Effect of chronic activation of 5-HT3 receptors on 5-HT3, 5-HT1A and 5-HT2A receptors functional activity and expression of key genes of the brain serotonin system

Among serotonin (5-HT) receptors, the 5-HT₃ receptor is the only ligand-gated ion-channel. Little is known about the interaction between the 5-HT₃ receptor and other 5-HT receptors and influence of 5-HT₃ chronic activation on other 5-HT receptors and the expression of key genes of 5-HT system. Chronic activation of 5-HT₃ receptor with intracerebroventricularly administrated selective agonist 1-(3-chlorophenyl)biguanide hydrochloride (m-CPBG) (14 days, 40 nmol, i.c.v.) produced significant desensitization of 5-HT₃ and 5-HT_1A receptors. The hypothermic responses produced by acute administration of selective agonist of 5-HT₃ receptor (m-CPBG, 40 nmol, i.c.v.) or selective agonist of 5-HT_1A receptor (8-hydroxy-2-(di-n-propylamino)tetralin) (8-OH-DPAT, 1 mg/kg, i.p.) was significantly lower in m-CPBG treated mice compared with the mice of control groups. Chronic m-CPBG administration failed to induce any significant change in the 5-HT_2A receptor functional activity and in the expression of the gene encoding 5-HT_2A receptor. Chronic activation of 5-HT₃ receptor produced no considerable effect on the expression on 5-HT₃, 5-HT_1A, and 5-HT transporter (5-HTT) and tryptophan hydroxylase-2 (TPH-2) genes – the key genes of brain 5-HT system, in the midbrain, frontal cortex and hippocampus. In conclusion, chronic activation of ionotropic 5-HT₃ receptor produced significant desensitization of 5-HT₃ and postsynaptic 5-HT_1A receptors but caused no considerable changes in the expression of key genes of the brain 5-HT system.     

Receptor-genes cross-talk: effect of chronic 5-HT1A agonist 8-hydroxy-2-(di-n-propylamino) tetralin treatment on the expression of key genes in brain serotonin system and on behavior

Dysfunction in brain serotonin (5-HT) system has been implicated in the psychopathology of anxiety, depression, drug addiction, and schizophrenia. The 5-HT_1A receptors play a central role in the control of 5-HTergic neurotransmission. There are some scarce data showing cross-regulation between 5-HT receptors. Here, we investigated whether interaction exists between 5-HT_1A receptor and genes encoding key members in brain 5-HT system. Chronic treatment with selective agonist of 5-HT_1A receptor 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) (1.0 mg/kg i.p., 14 days) produced considerable decrease in hypothermic response to acute administration of 8-OH-DPAT in CBA/Lac mice indicating desensitization of 5-HT_1A receptors. The decrease in 5-HT_1A gene expression as well as decrease in the expression of gene encoding key enzyme in 5-HT synthesis, tryptophan hydroxylase-2 (TPH-2) in the midbrain, and the expression of the gene encoding 5-HT_2A receptor in the frontal cortex was shown. There were no significant changes in 5-HT transporter mRNA level in the midbrain. Despite considerable decrease in the expression of the genes encoding tryptophan hydroxylase-2, 5-HT_1A and 5-HT_2A receptors, chronic 8-OH-DPAT treatment failed to produce significant changes in 5-HT_1A-linked behavior (intermale aggression, open-field behavior, light-dark box, and pinch-induced catalepsy), suggesting compensatory and adaptive effect of genes suppression. The obtained data on the effect of 8-OH-DPAT-induced desensitization of 5-HT_1A receptors on 5-HT_1A, 5-HT_2A and TPH-2 gene expression demonstrated the role of 5-HT_1A receptor as indirect regulator of gene expression. The results provide the first evidence of receptor-key genes interaction in brain 5-HT system and may have profound implications in understanding the functioning of the brain neurotransmitter systems.     

