Brain 5-HT receptor system in the stressed infant rat: implications for vulnerability to substance abuse

Abstract

Clinical and epidemiological studies have found an association between aversive experiences early in life and an increased risk for depression, anxiety and substance abuse. In order to elucidate the mechanisms by which adverse life events are translated into behavioral and psychological abnormalities, we used a rat model to study the impact of chronic injection and 24 h maternal deprivation on the developing rat brain. Specifically, we investigated the regulation of molecules related to the 5-HT (5-HT) system and studied the effect of desipramine administration on animals that were maternally deprived (DEP) on day 13 of life compared with non-deprived animals. We found that maternal deprivation caused an enhanced corticosterone response to an acute stress. Maternally deprived animals also showed a decrease in corticosteroid receptors and an increase in 5-HT 1A and 1B receptors restricted to the CA1 region of the hippocampus. Desipramine prevented the maternal deprivation induced up-regulation of the 5-HT 1B receptor and the enhanced adrenocortical response observed in these animals. Interestingly, non-deprived animals receiving chronic injections showed a decrease in hippocampal 5-HT1B receptor mRNA. At 80 days of age, a group of animals that were treated as infants were given the option of drinking from two identical water bottles, one bottle contained tap water, while the second contained ethanol at increasing concentrations. Animals that received chronic injections during the newborn period consumed more alcohol than those that were not injected. On the other hand, maternal deprivation did not have an impact on alcohol consumption. Alcohol preference has implications to the organism since studies of drug self-administration in laboratory animals have shown that ethanol ingestion is positively related to the use of other drugs, principally opioids and psychostimulants. Our findings suggest that the quality and/or chronicity of early life stressors can influence the neurobiological substrates that may trigger and/or predispose individuals to substance abuse in adulthood.

Introduction

It is widely accepted that both genetic and environmental factors contribute to the phenotypic manifestation of many psychiatric disorders. Among the environmental factors believed to play a key role in the development of psychopathology, aversive or stressful early life experiences have received a great deal of attention. For example, a number of clinical and epidemiological studies have found a strong association between psychosocial stressors early in life (e.g. parental loss, child abuse) and an increased risk for depression, anxiety, impulsive behavior, and substance abuse in adulthood (Kendler et al., 1992). However, aversive early life experiences not only have negative consequences for the adult, but also have more immediate repercussions in the child. It is known that abused children, as well as children who have experienced a recent loss of a parent, have early onset and high rates of depression, anxiety, and suicidal behavior (Lewis, 1992, Pelkovitz et al., 1994). An increased risk for substance abuse is described in populations that have been subjected to childhood neglect or abuse (Dinwiddie et al., 1992, Duncan, 1977, Gutierres et al., 1994). In addition, studies in the non-human primate have found a relationship between lack of maternal care, alcohol preference and impulsive behavior (Higley and Linnoila, 1997b). Understanding what neurobiological systems may be affected by aversive early life experiences may help us elucidate the mechanisms by which early life events are translated into behavioral and psychological abnormalities in the human.

Serotonin (5-HT) has been widely investigated as a key element in the pathophysiology of mood and anxiety disorders, as well as in suicidal and impulsive behavior (Mann et al., 1989, Meltzer, 1988, Meltzer and Lowy, 1987). 5-HT dysfunction has also been implicated in alcohol abuse as well as the reinforcing effects of psychostimulant drugs (Higley and Linnoila, 1997a, Higley and Linnoila, 1997b, Higley and Bennett, 1999). The actions of 5-HT are mediated by multiple receptors. These receptors are subdivided on the basis of pharmacological profile (Peroutka and Snyder, 1979) and there are at least seven major 5-HT receptor families or classes: 5-HT1 through 5-HT7, with some families containing multiple subtypes (Hoyer and Martin, 1996). Among the many components of the 5-HT system, the 5-HT molecules most closely associated with the neurobiology of mood and anxiety are the 5-HT transporter (5-HTt), the 5-HT 1A receptor (5-HT1A), and the 5-HT 2A receptor (5-HT2A, formerly 5-HT2). Most new (as well as older) antidepressants inhibit the re-uptake of 5-HT from the synapse, and alter 5-HTt protein and mRNA levels (López et al., 1994, Owens and Nemeroff, 1998). Animal studies have also demonstrated that chronic antidepressant administration affects the function and number of the 5-HT1A and 2A receptors (Blier and de Montigny, 1994, Bourin and Baker, 1996, Peroutka and Snyder, 1980a, Peroutka and Snyder, 1980b). Another receptor that may be relevant to psychiatric disorders, including substance abuse, is the 5-HT 1B (5-HT1B) receptor. It has been reported that knock-out mice lacking this receptor show an increase in aggressive behavior, impulsivity and cocaine self administration (Rocha et al., 1998, Saudou et al., 1994, Zhuang et al., 1999).

In the rat, the 5-HT neurotransmitter system shows a predominantly postnatal development (Dinopoulos and Dori, 1995, Hansson et al., 1998, Hedner and Lundborg, 1980, Murrin et al., 1985, Nelson et al., 1980, Stancheva et al., 1985). The 5-HT containing cell bodies are present at birth but increase in number and intensity of fluorescence and sprouting, reaching a maximum density by the end of the second week of life. After this maximal peak of biochemical and morphological development there is a slow decline to an adult serotonergic innervation pattern by puberty, at 35–45 days of age. The developmental pattern of the 5-HT1A, 5-HT2C and 5-HT2A receptors follows the morphological development of 5-HT containing neurons, suggesting that they might function to both aid and restrict neuronal development and synaptogenesis (Hellendall et al., 1993, Hillion et al., 1993, Morilak and Ciaranello, 1993).

