Abstract

Introduction

Five neuron types are commonly involved in cerebellar cortex microcircuitry, which include four associative interneurons and one projective neuron type.^1-6 These traditional neuron types include the inhibitory GABAergic stellate and basket neurons which map within the molecular layer,^7-9 the excitatory glutamatergic granule neuron, widely distributed within the granular layer,^10,11 the inhibitory GABAergic Golgi neuron, located within the external granular layer, and the inhibitory GABAergic projective Purkinje neuron, localized in the homonymous layer.^7-9 In addition, in a number of studies focusing on the cerebellar cortex granular layer, different neuron types were identified, apart from the granules and the Golgi neuron.^12-31 These cerebellar neuron types have been referred to as “non-traditional large neurons”, since they are not usually considered in the morphofunctional analysis of the cytoarchitectural structure of the cerebellar cortex, and volume of their cell bodies displayed a main diameter of 20-30 μm, in comparison with the one of granules cell bodies which only measures 5-8 μm.^16,17,21-23 Moreover, an excitatory glutamatergic neuron type should also be included among them, the unipolar brush neuron, also called monodendritic neuron, present only in the granular layer of the vestibulocerebellum, which plays a role in the intrinsic cerebellar cortex excitatory circuits.^13,19,27-29 Furthermore, at least nine inhibitory GABAergic large neuron types are distributed in three different zones of the layer and ubiquitously in the various cerebellar lobules, whose role in the cerebellar cortex circuitry is not completely known.^{16-18,21-23,30} In addition, among these large neuron types only the Lugaro neuron, the candelabrum neuron, the globular neuron and the perivascular neuron are involved in specific intrinsic GABAergic inhibitory circuitries of the cerebellar cortex.^{16-18,21-23,31} Against this backdrop, the synarmotic neuron must be considered differently, as it is the only inhibitory GABAergic non-traditional large neuron type to which a role in extrinsic (projective) cerebellar circuits is attributed.^{16,17,21-23,32-36}

In this minireview we analyze in detail the morphofunctional and neurochemical data on the synarmotic neuron, so far reported. Moreover, in this context, we evaluate the possible involvement of such neuron in an immune-mediated form of cerebellar ataxia.

Synarmotic neuron morphological data

Pioneering morphological studies on cerebellar cortex mentioned the presence, within the granular layer, of large neuron types among the traditional neuronal constituents, characterized by a long axon and extending in the subcortical white substance.^37-47

Passing through Nissl and Golgi staining studies, Landau⁴¹ first revealed the presence of a specific large neuron type, called synarmotic neuron, in the internal zone of the granular layer or in the subjacent white substance of the cerebellar cortex of several mammals including humans (Figures 1-3).^42-45 Morphological Golgi staining studies confirmed the presence of synarmotic neuron in the internal zone of the granular layer as well as in the subcortical white substance.^46,47 Since the first studies on the synarmotic neuron, it was tought to be implicated in cortico-cortical associative and in cortico-nuclear projective circuits, and recent studies confirmed the projective role of the synarmotic neurons.9,16,17,21-23,32-36,46,48,49

Even though the existence of a second projective neuron type in the cerebellar cortex attracted the interest of several neuroscientists^1,4,38-41,50 in most cases this aspect was neglected or not fully considered. Moreover, although in the last twenty-five years several morphological studies through different staining methods supported the presence of a second putative projective large neuron type in the internal zone of the cerebellar granular layer of the mammals including humans, the synarmotic neuron of Landau was not deeply mentioned.

Figure 1.

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The Anatomic Table by Eber Landau⁴⁴ on the synarmotic neuron in the human cerebellar cortex (modified by Paolo Flace, 2021). Table of transverse section of human cerebellar cortex according to the information provided by the Golgi and by Ramon y Cajal silver staining. Morphological structure of the cerebellar cortex: ML, molecular layer; PL, Purkinje neuron layer; GL, granular layer; WS, white substance. Cortico-cerebellar neurons present in the modified anatomic table: P, Purkinje neuron; G, Golgi neuron; S, synarmotic neuron. The cell bodies of different synarmotic neurons are distributed in the internal zone of the granular layer and in the subcortical white substance.

Figure 2.

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The Anatomic Table by Eber Landau⁴⁴ on the synarmotic neuron in the human cerebellar cortex (modified by Paolo Flace, 2021). Table of transverse section of human cerebellar cortex according to the information provided by the Golgi silver staining. Morphological structure of the cerebellar cortex: ML, molecular layer; PL, Purkinje neuron layer; GL, granular layer; WS, white substance. Cortico-cerebellar neurons present in the modified anatomic table: P, Purkinje neuron; G, Golgi neuron; S, synarmotic neuron. A synarmotic neuron cell body localized in the subcortical white substance interposed between two adjacent granular layers, its processes are distributed in both the granular layers.

Through a modified Golgi staining method, Braak and Braak¹² detected a large neuron type in the internal zone of the granular layer of the human cerebellar cortex, which displayed similar morphological features compared to the synarmotic neuron, and they included it in the type 2 of the classification.

