An Immunohistochemical Analysis on The Human Cerebellar Dopaminergic System

The cerebellum now it has not considered a dopaminergic region. Despite, is traditionally reported only the presence of dopaminergic afferents to the cerebellum. Currently, studies attribute to the cerebellum a critical role in motor and cognitive functions and suggest a cerebellar involvement in dopamine related neurologic and psychiatric disorders. In several studies has been demonstrated mainly in the cerebellum of rodents a widely distribution of all dopaminergic receptor subtypes (DRD 1 -DRD 5 ), a poor number of dopaminergic Purkinje neurons, and the presence of several dopaminergic neurons in the deep cerebellar nuclei. Data on an intrinsic dopaminergic neuronal system in the human cerebellum are lacking. presence neuronal


Abstract Background
The cerebellum now it has not considered a dopaminergic region. Despite, is traditionally reported only the presence of dopaminergic afferents to the cerebellum. Currently, studies attribute to the cerebellum a critical role in motor and cognitive functions and suggest a cerebellar involvement in dopamine related neurologic and psychiatric disorders.
In several studies has been demonstrated mainly in the cerebellum of rodents a widely distribution of all dopaminergic receptor subtypes (DRD 1 -DRD 5

Methods
The aim of the present study of chemical neuroanatomy was to investigate in human cerebellum on the presence of a dopaminergic neuronal system, through an immunohistochemical approach based on the use of speci c antibodies against the dopamine membrane transporter (DAT) and the dopamine receptor subtype 2 (DRD 2 ).

Results
The immunoreactions revealed the presence of DAT and DRD 2 positive neuronal cell bodies and processes of all the layers of the cerebellar cortex and in the dentate nucleus. These results are in agreement with previous studies, and suggest which the intrinsic cerebellar neuronal dopaminergic system may be involved in intrinsic and extrinsic (projective) cerebellar circuits.

Conclusions
This study open a new scenario on the interpretation of the cerebellar role in dopaminergic related brain disorders. Finally, this study may be an innovative critical element for the development of pharmacologic and non-pharmacologic therapeutic strategies for neurologic and psychiatric disorders related to dopamine.

Background
The cerebellum is traditionally not considered a dopaminergic area [1][2][3], in morphological studies has been detected only dopaminergic projection to the cerebellum, which presumably originating mainly by the ventral tegmental area (A 10 ) [4][5][6][7]. Although, the existence of an intrinsic cerebellar dopaminergic neuronal system in the human cerebellum is in part predictable. In the postmortem human cerebellum has been detected signi cant levels of dopamine (DA), homovanillic acid (HVA), the most catecholamine metabolite [8][9][10], tyrosine hydroxylase (TH), the rst enzyme involved in catecholamine biosynthesis, dopamine transporter (DAT), the membrane transporter involved in DA reuptake [11]. Positron emission tomography (PET) studies in monkey cerebellum and autoradiographic evaluations in human postmortem cerebellum has been detected the presence of selective dopamine transporter ligands (DAT-Ls) [12][13][14]. Moreover, in biochemical analysis has been revealed in rat and monkey cerebellum high levels of DA in the vermis lobules VII, VIII, IX, X, in the fastigial and dentate nuclei [11,[15][16][17].
In studies of chemical neuroanatomy on mammals cerebellar cortex, dopaminergic bers has been observed in the granular layer in form of mossy bers-like, fewer of them have been localized on the Purkinje neuron cell bodies and their dendritic arborizations, and only few bers were detected in the molecular layer [18,19]. In the adult rodents cerebellum by means of immunohistochemical, pharmacological, biochemical approaches has been demonstrated the presence of DAT immunoreactivity in Purkinje neurons and in several neurons of all deep cerebellar nuclei [20,21] and into cerebellar synaptosomes has been evidenced an active [3H]-DA transport and an endogenous release of DA [20,22]. Furthermore, in adult rodents cerebellum in the lobules I, VII, VIII, IX, X has been observed a poor number of Purkinje neurons immunoreactive to the vesicular monoamines transporter 2 (VMAT 2 ), the protein transporter that carries DA from cytosol to synaptic vesicles [23], TH [24] and to DAT [19][20][21][24][25][26]. Moreover, in clinical and experimental studies has been suggested a considerable role of the cerebellum in neurologic and psychiatric dopaminergic related disorders such as Parkinson's disease (PD) [27,28], ataxias (ATX) [21,30], schizophrenia (SCZ) [31][32][33], autism spectrum disorders (ASD) [33,34], bipolar disorders (BD) [32,35] and drug addiction (DGA) [36,37].
Despite these studies, the data on an intrinsic neuronal cerebellar dopaminergic system in non-primate mammals are still incomplete, and detailed data on the existence of a human cerebellar neuronal dopaminergic system are often neglected or denied. Therefore, the aim of this study of chemical neuroanatomy by means of an immunohistochemical approach using speci c polyclonal antibodies against DAT and dopamine receptor type 2 (DRD 2 ) is to evaluate the presence and the distribution of DAT and DRD 2 immunoreactive neuronal subpopulations in the human cerebellar cortex and in the dentate nucleus.

