Cannabinoid receptor CB2 ablation protects against TAU induced neurodegeneration.

Tauopathies are a group of neurodegenerative diseases characterized by the alteration/aggregation of TAU protein, for which there is still no effective treatment. Therefore, new pharmacological targets are being sought, such as elements of the endocannabinoid system (ECS). We analysed the occurrence of changes in the ECS in tauopathies and their implication in the pathogenesis. By integrating gene expression analysis, immunofluorescence, genetic and adeno-associated virus expressing TAU mouse models, we found a TAU-dependent increase in CB2 receptor expression in hippocampal neurons, that occurs as an early event in the pathology and was maintained until late stages. These changes were accompanied by alterations in the endocannabinoid metabolism. Remarkably, CB2 ablation in mice protects from neurodegeneration induced by hTAUP301L overexpression, corroborated at the level of cognitive behaviour, synaptic plasticity, and aggregates of insoluble TAU. At the level of neuroinflammation, the absence of CB2 did not produce significant changes in concordance with a possible neuronal location rather than its classic glial expression in these models. These findings were corroborated in post-mortem samples of patients with Alzheimer's disease, the most common tauopathy. Our results show that neurons with accumulated TAU induce the expression of the CB2 receptor, which enhances neurodegeneration. These results are important for our understanding of disease mechanisms, providing a novel therapeutic strategy to be investigated in tauopathies.

Globular glial tauopathy caused by MAPT P301T mutation: clinical and neuropathological findings.

OBJECTIVE: To describe the clinical, biochemical, and neuropathological findings of an autosomal dominant globular glial tauopathy caused by the P301T mutation at the MAPT gene. METHODS: Five patients from two unrelated pedigrees underwent clinical evaluation. Genetic analysis, brain pathological examination, and biochemical analysis of tau were performed. RESULTS: The patients studied were 3 men and 2 women with a mean age at onset of 52.2 years and mean disease duration of 5.2 years. Three patients presented a corticobasal syndrome, one patient an asymmetric pyramidal syndrome compatible with primary lateral sclerosis, and one patient a frontotemporal dementia. In both pedigrees (4 patients) Sanger sequencing showed the p.P301T mutation in exon 10 of the MAPT gene. Neuropathological findings consisted of atrophy of frontal and temporal lobes with marked spongiosis and astrogliosis, and abundant phosphorylated tau protein deposits in the frontal and temporal cortex, limbic area, basal ganglia, and brain stem. The most striking finding was the presence of oligodendroglial 4R phospho-tau globular positive inclusions in the white matter and cortex. Globose-type neurofibrillary neuronal tangles, and in particular astrocytic globular inclusions and coarse tufts, were present in the grey matter. Biochemical analysis of sarkosyl-insoluble fractions revealed two tau bands of 64 and 68 kDa and case-dependent bands of lower molecular weight. CONCLUSION: This is the first pathological and biochemical study of the MAPT p.P301T mutation showing variable clinical manifestation and neuropathological phenotype of globular glial tauopathy not only among different families but also within families.

Dopamine D2 and angiotensin II type 1 receptors form functional heteromers in rat striatum.

Identification of G protein-coupled receptors and their specific function in a given neuron becomes essential to better understand the variety of signal transduction mechanisms associated with neurotransmission. We hypothesized that angiotensin II type 1 (AT1) and dopamine D2 receptors form heteromers in the central nervous system, specifically in striatum. Using bioluminescence resonance energy transfer, a direct interaction was demonstrated in cells transfected with the cDNA for the human version of the receptors. Heteromerization did not affect cAMP signaling via D2 receptors but attenuated the coupling of AT1 receptors to Gq. A common feature of heteromers, namely cross-antagonism, i.e. the blockade of the signaling of one receptor by the blockade of the partner receptor, was tested in co-transfected cells. Candesartan, the selective AT1 receptor antagonist, was able to block D2-receptor mediated effects on cAMP levels, MAP kinase activation and ß-arrestin recruitment. This effect of candesartan, which constitutes a property for the dopamine-angiotensin receptor heteromer, was similarly occurring in primary cultures of neurons and rat striatal slices. The expression of heteromers in striatum was confirmed by robust labeling using in situ proximity ligation assays. The results indicate that AT1 receptors are expressed in striatum and form heteromers with dopamine D2 receptors that enable drugs selective for the AT1 receptor to alter the functional response of D2 receptors.

