Influence of lighting conditions on toxicity and genotoxicity of various PAH in the newt in vivo. We evaluated the influence of near-ultraviolet light (UVA) on the cytotoxicity and genotoxicity of 7 polycyclic aromatic hydrocarbons (PAH) in larvae of the amphibian Pleurodeles waltl. Benz[a]anthracene (BA), 7,12-benz[a]anthraquinone (BAQ) and anthracene (Ac) proved to be lethal at low doses (some ppb), and the following order of genotoxicity was observed: BA approximately BAQ > DMBA > DMA (9,10-dimethylanthracene). Ac, AQ (9,10-anthraquinone) and DBA (dibenz[a,h]anthracene) were not found to be clastogenic. In the larvae reared in normal conditions (subdued natural daylight/darkness alternation) or in continuous darkness, the BA derivatives were shown to be more genotoxic than BA itself: DMBA > BAQ > BA; BA (> or = 187.5 ppb) slightly increased the level of micronuclei in circulating erythrocytes, while DMBA was strongly clastogenic, in line with their reported carcinogenicity. In other experiments, rearing media alone (i.e., water containing BA, BAQ or DMBA) were UVA-irradiated for 24 h, and then tested on larvae in the dark ('IR-UV/dark' conditions). Photodegradation of BA (50 and 100 ppb) gave rise to clastogenic products. By contrast, DMBA (12.5, 25 or 50 ppb) was destroyed by UVA, and we suggested that any potentially mutagenic photoproducts formed were not in sufficient amounts to yield a positive response in the newt micronucleus test.


Genetic toxicology evaluation of commercial beers, III. SCE, chromosome aberrations, and forward mutation (HGPRT) of commercial beer products in CHO cells. Concentrated organic residues extracted from 5 blended aliquots of commercial beers were evaluated for their ability to induce sister chromatid exchange (SCE), chromosomal aberrations and forward mutation in Chinese hamster ovary (CHO) cells. Each extract was prepared by blending 4 commercial beers of similar ingredients and brewing method, passing the beer pool over XAD-2 resin, extracting the resin and concentrating the extract. Studies were performed both with and without metabolic activation using variable amounts of reconstituted residues from 225-fold concentrates of the blended samples. CHO cultures were treated with 0.75 microliters/ml through 10.0 microliters/ml of the concentrates in the SCE assays, 1.0 microliters/ml through 10.0 microliters/ml of the extracts in the aberration assays and 2.5 microliters/ml up to 20 microliters/ml for forward mutation assays. In preliminary screening for SCE as an indicator of potential DNA damage, a significant increase was observed for 3 of 5 concentrated samples; however, no increase in SCE was induced by any of the 5 samples when S9 was added as a source of exogenous metabolic activation. More definitive tests for induction of genetic events, i.e., chromosome aberrations and forward HGPRT mutations, were negative for all 5 extracts whether or not S9 mix was present. Since SCE were not induced in tests with metabolic activation and since there was no concordant aberration or point mutation induction, the preliminary indication of potential DNA damage shown by elevated SCE under conditions without metabolic activation appears to have little biological significance.


Mutagenic activity of oxathiolane steroids: structural requirement for the genotoxic activity in Salmonella and E. coli. Oxathiolanes and disulfonyl derivatives of steroids were tested for mutagenic activity in the Ames tester strains. The test compounds exhibited mutagenic activity without metabolic activation although metabolic activation markedly enhanced their activity. A significant decrease in the survival of the radiation-sensitive mutants recA, lexA and rer of E. coli was observed as compared to their wild-type counterpart in the presence of the test steroid. Structural features which appear to be crucial for the mutagenic activity in these steroidal drugs are: (i) an electron-donating group at position 3, and (ii) a bulky group anchored at the 5th and 6th positions. The test steroids appear to damage DNA which in turn initiates the SOS repair with the concomitant induction of mutation.


Assessment of the genotoxic potential of riboflavin and lumiflavin. A. Effect of metabolic enzymes. The mutagenic potential of riboflavin and its photodegradation product lumiflavin was evaluated using the umu test, SOS chromotest and Ames Salmonella assay. Both riboflavin and lumiflavin by themselves were found to be non-mutagenic. On treatment with rat liver microsomal enzymes (S9) or caecal cell-free extract (CCE), lumiflavin acquired mutagenicity, while the status of riboflavin remained unaffected. Activation of lumiflavin by metabolic enzymes was found to result in an alteration of its spectral characteristics.


Polymorphism in the circumsporozoite protein of the human malaria parasite Plasmodium vivax. The circumsporozoite (CS) protein that covers the surface of infectious sporozoites is a candidate antigen in malaria vaccine development. To determine the extent of B- and T-epitope polymorphism and to understand the mechanisms of antigenic variability, we have characterized the CS protein gene of Plasmodium vivax from field isolates representing geographically distant regions of Papua New Guinea (PNG) and Brazil. In the central repeat region of the CS protein, in addition to variation in the number of repeats, an array of mutations was observed which suggests that point mutations have led to the emergence of the variant CS repeat sequence ANGA(G/D)(N/D)QPG from GDRA(D/A)GQPA. Outside the repeat region of the protein, the nonsilent nucleotide substitutions of independent origin are localized in three domains of the protein that either harbor known T-cell determinants or are analogous to the Plasmodium falciparum immunodominant determinants, Th2R and Th3R. We have found that, with the exception of one CS clone sequence that was shared by one P. vivax isolate each from PNG and Brazil, the P. vivax CS protein types can be grouped into Papuan and Brazilian types. These results suggest that an in-depth study of parasite population dynamics is required before field trials for vaccine formulation based on polymorphic immunodominant determinants are conducted.


Inhibitory monoclonal antibodies recognise epitopes adjacent to a proteolytic cleavage site on the RAP-1 protein of Plasmodium falciparum. The low-molecular-weight rhoptry-associated protein (RAP) complex of Plasmodium falciparum consists of at least two gene products, RAP-1 and RAP-2, and has the ability to immunise Saimiri monkeys against experimental P. falciparum infection. Several monoclonal antibodies specifically recognise this complex and in this study we show that purified immunoglobulin derived from these monoclonals is capable of inhibiting parasite growth in vitro. It has previously been shown that RAP-1 initially appears as an 80-kDa protein (p80) in early schizogony and is processed to a 65-kDa protein (p65) in late schizogony. Several of the inhibitory monoclonals recognise both the 80- and 65-kDa proteins by Western blot analysis suggesting that they recognise linear epitopes on RAP-1. We have mapped these epitopes by testing the reactivity of the monoclonals against fragments of the rap-1 gene expressed as beta-galactosidase fusion proteins and subsequently against synthetic peptides. All of the epitopes map to a region 10-20 amino acids C-terminal to the proteolytic cleavage site for the processing of p80 to p65 at amino acid 190. We also show that the 65-kDa protein is not present in purified merozoites, suggesting that its generation is associated with merozoite release rather than erythrocyte invasion. These results are discussed with respect to possible inhibitory mechanisms for the monoclonals.


Surface epitope localization and gene structure of a Babesia bovis 44-kilodalton variable merozoite surface antigen. Variation of Babesia bovis merozoite surface antigens occurs among geographic strains of the parasite. In this and a concurrent report, we investigate this variation at the gene and protein level. Using a monoclonal antibody (mAb 23/70.174), B. bovis gene sequences were identified that encoded a surface epitope of a 44-kDa merozoite surface antigen (MSA-2). This epitope is variably expressed among geographic isolates of B. bovis. Here, we describe the MSA-2 protein gene sequence, localize this surface epitope to a repeated amino acid sequence, and investigate the genomic organization of the gene in B. bovis strains from Mexico and Australia. The predicted protein sequence had hydrophobic regions at its amino and carboxy termini consistent with a signal peptide and a membrane anchor via glycosylphosphatidyl inositol, respectively. The surface epitope recognized by mAb 23/70.174 was localized within a 24-amino acid sequence which is repeated twice in tandem. Six different EcoRI bands hybridized to the MSA-2 gene sequence with varying intensities in genomic Southern blots of the homologous strain. Two of these appear to be alleles of the MSA-2 gene. Whereas 5' and 3' sequences of the MSA-2 gene sequence were detected in an Australia strain of B. bovis, internal gene sequences encoding the surface epitope were not. The 3' sequences of the MSA-2 gene also had significant sequence similarity with the MSA-1 gene of the Mexico strain B. bovis and a gene from the previously described BabR locus. These data indicate that the MSA-2 protein gene belongs to the BabR locus which encodes variable merozoite surface antigens.


Neutralization-sensitive merozoite surface antigens of Babesia bovis encoded by members of a polymorphic gene family. Monospecific antibodies against native and recombinant versions of the major merozoite surface antigen (MSA-1) of Babesia bovis neutralize the infectivity of merozoites from Texas and Mexico strains in vitro. Sequence analysis shows that MSA-1 and a related, co-expressed 44 kDa merozoite surface protein (MSA-2) are encoded by members of a multigene family previously designated BabR. BabR genes, originally described in Australia strains of B. bovis, are notable because their marked polymorphism is apparently mediated by chromosomal rearrangements, but protein products of BabR genes have not previously been identified. The 3' terminal 173 nucleotides of the MSA-1 gene, including 60 nucleotides of untranslated sequence, are highly similar to the 3' terminal sequences of BabR 0.8 (84% identity) and MSA-2 (94% identity). Alignment of the predicted protein sequences demonstrates significant overall homology between MSA-1 and MSA-2, and between both proteins and the amino terminal BabR sequence. MSA-1 nucleic acid probes also hybridize weakly to genomic DNA from the Australia 'L' strain, even though this strain does not express merozoite surface epitopes cross-reactive with MSA-1 or MSA-2. Hybridization of these same probes to genomic DNA from the cloned Mexico strain reveals a pattern of bands compatible with two copies each of MSA-1 and MSA-2. Proteins encoded by this B. bovis gene family have been designated variable merozoite surface antigens (VMSA). The extent and mechanism of VMSA polymorphism among strains will be important when evaluating the role these surface proteins have in the host-parasite interaction, including immunity to blood stages.


Keratinolysis by Absidia cylindrospora and Rhizomucor pusillus: biochemical proof. Absidia cylindrospora and Rhizomucor pusillus causal agents of phycomycoses, were cultured on sterile natural keratins in a mineral solution and the keratin degradation products analyzed. The excess of sulphur was removed by oxidation to inorganic sulphate and thiosulphate, which were the main products of sulphitolysis of keratin. The proteolytic activity of the two fungi depended on the nature of the keratin substrate. Human scalp hair was the most favoured keratin substrate by both the fungi.


