G-proteins (Gi,Go) in the basal ganglia of control and schizophrenic brain

Summary We detected the existence of Gi (the inhibitory G-protein) or Go (a similar G-protein of unknown function) in the striatum of control and schizophrenic brains utilizing pertussis toxin-catalyzed ADP ribosylation. The level of Gi/Go was significantly decreased by 42% in the putamen of the left hemisphere in schizophrenics; caudate head and globus pallidus levels were unchanged. Decreased Gi or Go may underlie enhanced dopamine function in the schizophrenic brain.     

G proteins (Gi,Go) in the medial temporal lobe in schizophrenia: preliminary report of a neurochemical correlate of structural change

Summary We have measured the amount of Gi (the inhibitory G-protein) or Go (a similar G-protein of unknown function) in 5 areas of the medial temporal lobe of control and schizophrenic brains utilizing pertussis toxin-catalyzed ADP ribosylation. The material used has previously been shown to have asymmetrical structural abnormalities of the ventricular system. The amount of Gi or Go was reduced on the left side in the hippocampus, amygdala and parahippocampal gyrus, the difference reaching significance in the hippocampus. This data is the first report of a neurochemical correlate of the structural change in the brains of patients with schizophrenia. Decreased Gi or Go in hippocampus may relate to other reported neurochemical deficits or other transmembrane signalling abnormalities. Further investigations of these indices of secondary messenger function in relation to structural changes are indicated.     

Pertussis toxin blocks presynaptic glutamate receptors — a novel ‘glutamateB’ receptor in the lobster neuromuscular synapse

Topical application of L-glutamate to the neuromuscular synapse of the lobster walking leg induced K+-dependent hyperpolarization in the presynaptic membrane. This presynaptic glutamate potential (PGP) was insensitive to Joro spider toxin (JSTX), a spider toxin which specifically blocks the postsynaptic glutamate receptor, but was blocked by pertussis toxin island activating protein (IAP) in a dose-dependent manner. IAP had little effect on the resting conductance channels in pre- and postsynaptic membranes. GTP gamma S, a hydrolysis-resistant analogue of GTP, reduced the PGP supporting the involvement of G-protein in generation of K+ activation. The results suggest that a new type of glutamate receptor exists in the presynaptic membrane in the crustacean neuromuscular synapse.     

Both ETA and ETB receptors are involved in mitogen‐activated protein kinase activation and DNA synthesis of astrocytes: study using ETB receptor‐deficient rats (aganglionosis rats)

Endothelin (ET) is known to be a potent mitogen in astrocytes. However, the contribution and signalling pathway of ET_A and/or ET_B receptor to the proliferation of astrocytes remain unclear. We investigated ET-induced DNA synthesis in astrocytes using ET_B receptor-deficient mutant rats (aganglionosis rats: sl/sl). Western blotting with anti-ET receptor subtype-specific antibodies and Scatchard analysis of binding revealed that ET_B receptor expression in astrocytes depended on gene dosage (+/+: sl/+: sl/sl = 2: 1: 0), whereas ET_A receptor expression was unchanged among the three genotypes. ET-1 (10 nm ) stimulated [³H]thymidine incorporation and mitogen-activated protein kinase (MAP kinase) activity not only in +/+ via both ET_A and ET_B receptors, but also in sl/sl astrocytes via ET_A receptor with about half the extent of those observed in +/+ astrocytes. Treatment with pertussis toxin (PTX) suppressed the ET-1-induced increases in the incorporation and MAP kinase activity in +/+, but not sl/sl astrocytes, indicating that the ET_B receptor-, but not the ET_A receptor-, mediated pathway to DNA synthesis involves PTX-sensitive G proteins, e.g. G_i and/or G_o (G_i/o). In +/+ astrocytes, ET-1 (1 nm ) stimulated cAMP accumulation, and the ET_B receptor-selective agonist IRL 1620 (1 nm ) suppressed 10 μm forskolin-induced cAMP accumulation, suggesting G_s coupling to the ET_A receptor and G_i/o coupling to the ET_B receptor. On the other hand, ET-1 did not increase cAMP accumulation in sl/sl astrocytes, although ET-1 (1 nm ) suppressed the forskolin-induced response, suggesting G_i/o coupling to the ET_A receptor. Our results suggest the possibility that the selectivity of G protein for ET_A receptor is changed from G_s to G_i/o in ET_B receptor-deficient astrocytes.     

