Elena del Olmo

Chronic fluoxetine induces opposite changes in G protein coupling at pre and postsynaptic 5-HT1A receptors in rat brain.

Chronic treatment with the antidepressant fluoxetine may lead to changes in the properties of pre- and postsynaptic 5-HT(1A) receptors due to modifications in the receptor-G protein coupling process. We have evaluated, in rats, the effect of chronic fluoxetine (10 mg/kg/day) at brain 5-HT(1A) receptors using different techniques. The density of 5-HT(1A) receptors was unchanged in fluoxetine-treated rats vs. vehicle group. Stimulation of [(35)S]GTPgammaS binding induced by (+/-)8-OH-DPAT was significantly attenuated in dorsal raphe nucleus after fluoxetine (+3.7 vs. +31.2% in vehicle). The inhibition of dorsal raphe firing by (+/-)8-OH-DPAT (ED(50) in vehicle = 2.1 microg/kg, i.v.) was also attenuated in rats treated with fluoxetine (ED(50)=4.7 microg/kg). In contrast, a significant increase on (+/-)8-OH-DPAT-induced stimulation of [(35)S]GTPgammaS binding was observed in CA(1) (+53.4 vs.+20.2% in vehicle) and dentate gyrus (+105.7 vs. +52.6% in vehicle) but not in entorhinal cortex. Our data demonstrate that fluoxetine-induced desensitization of 5-HT(1A) autoreceptors occurs at G protein level. Moreover, a relevant finding is the region-specific hypersensitivity of postsynaptic 5-HT(1A) receptors, in the hippocampus but not in entorhinal cortex, following chronic fluoxetine. These differential adaptive changes in brain 5-HT(1A) receptors could underlie the mechanism of action of antidepressants and also contribute to their clinical effects.

[3H]Sumatriptan binding sites in human brain: regional-dependent labelling of 5-HT1D and 5-HT1F receptors

The general properties of [3H]sumatriptan binding sites in postmortem human brain tissue sections are described. High concentrations of autoradiographic grains were seen in globus pallidus = substantia nigra > cortex > putamen > hippocampus. While 5-HT (5-hydroxytryptamine) displaced in all regions more than 90% of [3H]sumatriptan binding, the level of binding inhibited by 5-CT (5-carboxamidotryptamine) varied in each region. Although the binding inhibited by 5-CT in some regions such as globus pallidus and substantia nigra was equivalent to that obtained with 5-HT, in cortical areas, such as frontal cortex and hippocampus, a substantial level of binding insensitive to 5-CT was seen. In addition, in membrane binding assays, 10 nM metergoline displaced most [3H]sumatriptan specific binding in striatum and only 16% in frontal cortex. In the human brain sumatriptan binds to at least two 5-HT1 receptors, 5-HT1D and 5-HT1F.

Early localization of mRNA coding for 5-HT1A receptors in human brain during development

The distribution of 5-HT1A receptor mRNA in the human brain was studied in neonatal, children and adult cases by means of in situ hybridization histochemistry, using an oligonucleotide derived from the coding region of the human receptor. A prenatal pattern of development was observed. The hippocampus, raphe nuclei and neocortex presented high levels of hybridization already at the fetal/neonatal stage, fully comparable to the adult. A high and transient hybridization signal was found in cerebellum. These results support a role for 5-HT1A receptors in the regulation of neural development.

Transient localization of 5-HT1A receptors in human cerebellum during development

The appearance and distribution of 5-HT1A receptors in the human cerebellum during the ontogenetic development was studied in samples from a group of fetal, neonatal and adult cases, by means of quantitative autoradiography and membrane binding. [3H]8-OH-DPAT was used as a ligand. High densities of 5-HT1A receptors were found during the fetal and neonatal stages over the whole cerebellar cortex, with a slight predominance in the external part, in a band which included the molecular layer. In contrast, the adult cerebellum was nearly devoid of such sites. This finding supports a role for 5-HT1A receptors in the regulation of development in the human cerebellum.

Regionally specific age-dependent decline in α2-adrenoceptors: An autoradiographic study in human brain

The effect of sex, postmortem delay and aging on alpha 2-adrenoceptor binding was studied in tissue sections from several representative regions of the human brain from 21 subjects using [3H]UK-14304 as a ligand. Sex and postmortem delay did not influence the density of alpha 2-receptors. Aging resulted in clear decreases in most forebrain areas examined (n. basalis greater than basal ganglia greater than hypothalamus greater than fronto-temporal cortex greater than hippocampus greater than visual cortex), whereas alpha 2-receptors did not significantly change with age in the amygdala and several infratentorial areas. We conclude that age-related, regionally specific decreases in the density of alpha 2-receptors occur in the human brain. The implications of these findings for age-dependent noradrenergic degeneration are discussed.

