Influence of prior experience with homotypic or heterotypic stressor on stress reactivity in catecholaminergic systems

Abstract

Here we review how prior experience with stress alters the response to a subsequent homotypic or heterotypic stressor, focusing on the catecholaminergic systems in the adrenal medulla and the locus coeruleus (LC). The changes in response to homotypic stress differ depending on the stressor applied. With immobilization stress (IMO), transcriptional responses in the adrenal medulla to a single exposure are pronounced and several of the transcription factors and signaling kinases induced or activated are reviewed and compared to the longer term alterations with repeated stress, consistent with persistent activation of gene expression of catecholamine (CA) biosynthetic enzymes. In the LC, transcriptional and post-transcriptional activation of gene expression are shown to be important. Repeated IMO stress triggers further activation of a number of signalling pathways. Neither adrenal medulla nor LC display habituation to long term repeated stress. In contrast, gene expression for CA biosynthetic enzymes habituates to prolonged cold stress in the adrenal medulla and LC, but displays an exaggerated response with exposure to a novel or heterotypic stressor such as IMO. Some of the transcriptional pathways displaying sensitization are described.

• Adrenal medulla
• cold stress
• immobilization stress
• locus coeruleus
• transcription factors
• novel stressor

Introduction

One of the important and complex issues of stress research is that the response to the same stressor is not always identical. It is influenced by many factors, including perception of the stress, personal behavior (diet, drugs, smoking), and individual differences, including genetic factors and prior experience with the same or other stressors (reviewed in McEwen 1998). In some cases, there is habituation to the effects of stress such that an individual becomes accustomed to a particular situation, which had been stressful initially.

Alternatively, there can be a sensitization or exaggerated response based on prior experience. There is “memory” of the stress, as indicated by an altered response to a repeated as compared to a single episode of the same stressor or to a novel stress. A number of excellent studies have concentrated on how prior history with the same or novel stress alters the response of the hypothalamo-pituitary–adrenal (HPA) axis (Akana and Dallman 1997; Armario et al. 2004a, b; Dallman et al. 2004; Herman and Seroogy 2006).

A series of elegant experiments has determined that exposure to stress at a critical stage of development leads to an exaggerated stress response throughout the lifetime (Meaney 2001; Sanchez et al. 2001; Plotsky et al. 2005). This appears to be modulated by epigenetic programming and has been associated with alterations in DNA methylation on the promoter of the glucocorticoid receptor gene at an Egr1 responsive site (Weaver et al. 2004; Meaney and Szyf 2005).

In this review we will focus on how prior experience with the same (homotypic) or different (heterotypic) stress alters the response to subsequent stress, focusing on the several genes in catecholaminergic systems in the adrenal medulla and the locus coeruleus (LC).

Prior experience with homotypic stressor

When an animal is initially exposed to stress, it does not know if, or when, the stress will be terminated. A similar circumstance may or may not hold true for the human situation. Thus, a person may know that the stress is of limited duration, for example by hearing a news report, or by being aware of a deadline. Alternatively, he may not know if or when the stress will be terminated, which is more comparable to the situation with experimental animals.

Immobilization stress

Adrenal medulla

Seminal studies by Richard Kvetnanksy and Irwin Kopin in the 1970's provided the groundwork for understanding the ability of the adrenal medulla to meet the demand of catecholamine (CA) release upon exposure to repetitive experience with homotypic stress (Kvetnansky et al. 1971a, 1978; Kvetnansky and Kopin 1972). They found that exposure to single immobilization (IMO) stress reduced adrenal epinephrine (Epi) levels, and had no significant effect on adrenal norepinephrine (NE). However, after prolonged daily exposure to IMO for 1 week or over 1 month, adrenal medullary NE and Epi attain new elevated levels. After 42 days of daily IMO, there is no habituation, rather adrenal Epi content is about 50% higher and NE content is nearly double the basal level in the adrenals of unstressed rats (Kvetnansky and Mikulaj 1970). This was subsequently shown to result from increased CA biosynthesis (Kvetnansky et al. 1970, 1971a).

