5-HT(1A) and 5-HT(2A) serotonin receptor turnover in adult rat offspring prenatally exposed to cocaine.

This study investigated the effects of prenatal exposure to cocaine on the intracellular kinetics (i.e. rate constant of receptor production and degradation) that govern the maintenance and regulation of cortical 5-HT(1A) and 5-HT(2A) receptor densities in offspring. Adult male rat offspring, prenatally exposed to saline or (-) cocaine (15 mg/kg, s.c., b.i.d, from gestational day 13 through 20), were injected with either vehicle or the irreversible receptor antagonist, EEDQ (10 mg/kg, s.c.), and sacrificed at various post-injection times to monitor the recovery of receptor densities in cerebral cortex. In both saline and cocaine exposed offspring, initial EEDQ-induced reductions (>80%) in 5-HT(1A) and 5-HT(2A) receptor densities were followed by a time-dependent repopulation that reached steady state ([B(max)](ss)) densities comparable to non-EEDQ treated controls by day 10 post-treatment. Calculation of 5-HT(1A) receptor kinetic parameters indicated that prenatal exposure to cocaine did not significantly alter: (1) the receptor production rate (saline: 0.809 fmol/mg protein/h; cocaine: 0.724 fmol/mg protein/h), (2) the receptor degradation rate constant (saline: 0.0063 h(-1); cocaine: 0.0062 h(-1)) or (3) the half-life (t(1/2)) of receptor repopulation (saline: 109.2 h; cocaine: 111.5 h). Similarly, 5-HT(2A) receptor rate constants for production (1. 550 fmol/mg protein/h) and degradation (0.0061 h(-1)) and consequently, t(1/2) (113.2 h), were not significantly altered by prenatal exposure to cocaine. These data suggest that within homogenates of cerebral cortex, prenatal exposure to cocaine did not alter the overall intracellular processes that underlie receptor production or degradation and determine steady state densities of 5-HT(1A) or 5-HT(2A) receptors.

Neurochemical changes in brain serotonin neurons in immature and adult offspring prenatally exposed to cocaine.

The present study investigates the age-dependent effects of prenatal cocaine exposure on changes in the neurochemical and functional status of brain serotonin neurons. Pregnant rats were administered either saline or (-)cocaine HCl (15 mg/kg, subcutaneously), twice daily from gestational days 13 through 20. Neurochemical changes in frontal cortex, hypothalamus, hippocampus, striatum and midbrain of prepubescent and adult offspring were determined by measuring: (1) the content of serotonin (5-HT) and its major metabolite 5-hydroxyindolacetic acid (5-HIAA), and (2) the ability of the serotonin releasing drug p-chloroamphetamine (PCA) to reduce brain serotonin levels. Brain catecholamine content was determined in progeny for comparative purposes. Prior to maturation, prenatal exposure to cocaine did not alter basal levels of brain 5-HT or 5-HIAA in any brain region examined. However, in adult progeny prenatally exposed to cocaine, basal 5-HT content was significantly reduced in the frontal cortex (-32%) and hippocampus (-40%), suggesting maturation-dependent effects of prenatal cocaine exposure on brain 5-HT neurons. Consistent with the maturational onset of changes in 5-HT, striatal dopamine was significantly reduced (-10%) by prenatal exposure to cocaine only in adult offspring. Reductions in 5-HT in most brain regions, produced by pharmacological challenge with p-chloroamphetamine (PCA), were comparable in prenatal saline versus cocaine offspring. One notable exception was the markedly greater reduction (-40%) in 5-HT in the midbrain of immature offspring prenatally exposed to cocaine, suggesting alterations in midbrain 5-HT neurons prior to maturation. Overall, these data demonstrate prenatal cocaine exposure produces region-specific changes in 5-HT neurons in offspring with some deficits occurring only following maturation.

Prenatal cocaine exposure potentiates 5-HT(2a) receptor function in male and female rat offspring.

