Notes on the combined use of V-VIP and DAB peroxidase substrates for the detection of colocalising antigens

The purpose of the present report was to investigate to what extent the new peroxidase substrate Vector VIP (V-VIP) can be used in combination with DAB chromogen for the unequivocal and permanent detection of colocalising antigens within a single neurone, according to a two-colour paradigm. With this aim, retrograde tract-tracing with cholera toxin B subunit (CTB) or fluoro-gold (FG) was performed to disclose individual, identified subpopulations of neurones in the primate substantia nigra projecting to the caudate nucleus or to the putamen, respectively. Each tracer was detected by means of a PAP procedure and finally stained brown using DAB as a chromogen. Subsequently, both series of sections were processed for the immunocytochemical detection of tyrosine hydroxylase (TH). TH-immunoreactive neurones were stained purple with the peroxidase substrate V-VIP. As a result of the present procedure, several cell bodies of projection neurones, stained brown, can easily be identified within the primate substantia nigra. Some of these neurones additionally displayed purple TH immunoreaction product located in the neuronal dendrites. By contrast, CTB- or FG-unlabelled neurones only show the typical purple precipitate that belongs to V-VIP substrate, both in the cell body as well as in the dendrites

Relationships between thalamostriatal neurons and pedunculopontine projections to the thalamus: a neuroanatomical tract-tracing study in the rat

The present study aimed to investigate whether the pedunculopontine projection to the thalamus overlaps with identified thalamostriatal neurons. These projections were studied using a dual tract-tracing procedure combining anterogradely transported biotinylated dextran amine (pedunculopontine projections) and retrogradely transported Fluoro-Gold (thalamostriatal projections). Overlapping thalamic territories between thalamostriatal neurons and the axon terminals arising from the pedunculopontine tegmental nucleus were observed in the midline (paraventricular) and in the intralaminar (centrolateral, central medial, paracentral and parafascicular) thalamic nuclei. Other thalamic nuclei, such as the ethmoid, intermediodorsal, mediodorsal, paratenial, posteromedian, ventromedian, ventrolateral and rhomboid thalamic nuclei, displayed a lesser degree of overlap. These observations suggest the existence of presumptive contacts between thalamostriatal neurons and axons emerging from the pedunculopontine tegmental nucleus, therefore supporting the possible existence of feedback circuits in the rat basal ganglia in which the tegmento-thalamic projection would play a major role

Re-examination of the thalamostriatal projections in the rat with retrograde tracers

Topographical arrangements of thalamostriatal projection neurons was examined in the rat by the retrograde tract-tracing method. After injecting Fluoro-Gold (FG) and/or cholera toxin beta-subunit (CTB) in different regions of the caudate-putamen (CPu), distribution of retrogradely labeled neurons was observed in the thalamus. The main findings were as follows: (1) Retrogradely labeled neurons were seen in the midline-intralaminar thalamic nuclei in all rats examined in the present study. Neurons in the ventral lateral and posterior thalamic nuclear groups were also labeled in the rats which were injected with the tracer into the dorsal part of Cpu, but not in the rats which were injected with the tracer into the nucleus accumbens (Acb) and its adjavent regions in the ventromedial part of the Cpu. (2) Topographical organization was observed in the projections from the midline-intralaminar thalamic nuclei to the CPu. After the tracer injection into the dorsal part of the CPu or the ventral part of the CPu (including the Acb), labeled neurons in the midline-intralaminar thalamic nuclei were distributed predominantly in the lateral part of the intralaminar nuclei or the midline nuclei, respectively. On the other hand, after the tracer injection into the medial or the lateral part of the CPu, labeled neurons in the midline-intralaminar nuclei were distributed mainly in the dorsal or the ventral part of these nuclei, respectively. (3) Topographical organization was also observed in the thalamostriatal projections from the ventral and Pos. After the tracer injection into the rostral part of the CPu, labeled neurons were distributed mainly in the rostral part of the ventral nuclear group. On the other hand, after the tracer injection into the caudal part of the CPu, labeled neurons were distributed mainly in the caudal part of the ventral nuclear group, as well as in the posterior nuclear group

