J. Vuillet

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 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.

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.

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.

Neuropeptide Y Neurons in the Striatal Network. Functional Adaptive Responses to Impairment of Striatal Inputs

Neuropeptide Y (NPY), a 36 amino acid peptide enriched with tyrosine residues first isolated from porcine brain extracts (Tatemoto, 1982; Tatemoto et al, 1982), is thought to be the neuroactive member of the pancreatic polypeptide family (Di Maggio et al, 1985). It is one of the most abundant and widely distributed peptides isolated so far within the mammalian central nervous system and it shows a distinctive pattern of distribution as compared with other neuropeptides. The most salient feature of its distribution is the high concentration centred on the striatum, especially in the human brain (Adrian et al, 1983; Allen et al, 1983). Immunohistochemical studies have shown that NPY is present in numerous cell bodies and fibers in both the dorsal striatum or caudate putamen (CP) and the ventral striatum or nucleus accumbens (NAcc) (Adrian et al, 1983; Allen et al, 1983; De Quidt and Emson,1986; Smith et al, 1985). Within these structures and in related cortical areas NPY has been reported to coexist extensively with somatostatin and the enzyme nicotinamide adenine dinucleotide phosphatediaphorase (NADPH-D) (Chronwall et al, 1984; Gaspar et al, 1987; Kowall et al, 1987; Smith and Parent, 1986; Vincent et al, 1982; 1983). The presence of high levels of NPY in the basal ganglia nuclei has suggested that this peptide may play a fundamental role in the control of sensorimotor function and has led to detailed investigations on the morphological features and cellular relationships of the NPY neurons within these structures. The purpose of the present report is to briefly review data from biochemical and immunohistochemical studies in an attempt to clarify the anatomical and functional position of NPY elements in the striatal network (dorsal striatum).

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. Synapse 34:208–221, 1999.

A Dual Immunoperoxidase Labelling Method to Study the Ultrastructural Relationships between Choline Acetyltransferase- and Neuropeptide Y-Containing Neurons in the Rat Striatum

In the striatum, neuropeptide Y (NPY) and choline acetyltransferase (ChAT) are to be found in populations of aspiny neurons with distinct morphological features. Indeed, immunocytochemical studies using selective antibodies have indicated that NPY-labelled neurons are medium-sized type IV aspiny neurons (Vuillet et al., 1989b), whereas ChAT-labelled neurons belong mainly to the category of large neurons in the striatum (Bolam et al., 1984). Neurochemical studies in combination with lesion experiments and morphological studies have provided evidence that both NPY- and ChAT- containing neuronal populations represent interneurons, receive afferents from extrinsic origin (Chang, 1988; Vuillet et al., 1989a; 1989b), and provide synaptic input to medium-sized spiny neurons that are predominantly striatal output neurons (Izzo and Bolam., 1988; Vuillet et al, 1989b). These data suggest that both cholinergic and NPY interneurons in the striatum may play an integrative role which may be of particular relevance in the control of sensorimotor function. The question of whether or not synaptic interactions exist between those NPY and cholinergic interneurons then appears of great interest for further functional resolution of the striatal network. Two major difficulties emerge to answer this question: both types of neurons represent less than one percent of the total striatal neuron population; in addition, besides the paucity of NPY terminals, both ChAT and NPY axonal boutons form predominantly symmetrical type synapses that are difficult to characterize.

Further Investigations on the Mechanisms Involved in Intrastriatal Mesencephalic Grafts in the Rat, with Special Reference to Dopamine-Neuropeptide Y Interactions

A number of transplantation studies on an animal model for Parkinson’s disease bearing a 6-hydroxydopamine (6-OHDA) lesion of the nigrostriatal dopaminergic pathway were carried out since the late 70’s (Bjorklund and Stenevi, 1979, Perlow et al., 1979). Implanting foetal dopamine (DA) neurons into the striatum of these rats was found to alleviate some Parkinson’s disease like symptoms in the DA deficient recipients. In rats with unilateral lesion, motor impairments such as drug-induced rotational asymmetry were found to be completely abolished after DA grafts, while in more complex conditioned behavior tests, rats remained severely impaired (Bjorklund et al., 1987). Despite these limitations, human foetal ventral mesencephalic tissue has been implanted into the brain of Parkinsonian patients with variable success. Although there is some ambiguity in the statement that brain grafts can have functional effects, because it contains spontaneously active neurons and releases neurotransmitter, or because it may trigger some adaptive behavioral responses in the host animals, the cellular mechanisms whereby ventral mesencephalic transplants act need to be exactly determined for research on neural transplantation to be able to progress.