An immunohistochemical study of glial fibrillary acidic (GFA) protein and S-100 protein in the colon affected by Hirschsprung's disease

Summary The supportive cells of the enteric nervous system were examined in gut tissues from 15 patients with Hirschsprung's disease by means of immunohistochemistry, utilizing antisera to glial fibrillary acidic (GFA) protein and S-100 protein. In the normoganglionic segment, GFA protein immunoreactivity was predominantly found in association with the myenteric plexus and to a lesser extent in the submucous plexus. On the other hand, the extrinsic, hypertrophic nerve fasciculi were selectively immunostained with GFA protein antiserum throughout the entire length of the aganglionic intestinal walls from all children studied. The large fasciculi were numerous in the distal aganglionic segment and commonly appeared in the intermuscular zone and submucosal connective tissue. Both small-and mediumsized nerve fasciculi with GFA protein immunoreactivity were also encountered within the circular muscle layer of the proximal aganglionic segment. A subpopulation of supportive cells within the hypertrophic nerve fasciculi showed immunoreactivity for GFA protein, while all supportive elements of these fasciculi were stained for S-100 protein. The intrinsic nerve fibers within the circular muscle layer of normoganglionic segments were stained for S-100 protein, but not for GFA protein. The present study supports our previous findings that two types of supportive cells can be differentiated by immunohistochemistry in the enteric nervous system, utilizing antisera to GFA protein and S-100 protein. It is also concluded that the demonstration of GFA protein by immunohistochemical methods favors the diagnosis of aganglionic colons with Hirschsprung's disease, since GFA protein immunoreactivity is confined to the extrinsic, hypertrophic nerve fasciculi characteristic of aganglionic bowels.     

The role of low-density receptor-related protein 1 (LRP1) as a competitive substrate of the amyloid precursor protein (APP) for BACE1

Cleavage of APP by BACE1 is the first proteolytic step in the production of amyloid-beta (Aβ), which accumulates in senile plaques in Alzheimer's disease. Through its interaction with APP, the low-density receptor-related protein 1 (LRP1) enhances APP internalization. Recently, BACE1 has been shown to interact with and cleave the light chain (lc) of LRP1. Since LRP1 is known to compete with APP for cleavage by gamma-secretase, we tested the hypothesis that LRP1 also acts as a competitive substrate for β-secretase. We found that the increase in secreted APP (sAPP) mediated by over-expression of BACE1 in APP-transfected cells could be decreased by simultaneous LRP1 over-expression. Analysis by multi-spot ELISA revealed that this is due to a decrease in sAPPβ, but not sAPPα. Interaction between APP and BACE1, as measured by immunoprecipitation and fluorescence lifetime assays, was impaired by LRP1 over-expression. We also demonstrate that APP over-expression leads to decreased LRP1 association with and cleavage by BACE1. In conclusion, our data suggest that - in addition to its role in APP trafficking - LRP1 affects APP processing by competing for cleavage by BACE1.     

Fast spiking cells in rat hippocampus (CA1 region) contain the calcium-binding protein parvalbumin

Fast spiking cells in the CA₁ region of the rat hippocampus were revealed as γ-aminobutyric acid (GABA)ergic non-pyramidal cells containing the calcium-binding protein parvalbumin by intracellular injection of Lucifer yellow in vitro in combination with postem-bedding parvalbumin immunohistochemistry.     

Intracellular GTPγS restores the ability of morphine to hyperpolarize rat locus coeruleus neurons after blockade by pertussis toxin

The hyperpolarizing effect of morphine on locus coeruleus (LC) neurons, recorded with standard intracellular electrodes, was blocked in brain slices from rats pretreated with pertussis toxin, an inactivator of certain G proteins. In the same slices, when electrodes contained the hydrolysis-resistant GTP analog GTP gamma S, the ability of morphine to rapidly hyperpolarize LC neurons was restored and responses were similar in magnitude to those in control slices. We conclude that there is sufficient residual coupling between opiate receptors and G proteins after pertussis toxin treatment to allow the agonist to be effective when the hydrolysis-resistant GTP analog GTP gamma S is present.     

