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Comparative Study
. 2008 Aug 10;509(5):449-73.
doi: 10.1002/cne.21735.

The neuronal organization of a unique cerebellar specialization: the valvula cerebelli of a mormyrid fish

Zhigang Shi  1 , Yueping Zhang, Johannes Meek, Jiantian Qiao, Victor Z Han
Affiliations
Comparative Study

The neuronal organization of a unique cerebellar specialization: the valvula cerebelli of a mormyrid fish

Zhigang Shi et al. J Comp Neurol. .

Abstract

The distal valvula cerebelli is the most prominent part of the mormyrid cerebellum. It is organized in ridges of ganglionic and molecular layers, oriented perpendicular to the granular layer. We have combined intracellular recording and labeling techniques to reveal the cellular morphology of the valvula ridges in slice preparations. We have also locally ejected tracer in slices and in intact animals to examine its input fibers. The palisade dendrites and fine axon arbors of Purkinje cells are oriented in the horizontal plane of the ridge. The dendrites of basal efferent cells and large central cells are confined to the molecular layer but are not planar. Basal efferent cell axons are thick and join the basal bundle leaving the cerebellum. Large central cell axons are also thick, and they traverse long distances in the transverse plane, with local collaterals in the ganglionic layer. Vertical cells and small central cells also have thick axons with local collaterals. The dendrites of Golgi cells are confined to the molecular layer, but their axon arbors are either confined to the granular layer or proliferate in both the granular and ganglionic layers. Dendrites of deep stellate cells are distributed in the molecular layer, with fine axon arbors in the ganglionic layer. Granule cell axons enter the molecular layer as parallel fibers without bifurcating. Climbing fibers run in the horizontal plane and terminate exclusively in the ganglionic layer. Our results confirm and extend previous studies and suggest a new concept of the circuitry of the mormyrid valvula cerebelli.

