conditioning, bilateral cannulas were attached with PE10 tubing to a Harvard syringe pump driving two Hamilton microliter syringes. The cannulas were filled (10 sec for amygdala cannulas and 12 sec for LC cannulas at rate of 0.5 μl/min) with either drug (described below) or control. During the first 20 min conditioning period, for both pups with amygdaloid cannulas or LC cannulas, drug or control was infused at 0.1 μl/min, for a total infusion volume of 2.0 μl as previously described (Sullivan et al., 1992; Sullivan et al., 2000b; Moriceau and Sullivan, 2004b). Following conditioning, pups were disconnected from the syringe pump and returned to the nest until testing the following day.

Amygdala infusions

Pups with bilateral cannulas into the amygdala received either CORT (50 ng or 100 ng, Sigma,) or cholesterol (Sigma).

LC infusions

Pups with bilateral cannulas implanted into the LC received either CRH (50 ng, 100 ng or 200 ng, Sigma) or saline.

Odor-0.5mA shock conditioning

On PN7, pups were randomly assigned to one of the three following conditioning groups: 1) paired odor-shock, 2) unpaired odor-shock, or 3) odor only. Pups were placed in individual 600 ml clear plastic beakers, and given a 10 min adaptation period to recover from experimental handling. During a 45 min conditioning session, pups received 11 presentations of a 30-sec peppermint odor (CS) and a 1 sec 0.5mA foot shock (US; Lafayette scrambled shock generator), with an intertrial interval of 4 min. Peppermint odor was delivered by a flow dilution olfactometer (2L/min flow rate) at concentration of 1:10 peppermint vapor. Paired odor-shock pups received a shock overlapping with the last sec of the 30 sec odor presentation. Unpaired odor-shock pups received the shock and odor presentation pseudorandomly. Odor and shock were never coincident in this group. Odor only pups received only the peppermint odor presentation.

During conditioning, the number of limbs moving was recorded (0 = no movement of the extremities; 5 = movement of all 5 extremities including head) 20 sec before presentation as well as the first 20 sec of the odor presentation and the shock delivery (Hall, 1979).

Behavioral testing: Y-Maze

The day following conditioning, pups were given a Y-maze test consisting of a start box (8.5×10×8cm) and two arms (8.5×24×8cm) separated via two sliding doors. This test required pups to choose between two arms of a Plexiglas Y-maze, one containing the peppermint odor CS (25 μl of peppermint odor placed on a Kim Wipe) and the other containing the familiar odor of pine shavings (20 ml of clean shaving in a petri dish, same bedding used in home cage). During 5 sec before the door to each arm was opened, a pup was placed in the start box. Each pup was given 60-sec to choose an arm. A response was considered a choice when a pup's entire body was past the entrance to the alley. Pups received 5 trials with 30 sec between trials and the floor was wiped clean between each trial (Sullivan and Wilson, 1991). The testing was done blind to the conditioning groups and no drugs were infused during testing.

Behavioral testing: Immobility/freezing

It should be noted that pups do not show the entire spectrum of behaviors associated with freezing in the adult rat. For example, there is no piloerection and crouching position in PN7-8 pups, and immobile/freezing was defined as the cessation of body movement (Takahashi, 1994; Hunt and Campbell, 1999; Wiedenmayer and Barr, 2001; Richardson et al., 2002; Moriceau et al., 2004). Odors were presented to pups the day following conditioning. Pups were placed in individual 600 ml plastic beakers and given 5 min adaptation period to recover from experimenter handling. They were then given five odor presentations (2L/min, 1:10 odor:air ratio, ITI 4 min). The odor was presented with an olfactometer as described above (same intensity and flow rate).

