Cell Morphology And Electrical Responses In Local Circuitry Of The Valvula In Mormyrid Cerebellum

Comparative anatomists have been interested in the cerebellum of mormyrid electric fish for more than 150 years because of its extraordinary size and unusual crystalline regularity of its histological structure. Actually, the extraordinary size of the mormyrid cerebellum gives these fishes a brain-to-body ratio that is higher than that of humans. Besides its unusually regular histology, two morphological features of the mormyrid cerebellum make this structure an attractive site for studying the functional circuitry of the cerebellum in general. One such feature is the clear spatial separation of climbing fiber and parallel fiber inputs to Purkinje cells, and the other feature is that the axons of most Purkinje cells, like that in the cerebellum of other ray-finned fishes known as Actinoptegii, do not leave the cerebellar cortex but instead terminate locally on nearby efferent neurons which do project out of the cerebellar cortex. The efferent cells of mormyrids are equivalent to the deep nuclei cells in cerebellum of mammals. Besides these anatomical features, the mormyrid cerebellum is also of interest for studying cerebellar function because of its involvement in the active electrosensory system, a special function, mostly being attributed to the part of valvula in cerebellum, that helps mormyrids to detect their environment and to communicate with others.There are three main parts in the mormyrid cerebellum, which are usually referred to as the caudal cerebellar lobe, the central cerebellar lobes (C1, C2, C3, and C4), and the valvula. The part known as the valvula in mormyrids is composed of a series of small ridges and covers the external surface of the brain, with anterior portion being folded back on itself. The central cerebellar lobes and the caudal cerebellar lobe lie beneath the valvula. Although the central lobes of the mormyrid cerebellum have been well studied previously with different techniques, the valvula has not been studied extensively with only a few Golgi observations by some investigators and the local circuitry for this structure is still uncompleted. The goal of the present study, therefore, was to reveal the detailed histology of the neuronal elements of the valvula and to establish a functional circuitry for this structure, and the observations were carried out in such a combined way: biocytin or neurobiocytin was injected intracellularly in different cells in valvula in in vitro slices for morphological examination, and responses were recorded by stimulating related afferent fibers or by injecting depolarization current into cells with either voltage clamp or current clamp modes; in some experiments, anterograde tracers were placed extracellularly in the inferior olive to labeling ascending climbing fibers in the valvula. Examples of the labeling were drawn and reconstructed using a camera lucid system or photographed with a confocal laser scanning microscope. The basic morphological and physiological features of different cell types and fibers obtained in these observations were summarized as follows:Purkinje cells. The cell bodies of Purkinje cells (5～15μm in diameter) are located in the ganglionic layer. Both the characteristic palisade dendrites and axons of Purkinje cells are oriented in the horizontal plane. The spiny dendritic trees of Purkinje cells show a striking reduction from the rostral portion (164～269 um) to the caudal portion (41～72 um). The thin axons of Purkinje cells arise from the soma and branches in the ganglionic layer. The axonal arbors extend 129±40μm in the horizontal plane and are minimal in the transverse plane. A few spines or thorns were observed in the initial part of the axon, and en passant boutons were present along the axons.Purkinje cells in the valvula discharged at least with two types of spikes to intracellular depolarization under voltage clamping: a small narrow spike and a large broad spike. The broad spike was used to distinguish Purkinje cell from other cell types during recordings because it could be observed only in Purkinje cells. Parallel fiber stimulation evoked graded EPSPs or EPSCs, whereas climbing fiber stimulation evoked large all-or-none EPSPs or EPSCs.Basal efferent cells. The large cell bodies (10～15μm in diameter) of basal efferent cells restrict exclusively within a small area at the base of the ridge between ganglionic and granular layers. They are arranged in a single row meandering continuously throughout each side of the ridge. Their dendrites are smooth and most of them carry small swellings. The dendritic trees extend 158±26μm (n=14) in the transverse plane, with a preference toward the top of the ridge due to the location of their somas, 120±17μm (n=9) in the horizontal plane. The thick axons of these cells enter the basal bundle and project out of the valvula without local collaterals.The basal efferent cells showed only one type of electrical response upon intracellular depolarization: a large, narrow spike with a prominent afterhyperpolarization. Parallel fiber stimulation evoked EPSCs or EPSPs that showed moderate but clear paired pulse facilitation.Type II efferent cells. The large cell bodies (10～15μm in diameter) of type II efferent cells are usually and randomly distributed in the ganglionic layer, except for the basal area of the ridge. Their smooth dendritic trees extend both horizontally and transversely in the molecular layer, with or without swelling. The major morphological feature of type II efferent cells is that their axons include one long and thick out-going axon with several local axon collaterals.Electrophysiologically, type II efferent cells showed similar features as basal efferent cells did, exhibiting a large narrow spike by intracellular current injection and graded EPSPs or EPSCs by parallel fiber stimulation.Stellate cells. The stellate cells have small cell bodies (6～8μm in diameter) in gangionic and molecular layers whereas with smooth and fine dendrites in the molecular layer. The axon arbors extend entensively in ganglionic and molecular layers. The axons of stellate cell have branches passing in the transverse plane toward the apex or base of the ridge.Stellate cell also showed only repetitive discharges responding to intracellular current injection and EPSPs responding to parallel fiber stimulation.Vertical cells. The dendritic tree of vertical cells span the entire height of the ridge. The thicker axon extend toward the base of the ridge, where it enters the basal bundle.Vertical cell also showed single type of large narrow spike. It fired repetitively to intracellular current injection.Golgi cells and Golgi-like cells. The Golgi cell has dendrites extending longitudinally in the molecular layer and axon arbors in the granular layer. Four cells examined have similar dendiritc arbors, but their axon arbors extend in both granular and ganglionic layers. These cells are named Golgi-like cells. Both cell types showed a large, narrow spikes repetitively responding to the intracellular current injection and EPSPs to the stimulation of parallel fibers in the molecular layer.Parallel fibers. Both anterograde labeled parallel fibers and retrograde labeled granule cells show that the axons of granule cells ascend and enter the molecular layer, then extend transversely toward the apex of the ridge without bifurcation. The axons of granule cells project exclusively to the molecular layer that is above the cell bodies.Climbing fibers. Anterograde labeling shows that the climbing fibers in the valvula are confined in the ganglionic layer and strongly oriented in the horizontal plane.In summary, the present study provides following new findings: 1) Axonal arbors of Purkinje cells are oriented in the horizontal plane; 2) Dendritic trees of Purkinje cells show a remarkable reduction from the rostral to the caudal portions of the valvula; 3) Type II efferent cells have local axon collaterals; 4) Axonal arbor of Golgi-like cells terminate in both granular and the ganglionic layers, some of them extend even into the deep molecular layer; 5) The origin of parallel fibers are confined in the granular layer underneath the molecular layer.These results together with those of previous studies indicate that the local circuitry of the valvula in the mormyrid cerebellum is similar to that in the mammalian cerebellum, but several unique morphological and physiological features of this circuitry suggest that this system can be used as a suitable model to study the function of the cerebellum in general.