5-HT2A receptor inactivation potentiates the acute antidepressant-like activity of escitalopram: involvement of the noradrenergic system

Evidence suggests that the serotonin 2A receptor (5-HT_2AR) modulates the therapeutic activity of selective serotonin reuptake inhibitors (SSRIs). Indeed, among the genetic factors known to influence the individual response to antidepressants, the HTR2A gene has been associated with SSRIs response in depressed patients. However, in these pharmacogenetic studies, the consequences of HTR2A gene polymorphisms on 5-HT_2AR expression or function are lacking and the precise role of this receptor is still matter of debate. This study examined the effect of 5-HT_2AR agonism or antagonism with DOI and MDL100907, respectively, on the serotonergic system and the antidepressant-like activity of the SSRI escitalopram in mouse. The 5-HT_2AR agonist DOI decreased the firing rate of 5-HT neurons in the dorsal raphe (DR) nucleus of 5-HT_2AR^+/+ anesthetized mice. This inhibitory response persisted in 5-HT_2CR^?/? but was completely blunted in 5-HT_2AR^?/? mutants. Moreover, the suppressant effect of DOI on DR 5-HT neuronal activity in 5-HT_2AR^+/+ mice was attenuated by the loss of noradrenergic neurons induced by the neurotoxin DSP4. Conversely, in 5-HT_2AR^+/+ mice, the pharmacological inactivation of the 5-HT_2AR by the selective antagonist MDL100907 reversed escitalopram-induced decrease in DR 5-HT neuronal activity. Remarkably, in microdialysis experiments, a single injection of escitalopram increased cortical extracellular 5-HT, but not NE, levels in awake 5-HT_2AR^+/+ mice. Although the addition of MDL100907 did not potentiate 5-HT neurotransmission, it allowed escitalopram to increase cortical NE outflow and consequently to elicit an antidepressant-like effect in the forced swimming test. These results suggest that the blockade of the 5-HT_2AR may strengthen the antidepressant-like effect of escitalopram by facilitating the enhancement of the brain NE transmission. They provide support for the use of atypical antipsychotics with SSRIs as a relevant antidepressant augmentation strategy.     

Changes in 5-HT2A-mediated behavior and 5-HT2A- and 5-HT1A receptor binding and expression in conditional brain-derived neurotrophic factor knock-out mice

Changes in brain-derived neurotrophic factor (BDNF) expression have been implicated in the etiology of psychiatric disorders. To investigate pathological mechanisms elicited by perturbed BDNF signaling, we examined mutant mice with central depletion of BDNF (BDNF^2L/2LCk-cre). A severe impairment specific for the serotonin 2A receptor (5-HT_2AR) in prefrontal cortex was described previously in these mice. This is of much interest, as 5-HT_2ARs have been linked to neuropsychiatric disorders and anxiety-related behavior. Here we further characterized the serotonin receptor alterations triggered by BDNF depletion. 5-HT_2A ([³H]-MDL100907) and 5-HT_1A ([³H]-WAY100635) receptor autoradiography revealed site-specific alterations in BDNF mutant mice. They exhibited lower 5-HT_2A receptor binding in frontal cortex but increased binding in hippocampus. Additionally, 5-HT_1A receptor binding was decreased in hippocampus of BDNF mutants, but unchanged in frontal cortex. Molecular analysis indicated corresponding changes in 5-HT_2A and 5-HT_1A mRNA expression but normal 5-HT_2C content in these brain regions in BDNF^2L/2LCk-cre mice. We investigated whether the reduction in frontal 5-HT_2AR binding was reflected in reduced functional output in two 5-HT_2A-receptor mediated behavioral tests, the head-twitch response (HTR) and the ear-scratch response (ESR). BDNF^2L/2LCk-cre mutants treated with the 5-HT_2A receptor agonist (±)-2,5-dimethoxy-4-iodoamphetamine (DOI) showed a clearly diminished ESR but no differences in HTR compared to wildtypes. These findings illustrate the context-dependent effects of deficient BDNF signaling on the 5-HT receptor system and 5-HT_2A-receptor functional output.     