Effective GDNF brain delivery using microspheres-A promising strategy for Parkinson's disease

Glial cell line-derived neurotrophic factor (GDNF) has shown promise in the treatment of neurodegenerative disorders of basal ganglia origin such us Parkinson\u27s disease (PD). In this study, we investigated the neurorestorative effect of controlled GDNF delivery using biodegradable microspheres in an animal model with partial dopaminergic lesion. Microspheres were loaded with N-glycosylated recombinant GDNF and prepared using the Total Recirculation One-Machine System (TROMS). GDNF-loaded microparticles were unilaterally injected into the rat striatum by stereotaxic surgery two weeks after a unilateral partial 6-OHDA nigrostriatal lesion. Animals were tested for amphetamine-induced rotational asymmetry at different times and were sacrificed two months after microsphere implantation for immunohistochemical analysis. The putative presence of serum IgG antibodies against rat glycosylated GDNF was analyzed for addressing safety issues. The results demonstrated that GDNF-loaded microspheres, improved the rotational behavior induced by amphetamine of the GDNF-treated animals together with an increase in the density of TH positive fibers at the striatal level. The developed GDNF-loaded microparticles proved to be suitable to release biologically active GDNF over up to 5 weeks in vivo. Furthermore, none of the animals developed antibodies against GDNF demonstrating the safety of glycosylated GDNF use

Ischemia induces cell proliferation and neurogenesis in the gerbil hippocampus in response to neuronal death

We studied hippocampal cellular proliferation and neurogenesis processes in a model of transient global cerebral ischemia in gerbils by labelling dividing cells with 5'-Bromo-2'-deoxyuridine (BrdU). Surrounding the region of selective neuronal death (CA1 pyramidal layer of the hippocampus), an important increase in reactive astrocytes and BrdU-labelled cells was detected 5 days after ischemia. A similar result was found in the dentate gyrus (DG) 12 days after ischemia. The differentiation of the BrdU+ cells was investigated 28 days after BrdU administration by analyzing the morphology, anatomic localization and cell phenotype by triple fluorescent labelling (BrdU, adult neural marker NeuN and DNA marker TOPRO-3) using confocal laser-scanning microscopy. This analysis showed increased neurogenesis in the DG in case of ischemia and triple positive labelling in some newborn cells in CA1. Seven brain hemispheres from gerbils subjected to ischemia did not develop CA1 neuronal death; hippocampus from these hemispheres did not show any of the above mentioned findings. Our results indicate that ischemia triggers proliferation in CA1 and neurogenesis in the DG in response to CA1 pyramidal neuronal death, independently of the reduced cerebral blood flow or the cell migration from subventricular zone (SVZ)

Multiple neuroanatomical tracing in primates

The present report deals with a multiple tract-tracing procedure in non-human primates enabling the simultaneous visualization of retrogradely transported Fluoro-Gold (FG) and cholera toxin B subunit (CTB) in combination with anterogradely transported biotinylated dextran amine (BDA). Two issues have played key roles on the achievement of this reliable procedure: first, the recent development of a commercial antiserum against FG that allows us to convert the original fluorescent signal of this dye in a permanent precipitate via standard peroxidase-anti-peroxidase methods; second, the introduction of the novel peroxidase substrate Vector(R) VIP (V-VIP), resulting in a purple precipitate. The combination of these neuroanatomical tracers in one and the same histological section opens a possibility for the permanent visualization of the convergence of inputs from a particular brain area onto identified, two different subsets of projection cells of another area. Furthermore, this combination of three tracers emerges as a powerful technical tool for obtaining broad amounts of complementary data regarding the monkey brain connectivity, thus significantly reducing the number of animals needed to complete a particular study

Expresión y purificación de GDNF para su microencapsulación y aplicación en la enfermedad de Parkinson