Microdialysis procedure

PN5 rat pups were anesthetized by isoflurane and placed in a stereotaxic frame adapted for rat pups surgery as described previously. A cannula was placed into the right LC (used to infused CRH) and a microdialysis guide cannula (8 mm long, 500 μm diameter acrylic resin; EICOM corp, Kyoto, Japan) was positioned into the right olfactory bulb; two additional holes were drilled for two skull screws (EICOM corp, Kyoto, Japan) and both were secured to the skull with dental cement. After surgery, pups were returned to the dam and littermates for a 1-day recovery period before experimentation.

On the day of the experiment, pups were placed in a 27 cm diameter acrylic circular cage (EICOM corp., Kyoto, Japan) and were able to move freely and kept at 27°C. The guide cannula was used for insertion of the microdialysis probe (A-I-8-02, 8 mm length, 2 mm membrane, 220 μm diameter; EICOM corp., Kyoto, Japan). The probes were perfused with artificial cerebrospinal fluid (ACSF; 147mM NaCl, 2.7mM KCl, 1.2mM CaCl2, 0.85mM MgCl2) at a flow rate of 1.5 μl/min. The dead volume of the collection apparatus is 4 μl. Dialysate was collected automatically in a refrigerated (4°C) microfraction collector (EICOM corp., Kyoto, Japan; EFC-82) in which every vial contained 2 μl of 12.5mM perchloric acid/ 250 μM EDTA. After completion of the experiment, dialysate samples were immediately stored at -80°C until HPLC analysis.

NE was assessed by high-pressure liquid chromatography with electrochemical Detection (HPLC-EC). HPLC-EC consisted of a 150 × 2.1mm SC-5ODS, 5μm particle column (EICOM corp., Kyoto Japan). Mobile phase (0.1M citric acid, 0.25mM octyl sulfate sodium salt, 0.5mM EDTA, 0.085 tryethylamine, and 6% acetonitrile, pH 2.4) was delivered at 0.23 ml/min by a EICOM EP-300 pump. Neurotransmitters were detected with a graphite carbon detector electrode maintained at +0.75 V relative to an Ag/AgCl reference electrode. Neurochemical concentrations were estimated using chromatographic peak areas and calibration curves obtained with standard mixtures of known monoamine compounds. During the course of dialysate autoinjection fractions, a standard mixture was injected every fifth sample to monitor and correct calibration curves.

Assessing neural correlates within the olfactory bulb and the amygdala

PN7 pups were injected with 14C 2-deoxyglucose (2-DG; 20 μCi/100g, sc) 5 min prior to the 45 min odor-shock conditioning. Immediately following conditioning, pups were decapitated and their brains quickly removed, frozen in 2-methylbutane (-45°C) and stored in a -70°C freezer. Then, brains were sectioned (20 μm) in a -20°C cryostat, and every other section was saved to be placed on a cover slip and exposed for 5 days along with standards (14C standards 10×.02 mCi, American Radiolabeled Chemicals Inc. St Louis, MO) to x-ray film (Coopersmith and Leon, 1986; Sullivan and Wilson, 1995).

Olfactory Bulb

The olfactory bulb does not require staining since anatomical landmarks are clearly visible with 2-DG, with odors producing an odor-specific pattern of 2-DG uptake within the glomerular layer of the olfactory bulb, which was expressed relative to the bulb's periventricular core (Greer et al., 1982). These odor-specific loci, along with the periventricular core, were measured using quantitative optical densitometry with NIH image software (Coopersmith and Leon, 1986; Sullivan and Leon, 1986). To quantify 2-DG uptake, the computer constructed a calibration curve that related the gray value of 14C standards that were exposed with the brain sections to that of determined value. The autoradiographs were observed for the presence of odor-specific glomerular layer foci, which are several times above the background uptake (Figure 2E). Five readings were taken from the periventricular core and the center of the odor-specific loci. Data were analyzed as the uptake within the odor-specific loci relative to the uptake in the periventricular core (Sullivan and Wilson, 1995). Analyses were made blind to conditioning groups.

Figure 2
Odor-0.5mA shock conditioning in PN7 pups with CORT injection vs. saline injection. (A) Number of choices toward the conditioned stimulus (CS) odor during the Y-maze test (5 trials). (B-D) 14C 2-DG autoradiography during odor-0.5mA shock conditioning ...