Another function linked to the development of the 5-HT system is the onset of stress responsiveness. During the first 2 weeks of life (day 4 to 14), the infant rat shows a markedly diminished adrenocortical response to specific stress challenges. This period was termed the stress-hyporesponsive-period (SHRP) based on the adrenocortical response to stress (Schapiro et al., 1962, Schoenfeld et al., 1980). Although adrenal responses are limited during this period, some brain elements appear to be responsive to stressors in a time and stimulus specific fashion unique to the developing animal (Smith et al., 1997, Walker et al., 1991). Interestingly, a more predictable increase of adrenocortical response to stress, which begins to be observed at 12 days of life in the infant rat, coincides with the appearance of increases in brain 5-HT levels following stress (Mitchell et al., 1990, Popova and Lobacheva, 1981, Popova and Lobacheva, 1982). However, it is also known that maternal behavior influences the relative stress responsiveness seen early in life (Rosenfeld et al., 1992). Following 24 h of maternal deprivation, the neonatal rat responds with significant increases in basal and stressed-induced corticosterone (CORT) secretion at a time when the stress axis is quiescent (Levine, 1994, Levine et al., 1991, Vázquez et al., 1996). The endocrine response observed is unique in that the animal has elevated basal CORT levels and in response to a new stress, the CORT response remains elevated for a prolonged time after the stressor. A hyper-responsive limbic–hypothalamic–pituitary–adrenal (LHPA) axis along with prolonged glucocorticoid responses during this stage of development can potentially affect the differentiation of multiple brain neurotransmitter systems.

Studies in adult rats have shown that corticosteroids can alter several elements of serotonergic neurotransmission. Removal of circulating corticosteroids by adrenalectomy (ADX) results in anatomically specific decreases in indices of 5-HT metabolism, while stressful procedures, which raise corticosteroid levels, produce corresponding increases in 5-HT turnover (Curzon et al., 1972, Van Loon et al., 1981). Corticosteroids may also act to directly modulate serotonergic neurotransmission by regulating 5-HT receptors (Biegon et al., 1985, Chalmers et al., 1992, Chalmers et al., 1994). Electrophysiological studies have shown a suppression of 5-HT induced hyperpolarizations within CA1 pyramidal cells after brief application of steroids in this area of the hippocampus (Joels and de Kloet, 1991), establishing a functional coupling for steroid–5-HT receptor interactions within this structure. Furthermore, the hippocampus contains a high abundance of the two types of corticoid receptors, which are thought to control negative feedback: glucocorticoid (GR) and mineralocorticoid (MR) receptors (De Kloet et al., 1998, McEwen, 1991). Therefore, it seems likely that the high concentration of corticosteroid receptors within the hippocampus underlies the sensitivity of 5-HT receptors to corticosteroid regulation in this region. As such, this area provides a unique anatomical environment in which to study the molecular interplay between serotonergic systems and corticosteroids during development.

Most clinically effective antidepressants have an effect upon both the 5-HT system and the LHPA axis (Brady et al., 1991, Holsboer and Barden, 1996, López et al., 1998). Animal studies have shown that administration of tianeptine, a tricyclic antidepressant, prevents the down-regulation of 5-HT1A receptor and dendritic atrophy observed in the hippocampus of chronically stressed rats (Watanabe et al., 1993). Chronic administration of the tricyclic antidepressant, desipramine, to adult rats has similar results. Desipramine prevents the stressed induced down-regulation of hippocampal 5-HT1A receptors, and normalizes the hippocampal mineralocorticoid receptor to glucocorticoid receptor ratio in chronically stressed animals (López et al., 1998).

In view of the relationship between stress, corticosterone secretion and 5-HT, the present article reports a series of studies that were designed to understand the impact of early life adversity and desipramine administration on specific components of the 5-HT brain system in the immediate lactation period and on drug seeking behavior once the animals reached adulthood. We hypothesized that both chronic injections and maternal deprivation would alter the expression of 5-HT related molecules in the hippocampus, where corticoid and 5-HT receptors are co-localized. We further hypothesized that desipramine would prevent the 5-HT receptor changes resulting from these stressors. Finally, based on the clinical literature (Pohorecky, 1990), we hypothesized that early life stress would increase the likelihood of alcohol use in adulthood. Thus, in our first study we asked:

• Are hippocampal, cortical and raphe 5-HT related receptor and transporter molecules altered by the chronic stress of injections?

• Are these altered by maternal deprivation?

• Does administration of the tricyclic antidepressant, desipramine prior to the maternal deprivation event prevent these abnormalities?

We measured 5-HT1A mRNA in hippocampus and raphe, 5-HT2A mRNA in cortex, 5-HT1B mRNA in hippocampus, and 5-HT transporter mRNA in raphe. We also quantified MR and GR gene expression in the hippocampus of these animals. In addition, we measured 5-HT2A and 1A receptor protein concentration using radioimmunocytochemistry and receptor autoradiography, respectively. In a second study, we assessed alcohol preference in a group of adult male and female rats exposed to the same treatments in early postnatal life.