La enfermedad de Parkinosn (EP) es un proceso neurodegenerativo del sistema nervioso central que afecta a las neuronas de dopamina de la sustancia negra, núcleo mesoencefálico del control motor. La perdida en el cerebro de este neurotransmisor vital causa los síntomas de la enfermedad. La EP afecta actualmente a 200 de cada 100.000 personas y a 2 de cada 100 entre los mayores de 60 años. En España hay unos 110.000 enfermos. Además, hoy por hoy no se conoce nada que pueda prevenir o curar la enfermedad, ni existe ninguna prueba de laboratorio que permita diagnosticarla. Recentiemente se ha demostrado que el GDNF, factor neurotrófico derivado de las células gliales, es capaz de proteger las neuronas dopaminérgicas e incluso inducir la regeneración del tejido dopaminérgico dañado in vivo. El objetivo del trabajo fue diseñar y desarrolar un método de expresión y purificación de GDNF bioactivo para su posterior microencapsulación y aplicación en la enfermedad de Parkison. El sistema escogido para expresar el GDNF fue el sistema de células eucariotas de mamífero. El vector utilizado para la producción del GDNF en células eucariotas fue el pDEST26 (Tecnología Gateway de Invitrogen). Como sistema de expresión de GDNF se utilizaron las líneas celulares eucariota BHK, 293 y COS 7. Estas células fueron cultivadas en medio D-MEM (Invitrogen) complementado con un 10% de suero fetal bovino (FBS) y Penicilina/Streptomicina (100u/ml) (Invitrogen). La transfección se realizó con Lipofectamine Plus (Invitrogen). Se analizó la expresión de GDNF a nivel de mRNA mediante PCR y a nivel de proteína mediante Western Blot del medio condicionado. Los clones positivos se crecieron en botellas de cultivo de 850 cm2 (Corning) y se realizaron ciclos de recolección del medio. Cada ciclo fue analizado por SDS-PAGE y Western Blot. Para evaluar la actividad de la proteína se ha desarrollado un ensayo de actividad en el que se demuestra la diferenciación morfológica de células PC-12 inducida por GDNF. La presencia de los receptores GFRa1 y RET, necesarios para que el GDNF ejerza su acción, fue determinada por PCR. Las conclusiones obtenidas de este estudio son la obtención de GDNF recombinante a partir de un sistema de expresión en células eucariotas, el desarrollo de un protocolo para su posterior purificación y la obtención de GDNF recombinante biológicamente activo

Use of peroxidase substrate Vector VIP for multiple staining in light microscop

The study of the distribution of a fiber input to a particular brain area and the visualization of the anatomical relationships of that input with both projection- and interneurons, requires a triple-staining that allows the unequivocal distinction of each of the three components in one and the same histological section. In this regard, we investigated the properties of a recently introduced peroxidase chromogen, VIP (V-VIP; Vector Labs) in combination with two traditional substrates, standard diaminobenzidine (DAB, brown precipitate) and nickel-enhanced DAB (DAB-Ni, black). In rats, the anterograde tracer biotinylated dextran amine (BDA) and the retrograde tracer fluorogold (FG) were injected in the perirhinal cortex and hippocampus, respectively. Transported BDA was detected with an avidin-biotin-peroxidase complex, whereas the transported FG was detected via a PAP method. Tracing with BDA and FG was combined with parvalbumin- or calbindin-immunocytochemistry. We compared various combinations and staining sequences. The best results were obtained with a staining sequence comprising first the BDA stain with DAB-Ni as chromogen, second the FG protocol with the chromogen DAB and finally, parvalbumin- or calbinding-immunocytochemistry using the chromogen V-VIP. The order with which the chromogens were applied appeared to be critical. Partial or even total loss of V-VIP reaction product has been observed after standard dehydration in ethanol. As an alternative, a quick dehydration procedure in toluene yields much better staining. Colour separation is excellent and the sensitivity is high. This procedure may also be used for detection of any other combination of three different labels, taking the usual care to avoid cross-reactivity between antibodies