JoséM. Palacios

Dopamine D3 receptor mRNA and binding sites in human brain

Dopamine D3 receptors (Sokoloff et al., 1990) have been shown to be related to dopamine D2 receptors and have been suggested to play a role in mediating the antipsychotic effects of neuroleptics. So far studies on the expression of D3 mRNA and of binding sites with pharmacological characteristics of D3 receptors have been restricted to rat brain. Using in situ hybridization histochemistry, we demonstrate that D3 mRNAs are enriched in human n, accumbens and in the islands of Calleja. In addition, D3 mRNA was detected at very low levels in anterior caudate and putamen with a rostro-caudally decreasing gradient and in hypothalamic mammillary nuclei. In receptor autoradiographic binding studies, the islands of Calleja were found to be labeled by [125I]iodosulpride and [3H]CV 205 502 but not by [3H]raclopride and [3H]YM 09151-2. Pharmacological analysis of binding of the D2/D3 ligand [3H]CV 205 502 in n. accumbens and caudate-putamen is consistent with the presence of D3 receptor sites in ventral striatum. Overall distribution and pharmacology of D3 sites in human and rat brain appear to be similar. Presence and distribution of D3 receptors in human brain are compatible with the notion that D3 receptors might be involved in mediating the clinical effects of antipsychotics.

Differential Visualization of Dopamine D2 and D3 Receptor Sites in Rat Brain. A Comparative Study Using In Situ Hybridization Histochemistry and Ligand Binding Autoradiography

At least five members of the dopamine receptor family have been characterized at the gene level. D2, D3 and D4 dopamine receptors are related pharmacologically. In order to visualize the differential expression of D1, D2 and D3 receptors in rat brain we have combined in situ hybridization histochemistry with receptor autoradiography. Regions enriched with D3 messenger RNA (mRNA) included the islands of Calleja (ioC) and nucleus accumbens. Very low or undetectable levels were present in the caudate–putamen. In contrast, no D2 transcripts were observed in the islands of Calleja, but there were high levels in the nucleus accumbens, caudate–putamen (CP) and pyramidal layer of the olfactory tubercle. A comparison of the binding pattern of six dopamine receptor radioligands hitherto regarded as D2 receptor‐selective showed that the islands of Calleja were intensely labelled by [125I]iodosulpride, [3H]CV 205 502 and [3H]SDZ 205 501, while the binding of [3H]spiperone, [3H]raclopride and [3H]YM 09151–2 was much lower or undetectable. Pharmacological analysis of the binding of D2/D3 ligands to the islands of Calleja and caudate–putamen suggests that binding sites in these two regions are of different pharmacology, consistent with the presence of D3 sites in the islands of Calleja and the predominance of D2 sites in the caudate. These results demonstrate the expression of D3 binding sites in the rat brain and provide a procedure to differentiate D2 and D3 receptor populations in binding studies.

Increased levels of the Kunitz protease inhibitor-containing βAPP mRNAs in rat brain following neurotoxic damage

Deposits of beta-amyloid are one of the main pathological characteristics of Alzheimers disease. The beta-amyloid peptide (or beta/A4) constituent of these deposits is derived from the beta-amyloid precursor protein (beta APP), which is expressed in several isoforms. It has been suggested that an imbalance in the normal ratio between the Kunitz protease inhibitor (KPI)-containing beta APPs versus the non containing forms could result in altered processing of beta APP and progressive beta/A4 deposition. We have studied the expression of four beta APP isoforms in the rat brain after intracerebroventricular application of kainic acid. Increased levels of the KPI-containing beta APP and GFAP mRNAs were observed in tissues surrounding areas of neuronal damage. A parallel increase of beta APP and GFAP immunoreactivity was observed in reactive astrocytes in these areas. These results suggest that the normal ratio of beta APP isoforms may be profoundly altered as a result of neuronal damage and that non-neuronal cells may respond to neuronal injury by increased expression of the KPI-containing beta APP isoforms.

Excitatory amino acid AMPA receptor mRNA localization in several regions of normal and neurological disease affected human brain. An in situ hybridization histochemistry study

In situ hybridization histochemistry was used to localize the mRNAs coding for four alpha-aminoisoxazole propionic acid-sensitive glutamate receptor subunits in human brain (age range 51-95 years, postmortem delay 4.5-10 h). High levels of the B receptor subunit mRNA were present in all the studied regions, followed by the A-subunit and the C-subunit. Only very low levels of the D-subunit mRNA were detected. In hippocampus, the mRNA coding for the B-subunits of the glutamate receptor was observed in granule cells of dentate gyrus and in the pyramidal cells of Ammons horn. In cortex, the highest levels of glutamate receptor subunit mRNAs were found in layer I and layers III-IV of entorhinal and temporal cortex, although significant levels were also observed in the other cell layers. A differential distribution was seen in cerebellum where the A-subunit mRNA is expressed mainly by Purkinje cells, while the B-subunit mRNA is present in the internal granule cell layer. These results correlate well with previous data from autoradiographic studies on the localization of excitatory amino acid binding sites in human brain and pinpoint the cells where these receptors are synthesized. In situ hybridization in the hippocampus of patients affected by Alzheimers disease (age range 77-82 years, postmortem delay 19-25.5 h) revealed a decrease on the content of the mRNAs coding for these excitatory amino acid receptors, while an increase was detected in surgically dissected epileptic human hippocampi. These results corroborate and extend the previous data from in vitro autoradiography and suggest alteration of the excitatory amino acid disfunction during these neurodegenerative processes.