We have reported previously prenatal cocaine-induced functional deficits in serotonergic terminals, and gender-specific supersensitivity of postsynaptic 5-HT(1A) receptor-mediated hormone responses in offspring. This study investigates the effects of prenatal exposure to cocaine on postsynaptic 5-HT(2A) receptor-mediated responses in prepubescent male and female offspring. Pregnant rats were administered saline or (-)cocaine (15 mg/kg, s.c., b.i.d) from gestational day 13 through 20. Changes in 5-HT(2A) receptor function in offspring were assessed by differences in the ability of DOI [4-iodo, 2,5-dimethoxyphenyl-isopropylamine; 2. 0 mg/kg, s.c.] to elevate plasma levels of the hormones ACTH, corticosterone and renin. Basal hormone levels in male and female progeny were unaffected by prenatal cocaine exposure. However, prenatal exposure to cocaine significantly potentiated the magnitude of the ACTH response to DOI in both male (+19%) and female (+43%) progeny. Similarly, the DOI-induced elevation of plasma renin was markedly potentiated in male (+51%) and female (+83%) cocaine-exposed offspring. Although DOI significantly elevated corticosterone levels in both male and female offspring, the magnitude of corticosterone responses was not altered by prenatal exposure to cocaine. Densities of agonist ((125)I-DOI)-labeled receptors in hypothalamus and cortex were unaltered by prenatal exposure to cocaine. These data indicate prenatal cocaine-induced supersensitivity of postsynaptic 5-HT(2A) receptor function in male and female offspring without changes in receptor density. Synapse: 35:163-172, 2000.

Two amino acids within the alpha4 helix of Galphai1 mediate coupling with 5-hydroxytryptamine1B receptors.

We previously reported that residues 299-318 in Galphai1 participate in the selective interaction between Galphai1 and the 5-hydroxytryptamine1B (5-HT1B) receptor (Bae, H., Anderson, K., Flood, L. A., Skiba, N. P., Hamm, H. E., and Graber, S. G. (1997) J. Biol. Chem. 272, 32071-32077). The present study more precisely defines which residues within this domain are critical for 5-HT1B receptor-mediated G protein activation. A series of Galphai1/Galphat chimeras and point mutations were reconstituted with Gbetagamma and Sf9 cell membranes containing the 5-HT1B receptor. Functional coupling to 5-HT1B receptors was assessed by 1) [35S]GTPgammaS binding and 2) agonist affinity shift assays. Replacement of the alpha4 helix of Galphai1 (residues 299-308) with the corresponding sequence from Galphat produced a chimera (Chi22) that only weakly coupled to the 5-HT1B receptor. In contrast, substitution of residues within the alpha4-beta6 loop region of Galphai1 (residues 309-318) with the corresponding sequence in Galphat either permitted full 5-HT1B receptor coupling to the chimera (Chi24) or only minimally reduced coupling to the chimeric protein (Chi25). Two mutations within the alpha4 helix of Galphai1 (Q304K and E308L) reduced agonist-stimulated [35S]GTPgammaS binding, and the effects of these mutations were additive. The opposite substitutions within Chi22 (K300Q and L304E) restored 5-HT1B receptor coupling, and again the effects of the two mutations were additive. Mutations of other residues within the alpha4 helix of Galphai1 had minimal to no effect on 5-HT1B coupling behavior. These data provide evidence that alpha4 helix residues in Galphai participate in directing specific receptor interactions and suggest that Gln304 and Glu308 of Galphai1 act in concert to mediate the ability of the 5-HT1B receptor to couple specifically to inhibitory G proteins.

Infraorbital nerve transection alters serotonin transporter expression in sensory pathways in early postnatal rat development.

The serotonin transporter MRNA has been found throughout the trigeminal sensory system late in gestation and during early postnatal development, a period known to be critical for maturation of the sensory circuitry. The purpose of the present study was to determine whether sensory denervation in newborn rat pups would alter either the density or pattern of expression of the 5-HT transporter (5-HTT) within the trigeminal system. We combined autoradiographic localization of 5-HT transporters and in situ hybridization techniques to visualize both the transporter protein and mRNA in thalamic sensory neurons and in the somatosensory cortex following unilateral infraorbital nerve transection at postnatal day 1. For comparative purposes, similar measurements were conducted in thalamic visual neurons as well as in the visual cortex. Lesion of the infraorbital nerve decreased the [3H]citalopram labelling of 5-HT transporters in the ventral basal and ventral medial areas of the thalamus contralateral to the lesion, while labelling of 5-HT transporters was decreased in both contralateral and ipsilateral sides of the lateral genicuate (visual thalamus). Citalopram labelling of 5-HT transporters was not significantly altered in somatosensory or in cingulate cortex, however a significant decrease was observed in the visual cortex. In contrast, there were no obvious changes in the intensity of the 5-HT mRNA hybridization signal in sensory or visual thalamic areas. Given that the serotonin transporter regulates extracellular concentrations of 5-HT, the present data suggest that altered peripheral innervation and thereby altered sensory inputs to the thalamus during fetal development can potentially influence 5-HT transporter densities and thus, may influence extracellular levels of 5-HT in thalamus and cortex during a critical period of synapse formation. In turn, modulation of 5-HT transporter levels may influence extracellular concentrations of 5-HT in thalamus and cortex during a critical period of synapse formation.

