Daniel Vergé

Quantitative autoradiography of multiple 5-HT1 receptor subtypes in the brain of control or 5,7-dihydroxytryptamine-treated rats

The distribution of the 2 main types (A and B) of 5-HT1 binding sites in the rat brain was studied by light-microscopic quantitative autoradiography. The 5-HT1A sites were identified using 3H-8-hydroxy-2- (N-dipropylamino)tetralin (3H-8-OH-DPAT) or 3H-5-HT as the ligand. In the latter case, it was shown that 3H-5-HT binding to 5-HT1A sites corresponded to that displaceable by 0.1 microM 8-OH-DPAT or 1 microM spiperone. The “non-5-HT1A” sites labeled by 3H-5-HT in the presence of 0.1 microM 8-OH-DPAT corresponded mainly to 5-HT1B sites. 5-HT1A binding was notably high in limbic regions (dentate gyrus, CA1 and CA3 hippocampal regions, lateral septum, frontal cortex), whereas 5-HT1B binding was particularly concentrated in extrapyramidal areas (caudate nucleus, globus pallidus, substantia nigra). Except in the latter regions, where only one class of 5-HT1 sites was found, both 5-HT1A and 5-HT1B sites existed in all areas examined. The selective degeneration of serotoninergic neurons produced by an intracerebral injection of 5,7- dihydroxytryptamine was associated only with a significant loss of 5- HT1A binding to the dorsal raphe nucleus (-60%) and of 5-HT1B binding to the substantia nigra (-37%). These results are discussed in relation to the possible identity of 5-HT1A and/or 5-HT1B sites with the presynaptic 5-HT autoreceptors controlling nerve impulse flow and neurotransmitter release in serotoninergic neurons.

Autoradiography of serotonin receptor subtypes in the central nervous system

The autoradiographic technique is the most relevant approach for the visualization at the light microscope level of the different classes (5-HT(1A), 5-HT(1B), 5-HT(1C), 5-HT(1D), 5-HT(2) and 5-HT(3)) of receptors for the monoamine neurotransmitter serotonin (5-HT) in the central nervous system of mammals, including man. The only exception is the 5-HT(4) subtype for which no satisfactory radioligand has been developed to date. Quantitative estimates of receptor labelling can be achieved by measurements of optical density on autoradiographic films of brain sections incubated with specific radioligands. This review summarizes the most significant contributions of quantitative autoradiography to the current knowledge of the respective regional distributions and differential regulations of the various classes of central 5-HT receptors.

Transient expression of 5-HT1A receptor binding sites in some areas of the rat CNS during postnatal development

The developmental evolution of 5‐HT1A receptor binding sites was examined in the rat CNS during the early postnatal period using quantitative autoradiography and binding assays with 3H‐8‐OH‐DPAT as the selective ligand. A progressive increase in the density of 5‐HT1A sites was observed in the hippocampus, septum and cerebral cortex, up to adult levels which were reached around the third postnatal week. In contrast, complex biphasic (increase then decrease) changes were noted in other structures (for instance the nucleus of the lateral lemniscus) and even a progressive decrease in the density of 5‐HT1A sites took place in the cerebellum during the first two postnatal weeks. The transient expression of 5‐HT1A receptor binding sites in a structure such as the cerebellum which develops exclusively for the postnatal period further supports that 5‐HT might play a trophic role during maturation of the CNS.

Pre- and postsynaptic localization of the 5-HT7 receptor in rat dorsal spinal cord: immunocytochemical evidence.