Differential regional distribution of AMPA receptor subunit messenger RNAs in the human spinal cord as visualized by in situ hybridization

The electrophysiological characteristics of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors vary with their subunit composition. The establishment of the subunit distribution is an essential step in the understanding of the function of these receptors. In the spinal cord, AMPA receptors are involved in normal and, possibly, pathological processes. Using in situ hybridization histochemistry with radiolabelled oligonucleotides as probes, we have studied the distribution of AMPA receptor subunit messenger RNAs (spliced flip and flop variants of glutamate receptor subunits A-D) in the human post mortem spinal cord. Transcripts for flip variants were preferentially expressed in the superficial dorsal horn, with a dorsoventral decreasing gradient of the signals. Transcripts for flop variants were also abundantly present in all layers of the gray matter, with the highest signal being observed for glutamate receptor subunit Bflop. Accordingly, flop forms were predominant in areas other than the superficial dorsal horn. This differential distribution of transcripts in the dorsal horn suggests that the subunit composition of AMPA receptors varies with the afferent inputs; AMPA receptors on neurons in the superficial dorsal horn, where terminals of thin primary afferents conducting noxious information are located, contain more flip forms, whereas neurons in the deep dorsal horn, where thick primary afferents mediating innocuous stimuli terminate, have AMPA receptors which are mainly composed of flop forms of glutamate receptor subunits A and B. The relatively high abundance of glutamate receptor subunit B transcripts in the superficial laminae of the dorsal horn indicates that AMPA receptors in these laminae have lower Ca2+ permeability. In addition, the relative abundance of glutamate receptor subunits Bflip and Dflop may show that AMPA receptors in the superficial dorsal horn have slow desensitization, while those of motor neurons have rapid desensitization.

Distribution of Galanin mRNA Containing Cells and Galanin Receptor Binding Sites in Human and Rat Hypothalamus.

The distribution of cells containing galanin mRNA and that of galanin receptor binding sites were investigated using in situ hybridization histochemistry and receptor autoradiography in male rat hypothalamus and in postmortem hypothalamic tissues from control human brains. Oligonucleotide probes labelled with 32P were used for hybridization experiments. The specificity of the hybridization signal was ascertained using several probes, competition assays and Northern blot analysis. High levels of hybridization were found in the paraventricular, supraoptic and arcuate nuclei of rat and human hypothalamus. Human intermediate nuclei and scattered cells of the posterior perifornical nucleus also contained galanin mRNA. Galanin mRNA was also found in the dorsomedial nucleus of the rat. The distribution of galanin receptor sites was investigated by receptor autoradiography using 125I‐labelled porcine galanin. The specificity of the binding was assessed by competition with different neuropeptides. While galanin blocked the binding at nanomolar concentrations, the other neuropeptides examined were ineffective at 10‐7 M concentrations. The highest densities of galanin binding sites were seen in the preoptic area, ventromedial and lateral nuclei, of rat and human hypothalamus. In contrast, very low densities of binding sites were observed in the paraventricular, supraoptic and arcuate nuclei. Our results show that the distribution of neurons expressing galanin is complementary to that of galanin receptors in the rat and human hypothalamus. This suggests that receptors for galanin are not located on the cell bodies of galaninergic neurons, but are probably presynaptic on or postsynaptic to the processes of these cells.

Differential regional and cellular distribution of β-amyloid precursor protein messenger RNAs containing and lacking the kunitz protease inhibitor domain in the brain of human, rat and mouse

The beta-amyloid precursor protein is the precursor of the main component of senile plaques (the beta-amyloid peptide or beta/A4) found in the brain of aged humans and, in higher amounts, in the brain of Alzheimers disease and Downs syndrome subjects. Four different forms of beta-amyloid precursor protein messenger RNAs have been described in humans and rodents: beta-amyloid precursor protein 695, beta-amyloid precursor protein 714, beta-amyloid precursor protein 751 and beta-amyloid precursor protein 770 messenger RNAs (numbers corresponding to the number of encoded amino acids). The two latter forms are characterized by containing in their sequence a region with high homology to the Kunitz family of serine protease inhibitors. We have used oligonucleotide probes to study the distribution of the different messenger RNAs encoding each of the four beta-amyloid precursor proteins by in situ hybridization histochemistry in human, rat and mouse brain. We found that beta-amyloid precursor protein 695, beta-amyloid precursor protein 714 and beta-amyloid precursor protein 751 messenger RNAs were widely distributed in the human, rat and mouse brain and that their distribution was roughly similar in most brain areas in these three species. The distribution of beta-amyloid precursor protein 770 messenger RNA was not so wide and differed among the three species studied. beta-amyloid precursor protein 751 and 770 messenger RNAs were the only forms present at significant levels in rodent choroid plexus and meninges, while beta-amyloid precursor protein messenger RNA isoforms containing and lacking the Kunitz domain were detected in the human choroid plexus. We also observed that the relative levels of beta-amyloid precursor protein 751 and 770 messenger RNAs in the rat cerebral white matter as well as in the mouse and human striatum were higher than those of the beta-amyloid precursor protein messenger RNAs lacking the Kunitz domain. While the most abundant beta-amyloid precursor protein messenger RNAs in the brain of all three species under study were, in descending order, beta-amyloid precursor protein 695 and beta-amyloid precursor protein 751 messenger RNAs, the least abundant form was not the same for all species: in human it was beta-amyloid precursor protein 714 messenger RNA and in rat and mouse brain it was beta-amyloid precursor protein 770 messenger RNA. Our results show differences both inter- and intraspecies of the relative abundance and distribution of four beta-amyloid precursor protein messenger RNAs in rat, mouse and human brain.(ABSTRACT TRUNCATED AT 400 WORDS)

