Differential expression of 5HT-1A, alpha 1b adrenergic, CRF-R1, and CRF-R2 receptor mRNA in serotonergic, gamma-aminobutyric acidergic, and catecholaminergic cells of the rat dorsal raphe nucleus.
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The dorsal raphe nucleus (DR) has a topographic neuroanatomy consistent with the idea that different parts of this nucleus subserve different functions. Here we use dual in situ hybridization to describe the rostral-caudal neurochemical distribution of three major cell groups, serotonin (5-hydroxytryptamine; 5-HT), gamma-aminobutyric acid (GABA), and catecholamine, and their relative colocalization with each other and mRNA encoding four different receptor subtypes that have been described to influence DR responses, namely, 5HT-1A, alpha(1b) adrenergic (alpha(1b) ADR), and corticotropin-releasing factor type 1 (CRF-R1) and 2 (CRF-R2) receptors. Serotonergic and GABAergic neurons were distributed throughout the rostral-caudal extent of the DR, whereas catecholaminergic neurons were generally restricted to the rostral half of the nucleus. These phenotypes essentially represent distinct cell populations, because the neurochemical markers were rarely colocalized. Both 5HT-1A and alpha(1b) ADR mRNA were highly expressed throughout the DR, and the vast majority of serotonergic neurons expressed both receptors. A smaller percentage of GABAergic neurons also expressed 5HT-1A or alpha(1b) ADR mRNA. Very few catecholaminergic cells expressed either 5HT-1A or alpha(1b) ADR mRNA. CRF-R1 mRNA was detected only at very low levels within the DR, and quantitative colocalization studies were not technically feasible. CRF-R2 mRNA was mainly expressed at the middle and caudal levels of the DR. At midlevels, CRF-R2 mRNA was expressed exclusively in serotonin neurons, whereas, at caudal levels, approximately half the CRF-R2 mRNA was expressed in GABAergic neurons. The differential distribution of distinct neurochemical phenotypes lends support to the idea of functional differentiation of the DR. (+info)
Acute treatment with the antidepressant fluoxetine internalizes 5-HT1A autoreceptors and reduces the in vivo binding of the PET radioligand [18F]MPPF in the nucleus raphe dorsalis of rat.
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Because 5-HT1A receptors located on the soma dendrites of serotonin (5-HT) neurons normally mediate an inhibition of 5-HT firing and release, the desensitization of these autoreceptors is essential for obtaining an enhancement of 5-HT transmission after treatment with 5-HT reuptake inhibitors (SSRIs). We have demonstrated previously, using immunoelectron microscopy with specific 5-HT1A antibodies, that an internalization of 5-HT1A autoreceptors is associated with their desensitization in rats given a single dose of the 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin. Here, we examined the subcellular distribution of 5-HT1A receptors in dendrites from nucleus raphe dorsalis (NRD) (autoreceptors) and hippocampus (heteroreceptors) after acute treatment with the antidepressant SSRI, fluoxetine (10 mg/kg, i.p.). In parallel experiments, the kinetics of in vivo binding of the 5-HT1A positron emission tomography radioligand 4,2-(methoxyphenyl)-1-[2-(N-2-pyridinyl)-p-fluorobenzamido]ethylpiperazine ([18F]MPPF) was measured in these two brain regions by means of stereotaxically implanted beta microprobes. One hour after treatment, there was a 36% decrease in 5-HT1A immunogold labeling of the plasma membrane of NRD dendrites, and a concomitant increase in their cytoplasmic labeling, without any change in hippocampal dendrites. In vivo binding of [18F]MPPF was reduced by 35% in NRD and unchanged in hippocampus. Both effects were blocked by pretreatment with the 5-HT1A receptor antagonist (N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl) cyclohexane-carboxamide) (1 mg/kg, i.p.). In brain sections of NRD and hippocampus, [18F]MPPF autoradiographic labeling did not differ between fluoxetine- and saline-treated rats. These immunocytochemical results confirmed that internalization of 5-HT1A autoreceptors may account for their desensitization, and the microprobe results suggest that this prerequisite for antidepressant treatment efficacy could be amenable to brain imaging in humans. (+info)
Role of hippocampal CaMKII in serotonin 5-HT(1A) receptor-mediated learning deficit in rats.