In a study made by using the modified Ehrlich’s methylene blue supravital staining technique, the presence of a large neuron type with a stellate-shaped cell body and with a long axon descending into the subcortical white substance was observed in the granular layer of the mouse cerebellar cortex.⁴⁷

The synarmotic neuron exhibits a pear or ovoidal-shaped cell body, with a main diameter ranging from 20 to 25 µm, localized in the internal zone of the granular layer or just below the subcortical white substance (Figures 1-3; Table 1). Its dendritic tree arises from various sites of the cell body and it mainly develops in the internal zone of the granular layer, although, in some cases, the dendrites can run across and radiate within the white substance or direct their extensions into the two granular layers on both sides of the folium white substance (Figures 1-3; Table 1). The axon of the synarmotic neurons originates through the axon hillock from the cell body and runs for a long distance through the white substance, intermingled with efferent and afferent axons to the cerebellar cortex. Subsequently, the axon undertakes different types of growth: it can reenter in the cerebellar cortex so as to associate two adjacent cortico-cerebellar regions of the same folium or different folia; alternatively it may play a role in projective circuits towards the intrinsic cerebellar nuclei (Figures 1-3; Table 1). In relation to the synarmotic neuron afferents, few data are available: it may receive excitatory glutamatergic inputs from mossy fibers (Figures 1-3; Table 1),^{2,16,17,21-23,42-45,48} hence the synarmotic neuron dendrites may contribute to the formation of the cerebellar glomeruli, the synaptic complex of the granular layer.^{2,16,17,21-23,50,51} It is also likely that its dendrites may receive inhibitory GABAergic afferents from axonal collaterals from other non-traditional large neuron types (e.g., globular neuron; Table 1).^{16,17,21-23,52,53} Although, in studies on the cerebellar cortex of several mammals including humans, the presence of the synarmotic neuron has been demonstrated,^{9,12,16,17,21-23,32,33,35,36,42-49,52,54} at present time, only few studies have evaluated the quantitative data of the synarmotic neuron. In a quantitative analysis it has been suggested that the synarmotic neuron represents a rare variant,⁵² while, in another quantitative evaluation, the synarmotic neuron to equal half of the number of Golgi neurons was found.² In a study of chemical neuroanatomy on the inhibitory GABAergic non-traditional large neuron types in the granular layer of the human cerebellar cortex by using an antibody raised against the glutamic acid decarboxylase isoforms 65/67 (GAD_65/67), the GABA biosynthetic enzymes, it was evaluated that the 19.8% of the GAD_65/67 immunoreactive large neurons localized in the internal zone of the granular layer or in the subcortical white substance corresponds to the synarmotic neurons.¹⁶

Figure 3.

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The Anatomic Table by Eber Landau⁴² on the synarmotic neuron in the dog cerebellar cortex (modified by Paolo Flace, 2021). Table of transverse section of dog cerebellar cortex according to the information provided by the Golgi and by Ramon y Cajal silver staining. Morphological structure of the cerebellar cortex: ML, molecular layer; PL, Purkinje neuron layer; GL, granular layer; WS, white substance. Cortico-cerebellar neurons present in the modified anatomic table: S, synarmotic neuron. A synarmotic neuron cell body localized in the internal zone of the granular layer, its processes are distributed in two adjacent granular layers.

Figure 4.

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GAD_65/67 immunoreactive synarmotic neurons in the granular layer of the human cerebellar cortex. GAD_65/67 immunoreactive synarmotic neuron cell body (arrow) localized in the granular layer in close proximity to the subcortical white substance (WS), from the cell body originates a thin axon-like process. GAD_65/67 immunoreactive Purkinje neuron cell body and primary dendritic trunk (asterisk); magnification 40x; scale bar: 15 μm. Method: streptavidin-biotin-peroxidase immunoenzymatic technique; chromogen: 3-amino-9-ethyl-carbazole; type of antibody: GAD_65/67 rabbit polyclonal antibody; Chemicon International, Temecula, CA, USA. Negative control: the sections are covered with normal rabbit serum or with anti-GAD antiserum absorbed with an excess of synthetic GAD (Chemicon International) in place of the primary antibody (original unpublished micrographs; for protocol details, see¹⁶).

Neurochemical data on the synarmotic neuron

Only few morphofunctional data on the neurotransmitters and neuromodulators expressed by synarmotic neurons are available. In chemical neuroanatomy studies mainly performed on the human cerebellar cortex, the inhibitory GABAergic nature of a subpopulation of synarmotic neuron localized in the internal granular layer (Figure 4) or in the subcortical white matter has been elucidated (Figure 4; Table 2).^16,17,21-23

