Wouterlood, F.G. (Floris G.)
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- A half century of experimental neuroanatomical tracing(Elsevier, 2011) Wouterlood, F.G. (Floris G.); Lanciego, J.L. (José Luis)Most of our current understanding of brain function and dysfunction has its firm base in what is so elegantly called the 'anatomical substrate', i.e. the anatomical, histological, and histochemical domains within the large knowledge envelope called 'neuroscience' that further includes physiological, pharmacological, neurochemical, behavioral, genetical and clinical domains. This review focuses mainly on the anatomical domain in neuroscience. To a large degree neuroanatomical tract-tracing methods have paved the way in this domain. Over the past few decades, a great number of neuroanatomical tracers have been added to the technical arsenal to fulfill almost any experimental demand. Despite this sophisticated arsenal, the decision which tracer is best suited for a given tracing experiment still represents a difficult choice. Although this review is obviously not intended to provide the last word in the tract-tracing field, we provide a survey of the available tracing methods including some of their roots. We further summarize our experience with neuroanatomical tracers, in an attempt to provide the novice user with some advice to help this person to select the most appropriate criteria to choose a tracer that best applies to a given experimental design.
- Neuroanatomical tract-tracing methods beyond 2000: what's now and next(Elsevier, 2000) Wouterlood, F.G. (Floris G.); Lanciego, J.L. (José Luis)
- Use of peroxidase substrate Vector VIP for multiple staining in light microscop(Elsevier, 1997) Wouterlood, F.G. (Floris G.); Lanciego, J.L. (José Luis); Witter, M.P. (Menno P.); Goede, P.H. (P. H.)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.
- Origin of calretinin-containing, vesicular glutamate transporter 2-coexpressing fiber terminals in the entorhinal cortex of the rat(wiley Blackwell, 2008) Zaborszky, L. (Laszlo); Hur, E.E. (E. E.); Wouterlood, F.G. (Floris G.); Aliane, V. (V.); Lanciego, J.L. (José Luis); Boekel, A.J. (A.J.); Barroso-Chinea, P. (P.); Härtig, W. (W.); Witter, M.P. (Menno P.)The entorhinal cortex of the rat (EC) contains a dense fiber plexus that expresses the calcium-binding protein calretinin (CR). Some CR fibers contain vesicular glutamate transporter 2 (VGluT2, associated with glutamatergic neurotransmission). CR-VGluT2 coexpressing fibers may have an extrinsic origin, for instance, the midline thalamic nucleus reuniens. Alternatively, they may belong to cortical interneurons. We studied the first possibility with anterograde and retrograde neuroanatomical tracing methods combined with CR and VGluT2 immunofluorescence and confocal laser scanning. The alternative possibility was studied with in situ hybridization fluorescence histochemistry for VGluT2 mRNA combined with CR immunofluorescence. In the anterograde tracing experiments, we observed many labeled reuniens fibers in EC expressing CR. Some of these labeled fibers contained immunoreactivity for VGluT2 and CR. In the complementary retrograde tracing experiments, we found retrogradely labeled cell bodies in nucleus reuniens of the thalamus that coexpressed CR. We also examined the colocalization of VGluT2 and CR in the entorhinal cortex by using in situ hybridization and CR immunofluorescence. In these experiments, we observed CR-immunopositive cortical neurons that coexpressed VGluT2. For the same sections, with CR as the principal marker and parvalbumin as a control marker, we found that parvalbumin neurons were negative for VGluT2 mRNA. Thus, CR-VGluT2-expressing axon terminals in EC belong to two sources: projection fibers from the thalamus and axon collaterals of local interneurons. VGluT2 expression is linked to the synaptic transmission of the excitatory neurotransmitter glutamate, so these thalamic CR-VGluT2 projection neurons and entorhinal CR-VGluT2 interneurons should be regarded as excitatory.
