Multiple brain disorders that show serotonergic imbalances have a developmental onset. Experimental models indicate a role for serotonin as a morphogen in brain development. To selectively study the effects of serotonin depletions on cortical structural development and subsequent behavior, we developed a mouse model in which a serotonin neurotoxin, 5,7-dihydroxytryptamine (5,7-DHT), is injected into the medial forebrain bundle (mfb) on the day of birth. Littermates with saline injections into the mfb and age matched mice served as controls. This study characterized the extent and duration of serotonergic denervation after the selective neonatal lesion and investigated effects on exploratory behavior, spatial learning and anxiety in mice of both sexes. We report significant decreases in the serotonergic (5-HT) innervation to cortex and hippocampus, but not to subcortical forebrain structures in 5,7-DHT-lesioned mice. The depletion of 5-HT fibers in cortical areas was long lasting in lesioned mice but autoradiographic binding to high affinity 5-HT transporters was only transiently reduced. Male but not female lesioned mice reduced their exploration significantly in response to spatial rearrangement and object novelty, suggesting increased anxiety in response to change but normal spatial cognition. Our data show that developmental disruptions in the serotonergic innervation of cortex and hippocampus are sufficient to induce permanent, sex specific, behavioral alterations. These results may have significant implications for understanding brain disorders presenting with cortical morphogenetic abnormalities and altered serotonin neurotransmission, such as autism, schizophrenia and affective disorders.

Elevating cortical serotonin (5-HT) in rats from postnatal day (P-) 0 to P-6 by administering the monoamine oxidase (MAO(A)) inhibitor, clorgyline, produces a dose-dependent spectrum of effects on rat somatosensory organization, ranging from enlarged with indistinct septa to a complete lack of vibrissae-related patterns. However, if clorgyline treatment is stopped on P-6, a qualitatively and quantitatively normal vibrissae-related pattern of thalamocortical afferents appears in somatosensory cortex (S-I) on P-10. We employed high performance liquid chromatography (HPLC), infraorbital nerve (ION) transection, N-methyl-D-aspartate (NMDA) receptor blockade, 1,1'-dioctadecyl-3,3,3"3'-tetramethylindocarbocyanine perchlorate (DiI) labeling of thalamic afferents, and CO histochemistry to determine whether peripheral nerve input and/or cortical NMDA receptor activity were required for the recovery of vibrissae-related patterns in clorgyline-treated animals. Clorgyline administration from P-0 to P-6 produced a 1589.4+/-53.3% increase in cortical 5-HT over control animals on P-6 and a 268.8+/-6.3% elevation over controls at P-10. Postnatal day 6 pups had significantly altered vibrissae-related patterns in S-I following 6 days of clorgyline treatment but by P-10, the characteristic vibrissae-related patterns were restored. Neither transection of the ION nor application of the NMDA antagonist, DL-2-amino-5-phosphonovaleric acid (APV), to the cortices of P-6 pups that were treated with clorgyline from birth had any significant effect on the recovery of the vibrissae-related patterns by P-10. These results indicate that neither peripheral nerve input nor cortical NMDA receptor activity are necessary for the restoration of cortical vibrissae-related patterns in rats that have sustained transient elevations of 5-HT.

Manipulation of cortical serotonin (5-HT) levels in perinatal rodents produces significant alterations in the development of the layer IV cortical representation of the mystacial vibrissae. Monoamine oxidase A (MAO(A)) knockout mice have highly elevated cortical 5-HT and completely lack barrels in somatosensory cortex (S-I). The present study was undertaken to determine whether the effects on thalamocortical development seen in MAO(A) knockout mice can be replicated in perinatal rats treated with an MAO(A) inhibitor and, second, to determine whether these effects persist with continued treatment or after discontinuation of the drug. Littermates were injected with either clorgyline (5 mg/kg) or sterile saline five times daily. Clorgyline administration from birth to postnatal day (P) 6, 8, or 10 produced increases of 1,589.4 +/- 53.3%, 1660.2 +/- 43.1% and 1,700.5 +/- 84.5 %, respectively, in cortical 5-HT as compared with controls. Serotonin immunocytochemistry, 1,1;-dioctadecyl-3,3,3", 3;-tetramethylindocarbocyanine perchlorate (DiI) labeling of thalamocortical afferents and Nissl and cytochrome oxidase staining of layer IV cellular aggregates demonstrated that clorgyline treatment from P0 to P6 produced a complete absence of any segmentation of vibrissae-related patches in S-I. However, continued treatment until P8 or P10 did not prevent the appearance of these patches. Animals treated with clorgyline from birth to P6 and killed on P8 or P10 had increases of 546.8 +/- 33.2% and 268.8 +/- 6.3% in cortical 5-HT and they had qualitatively normal vibrissae-related patterns in S-I. These results indicate that clorgyline treatment produces a transient disruption of vibrissae-related patterns, despite the continued presence of elevated cortical 5-HT.