Effects of 5-vinyloxazolidine-2-thione on lactating rats and their sucklings. 5-vinyloxazolidine-2-thione (VOT) administered orally to lactating rats was found to be efficiently transferred to the sucklings via the milk. In mothers the exposure to VOT resulted in an increased percentage of neutrophils, a decreased percentage of lymphocytes and increases in the relative weights of liver and thyroid. Suckling rats showed a decreased number of leucocytes, increases in the relative weights of liver and thyroid and structural changes in the thyroid. Male sucklings were more affected than female pups. The antithyroid effects were clearly related to the maternally administered VOT doses.


Signal-sequence recognition by an Escherichia coli ribonucleoprotein complex. Hydrophobic signal-sequences direct the transfer of secretory proteins across the inner membrane of prokaryotes and the endoplasmic reticulum membranes of eukaryotes. In mammalian cells, signal-sequences are recognized by the 54K protein (M(r) 54,000) of the signal recognition particle (SRP) which is believed to hold the nascent chain in a translocation-competent conformation until it contacts the endoplasmic reticulum membrane. The SRP consists of a 7S RNA and six different polypeptides. The 7S RNA and the 54K signal-sequence-binding protein (SRP54) of mammalian SRP exhibit strong sequence similarity to the 4.5S RNA and P48 protein (Ffh) of Escherichia coli which form a ribonucleoprotein particle. Depletion of 4.5S RNA or overproduction of P48 causes the accumulation of the beta-lactamase precursor, although not of other secretory proteins. Whether 4.5S RNA and P48 are part of an SRP-like complex with a role in protein export is controversial. Here we show that the P48/4.5S RNA ribonucleoprotein complex interacts specifically with the signal sequence of a nascent secretory protein and therefore is a signal recognition particle.


Vascular endothelial growth factor induced by hypoxia may mediate hypoxia-initiated angiogenesis. Inefficient vascular supply and the resultant reduction in tissue oxygen tension often lead to neovascularization in order to satisfy the needs of the tissue. Examples include the compensatory development of collateral blood vessels in ischaemic tissues that are otherwise quiescent for angiogenesis and angiogenesis associated with the healing of hypoxic wounds. But the presumptive hypoxia-induced angiogenic factors that mediate this feedback response have not been identified. Here we show that vascular endothelial growth factor (VEGF; also known as vascular permeability factor) probably functions as a hypoxia-inducible angiogenic factor. VEGF messenger RNA levels are dramatically increased within a few hours of exposing different cell cultures to hypoxia and return to background when normal oxygen supply is resumed. In situ analysis of tumour specimens undergoing neovascularization show that the production of VEGF is specifically induced in a subset of glioblastoma cells distinguished by their immediate proximity to necrotic foci (presumably hypoxic regions) and the clustering of capillaries alongside VEGF-producing cells.


Vascular endothelial growth factor is a potential tumour angiogenesis factor in human gliomas in vivo. Clinical and experimental studies suggest that angiogenesis is a prerequisite for solid tumour growth. Several growth factors with mitogenic or chemotactic activity for endothelial cells in vitro have been described, but it is not known whether these mediate tumour vascularization in vivo. Glioblastoma, the most common and most malignant brain tumour in humans, is distinguished from astrocytoma by the presence of necroses and vascular proliferations. Here we show that expression of an endothelial cell-specific mitogen, vascular endothelial growth factor (VEGF), is induced in astrocytoma cells but is dramatically upregulated in two apparently different subsets of glioblastoma cells. The high-affinity tyrosine kinase receptor for VEGF, flt, although not expressed in normal brain endothelium, is upregulated in tumour endothelial cells in vivo. These observations strongly support the concept that tumour angiogenesis is regulated by paracrine mechanisms and identify VEGF as a potential tumour angiogenesis factor in vivo.


Leukocyte accumulation promoting fibrin deposition is mediated in vivo by P-selectin on adherent platelets. The glycoprotein P-selectin is a cell adhesion molecule of stimulated platelets and endothelial cells, which mediates the interaction of these cells with neutrophils and monocytes. It is a membrane component of cell storage granules, and is a member of the selectin family which includes E-selectin and L-selectin. P-selectin recognizes both lineage-specific carbohydrate ligands on monocytes and neutrophils, including the Lewis x antigen, sialic acid, and a protein component. In inflammation and thrombosis, P-selectin may mediate the interaction of leukocytes with platelets bound in the region of tissue injury and with stimulated endothelium. To evaluate the role of P-selectin in platelet-leukocyte adhesion in vivo, the accumulation of leukocytes within an experimental thrombus was explored in an arteriovenous shunt model in baboons. A Dacron graft implanted within an arteriovenous shunt is thrombogenic, accumulating platelets and fibrin within its lumen. These bound platelets express P-selectin. Here we show that antibody inhibition of leukocyte binding to P-selectin expressed on platelets immobilized on the graft blocks leukocyte accumulation and inhibits the deposition of fibrin within the thrombus. These results indicate that P-selectin is an important adhesion molecule on platelets, mediating platelet-leukocyte binding in vivo, that the presence of leukocytes in thrombi is mediated by P-selectin, and that these leukocytes promote fibrin deposition.


Crystal structure of a Src-homology 3 (SH3) domain. The Src-homologous SH3 domain is a small domain present in a large number of proteins that are involved in signal transduction, such as the Src protein tyrosine kinase, or in membrane-cytoskeleton interactions, but the function of SH3 is still unknown (reviewed in refs 1-3). Here we report the three-dimensional structure at 1.8 A resolution of the SH3 domain of the cytoskeletal protein spectrin expressed in Escherichia coli. The domain is a compact beta-barrel made of five antiparallel beta-strands. The amino acids that are conserved in the SH3 sequences are located close to each other on one side of the molecule. This surface is rich in aromatic and carboxylic amino acids, and is distal to the region of the molecule where the N and C termini reside and where SH3 inserts into the alpha-spectrin chain. We suggest that a protein ligand binds to this conserved surface of SH3.


Control of CFTR chloride conductance by ATP levels through non-hydrolytic binding. Site-specific mutation and membrane reconstitution experiments provide compelling evidence that the product of the gene which is at fault in the disease cystic fibrosis, termed the cystic fibrosis transmembrane conductance regulator (CFTR), is a small-conductance chloride channel activated by phosphorylation. As transport of chloride ions is passive, the predicted presence of two nucleotide-binding domains in CFTR seems as puzzling as a report that ATP hydrolysis is essential to activate the channel. We now find that in the sweat duct, which expresses high levels of CFTR and has a very high Cl- conductance, intracellular concentrations of ATP must be about normal (5 mM) for activation of this conductance, apparently by a non-hydrolytic, perhaps allosteric, mechanism. This passive dependence on ATP should mean that even a modest depletion of cell energy levels will significantly lower the energy demands of electrolyte transport by decreasing chloride conductance. We believe this direct coupling between cellular ATP levels and chloride channel activity is an adaptive mechanism to protect the tissue from damage resulting from excessive energy depletion.


The protein kinase A-regulated cardiac Cl- channel resembles the cystic fibrosis transmembrane conductance regulator. Stimulation of beta-adrenoceptors in cardiac ventricular myocytes activates a strong chloride ion conductance as a result of phosphorylation by cyclic AMP-dependent protein kinase (PKA). This Cl- conductance, which is time- and voltage-independent, counters the tendency of the simultaneously enhanced Ca2+ channel current to prolong the ventricular action potential. Using inside-out giant patches excised from guinea-pig myocytes, we show here that phosphorylation by the PKA catalytic subunit plus Mg-ATP elicits discrete Cl- channel currents. In almost symmetrical Cl- solutions (approximately 150 mM), unitary current amplitude scales with membrane potential, and reverses sign near 0 mV, to yield a single channel conductance of approximately 12 pS. Opening of the phosphorylated channels requires hydrolysable nucleoside triphosphate, indicating that phosphorylation by PKA is necessary, but not sufficient, for channel activation. The properties of these PKA-regulated cardiac Cl- channels are very similar, if not identical, to those of the cystic fibrosis transmembrane conductance regulator (CFTR), the epithelial cell Cl- channel whose regulation is defective in patients with cystic fibrosis. The full cardiological impact of these Cl- channels and of their possible malfunction in patients with cystic fibrosis remains to be determined.


Engineering galactose-binding activity into a C-type mannose-binding protein. Calcium-dependent or C-type carbohydrate-recognition domains are homologous protein modules found in a variety of animal lectins. Selective binding of sugars by these domains is essential for glycoprotein clearance, cell-cell adhesion and pathogen neutralization. Although various C-type carbohydrate-recognition domains share sequence identity ranging from 20 to 55%, their sugar-binding characteristics vary widely. The structure of a mannose-binding carbohydrate-recognition domain in complex with a saccharide ligand suggests that two glutamic acid-asparagine pairs are essential determinants of ligand binding by this domain. In C-type lectins that bind galactose with higher affinity than mannose, one of these pairs is replaced by glutamine-aspartic acid. Here we shift the sequence of the mannose-binding protein to correspond to that found in galactose-binding domains in order to test the importance of these residues in sugar-binding selectivity. This simple switch in the position of a single amide group alters the binding activity of the domain so that galactose becomes the preferred ligand.


Pheromone binding to two rodent urinary proteins revealed by X-ray crystallography. The principal protein excreted in male rat urine, urinary alpha 2-globulin and the homologous mouse protein, major urinary protein, have been well characterized, although their functions remain unclear. Male rat urine affects the behaviour and sexual response of female rats, leading to the proposal that rodent urinary proteins are responsible for binding pheromones and their subsequent release from drying urine. Urinary alpha 2-globulin is also involved in hyaline droplet nephropathy, an important toxicological syndrome in male rats resulting from exposure to a number of industrial chemicals and characterized by the accumulation of liganded urinary alpha 2-globulin in lysosomes in the kidney, followed by the induction of renal cancer. We now report the three-dimensional structures of mouse major urinary protein (at 2.4 A resolution) and rat urinary alpha 2-globulin (at 2.8 A resolution). The results corroborate the role of these proteins in pheromone transport and elaborate the structural basis of ligand binding.


Crystal structure at 2.8 A resolution of a soluble form of the cell adhesion molecule CD2. The crystal structure of a soluble form of the T lymphocyte antigen CD2 provides the first complete view of the extracellular region of a cell adhesion molecule. The topology of the molecule, which comprises two immunoglobulin-like domains, is the same as that of the first two domains of CD4 but the relative domain orientation is altered by a fairly flexible linker region. The putative ligand-binding beta-sheet forms a flat surface towards the top of the molecule. Crystal contacts between these surfaces suggest a plausible model for the adhesive interaction.


