Ultrastructural and metabolic changes in the neuropeptide Y-containing striatal neuronal network after thermocoagulatory cortical lesion in adult rat.

This study examined the effects of unilateral thermocoagulatory cortical lesion on the pattern of neuropeptide Y immunostaining in the rat ipsilateral striatum at 4 and 21 days post-lesion. Light microscopic analysis showed a significant increase in the number of neuropeptide Y-positive neurons vs. control at both time points; paradoxically, the intraneuronal level of labelling significantly decreased at 4 days post-lesion but increased at 21 days post-lesion. Ultrastructural analysis in control condition showed a higher proportion of dendritic versus axonal labelled processes (3.5 ratio); all the neuropeptide Y synaptic terminals formed symmetrical contacts, mostly onto unlabelled dendrites. At 4 days post-lesion, the neuropeptide Y-positive axon density dramatically increased (+576%) without significant change in the labelled dendrite density, vs. control values; the density of neuropeptide Y synaptic terminals increased in parallel by 233%. In addition, a significant proportion of large neuropeptide Y boutons forming asymmetrical synapses onto unlabelled spines were observed. At 21 days post-lesion, densities of neuropeptide Y dendrites, axons, and synaptic terminals increased by 68, 246 and 125%, respectively, vs. control. But, the morphological features of the neuropeptide Y axonal processes and synaptic specializations of the boutons were similar to those observed in control condition. These data (1) raise an important issue regarding the origin of the terminals forming asymmetrical synapses in the striatum, (2) suggest that adaptative changes in the neuropeptide Y neuronal network may be a main component of striatal remodelling resulting from the progressive loss of cortical inputs, and (3) reinforce the view that neuropeptide Y and excitatory amino acid functions may be tightly linked in the striatum.

Relationships between striatin-containing neurons and cortical or thalamic afferent fibres in the rat striatum. An ultrastructural study by dual labelling.

Striatin, a recently isolated rat brain calmodulin-binding protein belonging to the WD-repeat protein family, is thought to be part of a calcium signal transduction pathway presumably specific to excitatory synapses, at least in the striatum. This study was aimed to specify the cellular and subcellular localization of striatin, and to determine the possible synaptic relationships between the two main excitatory afferent pathways, arising from the cerebral cortex and the thalamus, and the striatin-containing elements, in the rat striatum. Anterograde tract-tracing by means of biotinylated dextran amine injection in the frontoparietal cerebral cortex or the parafascicular nucleus of the thalamus was combined with immunogold detection of striatin. Striatin-immunoreactivity was confined to the neuronal somatodendritic compartment, including spines. Whereas 90-95% of the striatal neurons were striatin-positive, only about 50% of the sections of dendritic spines engaged in asymmetrical synaptic contacts exhibited striatin labelling. Among the sections of striatin-immunopositive dendritic spines, the number of immunogold particles ranged from one to more than seven, indicating an heterogeneity of the spine labelling. Moreover, within each class of spines presenting at least two silver-gold particles, the distribution of the particles varied from a clear-cut alignment under the postsynaptic densities (24-33% of spines) to a location distant from the synaptic area. In the cell bodies and dendrites, striatin labelling was usually not associated with the cytoplasmic membrane nor with the postsynaptic densities. In the striatum ipsilateral to the tracer injections, only 34.8% of the synaptic contacts formed by corticostriatal afferents involved striatin-positive elements (slightly labelled dendritic spines), whereas 56.7% of the synaptic contacts formed by thalamostriatal boutons were made on striatin-positive targets (mostly dendrites). In both cases, striatin labelling was usually not associated with the postsynaptic density. Most of the immunoreactive dendritic spines were in contact with unidentified afferents. These data reveal that striatin is expressed in the vast majority of the cell bodies of striatal spiny neurons, but is heterogeneously distributed among the dendritic spines of those neurons. Data also indicate a preferential relationship between striatin-containing structures and afferents from the parafascicular thalamic nucleus with respect to the frontoparietal cerebral cortex. But, at the dendritic spine level, striatin may be involved in signal transduction mechanisms involving as yet unidentified excitatory afferents to striatal neurons.