In collaboration with Richard Kvetnansky we have followed a similar stress model to ascertain at the molecular level the mechanisms responsible for the transition from brief exposure to prolonged stress. Exposure to IMO stress triggers persistent activation of the tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT) genes in the adrenal medulla, which should be crucial for adaptation (or maladaptation) to the stressful situation. Although a single episode of IMO stress is insufficient to elicit significant changes in TH, DBH and PNMT enzyme activities and protein levels, it triggers a robust elevation in expression of mRNAs for CA biosynthetic enzymes (McMahon et al. 1992; Nankova et al. 1994; Viskupic et al. 1994; Sabban and Kvetnansky 2001; Wong et al. 2002). Compared to values in unstressed controls, TH mRNA levels increase approximately 7-times, DBH mRNA levels about 2.5-times and PNMT mRNA levels increase about 5-times following a single IMO for 2 h. Elevation of gene transcription appears to be mediating this response (Osterhout et al. 1997; Nankova et al. 1999). Stress-triggered changes in transcription have been implicated, and a single exposure to IMO for even 5 min (shortest time examined) was shown to be sufficient to lead to increased TH and DBH transcription (Nankova et al. 1999).

One of the main features of changes in gene expression for CA biosynthetic enzymes with single exposure to IMO stress is that the elevation of transcription and increase in mRNA levels are transient. However, even with only a second exposure to the same stressor, there is “memory” of the first experience such that the increase in TH mRNA is now much more prolonged and is sustained for longer periods of time after termination of the stress. With prolonged repeated IMO stress, over 5–7 days, the elevation of mRNA levels in the adrenal medulla remains high for a long time following stress termination (Nankova et al. 1994; Viskupic et al. 1994). The transcription rate is also more sustained (Nankova et al. 1999) and TH transcription remains elevated for as long as 2 days following cessation of repeated daily IMO stress for 7 days (Sun et al. 2003). This is likely reflected by a sustained increase in TH protein and activity, since the excess TH activity in the adrenals of rats after 7 days IMO declines toward basal levels with first order kinetics and a half life of 3 days (Kvetnansky et al. 1970).

In attempts to delineate the mechanism of transition from rapid adaptive transient response to IMO, to more prolonged responses with repeated IMO stress, we have examined changes in expression or phosphorylation of several transcription factors, which are known to regulate gene expression of CA biosynthetic enzymes, as well as kinases involved in activating them (Table I). Five minutes of IMO stress is enough to increase phosphorylated CREB (P-CREB), and to activate MAP kinases, Erk 1/2 and JNK in the adrenal medulla (Nankova et al. 1998; Sabban et al. 2006a). With long exposure, c-Fos is induced after 30 min and elevation of Egr1 and Fra-2 are evident after 2 h of IMO (Nankova et al. 2000; Wong et al. 2002; Liu et al. 2005).

Table I.  Comparison of changes in adrenal medulla with single and repeated IMO stress.

Single IMO	Repeated IMO
Transcription factors
↑ P-CREB, transient	↑ P-CREB
↑ c-Fos
↑ Egr1	↑ Egr1
↑ Fra-2	↑ ↑ Fra-2
Activity of MAP kinases
↑ JNK	↑ JNK
↑ Erk (1/2)
Gene expression of CA biosynthetic enzymes
	↑ TH transcription, mRNA, protein ↑ DBH transcription, mRNA, protein

However, the repertoire of changes in transcription factors is different with repeated IMO. There is only slight increase in c-Fos and no change in activation of Erk 1/2. However, a greater induction of Fra-2, prolonged elevation of P-CREB, and continued activation of JNK are observed (Sabban et al. 2006b). Thus, at the end of a 6th daily 2 h IMO, Fra-2 level is at least 6-fold higher, and P-CREB about 10-fold over levels at the end of the first IMO. These results are based on western blots of adrenal medulla homogenates of both Epi and NE synthesizing chromaffin cells. However, immunocytochemistry reveals co-localization of Fra-2 and P-CREB with TH in the adrenal medulla after IMO stress (Liu et al. 2005). Thus, these greater changes in transcription factor expression may mediate the more persistent elevation of TH gene transcription with repeated stress.