GM-CSF and TNF-alpha cooperate in the generation of dendritic Langerhans cells. Dendritic cells comprise a system of highly efficient antigen-presenting cells which initiate immune responses such as the sensitization of T cells restricted by major histocompatibility complex molecules, the rejection of organ transplants and the formation of T-cell-dependent antibodies. Dendritic cells are found in many non-lymphoid tissues, such as skin (Langerhans cells) and mucosa, and they migrate after antigen capture through the afferent lymph or the bloodstream to lymphoid organs, where they efficiently present antigen to T cells. Dendritic cells are difficult to isolate and, although they originate from bone marrow their site of maturation and the conditions that direct their growth and differentiation are still poorly characterized. Granulocyte macrophage-colony stimulating factor (GM-CSF) favours the outgrowth of dendritic cells from mouse peripheral blood. Here we extend this finding to man and demonstrate that cooperation between GM-CSF and tumour necrosis factor-alpha (TNF-alpha) is crucial for the generation of human dendritic/Langerhans cells from CD34+ haematopoietic progenitors. The availability of large numbers of these cells should now facilitate the understanding of their role in immunological regulation and disorder.


Signalling through the MHC class II cytoplasmic domain is required for antigen presentation and induces B7 expression. Class II major histocompatibility complex (MHC) molecules function as antigen-presenting elements as well as signal transducers on B lymphocytes. We previously reported that a B lymphoma cell transfectant, 5C2, expressing genetically engineered I-Ak molecules with truncated cytoplasmic domains was severely impaired in both antigen presentation and in anti-Ia-induced intracytoplasmic signalling. These two functions could be restored by preculturing 5C2 cells with cyclic AMP analogues. Here we demonstrate that impaired signal transduction by truncated class II molecules results in a deficiency in induction of the newly defined B-cell accessory molecule B7 (ref. 8), which can be reversed by restoration of B7 expression. These data imply that contact of the T-cell antigen receptor with MHC/antigen ligand results in signal transmission through the class II cytoplasmic domain. This signal, which can be mimicked by dibutyryl cAMP, induces expression of B7, resulting in effective antigen presentation. The fact that crosslinking of surface class II MHC also induces B7 expression on normal resting human B cells supports this contention.


Streptozotocin-induced decreases in serotonin turnover are prevented by thyroidectomy. Although characterized as hypothyroid, streptozotocin-diabetic rats have reduced serotonin turnover (5-hydroxyindoleacetic acid/serotonin, 5-HIAA/5-HT) in brain stem, while hypothyroid rats have increased 5-HIAA/5-HT. In the present study the two treatments were combined to determine if they affected 5-HIAA/5-HT through the same mechanism. In addition, an alternative method was used to assess 5-HT activity in thyroidectomized (TX) rats, i.e. measurement of 5-HT disappearance after inhibition of tryptophan hydroxylase with p-chlorophenylalanine (PCPA). Adult male rats were first TX (experiment 1) or given methimazole (METH; experiment 3). Two weeks later, diabetes (DB) was induced with streptozotocin in hypothyroid rats and euthyroid controls. Two weeks later, functional measurements were taken. Rats were then killed, and spinal cord and brain stem serotonin turnover (5-HIAA/5-HT), as well as plasma T3, T4 and corticosterone (CORT) concentrations were measured. TX attenuated diabetic hyperphagia and weight loss. DB alone led to moderate reductions in T3 and T4, but the hormones were barely detectable in plasma of TX and METH rats. CORT was elevated in DB but was not affected by TX. Open field activity was not affected by DB or TX. TX and METH significantly increased 5-HIAA/5-HT in both spinal cord and brain stem. TX also led to enhanced disappearance of 5-HT after PCPA. DB significantly reduced 5-HIAA/5-HT, suggesting independent effects of the treatments. However, DB-TX rats still had significantly higher 5-HIAA/5-HT than control-sham surgery rats, while DB-METH rats had 5-HIAA/5-HT indistinguishable from controls. In both cases, prior induction of primary hypothyroidism interfered with the expected diabetes-induced reduction in 5-HT turnover.


Oxytocin nerve fibers innervate beta-endorphin neurons in the arcuate nucleus of the rat hypothalamus. Fine, varicose oxytocin-containing nerve fibers have been demonstrated in the hypothalamic arcuate nucleus in rats. Using Phaseolus vulgaris leukoagglutinin as an anterograde tracer, fine neuronal fibers of paraventricular nucleus origin could be seen throughout the arcuate nucleus. Using double immunostaining, oxytocin-immunoreactive varicose fibers were observed around or in the close vicinity of beta-endorphin-immunoreactive neurons. Silver-gold-labeled oxytocin-immunoreactive presynaptic boutons were shown to make synaptic contacts with diaminobenzidine-labeled beta-endorphin-immunoreactive neurons by electron microscopy. These findings provide morphological evidence for a possible influence of oxytocin on the activity of the brain beta-endorphin system at the hypothalamic level.


Comparison of norfluoxetine enantiomers as serotonin uptake inhibitors in vivo. Norfluoxetine, the N-desmethyl metabolite of fluoxetine, has been reported to resemble fluoxetine in being a potent and selective inhibitor of the serotonin uptake carrier. The enantiomers of norfluoxetine have now been compared as serotonin uptake inhibitors in vivo, based on their antagonism of p-chloroamphetamine-induced depletion of serotonin in brain and their lowering of concentrations of the metabolite of serotonin, 5-hydroxyindoleacetic acid (5-HIAA) in brain. In rats, S-norfluoxetine (ED50 3.8 mg/kg) was more potent than R-norfluoxetine (ED50 > 20 mg/kg) in blocking the depletion of serotonin by p-chloroamphetamine after intraperitoneal administration. The S enantiomer decreased concentrations of 5-HIAA in whole brain after doses of 2.5-20 mg/kg, whereas the R enantiomer did not. The concentrations of both enantiomers in brain increased in proportion to dose and the R enantiomer disappeared from the brain at a slightly slower rate than the S enantiomer. The relative inability of the R enantiomer to block the uptake of serotonin was therefore not a result of smaller concentrations of drug in the brain. In mice, S-norfluoxetine was also more potent than R-norfluoxetine in blocking depletion of serotonin by p-chloroamphetamine (ED50 values 0.82 and 8.3 mg/kg, respectively). Thus, in contrast to the relatively similar potencies of the enantiomers of fluoxetine in blocking the uptake of serotonin, the enantiomers of norfluoxetine have markedly different potencies as inhibitors of the uptake of serotonin.


The effect of 3,3-di-pyridyl-methyl-1-phenyl-2-indolinone on the nerve terminal currents of mouse skeletal muscles. The effects of 3,3-dipyridyl-methyl-1-phenyl-2-indolinone (DPMPI), a new cognition enhancer, on perineural waveforms were assessed on triangularis sterni nerve-muscle preparations in the mouse. The perineural waveforms were recorded with extracellular electrodes placed in the perineural sheaths of motor nerves. At 64.5 microM, DPMPI decreased the fast potassium current of the nerve terminal. The sodium current, calcium currents and calcium-dependent potassium current of the nerve terminal were not affected. At a greater concentration (215 microM), DPMPI decreased all of the components of the waveforms associated with sodium, potassium and calcium currents. It is concluded that DPMPI affects potassium, as well as sodium currents in the nerve terminal. The effect may contribute to its pharmacological actions on synaptic transmission.


Substance P augments the rate of vasodilation induced by calcitonin gene-related peptide in porcine ophthalmic artery in vitro. Peptides may function as neurotransmitters liberated antidromically by sensory nerve fibres, provoking vascular responses having potential importance in some neurological disorders. Dose-response relaxation curves induced by substance P (SP) and calcitonin gene related peptide (CGRP) have been studied in porcine ophthalmic arteries in vitro. Both peptides induced vasodilation when tested separately (CGRP much greater than SP). Because of the putative interactions between such peptides in this vascular territory, a computerised system was also used for analysing over time the response to a single addition of either 10(-8) M CGRP, 10(-8) M SP or a combination of 10(-8) M SP + 10(-8) M CGRP. SP did not augment the maximum relaxation induced by CGRP alone, but increased significantly the rate of relaxation during the initial phase of the response. The effect induced by the SP+CGRP combination was stronger than the sum of the individual SP and CGRP-induced relaxations during the first 4 min of the response, which suggests a SP-CGRP synergism in this artery.


Peptidergic modulation of G-protein coupled cyclic-AMP accumulation in the rat caudate nucleus. Somatostatin, substance P, and vasoactive intestinal polypeptide were incubated in an adenylate cyclase assay with a particulate fraction of caudate-putamen tissue of the rat in order to examine the effect of the neuropeptides on G-protein coupled adenylate cyclase in vitro. Somatostatin induced an enhancement of cyclic AMP formation in presence of guanine nucleotides and cholera toxin but inhibited pertussis toxin and forskolin enzyme stimulation. Pertussis toxin and cholera toxin also depressed forskolin-induced stimulation as described previously. Somatostatin was able to antagonize these inhibitory effects of both toxins. On the contrary, substance P reduced GTP and cholera toxin stimulated striatal adenylate cyclase, without affecting forskolin activation. In our preparation, VIP did not influence basal adenylate cyclase activity or the stimulation by guanine nucleotides, cholera toxin, and pertussis toxin. VIP potently inhibited the enhancement of cyclic AMP formation by forskolin and completely antagonized the inhibitory effect of cholera toxin on forskolin activation. These results suggest that neuromodulatory effects of somatostatin, substance P, and VIP are mediated by the inhibitory as well as stimulatory guanine nucleotide proteins G-i and G-s coupled to an adenylate cyclase system.


Management outcome for vertebrobasilar artery aneurysms by early surgery. Treatment of vertebrobasilar artery aneurysms remains fraught with complications, even in the present era of microneurosurgery. In a series of 1150 consecutive patients with cerebral aneurysms from a defined catchment area with 870,000 inhabitants, 93 with vertebrobasilar artery aneurysms were treated by two surgeons during a 14-year period. Sixty-three patients had surgery, 36 during the first week after bleeding. There was no surgical mortality among 33 good grade patients. Nine (14%) of the 63 surgical cases had died at 1 year. Forty-nine (53%) of the total group of 93 patients were functioning independently at 1 year. Overall management mortality was 37%. All 11 patients admitted in Grade V died. In spite of improvements in surgical techniques, we are far from achieving ideal results. Early diagnosis and surgery before rupture are urgently needed. Arteriosclerotic giant aneurysms remain untreatable.


The expression of the immediate-early genes c-fos, egr-1 and c-jun in the accessory olfactory bulb during the formation of an olfactory memory in mice. Female mice form a memory for the pheromones of the male with which they mate. It has been proposed that the site of the synaptic changes underlying this memory is the accessory olfactory bulb, at the first level of the accessory olfactory system. In this study we have examined the expression of the immediate-early genes c-fos, c-jun and egr-1 in the mitral and granule cells of the accessory olfactory bulb immediately after mating, during the period of memory formation. Transient increases were seen in the number of granule cell nuclei expressing c-fos and the number of granule and mitral cell nuclei expressing egr-1, during the period of memory formation. No changes were observed in the expression of c-jun during this period. The increase in the number of cells expressing c-fos and egr-1 required the association of mating and pheromonal exposure, conditions also required for memory formation. Large increases in the number of mitral and granule cell nuclei expressing c-fos and egr-1 were also observed following the infusion of the drug bicuculline into the accessory olfactory bulb in the absence of mating. This procedure has previously been shown to result in the formation of a nonspecific memory for male pheromones. These results associate the expression of c-fos and egr-1 in the accessory olfactory bulb with the conditions required for the formation of an olfactory memory for male pheromones.


