In this study, we have demonstrated that maternal intrauterine endotoxin administration leads to a significant decrease in multiple serotonergic markers in the offspring. Tryptophan metabolism to serotonin as measured by AMT SUV and serotonin levels in the frontal and parietal cortices of the neonatal rabbit brain were significantly decreased following intrauterine endotoxin exposure. In addition to the decreased serotonin content, there was a loss of serotonin-immunoreactive terminals and decreased expression of 5HTT measured in the parietal sensory cortex of endotoxin-exposed kits. Although serotonergic raphe nuclei cell bodies and TPH2-positive cortical fibers were intact in the endotoxin-treated kits, there was increased apoptosis of VB thalamic cells. These results indicate that the loss of serotonin-immunoreactive fibers in the cortex is most likely due to loss of thalamic neurons and thalamocortical afferents that transiently express 5HTT to uptake and store serotonin during development. Serotonin signaling has been shown to influence axonal outgrowths and thalamocortical pathfinding (Bonnin et al, 2007
). Diminished serotonin signaling during development may result in defects in thalamocortical circuit formation. These results are significant because they demonstrate that maternal intrauterine inflammation can disrupt the serotonergic developmental regulation of thalamocortical innervation in somatosensory cortex of newborn kits.
Tryptophan is an essential amino acid that is metabolized in several pathways of which the most common one is the serotonergic pathway. Serotonin is a monoamine neurotransmitter that is known to have a vital role in regulating brain development (Lauder and Krebs, 1978
). Decreased tryptophan metabolism resulting in a decrease in serotonin content in the cortex may be secondary to induction of IDO in the placenta and in activated microglia in the PVR of the newborn brain following intrauterine endotoxin exposure. Maternal inflammation results in induction of IDO in the placenta that would limit the tryptophan available for the fetal brain for serotonin synthesis. Similarly, microglia when activated, overexpress IDO that would shunt tryptophan away from serotonin formation along the kynurenine pathway resulting in serotonin depletion in the developing brain.
During development, serotonin-immunoreactive fibers in the cortex primarily consist of two populations, afferents of raphe serotonergic neurons and thalamocortical glutamatergic neurons transiently expressing 5HTT and vesicular monoamine transporter leading to uptake and storage of serotonin (D'Amato et al, 1987
; Lebrand et al, 1996
; Bennett-Clarke et al, 1997
; Persico et al, 2001
). Evidence from both pharmacological and gene knockout experiments demonstrates that serotonin has a role in thalamocortical development. Immunocytochemistry for serotonin and [3
H]citalopram binding to serotonin uptake sites both have demonstrated a transient serotonergic innervation of primary sensory cortex between postnatal days 2 and 14 during the period of synaptogenesis in rat cortex (D'Amato et al, 1987
). This actually represents transient expression of the high-affinity 5HTT (Lebrand et al, 1996
) and vesicular monoamine transporter (Persico et al, 2001
) by glutamatergic thalamocortical neurons. During the first 2 postnatal weeks in rats, these thalamocortical neurons take up and store serotonin, although they do not synthesize serotonin. Depletion of serotonin during this crucial period delays the development of the barrel fields of the rat somatosensory cortex, decreases the size of the barrel fields, and disrupts the synaptic connectivity in sensory cortices (Blue et al, 1991
; Bennett-Clarke et al, 1994
). In rabbits, the somatosensory barrels are not as clearly defined and distinct as in rats and mice, and the time course for the development of the somatosensory cortex differs. The cortical barrels appear earlier in the rabbit, with appearance at postnatal day 1 (31 to 32 days gestation), becoming indistinct and similar to that in adults by postnatal day 5 (PND5) (Rice et al, 1985
). As the time course for the development of somatosensory cortex occurs earlier in the rabbit, it is likely that the critical period for the trophic role of serotonin on thalamocortical development occurs in the perinatal period in rabbits instead of the first and second postnatal week as in rats and mice.