There does not appear to be loss, or habituation, of the response of CA biosynthetic enzymes in the adrenal medulla to prolonged repeated exposure to IMO. Although the mRNA levels are still high on the day following repeated IMO stress, exposure of chronically stressed animals to an additional IMO, even the 42nd daily exposure still elicits a further increase in TH and PNMT mRNA levels and triggers further induction of Fra-2 (Kvetnansky et al. 2002b).

Does this mean that there is no habituation, but rather a maladaptive response to repeated IMO stress which will lead to changed susceptibility to various stress-related disorders? This remains to be determined. However, with chronic restraint stress, repeated daily for 10 days, there is a gradual development of anxiety-like symptoms associated with enhanced synaptic connectivity in the basolateral amygdala (Mitra et al. 2005).

More recently, microarray profiling has been used to determine common and distinct global changes in gene expression with 2 h IMO stress exposure once or daily for 6 consecutive days (Liu et al. 2006). Among the defined transcripts elevated by IMO, a large percentage is for transcription factor and signaling related genes. The repertoire of transcription factors changed with IMO is greater than previously appreciated. Among the transcripts changed with IMO in the adrenal medulla, about 80% are uniquely induced by single IMO. Approximately half of the transcripts induced by repeated IMO are also responsive to single IMO stress. Clearly more work remains to be done to characterize the molecular mechanisms in the adrenal medulla mediating the transition from acute to chronic repeated exposure to IMO stress.

Locus coeruleus

Exposure to many types of physiological, social or pharmacological stressors, such as cold, restraint, footshock, isolation, forced walking and chronic social stress (but not insulin-induced hypoglycemia or chronic mild intermittent stress) elevate TH mRNA in the LC (Angulo et al. 1991; Smith et al. 1991; Mamalaki et al. 1992; Melia et al. 1992; Watanabe et al. 1995; Rusnak et al. 1998; Wang et al. 1998).

With regard to IMO stress, even a single exposure elicits about a 3–4-fold elevation in TH mRNA expression in the rat LC (Serova et al. 1999; Sun et al. 2004). This is reflected by elevated transcription (Serova et al. 1999; Osterhout et al. 2002). However, a single IMO is insufficient to elicit elevated TH protein content, although if sufficient time is given for subsequent gene expression, an elevation of TH protein is observed in the LC 1 day after a single IMO (Hebert et al. 2005). In addition, a single IMO stress for 2 h also increases mRNA levels and transcription of DBH and GTP cyclohydrolase (GTPCH) genes in the LC (Serova et al. 1999).

Repeated IMO also elicits a rapid elevation of TH, DBH and GTPCH transcription in LC that is evident immediately after the IMO. However the increased transcription, at least for TH, is not sustained afterwards, and post-transcriptional mechanisms are likely important to maintain the prolonged increase in mRNA levels in LC following repeated stress (Serova et al. 1999; Sun et al. 2004).

The changes in several signaling pathways and transcription factors in the LC that can be involved in the regulation of TH and DBH gene expression following different durations of single and repeated IMO stress have been examined (Hebert et al. 2005) and are summarized in Table II. Exposure to a single IMO stress is sufficient to elicit significant phosphorylation of CREB and induction of c-Fos. In contrast to the adrenal medulla, Egr1 is not induced in the LC. Activation of MAP kinases is undetectable following single IMO stress.

Table II.  Comparison of changes in LC with single and repeated IMO stress.

Single IMO	Repeated IMO
Transcription factors
↑ P-CREB	↑ P-CREB, ↑ CREB
↑ c-Fos	↑ ↑ c-Fos
No change in Fra-2	↑ Fra-2
Activity of MAP kinases
No phosphorylation	↑ Erk (1/2)
of MAP kinases	↑ JNK1/2/3
	↑ p38
Gene expression of CA biosynthetic enzymes
↑ TH transcription, mRNA	↑ TH transcription, mRNA, protein
↑ DBH transcription, mRNA	↑ DBH transcription, mRNA, protein

However, with repeated exposure to IMO there is also a robust elevation of Fra-2, a continued induction of P-CREB and an elevated expression of CREB. Repeated, but not single, exposure to IMO also triggers phosphorylation of the several upstream MAP kinases, Erk1/2, JNK2/3 and p38 (Nankova et al. 1998; Hebert et al. 2005). The phospho-Erk1/2 is co-localized with TH positive cells of the LC. The findings demonstrate differences in activation or expression of transcription factors and kinases, depending on the repetition of the stress, and implicate a variety of MAP kinase pathways in mediating potentially long-lasting neuronal remodeling and plasticity in response to prolonged repeated stress.