Transmitter content, origins and connections of axons in the spinal cord that possess the serotonin (5-hydroxytryptamine) 3 receptor

Recent evidence suggests that serotonin has pronociceptive actions in the spinal cord when it acts through 5-hydroxytryptamine (5-HT)₃ receptors. Cells and axon terminals which are concentrated in the superficial dorsal horn possess this receptor. We performed a series of immunocytochemical studies with an antibody raised against the 5-HT_3A subunit in order to address the following questions: 1) Are axons that possess 5-HT₃ receptors excitatory? 2) Are 5-HT₃ receptors present on terminals of myelinated primary afferents? 3) What is the chemical nature of dorsal horn cells that possess 5-HT₃ receptors? 4) Do axons that possess 5-HT₃ receptors target lamina I projection cells?Approximately 45% of 5-HT_3A immunoreactive boutons were immunoreactive for the vesicular glutamate transporter 2 and almost 80% formed synapse-like associations with GluR2 subunits of the AMPA receptor therefore it is principally glutamatergic axons that possess the receptor. Immunoreactivity was not present on myelinated primary afferent axons labeled with the B-subunit of cholera toxin or those containing the vesicular glutamate transporter 1. Calbindin (which is associated with excitatory interneurons) was found in 44% of 5-HT_3A immunoreactive cells but other markers for inhibitory and excitatory cells were not present. Lamina I projection cells that possessed the neurokinin-1 receptor were associated with 5-HT_3A axons but the density of contacts on individual neurons varied considerably.The results suggest that 5-HT₃ receptors are present principally on terminals of excitatory axons, and at least some of these originate from dorsal horn interneurons. The relationship between lamina I projection cells and axons possessing the 5-HT₃ receptor indicates that this receptor has an important role in regulation of ascending nociceptive information.     

Differential roles of hypothalamic serotonin receptor subtypes in the regulation of prolactin secretion in the turkey hen

In the turkey, exogenous serotonin (5-hydroxytryptamine, 5-HT) increases prolactin (PRL) secretion by acting through the dopaminergic (DAergic) system. In the present study, infusion of the 5-HT_2C receptor agonist, (R)(−)-DOI hydrochloride (DOI), into the third ventricle stimulates PRL secretion, whereas the 5-HT_1A receptor agonist, (+/−)-8-OH-DPAT hydrobromide (DPAT), inhibits PRL secretion. Using the immediate-early gene, c-fos, as an indicator of neuronal activity, in situ hybridization histochemistry showed preferential c-fos co-localization within tyrosine hydroxylase immunoreactive neurons (the rate limiting enzyme in DA synthesis) in the areas of the nucleus preopticus medialis (POM) and the nucleus premammillaris (PMM), in response to DPAT and DOI, respectively. To clarify the involvement of 5-HT_1A and 5-HT_2C receptors in PRL regulation, their mRNA expression was determined on hypothalamic tissue sections from birds in different reproductive stages. A significant difference in 5-HT_1A receptor was observed, with the POM of hypoprolactinemic short day and photorefractory birds showing the highest expression. 5-HT_2C receptors mRNA did not change during the reproductive cycle. The data presented support the notion that DA neurons in the PMM and POM mediate the stimulatory and inhibitory effects of 5-HT, respectively, on PRL secretion and the 5-HTergic system can both stimulate and inhibit PRL secretion.     

Functional Characteristics of Serotonin 5-HT<Subscript>2A</Subscript> and 5-HT<Subscript>2C</Subscript> Receptors in the Brain and the Expression of the 5-HT<Subscript>2A</Subscript> and 5-HT<Subscript>2C</Subscript> Receptor Genes in Aggressive and Non-Aggressive Rats

The functional activity of serotonin 5-HT_2A and 5-HT_2C receptors and the expression of the genes encoding them were studied in Norway rats bred for 60 generations for the presence and absence of high levels of stress-evoked aggression to humans. There were no significant differences in the levels of 5-HT_2A receptor mRNA in the midbrain, frontal cortex, and hippocampus and the extents of head twitching evoked by the 5-HT_2A agonist DOI in rats with and without genetically determined high levels of aggression. Administration of the selective 5-HT_2C agonist MK-212 weakened reflex startle in response to an acoustic signal (the acoustic startle response) in non-aggressive animals but had no significant effects on the response in aggressive animals. Increases in the level of 5-HT_2C receptor mRNA were seen in the frontal cortex and hippocampus in non-aggressive rats as compared with aggressive animals. Increases in the expression of the 5-HT_2C receptor gene and the functional state of 5-HT_2C receptors were seen in the brains of non-aggressive rats, without any changes in the 5-HT_2A receptor mRNA level or receptor sensitivity; this is evidence for the involvement of 5-HT_2C receptors in the mechanisms inhibiting fear-evoked aggressive behavior.     