Methods
The study was carried out on human postmortem cerebellum samples taken in accordance with the guidelines of the 'Codice di Polizia Mortuaria' (art. 40  The samples of human cerebellum were obtained from 4 healthy subjects aged between 20 and 63 years, 2 male and 2 females; (for each sex, one young and one senior) removed maximum of 36-40h after death. The standards for subject inclusion was the absence of neurological and psychiatric diseases in their medical history, and the absence of brain abnormalities at routine macroscopic examination.
From each cerebellum, were taken samples which corresponding to the left dentate nucleus and to the following cerebellar lobules: tonsilla (anterior lobe, left hemisphere), superior semilunar lobule (posterior lobe; left hemisphere) and inferior semilunar lobule (posterior lobe; left hemisphere).

Preparation of histological sections
From each lobules of cerebellar cortex, were obtained 3 fragments of 25-55 mm 3 , which included the entire cerebellar cortex and part of the underlying white matter; from each dentate nucleus were taken fragments that include its total extension surface considering the transverse axis of the nucleus. The fragments were xed by immersion for 3h at 4°C in Zamboni's uid [38]. We chose a xative solution composed by 10% formaldehyde and a satured concentration of picric acid, because in our previous experimental procedures the same xative solution had shown to be optimal and useful for postmortem nervous tissue xation, and for immunohistochemical visualization of different neuroactive molecules (i.e. enzymes, neuropeptides, calcium binding proteins) [For details see: [39][40][41][42][43][44].
The fragments after the xation procedure, were washed and dehydrated in series of ethanol and incorporated in a semi-synthetic para n. The para n blocks were serially cut into 5μm sections, respectively oriented orthogonally to the long axis of the cerebellar cortex and to the major axis of the dentate nucleus. Before the immunostaining, randomly chosen sections from each fragment were stained with toluidine blue and submitted to light microscopic histopathological analysis to ascertain the normality of the nervous cerebellar tissue. In any case selected for the study, the microscopic structure of the cerebellum showed no pathological changes.
Immunohistochemistry for DAT and DRD 2 Brie y, from each sample series, 15 sections were selected at intervals of 150µm and subjected to immunohistochemistry for DAT or for DRD 2 , consecutive sections were stained to toluidine blue. The sections were immunolabeled according to the following steps: rehydratation in a descendent ethanol series; 2) immersion in 3% hydrogen peroxide solution, for 10 min at room temperature (RT) to inactive the endogenous peroxidase; 3) rinsing in PBS, pH 7.6, for 3×10 min at RT; 4) pre-incubation with donkey

Qualitative analyses
The distribution pattern of DAT and DRD 2 immunoreactivity were analysed in all the immunostained section of cerebellar cortex and dentate nucleus; the qualitative morphological identi cation of the different immunoreactive cerebellar neuron types was carried out considering the following parameters: localization, cell body position, shape and cell body size, neuronal processes spatial arrangement, or through the comparison with the nearby stained toluidine blue sections [for further on the morphological parameters details see: [41][42][43][44][50][51][52][53][54]. Microscopical analysis were performed with the light microscope Olympus Vanox-T and with the Spot Insight Color V3, Diagnostic Instruments Inc., USA.