Amygdala

Specific amygdala nuclei (central, basolateral and lateral nuclei) were identified by counterstaining sections with cresyl violet and by making a template of that brain area for use with the autoradiographs. The 2-DG uptake was expressed relative to 2-DG uptake in the corpus callosum (which did not vary among conditioning groups) to control for differences in section thickness and exposure (Figure 2F) (Sullivan et al., 2000a; Sullivan et al., 2000b). Three readings were taken from the central, lateral and basolateral nuclei of the amygdala. Data were analyzed as the uptake within the central, lateral and basolateral nuclei of the amygdala relative to the uptake in the corpus callosum (Sullivan and Wilson, 1995). Analyses were made blind to conditioning groups.

Histology and drugs spread

After behavioral testing, brains were removed and frozen in 2-methylbutane (-45°C) and stored in a -70°C freezer. For analysis, brains were sectioned (20 μm) in a -20°C cryostat and cresyl violet staining was used to verify LC and amygdaloid complex cannulas placements. Cannula tracks are shown in Figures 3D (amygdala) and 4D (LC).

Figure 3
Odor-0.5mA shock conditioning in PN7 pups with intra-amygdala CORT infusion. (A) Number of choices toward the conditioned stimulus (CS) odor in a Y-Maze test, (B) Locations of cannula tips (solid circles) for rats used for CORT infusion into the amygdala, ...

In order to characterize the extent of drug diffusion within and outside of the LC and the amygdaloid complex, additional pups were used. On PN7, pups were anesthetized by urethane and placed in a stereotaxic apparatus. For the LC, holes were drilled through the skull at 1.4 mm posterior to lambda, and ±0.60 mm from the midline. A 10 μl Hamilton syringe was lowered 5.5 mm from the surface of the skull, which placed the tip near the LC. The pups were infused with 2 μl of a saline solution of [3H]CRH (0.37 μCi/μl; NEN Research Products). For the amygdaloid complex, holes were drilled through the skull at 0.80 mm posterior to bregma, and ±3.00 mm from the midline. A 10 μl Hamilton syringe was lowered 5.0 mm from the surface of the skull, which placed the tip near the amygdaloid complex. The pups were infused with 2 μl of a saline solution of [3H]CORT (1 μCi/μl; NEN Research Products). Twenty min after infusion, the brains were quickly removed and frozen in methyl butane at -45°C. Brains were sliced in 20 μm coronal sections. The slides were apposed to a tritium storage phosphor screen (Amersham Biosciences, USA). After 14 days exposure, the screen was scanned at a pixel density of 50 μm (5000 dots per cm2) with a STORM 820 Phosphor Imager (Molecular Dynamics, Sunnyvale, Calif). Phosphorimaging of the slides results in a TIFF image file (Tucker et al., 2002; Moriceau and Sullivan, 2004b; Moriceau et al., 2006).

Statistical analysis

Comparisons were made between groups using the analysis of variance test (ANOVA) followed by post hoc Fisher tests. In all cases, the level of significance was set at p < 0.05 level.

Results

Early life stress effect on learning and neural circuit

As shown in Figure 1A, at PN7, normally-reared paired odor-0.5mA shock pups learned an odor preference, while, stressed-reared paired pups learned an avoidance. However, CORT antagonist injection in stressed-reared paired pups reinstated the learned odor preference. The Y-maze ANOVA analysis revealed a main effect of rearing condition (F(1.46) = 24.939, p < 0.0005), a significant effect of conditioning group (F(3.46) = 36.157, p < 0.0001), and a significant interaction between conditioning group and rearing condition (F(3.46) = 24.939, p < 0.0001); post hoc Fisher tests revealed that the stressed-reared paired groups differed significantly from the normally-reared paired group and the stressed-reared paired pups receiving a CORT antagonist injection. Also, the normally-reared paired group, the stressed-reared paired pups receiving a CORT antagonist injection, and the stressed-reared paired groups each differed significantly from each of the control groups at the p < 0.05 level.

