Ultrastructural features of NPY-containing neurons in the rat striatum

In the present study, we examined the ultrastructure of striatal neurons containing neuropeptide Y (NPY) which were labeled by an immunohistochemical method using peroxidase-conjugated F(ab) fragments in the rat. Each of the 26 neurons identified had a deeply indented oval nucleus. The cytoplasm, which was mainly concentrated at the emergence of the dendrites, contained an abundant Golgi apparatus and a well-developed granular endoplasmic reticulum. Dendrites were poorly branched and rarely exhibited varicosities or dendritic spines. NPY-immunoreactive (Ir) axons were small in diameter and unmyelinated. These features corresponded to a subpopulation of striatal neurons classified as aspiny type IV in previous Golgi studies. Axon terminals forming symmetrical synapses were numerous on the NPY-Ir perikarya and proximal dendrites. On distal NPY-Ir dendrites, synaptic contacts were mainly of the asymmetrical type, suggesting that NPY neurons are contacted by at least 2 categories of afferent fibers. Several NPY-Ir axonal processes and boutons were found to form symmetrical synapses with dendrites, dendritic spines and perikarya belonging to spiny type neurons. These data were consistent with the view that NPY may act as a neurotransmitter of striatal interneurons. Moreover, the frequent observation of NPY axonal processes in the close vicinity of striatal vessels suggested that NPY might also play a role in the control of cerebral vasomotricity. Thirty hours after intranigral injection of 6-hydroxydopamine to induce a degeneration of nigrostriatal dopamine terminals, some characteristic degenerative boutons were observed in close apposition to NPY-Ir cell bodies, suggesting that NPY neurons are under a direct nigrostriatal dopaminergic influence.     

Pharmacological characterization of dopaminergic influence on expression of neuropeptide Y immunoreactivity by rat striatal neurons

Selective unilateral lesion of the nigrostriatal dopamine pathway by the cytotoxin 6-hydroxydopamine was previously shown to enhance the number and staining intensity of neurons expressing neuropeptide Y immunoreactivity in the ipsilateral striatum. This effect was completely reversed by treatment of the 6-hydroxydopamine-injected animals with the directly acting dopamine agonist apomorphine. This finding reinforces our previous hypothesis that changes in striatal neuropeptide Y staining subsequent to 6-hydroxydopamine lesions of this kind reflect changes in intraneuronal neuropeptide Y levels which are directly attributable to the suppression of a tonic dopaminergic control. In contrast to the effect of 6-hydroxydopamine lesion, non-destructive impairment of striatal dopamine transmission by treatments with either the dual dopamine D1/D2 receptor antagonist haloperidol or the dopamine synthesis inhibitor alpha-methylparatyrosine induced a decrease in both the number of neuropeptide Y striatal cells (-29.8% and -34.8%, respectively) and in their labeling intensity. The selective D2-antagonist sulpiride also showed a tendency to reduce the number of neuropeptide Y immunoreactive cells, whereas the selective D1 antagonist SCH 23390 induced a small but constant increase in this number. Taken as a whole, these results suggest that the dopaminergic D1 and D2 receptor subtypes play opposite roles in the dopaminergic control of the striatal neuropeptide Y neuronal system, which may account for the different changes in striatal neuropeptide Y immunostaining observed after 6-hydroxydopamine injury and after non-destructive impairment of nigrostriatal dopaminergic transmission.     

Projections from Areas 18 and 19 to Cat Striate Cortex: Divergence and Laminar Specificity

The results of electrical stimulation experiments [Bullier et al., (1988) Exp. Brain Res., 70, 90 - 98] demonstrated that afferents from areas 18 and 19 contact different functional types of neurons in area 17. We were therefore interested in examining whether these results could be explained by differences in the morphology of the terminals of these two groups of afferent connections to area 17. We also wanted to confirm, by a direct method, our earlier results [Salin et al. (1989) J. Comp. Neurol., 283, 486 - 512] that cortical afferents to area 17 in the cat present extensive divergences. We therefore placed small injections of anterograde tracers in areas 18 and 19 and examined the laminar distributions of terminals thus revealed and the extent of the surface of area 17 contacted by these terminals. Three tracers were used: wheat germ agglutinin - horseradish peroxidase (WGA - HRP), Phaseolus vulgaris leucoagglutinin (Pha-L) and biocytin. The results show that the divergence of these afferent connections are very extensive: 7 - 8 mm in the rotrocaudal direction and 3.5 - 6 mm in the mediolateral direction. In other words, neurons located in a region a few hundreds micron wide in areas 18 or 19 contact a region of area 17 covering several millimeters. Corticocortical connections are therefore not organized in a point-to-point fashion but are strongly divergent. The laminar distributions of terminals from areas 18 and 19 displayed a specific pattern. Area 19 projects most heavily to layers 5 and 6, also terminates in layers 1 - 3 and very little is present in layer 4. In contrast, the afferent terminals from area 18 are heaviest in layers 1, 2, 3, 4A and 5 and are rare in layer 6. Injections placed at different depths in area 18 revealed that upper layer neurons in that area mostly project to layers 1, 2, 3 and 5 in area 17, whereas lower layer neurons send their heaviest projections to layers 4A, 5 and 6 and hardly project to layers 1, 2 and 3.     