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Figures

Fig. 1
Fig. 1. Anatomical overview of mormyrid cerebellum
A. Dorsal view of the brain, showing that the entire brain is covered by the valvula. The smooth portions are the bottom of the valvular sheet folded up. The medio-lateral strips are the tops of the valvular ridges. B. Dorsal view of the brain with the valvula unfolded. Insertion shows transverse view of ridges. C. Sagittal view of the brain. All structures above the dashed line are cerebellum or cerebellar-like structures. Abbreviations: C1-4, central lobes C1-4 of the cerebellum; Cr, cerebellar crest; CLa, anterior caudal lobe; CLp, posterior caudal lobe; ELL, electrosensory lobe; ggl, ganglionic layer; gran, granule layer; LT, lobus transitorius of the cerebellum; mol, molecular layer; Va, valvula of the cerebellum. All figures are adapted with permission from Nieuwenhuys and Nicholson (1969a). Scale bars = 2 mm in A, B; = 1 mm in C.
Fig. 2
Fig. 2. Organization of the vauvular ridge
A. A schematic drawing of a valvular ridge, showing organization of cells and fibers in three dimensions. Insertion shows presumed connections among the different cell types. Figure is adapted with permission from Nieuwenhuys and Nicholson (1969a). B. Schematic drawings, showing the laminar organization of a valvular ridge in different planes, as defined in this study: a, transverse plane; b, sagittal plane; c, horizontal plane. C. Experimental arrangements. Schematic drawing of a Purkinje cell viewed in the horizontal plane. Purkinje cell axon is shown in blue, parallel fibers in green and climbing fiber in red. Stimulus electrodes are placed in the molecular layer (SM) and ganglionic layer (SG) to activate parallel fibers and climbing fibers, respectively. Abbreviations: B, basal cell; bb, basal bundle; cf, climbing fiber; dst, deep stellate cell; G, Golgi cell; ggl, ganglionic layer; gr, granule cell; gran, granular layer; mol, molecular layer; mf, mossy fiber; P, Purkinje cell; pf, parallel fiber; Rec, recording electrode; sc, stellate cell (molecular leyer); SG, stimulation of ganglionic layer; SM, stimulation of molecular layer; vc, vertical cell. Scale bar = 50 μm in C.
Fig. 3
Fig. 3. Morphology and physiology of Purkinje cells
A and B. Reconstructions of two Purkinje cells from horizontal (A) and transverse (B) sections, respectively. Note that the dendritic trees and axon arbors of Purkinje cells are complete in the horizontal plane but both were cut in the transverse plane. Also note that in this figure, as well as in the following figures, reconstructions were made from thin sections (40-50 μm thickness) after a DAB procedure. Insertions show the orientations of cells in the ridges. C. Typical responses of a Purkinje cell to a somatic voltage step (single trace): small narrow spikes and large broad spikes. D. Averaged EPSC response of a Purkinje cell to molecular layer stimulation (SM), showing paired-pulse facilitation. E. A Purkinje cell responds to ganglionic layer stimulation (SG) with large all-or-none EPSCs. The responses (overlay of single traces) show clear paired-pulse depression (ppd, black traces). The stimulus intensity was near the threshold, and if the first stimulus failed, the response to second stimulus was near the full size (gray trace). Abbreviations are the same as in Fig. 2. Scale bars = 10 μm in A, B.
Fig. 4
Fig. 4. Photomicrographs of Purkinje cells labeled with fluorescent dye
A. A Purkinje cell labeled in the horizontal plane. The soma, proximal dendrites and axon (ax) are confined to the ganglionic layer (ggl), and the palisade dendrites are confined to the molecular layer (mol). Note in this figure and following figures that fluorescent labeled cells were processed and photographed from 200 μm slices unless otherwise noted. B. Two Purkinje cells were labeled in a horizontal slice, with axons projecting in opposite directions in the ganglionic layer. C. A Purkinje cell labeled in a transverse plane. The dendritic tree is narrow and the axon was cut (arrow) during slicing. Nissl counterstaining is shown in green in this and the following figures. Abbreviations are the same as in Fig. 2. Scale bars = 20 μm in A, B; 50 μm in C. See magenta-green version of this figure in supplementary files.
Fig. 5
Fig. 5. Comparisons of dendritic tree sizes of Purkinje cells from different regions of the valvula
A-C. Representative cells reconstructed from horizontal sections, from the rostral (A), the middle (B) and the caudal (C) portions of the valvula, respectively. D. The proximal valvular regions from which the three groups of cells were sampled. Abbreviations are the same as in Fig. 2. Scale bars = 10 μm in A-C.
Fig. 6
Fig. 6. Morphology and physiology of basal efferent cells
A and B. Reconstructions of two basal efferent cells from horizontal (A) and transverse (B) sections, respectively. The axons of both cells are thick and have no collaterals. Insertions show cell orientations in the ridges. Note the symmetric (A) and asymmetric (B) dendritic trees. C. Response (single trace) of a basal efferent cell to a somatic current step of 200 pA. D. Averaged responses of a basal efferent cell to stimulation of molecular layer (SM). The EPSPs show a minimal paired-pulse potentiation. Abbreviations are the same as in Fig. 2. Scale bars = 10 μm in A, B.
Fig. 7
Fig. 7. Morphology and physiology of large central cells
A and B. Reconstructions of two large central cells in horizontal planes. Both cells have a thick axon that traverses long distances in this plane and gives off local collaterals which are largely confined to the ganglionic layer. C. Responses (single trace) of a large central cell to a somatic current step of 200 pA (top), and averaged responses to stimulation of molecular layer (SM, bottom). The EPSCs show moderate paired-pulse facilitation under voltage clamp. Abbreviations are the same as in Fig. 2. Scale bars = 50 μm in A, = 40 μm in B.
Fig. 8
Fig. 8. Photomicrograph of efferent cells labeled with fluorescent dye. Nissl counterstaining showed in green
A. A basal efferent cell labeled in a transverse slice, with a cut thick axon (ax). B. A basal efferent cell labeled in a sagittal slice, with a long thick axon (ax) in the granular layer. C. A basal efferent cell labeled in a horizontal slice, with a long thick in the ganglionic layer. D. A large central cell labeled in a horizontal slice, with a long thick axon and thin local collaterals in the ganglionic layer. E. A large central cell labeled in a transverse slice, with a cut thick axon and minimal local collaterals in the ganglionic layer. Abbreviations are the same as in Fig. 2. Scale bars = 50 μm in A, B, D; =25 μm in C, E. See magenta-green version of this figure in supplementary files.
Fig. 9
Fig. 9. Photomicrographs of dendritic arbors of different cell types. Cells were stained with DAB as chromagen in thin sections