Striatal neuropeptide Y neurones are not a target for thalamic afferent fibres.

This study examined at the ultrastructural level the putative relationships between afferent fibres coming from the parafascicular nucleus of the thalamus and neuropeptide Y (NPY)-containing neurones in the rat striatum. Experiments used a combination of anterograde transport of the biotin dextran amine to label the thalamo-striatal pathway and immunogold labelling to reveal the NPY-containing neurones at the electron microscopic level. Examination of sections from three animals failed to demonstrate thalamic terminals in synaptic contact with NPY-immunoreactive dendrites or cell bodies, although both types of labelled elements were frequently involved in synaptic complex with unlabelled profiles. These results strongly suggest that striatal NPY interneurones are not under the direct influence of the main component of the thalamo-striatal system.

Emergence of a synaptic neuronal network within primary striatal cultures seeded in serum-free medium.

In order to investigate the basic cellular mechanisms involved in neuronal interactions within the striatum, we prepared a primary striatal cell culture from rat fetal brain in chemically defined medium. Using morphological and whole-cell recording methods, we observed that an intensive neuritic elongation with a progressive build up of a sodium-dependent electrogenesis occurred during the first week of culture. Morphologically mature synapses began to develop after 10 days in vitro. By this time, most of the neurons (82 +/- 9%) received spontaneously synaptic potentials, which led them to fire (71 +/- 11%). The spontaneous firing was prevented by cadmium (200 microM) and tetrodotoxin (5 microM), which suggested that a Ca(2+)-dependent release of neurotransmitters was involved in the synaptic activation. We further obtained evidence that GABA, and to a lesser extent acetylcholine, contributed to these spontaneous synaptic potentials. At 15 days in vitro, it was possible to observe up to four synaptic contacts on a given dendrite. By this time, whole-cell recordings performed on pairs of neurons showed that the mature neurons were interconnected by excitatory synapses. As the number of synapses increased, the striatal neurons gradually formed a large network in which spontaneous activity developed, which tended to be organized into synchronized bursting patterns.

Ultrastructural analysis of graft-to-host connections, with special reference to dopamine-neuropeptide Y interactions in the rat striatum, after transplantation of fetal mesencephalon cells.

In a previous study we demonstrated that grafted dopamine (DA) neurons are able to induce an early and widespread normalization of DA-neuropeptide Y (NPY) interactions in the host striatum previously deprived of its DA input. Since similar recoveries were found to occur in striatal areas densely or poorly reinnervated by the graft, the question was raised as to what mechanisms (synaptic or volumic release) were involved in these functional effects. Ultrastructural analysis of graft-to-host relationships was performed using single--and double--immunolabelling techniques to detect neurons containing tyrosine hydroxylase (TH) and NPY, with a view to analysing the early establishment of synaptic connectivity in various areas of the host striatum. Within 1 month of the grafting, TH-immunoreactive (TH-IR) neurons showed most of the normal intrinsic morphological features characteristic of adult rat neurons and were found to have established direct relationships with various striatal neuronal populations. TH-NPY relationships were observed only in the area most densely reinnervated by the graft, and their relative frequency was found to be roughly the same as that determined in the intact striatum. Three months after the grafting, this percentage decreased, probably owing to the further elongation in TH-IR axons resulting in a wider distribution of the TH-NPY associations over the host striatum. In the zones distal from the graft, the reinnervation was far from complete and the few TH-IR fibres projected only to some unlabelled elements, mainly of the spiny type, which have been shown to interact normally with both DA afferents and NPY cells and therefore may relay the DA action over the whole striatum on the NPY population. It can be concluded from these data that the rapid and extensive functional normalization of the TH-NPY interactions previously found to occur in the entire striatum may depend on the restoration of direct and indirect synaptic relationships. A diffuse action of DA through non-synaptic mechanisms may also account for the fact that the amine has access to broader striatal populations than to those presumably reached by DA fibres arising from the graft.