Recent studies have suggested that 5-HT may modulate thalamocortical development in somatosensory cortex (S-I) of rats and mice, and that the 5-HT(1B) receptor may play a critical role in this process. Analysis of CO-stained sections through lamina IV of S-I in perinatal and adult 5-HT(1B) knockout mice revealed a normal vibrissae-related pattern, indicating that activation of the 5-HT(1B) receptor is not necessary for the normal development of the vibrissae representation in S-I.

Immunocytochemical and autoradiographic techniques were employed to determine the time course of expression of the serotonin (5-HT) transporter (SERT) on thalamocortical afferents in the rat's primary somatosensory cortex (S-I), and to correlate this expression to the transient vibrissae-related patterning of 5-HT immunostaining previously described. In additional in vivo and in vitro experiments, 5-HT and 3 H-5-HT were applied directly to the cortices of untreated and 5,7-dihydroxytryptamine-treated (5,7-DHT) rats in order to determine the period during which SERT functions on thalamocortical axons to take up 5-HT. In postnatal rats, SERT immunohistochemistry revealed a somatotopic patterning in S-I that persisted until P-15, which is 6 days after the disappearance of the vibrissae-related 5-HT immunostaining. 3 H-citalopram autoradiography revealed a vibrissae-related pattern in layer IV of S-I until at least P-30. Following destruction of raphe-cortical afferents with 5,7-DHT on the day of birth, this binding pattern remained visible until at least P-25, indicating that SERT located on thalamocortical axons is responsible for the 3 H-citalopram patterning observed in S-I. Tissue from 5,7-DHT-treated rats that had 5-HT applied directly to their cortices revealed a normal vibrissae-related pattern of 5-HT immunostaining in S-I at P-7 and P-11 but only a faint pattern at P-13 and none at P-14. In addition, 3 H-5-HT injected directly into S-I labeled layer IV barrels at P-6 and P-12 but not at P-18. The results of these experiments demonstrate that SERT is expressed by thalamocortical afferents and remains functional long after the vibrissae-related 5-HT immunostaining in cortex disappears.

Previous studies in adult animals have suggested that the peptides galanin and neuropeptide Y (NPY) may be upregulated in the same primary afferent neurons after peripheral axotomy. The present study was undertaken to determine whether such upregulation occurred in vibrissae-related primary afferent neurons and their axons after damage to the infraorbital nerve [ION; the trigeminal (V) branch that innervates the vibrissae follicles]. Double-labelling experiments demonstrated that approximately 75% of axotomized V ganglion cells and the central arbors of vibrissae-related primary afferents expressed both galanin and NPY after perinatal, but not adult, nerve damage. However, additional experiments demonstrated that the sensitive periods for lesion-induced upregulation of the two peptides and the period over which they were expressed after neonatal ION transection differed substantially. Staining for both peptides was increased after ION damage on P-0 through P-14, but only galanin staining was increased in vibrissae-related primary afferents after lesions on P-21. Galanin expression was elevated in vibrissae-related primary afferents in rats killed 3, 8, and 15 days after neonatal ION transection, while increased NPY was observed at only the middle time point. The lesion-induced increases in galanin and NPY in vibrissae-related ION primary afferents suggest that these peptides may modulate central V reorganization after such damage.