Reduced 5-HT2A receptor binding in patients with mild cognitive impairment

Previous studies of patients with Alzheimer's disease (AD) have described reduced brain serotonin 2A (5-HT_2A) receptor density. It is unclear whether this abnormality sets in early in the course of the disease and whether it is related to early cognitive and neuropsychiatric symptoms.We assessed cerebral 5-HT_2A receptor binding in patients with mild cognitive impairment (MCI) and related 5-HT_2A receptor binding to clinical symptoms. Sixteen patients with MCI of the amnestic type (mean age 73, mean MMSE 26.1) and 17 age and sex matched control subjects were studied with MRI and [¹⁸F]altanserin PET in a bolus–infusion approach. A significant global reduction of 20–30% in 5-HT_2A binding (atrophy corrected) was found in most neocortical areas. Reduced 5-HT_2A binding in the striatum correlated significantly with Neuropsychiatric Inventory depression and anxiety scores. We conclude that widespread reductions in 5-HT_2A receptor binding were found in amnestic MCI, pointing at the presence of serotonergic dysfunction in prodromal AD. This may provide some of the pathophysiological background for the neuropsychiatric symptoms found in early AD.     

Serotonin regulates osteoblast proliferation and function in vitro

The monoamine serotonin (5-hydroxytryptamine, 5-HT), a well-known neurotransmitter, also has important functions outside the central nervous system. The objective of this study was to investigate the role of 5-HT in the proliferation, differentiation, and function of osteoblasts in vitro. We treated rat primary calvarial osteoblasts with various concentrations of 5-HT (1 nM to 10 µM) and assessed the rate of osteoblast proliferation, expression levels of osteoblast-specific proteins and genes, and the ability to form mineralized nodules. Next, we detected which 5-HT receptor subtypes were expressed in rat osteoblasts at different stages of osteoblast differentiation. We found that 5-HT could inhibit osteoblast proliferation, differentiation, and mineralization at low concentrations, but this inhibitory effect was mitigated at relatively high concentrations. Six of the 5-HT receptor subtypes (5-HT_1A, 5-HT_1B, 5-HT_1D, 5-HT_2A, 5-HT_2B, and 5-HT_2C) were found to exist in rat osteoblasts. Of these, 5-HT_2A and 5-HT_1B receptors had the highest expression levels, at both early and late stages of differentiation. Our results indicated that 5-HT can regulate osteoblast proliferation and function in vitro.     

Serotonin 5-HT2 and 5-HT1A-like receptors differentially modulate aggressive behaviors in Drosophila melanogaster

Aggressive behavior is widespread throughout the animal kingdom, and is a complex social behavior influenced by both genetics and environment. Animals typically fight over resources that include food, territory, and sexual partners. Of all the neurotransmitters, serotonin (5-HT) has been the most implicated in modulating aggressive behaviors in mammalian systems. In the fruit fly, Drosophila melanogaster, the involvement of 5-HT itself in aggressive behaviors has been recently established, however, the underlying mechanisms have largely remained elusive. Here we describe the influence of different 5-HT receptor subtypes on aggressive behaviors in Drosophila. Drosophila express homologs of three mammalian 5-HT receptors: the 5-HT_1A, 5-HT₂, and 5-HT₇ receptors. Significantly, these receptors mediate important behaviors in mammalian systems ranging from feeding, aggression, and sleep, to cognition. To examine the role of the 5-HT₂Dro receptor, we utilized the selective 5-HT₂ receptor agonist (R)-1-[2,5-dimethoxy-4-iodophenyl]-2-aminopropane (DOI), and the 5-HT₂ receptor antagonist, ketanserin. To examine the role of 5-HT_1A-like receptors we used the 5-HT_1A receptor agonist 8-hydroxy-2-dipropylaminotetralin hydrobromide (8-OH-DPAT), and the 5-HT_1A receptor antagonist WAY100635. We find that activation of 5-HT₂ receptors with (R)-DOI appears to decrease overall aggression, whereas activation of 5-HT_1A-like receptors with 8-OH-DPAT increases overall aggression. Furthermore, the different 5-HT receptor circuitries appear to mediate different aspects of aggression: 5-HT₂ receptor manipulation primarily alters lunging and boxing, whereas 5-HT_1A-like receptor manipulation primarily affects wing threats and fencing. Elucidating the effects of serotonergic systems on aggression in the fly is a significant advancement not only in establishing the fly as a system to study aggression, but as a system relevant to elucidating molecular mechanisms underlying aggression in mammals, including humans.     