Prenatal exposure to fluoxetine (Prozac) produces site-specific and age-dependent alterations in brain serotonin transporters in rat progeny: evidence from autoradiographic studies.

The present study provides the first autoradiographic evidence of age-dependent regional changes in the density of serotonin (5-HT) transporters in offspring following prenatal exposure to fluoxetine. Pregnant rats received either saline or fluoxetine (10 mg/kg, s.c.) daily from gestational day 13 through 20. The density of [3H]citalopram-labeled 5-HT transporters was determined in forebrain regions and in midbrain raphe nuclei of prepubescent and adult male offspring. Brain regions representing integral components of the limbic system were particularly sensitive to the prenatal treatment. For example, prenatal fluoxetine exposure significantly altered the density of 5-HT transporters in subregions of the hypothalamus (dorsomedial nucleus, -21%; lateral hypothalamus, +21%), hippocampus (CA2, +47%; CA3, +38%), and amygdala (basolateral nucleus, +32%; medial nucleus, +44%) in prepubescent offspring. However, 5-HT transporter density in the dorsal and median raphe was unaltered in this same group of offspring. In adult offspring, 5-HT transporter densities, in all brain regions examined, were not significantly altered by prenatal exposure to fluoxetine. The present study also identifies significant age-related differences in 5-HT transporter densities between prepubescent and adult control offspring. For example, in adult control offspring, densities of 5-HT transporters were significantly greater in the cingulate cortex (+33%), basolateral amygdala (+58%), and CA1 area of the hippocampus (+78%); but significantly lower in the temporal cortex (-65%) and median raphe (-25%). The age-dependent and site-specific alterations in the density of 5-HT transporters suggests that either 5-HT innervation and/or 5-HT neuron function in various forebrain regions may be altered by prenatal exposure to fluoxetine.

Effect of prenatal fluoxetine (Prozac) exposure on brain serotonin neurons in prepubescent and adult male rat offspring.

The present study examines the consequences of prenatal fluoxetine exposure on brain serotonin [5-hydroxytryptamine (5-HT)] neurons in male offspring. Pregnant rats were administered either saline or fluoxetine (10 mg/kg s.c.) daily from gestational day 13 through gestational day 20. The biochemical status of brain 5-HT neurons was assessed in prepubescent and adult offspring by measuring 1) the 5-HT and 5-hydroxyindoleacetic acid content, 2) the density of [3H]paroxetine-labeled 5-HT uptake sites and 3) the ability of the 5-HT-releasing drug p-chloroamphetamine to reduce 5-HT content. Biochemical parameters were assessed in the frontal cortex, hypothalamus, hippocampus, striatum and midbrain. Comparative effects on dopamine and norepinephrine content in selected regions were also determined. Prenatal exposure to fluoxetine significantly reduced (-28%) 5-HT content in the frontal cortex of prepubescent but not adult male offspring. In contrast, in adult progeny prenatal fluoxetine exposure produced a significant decrease only in midbrain 5-HT content (-28%). In addition, p-chloroamphetamine markedly reduced 5-HT content in all brain regions examined, but the ability of p-chloroamphetamine to reduce 5-HT content was significantly attenuated only in the midbrain of adult progeny prenatally exposed to fluoxetine. No significant differences were observed between control and fluoxetine-exposed progeny with respect to brain 5-hydroxyindoleacetic acid content, the 5-hydroxyindoleacetic acid/5-HT ratio or the density of 5-HT uptake sites, regardless of the brain region examined or the age of the offspring. These data provide additional evidence that prenatal exposure to fluoxetine can produce limited, rather than global, changes in brain 5-HT neurons in male rat offspring and that the effects observed are region-specific and age-dependent. The potential functional consequences and clinical implications of these alterations in brain 5-HT systems remain to be elucidated.