Ultrastructural features of the choline acetyltransferase-containing neurons and relationships with nigral dopaminergic and cortical afferent pathways in the rat striatum.

The aim of this study was first to specify the morphology and neuronal environment of the large cholinergic neurons, and second to determine the distribution and mode of termination of the corticostriatal and dopaminergic inputs on these neurons in the rat striatum. Immunocytochemical procedures with a monoclonal antibody against choline acetyltransferase, Golgi staining and standard electron microscopic techniques were used to specify the ultrastructural features of the putatively cholinergic classical large neurons. The large/choline acetyltransferase-positive neurons are characterized by a voluminous, eccentric, and deeply indented nucleus leaving a large cytoplasmic area, and by the presence of an abundant granular endoplasmic reticulum and of many polysomes and free ribosomes. Serial ultrathin sectioning further indicated the presence of nematosomes or nucleolus-like bodies within the nucleus and the cytoplasm of the large neurons. In addition, these neurons were found to be in direct apposition with up to four surrounding neurons showing features typical of medium-sized spiny neurons. These data support the view that the putatively cholinergic neurons may have an intense metabolic activity and may be involved in striatal clusters. When choline acetyltransferase immunostaining was coupled with the identification of degenerating corticostriatal afferents after lesion of the cerebral cortex, degenerating terminals were seen to form synapses of an asymmetrical type on distal labelled dendrites, but these contacts were very rare. On the other hand, nigrostriatal dopaminergic axons, identified by means of either the degeneration method or tyrosine hydroxylase immunostaining, were often found to run directly for long distances around the choline acetyltransferase-positive cell bodies. Occasionally, dopaminergic terminals formed possible symmetrical synapses on choline acetyltransferase-positive cell bodies or proximal dendrites. These data provide evidence that the putatively cholinergic neurons are directly contacted by corticostriatal and dopaminergic nigrostriatal afferents. The respective positions and nature of the two types of contacts further provide morphological support for the hypothesis that postsynaptic interactions may occur between the corticostriatal and dopaminergic nigrostriatal afferents at the level of the cholinergic neurons.

Ultrastructural relationships between choline acetyltransferase- and neuropeptide y-containing neurons in the rat striatum.

The relationships between cholinergic and neuropeptide Y-containing neuronal systems in the rat striatum were examined using a dual immunoperoxidase labelling method. These neurons were identified by their immunoreactivity to choline acetyltransferase and neuropeptide Y, respectively, and were visualized on the same sections using 3,3'-diaminobenzidine and benzidine dihydrochloride as distinct chromogens under two conditions: (i) neuropeptide Y detection by the 3,3'-diaminobenzidine diffuse brown reaction product and choline acetyltransferase detection by the benzidine dihydrochloride blue, granular reaction product; (ii) choline acetyltransferase detection by 3,3'-diaminobenzidine and neuropeptide Y detection by benzidine dihydrochloride. Although both neuropeptide Y- and choline acetyltransferase-immunoreactive cell bodies were simultaneously detected and were easily distinguishable whatever the conditions used, neuropeptide Y- and choline acetyltransferase-immunoreactive dendrites and axons could not be visualized on the same sections, since only the diaminobenzidine-labelled processes were detectable. Light microscopic observations on sections dual labelled with either method confirmed that choline acetyltransferase and neuropeptide Y immunoreactivities were localized in morphologically different populations of striatal neurons scattered throughout the striatum, choline acetyltransferase immunoreactivity being associated with large neurons and neuropeptide Y immunoreactivity with medium-sized neurons. In addition, the choline acetyltransferase-immunoreactive neurons were found to be more numerous than the neuropeptide Y-immunoreactive neurons and to be prevalent in the dorsolateral areas of the striatum, whereas neuropeptide Y-immunoreactive neurons were preferentially found in the ventromedial areas of this structure. Electron microscopic observations on sections processed under either condition revealed that choline acetyltransferase-positive terminals form synaptic contacts of the symmetrical type with neuropeptide Y-positive somata and proximal dendrites and that choline acetyltransferase-positive neurons are contacted by neuropeptide Y-positive terminals. These data show that the striatal neuropeptide Y- and choline acetyltransferase-containing neuronal systems have reciprocal synaptic interactions and provide morphological support for the hypothesis that striatal cholinergic and neuropeptide Y interneuron activities may be functionally linked.