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 ne and diffuse DAT immunoreactive puncta (referable to sectioned dendritic or axon processes, or axon terminals) were also widely distributed within the neuropil immunoreactivity was also detected in the cytoplasm of the Purkinje neurons primary, secondary dendritic trunks and in their distal rami cations (Figs. 9A, 12A). In the neuropil of the layer a DRD 2 immunoreactivity in form of diffuse and ne puncta (referable to sectioned dendrites or axon processes, or axon terminals) were also observed (Figs. 9A, 9B, 12A).

Purkinje neuron layer
The DRD 2 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 DRD 2 immunoreactivity in form of ne granular densely packed deposits was detected; the same DRD 2 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 pro le surrounded by DRD 2 positive puncta were also observed (Fig. 12B).

Granular layer
In the GL the DRD 2 immunoreactivity was detected in cell bodies and processes of different neuron types,  (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 DRD 2 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 DRD 2 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 DRD 2 moderate immunoreactivity variously oriented, throughout the layer, were observed (Fig. 11D). In addition, a DRD 2 immunoreactivity in form of small clusters and nely 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 DRD 2 immunoreactive clusters of puncta, corresponding to the axon terminals of mossy bers at cerebellar glomeruli (Fig. 11D).

Subcortical white matter
In the underlying white matter, moderate DRD 2 immunoreactive nerve bers were observed (Fig. 13E).