Hypothalamic serotonin-1A receptor binding measured by PET predicts the plasma level of dehydroepiandrosterone sulfate in healthy women

Serotonin modulates the activity of the hypothalamic–pituitary–adrenal (HPA) axis particularly via the serotonin-1A receptor (5-HT_1A). Therefore, the rationale of this positron emission tomography (PET) study was to investigate the influence of the 5-HT_1A receptor distribution in the human brain on plasma levels of dehydroepiandrosterone sulfate (DHEAS) and cortisol in vivo. Eighteen healthy female were measured with PET and the selective 5-HT_1A receptor radioligand [carbonyl-¹¹C]WAY-100635. Nine a priori defined brain regions (hypothalamus, orbitofrontal cortex, amygdala, hippocampus, anterior and posterior cingulate cortices, dorsal raphe nucleus, retrosplenial cortex, and insula) and the cerebellum (reference region) were delineated on coregistered MR images. DHEAS and cortisol plasma levels were collected by blood sampling in the morning of the PET day. Linear regression analysis of DHEAS plasma level as dependent variable and hypothalamic 5-HT_1A receptor binding potential (BP) as independent variable showed a highly significant association (r = .691, p = .002). The hypothalamic 5-HT_1A BP predicted 47.7% of the variability in DHEAS plasma levels. Regressions were borderline significant (p < .01, Bonferroni corrected threshold <.0056) between 5-HT_1A BP in the anterior cingulate and orbitofrontal cortices and free cortisol levels. No significant associations between DHEAS or cortisol and the 5-HT_1A receptor BP in other investigated brain regions were found. In conclusion, the serotonergic system may influence the DHEAS plasma level by modulating CRH and ACTH release via hypothalamic 5-HT_1A receptors as reported for cortisol before. As disturbances of the HPA axis as well as changes of the 5-HT_1A receptor distribution have been reported in affective disorders, future studies should focus on these interactions.     

Elevated cyclic stretch and serotonin result in altered aortic valve remodeling via a mechanosensitive 5-HT2A receptor-dependent pathway

IntroductionSerotonin/5-hydroxytryptamine (5-HT) has been implicated in valve disease and in the modulation of valve mechanical properties. Several 5-HT receptor subtypes are also known to be mechanosensitive in other cell types, but this has not been studied in the context of the valve. In this study, we sought to understand the effects of elevated 5-HT levels and stretch overload on aortic valve remodeling and the dominant 5-HT receptor subtype that regulates these processes.Methods and resultsCollagen biosynthesis and tissue mechanical properties of porcine aortic valve cusps were evaluated after 10% (physiologic) and 15% (pathologic) dynamic stretch. These studies were performed in normal medium or medium supplemented with 5-HT (1, 10, 100 μM) in the absence and presence of 5-HT_2A or 5-HT_2B receptor antagonists. Fresh valves served as controls. Valve collagen content was maximal at the 10-μM 5-HT concentration for both 10% and 15% stretch. The 5-HT_2A receptor antagonist reduced collagen synthesis, cell proliferation, and hsp47 expression under elevated and normal stretch, whereas the 5-HT_2B receptor antagonist was effective only at normal stretch. The pretransition stiffness of the valve cusps was also increased in response to 5-HT via a stretch-sensitive 5-HT_2A mechanism, with the post-transition stiffness unaltered.ConclusionsCombined elevated stretch and 5-HT resulted in increased valve collagen biosynthesis, cell proliferation, and tissue stiffness. These responses were inhibited by a 5-HT_2A antagonist. This strongly suggests that the 5-HT_2A receptor subtype is sensitive to elevated stretch.     