Striatal NPY-Containing Neurons Receive GABAergic Afferents and may also Contain GABA: An Electron Microscopic Study in the Rat.

Dual labelling methods were applied to localize simultaneously neuropeptide Y (NPY) and glutamate decarboxylase (GAD) immunoreactivities on ultrathin sections of the rat caudate-putamen (CP). By means of a double peroxidase-anti-peroxidase technique, using 3,3'-diaminobenzidine and benzidine dihydrochloride as chromogens in animals with no colchicine pretreatment, GAD immunoreactivity was found to be present in terminals only whereas NPY immunoreactivity was detected in neurons displaying the features of aspiny type cells and processes. With this approach, we observed numerous synaptic associations of the symmetrical type between GAD-immunoreactive (-Ir) axonal boutons and NPY-Ir cell bodies and dendrites. By combining immunoperoxidase and radioimmunocytochemical labelling in animals pretreated with colchicine, NPY was again detected in a single population of aspiny type neurons whereas GAD immunoreactivity was observed in neurons which could be classified as aspiny and spiny on the basis of their ultrastructural characteristics. All the cells of the aspiny type displaying clear-cut NPY immunoreactivity were also found to be GAD-positive. Some other neurons of both the aspiny and the spiny type were found to be immunoreactive to GAD alone. GAD/NPY dually labelled terminals were also observed and some axo-axonic appositions between GAD- and NPY-Ir terminals were also detected. All in all, these data show that NPY aspiny type neurons of the rat CP receive GABAergic afferents and provide morphological support for two hypotheses: that NPY is co-localized with GABA in some cell bodies, dendrites and axons, and that presynaptic interactions may occur between NPY and GABAergic neuronal systems.

Ultrastructural correlates of functional relationships between nigral dopaminergic or cortical afferent fibers and neuropeptide Y-containing neurons in the rat striatum.

This study examines the ultrastructural relationships established by the nigrostriatal dopaminergic and the corticostriatal afferent fibers with neuropeptide Y (NPY)-containing neurons in the rat striatum. By means of dual immunolabeling procedures using peroxidase conjugated F(ab) fragments and 125I-labeled protein A, direct appositions and morphologically defined synaptic contacts of the symmetrical type were visualized between tyrosine hydroxylase-labeled nerve terminals and NPY-labeled neurons. After deafferentation of the striatum from its cortical input direct appositions and asymmetrical synaptic contacts were evidenced between characteristic degenerative boutons and NPY-positive neurons in the striatum. These results suggest that striatal NPY interneurons undergo direct influence from both nigrostriatal dopaminergic and corticostriatal neuronal systems.

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.

Region-specific neuro-astroglial interactions: ultrastructural study of the in vitro expression of neuronal polarity.

Mesencephalic neurons were cultured for 2 days on mesencephalic or striatal astrocyte monolayers. The morphology of these neurons was studied in electron microscopy. The number of dendritic profiles was higher on mesencephalic astrocytes (homotopic neuro-astroglial co-cultures) than on striatal astrocytes (heterotopic co-cultures). This increase in the number of dendrites correlated with a more mature aspect of the neurons. Striatal neurons were also cultured on the astrocytic monolayers. The state of maturation of these neurons was more advanced, and the number of their dendrites was higher on striatal than on mesencephalic astrocytes. These results confirm and extend the fact that neuronal maturation and dendritic growth can be regulated through region-specific neuro-astroglial interactions (Denis-Donini et al., 1984; Chamak et al., 1987).