CB1 and GPR55 receptors are co-expressed and form heteromers in rat and monkey striatum.

Heteromerization of G-protein-coupled receptors is an important event as they integrate the actions of extracellular signals to give heteromer-selective ligand binding and signaling, opening new avenues in the development of potential drug targets in pharmacotherapy. The aim of the present paper was to check for cannabinoid CB1-GPR55 receptor heteromers in the central nervous system (CNS), specifically in striatum. First, a direct interaction was demonstrated in cells transfected with the cDNA for the human version of the receptors, using bioluminescence resonance energy transfer and in situ proximity ligation assays (PLA). In the heterologous system, a biochemical fingerprint consisting on cross-antagonism in ERK1/2 phosphorylation was detected. The cross-antagonism was also observed on GPR55-mediated NFAT activation. Direct identification of GPR55 receptors in striatum is here demonstrated in rat brain slices using a specific agonist. Moreover, the heteromer fingerprint was identified in these rat slices by ERK1/2 phosphorylation assays whereas PLA assays showed results consistent with receptor-receptor interactions in both caudate and putamen nuclei of a non-human primate. The results indicate not only that GPR55 is expressed in striatum but also that CB1 and GPR55 receptors form heteromers in this specific CNS region.

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's 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.

Sustained release of bioactive glycosylated glial cell-line derived neurotrophic factor from biodegradable polymeric microspheres.

Glial cell-line derived neurotrophic factor (GDNF), a potent neurotrophic factor for dopaminergic neurons, appeared as a promising candidate for treating Parkinson's disease. GDNF microencapsulation could ensure protection against degradation due to the fragile nature of the protein. Poly(lactide-co-glycolide) (PLGA) microparticles loaded with recombinant glycosylated GDNF obtained in a mammalian cell line were prepared by TROMS, a semi-industrial technique capable of encapsulating fragile molecules maintaining their native properties. The effects of several parameters as PLGA copolymer type, PEG 400 quantity co-encapsulated with GDNF or drug loading, on the properties of the particles were investigated. Microparticles showed a mean diameter between 8 and 30 microm, compatible with their stereotaxic implantation. The drug entrapment efficiency ranged from 50.6% to 100% depending on the microsphere composition. GDNF was better encapsulated using hydrophilic polymers with high molecular weight such as RG 503H. In vitro drug release was influenced by the polymer type as well as by the amount of PEG 400 co-encapsulated with GDNF. Microparticles prepared using PLGA RG 503H released 67% of the total protein content within 40 days. Moreover, very low concentrations of poly(vinyl alcohol) were detected after microparticles washing and freeze-drying. Finally, a PC-12 bioassay demonstrated that the in vitro GDNF released was bioactive.

Purification of bioactive glycosylated recombinant glial cell line-derived neurotrophic factor.

Glial cell line-derived neurotrophic factor (GDNF) neuroprotective effect on dopaminergic neurons has been described in vitro and in vivo, turning up as a promising drug for the treatment of Parkinson's disease. Unglycosylated bacteria-obtained GDNF has already been successfully delivered for a long period of time through an infusion pump directly to the putamen of Parkinsonian patients. Nevertheless, improved distribution and safety issues need to be solved and alternative strategies to long-term delivery seem necessary. The use of glycosylated GDNF could eliminate some safety concerns regarding the presence of antibodies against exogenous unglycosylated GDNF used for the treatment. Therefore, we have chosen a mammalian expression system as a source of glycosylated GDNF. In the present work, we describe the purification of recombinant rat GDNF from the culture media of baby hamster kidney (BHK) cells through several purification steps. Highly pure N-glycosylated recombinant GDNF has been obtained similar to the endogenous protein. Furthermore, the purified protein is biologically active when tested its ability to induce PC12 neurite outgrowth.