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The serotonin 5-HT(1A) receptor agonist, 8-OH-DPAT (8-hydroxy-2-di-n-propylamino-tetralin), impairs retention performance in a passive avoidance learning task in rats. In the hippocampus of rats trained on this procedure and killed 1 h after the acquisition trial, an increase in the membrane levels of both Ca2+/calmodulin-dependent protein kinase II (CaMKII) and phosphorylated CaMKII, as well as in total and Ca2+-independent enzyme activity in tissue lysates was found. These effects were learning-specific as no changes in CaMKII levels or activity were found in rats receiving a footshock identical to the trained rats. The effect of training on CaMKII was prevented by a low 8-OH-DPAT dose. The 5-HT(1A) agonist also reduced protein kinase A (PKA) activity and increased the membrane levels of phosphatase 1 (PP1) and PP1 enzyme activity in the hippocampus. All of the changes induced by 8-OH-DPAT were reversed by the selective 5-HT(1A) antagonist WAY-100635, indicating receptor-specific effects. We suggest that 5-HT(1A) receptor-mediated disruption of retention performance is a consequence of the reduced PKA activity and the ensuing enhancement in PP1 activity, possibly through decreased phosphorylation/activation of endogenous PP1 inhibitors, that cause a reduced activity of phosphorylated CaMKII, a key enzyme in early stages of memory formation. This study provides an in vivo molecular basis for the cognitive deficits induced by stimulation of hippocampal 5-HT(1A) receptors. (+info)
The influence of repeated administration of clozapine and haloperidol on the effects of the activation of 5-HT(1A), 5-HT(2) and 5-HT(4) receptors in rat frontal cortex.
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The effects of a repeated treatment with antipsychotic drugs, clozapine and haloperidol, on the modulation of network activity ex vivo by 5-HT receptors were examined in rat frontal cortical slices using extracellular recording. Rats were treated for 21 days with clozapine (30 mg/kg p.o.), or haloperidol (1 mg/kg p.o.). Spontaneous bursting activity was induced in slices prepared 3 days after the last drug administration by perfusion with a medium devoid of Mg(2+) ions and with added picrotoxin (30 mM). The application of 2-3 microM 8-OH-DPAT, acting through 5-HT(1A) receptors, resulted in a reversible decrease of bursting frequency. In the presence of 1 microM DOI, the 5-HT(2) agonist, or 5 microM zacopride, the 5-HT(4) agonist, bursting frequency increased. Chronic clozapine treatment resulted in an attenuation of the effect of the activation of 5-HT(2) receptors, while the effects related to 5-HT(1A) and 5-HT(4) receptor activation were unchanged. Treatment with haloperiol did not influence the reactivity to the activation of any of the three 5-HT receptor subtypes. These data are consistent with earlier findings demonstrating a selective downregulation of 5-HT(2A) receptors by clozapine and indicate that chronic clozapine selectively attenuates the 5-HT-mediated excitation in neuronal circuitry of the frontal cortex while leaving the 5-HT-mediated inhibition intact. (+info)
Variability in the benzodiazepine response of serotonin 5-HT1A receptor null mice displaying anxiety-like phenotype: evidence for genetic modifiers in the 5-HT-mediated regulation of GABA(A) receptors.
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Benzodiazepines (BZs) acting as modulators of GABA(A) receptors (GABA(A)Rs) are an important group of drugs for the treatment of anxiety disorders. However, a large inter-individual variation in BZ sensitivity occurs in the human population with some anxiety disorder patients exhibiting diminished sensitivity to BZ and reduced density of GABA(A)Rs. The mechanism underlying BZ treatment resistance is not known, and it is not possible to predict whether an anxiety patient will respond to BZ. 5-hydroxytryptamine1A receptor (5-HT1AR) null mice (R-/-) on the Swiss-Webster (SW) background reproduce several features of BZ-resistant anxiety; they exhibit anxiety-related behaviors, do not respond to BZ, have reduced BZ binding, and have decreased expression of the major GABA(A)R subunits alpha1 and alpha2. Here, we show that R-/- mice on the C57Bl6 (B6) background also have anxiety phenotype, but they respond to BZ and have normal GABA(A)R subunit expression. This indicates that the 5-HT1AR-mediated regulation of GABA(A)R alpha subunit expression is subject to genetic modification. Hybrid SW/B6-R-/- mice also exhibit BZ-resistant anxiety, suggesting that SW mice carry a genetic modifier, which mediates the effect of the 5-HT1AR on the expression of GABA(A)Ralpha subunits. In addition, we show that this genetic interaction in SW mice operates early in postnatal life to influence the expression of GABA(A)R alpha subunits at the transcriptional level. These data indicate that BZ-resistant anxiety results from a developmental arrest of GABA(A)R expression in SW-R-/- mice, and a similar mechanism may be responsible for the BZ insensitivity of some anxiety patients. (+info)
Ro 64-6198 [(1S,3aS)-8-(2,3,3a,4,5,6-Hexahydro-1H-phenalen-1-yl)-1-phenyl-1,3,8-triaza-spiro [4.5]decan-4-one] acts differently from nociceptin/orphanin FQ in rat periaqueductal gray slices.