A and B. A Purkinje cell, showing the typical palisade dendritic tree in the molecular layer (A) and spiny dendrites at higher magnification (B). C and D. A basal efferent cell, showing swellings along the dendrites in the molecular layer. E. A basal efferent cell, showing smooth dendritic arbors. Scale bars = 20 μm in A, C; 10 μm in B, D, E.
Fig. 10
Fig. 10. Photomicrographs of a vertical cell and a small central cell labeled with fluorescent dye
A. A vertical cell labeled in a slice that was cut obliquely. Its dendrites occupy much of the molecular layer (mol) of the ridge, and its axon arbors are largely confined to the ganglionic layer (ggl). B. A small central cell labeled in a sagittal slice. Its dendrites are largely confined to the ganglionic layer (ggl) where its thick axon with local collaterals are also located. C. Responses of a vertical cell (same cell as shown in A) to somatic current injection. Abbreviations are the same as in Fig. 2. Scale bars = 20 μm in A, B. See magenta-green version of this figure in supplementary files.
Fig. 11
Fig. 11. Morphology and physiology of a Golgi cell
A. A Golgi cell labeled with fluorescent dye in a sagittal slice, as shown schematically in the insertion. This cell has a large multipolar soma in the ganglionic layer (ggl). Its smooth and none-planer dendritic tree is confined to the molecular layer (mol), while its axon branches extensively in the granular layer (gran). B. A Golgi cell (same cell as shown in A) fired a single type of spikes in response to a somatic current injection of 200 pA (left, single trace), and responded to stimulation of the molecular layer (SM) with EPSCs under voltage clamp (right, average). The responses showed a moderate paired-pulse facilitation. Abbreviations are the same as in Fig. 2. Scale bars = 40 μm in A. See magenta-green version of this figure in supplementary files.
Fig. 12
Fig. 12. Morphology and physiology of a Golgi-like cell
A. A Golgi-like cell labeled with fluorescent dye in a transverse slice, as shown schematically in the insertion. The characteristic morphological feature of this cell is the way its axon arbors project into both the granular layer (gran) and the ganglionic layer (ggl). B. Similar to Golgi cell shown in Fig. 11, this Golgi-like cell fired a single type of spikes with adaptation in response to a somatic current injection of 200 pA (left), and responded to stimulation of molecular layer (SM) with an EPSP under current clamp (right). Abbreviations are the same as in Fig. 2. Scale bars = 20 μm in A. See magenta-green version of this figure in supplementary files.
Fig. 13
Fig. 13. Morphology and physiology of deep stellate cells. Cells were labeled with fluorescent dye and histology was carried out in resectioned sections (A) or whole slice (B)
A. A deep stellate cell was photographed from horizontal sections of 40 μm. Its small soma (arrow) was located in the upper ganglionic layer, and the fine dendrites and axon arbors are hardly distinguishable. Note that one cell was recorded and injected, and two small cells nearby were also labeled, with minimal processes. B. Another deep stellate cell was labeled and photographed from a transverse slice of 200 μm. Its dendrites were clearly cut and axon arbors were confined to the ganglionic layer (gran). C. Recording from a deep stellate cell (same cell as shown in A) under current clamp shows a rapid adaptation of spikes evoked by somatic current step (top), and minimal paired-pulse facilitation of parallel fiber EPSCs (bottom). Abbreviations are the same as in Fig. 2. Scale bars = 20 μm in A, =50 μm B. See magenta-green version of this figure in supplementary files.
Fig. 14
Fig. 14. Photomicrographs of labelings in slice preparation. Tracer was injected in transverse slice and labelings were revealed with red fluorescent dye after resection. Nissl counterstaining is shown in green
A. Injection site was located in the molecular layer (mol), and all retrogradely labeled granule cells were confined to the granular layer (gran) directly beneath the molecular layer where the injection site was located (arrow). B. Another molecular layer injection that shows all axons of labeled granule cells as combining into a small bundle (arrow) before entering the molecular layer (mol). Note that at least one Purkinje cell (arrowhead) was labeled with cut dendrites in this plane. C. An adjacent section to the one showed in B, showing the same parallel fiber bundle running in the proximal molecular layer (mol), above the ganglionic layer (ggl). Note at least one Purkinje cell (P) with cut flat dendrites and a basal efferent cell (B) with broad dendrites in the basal region of the ridge. D. An enlarged image, showing single parallel fibers with en passant buttons in the molecular layer (mol). E. Retrogradely labeled granule cells, with short dendrites that end with knobs. F. Mossy fibers labeled by injection of tracer into the granular layer (gran). Note the branching of the thick fibers and the large en passant buttons of the thin fibers. Abbreviations are the same as in Fig. 2. Scale bars = 100 μm in A, = 40 μm in B; = 50 μm in C; = 20 μm in D; = 10 μm in E; = 24 μm in F. See magenta-green version of this figure in supplementary files.
Fig. 15
Fig. 15. Photomicrographs of climbing fibers in intact animals. Tracer was injected to the inferior olive and labeling was revealed with red fluorescent dye. Nissl counterstaining is shown in green
A. Transverse view, showing that labeled climbing fibers are confined in the ganglionic layer (ggl). The thick fibers were usually cut and thin fibers have en passant and terminal buttons. Note climbing fibers are only present in one ganglionic layer (left), but not in the other. B. Horizontal view, showing that climbing fibers are confined to the ganglionic layer (ggl). Thick fibers traverse along the ganglionic layer and give off branches, and thin fibers have terminal buttons. C. Transverse view, showing that climbing fibers traverse long distance in the ganglionic layer (ggl) in this plane. Abbreviations are the same as in Fig. 2. Scale bars = 20 μm in A; = 30 μm in B; = 50 μm in C. See magenta-green version of this figure in supplementary files.
Fig. 16
Fig. 16
Summary diagram, showing the cell types and their connections identified in this study. Opened circles represent excitatory terminals, filled circles represent inhibitory terminals, and dashed lines represent presumed connections. Noted that axon of the granule cell projects to the molecular layer without a T bifurcation. Abbreviations: B, basal cell; cf, climbing fiber; dst, deep stellate cell; G, Golgi cell; ggl, ganglionic layer; gr, granule cell; gran, granular layer; L, large central cell; mol, molecular layer; mf, mossy fiber; P, Purkinje cell; pf, parallel fiber.
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