Cellular localization of serotonin2A (5HT2A) receptors in the rat brain

The serotonin_2A (5HT_2A) receptors have been shown to play an important role in several psychiatric disorders, including depression, schizophrenia, and alcoholism. This immunohistochemical study examined the cellular localization of 5HT_2A receptors in various rat brain structures (olfactory, striatum, cortex, hippocampus, and amygdala). The colocalization of 5HT_2A receptors in astrocytes was performed by double-immunofluorescence staining of 5HT_2A receptors and of glial fibrillary acidic protein (GFAP) using confocal laser microscopy. 5HT_2A receptor immunolabeling was observed in olfactory bulbs, neostriatum, hippocampus, amygdala, and neocortex. Somata and dendrites of pyramidal cells in the frontal cortex (layer V) were densely labeled with 5HT_2A receptors. In several other brain structures (hippocampus, amygdala, striatum, olfactory structures), 5HT_2A receptor immunolabeling was found in cell bodies and processes of neurons. 5HT_2A receptor immunolabeling was also observed in GFAP-positive cells of the various brain structures we investigated (layers I/VI of the neocortex, corpus callosum, hippocampal fissure and hilus, and amygdala). These results indicate that 5HT_2A receptors are expressed in neurons and astrocytes and suggest the possibility that not only neuronal but also glial 5HT_2A receptors have functional implications in psychiatric disorders.     

OFF pathway is preferentially suppressed by the activation of GABAA receptors in carp retina

This study examines the effect of γ-aminobutyric acid (GABA) on the ON and OFF pathways in isolated, superfused carp retina. In most (76%) of amacrine cells bath-applied GABA preferentially suppressed the OFF response. The effect of GABA was blocked by bicuculline. Baclofen did not cause a similar effect. Furthermore, GABA produced a substantial suppression of the OFF bipolar cell response. We conclude that the preferential suppression of the OFF pathway may be due to a presynaptic inhibition mediated via GABA_A receptors at the terminals of OFF bipolar cells.     

Humoral immune recognition of proteolipid protein (PLP)-specific encephalitogenic epitopes in the SJL/J mouse

SJL/J mice were immunized with human PLP as well as encephalitogenic PLP peptides 139–151 and 178–191 and the resulting antibody responses examined for immunochemical specificity employing a panel of 17 synthetic PLP peptide ligands. All animals had demonstrable circulating titers of antibodies early in the humoral immune response to their respective encephalitogens, however, there was no clear qualitative correlation between antibody responses and the induction of EAE. In the majority of PLP immunized animals, determinant-specific antibody populations, including those against encephalitogenic centers, were not detectable in the presence of an anti-PLP antibody response. Antiencephalitogenic peptide antibodies were present in both 139–151 and 178–191 immunized animals regardless of clinical/histologic status. Neither group produced cross-reactive anti PLP antibodies as detected by ELISA. In animals immunized with peptide 139–151, only anti-139–151 antibody specificities were noted. In contrast, all animals immunized with peptide 178–191 had an antibody population cross-reactive with three other PLP peptides: 97–110, 209–217, and 215–228. As humoral immune responses can be demonstrated against PLP-specific encephalitogenic epitopes, the significance of these B cell responses should be considered in the context of their potential role in the development, modulation, and/or potentiation of EAE. © 1994 Wiley-Liss, Inc.     

Autoradiographic analysis of mu1, mu2, and delta opioid binding in the central nervous system of C57BL/6BY and CXBK (opioid receptor-deficient) mice