Figure 1
PN7 pups stressed-reared vs. normally-reared and odor-0.5mA shock conditioning. (A) Number of choices toward the conditioned stimulus (CS) odor during the Y-maze test (5 trials), (B) Freezing to the conditioned odor presentations (Cue test, 5 odor presentations), ...

Furthermore, as illustrated in Figure 1B, stressed-reared paired pups showed freezing compared to normally-reared paired pups or stressed-reared paired pups receiving a CORT antagonist injection, which do not show freezing behavior. The freezing behavior ANOVA analysis revealed a main effect of rearing condition (F(1.32) = 66.016, p < 0.0001), a significant effect of conditioning group (F(3.32) = 81.926, p < 0.0001), and a significant interaction between conditioning group and rearing condition (F(3.32) = 66.016, p < 0.0001); post hoc Fisher tests revealed that the stressed-reared paired groups differed significantly from each of the control groups at the p < 0.05 level.

Also, Figure 1C shows that paired, unpaired and odor only pups exposed to stressed rearing had increased levels of CORT compared to normally-reared pups. ANOVA analysis for the CORT levels revealed a significant main effect of rearing condition (F(1,22) =54.060, p < 0.0005), a main effect of conditioning group (F(1,22) =4.298, p < 0.05), and a significant interaction between conditioning group and rearing condition (F(2.22) = 6.873, p < 0.005); post hoc Fisher tests revealed that the paired, unpaired and odor only stressed-reared pups were significantly different from the normally-reared groups, although the paired, unpaired and odor only stressed-reared groups were also significantly different from one another at the p < 0.05 level.

Figure 1D illustrates an increase 2-DG uptake within the glomerular layer of the olfactory bulb of pups learning either an odor aversion (stressed-reared paired pups) or an odor preference (normally-reared paired pups and stressed-reared paired pups receiving a CORT antagonist injection). No changes were observed in control groups. ANOVA revealed a main effect of conditioning group (F(3,32) = 22.171, p < 0.0001); post-hoc Fisher tests revealed that each of the paired groups were significantly different from each of the control groups.

As illustrated in Figure 1E, 1F and 1G, stressed-rearing, which results in pups learning an odor aversion rather than the age-typical odor preference, produced odor-shock induced enhance 2-DG uptake in the lateral, basolateral and central amygdala nuclei. CORT antagonist injection prevented the enhance amygdala 2-DG uptake. Specifically, central amygdala nucleus ANOVA analysis revealed a significant main effect of conditioning group (F(3,32) = 11.156, p < 0.0001), a main effect of rearing condition (F(1,32) = 6.243, p < 0.05), and a significant interaction between conditioning group and rearing condition (F(3,32) = 8.547, p < 0.0005). The basolateral amygdala nucleus ANOVA analysis revealed a significant main effect of conditioning group (F(3,32) = 7.975, p < 0.0005), a main effect of rearing condition (F(1,32) = 10.215, p < 0.005), and a significant interaction between conditioning group and rearing condition (F(3,32) = 8.358, p < 0.0005). The lateral amygdala nucleus ANOVA analysis revealed a significant main effect of conditioning group (F(3,32) = 8.827, p < 0.0005), a main effect of rearing condition (F(1,32) = 9.375, p < 0.005), and a significant interaction between conditioning group and rearing condition (F(3,32) = 9.484, p < 0.0001); post hoc Fisher tests revealed that the basolateral and the lateral nucleus of the amygdala of stressed-reared paired pups differed from each of the other groups at the p < 0.05 level.