Overall, the findings indicate that there is no habituation in the response of the LC to repeated IMO stress, and that additional pathways are recruited with repeated stress.

Cold stress

Adrenal medulla

Cold stress triggers strong activation of the sympathetic nervous system. In the adrenal gland, elevated TH mRNA, protein and enzyme activity are observed upon exposure of rats to cold stress at 4°C for a number of days (Kvetnansky et al. 1971b; Chuang and Costa 1974; Hoeldtke et al. 1974; Stachowiak et al. 1985; Baruchin et al. 1990; Cheng et al. 2005). However, if the cold stress is very prolonged (28 days), the levels return to baseline as in untreated controls (Kvetnansky et al. 2002a). Similarly, adrenal PNMT mRNA expression is elevated with cold exposure for 1 day, but this returns to control values with chronic continued exposure to cold stress (Kvetnansky 2004). Thus, the adrenal medulla displays habituation to prolonged cold stress with respect to induction of TH and PNMT gene expression.

Locus coeruleus

Chronic cold stress results in persistent alterations in activity of ascending NE afferents from the LC (Jedema et al. 2001; Jedema and Grace 2003), and increased number of axons in the prefrontal cortex containing TH and membrane-associated NE transporter (Miner et al. 2006). Short term exposure to cold stress (5 h) significantly elevates TH mRNA expression in the LC (Rusnak et al. 2001). When mice are exposed to 3 days of cold stress (4°C) they display elevation of TH activity and mRNA levels, although these changes apparently depend primarily on post-transcriptional mechanisms, since activation of a reporter gene (human alkaline phosphatase) is not pronounced (Osterhout et al. 2005). We found that after 24 h of cold stress outbred Sprague-Dawley male rats display a dichotomous response to cold stress (Cheng et al. 2005), with some of the animals displaying increased, and some a decline in TH and DBH mRNA expression. These results may emphasize individual differences in the stress reactivity of the LC to cold.

Long term cold appears to lead to habituation also in the LC. TH activity, elevated in LC within the first week of cold stress, declines to levels which do not differ from unstressed controls after 2 and 3 weeks of continuous cold (Nisenbaum et al. 1991). In this regard, chronic cold stress (5°C) for 21 days leads to a significant reduction of TH mRNA levels in the LC (Featherby and Lawrence 2004). Whether there is also a habituation in the response of DBH and the mechanisms involved are still unclear.

Exposure to novel stressors

An important example of the influence of prior experience with stress on subsequent stress reactivity is evident following prolonged cold stress. When cold-acclimated rats are exposed to a new acute stressor, the increase in plasma Epi and NE concentrations in response to the novel stressor is substantially greater than when naive animals are exposed to the same stressor (Konarska et al. 1989; Dronjak et al. 2004). Importantly, the Epi level in cold pre-exposed rats remains increased for a longer period than in previously unstressed animals.

Adrenal medulla

An exaggerated response to a novel stressor is also found for expression of the TH and PNMT genes in the adrenals of chronically cold stressed rats. As indicated above, the adrenal medulla displays habituation to prolonged cold stress with respect to induction of the TH and PNMT genes. Despite this habituation, there is “memory” of the stress as indicated by an enhanced or exaggerated response for gene expression to a different or a novel stressor. Exposure of the rats after 28 days of continuous cold to the novel stress of IMO induces an exaggerated increase in TH and PNMT mRNA levels compared to the response of previously unstressed rats (Kvetnansky et al. 2002a; Kvetnansky 2004). A similar exaggerated response is also observed when the cold pretreated rats are exposed to novel metabolic stressors, such as insulin induced hypoglycemia or 2-deoxyglucose-induced glucopenia.

Another example of the influence of prior experience is revealed in a study comparing the induction of TH mRNA levels in the adrenals of rats with or without prior exercise. In this study TH mRNA levels were elevated by shaking stress only in exercised rats (Levenson and Moore 1998). Similarly, rats acclimated to high altitude conditions living outdoors for 1 year display a larger increase in adrenal TH activity and higher levels of plasma CA in response to IMO than rats that are not adapted to high altitude (Balaz et al. 1980).