The recent development of in vitro autoradiography techniques has enabled investigators to determine the distribution and relative levels of multiple ligand binding sites in discrete anatomical areas. In this study we used semi-quantitative in vitro autoradiography to compare the levels of binding to central mu₁, mu₂, and delta opioids sites in two strains of mice, C57BL/6BY and CXBK. The CXBK strain is known to be deficient in whole brain opioid binding sites and to be less sensitive than the C57 strain to the analgesic and locomotor stimulatory effects of opiates and opioids. Delta sites were visualized using [³H][d-Ala²-d-Leu⁵]-enkephalin (DADL) plus a low concentration of morphine, total mu sites (mu₁ and mu₂) were visualized using [³H]dihydromorphine (DHM), and mu₂ sites were visualized using [³H]DHM plus a low concentration of DADL. Binding to mu₁ sites was determined by subtracting mu₂ binding from total mu binding. We found that the two strains did not consistently differ in the levels of delta sites; in some areas the CXBKs had lower levels but in many areas they had levels equal to or greater than those for the C57s. The CXBK strain, however, either had less or the same amount of mu binding as the C57 strain in all areas studied. The CXBK strain was especially deficient in mu₁ binding, particularly in areas involved in pain processing.     

Expression of utrophin (dystrophin-related protein) and dystrophin-associated glycoproteins in muscles from patients with Duchenne muscular dystrophy

We examined whether the dystrophin-associated glycoprotein complex (GPC), which serves to fix dystrophin to cell membranes, is present at the sarcolemma in Duchenne muscular dystrophy (DMD) muscles using an immunohistochemical method. Antibodies against 50DAG (A2) and 43DAG (A3a). the components of GPC, were used for the detection of GPC. We found that, although the amount of GPC was reduced in DMD muscles where ulrophin but not dystrophin was distinctly present, 43DAG (A3a) was fairly heavily and 50DAG (A2) was lightly but distinctly stained on the cell surfaces. It is likely that the capability of utrophin to preserve 50DAG (A2) is less than that of dystrophin, although utrophin has been reported to bind to GPC. We also found that 43DAG (A3a) but not 50DAG (A2) was detected in the peripheral nerves where utrophin was detected. Therefore, it is likely that 43DAG (A3a) is essential for the fixation of utrophin to cell membranes, as in the case of dystrophin. 50DAG (A2) may play other important roles in the pathogenesis of DMD. © 1994 John Wiley & Sons, Inc.     

Neuronal modulation of Schwann cell glial fibrillary acidic protein (GFAP)

Adult rat sciatic nerves contain cytoskeletal peptides that resemble CNS glial fibrillary acidic protein (GFAP) in immunoreactivity and molecular weight. Immunohistological examination of teased nerve fascicles indicated that these peptides are expressed selectively by Schwann cells related to small axons. Radiolabelled mouse and rat CNS GFAP cDNA probes hybridized with a single, 2.7 kb RNA band in Northern blots prepared from total RNA from both rat sciatic nerve and rat brain. Sciatic nerve GFAP mRNA was detectable by this means in adult, 2 month, or 21 day postnatal rats, but not in 3,6, or 10 day postnatal rats. Sciatic nerve transection caused a marked reduction in the level of GFAP mRNA in the axotomized distal stump. We conclude that Schwann cell synthesis of GFAP is developmentally regulated and that Schwann cells, unlike astroglia, require continued trophic input from small axons in order to express GFAP.     

Ontogeny of cholinergic amacrine cells in the opossum (Didelphis aurita) retina

Immunocytochemistry for choline acetyltransferase (ChAT), the synthesizing enzyme for acetylcholine, was used to determine the onset and to follow the maturation of the cholinergic cells in the retina of a marsupial, the South American opossum (Didelphis aurita). ChAT-immunoreactivity was first detected in amacrine cells in the ganglion cell layer by postnatal day 15 (P15) and in the inner nuclear layer by P35. Much later, at P50 a second sub-population of ChAT-immunoreactive cell bodies was evident in the inner nuclear layer. Processes from ChAT-immunoreactive amacrine cells were detected in the two bands of the inner plexiform layer before synaptogenesis. In the adult retina, these two bands correspond to sublamina 2 and 4 of the inner plexiform layer. In flat whole-mounted preparations, cholinergic cell density was 263±13 cells/mm[2] in the ganglion cell layer and it was estimated a total of 24,000 cholinergic neurons. ChAT-immunoreactive somata showed a random pattern of distribution.     