Identification and characterization of a new serotonergic recognition site with high affinity for 5-carboxamidotryptamine in mammalian brain.

Abstract: We analyzed the existence of an additional serotonin (5‐HT) receptor subtype, sensitive to 5‐carboxamidotryptamine, in the mammalian brain. Radioligand binding studies with [3H]5‐HT were carried out in rat, guinea pig, and human brain membranes, in the presence of unlabeled drugs to mask the binding to all known 5‐HT receptors, with the exception of 5‐HT1E sites. Under these conditions, unlabeled 5‐carboxamidotryptamine still showed a biphasic competition curve with a nanomolar affinity component. Saturation studies with 5‐[3H]carboxamidotryptamine were carried out in the presence of (±)‐8‐hydroxy‐2‐(di‐n‐propylamino)tetralin, mesulergine, and ergotamine, to mask the binding to all receptors known to be labeled by 5‐carboxamidotryptamine. These studies showed the existence in cortex and hippocampus from guinea pig and human brain of a remaining binding site with high affinity (pKD = 7.8–8.1) and a unique pharmacological profile. 5‐HT and 5‐carboxamidotryptamine showed nanomolar affinity, whereas 5‐methoxytryptamine recognized this binding site with intermediate affinity. Other drugs exhibited low or very low potency in inhibiting this binding. The addition of 5′‐guanylylimidodiphosphate significantly reduced the number of binding sites labeled by 5‐[3H]carboxamidotryptamine, in the presence of the masking drugs described above, indicating the interaction with a GTP‐binding protein. Preliminary autoradiographic studies in human brain appear to indicate that this 5‐HT binding site is present in areas such as the globus pallidus, neocortex, and hippocampus, among others.

In vitro and in vivo characterization of F-97013-GD, a partial 5-HT1A agonist with antipsychotic- and antiparkinsonian-like properties

In order to better define the role of 5-HT(1A) receptors in the modulation of extrapyramidal motor functions, we investigated the effect of 5-HT(1A) agonists on tacrine-induced tremulous jaw movements (TJM) in rats, a putative model of parkinsonian tremor. Acute injection of 5-HT(1A) agonists 8-OH-DPAT and buspirone dose-dependently counteracted the tacrine-induced oral movements (ED(50)=0.04 and 1.0mg/kg, respectively), an effect reversed by the selective 5-HT(1A) antagonist WAY 100,635. In contrast to classical antipsychotics, the atypical antipsychotics risperidone (ED(50)=0.3mg/kg) and clozapine (ED(50)=1.5mg/kg) blocked the oral movements induced by the cholinomimetic agent at or below the doses required for suppression of conditioned avoidance response. The compound F-97013-GD (6-methyl-2-[4-(naphtylpiperazin-1-yl)butyl]-3-(2H)-pyridazinone), a putative antipsychotic drug that in functional in vitro and in vivo assays behaved as a mixed dopamine D(2)-antagonist and 5-HT(1A)-partial agonist, also displayed a potent antitremorgenic effect in this paradigm (ED(50)=0.5mg/kg). Interestingly, pretreatment with WAY 100,635 blocked the inhibitory effect of F-97013-GD but not that of clozapine. The 5-HT depleting agent para-chlorophenylalanine (PCPA) partially attenuated tacrine-induced TJM but did not block the suppressive effect of 5-HT(1A) agonists. In addition, only high doses of F-97013-GD induced catalepsy in rodents and, like 8-OH-DPAT and clozapine, the compound reversed the haloperidol-induced catalepsy in rats. These results show that 5-HT(1A) receptors play a role in the regulation of tacrine-induced TJM and suggest that their activation by novel antipsychotics may not only reduce the extrapyramidal side effects EPS liability, but also be effective in the treatment of parkinsonian tremor.

WAY100635 prevents the changes induced by fluoxetine upon the 5-HT1A receptor functionality.