Terapias neuroprotectoras y neurorestauradoras en el tratamiento de la enfermedad de Parkinson / Neuroprotective and neurorestorative therapies for the treatment of Parkinson’s disease

La enfermedad de Parkinson es la segunda enfermedad neurodegenerativa más común después del Alzheimer. Actualmente se dispone únicamente de terapias sintomáticas que, aunque son muy eficaces en las primeras etapas de la enfermedad poseen a largo plazo considerables efectos secundarios. La terapia ideal sería aquella que permitiese frenar o detener la progresión de la enfermedad. Este es el caso de las terapias neuroprotectoras y neurorestauradoras. De entre todas ellas, la terapia celular y la terapia con factores tróficos tipo GDNF son las que mayores expectativas han generado en la comunidad científica. Aunque ya se ha planteado el uso de GDNF para el tratamiento de la enfermedad de Parkinson, es necesario buscar nuevas estrategias que permitan administrar dicho factor neurotrófico en las zonas concretas del cerebro donde vaya a ejercer su acción. Aquí se discute el uso de micropartículas como el sistema más apropiado para la administración de dicho factor neurotrófico

Parkinson´s disease is the second most common neurodegenerative disorder after Alzheimer´s disease. Current therapies are symptomatic and, although these therapies are efficacious during the early stages of the disease, they present important side effects when they are used for a long time. The ideal therapy would be the one that would slow down or stop the progression of the disease. This can be achieved, for instance, with neuroprotective and neurorestorative therapies. Among them, cell therapy and therapy with trophic factors such as glial cell line derived neurotrophic factor (GDNF) are the most challenging and promising ones for the scientific community. Although the use of GDNF as a treatment for Parkinson ´s disease was proposed several years ago, it is necessary to develop alternative strategies to deliver GDNF appropriately to concrete areas of the brain. Here, the use of microspheres as the most suitable tool for the administration of this neurotrophic factor is discussed

Consequences of unilateral nigrostriatal denervation on the thalamostriatal pathway in rats.

The position of the caudal intralaminar nuclei within basal ganglia circuitry has largely been neglected in most studies dealing with basal ganglia function. During the past few years, there has been a growing body of evidence suggesting that the thalamic parafascicular nucleus in rodents (PF) exerts a multifaceted modulation of basal ganglia nuclei, at different levels. Our aim was to study the activity of the thalamostriatal pathway in rats with unilateral dopaminergic depletion. The experimental approach comprised first unilateral delivery of 6-OHDA in the medial forebrain bundle. Thirty days post-lesioning, animals showing a clear asymmetry were then subjected to bilateral injection of Fluoro-Gold (FG) within the striatum. Subsequently, expression of the mRNA encoding the vesicular glutamate transporter 2 (vGLUT2) was detected within thalamostriatal-projecting neurons (FG-labeled) by in situ hybridization and the results were confirmed by laser-guided capture microdissection microscopy followed by real-time PCR. The data showed that there was a marked neuronal loss restricted to PF neurons projecting to the dopamine-depleted striatum. Moreover, PF neurons innervating the dopamine-depleted striatum were intensely hyperactive. These neurons showed a marked increase on the expression of vGLUT2 mRNA as well as for the mRNA encoding the subunit I of cytochrome oxidase as compared with those neurons projecting to the striatum with normal dopamine content. Thus, the selective neurodegeneration of PF neurons innervating the striatum together with the increased activity of the thalamostriatal pathway coexist after nigrostriatal denervation.