- Proceedings of the workshop "Neuroanatomical Tracing and Systems Neuroscience: the State of the Art", 7th FENS meeting, Amsterdam, The Netherlands, July 3, 2010(Elsevier, 2010) Wouterlood, F.G. (Floris G.); Lanciego, J.L. (José Luis)
- Complex brain circuits studied via simultaneous and permanent detection of three transported neuroanatomical tracers in the same histological section.(Elsevier, 2000) Arribas, J. (J.); Wouterlood, F.G. (Floris G.); Erro-Aguirre, M.E (María Elena); Gonzalo, N. (Nancy); Lanciego, J.L. (José Luis); Cervantes-Ibáñez, S. (Sebastián); Gimenez-Amaya, J.M. (José Manuel); Urra, X. (X.)Experimental neuroanatomical tracing methods lie at the basis of the study of the nervous system. When the scientific question is relatively straightforward, it may be sufficient to derive satisfactory answers from experiments in which a single neuroanatomical tracing method is applied. In various scientific paradigms however, for instance when the degree of convergence of two different projections on a particular cortical area or subcortical nucleus is the subject of study, the application of single tracing methods can be either insufficient or uneconomical to solve the questions asked. In cases where chains of projections are the subjects of study, the simultaneous application of two tracing methods or even more may be compulsory. The present contribution focuses on combinations of several neuroanatomical tract-tracing strategies, enabling in the end the simultaneous, unambiguous and permanent detection of three transported markers according to a three-color paradigm. A number of combinations of three tracers or of two tracers plus the immunocytochemical detection of a neuroactive substance can be conceived; we describe several of these combinations implemented by us using the present multitracer protocol.
- Multiple axonal tracing: simultaneous detection of three tracers in the same section(Springer Verlag, 1998) Wouterlood, F.G. (Floris G.); Erro-Aguirre, M.E (María Elena); Lanciego, J.L. (José Luis); Gimenez-Amaya, J.M. (José Manuel)Multiple neuroanatomical tract-tracing methods are important tools for elucidating the connectivity between different populations of neurons. Evaluation of the question as to whether two specific fiber inputs converge on a particular, identified population of projection neurons requires the application of a triple-staining procedure that allows the unequivocal detection of three markers in a single section. The present report deals with a combination of tracing methods using anterogradely transported Phaseolus vulgaris leucoagglutinin and biotinylated dextran amine in conjunction with retrogradely transported Fluoro-Gold. These tracers were simultaneously detected according to a three-color paradigm, which includes the use of three different peroxidase substrates (nickel-enhanced diaminobenzidine, diaminobenzidine, and Vector VIP), thus resulting in three distinct precipitates: black, brown, and purple. We illustrate this method by showing convergence of projections arising from neurons located in two separate basal ganglia-related nuclei onto identified thalamostriatal projection neurons.
- Neuroanatomical tracing combined with in situ hybridization: analysis of gene expression patterns within brain circuits of interest(Elsevier, 2010) Wouterlood, F.G. (Floris G.); Conte-Perales, L. (L.); Lanciego, J.L. (José Luis); Rico, A.J. (Alberto J.); Roda, E. (Elvira); Lopez, I.P. (Iciar P.); Barroso-Chinea, P. (P.); Gomez-Bautista, V. (V.)Most of our current understanding of brain circuits is based on hodological studies carried out using neuroanatomical tract-tracing. Our aim is to advance one step further by visualizing the functional correlate in a given circuit. In this regard, we believe it is feasible to combine retrograde tracing with fluorescence, non-radioactive in situ hybridization (ISH) protocols. The subsequent detection at the single-cell level of the expression of a given mRNA within retrograde-labeled neurons provides information regarding cellular function. This may be of particular interest when trying to elucidate the performance of brain circuits of interest in animal models of brain diseases. Several combinations of retrograde tracing with either single- and double-ISH are presented here, together with some criteria that influence the selection of the tracer to be used in conjunction with the strong demands of the ISH.
- Neuroanatomical tract-tracing techniques that did go viral(Springer, 2020) Wouterlood, F.G. (Floris G.); Lanciego, J.L. (José Luis)Neuroanatomical tracing methods remain fundamental for elucidating the complexity of brain circuits. During the past decades, the technical arsenal at our disposal has been greatly enriched, with a steady supply of fresh arrivals. This paper provides a landscape view of classical and modern tools for tract-tracing purposes. Focus is placed on methods that have gone viral, i.e., became most widespread used and fully reliable. To keep an historical perspective, we start by reviewing one-dimensional, standalone transport-tracing tools; these including today’s two most favorite anterograde neuroanatomical tracers such as Phaseolus vulgaris-leucoagglutinin and biotinylated dextran amine. Next, emphasis is placed on several classical tools widely used for retrograde neuroanatomical tracing purposes, where Fluoro-Gold in our opinion represents the best example. Furthermore, it is worth noting that multi-dimensional paradigms can be designed by combining diferent tracers or by applying a given tracer together with detecting one or more neurochemical substances, as illustrated here with several examples. Finally, it is without any doubt that we are currently witnessing the unstoppable and spectacular rise of modern molecular-genetic techniques based on the use of modifed viruses as delivery vehicles for genetic material, therefore, pushing the tract-tracing feld forward into a new era. In summary, here, we aim to provide neuroscientists with the advice and background required when facing a choice on which neuroanatomical tracer—or combination thereof—might be best suited for addressing a given experimental design.