5-HT1A receptor-mediated signalling in rat brain was evaluated after chronic administration (14 days; s.c.) of the selective serotonin reuptake inhibitor (SRRI) fluoxetine (10 mg/kg/day) alone, or in combination with the 5-HT1A receptor antagonist WAY100635 (0.1 mg/kg/day). The density of 5-HT1A binding sites was unchanged following fluoxetine, WAY100635, or the combination of fluoxetine and WAY100635. However, the net stimulation of [35S]GTPgammaS binding induced by the 5-HT1A agonist 8-OH-DPAT was significantly attenuated in dorsal raphe nucleus (DRN), but not in hippocampus, after chronic fluoxetine. Moreover, depending of the area analysed, the basal binding of [35S]GTPgammaS was differentially affected by this treatment: increased in DRN and decreased in hippocampal dentate gyrus. Interestingly, the changes in [35S]GTPgammaS basal binding and on 5-HT1A receptors functionality were prevented by the concomitant administration of WAY100635. The inhibition of dorsal raphe firing by 8-OH-DPAT was also attenuated in fluoxetine-treated rats (ED50 = 2.12 +/- 0.32 microg/kg and 4.34 +/- 0.09 microg/kg, for vehicle and fluoxetine respectively), an effect which was also prevented by the concomitant administration of WAY100635 (ED50 = 2.10 +/- 0.58 microg/kg). Chronic administration of WAY100635 alone did not affect the 5-HT1A receptor-induced stimulation of [35S]GTPgammaS binding, nor the 8-OH-DPAT-induced inhibition of 5-HT neuron firing. These results demonstrate that the concomitant blockade of 5-HT1A receptors when administering fluoxetine prevents those adaptive changes of 5-HT1A receptor function associated with the chronic administration of this antidepressant. These findings could be relevant from the therapeutic point of view, and further support the potential benefit of treatments with a SSRI/5-HT1A receptor antagonist combination.

Transmembrane signaling through phospholipase C‐β in the developing human prefrontal cortex

To investigate changes in muscarinic receptor‐stimulated phospholipase C‐β (PLC‐β) activity during brain development, we examined the functional coupling of each of the three major protein components of the phosphoinositide system (M1, M3, and M5 muscarinic receptor subtypes; Gq/11 proteins; PLC‐β1–4 isoforms) in membrane preparations from post‐mortem human prefrontal cerebral cortex collected at several stages of prenatal and postnatal development. In human prenatal brain membranes, PLC was found to be present and could be activated by calcium, but the ability of guanosine‐5′‐o‐3 thiotriphosphate (GTPγS) or carbachol (in the presence of GTPγS) to modulate prenatal PLC‐β was significantly weaker than that associated with postnatal PLC‐β. Western blot analysis revealed that the levels of Gαq/11 did not change significantly during development. In contrast, dramatically higher levels of expression of PLC‐β1–4 isoforms and of M1, M3, and M5 muscarinic receptors were detected in the child vs. the fetal brain, a finding that might underlie the observed increased activity of PLC. Thus, inositol phosphate production may be more efficiently regulated by altering the amount of effectors (PLC‐β1–4) and receptors (M1,3,5 subtypes) than by altering the level of Gαq/11 subunits. These results demonstrate that different PLC isoforms are expressed in the prefrontal cortex of the developing human brain in an age‐specific manner, suggesting specific roles not only in synaptic transmission but also in the differentiation and maturation of neurons in the developing brain.

Aminergic receptors during the development of the human brain: the contribution of in vitro imaging techniques

The development of the human brain is a complex process and, in this regard, the maturation of neurotransmitter systems and their receptors is of special interest. The study of these systems requires methodological approaches with powerful anatomical resolution. In this paper we review the application of visualization procedures to the fine localization, pattern of appearance and functional relevance of monoaminergic receptors in postmortem human brain samples corresponding to different stages of development (fetal, neonatal, infant). Data obtained by using mostly in vitro autoradiography but also in situ hybridization and, very recently, second messenger labeling, are discussed, including the methodological limitations inherent in working with inmature human tissue. From these studies, several conclusions were made. (1) It is possible to visualize, in the human brain with high resolution, the presence of neuroreceptors at early prenatal stages. (2) The anatomical distribution of monoaminergic receptors in the developing human brain is, in general terms, comparable to that found in the adult. (3) During the developmental process, some receptors, which are early and sometimes transiently expressed, play important thophic roles in the regulation of neuronal development: this is the case with the serotonin 5-HT1A receptors, which attain peak levels of hyperexpression over the hippocampus (dentate gyrus, dendritic areas of CA fields) and the raphe nuclei and show a transient expression in the cerebellum, around the 25 week of gestational age. (4) Different patterns of ontogenetic appearance for human receptors have been identified: dopamine D2-like (caudate, putamen, nigra) and 5-HT1A receptors are good examples of prenatal development, while 5-HT1B sites (basal ganglia, neocortex) present a mainly postnatal pattern of appearance. (5) Neurotransmitter receptors at human fetal stages are already functional from the point of view of transducing response.