Systematic screening for mutations in the promoter and the coding region of the 5-HT1A gene

In the present study we sought to identify genetic variation in the 5-HT_1A receptor gene which through alteration of protein function or level of expression might contribute to the genetic predisposition to neuropsychiatric diseases. Genomic DNA samples from 159 unrelated subjects (including 45 schizophrenic, 46 bipolar affective, and 43 patients with Tourette's syndrome, as well as 25 healthy controls) were investigated by single-strand conformation analysis. Overlapping PCR (polymerase chain reaction) fragments covered the whole coding sequence as well as the 5′ untranslated region of the 5-HT_1A gene. The region upstream to the coding sequence we investigated contains a functional promoter. We found two rare nucleotide sequence variants. Both mutations are located in the coding region of the gene: a coding mutation (A→G) in nucleotide position 82 which leads to an amino acid exchange (Ile→Val) in position 28 of the receptor protein and a silent mutation (C→T) in nucleotide position 549. The occurrence of the Ile-28-Val substitution was studied in an extended sample of patients (n = 352) and controls (n = 210) but was found in similar frequencies in all groups. Thus, this mutation is unlikely to play a significant role in the genetic predisposition to the diseases investigated. In conclusion, our study does not provide evidence that the 5-HT_1A gene plays either a major or a minor role in the genetic predisposition to schizophrenia, bipolar affective disorder, or Tourette's syndrome. © 1995 Wiley-Liss, Inc.     

Chronic Actions of Thyroxine on Behavior and Serotonin Receptors in Mouse Strains with Contrasting Predispositions to Catalepsy

The studies reported here addressed the effects of chronic administration of thyroxine (2 mg/liter for 60 days) on catalepsy and the functional activity and expression of the 5-HT_1A and 5-HT_2A receptor genes in the brains of adult male mice of the cataleptic ASC strain and the catalepsy-resistant AKR strain. Thyroxine induced cataleptics in AKR mice but had anticataleptic activity in ASC animals. Chronic thyroxine administration increased the functional activity and expression of 5-HT_2A receptors in the frontal cortex in AKR mice but not in ASC mice. In ASC mice, the hormone significantly weakened the hypothermic effect of the 5-HT_1A receptor agonist 8-OH-DPAT, though it did not alter the expression of these receptors. These results suggest that 5-HT_2A receptors are involved in the cataleptogenic while 5-HT_1A receptors are involved in the anticataleptic effects of the hormone in mice.     

Subtype specific roles of serotonin receptors in the spine formation of cortical neurons in vitro

Dendritic spines are postsynaptic structures which are formed from filopodia. We examined roles of serotonin (5-HT) receptors in the spine formation. Embryonic rat cortical neurons were cultured for 10 or 14 days and treated by 5-HT receptor agonists for 24 h. At 11 days in vitro, 5-HT_1A agonist increased filopodia density, whereas 5-HT_2A/2C agonist increased the density of puncta and spines. At 15 days in vitro, 5-HT_1A agonist decreased the density of puncta and spines, whereas 5-HT_2A/2C agonist decreased filopodia density with increase of spines. In conclusion, the present study shows 5-HT receptors have subtype-specific effects on the spine formation.     

Polymorphism of Serotonin 5-HT Receptors as the Basis of the Multifunctionality of Serotonin

