Expression of mitogen-activated protein kinase pathways during postnatal development of rat heart

The loss of ability to proliferate (terminal differentiation) and reduction in capability to resist ischemia are key phenomena observed during postnatal development of the heart. Mitogen-activated protein kinases (MAPKs) mediate signaling pathways for cell proliferation/differentiation and stress responses such as ischemia. In this study, the expression of these kinases and their associated kinases were investigated in rat heart ventricle. Extracts of 1-, 10-, 20-, 50-, and 365-day-old rat heart ventricles were probed with specific antibodies and their immunoreactivities were quantified by densitometry. Most of the mitogenic protein kinases including Raf1, RafB, Mek1, Erk2, and Rsk1 were significantly down-regulated, whereas the stress signaling kinases, such as Mlk3, Mekkl, Sekl, Mkk3, and Mapkapk2 were up-regulated in expression during postnatal development. Most MAP kinases including Erk1, JNKs, p38 Hog, as well as Rsk2, however, did not exhibit postnatal changes in expression. The proto-oncogene-encoded kinases Mos and Cot/Tpl 2 were up-regulated up to two- and four-fold, respectively, during development. Pakl, which may be involved in the regulation of cytoskeleton as well as in stress signaling, was downregulated with age, but the Pak2 isoform increased only after 50 days. All of these proteins, except RafB, were also detected in the isolated adult ventricular myocytes at comparable levels to those found in adult ventricle. Tissue distribution studies revealed that most of the protein kinases that were up-regulated during heart development tended to be preferentially expressed in heart, whereas the downregulated protein kinases were generally expressed in heart at relatively lesser amounts than in most of other tissues.     

Sodium current expression during postnatal development of rat outer hair cells

Outer hair cells of the cultured organ of Corti from newborn rats (0-11 days after birth) were studied in the whole-cell patch-clamp configuration. A voltage-activated sodium current was detected in 97% (n = 109) of the cells at 0-9 days after birth. The properties of this current were: (1) its activation and inactivation kinetics were fast and voltage-dependent, (2) the voltage at half-maximum activation was -45.0 mV, (3) its steady-state inactivation was temperature-sensitive (the half-inactivating voltage was -92.6 mV at 23 degrees C and -84.8 mV at 37 degrees C), (4) the reversal potential (80 mV) was close to the sodium equilibrium potential and currents could be abolished by the removal of extracellular sodium, and (5) tetrodotoxin blocked the current with a Kd of 474 nmol/l. Current amplitudes were up to 1.7 nA at room temperature. Mean current amplitudes showed a developmental time course with a maximum at postnatal days 3 and 7 for outer hair cells from the basal and apical part of the cochlea, respectively. In current-clamp mode cells had membrane potentials of -59.7 +/- 11.7 mV (n = 9). When cells were hyperpolarized by constant current injection, depolarizing currents were able to trigger action potentials. At 18 days after birth, sodium currents were greatly reduced and barely detectable. The results show that, unlike adult outer hair cells, immature outer hair cells regularly express voltage-gated sodium channels. However, due to mismatching of the sodium current inactivation range and membrane potential in vitro, a physiological function appears questionable.     

The organization and postnatal development of the commissural projection of the rat somatic sensory cortex