Expresión y purificación de GDNF para su microencapsulación y aplicación en la enfermedad de Parkinson / Expression and Purification of GDNF for its microencapsulation in drug delivery systems and application in Parkinson’s disease

La enfermedad de Parkinosn (EP) es un proceso neurodegenerativodel sistema nervioso central que afecta a las neuronasde dopamina de la sustancia negra, núcleo mesoencefálico delcontrol motor. La perdida en el cerebro de este neurotransmisorvital causa los síntomas de la enfermedad. La EP afectaactualmente a 200 de cada 100.000 personas y a 2 de cada100 entre los mayores de 60 años. En España hay unos110.000 enfermos. Además, hoy por hoy no se conoce nadaque pueda prevenir o curar la enfermedad, ni existe ningunaprueba de laboratorio que permita diagnosticarla. Recentiementese ha demostrado que el GDNF, factor neurotrófico derivadode las células gliales, es capaz de proteger las neuronasdopaminérgicas e incluso inducir la regeneración del tejidodopaminérgico dañado in vivo.El objetivo del trabajo fue diseñar y desarrolar un método deexpresión y purificación de GDNF bioactivo para su posteriormicroencapsulación y aplicación en la enfermedad de Parkison.El sistema escogido para expresar el GDNF fue el sistema decélulas eucariotas de mamífero. El vector utilizado para la produccióndel GDNF en células eucariotas fue el pDEST26 (TecnologíaGateway de Invitrogen). Como sistema de expresión deGDNF se utilizaron las líneas celulares eucariota BHK, 293 yCOS 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). Latransfección se realizó con Lipofectamine Plus (Invitrogen). Seanalizó la expresión de GDNF a nivel de mRNA mediante PCR ya nivel de proteína mediante Western Blot del medio condicionado. Los clones positivos se crecieron en botellas de cultivode 850 cm2 (Corning) y se realizaron ciclos de recolección delmedio. Cada ciclo fue analizado por SDS-PAGE y Western Blot.Para evaluar la actividad de la proteína se ha desarrollado unensayo de actividad en el que se demuestra la diferenciaciónmorfológica de células PC-12 inducida por GDNF. La presenciade los receptores GFRa1 y RET, necesarios para que el GDNFejerza su acción, fue determinada por PCR.Las conclusiones obtenidas de este estudio son la obtenciónde GDNF recombinante a partir de un sistema de expresión encélulas eucariotas, el desarrollo de un protocolo para su posteriorpurificación y la obtención de GDNF recombinante biológicamenteactivo

Parkinson’s disease (PD) is a slowly progressive neurodegenerativedisorder caused by decreased levels of dopamine in thesubstantia nigra, brain region responsible for movement. Thelack of dopamine is believe to be responsible of the symptomsof PD. Parkinson’s affects 200 in 100000 people and 2 inevery 100 persons over 60 years old. In Spain, there areabout 110000 people with PD. There is no cure at this time,and there are no prevention techniques or therapies. Recently,it has been demonstrated that GDNF, a glial-derived neurotrophicfactor is able to protect the dopaminergic neurons of thesubstancia nigra and it can also induce regeneration of injuredneurons in the central nervous system in vivo.The aim of this work was to develop a procedure for theexpression and purification of bioactive GDNF in view of itsmicroencapsulation for the treatment of PD.The cDNA of the GDNF gene was cloned in the expressionvector pDEST26 (Invitrogen) using the Gateway® Technology.Several eukaryotic mammalian cell lines (BHK, COS-7, and293) were stably transfected with the construction with LipofectaminePlus (Invitrogen). In those clones in which the presenceof the mRNA of the GDNF was confirmed by PCR studies,the expression of the recombinant protein in the serumfree medium was analyzed by Western Blot studies. The highestGDNF-producing clone, obtained from the BHK cell line,was cultured in 850 cm2 roller bottles (Corning) alternatingthe presence or the absence of FBS in the medium every 24hours, being collected only the serum free medium for theproduction of the recombinant GDNF. Each cycle protein expression was analyzed by SDS-PAGE and by Western Blot.The secreted protein was purified by several chromatographysteps. After each step of the purification procedure the fractionsobtained were analyzed by SDS-PAGE, Western Blotand silver staining analysis. A neuronal differentiation PC-12cell-based bioassay was also developed to confirm the biologicalactivity of the purified recombinant protein. Previously,the presence of GFRa1 and RET, receptors required forGDNF activity were confirmed by PCR.In conclusion, recombinant GDNF was produced in an eukaryoticmammalian cell line-based system. The protein purificationprocedure developed, allowed to obtain a highly purifiedrecombinant GDNF. Furthermore, the recombinant protein isbioactive

[Neuroprotective and neurorestorative therapies for the treatment of Parkinson's disease]. / Terapias neuroprotectoras y neurorestauradoras en el tratamiento de la enfermedad de Parkinson.

