Developmental lead exposure impairs extinction of conditioned fear in young adult rats

Abstract

Pavlovian fear conditioning is a model of emotional learning in which a neutral stimulus such as a tone is paired with an aversive stimulus such as a foot shock. Presentation of a tone with a foot shock in a context (test box) elicits a freezing response representative of stereotypic fear behavior. After conditioning has occurred, presentation of the context (test box) or tone in the absence of the unconditioned stimulus (shock) causes extinction of the fear response. Rats chronically exposed to environmentally relevant levels of lead (Pb²⁺) and controls were tested in a fear-conditioning (FC) paradigm at 50 days of age (PN50). Littermates to FC rats received an immediate shock (IS) when place in the test box with no tone. Blood Pb²⁺ levels in control and Pb²⁺-exposed animals were (mean ± sem): 0.76 ± 0.11 (n=15) and 25.8 ± 1.28 μg/dL (n=14). Freezing behavior was recorded during acquisition (day of training) or during 4 consecutive extinction days. Control and Pb²⁺-exposed FC rats exhibited the same level of freezing time on the acquisition day. No freezing behavior occurred in IS rats regardless of treatment. Presentation of context 24 hrs later produced a freezing response on both control and Pb²⁺-exposed FC rats but not in IS rats. When tested in the extinction phase, Pb²⁺-exposed FC rats exhibited deficits in extinction compared to control FC rats. That is, when presented with context on 4 consecutive days after acquisition of the fear response, Pb²⁺-exposed FC rats exhibited a greater freezing response than control FC rats. These findings indicate that chronic Pb²⁺ exposure produces a deficit in extinction learning and the animals remain more fearful than controls.

Introduction

Plasticity of the central nervous system is essential for the acquisition and consolidation of new information. These processes are thought to be dependent upon strengthening of synaptic connections and the N-methyl-d-aspartate subtype (NMDAR) of excitatory amino acid receptors has been shown to play a key role in synaptic plasticity in the mammalian brain (Malenka and Nicoll, 1993). For example, synaptic plasticity in the form of long-term potentiation (LTP) is thought to represent a cellular model of learning and memory and NMDAR activation is essential to this cellular phenomenon (Zalutsky and Nicoll, 1990; Bliss and Collingridge, 1993; Malenka and Nicoll, 1993, 1999).

Previous work from several laboratories has shown that lead (Pb²⁺) is a potent inhibitor of the NMDAR (Alkondon et al., 1990; Guilarte and Micelli, 1992; Omelchenko et al., 1996; Marchetti and Gavazzo, 2005). Exposure to Pb²⁺ during brain development impairs spatial learning and LTP in the hippocampus (Altman et al., 1993; Gilbert et al., 1996; Ruan et al., 1998; Nihei et al., 2000), both of which are dependent upon NMDAR activation. Animals exposed to Pb²⁺ during development exhibit alterations in the expression of NMDAR subunits in the hippocampus (Guilarte and McGlothan, 1998; Nihei and Guilarte, 1999; Guilarte et al., 2000; Zhang et al., 2002), altering their subunit composition and coupling to signaling transduction pathways (Toscano et al., 2002; 2003). We have proposed that the deficits in spatial learning and LTP documented in Pb²⁺-exposed rats are mediated by Pb²⁺ inhibition of NMDAR and subsequent changes in their subunit composition and signaling (Toscano and Guilarte, 2005). To further study the effects of Pb²⁺ on associative learning, we examined the effect of developmental Pb²⁺ exposure on fear conditioning in young adult rats.

Fear conditioning is a model of emotional learning in which a neutral conditioned stimulus (CS) such as a tone or the context in which the animal was present (test box) when paired with an aversive unconditioned stimulus (US) such as a foot shock elicits a freezing response (or conditioned response) representative of stereotypic fear behavior. The conditioned fear response diminishes if the condition stimulus or the context is presented in the absence of a shock and this process is known as “Extinction”. Extinction of conditioned fear is thought to involve the formation of a “new memory” rather than the erasure of the fear conditioning (Quirk, 2002; Bouton et al., 2006; Myers and Davis, 2007). This is supported by evidence, as with many types of learning, that extinction requires activation of NMDAR (Falls et al., 1992; Baker and Azorlosa, 1996; Santini et al., 2001; Walker et al., 2002) and a switch from long-term depression (LTD) to LTP (Herry and Garcia, 2002; Herry and Mons, 2004). Based on this information, we tested the hypothesis that extinction of fear conditioning may be altered in young adult rats that were exposed to environmentally relevant levels of Pb²⁺ during gestation and postnatally.