Anterograde and retrograde tracing experiments have been used to demonstrate the origin and terminal distribution of commissural fibers in the first somatosensory cortex (SI) of the rat. The commissural fibers originate from pyramidal cells of all layers, but predominantly from layers III and V. The fibers terminate in a series of approximately vertical bands. In each of these there are concentrations of terminals extending from the inner portion of the molecular layer to the deep portion of layer III as well as in the superficial part of layer V, and in layer VI. Discrete vertical bands of cortex are reciprocally connected across the midline to give both the origin and terminal regions of the projection a patchy or "columnar" appearance. The commissural fibers arise from and terminate in areas of the cortex that lie between and alongside the aggregations of granule cells that distinguish SI of the rat. No commissural fibers terminate within the aggregations of layer IV cells themselves but the more superficial terminal ramifications may come to overlie these aggregations. A heterotopic projection to the contralateral second somatosensory cortex has been observed and is similar in form to the homotopic projection to SI. Many commissural fibers have crossed the midline in the corpus callosum by the day of birth but lie in the underlying white matter and do not enter the cortical plate until at least the third postnatal day. During the first postnatal week these fibers grow somewhat diffusely into the maturing cortex and their topographic and laminar pattern of distribution attains its adult characteristics by the end of the first week. Commissural axons, thus, arise from immature cells but the maturation of cell form seems to precede the ingrowth of these axons and the acquisition of commissural synapses.     

Polarized expression of the antidepressant-sensitive serotonin transporter in epinephrine-synthesizing chromaffin cells of the rat adrenal gland

Antidepressant-sensitive serotonin (5-hydroxytryptamine, 5HT) transporters (SERTs) clear the amine from extracellular spaces in the CNS and periphery as a mechanism for transmitter inactivation and recycling. Although it is known that SERTs are preferentially expressed on basolateral domains in transfected epithelial cells, details of the transporter's membrane localization in vivo are lacking. 5HT and 5HT receptors have been identified in the rodent adrenal gland. Using SERT antagonist autoradiography, we establish the presence of antidepressant-sensitive transport sites in the rat adrenal medulla. Immunofluorescence experiments using antibodies specific for the SERT COOH and NH2 termini, for 5HT, or for catecholamine biosynthetic enzymes suggest that SERT mediates intra-cellular 5HT accumulation by epinephrine-secreting chromaffin cells. Using confocal microscopy, we establish that SERT expression is nonuniformly distributed along the plasma membrane of chromaffin cells. Notably, SERT immunoreactivity is largely absent from plasma membranes bordering smooth muscle that surrounds vascular sinusoids. Rather, SERT is highly expressed in membranes adjoining other chromaffin cells, consistent with a role for 5HT and SERT in autocrine or paracrine control of chromaffin cell physiology. SNAP-25, a t-SNARE protein implicated in neurotransmitter release, was found to colocalize with SERT. In contrast, Na,K ATPase and NCAM are uniformly distributed along the entire perimeter of chromaffin cell membranes. These findings underscore a role for 5HT and SERT in adrenal physiology, reveal unrecognized polarity of chromaffin cell plasma membranes, and warrant a consideration of common targeting mechanisms localizing amine transporters near release sites.     

GAP-43 expression in the medulla of macaque monkeys: changes during postnatal development and the effects of early median nerve repair

Expression of GAP-43, a neuronal specific growth associated phosphoprotein, has been highly correlated with the growth and remodeling of the nervous system during development and regeneration. As part of an effort to understand mechanisms of developmental plasticity in the somatosensory system, we determined how the expression of GAP-43 is affected by prenatal and early postnatal nerve cut and repair in macaque monkeys. We also observed normal developmental changes in the expression of GAP-43 during early postnatal life in macaque monkeys. The normal cuneate nucleus, as well as other nuclei of the ascending somatosensory pathways, had low levels of GAP-43 at birth that increased by 3 months and declined thereafter to reach adult levels between 8 and 15 months of age. Fiber tracts expressed low levels of GAP-43 at all postnatal ages, except the pyramidal tract which demonstrated high levels a birth that decreased over the first year. These observations suggest a gradual but differential synaptic maturation in lower brain stem nuclei as macaque monkeys mature. Greatly increased levels of GAP-43 were observed at the time of birth in the cuneate nucleus of two macaque monkeys with prenatal (E94 and El 14) nerve repair. Such an increase was not found after prenatal nerve repair with a postnatal survival time of 15 months, or after early postnatal nerve repair with short (80 days) or long (20 months) survivals. The results suggest that reorganization mechanisms at central terminals of peripheral nerves are very different following prenatal than postnatal nerve damage.     