Still in developing as well as in the adult human cerebellar cortex, a subpopulation of calbindin D-28k, immunoreactive synarmotic neurons has been detected (Table 2).^48,51 Moreover, in the cerebellar cortex of dromedary camel, calretinin immunoreactive subpopulation of synarmotic neurons has been observed (Table 2).⁵⁴ In addition, the presence of different neuropeptidergic subpopulations of synarmotic neurons immunoreactive for vasoactive intestinal polypeptide and motilin (Table 2),^23,55,56 neurotensin and the neurotensin receptor type 1 (NTR₁) has been demonstrated (Table 2).^21-23,34,35 Moreover, recently, the monoaminergic nature of the synarmotic neuron has been also demonstrated. In several studies the presence of two distinct subpopulations of monoaminergic synarmotic neurons has been detected, respectively indolaminergic synarmotic neurons, serotonin (5-HT) immunoreactive (Figure 5; Table 2).^21-23,34,35 and dopaminergic synarmotic neurons, dopamine transporter (DAT) immunoreactive (Table 2).^{21-23,32-35,56-58} Ultimately, in the adult mouse cerebellar cortex, a subpopulation of synarmotic neurons is immunoreactive for Contactin-1, a cell adhesion glycoprotein involved during the cerebellar development in cellular proliferation and differentiation mechanisms, while, in the adult it plays a role in the regulation of trans-synaptic mechanisms (Figure 6; Table 2).^34,35,59-61

Figure 5.

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5-HT immunoreactive synarmotic neuron in the granular layer of the human cerebellar cortex. 5-HT immunoreactive synarmotic neuron cell body localized in the internal zone of the granular layer (arrow) among immunonegative granules, in proximity to the subcortical white substance (WS); magnification 40x; scale bar: 20 μm. Method: streptavidin-biotin-peroxidase immunoenzymatic technique; chromogen: 3,3′-Diaminobenzidine; type of antibody: 5-HT rabbit polyclonal antobody, Covance Lab., Inc., Princeton, NJ, USA. Positive control: fragments of rat duodenum and brainstem are subjected to the same experimental procedures (original unpublished micrographs; for protocol details, see²³).

Figure 6.

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Contactin-1 immunoreactive synarmotic neuron in the mouse cerebellar cortex. Contactin-1 immunoreactive synarmotic neuron cell body (arrow) localized in the internal zone of the granular layer, in close proximity to the subcortical white substance (WS). Contactin-1 immunoreactive Purkinje neuron cell body (asterisk); magnification 40x; scale bar: 20 μm. Method: streptavidin-biotin-peroxidase immunoenzymatic technique; chromogen: 3-amino-9-ethyl-carbazole; type of antibody: Contactin-1 rabbit polyclonal antibody (original unpublished micrographs; for protocol details, see⁵⁹).

Potential significance of synarmotic neuron in immune-mediated cerebellar ataxias

Recently, an immune-mediated cerebellar ataxias characterized by the presence of autoantibodies (Auto-Abs) against the GABA biosynthetic enzymatic isoform, glutamic acid decarboxylase isoform 65 (GAD₆₅-Auto-Abs),^62-67 has been described. In this form of cerebellar ataxia, the GAD₆₅-Auto-Abs^{62,63,65,68-74} cause a damage of the cerebellar cytoarchitecture and a dysregulation of the inhibitory signaling of the traditional GABAergic corticocerebellar neurons (e.g., stellate neurons, basket neurons, Purkinje neurons) involved in associative and in projective cerebellar inhibitory circuits.63,64,66,70-72

In this regard, a dysregulation of the inhibitory GABAergic synarmotic neurons circuitries (Figure 7),^16,17,21-23 could be involved in the impairment of the inhibitory GABAergic signaling implied in this GAD₆₅-Auto-Abs mediated form of cerebellar ataxia^{62,63,68,71,73} and could play a foundamental role in the symptomatic manifestations of this subtype of cerebellar ataxia.^62,63,68-73

Figure 7.

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GAD₆₅-auto-antibodies in the cerebellum. Scheme on the distribution of the autoantibodies against the GABA biosynthetic enzymatic isoform, glutamic acid decarboxylase isoform 65 (GAD₆₅-Auto-Abs) related to the GABAergic synaptic sites of the synarmotic neuron in the cerebellum. BN, basket neuron; PN, Purkinje neuron, SN, stellate neuron; GN, Golgi neuron; GrN, granule; Syn, synarmotic neuron; CNu, cerebellar nuclei; CB, climbing fibers; MF, mossy fibers.

Concluding remarks

This minireview supplies, for the first time, several morphofunctional aspects on the assumptive projective neuron type of the cerebellar cortex, the synarmotic neuron also called the Landau neuron,^41-46 one of the neglected inhibitory GABAergic non-traditional large neuron types of the cerebellar cortex.^16,17,21-23 In addition, the neurochemical data of the synarmotic neuron and its possible involvement in the impairment of GABAergic neurotransmission mechanisms of the anti-GAD₆₅-Auto-Abs immune-mediated cerebellar ataxia has been also analized.

Conclusively, in this minireview the presence of the subpopulation of synarmotic neurons in the mammal cerebellar cortex (including humans), has been established, and various neurochemical aspects have been also analyzed. However, yet further detailed research is needed for understanding data on the development of the synarmotic neuron, and on its functional role in neurotransmission mechanisms and in cerebellar disorders.

Acknowledgments

Funding Statement

Funding: this review was funded by Paolo Flace and Diana Galletta, with personal funds.

References