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Given the paucity of data on the distribution of serotonin (5-HT) receptors of type 6 (5-HT(6)) in the human brain, the aim of this study was to investigate their distribution in postmortem human prefrontal cortex, striatum and hippocampus by either immunohistochemical or immunofluorescence techniques. The brain samples were obtained from 6 subjects who had died for causes not involving primarily or secondarily the CNS. The 5-HT(6) receptor distribution was explored by the [(125)I]SB-258585 binding to brain membranes followed by immunohistochemical and immunofluorescence evaluations. A specific [(125)I]SB-258585 binding was detected in all the regions under investigation, whilst the content in the hippocampus and cortex being about 10-30 times lower than in the striatum. Immunohistochemistry and double-label immunofluorescence microscopy experiments, carried out in the prefrontal cortex and hippocampus only, since data in the striatum were already published, showed the presence of 5-HT(6) receptors in both pyramidal and glial cells of prefrontal cortex, while positive cells were mainly pyramidal neurons in the hippocampus. The heterogeneous distribution of 5-HT(6) receptors provides a preliminary explanation of how they might regulate different functions in different brain areas, such as, perhaps, brain trophism in the cortex and neuronal firing in the hippocampus. This study, taking into account all the limitations due to the postmortem model used, represents the starting point to explore the 5-HT(6) receptor functionality and its sub-cellular distribution.

The stereoselectivity of the serotonin1A (5-HT1A) receptor compound 8-hydroxy-2(di-N-propylamino)tetralin (8-OH-DPAT) on forskolin-stimulated adenylyl cyclase activity was investigated in membranes from human 5-HT pre-synaptic (raphe nuclei) and post-synaptic (hippocampus and prefrontal cortex) regions of autopsy brains. After sample incubation with agonists and antagonists, results showed that both the racemic mixture of 8-OH-DPAT or its (+) and (-) enantiomers behaved as full agonists in the tested brain regions. Enantiomer potency (EC50, nM) and efficacy (percentage of maximal inhibition, %) values were similar in all regions under investigation. However, some inter and intra-region variations in racemic 8-OH-DPAT potency and efficacy have been observed. In particular, the potency of racemic 8-OH-DPAT was higher in the prefrontal cortex and raphe nuclei than in the hippocampus, where it was in fact lower than either single enantiomers. Agonist effects were competitively reversed by 5-HT1A antagonists, although once again a different profile was revealed in the hippocampus. The data underscores the lack of stereospecificity of 8-OH-DPAT-mediated inhibition of adenylyl cyclase activity in either pre- or post-synaptic human brain regions. Moreover, such results have significant implication, as they support the notion that human 5-HT1A receptors might vary from one brain region to the other.

The saturation parameters and the pharmacological characteristics of the binding of the serotonin 1A (5-HT 1A ) receptor agonist [ 3 H]8-hydroxy-2-(di-N-propylamino)tetralin ([ 3 H]8-OH-DPAT), as well as the effects of nucleotides and divalent cations (Mg 2+ , Mn 2+ ) on it, were compared in some human postmortem brain regions: the main cortical areas, hippocampus and striatum. [ 3 H]8-OH-DPAT labelled a single population of recognition sites with the highest maximal capacity (B max ) in the hippocampus and the lowest affinity in the striatum. Among the various cortical areas, the frontal cortex exhibited the highest B max . The pharmacological profile of the [ 3 H]8-OH-DPAT binding sites was consistent with the labelling of the 5-HT 1A receptor in the hippocampus and cortex, whereas the striatal site shared strong similarity to the presynaptic serotonin transporter. Modulation of [ 3 H]8-OH-DPAT binding by divalent cations and nucleotides was detectable and stable in autopsy brains. In particular, nucleotide effects were area-dependent: guanosine thiotriphosphate (GTPγS) reduced [ 3 H]8-OH-DPAT binding to the same extent in the hippocampus and frontal cortex, while having no effect in the striatum. Divalent cation effects depended also upon the brain area: in the striatum, they inhibited [ 3 H]8-OH-DPAT binding, while stimulating it in the hippocampus and, with less extent, in the frontal cortex. In summary, these findings suggest that the [ 3 H]8-OH-DPAT binding and its modulatory parameters in human brain tissues seem to show similarities but also some differences with respect to those determined in the rat brain. Furthermore, postmortem stability of GTP and divalent cation sensitive 5-HT 1A receptors underlines the need for further studies on the regulatory and functional properties of this receptor in the human brain.

The first effects of 3,4-methylen-dioxy-metamphetamine (MDMA, "ecstasy"), on serotonin 1A (5-HT(1A)) receptors in rat hippocampus were determined by means of [(3)H]-8-hydroxy-dipropylamino-tetralin ([(3)H]-8-OH-DPAT) and 5'guanosine-(gamma-[(35)S]-thio)triphosphate ([(35)S]-GTPgammaS) binding as well as inhibition of forskolin (FK)-stimulated adenylyl cyclase (AC) activity. The study was completed by [(35)S]-GTPgammaS functional autoradiography experiments carried out in frontal sections of rat brain, including the hippocampal region. Results showed that MDMA was either able to displace [(3)H]-8-OH-DPAT binding (K(i) congruent with 500 nM) or to reduce the number of specific sites (B(max)) without affecting K(d). The drug also failed to change the [(35)S]-GTPgammaS binding or to inhibit AC velocity, underlying its behavior as a non-competitive 5-HT(1A) receptor antagonist. Further, MDMA (1 or 100 microM), partially antagonized either [(35)S]-GTPgammaS binding stimulation of the agonists 5CT and 8-OH-DPAT or the AC inhibition induced by 5CT and DP-5CT. However, in contrast to binding studies, in AC assays the amphetamine displayed an effect also on EC(50), always being less potent than the reference antagonist WAY100,635. In functional autoradiography, MDMA behaved either as a partial 5-HT(1A) antagonist in limbic areas or, added alone, as an agonist, increasing the coupling signal presumably through 5-HT release from synapses. Interestingly, the selective 5-HT re-uptake inhibitor (SSRI) fluoxetine had no effect on MDMA [(35)S]-GTPgammaS binding activation. This latter finding indicates that the amphetamine can release 5-HT via alternative mechanisms to 5-HT transporter binding, probably via membrane synaptic receptors or vesicular transporters. The release of other transmitters is not excluded. Therefore, our results encourage at extending the study of MDMA biochemical profiles, in the attempt to elucidate those amphetamine-induced pathways with a potential for neurotoxicity or psycho-stimulant activity.