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.

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.

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.

Thalamic interaction between the input and the output systems of the basal ganglia.

The striatal return through the thalamus is largely neglected in current studies dealing with basal ganglia function, and its role within this circuitry remains obscure. In this contribution the thalamus is regarded as an important place of interaction between the input and the output organization of the basal ganglia. In support of this idea, a brief overview is provided of some of the most recent findings concerning the thalamus in relation to the basal ganglia circuitry. In particular, we have focused on the thalamostriatal projections themselves, on the output of the basal ganglia to the thalamus and also on the overlapping territories between the thalamic projection of the output nuclei and the thalamostriatal neurons. These data support the existence of several thalamic feedback circuits within the basal ganglia neural system. Finally, some considerations are provided upon the functional significance of these thalamic feedback circuits in the overall organization of the basal ganglia.

[New neuronal tracers and their combined use]. / Nuevos trazadores neuronales y su utilización combinada.

Neuroanatomical tract-tracing methods are powerful tools for the study of brain circuits. The use of axonal tracers has become very popular during the past few years. Tract-tracing allows us to study the way in which two or more brain areas are connected and can be used to obtain detailed data on the processing of information within a particular area. The recent development of protocols combining several tracers has resulted in an important breakthrough. Although technically very demanding, these multitracer procedures have become state of the art protocols in several laboratories, rendering a broad range of possibilities for their application in Neurobiology.

[Pedunculopontine tegmental nucleus. Anatomy, functional considerations and physiopathological implications]. / El núcleo tegmental pedunculopontino. Anatomía, consideraciones funcionales e implicaciones fisiopatológicas.

Pedunculopontine tegmental nucleus is formed by an ensemble of cholinergic and non-cholinergic neurons located in the caudal pontomesencephalic tegmentum, surrounding the superior cerebellar peduncle. It is an integral part of the reticulate formation of the brain stem, with extensive anatomical connections and highly varied functions. By means of ascendant projections that it sends to the thalamus, it intervenes in the waking-sleep cycle. Besides, it constitutes the most caudal nucleus of the neuroaxis, receiving connections from the basal ganglia, for which reason it has attracted the interest of those researchers concerned with the study of these structures. Thanks to its reciprocal connections with the basal ganglia, as well as to its descending projections to different structures of the pons, medulla and spinal cord; it has been related to the control of locomotion. Recently, it has also been considered as a possible centre for the integration of the motor information provided by the dorsal striatum with the motivational or limbic information proceeding from the ventral striatum, to permit its direct access to bulbar or spinal motor centres. In this work we will review its anatomical and functional characteristics, as well as its implication in some diseases of the nervous system such as narcolepsy, progressive supranuclear paralysis, schizophrenia and Parkinson's disease.

Multiple axonal tracing: simultaneous detection of three tracers in the same section.

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.

[Preliminary results of the study of neuronal death and the expression of bcl-2 protein in Alzheimer's disease]. / Resultados preliminares del estudio de la muerte neuronal y de la expresión de la proteína bcl-2 en la enfermedad de Alzheimer.

Alzheimer's disease (AD) is a degenerative central nervous system disorder where beside the histopathologic features of senile plaques and neurofibrillary tangles there is an important neuronal loss. It has been suggested that this neuronal death occurs via an apoptotic mechanism. Recognition of apoptotic cells is possible by an in situ end-labeling technique which identify the 3'-OH termini of DNA strands breaks through the incorporation of labeled nucleotides with the enzyme terminal-deoxinucleotidyl transferase (Tdt). We have applied this technique and high densities of apoptotic cells were found in 5 AD brains compared to 5 age-matched normal samples. We studied by immunohistochemical analyses the expression of the antiapoptotic protein bcl-2. We have not found neuronal bcl-2 immunoreactivity and we found an increased expression of bcl-2 by astrocytes compared to controls, this fact may aid glial survival or may have a deleterious effect on neuronal viability.