Differential expression of Fos protein after transection of the rat infraorbital nerve in the trigeminal nucleus caudalis

To determine the effects of nerve injury on Fos expression, temporal and spatial distributions of Fos-positive neurons in the trigeminal nucleus caudalis were examined after tissue injury for isolation of the infraorbital nerve as controls and transection of this nerve as well as noxious chemical stimulation by formalin injection in adult rats. Fos immunoreactivity was markedly elevated in laminae I and II of the only ipsilateral nucleus caudalis 2 h after these surgical procedures and noxious chemical stimulation. The distributions of Fos-positive neurons were restricted rostro-caudally following formalin injection and tissue injury compared to transection of the infraorbital nerve. One day after tissue injury and nerve transection, however, Fos-positive neurons were distributed bilaterally in laminae III and IV extending rostro-caudally and medio-laterally in this nucleus, and this persisted over the 2-week study period. The number of Fos-positive neurons in the side ipsilateral to nerve transection was markedly less than that in the contralateral side whereas positive neurons in the tissue injured rats were distributed symmetrically along the rostro-caudal axis. There was no difference in the contralateral sides between nerve transection and tissue injury groups. The rostro-caudal level showing reduction in Fos expression corresponded roughly to the sites of central termination of the injured nerve in this nucleus, suggesting a role for the primary afferents in the reduction of Fos expression in laminae III and IV neurons of the ipsilateral nucleus caudalis.     

Dopamine transporter development in postnatal rat striatum: an autoradiographic study with [3H]WIN 35,428

The dopamine transporter mediates the reinforcing effects of cocaine, thus playing a central role in human cocaine addiction, and perhaps providing the mechanism for inducing the effects of prenatal cocaine exposure. This possibility has stimulated growing interest in the normal and abnormal development of this transporter. [3H]WIN 35,428 is a cocaine analog that is useful for studying the distribution and density of the dopamine transporter in striatum and other brain regions. The postnatal development of the dopamine transporter in the rat striatum was measured by quantitative autoradiography with [3H]WIN 35,428. Dopamine transporter levels were low at birth, increased through day 15, followed by much more rapid growth in late postnatal development. The majority of the transporter sites appeared after day 15. Lateral to medial and anterior to posterior gradients in transporter density were established early during development, and there was also an early concentration of transporter in striosomes that became difficult to identify by day 15. Differences between the developmental patterns described here and studies using other ligands for the dopamine transporter suggest there are significant differences in the transporter binding sites for these drugs. These differences in transporter ligand binding characteristics may reflect developmental changes in post-translational modification of the transporter and/or changes in the functional activity rather than simply the presence of the transporter.     

Cellular localization and expression of the serotonin transporter in mouse brain

The high-affinity serotonin (5-HT) transporter (5-HTT) plays an important role in the removal of extracellular serotonin, thereby modulating and terminating the action of this neurotransmitter at various pre- and post-synaptic serotonergic receptors and heteroreceptors. In order to characterize the anatomical distribution of the 5-HTT in mouse brain, in situ hybridization histochemistry using 35S-labeled riboprobes was performed. These results were compared with 5-HTT binding site distribution as evaluated by [125I]RTI-55 autoradiography. High levels of 5-HTT mRNA were detected in all brain stem raphe nuclei, with variations in labeling among the various subnuclei. Those brain areas known to possess serotonergic cell bodies stained intensely for both 5-HTT mRNA and 5-HTT binding sites. In contrast to previous findings in rat brain, the highest densities of 5-HTT sites were found in areas outside the raphe complex, particularly in the substantia nigra, globus pallidus, and superior colliculi.     