Parkinson's disease is the second most common neurodegenerative disorder after Alzheimer's disease. Current therapies are symptomatic and, although these therapies are efficacious during the early stages of the disease, they present important side effects when they are used for a long time. The ideal therapy would be the one that would slow down or stop the progression of the disease. This can be achieved, for instance, with neuroprotective and neurorestorative therapies. Among them, cell therapy and therapy with trophic factors such as glial cell line derived neurotrophic factor (GDNF) are the most challenging and promising ones for the scientific community. Although the use of GDNF as a treatment for Parkinson s disease was proposed several years ago, it is necessary to develop alternative strategies to deliver GDNF appropriately to concrete areas of the brain. Here, the use of microspheres as the most suitable tool for the administration of this neurotrophic factor is discussed.

The origin of motor fluctuations in Parkinson's disease: importance of dopaminergic innervation and basal ganglia circuits.

The severity of dopamine depletion and the consequent pathophysiologic changes that occur in basal ganglia circuits determine the severity of parkinsonian signs. Restoring the dopamine deficit or the downstream physiologic abnormalities improves Parkinson's Disease (PD) main motor features and as a result, attenuates the short-duration response (SDR). Therefore, both the magnitude and duration of the motor response are a function of the degree of motor severity, which is primarily governed by the loss of tonic dopaminergic activity and disruption of basal ganglia homeostatic mechanisms among which the STN-GPe/GPi circuits play a fundamental role. As neurodegeneration advances, standard levodopa administration give rises to wider oscillations in striatal dopamine availability and "pulsatile" stimulation of striatal dopamine receptors becomes predominant. This induces molecular and physiologic changes that further accentuate and aggravate the SDR that sustains motor fluctuations. Treatments capable of providing and restoring more tonic and physiologic dopaminergic stimulation may avoid many of these abnormalities and lead to better clinical outcomes.

["Neurorestorative" cell therapy in Parkinson's disease: an unresolved debate]. / Terapia celular "neuro-restauradora" en la enfermedad de Parkinson: un debate pendiente.

At present there is great enthusiasm over the perspectives deriving from so-called cell therapy in Parkinson's disease. This enthusiasm has spread beyond the ambit of the medical community, reaching the general public, and has been fuelled by a considerable ethical and political debate, sidestepping the need for a really scientific analysis of the real qualities and limitations of treatment with stem-cells in neurodegenerative diseases. Parkinson's disease is frequently observed from a simplistic perspective, as a mere neurodegeneration of the nigrostriatal dopaminergic pathway. This viewpoint encompasses different designs that tend to replace the lack of dopamine in the striatum through the use of different types of cell therapy. In this respect, it is important to indicate, on the one hand, the multisystemic and generalised nature of the disease and, on the other, the progressive character of the neurodegenerative process of Parkinson's disease. With this approach, to claim that the mere replacement of striatal dopamine through replacement cell therapy can correct the generalised and progressive character of the disease is a fanciful aspiration, which can only contribute to generating unfounded expectations in the general public. This article attempts to set out from a purely scientific point of view the doubts over the expectations created by these new therapeutic designs.

A sequential protocol combining dual neuroanatomical tract-tracing with the visualization of local circuit neurons within the striatum.