Transient expression of 5HTT during postnatal development was noted in 70% to 80% of the neurons of the VB thalamic nucleus projecting to the sensory cortex (Lebrand et al, 1996
). In our model, there was a significant decrease in the expression of 5HTT in the parietal cortex along with apoptosis of the neurons in the VB thalamus in the endotoxin-treated animals. Moreover, there was no difference in the number of serotonergic neurons in the raphe nucleus or in the presence of TPH2-staining fibers in the cortex between the groups. As serotonergic neurons in the raphe nucleus and their projections develop very early in gestation (Rubenstein, 1998
), an inflammatory insult closer to term may not influence these cells, although an arrest in the development or maturation of the fibers cannot be ruled out. Taken together, our data suggest that the decrease in serotonin-immunoreactive fibers in the cortex may primarily be due to a loss of thalamocortical afferents. This is in accordance with studies that have shown that experimental thalamic lesions involving the VB thalamus in postnatal rats resulted in the loss of 5HT-immunoreactive fibers in the cortex (Lebrand et al, 1996
). We hypothesize that maternal inflammation may either lead to a direct cytokine-mediated injury to the thalamocortical afferents with retrograde involvement of the VB neurons, or may result in direct injury to the VB neurons leading to loss or impaired development of thalamocortical afferents. Future studies directed at evaluating the development of the serotonergic system at different time points following a maternal inflammatory exposure may help elucidate this further.
We propose that one link between maternal intrauterine inflammation and fetal brain development may be the transport and metabolism of serotonin and its precursor tryptophan in the placenta. Studies in mice deficient in peripheral serotonin synthesis have shown that maternal serotonin production is crucial for normal fetal neurogenesis and development (Cote et al, 2007
). The placenta also expresses TPH2 (Correa et al, 2009
), resulting in serotonin production that would be available for transport to the fetus. Induction of IDO in inflammatory conditions with increased tryptophan metabolism along the kynurenine pathway can lead to decreased tryptophan availability for serotonin synthesis in the placenta and in the fetus. Thus, by the regulation of tryptophan levels, IDO activity may influence availability of serotonin in the fetus. Furthermore, several kynurenine pathway metabolites (e.g., quinolinic acid, kynurenine, 3-hydroxykynurenine) are neurotoxic (Stone, 2001
). These tryptophan metabolites might also have a role in neurodegeneration of thalamocortical neurons in this model.
Maternal intrauterine inflammation resulting in immune dysfunction during development has been implicated in the development of neurodevelopmental disorders such as autism, schizophrenia, and cerebral palsy (Fatemi et al, 2008
). A common link among these disorders appears to be the presence of activated microglia and evidence for immune dysregulation in the developing brain (Patterson, 2009
). Brain tissues of autistic patients were found to have increased number of activated microglia and astrocytes along with an increase in the levels of proinflammatory cytokines (Vargas et al, 2005
). Animal models of maternal inflammation have been shown to result in motor deficits, behavioral abnormalities, and neuroglial activation in the offspring (Meyer et al, 2006
; Saadani-Makki et al, 2008
). Viral infections in the prenatal period have been associated with alterations in serotonin synthesis in the cerebellum of P14 mice (Winter et al, 2008
). We have previously shown that maternal intrauterine endotoxin administration results in motor deficits suggestive of cerebral palsy in the newborn rabbit along with microglial activation in the PVR (Saadani-Makki et al, 2008
). Using the same model, we have shown in this study that intrauterine endotoxin administration results in decreased tryptophan metabolism to serotonin and loss of serotonin containing thalamocortical terminals in the parietal cortex of the neonatal rabbit.
The decreases in serotonin synthesis in this rabbit model of maternal inflammation induced perinatal brain injury show some similarity to those found in children with autism. In nonautistic children, serotonin synthesis capacity was high with values >200% of that in adults until the age of 5 years, subsequently declining toward adult levels. In contrast, serotonin synthesis capacity in autistic children increased gradually between the ages of 2 and 15 years to values 1.5 times normal adult values (Chugani et al, 1999
). These data suggest that humans undergo a period of high brain serotonin synthesis capacity during early childhood, and that this developmental process is disrupted in autistic children.
Impairment in sensory perception and integration are not only documented in patients with autism and autism spectrum disorders (Molloy et al, 2003
) but are also known to be associated with motor deficits in patients with cerebral palsy (Wingert et al, 2008
). Indeed, numerous reports have associated the degree of motor impairment to the extent of somatosensory deficits in cerebral palsy (Gordon and Duff, 1999
; Wingert et al, 2008
). Diffusion tensor imaging studies have shown that patients with cerebral palsy have severe disruption of the thalamocortical connections to the somatosensory cortex, along with injury to the descending corticospinal pathways leading to the hypothesis that the injury to the ascending somatosensory pathways would define the extent of clinical impairment (Hoon et al, 2009
). We have demonstrated that maternal intrauterine exposure to endotoxin results in decreased serotonin synthesis along with the loss of thalamocortical afferents in somatosensory cortex of the newborn rabbit. This may disrupt the normal development of neocortical circuitry in the somatosensory cortex, resulting in altered sensory processing and sensorimotor integration adding to the motor deficits in patients with cerebral palsy. This may also provide a link between prenatal exposure to inflammation and the development of autism.