Effects of trichothecenes (T-2 toxin) on protein synthesis in vitro by brain polysomes and messenger RNA

The effects of T-2 toxin on protein synthesis were tested in two reticulocyte lysate in vitro systems pretreated with micrococcal nuclease. One of the test systems contained purified globin mRNA and was initiation dependent. The other contained rat brain polysomes and incorporated amino acids by an elongation dependent process. T-2 toxin inhibited the translation of globin mRNA at all concentrations tested, from 10(-8) M to 10(-4) M. Rat brain polysomes were much less sensitive to T-2 toxin than globin mRNA. While high concentrations of the toxin (10(-4) M) led to partial inhibition of protein synthesis by polysomes, low concentrations (10(-8) M and 10(-6) M) stimulated protein synthesis. Comparison of the above results with those obtained by other workers suggest that the T-2 toxin may inhibit not only the initiation step of translation, but also elongation and termination, depending upon the concentration of the toxin and the nature of the translation system. A similar mechanism may operate for all the trichothecene toxins that exert their effect through binding to ribosomal peptidyl transferase.     

The role of the low-density lipoprotein receptor-related protein (LRP1) in Alzheimer's A beta generation: development of a cell-based model system

The clearance and degradation of extracellular Aβ is critical for regulating β-amyloid deposition, a major hallmark of brains of patients with Aβ in Alzheimer’s Disease. The low-density lipoprotein receptor-related protein, LRP1, is a large endocytic receptor that significantly contributes to the balance between degradation and production of Aβ. An extracellular portion of the LRP, known as the cluster II region can bind to the secreted form of APP (sAPP-KPI). We show here that a GST fusion protein containing the cluster II region of LRP can be used as a ‘mini-receptor’ that specifically binds to sAPP-KPI from conditioned cultured medium. The binding between the GST-LRP-cluster II fusion protein and sAPP-KPI can be inhibited with the strong binding ligand of LRP1, called receptor-associated protein (RAP). Furthermore, a cell-based in vitro assay system has been developed to monitor the production of total Aβ and Aβ_1–42 in the presence and absence of RAP in Chinese hamster ovary (CHO) cell lines both deficient in LRP and expressing LRP. A 3-day treatment of the L2 (CHO cells deficient in LRP and overexpressing APP751) and L3 (CHO cells expressing LRP and overexpressing APP751) cell lines with RAP showed a decrease in total Aβ and, interestingly, also a decrease in the ratio of Aβ₄₂/Aβ_total. This cell-based model system and LRP-cluster II mini-receptor will be very useful for screening novel compounds that can reduce Aβ accumulation by inhibiting binding of APP-KPI to LRP1.     

Activation of 5-hydroxytryptamine1A receptors increases the affinity of galanin receptors in di- and telencephalic areas of the rat

Since galanin in vitro selectively increases theK_D value of 5-HT_1A receptors without altering the binding of 5-HT_1B or 5-HT₂ receptors, we have studied whether 5-HT_1A receptor activation in turn may affect galanin binding in the ventral di- and telencephalon and the substantia nigra of the rat. As analyzed by autoradiography, the binding of¹²⁵I-galanin was increased by about 55% in the presence of 3–30 nM of 8-OH-2-(di-npropylamino)-tetralin (DPAT) in the paraventricular thalamic nucleus, the nucleus reuniens and rhomboideus, the zona incerta, the medial and the lateral hypothalamus, and the medial and the lateral amygdaloid area, but not in the pars compacta of the substantia nigra, which lacks 5-HT_1A binding sites. DPAT (10 nM) reduced the IC₅₀ values of galanin at¹²⁵I-galanin binding sites by approximately 55% within all the analyzed di- and telencephalic regions. The overall increase inB_O values was50 ± 11%. Using the filter wipe technique in cryostat sections at Bregma -2.8 mm covering all the brain regions at this level, DPAT (10 nM) decreased the IC₅₀ values of galanin from21.6 ± 1.1nM (control) to15.5 ± 0.9nM, and increased theB_O values by19.4 ± 4.1%. In membrane preparations from the ventral di- and telencephalon, DPAT decreased the IC₅₀ values of galanin binding sites by20 ± 3% at 100 nM of DPAT. This effect could be completely blocked by the specific 5-HT_1A receptor antagonist 1-(2-methoxyphenyl)-4-[4-(2-pthalimido)butyl]piperazine. GTP (0.1 nM) produced a17 ± 5% increase in the IC₅₀ value of galanin and a23 ± 4% decrease in theB_O value of¹²⁵I_galanin binding sites. However, DPAT (100 nM) was still able to decrease the IC₅₀ values of galanin in the presence of GTP (-8 ± 3%;control-10 ± 3%). TheB_max value of¹²⁵I-galanin binding was not affected by DPAT. The increased affinity of galanin binding sites by DPAT seems to reflect a G-protein-independent intramembrane receptor-receptor interaction between 5-HT_1A and galanin receptors. This interaction may represent an intramembrane inhibitory feed-back mechanism of 5-HT_1A receptor sensitivity, and may be important both under normal conditions and in 5-HT-mediated mental disorders.     