Several lines of evidence indicate that 5‐HT7 receptors are involved in pain control at the level of the spinal cord, although their mechanism of action is poorly understood. To provide a morphological basis for understanding the action of 5‐HT on this receptor, we performed an immunocytochemical study of 5‐HT7 receptor distribution at the lumbar level. 5‐HT7 immunolabelling is localized mainly in the two superficial laminae of the dorsal horn and in small and medium‐sized dorsal root ganglion cells, which is consistent with a predominant role in nociception. In addition, moderate labelling is found in the lumbar dorsolateral nucleus (Onufs nucleus), suggesting involvement in the control of pelvic floor muscles. Electron microscopic examination of the dorsal horn revealed three main localizations: 1) a postsynaptic localization on peptidergic cell bodies in laminae I–III and in numerous dendrites; 2) a presynaptic localization on unmyelinated and thin myelinated peptidergic fibers (two types of axon terminals are observed, large ones, presumably of primary afferent origin, and smaller ones partially from intrinsic cells; this presynaptic labelling represents 60% and 22% of total labelling in laminae I and II, respectively); and 3) 16.9% of labelling in lamina I and 19.8% in lamina II are observed in astrocytes. Labeled astrocytes are either intermingled with neuronal elements or make astrocytic “feet” on blood vessels. In dendrites, the labelling is localized on synaptic differentiations, suggesting that 5‐HT may act synaptically on the 5‐HT7 receptor. This localization is compared with other 5‐HT receptor localizations, and their physiological consequences are discussed. J. Comp. Neurol. 490:256–269, 2005.

The 5-HT2A receptor is widely distributed in the rat spinal cord and mainly localized at the plasma membrane of postsynaptic neurons.

Serotonin (5‐HT) plays a major role at the spinal level by modulating most spinal functions through several receptor subtypes including the 5‐HT2A receptor. To gain further insight into the cellular role of this receptor, we performed an immunocytochemical study of 5‐HT2A receptors in the rat spinal cord, at light and electron microscope levels. The results showed that 5‐HT2A receptors were widely distributed in the spinal cord at all segmental levels. Immunolabeling was particularly dense in lamina IX and in the dorsal horn lamina IIi. Immunoreactive cell bodies were numerous in lamina IX, where many but not all motoneurons were labeled, as shown by double labeling with choline acetyltransferase antibodies. Stained cell bodies were also observed in the gray matter. The study at the ultrastructural level focused on the lumbar dorsal horn (laminae I–II) and ventral horn (lamina IX). At both levels, 5‐HT2A immunoreactivity was mainly postsynaptic on dendrites and cell bodies. However, a little presynaptic labeling was also observed in axon and axon terminals, some of them containing large granular vesicles attesting to their peptidergic nature. The main result of our study was the “nonsynaptic” plasma membrane localization of 5‐HT2A receptors covering a large surface of cell bodies and dendrites, suggesting a paracrine form of action of serotonin. These observations are consistent with a double role (pre‐ and postsynaptic) for serotonin on these receptors on various cellular targets. J. Comp. Neurol. 472:496–511, 2004.

Production and characterization of polyclonal antibodies recognizing the intracytoplasmic third loop of the 5-hydroxytryptamine1A receptor

The portion of the complementary DNA encoding the third intracellular loop of the rat 5-hydroxytryptamine1A (serotonin) receptor was subcloned into the vector pGEX-KG and expressed in Escherichia coli as a fusion protein coupled with the glutathione S-transferase of Schistosoma japonicum. The fusion protein was purified on a glutathione-agarose affinity column and used to immunize rabbits for the production of polyclonal anti-5-hydroxytryptamine1A receptor antibodies. Enzyme-linked immunosorbent assay revealed that antibodies were produced as early as one month after the first injection of the fusion protein, and immune response plateaued at a maximum after the third (monthly) booster injection. These antibodies only marginally affected the specific binding of [3H]8-hydroxy-2-(di-n-propyl-amino) tetralin to solubilized and membrane bound 5-hydroxytryptamine1A receptors, and did not interfere with serotonin-induced inhibition of forskolin-stimulated adenylate cyclase negatively coupled to 5-hydroxytryptamine1A receptors in rat hippocampal membranes. However, antibodies were able to immunoprecipitate 5-hydroxytryptamine1A receptor binding sites solubilized from rat hippocampal membranes. The distribution of immunoautoradiographic labelling and immunohistochemical staining of rat brain sections exposed to the antibodies raised against the fusion protein superimposed to that of 5-hydroxytryptamine1A receptor binding sites labelled by specific radioligands, with marked enrichment in the limbic areas (dentate gyrus and CA1 area in the hippocampus, lateral septum, entorhinal cortex) and the anterior raphe nuclei. The differential cellular location of immunoreactivity within the hippocampus (where dendritic fields but not pyramidal cell somas were immunostained) and the median raphe nucleus (where the plasmic membrane of somas was strongly immunoreactive) suggests that the addressing of 5-hydroxytryptamine1A receptors might differ from one neuronal cell type to another.