Platelet-derived growth factor B-chain expression in the rat retina and optic nerve: distribution and changes after transection of the optic nerve

To test the possible involvement of platelet-derived growth factor B-chain (PDGF-B) in anterograde and retrograde degenerations of the CNS neurons, we studied the changes of PDGF-B localization and its mRNA expression in the rat retina and optic nerve (ON) after unilateral ON transection, using immunohistochemistry and in situ hybridization. In the control retinas immunoreactivity for PDGF-B and its mRNA expression were localized in the retinal ganglion cells (RGCs) and the nerve fiber layer. After ON transection PDGF-B immunoreactivity in the nerve fiber layer started to decrease on post-injury day 3 or 4. Atrophic changes in the RGCs started on day 5 just after the decrease of PDGF expression, and thereafter the RGC number decreased. In the longitudinal section of the ON rostral to the transected site, swollen axons showed intense PDGF-B immunoreactivity and macrophages, and some glial cells revealed a significant increase in both immunoreactivity and hybridization signals. Based on these findings, we hypothesized that the decrease in PDGF-B in RGCs after axotomy causes the loss of RGCs, and that increased PDGF-B expression in the ON plays a role in the cascade of tissue reactions following ON transection.     

Neonatal chorda tympani transection alters adult preference for ammonium chloride in the rat.

The immature gustatory system of the neonatal rat is characterized by sensitivity to disruption by early interventions such as receptor or nerve damage. The present studies examined the effect of chorda tympani transection (neoCTX) of neonates on adult preference for salt and nonsalt stimuli. NeoCTX at 10 days of age led to a striking change in adult rats' preference for NH?C1 solutions but little change in preference for other solutions, including NaC1 and KC1. Permanent anatomical effects of neoCTX included failure of the nerve to regenerate and a loss of all fungiform taste buds. Preference for NH?C1 was not due to an inability to discriminate it from NaC1. Following taste aversion conditioning to NaC1, neoCTX rats clearly distinguished between NaC1 and NH?C1. The effects on NH?C1 preference reflect a sensitive period during development because adult rats receiving similar surgery did not show any change in NH?C1 preference. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Quantitative data on serotonin nerve terminals in adult rat neocortex

Serotonin (5-HT) nerve terminals, specifically labeled with [3H]5-HT have been counted in light microscope radioautographs from the fronto-parietal neocortex of adult rats, following prolonged superfusions with relatively high concentrations of tracer (10(-5) M or 10(-4) M), and after 15 or 30 days of radioautographic exposure. Comparative analysis of the results indicated that all 5-HT varicosities did not possess the same capacity to accumulate the exogenous amine. Nevertheless, superfusions with 10(-4) M[3H]5-HT provided a sufficient loading of 5-HT boutons to allow their complete detection within any given layer of the neocortex. The labeled varicosities were found to be present within all cortical layers, except layer VI. Their total number was extrapolated to be approximately 1 X 10(6)/cu.mm of cortex. This figure represented a mean incidence of one 5-HT nerve terminal/850-1400 cortical synapses (0.07-0.12%). The intralaminar density of 5-HT innervation increased progressively from layer V to layer I, in a distribution pattern suggestive of unspecific afferents. Based on endogenous 5-HT levels of 346 +/- 32 ng/g for the fronto-parietal neocortex, the mean 5-HT content per varicosity could be estimated to be 0.35 X 10(-3) pg, and the concentration 1900 ng/g wet weight of terminal or 0.2%. In view of their particular ultrastructural features7, ubiquitous repartition and endogenous amine content, 5-HT nerve endings may be considered as capable of exerting a rather widespread influence in the neocortex.     

Ontogenic expression of natriuretic peptide mRNAs in postnatal rat brain: implications for development?