Two simian virus 40 (SV40)-transformed cell lines from the mouse striatum and mesencephalon presenting astrocytic characters. III. A light and electron microscopic study.

In two preceding papers we described the cloning of two astrocytic cell lines by simian virus 40 (SV40) transformation of embryonic mouse mesencephalon (F7-Mes) and striatum (F12-Str). The characterization of these lines as belonging to the astrocytic lineage is based on pharmacological, immunocytochemical and physiological data. Here we present quantitative and qualitative data on the morphological aspects of these two astrocytic clones observed under light and electron microscopy. We show that the clones present ultrastructural characters reminiscent of the morphology of young astrocytes. On one hand, they are rather similar to primary astrocytes in culture; on the other, they differ both from a clonal fibroblastic cell line (BT2) and from embryonic mouse fibroblasts in primary culture. These astroblastic clones display 4 morphologically different cell populations which we called types I, II, III and IV. Types II and III are very similar and represent the most predominant cells; their morphologies strongly remind of that of astroblasts. Type I corresponds to glioblasts and does not account for more than 15-20% of the total population. Type IV, which is very similar to differentiated velamentous astrocytes, normally represent ca. 5% of the cells. However, when the transformed cells are treated with mitomycin or mitomycin + dibutyryl cyclic AMP (dbcAMP), the proportion of type IV cells increases very much (up to more than 50% of the cells) while types I, II and III become less numerous. Morphological analysis therefore confirms that the two cell lines derived from the SV40 transformation of 14-day-old embryonic mesencephalic and striatal cells belong to the astrocytic lineage. Moreover, it seems that they can differentiate in vitro in cell culture conditions either spontaneously or under the action of pharmacological treatments known to enhance normal astrocyte maturation.

A combined light and electron microscopic method for the visualization of the same in vitro neuron by radioautography and serial sections.

We report here on a technical improvement which makes it possible to study, at the ultrastructural level, a dopaminergic neuron which has been previously identified by light microscopy. Primary cultures of virtually pure mesencephalic neurons from mouse embryos were obtained. These cultures were kept for 6 days, then incubated with tritiated dopamine, fixed and embedded in Epon. The dopaminergic neurons were firstly visualized by radioautography directly through Epon blocks in toto by light microscopy. In a second step, ultrathin sections of the identified dopaminergic cells were prepared and the neurons observed at the electron microscopy level. The dopaminergic nature of these neurons was regularly checked by radioautographic control on some selected ultrathin sections.

The axon of the giant neuron in the rat neostriatum.

The axon of the giant cells in the rat neostriatum was studied by serial sections on Golgi and electron microscopic preparations and with the combined Golgi-electron microscopic technique (Fairen, Peters and Saldanha, 1977). The ultrastructural characteristics of the initial part are given. It was established that the axon was coated with myelin and gives off myelinated collaterals. The present observations are discussed with regard to the problem of striato-cortical connections.

[Mechanism of the appearance of nuclear inclusions with microfilaments and microtubules in sympathetic neurons. Influence of dibutyryl-adenosine 3'-5' monophosphate and theophylline]. / Recherches sur le mécanisme d'apparition des inclusions nucleaires à microfilaments et à microtubules des neurones sympathiques. Influence du dibutyryl-adénosine 3'-5' monophosphate cyclique et de la théophylline

As we have shown previously the nucleus of sympathetic neurons contains microtubules and microfilaments giving rise to highly organized inclusions. Present in vivo experiments on cat stellate ganglia have shown that dibutyryl cyclic AMP and theophyllin induce an increase of their frequency (up to 8.5 fold). These and earlier observations lead us to conclude that cyclic AMP mediates the process of nuclear microtubules and microfilaments assembly from preformed protein subunits.