Limbic seizures cause pronounced changes in the expression of neurokinin B in the hippocampus of the rat. Immunohistological and in situ hybridization techniques were used to study the influence of kainic acid-induced seizures and of pentylenetetrazol kindling on neurokinin B immunoreactivity and neurokinin B mRNA in the rat hippocampus. Pronounced increases in neurokinin B immunoreactivity were observed in the terminal field of mossy fibres 10-60 days after intraperitoneal injection of kainic acid. These slow but persistent increases in immunoreactivity were accompanied by markedly enhanced expression of neurokinin B mRNA in the granule cells and in hilar interneurons adjacent to the granule cell layer. These changes were preceded by transient increases in neurokinin B mRNA and immunoreactivity in CA1 pyramidal cell layer two and 10 days after kainic acid, which, however, subsided later on. Pentylenetetrazol kindling caused similar increases in neurokinin B mRNA expression in granule cells and in CA1 pyramidal cells, but not in hilar interneurons. In CA1, increased neurokinin B message was present two days after termination of the kindling procedure but not after 10 days. Sixty days after kainic acid injection, neurokinin B immunoreactivity extended to the inner-third of the molecular layer of the dentate gyrus. After pentylenetetrazol kindling, a neurokinin B-immunoreactive band was observed in the infrapyramidal region of CA3. Lesions of the dentate granule cells by local injection of colchicine in kainic acid-treated rats abolished the supragranular neurokinin B-positive staining, whereas it was almost unchanged after transection of the ventral hippocampal commissure. These observations suggest that neurokinin B immunoreactivity may be located in ipsilateral mossy fibres undergoing collateral sprouting to the inner molecular layer or to the infrapyramidal region in CA3, respectively. Preprotachykinin A mRNA, which encodes for neurokinin A and substance P, and substance P immunoreactivity were not changed in the hippocampus of epileptic rats compared with untreated animals. The observed changes in neurokinin B immunoreactivity and mRNA indicate that specific functional and morphological changes may be induced in hippocampal neurons by recurrent limbic seizures.


Mapping of the motor pathways in rats: c-fos induction by intracortical microstimulation of the motor cortex correlated with efferent connectivity of the site of cortical stimulation. The general goal of the present study was to investigate structural components of a neural system anatomically as well as functionally. The rat motor system, which is reasonably well understood, was selected and a new procedure was developed to combine a functional marker with axonal tracing methods (in the same animal). This was achieved by mapping c-fos induction immunocytochemically as a result of intracortical microstimulation in the distal forelimb area of the motor cortex. The anterograde tracers Phaseolus vulgaris-leucoagglutinin or biocytin were deposited at the site of intracortical microstimulation, the former three weeks and the latter two to three days before stimulation. Neuronal nuclei, labeled for the expressed c-fos protein, were present and mapped in the following structures: motor cortex; basal ganglia (caudate-putamen, globus pallidus); thalamus (reticular, ventromedial and posterior nuclei); subthalamic nucleus; substantia nigra; tectum; red nucleus; pontine nuclei; inferior olive; external cuneate nucleus; cerebellar cortex; deep cerebellar nuclei. Labeling was often bilateral but generally more substantial ipsilaterally, except in the cerebellum where it was mainly contralateral. Axonal labeling, including terminal branches and boutons, was also found in most of the above structures with the exception of the globus pallidus, deep cerebellar nuclei, cerebellar cortex and external cuneate nucleus. These expected exceptions demonstrate that activity changes in these latter structures, as revealed by c-fos labeled neurons, were induced over more than one synapse. This combined procedure might, therefore, be useful in deciding whether two structures in a given system are linked directly (monosynaptically) or indirectly (polysynaptically) to each other. In contrast to the 2-deoxyglucose technique, functional mapping by means of c-fos induction provides cellular resolution, making it possible to establish fine details of axonal contacts with target neurons: boutons in close apposition to c-fos labeled neurons were clearly observed here, for instance in the cerebral cortex, caudate-putamen, thalamus, subthalamic nucleus and pontine nuclei. Surprisingly, the ventrolateral and ventrobasalis nuclei of the thalamus contained numerous and dense axon terminals labeled with Phaseolus vulgaris-leucoagglutinin or biocytin, but the contacted neurons in the ventrolateral and ventrobasalis nuclei were not marked with c-fos. However, with respect to directly connected structures, there was, in general, a good correlation between structures with axonal labeling and those with c-fos labeled neurons.


Calbindin D28k-containing nonpyramidal cells in the rat hippocampus: their immunoreactivity for GABA and projection to the medial septum. Calbindin D28k-containing non-pyramidal cells were found in all layers and subfields of the hippocampus, with the highest frequency in stratum radiatum of the CA1-CA3 subfields. A large number of these neurons had a vertically oriented dendritic tree, often restricted to to stratum radiatum. In stratum oriens and near to the border of strata radiatum and lacunosum moleculare cells with horizontally running dendrites were also found. Multipolar cells were most common in stratum radiatum of the CA3 region. The GABAergic nature of the calbindin D28k-containing non-pyramidal cells was studied using the "mirror" technique. Adjacent thick sections were immunostained for calbindin D28k and GABA, and halved neurons were identified on the common surfaces. The majority of calbindin D28k-containing non-pyramidal cells were shown to be GABAergic. The GABA-negative calbindin cells were found in relatively large numbers in stratum oriens of the CA1-CA3 region, and occasionally in strata radiatum and pyramidale of CA2, and in stratum radiatum of the CA3c region near to the border of the dentate hilus. However, even in these cells a weak immunostaining, only slightly but consistently above background level, was always observed. Earlier studies have demonstrated that the somata of GABAergic neurons with distant projections may contain a level of GABA that is below the detection threshold of immunocytochemistry. Here we provide direct evidence that the calbindin-containing non-pyramidal cells were among those projecting to the medial septum. Following horseradish peroxidase injections into the medial septum 80% of the retrogradely labelled non-pyramidal cells were found to be immunoreactive for calbindin D28k, and 20% contained neuropeptide Y. These results suggest that the calbindin D28k-containing and apparently GABA-immunonegative non-pyramidal cells in stratum oriens of the CA1-CA3 regions may also be GABAergic, but have a distant projection, that is, to the medial septum.


Regional stimulatory and inhibitory effects of guanine nucleotides on [125I]galanin binding in rat brain: relationship with the rate of occupancy of galanin receptors by endogenous galanin. Galanin has been shown to stimulate feeding or modulate neuroendocrine secretions when administered centrally. In the present work, using quantitative autoradiography, we documented the existence of [125I]galanin specific binding sites in several hypothalamic nuclei expected to mediate these effects. In standard binding conditions, [125I]galanin specific binding can be visualized in the hypothalamic ventromedial nucleus, stria terminalis, piriform cortex, central amygdaloid nucleus and medial amygdaloid nucleus, while it is almost undetectable in most neuroendocrine or autonomic hypothalamic areas. We hypothesized that high endogenous galanin levels in these regions might mask galanin receptors. We first showed that a high ionic strength/acid wash of brain slices is effective in removing more than 80% of specifically prebound [125I]galanin in all tested regions. After such treatments, specific binding sites could be revealed in the hypothalamus namely in the parvocellular paraventricular nucleus, periventricular nucleus, arcuate nucleus and median eminence. In contrast, regions already labeled in standard conditions exhibited a slight decrease in [125I]galanin binding. Thus, regions were ranked from low to high rate of occupancy of galanin receptors by endogenous galanin, the rate of occupancy of galanin receptors being maximal in median eminence (greater than 90%). We thus studied the regional effect of guanine nucleotides on [125I]galanin specific binding. A high concentration (100 microM) of guanyl 5'-yl imidodiphosphate, a nonhydrolyzable analog of GTP directly added to the incubation medium, inhibited [125I]galanin binding in all telencephalic regions. On the same sections and only in regions of high index of galanin receptor occupancy (arcuate nucleus, median eminence, dorsomedial nucleus, paraventricular nucleus, and periventricular hypothalamic nucleus), guanyl 5'-yl imidodiphosphate paradoxically enhanced [125I]galanin binding. The effects of acid preincubation and guanyl 5'-yl imidodiphosphate incubation on [125I]galanin binding were strongly correlated in these hypothalamic areas (r = 0.97). In all regions, guanyl 5'-yl imidodiphosphate increased the rate of dissociation of [125I]galanin. In competition studies, guanyl 5'-yl imidodiphosphate decreased the IC50 s of unlabeled galanin which were homogenized around 4 nM in most telencephalic and hypothalamic regions. Thus, the guanyl 5'-yl imidodiphosphate-induced stimulation of [125I]galanin specific binding measured in the neuroendocrine and autonomic hypothalamus is linked to an increase in receptor capacity and not to a rise in receptor affinity. Both inhibitory and stimulatory guanyl 5'-yl imidodiphosphate effects observed in [125I]galanin equilibrium binding studies were dose-dependent and guanine nucleotide-specific with guanyl 5'-yl imidodiphosphate more potent than GTP or GDP.(ABSTRACT TRUNCATED AT 400 WORDS)


Chronic exposure to morphine does not induce dependence at the level of the calcium channel current in human SH-SY5Y cells. mu-Opioid receptors mediate inhibition of the N-type calcium channel current in the human neuroblastoma cell line SH-SY5Y. We have previously shown that chronic exposure to morphine induces homologous tolerance to this effect. Here we show that chronic incubation with morphine (1 microM for three to seven days) does not, however, induce physical dependence at the level of the calcium channel current. Initial experiments were performed using the whole cell voltage-clamp technique. Chronically treated cells were bathed in superfusate which also contained morphine (1 microM). On washout of morphine the current amplitude increased by 12% and this was reversed by re-addition of morphine. Naloxone (1 microM) elicited a similar increase. However, this increase is most likely due to a reversal of the residual inhibitory effect of morphine on the calcium channel current rather than being a novel withdrawal response. Chronic exposure to morphine did not change the voltage-sensitivity of the calcium channel current or induce the appearance of a current sensitive to the L-type calcium channel agonists Bay K 8644 (3 microM) and S(+)-PN 202-791 (1 microM). In a further series of experiments the nystatin-perforated patch technique was employed in order to prevent washout of any L-type current in these cells. Under these conditions a Bay K 8644-sensitive, L-type current was unmasked following treatment with omega Conus Toxin GVIA. The peak current was depressed by omega Conus Toxin GVIA (1 microM) by approximately 90% both in control cells and cells chronically exposed to morphine. Now Bay K 8644 (3 microM) almost doubled the remaining current but the effect was equal in both groups of cells. It is concluded that chronic exposure to morphine does not induce physical dependence and a withdrawal syndrome in the human SH-SY5Y neuroblastoma cell line by changing either N-type or L-type calcium channel activity.