Characterization of zinc oxide and zinc ferrite doped with Ti or Cu as sorbents for hot gas desulphurization

Three series of samples based on zinc oxide and zinc ferrite doped with copper or titanium oxides have been prepared in order to improve their performance as regenerable sorbents for hot gas desulphurization. In each series the oxide concentration was varied over a broad range to enhance the formation of different chemical species. The stability against reducing agents and the performance of these sorbents were studied elsewhere. The characterization of fresh, sulphided and regenerated samples was undertaken using XRD, FT-Raman and XPS techniques. The addition of Ti increased the stability of ZnO against reducing agents such as H2, up to an atomic ratio Ti/Zn= 0.5 through the formation of Zn2TiO4. Furthermore, the Ti excess is segregated as TiO2. The addition of Cu to zinc ferrite did not affect the stability but improved the sorbent performance enhancing the ferrite formation and migrating to the sorbent surface during the calcination and regeneration steps. The addition of Ti to zinc ferrite prevented its decomposition into the two component oxides below 600°C stabilizing the structure through the inclusion of Ti in the ferrite lattice. In the sulphiding process Fe, Zn and Cu oxides were converted into the lowest oxidation state sulphides that facilitated the sorbent regeneration during the regeneration process.

β-Amyloid precursor protein localization in the Golgi apparatus in neuron and oligodendrocytes. An immunocytochemical structural and ultrastructural study in normal and axotomized neurons

We have used a polyclonal antibody raised against a synthetic peptide from the carboxyl terminal of the beta-amyloid precursor protein (APP) to examine the cellular and subcellular localization of this protein in the rat brain. Light and electron microscopic immunocytochemical techniques were used. Immunoreactivity was found throughout the brain in all the neurons examined as well as in oligodendrocytes. At the light microscopic level, a perinuclear filamentous distribution was seen in neurons, suggesting a concentration of the protein to the Golgi apparatus. Axotomy of motor neurons of the facial nucleus produced a decrease in choline acetyltransferase (ChAT) activity and an increase in the perineuronal microglial nucleoside diphosphatase (NDPase)-positive cells in addition to a hypertrophy of the GFAP immunoreactive astrocytes. On the other hand, increased APP-like immunoreactivity all over the neuronal cell bodies accompanied by a dispersion (rete dispersion) of the Golgi apparatus labeling was demonstrated. In contrast, reactive microglia and hypertrophic astrocytes in axotomized facial nucleus were not immunolabeled. Oligodendrocytes showed a punctate APP immunoreactivity corresponding to the Golgi apparatus in both operated and control facial nucleus. This was further demonstrated by electron microscopic immunolabeling. These results show that the main localization of the C-terminal containing forms of the APP in the rat brain is the Golgi apparatus in both neurons and oligodendroglia and further supporting the secretory nature of these proteins. The increased synthesis of this protein after axotomy is suggestive of a role of the APPs in growth and/or regeneration.

Characterization of Mn and Cu oxides as regenerable sorbents for hot coal gas desulfurization

To enhance the formation of different mixed oxides, which potentially might stabilize copper in oxidation states 2+ and/or 1+, regenerable sorbents for hot coal gas desulfurization have been prepared by calcination at 950°C of MnO2 and CuO powders in different mole ratios. The fresh sorbents were sulfided at 600°C using a simulated coal gas from cylinders, reduced in identical operating conditions compared to sulfidation in the absence of H2S, and regenerated at 710°C in oxidising atmosphere. Successive sulfidation–regeneration cycles were carried out in a quartz fixed-bed reactor. The fresh, reduced, sulfided or regenerated sorbents were characterized by SEM-EDX, XRD, TPR, XPS and FTIR spectroscopy. The study shows that, under the reducing power of coal gas at the operating conditions used, manganese oxides cannot prevent the reduction of copper oxides and, consequently, the expected beneficial effect derived from copper oxide addition, reducing the equilibrium H2S concentration in the outlet gas to very low levels cannot be achieved. In addition, copper oxides do not prevent the tendency of manganese-based sorbents to form sulfate species in an oxidative atmosphere and the regeneration of these sorbents has to be carried out at higher temperature.