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Ro 64-6198 [(1S,3aS)-8-(2,3,3a,4,5,6-hexahydro-1H-phenalen-1-yl)-1-phenyl-1,3,8-triaza-spiro [4.5]decan-4-one] was developed as a nonpeptide agonist of nociceptin/orphanin FQ (N/OFQ) peptide (NOP) receptors, using bioassays at cloned receptors expressed in cell cultures. We have investigated the actions of Ro 64-6198 at native NOP receptors of the ventrolateral periaqueductal gray (PAG), a crucial site for N/OFQ-induced reversal of opioid analgesia, using the patch-clamp recording technique in rat brain slices. Ro 64-6198, like N/OFQ, activated G protein-coupled inwardly rectifying K(+) channels (GIRK) in ventrolateral PAG neurons but displayed only 60% efficacy and 22% potency of N/OFQ. Unlike N/OFQ that activated GIRK through NOP receptors in almost all tested neurons, Ro 64-6198 affected only 62% (114/185) of the neurons recorded, among which 57% were sensitive to CompB (J-113397), a selective NOP receptor antagonist. The effect of Ro 64-6198 was not affected by naloxone (1 microM), sulpiride (10 microM), and [1-(2-methoxyphenyl)-4-[4-2-phthalimido)butyl]piperazine (NAN-190) (1 microM), respectively, the antagonist of opioid, dopamine D(2), and 5-HT(1A) receptors. In Ro 64-6198-unresponsive neurons, N/OFQ activated GIRK through NOP receptors. It is concluded that Ro 64-6198 is a weak agonist of NOP receptors both in terms of potency and efficacy in ventrolateral PAG neurons. Heterogeneity of NOP receptors has been proposed from binding studies and in vivo functional studies. The possibility was discussed that two subsets of NOP receptors exist in ventrolateral PAG neurons, and Ro 64-6198 activates only one subset but N/OFQ activates both of them. (+info)
G protein-coupled receptors: in silico drug discovery in 3D.
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The application of structure-based in silico methods to drug discovery is still considered a major challenge, especially when the x-ray structure of the target protein is unknown. Such is the case with human G protein-coupled receptors (GPCRs), one of the most important families of drug targets, where in the absence of x-ray structures, one has to rely on in silico 3D models. We report repeated success in using ab initio in silico GPCR models, generated by the predict method, for blind in silico screening when applied to a set of five different GPCR drug targets. More than 100,000 compounds were typically screened in silico for each target, leading to a selection of <100 "virtual hit" compounds to be tested in the lab. In vitro binding assays of the selected compounds confirm high hit rates, of 12-21% (full dose-response curves, Ki < 5 microM). In most cases, the best hit was a novel compound (New Chemical Entity) in the 1- to 100-nM range, with very promising pharmacological properties, as measured by a variety of in vitro and in vivo assays. These assays validated the quality of the hits as lead compounds for drug discovery. The results demonstrate the usefulness and robustness of ab initio in silico 3D models and of in silico screening for GPCR drug discovery. (+info)
Glucocorticoid receptor overexpression in forebrain: a mouse model of increased emotional lability.
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The molecular mechanisms that control the range and stability of emotions are unknown, yet this knowledge is critical for understanding mood disorders, especially bipolar illness. Here, we show that the glucocorticoid receptor (GR) modulates these features of emotional responsiveness. We generated transgenic mice overexpressing GR specifically in forebrain. These mice display a significant increase in anxiety-like and depressant-like behaviors relative to wild type. Yet, they are also supersensitive to antidepressants and show enhanced sensitization to cocaine. Thus, mice overexpressing GR in forebrain have a consistently wider than normal range of reactivity in both positive and negative emotionality tests. This phenotype is associated, in specific brain regions, with increased expression of genes relevant to emotionality: corticotropin-releasing hormone, serotonin, norepinephrine and dopamine transporters, and 5-hydroxytryptamine(1A) receptor. Thus, GR overexpression in forebrain causes higher "emotional lability" secondary to a unique pattern of molecular regulation. This finding suggests that natural variations in GR gene expression can contribute to the fine-tuning of emotional stability or lability and may play a role in bipolar disorder. (+info)