Evidence that the masticatory muscles receive a direct innervation from cell group k in the rabbit. These experiments have shown that a group of neurons lateral to the trigeminal motor nucleus innervates the muscles of mastication. The work began to describe the location of digastric last-order interneurons, using the technique of transneuronal labeling with wheatgerm agglutinin-conjugated horseradish peroxide injected into the left digastric muscle of rabbits under general anaesthesia. Four to eight days later, the animals were killed with an overdose of anaesthetic and perfused. Coronal sections of the frozen brainstem were cut at 20 microns thickness and processed for peroxidase activity. Motoneurons in the ventral and caudal divisions of the trigeminal motor nucleus were labeled in all animals as expected. An additional population of neurons located ventrolaterally to the motor nucleus in cell group k were also found to be labeled if the survival time was five days or more. In an attempt to determine whether cell group k neurons were labeled transynaptically, two series of control experiments were carried out. In the first, crystals of fluorescein- and rhodamine-conjugated dextran amines and horseradish peroxidase were applied directly to central ends of cut digastric nerves. In the second, central ends of cut digastric nerves were enclosed in cuffs containing 40-60% horseradish peroxidase solutions. Again, neurons in both the trigeminal motor nucleus and cell group k were labeled suggesting that neurons within cell group k project to the digastric muscle. Similar experiments using dextran amines and wheatgerm peroxidase were carried out on the masseter muscle. Motoneurons in the dorsomedial and rostral half of the trigeminal motor nucleus, as well as primary afferent cell bodies in the mesencephalic nucleus of the trigeminal nerve, were labeled in all experiments. In addition, a population of neurons in cell group k, dorsal to those associated with the digastric muscle, were found to contain each one of the reaction products. Since it is thought that only the wheatgerm agglutinin-conjugated horseradish peroxidase transferred from one neuron to another, we conclude that cell group k neurons provide an additional innervation to the digastric and masseter muscles.


Light and electron microscopic localization of retrogradely transported neurotensin in rat nigrostriatal dopaminergic neurons. We previously demonstrated the existence of a retrograde axonal transport of radioactivity to the substantia nigra pars compacta following injection of mono-iodinated neurotensin in rat neostriatum. In the present study, the topographical and cellular distribution of this retrogradely transported material was examined by light and electron microscopic autoradiography. Four and a half hours after unilateral injection of [125I]neurotensin in the caudoputamen, retrogradely labelled neuronal cell bodies were detected by light microscopic autoradiography throughout the ipsilateral substantia nigra pars compacta as well as within the ventral tegmental area and retrorubral field. In semithin sections, silver grains were prevalent over the perinuclear cytoplasm of neuronal cell bodies but were also detected over neuronal nuclei. Analysis of electron microscopic autoradiographs revealed that the vast majority (greater than 85%) were associated with neuronal perikarya, unmyelinated and myelinated axons, dendrites and terminals. Within the soma, a significant proportion of silver grains (16% of somatic grains) was detected over the nucleus. However, the majority were identified over the cytoplasm where they often encompassed cytoplasmic organelles, including rough endoplasmic reticulum, mitochondria, Golgi apparatus, lysosomes, and multi-vesicular bodies. In dendrites and axons, a substantial percentage of silver grains (63-89%) was localized over the plasma membrane. A minor proportion (13% of total) of the autoradiographic labelling was detected over myelin sheaths, astrocytes, and oligodendrocytes. The present results are consistent with previous light-microscopic evidence for internalization and retrograde transport of intrastriatal neurotensin within nigrostriatal dopaminergic neurons. They further suggest that retrogradely transported neurotensin may be processed along a variety of intracellular pathways including those mediating degradation in lysosomes and recycling in rough endoplasmic reticulum. The detection of specific autoradiographic labelling in the nucleus supports the concept that neurotensin alone, or complexed to its receptor, might be involved in the regulation of gene expression through direct or indirect interactions with nuclear DNA. Consequently, the retrograde transport of neurotensin in nigrostriatal dopaminergic neurons might provide a vehicle through which events occurring at the level of the axon terminal may initiate long-term biological responses.


Substance P opens cation channels and closes potassium channels in rat locus coeruleus neurons. Whole-cell recordings were made from neurons of the rat locus coeruleus in a tissue slice removed from rat brain. Substance P caused an inward current in cells voltage-clamped at -60 mV. The effect of substance P was concentration-dependent (30 nM-3 microM) and was mimicked by similar concentrations of substance K and neuromedin K. The inward current resulted predominantly from an increase in membrane cation conductance; in potassium-free solutions it reversed polarity at about 12 mV. Substance P also reduced the conductance of an inwardly rectifying potassium current; this action was studied with low external sodium concentration. It is concluded that substance P excites rat locus coeruleus neurons by activating an intracellular transduction pathway leading to both cation conductance increase and potassium conductance decrease.


Topography and functional role of dopaminergic projections from the ventral mesencephalic tegmentum to the ventral pallidum. A dopaminergic projection from the ventral tegmental area to the ventral pallidum was identified in the rat using anterograde tract tracing and combined retrograde tracing-immunocytochemistry. The projection was found to be topographically organized such that fibers innervating the ventromedial ventral pallidum arose from neurons located along the midline nuclei of the ventral mesencephalon, including the nucleus interfascicularis and nucleus linearis caudalis. Ventral tegmental neurons situated more laterally, in the nucleus parabrachialis pigmentosus and nucleus paranigralis, projected to the ventromedial and dorsolateral ventral pallidum. The substantia nigra did not supply a major contribution to this projection. The proportion of ventral tegmental area dopaminergic neurons projecting to the ventral pallidum ranged from approximately 30% to 60%. The functional significance of the projection is indicated since intra-ventral pallidum microinjections of dopamine elicited a dose-dependent increase in locomotor activity. Furthermore, whereas pretreatment of the ventral pallidum with the GABAA agonist muscimol has been shown to attenuate opioid-induced locomotor activity elicited from the ventral pallidum, it did not attenuate the dopamine-induced motor response. Thus, while mu-opioids in the ventral pallidum may presynaptically regulate GABAergic efferents from the nucleus accumbens, it appears that the dopaminergic input directly influences the ventral pallidal output neuron which is involved in locomotion.


Afferent connections of the parvocellular reticular formation: a horseradish peroxidase study in the rat. The afferent connections of the parvocellular reticular formation were systematically investigated in the rat with the aid of retrograde and anterograde horseradish peroxidase tracer techniques. The results indicate that the parvocellular reticular formation receives its main input from several territories of the cerebral cortex (namely the first motor, primary somatosensory and granular insular areas), districts of the reticular formation (including its contralateral counterpart, the intermediate reticular nucleus, the nucleus of Probst's bundle, the dorsal paragigantocellular nucleus, the alpha part of the gigantocellular reticular nucleus, the dorsal and ventral reticular nuclei of the medulla, and the mesencephalic reticular formation), the supratrigeminal nucleus and the deep cerebellar nuclei. Moderate to substantial input to the parvocellular reticular formation appears to come from the central amygdaloid nucleus, the parvocellular division of the red nucleus, and the orofacial and gustatory sensory cell groups (comprising the mesencephalic, principal and spinal trigeminal nuclei, and the rostral part of the nucleus of the solitary tract), whereas many other structures, including the substantia innominata, the field H2 of Forel, hypothalamic nuclei, the superior colliculus, the substantia nigra pars reticulata, the retrorubral field and the parabrachial complex, seem to represent relatively modest additional input sources. Some of these projections appear to be topographically distributed within the parvocellular reticular formation. From the present results it appears that the parvocellular reticular formation receives afferents from a restricted group of sensory structures. This finding calls into question the traditional characterization of the parvocellular reticular formation as an intermediate link between the sensory nuclei of the cranial nerves and the medial magnocellular reticular districts, identified as the effector components of the reticular apparatus. Some of the possible physiological correlates of the fiber connections of the parvocellular reticular formation in the context of oral motor behaviors, autonomic regulations, respiratory phenomena and sleep-waking mechanisms are briefly discussed.


Synaptic organization of GABAergic inputs from the striatum and the globus pallidus onto neurons in the substantia nigra and retrorubral field which project to the medullary reticular formation. Anatomical tract-tracing and immunohistochemical techniques involving correlated light and electron microscopy were used to determine whether the descending striatal and pallidal afferents to the substantia nigra pars reticulata converge onto individual neurons projecting to the pontomedullary and medullary reticular formation in the rat. Injections of biocytin into the ventrolateral region of the striatum and Phaseolus vulgaris-leucoagglutinin into the ventrolateral and caudal regions of the globus pallidus led to overlapping anterogradely labelled terminal fields within the dorsolateral substantia nigra pars reticulata. These terminal fields were punctuated by neurons which had been retrogradely labelled following injections of wheatgerm agglutinin conjugated to horseradish peroxidase into the lateral pontomedullary reticular formation. The anterogradely labelled striatal and pallidal terminals displayed different morphological characteristics; the striatal terminals were small and diffusely distributed throughout the neuropil without any particular neuronal association whereas the pallidal terminals were large and formed pericellular baskets around the perikarya of retrogradely and non-retrogradely labelled nigral neurons. In areas of the substantia nigra where there was an overlap between the two terminal fields, individual retrogradely labelled nigroreticular neurons were found to be apposed by both sets of anterogradely labelled terminals. Electron microscopic analysis revealed that the striatonigral and pallidonigral terminals displayed different ultrastructural features, the striatal terminals were small, contained few mitochondria and formed symmetric synaptic contacts predominantly with the distal dendrites of nigroreticular neurons whereas the pallidal terminals were large, contained numerous mitochondria and formed symmetric synaptic contacts preferentially with perikarya and proximal dendrites of nigroreticular neurons. Post-embedding immunohistochemical staining revealed that both striatonigral and pallidonigral terminals, some which formed synaptic contact with nigroreticular neurons, displayed GABA immunoreactivity. Examination of twelve retrogradely labelled neurons in the electron microscope revealed that all received synaptic inputs from both sets of anterogradely labelled terminals. In addition to the substantia nigra pars reticulata, neurons of the retrorubral field were also retrogradely labelled following injections of wheatgerm agglutinin conjugated to horseradish peroxidase into pontomedullary reticular formation. These retrorubroreticular neurons were part of a continuum of labelled cells which extended from the dorsolateral substantia nigra pars reticulata caudally into the retrorubral field. When combined with anterograde tracing methods it was found that the retrorubroreticular neurons received synaptic inputs from pallidal terminals which were morphologically similar to the pallidonigral terminals and formed symmetric synapses with the neuronal somata and proximal dendrites. In contrast to nigroreticular neurons, the stratonigral terminals were not seen in contact with retrorubroreticular cells.(ABSTRACT TRUNCATED AT 400 WORDS)


