DAT immunoreactive neuronal bodies and processes in the cerebellar cortex
In the cerebellar cortex the DAT immunoreactivity were detected in neuronal cell bodies and processes distributed in the molecular layer (ML), in the Purkinje neuron layer (PL), in the granular layer (GL) and in the subjacent withe matter (WM) (Fig. 1).
Molecular layer
In the ML were observed DAT immunonegative stellate neurons (Figs. 1, 2A-E), and, a diffuse DAT immunoreativity in few perikarya of basket neurons localized in the deep zone of the layer or at the border with the Purkinje neuron layer was occasionally detected (Figs. 2A, 2B). A strong DAT immunoreactivity in form of densely packed granular deposits, in the cytoplasm in variously oriented primary and secondary dendritic trunks of the Purkinje neurons was observed (Figs. 2C, 2D). Moreover, intensely DAT immunoreactive puncta (referable to axon terminals) were localized in the close relationship with the wall of the microvessels (Fig. 2E). In the ML fine and diffuse DAT immunoreactive puncta (referable to sectioned dendritic or axon processes, or axon terminals) were also widely distributed within the neuropil (Figs. 2C-E).
Purkinje neuron layer
A great number of the Purkinje neuron cell bodies presented a DAT immunoreactivity in form of grains dispersed within the perikarya (Figs. 1, 2A, 2C, 3A, 3B); in particular, the DAT immunoreactivity in form of grains within the primary dendritic trunks that extend into the ML were also detected (Figs. 1, 2A, 3A), and only occasionally DAT immunonegative Purkinje neurons were observed, and their cell bodies profile was surrounded by DAT immunoreactive puncta (Fig. 3B).
Granular layer
In the protoplasmic spaces of Held, where they are localized the synaptic glomeruli complex, DAT immunoreactive small and finely grains were localized among granules (Figs. 2A, 3B). Moreover, DAT immunoreactive clusters of puncta referred such as terminals of mossy fibers at cerebellar glomeruli complex were also observed (Figs. 4B). In addition, within the GL among the numerous immunonegative granules, few intensely DAT immunoreactive cell bodies of granules were also observed (Figs. 2A, 4B).
In addition, DAT immunoreactivity was also observed in a non-traditional large neuron type, it was identified on the basis of the localization, cell body position, cell body shape and size, spatial arrangement of neuronal processes as the synarmotic neuron [For further details: 41, 43, 44, 56, 57] (Fig. 4A).
Subcortical white matter
In the subcortical WM, numerous intensely DAT immunoreactive nerve fibers variously oriented, often in continuity with the positive processes located in the overlying GL were observed (Figs. 1, 2A, 4B).
DAT immunoreactive neuronal cell bodies and processes of the dentate nucleus
Numerous DAT immunoreactive neuronal cell bodies have been observed within the dentate nucleus (DN) and at the bordering to the nearby WM (Figs. 5, 6A, 6B); the DAT immunoreactive neurons on the basis of their morphological features were principally identified and subdivided in small, medium and large neuron types which respectively displayed a main diameter ranging of 6 to 9 μm, 18 to 25 μm and 18 to 40 μm [For further details see: 50-54].
The DAT immunoreactive small neuron types were scattered thorough within the DN (Figs. 6A, 6B, 7A, 7B), while, the DAT immunoreactive and immunonegative medium neuron types were preferentially localized in the intermediate zone of the DN (Figs. 5, 6A, 6B); in addition, both DAT immunoreactive neuron types showed in the cell bodies and processes an intense positivity in form of densely packed granular deposits.
In the DN, numerous DAT immunoreactive large neuron types characterized by a different distribution pattern, cell body shape and spatial organization of processes, were observed (Figs. 5, 6A, 6B, 7A, 7B). A first large neuron type showing a roundish cell body, was predominantly distributed in the internal zone of the DN (Figs. 5, 6A, 6B); a second large neuron type characterized by an ellipsoidal cell body was concentrated at the boundary of the DN (Figs. 6A, 6B); a third large neuron type presenting a large ellipsoidal cell body was localized in the external zone of the DN, characteristically from the internal pole of the cell body give rise a thickness dendritic trunk (Figs. 6A, 6B); a fourth large neuron type showed an elongated fusiform cell body were distributed within the DN (Figs. 6A, 6B). In addition, a large neuron type characterized by the cell body and neuronal processes in close relationship with the wall of microvessels was also observed (Figs. 7A, 7B) [For further details see: 41, 43, 44, 55, 57]. Moreover, in the neighboring white matter of the DN clusters of intensely DAT immunoreactive puncta was observed (referable to sectioned nerve fibers) (Figs. 5, 6A, 6B).
DRD2 immunoreactive neuronal bodies and processes in the cerebellar cortex
In the cerebellar cortex the DRD2 immunoreactivity was observed in neuronal cell bodies and processes distributed in all the three layers of the cerebellar cortex (Figs. 8A, 8B) and in the underlying WM (Fig. 11E).