We describe here an experimental approach designed to aid in the identification of complex brain circuits within the rat corpus striatum. Our aim was to characterize in a single section (i) striatal thalamic afferents, (ii) striatopallidal projection neurons and (iii) striatal local circuit interneurons. To this end, we have combined anterograde tracing using biotinylated dextran amine and retrograde neuroanatomical tracing with Fluoro-Gold. This dual tracing protocol was further implemented with the visualization of different subpopulations of striatal interneurons. The subsequent use of three different peroxidase substrates enabled us to unequivocally detect structures that were labeled within a three-color paradigm.

Striatal input from the ventrobasal complex of the rat thalamus.

We have analyzed whether caudal regions of the caudate putamen receive direct projections from thalamic sensory relay nuclei such as the ventrobasal complex. To this aim, the delivery of the retrograde neuroanatomical tracer Fluoro-Gold into the caudal caudate putamen resulted in the appearance of retrogradely labeled neurons in the ventral posteromedial and ventral posterolateral thalamic nuclei. These projections were further confirmed with injections of the anterograde tracers biotinylated dextran amine or Phaseolus vulgaris leucoagglutinin into these thalamic nuclei, by showing the existence of axonal terminal fields located in the caudal striatum. These results support the existence of direct projections linking the thalamic ventrobasal complex and the caudal striatum in the rat, probably via collateralization of thalamocortical axons when passing through the caudate putamen, and therefore supporting the putative involvement of the caudal striatum in sensory-related functions.

Complex brain circuits studied via simultaneous and permanent detection of three transported neuroanatomical tracers in the same histological section.

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.

Pathophysiology of the basal ganglia in Parkinson's disease.

Insight into the organization of the basal ganglia in the normal, parkinsonian and L-dopa-induced dyskinesia states is critical for the development of newer and more effective therapies for Parkinson's disease. We believe that the basal ganglia can no longer be thought of as a unidirectional linear system that transfers information based solely on a firing-rate code. Rather, we propose that the basal ganglia is a highly organized network, with operational characteristics that simulate a non-linear dynamic system.

Considerations on the thalamostriatal system with some functional implications.

The thalamostriatal projections are largely neglected in current reviews dealing with basal ganglia function. In the past few years, however, several studies have re-evaluated these projections and have postulated their implication in more complex tasks within the basal ganglia organization. In this review, we try to focus on the morphological and functional importance of this system in the basal ganglia of the rat, cat and monkey. Special attention is paid to the thalamus as an important place for interaction between the input and the output systems of the basal ganglia through the thalamostriatal projections. Thus, we stress on the overlapping thalamic territories between the thalamic projection of the output nuclei of the basal ganglia and the thalamostriatal neurons. Our experimental data support the existence of several thalamic feedback circuits within the basal ganglia functional design. Finally, some considerations are provided upon the functional significance of these thalamic feedback circuits in the overall organization of the basal ganglia in health and disease.

Current concepts in neuroanatomical tracing.

The development of new axonal tract tracing and cell labelling methods has revolutionised neurobiology in the last 30 years. The aim of this review is to consider some of the key methods of neuroanatomical tracing that are currently in use and have proved invaluable in charting the complex interconnections of the central nervous system. The review begins with a short overview of the most frequently used tracers, including enzymes, peptides, biocytin, latex beads, plant lectins and the ever-increasing number of fluorescent dyes. This is followed by a more detailed consideration of both well established and more recently introduced neuroanatomical tracing methods. Technical aspects of the application, uptake mechanisms, intracellular transport of tracers, and the problems of subsequent signal detection, are also discussed. The methods that are presented and discussed in detail include: (1) anterograde and retrograde neuroanatomical labelling with fluorescent dyes in vivo, (2) labelling of post mortem tissue, (3) developmental studies, (4) transcellular tracing (phagocytosis-dependent staining of glial cells), (5) electrophysiological mapping combined with neuronal tract tracing, and (6) simultaneous detection of more than one axonal tracer. (7) Versatile protocols for three-colour labelling have been developed to study complex patterns of connections. It is envisaged that this review will be used to guide the readers in their selection of the most appropriate techniques to apply to their own particular area of interest.

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.