Mimicking stressed-rearing by systemic CORT injection switches odor preference to odor aversion learning and alters the neural circuitry

We have previously demonstrated that systemic CORT injection during odor-0.5mA shock conditioning switches odor preference learning to odor aversion learning (Moriceau et al., 2006). Our goal here was to assess the effect of systemic CORT injection on the olfactory bulb and amygdala's (basolateral and lateral nuclei) participation in learning and verify if CORT injection was able to mimic the effect of early life stress.

At PN7, paired odor-shock pups injected with saline learned an odor preference (same as normally-reared pups, Exp 1). However, systemic CORT injection permitted pups to learn odor avoidance instead (similarly to stressed-reared pups, Exp 1) (Figure 2A). The Y-maze ANOVA analysis revealed a main effect of drug treatment (F(1.34) = 22.514, p < 0.0001), and a significant interaction between conditioning group and drug treatment (F(2.34) = 23.849, p < 0.0001); post hoc Fisher tests revealed that the saline-paired group and the CORT-paired groups each differed significantly from each of the control groups and also from one another at the p < 0.05 level.

Figure 2B depicted an increase 2-DG uptake within the glomerular layer of the olfactory bulb of pups learning either an odor aversion (CORT injected pups) or an odor preference (saline injected pups). No changes were observed in control groups. ANOVA revealed a main effect of conditioning group (F(5,22) = 23.507, p < 0.0001); post-hoc Fisher tests revealed that each of the paired groups were significantly different from each of the control groups.

As illustrated in Figure 2C and D, CORT injection, which causes pups to learn an odor aversion rather than the age-typical odor preference, produced odor-shock induced enhancement in basolateral and lateral amygdala nuclei 2-DG uptake. Specifically, basolateral amygdala nucleus ANOVA analysis revealed a significant main effect of conditioning group (F(2,23) = 12.597, p = 0.0005), a main effect of drugs treatment (F(1,23) = 9.086, p < 0.05), and a significant interaction between conditioning group and drug treatment (F(2,23) = 6.287, p < 0.05). The lateral amygdala nucleus ANOVA analysis revealed a significant main effect of conditioning group (F(2,23) = 12.304, p = 0.0005), a main effect of drugs treatment (F(1,23) = 7.039, p < 0.05), and a significant interaction between conditioning group and drug treatment (F(2,23) = 3.927, p < 0.05); post hoc Fisher tests revealed that the basolateral and the lateral nuclei of the amygdala of paired CORT injected pups differed from each of the other groups at the p < 0.05 level.

CORT infusion into the amygdaloid complex and CRH infusion into the LC permits odor aversion learning

Here we assess CORT action on the amygdala-LC axis during odor aversion learning with odor-shock conditioning. Specifically, we mimic the activation of the amygdala by CORT and the subsequent activation of the LC by central amygdala CRH afferents to permit the release of NE into the olfactory bulb.

As shown in Figure 3A, paired pups that received amygdala CORT infusions (50 or 100ng) during odor-shock conditioning exhibited a subsequent odor aversion. ANOVA analysis revealed a significant main effect of conditioning group (F(2.67) = 10.525, p = 0.0001), a main effect of drug treatment (F(2.67) = 24.899, p < 0.0001), and a significant interaction between conditioning group and drug treatment (F(4.67) = 17.966, p < 0.0001); post hoc Fisher tests revealed that paired pups infused with 50ng or 100ng CORT and paired pups infused with cholesterol into the amygdala each differed significantly from each of the control groups at the p < 0.05 level. Amygdala cannula tip placements are shown in Figure 3B. All tip placements were less than 1 mm from the basolateral complex of the amygdala. As demonstrated in Figure 3C, most of the drug diffusion was limited to the amygdala, most notably the lateral and central nuclei. However, some drug spread was found approximately 1mm outside the amygdala.