Molecular layer
In the ML the DRD2 immunoreactivity were detected in neuronal bodies and processes distributed throughout the layer (Fig. 8A). The DRD2 immunoreactivity were observed in stellate neurons characterized by polygonal or spheroidal cell bodies distributed in the external zone of the layer (Figs. 9A, 9B), and in basket neurons with fusiform or roundish cell bodies localized in the internal zone of the layer (Figs. 8B, 9A). In stellate and basket neurons the DRD2 immunoreactivity with a different degree of intensity, in form of densely packed granular deposits were observed in the perikaryon and in the cytoplasm of the proximal part of the processes (Figs. 8B, 9A, 9B). In addition, a strong DRD2 immunoreactivity was also detected in the cytoplasm of the Purkinje neurons primary, secondary dendritic trunks and in their distal ramifications (Figs. 9A, 12A). In the neuropil of the layer a DRD2 immunoreactivity in form of diffuse and fine puncta (referable to sectioned dendrites or axon processes, or axon terminals) were also observed (Figs. 9A, 9B, 12A).
Purkinje neuron layer
The DRD2 immunoreactivity were detected in the cell bodies of numerous Purkinje neurons (Figs. 8B, 9A, 10, 11A, 12A). In the perikarya of the Purkinje neurons an intense and diffuse DRD2 immunoreactivity in form of fine granular densely packed deposits was detected; the same DRD2 immunoreactivity was also observed in the cytoplasm of the primary dendritic trunks which ascends into the ML (Figs. 9A, 10, 11A, 12A). In addition, immunonegative Purkinje neurons with a cell bodies profile surrounded by DRD2 positive puncta were also observed (Fig. 12B).
Granular layer
In the GL the DRD2 immunoreactivity was detected in cell bodies and processes of different neuron types, which include traditional neuron types of the layer, as the granules and the Golgi neuron (Figs. 10, 11A, 11B, 11E, 12A), and some non-traditional large neuron types of the GL (Figs. 10A, 11, 12A-D, 13A-C, 14A, 14B). The DRD2 immunoreactive granules which present an intense and compact immunoreactivity, were detected in the external zone of the GL, beneath the cell bodies of the Purkinje neurons (Fig. 10) or in the internal zone of the layer at the boundary with the subjacent WM (Fig. 13E). The cell body of the Golgi neuron were localized in the external zone of the layer presented a strong and diffuse DRD2 immunoreactivity in the perikaryon and in the axon-like process (Figs. 11A, 11B, 12A). The DRD2 immunoreactivity were also detected in some non-traditional large neuron types, they were identified through accurate morphological analysis of their position in the zones of the GL, cell body shape and size, orientation, spatial arrangement of the processes [for details see: 41, 43, 44, 55-57]. The non-traditional large neuron types identified were the Lugaro neuron (Figs. 12A, 13A), the candelabrum neuron (Fig. 13B), the triangular neuron (Fig. 14A), the ellipsoidal neuron (Fig. 13D), the globular neuron (Fig. 14B) and the perivascular neuron (Fig. 13C). In addition, within the GL were observed a DRD2 immunoreactive neuron type characterized by spheroidal or ovoidal cell body, which displayed a main diameter ranging from 12 to 20 μm and presented a compact and heavy and DRD2 immunoreactivity. The axon-like process originates from the cell body and is observable for part of its course (Figs. 11B, 11C). Fibers characterized by a DRD2 moderate immunoreactivity variously oriented, throughout the layer, were observed (Fig. 11D). In addition, a DRD2 immunoreactivity in form of small clusters and finely grains were localized within the space among granules in the protoplasmic spaces of Held, the sites in which are localized the synaptic glomeruli complexes (Figs. 8B, 12B). Here, were also detected DRD2 immunoreactive clusters of puncta, corresponding to the axon terminals of mossy fibers at cerebellar glomeruli (Fig. 11D).
Subcortical white matter
In the underlying white matter, moderate DRD2 immunoreactive nerve fibers were observed (Fig. 13E).
DRD2 immunoreactive neuronal bodies and processes of the dentate nucleus
Numerous DRD2 immunoreactive neuronal elements were observed within the DN and in the neighboring WM (Fig. 15); DRD2 immunoreactive neurons on the basis of their morphological parameters were classified as small, medium and large neuron types that respectively displayed a main diameter ranging of 6 to 9 μm, 18 to 25 μm and 18-35 μm [For further details see: 50-54]. The DRD2 immunoreactive small neuron types were scattered through the nucleus, they presented an intense immunoreactivity in form of densely packed granular deposits (Figs. 15, 16A). The DRD2 immunoreactive medium neuron types were localized in the external and intermediate zones of the nucleus, their perikaryon presented a diffuse DRD2 immunoreactivity in form of fine granules (Figs. 15, 16A, 16B).
Moreover, in the DN different DRD2 immunoreactive large neurons types distinguished by distribution pattern, cell body shape and spatial organization of processes were observed (Figs. 16A, 16B). A large neuron type characterized by an ellipsoidal cell body was detected at the boundary of the DN (Figs. 15, 16A). A second large neuron type characterized by a large ellipsoidal cell body and a third large neuron type with an elongated fusiform cell body were both localized throughout within the DN (Figs. 15, 16A, 16B). Furthermore, in the neuropil of the DN and in the neighboring WM clusters of intensely DRD2 immunoreactive puncta (referable to sectioned dendrites, axons, axon terminals, or nerve fibers) were observed (Figs. 15, 16A, 16B).