Materials and Methods

Animal Husbandry

Adult female Long-Evans rats were purchased from Charles River, Inc. (Wilmington, MA) and fed 0 (control) or 1500 ppm lead acetate (PbAc) in the diet (Dyets, Bethlehem, PA). The level of Pb²⁺ in each diet was confirmed by analysis from an independent laboratory (ESA Laboratories, Inc., Chelmsford, MA). Female rats were bred to normal Long-Evans males following an acclimation period of 10 days. Litters were culled to 10 on postnatal day 1 (PN1). Pups were weaned on PN21 and fed the same diet as their respective dams. Rats were individually housed in plastic cages at 22 ± 2°C on a 12/12 light:dark cycle, and food and water were allowed ad libitum. At PN50, two male rats from each control and Pb²⁺-exposed litter were randomly assigned to two behavioral experimental groups: fear conditioning (FC) or immediate shock (IS). Blood Pb²⁺ analysis was conducted using the LeadCare system (ESA Laboratories, Inc, Chelmsford, MA). Litters of rats were considered a single experimental unit for statistical purposes so that for each experiment, one animal was used from a single litter for one data point. All animal studies were reviewed and approved by the Johns Hopkins University Animal Care and Use Committee.

Conditioning Chambers

Operant test boxes were used for the fear conditioning and freezing behavior observations. Rat Test Cages (Coulbourn Instruments, Allentown, PA) were fitted with grid shock floors (Modular Shock Floor, Coulbourn Instruments) containing 18 stainless steel bars connected to a shock generator (Precision Programmable Animal Shocker, Coulbourn Instruments). These cages were placed inside of light- and sound-proof Isolation Cubicles with a white noise generator, light, tone generator, and a monochrome minivideo camera mounted in the center of one side wall of the test box (Coulbourn Instruments). The delivery of the footshock was controlled by the H-Series Habitest Control System (Graphic State Software, Coulbourn Instruments). Cameras were connected to monitors with VHS digital tracking recorders so that all experiments were recorded. Both waste trays and grid floors were washed with soap and water between each experimental animal.

Acquisition and extinction of fear conditioning protocols

On the training day, FC rats were transported to the experimental room and individually placed in the conditioning chambers. After 3 minutes of adaptation, a 30 second tone of 85 dB was presented with a 1 mA foot shock delivered during the last 2 seconds of the tone. Each FC experimental animal remained in the chamber for 2 additional minutes and then were removed and returned to their home cages. The IS rats were transported to the experimental room and individually placed into the conditioning chambers where they immediately received a 2 second 1 mA footshock. Each IS experimental animal remained in the chamber for 2 additional minutes and then were removed and returned to their home cages.

Extinction trials were performed 24 hrs after the conditioning and consisted of non-reinforced exposure to context. Freezing behavior was measured for 3 minutes during 4 consecutive extinction days. Freezing was defined as the absence of all visible movements of the body and vibrissae aside from respiration and heartbeat. Only animals that exhibited freezing behavior greater than 50% of the time on extinction day 1 were included in the analysis. All procedures were monitored and recorded using VHS tapes.

Data Analysis

Freezing data from each day were collected for each animal and converted to a percentage of total observation time. Extinction data were analyzed by two-factor repeated measures ANOVA. Data are represented as the mean ± sem.

Results

Blood lead levels and body weight of animals

Table 1 shows that blood Pb²⁺ levels measured at the termination of the study in the Pb²⁺ exposed group are environmentally relevant since they range from 18.1–35.1 μg/dL with a mean of 25.8 μg/dL. There were no significant differences in body weight between the experimental groups (Table 2).

Table 1.

Blood Pb²⁺ levels in control and Pb²⁺-exposed rats measured immediately after Extinction Day 4 observations (PN54).

	Blood Pb2+ Values (μg/dL)
Control (n=15)	0.75 ± 0.11	Range: 0.1 – 1.5
Pb2+-exposed (n=14)	25.8 ± 1.28*	Range: 18.1 – 35.1

Data from each behavioral group are combined and expressed as means ± s.e.m. and as the range of values for each exposure group.

^*p<0.05 relative to control values.

Table 2.

Body weights for control and Pb²⁺-exposed rats in each behavioral group measured on the Conditioning Day (PN50) and on Extinction Day 4 (PN54).

	Body Weight (g)
	Conditioning Day	Extinction Day 4
CFC-Control (n=10)	273.9 ± 7.3	308.8 ± 8.0
CFC-Pb (n=8)	245.0 ± 10.5	275.5 ± 12.1
IS-Control (n=9)	254.3 ± 9.2	285.1 ± 9.7
IS-Pb (n=11)	264.8 ± 7.1	297.6 ± 7.3

Data are expressed as means ± s.e.m. No differences were observed in body weights amongst the groups.