As shown in Figure 4A, paired pups that received 100 or 200ng CRH infused into the LC during odor-shock conditioning exhibited an odor aversion whereas 50ng CRH infusion prevented the odor-preference learning in paired pups. ANOVA analysis revealed a significant effect of drug treatment (F(3.76) = 6.182, p < 0.001), and a significant interaction between conditioning group and drug treatment (F(6,76)=10.537, p< 0.001); post hoc Fisher tests revealed that LC CRH 100 and 200ng paired pups and LC paired saline pups each differed significantly from each of the control groups at the p < 0.05 level. Cannula tip placements directed at the LC, which were all less than 1 mm from the LC, are shown in Figure 4B. Among our cannulated animals, 2 paired pups that received 100ng CRH had misplaced cannula and they did not show the aversive behaviors. As demonstrated in Figure 4C, the volume of drugs infused into the LC diffused less than 1 mm from the LC.

Figure 4
Odor-0.5mA shock conditioning in PN7 pups with intra-LC CRH infusion. (A) Number of choices toward the conditioned stimulus (CS) odor in a Y-Maze test, (B) Locations of cannula tips (solid circles) for rats used for CRH infusion into the LC, (C) Brain ...

CRH infusion into the LC increases olfactory bulb NE

Figure 5 showed that CRH infusion alone into the LC increases NE levels in the olfactory bulb while saline infusion alone does not change NE levels. ANOVA analysis for NE levels revealed a significant main effect of drug treatments (F(1,42) = 96.956, p <0.0001), a main effect of time (F(7,42) = 50.938, p < 0.0001), and a significant interaction between drug treatments and time (F(7,42) = 41.414, p < 0.0001); post hoc Fisher tests revealed that the CRH infusion differed significantly from saline infusion and from the baseline levels at the p < 0.05 level.

Figure 5
Effect of CRH infusion into the LC on the level of NE measured by microdialysis and HPLC in the OB of sensitive period pups (PN7). Asterisk represents a significant difference from all other groups (p<0.05).

No significant difference in NE metabolites were found; also no difference in basal level of NE were found between groups over the 2 hour baseline.

Discussion

Our data suggests that early life stress compromises social behavior and attachment via a unique cascade of neural events. Specifically, the attachment odor learning combined with stress produces dual activation of the attachment circuit (LC-olfactory bulb) together with the fear circuit (amygdala), which results in behavioral aversion to an odor, rather than the age appropriate approach. We suggest a neural pathway (Figure 6) to accommodate the dual activation of the attachment and fear systems. The potential pathway to support this combined fear/attachment learning circuit involves a cascade of events beginning with CORT release in response to stress, which will activate the amygdala supporting aversion/fear learning, that will excite the LC via CRH afferents from the amygdala and increases olfactory bulb NE that would typically support preference learning. Each step in this cascade is described in more detail below. Importantly, this dual activation of the attachment and fear circuit was also activated in pups reared by a stressed-mother that handled pups roughly during mother-infant interactions (i.e. mother steps on pups leaving/entering the nest). While pups within the nest do not avoid the mother and show normal weight gain, they do spend less time in contact with the mother and nursing (see table 1).

Figure 6
Schematic illustrating the normal pathway activated by odor preference learning and the pathway activated by chronic stress. Specifically, chronic stress activates the hypothalamus-pituitary-adrenal (HPA) axis to release CORT. Consequently, this blood ...