Topographical organization of the nigrotectal projection in rat: evidence for segregated channels. Recent evidence suggests that projections from the superior colliculus to the brainstem in rat are organized into a series of anatomically segregated output channels. To understand how collicular function may be modified by the basal ganglia it is important to know whether particular output modules of the superior colliculus can be selectively influenced by input from substantia nigra. The purpose of the present study was, therefore, to examine in more detail topography within the nigrotectal system in the rat. Small injections (10-50 nl) of a 1% solution of wheatgerm agglutinin conjugated with horseradish peroxidase were made at different locations within substantia nigra and surrounding structures. A discontinuous puff-like pattern of anterogradely transported label was found in medial and caudal parts of the ipsilateral intermediate layers of the superior colliculus. In contrast, the rostrolateral enlargement of the intermediate layers contained a greater density of more evenly distributed terminal label. Injection sites associated with this dense pattern of laterally located label were concentrated in lateral pars reticulata, while the puff-like pattern was produced by injections into ventromedial pars reticulata. Retrograde tracing experiments with the fluorescent dyes True Blue and Fast Blue revealed that injections involving the rostrolateral intermediate layers were consistently associated with a restricted column of labelled cells in the dorsolateral part of ipsilateral pars reticulata. Comparable injections into medial and caudal regions of the superior colliculus produced retrograde labelling in ventral and medial parts of the rostral two-thirds of pars reticulata. Both anterograde and retrograde tracing data indicated that contralateral nigrotectal projections arise from cells located in ventral and medial pars reticulata. The present results suggest that the main ipsilateral projection from substantia nigra pars reticulata to the superior colliculus comprises two main components characterized by regionally segregated populations of output cells and spatially separated zones of termination. Of particular interest is the apparent close alignment between terminal zones of the nigrotectal channels and previously defined populations of crossed descending output cells in the superior colliculus. Thus, the rostrolateral intermediate layers contain a concentration of terminals specifically from dorsolateral pars reticulata and output cells which project to the contralateral caudal medulla and spinal cord. Conversely, the medial and caudal intermediate layers receive terminals from ventral and medial pars reticulata and contain cells which project specifically to contralateral regions of the paramedian pontine and medullary reticular formation.(ABSTRACT TRUNCATED AT 400 WORDS)


Peripheral injury and anterograde transport of wheat germ agglutinin-horse radish peroxidase to the spinal cord. Previous observations have revealed labeling in the extracellular space surrounding boutons and unmyelinated fibers in superficial laminae of the spinal cord after injection of the tracer wheat germ agglutinin conjugated to horseradish peroxidase in dorsal root ganglia. The degree of extracellular labeling appeared related to the extent of the damage to the ganglia at the time of the injection. To determine whether injury might produce extracellular labeling, we investigated the effects of unilateral nerve crush or transection on spinal labeling after bilateral injections of the tracer into sciatic nerves. Confirming previous reports, labeling was confined to small dorsal root ganglion cells and to spinal laminae I and II, suggesting a selective affinity of this tracer for unmyelinated fibers. Labeling of both ganglion neurons and superficial spinal laminae was increased on the injured side, probably as a result of increased efficiency of receptor-mediated endocytosis. Electron microscopical observations revealed that the tracer was largely confined to unmyelinated dorsal root fibers bilaterally; a higher percentage of these fibers were labeled on the injured side. In the dorsal horn, the tracer was predominantly within unmyelinated axons and their terminals on the control side, whereas most of the labeling was extracellular and transneuronal on the injured side. The extracellular labeling surrounded unmyelinated fibers and their terminals in the spinal cord, but was excluded from the synaptic cleft. The demonstration that injury is accompanied by significantly increased release of this tracer from the terminals of unmyelinated fibers into the extracellular space suggests that endogenous substances may be released after peripheral lesions as a central signal of injury.


Sensory neuropeptide interactions in the production of plasma extravasation in the rat. We used an experimental model of neurogenic inflammation to study the contribution of the primary afferent peptides substance P, calcitonin gene-related peptide, galanin and somatostatin to plasma extravasation in rat synovium. Perfusion of the C-fiber excitotoxin, capsaicin (1.6 mM), through the knee joint of the pentobarbital anesthetized rat, increased plasma extravasation transiently (< 30 min). Perfusion of substance P (1 microM) or calcitonin gene-related peptide (100 nM), two primary afferent neuropeptides that are released by acute capsaicin administration, had no significant effect on plasma extravasation. Co-perfusion of these two neuropeptides, however, evoked an increase in plasma extravasation that was greater than that produced by capsaicin remaining above 250% of the baseline level by the end of the perfusion period (55 min). Capsaicin co-perfused with either galanin (100 nM) or somatostatin (1 microM) failed to increase plasma extravasation. Neither galanin nor somatostatin significantly affected increase in plasma extravasation induced by co-perfusion of substance P plus calcitonin gene-related peptide. Therefore, we suggest that galanin and somatostatin inhibit, presynaptically, the release of substance P and calcitonin gene-related peptide from primary afferent terminals. The interactions among these four neuropeptides provide a novel mechanism for the regulation of primary afferent neurogenic inflammation.


[Postoperative hyperamylasemia: the evaluation of the efficacy of ranitidine in prevention]. The paper examines the role of ranitidine in the prevention of postoperative hyperamylasemia, a complication following major biliary tract surgery, especially if associated with intraoperative cholangiography. Forty patients underwent cholecystectomy and intraoperative cholangiography: calculosis of the choledochus was observed in 11 patients and a revision of the VBP was therefore carried out involving the transcystic insertion of Kehr's tube. Patients were randomly subdivided into two groups: group A (treated with 50 mg ranitidine 4 times a day e.v.) and group B (no anti H2 treatment). No significant differences were noted between the two groups with regard to the onset of hyperamylasemia, although it is worth reporting that ranitidine virtually eliminated all hemorrhagic complications due to ulcer or postoperative gastritis.


[Duodenal adenocarcinoma. A case report]. The duodenal carcinoma is a rare malignant neoplasm (1-6% of all the tumors of the digestive tract). In this paper the authors review the clinical and diagnostic approach to the disease and discuss both the palliative and curative surgical therapy.


Dystrophin-like immunoreactivity in monkey and human brain areas involved in learning and motor functions. Two antidystrophin antibodies against different fragments of dystrophin were used to detect this polypeptide in monkey and human brains. Dystrophin was revealed by immunoperoxidase amplified with the biotin/avidin system and by immunoblotting. A dystrophin-like immunoreactivity was uniformly expressed in several brain regions implicated in learning and motor functions. Dystrophin function is not clear but our results raise the possibility that this protein may be involved in the cognitive impairment observed in several Duchenne muscular dystrophy (DMD) patients.


Quaking phenotype influences brain lipid-related mRNA levels. Although lipids compose almost 80% of myelin, the influence of quaking on mRNAs encoding lipid biosynthetic enzymes and transport proteins has not been previously reported. Understanding the influence of quaking on myelin-specific and lipid-related mRNAs will be useful in determining the mechanism of the quaking defect. Stearoyl CoA desaturase (SCD) catalyzes a key step in the biosynthesis of oleic acid (C18:1, n-9), a major fatty acid in myelin. SCD, LDL receptor (LDLR) and apolipoprotein E (Apo E) mRNA levels are all reduced in neonatal quaking brains. In contrast to brain, quaking hepatic LDLR and Apo E mRNA levels are normal. These results indicate that lipid-related mRNAs are reduced in neonatal quaking brain, but the quaking liver is unaffected. The quaking defect influences gene expression in multiple cell types of glial lineage in the developing CNS.


Single-channel currents of NMDA type activated by L- and D-homocysteic acid in cerebellar granule cells in culture. The whole-cell and the outside-out configurations of the patch-clamp technique were used to study macroscopic and single-channel currents evoked by the enantiomers of homocysteic acid, the sulphur containing analogue of glutamate, in cerebellar granule cells in culture. L-Homocysteic acid (L-HC, 15 microM) and D-homocysteic acid (D-HC, 50 microM) induced whole-cell currents of comparable amplitude, that were abolished by the N-methyl-D-aspartate (NMDA) receptor antagonist 2-amino-5-phosphonovalerate (AP-5) (20 microM). AMPA (100 microM) induced whole-cell currents which were not modified by AP-5 (20 microM) but were blocked by CNQX. In the outside-out configuration, both L-HC and D-HC (15 microM) elicited single-channel currents of the same conductance, mean open time and reversal potential as the NMDA-induced single-channel events. In the presence of Mg2+ (2 mM), D- and L-HC-induced single-channel currents were voltage-dependent. These data suggest that in cerebellar granule cells in culture, both L-HC and D-HC activate the same NMDA receptor channel complex.


The substance P (NK1) receptor antagonist (+/-)-CP-96,345 causes sedation and motor impairment in Swiss albino mice in the black-and-white box behavioral paradigm. In order to test the suggested involvement of substance P (NK1) receptors in anxiety, the non-peptide NK1 antagonist (+/-)-CP-96,345 was tested in Swiss albino mice using the black-and-white box behavioral paradigm. Intraperitoneal (+/-)-CP-96,345 dose-dependently decreased the motor activity and the number of exploratory rearings in both the brightly lit and dark compartment as well as the transitions between the compartments, whereas it increased the latency of the initial movement into the dark compartment as well as the time spent in the light section. ED50 or ID50 values ranged from 1.9 to 3.6 mg/kg and Hill slopes from 1.4 to 5.0. (+/-)-CP-96,345 also produced rotatory behavior of no preferred lateralization as well as the Straub phenomenon in some animals. The effects of (+/-)-CP-96,345 (5 mg/kg) were not affected by 2 mg/kg naloxone (i.p.) which was also ineffective when given alone. Thus, (+/-)-CP-96,345 does not display any anxiogenic effect but causes dose-dependent sedation and motor impairment.


Capsaicin prevents the adrenocorticotropin-induced improvement of cardiovascular function and survival in hemorrhage-shocked rats. A volume-controlled hemorrhagic shock was produced in anesthetized rats by intermittent bleeding from an iliac vein over a period of 20-30 min, until the carotid mean arterial pressure (MAP) stabilized around 20-24 mmHg. In this condition, which caused the death of all saline-treated animals within 25-30 min, the intravenous (i.v.) bolus injection of the adrenocorticotropin fragment 1-24 (ACTH(1-24)) at a dose of 160 micrograms/kg promptly restored MAP, as well as pulse pressure, heart rate and respiratory function, and greatly prolonged the survival time. Capsaicin (125 mg/kg cumulatively, s.c., 1 week before) completely prevented the anti-shock effect of ACTH(1-24), which, on the other hand, was shared by i.v. [Nle11]-substance P (SP) (200-300 micrograms/kg). Finally the SP-antagonist [D-Arg1,D-Pro2,D-Trp7,9,Leu11]-SP prevented the effect of ACTH(1-24). These results suggest that SP-containing nerve fibers are required for the effect of ACTH in hemorrhagic shock.


