Selective serotonin reuptake inhibitors (SSRIs) are the pharmacological treatment of choice for depression, anxiety, and obsessive–compulsive disorder. Moreover, these drugs are specifically recommended for treatment of these disorders during pregnancy and lactation (Wisner et al, 2000
; Cohen et al, 2004
). This is largely due to their low perceived toxicity to both mother and fetus or infant. As a consequence, there is a significant and increasing likelihood that children will be exposed to SSRIs either in utero
or via breast milk. The long-term consequences of this early exposure to SSRIs are largely unknown.
A large fraction of children exposed in utero
to SSRIs have been reported to display signs of antidepressant withdrawal in the first week or two of life (Nordeng et al, 2001
; Laine et al, 2003
; Zeskind and Stephens, 2004
; Sanz et al, 2005
), indicating that the fetus can be exposed to neurobiologically relevant doses of these drugs. In fact, significant SSRI and metabolite concentrations have been detected in both umbilical cord blood and amniotic fluid in women taking these medications during pregnancy. The mean ratios of umbilical cord to maternal serum concentrations ranged from 0.29 to 0.89 (Hostetter et al, 2000
; Hendrick et al, 2003
Children exposed to SSRIs in utero
have been followed for up to 72 months postnatally and have not been reported to display increased behavioral abnormalities compared to unexposed children although they have been reported to display subtle changes in motor development and in motor movement control (Costei et al, 2002
; Nulman et al, 2002
; Casper et al, 2003
). However, no study has followed such children beyond early childhood. Thus, it remains possible that such exposure may result in later neurobehavioral abnormalities.
In neonatal rodents, chronic administration of serotonin reuptake inhibitors (clomipramine, fluoxetine, zimeldine, LU-10-134C) as well as some other tricyclic antidepressants but not the atypical antidepressants iprindole or nomifensine during the early life period from postnatal day 8 (PN8) to PN21 results in a pattern of maladaptive behaviors that are evident long after drug discontinuation and persist into adulthood (Mirmiran et al, 1981
; Hilakivi et al, 1984
; Hilakivi and Hilakivi, 1987
; Hansen et al, 1997
; Ansorge et al, 2004
). These behavioral changes, described here as the ‘neonatal antidepressant exposure syndrome (NADES)’, in rats include alterations in locomotor activity, reduced male sexual activity and competence, increased ethanol consumption, dysregulation of the hypothalamic–pituitary–adrenal axis, increased rapid eye movement (REM) sleep time and reduced latency to enter the REM sleep phase, and increased immobility in the forced swim test (Mirmiran et al, 1981
; Hilakivi et al, 1984
; Hilakivi and Hilakivi, 1987
; Hartley et al, 1990
; Hansen et al, 1997
). In contrast, adults exposed to similar doses and durations of antidepressants exhibit no persistent behavioral effects after drug discontinuation, indicating that the neurobiological response to long-term antidepressant administration differs markedly between early life and adulthood.
Similarly, serotonin transporter (SERT) knockout mice have been reported to display increased anxiety- and depression-like behaviors, reduced aggressive behavior, and exaggerated reponse to environmental stress (Holmes et al, 2003
; Lira et al, 2003
; Ansorge et al, 2004
). Moreover, there is evidence from these studies that the absence of SERT expression is accompanied by reductions in the density of 5HT1A
receptors in dorsal raphe (DR) as well as increases in the density of 5HT2A
receptors in amygdala and choroid plexus (Li et al, 2000
). However, it is not known whether these effects are the result of adult adaptation to the absence of SERT or are a function of alterations in extracellular serotonin levels during critical periods of development. Nonetheless, it might be predicted from these studies that disruption of SERT function by SSRIs would be accompanied by alterations in aggressive and conflict-related behaviors.