5-HT5A receptor localization in the rat spinal cord suggests a role in nociception and control of pelvic floor musculature.

The 5‐HT5A receptor is a seven‐transmembrane receptor negatively coupled to adenylate cyclase, whose activation opens K+ channels. The 5‐HT5A receptor may thus exert an inhibitory effect on neuronal activity. However, the function of this receptor is still largely unknown, in particular at the spinal level, and this is partly due to lack of specific ligands. Immunocytochemistry using specific anti‐5‐HT5A antibodies reveals a particularly dense labeling in the two superficial layers of the dorsal horn, suggesting that the 5‐HT5A receptor may be involved in the spinal modulation of pain. In addition, a very intense staining in the lumbar dorsolateral nucleus (Onuf nucleus) in both males and females suggests that the 5‐HT5A receptor is also involved in micturition through the control of urethral sphincter muscles. Colchicine pretreatment allows the staining of numerous cell bodies in lamina II. Fewer labeled cell bodies are seen in laminae I and III–VI, in the lateral spinal nucleus, and in lamina X. Electron microscope examination of 5‐HT5A receptor immunoreactivity in spinal cords from untreated animals confirmed the postsynaptic labeling in all regions studied (dorsal horn, dorsolateral nucleus, and lamina X). The morphological heterogeneity of labeled dorsal horn cell bodies suggests that they belong to functionally distinct neurons (projection neurons and interneurons). In the lumbar dorsolateral nucleus, the labeling is preferentially localized on dendrites, suggesting that in this nucleus 5‐HT preferentially acts at the dendritic level. Finally, the dense labeling of postsynaptic specializations suggests that the receptor may be in stock before being addressed to the synaptic differentiation. J. Comp. Neurol. 476:316–329, 2004.

Quantification of 5-hydroxytryptamine1a receptors in the cerebellum of normal and X-irradiated rats during postnatal development

5-Hydroxytryptamine1A receptors were studied in rats during the first postnatal month in the normal cerebellum and in the granule cell-deprived cerebellum produced by X-irradiation at postnatal day 5. Quantitative autoradiographic studies on sagittal sections of cerebellar vermis, using [1251]BH-8-MeO-N-PAT as radioligand or specific anti-receptor antibodies, revealed that 5-hydroxytryptamine1A receptors existed in the molecular/Purkinje cell layer but at variable density from one lobule to another. Thus, in both normal and X-irradiated rats, the posterior lobules were more heavily labelled than the anterior ones, and the density of 5-hydroxytryptamine1A sites decreased progressively in all the cerebellar folia down to hardly detectable levels at postnatal day 21. However, the intensity of labelling remained higher at postnatal day 8 and postnatal day 12 in X-irradiated rats than in age-paired controls. Measurements of [3H]8-OH-DPAT specific binding to membranes from whole cerebellum confirmed that the density of 5-hydroxytryptamine1A sites per mg membrane protein (Bmax) was higher in X-irradiated animals than in age-paired controls. However, on a per cerebellum basis, no significant difference could be detected between the total number of 5-hydroxytryptamine1A sites, which progressively increased in both control and X-irradiated animals during the first postnatal month. These results therefore show that 5-hydroxytryptamine1A receptors are not located on developing granule cells. The progressive decrease in 5-hydroxytryptamine1A receptor density during the first postnatal month did not reflect a transient expression of 5-hydroxytryptamine1A receptors in the cerebellum of newborn rats, but resulted from the progressive dilution of these sites in this growing structure. The higher density of 5-hydroxytryptamine1A sites in X-irradiated rats simply reflected a lower dilution due to the delayed growth of the cerebellum in these animals.