The central natriuretic peptide system is composed of at least three structurally homologous and uniquely distributed peptides and receptors which are thought to be involved in the central regulation of cardiovascular and autonomic function and more recently been shown to affect cellular growth and proliferation, processes pertinent to mammalian development. As such, following our initial mapping of preproatrial natriuretic peptide (ppANP) mRNA in adult brain [M.C. Ryan, A.L. Gundlach, Anatomical localization of preproatrial natriuretic peptide mRNA in the rat brain by in situ hybridization histochemistry: in olfactory regions, J. Comp. Neurol., 356 (1995) 168-182], it was of interest to determine the ontogenic expression of natriuretic peptide mRNAs in the developing rat brain. Using in situ hybridization histochemistry of specific [35S]- or [33P]-labeled oligonucleotides, ppANP and preproC-type natriuretic peptide (ppCNP) mRNAs were detected in the developing rat brain from postnatal day 4 to day 60 (adult). PpANP mRNA was observed in many hindbrain, but only some forebrain, regions at postnatal day 4. Regional differences in the temporal expression of ppANP mRNA were apparent with ppANP mRNA detected in the medial preoptic area, mammillary nuclei and medial habenular nucleus at postnatal day 4 and in other areas including the arcuate and dorsomedial hypothalamic nuclei and in olfactory and limbic regions at postnatal day 10. A number of regions also exhibited transient expression of ppANP mRNA such as the bed nucleus of the stria terminalis and the medial cerebellar nucleus. In contrast, ppCNP mRNA was detected at relatively high levels in several regions on postnatal day 4 including olfactory nuclei, the hippocampus and particularly the pontine nucleus. The level of expression appeared to increase markedly in most regions including forebrain olfactory and hippocampal areas and in brainstem regions including the pontine nucleus, the parvocellular and lateral reticular and spinal trigeminal nuclei by postnatal days 10 and 13, but decreased from this peak to equivalent to adult levels by postnatal day 28. The differential and transient expression of the natriuretic peptides during postnatal development, together with previous reports of the ontogenic regulation of natriuretic peptide receptor expression and binding patterns, further suggests their involvement in developmental processes in the rat CNS and provides information relevant to the likely functional development of natriuretic peptide-utilizing pathways.     

Calcitonin gene-related peptide and alpha-CGRP mRNA expression in cranial motoneurons after hypoglossal nerve injury during postnatal development

Changes in calcitonin gene-related peptide (CGRP) immunoreactivity and alpha-CGRP mRNA expression were determined in the hypoglossal nucleus after the nerve was crushed or transected in rats at 10, 14 and 21 days postnatal. alpha-CGRP mRNA expression was determined in normal, noninjured, hypoglossal nuclei at the three ages and after both injuries in 10 and 21 days postnatal rats. Reinnervation and neuronal survival were assayed. Although the three age groups expressed comparable levels of alpha-CGRP mRNA and its peptide in intact, hypoglossal nuclei, axonal injury produced age-dependent alterations in alpha-CGRP mRNA and CGRP. In the 21 days postnatal rats, changes in alpha-CGRP mRNA and peptide mimicked those reported in adult motoneurons after the same injuries. CGRP was elevated until reinnervation after nerve crush, whereas biphasic elevations occurred after nerve transection. In 21 days postnatal rats, increases in alpha-CGRP mRNA preceded elevations of the peptide but a greater increase resulted initially after nerve transection. An upregulation of alpha-CGRP mRNA also developed initially after both injuries in 10 days postnatal rats but subsequent elevations of alpha-CGRP mRNA did not materialize. In contrast, CGRP immunoreactivity did not increase after either injury in 10 days postnatal rats and, in fact decreased. Levels of CGRP immunoreactivity did not differ from normal amounts after either nerve injury in 14 days postnatal rats. Substantial neuronal cell loss occurred after each injury in 10 and 14 days postnatal rats but was not found in 21 days postnatal rats. Tongue reinnervation by surviving motoneurons was established after all injury paradigms except 10 days postnatal transection. The current findings demonstrate an age-dependent correlation between injury-induced expression of CGRP and hypoglossal motoneuron survival.     