Brain growth (mass) in rodents begins antenatally, peaks at 5–10 days postnatally (PN5–PN10) and continues to PN21–PN28 days with adult mass attained after that time. By comparison, human brain growth begins antenatally, peaks at birth and continues beyond 30 months postnatally (Dobbing, 1974
). In addition to simple increases in brain mass, synaptogenesis and glial proliferation begin during late antenatal periods and continue through postnatal day (PN)40 in the rat (4–6 years old in humans). Likewise, neurotransmitter receptor development continues well into adolescence in both rats and humans (PN50+ in rats, 12–17 years old in humans). For both rat and human, the competence of the blood–brain barrier is not mature until well after birth. Thus, the developing CNS during the prenatal and early postnatal periods is uniquely vulnerable to neurobiological teratogenicity due to exogenous drug exposure (Lauder, 1990
; Whitaker-Azmitia, 1991
In the rat, monoaminergic neurons appear relatively early in prenatal development (10–14 days of gestation) (Lauder and Bloom, 1974
). Synaptogenesis, while beginning in late prenatal development, undergoes its most extensive development between postnatal days 5–20 (Lauder and Bloom, 1975
). This is accompanied by increases in synthetic enzyme (tyrosine and tryptophan hydroxylase) activity up to PN30–PN40 (Johnston and Coyle, 1980
). Likewise, monoaminergic transporter proteins are poorly expressed prenatally, but undergo a massive proliferation and subsequent ‘pruning’ in the 5–6 weeks after birth (Hansson et al, 1998
; Zhou et al, 2000
). Thus in the rat, extensive development of neurotransmitter systems affected by antidepressant treatments occurs up to 5–6 weeks postnatally. It therefore appears that the NADES paradigm in rats closely parallels the exposure of children to antidepressants during the late stages of in utero
development and the first 3 years of postnatal life.
The neurobiological events that produce NADES are unknown. The paradigm was introduced by Mirmiran et al (1981)
using clomipramine as a pharmacological means of suppressing active sleep during development and this group first reported that neonatal clomipramine exposure resulted in reductions in cortical and medullary weight, total protein, and total DNA (Mirmiran et al, 1983
). Since these initial studies, a number of investigators have reported that neonatal clomipramine exposure results in reductions in basal monoamine concentration and turnover in subcortical regions (striatum, hypothalamus, limbic structures) (Hilakivi et al, 1987a
; Feenstra et al, 1996
; Vijayakumar and Meti, 1999
; Yannielli et al, 1999
). Similarly, animals exposed to clomipramine neonatally display reduced firing of neurons in the DR nuclei (Yavari et al, 1993
; Kinney et al, 1997
). Finally, two groups have reported that neonatal clomipramine impairs hypothalamic–pituitary axis responsiveness as marked by increased circulating levels of corticosterone and reduced suppression of corticosterone secretion in response to dexamethasone treatment (Ogawa et al, 1994
; Prathiba et al, 1998
). However, the mechanism(s) by which neonatal but not adult antidepressant exposure produce these effects is/are unknown.
Serotonergic cell bodies in the raphe nuclei project throughout the CNS. Since the neonatal treatment period associated with NADES coincides with the period of rapid development of monoaminergic systems, it is reasonable to suspect that alterations in normal synaptic concentrations of serotonin during critical periods of neuronal interaction would have far-reaching consequences throughout the CNS. By changing the performance of cognitive, sensory, and motor circuits, such alterations would be expected to distort the way information is received, interpreted, and acted upon. Xu et al (2004)
have provided an anatomical basis for this assertion by showing that neonatal administration of the SSRI, paroxetine, disrupts the organization of barrel field cortex via interference with the refinement of thalamocortical afferents.
In fact, the early genesis of the central monoaminergic neurons in mammals has repeatedly led to the postulation of a trophic role of monoamines on brain morphogenesis. Serotonin is one of the first neurotransmitters to appear in the CNS and has been proposed to act as a developmental signal in cell proliferation, differentiation, and apoptosis (Lauder, 1990
; Azmitia, 2001
; Verney et al, 2002
). Tryptophan hydroxylase (TPH) is the rate-limiting enzyme in the production of serotonin, and during development, brain serotonin levels and TPH mRNA increases 35-fold in DR between embryonic day 18 and PN22 in rat (Rind et al, 2000
). Likewise, the level of SERT mRNA, the primary means of removing serotonin from the synapse and terminating serotonin signaling, has been found to be related to synaptogenesis (Ivgy-May et al, 1994
; Hansson et al, 1998
). In human brain, serotonergic neurons have been detected by 5 weeks of gestation and synaptic levels of serotonin increase during the first 2 years after birth and then decrease to adult levels after the age of 5 years (Sodhi and Sanders-Bush, 2004
Given that the development of maladaptive behaviors after early life exposure to antidepressants is seen in animals and humans alike, we hypothesized that transient SSRI treatment alters the long-term chemical profile of the raphe cortical projection system. In order to reveal cellular substrates of serotonergic dysfunction, which may correlate with stereotyped behavioral repertoires of NADES rats, we examined TPH and SERT immunoreactivity in male rats at PN22 and PN130 after they had received neonatal antidepressant treatment (PN8–PN21). For these studies, we employed the highly selective SSRI, citalopram, and clomipramine, a tricyclic compound that preferentially inhibits serotonin reuptake and is the antidepressant first reported to induce NADES.