Dysfunctional regulation of alphaCaMKII and syntaxin 1B transcription after induction of LTP in the aged rat

Syntaxin 1B and alphaCaMKII are two genes that are upregulated after the induction of LTP and appear to underlie different mechanisms of synaptic plasticity. alphaCaMKII is directly implicated in strengthening the synapses that have been modified, whereas syntaxin 1B has been implicated in a mechanism for the propagation of synaptic plasticity within neural circuits. In these experiments we have investigated whether the regulation of these genes is altered after the induction of LTP in aged rats. We found, three hours after the induction of LTP in the dentate gyrus, that aged rats could be subgrouped into those in which LTP was maintained and those in which LTP had decayed back to basal levels. Both genes were upregulated in young adult rats, whereas there was a differential pattern of LTP-induced expression in the aged rats. Dendritic alphaCaMKII was upregulated in aged rats only when LTP was maintained. In contrast, regulation of syntaxin 1B and alphaCaMKII was absent in the granule cell bodies of the aged rats regardless of whether LTP was maintained or not. These results suggest that molecular mechanisms implicated in two aspects of hippocampal synaptic plasticity malfunction during normal ageing and therefore may have some contributory role in the decline in memory function routinely observed in ageing.     

Dissipation Kinetics of Chlorantraniliprole in Soils of Sugarcane Ecosystem

Dissipation kinetics of chlorantraniliprole was studied in sandy loam soils of sugarcane ecosystem by adopting a rapid analytical method. The recovery of chlorantraniliprole was 91.67 % when extracted with ethyl acetate as against only 65.58 % in acetonitrile-based extraction. An additional cleanup step with primary secondary amine did not enhance the recovery significantly over the no-cleanup method. The ethyl acetate-based extraction followed by direct quantification in HPLC (High-performance liquid chromatography) without any cleanup facilitated rapid quantification of chlorantraniliprole residues. The LOQ (limit of quantification) of the method was 0.01 μg/g. The half-life of chlorantraniliprole was 6.50 and 6.81 days for the recommended and double the recommended doses, respectively.     

H5N1 Oseltamivir-resistance detection by real-time PCR using two high sensitivity labeled TaqMan probes

A single amino acid substitution, from histidine to tyrosine at position 274 of the neuraminidase gene has converted Oseltamivir sensitive H5N1 influenza A virus into a resistant strain. Currently, Oseltamivir is being stockpiled in many countries potentially affected by the influenza A virus subtype H5N1 epidemic. To identify this change in Oseltamivir-treated patients, a method based on real-time PCR using two labeled TaqMan probes was developed for its rapid detection. In order to validate the method, Oseltamivir specimen from treated (Oseltamivir-resistant strain from a Vietnamese patient, two Oseltamivir-treated tigers) and untreated subjects have been used for this study. The results thus obtained as well as those derived from clone selection and sequencing showed that TaqMan probes could clearly discriminate wild type H274 from the mutant 274Y variant. The sensitivity of this assay was as low as 10 copies/microl and allowed the detection of the mutation in a mixture of wild type and mutant. Overall, the assay based on real-time PCR with two labeled TaqMan probes described here should be useful for detecting Oseltamivir-resistant H274Y H5N1 influenza A virus in many species and various sources of specimens with high sensitivity and specificity. Such studies can address potential differences in the diagnostic outcomes between patients who develop detectable Oseltamivir resistance and those who retain only the wild type strain of H5N1.     

Complete coding sequences and phylogenetic analysis of Human Bocavirus (HBoV)

Human Bocavirus (HBoV) is a novel virus which can cause respiratory tract disease in infants or children. Recently, the prevalence of this virus has been studied worldwide. In this study, 18 of 252 (7.14%) nasopharyngeal suctions from infants or children between 1 month and 9 years of age with respiratory tract illness were HBoV-positive by PCR. Three positive samples were selected for sequencing the entire coding sequences using a new conserved set of primers. The results showed that the most conserved regions of HBoV are the NS1 and NP1 genes, whereas VP1 and VP2 showed frequent variations. However, the complete coding sequences showed that the variations did not depend on the origin of virus found. The complete coding sequences determined in this study can resolve the problem of an HBoV detection method, which can be advantageously implemented in laboratory detection.     

Glutamate transporter 1-expressing glia in the rat substantia nigra—Morphometric analysis and relationships to synapses

Glial cells have a major role in protecting neurons against various forms of stress. Especially, astrocytes mediate the bulk of glutamate clearance in the brain via specific membrane transporters (GLAST and GLT1), thereby preventing the occurrence of excitotoxic events. Although glutamate-mediated mechanisms are thought to contribute to nigral dopaminergic neuron degeneration in Parkinson's disease, detailed information on the organization of glia in the substantia nigra is still lacking. The present study was performed to provide quantitative information on the organization of astroglia and on the relationships between astrocytes and excitatory synapses in the rat substantia nigra. Using immunolabeling of GLT1 and confocal imaging, we found that the substantia nigra was filled with a dense meshwork of immunoreactive astrocyte processes. Stereological analysis performed on electron microscope images revealed that the density of immunoreactive astrocyte plasma membranes was substantial, close to 1 μm²/μm³, in the substantia nigra neuropil, both in the pars compacta and the pars reticulata. Excitatory synapses had on average two thirds of their perimeters free from glia, a disposition that may favor transmitter spillover. The density of glutamatergic synapses, as quantified on confocal images by the simultaneous detection of bassoon and of vesicular glutamate transporter 1 or 2, was very low (0.01 and 0.025 per μm³ in the reticulata and compacta subdivisions, respectively). Thus the ratio of GLT1-expressing glial membrane surface to glutamatergic synapses was very high (40–100 μm²), suggesting an efficient regulation of extracellular glutamate concentrations.