Release of calcitonin gene-related peptide-like (CGRP-LI) immunoreactivity from rat isolated soleus muscle by low pH, capsaicin and potassium. Capsaicin (10 microM), KCl (80 mM) or superfusion with a low pH medium (pH 5 or 6) produced a significant increase of calcitonin gene-related peptide-like immunoreactivity (CGRP-LI) outflow from the superfused rat isolated soleus muscle. CGRP-LI outflow produced by capsaicin or pH 5 medium was totally abolished in a calcium free medium containing EDTA (1 mM) and the effect of pH 5 medium was prevented by a previous application of capsaicin. Ruthenium red (10 microM) produced a marked inhibition of CGRP-LI release produced by capsaicin or pH 5 medium (69 and 84%, resp.), without affecting that evoked by KCl. These findings demonstrate that protons activate capsaicin-sensitive primary afferents in rat skeletal muscle through a Ruthenium red-sensitive mechanism. Proton-induced CGRP-LI release in skeletal muscle could be of relevance during exercise and/or skeletal muscle ischemia.


Decreased cerebrospinal fluid neuropeptide-converting enzyme activity in monoarthritic rats. The activity in rat cerebrospinal fluid (CSF) of dynorphin-converting enzyme (DCE) and substance P endopeptidase (SPE) was determined in control animals and in rats with monoarthritis. Enzymatic activities were measured with specific radioimmunoassays toward the N-terminal products Leu-enkephalin-Arg6 and substance P1-7, respectively. A monoarthritis stable during weeks 2-6 post-injection was induced by injection (0.05 ml) into one joint with Freund's adjuvant. Both SPE and DCE were significantly decreased 15 days after the intraarticular injection. Despite the degree of arthritis that was sustained equally at four weeks after inoculation, both DCE and SPE were back to control levels at that time. It can therefore be concluded that arthritis from a single joint is sufficient to elicit changes in CSF convertase activities, and that these effects disappear between 2 and 4 weeks after injection, although the arthritis persists.


Regional differences in the distribution of capsaicin-sensitive target-identified adult rat dorsal root ganglion neurons. A retrograde labelling technique combined with a cobalt uptake assay in cultured adult rat dorsal root ganglion (DRG) neurons were applied to study the distribution of capsaicin sensitivity in relation to different peripheral targets. This study shows that there are regional differences between skin, skeletal muscle and urinary bladder; 20-30% of skin afferents, 40% of muscle afferents and 60% of bladder afferents were found to be capsaicin-senditive. This may reflect differences in the proportion of chemosensitive afferents innervating different peripheral tissues.


Blockade of nitric oxide formation does not prevent glutamate-induced neurotoxicity in neuronal cultures from rat hippocampus. This study examined the role of nitric oxide (NO) in glutamate-induced, N-methyl-D-aspartate (NMDA) receptor-mediated neurotoxicity in rat hippocampal neuronal cultures grown under serum-free conditions. Formation of cGMP was used as an indirect measure of NO formation. Neuronal cell degeneration was monitored by measuring the release of lactate dehydrogenase (LDH). Neuronal cells showed a 4-fold increase in cGMP formation and release of LDH upon exposure to 30 microM glutamate. cGMP formation was fully inhibited by 1 microM nitro-arginine (N-Arg), 100 microM hemoglobin or 1 microM MK-801. In the presence of 1 microM MK-801, glutamate induced neither cGMP formation nor neuronal cell degeneration. However, when NO formation was inhibited by means of 100 microM N-Arg, glutamate still induced neurotoxicity. Therefore, in serum-free hippocampal cultures glutamate neurotoxicity occurs notwithstanding complete inhibition of the NO-synthase enzyme by N-Arg. Our data provide evidence that NO, synthesized upon glutamate exposure, has not a primary toxic action in pure hippocampal neuronal cultures.


Colocalization of nitric oxide synthase and NADPH-diaphorase in the myenteric plexus of the rat gut. The pattern of distribution and colocalization of nitric oxide-synthase (NOS) and NADPH-diaphorase in the myenteric plexus of whole-mount preparations of the antrum, duodenum, ileum, caecum, proximal colon and distal colon of the rat were investigated using immunohistochemical and histochemical staining techniques. Almost all the myenteric neurons that were NOS-positive in all regions of the gut examined were also stained for NADPH-diaphorase. However, in the stomach, duodenum and ileum, only a few of the NOS-positive nerve fibres in the tertiary and secondary plexuses and circular muscle layer were also stained for NADPH-diaphorase, whereas in the caecum and distal colon almost all the NOS-positive nerve fibres were also stained for NADPH-diaphorase. The results in the present study are consistent with the view that nitric oxide (NO) has a mediating role in gastrointestinal neurotransmission.


Nitric oxide synthase immunoreactivity and NADPH-diaphorase activity in a subpopulation of intrinsic neurones of the guinea-pig heart. This is the first report of the presence of nitric oxide synthase (NOS) immunoreactivity and NADPH-diaphorase (NADPH-d) activity in a subpopulation of the intrinsic neurones that innervate the heart. A cytochemical technique to detect NADPH-d and antisera raised against purified rat cerebellar NOS were employed to examine the expression of these enzymes by cells in a dissociated cell culture preparation from newborn guinea-pig atria and interatrial septum. Comparison of the results obtained by these two techniques and double-labelling experiments indicate that a subpopulation of intracardiac neurones contain both NADPH-d and NOS. These results indicate that some intracardiac neurones are capable of synthesizing nitric oxide. This raises the possibility that nitric oxide plays a role in the neural control of the heart.


The polyamine diaminodecane (DA-10) produces a voltage-dependent flickery block of single NMDA receptor channels. Receptor binding assays have shown that diaminodecane (DA-10) reduced binding of open channel blockers to the N-methyl-D-aspartate (NMDA) subtype of postsynaptic glutamate receptor through an interaction with the polyamine regulatory site. Because the action of DA-10 was opposite to that of the polyamine agonist spermine and was reversed by polyamine antagonists, DA-10 has been classified as an inverse agonist at the polyamine site. Using whole-cell voltage-clamp and single-channel recordings from cultured rat cortical neurons, we show that at negative holding potentials DA-10 (1-300 microM) reduced NMDA receptor whole cell current (IC50 = 34 microM) and produced a flickery block of NMDA single-channel currents. The flickery block of NMDA single channels was voltage-dependent and not reversed by the polyamine antagonist diethylenetriamine (DET). Potential mechanisms for the flickery block of NMDA single channel currents are discussed.


Selective potentiation of GABA-mediated Cl- current by lanthanum ion in subtypes of cloned GABAA receptors. The effect of lanthanum ion (La3+) on gamma-aminobutyric acid (GABA)-mediated Cl- currents was examined in the alpha 1 beta 2 or alpha 1 beta 2 gamma 2 subtype of GABAA receptors expressed in a human kidney cell line (A293), using a whole-cell configuration of patch-clamp techniques. La3+ dose-dependently stimulated the Cl- currents in the alpha 1 beta 2 gamma 2 subtype with an EC50 of 21.3 +/- 3.5 microM with a maximal potentiation of 240 +/- 16% as normalized to the GABA response at 5 microM. In the alpha 1 beta 2 subtype, however, the ion marginally potentiated GABA response, a maximal stimulation being less than 70% with an EC50 for La3+ near 200 microM. The stimulation of GABA response by La3+ in the alpha 1 beta 2 gamma 2 subtype was due to a decrease in the half maximal concentration for GABA and was more pronounced at the negative membrane potentials. This selectivity of La3+ toward the subtypes of GABAA receptors contrasts to that of Zn2+ which inhibits the currents in the alpha 1 beta 2, but not in the alpha 1 beta 2 gamma 2 subtype (Neuron, 5: (1990) 781-788). It appears that these polyvalent cations are useful in understanding the molecular basis for the functional diversity and in characterizing the molecular organization of native GABAA receptors.


Modulation of AMPA and NMDA responses in rat spinal dorsal horn neurons by trans-1-aminocyclopentane-1,3-dicarboxylic acid. In freshly isolated spinal dorsal horn (DH) neurons (laminae I-IV) of the young rat the effects of 25-100 microM of (+/-)-trans-1-aminocyclopentane-1,3-dicarboxylic acid (trans-ACPD), 1S,3R-ACPD and 1R,3S-ACPD, a metabotropic glutamate receptor (mGluR) agonist, on inward currents induced by glutamate (Glu), alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), N-methyl-D-aspartate (NMDA) and kainate were studied under whole-cell voltage-clamp conditions. When the cells were clamped to -60 mV, the racemic mixture and both stereo isomers of trans-ACPD increase the responses elicited by Glu, AMPA, and NMDA, but little those of kainate. In addition, quisqualate (10-50 microM), in the presence of CNQX (5-20 microM) or NBQX (5 microM), potentiated NMDA-induced currents. The enhancing effect lasted 10-75 min, depending upon both dose and length of application. In a smaller proportion of dorsal horn neurons, the enhancing effect was preceded by a transient depression of the responses to Glu, AMPA, and NMDA. 2-Amino-3-phosphonopropionic acid (L-AP3), a putative antagonist of mGluR exerted little effect on responses to AMPA itself, but reduced or prevented the enhancing effect of 1S,3R-ACPD. It is concluded that activation of a metabotropic glutamate receptor by trans-ACPD, and its two enantiomers, may mediate the enhancement of AMPA and NMDA responses in acutely isolated rat spinal dorsal horn neurons. These results are consistent with the possibility that the activation of metabotropic glutamate receptor may contribute to the regulation of the strength of excitatory amino-mediated primary afferent neurotransmission, including nociception.


Patterns of termination of cerebellar and basal ganglia efferents in the rat thalamus. Strictly segregated and partly overlapping projections. There is a widely held view that the cerebellum and basal ganglia act via separate subcortical channels. In rodent, however, electrophysiological evidence suggests that the output of these two systems is partly sent to a common set of thalamic neurons. In this study, the pattern of thalamic innervations provided by the deep cerebellar nuclei, the entopeduncular nucleus, and the substantia nigra pars reticulata was reinvestigated in the rat using the anterograde tracers Phaseolus vulgaris leucoagglutinin and wheat germ agglutinin. Although the results confirm the existence of some overlap in the cerebellar and basal ganglia projection fields, they also show that in such convergent areas the cerebellar innervation is modest and consists of sparsely distributed fibers of thin diameter that provide a few scattered terminal boutons. These observations are consistent with the view that, in rodent as in higher mammalian species, the cerebellum and the basal ganglia act mainly via distinct thalamo-cortical channels.