This review addresses a question at the junction of neurophysiology and neurogenetics – the multiplicity of types and subtypes of serotonin (5-HT) receptors and their roles in the physiological effects of the brain neurotransmitter serotonin. Particular attention is paid to comparison of the behavioral effects of related subtypes of 5-HT receptors – 5-HT₁ (5-HT_1A and 5-HT_1B) and 5-HT₂ (5-HT_2A and 5-HT_2C) receptors – which are involved in the mechanisms regulating behavior and psychopathology. Published data are presented, along with results obtained at the Laboratory for Behavioral Neurogenics, providing evidence that different types of 5-HT receptors can act in different directions as well as similarly, supplementing each other, in the control of physiological functions and behavior. The roles of 5-HT_1A and 5-HT_1B receptors in regulating aggressive behavior, sexual arousal, drinking behavior, and food consumption are discussed. Experimental results demonstrating reciprocal relationships between 5-HT_2A and 5-HT_2C receptors and interactions between 5-HT_1A and 5-HT₃ receptors are presented. These data provide evidence of a complex system for these interactions not only at the receptor level, but also with the involvement of genes controlling the main elements of the serotonin system. Interaction of receptors evidently underlies neuroplasticity and plays a significant role in compensatory processes and in adaptive mechanisms.     

Gender and the use of hormonal contraception in women are not associated with cerebral cortical 5-HT 2A receptor binding

Gender influences brain function including serotonergic neurotransmission, which may play a role in the well-known gender variations in vulnerability to mood and anxiety disorders. Even though hormonal replacement therapy in menopause is associated with globally increased cerebral 5-HT_2A receptor binding it is not clear if gender or use of hormonal contraception exhibits associations with 5-HT_2A receptor binding. We found no significant effect of gender on cortical 5-HT_2A receptor binding (P=0.15, n=132). When adjusting for the personality trait neuroticism, known to be positively correlated to frontolimbic 5-HT_2A receptor binding and to be more pronounced in women, again, the effect of gender was not significant (P=0.42, n=127). Also, the use of hormonal contraception (n=14) within the group of pre-menopausal women (total n=29) was not associated with cortical 5-HT_2A receptor binding (P=0.31). In conclusion, neither gender, nor the use of hormonal contraception in premenopausal women was associated with cortical 5-HT_2A receptor binding.     

Immunolabeling of the rat central nervous system with antibodies partially selective of the short form of the 5-HT3 receptor

Polyclonal antibodies were raised against a synthetic hexadecapeptide corresponding to the portion of the second intracytoplasmic loop of the short form of the mouse 5-hydroxytryptamine-3_A receptor subunit (5-HT₃A-S), which differs from the long form (5-HT₃A-L) by the removal of six amino acids. Antibodies were detected by enzyme-linked immunosorbent assay as soon as two months after the first injection to rabbits of the peptide coupled to keyhole limpet hemocyanin. Immunoblot detection of fusion proteins comprising glutathione-S-transferase and the second intracellular loop of 5-HT₃A-S or 5-HT₃A-L, and immunoprecipitation of cloned receptors showed that antibodies exhibited some selectivity for the short variant. Affinity chromatography allowed the purification of selective anti-5-HT₃A-S antibodies which yielded a strong positive labeling of plasma membrane, reticulum and Golgi apparatus of COS-7 cells expressing murine 5-HT₃A-S. In contrast, COS-7 cells expressing similar levels of 5-HT₃A-L exhibited only a very weak labeling. Selectivity was also observed on immunoblots of cloned receptors transiently expressed in COS-7 cells, or stably expressed in CHO cells, both systems showing an immunolabeled component at 53,000–54,000 mol. wt. Immunoautoradiographic labeling of central nervous system sections showed that 5-HT₃A-S-like immunoreactivity was found mostly within the nucleus of the solitary tract, the nucleus of the spinal tract of the trigeminal nerve, and the dorsal horn of the the spinal cord in the rat. After unilateral ablation of the nodose ganglion, 5-HT₃A-S-like immunoreactivity decreased markedly in the ipsilateral part of the nucleus of the solitary tract, as expected of the presynaptic localization of 5-HT₃ receptors. Finally, immunohistochemistry at the light and electron microscope levels revealed that 5-HT₃A-S-like immunoreactivity was associated essentially with terminals and axonal profiles.All these results demonstrate that the immunolabeling exhibited by these antibodies is consistent with a specific and partially selective recognition of the short isoform of the 5-HT₃A subunit. Because the pattern of immunoautoradiographic labeling matches the distribution previously established with selective radioligands, it can be inferred that these antibodies probably recognized the same fully assembled form of the 5-HT₃A-S receptor subunit.