Activation of the fear circuit

During the sensitive period, pups exhibit attenuated odor aversion learning supported by the failure of the amygdala to participate in learning (Sullivan et al., 2000a; Moriceau and Sullivan, 2006; Moriceau et al., 2006). Indeed, the infant amygdala does not exhibit learning-induced plasticity, nor does temporary suppression of the amygdala influence learning until PN10. However, CORT injection permitted precocious odor aversion learning concurrently with increase amygdala 2-DG uptake (Moriceau and Sullivan, 2006; Moriceau et al., 2006). This is in sharp contrast to older pups and adult animals, where the amygdala has a critical role in fear conditioning (Cousens and Otto, 1998; Cahill et al., 1999; Doron and Ledoux, 1999; Fanselow and LeDoux, 1999; Sullivan et al., 2000a; Schafe et al., 2001; Wallace and Rosen, 2001; Fanselow and Gale, 2003; Maren, 2003; McGaugh, 2004; Moriceau and Sullivan, 2006; Moriceau et al., 2006; Sevelinges et al., 2007; Rodrigues et al., 2009). Furthermore, systemic CORT injection, direct administration of CORT into the amygdala or exposure to a psychological stressor increases the expression of CRH mRNA into the central amygdala in both adult and developing rats and increases the fear conditioned response in adult (Grino et al., 1989; Makino et al., 1994; Hatalski et al., 1998; Hsu et al., 1998; Merali et al., 1998; Shepard et al., 2000; Schmidt et al., 2004; Thompson et al., 2004; Myers et al., 2005; Vazquez et al., 2006; Korosi and Baram, 2008). While CRH is a neuropeptide displaying a broad extrahypothalamic distribution (Bittencourt and Sawchenko, 2000) and CRH interacts with noradrenergic mechanisms in the basolateral complex of the amygdala (Roozendaal et al., 2008), a CRH connection also exists between the amygdala and the LC as shown by CRH neurons from the central nucleus of the amygdala projecting directly to the rostrolateral peri-LC (Valentino et al., 1992; Lehnert et al., 1998; Van Bockstaele et al., 1998; Koob and Heinrichs, 1999; Lechner and Valentino, 1999; Van Bockstaele et al., 1999; Van Bockstaele et al., 2001; Bouret et al., 2003; Reyes et al., 2006; Valentino and Van Bockstaele, 2008). Here, we showed that during the sensitive period, early life stress (stressed-reared or CORT injected pups) permits precocious activation of the amygdala.

Activation of the attachment circuit

Sensitive Period pups exhibit enhanced odor-preference learning supported by a hyperfunctioning LC resulting in elevated levels of olfactory bulb NE. Indeed, NE from the LC is required for the olfactory bulb's neural plasticity and attachment learning. Specifically, in pups, pairing an odor with a moderate level of NE (2mg/kg) supports odor preference learning while a higher dose (4mg/kg) produces an odor aversion (Sullivan and Leon, 1986; Sullivan et al., 1989; Sullivan et al., 1991; Yuan et al., 2000; Harley et al., 2006; Christie-Fougere et al., 2009).

Attachment circuit activation presumably involves the unique neonatal LC response characteristics. Indeed, developmental differences in LC activity are reflected during both noxious (electric shock) and nonnoxious (stroking) stimulus-evoked NE release in the olfactory bulb, with neonatal pups releasing significantly more NE than older pups/adult (Kimura and Nakamura, 1987; Nakamura et al., 1987; Nakamura and Sakaguchi, 1990; McLean and Shipley, 1991; Sullivan et al., 1992; Sullivan and Wilson, 1994; Langdon et al., 1997; Sullivan et al., 2000b; Yuan et al., 2000). The mechanism for the prolonged neonatal LC response appears due to the lack of functional α2 inhibitory noradrenergic autoreceptors that terminate the LC response in older pups/adult (Nakamura et al., 1987; Pieribone et al., 1994; Scheinin et al., 1994; Winzer-Serhan et al., 1999).