Selective irreversible blockade of 5-hydroxytryptamine1A and 5-hydroxytryptamine1C receptor binding sites in the rat brain by 8-MeO-2'-chloro-PAT: a quantitative autoradiographic study.

The possible irreversible blockade of 5-hydroxytryptamine1 receptor subtypes 5-hydroxytryptamine1A, 5-hydroxytryptamine1B/5-hydroxytryptamine1D and 5-hydroxytryptamine1C by the chloramine 8-methoxy-2-(N-2-chloropropyl,N-propyl)aminotetralin (8-MeO-2-chloro-PAT) was investigated in rat brain sections by quantitative autoradiography using [3H]8-hydroxy-2-(di-n-propylamino)tetralin [( 3H]8-OH-DPAT), [3H]5-hydroxytryptamine, [125I]BH-8-MeO-N-PAT and [125I]cyanopindolol as radio-ligands. A marked reduction (-50% to -75%) of [3H]8-OH-DPAT and [125I]BH-8-MeO-N-PAT specific binding to 5-hydroxytryptamine1A sites in the hippocampus (CA1 area) and the dorsal raphe nucleus, and of [3H]5-hydroxytryptamine specific binding to 5-hydroxytryptamine1C sites in the choroid plexus was found in sections exposed to 1 microM 8-MeO-2-chloro-PAT and then washed extensively. In contrast the specific binding of [3H]5-hydroxytryptamine to 5-hydroxytryptamine1B/5-hydroxytryptamine1D sites and of [125I]cyanopindolol to 5-hydroxytryptamine1B sites in the substantia nigra and dorsal subiculum remained unaltered by this treatment. Similarly [125I]cyanopindolol binding to beta-adrenergic receptors was not affected by 8-MeO-2-chloro-PAT. Prior occupancy of 5-hydroxytryptamine1A sites by 10 microM 5-hydroxytryptamine or 8-OH-DPAT, and of 5-hydroxytryptamine1C sites by 10 microM 5-hydroxytryptamine prevented any subsequent blockade by 8-MeO-2-chloro-PAT. These data indicate that 8-MeO-2-chloro-PAT should be a useful alkylating agent for achieving selective irreversible blockade of central 5-hydroxytryptamine1A and 5-hydroxytryptamine1C receptors in vivo in the rat.

Functional interactions between subunits of aspartate aminotransferase: Formation of monoliganded dimers during titration of the apoenzyme by pyridoxal 5′-phosphate☆☆☆

Abstract The interaction between apoaspartate aminotransferase and pyridoxal 5′-phosphate at either pH 8.3 (active form of holoenzyme) or pH 5.0 (inactive form) corresponds to a strong quenching of tryptophan fluorescence. The hybrid molecule containing one pyridoxal 5′-phosphate bound per dimer has been prepared both by electrofocusing and by ion exchange chromatography. At both pH values, the fluorescence of the hybrid is 80 to 85% of the arithmetic mean between the fluorescence of the symmetrical holoenzyme and apoenzyme. This is direct evidence of energy transfer from tryptophan residues of the subunit of apoenzyme to the coenzyme of the other subunit. Fluorescence intensity was used to determine the quantity of hybrid holoapoenzyme formed during titration of the apoenzyme by pyridoxal 5′-phosphate. At pH 8.3 a non-linear decrease in the fluorescence is observed, corresponding to 60% of hybrid for the point of half reactivation; this value corresponds to the percentage obtained by electrofocusing (Schlegel & Christen, 1974). At pH 5.0, the decrease in fluorescence is linear during pyridoxal binding; this indicates that at this pH the hybrid is never obtained at detectable concentrations. These results indicate strong interactions between subunits of aspartate aminotransferase corresponding to a weakly negative co-operativity at alkaline pH and a positive cooperativity at acidic pH for the binding of the coenzyme.