DRD 2 immunoreactive neuronal bodies and processes of the dentate nucleus
Numerous DRD 2 immunoreactive neuronal elements were observed within the DN and in the neighboring WM (Fig. 15); DRD 2 immunoreactive neurons on the basis of their morphological parameters were classi ed 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][51][52][53][54]. The DRD 2 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 DRD 2 immunoreactive medium neuron types were In addition, in the current study in the PN were observed DAT and DRD 2 immunoreactive cell bodies and processes of Purkinje neurons in the hemispheric lobules VII and IX, these ndings are in accordance to immunohistochemical studies which reported in the cerebellar lobules VI-X an inconstant presence of dopaminergic Purkinje neurons [19][20][21]26]. Furthermore, in the present study were also occasionally detected immunonegative Purkinje neurons, whose cell bodies pro le were surrounded by DAT and DRD 2 immunoreactive 'puncta' (referable to axon terminals). Moreover, in previous studies were detected the presence of lobule IX of VMAT 2 , TH and DAT immunoreactive axon terminals in close relationship to the cell bodies of the Purkinje neurons in the cerebellum of rodents and in non-human mammals primates cerebellum [19][20][21]. These last ndings, were in some ways in accordance to the previous demonstrations in the PN of extrinsic dopaminergic bers, a poor number of dopaminergic cell bodies and processes of Purkinje neurons localized in several cerebellar lobules, and by the presence of all the dopamine receptor subtypes (DRD 1 -DRD 5 ) mainly described in the layers of the cerebellar cortex [4, 5, 11, 19-21, 49, 60-62]. In addition, by means of a physiological and pharmacological combined approach has been demonstrated the presence of DAT immunoreactive Purkinje neurons and in which, DAT inhibitors, dopamine antagonists or DA exert a modulation of depolarization-induced slow currents (DISCs) [19]. Therefore, it is possible to hypothesize an involvement of the immunoreactive DAT and DRD 2 Purkinje neurons in dopaminergic signaling mechanisms in the cortico-nuclear projective circuits of the cerebellar cortex.
In the GL, were observed DRD 2 immunoreactive Golgi neurons and DAT and DRD 2 immunoreactive granules. Moreover, in the GL were also observed different DRD 2 immunoreactive non-traditional large neuron types [41,44,55], distributed in three zones of the GL; such as Lugaro neurons, candelabrum neurons, perivascular neurons in the external zone, triangular neurons in the intermediate zone; ellipsoidal neurons and globular neurons in the internal zone [41,43,44,[55][56][57]. In the GL, were also detected the presence of DAT immunoreactive synarmotic neurons, the non-traditional large neuron type characteristically localized in the internal zone of the layer or in the subjacent withe matter of the folium, which could be involved in corticocerebellar and/or corticonuclear projective circuits [41,43,44,[55][56].
In addition, in the GL an intensely DRD 2 positivity were also observed in the cell body and processes of a neuron type has intermediate dimensions between the granules and the non-traditional large neuron types [41,44,55,56,64]. Although, this neuron type which we called 'big granule', was similar to the monodentric neuron [65] and to the unipolar brush neuron [66,67], it was mainly observed throughout within the GL of the cerebrocerebellum lobules, instead, studies on the distribution of the glutamatergic unipolar brush neuron demonstrated an it exclusively distribution in the vestibulocerebellum [67,68]. Though, this initial morphological datum on the big granule constitutes an element of novelty, it needs further future morphofunctional insights.
Furthermore, in the DN has been found a widely DAT and DRD 2 imunoreactivity in cell bodies and processes of different neuron types. In many cases, it was possible to recognize them, through the evaluation of morphological parameters, as the small neuron type involved in intrinsic circuits, the medium neuron type mainly involved in extrinsic circuits of the DN [49,50,[69][70][71][72], and at least 4 different large neuron types that include the central neuron, the border neuron, the intermediate asymmetrical In addition, the presence of DAT and DRD 2 immunoreactive ne puncta in the neuropil of the ML and in the space of Held of the GL, the sites of the cerebellar glomeruli complex and in the neuropil of the DN suggested a role of DA to the cerebellar mechanisms of synaptic plasticity [73,74]. may be through the dopamine and cAMP-regulated neuronal phosphoprotein (DARPP-32), widely expressed in the neurons of the cerebellum [75][76][77], that play a role in dopaminergic neuronal synaptic signaling [78].
In addition, studies attributed to the dorsal portion of DN a motor role and to the ventral portion an involvement in non-motor functions [79][80][81][82]. The ndings of dopaminergic projective neuron types in the both portions of the DN, may suggest the existence of dopaminergic interconnections between the DN and the midbrain dopaminergic nuclei chie y to the substantia nigra (A 9 ) and to the ventral tegmental area (A 10 ) and may also indicate the existence of direct and indirect dopaminergic interconnections to other basal ganglia nuclei, mainly to the subthalamic nucleus and the globus pallidus [83][84][85][86][87].
Moreover, the dopaminergic projective neuron types of the DN, may be involved in direct or indirect pathways to the regions of the limbic system such as the hypothalamus, amygdala and hippocampus [83, [85][86][87][88]. Furthermore, a recent study in mice suggested direct interconnections between the DN and the nucleus accumbens [89], this last data may evidenced a role of the dopaminergic projective neuron types of the DN in the modulation mechanisms of DA release in the mesolimbic system.
Moreover, the presence of dopaminergic perivascular neurons in the GL of the cerebellar cortex and in the DN, and presence of dopaminergic putative perivascular axon terminals, in close relationship to the wall of microvessels in the cerebellar cortex and in the DN of could represent the morphofunctional demonstration in the cerebellum of speci c dopaminergic neuronal elements involved in regulatory mechanisms of the blood ow and of the blood brain barrier [90][91][92][93][94]. Furthermore, we can also suggest which dopaminergic cerebellar perivascular neurons may be involved in the ampli cation of the extrasynaptic dopaminergic signaling (volume transmission) [95][96][97].
In conclusion, the nding of an intrinsic neuronal cerebellar dopaminergic system may provide the morphological basis of considerable interest for its applications in clinical neurology and psychiatry. In fact, we cannot exclude an involvement of the cerebellar dopaminergic neurons demonstrate in this study in DA related neurologic and psychiatric disorders such as PD, SCZ ASD, and DGA. Recently, studies demonstrate a role of the cerebellum in the pathophysiology of motor and non-motor symptoms of PD [118][119][120][121], and as a target for the non-pharmacological treatment of the side effects caused by traditional dopaminergic therapies [122,123]. Furthermore, studies suggest in SCZ and in ASD a role of the cerebellum, and in particular of the DN in the modulation of prefrontal circuitry involved in the dopaminergic release in the prefrontal cortex and in cognitive abnormalities [126][127][128] and suggest a role of the cerebellum in innovative therapeutic non-invasive approaches for SCZ [129] and for ASD [130].          Distribution pattern of the dopamine receptor type 2 (DRD2) immunoreactivity in the dentate nucleus gray matter and in the neighboring withe matter. (Scale bar: 40μm).