It is important to understand the mechanism whereby prior experience with one type of stress increases the sensitivity to a heterotypic stressor or novel stressor. To begin to answer this question we examined the influence of cold stress on induction and phosphorylation of several transcription factors that could regulate TH and/or PNMT gene expression (Liu et al. 2005). Rats with or without pre-exposure to cold stress for 28 days were subjected to single IMO stress for changes in the adrenal medulla examined at various times of IMO (up to 2 or 3 h afterwards). As shown in Table III, phosphorylation of CREB after 30 min IMO is greater in cold pre-exposed rats. Induction of Egr1 is three times higher in cold pre-exposed rats and remains significantly elevated even 3 h after cessation of IMO. Exposure to IMO triggers a 10–20-fold elevation in Fra-2 in both groups, which is even higher 3 h after the IMO. However, Fra-2 is more heavily phosphorylated following IMO stress in cold pre-exposed animals. The results reveal that sensitization to novel stress in cold pre-exposed animals is manifested by an exaggerated response of induction or activation of several transcription factors and thus is likely to have broad physiological consequences.

Table III.  IMO triggered changes in several factors in adrenal medulla in cold pretreated animals.

	Prolonged cold (28 days)	Single IMO	Single IMO after prolonged cold
P-CREB (ser-133)	No effect	Transient rise up 3-fold with 30 min IMO	Larger rise, up 6-fold after 30 min
Egr1	No effect	Induction, up 2–3-fold with 30 min IMO	Larger more sustained rise, up 6-fold with 2 h IMO still high 3 h later
Fra-2	Up 2–3-fold	Induction up>10-fold with 2 h IMO and 3 h later rise in phospho Fra-2	Induction after 30 min, up > 10-fold with 2 h IMO and 3 h later greater rise in phospho- Fra-2

Novel stress and noradrenergic pathways

The increased sensitivity of cold pre-exposed rats to a novel stressor is not restricted to the adrenal medulla. Chronic cold stress also triggers a greater activation of the HPA axis, increased extracellular NE in hippocampus and prefrontal cortex and altered cardiovascular responses to a novel or a heterotypic stressor (Nisenbaum et al. 1991; Gresch et al. 1994; Bhatnagar and Dallman 1998; Bhatnagar et al. 1998). The elevation of hippocampal NE with novel stress results from increased TH activity (Nisenbaum et al. 1991). It is still unclear if the increased extracellular NE in the hippocampus and prefrontal cortex may reflect an exaggerated response of increased gene expression of NE biosynthetic enzymes in the LC. However, the novel stressors of cold or 2-deoxyglucose-mediated glucopenia elicit an exaggerated elevation of TH mRNA levels in the LC of rats following long term repeated exposure to IMO stress (Rusnak et al. 2001).

Sensitization of LC neurons to excitatory inputs appears to be elevated after chronic cold. Chronic cold potentiates the increase in electrophysiological activity of LC neurons evoked by corticotrophin-releasing hormone (Jedema et al. 2001). The sensitization of NE in the medial prefrontal cortex is influenced by different stress protocols as well as the nature of the stress. It does not develop with continual footshock or intermittent cold (Jedema et al. 1999).

Conclusion

Prior experience of stress has a profound influence on subsequent stress reactivity of catecholaminergic systems of the adrenal medulla and the LC. The changes in response to a homotypic stressor differ depending on the stressor applied. The adrenal medulla habituates to prolonged cold stress, but not to repeated IMO stress with respect to ability to induce gene expression of at least some CA biosynthetic enzymes. However, there are marked differences in the profiles of changes in gene expression triggered by repeated compared to single exposure to IMO stress, and the increase in gene expression of CA biosynthetic enzymes is much more persistent even following termination of the stress. In the LC, additional signaling pathways are expressed with repeated, than with a single IMO stress.

Stress reactivity to novel stressors of animals adapted to a particular stress, such as cold is dramatic. It is reflected in exaggerated responses in induction or activation of several transcription factors and thus is likely to have broad physiological consequences. Much work remains to be done to elucidate the mechanisms involved.