Characterization of gonadotropin-releasing hormone (GnRH)-immunoreactive protein in the rat pineal gland

The aim of the present study was to characterize GnRH-like substance(s) in the rat pineal gland using a monoclonal antibody, LRH13, as a probe. The epitope of LRH13 is between 2nd and 5th amino acid residues of the mammalian GnRH, and its immunological characters were previously defined by us. LRH13 could show strong immunological signal on the rat pineal gland. Immunoblot after SDS-PAGE of the pineal gland preparations showed a LRH13 immunoreactive band with apparent mol wt 52 kiloDalton (kD), which is much bigger than that of hypothalamic GnRH precursor (10 kD). The 52 kD protein, however, was detected from insoluble fraction of the pineal homogenate and liberated from the fraction by Triton X-100 (2%) treatment. On the other hand, NaCl (140 mM and 500 mM) or EDTA (10 mM) treatment failed to liberate. Two-dimensional gel electrophoresis showed that the 52 kD protein is a mixture of two proteins with different isoelectric points (pI approximately 6.8 and 7.0). Both proteins showed identical patterns of peptide mapping by V8 protease digestion, and they might be originated from the same peptide. These results suggest that the rat pineal GnRH-immunoreactive substance has a unique property as a membrane associate protein. © 1995 Wiley-Liss, Inc.     

Non-glial specificities of immunocytochemistry for the glial fibrillary acidic protein (GFAP)

Summary In an extensive immunocytochemistry study for glial fibrillary acidic protein (GFAP) of human neuropathological biopsy or autopsy tissue specimens examined for diagnostic or research purposes, rare non-glial specificities of the GFAP immunostain were observed: Schwann cells of some small nerves in salivary gland, renal capsule, and in epidural fat adjacent to a metastatic carcinoma, Schwann and satellite cells in a spinal ganglion invaded by tumor, chondrocytes of epiglottic cartilage, few cells of a malignant pleomorphic adenoma of salivary gland, most cells of a recurrent papillary meningioma with areas similar to the hemangiopericytic variant, and many cells of a renal carcinoma metastatic to brain; the primary renal tumor had been operated 4 years earlier and focally contained some GFAP-positive cells. To ascertain the specificity of such unexpected immunoreactivities for GFAP and to exclude possible crossreactivities with other intermediate filament (IF) proteins, a panel of different antibodies was used for immunocytochemistry with the peroxidase-antiperoxidase (polyclonal antisera) or labeled biotin-avidin (monoclonal antibodies) techniques: two monoclonal and four polyclonal anti-GFAP, three monoclonal and one polyclonal anti-cytokeratins (CK), and two monoclonal anti-vimentin (VIM) antibodies. Triple expression of GFAP, VIM and CK was found in the papillary meningioma (in patterns suggesting frequent co-localization), in the metastatic carcinoma (in patterns suggesting little co-localization), and in the pleomorphic adenoma (only few GFAP-positive cells). Co-expression of GFAP and VIM was seen in epiglottic chondrocytes and reactive astroglia; another metastatic carcinoma was labeled only for CKs. In the light of previous reports on non-glial specificities of the GFAP immunostain, and of the consistency of our immunostaining results obtained by all monospecific anti-GFAP antibodies as well as the lack of immunocytochemically evident crossreactivity with other IF proteins, authentic GFAP production by some rare non-glial tissues and tumors is suggested.Dedicated to Prof. F. Seitelberger on the occasion of his seventieth birthdaySupported by a project fund of the Austrian Ministry for Science and Research