Immunohistochemical localization of serotonin transporter in normal and colchicine treated rat brain

The activity of liver microsomal CYP2E1 is commonly measured as the rate of 5-chloro-2-benzoxazolone (chlorzoxazone) 6-hydroxylation, which requires separation of 6-hydroxychlorzoxazone and chlorzoxazone by high pressure liquid chromatography (HPLC). In the present study, we describe a solvent extraction (non-HPLC) assay for measuring CYP2E1 activity, based on the 6-hydroxylation of [14C]chlorzoxazone. When [14C]chlorzoxazone was incubated with human or rat liver microsomes in the presence of NADPH, the major product formed was 6-[14C]hydroxychlorzoxazone. Unreacted [14C]chlorzoxazone was quantitatively extracted from the incubation mixture with dichloromethane under conditions that resulted in approximately 45% extraction of 6-[14C]hydroxychlorzoxazone. The amount of 6-[14C]hydroxychlorzoxazone remaining in the aqueous incubation mixture ( approximately 55% of the total amount formed) was quantified by liquid scintillation spectrometry. The limit of detection for this assay was 100 pmol of 6-[14C]hydroxychlorzoxazone. The solvent extraction procedure was validated by comparing the rates of formation of 6-[14C]hydroxychlorzoxazone with those determined by HPLC under a variety of experimental conditions. The close correspondence between the two analytical methods suggests that the extraction procedure for measuring 6-[14C]hydroxychlorzoxazone provides a simple, sensitive, and rapid alternative to the HPLC procedure for measuring CYP2E1 activity. In rats, the assay is not specific for CYP2E1 because CYP1A1 also catalyzes the 6-hydroxylation of chlorzoxazone. Recombinant human CYP1A1 also catalyzed the 6-hydroxylation of chlorzoxazone (at (1)/(5) the rate of CYP2E1), although CYP1A1 is not expressed in human liver microsomes. The non-HPLC assay was used to investigate the postulated role of CYP1A2 in the 6-hydroxylation of chlorzoxazone by human liver microsomes. Recombinant CYP1A2 did not catalyze the 6-hydroxylation of chlorzoxazone, and studies with 1-[(3,4-dimethoxyphenyl)methyl]-6,7-dimethoxyisoquinoline, which inhibits CYP1A2 but not CYP2E1, indicated that, in human liver microsomes, the 6-hydroxylation of chlorzoxazone is catalyzed by CYP2E1 with little or no contribution from CYP1A2 enzymes over a wide range of substrate concentrations.     

Adipose tissue development during early postnatal life in ewe-reared lambs

This study examines the precise time course that brown adipose tissue (BAT) takes to adopt the characteristics of white adipose tissue in postnatal lambs. Perirenal adipose tissue was sampled from ewe-reared lambs within 1 h of birth and at 1, 2, 4, 7, 14, 21 and 30 days of age and analysed for the amount of mRNA for uncoupling protein (UCP), the amount and activity of UCP, and protein, mitochondrial protein and lipid content. This was combined with measurements of colonic temperature and jugular venous plasma concentrations of thyroid hormones and insulin-like growth factor-1 (IGF-1). Over the first 4-7 days of age, large quantities of UCP mRNA were associated with a peak in plasma triiodothyronine concentration at 2 days of age followed by a maximal amount and activity of UCP at 4 days and a basal colonic temperature of 39.3 degrees C. Between 7 and 30 days there was a large increase in lipid deposition as the amount and activity of UCP and the amount of UCP mRNA declined to basal values and colonic temperature was maintained at 40 degrees C. A significant positive relationship between perirenal adipose tissue lipid content and plasma IGF-1 concentration was observed throughout the study period. It is concluded that ovine adipose tissue maturation occurs in two distinct phases over the first month of life. The precise time scale of this process could be regulated in part by the lamb's body temperature which determines whether adipose tissue is required for heat production (i.e. BAT) or as an endogenous energy source (i.e. white adipose tissue).     