In contrast, in older pups and adult, the LC has different characteristics limiting NE release that is associated with α2 inhibitiory autoreceptors functional emergence (Nakamura et al., 1987; Nakamura and Sakaguchi, 1990; Scheinin et al., 1994). This NE levels decrease is responsible for the termination of the sensitive period in pups' NE-dependent rapid odor preference learning (Moriceau and Sullivan, 2004b). Furthermore, as described above, the LC is activated via CRH afferent from the amygdala during stress in adults. Indeed, CRH antagonist application into the LC prevents fear conditioning (Bouret et al., 2003) while CRH administered locally into the adult LC is able to increase the fear response (Valentino et al., 1983; Dunn and Everitt, 1987; Butler et al., 1990; Emoto et al., 1993; Borsody and Weiss, 1996; Curtis et al., 1997; Page and Abercrombie, 1999; Bouret et al., 2003; Dunn et al., 2004; Jedema and Grace, 2004; Reyes et al., 2006; Dunn and Swiergiel, 2008). This increase in fear response is associated with a firing rate increase of the LC-NE neurons and application of CRH antagonist into the LC blocked this effect. Here, we showed that during the sensitive period, early life stress (stressed-reared or CORT injected paired pups) increases olfactory bulb 2-DG uptake. Furthermore, CRH infusion directly into the LC permits odor aversion learning associated with a large NE increase into the olfactory bulb, probably the consequence of too much NE into the bulb due to the unique neonatal LC response characteristics. Therefore, our data suggest that the 2-DG increase is not due to the activation of the attachment circuit per se but to the infant's unique hyperfunctioning LC being stimulated by CRH. However, due to the incompletely defined NE pathway between the LC, amygdala and A1/A2 noradrenergic nuclei, other potential pathways cannot be eliminated including NE feedback to the basolateral nucleus of the amygdala.

Pup behavior and neural changes: Early life stress and elevated CORT

The present results, together with results from the Baram lab (Gilles et al., 1996; Avishai-Eliner et al., 2001), suggest that elevation of CORT on pups' behavior leads to disruption of social interactions with the mother, as well as learning about the mother. These disruptions in behavior are presumably due to neural changes within the amygdala and LC (Avishai-Eliner et al., 2001; Brunson et al., 2005; Levine, 2005; Guijarro et al., 2007; Champagne et al., 2008; McEwen, 2008; Cirulli et al., 2009; Lupien et al., 2009). The present data suggests the effects of early stress on the infants' LC and amygdala have immediate consequences for pups' interactions with the mother via disruption in attachment.

As demonstrated previously, early life stress alters the developmental trajectory of myriad brain areas, including the amygdala and LC, with elevated CORT implicated as a causal factor (Caldji et al., 1998; Dent et al., 2001; Sanchez et al., 2001; Plotsky et al., 2005; Champagne et al., 2008; McEwen, 2008; Cirulli et al., 2009; Lupien et al., 2009). The maternal stress procedure used here, which is based on the insufficient nest/bedding paradigm developed in the Baram laboratory, also produces an increase in pups' CORT through maternal rough handling of pups (Gilles et al., 1996; Avishai-Eliner et al., 2001). This precocious increase in CORT is significant because it indicates that early life stress prematurely ends the stress hyporesponsive period (SHRP), which is characterized by pups' low basal CORT levels and the failure to mount the stress-induced CORT release (Rosenfeld et al., 1992; Grino et al., 1994; Levine, 2001).

Implications

Early life stress seems to manipulate the precise timing for the participation of specific brain regions in learning. Specifically, these data suggest the fear circuit (amygdala/CORT-LC/CRH) is co-activated with the attachment circuit (LC-NE-olfactory bulb). Since early life activation of LC-CRH has been linked with later life behavioral and neural deficits, these data may suggest a very specific route of activation during early life pain, which is blocked without stress. Furthermore, this attachment/fear circuit overlaps with neural correlates of compromised mental health, including depression and anxiety in humans (Heim et al., 2001; Nestler et al., 2002; Teicher et al., 2003; Gunnar et al., 2009; Lupien et al., 2009). Overall, there is remarkable convergence between this new early life stress paradigm and other paradigms and species, as well as the clinical literature (Kaufman, 1991; Kaufman et al., 1997; Caldji et al., 1998; Lehnert et al., 1998; Glaser, 2000; Dent et al., 2001; Kaufman and Charney, 2001; Sanchez et al., 2001; Grossman et al., 2003; Teicher et al., 2003; Plotsky et al., 2005; Gunnar and Quevedo, 2007; Cirulli et al., 2009).

Acknowledgments

This work was funded by grants NIH DC003906 and DC009910, NSF IOB0850527, Leon Levy Foundation, Hope for Depression Foundation to RMS.

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