Acquisition of fear conditioning and extinction

There were no significant differences between control and Pb²⁺-exposed groups in the percentage of total rats tested in the FC protocol that acquired fear conditioning (data not shown). There was a significant increase in the freezing time of the FC rats on conditioning day after the presentation of the CS when compared to the IS (Figure 1). There were no significant differences in the degree of freezing between control and Pb²⁺-exposed rats. Therefore, chronic Pb²⁺ exposure had no effect on the acquisition of FC when compared to controls (Figure 1).

Figure 1.

Percentage of freezing behavior observed after the tone/shock on conditioning day in Control and Pb²⁺-exposed rats in both the Immediate Shock (IS) and Fear Conditioning (FC) behavioral groups. There were no differences in the observed freezing time between Control and Pb²⁺-exposed groups. There was an obvious increase in the freezing time for the FC group (allowed exploration of the test box and received a CS and US) when compared to the IS group (no exploration of the test box and only US). Data are expressed as mean ± s.e.m.

During the Extinction Phase, the control and Pb²⁺-exposed IS groups froze less than 10% of the observation time on all 4 days. The percent freezing time of the control FC group steadily decreased from approximately 65% to less than 15% by day 4 of extinction (Figure 2). The percent freezing time of the Pb²⁺-exposed FC group remained significantly higher than the control FC group, decreasing from approximately 75% to only about 40% by day 4 of extinction (Figure 2) at which time the differences between Pb²⁺-exposed and control rats was significantly different (p < 0.05). These findings suggest that young adult rats chronically exposed to Pb²⁺ have the ability to acquire a normal fear response, but have deficits during the extinction of the learned response.

Figure 2.

Percentage of freezing behavior observed on 4 days of extinction observations in Control and Pb²⁺-exposed rats in each of the behavioral groups. There were low levels of freezing behavior observed in the Control and Pb²⁺-exposed IS group on all 4 Extinction Days. The Control FC group had a steady decrease in freezing time over the 4 days until they nearly reached the levels of the IS group. However, the freezing time of the Pb²⁺-exposed FC group remained significantly higher (F_3,102=34.7; p=0.0001) than all other groups on day 4. A repeated measures ANOVA showed significant differences across the days of extinction observations. Data are expressed as mean ± s.e.m.

Discussion

In the present study we show that chronic Pb²⁺ exposure during brain development has no significant effect on the acquisition of fear conditioning but it impairs the extinction of fear. Our findings are consistent with a previous study (Salinas and Huff, 2002) that showed that chronic exposure to Pb²⁺ that resulted in blood Pb²⁺ levels higher than those in our present study did not alter the acquisition of contextual or cue conditioning but inhibited the extinction phase. Taken together, these findings suggest that chronic Pb²⁺ exposure impairs the extinction of fear conditioning.

Extinction of conditioned fear is believed to involve the formation of a new memory rather than the erasure of the conditioned response. This is supported by studies showing that extinction of conditioned fear depends upon NMDA receptor-mediated plasticity. Thus, as with many types of learning, long-term retention of extinction to fear requires activation of NMDA receptors. This is based on studies in which systemic administration of NMDA receptor antagonists or their direct administration into the amygdala (Falls et al., 1992) prevents extinction of conditioned fear. Conversely, facilitation of NMDA receptor activation by direct injection of the partial agonist D-serine into the amygdala, strengthens extinction of fear-potentiated startle in rats (Walker et al., 2002).

Activation of NMDA receptors has also been shown to be essential in the medial prefrontal cortex (mPFC) during consolidation of extinction learning but not during the acquisition of fear extinction (Santini et al., 2001). Other studies have shown that in the prefrontal cortex a switch from LTD to LTP is also required for consolidation of long-term extinction memory (Herry and Garcia, 2002; Herry and Mons, 2004). Our current findings indicate that chronic developmental Pb²⁺ exposure produces a deficit in extinction learning and the animals remain “more fearful” than controls. Since long-term retention of extinction involves a transfer from NMDA receptor-independent to NMDA receptor-dependent memory (Santini et al., 2001), our present findings provide additional evidence that Pb²⁺-exposed rats have deficits in NMDA receptor-dependent learning.

Our present findings show that chronic exposure to Pb²⁺ during brain development leads to the dysfunction of neuronal systems associated with extinction of conditioned fear, leaving the animals resistant to extinction of fear. These findings could have important implications to the treatment of anxiety disorders such as panic disorders and post-traumatic stress.