Multiple subtypes of serotonin receptors are expressed in rat sensory neurons in culture

[3H]5-HT revealed the presence of serotonin receptors in cultured rat sensory neurons. [3H]5-CT binding was inhibited by cyanopindolol with an IC50 of 0.87 +/- 0.30 nM, suggesting the expression of the 5-HT1B receptor in these neurons. The presence of 5-HT1B receptors was confirmed by the displacement of [125I]Iodocyanopindolol binding by cyanopindolol with an IC50 of 2.43 +/- 0.81 nM. 5-HT1B receptors are the predominant type of serotonin receptors labeled by [3H]5-HT in cultured DRG neurons, representing approximately 60% of the specific [3H]5-HT binding sites. In addition, 5-HT1D and 5-HT2A receptor binding was also found in these neurons. RT-PCR analysis of RNA isolated from embryonic sensory neurons in culture confirmed the expression of 5-HT1B, 5-HT1D and 5-HT2A receptor mRNA. It also demonstrated the presence of 5-HT1F, 5-HT2C, 5-HT3, 5-HT4, 5-HT5A and 5-HT5B receptor mRNA and the absence of 5-HT1A, 5-HT1E, 5-HT2B, 5-HT6 and 5-HT7 mRNA. The identification of multiple subtypes of serotonin receptors expressed in cultured embryonic sensory neurons suggests that DRG neuronal cultures may be an excellent model to examine the direct effects of serotonin on the activity of these sensory neurons.     

Serotonin transporter messenger RNA in the developing rat brain: early expression in serotonergic neurons and transient expression in non-serotonergic neurons

Serotonin has been shown to affect the development of the mammalian nervous system. The serotonin transporter is a major factor in regulating extracellular serotonin levels. Using in situ hybridization histochemistry the rat serotonin transporter messenger RNA was localized during embryogenesis, the first four weeks postnatally and adulthood. Three general classes of serotonin transporter messenger RNA expression patterns were observed: (i) early detection with continued expression through adult age, (ii) transient expression colocalized with vesicular monoamine transporter 2 messenger RNA but with no detectable tryptophan hydroxylase immunoreactivity, and (iii) transient expression in the apparent absence of both vesicular monoamine transporter 2 messenger RNA and tryptophan hydroxylase immunoreactivity. For example, hybridization for serotonin transporter messenger RNA was strong in serotonin cell body-containing areas beginning early in gestation, and remained intense through adulthood. Immunoreactivity for tryptophan hydroxylase, the rate-limiting enzyme in serotonin synthesis, was completely overlapping with the presence of serotonin transporter messenger RNA in raphe nuclei postnatally. Sensory relay systems including the ventrobasal nucleus (somatosensory), lateral and medial geniculate nuclei (visual and auditory, respectively) as well as trigeminal, cochlear and solitary nuclei were representative of the second class of observations. In general, the limbic system expressed serotonin transporter messenger RNA in the third pattern with various limbic structures differing in the timing of expression. Septum, olfactory areas and the developing hippocampus contained serotonin transporter messenger RNA early in the developing brain. Other regions such as cingulate and frontopolar cortex exhibited hybridization peri- and postnatally, respectively. Several hypothalamic nuclei and pituitary transiently expressed serotonin transporter messenger RNA either postnatally or perinatally, respectively. If the observed patterns correlate with functional protein expression, distinct classes of serotonin transporter messenger RNA expression may reflect different functional roles for the serotonin transporter and serotonin, itself. Since the serotonin transporter is a target for a number of addictive substances including cocaine and amphetamine derivatives as well as antidepressants, transient expression of the serotonin transporter might suggest a window of vulnerability of associated cells to fetal drug exposure. Re-uptake, storage and re-release from non-serotonergic neurons might serve as a feedback mechanism from target neurons to serotonergic neurons. Alternatively, the transient expression of serotonin transporter messenger RNA may reflect critical periods important for tight regulation of extracellular serotonin in several brain regions, and may indicate previously unappreciated roles for serotonin as a developmental cue.