Adenosine inhibits action potential-dependent release of calcitonin gene-related peptide- and substance P-like immunoreactivities from primary afferents in rat spinal cord. Electrical field stimulation (5 Hz) evoked a prompt outflow of calcitonin gene-related peptide- and substance P-like immunoreactivities (CGRP-LI and SP-LI, respectively) from superfused slices of the dorsal but not ventral half of the rat spinal cord. The evoked outflow was abolished by tetrodotoxin, calcium-free medium or previous exposure to capsaicin, indicating that it is produced through action potentials invading the central terminals of capsaicin-sensitive primary afferents. Adenosine as well as gamma-aminobutyric acid (GABA) or the GABAB receptor agonist (-)-baclofen produced a concentration-dependent inhibition of the evoked CGRP-LI outflow. Adenosine also inhibited the evoked SP-LI outflow. These findings demonstrate that inhibition of transmitter release from primary afferent neurons should be considered as a possible mechanism of the antinociceptive action of adenosine and adenosine analogs.


A-current in rat globus pallidus: a whole-cell voltage clamp study on acutely dissociated neurons. Globus pallidus neurons were dissociated from adult rats and recorded in whole-cell voltage-clamp mode. In tetrodotoxin- and cadmium-added bath, outward potassium conductances were evaluated. Depolarization activated large and predominantly sustained currents, with little inactivation. The transient outward current was isolated through its voltage-dependency, kinetics and pharmacology. A-like current activated around -45 mV and decayed exponentially with a time-constant of about 50 ms at 0 mV. Steady-state half-inactivation voltage was about -60 mV, suggesting that A-current was not fully inactivated at rest. 4-Aminopyridine sensitivity of the transient outward current was in the low millimolar range. A-current might play a relevant role in shaping firing properties of pallidal neurons.


Normal serotonin but elevated 5-hydroxyindoleacetic acid concentration in cerebellar cortex of patients with dominantly-inherited olivopontocerebellar atrophy. Beas-Zarate and coworkers (Eur. J. Pharmacol., 198 (1991) 7-14) recently reported markedly reduced concentration of presynaptic serotonin neurotransmitter markers in cerebellum of rodents which had suffered destruction of the inferior olivary-cerebellar (climbing fibre) projections by the neurotoxin 3-acetylpyridine; these experimental animal data suggested that serotonin might be one of the neurotransmitters released by climbing fibres. We measured the concentration of serotonin and its major metabolite 5-hydroxyindoleacetic acid (5-HIAA) in autopsied cerebellar cortex of 14 patients with dominantly-inherited olivopontocerebellar atrophy (OPCA) who all had near-total degeneration of the inferior olivary climbing fibres. As compared with the controls, mean concentration of serotonin in cerebellar cortex of the OPCA patients was normal whereas 5-HIAA levels (+79%, P less than 0.02) and 'turnover' ratio 5-HIAA/serotonin (+148%, P less than 0.05), on average, were significantly elevated. These data do not support the notion that serotonin is a predominant neurotransmitter of the human climbing fibre. However, the markedly elevated serotonin turnover ratio suggests the possibility of increased serotonergic neuronal activity, which might alter, and perhaps improve, the functioning of the preserved cerebellar cortical neurones in OPCA.


Tetanus toxin alters 5-hydroxytryptamine, dopamine, and their metabolites in rat hippocampus measured by in vivo microdialysis. Tetanus toxin is a potent neurotoxin, the mode of action of which is unknown, although it is widely considered to be mediated by loss of inhibitory neurotransmission. In the present study we have investigated the effect of a single unilateral injection of tetanus toxin in the hippocampus on extracellular concentrations of 5-hydroxytryptamine (5-HT), dopamine and their metabolites in freely moving rats. Tetanus toxin treatment caused a progressive decline in hippocampal dialysate 5-hydroxytryptamine over the 3-day period following the injection. After 3 days the level of 5-HT on the injected side was only 20% of the control side. On the second day of observation the level of dopamine was found to be elevated by more than 2-fold but this was not the case for either of the other 2 days studied. The 5-hydroxytryptamine metabolite, 5-hydroxyindoleacetic acid declined in a manner similar to that of the parent transmitter, but to a lesser extent. Of the dopamine metabolites, 3,4-dihydroxyphenylacetic acid was decreased on the second and third days of observation, whilst homovanillic acid was not significantly altered during the period of observation.


Effects of chlordecone on food intake and body weight in the male rat. The effects of the chlorinated pesticide chlordecone on food intake, body weight, and water intake were examined in adult male rats. Chlordecone treatment produced a dose dependent suppression of food intake. Loss of body weight accompanied the reduced food intake. However, chlordecone did not suppress water intake. Chlordecone treated animals maintained on a liquid diet also demonstrated reduced food intake, suggesting that chlordecone has a specific effect on feeding behavior and not a general effect on ingestive behaviors. The potential contribution of chlordecone-induced tremor to the suppressed food intake and the loss of body weight was considered. When treatment occurred immediately before a 24 hr fast, controls and animals given 75 mg/kg showed no differences in the body weight decline, even though tremor occurred in the pesticide-treated rats. Thus, it is unlikely that tremor alone produced the body weight loss observed in the present experiment. Similarly, animals were capable of initiating eating behavior even though tremor was present. In addition, chlordecone treatment inhibited food intake within 2 hr. Consequently, these results suggest that chlordecone suppresses food intake which in turn produces the decline in body weight.


Changes of fast axonal transport by taxol injected subepineurally into the rat sciatic nerve. In contrast to the complete and long-lasting inhibition of tubulin transport, taxol has no effect on fast axonal transport examined immediately after its sub-epineural application to rat sciatic nerve. However, a significant accumulation of rapidly migrating radioactivity appears at the site proximal to the injection when examined a few weeks after treatment, probably due to mechanical obstruction caused by abnormal aggregation of a huge number of intra-axonal microtubules. It also decreases slightly in amount within a few weeks post-treatment, which may be due to reduction of the number of axons caused by degeneration.


The efficacy of serum prostate specific antigen as a tumour marker in prostatic carcinoma: a comparison with serum acid phosphatase. To assess the efficacy of serum prostate specific antigen (PSA) in the diagnosis of prostatic cancer. To compare this tumour marker with serum acid phosphatase (ACP) in order to define the more effective diagnostic test. Serum samples from 349 patients attending a urology department were assayed for PSA and ACP. Histological assessment of prostatic biopsy samples was used as the standard by which the diagnostic effectiveness of the tumour markers was determined. Mean serum PSA results from patients with prostatic carcinoma (159 (SEM 35) ng/mL) were significantly different to those from patients with benign prostatic hyperplasia (4.0 (0.53) ng/mL). As there was considerable overlap of results, test sensitivities and specificities were calculated for various decision points. The sensitivity and specificity of PSA at a level of 10 ng/mL were 61.2% and 93.0% respectively, while for ACP at a level of 0.8 U/L they were 47.6% and 89.9%. The areas beneath receiver-operator curves (0.81 for PSA and 0.72 for ACP) indicated that PSA gives better diagnostic information than ACP. For a stated incidence of cancer, posttest probabilities following a given PSA result have been calculated. PSA is a more effective serum marker for prostatic carcinoma than ACP. We present a table so that for similar patient groups posttest probabilities for prostatic cancer can be assessed for a given serum PSA level.


Upper gastrointestinal endoscopy in patients with human immunodeficiency virus infection: is it worthwhile? To determine the diagnostic accuracy of upper gastrointestinal endoscopy in a selected group of patients with HIV infection, who had severe symptoms unresponsive to empirical treatment. We reviewed all patients with HIV infection, who had undergone upper gastrointestinal endoscopy at Auckland Hospital. Specific diagnoses were based on the endoscopic appearances and on the histological and microbiological examination of endoscopic biopsies taken from the oesophagus, stomach and duodenum. A definitive diagnosis was reached in 16 of the 21 patients endoscoped, of whom 13 had a good clinical response to treatment. The median survival was three months. The most common diagnosis was oesophageal candidiasis seen in six patients, all of whom responded to treatment with a median survival of six months. Invasive cytomegalovirus gastrointestinal disease was seen in three patients, none of whom had positive blood cultures for cytomegalovirus. All responded to intravenous ganciclovir. Three patients with severe diarrhoea had opportunistic infection of the small bowel. All three had advanced AIDS with a median survival of less than one month. Upper gastrointestinal endoscopy has a high diagnostic yield in patients with HIV infection with oesophageal symptoms which fail to respond to antifungal therapy, or with severe weight loss and diarrhoea and it should be considered in these patients because excellent palliation may be possible.


Conjunctival mast cells and the allergic late phase reaction. An active model of ocular anaphylaxis was developed in guinea pigs to evaluate the histopathology of the early (EPR) and late (LPR) phase reaction, focusing on the role of mast cells. Five groups (n = 6) of animals were actively immunized by first injecting into each of the axillary and inguinal lymph node areas, 0.25 ml of an emulsion containing 1 mg dinitrophenyl bovine gamma-globulin (DNP-BCG) with 0.5 ml complete Freund's adjuvant. After two weeks, an intramuscular injection of 0.5 ml of an emulsion containing 1 mg DNP-BGG with 0.5 ml of incomplete adjuvant was administered. One month after the first injection, animals were sacrificed after topical ocular challenge with 10 microliters of 1 mg/ml divalent hapten, di-DNP-lysine, in one eye and phosphate buffered saline (PBS) in the fellow eye as control. Clinical reactions were graded over time and histology evaluated at the endpoint (time 0, 0.5, 3, 9, and 24 h). Results showed that all animals clearly developed both an EPR and an LPR, as either a biphasic, multiphasic or prolonged clinical response. A small percentage of mast cells were degranulated at baseline, whereas, at 0.5 h, 95% of mast cells were degranulated in the eyes treated with specific hapten and 25% in the control eyes treated with PBS. At 3 h, 84% of the mast cells were degranulated. This value rose to 89% at 9 h, and remained unchanged at 24 h.(ABSTRACT TRUNCATED AT 250 WORDS)


Histopathology of smokeless tobacco lesions in professional baseball players. Associations with different types of tobacco. We examined 142 biopsy specimens of smokeless tobacco-associated oral mucosal lesions from 133 professional baseball players. Four types of epithelial change were observed in the specimens: hyperparakeratosis, hyperorthokeratosis, pale surface staining, and basal cell hyperplasia. These types of epithelial change were associated with the type of smokeless tobacco used (snuff or chewing tobacco) but not with the duration (years) or amount (hours per day) of use. The thickness of hyperkeratosis in a specimen correlated directly with the amount of smokeless tobacco use. The use of snuff was more frequently associated with development of oral mucosal lesions than was the use of chewing tobacco, and snuff appeared to cause a greater variety and severity of epithelial change than did chewing tobacco.